Enhancing biological control of basal stem rot disease (
Ganoderma
boninense
) in oil palm plantations
A. Susanto, P.S. Sudharto & R.Y. Purba
Indonesian Oil Palm Research Institute, Jl. Bigjen Katamso No. 51, Medan 20158, Indonesia
Abstract
Basal Stem Rot (BSR) disease caused byGanoderma boninenseis the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that
Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride,
Pseudomonas fluorescens, andBacillussp. A glasshouse and field trial forGanodermacontrol indicated that treatment withT. harzianumandG. viridewas superior toBacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.
Key words: biological control,Ganoderma boninense, oil palm,Trichodermaspp.
Introduction
During the last two decades there has been a rapid expansion in the areas planted with oil palm. Major oil palm developments in Indonesia include not only Sumatra, but also Borneo, Celebes, Pa-pua, and Banten. This expansion has involved both the use forest land and the conversion of existing plant plantations. One of the major con-straints to oil palm cultivation is the presence of disease. Of the diseases that occur in oil palm plantations, Basal Stem Rot caused byGanoderma boninenseis the most destructive [1, 2].
In several oil palm plantations in Indonesia, BSR has caused large losses where 50%or more of the productive plants have died [3], and currently
may be biological control and the utilisation of resistant oil palms. Breeding for resistance is a long-term activity, whereas biological control may be developed over a shorter time scale.
Materials and methods
Collection of candidate fungal and bacterial biocontrol agents
Isolation of biocontrol agent candidates was undertaken from oil palm rhizospheres in North Sumatra, West Sumatra, Lampung, and Banten. The choice of plantation area was based on the stage of development of the plants, the plantation age, the plant condition and the history of the plantation. Plant development stages that were
considered were immature plant, productive plant, and old plant. The age of the planta-tion included first, second, third, and fourth gen-eration plantings. The history of the plantation considered its previous usage, and whether it had been used for forest, tea, cocoa, rubber, and coffee. All of the observations were conducted on healthy and infected plants where possible. Isolation of candidate fungus biocontrol agents was by dilution plating on Martin Agar + chloramphenicol medium as described by Johnson and Curl [5]. The fungi isolated were counted and the population density was calculated after 1 week. The fungi obtained were purified and identified. A similar method was also applied to the isolation of bacterial biocontrol agents, but with serial dilutions to 10)7plated on nutrient agar
(Table 1).
Table 1. Population and inhibition capacity of candidate biocontrol agents
No. Isolate Population/g soil Inhibition (%) Plantation area
1 T. harzianum-1 1.3·104 85.6 jk Banten Sehat TBM 2 T. harzianum-9 5.4·104 92.0 defg Banten Sehat TM 3 T. harzianum-11 1.7·105 83.8 k Banten Sakit TM 4 T. harzianum-21 5.3·104 91.1 efg Lampung Sehat T III 5 T. harzianum-26 1.3·104 87.0 hijk Lampung Sakit T III 6 T. harzianum-29 2.2·106 84.0 k Sumbar Sehat TM I
7 T. harzianum-34 3.5·105 87.7 hij Sumbar Sakit TM I
8 T. harzianum-39 2.3·105 93.0 bcdef Sumut Sehat TBM IV
10 T. harzianum-45 4.0·104 96.0 abc Sumut Sakit TBM IV
11 T. harzianum-58 1.3·104 92.6 cdef Sumut Hutan Sehat T
14 T. harzianum-88 1.3·104 94.6 abcd Sumut Sehat II TBM
15 T. harzianum-91 1.3·104 96.7 a Sumut Sakit II TBM
16 T. harzianum-95 1.5·105 93.0 bcdef Sumut Sehat II TM
20 T. harzianum-107 4.1·105 96.1 ab Sumut Sakit II T
21 T. harzianum-112 2.7·104 92.7 bcdef Sumut Sehat III TBM 22 T. harzianum-119 4.0·104 94.5 abcde Sumut III TBM 24 T. harzianum-131 4.0·104 97.8 a Sumut Sakit III TM 9 G. viride-44 6.7· 104 92.0 defg Sumut Sakit TBM IV 17 G. viride-98 1.3·104 87.6 hij Sumut Sakit II TM 19 G. viride-105 1.3·104 85.9 ijk Sumut Sehat II T 25 G. viride-136 4.0·104 84.0 k Sumut Sehat III T 12 T. viride-70 8.4·105 84.0 k Sumut Teh Sehat TBM 13 T. viride- 82 1.3·104 90.0 fgh Sumut Karet Sehat T 18 T. viride-102 2.4·105 89.1 ghi Sumut Sehat II T 23 T. viride-123 4.0·104 90.0 fgh Sumut Sehat III TM
26 T. viride-138 6.7·104 92.1 defg Sumut Sakit III T
27 Bacillussp.-10 5.6·108 54.2 l Lampung Sakit TBM IV
28 Pseudomonas fluorescens-1
1.8·108 49.6 m Banten Sehat TBM I
29 P. fluorescens-58 4.6·108 42.2 n Sumut Sakit II T
30 P. fluorescens-63 4.1·108 50.4 m Sumut Sakit III TBM
Inhibition effectiveness of candidate biocontrol agents
A dual culture method was used to evaluate the effectiveness of the candidate biocontrol agents. After 4 days incubation this was calculated as a percentage inhibition from the formula:
KP¼
ab
a 100%
where KP = inhibition effectiveness, a = the radius of the G. boninense colony without the biocontrol agent,b = the radius of theG. bonin-ensecolony with the biocontrol agent (Table 2).
Efficacy of biocontrol agents in glasshouse trials
Plant materials used were BO909 T· BO 936 T,
BO 906 T · BO 796 P, M1314 D · BO 932 T,
BO2581 D·BO2581 D, BO 5462 D· BO 358 P,
and BO 5585 D · BO 5510 D. Sawdust + PDA
was used for the production of the G. boninense
inocula. Inocula were divided in to smaller aliqu-ots of 163 cm3 in plastic measuring cylinders. Seedlings were inoculated when they were trans-planted from the pre-nursery to the main nursery. One inoculated root of each oil palm seedling was put into a plastic bag and tied, and this was then covered with soil in the main nursery plastic bag. The experimental design was a completely random block design factorial of 6· 5 ·2 · 2 with three
replications for each combination. The first factor was type of cross used for the oil palm material (where V1 = T·T, V2 = T·P, V3 = D·T, V4
= D·D selfing, V5 = D·P, V6 = D·D). The
second factor was the biocontrol agent used (T0 = without biocontrol agents, T1 =T. harzianum, T2 =G. viride, T3 =Bacillussp., T4 =T. harzianum
+G. viride+ Bacillussp.). The third factor was
the G. boninense inoculation (G0 = without inoculation and G1 = inoculation with G. bonin-ense), and the fourth factor was the addition of chitin (K0 = without chitin and K1 = plus chitin). The biocontrol agents were added as 10 g formulation for fungal biocontrol agents and 10 ml formulation for bacterial biocontrol agents. For the T4 treatment, 5 gT. harzianum, 5 gG. vi-ride, and 5 ml Bacillus sp. were used respec-tively. Chitin was added at 1 g per polybag. The conidial density of T. harzianum and G. viride
preparations were 4·106 conidium/ml, whereas
Bacillus sp. was used at 7·108cfu/ml. The
vari-ables recorded were disease incidence of basal stem rot, plant height and number of leaves, plant vig-our, and development of the biocontrol agent population. Observations were made monthly for 1 year.
Results and discussion
Collection of candidate biocontrol agents
One hundred and forty fungi were isolated from various oil palm rhizospheres and these included 18 isolates ofT. harzianum, 5 isolates ofT. viride, 4 isolates of Gliocladium viride, 28 isolates of
A. flavus, 4 isolates of A. niger, 13 isolates of A. fumigatus, 14 isolates of Penicillium citrinum, 4 isolates ofRhizopus, 6 isolates ofP. chrysogenum, 12 isolates of P. commune, and 32 isolates of
P. funiculosum. The majority of the isolates were considered as candidate biocontrol agents. The population densities of each species were similar between plantations and were about 104–105cfu/g soils. Seventy two isolates of bacteria were ob-tained and these included isolates ofPseudomonas fluorescensandBacillussp.
Table 2. Percentage of oil palm seedling death at 12 months after inoculation
Cross Disease incidence (%) Plant height (cm) Frond numbers Vigour
BO909 T·BO 936 T 0.00 b 88.52 c 9.08 d 2.98 a
Inhibition activity of candidate biocontrol agents
Twenty six fungi and four bacteria showed po-tential biocontrol activities in dual culture analy-sis. Fungi were categorised as biocontrol agents if they showed an inhibition capacity >80%, and
bacteria were considered biocontrol agents if they showed an inhibition capacity of >40%. The
iso-late that gave the highest inhibition capacity was
T. harzianum (97.8%). This was isolated from
a third generation oil palm planting in North Sumatra. The isolate was similar to another
T. harzianum isolate from a BSR infected immature fourth generation planting in North Sumatra, to an isolate from healthy immature second generation planting in North Sumatra, to an isolate from infected immature second genera-tion planting in North Sumatra, and to an isolate from infected productive first generation planting in West Sumatra. T. harzianum was the most common species among the candidate biocontrol agents.
Only four bacteria showed potential biocontrol properties, all of the bacteria gave lower inhibition values than the fungi. The highest inhibition capacity was shown by an isolate of Bacillus sp. from Lampung (54.2%). The remaining bacterial biocontrol agents all had inhibition capacities of between 40 and 50%.
The presence of Trichoderma and Gliocladium
was influenced by soil porosity, soil pH, and other chemical properties of the soil. The abundance of
Trichoderma in soil depends on its ability to de-grade organic matter and its resistance to other microorganisms. The differences in the abundan-cies of the biocontrol agents may also be influ-enced by the climatic conditions at each site.
Efficiency of biocontrol agents in glasshouse trial
The disease incidence of Basal Stem Rot was not consistent for all oil palm crosses. The cross with the highest disease incidence was the selfed D·D
at 6.11%, compared to the other crosses where the disease incidence ranged from 1.11 to 2.78%. The T ·T had not shown any disease symptoms
by the end of the 1 year period. This does not indicate that T·T was resistant to G. boninense,
as this cross is known to develop BSR in the field, and one possibility is that the 1 year period was not enough for the pathogen to infect T · T.
The D · D cross may have the highest disease
incidence, due to selfing leading to an accumula-tion of susceptible traits, as selfing apparently did not result in an accumulation of resistance traits.
The addition of candidate biocontrol agents (T. harzianumandG. viride) was found to signifi-cantly reduce Basal Stem Rot incidence (Table 3). Disease was only seen in the negative controls and with the Bacillus sp. and mixed inoculum. The ability of Bacillus sp. to inhibit G. boninense ap-peared to be lower than either of the fungal bio-control agents, and every test involvingBacillussp. gave higher disease incidences. In the mixed inoc-ula it may be that theBacillus sp. competed with theT. harzianum andG. virideisolates.
The oil palms treated with T. harzianum and
G. viridewere significantly taller than the negative controls and the palms treated with Bacillus sp. and the mixed treatments. Adding ofT. harzianum
and G. viride also appeared to give significantly higher plant vigour than the control, Bacillus sp. and mixed species treatments.
Conclusions
This study has isolated 30 candidate biocontrol agents consisting of 17 isolates ofT. harzianum, 4 isolates of G. viride, 5 isolates of T. viride, one isolate of Bacillus sp., and 3 isolates of Pseudo-monas fluorescents. After 1 year of inoculation,
T. harzianum and G. viride appeared to prevent Basal Stem Root in glasshouse trials, whereas,
Bacillus sp. had very little capacity to prevent infection byG. boninense.
Table 3. The effect of biocontrol agents on Basal Stem Rot incidence and oil palm vigour
Treatment Disease incidence (%) Plant vigour
Control 18.06 a 2.67 b
T. harzianum 0.00 c 3.00 a
G. viride 0.00 c 3.00 a
Bacillussp. 9.72 b 2.71 b
T. harzianum+
G. viride+
Bacillussp.
8.33 bc 2.82 b
References
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3. Turner PD. Oil Palm Diseases and Disorders. Oxford: Oxford University Press, 1981: 280.
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Address for correspondence: A. Susanto, Indonesian Oil Palm Research Institute, Jl. Bigjen Katamso No. 51, Medan 20158, Indonesia
