GANODERMA DISEASE OF THE OIL PALM:
IMPLEMENTATION OF AN EARLY ARTIFICIAL INOCULATION TEST TO
SCREEN OIL PALM PROGENIES FOR THEIR LEVEL OF RESISTANCE
AND HYPOTHESIS ON NATURAL INFECTION
F. BRETON, R. MIRANTI, Z. LUBIS, Z. HAYUN, S. UMI, A. FLORI, S.P.C. NELSON, T. DURAND-GASSELIN, J.C. JACQUEMARD and H. DE FRANQUEVILLE
PT Socfin Indonesia Sumatra Bioscience (Lonsum) Oil Palm Breeding Unit
INTRODUCTION
• Three major diseases seriously limit oil palm cultivation in each of its
main growing areas:
– Bud rot disease(s) or PC in Latin America, which can destroy
plantations of several thousand hectares. Recent findings on the possible
role of Phytophthora palmivora are promising in terms of research and
disease management
– Vascular wilt in Africa, caused by Fusarium oxysporum f.sp. elaeidis, a
soil-borne fungal pathogen. Losses increase with the successive crop
cycles (generation), if a non resistant material is planted. Fusarium wilt, as
an example, will be briefly evoked in some following slides
– Basal stem rot (BSR), mainly caused by Ganoderma boninense in
Southeast Asia, another soil-borne fungal pathogen. It starts to induce
damages in Central Africa and to a lesser extent in South America. The
number of successive generations is also of importance
Incidence of Ganoderma (BSR)
•
In Southeast Asia, specially in North Sumatra (Indonesia), many oil palm
plantations are planted in their 4th generation. 80% of the palms can have
been affected by the end of the 3rd generation
•
In (Central) Africa, most of the plantations are old second generations. It
is frequent that BSR appear when the plantations are older than their
normal economic life. Ganoderma can interact with Fusarium wilt and then
install before the next replanting
•
In South America, where most of the plantations are at the end of the first
generation - early second generation, Ganoderma is still contained at a
Part B:
A Lignified Star-Shape Cavity at Root-Bole Interface:
An Appropriate Culture Chamber for Ganoderma boninense
Part A:
Early Screening Test to Evaluate Resistance/Susceptibility Level of Oil Palm
Progenies to Basal Stem Rot Disease
Leaf symptom Rotten tissue Sporophores
Three kinds of disease symptoms can be observed “independently”
Some recommendations
•
Elimination of trees affected by Ganoderma
•
Bole extraction (do not cover with soil and weeds)
•
Dig a sanitation hole :1,5 x 1,5 x 1m (hxLxd)
Replanting at 160 palms/ha
Red circles:
potential focuses
Part A:
Early Screening Test to Evaluate Resistance/Susceptibility Level of Oil Palm
Progenies to Basal Stem Rot Disease
Two types of symptoms on
mature palms:
Acute, generally killing the palm
six months after they appear
Chronic, stunting the growth of
the oil palm, which only produces
a few lightweight bunches
Resistance to Fusarium wilt
Substantial differences in performance exist in the field depending on
planting material origin
This differences can be detected through artificial inoculations of the
pathogen in prenursery and Cirad has implemented an early screening
test since several decades
Each
test
usually
compares
200
crosses
,
each
represented
by
8
replicates of 20 seedlings
Results are obtained
5 months
after
inoculation
The results are usually well correlated
to field results
They are expressed as a
“vascular wilt
index”
Screening for Fusarium wilt resistance
Dabou experimental prenursery, CNRA,
•
Base 100: Mean of the test
100
90
80
110
120
Susceptibility
Resistance
Fusarium wilt index
0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40-49 50-59 60-69 70-79 80-89 90-99 100-109 110-119 >120 Index prenursery % V as cu lar w il t in f ie ld
• High differences between both pathogens, Fusarium and Ganoderma
• Are there differences of susceptibility to Ganoderma within the planting
or breeding material ?
• About Ganoderma, let us quote R.H.V. Corley & P.B. Tinker (2003), in
“The Oil Palm”, 4th Edition, p. 413 :
“It appears to us that, in the longer term, the best approach to
controlling the disease in areas where it is prevalent may be to
develop tolerant material, using nursery inoculation for screening,
in much the same way as has been done for Fusarium wilt.”
• We share this point of view, breeding being a major component of an
integrated control. A research program has started in 2002-2003.
0 5 10 15 20 25 30 35 40 1992 1993 1994 1995 1996 1997 1998 1999 2000 Years % G a no de rma C 2501 (b) SOC 0903 (a) SOC 0901 LMC 063 (b) LMC 043 (a,b) LMC 044 LMC 051 (b) LMC 074
BB CL 4 (1989 planting, 3 rdplanting cycle)
Thus possible to identify
susceptible/resistant material soon after
field planting
Genetic trials have led to the detection of significant differences in
susceptibility to Ganoderma, in relation to the genetic origin of the material
exposed to the disease (de Franqueville et al., 2001, Durand-Gasselin et al.,
2005)
But
How to identify early (before planting) the sources of resistance used in
breeding programmes ?
Scientific collaboration
on the oil palm Ganoderma disease (BSR) in North Sumatra
(PT Socfindo – Sumbio/Lonsum - Cirad)
OBJECTIVE
To implement an early screening method for oil palm “resistance/tolerance” to Ganoderma
Characteristics of the method to set up :
1) Discriminating (susceptibility/resistance)
2) Closely correlated to field observations (in natural infection) 3) Reproducible (standardisation)
In Previous Study
(Breton et al., 2005)
Characterisation and standardisation of several major parameters ensuring a successful prenursery screening test, based on artificial inoculation of the Ganoderma
Preparation of inoculum (laboratory) :
-Nature of substrate source (RWBs : Rubber Wood Block) -size of the inoculum source (6x6x3cm)
-Preparation of the RWBs prior to inoculation with Ganoderma isolates -incubation time of Ganoderma inoculated RWBs (12 to 14 weeks) -Conditions of incubation (dark, 27°C, 34% relative humidity )
Artificial inoculation of plant material (nursery) :
-the physiological stage of the planting material (germinated seed, GS) -Volume of prenursery polybag (20x30cm)
-Distance within polybag between GS with inoculum source (5cm) -prenursery shade and temperature (80% light filtration)
Symptoms obtained after artificial
inoculation
0 10 20 30 40 50 60 70 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Weeks % o f in fe c te d s e e d li n g s 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Weeks % o f in fe c te d s e e d li n g s
BSR disease symptoms obtained in nursery 3-4 months after germinated seeds inoculation
Index Evolution During Recording Period
Progenies code tested in Trial 1
Index (based on external symptoms) 16W 18W 20W 22W 24W 26W 28W 12675 164 152 135 127 121 117 116 12700 78 95 82 81 78 89 88 15270 267 210 178 167 158 154 143 12751 60 62 82 89 96 96 97 12752 78 86 96 97 89 94 95 15271 86 95 109 108 117 121 113 12773 103 119 119 116 119 114 118 12276 52 57 76 78 78 81 82 15272 138 119 112 110 114 112 115 15273 112 100 102 110 112 104 103 11672 95 105 86 89 85 83 88 12444 60 57 76 75 80 77 84 11143 52 67 79 83 89 96 97 15274 26 38 36 43 41 48 46 15275 129 138 132 127 123 114 115 Trial mean (% infection) 11.6 21.0 30.3 37.1 43.7 48.1 52.4 Correlation with index 28W 0.83 0.86 0.95 0.96 0.98 0.98 1.00
Progenies code tested in Trial 4
Index (based on external symptoms) 16W 18W 20W 22W 24W 26W 28W 12675 236 231 199 171 153 140 135 12700 41 30 50 68 78 78 77 12752 82 82 99 100 104 103 104 15271 216 187 163 157 147 142 128 12271 103 97 81 86 89 91 90 12778 164 149 127 114 104 106 108 13790 144 134 163 157 141 132 128 15278 62 60 81 82 95 98 97 15279 21 45 50 57 63 62 77 15280 82 90 72 86 95 109 113 15281 92 119 104 107 101 93 101 15282 10 22 36 54 55 62 59 15283 92 97 122 104 112 114 110 15284 41 52 68 68 69 75 79 15285 113 104 86 89 95 96 95 Trial mean (% infection) 9.7 13.4 22.1 27.9 34.7 38.7 44.4 Correlation with index 28W 0.88 0.88 0.92 0.93 0.96 0.97 1.00
An infection mean of 30% provided useful information on the level of resistance/susceptibility of tested crosses
Standards crosses Index distribution between independent tests (base 100) Categories Codes
Number of independent tests performed
Lower than 100 higher than 100
Average Index (30%) Data summary < 100 < S1 3 0 3 156 0-3 S2 11 3 8 123 3-8 S3 8 0 8 142 0-8 S4 3 0 3 156 0-3 S5 4 0 4 134 0-4 S6 3 0 3 154 0-3 S7 4 0 4 155 0-4 S8 7 3 4 120 3-4 S9 11 3 8 114 3-8 Susceptible S10 9 3 6 114 3-6 M1 4 2 2 97 2-2 M2 6 2 4 109 2-4 M3 3 2 1 94 2-1 M4 4 2 2 98 2-2 M5 14 8 6 95 8-6 M6 20 10 10 100 10-10 M7 4 2 2 108 2-2 M8 6 3 3 99 3-3 M9 6 3 3 102 3-3 Medium M10 5 3 2 96 3-2 T1 5 5 0 45 5-0 T2 7 6 1 75 6-1 T3 7 6 1 78 6-1 T4 4 4 0 77 4-0 T5 16 14 2 81 14-2 T6 10 10 0 56 10-0 T7 6 5 1 66 5-1 T8 9 9 0 62 9-0 T9 11 10 1 63 10-1 Tolerant T10 8 7 1 80 7-1
A set of standard crosses allows to link trials between each others and the combined analysis of trial results
Standard Crosses
Results demonstrate also the reproducibility and the consistency of the test
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4 8 12 16 20 24 28
Weeks after germinated seeds inoculation
F -Va lu e o f Pr o g e n y Eff e c t o n I n fe c ti o n R a te TG18 TG19 TG21
Evolution of F-value for three screening tests during the period of recording the external disease symptoms
With an average percentage of infected standard crosses of around 30%, F-values do not vary extensively and relative variation between progenies has stabilized
0 140 0-5 5-10 10-15 15-20 20-25 25-30 30-35 35-40 40-45 45-50 50-60 Frequency (% infection) N o . o f Pr o g e n ie s T e s te d
Distribution of the Resistance/Susceptibility to Ganoderma
Within a Genetic Population of Oil Palm
The distribution of the resistance/susceptibility to Ganoderma is continuous and suggests that more than one or two major genes are involved in providing tolerance/resistance
General Procedure for Routine Screening Work
at PT Socfindo and Sumatra Bioscience
• 100 crosses tested every month
• 5 replicates of 20 germinated seeds per tested cross
• 20 additional seeds per cross are inoculated at the same time for use as replacements four weeks after initial inoculation
• External symptoms are recorded every month then every two weeks when infection level of trial reaches 25%
• Splitting seedlings and final record when average trial is around 30% infection (between 22 to 24 weeks after inoculation)
Preparation of RWBs
Inoculation of RWBs
Storage of RWBs
Germinated seeds inoculation
Recording of disease symptoms
Splitting of seedlings then recording symptoms 1 month 3 months 5 to 6 months
Female Origin Binga x Lobe 75 75 AVROS 72 85 80 79 (MRS x MRS) x Lobe 90 90 Lobe 85 97 91 Binga x Binga 75 81 92 97 77 117 109 116 100 96 Lobe x Lobe 87 108 97
(AVROS x UR Deli) x Binga 102 92 103 99
SF Deli x GM Deli 47 85 108 121 110 94 152 102 GM Deli 109 104 87 117 104 AVROS x Binga 107 106 91 106 111 104 Lobe x GM Deli 93 99 104 123 97 112 118 107 Binga x Pobe 86 84 115 144 107 GM Deli F1 85 137 106 109 DM Deli 89 96 92 111 114 118 151 110 Lobe x SF Deli 107 101 135 114 (GM Deli x MRS) x Lobe 101 125 101 167 124 Index Average 70 86 87 93 97 97 97 98 101 104 105 106 111 111 112 114 118 134 134 152 103 Lo b e x L o b e D M D e li x A V RO S (A V RO S x U R D e li ) x Bi n ga Bi n ga x L o b e SP 5 4 0 A V RO S x A V RO S Bi n ga x P o b e Ya n ga m b i x Ya n ga m b i A V RO S Lo b e x S F D e li Bi n ga x Bi n ga A V RO S x Bi n ga (C o w an x C o w an ) x A V RO S Index Average Male Origin (C o w an x C o w an ) x Lo b e D M D e li (M RS x M RS ) x Lo b e Lo b e x G M D e li A V RO S x La M e (G M D e li x M RS ) x A V RO S (G M D e li x M RS ) x Lo b e Sources of resistance and sources of susceptibility
Female Origin
Deli Dabou x Deli Dabou 64 64
Deli Socfindo x Deli Socfindo 85 85
Deli Dabou x Deli Socfin 86 86
Deli Dabou x Deli Socfin 94 95 84 91
Deli Socfindo 80 90 106 104 95
Deli Dabou x Deli Dabou 95 103 99
Deli Dabou x Deli Socfin 101 101
Deli Dabou x Deli Socfin 96 103 120 106
Deli Socfin 83 109 126 110 107
Deli Dabou 96 113 115 108
Deli Dabou x Deli Dabou 111 107 109
Deli Dabou x Deli Dabou 109 109
Deli Socfin x Deli Dabou 105 114 109
Deli Socfin x Deli Socfin 110 110
Angola x Deli Dabou 119 119
Index Average 85 86 86 90 95 101 103 105 109 111 111 101 (L a M é x La M é) x ( La M é x La M é) La M é x La M é La M é Ya n ga m b i Index Average La M é x La M é Ya n ga m b i x Y an ga m b i Male Origin Ya n ga m b i x Y an ga m b i La M é La M é x La M é La M é x La M é La M é x La M é Sources of resistance and sources of susceptibility
GROUPE B
(male) org2 parent2
P O y y y y P O y y y y P O y y y y S L y y y S L y y y S L y y y S L y y y S L y y y S L y y y S L y y y S L y y y S L y y y S L y y y y 1 131 134 2 148 99 3 129 106 4 5 81 106 6 127 148 7 8 127 134 1 93 2 47 93 3 41 104 4 60 147 5 78 73 6 135 106 7 SL xxxx SL xxxx X GROUP A (female) Y
123
89
Number Progeny
code Field status 1
Number of trials 2 Index average Index distribution Test status Id 3<100 Id>100
1 12658 Intermediate 4 86 3 1 Resistant to intermediate 2 12675 Susceptible 8 126 2 6 Susceptible
3 15278 - 4 79 3 1 Resistant
4 12700 Resistant 5 75 5 0 Resistant
5 15284 - 4 75 3 1 Resistant
6 12751 Intermediate 4 97 2 2 Intermediate 7 12752 Intermediate 6 109 2 4 Intermediate to susceptible
8 15271 - 8 141 0 8 Susceptible
9 12773 Intermediate 4 101 2 2 Intermediate 10 12276 Resistant 5 46 5 0 Resistant 11 12778 Intermediate 5 91 4 1 Resistant to intermediate 12 2615580 Exp. susceptible 7 113 2 5 Susceptible 13 2648858 Exp. susceptible 8 100 4 4 Intermediate
14 2654198 - 5 74 4 1 Resistant
15 2655003 Susceptible 5 105 2 3 Intermediate to susceptible
16 2648147 - 6 127 2 4 Susceptible
17 2653769 - 5 76 5 0 Resistant
18 2641311 Exp. resistant 4 65 4 0 Resistant
19 2660428 - 4 112 1 3 Susceptible
20 2641666 Exp. resistant 5 77 5 0 Resistant
21 2632316 - 4 95 2 2 Intermediate
CONCLUSION (1)
1) An early and rapid screening test of oil palm progenies to BSR disease has
been developed and validated by using planting and breeding materials from two
Indonesian private companies in a partnership with Cirad:
Sumatra Bioscience
(Lonsum) and PT Socfin Indonesia (Socfindo)
2) The results are both consistent and reproducible and correlated to field
observations.
3) This prenursery test using germinated seed allowed the rapid screening of
several hundreds of crosses per year and is highly asset valuable to plant
breeders.
This screening test provides :
1) a method to ensure no highly-susceptible progenies are transferred to the field
for commercial planting,
2)
a breeding tool,
-
to develop more Ganoderma resistant high yielding planting material,
-
to understand the genetics of Ganoderma resistance/susceptibility,
-
to investigate Ganoderma infection,
-
and as a more general tool to test other methods to control and
prevent Ganoderma.
Part B:
A Lignified Star-Shape Cavity at Root-Bole Interface:
An Appropriate Culture Chamber for Ganoderma boninense and
Stromatic-Like Structure Development
A LIGNIFIED STAR-SHAPE CAVITY AT ROOT-BOLE INTERFACE
This lignified scar will form the future star-shape cavity observed in mature palms.
At the nursery stage, a lignified scar is clearly observed
Lignified Star-Shape Cavity an Appropriate Culture Chamber
for Ganoderma boninense
Suitable conditions for Ganoderma development and infection:
- dark cavity
- lower than ambient temperature - high humidity
- dense subtrate (lignified oil palm tissues)
- less antagonists than in the open soil (Ganoderma poor competitor) - attached roots
Production of Stromatic-Like Structure or Pseudo-Sclerotium
Stromatic-Like Structure (SLS) or Pseudo-Sclerotium
No SLS No infection
SLS Infection
This close contact root-SLS is the preliminary step of the internal invasion
Development of SLS occurred only on a dense subrate
the presence of this melanised mycelium mass is necessary to induce BSR symptoms
Rhizotron method
0 10 20 30 40 50 60 12 16 20 24 28 32 36 40
Weeks after germinated seeds inoculation
% of i nf e c te d s e e dl ing s RWB+SLS RWB-SLS SLS a b c
Stromatic-Like Structure (SLS) or Pseudo-Sclerotium
The infection observed for the treatment SLS-free RWB correspond to a production
de novo of the SLS
This fungal melanised mass (SLS) need a hardened food base to survive in the soil and to express its high infectious potential
Stromatic-Like Structure (SLS) in Mature Oil Palm Recently Infected
Stromatic-Like Structure (SLS) localised only at the external surface of the lignified star-shape cavity (root-bole
interface)
In nursery trials the RWB provides a dense substrate to reproduce or simulate the role of the lignified star-shape cavity observed in mature palms
Longitudinal and Transverse Sections of Root-Bole Interface
from Recently Infected Mature Oil Palms
Arrows show the progression of the rotten tissues from the basal lignified cavity
Indicating
a radial and a centrifugal colonisation of the bole by Ganoderma
CONCLUSION
- Melanised stromatic-like structure (SLS) or pseudo-sclerotium produced by Ganoderma only on dense substrates seems to be an essential physiological stage prior the invasion of the oil palm bole (nursery-field).
- For mature palms, the presence of a lignified star-shape cavity at root-bole interface plays the role of Ganoderma culture chamber for SLS development.
- Transverse and longitudinal sections of the root-bole interface from recently infected young palm confirmed a centrifugal and radial initial invasion of the bole from this lignified cavity (major location for the initial invasion of the fungus inside the bole).
- If no initial penetration points were found beyond the periphery of this lignified area, multiple penetration points were observed from this cavity which probably involved more than one isolate.
The melanised SLS could be considered as an aggressive or pathogenic stage
in the physiological life cycle of the fungus
PERSPECTIVES
- Insufficient field observations have yet been conducted to establish any relationship between cavity size with genetic origin of oil palms or environmental factors (soil characteristics).
- Work is in progress to investigate the relationship between the size of the basal lignified star-like cavity with the resistance/susceptibility of oil palm or with the kinetic of the bole invasion by Ganoderma.
- In parallel, localised injections (fungicides and antagonists microorganisms) in and around this lignified star-shape cavity are being tested.
GENERAL CONCLUSIONS
• Like for Fusarium wilt, it is possible now to screen at an early stage oil
palm progenies for their degree of resistance to Ganoderma
• The high number of tested progenies (100/month) is a recent step and
more and more data will be available
• But, planters must not expect “zero-Ganoderma” progenies, due to the
nature of the fungus, the unknown mechanisms of (partial) resistance
• Elementary cultural practices must be implemented (sanitation, for
example)
• The star-shape cavity might be a very important point to perform very
localized treatments (fungicides or antagonists)
• An integrated disease management is therefore highly possible,
specially when the disease is still at a low level
PT Socfin Indonesia Sumatra Bioscience (Lonsum) Oil Palm Breeding Unit
