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