Novel approaches to the management of soilborne

diseases in vegetable crops

Presented on behalf of:

Horticulture Australia Limited

DPI VictoriaIan Porter

Plant volatilesCassie Scoble

Caroline DonaldDenise Wite

Oscar Villalta

Fungal volatilesRoss Mann

Scott Mattner

Systemic Acquired Resistance

Arati AgarwalCaroline Donald

Northern Territory Dept. of Resources

GraftingBarry Condè

Mark Traynor

With thanks to:

Disease control strategies

Inoculum load

Pathogen lifecycle

Host resistance

Direct effects on the pathogenI) Plant volatiles• Secondary metabolites

ie. essential oilsvolatile compounds

• Rich in bioactive chemicals terpenoids and phenolics

• Strong antimicrobial activity

Aim: Screen efficacy of essential oils against key soilborne pathogens

Contact bioassaysVapour phase

(volatile) bioassays

Pot trials Preliminary field trials

Essential oilsInhibited growth & biocidal

– Thyme, Clove bud, Origanum

Inhibited growth but not biocidal– Geranium, Peppermint, Pine,

Tea tree, Basil, Fennel Sweet

Did not inhibit growth– Eucalyptus, Rosemary,

Black pepper, Orange sweet, Cardamom

Pure compounds– Terpenoid (thymol, carvacrol & eugenol) and phenolic (geraniol) compounds - NO growth &

biocidal against all pathogens at lowest rate tested 500 ppm

Commercial blends– Effective only at the higher concentrations and only against some pathogens – eg. ECO-V (mustard oil & allyl isothiocyanates) and Vigor (mustard & chilli pepper extracts)

biocidal against R. solani and inhibitory to F. oxysporum at 2500ppm

Summary of in-vitro (contact bioassay) screening% growth compared to control

Essential OilRate ppm S.minor F.oxysporum R.solani T.hamatum

Origanum 1 118.49 49.93 51.9 80.6910 96.28 54.64 75.44 80.31100 61.47 24.22 0.00 38.03500 0.00 0.00 0.00 0

Thyme 1 96.76 26.81 101.15 85.0610 96.32 25.99 90.49 78.37100 68.96 5.12 0.00 32.91500 0.00 0.00 0.00 0

Clove bud 1 98.25 21.20 91.54 98.2210 87.42 21.40 76.78 112.41100 66.79 7.29 0.00 59.93500 0.00 0.00 0.00 0

Red= Biocidal activity (plugs did not regrow on umamended agar)

400 kg/ ha 200 kg/ ha

Basamid® 100% emergence

0 %

0 %

10% 5 % 1%

Origanum 32% emergence

Uninoc Inoc

Treatment Active ingredient

Disease incidence

(%)

Disease severity

(0-5)Perlka® calcium

cyanamide100 b 4.0 b

Fumafert® mustard meal & neem cake

99 b 3.9 b

Bioweed™ pine oil 100 b 3.8 b

Promax™ thyme oil 100 b 3.4 b

Vigor® mustard & chilli pepper extracts

100 b 3.3 a

ECO-V mustard oil & allyl isothiocyanates

98 b 3.3 a

Basamid® dazomet 80 a 2.9 a

Untreated 100 b 4.0 b

Root rot incidence and severity on green beans grown in soil treated with formulations of plant products

compared to Perlka® & Basamid® at Lindenow, Victoria.

56% emergenceThyme

5%10% 1%

Direct effects on the pathogenII) Fungal volatiles

• Fungi that live in native flora (endophytes)- volatile metabolites

• Mycofumigation- Incorporation of endophyte into soil where it

releases volatile biocidal compounds

Aim - Screen native flora for volatile producing endophytes and evaluate efficacy against key pathogens

Percent inhibition of three pathogens of vegetable crops (Pythium sulcatum, Fusarium oxysporum and Sclerotinia minor) following exposure (5 days) to volatile secondary metabolites produced by isolates of Genus 1 (blue – industry standard).

Pythium Fusarium Sclerotinia Isolate 1.1 100.0 % 46.4 % 100.0 % Isolate 1.2 100.0 % 6.9 % 100.0 % Isolate 1.3 100.0 % 29.4 % 100.0 % Isolate 1.4 89.0 % 21.6 % 95.0 % Isolate 1.5 100.0 % 20.6 % 100.0 % Isolate 1.6 100.0 % 10.2 % 95.2 % Isolate 1.7 100.0 % 36.1 % 100.0 % Isolate 1.8 100.0 % 28.9 % 100.0 % Isolate 1.9 54.5 % 2.9 % 44.7 % Isolate 1.10 100.0 % 27.2 % 100.0 % Isolate 1.11 100.0 % 29.2 % 100.0 % Isolate 1.12 100.0 % 25.9 % 100.0 % Isolate 1.13 100.0 % 29.0 % 100.0 % Isolate 1.14 100.0 % 0.4 % 69.5 % Isolate 1.15 100.0 % 29.4 % 98.4 % Isolate 1.16 54.2 % 0.0 % 62.5 % Isolate 1.17 68.8 % 5.9 % 64.4 % Isolate 1.18 100.0 % 9.8 % 96.3 %

Muscodor albus (CZ620) 100.0 % 32.3 % 100.0 % LSD (5 %) 12.6 7.4 13.5

P Value 0.001 0.001 0.001

Antimicrobial activity•18 endophytes (from one genus) demonstrating strong antimicrobial activity in vitro (septum plate bioassays)

GC/MS profile of volatile compounds produced by Isolate 1.1 over a 12 day period (peaks relate to individual compounds; y axis relates to concentration of those compounds).

Chromatogram Plots

5 10 15 20 25 30 35minutes

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10-2-A_Rep2.xms 10.0:250.0> 10.0:250.0>

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2 days

3 days

4 days

5 days

10 days

11 days

12 days

Pinene (terpenes)

Eucalyptol

Cyclohexane

derivatives

Ethanol

Mycofumigation pot trial

Substrate Pythium spp. Rhizoctonia solani (AG 2.1) (pg DNA / g soil) (pg DNA / g soil)

Untreated 179 10,581 Bran : Sand (1:1) 271 11,986

Vermiculite 285 15,846 Wheat 65 4

Basamid® 4 0 P Value 0.004 <0.001

LSD (5 %) 131.5 5571.3

Effect of mycofumigation with isolate 1.1 (200g/L) on populations of Pythium spp. and Rhizoctonia solani (AG2.1) in field soil (ex. Cranbourne).

Host resistance

I) Grafting

Vegetable crops extensively grafted worldwide:

•Watermelon,cucumber, melon, bitter melon, tomato, eggplant, pepper

Countries where grafting is common:

•Japan, Korea, China, Taiwan, USA, Spain, Italy, France, Netherlands

•75% cucumber plants grown in Korea and Japan are grafted

•50% of commercial seedling growers in Korea produce grafted seedlings – the largest of these produced 9 million grafted seedlings in 2008

Australian experience

•Limited!

•Fusarium wilt of Snake bean (NT)

•Bacterial wilt of eggplant and tomato (QLD, NT)

•Improved vigour of tomatoes

Use of grafting to manage Fusarium wilt of snake beans - NT

Host resistanceII) Salicylic acid mediated Systemic Acquired Resistance

SYSTEMIN (wounding/insects)

OLIGOURONIDES(pathogen attack)

CHITOSAN

LINOLENIC ACID

JASMONIC ACID

GENE ACTIVATION

mRNA

PROTEINASE INHIBITORS

XPCMBS

XDIECASALICYLIC

ACID

X

OCTADECANOID SIGNALLING

PGPRPATHOGEN INVASION

PHENYLALANINEPHENYLALANINE

AMMONIUM LYASE (PAL)TRANS-CINNAMIC ACID

BENZOIC ACID

SALICYLIC ACID

GENE ACTIVATION

BTHINA

PATHOGENESIS RELATED PROTEINSSALICYLIC ACID SIGNALLING

BENZOIC ACID 2-HYDROXYLASE (BA2H)

SA CONJUGATESEG SA β-GLUCOSIDE(AND OTHER STORAGE OR TRANSPORT FORMS)

REDUCTION OF NPR1 FROM OLIGOMERIC TO MONOMERIC FORM

Plant defence responses mediated by jasmonic acid or Salicylic acid. (Based on Doares et al. 1995; Mauch-Mani and Métraux 1998; Ryals et al. 1996).

0.5mM salicylic acid dip applied for 1 minute.

Inoculated 4 hours later with 200µL of resting spore suspension (108 spores/mL).

Control plants

Disease index = 0

Infection rate = 0%

P. brassicae inoculated, not treated

Disease index = 81.5

Infection rate = 100%

P. brassicae inoculated

Salicylic acid pretreatment

Disease index = 20

Infection rate = 50%

- SA

+ SA

Cont 104 105 106 107

SAR in broccoli

0.1mM SA dip applied for 15 mins, 24hrs before inoculation

Gene Root Leaf

Actin 8 No change No change

PR-1 38 fold change 4 fold change

PR-2 Not induced 60 fold change

Chitinase Not induced 23 fold change

Actin 133 bp

PR-1105 bp

PR-2111 bp

Chitinase133 bp

ROOT

LEAF

Gene expression studies(plants treated with triple dip 0, 24 & 48 hrs before analysis at 72 hrs)

Where to now?Plant volatiles•Key issue: application - retention of volatiles in soil

Fungal volatiles•Work ongoing PhD Ross Mann.

Grafting•Commercial acceptance in NT snake bean industry•Seek to expand into other high value, multiple pick commodities (eg. cucumber, eggplant, capsicum)

Systemic Acquired Resistance•Work ongoing PhD David Lovelock.•Understand the biochemistry of the observed effects.•Issues - variability of response, application to older seedlings, wider use across a range of brassica hosts and pathogens.