Insects and fungi: ecological interactions and functional ... · Summary •About 15% of identified...

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Insects and fungi: ecological interactions and functional biodiversity

PhD candidate Antonino MALACRINÒ

Tutor

Prof. Leonardo SCHENA

Co-tutor Prof. Virgilio CALECA

Interactions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Fungi

Bacteria

Insects

Plants

Other Invertebrates and animals

Soil and organic debris

Viruses

Micro-organisms

Interactions with micro-organisms

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Fungi

Bacteria

Insects

Micro-organisms

Bacteria: • Symbiosis

• Feeding • Reproduction • Protection • Others…

• Pathogens

Fungi: • Feeding activity (e.g. bark

beetles or harvester ants) • Insect pathogens

• Other interactions (?)

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Aims

• Which is the fungal community associated to insect pests?

• Can international harbours be a point-of-entry of known and potential fungal pathogens carried by scolytid beetles?

• Can insects impact on soil microbial community? Is it possible to have a vice-versa effect?

What’s metabarcoding?

Sample DNA PCR Sequencing Bioinformatics

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Sequencing Data handling OTU table Data analysis Insights

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

PART ONE

FUNGI ASSOCIATED TO BACTROCERA OLEAE

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Aims and Scope

Bactrocera oleae (Rossi) damages:

o For table olives growers

o For oil producers:

- Increase of oil acidity

- Decrease of oil quality

- Decrease of income

• Study the fungal community of B. oleae

• Among these fungi, are there species that are causal agents of plant diseases?

Metabarcoding

DNA extraction

PCR ITS2 region

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Amplicons Cloning Sanger sequencing

Amplicons with MID tags Pyrosequencing

454 FLX Titanium

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Coordinate1(26.26%)

Coordinate2(16.37%

)

0

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10 500 1000 1500 2000

NumberofobservedOTU

S

Numberofsequences

Males

Females

Results

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Persistence(nr.ofsamplescontainingeachOTU)

log10ofm

axim

umabundance

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FrequencyofO

TUs

foreachabundanceclass

AbundanceclassesofcoreOTUs

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103

104 A) B)

0 2 4 6 8 10 12

Persistence(nr.ofsamplescontainingeachOTU)

log10ofm

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umabundance

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FrequencyofO

TUs

foreachabundanceclass

AbundanceclassesofcoreOTUs

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104 A) B)

Results

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Cladosporium 55.60%

NPPF 21.58% Alternaria

12.60%

Auerobasidium 7.92%

Colletotrichum 0.89%

Pseudocercospora 0.78%

Pheomoniella 0.30%

Fusarium 0.25%

Neofisucoccum 0.05%

Phellinus 0.03%

Altra 2.30%

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

• NGS→ 57,964 sequences (after denoising) → 108 OTUs

Results

Cladosporium 55.6% Sooty moulds

Alternaria 12.60% Fruit – Sooty moulds

Aureobasidium 7.92% Sooty moulds

Colletotrichum 0.89% Fruit

Pseudocercospora 0.78% Fruit - Leaves

Phaeomoniella 0.30% Wood

Fusarium 0.25% Fruit

Neofusicoccum 0.05% Fruit

Phellinus 0.03% Wood

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Comparison with data from olive leaves and fruits

Female flies Male flies Olive leaves Olive fruits

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Comparison with data from olive leaves and fruits

Female flies Male flies Olive leaves Olive fruits

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Comparison with data from olive leaves

Genus Bactrocera Olive leaves

Cladosporium 55.6% + 10.5%

Alternaria 12.6% + 1.5%

Aureobasidium 7.9% - 27.3%

Pseudocercospora 0.8% - 10.1%

Colletotrichum 0.9% - 18.6%

Summary

• About 15% of identified OTUs are fungi that cause diseases to olive trees or fruits • Colletotrichum acutatum and C. gleosporioides → causal agent of olive

anthracnosis

• Other plant pathogenic fungi (e.g. Alternaria spp.)

• New insights about the epidemiology of these fungi and the role of B. oleae, not only as direct source of damage, but also as potential carrier of plant pathogenic fungi

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

PART TWO

FUNGI ASSOCIATED TO SCOLITID BEETLES

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Bark and Ambrosia beetles

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Ver

tica

l tra

nsm

issi

on

Horizontal transmission

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Ver

tica

l tra

nsm

issi

on

Horizontal transmission

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Ver

tica

l tra

nsm

issi

on

Horizontal transmission

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Seebens et al. 2013 Ecol. Lett.

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Aims and Scope

• Study the fungal community associated to both beetles, collected at international harbours, with a high-throughput culture-independent approach

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Insects

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Insects

1. Commonly trapped beetles in italian harbours

2. Native to Europe and introduced in several other Countries

3. Wide host and climatic range

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Insects

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Insects

Orthotomicus erosus

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Insects

Xyleborinus saxesenii

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Results • ≃60,000 sequences

• 294 OTUs (0.99 cut-off)

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

-2.0 -1.6 -1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 1.6

C1 (35.75%)

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

0.3

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C2 (

30.7

0%

)

PseudoF = 8.295; P = 0.01

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Number of sequences

X. saxesenii O. erosus

A) B)

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Number of sequences

Ravenna Salerno Marghera

Ravenna Salerno Maghera

X. saxesenii

O. erosus

B)

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Number of sequences

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A) B)

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Number of sequences

Ravenna Salerno Marghera

Ravenna Salerno Maghera

X. saxesenii

O. erosus

B)

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O. erosus X. saxesenii

Observed OTUs

**

Results

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Aspergillus ruber

Uncultured

Devriesia xanthorrhoeae Geosmithia sp.

Pleospora herbarum

Fusarium equiseti

Ophiostoma ips

Aspergillus spelunceus

Alternaria tenuissima

Botrytis cinerea Fusarium incarnatum Boeremia hedericola

Altra

Core OTUs – Ortotomichus erosus

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Core OTUs – Xyleborinus saxesenii

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Uncultured

Botryotinia fuckeliana

Cladosporium cladosporioides Aspergillus candidus

Aureobasidium pullulans

Aspergillus tritici

Aspergillus ruber

Alternaria tenuissima

Yeasts

Altra

104 3052

O. erosus X. saxesenii

18

5525

7 20

27

34

Marghera

RavennaSalerno

A) B)

X. saxesenii O. erosus

PC1 (35.75%)

PC

2 (

30

.7%

)

Results

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Discussion

• First study that used NGS metabarcoding to assess the whole fungal community associated to bark and ambrosia beetles trapped at international harbors

• Main taxa • Ophiostoma

• Geosmithia

• Acremonium

• Fusarium

• Aspergillus

• Alternaria

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Discussion

• New associations with fungi that could represent agents of plant diseases

• Food sources in non-native environments

• High number of unknown sequences

• In-out travelling populations – horizontal transfer

• Extend this approach to other pests

• Illumina SBS

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Conclusions

• New scenario:

Bark and Ambrosia beetles not only direct source of damages, but also potential carriers of known/unknown plant pathogens

• Surveillance should be extended also to microorganisms associated to wood-associated insect

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

PART THREE

INTERACTIONS BETWEEN APHIDS AND SOIL MICROBIOTA

Above- belowground interactions

• Aboveground and belowground communities interact • Spatial scale

• Temporal scale

• Ecological effects

• Plant growth

• Microbial ecology

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

1. Can aphids and/or aphid’s endosymbionts

impact the soil microbial community?

(Top – Down effects)

2. Can soil microbial community impact the aphid

microbiota? (Bottom – Up effects)

Questions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Solanum tuberosum Solanum vernei

Plants Aphids Soil

Aphids Leaves

Roots Soil

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Bacteria

Fungi

AM Fungi

16S

ITS2

18S

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

1. Can aphids or aphid’s endosymbionts impact the

soil microbial community?

(Top – Down effects)

2. Can soil microbial community impact the aphid

microbiome? (Bottom – Up effects)

Questions

Soil microbial community

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Aphids impact on soil bacterial community

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

1. Can aphids or aphid’s endosymbionts impact the

soil microbial community?

(Top – Down effects)

2. Can soil microbial community impact the aphid

microbiome? (Bottom – Up effects)

Questions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Soil microbial community impact on aphids’ microbiome

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

1. Does plant species matter?

2. Can belowground microbial community shape

the plant microbiota?

Further questions

Plant species structures microbial community

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Belowground microbial community shapes plant microbial assembly

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

16S

PLANT SPECIES APHID PRESENCE Aphid genotype

PLANT SPECIES APHID PRESENCE

-

LEA

VES

SOIL SOIL

PLANT SPECIES SOIL

PLANT SPECIES SOIL

SOIL PLANT SPECIES

SOIL PLANT SPECIES

SOIL PLANT SPECIES

RO

OTS

ITS 18S

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Future work

• Identify the microbial actors of these interactions

• Identify the effectors that lead to these effects

• Extend the analysis to other plant species

• Disclose the role of H. defensa

• Networks

With aphids

Network analysis

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Without aphids

Concluding remarks

• Fungal community associated to B. oleae

• Scolytid beetles trapped at harbors carry unknown fungi

• Aboveground-Belowground insect/fungi interactions

• Novel and unexpected answers to this ecological topic still poorly understood.

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Thank you! Any question?