U N I V E R S I T Y O F C O P E N H A G E N U N I V E R S I T Y O F C O P E N H A G E N

PhD thesis

Chad Alton Keyser

Protecting plants against pests and pathogens with

entomopathogenic fungi: The biocontrol agent Metarhizium, its distribution, application, and interaction with other

beneficial fungi

Academic Advisor: Nicolai Vitt Meyling

Submitted: 30 January, 2015

This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen

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Institution: University of Copenhagen, Faculty of Science

Department: Department of Plant and Environmental Sciences (PLEN)

Author: Chad Alton Keyser

Title: Protecting plants against pests and pathogens with entomopathogenic fungi:

the biocontrol agent Metarhizium, its distribution, application, and interaction

with other beneficial fungi.

Academic advisor: Nicolai Vitt Meyling

Co-advisor: Kristian Thorup-Kristensen

Submitted: 30 January 2015

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Acknowledgments

“It is only with the heart that one can see clearly, for the most essential things are invisible to the eye."

― Hans Christian Andersen, The Ugly Duckling

Through this acknowledgment I would like to express my profound appreciation to the many

individuals that have supported and encouraged me during the course of my PhD program. Throughout the

last three years I have grown significantly as a scientist and as a person – this growth is due mainly to the

collaborations, friendships and nurturing influence of the many people I have had the opportunity to interact

with.

I first met Nicolai Meyling in 2009 at a scientific conference in Park City, Utah, as we stood in a

crowded hall waiting for seating to begin. During our short conversation I was impressed by his attentive

interest as I explained what I was working on as a Masters student. As my supervisor, Nicolai has continued

to support and facilitate my efforts and growth. He has patiently steered my ideas in scientifically-relevant

directions and ensured that my project was progressing. I am very grateful to Nicolai for his relaxed manner,

thoughtful comments and suggestions, and the diligence and effort he has put into mentoring me.

The Section for Organismal Biology (SOBI) has assembled an exemplary team of insect

pathologists. It has been a pleasure to work alongside such a cohesive group, to share ideas, experience and

encouragement – my experience here will always serve as a paragon for future collaborations. As the head

of this research group, I am grateful to Jørgen Eilenberg for his infectious enthusiasm, his example of

efficiency and his effort to make me feel part of the team. Also, I am very grateful to Bernhardt Steinwender

for both his friendship and constant willingness to listen and discuss even the most ridiculous of ideas. I am

also thankful to Henrik de Fine Licht for his patients in instructing me in art of AFLP analysis. The Team

consists of many more PhD fellows, post-docs and professors who have each individually encouraged,

inspired, taught and helped me in many ways and for which I am truly appreciative.

It has been a great pleasure to work alongside many skilled technicians and assistants. I am

especially grateful to Louise Munk Larsen for her ample technical skills and continued willingness to drop

what she is doing to help. I thank Sylvia Mathiasen and Vinnie Deichmann for their assistance with my

molecular work. I am also grateful to Line Lykke, Lærke Thordsen, Martina Falagiarda, Jesper Anderson,

Azmi Mahmood, Darren Thomsen and the many other student helpers that have assisted me in many aspects

of my experiments – I would not have been able to accomplish what I have needed to do without their

diligent effort and skills.

I have also been fortunate to have Kristian Thorup-Kristensen as a co-advisor. I am thankful to him

for sharing his expertise in working with plants and in experimental design, as well as including me as part

of his team. I also thank Birgit Jensen for her assistance in working with Fusarium and Clonostachys and her

interest and willingness to train and assist me throughout the study for the second Manuscript.

Living in Denmark and attending the University of Copenhagen has been the experience of a

lifetime. I am thankful to Plant Biosystems Elite Environment at the University of Copenhagen for funding

my PhD research. I am also grateful to the Department of Plant and Environmental Sciences and the Section

for Organismal Biology for hosting and providing necessary facilities for my studies.

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I am also supremely grateful for the support and encouragement I have received from my families. I

recognize that living overseas has been difficult for both my family and my wife’s family; I sincerely

appreciate their willingness to accept my decision to pursue this degree and the support and love they have

shown which has been an enabling power to finish. I also appreciate the family and friends that have found

the opportunity to visit us and share in this experience – their refreshing visits have made the distance more

bearable.

Most importantly, I would like to express sincere gratitude to my beautiful wife Shannon and three

energetic children: Myra, Alexys and Noah. They willingly left behind their friends, families, job and

comforts to follow me on an unknown path. My children have worked hard, learned the difficult language

and adjusted quickly to the Danish lifestyle – their adaptability and bravery has given me strength. Shannon

has also thrived and grown to love European living – her infectious eagerness to explore and discover the

world around us has lifted and strengthened our whole family. I could not have succeeded in completing this

program if not for her. Having my best friend by my side and knowing that she supports my ambitions has

made this experience truly enjoyable. It is to my wife and children I dedicate this work and my life.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS ....................................................................................................................................... 3

I. LIST OF INCLUDED MANUSCRIPTS ........................................................................................................... 6

II. SUMMARY .................................................................................................................................................. 7

III. DANSK RESUMÈ ......................................................................................................................................... 9

IV. THESIS OBJECTIVES ................................................................................................................................ 11

1. INTRODUCTION ........................................................................................................................................... 12

2. THE ENTOMOPATHOGENIC FUNGAL GENUS METARHIZIUM ................................................................... 13

2.1 Phylogeny and Taxonomy ..................................................................................................................... 15

2.2 Ecology .................................................................................................................................................. 18

2.2.1 Abundance and distribution ............................................................................................................ 18

2.2.2. Abiotic factors that affect survival and growth ............................................................................. 21

2.2.3 Environmental dissemination pathways ......................................................................................... 22

3. TROPHIC INTERACTION .............................................................................................................................. 23

3.1 Metarhizium ↔ Insects .......................................................................................................................... 24

3.2 Metarhizium ↔ Plants ........................................................................................................................... 26

3.3 Metarhizium ↔ Other microorganisms ................................................................................................. 29

3.4 Multi-trophic interactions with Metarhizium ........................................................................................ 31

3.4.1 Metarhizium ↔ Other microorganisms ↔ Insects ......................................................................... 31

3.4.2 Metarhizium ↔ Other microorganisms ↔ Plants .......................................................................... 32

3.4.3 Metarhizium ↔ Plants ↔ Insects ................................................................................................... 32

3.4.4 Metarhizium ↔ Other microorganisms ↔ Plants ↔ Insects ......................................................... 33

4. METHODOLOGY ......................................................................................................................................... 34

4.1 Selective media ...................................................................................................................................... 34

4.2 Bioassay statistics .................................................................................................................................. 37

4.2.1 Experimental design ....................................................................................................................... 37

4.2.2 Types of Statistical Analyses .......................................................................................................... 38

V. CONCLUSION AND FUTURE PERSPECTIVES .............................................................................................. 42

VI. REFERENCES ............................................................................................................................................ 44

VII. APPENDIX ................................................................................................................................................ 52

Manuscript 1 ................................................................................................................................................ 52

Manuscript 2 ................................................................................................................................................ 63

Manuscript 3 ................................................................................................................................................ 90

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i. List of included Manuscripts

Manuscript 1

METARHIZIUM SEED TREATMENT MEDIATES FUNGAL DISPERSAL VIA ROOTS AND INDUCES

INFECTIONS IN INSECTS

Chad A. Keyser, Kristian Thorup-Kristensen, & Nicolai V. Meyling

Status: Published in Fungal Ecology, October 2014, Vol. 11, pg. 122-131

License Number: 3531750730534

Manuscript 2

BEST OF BOTH WORLDS: DUAL EFFECTS OF METARHIZIUM SPP. AND CLONOSTACHYS ROSEA

AGAINST AN INSECT AND A SEED-BORNE PATHOGEN IN WHEAT

Chad A. Keyser, Birgit Jensen & Nicolai V. Meyling

Status: Under Review - Pest Management Science, submitted 22 Dec, 2014

Manuscript 3

DIVERSITY OF METARHIZIUM FLAVOVIRIDE POPULATIONS ASSOCIATED WITH ROOTS OF CROPS IN

DENMARK

Chad A. Keyser, Henrik H. de Fine Licht, Bernhardt M. Steinwender & Nicolai V. Meyling

Status: Manuscript

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ii. Summary

Background: Insect-pest management is an increasingly important area of research. Efforts

to maximize agricultural output are significantly dependent on reliable means for pest suppression.

Biological control, or the use of living organisms to suppress a pest population, is among one of the

leading alternatives to traditional chemical-based pesticides for crop protection. For the past 130

years several isolates of the fungal genus Metarhizium has been lead candidates among potential

fungal-based biological control agents (BCAs) for insect pest control in agriculture. However, the

majority of Metarhizium research has emphasized product development and application, largely

neglecting the ecological and fundamental aspects. Inconsistent field reliability and economic

viability have limited wider implementation of many BCAs, including Metarhizium-based products;

an increased understanding of the fundamental ecology and environmental interactions has

substantial potential to improve biological control efforts.

The overall aim of this thesis was to improve the current understanding of how members of

the fungal genus Metarhizium naturally occur in association with roots of crops in Denmark and

interact with other organisms in relation to plant roots when applied as BCAs. Several scientific

studies were conducted to answer important ecological questions regarding Metarhizium spp.

interactions and their use as BCAs. The results of these studies are presented in three manuscripts.

Manuscript 1: Recent research has revealed that many Metarhizium spp. interact with plants

in the rhizosphere and have been shown to increase nutrient uptake and promote plant growth. In

Manuscript 1 we investigate how might the fungus benefit from a plant association; namely,

whether the plant provides a means of dispersal for the otherwise immobile fungus; as well as if the

fungus maintains pathogenicity to insects while interacting with the plant. We found that when

Metarhizium spp. were inoculated as conidia on wheat seeds they were able to disperse through the

soil with the growing root and be re-isolated from lower portions of the root. Furthermore we

observed that when washed roots were placed with Tenebrio molitor larvae, the larvae would

succumb to Metarhizium spp. infection.

Manuscript 2: Agricultural yields are threatened by multiple pests including insects and

plant pathogens. Often the control of these pests requires the application of multiple biological

control agents. In Manuscript 2 we investigate whether the mycoparasite Clonostachys rosea,

commonly used to control plant-fungal pathogens, can be applied jointly with Metarhizium spp. to

control both a plant pathogen and an insect pest. In this study we observed that C. rosea was highly

efficacious at controlling Fusarium culmorum alone and in combination with Metarhizium – when

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applied as a conidial seed treatment to wheat seeds. Additionally, we observed that while a

significant level of T. molitor were infected with Metarhizium spp. after a combined treatment,

there was a slight reduction in virulence when either C. rosea or F. culmorum were also present

when compared to Metarhizium spp. only seed treatments. Based on the result of the direct

inoculation bioassay of T. molitor larvae in which we did not observe a reduction in virulence when

comparing combination treatments to individual treatments, we suspect that the virulence reduction

is the result of resource competition on the growing root and not direct mycoparasitism.

Manuscript 3: An awareness of the composition and distribution of naturally occurring

Metarhizium spp. communities is important to understanding their role to insect host regulation.

However there is an acute lack of ecological studies that assess the occurrence and community

structure of entomopathogenic fungi. The objective of Manuscript 3 was to evaluate the

occurrence, diversity and community structure of Metarhizium spp. isolates obtained from different

crops at geographically separated agricultural fields in Denmark. Root and root-associated soil was

sampled from wheat, oilseed rape, and bordering uncultivated grass fields at three different

locations; 132 new Metarhizium isolates were obtained. Morphological data and sequencing of the

rDNA intergenic spacer region (IGS) revealed that 118 of the isolates belonged to Metarhizium

flavoviride, 13 M. brunneum and one M. majus. We then further characterized the intraspecific

variability within M. flavoviride by unspecific markers (i.e., AFLP identification) to evaluate

diversity and potential crop and/or area associations. We found there was a high level of diversity

among the M. flavoviride isolates indicating that the isolates were not of the same clonal origin,

however due to insufficient loci in the AFLP analysis we were not able to determine haplotype

groupings or confirm any habitat associations. We suggest that the development of more specific

markers would greatly improve our ability to evaluate M. flavoviride diversity. This represents the

first time that an in-depth analysis of the molecular diversity within a large isolate collection of the

species M. flavoviride has been reported.

Overall the scientific studies presented in this thesis are both important and novel to the field

of Metarhizium research; these studies advance the current knowledge of the ecological significance

of Metarhizium spp. as a naturally occurring microorganism and increase our understanding of their

interactions as biological control agents with other organisms. Furthermore, this thesis presents the

background literature and motivation for the research and their implication to the field of insect

pathology.

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iii. Dansk resumè

Baggrund: Bekæmpelse af skadedyr er et vigtigt forskningsområde. Mulighederne for at øge

landbrugsudbyttet er afhængigt af pålidelige bekæmpelsesmetoder. Biologisk bekæmpelse, brugen

af levende organismer til at begrænse skadedyrspopulationer, er en af de væsentligste alternativer til

kemisk baseret plantebeskyttelse. I løbet af de seneste 130 år har flere isolater af den insektpatogene

svampeslægt Metarhizium blevet førende kandidater til svampebaseret biologisk bekæmpelse af

skadedyr. Dog har hovedparten af forskning i Metarhizium fokuseret på produktudvikling og

udbringning, hvor forståelse af grundlæggende økologiske aspekter har blevet negligeret.

Varierende pålidelighed har begrænset implementeringen af mange biologiske

bekæmpelsesprodukter, inklusiv flere baseret på Metarhizium. En øget forståelse af de

fundamentale økologiske og miljømæssige interaktioner, hvori Metarhizium indgår, har stort

potentiale til at forbedre brugen af biologisk bekæmpelse.

Det overordnede formål med denne afhandling var at øge forståelsen af hvordan medlemmer

af svampslægten Metarhizium indgår i naturlig associering med rødder af afgrøder i Danmark og

hvordan de interagerer med andre organismer i relation til planterødder når de tilføres ved biologisk

bekæmpelse. Flere videnskabelige studier blev gennemført for at svare på dette, og resultaterne er

præsenteret i tre manuskripter.

Manuskript 1: Det er for nyligt blevet videnskabeligt vist at flere arter af Metarhizium

interagerer med planter i rhizosfæren og at dette kan øge næringsstofoptag og vækst hos planten. I

Manuskript 1 undersøges det om svampen kan have fordel af denne interaktion ved at bruge

plantens rødder til transport i jorden, og om svampen bevarer sin evne til at inficere insekter ved at

interagere med planten. Det blev fundet, at når Metarhizium spp. blev inokuleret som konidier på

frø af hvede, kunne svampen sprede sig gennem jord med de voksende rødder og blive genisoleret

fra den nedre del af rødderne. Desuden var vaskede rødder infektive mod larver af melorme

Tenebrio molitor, som efter inkubering sammen med rødder døde af Metarhizium infektion.

Manuskript 2: Afgrøder bliver ofte angrebet af flere organismer, primært insekter og

plantepatogener, hvilket kræver samtidig brug af flere biologiske bekæmpelsesmidler. I Manuskript

2 blev det undersøgt, om nyttesvampen Clonostachys rosea, som normalt bruges til at bekæmpe

plantepatogener, kan blive anvendt sammen med Metarhizium spp. til at bekæmpe både insekter og

plantesygdomme på samme tid. Det blev observeret at C. rosea var meget effektiv til at bekæmpe

Fusarium culmorum alene og sammen med Metarhizium spp., når de to typer af svampe blev tilført

som konidier på frø af hvede. Desuden blev larver af melorme inficeret af Metarhizium spp. både

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når denne svamp blev tilført alene og sammen med C. rosea eller F. culmorum, men infektionen var

en smule nedsat i kombinationerne. Ved direkte inokulering af larverne med sammen

svampekombinationer sås ikke denne reduktion. Reduktionen observeret ved planteinokuleringen

kunne derfor skyldes en indirekte interaktion på planteroden og ikke direkte interaktion som

mykoparasitisme mellem svampene.

Manuskript 3: Det er afgørende af kende til den naturlige forekomst og udbredelse af

Metarhizium spp. for at forstå deres bidrag til regulering af insektpopulationer. Der findes dog

fortsat kun få studier af den naturlige forekomst af insektpatogene svampe. Formålet med studiet

præsenteret i det tredje manuskript var at beskrive forekomst, udbredelse og diversitet af

Metarhizium spp. i forbindelse med rødder af forskellige afgrøder i Danmark på tre geografisk

afgrænsede lokaliteter. Rødder og rod-associeret jord blev indsamlet på hver lokalitet fra

vinterhvede, raps, og fra et uforstyrret græsningsareal. 132 Metarhizium isolater blev indsamlet.

Morfologisk og DNA sekventering viste, at 118 af isolaterne tilhørte arten Metarhizium flavoviride,

13 var M. brunneum, og et var M. majus. Ved yderligere karakterisering af intraspecifik variation i

M. flavoviride med uspecifikke markører (AFLP) blev diversitet og potentiel lokalitet- eller

afgrødespecificitet undersøgt. En høj grad af diversitet blev fundet med de anvendte markører,

hvilket indikerer at de ikke alle har samme klonale ophav, men metoden var ikke tilstrækkelig til at

identificere lokalitet- eller afgrødespecificitet. Specifikke markører udviklet til M. flavoviride vil

være nødvendige for at undersøge dette. Dette studie er det første hvor arten M. flavoviride blev

undersøgt mht. molekylær diversitet ved brug af en større samling af isolater.

De videnskabelige studier præsenteret i denne afhandling er både vigtige og nye indenfor

forskningen i Metarhizium. Studierne øger den nuværende viden om økologisk betydning af

Metarhizium spp. som naturlig forekommende mikroorganisme samt ved interaktioner med planter

og med andre mikroorganismer. Desuden giver denne afhandling en gennemgang af litteraturen om

emnet og betydningen af den præsenterede forskning for insektpatologi.

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iv. Thesis Objectives

The aim of this thesis is to advance our understanding of how members of the entomopathogenic

fungal genus Metarhizium interact with other organisms, with an emphasis placed on its role as a

biological control agent. To accomplish this goal the following research questions were

investigated:

■ Can plant roots act as a vehicle to disseminate Metarhizium in soil? Additionally, while

associating with plants, do Metarhizium spp. maintain their pathogenicity to insects?

■ In a seed-treatment biocontrol context, what interactions occur between other organisms,

namely: the mycoparasitic fungus Clonostachys rosea, and the plant pathogenic fungus

Fusarium culmorum?

■ What is the prominence and species composition of naturally occurring Metarhizium in roots

and soil within different agro-ecosystems in Sjælland, Denmark?

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1. Introduction

Agricultural production is incalculably important to human existence both from an

economical as well as a nutrient-resources perspective. Worldwide estimates suggest that currently

38% of the Earth’s terrestrial surface is dedicated to agricultural endeavors (Foley et al., 2011).

Furthermore, it has been projected that from 2005 to 2050 global crop production will need to

increase as much as 110% to meet the caloric and protein demands of the world population (Tilman

et al., 2011). We cannot expect to meet the growing agricultural needs by merely dedicating more

land to food production; increasing the productivity of the current agricultural systems is vital to our

future (Tilman et al., 2011). A significant limiting factor to agriculture yields are pests – this

includes herbivores (e.g., arthropods), weeds and plant pathogens. Over the last century advances

in chemical pesticide development have greatly mitigated pest related crop losses. However, due to

pesticide resistance and an increased awareness of deleterious effects on non-target organisms,

including humans, reliable alternative methods for controlling pests are desperately needed.

Biological control is an important alternative which has potential to effectively control pest

populations with limited risk (Hajek, 2004).

Biological control can be defined as “the use of living organisms to suppress the population

of a specific pest organism, making it less abundant or less damaging then it would otherwise be”

(Eilenberg et al., 2001). There are several types of organisms that have been identified as biological

Glossary of Terms

Agroecosystem Agricultural ecosystem - Specialized ecosystem which has been manipulated by human activities with the aim to produce high levels of organic output. Includes living and non-living components and their interactions.

Anamorph The asexual (conidial or imperfect) stage in the life history of a fungus.

BCA Biological control agent – the organism used for Biological control

Bioassay The measurement of the potency of any BCA, by means of the response which it produces in a living host.

Biological control The use of living organisms to control (usually meaning to suppress) undesirable animals and plants.

Entomopathogen A microbe affecting insects (or in a more general sense, other terrestrial arthropods including arachnids), usually causing mortality in the host (as opposed to a more benign relationship).

Fungal endophyte An asymptomatic plant-colonizing fungus that lives a portion of its life cycle inside the plant.

Pathogenicity The quality or state of being pathogenic. The potential ability to produce disease. Pathogenicity is qualitative, an all-or-none concept

Rhizosphere The narrow region around the plant root that is directly influenced by root secretions and associated soil microorganisms.

Teleomorph The sexual stage in the life history of a fungus.

Virulence The disease-producing power of a microorganism. Virulence can be quantified.

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control agents (BCAs), including: predators, parasitoids, parasitic nematodes, bacteria, viruses,

fungi and microsporidia (Hajek, 2004), the latter group is now recognized and a basal fungal group

(James et al., 2006). Each BCA has different characteristics which determine their effectiveness in

a particular circumstance. Understanding the environmental factors that contribute to effectiveness

will largely determine the success of a BCA. For many years research regarding the fundamental

ecology of these organisms has had far less priority than product development; this has likely led to

inconsistent results in the field (Vega, 2008). The present thesis contributes in part, to increasing

our understanding of the complex interactions involved between a particular fungal BCA and other

important organisms in its environment, within a biological control context.

2. The entomopathogenic fungal genus Metarhizium

Entomopathogenic fungi are fungal organisms that have the ability to infect and cause

disease in an arthropod. The kingdom Fungi is estimated to contain more than 5.1 million species

(O'Brien et al., 2005), of those, 750-1000 species are pathogenic to insects (Vega et al., 2012).

Fungi of the Ascomycota genus Metarhizium (Hypocreales: Clavicipitaceae) are among one

of the most studied groups of entomopathogenic fungi, in fact in the last decade the number of peer-

reviewed publications has increased substantially (Figure 1). Metarhizium spp. are ubiquitously

found in terrestrial ecosystems worldwide, having been isolated from every continent except

Antarctica (Roberts and St. Leger, 2004). Many species of Metarhizium have a wide host range,

like M. brunneum and M. robertsii, infecting at least seven insect orders (Veen, 1968;

Zimmermann, 1993); however a few species are known to be host specific like M. acridum which

only infects some taxa in the order Orthoptera (Driver et al., 2000; Wang et al., 2011). In addition

to their host range there are several other key attributes that make Metarhizium spp. ideal candidates

for BCAs, including: the infection process is topical (does not need to be ingested by host),

infectious conidia can be mass produced on artificial media, remain viable and can be easily

0

300

600

900

1200

1500

1975-1979 1980-1984 1985-1989 1990-1994 1995-1999 2000-2004 2005-2009 2010-2014

Nu

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er

of

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blicati

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s

Figure 1. The number of peer-reviewed publications published of 5 year periods for the last 40 years according to a search on web of science with the key word “Metarhizium”.

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disseminated, and many species of Metarhizium produce secondary metabolites known as

destruxins which are toxic to invertebrates and help the pathogen overcome the host immunity

quickly (Dorta et al., 1996; Zimmermann, 2007).

The infection process of Metarhizium spp. to a suitable host was reviewed by Zimmermann

(2007) and is summarized in Figure 2. He describes the process in 6 steps including: 1. Attachment,

in which the conidia adheres to the cuticle using a combination of hydrophobic interaction and

specialized adhesion proteins; 2. Germination and appressoria formation; 3. Penetration through the

cuticle, which is mechanical but aided by the production of enzymes including proteases, chitinases

and lipases; 4. Overcoming host defenses, often by the production of novel destruxins; 5.

Proliferation within the host, generally via the production of blastospores or hyphae; and lastly, 6.

Outgrowth and production of new infective conidia.

Figure 2: The infection process, an illustration showing the 6 stages of fungal infection in an insect: 1. Attachment; 2. Germination and appressoria formation; 3. Penetration through the cuticle; 4. Overcoming host defenses; 5. Proliferation within the host; 6. Outgrowth and production of new infective conidia. Illustration by C. A. Keyser.

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2.1 Phylogeny and Taxonomy

The taxonomy of the Genus Metarhizium (Family: Clavicipitaceae, Order: Hypocreales,

Class: Sordariomycetes Phylum: Ascomycota, Kingdom: Fungi) has been revised many times over

the years. In 1883 Russian mycologist Sorokin first introduced the name Metarhizium as the genus

name for fungi that are the causal agent of insect disease “green muscardine”, which had originally

been called Entomophthora anisopliae by Elia Metchnikoff in 1879 (Zimmermann et al., 1995).

For the last 130 years the genus name Metarhizium has persisted, however many of the species

names belonging to this genus have changed.

Many fungi are pleomorphic, i.e., they have different life stages which are morphologically

distinct – often the different stages have been identified as different organisms. To help standardize

the descriptions the term teleomorph is used to describe the sexual stage, anamorph to describe the

asexual stage, and holomorph when both are present (Hennebert and Weresub, 1977). Species of

the genus Metarhizium have historically been considered anamorphic fungi (i.e., only exhibiting the

asexual stage and reproduce clonally by mitosporic conidia production) and placed in the former-

division Deuteromycota; however the discovery of a sexual and asexual stage (Liang et al., 1991),

as well as molecular analysis have facilitated their placement in the phylum Ascomycota (Kepler et

al., 2014). In this thesis only the anamorphic stage of Metarhizium is discussed.

Another challenge for Metarhizium taxonomy is that many species of Metarhizium are

morphologically similar, so identification based on morphological attributes is difficult. Tulloch

(1976) made a major revision of the genus Metarhizium based on drawings and the morphological

descriptions available, she reduced all known taxa to two speices, M. flavoviride and M. anisopliae.

The next major revision of the genus was performed by Driver et al. (2000) using ITS sequence data

for phylogenetic analyses; they observed a high level of genetic diversity and were able to identify

ten distinct clades, however they restricted their descriptions to varieties rather than species due to

limited resolution and support in the sequence analysis. Bischoff et al. (2009) used a multigene

phylogenetic approach to resolve the M. anisopliae group which at the time consisted of 4 varieties

as defined by Driver et al. (2000); i.e., M. anisopliae var. acridum, M. anisopliae var. anisopliae,

M. anisopliae var. lepidiotae, and M. anisopliae var. majus. They describe ten species within the

M. anisopliae complex (viz., M. anisopliae, M. acridum, M. brunneum, M. globosum, M.

guizhouense, M. lepidiotae, M. majus, M. pingshaense, and M. robertsii) (Bischoff et al., 2009).

The taxonomic clarification of the Metarhizium genus was continued by Kepler et al. (2014) and the

M. flavoviride complex was resolved into four species (viz., M. flavoviride, M. koreanum, M.

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minus, and M. pemphigi). Additionally, in this revision an effort was made to reevaluate fungal

nomenclature with the intention to give one name to one fungus, regardless of life stage (Taylor,

2011). In doing this Kepler et al. (2014) transferred many taxa from Chamaeleomyces,

Metacordyceps and Nomuraea to Metarhizium. The genus Metarhizium now includes 36 species

(Table 1). Isolates described as “M. anisopliae” or “M. flavoviride” in studies published prior to

(and sometimes after) the 2009 and 2014 taxonomic revisions are now designated as “M. anisopliae

sensu lato” (s.l.) or “M. flavoviride s.l.” if the new taxonomic identity according to Bischoff et al.

(2009) or Kepler et al. (2014) is unknown or not specified, M. anisopliae and M. flavoviride

identified using the current taxonomic criteria are labeled using the sensu stricto (s.s.) designation.

As noted by Steinwender (2013) “taxonomy is not set in stone but rather a snapshot of a given

moment”, it is likely the genus Metarhizium will continue to develop and change as our

understanding of the complexity of life improves.

17

Table 1: Current Species of the Genus Metarhizium, the authorities and year of description – arranged alphabetically.

Species name Authorities, year of description

M. acridum (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber, 2009

M. album Petch, 1931

M. anisopliae (Metsch.) Sorokin, 1883

M. atrovirens (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014

M. brasiliense Kepler, S.A. Rehner & Humber, 2014

M. brittlebankisoides (Zuo Y. Liu, Z.Q. Liang, Whalley, Y.J. Yao & A.Y. Liu) Kepler, S.A. Rehner & Humber, 2014

M. brunneum Petch, 1935

M. campsosterni (W.M. Zhang & T.H. Li) Kepler, S.A. Rehner & Humber, 2014

M. carneum (Duché & R. Heim) Kepler, S.A. Rehner & Humber, 2014

M. flavoviride W. Gams & Rozypal, 1973

M. frigidum (Driver & Milner) J.F. Bisch. & S.A. Rehner, 2006

M. globosum J.F. Bisch., S.A. Rehner & Humber, 2009

M. granulomatis (Sigler) Kepler, S.A. Rehner & Humber, 2014

M. guizhouense Q.T. Chen & H.L. Guo, 1986

M. guniujiangense (C.R. Li, B. Huang, M.Z. Fan & Z.Z. Li) Kepler, S.A. Rehner & Humber, 2014

M. indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014

M. khaoyaiense (Hywel-Jones) Kepler, S.A. Rehner & Humber, 2014

M. koreanum Kepler, S.A. Rehner & Humber, 2014

M. kusanagiense (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014

M. lepidiotae (Driver & Milner) J.F. Bisch., S.A. Rehner & Humber, 2009

M. majus (J.R. Johnst.) J.F. Bisch., S.A. Rehner & Humber, 2009

M. marquandii (Massee) Kepler, S.A. Rehner & Humber, 2014

M. martiale (Speg.) Kepler, S.A. Rehner & Humber, 2014

M. minus (Rombach, Humber & D.W. Roberts) Kepler, S.A. Rehner & Humber, 2014

M. novozealandicum Kepler, S.A. Rehner & Humber, 2014

M. owariense (Kobayasi) Kepler, S.A. Rehner & Humber, 2014

M. owariense f. viridescens (Uchiy. & Udagawa) Kepler, S.A. Rehner & Humber, 2014

M. pemphigi (Driver & R.J. Milner) Kepler, S.A. Rehner & Humber, 2014

M. pingshaense Q.T. Chen & H.L. Guo, 1986

M. pseudoatrovirens (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, 2014

M. taii Z.Q. Liang & A.Y. Liu, 1991

M. rileyi (Farl.) Kepler, S.A. Rehner & Humber, 2014

M. robertsii J.F. Bisch., S.A. Rehner & Humber, 2009

M. viride (Segretain, Fromentin, Destombes, Brygoo & Dodin ex Samson) Kepler, S.A. Rehner & Humber, 2014

M. viridulum (Tzean, L.S. Hsieh, J.L. Chen & W.J. Wu) B. Huang & Z.Z. Li, 2004

M. yongmunense (G.H. Sung, J.M. Sung & Spatafora) Kepler, S.A. Rehner & Humber, 2014

18

2.2 Ecology

Despite having more than 100 years’ worth of research interest, we are only beginning to

understand the ecology of Metarhizium spp. and the important role they play in the ecosystem.

There are two main reasons why the natural ecology of Metarhizium is important to biological

control: first, as a ubiquitous organism infectious to insects, understanding the natural occurrence

and distribution, and the contributions of Metarhizium in regulating insect populations is highly

relevant; and second, understanding how Metarhizium interacts with other organisms and is affected

by abiotic factors will help optimize how to most effectively use them as BCAs.

Bruck (2010) pointed out that in plant pathology a concept known as the “disease triangle”

is often used to describe the interaction between a host, a pathogen, and the environment; he

suggested that this same concept should also be applied to biological control. By emphasizing a

total ecological approach to Metarhizium spp. research, which focuses on both the direct and

indirect effects of biotic and abiotic factors in the environment, we gain greater clarity of the role

Metarhizium spp. play in the ecosystem. In this section I will discuss with regard to Metarhizium:

2.2.1. Abundance and distribution; 2.2.2. Abiotic factors that affect survival and growth; and 2.2.3.

Environmental dissemination pathways. In section 3 (below) I will discuss biotic interaction that

occur between Metarhizium and other organisms.

2.2.1 Abundance and distribution

The United States Department of Agriculture’s (USDA) Agricultural Research Service

Collection of Entomopathogenic Fungal Cultures (ARSEF), Ithaca, NY, hosts one of the largest

libraries of entomopathogenic fungal isolates collected from all over the world. This collection has

over 1500 different isolates of Metarhizium, however most of these isolates, as well as the many

others that have been collected over the years, were collected not with the intention to understand

abundance and distribution but rather to find new potential products for commercialization:

examples include the LUBILOSA project (Roberts and St. Leger, 2004), in which researchers

scoured the African and Australian continents searching for entomopathogenic fungi with the intent

to develop a BCA for locust control. More recently, an 8 year USDA-APHIS project which

surveyed 30,000 soil samples in the western US and collected more than 2,000 new isolates of

Metarhizium with a goal to find M. acridum in USA soil (C. A. Keyser, unpublished data). While

these “goal-orientated” types of surveys often produce useful information about the ecology and

distribution of Metarhizium in nature; they are not designed as ecological studies and so many of

the conclusions they can provide are incomplete.

19

There have, however, been several studies intended to investigate Metarhizium occurrence

in both natural habitats and agroecosystems (Table 2); it is noteworthy that there are a lack of

studies from latitudes below 40º (e.g., tropical regions). Many aspects differ between these studies

(e.g., sampling and isolation method) which makes direct comparison difficult; however, when

viewed as a group we are able to make some generalizations about the abundance and distribution

of Metarhizium spp. in different environments, several of which I would like to briefly outline:

First, Metarhizium spp. tend to be less abundant in colder regions than in temperate regions (Inglis

et al., 2008; Klingen et al., 2002; Vanninen, 1995), this observation is also supported by laboratory

work which has demonstrated that most species of Metarhizium , with the exception of M. frigidum,

do not grow at cold temperatures (Fernandes et al., 2010a; Fernandes et al., 2008). Second,

Metarhizium is more abundant than other entomopathogenic fungi in cultivated fields and open

meadows (Bidochka et al., 1998; Quesada-Moraga et al., 2007; Sun et al., 2008; Vanninen, 1995).

Third, Metarhizium is primarily found in the soil environment and not on above ground substrates

(Meyling et al., 2011; Vega et al., 2012). Lastly, Metarhizium spp. distribution tends to associate

with habitats and not with host insects (Bidochka et al., 2001; Fisher et al., 2011; Wyrebek et al.,

2011). Traditionally, insect association was thought to be the key factor in determining population

structure (Bridge et al., 1997; St. Leger et al., 1992), this shift away from the traditional paradigm

has led to many questions about what role these fungi truly play in the environment (Bidochka et

al., 2001; Vega, 2008).

Another interesting observation we can glean from viewing these studies together is that

Metarhizium spp. composition and dominance appears to be location specific. For example, a study

in the mid-east part of Canada found M. robertsii to be the most common species of Metarhizium

(Wyrebek et al., 2011), while a study in the western part of Canada found M. anisopliae s.l. to be

most common (Inglis et al., 2008). Even in Denmark survey studies have yielded inconsistent

species compositions. In 1995 Steenberg reported that M. anisopliae s.l. dominated cultivated soils

(Vega et al., 2012), also Steinwender et al. (2014) primarily found M. brunneum (a species within

the M. anisopliae complex) to be most often isolated from agricultural soil in Denmark. However,

Meyling and Eilenberg (2006) found that M. anisopliae s.l. was almost absent in a single organic

agroecosystem sampled. Also, as reported in Manuscript 3, we found that in three separate

agricultural areas, M. flavoviride was the most frequently isolated species. While further sampling

is needed to confirm these observations, it is clear that Metarhizium spp. occurrence is neither

random nor ubiquitous.

20

Table 2: Metarhizium abundance and distribution studies.

Country

(latitude) Reference

Isolation

method

Samples

taken

Habitat

types

Metarhizium

isolates

obtained General Metarhizium results

Finland

(62º N)

(Vanninen,

1995)

Insect soil

baiting

590 soil

samples

from 347

sites

Forests,

agricultural

fields

92 isolates Found in lower latitudes, not affected by

cultivation

Canada

(45 ºN)

(Bidochka et

al., 2001,

Bidochka et al.,

1998)

Insect soil

baiting

266 soil

samples

from 133

sites

Natural

(forests) and

agricultural

357 isolates Most abundant entomopathogen isolated,

most frequently recovered from soil baiting at

25ºC, more often in Agricultural soil.

Genotype association with habitat, no

association with insect host.

Norway

(66ºN)

(Klingen et al.,

2002)

Insect soil

baiting

200 samples Conventional

and organic

farms

9 isolates Not commonly found. No difference between

fields and field margins.

Denmark

(55 ºN)

(Meyling and

Eilenberg,

2006)

Insect Soil

baiting

544 soil

samples over

two years

experimental

research farm

and hedgerow

134 isolates Surprisingly low occurrence of M. anisopliae

s.l.

Spain

(40ºN)

(Quesada-

Moraga et al.,

2007)

Insect soil

baiting

244 samples Natural and

cultivated areas

71 isolates Most common in cultivated areas. Preferred

soil with low clay content.

Canada

(45 ºN)

(Inglis et al.,

2008)

Insect soil

baiting and

selective media

250 soil

samples

Urban,

agricultural and

forest

250 isolates Metarhizium very widespread and diverse

however one genotype dominated.

China

(40ºN)

(Sun et al.,

2008)

Insect soil

Baiting

>2300 soil

samples

cultivated fields

and orchards

60% of soil

samples

More frequent in cultivated fields than

orchards.

South

Africa

(33ºS)

(Goble et al.,

2010)

Insect soil

baiting

288 soil

samples

Conventional

and organic

citrus farms

16 isolates No difference between organic and

conventional.

USA

(44 ºN)

(Fisher et al.,

2011)

Insect baiting

with roots

339 root

samples

Strawberries,

blueberry,

grapes, and

Christmas trees

(roots)

94 isolates Species/ plant-root association; M. brunneum

= strawberries and blueberries, M.

guizhouense = Christmas trees, and M.

robertsii = Christmas trees

Canada

(45 ºN)

(Wyrebek et al.,

2011)

Washed root

homogenate on

selective media

200 root

samples

Natural

meadows, and

forests (roots)

102 isolates Species/ plant-root association; M. brunneum

= shrubs and trees, M. guizhouense = tree

roots, and M. robertsii = grasses and

wildflowers

Denmark

(55 ºN)

(Steinwender et

al., 2014)

Insect soil

baiting

53 soil

samples

Agricultural

field and

boarding

hedgerow

123+ isolates Five genotypes of M. brunneum , 6

genotypes of M. robertsii. One M. brunneum

genotype most dominant.

Denmark

(55 ºN)

Keyser. et al.

(2015) –

Manuscript 3

Insect soil

baiting and

selective media

450 soil and

root samples

Winter wheat,

Oilseed rape

and natural

meadow

132 isolates 118 of the isolates were M. flavoviride, 13 M.

brunneum and 1 M. majus. AFLP analysis

revealed high level of diversity with the M.

flavoviride species

21

2.2.2. Abiotic factors that affect survival and growth

Three of the most important abiotic factors that affect entomopathogenic fungal

performance are: temperature, humidity, and UV-radiation. Temperature has been shown to affect

Metarhizium spp. germination, hyphal growth and infection rates (Keyser, 2010; Keyser et al.,

2014a). Growth at high and low temperatures has even been used as phenotypic traits to distinguish

certain species of Metarhizium (Fernandes et al., 2010a). Fluctuating ambient temperatures have

also been indicted as one of the primary limiting factors in field success (Foster et al., 2010; Foster

et al., 2011). Likewise, relative humidity is an important factor in determining growth, infection,

sporulation and conidial longevity (Arthurs and Thomas, 2001; Daoust and Roberts, 1983; Milner et

al., 1997; Vega et al., 2012). UV radiation, especially UV-B radiation can be highly detrimental to

conidia survival (Braga et al., 2001; Rangel et al., 2005) and even sub-lethal UV-B radiation

exposure was observed to delay conidial germination (C.A. Keyser, unpublished data). These

factors are also highly relevant in understanding the natural distribution and abundance in the field.

Some species of Metarhizium have been shown to be less cold tolerant then Beauveria spp.

(Fernandes et al., 2008), which may explain why Vanninen (1995) found Metarhizium primarily in

the southern Finland areas while Beauveria was isolated all over Finland. In one area of southern

Alberta, Canada, Inglis et al. (2008) did not find any Metarhizium isolates, they suggested that a

possible explanation for the lack of isolates is that the area surveyed was semi-arid, had very cold

winters and short summers. Bidochka et al. (2001) also observed that isolates found in forested

areas were more likely to grow at low temperatures while those found in open fields showed a

propensity for growth at higher temperatures and UV-B tolerance. Abiotic factors have strong

influence on Metarhizium population structure and biological success.

One of the challenges that has slowed BCA implementation is that they are sometimes

viewed (especially by the end user) to be one-to-one substitutions for chemical pesticides (Cook,

1993). This leads the expectation that they will have a similar shelf life, can be applied in similar

conditions, and will have a similar mode of action and time to kill. Distinguishing the differences

between applying a living organism and a chemical is vital to successfully integrate BCAs as a

viable treatment option. One of the limitations is BCA susceptibility to abiotic environmental

factors (Jaronski, 2010; Starnes et al., 1993). Future research that uncovers the mechanisms and

limitation regarding responses to abiotic factors will greatly aid in improving BCA effectiveness.

22

2.2.3 Environmental dissemination pathways

Fungi are non-motile organisms, in nature the infection propagules (e.g., conidia) of

Metarhizium are dispersed passively. Generally dispersal is thought to occur by abiotic factors such

as wind and rain (Hajek, 1997; Inglis et al., 2001; Meyling and Eilenberg, 2007). Additionally, it is

possible that insects as well as other animals may act as vectors for conidia (Meyling et al., 2006).

In Manuscript 1 we show that plants also act as a vector in aiding conidia dispersal through the soil

environment (Keyser et al., 2014b), and we speculate that this may explain why entomopathogenic

fungi have evolved to associate with plants (see also section 3). Historically, biological control

programs utilizing Metarhizium as a bio-pesticide have tried to adapt chemical-pesticide application

methods for conidial dispersal (Bateman, 1997) (Figure 3). Often formulating conidia for

application in liquid substrate or as a powder, this is then applied topically to vegetation (Booth et

al., 2000; Caudwell and Gatehouse, 1996; Griffiths and Bateman, 1997).

The effectiveness of biological control programs using Metarhizium have been inconsistent,

one factor that may be contributing to the unpredictability of Metarhizium as a BCA is the

application method or delivery system. Several studies have shown that application method does

greatly influence infection rates and environmental persistence (Farenhorst et al., 2008; Fargues et

al., 1997; Jenkins and Thomas, 1996; Kanga et al., 2003); however nearly all of these studies are

focused solely on the BCA implementation and thus neglect further investigations to understand the

underlying ecological principles contributing to increased infectivity. Understandably, designing

Figure 3. Methods of Metarhizium application. (A) In China mortar shells filled with powdered conidia were launched above a forested area using a grenade launcher (photo by Richard Soper), arrow indicates conidial cloud. (B) Conidia formulated in oil is applied to grasshopper infected rangeland in Utah, USA, using a rear-mounted sprayer (photo by C. A. Keyser).

23

BCAs to fit seamlessly into insecticide application methods greatly enhances their usability and

facilitates an easy transition away from chemical pesticides. Unfortunately, without an

understanding of the natural dispersal mechanisms which contribute to efficacy, consistent

biological control results will be more a matter of luck than design. Only by investigating the

ecological principles can we hope to improve reliability. For example, in a study to evaluate the M.

brunneum biocontrol product Met52 for control of Black vine weevil, Otiorhychus sulcatus (Col.

Curculionidae) in field grown strawberries, Ansari and Butt (2013) tested several application

methods using the granular formulation of the commercial product. The methods of application

included: 1. Premixed in soil; 2. Drench application to the base of each plant; and 3. Plant roots

being dipped in an aqueous suspension of the conidial product and then planted. They found that

Black vine weevil control was more efficient when the drench application method was used.

Further studies revealed that plant roots were colonized by significantly more conidia than when the

drench method was used (Ansari and Butt, 2013). This additional information allowed them to

conclude that the conidia concentration in the rhizosphere was a key component to improving

infectivity. It was recently shown, however that M. brunneum population dynamics in the

rhizosphere may depend on adaptations to the local environmental conditions; a Norwegian M.

brunneum isolate proliferated more in strawberry rhizopheres at ambient temperatures in Norway

than an isolate similar to Met52 which originated from Austria (Klingen et al., 2015). Clearly,

understanding how infectious propagules are dispersed naturally can improve BCA performance in

the field.

3. Trophic interaction

One of the greatest challenges to fully understanding the ecology of the world we live in is

accounting for all the trophic interactions that influence an organism; however for BCAs it is

crucial that we account for these interactions as they will greatly affect efficiency. If we truly hope

to exploit biological control to suppress pests, we must view insect pathology not as one organism

acting on another, but rather as a node in a complex web of intertwined organisms and response

variables which have co-evolved and adapted to each other. This can only be achieved by studying

the organisms in combination and not individually and valuing both direct and indirect interactions.

The following sections will highlight several key interactions that occur between

Metarhizium and other important organisms (Figure 4). Some of these interactions have been well

studied (e.g., Metarhizium ↔ Insects), while for others very little is known (e.g., Metarhizium ↔

Other microorganisms ↔ Plants). The overall message that I hope to convey is the importance of

24

not only understanding the various responses associated with individual bi-trophic interactions, but

also a broad perspective of how they affect each other.

Figure 4: Illustration depicting several trophic interactions that may occur between Metarhizium

spp. and other organisms. Blue arrows represent direct interactions and orange arrows represent

indirect interactions. Diagram by C. A. Keyser

3.1 Metarhizium ↔ Insects

Among the millions of fungal species in the world, Metarhizium spp. have garnered

significant scientific and economic interest primarily because of how they interact with arthropods.

A large portion of the research involving Metarhizium centers on its use as a BCA and consequently

25

deals with its relationship to various insects. As a generalist, many Metarhizium spp. are able to

infect a wide range of hosts (Veen, 1968; Zimmermann, 1993), however the virulence can vary

considerably between different host species (Butt et al., 1992), making it necessary to test virulence

towards each insect species of interest. Furthermore, virulence varies between isolates even within

the same fungal species (Keyser, 2010), which further complicates the selection of appropriate

isolates for BCA use. The variation in virulence should not necessarily be looked at as a negative

aspect for a potential BCA; it is important to remember that a very small percentage of all insect

species are important pests – many herbivores do not cause any economic damage and some

predators, parasitoids and pollinators are important beneficial insects. The variation in host

virulence allows selection for a BCA that is highly virulent to a particular pest insect while being

less virulent to non-target insects. For example, Falagiarda (2014) observed that a commercial

isolate of M. brunneum, the same isolate used in Manuscript 1 and 2, to be highly virulent to the

grain-pest insect T. molitor but having very low virulence to the beneficial coleopteran, Atheta

coriaria (Figure 5). Her results suggest that this isolate of M. brunneum could therefore be

considered a “low risk” substance under the proposed EU legislation. Further testing might

investigate whether there is an additive or synergistic effect when M. brunneum and A. coriaria are

used in combination as BCAs.

Many factors from both the host and the pathogen contribute to virulence; it is a give and

take relationship with each attempting to maximize their own fitness. Each step in the infection

process (see Figure 2) is controlled by specific cues and responses which determine the success of

the infection. For example, ambient pH levels on and around the insect cuticle regulate the

secretion of proteolytic and chitinolytic enzymes which aid in the penetration process (St. Leger et

al., 1998). In response to the infections, insects have evolved different immune responses, both

physiological and behavioral, to mitigate the pathogenic effects. For example, after exposure to

Metarhizium there are changes in insect biochemistry, including melanization of the cuticle and

antimicrobial defenses of the haemolymph (Dubovskiy et al., 2013; Gillespie et al., 2000). Also,

behavioral adaptations have been noted, including: increased grooming among infected ants and

termites (Hughes et al., 2002; Qiu et al., 2014; Yanagawa et al., 2008), or basking in the sun in

order to elicit a “behavioral fever” which restricts or kills the infecting pathogen (Blanford and

Thomas, 2001; Kemp, 1986; Ouedraogo et al., 2004). These immune responses can severely limit

the effects of a pathogen; however some species of Metarhizium have developed traits to more

efficiently overcome insect immunity. For example, to increase virulence many of the generalist

26

species of Metarhizium produce cyclic peptides known as destruxins (Roberts and St. Leger, 2004);

these insecticidal toxins cause muscle paralysis and suppress immunoresponses in the host (Pedras

et al., 2002; Roberts, 1981).

These examples represent only a small fraction of the many interactions that take place

between insects and Metarhizium; however, they begin to illustrate the complex dance that occurs

between host and pathogen. The level of complexity can be even greater when multiple trophic

levels of insects are considered. For example, Rännbäck et al. (2015) observed a reduction in

parasitoid egg laying when hosts were infected with Metarhizium. While the complexity of these

interactions can seem overwhelming, it is highly relevant that we understand what is occurring if we

hope to successfully implement BCA to control pest insects.

3.2 Metarhizium ↔ Plants

Most land plants form symbiotic relationships with soil fungi (van der Heijden et al., 1998).

It is therefore not surprising that several species of Metarhizium have been observed to interact with

some plants. A fungal endophyte is defined as an asymptomatic plant-colonizing fungus that lives a

portion of its life cycle inside the plant (Behie and Bidochka, 2014a). Metarhizium spp. have a dual

life cycle, persisting in the environment both as an insect pathogen and as a facultative saprophyte

(Wang et al., 2005). While soil has long been considered a reservoir for naturally occurring

Metarhizium, it has not been clear whether the recovered isolates were from dormant conidia in the

soil or hyphae actively growing on plant material (St Leger, 2008). Metarhizium has been viewed

for many years as only an insect pathogen – the realization that it also actively interacts with plants

a b

Figure 5. Bioassay survival curves after exposure to Metarhizium brunneum or Beauveria bassiana of

(a) Tenebrio molitor larvae and (b) Atheta coriaria after exposure to M. brunneum (Falagiarda, 2014).

27

is both novel and exciting for the field of Metarhizium research. For this reason and because it is of

special interest to the research contained in this thesis, I will give a more comprehensive outline of

the current status on Metarhizium ↔ Plants interactions.

As mentioned in section 2.2.1, specific species of Metarhizium have been shown to associate

closely with different plants. In a Canadian-field survey Wyrebek et al. (2011) observed that M.

robertsii was the only species associating with grass roots, while M. guizhouense tended to

associate with roots of trees and M. brunneum was found in the roots of shrubs and trees. In a

similar study Fisher et al. (2011) found that in Oregon, M. brunneum associated strongly with roots

of Strawberry and Blueberry plants while M. robertsii and M. guizhouense were isolated

predominantly from Christmas tree roots. The plant specificity observed in these two studies could

indicate a history of co-evolution between Metarhizium and certain plants.

The rhizosphere is a region of soil where plant-root exudate influences soil microorganisms.

M. anisopliae s.s was first discovered to be a rhizosphere competent isolate by Hu and St Leger

(2002) in a field study designed to evaluate the fate of the BCA after application. In a cabbage field

soil samples were collected 4-5 cm from the plant as well as directly next to the taproot. The

researchers observed that even after a year the population of M. anisopliae s.s. remained high in the

rhizosphere area while it declined over time in the bulk soil (Hu and St Leger, 2002). M. brunneum

was also observed to be rhizosphere competent in soilless potting media (Bruck, 2005) and several

Metarhizium spp. isolates have been shown to germinate and grow in root exudates (Pava-Ripoll et

al., 2011; Wang et al., 2005). Additionally, specialized genes which become active when

Metarhizium interacts with plants or plant compounds have been identified; like, the Mad2 gene

which is involved in adhesion to plants during colonization (Barelli et al., 2011; Wang and St

Leger, 2007), or the Mrt and Mlrnv genes which transport oligosaccharides found in root exudate

(Fang and St Leger, 2010; Liao et al., 2013). These studies clearly show that Metarhizium is more

than just an entomopathogen but that it has evolved to propagate while interacting with plants.

In an effort to determine if Metarhizium had a localized plant-tissue preference when

associating with plants in the field Behie et al. (2015) performed a field survey of grasses and forbs

in Canada. They found that endophytic Metarhizium spp. (95% of which were M. robertsii)

associations were exclusively with plant roots and not with hypocotyl or the stem and leafs of a

plant. In vitro laboratory studies with Haricot beans also showed endophytic root preference of

Metarhizium spp. (Behie et al., 2015). However, Batta (2013) was able to re-isolate M. anisopliae

s.s from untreated leaves, petioles and stems of the oilseed rape plants when other leaves were

28

sprayed with a high dose of M. anisopliae. Furthermore, Golo et al. (2014) observed that cowpea

and cucumber plants resulting from M. robertsii or M. acridum inoculated seeds had endophytic

association in both the roots and leaves of the plants after 12 days. They further observed the

production of destruxins by M. robertsii in the cowpea plants.

Akello and Sikora (2012) showed that M. anisopliae s.s. could live for at least a month

inside V. faba roots as an endophyte after seeds were soaked for 4 hours in a conidial suspension.

Interestingly, in a similar study Akutse et al. (2013) did not observe M. anisopliae s.s. colonizing

any part of V. faba plant when seeds were inoculated with a conidial suspension even though the

same fungal isolate and plant were used as well as similar methods as Akello and Sikora (2012);

although in addition to treating the seeds with conidia (as was done in Manuscript 1), Akello and

Sikora (2012) also drenched soil with a conidial suspension, the additional load of conidia in the

soil might explain why an endophytic interaction was observed in one and not the other.

Further implications regarding the importance of Metarhizium ↔ Plants interactions were

suggested when, in a proof of concept study, Behie et al. (2012) showed that nitrogen from a M.

robertsii-infected insect can be transferred to a plant via an endophytic hyphal connection. In a

follow-up, in-depth study Behie and Bidochka (2014b) tested the insect-derived nitrogen-

transferring abilities of five species of Metarhizium [i.e., M. acridum, M. brunneum, M. pemphigi

(=flavoviride var. pemphigi), M. guizhouense, and M. robertsii] to four types of plants (haricot

bean, soybean, switchgrass and wheat). They showed that all five Metarhizium species had the

capacity to transfer nitrogen to plants, although in varying degrees. In addition, they showed that

nitrogen was also transferred in the field, despite other competing microorganisms (Behie and

Bidochka, 2014b).

Metarhizium-plant associations have also been observed to promote plant growth. Sasan

and Bidochka (2012) observed that after inoculation with M. robertsii, switchgrass and haricot bean

plants both had increased root hair growth. Also, increased plant growth has been observed in

several agricultural crops, including soy bean (Khan et al., 2012), tomato (Elena et al., 2011), and

corn (Liao et al., 2014). In soy bean, Khan et al. (2012) observed endophytic interaction with M.

anisopliae s.l. increased biomass, chlorophyll contents, transpiration rate, photosynthetic rates and

leaf area compared to untreated control plants. While wild type M. robertsii, M. brunneum and M.

anisopliae s.s. increased many aspects of corn growth, Liao et al. (2014) observed that when

Metarhizium genes that are associated with either adhesion to the plant or the utilization of plant

exudates were knocked out, no plant growth promotion was observed.

29

The ecological significances of delving in to the complex systems of the Metarhizium ↔

Plants interactions are clear. Furthermore, these fundamental research studies also have important

implications for biological control. Nearly all the studies so far have described either beneficial or

neutral effects on the plant resulting from Metarhizium associations. Based on the results of these

studies, Metarhizium based BCA may do more than just protect crops from insect pests, they may

also aid in nutrient acquisitions and plant-growth promotion. However, more research is necessary

to illuminate the mechanisms involved in these processes and whether the benefits observed in the

laboratory are also seen in the field.

Thus far, the majority of the published studies involving Metarhizium ↔ Plants interaction

have focused on responses exhibited by the plant. This is most likely because traditionally

underlying justification for Metarhizium research has been in the interest of plant protection,

however, research into beneficial or deleterious effects to the fungal organism should also be of

interest. In Manuscript 1, after demonstrating that species of Metarhizium will disperse along a

growing root though soil, I suggest that a possible benefit that Metarhizium derives from associating

with plants is mobility and proximity to potential hosts. It is likely that the fungus is also affected

in other ways while interacting with plants so it is important that questions like, “Why would a

Metarhizium organism give up a limited resource such as nitrogen and what does it receive in

return”, continue to be investigated. Metarhizium ↔ Plants interactions are an important part of the

future of biological control research.

3.3 Metarhizium ↔ Other microorganisms

Very few studies have been conducted to investigate the interaction between Metarhizium

and other microorganisms. This is an important aspect of Metarhizium ecology that should be

addressed more thoroughly. Metarhizium spp. are abundantly found in the soil environment

(Tkaczuk et al., 2014). It is likely that an actively growing saprophyte will have developed

antimicrobial strategies to survive and compete in an environment teaming with natural

microorganisms. I observed evidence of this while performing isolation on nutrient agar.

Inadvertently, a petri plate which had been inoculated with a new isolate of M. flavoviride also

became contaminated with a fast growing unknown fungus, however a very clear inhibition zone

was present between the M. flavoviride and the contaminate (Figure 6a). Additionally, while

developing the methodology for Manuscript 2, media plates with M. brunneum, M. robertsii and M.

flavoviride were also inoculated with plugs of Fusarium culmorum. A small zone of inhibition was

observed on the plates with M. brunneum and M. robertsii and a much larger zone was present on

30

Figure 6: Inhibition zones observed between Metarhizium spp. isolates and other fungi on PDAY media. (A) M. flavoviride isolate from bait insect on petri plate with un-known fungal contaminant. (B) M. brunneum, M. robertsii, and M. flavoviride on petri plate with Fusarium culmorum.

those with M. flavoviride (Figure 6b). Sasan and Bidochka (2013) also observed a zone of

inhibition when M. robertsii and Fusarium solani were cultured on the same petri plate and they

noted a significant reduction of the colony size of the F. solani. Furthermore, Sasan and Bidochka

(2013) showed that liquid media, which had had M. robertsii growing in it but was then passed

through a filter to remove all fungal material suppressed F. solani germination. They suggested that

this indicates that M. robertsii secretes an anti-fungal compound that inhibits F. solani growth.

Krauss et al. (2004) tested the interaction between several entomopathogenic fungi,

including two strains of M. anisopliae s.l., and mycoparasitic fungi by completely colonizing a petri

plate with the entomopathogen and then placing an agar plug of the mycoparasite on top of the

entomopathogen colony. They claimed that because the mycoparasite was not in contact with the

media any growth indicated it was receiving nutrients from the fungus and not the media. They

found that of the isolates they tested M. anisopliae s.l. was the most susceptible to mycroparasitism

and allowed growth of all the mycoparasites tested including Clonostachys byssicola, C. rosea and

Lecanicillium lecanii (Krauss et al., 2004). These two studies demonstrate that Metarhizium spp.

are both affected by and effectors of other microorganisms. Further studies with regard to the

mechanisms involved in these interactions would greatly expand our understanding of the ecology

of Metarhizium spp. in the field and identify potential benefits and challenges to their use as BCAs.

31

3.4 Multi-trophic interactions with Metarhizium

Each additional trophic level included in a study greatly increases the size and complexity of

the experiment necessary to evaluate all the variable permutations. It is therefore not surprising that

most studies focus on bi-trophic interactions. Nevertheless, multi-trophic interactions which

include three or more levels are necessary to both better grasp what occurs in nature and provide

greater predictive power to BCA employment. Generally bi-trophic interactions are concerned with

direct effects while multi-trophic interactions must account for both direct and indirect effects. In

the following section I will review several Metarhizium studies which evaluate multi-trophic

interactions. The trophic levels that I focus on are those of different types of organisms (i.e., other

microorganisms, insect and plants); however multi-trophic interactions that involve multiple species

of the same type of organism (e.g., insect ↔ insect ↔ microorganism) – as was seen above in the

study by Rännbäck et al. (2015) in which interaction between a parasitoid insect as pest insect and

M. brunneum were evaluated – should not be neglected.

3.4.1 Metarhizium ↔ Other microorganisms ↔ Insects

There are a few examples of tri-trophic interaction involving Metarhizium, other microbes

and insects; generally they are geared towards evaluating how the biological control capacity of

Metarhizium is affected when other microorganisms are present. Hughes and Boomsma (2004)

found that the normally avirulent, opportunistic fungal pathogen Aspergillus flavus would out-

compete a highly virulent isolate of M. anisopliae s.l. when applied as a mixture on a leaf-cutting

ant host. They explained that alone A. flavus is generally unable to overcome the host immune

defenses, however, when coupled with a second pathogen like Metarhizium, which is more adept at

suppressing the immune responses, the avirulent fungus is then able to grow much faster and better

utilize the pilfered resources. Geetha et al. (2012) demonstrated that co-application of multiple

fungi on the same insect host negatively influence the virulence and sporulation of M. anisopliae s.l.

Also, Krauss et al. (2004) found that when applied in combination with mycoparasitic fungi on

three different species of insects, the virulence of M. anisopliae s.l. was not significantly affected,

despite the authors having found that M. anisopliae s.l. was highly susceptible to mycroparasitism.

In Manuscript 2, an insect bioassay was conducted in which Tenebrio molitor larvae were

inoculated with one of three species of Metarhizium (M. brunneum, M. flavoviride s.s., or M.

robertsii) combined with either the mycoparasite Clonostachys rosea or the plant pathogen

Fusarium culmorum or both. We observed that, when compared to the individual treatments, for

some of the combined treatments there was a slight reduction in virulence; however, in general,

32

virulence remained similar to the individual Metarhizium treatments. Based on this study and the

others mentioned it is obvious that interaction which affect the BCA performance of Metarhizium

occur when other microorganisms are present. Since microbes are always present in the field these

interactions are highly relevant – especially those with other BCA like C. rosea which are also often

applied for crop protection.

3.4.2 Metarhizium ↔ Other microorganisms ↔ Plants

In light of the recent emphasis placed on Metarhizium ↔ plant interactions, the tri-trophic

interaction between Metarhizium, other microorganisms and plants are highly relevant. There have

been very few studies which have looked at these interactions. Sasan and Bidochka (2013) found

Haricot bean seeds had higher germination when in the presence of M. robertsii and F. solani than

when only F. solani was present. The resulting plants also showed significantly less F. solani

infection symptoms when M. robertsii was also present. In Manuscript 2 we also evaluate the

interactions between Metarhizium (M. brunneum or M. flavoviride), C. rosea, F. culmorum and

Winter wheat. We observed that the presence of either of the Metarhizium species did not reduce

the F. culmorum infection or hinder the biocontrol efficacy of C. rosea. The two beneficial BCAs

may therefore potentially be used in concert against both insect pests and plant pathogens without

obstructing each other’s effects. But no additive nor synergistic effects should be expected

(Manuscript 2). Several important differences exist between Manuscript 2 and the study by Sasan

and Bidochka (2013) which might account for the different observations. For example, the species

of Metarhizium and Fusarium that were used are different, as was the plant species and inoculation

method; also Sasan and Bidochka (2013) allowed an establishment period for M. robertsii and F.

solani in the soil before introducing the bean seeds, where as in Manuscript 2, un-germinated seeds

were exposed to F. culmorum first and then Metarhizium spp. before germination.

3.4.3 Metarhizium ↔ Plants ↔ Insects

The indirect effects to plants are nearly always implied in Metarhizium ↔ insect studies

which have a biological control aspect to them. Nevertheless these indirect effects (i.e. increased

plant productivity caused by reduced effects of pest insects after BCA application) are often not

measured. One example is a study by Kabaluk and Ericsson (2007) in which they found that corn

seeds treated with the M. brunneum BCA F52 (called M. anisopliae by the authors) resulted in an

increased yield in comparison to untreated control plants when planted in fields with wireworm. On

the other hand, indirect effects may also be observed to occur to the insect when the plant interacts

with Metarhizium. For example, Akello and Sikora (2012) showed that aphids feeding on V. faba

33

plants which had been treated as seeds with different fungi including Metarhizium spp. had reduced

survival, offspring fitness, development and fecundity compared to untreated control. They

demonstrated that Metarhizium spp. isolates along with other endophytic isolates indirectly

protected the plant from aphid damage. However, the mechanisms behind this remain unknown.

3.4.4 Metarhizium ↔ Other microorganisms ↔ Plants ↔ Insects

The last type of interaction that I would like to mention is that which involves four or more

trophic levels. Very few studies have attempted to incorporate so many different trophic levels; in

fact I was unable to identify any other studies, involving Metarhizium, which evaluated response

variables for four trophic levels. One example, which uses another entomopathogenic fungus (B.

bassiana) tested whether insect pollinators could be utilized as delivery vehicles to vector two

BCAs to plants in order to control both an insect pest and a plant pathogen (Kapongo et al., 2008).

They found in laboratory greenhouse trials that the bees successfully vectored the BCAs to the

flowering plants and they observed a reduction in both plant pathogen and insect pest occurrence.

Manuscript 2 also describes a system with up to five trophic levels are evaluated. F.

culmorum infected seeds were treated with C. rosea, a mycoparasitic fungus which can be used as a

BCA against plant disease, and two Metarhizium spp. isolates. After the plants had grown for 14

days the roots were feed to T. molitor larvae which were then monitored for mortality and fungal

disease. There were two main response variables assessed in this study, plant disease incidence and

insect mortality. Combination effects could be assessed by comparing the data of the combination

treatments with those of the non-combined treatments.

Prior to this experiment we had several hypotheses on what combination effects we might

expect. Metarhizium has been shown to have antifungal properties (Sasan and Bidochka, 2013) and

therefore might aid in Fusarium disease suppression. As a potential mycoparasite of Metarhizium

spp. (Krauss et al., 2004), C. rosea may severely reduce the ability of Metarhzium to disperse along

the root and subsequently infect the larvae. On the other hand, isolates of Fusarium spp. and C.

rosea have been observed to be pathogenic to insects (Teetor-Barsch and Roberts, 1983; Vega et al.,

2008), and may therefore have an additive effect on insect virulence when in combination with

Metarhizium spp. As reported in Manuscript 2, no effect on plant-disease reduction by C. rosea

was observed when Metarhizium was added, however when C. rosea was present, alone or

combined with Metarhizium spp. there was little to no disease symptoms in the plants; the high

efficiency of the C. rosea treatment may however have masked any positive effects of Metarhizium.

In most of the treatments there was a significant reduction in virulence to the insect when the seeds

34

had been treated with Metarhizium spp. combined with another fungus. This indicates that there is

an indirect effect on insect virulence when other fungi are present. Future studies should evaluate if

this effect is due to direct interaction between the combined fungi or due to an indirect effect like

resource competition. It is clear however, that multi-trophic interactions are important and may

yield unexpected results.

4. Methodology

In fulfilling the research requirements of this thesis it was necessary that I develop and test

various experimental methodologies and expand my analytical skills. The following section will

briefly summarize and describe a few important methodological techniques I worked with.

4.1 Selective media

Selective media is often used to assist in the isolation of entomopathogenic fungi. The basic

principle of selective media is to provide a substrate that allows the growth of a desired organism

while discouraging the growth of others. This can be accomplished in several ways, for example,

many bacteria are inhibited by low pH levels while fungi are not. Also the nutrient composition of

many growth media favor particular organisms, e.g., Potato dextrose media and Sabouraud dextrose

media are better suited for fungal growth than bacterial. While usually not included as a selective

medium, insects used for the soil baiting method (Zimmermann, 1986) are essentially a “ready-

made” selective medium that screens out any non-pathogenic organisms and selects for those that

can infect the host.

Media are often amended with more specific growth inhibiting components to further select

for desired organisms. Antibiotics, like streptomycin or chloramphenicol, can be added to help to

reduce contaminating bacteria. Contaminating fungi are more problematic. For many species of

Beauveria and Metarhizium some fungicides, like dodine or cyclohexamide, in low concentration

help to eliminate the fast growing contaminants like Rhizopus or Trichoderma while allowing

Metarhizium and Beauveria growth (Fernandes et al., 2010b; Rangel et al., 2010) (Figure 7). Many

different media have been developed for the isolating of Metarhizium spp.; Table 3 lists some of the

major component combinations that have been found effective as well as the studies in which they

were presented.

35

Figure 7: Selective media test with several types of media (columns) to evaluate effectivness at

supressing naturally occuring microbes while allowing the growth of entomopathogenic fungi.

Both natural and Metarhizium “spiked soil” was used (rows). Soil was diluted in ddH2O and then

200µl was pipetted onto each plate. Plate incubated for 21 days at ~21ºC.

36

Table 3: Selective media ingredients that have been observed to be effective for the isolation of

Metarhizium spp isolates from soil. All concentratoins represent grams/liter unless otherwise

specified.

Media Ingredients Ve

en

& F

err

on

, (1

966

)

Be

ilharz

et

al.,

(198

2)

Ch

ase

et

al.,

(19

86

)

Liu

et

al.,

(199

3)

Sh

imazu

& S

ato

, (1

996

)

Str

asse

r e

t a

l., (1

997

)

Hu

gh

es e

t a

l.,

(20

04

)

Sh

in e

t a

l.,

(201

0)

Re

ng

al e

t al.,

(20

10

)

Fe

rna

nd

es e

t al.,

(20

10b

)

Wyre

be

k e

t a

l.,

(20

11

)

Ro

ch

a &

Luz,

(201

2)

Ste

inw

end

er,

(2

013

)

Ke

yse

r e

t a

l.,

(20

14

b)

Se

lec

tive

In

gre

die

nts

Dodine 0.065 0.46* 0.01* 1ml* 0.1 0.05 0.02 0.325 0.300 0.29 0.2

Cycloheximide 0.25 0.25 0.05 0.25 0.5 0.06 0.25

Chloramphenicol 0.5 0.5 0.05 0.5 0.2 0.014 0.5

Thiabendazole 0.001

Chlortetracycline 0.005 0.005

Copper chloride (CuCl2) 0.02

Gentamicin

Streptomycin 0.6 0.1 1

Penicillin 0.4

Tetracycline 0.05

Crystal violet 0.01 0.01 0.01 0.01

Nu

trie

nt

Ing

red

ien

ts

PDA X X X X

SDA 30% X X X

Yeast extract 1 1 X

Glucose 10 10 20

Peptone 10 10 10

Oxgall 15 15

Oatmeal infusion 20 20 20

Agar 35 20 20 35 18 20

pH 10+ 6.9 6.9

37

4.2 Bioassay statistics

A bioassay is a scientific study with the aim to determine the biological activity or potency

of a substance by testing its effect on the growth of an organism. In insect pathology an infection

bioassay is a study which evaluates the infectivity of a particular pathogen on a host insect. The

ability to cause infection in insects is one of the most basic and interesting traits of

entomopathogenic fungi like Metarhizium spp. It is for this reason that many studies of these fungi,

among other things include infection bioassays. Infection bioassays are often designed to evaluate

pathogenicity, the ability to cause infection, and virulence, the killing power of a pathogen.

The most common type of studies that include infection bioassays are those aimed at

determining the biological control potential of a pathogen for a particular host. However, infection

bioassays are regularly included in other types of studies as a phenotypic trait that can be used to

compare different genotypes to each other or measure changes within the same isolate after a

mutation or stress. For example Jin et al. (2012) used infection bioassays to show the effects of the

Hog1-type mitogen-activated protein kinase gene in M. acridum, or Lopes et al. (2013) who while

performing a diversity study of Beauveria spp. and Metarhizium spp. in banana fields included a

bioassay and interpreted low virulence to explain the low occurrence of epizootics. For any insect

pathologist an understanding of how to appropriately design and execute an infection bioassay is a

vital skill.

4.2.1 Experimental design

There are several factors that influence the outcome of an infection bioassay; however, the

two most important factors are time and dose. Therefore, experiments are usually designed around

these factors either as dose-mortality or time-mortality experiments. In a dose-mortality experiment

the pathogen dose or number of conidia administered to each host is varied while the time at which

the response is evaluated is kept constant. Often in a dose-mortality experiment the response is

reported as a value of LD50 or LD90 (dose at which 50% or 90% of individuals exhibit the desired

response, i.e., mortality) for a particular treatment. The most important factors are sample size and

dose range. It has been recommended that sample size of 120 to 240 insects is necessary to obtain a

reliable response (Robertson et al., 1984). Ideally the dose range will include three to eight doses

that result in 25 to 75% responses at the time of observations (Robertson et al., 1984), however, the

experiment can be checked at several time periods and then the most appropriate observation time

for the analysis can be selected afterwards.

38

In a time-mortality experiment, dose is kept constant (or multiple doses are considered

separately) and the response is measured over a period of time, often results are reported as LT50 or

LT90. To avoid data being correlated in a time-mortality experiment, either different groups of

insects must be used for each time measurement so that the data are not correlated or the method of

analysis must allow for correlated data. The bioassay performed in Manuscript 1 and 2 were

designed as time-mortality experiments with the data for each dose being analyzed separately.

I conducted a survey of 30 randomly selected scientific publications selected from a Google

Scholar search with the key words “Metarhizium” + “Bioassay” and limited to papers published

between Jan 2010 and April 2014. Of these 30 papers, 15 included time-mortality bioassay designs,

7 dose-mortality experiments and 8 with both (Table 4).

4.2.2 Types of Statistical Analyses

4.2.2.1 Abbot correction: Random mortality not related to a treatment but rather

experimental conditions being tested in an infection bioassay can be adjusted for in the analysis

using the control group. The most common method employed in insect pathology is the Abbot’s

formula (Abbot, 1925). The following formula is used to estimate the percentage of insects killed

by a particular treatment: 𝑃 = (𝐶−𝑇

𝐶) ∗ 100. Where P is the estimated percentage of insect mortality

due to treatment, C is the percentage dead in the control and T is the percentage of dead in

treatment. A few considerations before implementing an Abbot adjustment include: First, the

Abbot formula assumes pathogenicity of a treatment, and therefore should not be used if the

researcher wishes to determine if a particular treatment differs from the control. Second, if a

control has greater mortality than a treatment an Abbot correction will result in negative percent

mortality and is not appropriate. Lastly, an Abbot correction cannot be used when performing a

survival analysis because the binomial data used for the analyzed is of the individual and not the

group.

4.2.2.2 Probit analysis: Infection bioassays are typically analyzed in one of two ways, the

first is using a probit analysis model. A probit analysis is most appropriate for dose-response

designs but can be used in a time-response design if different groups of insects are used for each

time measurement otherwise the data are correlated and the model is not valid; however, examples

have been shown where correlated data can be analyzed using a probit transformation of proportion

of insects killed (Goettel and Inglis, 1997).

Probit analysis is a type of regression used to analyze binomial response variables. It

transforms the sigmoid dose-response curve to a straight line that can then be analyzed by linear

39

regression analysis. Probit analysis was first published in 1934 by Chester Bliss, an entomologist

working with pesticides and their effectiveness for pest control (Bliss, 1934). Later in 1952 David

Finney, a professor of statistics, published a book called Probit Analysis (Finney, 1952). Probit

analysis continues to be one of the preferred statistical methods in understanding dose-response

relationships.

4.2.2.3 Survival analysis: The survival analysis is historically one of the oldest fields of

statistics dating as far back as the 17th

century (Aalen et al., 2009). As suggested in the name, a

survival analysis traditionally is used to analyze survival or death rates, however today it is often

used in engineering to predict failure times of machines. A survival analysis is used to analyze data

in which time duration until an event is of interest occurs. The response is often referred to as a

failure time, survival time, or event time. One of the primary advantages of using a survival model

is that it has the ability to account for censored data, most statistical models don’t. Censored data is

data in which the response is unknown in the window of observation, for example, if an experiment

ends before all the individuals are dead those individuals are considered censored. An ordinary

linear regression model cannot handle censored data. Despite its early beginnings, it was not until

the late 1950s that the field of survival analysis was significantly advanced. A publication in 1958

by Kaplan and Meier which allowed a survival curve estimation was presented and then later in

1972 Cox published a method of comparing survival curves (Aalen et al., 2009). Both of these

modeling approaches advanced the usefulness and the applicability of survival analysis to the

current state. In Manuscripts 1 and 2 bioassays were performed. The experimental design of these

bioassays in each study was based on a survival analyses. A Cox proportional hazard model was

used to compare the survival curves between the various treatments. This model was most

appropriate for the type of data that I collected because it reduced the number of dose-treatments

needed, allowed for the same batch of insects to be checked daily and accounted for censored data

(insects surviving beyond the time frame of the experiment).

Of the 30 studies surveyed included in table 4, 13 used a probit analysis, 8 a survival

analysis, 3 used both, and 6 used either another type of analysis or did not specify.

40

Table 4: A survey of experimental design and statistical methods used in 30 studies; selected from

a Google Scholer search with key words “Metarhizium + Bioassay”, limited to 2010-2014.

Time – Response

Method of analysis

Host Treatment method

Gao et al. (2013)

Survival analysis Bombyx mori Larvae immersed in 5 ×106 suspension for 1 min or injected

with 10µl of 106 conidia/ml. 15 insect (×3) checked every

12h.

Garza-Hernandez et al. (2013)

Survival analysis Aedes aegypti

Infection of mosquitos co-infected with virus. 25 adults exposed to filter paper impregnated with 1 × 10

8 conidia/ml.

Checked daily until all dead.

Goble et al. (2014)

Survival analysis Anoplophora glabripennis

For 36 individuals the ventral surfaces were pressed on to two agar plugs with conidia for 5 sec. Also, for 24 beetles were submerged for 10 sec in a 15ml suspension of 1 × 10

7

conidia/ml. Checked daily for 50 days.

Howard et al. (2010)

Survival analysis Anopheles gambiae

25 adult mosquitos were placed in a tube with netting that had been treated in a fungus. Checked daily until all fungal treated individuals died.

Jin et al. (2012) Method not specified

Locusta migratoria

Adults were either: dipped up to the head-thorax junction in a soybean-oil suspension containing 1×10

7 conidia/ml; or,

injected with 5 µl of a 1 × 106 conidia/ml in the haemocoel

cavity through. Twenty insect per rep, checked every 12h.

Lopes et al. (2013)

Survival analysis Cosmopolites sordidus

Adults were submerged in conidial treatment of 1 × 108

conidia/ml for 90 sec. Sixteen-eighteen insects per treatments, mortality assessed every other day for 14 or 16 days.

Orduno-Cruz et al. (2011)

Logistic regression

Metamasius spinolae

Twenty adults were immersed in 500ml suspension with 1 ×10

8 conidia/ml for 10 second. Checked for 41 days.

Peng et al. (2011)

Survival analysis Anoplophora glabripennis

Females exposed to fiber bands impregnated with fungi. Males held on conidia-covered surface for 30 seconds. Checked daily for 50 days.

Quinelato et al. (2012)

Probit analysis & non-parametric Kruskal–Wallis test

Rhipicephalus microplus

Larvae (~100/tube) were immersed in 1.2 × 108, 10

7, 10

6 and

105 conidia/ml suspensions for 3 min. The suspension was

absorbed out of test tube, mortality checked every 5 days for 30 days. Mortality was estimated as a percentage.

Quintela et al. (2013)

Probit analysis and Survival analysis

Tibraca limbativentris

Adults were inoculated with 10µl of 5 × 107 conidia/mL

suspension on the dorsal region. Checked daily for 12 days.

Reyes-Villanueva et al. (2011)

Survival analysis Aedes aegypti

Females exposed to filter paper impregnated with 6 × 108

conidia/ml for 48 hours and checked for survival. Also, females exposed to exposed males that had been exposed to impregnated filter paper for 48h and watched for survival.

San Andrés et al. (2014)

Probit analysis Ceratitis capitata

Adults exposed for 30s to an “infective dish”, a dish with 3.1 × 10

8 conidia spread uniformly over the dish. 10 host per

treatment checked daily for 10 days.

Tavassoli et al. (2012)

General linear model and repeated measure analysis

Ornithodoros lahorensis

Eggs, larvae, and adult ticks were immersed in either 1 × 105

or 1 × 107 conidia/ml for 5s. Survival was checked every 3

days for 21days.

Wang et al. (2012)

Survival analysis Bombyx mori & Locusta migratoria

Silkworms injected with 10 μL of suspension of 5 × 105

conidia/ml, locust injected with 10 µl of 1 × 107. Ten insects

in each repetition. Mortality checked every 12 hours.

Wang et al. (2014)

Probit analysis Galleria mellonella & Tenebrio molitor

Each individual larva (30 per repetition) was sprayed with 1 ml of suspension of 10

7 conidia/ml. Monitored daily for

mortality.

41

Dose – Response

Ansari et al. (2010)

Probit analysis Culicoides nubeculosus

Eggs suspended in 108 conidia/ml until hatch. Larvae

suspended in 104, 10

5, 10

6, 10

7, or 10

8 conidia/ml – checked

for 3 days.

Contreras et al. (2014)

Probit analysis Tuta absoluta Eight pupae suspended in one of six concentrations (0.34-11.15 × 10

9 viable conidia per liter), checked every 3 days

for up to 3 weeks.

Leemon and Jonsson (2012)

Probit analysis Rhipicephalus microplus & Lucilia cuprina

Six fungal concentrations ranging from 1 × 109 to

1 × 104 conidia/ml. Ticks were either topically inoculated with

2µl or submerged in inoculum. Blowflies were either topically treated with 2µl suspension or fungi was mixed with food source. 20 insects per treatment, checked for 10 days.

Luz et al. (2011)

Probit analysis Anopheles gambiae and A. arabiensis

Eggs were topically treated with 5 × 106 conidia/cm

2. Also,

eggs in soil treated with oil formulation of 105, 3.3 × 10

5, 10

6,

3.3 × 106, and 10

7 conidia/cm

2. Checked every 5 d for 30 d.

Nussenbaum and Lecuona (2012)

Probit analysis Anthonomus grandis

Lots of isolates tested by submerging adults for 15 sec in a 5 × 10

8 conidia/ml, checked daily for 20 d. Also, 40 individuals

immersed treatments of: 1 × 106, 5 × 10

6, 1 × 10

7, 5 × 10

7,

1 × 108 and 5 × 10

8 conidia/ml; checked daily for 15 d.

Zayed et al. (2013)

Probit analysis Phlebotomus papatasi

Suspension of 1 × 106, 5 × 10

6, 1 × 10

7, 5 × 10

7, 1 × 10

8, and

5 × 108 conidia/ml prepared. 0.5g of each was mixed with 0.15g finely ground larval diet. 10 larvae added to each vile. 9 replicates. Mortality based on failure for adult to emerge.

Behle and Jackson (2014)

Method not specified

Alphitobius diaperinus

Larvae were exposed to treated soil. Conidial treatment ranged from 1.69 × 10

7 to 2.08 × 10

5 conidia/ml. 30 larvae

per dosage was used. Mortality was monitored for 14 days.

Both Dose and Time Response

Jin et al. (2011) Method not specified

Nilaparvata lugens

Thirty-forty nymphs were sprayed at 5 different rates on with one of three fungal concentrations (2 × 10

8, 2 × 10

7 and 2 ×

106 conidia/mL). Checked daily.

Kirubakaran et al. (2013)

Probit analysis and ANOVA

Cnaphalocrocis medinalis

Leaves sprayed with 5ml of 1 × 103, 1 × 10

4, 1 × 10

5, 1 × 10

6,

1 × 107 and 1 × 10

8 conidia/ml in water or oil formulation, 20

larvae exposed to treatments, checked daily for 8 days.

Maldonado-Blanco et al. (2013)

Probit analysis Aedes aegypti Submersion of 25 larvae in fungal concentrations of 1 × 105,

3 × 105, 5 × 10

5, 8 × 10

5 or 1 × 10

6 submerged spores/ml.

Insects checked daily for 5 days.

Mishra et al. (2011)

Probit analysis Musca domestica

One ml fungal suspension of 103, 10

5, 10

6, 10

7, and

109 conidia/ml were applied to fly diet. Checked daily for 5

days. Also done in an arena setup.

Ortiz-Urquiza et al. (2013)

Probit analysis and Survival analysis

Ceratitis capitata & G. mellenella

Adult flies were treated with 1 of 6 spore concentrations from 10

3 - 10

8 conidia/ml by spraying. Galleria larvae treated by

injecting 8µl of suspension. 10 insect per treatment.

Shan and Feng (2010).

Method not specified

Myzus persicae A leaf with 40 adult aphids was sprayed with 1 ml suspension, 1 × 10

6, 1 × 10

7 and 1 × 10

8 conidia/ml),

mortality was checked daily for 8 days.

Vázquez-Martínez et al. (2013)

Probit analysis Anopheles albimanus

Larvae were placed in cups with 200 ml of 2.6×107

conidia/mL. Checked daily until dead or adult emergence. Adults were immobilized with cold and inoculated with 5µl 2.7×10

8 conidia/mL suspension. Checked daily for 7 days.

Yousef et al. (2013)

Probit analysis and Survival analysis

Bactrocera oleae

Adults were treated with 1 ml of a 1.0 × 108 conidia/ml by

spraying. Checked daily for 12 d. Two assays for puparia. 1. 1 ml of a10

8 conidia/ml added to 30g soil with 63 puparia; 2.

Puparia immersed in fungal suspension for 10 seconds.

42

v. Conclusion and future perspectives

The use of living organisms to control pest insects is an important part of current and future

crop protection. Understanding the fundamental ecology of these organisms is vital to their success

as BCAs. For example, research regarding their natural occurrence and effect on host populations

greatly enhances their potential for more efficient utilization in pest regulation; e.g., conservation

biological control. Additionally, research which investigates how these organisms interact with

other organisms, viz. plants, plant pathogens and other BCAs, is crucial to predicting their

effectiveness and maintaining consistency as BCAs. This thesis advances the current scientific

knowledge regarding the ecology and biological control use of Metarhizium spp. fungi in several

areas, namely:

M. flavoviride is the dominant species of the Metarhizium community found in some

agroecosystems in Denmark.

Significant intra-species diversity exists within the understudied species M. flavoviride.

M. brunneum, M. flavoviride and M. robertsii, applied as a seed treatment of conidia, will

disperse through soil with a growing plant root. Furthermore, these species of Metarhizium

will maintain pathogenicity to insects while interacting with plant roots.

Dual BCA seed treatments of C. rosea and M. brunneum or M. flavoviride will control F.

culmorum infection in wheat seedlings.

A reduction in virulence occurs when M. brunneum or M. flavoviride and C. rosea are

applied as a seed treatment on F. culmorum infected plants. However, virulence to T.

molitor was still observed at a significant level when compared to untreated controls.

Perhaps one of the most valuable components of any research is not the conclusion provided

but rather the new questions that remain unanswered. Based on the observations of this thesis there

are several research questions that I think should be addressed, including:

Worldwide survey studies that endeavor to elucidate the distribution and occurrence of

Metarhizium spp. in agriculture. These studies should emphasize habitat associations as

well seeking to understand what characteristics promote abundance in particular areas.

Highly important to these studies will be the continued development of molecularly based

ecological tools, like isolating SSR markers (microsatellites) that can explicitly discriminate

genotypic diversity within the M. flavoviride species.

43

Field studies to evaluate the effectiveness of Metarhizium seed treatment in protecting crops

from pest insects. These studies will not only evaluate infectivity, but also the longevity of

fungal survival in field, and effects of crop type.

Investigation into the mechanisms that resulted in reduced virulence when Metarhizium was

co-treated with another fungus. This study will investigate both direct interactions such as

antifungal metabolite production and mycoparasitism, as well as indirect interaction such as

resource competition.

Additional studies into the synergistic or combined effects of using multiple BCA to control

different plant pests and disease. There was some indication that Metarhizium spp.,

especially M. flavoviride, has antifungal properties; however, no control of F. culmorum

disease was observed on the plant. Effects may be realized with different treatment

methods, plant types or fungal concentrations.

44

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vii. Appendix

Manuscript 1

METARHIZIUM SEED TREATMENT MEDIATES FUNGAL DISPERSAL VIA ROOTS AND INDUCES

INFECTIONS IN INSECTS

Chad A. Keyser, Kristian Thorup-Kristensen, & Nicolai V. Meyling

Status: Published in Fungal Eclology, October 2014, Vol. 11, pg. 122-131

Printed version reproduced with permission from Elsevier – License Number: 3531750730534

Metarhizium seed treatment mediates fungaldispersal via roots and induces infections in insects

Chad A. KEYSERa,*, Kristian THORUP-KRISTENSENb, Nicolai V. MEYLINGa

aDepartment of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40,

1871 Frederiksberg, DenmarkbDepartment of Plant and Environmental Sciences, University of Copenhagen, Hojbakkeg�ard Alle 13,

2630 Taastrup, Denmark

a r t i c l e i n f o

Article history:

Received 28 March 2014

Revision received 1 May 2014

Accepted 5 May 2014

Available online

Corresponding editor:

Duur Aanen

Keywords:

Below ground interactions

Biological control

Entomopathogenic fungi

Plant protection

a b s t r a c t

The study aimed to evaluate the extent to which Metarhizium spp. applied as conidia to

seeds will disperse with the growing root system and maintain pathogenicity after dis-

persion. Tenebrio molitor larvae were exposed to roots of wheat plants that had been grown

from seeds inoculated with conidia of either M. brunneum (KVL 04-57 and KVL 12-37) or M.

robertsii (ARSEF 2575 and KVL 12-35) in both laboratory and greenhouse settings. All four

Metarhizium isolates tested maintained pathogenicity towards T. molitor larvae for up to 4

weeks after being dispersed by roots through both an artificial growth substratum and non-

sterile soil. Based on these results we propose that a planteroot association benefits

entomopathogenic fungi with mobility in the soil and an increased likelihood of encoun-

tering a susceptible insect host.

ª 2014 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction

The soil-inhabiting mitosporic entomopathogenic fungal

genus Metarhizium (Hypocreales: Clavicipitaceae) has a cos-

mopolitan distribution and a wide range of arthropod hosts

(Zimmermann, 2007). As early as 1888 the potential for a

biological control agentwas recognized and aMetarhizium spp.

isolate wasmass produced and applied to control a pest insect

(Krassilstschik, 1888; Roberts and St. Leger, 2004). Both the

need and hope for a reliable biological control agent have

fueled Metarhizium research for more than a century.

Emphasis placed on developing a biological control agent has,

however, resulted in a neglect of research concerning basic

Metarhizium ecology which may be essential to its success for

biological control (Bruck, 2005; Meyling and Eilenberg, 2007;

Vega et al., 2009).

To fill this gap of knowledge, several studies have recently

focused on the fundamental ecology of these fungi both in

their natural habitats and in the ecosystems where they are

applied. These have shown that genotypic groups of Meta-

rhizium spp. can associate closely with habitat and plant

species (Bidochka et al., 2001; Fisher et al., 2011; Wyrebek

et al., 2011). Rhizosphere competence, the ability of a micro-

organism to proliferate and function in the rhizosphere

* Corresponding author. Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Stair 2, 3rdFloor, B321, 1871 Frederiksberg, Denmark.

E-mail address: chadaltonkeyser@gmail.com (C.A. Keyser).

available at www.sciencedirect.com

ScienceDirect

journal homepage: www.elsevier .com/locate/ funeco

http://dx.doi.org/10.1016/j.funeco.2014.05.0051754-5048/ª 2014 Elsevier Ltd and The British Mycological Society. All rights reserved.

f u n g a l e c o l o g y 1 1 ( 2 0 1 4 ) 1 2 2e1 3 1

(St Leger, 2008), was observed in Metarhizium spp. isolates

(Bruck, 2005; Hu and St Leger, 2002). Furthermore,Metarhizium

spp. applied to the rhizosphere have been shown to benefit

plant growth and aid in plant-nutrient acquisition (Behie and

Bidochka, 2014; Behie et al., 2012; Khan et al., 2012; Sasan and

Bidochka, 2012), and it is now clear that the ecological com-

plexity and importance of the fungal genus Metarhizium

extend beyond that of ‘entomopathogen’.

One central question that remains to be explored is what

benefitsMetarhizium derives from plant associations. Thus far

the focus of endophytic interaction between plants and Met-

arhizium has been characterized by how the plant has bene-

fitted, eg. through nutrient acquisition (Behie et al., 2012),

while if and how the fungus benefits have largely been

ignored. Metarhizium spp. can process and utilize plant-

derived carbohydrates available in the rhizosphere for

growth (Fang and St Leger, 2010; Pava-Ripoll et al., 2011) but it

is possible that Metarhizium spp. acting as plant symbionts

might also increase their exposure to prospective host insects.

For example, actively growing plant roots could provide non-

mobile microorganisms, such as Metarhizium spp. with a

means of dispersal, thus increasing their chance of finding a

host insect. It can be assumed that there is an increased

likelihood of encountering a root feeding herbivore near its

food source (Park and Tollefson, 2005; Prystupa et al., 1988).

The first step towards answering these questions is to

determine if Metarhizium retains its pathogenicity while

associating with plant roots. Bruck (2005) found that root

cuttings inoculated with Metarhizium conidia (actually M.

brunneum) resulted in successful infections of root feeding

black vine weevil (Otiorhynchus sulcatus) larvae. Kabaluk and

Ericsson (2007) discovered Metarhizium infected wire worm

larvae (Agriotes obscurus) after sowing Metarhizium-treated (M.

brunneum) corn seeds in a field trial. They also observed an

increased yield in corn production when the seeds were

treated with the fungus and speculated that the observed

yield increase was due to seedlings being protected from

insect attack either by insects being infected or repelled by the

fungus. However, further studies are needed to determine the

effectiveness of seed treatments in infecting insects.

The hypothesis tested in this study was that Metarhizium

spp. applied as seed treatment will disperse with roots in non-

sterile soil and retain the ability to infect insects. To test this

hypothesis, Tenebrio molitor larvaewere exposed to the roots of

wheat plants that had been grown from seeds inoculated with

Metarhizium spp. conidia in both laboratory and greenhouse

settings. In addition a traditional infection bioassay was per-

formed to determine the virulence of the fungal isolates

included in this study.

Materials and methods

Fungal preparation

Four isolates of Metarhizium spp. were included in this study.

Two isolates ofM. robertsii (Mr): ARSEF 2575 obtained from the

USDA-ARS Collection of Entomopathogenic Fungal Cultures

(ARSEF) (USDA Plant, Soil and Nutrition Laboratory, Ithaca,

NY, USA) and KVL 12-35, isolated from agricultural soil in

Denmark (Steinwender, 2013); and two isolates ofM. brunneum

(Mb): KVL 04-57, same genotype as the active ingredient of the

commercial isolate Met52 (Novozymes, Salam, Virginia), and

KVL 12-37, isolated from agricultural soil in Denmark

(Steinwender, 2013). All four isolates are maintained at �80 �Cat the University of Copenhagen andwere identified to species

using DNA sequencing following Bischoff et al. (2009).

Fresh cultures of each isolate were prepared for experi-

ments from a stock culture, each grown for 2e3 weeks on

Sabouraud Dextrose Agar (SDA) (Merck KGaA, Darmstadt,

Germany) at 26 �C. Conidia were then harvested with a sterile

spatula and suspended in 0.005 % Triton X-100 (Sigma

Chemical, MO, USA), the suspension was then passed

through glass wool to remove hyphae, conidial clumps and

loose agar. Each suspension was vortexed and conidial con-

centrations estimated by counting in a hemocytometer

(Fuchs-Rosenthal).

Conidial viability was checked by transferring 20 ml of the

suspension onto agar medium [4-ml SDA with 0.002 %

Benomyl active ingredient (SigmaeAldrich, MO, USA)] and

counting conidia germination after 48 hr at 26 �C; suspensionswere only used if germination was greater than 90 %.

Seed treatment assay

Seed treatmentsFor each isolate of Metarhizium a 1 � 108 conidia ml�1 sus-

pension was prepared and then diluted to create a second

suspension of 1 � 107 conidia ml�1. Wheat seeds (Triticum

aestivum) were pre-germinated to ensure viability and then

soaked for 1 hr in a fungal treatment or distilled water (con-

trol); they were then removed and allowed to dry on filter

paper at room temperature. A prescreening of several vari-

eties ofwheat indicated that surface sterilizing seeds removed

some but not all natural occurrence ofmicrobes; tomaintain a

realistic seed-borne microbial community the wheat seeds

were not surface sterilized.

Paper roll setupTen treated seeds were evenly spaced 2 cm from the top edge

of 2-ply filter paper (58 � 29 cm) (Frisenette ApS, Denmark)

which had been moistened to saturation with ddH2O. Seeds

were then secured by placing a strip of filter paper (7 � 58 cm)

along the top of the paper, whichwas rolled up into a tube and

placed standing upright with seeds in the top in a 600 ml

beaker with 200 ml ddH20, placing the seed approx. 22 cm

from the surface of thewater (Supplementary Fig S1). Each roll

was wrapped in a piece of aluminum foil to keep seeds in the

dark and reduce evaporation and then placed under a growth

lamp with 12/12 hr light/dark regime at room temperature

(21 �C � 2) for 14 d. The resulting data from this setup will be

referred to as either Paper 108 or Paper 107 according to the

spore concentration. The setup was repeated on four separate

occasions.

Soil-pot experimentIn the greenhouse, pots were prepared with sandy non-sterile

subsoil taken from the lower horizon (20e60 cm) of an Agru-

dalf soil at the University research farm in Taastrup, Den-

mark. The texture of the soil was 3.9 % clay, 4.7 % silt, 33.3 %

Metarhizium dispersal via roots 123

fine sand, 57.1 % coarse sand and 1 % humus. Forty treated

seeds were distributed over the surface of the soil in each pot

and allowed to grow in a greenhouse at 20 �C day/15 �C night

temperature regime with 12/12 hr light/dark. The soil was

screened for entomopathogenic fungi before the experiment

using insect baiting and plating suspensions on selective

media [CTC media (Fernandes et al., 2010) amended with

0.02 g dodine (Agriphar, Belgium)]. No Metarhizium or other

entomopathogenic fungal taxa were recovered, suggesting

that naturally occurring Metarhizium spp. should not be

expected in the experimental soil used. Roots, from the same

growing pot, were harvested at 2 and 4 weeks after planting

(w20 randomly selected plants per harvest) by gently remov-

ing the plant from the soil and shaking off loose soil. Roots

were placed in 50 ml centrifuge tubes and kept at 5 �C until

they were used for the bioassay the following day. The data

resulting from this setupwill be labeled: Pot2w 108, Pot2w 107,

Pot4w 108 or Pot4w 107 according to the time of harvest and

spore concentration. The setup was repeated on four separate

occasions.

Root/insect assayPetri plates (60 � 15 with vents, Greenbio One, Hungary) were

lined with a single sheet of filter paper (Size 55, VWR inter-

national, France) and moistened with 200 ml of ddH20. For

roots from the potted plants 0.2e0.3 g of root material was cut

2 cm below the seed, washed with distilled water to remove

loose soil, and added to each Petri plate (three replicate plates

per treatment were prepared). For root material from the

paper roll setup, 0.1e0.2 g of root material was cut 2 cm below

the seed and added to a Petri plate (three replicates per

treatment). These root portions were representative of a plant

root system starting 2 cm below the seed and extending

w10 cm down for a single plant, however, root material used

for the experiment consisted of several root laterals randomly

selected from different plants of the same harvest. After

adding the cut-root section to each Petri plate ten T. molitor

(Coleoptera: Tenebrionidae) larvae (Avifauna, Copenhagen,

Denmark) were added to each Petri plate. Further, a Petri plate

with 10 T. molitor larvae was prepared with 10e14 g of soil

collected from the potted plants setup, the soil was collected

immediately after the roots were harvested. All plates with

larvae were checked regularly and any dead insects were

removed, washed in ddH2O and placed separately in medicine

cups to check for mycosis.

Additionally a sample of root (of the sameweight as used in

the insect assay) was cut from each treatment group (those

originating from the potted plants were washed), homogen-

ized in 5 ml of ddH20, and 200 ml of this homogenate was

pipetted on to selective media (see above) and incubated for 2

weeks at 22 �C. Metarhizium colonies were identified and the

number of colony forming units (CFUs) assessed.

These procedures were repeated four times with the two

experimental setups on different weeks using new fungal

cultures, soil, seeds and insects.

Virulence bioassay

For each of the four isolates of Metarhizium spp. a

1 � 107 conidia ml�1 suspension was prepared and then serial

diluted to prepare a 1 � 104 conidia ml�1 suspension. As in

the Root/insect assay experiment, three Petri plates of ten

larvae (with moistened filter paper) were prepared for each

fungal treatment plus two controls (the two controls are

exactly the same and the results were combined for the

analysis). Ten T. molitor larvae were individually treated with

5 ml of the designated suspension by pipette and placed

together in a single Petri plate (30 treated larvae per treat-

ment). The Petri plates were then stacked as a treatment

group, secured with an elastic band and placed in a plastic

box with moist paper towels on the bottom to maintain high

humidity. The treated insects were then incubated at 26 �C in

the dark. Mortality was checked daily, dead insects were

removed and surface sterilized by dipping each cadaver for

2 s in 70 % ethanol, 2 s in 7e5 % sodium hypochlorite and

then rinsed three times in ddH2O. The cadavers were then

placed in medicine cups with moist filter paper in order to

observe mycosis. The infection bioassays were repeated

three times on different weeks with new fungal cultures and

insects. This study will be referred to as Bioassay 107 or

Bioassay 104 depending on dilution.

Statistical analysis

Mortality and mycosis data from the root/insect assay

experiment were analyzed using a mixed model (with nlme

library for R) with the fungal isolate as the effects factor and

trial as the random factor. Prior to the analysis, data were split

into groups according to growth conditions, harvest time

and fungal dose (Paper 108, Paper 107, Pot2w 108, Pot2w 107,

Pot4w 108 or Pot4w 107) and analyzed separately. Assump-

tions of normality and homogeneity were met without

transformations.

A Pearson correlation analysis was performed with the

root/insect assaymortality ormycosis data and the CFUs from

root homogenate plated on selective media. The analysis was

performed in RStudio (version 0.97.551) using the cor.test

procedure.

For the virulence bioassay, mortality of the fungal isolate

treatments relative to the control were calculated as hazard

ratios (HR; relative average daily risk of death) fit to the sur-

vival data using a mixed Cox Proportional Hazards (PH)

regression model (Cox, 1972). The Cox PH model is based on

the assumption that while the risk of death is different from

one group to another, it remains constantly proportional over

time. This proportion is expressed as the hazard ratio, where

a value of 1.0 indicates that the two treatments have the

same risk of death at any given time. Pairwise comparisons

between the fungal treatments were performed by setting

each isolate as the reference group in the mixed model and

generating HR values. The risk of death of one treatment was

considered significantly different from another if the p-value

was less than 0.01 (0.05 divided by five pairwise compar-

isons). These analyses were performed in Rstudio using the

coxme package.

Post-hoc comparisons for both the virulence bioassay sur-

vival data and the root/insect assay data were performed

using the ghlt function in the multcomp package for R. P-

values less than 0.05 were considered significant. All of the

analyses were performed in RStudio (version 0.97.551).

124 C.A. Keyser et al.

Results

Seed treatment assay

Paper rollsA significant effect of fungal seed treatment on T. molitor

larvae mortality after exposure to washed roots 14 d after

inoculation was observed in both Paper 107 and Paper 108

experiments (F ¼ 7.4, df ¼ 4, p < 0.0001, F ¼ 12.5, df ¼ 4,

p < 0.0001, respectively; Fig 1) with higher mortalities in

fungal treatments than controls except for isolate KVL 12-37

(Mb) in Paper 107 (Fig 1A). No mycosis was observed in the

dead insects from the control treatments (Table 1). For

mycosis a significant effect of fungal treatment was noted in

both Paper 107 and Paper 108 (F ¼ 5.5, df ¼ 4, p ¼ 0.0007,

F ¼ 11.6, df ¼ 4, p < 0.0001, respectively). In Paper 108 sig-

nificantly more mycosis than in the control was observed in

all isolates except ARSEF 2575 (Mr); while for Paper 107 only

KVL 04-57 (Mb) had significantly more mycosis than the

control (Table 1). It is noteworthy, however, that many of

the dead insects from the fungal-treated groups showed

signs of fungal infection (melanization spots on the cuticle;

C.A. Keyser, pers. obs.) but fungi did not emerge from

cadavers.

Soil-pot assayTwo weeks after planting, the effect of fungal treatment on

larval mortality for Pot2w 107 was not significant (F ¼ 2.4,

df ¼ 4, p ¼ 0.06) despite all of the fungal treatments resulting

in mean mortalities apparently higher than in the control

(Fig 2A). Larvae exposed to soil collected from the pots with

wheat plants grown from seed inoculated with

107 conidia ml�1 showed significant effect of treatment

(F ¼ 3.05, df ¼ 4, p ¼ 0.04), but only KVL 12-37 (Mb) showed

significantly higher mortality than the control (Fig 2B). For

Pot2w 108 a significant effect of fungal treatment was found

(F ¼ 5.5, df ¼ 4, p ¼ 0.0007); T. molitor larvae exposed to roots

grown from wheat seeds treated with either of the Danish

isolates [KVL 12-35 (Mr) and KVL 12-37 (Mb)] had significantly

higher mortality than the control (Fig 2C). Mortality of larvae

placed with soil from the same experiment was also sig-

nificantly affected by treatment (F ¼ 5.92, df ¼ 4, p ¼ 0.004)

with higher mortalities for both M. brunneum isolates and M.

robertsii isolate KVL 12-35 (Fig 2D). No mycosis was observed

in any of the control treatment groups for larvae exposed to

either roots or soil (Table 1); the effect of fungal treatment on

mycosis was significant for both larvae exposed to roots and

soil at the low and high doses (F ¼ 5.36, df ¼ 4, p ¼ 0.0011;

F ¼ 3.02, df ¼ 4, p ¼ 0.049; F ¼ 6.95, df ¼ 4, p ¼ 0.0001; F ¼ 6.95,

df ¼ 4, p ¼ 0.0019, respectively).

After 4 weeks of plant growth a significant effect of fungal

treatment for Pot4w 107 on mortality was observed when

larvae were exposed to washed roots (F ¼ 3.4, df ¼ 4, p ¼ 0.01).

Both isolates KVL 12-35 (Mr) and KVL 04-57 (Mb) showed sig-

nificantly more mortality than the control (Fig 3A). In the soil

experiment, the effect of fungal treatment was almost sig-

nificant (F ¼ 2.9, df ¼ 4, p ¼ 0.05) with KVL 04-57 (Mb) causing

higher mortality than the control (Fig 3B). For the Pot4w 108

experiment a significant effect was observed among the

fungal treatments (F ¼ 8.7, df ¼ 4, p < 0.0001) where KVL 12-35

(Mr) and KVL 04-57 (Mb) showed higher mortality than the

control (Fig 3C). In the soil treatment all of the fungal isolates

caused higher mortality than the control (F ¼ 8.46, df ¼ 4,

p > 0.0001; Fig 3D). Mycosis was not detected in any of the

control treatment groups for larvae exposed to either roots or

soil after 4 weeks (Table 1). The effect of fungal treatment on

mycosis was significant for both larvae exposed to roots at

low and high dose and soil at the high dose (F ¼ 2.96, df ¼ 4,

p ¼ 0.026; F ¼ 9.65, df ¼ 4, p < 0.0001; F ¼ 10.42, df ¼ 4,

p < 0.0001, respectively); no significant effect was observed

Fig 1 e Mean percent mortality (DSE) of T. molitor larvae

after exposure to roots of wheat plant grown from

Metahizium inoculated seeds for 14 d. Seeds were

inoculated by soaking for 1 hr in suspensions of either

1 3 107 (A) or 1 3 108 (B) conidia mlL1 or water (control).

Mortality was based on 30 larvae per treatment and each

treatment was repeated four times. Different letters on

bars indicate significant differences within each treatment

group.

Metarhizium dispersal via roots 125

for mycosis in the soil at the low dose (F ¼ 2.21, df ¼ 4,

p ¼ 0.112).

CFU counts on plates correlationsNo Metarhizium CFUs were observed on the root homogenates

from control plants when plated on selective media. Positive

correlations between the number of CFUs on selective media

and the mortality in the root/insect assay (Pearson r ¼ 0.38,

df ¼ 88, p ¼ 0.0002) and between CFUs and mycosis was

observed (Pearson r ¼ 0.42, df ¼ 88, p < 0.0001; data not

shown).

Virulence assay

A significant effect of treatment was observed in both Bio-

assay 107 and Bioassay 104 (c2 ¼ 641.84, df ¼ 4, p < 0.0001;

c2 ¼ 123.14, df ¼ 4, p < 0.0001, respectively). Higher mortality

was observed among all larvae treated with a fungal isolate as

compared to control treatments in both Bioassay 107 and

Bioassay 104 (HR ¼ 100.45, p < 0.001 and HR ¼ 5.8, p < 0.001,

respectively). The median survival time for the four fungal

treatments for Bioassay 107 was 5 d (Fig 4A); however, isolate

KVL 12-37 (Mb) was significantly less virulent than the other

isolates (Fig 4B). For Bioassay 104 the median survival time

could only be established for KVL 04-57 (Mb) for which it was

14 d while the other fungal treatments did not cause 50 %

mortality within the course of the experiment (Fig 4C). How-

ever, all of the fungal treatments were more virulent than the

control; KVL 04-57 (Mb) was statistically the most virulent

(Fig 4D).

No mycosis of Metarhizium was detected on insects from

the control group. Mycosis was observed on dead larvae from

all four fungal-treated groups (Table 2). Significantly more

mycosis was observed on larvae from Bioassay 107 than Bio-

assay 104 (F ¼ 20.31, df ¼ 1, p < 0.0001). For both Bioassay 104

and Bioassay 107 the effect of treatment on mycosis was

significant (F ¼ 23.6, df ¼ 4, p < 0.0001; F ¼ 165.1, df ¼ 4,

p < 0.0001, respectively).

Discussion

All of the Metarhizium isolates tested in this study maintained

pathogenicity towards T. molitor larvae after being dispersed

by roots through both an artificial growth substratum and

non-sterile soil. The ability of seed-treated Metarhizium to

disperse through soil via a root system is highly relevant for

biological control programs. Bruck (2005) stated that “by

developing techniques to use the roots as a delivery system for

entomopathogenic fungi, the cost and logistics of biological

control would be much more favorable”. In the current study

the relatively small quantity of conidia applied to each seed

was sufficient to inoculate both the washed root and the

surrounding soil with sufficient Metarhizium propagules to

initiate lethal infections.

In the experiment Bioassay 104, M. brunneum KVL 04-57

was the most virulent isolate tested. Several studies have

shown that the commercial product Met52 (¼F52), the same

genotype as KVL 04-57, has good efficacy against a wide range

of soil-dwelling insect pests (Bruck et al., 2005; Liu and Bauer,

2006; Jaronski and Jackson, 2008; Ansari and Butt, 2013).

However, in this study, none of the isolates were consistently

more virulent when applied as a seed treatment. Foster et al.

(2010) also observed that in laboratory studies the F52 strain

was highly virulent, however, in field trials the effectiveness

was reduced. While virulence observed in the laboratory is a

useful trait when comparing isolates it often does not trans-

late to field efficacy. The present results demonstrated that

isolates retained their pathogenicity even in non-sterile soil,

suggesting that fungal presence can potentially protect roots

from damage by herbivores.

CFU counts from the homogenized root samples indicated

that, for all three 108 conidia ml�1 root assays (Paper 108,

Pot2w 108, and Pot4w 108), the infection dose supplied to each

Petri dish of T. molitor larvae was equal to that administered to

the insects in Bioassay 104 (w500 propagules/10 larvae).

Although the results are not directly comparable, in Bioassay

104 the averagemortality ranged from 26 to 62% after 2weeks.

Table 1eMeanpercent of observedmycosis (±SE) byMetarhizium spp. among dead T.molitor larvae after exposure towheatroots or soil for 14 d. The seeds were grown in one of three conditions: paper rolls for 2 weeks, soil pots for 2 weeks, or soilpots for 4 weeks. Letters designate significant differences within column and treatment conditions

2 weeks 4 weeks

Paper Roots from soil Soil Roots from soil Soil

1 3 107 conidia mlL1

Control 0.0a 0.0a 0.0a 0.0a 0.0a

ARSEF 2575 (Mr) 9.2(�4.8)a 0.0(�0.0)ab 7.5(�7.5)ab 1.7(�1.7)a 25(�25.0)ab

KVL 12-35 (Mr) 15.0(�5.2)ab 8.3(�5.5)b 20(�11.5)ab 11.7(�6.7)b 20(�20.0)ab

KVL 12-37 (Mb) 9.2(�7.1)a 2.5(�2.5)ab 22.5(�14.3)b 0.0(�0.0)a 12.5(�7.5)ab

KVL 04-57 (Mb) 30.8(�17.1)b 8.3(�3.5)b 10(�5.7)ab 7.5(�5.5)ab 52.5(�21.3)b

1 3 108 conidia mlL1

Control 0.0a 0.0a 0.0a 0.0a 0.0a

ARSEF 2575 (Mr) 8.3(�7.3)ab 1.7(�1.7)ab 10(�10)ab 0.0(�0.0)a 37.5(�22.5)ab

KVL 12-35 (Mr) 32.5(�14.1)bc 31.7(�21.8)c 77.5(�16.5)c 46.7(�19.8)b 92.5(�4.7)c

KVL 12-37 (Mb) 29.2(�7.6)bc 20.8(�14.2)bc 37.5(�22.5)abc 20.8(�15.8)b 47.5(�15.4)bc

KVL 04-57 (Mb) 49.2(�3.7)c 3.3(�3.3)ab 55(�21.0)bc 29.2(�13.7)b 72.5(�24.2)bc

126 C.A. Keyser et al.

In Pot4w 108 the average mortality similarly ranged between

16 and 54 % after 2 weeks exposure to roots. These data

indicate that despite the complexity of the growing conditions

during the 4 weeks in non-sterile soil in greenhouse settings

(e.g., variable temperature, fluctuating moisture levels, com-

petition with other microorganism for resources, etc.), path-

ogenicity was not obviated in the soil.

Nitrogen acquisition by plants from an otherwise

unavailable source (insects) has been demonstrated as a result

of association with several entomopathogenic fungal species

(Behie et al., 2012; Behie and Bidochka, 2014) obviously to the

benefit for the plant. It is likely that the funguswill also benefit

from this association, and Metarhizium spp. have been shown

to grow in root exudate (Fang and St Leger, 2010; Pava-Ripoll

et al., 2011), suggesting that it is likely that the fungus profits

from the carbohydrate rich environment in the rhizosphere to

buildup biomass. However, we expect that the fungus ulti-

mately benefits from the association with the root by being

able to locate and infect new insect hosts where buildup of

biomass and infective units, i.e., conidia, is likely to be much

greater than in the rhizosphere. Since soil-dwelling insects are

likely to be more abundant in the nutrient rich rhizosphere

Fig 2 e Mean percent mortality (DSE) of T. molitor larvae after exposure to roots of wheat plants grown from Metarhizium

inoculated seeds (Roots) or experimental soil (Soil) for 14 d. Roots and soil were collected 14 d after planting. Seeds were

inoculated by soaking for 1 hr in suspensions of either 1 3 107 (A and B) or 1 3 108 (C and D) conidia mlL1 or water (control).

Mortality was based on 30 larvae per treatment and each treatment was repeated four times. Different letters on bars

indicate significant differences within each treatment group.

Metarhizium dispersal via roots 127

than elsewhere in the soil (Walker et al., 2003) the root asso-

ciation of Metarhizium can be regarded as a vehicle to

encounter suitable insect hosts by an otherwise immobile

entomopathogen.

In general, dispersion of hypocrealean fungi is limited and

largely depends on passive dispersal by abiotic means such as

wind or rain (Meyling and Eilenberg, 2007). One of the benefits

a root-associating fungus derives from the plant interaction is

thus below ground mobility. This study clearly demonstrated

that infectious Metarhizium material was dispersed by roots.

Future studies evaluating both the effects on the plant and the

fungus would be particularly interesting and helpful in

determining if the rootefungus interaction should be con-

sidered as mutualistic. Dispersal by roots has been shown for

othermicroorganisms aswell, while percolatingwater is often

an important factor in ground dispersal it is noted that root

systems are either vital to, or greatly improve dispersal

(Bashan and Levanony, 1987; Elsas et al., 1991; Liddell and

Parke, 1989; Trevors et al., 1990). In the current study it is

important to note that the traditional watering system

employed in the greenhouse-pot experiment possibly aided

fungal dispersal, however, in the paper roll study moisture

was from below the growing plant. A further advantage of the

system utilized in this study is that it provides a platform to

A B

C D

Fig 3 e Mean percent mortality (DSE) of T. molitor larvae after exposure to roots of wheat plant grown from Metarhizium

inoculated seeds (Roots) or experimental soil (Soil) for 14 d. Seeds were inoculated by soaking for 1 hr in suspensions of

either 13 107 (A) and (B) or 13 108 (C) and (D) conidia mlL1 or water (control). Mortality was based on 30 larvae per treatment

and each treatment was repeated four times. Different letters on bars indicate significant differences within each treatment

group.

128 C.A. Keyser et al.

screen entomopathogens for effectiveness. While none of the

four isolates tested in the present plant interaction studies

were consistently more virulent than the others, the potential

to compare isolates to each other in a more realistic and

complex setting than a typical infection bioassay is evident.

Effective and cost efficient biological control agents such as

Metarhizium are an important aspect of current and future pest

management practices. A concerted effort to improve the

ecological understanding of these fungi will both improve

efficient application (e.g., seed treatments vs. field cover) and

ensure responsible usage. While much is still unknown, it is

clear thatMetarhizium spp. play both a complex and important

ecological role in the environment. The holdover paradigm,

from conventional pesticide practices, of only considering the

host/pathogen relationship is too limited in a biological con-

trol context. Wemust continue to increase our understanding

of the interactions between the biocontrol agent and themany

other organisms in those systems; such as, the plants we

intend them to protect, naturally occurring biota, and other

Fig 4 e Infection bioassay with four strains of Metarhizium. Mean survival of T. molitor larvae over 14 d after treatment with

5 ml of triton X (control) or a fungal suspension of either 1 3 107 or 1 3 104 conidia mlL1 (A, C). Pairwise comparisons of all

treatments within dose were determined using cox PHmodel and hazard ratios (relative risk of death) were calculated (B, D).

Values within each box indicate hazard ratios of the treatment on the y-axis in comparison to the treatment on the x-axis.

Two treatments that share a black square have a hazard ratio in relation to each other that is significantly different (D or L)

from one.

Table 2 e Mean percent of observed mycosis (±SE) byMetarhizium spp. among dead T. molitor larvae. Larvaewere treated with 5 ml of Triton X (control) or a fungaltreatment of either 1 3 104 or 1 3 107 conidia mlL1.Different letters designate significant differences withintreatment dose

Bioassay 104 Mycosis

Control 0(�0.0)a

ARSEF 2575 (Mr) 16.1(�16.2)a

KVL 12-35 (Mr) 68.3(�25.2)b

KVL 12-37 (Mb) 71.3(�20.0)b

KVL 04-57 (Mb) 83.4(�12.3)b

Bioassay 107 Mycosis

Control 0(�0.0)a

ARSEF 2575 (Mr) 84.4(�16.9)b

KVL 12-35 (Mr) 98.9(�1.9)b

KVL 12-37 (Mb) 94.4(�4.2)b

KVL 04-57 (Mb) 93.3(�4.1)b

applied biological agents e.g., predators, parasitoids, or

antagonists of plant pathogens.

Acknowledgments

We would like to give thanks to Jesper Andersen, Azmi Mah-

mood and Louise Lee Munk Larsen for their technical assis-

tance in this study and to Dr. Bernhardt M. Steinwender for

fruitful discussions. We also express gratitude to Richard

Humber for sending us several fungal cultures from the ARSEF

collection. This research was funded by a PhD grant to C.A.K

from the Plant Biosystems Elite Environment at the University

of Copenhagen.

Supplementary data

Supplementary data related to this article can be found at

http://dx.doi.org/10.1016/j.funeco.2014.05.005.

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Metarhizium dispersal via roots 131

63

Manuscript 2

BEST OF BOTH WORLDS: DUAL EFFECTS OF METARHIZIUM SPP. AND CLONOSTACHYS ROSEA

AGAINST AN INSECT AND A SEED BORNE PATHOGEN IN WHEAT

Chad A. Keyser, Birgit Jensen & Nicolai V. Meyling

Status: Under Review - Pest Management Science, submitted 22 Dec, 2014

64

Best of both worlds: Dual effects of Metarhizium spp. and Clonostachys rosea against an insect

and a seed borne pathogen in wheat

Chad A. Keyser1*, Birgit Jensen

2, and Nicolai V. Meyling

1

1Department of Plant and Environmental Sciences, Section for Organismal Biology, University of

Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

2Department of Plant and Environmental Sciences, Section for Genetics and Microbiology,

University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

*Corresponding author: Chad A. Keyser, Department of Plant and Environmental Sciences,

University of Copenhagen, Thorvaldsensvej 40,

1871 Frederiksberg, Denmark

Email: chadaltonkeyser@gmail.com

Running title

Dual effects of Metarhizium spp. and Clonostachys rosea

Keywords

Seed treatment; biological control, Fusarium culmorum, entomopathogenic fungi, mycoparasite

SUBMITED: PEST MANAGEMENT SCIENCE – 22 December 2014

65

Abstract

BACKGROUND: Crops are often prone to both insect herbivory and disease which necessitate

multiple control measures. Ideally an efficacious biological control agent must adequately control

the target organism and not be inhibited by other biological control agents when applied

simultaneously. Wheat seeds infected with the plant pathogen Fusarium culmorum were treated

with Metarhizium brunneum or M. flavoviride and Clonostachys rosea individually and in

combination with the expectation to control both root-feeding insects and the pathogen,

respectively. Roots emerging from seeds were evaluated for disease and then placed

with Tenebrio molitor larvae which were monitored for infection.

RESULTS: Plant disease symptoms were nearly absent for seeds treated with C. rosea, both

individually and in combination with Metarhizium spp. Furthermore, roots grown from seeds

treated with Metarhizium spp. caused significant levels of fungal infection in larvae when used

individually or combined with C. rosea. However, co-treated seeds showed reduced virulence

towards T. molitor as compared to treatments with Metarhizium spp. only.

CONCLUSIONS: This study clearly shows that seed treatments with both the entomopathogenic

fungus M. brunneum and the mycoparasitic fungus C. rosea can protect plant roots from insects and

disease. The dual-treatment approach to biological control presented here harmonizes well with the

ideals of IPM strategies.

66

1. INTRODUCTION

Crop production is consistently under pressure from various harmful organisms, including

plant pathogens and arthropod pests. Wheat is the most important cereal crop in the northern

hemisphere, Australia and New Zealand.1 Although current breeding and treatment programs have

greatly reduced the potential loss growers are in need of new and effective means for crop

protection.2 While use is continually increasing, in general, biological control agents (BCAs) of

pests and pathogens in agriculture have yet to become a mainstream option for crop producers.3

Many factors contribute to the reticence of the agricultural industry to turn full-heartedly to

biocontrol, including a lack of efficacy in consistent field performance and lack of economic

viability.3 An increased emphasis on fundamental research, especially in the areas of biology and

ecology of the beneficial organisms, has the potential to greatly improve biocontrol.3, 4

One

important question that needs to be addressed is whether BCAs of plant pests and pathogens can be

used simultaneously without mutually inhibiting the efficacy of the other BCA.5, 6

For biological control of arthropod pests members of the entomopathogenic fungal genus

Metarhizium (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) are among the most promising

candidates.7 Metarhizium spp. have a worldwide distribution, a wide host range and have been

found to be effective against many important agricultural pests, including: grasshoppers, locusts,

mosquitoes, spittlebugs, thrips, white flies, and even ticks.7, 8

Furthermore, recent research has

highlighted their important role as root-colonizers with potential to aid in nutrient acquisition and

plant-growth promotion.9-11

For many important plant diseases Clonostachys rosea (Link:Fr.) Schroers, Samuels, Seifert

and Gams (syn. Gliocladium roseum Bainier) is a highly effective BCA; like Metarhizium, C.

rosea has a cosmopolitan distribution and is often naturally found in agricultural soil.12

C. rosea is

a broad-spectrum mycoparasite effective at reducing the growth and associated diseases of several

plant pathogenic fungi including: Alternaria spp., Bipolaris sorokiniana, Botrytis cinerea,

Fusarium culmorum, Sclerotinia sclerotiorum, and Verticillium dahlia.13-18

Several modes of

action towards other fungi have been attributed to C. rosea, including: nutrient competition,

mycoparasitism, induced resistance of the plant, and antimicrobial metabolite production and

detoxification of mycotoxins.12, 15, 19-21

Furthermore, many studies have prescribed strategies for

implementing C. rosea as a BCA.18, 22, 23

Clearly Metarhizium spp. and C. rosea have great

potential for use as BCAs in agriculture.

67

It is often necessary for agricultural producers to combat arthropod pest and plant pathogens

simultaneously. As Metarhizium spp. and C. rosea based products become more common it is

likely that they will be applied against different harmful organism (i.e., pests and diseases) on the

same crop; it is thus becoming increasingly important to understand how they might interact. As a

general mycoparasite C. rosea could potentially have deleterious effects on Metarhizium spp.

efficacy. In fact one study observed that M. anisopliae sensu lato was highly susceptible to several

mycoparasites including Clonostachys spp. in an in vitro host-range test.24

However, they also

found that co-application with M. anisopliae s.l. generally enhanced mycoparasite recovery from

the host insect and that virulence of the entomopathogenic fungi was not significantly altered.24

Additionally, C. rosea has been reported as an entomopathogenic fungus,25

and therefore may

contribute to biological control of arthropod pests. Also, at least one study reported that M.

robertsii has antifungal properties and was able to reduce Fusarium solani f. sp. phaseoli infections

in bean.26

Co-application of Metarhizium spp. and C. rosea could therefore have either

antagonistic, neutral or additive effects on their respective biological control efficiency;

undoubtedly investigations in to how these fungi interact when co-applied are highly important.

The aim of this study was to assess the effectiveness of two BCAs applied in combination as

seed treatments to control an insect pest and a plant pathogen. This was tested by treating

pathogen-infected seeds of two wheat varieties with a combination of C. rosea and Metarhizium

spp. (either M. brunneum or M. flavoviride). The seeds were grown under laboratory conditions

and the resulting plants were evaluated for disease development and then the roots were fed to

insect larvae which were monitored for mortality and fungal infection. Additionally, direct-

inoculations of insect larvae with the various individual and combined fungal treatments were

performed to evaluate the entomopathogenic potential by the fungi in isolation and together.

2. EXPERIMENTAL METHODS

2.1 Organisms

Five species of fungi were used in various experiments throughout this study. Three species

from the genus Metarhizium were included: M. brunneum (Mb) isolate KVL 04-57 which has the

same origin as the active ingredient of the commercial isolate Met52 (Novozymes, Salam,

Virginia);27

M. flavoviride (Mf) KVL 14-112, isolated in 2013 from Danish soil closely associated

with wheat roots using Tenebrio molitor L. (Coleoptera: Tenebrionidae) as bait insect; and M.

robertsii (Mr) KVL 12-35, isolated from Danish agricultural soil using T. molitor as a soil bait

insect.28

Additionally, one isolate of Clonostachys rosea (Cr) IK726 isolated from barley root,13

68

and one isolate of the wheat pathogen Fusarium culmorum (Fc) IK5 isolated from barley seed was

included.29

All fungal isolates are stored at -80 oC at the University of Copenhagen.

Two varieties of wheat (Triticum aestivum L.) were used in this study, ‘Jensen’ and

‘Nakskov’ supplied by Nordic Seed, Denmark. The seed lots were stored at -20 ºC. Before use

seeds were surface sterilized by soaking for 30 s in 70% ethanol and then 10 min in 4% sodium

hypochlorite (Sigma Chemical, MO, USA), followed by rinsing 3 times in ddH2O and air drying

overnight in a ventilation hood.

Insect infection assays were performed using T. molitor larvae (Avifauna, Copenhagen,

Denmark).

2.2 Fungal suspensions

Fungal suspensions were prepared for each of the five fungal species using fresh cultures of

each isolate which had been grown for 2-3 weeks on Potato Dextrose Agar (PDA) (Sigma

Chemical, MO, USA) at 24º C. Conidia were harvested with a sterile spatula and suspended in

0.05% Triton X-100 (Sigma Chemical, MO, USA), the suspension was then passed through cheese

cloth to remove hyphae, conidial clumps and loose agar. Each suspension was vortexed and

conidial concentrations estimated by counting in a haemocytometer (Fuchs-Rosenthal). Conidial

viability was determined by pipetting 20 µl of a diluted portion of the suspension onto agar medium

[4-ml SDA with 0.002% Benomyl active ingredient (Sigma-Aldrich, MO, USA)] and counting

conidia germination after 48 hours at 24ºC; all fungal isolates were observed to have greater than

95% viability.

2.3 Seedling disease and root-fed insect assay

A F. culmorum suspension of 1.5 × 105 conidia/ml was used to inoculate about 200 surface-

sterilized wheat seeds; the seeds were soaked for 30 min at 150 rpm in the F. culmorum suspension

(1:2 w/v). This was done for both ‘Jensen’ and ‘Nakskov’ varieties of wheat. Additionally, ~200

seeds of both varieties were soaked for 30 min in ddH20 as a non-infected control group. The seeds

were then removed from the suspension and allowed to air dry in a ventilation hood, after which

they were kept at 5 ºC and used within 10 days of preparation; this same batch of seeds was used for

both repetitions below.

The following 6 treatments of M. brunneum, M. flavoviride and C. rosea were prepared:

0.05% Triton X (control – no fungus), Mb, Mf, Cr, Mb+Cr, and Mf+Cr. Each fungal suspension

was prepared so that the final concentrations of Metarhizium spp. conidia were 1 × 108 conidia/ml

and the final concentration of C. rosea was 1 × 107 conidia/ml. These concentrations of

69

Metarhizium spp. and C. rosea were selected based on previous work showing effectiveness at

controlling their target insect or pathogen.13, 30

A surplus of both Fc infected and uninfected seeds

were soaked separately in each of the fungal suspension (or ddH2O for untreated control) for 5 min,

giving 12 separate treatments for each wheat variety. Twelve treated seeds were then evenly spaced

2 cm from the top edge of a 2-ply filter paper (58 × 29 cm) (Frisenette ApS, Denmark) which had

been moistened to saturation with ddH2O. The seeds were secured by placing a strip of filter paper

(7 × 58cm) along the top of the paper, which was rolled up in to a tube and placed standing upright

in a shallow reservoir of water, which was maintained throughout the experiment at approx. 5 cm in

depth. Each roll was wrapped in a piece of aluminum foil to keep seeds in the dark and reduce

evaporation and then placed under a growth lamp with 12/12 h light/dark regime at room

temperature (21±2 ºC) for 14 days. For each treatment combination (Fc/No-Fc, Mb/Cr, Mf/Cr

combinations, and wheat varieties), two pseudo-replicate paper rolls were prepared resulting in 48

paper rolls prepared for each experimental repetition. Additionally, 5-6 seeds from each treatment

were placed on PDAY media to check for seed viability and the presence of the applied fungus

(Supplemental Figure 1).

On day 13 each paper roll was unrolled and plants were assessed for disease severity using

an index from 0 to 4 as previously suggested,13

viz.: 0=healthy plant, 1= slightly brown

coleoptile/roots, 2=moderately brown coleoptile and/or roots, 3=severe browning of the coleoptile

and/or roots, 4=dead plant (Supplemental Figure 2). All disease assessments were done by the

same person to avoid subjective inconsistencies; also the treatments were assessed randomly and

blindly to avoid any treatment expectations. After the assessment the paper rolls were re-rolled and

incubated for one additional day. This entire procedure was repeated twice at different times with

new plants and fungal suspensions.

Insect assay were performed using the methods described by Keyser et al.30

In short, petri

plates (60×15 with vents, Greenbio One, Hungary) were lined with a single sheet filter paper (Size

55, VWR international, France) and moistened with 200 µl of ddH20; for each treatment 3 replicate

plates were prepared. The top 10 cm, starting just below the seed, for all of the roots from a single

treatment (both pseudo-replicate rolls) were combined, cut into 1-2 cm pieces, mixed together, and

0.2-0.3 g of root material was added to each of the three replicate petri plate per treatment. After

adding the cut-root sections, ten T. molitor larvae were added to each plate. Each treatment set of 3

petri plates were stacked with a forth empty plate below, rubber banded together and placed upright

in a plastic container containing moist paper towels for humidity. Mortality was assessed daily and

70

the order of the petri plates in each stack was cycled each day with the empty plate always on the

bottom. Dead insects were removed and placed in medicine cups with moist filter paper to observe

for mycosis. The dual concept of the experimental design is outlined in Supplemental Figure 3.

2.2 Insect infection bioassay

Two separate infection bioassay experiments were performed: a low dose (1 × 105

conidia/ml) and a high dose (1 × 107 conidia/ml). For each experiment the following 16 treatments

were prepared: 0.05% Triton X (control), Mb, Mf, Mr, Cr, Mb+Cr, Mf+Cr, Mr+Cr, Fc, Mb+Fc,

Mf+Fc, Mr+Fc, Cr+Fc, Mb+Cr+Fc, Mf+Cr+Fc, and Mr+Cr+Fc. For each bioassay the fungal

treatments were prepared so that each fungal species included in the particular treatment had the

same number of conidia per ml; e.g., in the low dose treatment Mb+Cr had 1 × 105 conidia/ml of M.

brunneum and 1 × 105 conidia/ml of C. rosea. Suspensions were kept at 5 ºC until use which was

always within 24 h of preparation.

Thirty T. molitor larvae were prepared in three petri plates as in the root-insect assay above.

In each petri plate the 10 larvae were individually inoculated by pipetting 5 µl of a designated

suspension. Mortality was assessed daily and the order of the petri plates in each stack was cycled

each day. Dead insects were removed and placed in medicine cups with moist filter paper in order

to observe mycosis. This entire procedure was repeated two or three times for the 1 × 105

conidia/ml and for the 1 × 107 conidia/ml doses, respectively.

2.4 Statistics

For the root assay data and the infection bioassay data the virulence of the treatment groups

to T. molitor was estimated using a survival analysis.31

A comparison of the survival curves was

performed for biologically distinct groups of treatments. For the infection bioassay each conidial-

concentration bioassay was analyzed separately, for each concentration there were four distinct

groups, including, 1 (no Metarhizium): Untreated control, Cr, Fc, and Cr+FC; 2 (M. brunneum):

Mb, Mb+Cr, Mb+Fc, and Mb+Cr+Fc; 3 (M. flavoviride): Mf, Mf+Cr, Mf+Fc, and Mf+Cr+Fc; and 4

(M. robertsii): Mr, Mr+Cr, Mr+Fc, and Mr+Cr+Fc. For the root assay three groups were evaluated

separately, these were the same as the above groups 1, 2 and 3. The following procedures were

used to analyze each of the groups: first, a cox proportional hazard (PH) model was performed on

the survival curves; next, a chi-square test of the PH model was used to determine if there was

significant variation between the treatments within the model; lastly, if the chi-squared test yielded

a p-value less than 0.05 a pairwise comparisons was done using a cox mixed model with trial as the

random factor, and the p-values were adjusted using the Bonferroni method.

71

The data from the disease index was viewed as categorical; therefore each seed represented

an individual data point and was not averaged with other seed of the same treatment. To simplify

the analysis of the disease severity data was divided into two classes, plants receiving an index

value of 0 or 1 and plants designated as 2 or greater on the disease index scale. A chi-squared test

for independence was performed to determine whether there were differences in among the 12

fungal-treatment combinations. Further analyzes were then performed on two specific treatment

groups; viz. group 1: untreated, Mb, Mf, Cr, Mb + Cr, Mf + Cr, Cr + Fc, Mb + Cr + Fc, and Mf + Cr

+ Fc; and group 2: Fc, Mb + Fc, and Mf + Fc. This additional analysis was done using a

generalized linear mixed model (glmer) in R.

Fungal mycosis on dead larvae between treatments was evaluated using a generalized linear

model for binomial data. The proportions of dead insects with mycosis to dead insect with no

mycosis between treatments were compared. All of the analyses were performed in RStudio

(version 0.97.551).

3. RESULTS

3.1 Root-insect assay

When comparing the survival curves for ‘Jensen’ and ‘Nakskov’ wheat varieties separately,

Cr and Fc treatments which did not include Metarhizium spp. were not significantly different from

each other and the untreated control (2 = 6.9, df = 3, p = 0.07; and

2 = 0.45, df = 3, p = 0.9,

respectively) (Figs. 1a and 1b). In contrast, there was a significant effect of treatment for ‘Jensen’

and ‘Nakskov’ when comparisons included the untreated control and Cr and Fc treatments

combined with M. brunneum (2 = 133.6, df = 4, p < 0.0001; and

2 = 144.5, df = 4, p < 0.0001,

respectively); for both varieties the untreated control had significantly more survival (>95%) than

the other treatments. For both varieties the highest virulence was observed when the seeds were

treated with M. brunneum alone (Figs. 1c and 1d).

A significant effect of treatment was observed for both ‘Jensen’ and ‘Nakskov’ wheat in

comparisons which included the untreated control and Cr and Fc in combination with M.

flavoviride (2 = 49.6, df = 4, p < 0.0001; and

2 = 38.5, df = 4, p < 0.0001, respectively).

However, the treatments including F. culmorum did not reduce survival in comparison to the

untreated control of ‘Jensen’ (Fig. 1e) while all treatments that included M. flavoviride differed

from the control for the ‘Nakskov’ variety (Fig. 1f).

An analysis of the proportion of dead insects with observed mycosis to the total number of

dead insects in ‘Jensen’ indicated that there was an effect of treatment when either M. brunneum (2

72

= 9.6, df = 3, p = 0.02) or M. flavoviride (2 = 54.6, df = 3, p = 0.006) were included in the

treatments. However, an adjusted pairwise comparison of the treatments that included M.

brunneum was unable to identify significant differences among the treatments (Fig. 2a). For M.

flavoviride a significantly greater proportion of dead insects with observed mycosis were detected

when exposed to M. flavoviride alone than for the combined Mf+Cr treatment (Fig. 2b).

Mycosis on dead insects exposed to the roots of ‘Nakskov’ had a significant effect of fungal

treatment when M. brunneum (2 = 10.4, df = 3, p = 0.01) and M. flavoviride (

2 = 10.9, df = 3, p =

0.01) were included. A pairwise comparison revealed that for the M. brunneum treatments a

significantly greater proportion of dead insects had mycosis when treated with M. brunneum alone

than in the combination Mb+Cr (Fig. 2c). When the treatment included M. flavoviride only, a

significantly greater proportion of dead insects had mycosis than when combined with F. culmorum

(Fig. 2d).

3.2 Root disease

There was a significant effect of treatment on the root disease severity (2 = 679.8, df=11,

p<0.0001, Fig. 3). However, a subsequent analysis revealed that there were no differences between

the three treatments that included F. culmorum but did not include C. rosea (i.e., Fc, Mb + Fc, and

Mf + Fc) (2 = 184.3, df=2, p=0.85). Furthermore, the six treatments without F. culmorum and the

three treatments including both F. culmorum and C. rosea did not differ significantly (i.e., Mb, Mf,

Cr, Mb+Cr, Mf+Cr, Cr+Fc, Mb+Cr+Fc, and Mf+Cr+Fc) (2 = 59.6, df=8, p=0.26).

3.3 Infection bioassay of directly inoculated larvae

3.3.1 Low dose: No effects on T. molitor survival of Cr and Fc treatments were observed

when Metarhizium spp. were not included (2 = 2.2, df = 3, p = 0.52) (Fig. 4a). An analysis

evaluating the untreated controls with the treatments that included M. brunneum, M. flavoviride or

M. robertsii showed a significant decrease in survival for all three Metarhizium species (2 = 179.1,

df = 4, p < 0.0001; 2 = 48, df = 4, p < 0.0001;

2 = 145.1, df = 4, p < 0.0001, respectively) (Figs.

4c, 4e, and 4g). An analysis of the treatment combinations that included either of the Metarhizium

spp. isolates (excluding the untreated control) revealed no significant effect of treatments that

included M. brunneum (2 = 0.34, df = 3, p = 0.95) (Fig. 4c) as well as the treatments that included

M. flavoviride (2 = 5.81, df = 3, p = 0.12) (Fig. 4e). However, a significant effect was observed for

the treatments which included M. robertsii (2 = 9.1, df = 3, p = 0.02) where M. robertsii in

combination with F. culmorum had reduced virulence as compared to M. robertsii when used alone

(Fig. 4g).

73

3.3.2 High dose: An effect of treatment on T. molitor survival was observed when

Metarhizium spp. isolates were not included (2 = 13.8, df = 3, p = 0.003), and pairwise

comparisons revealed that the untreated control was significantly different from the Cr+Fc

treatment (z = 2.9, p = 0.017); after 14 days the average mortality of the Cr+Fc treatment was

18.9% whereas the average mortality of the control group was 3.3% (Fig. 4b). An effect of

treatment was observed in all groups in an analysis evaluating the untreated controls and treatments

including either M. brunneum, M. flavoviride, or M. robertsii (2 = 253.3, df=4, p < 0.0001;

2 =

187.6, df = 4, p < 0.0001; 2 = 277.9, df = 4, p < 0.0001, respectively). Pairwise comparisons

among these treatments revealed that the untreated control differed significantly from all treatments

that contained either Metarhizium species (Figs. 4d, 4f, and 4h). An additional analysis was

performed comparing the treatment combinations with each Metarhizium spp. isolate but excluding

the untreated control group. No significant difference was observed for those with M. brunneum or

M. robertsii (2 = 1.1, df = 3, p = 0.77;

2 = 0.7, df = 3, p = 0.86, respectively). However, an effect

of treatment was observed for those with M. flavoviride (2 = 14.3, df = 3, p = 0.002); in a pairwise

comparison of the treatments M. flavoviride alone was more virulent than when F. culmorum was

included in the treatments (z = 3.8, p = 0.0007; and z = 4.1, p = 0.0002, respectively) (see fig. 4f).

3.3.3 Mycosis: No significant effect of treatment on the proportion of dead insects with

mycosis to all dead insects was observed for M. brunneum or M. robertsii treatments at the low

dose (2 = 2.14, df = 3, p = 0.54;

2 = 7.53, df = 3, p = 0.056, respectively (Figs. 5a and 5c). At the

low dose for the M. flavoviride treatments a significant effect for treatment was observed (2 = 7.97,

df = 3, p = 0.046), however a pairwise comparison with adjusted p-values failed to identify the

differences (Fig. 5b). For the high dose when the treatments that included M. brunneum were

compared to each other a difference in the proportions for the overall model was observed (2 =

49.9, df = 3, p = 0.03), however a pairwise comparison with adjusted p-values failed to identify the

differences (Fig. 5d). There was an effect of treatment for the proportion of dead insects with

mycosis for those treated with M. flavoviride (2 = 90, df = 3, p < 0.0001), with the M. flavoviride

only treatment showing higher proportion of mycosis than when combined with other fungi (Fig.

5e). No significant effect of treatment on the proportion of dead insects with mycosis to dead

insects was observed for the treatments with M. robertsii (2 = 4.36, df = 3, p = 0.224) (Fig. 5f).

4. DISCUSSION

An integrated biological control treatment of the entomopathogenic fungi M. brunneum or

M. flavoviride combined with the mycoparasite C. rosea on wheat seeds resulted in almost complete

74

reduction of F. culmorum plant infections; and, in the case of the M. brunneum/C. rosea treatment,

insect mortality after exposure to wheat roots as high as 80%. These data clearly show that

cooperative biological control of two very different harmful organisms can be achieved utilizing the

combination of M. brunneum and C. rosea as wheat seed treatment. Others studies have also shown

that mixtures of BCAs can be effectively used against pests and pathogens,24, 32, 33

but this is the

first to focus on a seed borne pathogen and insects feeding on roots.

Survival of insects exposed to roots from treatments of M. brunneum combined with other

fungi was greater than those in the M. brunneum only treatment. However, when the same fungi

were exposed directly to the insects the virulence of M. brunneum was not affected by combination

with other fungi. These results could suggest that the reduced virulence seen in the plant assay was

either due to an indirect interaction (e.g., competition for resources) or a direct antagonism by the

other fungi as consequence of lower acquired dose of M. brunneum by the insects in the plant assay

allowing for the manifestation of subtle effects. The first hypothesis is supported by the observation

that the reduced virulence was seen in all combinations not just those with C. rosea, indicating that

the response is not specifically associated with the mycoparasitic ability of C. rosea. Krauss et. al

found that two isolates of M. anisopliae s.l. were susceptible to C. rosea mycoparasitism,24

however

this was tested in vitro by observing whether C. rosea was able to grow on agar media already

colonized by M. anisopliae s.l. While this method does indicate mycoparasitism against M.

anisopliae s.l., it is unlikely to effectively predict how the two fungi will interact when neither is yet

established and/or under more natural conditions. In fact in crop production systems there will be a

strong competition from the native soil microbiota. As in the current study, Krauss et al. observed

that co-inoculation of the two fungi did not reduce Metarhizium spp. virulence to insects.24

In the plant-insect assay with M. flavoviride some of the combined fungal treatments had

greater survival than the M. flavoviride only treatment; in fact on the wheat variety ‘Jensen’,

combinations of M. flavoviride with F. culmorum and these two fungal species combined with C.

rosea did not differ significantly from the untreated controls. We also observed comparable trends

for the same fungal treatments in the direct insect inoculation assay at the high dose. The isolate of

M. flavoviride used in this study was selected because it was observed to form inhibition halos

when grown on agar media with F. culmorum (C.A. Keyser, pers. obs.), indicating that there might

be some antifungal compounds produced by one or both of the species which could account for the

reduced virulence observed in the combined treatments. However, there was still a relatively high

level of infection of the directly-inoculated insects in the Mf+Cr, Mf+Fc and Mf+Fc+Cr high-dose

75

treatments suggesting that any direct interaction between the fungal isolates was likely minimal.

The particular isolate of M. flavoviride was previously found to be less virulent than the isolate of

M. brunneum against T. molitor larvae;30

a relatively low dose resulting in limited infection in the

host mortality is likely to be sensitive to effects of other fungi which might account for the reduced

virulence in the combination treatments.

Isolates of C. rosea and Fusarium spp. have been observed to infect insects,25, 34

however in

this study there was no indication that any of the C. rosea or F. culmorum isolates used were

pathogenic towards T. molitor. Although the direct inoculation assay at the high dose showed that

insects exposed to C. rosea and F. culmorum had significantly lower survival than the untreated

control, no fungal mycosis from the dead insects was detected suggesting that the slightly lower

survival was not due to fungal pathogenicity as defined by Koch’s postulates.35

Furthermore, this

observation could not be ascribed to inability of the two fungi to colonize wheat roots since dilution

plating on semi-selective media of root subsamples from the feeding experiments confirmed that

both fungi were present on roots grown from seed treated with the respective fungi (data not

shown). In a study with leaf-cutting ants it was shown that a combination of a virulent isolate of M.

anisopliae s.l. with an avirulent isolate of Aspergillus flavus resulted in the same level of virulence

as a M. anisopliae s.l. only treatment despite the combined treatment having only half as many M.

anisopliae s.l. conidia as the latter.36

They also observed that in the combined treatment more dead

insects showed mycosis by A. flavus than M. anisopliae s.l. and concluded that the avirulent A.

flavus, once inside the host, was able to out-compete the M. anisopliae s.l. isolate when the host

immune responses were compromised.36

In the present study, both C. rosea and F. culmorum

isolates grow more rapidly on artificial media than Metarhizium spp. isolates at equal conditions

(C.A. Keyser, pers. obs.); however, no C. rosea or F. culmorum mycosis was observed in any

insects, suggesting that the same mechanism of co-infection observed by Hughes and Boomsma is

not applicable to our fungal combinations.36

It could be speculated that the two fungal isolates

produce metabolites being toxic to the larvae.

The isolate of C. rosea was a highly effective BCA of F. culmorum in the current study,

corroborating results of other studies using the same isolate.37

Combination with either of the

Metarhizium spp. isolates did not affect the efficacy of C. rosea in reducing F. culmorum disease

manifestations. However, since the dose of C. rosea used in this study was sufficient to almost

completely control the plant pathogen any potential additive effects derived from Metarhizium spp.

would likely not be observed. Furthermore, additive effects would be unlikely to expect using the

76

current Metarhizium spp. isolates, since neither of the isolates tested in the plant assay were

observed to have an effect against F. culmorum alone. Antagonism against F. solani in bean plants

by an isolate of M. robertsii has been reported.26

In that study a significant reduction in F. solani

infection on bean roots was observed when sterile soil was pre-inoculated with F. solani and M.

robertsii compared to F. solani only treatments. This study also reported reduced germination of F.

solani conidia when grown in M. robertsii cell-free culture media; from this they concluded that the

actively growing M. robertsii isolate produced compounds that inhibited F. solani growth.26

While

no indication of Metarhizium spp.-mediated antagonism against F. culmorum was observed in the

current study, several important differences exist between the two studies which could have

accounted for the different results, including: species of Metarhizium and Fusarium used,

experimental plant species, inoculation method, and the fact that Sasan and Bidochka allowed an

establishment period for M. robertsii and F. solani in the soil before introducing the bean seeds.26

Biological control of plant pests and pathogens has enormous potential to improve

sustainable agricultural production and harmonizes with the ideals of IPM strategy. Nevertheless,

in order for BCAs to be used effectively and consistently we must improve our basic understanding

of how they interact in the field - especially how they work when applied in combination. While

further studies in soil microcosms and under field conditions are necessary, the current study clearly

demonstrated that entomopathogenic fungi from the genus Metarhizium and the plant pathogen

mycoparasite C. rosea can potentially be used in concert to control both belowground insects and

seed borne pathogens in a single treatment.

5. ACKNOWLEDGEMENTS

We would like to give special thanks to Line Lykke, Lærke Thordsen, Martina Falagiarda,

Louise Lee Munk Larsen for their technical assistants in this study. We would also like to give

thanks to Dr. Bernhardt M. Steinwender for his theoretical input and contribution into the

experimental design. This research was funded by a PhD grant to C.A.K from the Plant Biosystems

Elite Environment at the University of Copenhagen.

77

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79

Table

Table 1. Treatment combination with respective abbreviation and experiments in which they were

included.

Abbreviation Treatment See

d t

rea

tmen

t/

roo

t ass

ay

Dir

ect

ino

cula

tio

n

1 ×

10

5

Dir

ect

ino

cula

tio

n

1 ×

10

7

Untreated Treated with ddH2O as a control x x x

Cr Clonostachys rosea x x x

Fc Fusarium culmorum x x x

Cr + Fc Clonostachys rosea + Fusarium culmorum x x x

Mb Metarhizium brunneum x x x

Mb + Cr Metarhizium brunneum + Clonostachys rosea x x x

Mb + Fc Metarhizium brunneum + Fusarium culmorum x x x

Mb + Cr + Fc Metarhizium brunneum + Clonostachys rosea + Fusarium culmorum x x x

Mf Metarhizium flavoviride x x x

Mf + Cr Metarhizium flavoviride + Clonostachys rosea x x x

Mf + Fc Metarhizium flavoviride + Fusarium culmorum x x x

Mf + Cr + Fc Metarhizium flavoviride + Clonostachys rosea + Fusarium culmorum x x x

Mr Metarhizium robertsii x x

Mr + Cr Metarhizium robertsii + Clonostachys rosea x x

Mr + Fc Metarhizium robertsii + Fusarium culmorum x x

Mr + Cr + Fc Metarhizium robertsii + Clonostachys rosea + Fusarium culmorum x x

80

Figure Legends

Figure 1. Mean daily survival (%) of T. molitor larvae over a 14 day period after exposure to wheat

roots. Roots were grown for two weeks from seeds of two wheat varieties that had been exposed

separately to one of 12 various treatments which included untreated seeds and all combinations of

Metarhizium sp., Clonostachys rosea (Cr) and Fusarium culmorum (Fc). Survivorship curves

represent each wheat variety having received treatments without Metarhizium spp. (a, b), with M.

brunneum (c, d) or M. flavoviride (e, f). Within each graph the legend box contains a figure key

designating treatments and significance levels. Treatments with different letters are significantly

different while “ns” indicates no significant differences among treatments.

Figure 2. Total larval mortality of T. molitor and proportion of Metarhizium spp. mycosis observed

on dead larvae (black) after exposure to wheat roots for 14 days. Dead insects without mycosis are

represented by grey bar for all treatments. Mycosis was only observed on insects exposed to roots

treated with either M. brunneum (a, c) or M. flavoviride (b, d) (n=90 exposed larvae per treatment)

with the two varieties of wheat tested separately. Within each graph, statistical analyses of data

apply only to the proportion of dead larvae with mycosis. Lower case letters above the bars

represent significance levels in relation to the other treatments.

Figure 3. Proportions of wheat plants in each disease-severity rank grouped by treatment across

both experimental repetitions assessed after 13 days. Data from both wheat varieties of wheat were

combined. Only germinating seeds were assessed for disease thus number of observations varied

among treatments.

Figure 4. Mean daily survival (%) of T. molitor larvae over a 14 day period after treatment. Larvae

were directly inoculated with 5 µl of a designated fungal suspension of either ‘low’ (a, c, e, g) or

‘high’ dose (b, d, f, h) which included control inoculation and all combinations of Metarhizium sp.,

Clonostachys rosea (Cr) and Fusarium culmorum (Fc). Each individual graph contains a figure

key which designates the treatment; within each graph significantly different treatments are

presented by different letters while “ns” indicates no significant effect of treatment.

Figure 5. Total larval mortality of T. molitor and proportion of mycosis of Metarhizium spp.

observed on dead larvae (black) after direct inoculation with a fungal treatment or untreated control.

Dead insects without mycosis are represented by grey bars for all the treatments. Mycosis was only

observed on insects treated with either M. brunneum (a, d), M. flavoviride (b, e) or M. robertsii (c,

f) (n=90 exposed larvae per treatment); for the high dose experiment mycosis on dead insects was

only checked for two of the three repetitions, for the low dose mycosis was checked on both

repetitions. Lower case letters above the bars represent significance levels in relation to the other

treatments within the same graph.

81

Figure 1

20

40

60

80

100

20

40

60

80

100

Su

rv

iva

l o

f T

. m

oli

tor

larv

ae (

%)

Wheat variety Jensen Wheat variety Nakskov

b

e f

dc

a

nsnsns

CrFcCr+Fc

ns Untreated

nsnsns

CrFcCr+Fc

ns Untreated

bbaa

Mf

Mf+CrMf+FcMf+Cr+Fc

a Untreatedcbcbbc

Mf

Mf+CrMf+FcMf+Cr+Fc

a Untreated

cbbb

Mb

Mb+CrMb+FcMb+Cr+Fc

a Untreatedcbbb

Mb

Mb+CrMb+FcMb+Cr+Fc

a Untreated

2 4 6 8 10 12

0

20

40

60

80

100

Time (d)

2 4 6 8 10 12 14

82

Figure 2

83

Figure 3

Treatment

Pla

nts

wit

hin

ea

ch

dis

ease

-sev

erit

y c

lass

(%

)

0

20

40

60

80

100

Healthy Plant

Slightly brown

Moderately brown

Severe browning

Dead

Un

trea

ted

Mb

Mf

Cr

Mb

+ C

r

Mf

+ C

r

Fc

Mb

+ F

c

Mf

+ F

c

Cr

+ F

c

Mb

+ C

r +

Fc

Mf

+ C

r +

Fc

84

Figure 4

Time (d)

2 4 6 8 10 12

0

20

40

60

80

100

Su

rviv

al

of

T.

moli

tor

larvae (

%)

1 x 105 conidia/ml 1 x 107 conidia/ml

20

40

60

80

100

20

40

60

80

100

20

40

60

80

100

2 4 6 8 10 12 14

b

e f

dc

a

g h

cbcbb

Mf

Mf+CrMf+FcMf+Cr+Fc

a Untreatedbbbb

Mf

Mf+CrMf+FcMf+Cr+Fc

a Untreated

bbbb

Mb

Mb+CrMb+FcMb+Cr+Fc

a Untreated

nsnsns

CrFcCr+Fc

ns Untreated

dcdbcb

Mr

Mr+CrMr+FcMr+Cr+Fc

a UntreatedMr

Mr+CrMr+FcMr+Cr+Fc

Untreatedbbbb

a

ababb

CrFcCr+Fc

a Untreated

bbbb

Mb

Mb+CrMb+FcMb+Cr+Fc

a Untreated

85

Figure 5

86

Supplemental Figures

Supplemental figure 1: Treated wheat seeds were placed on PDAY media to assess viability of the

seeds and fungal treatments. Pictures of ‘Nakskov’ (top) and ‘Jensen’ (bottom) wheat varieties

after 3 (left) and 10 (right) days.

Supplemental figure 2: Disease index used to asses severity of F. culmorum infection in wheat

plants after 13 days growth in filter paper rolls with 12/12 h light/dark.

Supplemental figure 3: An illustration describing the experimental design of the wheat-seed

treatment and root-insect infection assay.

87

Supplemental figure 1

C. ro

sea

F. cu

lmoru

m

C. ro

sea a

nd

F. cu

lmoru

m

M. brunneum M. flavoviride M. brunneum M. flavoviride C

. ro

sea

F. cu

lmoru

m

C. ro

sea a

nd

F. cu

lmoru

m

88

Supplemental figure 2

0 =

Healthy

plant

1 =

Slightly

brown

coleoptile/

roots

2 =

Moderately

brown

coleoptile

and roots

3 =

Severe

browning

of

coleoptile

and roots

4 =

Dead plant

89

Supplemental figure 3

Symptoms of

Fusar ium

infection

1. Fusarium infected

and control seeds are

treated with biocontrol

agents

2. Seeds are allowed

germinate and grow for

14 days

3. Plants are assessed

for disease and roots

are fed to insect larvae

90

Manuscript 3

DIVERSITY OF METARHIZIUM FLAVOVIRIDE POPULATIONS ASSOCIATED WITH ROOTS OF CROPS IN

DENMARK

Chad A. Keyser, Henrik H. de Fine Licht, Bernhardt M. Steinwender & Nicolai V. Meyling

Status: Manuscript

91

Diversity of Metarhizium flavoviride populations associated with roots of crops in Denmark

Chad A. Keyser*, Henrik H. de Fine Licht, Bernhardt M. Steinwender, and Nicolai V. Meyling

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40,

1871 Frederiksberg, Denmark

*Corresponding author: Chad A. Keyser, Department of Plant and Environmental Sciences,

University of Copenhagen, Thorvaldsensvej 40, stair 2, 3rd floor, B321,

1871 Frederiksberg, Denmark

Email: chadaltonkeyser@gmail.com

Keywords

AFLP, Entomopathogenic fungi, soil baiting, rhizosphere association, endophyte

92

Abstract

Ecological studies assessing the natural occurrence and community structure of

entomopathogenic fungi are important to understanding their role in insect host regulation;

however, there has been an acute lack of studies on the distribution of fungi in the genus

Metarhizium. The objective of the present research was to evaluate the occurrence, diversity and

community structure of Metarhizium spp. isolates obtained from roots and root-associated soil of

wheat, oilseed rape, and bordering uncultivated grazed pastures at three geographically separated

agricultural fields in Denmark. Of the132 Metarhizium isolates obtained, morphological data and

sequencing of the rDNA intergenic spacer region (IGS) revealed that 118 belonged to Metarhizium

flavoviride, 13 to M. brunneum and one M. majus. Further characterization of intraspecific

variability within M. flavoviride by unspecific markers (amplified fragment length polymorism,

AFLP) was used to evaluate diversity and potential crop and/or area associations. A high level of

diversity among the M. flavoviride isolates was observed, indicating that the isolates were not of the

same clonal origin. However, due to insufficient loci in the AFLP analysis we were not able to

determine significant haplotype groupings or confirm any habitat associations of these isolates. We

suggest that the development of more specific molecular markers would greatly improve the ability

to evaluate M. flavoviride diversity which has largely been ignored. This study represents the first

in-depth analysis of the molecular diversity within a large isolate collection of the species M.

flavoviride.

93

1. Introduction

The predominantly entomopathogenic fungal genus Metarhizium (Hypocreales:

Clavicipitaceae) has a global distribution (Kepler et al., 2014; Roberts and St. Leger, 2004;

Zimmermann, 2007) and several species have been intensely researched for their pest control

potential. Discordantly, limited focus has been given to the assessment of natural occurrence and

distribution of this widespread fungal genus. Knowledge of the compositions of entomopathogenic

fungal communities and structure of their populations is important to understand their contribution

to host regulation and potential for conservation biological control (Meyling and Eilenberg, 2007).

Evaluation of fungal diversity and community structure depends heavily on criteria for

species identification. With few morphological distinct features, species referred to as M. anisopliae

in the literature could belong to several different species if no explicit molecularly based

identification has been conducted. Bischoff et al. (2009) revised the taxonomy of the M. anisopliae

lineage based on a multigene phylogeny and resolved nine species within what could

morphologically be considered as M. anisopliae sensu lato. Recently, the taxonomy of the whole

genus Metarhizium was revised leading to the inclusion of other genera as well as elevation of some

of the former M. flavovoride variants to species level (Kepler et al., 2014). Kepler and Rehner

(2013) further specified additional genomic regions for species identification. Molecularly based

evaluation of the diversity of Metarhizium communities should therefore be included in studies of

natural occurrence and distribution to provide full recognition of diversity.

In temperate climatic regions, Metarhizium spp. are predominantly isolated from the soil

environment (Vega et al., 2012). Surveys of entomopathogenic fungi from soil samples conducted

at different geographical locations have shown that M. anisopliae s.l. can be abundantly isolated

from managed ecosystems (e.g. Bidochka et al., 1998; Goble et al., 2010; Inglis et al., 2008;

Klingen et al., 2002; Meyling et al., 2011; Steinwender et al., 2014; Sun et al., 2008). However,

few studies have evaluated Metarhizium community structure using explicit molecular

characterization. In Denmark, Meyling et al. (2011) isolated M. anisopliae s.l. from an agricultural

field and Steinwender et al. (2014) subsequently showed that the Metarhizium community was

composed of several species, predominated by M. brunneum. Further characterization with

microsatellite markers revealed several co-occurring multilocus genotypes within both M.

brunneum and M. robertsii (Steinwender et al., 2014).

Naturally occurring Metarhizium spp. were recently isolated from roots of different plants

(Fisher et al., 2011; Wyrebek et al., 2011). Some level of plant association was indicated by a

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predominance of M. robertsii found on roots of grasses as compared to M. brunneum which was

mostly recovered from roots of woody plants (Wyrebek et al. 2011). Sampling roots of different

plants from an ecosystem might therefore reveal the Metarhizium diversity of an area since similar

M. brunneum and M. robertsii multilocus genotypes can be repeatedly isolated from roots and soil

samples at the same site (Steinwender, 2013). However, the entomopathogenic fungal community

structure of the soil can differ markedly between agricultural fields within relatively close

proximity. Although Meyling et al. (2011) mostly isolated M. anisopliae s.l. (predominantly M.

brunneum) another agricultural field in Denmark showed low occurrence of M. anisopliae s.l.

(Meyling and Eilenberg, 2006). At the latter site, M. flavoviride was isolated more frequently than

M. anisopliae s.l. indicating location specific fungal communities.

The aim of the current study was to evaluate the occurrence, diversity and community

structure of Metarhizium spp. isolates obtained from roots and root associated soil of different

common crops at geographically separated agricultural fields in Denmark. Using such a sampling

scheme we expected to reveal potential crop and/or location association. Based on recent evidence

(Steinwender et al., 2014) we expected to isolate M. brunneum most frequently, however the study

resulted in a predominance of M. flavoviride isolates. Since the available microsatellite markers

developed for M. anisopliae s.l. are not specific for M. flavoviride (Steinwender, 2013) we chose to

characterize the intraspecific variability within M. flavoviride by unspecific markers to evaluate

diversity and potential crop and/or area associations.

2. Material and Methods

2.1 Field sampling

Three geographically separated areas were located in Sjælland, Denmark, including: Fårevejle

(55.78º N, 11.43 ºE), Skibby (55.75 º N, 11.99 ºE), and Taastrup (55.67 º N, 12.3 ºE). Within each

sampling site three agricultural fields were identified resulting in a 2-level nested design. The fields

included one field of winter wheat (Triticum aestivum), one with oilseed rape (Brassica napus), and

a permanent grass field that had been without cultivation for more than 20 years.

From each of the nine fields, 50 root samples were collected by walking from one edge, a

transect of the field towards the center; a plant/soil sample was collected every 3-4 meters using a

long-handled weeding fork. Each sample was placed individually in a pre-labeled 10 liter plastic

bag along with about 500 g of root-associated soil adhering to the root system. The three fields

from each geographic area were sampled on the same day; collections were made on three

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consecutive days from July 23 – July 25, 2013. After collection samples were placed at 5 ºC until

they were processed within 30 days.

2.2 Fungal isolation

2.2.1 Selective media: From each sample a section of the root was removed and washed with

ddH2O to remove loose soil. A portion (~0.5 ± 0.25 g) of the washed root was then cut in to small

pieces (~2 mm) and placed in 10 ml ddH2O and homogenized using a rotary homogenizer

(Wyrebek et al., 2011). 200 µl of homogenate was then spread onto a selective agar media adapted

from Fernandes et al. (2010b), which consisted of: 39 g potato dextrose agar (Sigma Chemical, MO,

USA), 1g yeast extract (Merck KgaA, Darmstadt Germany), 0.25 g cyclohexamide (Sigma

Chemical, MO, USA), 0.5 g chloramphenicol (Sigma Chemical, MO, USA), 0.02 g dodine

(Agriphar, Belgium) (all weights per liter); two plates for each root sample were prepared. After

inoculation the plates were incubated for 28 days at 22 ºC in darkness and checked on day 7, 14, 21

and 28. Colonies morphologically identified as Metarhizium were transferred to a clean plate of

potato dextrose agar supplemented with 1% yeast extract (PDAY) using a sterile inoculating needle

to obtain pure cultures.

2.2.2 Soil baiting: The soil baiting method used in this study was adapted from Zimmermann

(1986). The soil from each sampling site was allowed to air dry over night at room temperature and

then re-moistened with ddH2O so that it was “slightly damp”. Soil (~120 ml) was then placed in a

plastic cup (155 ml) leaving 1 cm of airspace at the top. Prior to filling the cup large debris and

clumps were removed or broken up. Ten healthy 4-5th

instar Tenebrio molitor L. (Coleoptera:

Tenebrionidae) larvae were then added to each cup. A lid with ventilation holes was placed on top

of each sample and the cups were stacked upside down – so that the larvae were beneath the soil –

in a box. Every 1-2 days the box was rotated so that the orientation of the cups shifted, forcing the

T. molitor larvae to move through the soil substrate increasing the likelihood they would come in

contact with pathogenic microorganism in the soil. Insect survival was checked weekly; dead

insects were removed, washed with ddH2O and placed in a medicine cup with a moist piece of filter

paper to check for mycosis. Using a sterile inoculation needle, fungal isolations were made from

insect cadavers with mycosis and plated on PDAY media. After 14 days growth colonies were

evaluated, morphologically identified and Metarhizium spp. isolates were kept for further studies.

The number of isolate from each location and field type were assessed with a one-way

analysis of variance using the analysis of variance (AOV) command in R. All of the analyses were

performed in RStudio (version 0.97.551).

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2.3 PCR amplification and sequencing

For each Metarhizium isolate, lyophilized mycelium was prepared by growing cultured

isolates in liquid media (Steinwender et al., 2014) for four days at 22 ºC on a stirring table at 170

rpm. Mycelium was separated using a vacuum filtration system, frozen over night at -20 ºC and

lyophilized. DNA was extracted from the lyophilized mycelia using the DNeasy Plant Mini Kit

(QIAGEN, Hilden, Germany) following the manufacturer’s instructions.

The PCR amplification and sequencing methods in this study were according to Kepler and

Rehner (2013). The master mix reagents for each PCR reaction consisted of: 32 µl milli Q H2O, 5

µl Taq DNA buffer, 1 µl dNTP, 1 µl Taq DNA polymerase, 5 µl forward primer and 5 µl reverse

primer, and 1 µl DNA sample. The primers used were MzFG543igs_1F (5’-ATT CAT TCA GAA

CGC CTC CAA-3’) and MzFG543igs_4F (5’-GGT TGC GAC TCA CAA TCC ATG-3’). PCR

amplification was initiated by denaturation at 95 ºC for 2 min followed by 40 cycles of three steps

including: 95 ºC denaturation for 30 s, 62 ºC annealing for 30 s, and 72 ºC extension for 60 sec;

after the 40 cycles the final step was 72 ºC extension for 15 min. The PCR products were

visualized on 1.5% agarose gel to ensure strong single bands and purified using the Illustra™

GFX™ PCR DNA and Gel Band Purification Kit. The purified PCR products were sent to

Beckman Coulter Genomics (United Kingdom) for sequencing with the PCR primers. The sequence

chromatograms were manually corrected and aligned using ClustalW (Larkin M.A., 2007) with

default settings as implemented in BioEdit 7.1.3 (Hall, 1999). Additionally, sequences of known

Metarhizium spp. isolates from Kepler et al. (2014) in addition to three isolates form the ARSEF

collection (i.e., ARSEF nos. 1184, 1271, and 9358) were included in the the phylogenetic analyses.

The ~900 base pair multiple alignment was analyzed using ModelTest to determine the optimal

DNA substitution model and evaluated with AIC scores as implemented in Topali (Milne et al.,

2009). The phylogeny was inferred with maximum likelihood estimation using RaxML with 500

bootstrap replicates and Bayesian analyses using MrBayes ver3.1 and executed from within Topali.

The final dendrogram was depicted suing MEGA5 (Tamura et al., 2011).

2.4 Intraspecific variation of M. flavoviride

New DNA extractions with a greater quantity of DNA were made from the lyophilized

mycelium prepared in section 2.3. This was accomplished using the Qiagen DNeasy Plant Mini Kit

with the following protocol modifications: A small amount of lyophilized material (~2-3

“matchhead” size pieces) was placed in a 2 ml eppendorff vial, which had been prepared with 1.0

mm and 2.5 mm dia. zirconia-glass beads (Biospec Products). Next, 600 µl buffer AP1 from the

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Qiagen DNeasy Plant Mini Kit was added and the samples were placed in a Fast Prep machine and

shaken twice for 35 sec at 5.0 speed. The samples were then removed and 6 µl of proteinase K and

6 µl RNase A were added and incubated at 65 ºC for 1 hour during which they were shaken every

15 min. After incubation, 195 µl buffer P3 was added and they were incubated on ice for 5 min.

The remaining steps were according to the protocol and the DNA was eluted in 50 µl buffer AE.

The quantity and quality of DNA available in the extracted material was quantified using a

NanoDrop 1000 (Thermo Scientific).

For the Amplified Fragment Length Polymorphism (AFLP) procedures Vos et al. (1995), the

amount of DNA per reaction was standardized to 100 ±20 ng. Initially the DNA was digested by

two restriction enzymes and oligonucleotide adapters attached to the resulting ends (cut-ligation

step). Two master mixes were prepared, master mix A contained: 0.1µl MSE (10 u/µl), 0.25 µl Eco

RI (20 u/µl), 0.1 µl T4 ligase (6 Weiss u/µl), 0.1 µl T4 ligase buffer, 0.05µl diluted (1 mg/ml) BSA,

0.1 µl 0.5 M NaCl, and 0.3 µl milliQ H2O, for each reaction. Master mix B contained: 1 µl 10x T4

ligase buffer, 1 µl 0.5 M NaCl, 0.5 µl diluted BSA (1 mg/ml), 1 µl MseI adaptor pair, 1 µl EcoRI

adaptor pair, for each reaction. Prior to preparing master mix B, the MseI and EcoRI adaptor pairs

were heated to 95 ºC for 5 min and then allowed to cool at room temperature for 10 min; the MseI

adaptor pair consisted of a 1:1 combination of M-ADAP I (5´-GAC GAT GAG TCC TGA G-´3)

and M-ADAP II (5´-TAC TCA GGA CTC AT-´3) at a concentration of 20 µM each (so after

combination the concentration of each is 10 µM). After preparation master mix B was added to

master mix A and mixed before 5.5 µl of DNA was added so that the total volume was 11 µl. This

was then incubated at 37 ºC for 2 hours, after which 189 µl of milliQ H2O was added.

A PCR preamplification of the restriction-ligation products consisted of: 12.55 µl milliQ H2O,

4 µl Phusion buffer (Finnzymes), 0.4 µl dNTP (10µM), 0.25 µl Phusion polymerase (Finnzymes),

0.4 µl Eco pre-amp primer (5´-GAC TGC GTA CCA ATT CA-´3), and 0.4 µl Mse pre-amp primer

(5´-GAT GAG TCC TGA GTA AC-´3). To this mixture 2 µl of the product from the cut ligation

above was added. The DNA fragments were then amplified using the following PCR program: 98

ºC denaturation for 30 s, followed by 35 cycles next three steps, 98 ºC denaturation for 30 s, 56 ºC

annealing for 30 s, and 72 ºC extension for 1 min; after the 35 cycles the final step was 72 ºC

extension for 120 sec. A 1:10 dilution of the preamp product was then made for the selective

amplification step.

A selective amplification mixture was prepared which consisted of: 11.85 µl milliQ H2O, 4 µl

Phusion buffer, 0.4 µl dNTP (10µM), 0.25 µl Phusion polymerase, 1 µl Eco sel-amp primer with

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FAM labeled (10µM) (5´-GAC TGC GTA CCA ATT CAC C-´3), 0.5 µl Mse sel-amp CAC primer

(20µM) (5´-GAT GAG TCC TGA GTA ACA C-´3), and 0.5 µl Mse sel-amp CAT primer (20µM)

(5´-GAT GAG TCC TGA GTA ACA T-´3). To this 1.5 µl of the diluted pre-amplification product

was added to bring the total volume to 20 µl. The following PCR program was performed: for 8

cycles, 98 ºC denaturation for 30 s, 65 ºC annealing for 30 s, and 72 ºC extension for 90 s, reducing

the annealing temperature 1 ºC each cycle; then 24 cycles of: 98 ºC denaturation for 30 s, 56 ºC

annealing for 30 s, and 72 ºC extension for 90 s, followed by 72 ºC for 7 min. This was then

visualized on 1.5% agarose gel to insure the presence of a distinct smear and banding pattern.

The selective amplification products were prepared for fragment length analysis by mixing:

0.1 µl sample in 8.7 µl formamide and 0.3 µl Genescan Rox500 (Applied Biosystems). The

samples were then analyzed on ABI Prism® 3100 Genetic Analyzer (Applied Biosystems, Foster

City, CA). The entire AFLP procedure was performed twice using the same extracted DNA

resulting in two technical replicates for each isolate. Because the samples were run on more than

one 96-well plate, a specific isolate was included on all plates to control for plate-specific variation.

Presence of bands were scored as follows using GeneMapper® software 5 (Applied Biosystems)

and ROX500 as internal size standard: AFLP fragments were scored as present (1) if they had a

signal intensity greater than 100 in duplicate samples, or if there was a disagreement between the

two replicates and one was above 100 and other was above 50. In contrast, if an AFLP fragment

had signal intensity less than 100 for both isolate replicates or if one was below 50 then it was

considered absent (0). A cluster analysis of the resulting bionomial data set was performed by

calculating the pairwise distance measure using Dice coefficient with 1000 bootstrap replicates

using DistAFLP (available at: http://pbil.univ-lyon1.fr/ADE-4/microb/) (Mougel et al., 2002).

These bootstrap trees were then analyzed using the Neighbor and Consensus executables in the

Phylip Package (Felsenstein, 2005) to produce a tree with bootstrap support based on the unrooted

unweighted pair group method with arithmetic mean (UPGMA) (Sneath, 1973).

3. Results

3.1 Metarhizium distribution and species diversity

A total of 132 Metarhizium isolates were acquired from the collected samples representing

nine agricultural fields (Table 1). Fourteen of the isolations were made using selective media; the

remaining 118 isolates were attained using the soil baiting method. A total of 34 isolates were from

the Fårevejle area, 71 isolates from Skibby, and 27 isolates from Taastrup. Geographical area was

not a significant factor in determining the number of isolations made (F=1.04, df=2, p =0.43). A

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total of 25 isolates were associated with oilseed rape, 61 with Winter wheat, and 46 were recovered

from permanent grass fields; crop type was also not a significant contributing factor to the observed

distribution pattern (F=0.61, df=2, p =0.59).

Of the 132 isolates, 118 were morphologically identified as M. flavoviride based on their

characteristic bright green colony color and conidia dimensions. The remaining 14 isolates were

morphologically classified as belonging to the M. anisopliae complex. M. flavoviride isolates were

collected from all fields except the undeveloped field in Fårevejle (Fig. 1) which was wetter than at

the other areas. No significance was observed with regard to M. flavoviride isolation of

geographical area or crop type (F=1.72, df=2, p =0.29; F=0.37, df=2, p =0.71; respectively).

To verify species identity, 130 of the isolates collected were sequenced for the rDNA

intergenic spacer region (IGS) (Fig. 2). Of the 14 M. anisopliae s.l. isolates, 13 clustered with M.

brunneum while 1 was identified as M. majus. Among the M. brunneum isolates, 8 isolates

representing all three areas clustered with a clade previously found in Denmark, while the

remaining 5 belonged to two separate clades. The 116 M. flavoviride isolates collected from this

study and sequenced shared the same sequence and grouped with the other M. flavoviride isolates

from Denmark, Poland and Sweden as well as a reference M. flavoviride originating from France.

3.2 Intraspecific variation of M. flavoviride

The result of the AFLP analysis is presented in Figure 3. The AFLP method was performed

using 93 of the M. flavoviride isolates collected in this study as well as 13 isolates collected from

different geographical locations, including: Jutland strawberry fields, Denmark (Js: n=9), Århslev,

Denmark (Ars: n=2) (Steinwender et al., 2014), Poland (Pol: n=1), and Sweden (Swe: n=1). The

AFLP analysis produced 230 polymorphic loci; however, based on the scoring criteria and

inconsistency between dual replicates, only 30 repeatable and consistent loci were selected for

inclusion in the analysis. Using the UPGMA method, intraspecific variation among the M.

flavoviride isolates was quite high identifying 78 haplotypes among the 93 analyzed isolates, of

which 11 were shared among 2-4 isolates from different areas while the remaining were unique.

However only three significant pairwise clusters could be identified by bootstrapping, thus grouping

associated with area or crop could not be established.

4. Discussion

The present study revealed that a significant level of Metarhizium spp. is extant in the

investigated agricultural and undisturbed grass areas of Denmark. Furthermore, we observed that,

in the fields sampled, M. flavoviride was the predominant Metarhizium species. The AFLP analysis

100

of the M. flavoviride isolates revealed that a significant level of diversity is present among the

isolates of this species. Unfortunately the non-specificity of the AFLP method does not allow us to

identify any explicitly defined genotypes within M. flavoviride, but some AFLP haplotypes appears

to be widespread in Denmark and in neighboring countries. This is the first time that an in-depth

analysis of the molecular diversity within a large isolate collection of the species M. flavoviride has

been reported.

The majority of Metarhizium related research has favored species of the M. anisopliae

complex; the lack of research interest may suggest that M. flavoviride are less abundant or less

frequently isolated and has therefore likely been ignored. This proposition tend to be supported in

the literature as most Metarhizium spp. occurrence studies report of few to no M. flavoviride s.l.

isolates (Bidochka et al., 1998; Fisher et al., 2011; Goble et al., 2010; Inglis et al., 2008; Quesada-

Moraga et al., 2007; Sun et al., 2008; Vanninen, 1995; Wyrebek et al., 2011). Only Meyling and

Eilenberg (2006) have reported significant occurrence of M. flavoviride isolation within an

agroecosystem located in Denmark. However, other Danish studies have observed a predominance

of M. anisopliae complex isolates (Steinwender et al., 2014; Vega et al., 2012). The present study

surveyed three geographically separate areas and found similar species composition between all

three, indicating that the relatively high frequency of M. flavoviride reported by Meyling and

Eilenberg (2006) was not an isolated event. Clearly the species occurrence and composition of

Metarhizium is not ubiquitous or random; further in-depth ecology studies are necessary to identify

the driving forces that determine Metarhizium spp. distribution in nature.

The predominance of M. flavoviride we observed was an unexpected; our initial intention

was to analyze the within-species diversity using microsatellites or simple sequence repeat (SSR)

markers, which have been shown to be highly suitable for explicit multilocus genotyping (Enkerli

and Widmer, 2010). However the SSR markers, developed for M. anisopliae s.l., were observed to

have a high frequency (>40%) of null alleles when tested on M. flavoviride isolates and almost no

variability (Steinwender, 2013; Steinwender et al., 2014), and were therefore unsuitable for

diversity evaluation of this species. Thus, to characterize the intraspecific variability within M.

flavoviride and evaluate the diversity we chose to use unspecific markers. Diversity assessment

using AFLP markers has been used previously for other Metarhizium spp. (e.g., Fernandes et al.,

2010a; Inglis et al., 2008). Inglis et al. (2008) used AFLP markers to determine the diversity within

M. anisopliae s.l. from western Canada, and they were able to identify several distinct haplotypes.

It is important to note however that this was done prior to the Bischoff et al. (2009) taxonomic

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revision and the material evaluated by Inglis et al. (2008) has later been split into separate species,

predominantly M. brunneum (T. Kabaluk and M. Goettel, pers. comm.). Fernandes et al. (2010a)

used AFLP markers to evaluate inter-species variability among several species of Metarhizium; they

found the methods clearly distinguish between species but did not assess intra-species variability.

Reliable species identification when aligning with a molecular phylogeny as in the present study

will be important when applying unspecific markers such as AFLP markers to evaluate intraspecific

diversity of Metarhizium spp.

All M. flavoviride isolates sequenced for the rDNA intergenic region shared the same

sequence, even among distantly sampled isolates, indicating that this species may not be composed

of phylogenetically distinct clades as is evident for at least M. brunneum (Steinwender et al. 2014)

which was also shown in the present data set. All 93 M. flavoviride isolates subjected to the AFLP

characterization were repeated twice to ensure repeatability of the markers included in diversity

evaluation. While a high level of polymorphic loci were identified, the repeatability was low

among the repetitions for each isolate, which resulted in the inclusion of only 30 highly conserved

loci for the analysis. Reproducibility can be a problem with unspecific markers (Enkerli and

Widmer, 2010). Insufficient loci in the AFLP analysis may have resulted in the lack of support for

the haplotype groupings. However the UPGMA method clearly shows that there is diversity within

the species indicating that the isolates are not of the same immediate clonal origin. Development of

specific markers (i.e., SSR markers) would greatly improve our ability to evaluate the M.

flavoviride diversity, and determine if any habitat associations can be identified.

As in the present study it has been observed that a particular species of Metarhizium will be

the most prevalent in a given area (Fisher et al., 2011; Inglis et al., 2008; Steinwender et al., 2014;

Wyrebek et al., 2011), however it is unclear what factors lead to this high frequency in the

community. Metarhizuim spp. are able to acquire nutrients as an insect pathogen or as a plant

endophyte/saprophyte – it is reasonable to suggest that an aptitude at either life style would favor a

particular species. It has been observed that M. flavoviride had lower virulence than M. brunneum

or M. robertsii towards T. molitor larvae (Keyser et al. Manuscript 2), suggesting that M. flavoviride

is less efficient at infecting host insects. However, Meyling et al. (2011) reported of relatively

frequent natural infections by M. flavoviride in aboveground beetles suggesting that the species

plays a role in regulation of insect populations. At the site investigated by Meyling et al. (2011) M.

anisopliae s.l. [later shown to be predominantly M. brunneum by Steinwender et al. (2014)] was

most frequently isolated by insect baiting in the soil, but this fungal taxon was absent as infections

102

in aboveground hosts. We implemented two standard methods for the isolation of

entomopathogenic fungi in this study, viz., soil bait method and selective media method. The soil

bait method was more effective in recovering isolates of M. flavoviride. The selective media

method used was also employed by Wyrebek et al. (2011) to isolate Metarhizium spp. from roots of

different plants. Although Wyrebek et al. (2011) did not isolate M. flavoviride from roots, Behie

and Bidochka (2014) reported that M. flavoviride was able to associate with plant roots in the

rhizosphere. However, by sequencing in the present study the isolate used by Behie and Bidochka

(2014) (ARSEF 9358) appeared to be M. pemphigi (previously M. flavovoride var. pemphigi), and it

thus remains undetermined if M. flavoviride can associate with plant roots. The reduced number of

isolates of M. flavoviride collected on selective media from root homogenate in the present study

might indicate that, while present in the soil environment, M. flavoviride was not associating closely

with the roots of the crop plants. Further studies are needed to confirm these observations and

investigate what might be driving the M. flavoviride predominance at the investigated field sites.

However, the current data and that of Meyling et al. (2011) indicate that entomopathogenicity is an

important ecological trait for M. flavoviride occurrence and distribution.

Acknowledgements

We would like to give special thanks to Line Lykke, Lærke Thordsen, Azmi Mahmood,

Darren Thomsen, Jesper Andersen, Sylvia Mathiasen and Louise Lee Munk Larsen for their technical

assistance during several phases of this study. We would also like to thank the farmers at each

location for allowing us to sample their fields. This research was funded by a PhD grant to C.A.K

from the Plant Biosystems Elite Environment at the University of Copenhagen.

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Table and Figures

Table 1: Total number of new Metarhizium spp. isolations obtained using insect baiting and

selective media from various geographical location in Denmark and different field types of three

species of Metarhizium. Total number of new isolates (number of isolations made with selective

media).

Location / Field Mb Mf Mm Total Fårevejle

Oilseed rape 0 17 0 17

Winter wheat 0 17 (1) 0 17

Undeveloped meadow 0 0 0 0

Skibby

Oilseed rape 1 5 0 6

Winter wheat 2 19 (1) 0 25

Undeveloped meadow 6 (5) 37 (5) 1 (1) 40

Taastrup

Oilseed rape 1 1 0 2

Winter wheat 3 16 (1) 0 19

Undeveloped meadow 0 6 0 6 Total 13 118 1 132

106

Figure legends

Figure 1: Map depicting the geographic location of the three sites where samples were collected in

Sjælland, Denmark. At each location 150 root/soil samples were collected (50 from each field

type). Pie chart provides the number of new M. flavoviride isolates obtained from each field type at

each location.

Figure 2: Phylogenetic tree based on Bayesian analysis with branch support shown for both

Bayesian and Maximum-likelihood analyses. Bayesian (100=**, 95<*)/Maximum-likelyhood

(100=**, 95<*, # branching topology did not agree between Bayesian and Maximum-likelihood

analysis). 130 Metarhizium spp. isolates representing 3 species were sequences using the rDNA igs

region. Symbols indicate location and field type from which the isolate was acquired [F = Fårevejle

(square), S = Skibby (circle), T = Tåstrup (triangle), R = oilseed rape (yellow), U = un-cultivation

field (blue), and W = Wheat (green)].

Figure 3: AFLP dendrogram of 106 Metarhizium spp. isolates generated with the Phylip Package

using Dice coefficient with 1000 strap replicates. Bolded branches represent significant pairwise

cluster based on bootstrapping method. Symbols indicate location and field type from which the

isolate was acquired [F = Fårevejle (square), S = Skibby (circle), T = Tåstrup (triangle), R = oilseed

rape (yellow), U = un-cultivation field (blue), W = Wheat (green) and Other (star red): Js = Jutaland

strawberry field, Swe = Sweden, Ars = Årslev, and Pol = Poland].

107

Figure 1

108

Figure 2

109

Figure 3