Sustainable Agriculture –

An Insight Into Ganoderma

24 February 2011

Le Meridien Hotel, Kuala Lumpur

MD’s Opening Address

Good 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

AGRINOS COMPANY OVERVIEW – FEBRUARY , 2011

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

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

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

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

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

India - Trials and product

registration underway - Broadacre focus

EU - Cash crop and greenhouse focus

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 doesn‟t 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 Lunch & Registration of participants

2.30pm – 2.40pm Welcome address & Opening Speech

MD of Agrinos Sdn Bhd

Mohan Ramalingam

2.40pm – 3.10pm 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

3.10pm – 3.20pm Q & A

3.20pm – 3.50pm Overview and Research Updates of Ganoderma

Incidence in Oil Palm

Plant Pathologist, Universiti Putra Malaysia

Dr Wong Mui Yun

3.50pm – 4.00pm Q & A

4.00pm – 4.20pm TEA BREAK

4.20pm – 4.50pm Management of Ganoderma in Oil Palm: A Commercial Perspective

Advisor in Crop Protection

Mr Chung Gait Fee

4.50pm – 5.00pm Q & A

5.00pm – 5.30pm Soil-Plant System , Integrated Management

Chief Technical Officer, Agrinos Mexico

Mr Karl Fick

5.30pm – 6.30pm Panel Discussion and Q & A Session

6.30pm Closing & Thank You

MD of Agrinos Sdn Bhd

Mohan Ramalingam

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.

Sustainable Agriculture -

An Insight on Ganoderma 24 February 2011

Sultan’s Ballroom

Le Meridien Hotel, Kuala Lumpur

Presents…

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 co-

evolution. It is ironic though that study of one of the decidedly non-model

systems (cacao wilt) led to the unexpected and exciting discovery of man’s

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

TS

LS

Lesion extending

from infected root(s)

Natural infection can occur through

multiple roots

Uninfected 13 yr

Root-bole interface

Ganoderma progresses

from degraded root

TS trunk base:

Lesions on 1 side

indicate 1 or more

roots infected

Symptomless, infected 15 yr:

Multiple root infections

These

observations

strongly implicate

root infection as

one cause of BSR

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

[1] Degradation of root

cortical cell walls

[2] “Endophytic”intracellular growth in

bole; starch rapidly depleted

Microscopy

of infection

phases

[3] Melanized, aggregated

mycelium;Note very thick

cell walls

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

Dry wt Lignin

Cellulose

46%

26%

10%

0%

18%

Cellulose Lignin

Pectin Starch

Hemicellulose + Others

56%

18%

4%

2%

11%

9%

% Polymer Content of

Degraded Oil Palm in vivo

% Polymer Content of

control Oil Palm Wood

Lignin Peroxidase activity in infected wood

Laccase production on GSM

Activity shown by discoloration

due to oxidation of tannic acid

Ganoderma produces key extracellular wall-

degrading enzymes in culture (semi-solid) and in

infected wood:

Cellulases; lignase; pectinases; xylanase, glycosidases; also

amylase.

Screening for Ganoderma Resistance

Small rubber-wood blocks, attached,

gave reproducible & relatively rapid infection

Palm wood

Rubber wood

Root invasion: faster growth than

previously reported: ≤4.4 cm/month

Effect of Palm Shading on Infection

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10

Months after treatment

% i

nfe

cti

on

Shaded Unshaded

Soil Temperature has a

Dramatic Effect on Infection

•Increased shading of seedlings

increases dramatically infection: 90% vs

20% of palms infected after 10 months

•Temps of non-shaded soil often reach

>40ºC during the hottest part of the day

(North Sumatra trial)

•This finding will:

[1] improve testing isolates and screening

palm lines,

[2] may explain why disease appears late

(eg >10 years) in plantations after canopy

forms

Effect of Shade on Soil Temperature

20

25

30

35

40

45

8h 10h 12h 14h 16h

Time

Tem

pera

ture (

°C)

Shaded

Unshaded

Inhibition in unshaded soil is because:

Ganoderma boninense has optimal growth 25-30C. Likewise Fusarium oxysporum.

They probably requires canopy cover to function

0

1

2

3

4

5

6

25 30 37 40 45

Temperature (ºC)

Hy

ph

al

ex

ten

sio

n/d

ay

(m

m)

BLRS1

GMR3

Hyphal extension of 2

isolates 25C 30C

35C 40C 28C

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

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11 12

Months after treatment

% in

fectio

n

Monokaryon Dikaryon

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 (2³-10³/m³) 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).

S=sterile

NS=non-sterile

FD

SOIL

4 days 10 days

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?

Cut xylem exerts negative

tension and will suck in spores

to length of xylem vessels

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

TS cut petiole showing dye uptake at ≤ 20 cm from wound Spores can only travel the length of a xylem vessel

Possible direct entry and mating site in xylem of cut petioles and peduncles?

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

xylem

peduncle

Trunk wound

Cryo-Scanning Electron Microscopy

Spores in xylem should be

protected from:

[1] microbial competition

[2] UV

[3] dehydration

**

** ** 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.

Ganoderma inhibition in vitro

Trichoderma

Streptomyces

Inhibition of root infection by

3 Trichoderma isolates

Ganoderma established in blocks 2 weeks;

Trichoderma 3 weeks. Blocks attached to roots

Trichoderma Inhibition of Root Infection

Two isolates completely inhibited root infection

Blocks post-inoculation show thick

melanized Ganoderma hyphae (left),

prevented by Trichoderma treatment

(right)

Trichoderma inhibition of infection

by soil drench

Appearance of blocks after 2 months shows removal of

Ganoderma (right side) in many cases by Trichoderma

Trichoderma spores applied to soil/roots

Ganoderma-infested blocks attached to roots

Infection after 2 months analysed:

Significant but incomplete control

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 wood-

degraders.

Best application might be in composting palm debris?

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)

Future Research

GANODERMA VIRULENCE Genome of Ganoderma boninense isolates soon to be

added (MPOB) to other basidiomycetes: Phanerochaete (white rot), Heterobasidion

(tree pathogen)

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.

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

INFECTION Can basidiospores infect via cut frond bases?

ROBERT REES, U Bath

Julie Flood, CABI

Yonnes Hassan, Lonsum

Hugh Foster

Steve Nelson

Acknowledgements

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: Why?

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).

macro-

micro-

chlamydospores

conidia

Fusarium wilt

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.

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 Fusarium passes vessel-vessel via pits.

[where secondary wall not deposited

leaving thin primary wall]

Tyloses Hyphae & conidia in vessels

Browning

Conidia trapped

at end 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.

Aerial views of Foe

affected Zaire plantations.

The decline in production in some African countries

can be partly explained by Fusarium wilt (Flood et al., 1989)

H. Corley

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).

Also aerial spread? F. oxysporum sporulates on male inflorescences;Foe could also

be aerially dispersed. 96 viable spores m-³ 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.

Fusarium

on male

inflorescences

Dead

groups

Ghana-disease clusters

Disease epidemiology in 4 Ghana oil palm plantations

Chronic Acute Dead

Vascular wilt disease symptoms

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 ≤ 5x10³ 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

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 40°C 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

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.

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

500bp

1000bp

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

F. g

ram

inearu

m F

oxy p

isi

Foxy p

isi

Foxy ly

copers

ici

Foxy ly

copers

ici

Foxy v

asin

fectu

m F

oxy tu

lipae

Foxy p

haseoli

Foxy n

arc

issi

Foxy c

ube

nse

Foxy c

ube

nse

Foe (B

razil)

Foe (C

ongo)

Tric

hoderm

a s

p.

Vertic

illium

sp.

Asperg

illus s

p.

Scle

rotin

ia s

cle

rotio

rum

L

adder

Foe (G

hana)

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

500bp

1000bp

Ladder

Foxy e

laeid

is** F

oxy p

isi

Foxy p

isi

Foxy ly

copers

ici

Foxy ly

copers

ici

Foxy v

asin

fectu

m F

oxy tu

lipae

Foxy p

haseoli

F. g

ram

inearu

m

F. c

ulm

oru

m

F. fu

jikuro

i F

. redole

ns

F. fo

ete

ns

Tric

hoderm

a s

p.

Vertic

illium

sp.

Asperg

illus s

p.

Scle

rotin

ia s

cle

rotio

rum

L

adder

F. s

ola

ni

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).

Specific detection of Foe: problems and current strategy

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 No symptom

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

2

Necrosis / chlorosis over one quarter of leaves plant and some shortening of the

youngest leaves

3

Severe necrosis / chlorosis over one half of the leaves of the plant. Extensive

leaf desiccation and stunting

4

Severe necrosis / chlorosis over three quarters of the leaves of the plant.

Extensive leaf desiccation and stunting

5 Dead

Disease wilt index

1 2 3 4 5

0

0

1

2

3

4

5

0 5 10 15 20 25

Mean

wilt in

dex

Time (weeks) after inoculation

PK 5506 (F3)

PK 5506 (16F)

0

1

2

3

4

5

0 5 10 15 20 25

Mean

wilt in

dex

Time (weeks) after inoculation

PK 5463 (16F)

PK 5463 (F3)

0

1

2

3

4

5

0 5 10 15 20 25

Mean

wilt in

dex

Time (weeks) after inoculation

PK 5493 (16F)

PK 5493 (F3)

0

1

2

3

4

5

0 5 10 15 20 25

Mean

wilt in

dex

Time (weeks) after inoculation

PK 5525 (16F)

PK 5525 (F3)

All palm genotypes are susceptible to Foe: Disease index

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.

•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

Ongoing Research

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

(University of Bath)

HEFNI RUSLI

Dr Alan Wheals

Dr. Sweta Sharma Dr Idris Abu Seman (MPOB)

Dr Alastair Muir Dr Julie Flood (CABI)

Dr Mathew Wills Ghana: OPRI

Plantations: GOPDC, NORPALM

Christophe Lambing Unilever BOPP, TOPP

Emma Woodland

Caroline Roger

Vicki Wright

Cultures & vectors

Prof. David M. Geiser

(Pennsylvania State University; Fusaria isolates )

Prof. Kerry O‟Donnell (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 (Københavns Universitet, red fluoro protein vector )

Funding:

MPOB: Drs M. Basri Wahid;

A Kushairi Din;

Idris Abu Seman

Acknowledgements: current work

Soils:

FELDA & MPOB

FASSB

NORPALM

Plantation

Oil Palm

Research

Institute (OPRI)

BOPP

TOPP

Dr. Sheila Tagoe and Mr. K. Boafo Afrim

GOPDC Plantation

Ghananian Colleagues

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.

1 Foe

clade

Clade 2

Clade 1

Outgroup

Tax: 17

Characters: 204 Tax: 17

Characters: 145

Clade 3

RAMPs RAPD

Analysis showed the high level of genetic similarity

of Foe isolates ie they belong to the same unique

clonal lineage. RAPD analysis showed more genetic diversity of

Foe isolates which formed into 3 different clades.

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).

Foe origin, pathogenicity, VCG group, RFLP group

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

28S 18S 5.8S

ITS 1 ITS 2

Ribosomal DNA region

FUS-1f

FUS-1r

Development of genus Fusarium specific

primers

Gibberella (Fusarium)

fujikuroi Species Complex

Fusarium oxysporum SC

Fusarium incarnatum/

equiseti SC

F. chlamydosporum SC

F. solani SC

F. dimerum SC

)

Development of F. oxysporum species specific primers Molecular phylogeny of Fusarium species complex (SC) based on RNA

polymerase II second largest subunit (RPB2) (O‟Donnell et al., 2007

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)

Exon 2 Exon 3 Exon 4

Exon 1

Intron 2 Intron 3 Intron 1

*ef 1

*ef 2

Foxy F2