Sustainable Agriculture An Insight Into Ganoderma 24 February 2011 Le Meridien Hotel, Kuala Lumpur MDs Opening Address Good

d afternoon, ladies and gentlemen. On behalf of the Agrinos Team, welcome to our 2nd Seminar cum Dialogue entitled, SUSTAINABLE AGRICULTURE AN INSIGHT INTO GANODERMA. Our first seminar Sustainable Agriculture BACK TO BASICS, THE ROLE OF MICROBES was held on 18th September 2010 in Sandakan, Sabah. We are happy to have in the audience the Co-founder of Agrinos, Mr Kjetil Bohn from Norway, the developer of two of our three products Mr Karl Fick from Mexico and our Senior Agronomist Ms Daniela Garcia from USA. Thank you all for taking time to attend this seminar and we trust you all would contribute to this seminar and dialogue today, and benefit from it too. A very big thank you to the speakers and moderator: Mr Chung Gait Fee Dr Richard Cooper Dr Gurmit Singh Dr Wong Mui Yun Mr Karl Reiner Fick A very special thanks to Mr Chung who agreed to present a paper at the 11th hour as Mr Teh Chong Lay could not attend the seminar and present the paper due to unforeseen circumstances. I would also like to thank Mr Chew Poh Soon for giving us the names of some very important people who are interested on this subject (Ganoderma), some of whom are present in this hall.

Let me take a couple of minutes to tell you about who we are (Agrinos);
- Agrinos is an agriculturally focused, green technology company. - We currently are present in Norway, Mexico, Columbia, USA, China, Malaysia, Indonesia, India, EU and Ghana.

Agrinos group overview

Global development initiatives
Norway Agrinos AS - HQ Global management, R&D - Hub for Europe/Middle East & N Africa China Agrinos China Ltd. - Sales & distribution - Initial greenhouse focus - 450 distributors ready EU - Cash crop and greenhouse focus

US Agrinos Inc. - Sales and distribution - Cash crop focus - Hub for North America

Mexico Agrinos S.A. de C.V. - Production/R&D facilities - Cash crop focus - Hub for S America

Sub-Saharan Africa - Entry markets - Export agriculture focus - Research cooperation

India - Trials and product registration underway - Broadacre focus

Malaysia/Indonesia Agrinos Sdn Bhd - Sales & distribution - Oil palm focus - Hub for SE Asia

AGRINOS COMPANY OVERVIEW FEBRUARY , 2011

Agrinos owns, manufactures and distributes its propriety High Yield Technology (HYT) products. HYT products are: * Microbial-based and * Provide a basis for high yield sustainable agriculture.
- HYT products have been developed and tested for more than 15 years. - We are currently carrying out R&D activities in the USA, Mexico, Malaysia, Norway, China and India. When we hear the word sustainable the immediate perception to most people is lower ROI or higher cost or lower yield etc. Now here is a big difference HYT provides a basis for a high yield, sustainable crop with a higher ROI. The key objective today is for all of us to get a better idea of what has been done todate, what seems to work and what doesnt and possibly find new pathways to look into to solve this problem of Ganoderma. We at Agrinos are starting a five-year study with four researchers (Dr Wong Mui Yun, Dr Ganesan Vadamalai, Assoc. Prof. Dr Ahmad Husni Mohd Hanif & Dr Siva K Balasundram) from UPM and 5 plantation partners this month and we also hope to learn from our dialogue today on the avenues to explore. We believe that our Integrated Approach to solving this problem holds good promise in the long term. I will not go into the details about the Integrated Approach as I will leave this to my friend Karl Fick to talk about this in his presentation. Ladies and gentlemen, as you know the programme is as follows:

Programme
1.00pm 2.30pm 2.30pm 2.40pm Lunch & Registration of participants Welcome address & Opening Speech MD of Agrinos Sdn Bhd Mohan Ramalingam Ganoderma basal and upper stem rots of oil palm: Epidemiology; Infection; Resistance; Biological control; Future directions & The threat of Fusarium wilt President of the British Society of Plant Pathology, University of Bath, England Dr Richard Cooper Q&A Overview and Research Updates of Ganoderma Incidence in Oil Palm Plant Pathologist, Universiti Putra Malaysia Dr Wong Mui Yun Q&A TEA BREAK Management of Ganoderma in Oil Palm: A Commercial Perspective Advisor in Crop Protection Mr Chung Gait Fee Q&A Soil-Plant System , Integrated Management Chief Technical Officer, Agrinos Mexico Mr Karl Fick Panel Discussion and Q & A Session Closing & Thank You MD of Agrinos Sdn Bhd Mohan Ramalingam

2.40pm 3.10pm

3.10pm 3.20pm 3.20pm 3.50pm

3.50pm 4.00pm 4.00pm 4.20pm 4.20pm 4.50pm

4.50pm 5.00pm 5.00pm 5.30pm

5.30pm 6.30pm 6.30pm

Please, please participate in the Q & A and the Panel Discussion later on. Our objective is also for all of us to gain as much as possible from the next four hours or so. And we can only do this if we all participate. Thank you once again for your presence.

Presents

Sustainable Agriculture An Insight on Ganoderma

24 February 2011 Sultans Ballroom Le Meridien Hotel, Kuala Lumpur
Come, join us for a day of dialogue with the experts in the field of Ganoderma

Dr Richard Cooper Richard Cooper obtained his MSc and PhD from the Imperial College London in the mid 70s. In 1984 he was awarded by the Royal College of Science the Huxley Memorial Medal for research in Natural Sciences. He obtained a lectureship at University of Bath and is a now a Reader there. This long run was broken by a sabbatical in 1981 while at the University of Missouri, as a Leverhulme visiting research professor to the University of West Indies, and by various overseas field work on diseases of certain tropical crop. His interests centre on mechanisms of plant-pathogen interactions and involve both attack and defence, because the two are inextricably linked through coevolution. It is ironic though that study of one of the decidedly non-model systems (cacao wilt) led to the unexpected and exciting discovery of mans first fungicide, elemental sulphur, already being used by plants as an induced phytoalexin in this and later in other diverse crop species. Richard had been a member of BSPP for as long as he can remember and is an editor for Plant Pathology and Molecular Plant Pathology. He teaches plant pathology to year two undergraduates and plant-microorganism interaction to years 3 and 4.

Two major diseases of oil palm: Ganoderma boninense- basal & upper stem rot and a potential threat: Fusarium oxysporum- vascular wilt Richard M Cooper Dept Biology & Biochemistry University of Bath, UK

Ganoderma Basal and Upper Stem Rot of Oil Palm

Epidemiology Infection Resistance Biological control

Ganoderma Research at U Bath

Epidemiology: mycelium and/or spores? Mode of infection & pathogenicity Screening for resistance Biological control

Papers in prep: [1] Role of basidiospores (about to be submitted) [2] Cell wall degradation [3] Biological Control

Epidemiology: mycelium or spore infection? Infection of intact roots & subsequent bole infection (from infested wood block inoculum)

Wounding roots increases infection but not required by all isolates Method discriminates between isolate virulence. Infection requires intimate association

Type of inoculum influences infection: rubber wood > oil palm wood

Oil palm root Structure. Ganoderma must penetrate outer tough layers

Disease Progression: Root-Bole-Symptoms

Clearly infection via roots leads to typical BSR

LS Lesion extending from infected root(s)

TS

Natural infection can occur through multiple roots

These observations strongly implicate root infection as one cause of BSR Uninfected 13 yr Root-bole interface Ganoderma progresses from degraded root Symptomless, infected 15 yr: Multiple root infections

TS trunk base: Lesions on 1 side indicate 1 or more roots infected Root

Mode of root/stem infection: Developmental switches

Ganoderma seems to undergo developmental switches: Biotrophic then necrotrophic* invasion of root cortex.

Biotrophic then necrotrophic intracellular invasion of cells in basal palm stem (bole); rapid starch depletion.
Massive hyphal aggregation culminating in formation of basidiocarps * Hemibiotrophy
Tough mycelial crust

[2] Endophyticintracellular growth in bole; starch rapidly depleted

[3] Melanized, aggregated mycelium;Note very thick cell walls

[1] Degradation of root cortical cell walls

Microscopy of infection phases

Ganoderma degrades all major components of oil palm cell wall: Polymer and enzyme analyses showing removal of all main structural components; also starch from stem base
% Polymer Content of control Oil Palm Wood
9% 11% 2% 4% 56%

Dry wt

Lignin

18%

% Polymer Content of Degraded Oil Palm in vivo

18%

Cellulose

0% 10% 46%

26%

Cellulose Pectin Hemicellulose + Others

Lignin Starch

Ganoderma produces key extracellular walldegrading enzymes in culture (semi-solid) and in infected wood:
Cellulases; lignase; pectinases; xylanase, glycosidases; also amylase.

Lignin Peroxidase activity in infected wood

Laccase production on GSM Activity shown by discoloration due to oxidation of tannic acid

Screening for Ganoderma Resistance Small rubber-wood blocks, attached, gave reproducible & relatively rapid infection
Rubber wood

Palm wood

Root invasion: faster growth than previously reported: 4.4 cm/month

Soil Temperature has a Dramatic Effect on Infection

Effect of Palm Shading on Infection
100
Shaded Unshaded

80
% infection

60 40 20 0 1 2 3 4 5 6 7 8 9 10

Increased shading of seedlings increases dramatically infection: 90% vs 20% of palms infected after 10 months Temps of non-shaded soil often reach >40C during the hottest part of the day (North Sumatra trial)

Months after treatment

Effect of Shade on Soil Temperature

45 Shaded
Temperature (C)

This finding will:

[1] improve testing isolates and screening palm lines,

40 35 30 25 20 8h

Unshaded

10h

12h Time

14h

16h

[2] may explain why disease appears late (eg >10 years) in plantations after canopy forms

Inhibition in unshaded soil is because: Ganoderma boninense has optimal growth 25-30C.
Likewise Fusarium oxysporum. They probably requires canopy cover to function

6
Hyphal extension/day (mm)

Hyphal extension of 2 isolates

BLRS1 GMR3

25C

5 4 3 2 1 0 25 30 37 40

30C

45

35C

40C

28C

Temperature (C)

The Role of Basidiospores?

So far all attempts by various groups to induce infection using spores have failed; dikaryon is required However, consider: occurrence of Upper Stem Rot (USR) unlinked to BSR, considerable isolate variation within plantations vast nos. of basidiospores produced
Infection Using Dikaryon and Monokaryon Fungi
100 80
% infection

Monokaryon

Dikaryon

60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 Months after treatment

Sources of high Ganoderma spore inoculum levels within plantations

Windrows, from 25 year old toppled palms become infested rapidly with Ganoderma, or already contain the pathogen Also basidiocarps on infected trees

Ganoderma basidiocarps produce vast numbers (2-10/m) of basidiospores in plantations. *First quantitative data on aerial inoculum* Number is influenced by [1] time of day [2] plantation history/location

Rees et 2011 Plant Pathology

About to be submitted

Molecular analysis of Ganoderma genetic variability to reveal possible:

Mycelial spread, tree to tree? BSR-USR connections? Pathogen variation within & between plantations The importance of spores & outcrossing

RAMS (Randomly Amplified Microsatellites)

used to study epidemiology RAMS gave 6-12 clear bands 400-1500bp. Showed differences inseparable by ITS sequencing

Molecular analysis of Ganoderma genetic variability : RAMS profiles

Results: High genetic variation within fallen palms (FP); no link to adjacent BSR palms Some BSR trees (3/7) contained several isolates. Implies multiple infection of palms. Concurs with multiple root infection USRs only contained single isolate; suggests single infection event. Not related to BSR isolates. Not related to other USRs Neighbouring infected palms: only one plot had identical isolates=mycelial spread, not spores. Others all differed. Cluster analysis showed no more clustering within plantations than between them. Ganoderma from distant plantations (23 kms) clustered as much as within plantations. ie great diversity.

Conclusion: Basidiospores must play a key role.

The high genetic diversity also reported by Miller et al 1999 & Ariffin et al. 1996 in Malaysia & Pilotti & Sanderson 2003in PNG (using mitochondrial DNA markers; RAPDS; mating alleles; VCGs) must arise from sexual recombination & dispersal.
Outcrossing is forced because Ganoderma is heterothallic, with multiple alleles at both mating loci. Spores mate readily & anastomosis was evident from our observations on inoculated fronds (later images).** USR infections are unique genetically and also logically would derive from airborne spores

**Commercial palm is tenera seed from dura X pisifera & presents segregating populations and a
heterogeneous host. Ganoderma is ideally suited to cope with this selection pressure through outcrossing & prolific spore production to adapt for aggressiveness traits.

Contradictions! Spore infection never achieved by several labs How/where do spores establish infection dikaryotic mycelium?

How/where would the spores establish mycelium?

Ganoderma has very low competitive ability in soil & organic debris (which accumulates at frond bases).

4 days

10 days

FD

SOIL

S=sterile NS=non-sterile

Why is Ganoderma significant is some areas but virtually absent in others?

Soil type? (Physicochemical? Microbial?) Escape? (but wind-blown spores should ensure wide distribution*.)

*Spore longevity? Dehydration? UV?

Possible direct entry and mating site in xylem of cut petioles and peduncles? Cut xylem exerts negative tension and will suck in spores to length of xylem vessels

TS cut petiole showing dye uptake at 20 cm from wound

Spores can only travel the length of a xylem vessel

LS petiole. Eosin dye shows rapid uptake post-wounding

Vessel end wall will trap spores

Petiole vessels took up max 10 cm, Shown with fluorescent particles to simulate spores

Possible direct entry and mating site in xylem of cut petioles and peduncles? Germination under field conditions on fresh wound sites, 48 h pi

**
Frond parenchyma Spores in xylem should be protected from: [1] microbial competition [2] UV [3] dehydration

peduncle xylem

Cryo-Scanning Electron Microscopy

**
Trunk wound

** Anastomosis to form
heterokaryons

Is spore infection via cut fronds?

Extensive and frequent wound sites are created during routine pruning and harvesting

Mostly indirect evidence

Thompson 1931 first to conclude infection of fronds with spores.

Sanderson & Pilotti 1997 cut decayed frond base & followed lesions back into stem base. Initial infection would be left near centre of stem & appear to have originated from the base . Panchal & Bridge 2005 detected Ganoderma in recently cut frond bases especially near stem base (using PCR primer GanET) Lim et al 1992 infected frond surfaces (NOT using spores); Hasan et al 2005 using many methods including spores, failed.

Summary of infection studies:

Evidence (indirect) for basidiospore infection; via cut fronds Root infection after contacting colonized debris Infection by clonal vegetative mycelium in root to root spread

Implications of basidiospore involvement for control. In PNG zero tolerance of basidiocarps. In Malaysia perhaps harvesters could remove basidiocarps routinely, unless in heavily affected areas where impracticable. Protection of surfaces of newly cut fronds. Likely adaptability of Ganoderma to host resistance; use polygenic resistance not monogenic Implications for disease resistance screening and possibly disease resistance expression (roots currently used).

Biological Control? [1] Antagonists

[2] Competitors

Antagonistic fungi isolated from felled palms in North Sumatra Also commercial Streptomyces (Mycostop ) tested Some Trichodermas inhibit and even eliminate Ganoderma from infested wood. Streptomyces too, if inoculum boosted.

Trichoderma

Ganoderma inhibition in vitro

Streptomyces

Trichoderma Inhibition of Root Infection

Two isolates completely inhibited root infection

Inhibition of root infection by 3 Trichoderma isolates Ganoderma established in blocks 2 weeks; Trichoderma 3 weeks. Blocks attached to roots

Blocks post-inoculation show thick melanized Ganoderma hyphae (left), prevented by Trichoderma treatment (right)

Trichoderma inhibition of infection by soil drench

Trichoderma spores applied to soil/roots Ganoderma-infested blocks attached to roots Infection after 2 months analysed:

Significant but incomplete control

Appearance of blocks after 2 months shows removal of Ganoderma (right side) in many cases by Trichoderma

Persistence of Trichoderma in soil is limited.

Some increase in soil then decline

Root colonization might hold the key.

Light microscopy shows invasion of epidermis and outer cortex by Trichoderma

Implications?
Long term persistence to counter root infection Possible induction or priming of host defences. Application to wounds (petioles, peduncles?) vs basidiospores?

Biological Control?
[1] Antagonists

[2] Competitors
Wood-degrading fungi isolated from decaying palms & selected for ability to degrade palm wood in vitro. Strategy: remove nutrient base/outcompete Ganoderma potential inoculum in the field (application to windrows/debris).
Pre-emptive application through niche exclusion used in control of root rot of pine by Phlebiopsis gigantea vs Heterobasidion annosum
Wood dry wt loss by selected degraders cf. two Ganoderma isolates

Lignin degradation kinetics compared

Investigation of enhanced wood decay and inhibition of infection:

Preliminary trial shows inoculated palm trunk discs were not degraded faster with inoculated (infested corn chips) wood decay fungi

Requires more extensive study; possibly best applied to fragmented trunks

Infection not significantly reduced by wood degraders

Ganoderma blocks were surrounded by corn chip inoculum of wooddegraders. Best application might be in composting palm debris?

Future Research
GANODERMA VIRULENCE Genome of Ganoderma boninense isolates soon to be added (MPOB) to other basidiomycetes: Phanerochaete (white rot), Heterobasidion (tree pathogen)
RESISTANCE Is there substantial natural resistance to Ganoderma to be exploited?. Ganoderma generates great diversity through outcrossing. Screen diverse OP genotypes. Diversity in W & C Africa. Natural seedling variation within DxP crosses. Differences in susceptibility have been detected within the two Elaeis species, guineensis and oleifera. Also MPOB breeding programme e.g. tolerant PK 2567 (DxP) reported by Idris et al 1994; FELDA progenies; Durand-Gasellin, pers com)
If there is significant resistance, use Marker Assisted Breeding (ongoing) If not significant resistance, use Transgenes? But which? Possible synergy/synteny with other monocots eg coconut; rice cDNA-AFLP to ID candidate genes. Oil Palm microchip needs expanding & exploiting for understanding defence responses.

INFECTION Can basidiospores infect via cut frond bases?

BIOCONTROL Root-invading Trichoderma? Exclude Ganoderma and stimulate host defences Treat cut petioles & peduncles with Trichoderma? Is this the major invasion route? Treat windrows/debris with degraders REDUCE INOCULUM & PATHOGEN VARIATION Remove basidiocarps & fallen palms

Acknowledgements

ROBERT REES, U Bath Julie Flood, CABI Yonnes Hassan, Lonsum Hugh Foster Steve Nelson

Fusarium Vascular Wilt

Epidemiology; Detection; Prevention; Pathogen variation; Resistance; Malaysian situation

Fusarium wilt is perhaps the most serious disease of oil palm, especially in replantings. Affected areas: Nigeria, Congo, Cameroon, Ivory Coast, Ghana and other locations in C & W Africa. Also on localized plantations in Brazil and Ecuador. Absent from S E Asia: Fusarium wilt

Why?
chlamydospores

Causal agent: Fusarium oxysporum f. sp elaeidis Soil-borne fungus; produces macro- & microconidia & long-lived chlamydospores. Vigorous saprotroph; contrast with Ganoderma -a weak competitor in plantation soil and organic debris (Rees et al., 2007).

conidia

micromacro-

Internal symptoms:
Browning of vascular (xylem) tissue diagnostic, distinguishes from Ganoderma Pathogen as hyphae or conidia in xylem vessels Host responses: vessels may be occluded by gels and tyloses-defence response.

Browning Hyphae & conidia in vessels

Tyloses

Colonization: systemic, by rapid movement of conidia in transpiration stream. Xylem vessel end walls are breached at pits by conidial germ tubes or hyphae.

Vessel end wall

Conidia trapped at end wall

Fusarium passes vessel-vessel via pits. [where secondary wall not deposited leaving thin primary wall]

ECONOMIC IMPORTANCE
Dumortier et al. (1992): yield from palms with acute wilt in year before death as c. 54% and from palms with chronic wilt as 30% that of healthy palms. Renard et al. (1993): yield losses as 15% in a susceptible cross and 6% in a tolerant cross.

Renard and de Franqueville (1989): 6-16% yield reduction in young palms of which 5.5% showed external symptoms; this was only 6 years post-planting.

H. Corley

The decline in production in some African countries can be partly explained by Fusarium wilt (Flood et al., 1989)
Aerial views of Foe affected Zaire plantations.

P. D. Turner

Epidemiology
Root Infection: Foe first infects intact roots (Cooper et al., 1989). Elongating roots probably contact infected roots or debris containing chlamydospores; germination is induced by root exudates. Model of tree-tree spread is supported by occurrence of infected palms in pairs or groups (Rusli & Cooper, see fig below) & greater infection of palms with missing neighbours
(Dumortier et al., 1992).

Dead groups

Fusarium on male inflorescences

Ghana-disease clusters

Also aerial spread? F. oxysporum sporulates on male inflorescences;Foe could also be aerially dispersed. 96 viable spores m- of F. oxysporum resp. (Cooper et al., 1989).

50% of pollen samples (Zaire) contained F. oxysporum 4x105/g. Some isolates were pathogenic (Flood et al.,1990). NB Quarantine implications.

Disease epidemiology in 4 Ghana oil palm plantations

Vascular wilt disease symptoms

Chronic

Acute

Dead

Field assessment: Pathogen presence in trunks confirmed by isolation using auger technique (later slide)

Spread via seed? Implications for Malaysia

Foe can also contaminate outside & inside of seeds (Flood Cooper et al., 1990). About 50% of batches (Zaire/DRC) contaminated with 5x10 cfu (colony forming units) per seed; contamination of kernels in 30% of these samples was up to 100 cfu. Contamination is possibly post-harvest, eg retting to remove the pericarp, when Foe can proliferate (Cooper et al., 1989). Artificial infestation with Foe leads to a small proportion (3%) of infected plants

Foe penetrates seed via germ pore?

Genetic analyses reveal: (1) Foe can be spread between continents.

Contamination with Foe of breeding materials has serious implications for prevention of world-wide spread. Trans-continental spread appears already to have occurred. Limited, single plantation outbreaks in Brazil (Van de Lande, 1984)) and Ecuador (Renard and de Franqueville, 1989) in the 1980s. Foe isolates had identical RFLP patterns and were vegetatively compatible (same VCG group) with isolates from Ivory Coast (Flood Cooper et al., 1992; Mouyna et al., 1994). This suggests exported, contaminated seed was responsible.

VCG analysis Heterokaryon formation with left pairing of nitrate non-utilizing (nit) mutants

More detailed analysis later from our recent work

Control 1

Breeding for Resistance or Exclusion:

only practical solutions.

Resistance screening:
NB risk of only assessing disease-free palms in infested plantations-distribution & amount of Foe will vary. Symptomless individuals may be escapes. Method Seedlings; defined isolate; defined inoculum; Foe spore suspension onto roots. Shade seedlings/containers to mimic canopy cover. Analysis: wilt index; bulb browning; statistics. Defined conditions and high inoculum reduces no. of reps (eg 40 to 12) and time (9 months to 6 months or less (4.5 mos Flood et al, 1993)), but it remains SLOW

Field testing of resistant/tolerant palm crosses & clones: Symptoms & yield; but need for internal analysis
Vascular browning and easy re-isolation in vitro of Foe allow critical evaluation of putative tolerant or resistant genotypes in breeding programmes. Non-destructive sampling (Zaire) by removing trunk cylinders with an auger showed 25% healthy palms had internal symptoms (Mepsted et al., 1991). Also in Zaire, 54% healthy palms were infected; yet 40% with symptoms (probably induced by other pathogens) were not infected with Foe
(Buchanan, 1999).

Auger
Healthy xylem

Infected xylem

Inheritance and level of resistance

Resistance is partial (other than near-immunity of Dumpy Deli dura (Rosenquist, 1990)). But sustained breeding programmes have markedly reduced losses e.g. from 20-30% to <3% in Ivory Coast (deFranqueville & Renard, 1990)

How variable is Foe? Faced with single R genes, F.oxysporum evolves new races. e.g. F.o. lycopersici /tomato; ciceri/chickpea; dianthi/Dianthus. Can palms bred for resistance in one area, succumb in another? Examples of this e.g Ivory Coast progenies in Nigeria; Nigerian progenies in Ivory Coast and Zaire lines to a Brazilian isolate (Flood, Cooper et al., 1993). Isolate-clone/progenies inoculations suggest unlikely: no significant interactions. But, ranking of some Foe isolates by clones varied markedly and might explain occasional resistance breakdown (Mepsted, Cooper et al., 1994)

Control 2: Exclusion
Prevention of importation of Foe to unaffected areas is clearly the most effective control measure. But continued exploitation of genetic diversity from African centre of diversity is essential, so movement will continue.
Quarantine for seed: Artificially infested seed under glasshouse conditions at Bath gave c. 3% infection
(Flood et al., 1994).

Standard dormancy-breaking heat treatment at 40C reduces Foe contamination but does not eradicate it. We developed a method of vacuum infiltration with fungicide (Sportak Alpha): eradicates Foe from seed coat & from within seeds (Flood et al., 1994). Method used commercially & for seed entering Malaysia & Indonesia. Also equipment just introduced by us to Ghana A decontamination method for pollen has yet to be developed Seed soaked in fungicide plus dye -no penetration
?

Seed vacuum-infiltrated; kernel is coated

Commercial advantage of seed decontamination

DETECTION
Detection and specific ID of Foe is required for: Field testing of resistant palm lines, Presence in palms and plantation soils for epidemiological studies Key role in quarantine for seed &pollen

Reisolation is easy onto Fusarium-selective medium (Papavizas, 1967) and detection from xylem in auger cores should lead yield only Foe, but contamination does occur (unpub data).

To date only the species F. oxysporum can be diagnosed by morphology and by PCR. Thus quality seed & pollen batches will be discarded at quarantine if non-Foe F. oxysporum, a very common contaminant, is present. Only proof of Foe is lengthy inoculation of oil palms (>6 months): not practical.

STRATEGY:

Genus Fusarium specific primers F. oxysporum species specific primers Pathotype (Foe) specific primers

1000bp

500bp 200bp

PCR assays and validation for genus specific primers (Fus f1 and Fus r1)

Primers amplified all Fusarium isolates & excluded phylogenetically closely related genera

Ladder
Sclerotinia sclerotiorum Aspergillus sp.

Verticillium sp.
Trichoderma sp. Foe (Ghana) Foe (Congo) Foe (Brazil) Foxy cubense Foxy cubense Foxy narcissi Foxy phaseoli

Foxy tulipae
Foxy vasinfectum Foxy lycopersici Foxy lycopersici Foxy pisi

Foxy pisi
F. graminearum Ladder

1000bp

500bp

Specificity of primer pair FoxyF2 and EF2 as potential F. oxysporum species specific primers

Note exclusion of all other Fusarium spp & closely related fungi

200bp

Ladder Sclerotinia sclerotiorum Aspergillus sp. Verticillium sp. Trichoderma sp. F. solani F. foetens F. redolens F. fujikuroi F. culmorum F. graminearum Foxy phaseoli Foxy tulipae Foxy vasinfectum Foxy lycopersici Foxy lycopersici Foxy pisi Foxy pisi Foxy elaeidis** Ladder

Specific detection of Foe: problems and current strategy

Problematic because host specificity in F. oxysporum can evolve multiple times. e.g. formae speciales cubense (banana), gladioli (gladiolus) and lycopersici (tomato) are polyphyletic (Lievens et al., 2008).

Housekeeping genes & random markers do not enable specific pathotype detection. In many plant-pathogen interactions virulence effector gene products dictate host-pathogen specificity. Protein effectors target host defences. We are developing Foe specific primers based on these.

Why no Fusarium wilt in Malaysia and Indonesia?

Anomaly that SE Asia has escaped Foe, based on:
[1] no symptoms [2] soil isolates non-pathogenic [3] auger sampling revealed no infection (Mepsted, Cooper et al., 1991) Much importation in past of African (often contaminated) seed & pollen pre-quarantine rules. Oil palm genotypes were mainly Foe susceptible (current lines?)**** Climate should be conducive to disease (Ho et al., 1985)

****Recent test in UK of Malaysian palms to African isolates of Foe

Malaysian oil palm progenies are susceptible to Foe; it remains as a future threat?

0
0 1 No symptom

Disease wilt index

Slight necrosis/chlorosis on 1-2 leaf tips -usually oldest leaves

Necrosis / chlorosis over one quarter of leaves plant and some shortening of the youngest leaves
Severe necrosis / chlorosis over one half of the leaves of the plant. Extensive leaf desiccation and stunting Severe necrosis / chlorosis over three quarters of the leaves of the plant. Extensive leaf desiccation and stunting Dead

4
5

All palm genotypes are susceptible to Foe: Disease index

Mean wilt index

PK 5506 (F3)
PK 5506 (16F)

Mean wilt index

PK 5463 (16F)
3

0 0 5 10 Time (weeks) after inoculation 15 20 25

PK 5463 (F3)
0
0 5 10 15 20 25

Time (weeks) after inoculation

4
Mean wilt index

PK 5493 (16F)
3 2

Mean wilt index

PK 5525 (16F)
2

PK 5493 (F3)

PK 5525 (F3)
1 0

0 0 5 10 15 20 25

10

15

20

25

Time (weeks) after inoculation

Time (weeks) after inoculation

Previous data reveal Foe is variable. But how variable?

Ongoing investigation* of genetic diversity of Foe isolates between & within countries *gene sequencing; RAMs; RAPDs
Disease control is through resistant varieties Resistance could be vulnerable to pathogen diversity Foe genetic profiling might reveal its epidemiology & spread

Conclusions
By continuing to exclude Foe, SE Asian palm oil-producing countries can direct efforts towards productivity, new products and quality. However Fusarium remains a distinct threat. Development of a specific Foe probe would be highly beneficial.

Ganoderma remains a key problem here and involves even greater challenges than working with and controlling Fusarium wilt.

Pathologists trained in both diseases and preferably with access to specialists need to be part of the continued protection from Fusarium. Advances with understanding of Ganoderma infection and its control are still required.

Ongoing Research
Development of Foe specific primers Investigation of Trichoderma as a potential biological control agent. Fungal interactions in roots using transformed isolates expressing fluorescent proteins GFP & RFP.

Pathogenicity study of Ghanaian F. oxysporum isolates from acute, chronic and symptomless palms.
Defence gene expression during Foe infection of resistant & susceptible palm genotypes Study of potential suppressive soil towards Foe in Malaysia Test of efficacy of Agrinos HYT A_C vs Fusarium

Acknowledgements
University of Bath:
Early work: Drs Andrew Buchanan; Roger Mepsted Andrew Buchanan; Tabu Paul; Julie Flood

Many Colleagues in West Africa Funding: Unilever Plantations Group: Hereward Corley

BBSRC and Plant Breeding International

Acknowledgements: current work

(University of Bath) HEFNI RUSLI Dr Alan Wheals Dr. Sweta Sharma Dr Alastair Muir Dr Mathew Wills Christophe Lambing Emma Woodland Caroline Roger Vicki Wright
Cultures & vectors

Dr Idris Abu Seman (MPOB) Dr Julie Flood (CABI) Ghana: OPRI Plantations: GOPDC, NORPALM Unilever BOPP, TOPP
Funding: MPOB: Drs M. Basri Wahid; A Kushairi Din; Idris Abu Seman Soils: FELDA & MPOB
FASSB

Prof. David M. Geiser (Pennsylvania State University; Fusaria isolates ) Prof. Kerry ODonnell (USDA; Fusaria isolates) Prof. Baharuddin Salleh (USM; Fusaria isolates) Prof. Corby Kistler (University of Minnesota; Fusaria isolates) Dr. Christopher Thornton (Uni. Of Exeter; GFP vector)

Thomas Sundelin (Kbenhavns Universitet, red fluoro protein vector )

Ghananian Colleagues
Oil Palm Research Institute (OPRI)

Dr. Sheila Tagoe and Mr. K. Boafo Afrim

NORPALM Plantation

TOPP

BOPP
GOPDC Plantation

Comparing DNA fingerprinting techniques to determine genetic relationship between Foe isolates between and within countries using RAMP & RAPD. Ongoing study to obtain more robust analysis. Data will be combined to that effect.

RAMPs
Tax: 17 Characters: 204

RAPD
Tax: 17 Characters: 145

1 Foe clade

Clade 3

Clade 2 Clade 1

Analysis showed the high level of genetic similarity of Foe isolates ie they belong to the same unique clonal lineage.

Outgroup

RAPD analysis showed more genetic diversity of Foe isolates which formed into 3 different clades.

Genetic analyses reveal: (2) Local populations are similar.

Foe origin, pathogenicity, VCG group, RFLP group

Local Foe populations have evolved to be similar. All pathogenic isolates from Zaire in the same VCG & separate from Brazilian isolates. In contrast, non-pathogenic F. oxysporum isolates from soil & roots of healthy palms in Zaire & Malaysia had high VCG diversity; supported by RFLP analysis (Flood et al.,1992).

Development of genus Fusarium specific primers

FUS-1f 18S 5.8S FUS-1r ITS 1 ITS 2 28S

Ribosomal DNA region

Development of F. oxysporum species specific primers

Molecular phylogeny of Fusarium species complex (SC) based on RNA polymerase II second largest subunit (RPB2) (ODonnell et al., 2007

Gibberella (Fusarium) fujikuroi Species Complex Fusarium oxysporum SC Fusarium incarnatum/ equiseti SC

F. chlamydosporum SC

F. solani SC

F. dimerum SC
)

*ef 1 Intron 1 Exon 2 Exon 1

Foxy F2 Intron 2 Exon 3 Intron 3 Exon 4 *ef 2

Fig 2: Map of the TEF gene region in Fusarium used in FUSARIUM-ID, with primer location.

The translation elongation factor 1-a (TEF) gene:

(i) highly informative at species level in Fusarium (ii) non-orthologous copies of the gene have not been detected in the genus (iii) universal primers designed that work across phylogenetic genus breadth
(*Geiser et al., 2004)