Ganoderma in Oil Palm in Indonesia: Current Status and Prospective Use of Antibodies for the Detection of Infection

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1 T.W. Use 19 of Darmono Antibodies for Detection of Ganoderma Infection of Oil Palm Ganoderma in Oil Palm in Indonesia: Current Status and Prospective Use of Antibodies for the Detection of Infection T.W. Darmono 19 Biotechnology Research Unit for Estate Crops, Jl. Taman Kencana No. 1, Bogor, Indonesia Economic Importance of Basal Stem Rot (BSR) Disease Oil palm was introduced to Asia through Indonesia 150 years ago and then spread to other countries in the region (Pamin, 1998). In 1997, the total area of oil palm in Indonesia reached 2,463,823 ha and approximately 80% of this is located in Sumatra. In North Sumatra and Central Lampung, oil palm has been cultivated for several replanting generations, each of which takes between 25 and 30 years. Oil palms found in Kalimantan, Sulawesi and Irian Jaya are only recently cultivated. The 1997production of crude palm oil (CPO) was 5,904,175 t, valued at US$2,952,087,500, and that of palm kernel oil (PKO) was 1,189,603 t, valued at US$832,722,100. The total value of both CPO and PKO was US$3,784,809,600. Basal stem rot incited by Ganoderma spp. is one of the most important diseases in oil palm. The annual capital loss at 1% disease incidence, calculated on the basis of the export value of palm oil in 1996, reaches US$38,230,400. As the disease is difficult to control, the infected trees are usually left to deteriorate and die. In some cases the infected tree looks healthy although more than half of its base has been degraded by the pathogen. The magnitude of yield loss is greater if infection occurs at an early stage of tree maturity, when aged between 5 and 15 years. The disease incidence at the same site in a plantation tends to increase from year to year and from generation to generation. A survey in a plot of 10.5 ha of 23-year-old oil palms of the third planting generation conducted in July 1998 at Bekri Plantation, PTP Nusantara VII, in Central Lampung, Sumatra, revealed the occurrence of disease incidence to be up to 51% (Darmono, 1998). CAB International Ganoderma Diseases of Perennial Crops (eds J. Flood, P.D. Bridge and M. Holderness) 249

2 250 T.W. Darmono Current Status of Research on Ganoderma Detailed information of BSR in oil palm can be found in Turner (1981). This summarizes his findings from his own research and observations on the disease in Indonesia prior to Although this gives a better understanding of the disease, it does not provide clear guidance on how to control the disease effectively, which can be incorporated in the whole system of oil-palm management. Prior to 1980, there was no local research scientist in the country actively involved in research on basal stem rot disease in oil palm. This was probably due to two main reasons. First, there was no pressure from the oil-palm industry, which was unaware that Ganoderma was a significant problem. It was assumed that losses were not economically significant until more than 20% of the stand had been lost. That assumption was lately proven to be incorrect (Hasan and Turner, 1994) and the disease currently occurs at a high incidence. The second reason was that working with higher fungi such as Ganoderma spp. is generally difficult, slow and very long term. With the increase in the incidence of the disease, the pressure from the growers has increased, encouraging research institutions to speed up their study on Ganoderma. Institutions currently engaged in research on Ganoderma as an oil-palm pathogen in Indonesia include Biotechnology Research Unit for Estate Crops (BRUEC) in Bogor, the Indonesian Oil Palm Research Institute (IOPRI) in Medan, and Bah Lias Research Station (BLRS) of P.T.P.P. London Sumatra in Pematang Siantar. SEAMEO Bio-Tropical in Bogor was also involved in research between1986 and Research at SEAMEO Bio-Tropical and IOPRI had emphasized the understanding of the biology and ecophysiology of the pathogen as well as the evaluation of potential biological and chemical control assays in the laboratory. Under laboratory conditions, the pathogen could grow at a wide range of ph, from 3.0 to 8.5, and the optimum temperature for growth was 30 C (Abadi et al., 1989; Dharmaputra et al., 1990). In the field, this may represent a wide range of soil types and oil-palm growing conditions at low elevations. Based on field observations, there was no correlation between disease incidence and the distance of the plantation to the coast, elevation, soil ph, or the density and type of legume cover crops (Abadi et al., 1989). Later, it was also stated by Hasan and Turner (1994) that there were few differences in BSR incidence between plantings on coastal and most inland sites in Indonesia. Although under field conditions, density and type of legume cover crops did not seem to affect disease development, laboratory studies revealed that supplementation of the agar medium with stem and leaf extracts of three legume cover crops, i.e. Centrosema pubescens, Calopogonium mucunoides and Pueraria javanica, commonly enhanced mycelial growth of the pathogen (Mawardi et al., 1987; Dharmaputra et al., 1989). In this particular case, growth enhancement may have occurred due to nutritional enrichment of the medium. Legume cover crops are commonly established just after plantingline preparation at the time of planting of oil-palm seedlings. After reaching a

3 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 251 peak of vigour at 2 3 years after planting, these covers eventually die out under the shade of the developing trees. Although the use of ground covers in the plantation has been a subject of controversy, their use is beneficial in the control of Rigidoporus microporus in Hevea rubber (Fox, 1977; Soepadmo, 1981). This has been suggested to be largely due to the enhanced rate of decay of woody residues in the soil caused by the moist conditions and the high nitrogen status of the cover and its litter (Wycherley and Chandapillai, 1969). Although cover crops were commonly used in oil-palm plantations at the time when slash and burn was still allowed, their effect on the rate of decomposition of unburned, felled oil-palm stems has not been thoroughly investigated. At present, slash and burn techniques have been banned in the country under the blue sky programme enforced by the government for protecting the environment, particularly through the control of fire hazards. Quick decomposition of felled oil-palm stem is needed to prevent its colonization by Ganoderma which may subsequently act as an inoculum source for the disease. Research on the use of chemicals has been confined to laboratory studies and results have shown that triadimenol at a concentration of 1.00 µg ml 1 was able to kill the mycelia of the pathogen, but this concentration also inhibited a fungal antagonist (Dharmaputra et al., 1991). Preliminary results from a field experiment have shown that triadimenol application by root absorption was more effective in suppressing the disease than that applied by soil drenching (Puspa et al., 1991). Using the same technique, Hasan (1998) has shown that phosphonic acid application was capable of protecting seedlings from infection. However, although these studies gave promising results, the use of chemicals in the control of Ganoderma in the field on a commercial scale will be impractical and economically infeasible until a reliable technique of application has been developed. Also, even if a reliable application technique was found, the beneficial use of chemicals is still questionable since their effect can diminish rapidly. It has been shown that the effect of triadimefon on Ganoderma cultured on rubber wood vanished within 3 weeks (Darmono, 1996). Research on the use of biological control agents for BSR has also been initiated at SEAMEO-Biotrop in Bogor (Dharmaputra et al., 1994). Other research institutions, including IOPRI (Soepena, 1998), BRUEC (Darmono, 1998), and BLRS (Hasan, 1998), have more recently become involved in the same research subject. Studies conducted at these institutions have shown that Trichoderma harzianum gave better control than that of other species of Trichoderma. The use of a biological control agent in the control of Ganoderma has been seen to be more promising than that of chemical control. The capability of a biological control agent to grow and reproduce in the field and that will allow the destruction of the pathogen in the soil, are some of the advantages and attractiveness of its use. Biological control is also considered to be less hazardous to the environment. Research to investigate whether Trichoderma sp. can actively grow along the root needs to be conducted. This would reveal the potential use of the agent as a root protectant.

4 252 T.W. Darmono However, one problem with the application of chemical and biological control agents is that the pathogen is capable of forming brown layers (Darmono, 1998) that provide a barrier against the chemical or the antagonist. These agents have to penetrate this barrier before being able to kill the sensitive mycelium of the pathogen. The brown layers, composed of melanized mycelium, also termed the sclerotium plate, are formed in the vicinity of the interaction zones and at any sites in the decayed tissue of basal stem. Sclerotium plates cover white masses of mycelium, forming pockets of Ganoderma. These pockets of mycelium are commonly found in the decaying oil-palm tissue. Sclerotium-like bodies of various sizes, from 2 to 5 cm in diameter (Fig. 19.1), can be found easily, embedded in broken, dry tissue particles in the decomposed tissue of oil-palm stem. This structure can be considered as a resting body of Ganoderma sp. It is different from true sclerotium in that, in addition to mycelium, the resting body of Ganoderma also contains degraded plant tissue intermingled with the mycelium. These resting bodies are capable of forming fruiting bodies and are capable of infecting oil-palm seedlings. Molecular analysis has revealed that cultures obtained from inside the resting bodies were identical to those obtained from the fruiting bodies developed from the associated resting bodies. This result indicates that the resting bodies found in decomposed oil-palm stems may be derived from the pathogen. Direct transfer of the internal tissue of resting body into malt extract agar medium produced pure culture, indicating that the fungus remained viable in oil-palm logs under diverse environmental conditions in the field. The formation of brown mycelium layers and resting bodies in Ganoderma might function to protect the food resources acquired after invasion, to Fig Resting bodies of Ganoderma found embedded in the decomposed tissue of oil palm infected by the pathogen.

5 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 253 allow survival from one plant generation to another and to initiate a primary infection. Deposition of melanin in fungal mycelium and spores has been suggested to be important for resistance to environmental stress, including protection against ultraviolet irradiation, radio waves, desiccation and temperature extremes (Bell and Wheeler, 1986). Melanins in fungi have also been suggested to be essential for resistance to microbial attack. Good field sanitation is believed to be one of the best possible ways to control the disease effectively (Hasan and Turner, 1994; Darmono, 1998). Research on field sanitation has been conducted intensively at BLRS. A recommended technique for point sanitation was to remove all diseased material by digging a pit 1.5 m square and 1 m deep, centred on the point of planting spot (Hasan and Turner, 1994). The disease remnants raised to the soil surface are disrupted, the simplest way being by cutting them into four or more pieces, to allow enhanced biological control. Darmono (1998) suggested that field sanitation should be conducted before planting (pre-planting sanitation activities) and regularly after planting during the entire life of the plant (post-planting sanitation activities). In areas with a high disease incidence, pre-planting sanitation can be conducted by removing all remaining boles and root clumps. Root clumps up to 20 cm thick are usually found attached to the boles. Special attention should be given to boles and roots of newly infected trees that, in the new planting, will certainly form a potential source of inoculum. Boles and root clumps of healthy trees left in the ground can be more easily colonized by the pathogen than healthy roots of newly established plants. In the long term, the removal of these tissue remains will help in reducing the risk of greater Ganoderma infestation in the following replantings. In post-planting sanitation, all infected trees that no longer have economic value will be uprooted and sanitized. The action of sanitation should be based on the observation of disease incidence previously determined. Darmono (1998) generated a formula for calculating disease incidence and scoring the grade of sanitation, as follows. S E I = 100% N where I is the disease incidence; S, the number of standing trees infected by Ganoderma; E, the number of empty planting spots due to Ganoderma; and N, the total number of planting spots observed. R G = S E where G is the grade of sanitation; R, the number of sanitized planting spots; and S and E, as described above. It has been a common practice in the past, or even currently, to base the score of disease incidence merely on the number of empty planting spots or plant mortality, due to Ganoderma in the plantation. Such a form of scoring gives an impression that the infected standing trees do not have a significant

6 254 T.W. Darmono role for disease development, and they have since been neglected during land preparation for new planting. Detailed notes on the category of disease severity in each tree should be made during observations. Categories of disease severity proposed by Darmono (1998) are presented in Table The felling of old oil palms before land preparation for replanting was usually conducted by pushing individual trees over with a bulldozer. By this action, the healthy trees are usually uprooted along with their boles and root clumps. If the tree is diseased (category R and Y), the pushing action usually causes it to break off at the base and the boles and roots are left behind in the ground. If not removed or sanitized, these remains will become potential infection foci. In a long-term programme, research activities at IOPRI and BRUEC are currently undertaking the production of resistant oil-palm material by means of conventional breeding and molecular biology techniques. At BRUEC, chitinase and glucanase genes obtained from local strains of microbes will be transformed into the plant genome and specifically expressed in the root system so that, hopefully, the palm will become resistant to Ganoderma infection. Atransformation system in oil palm mediated with Agrobacterium tumefaciens has also been developed (Chaidamsari et al., 1998) and a propagation system for oil palm using tissue-culture techniques has been acquired (Tahardi, 1998). Development of resistant planting materials needs knowledge of the genetic variability in the pathogen. Studies on genetic variability of Ganoderma associated with oil palm showed variation among isolates from the same plantation and among those from different plantations (Darmono, 1998). Table Categories of disease severity caused by Ganoderma in oil palm (Darmono, 1998). Mark colour Colour abbreviation Description Green Yellow Red Black White G Y R B W Plant looks healthy with no disease symptom or sign of infection; or plant recovers from infection with no sign of Ganoderma activities. This may include plants with basal cavity due to previous Ganoderma Plant looks healthy, but a fruiting body of Ganoderma or brown discolouration can be observed at the base of the stem Plant looks as if it is suffering from the disease and shows typical symptoms and signs of infection Empty planting spot with infected boles and roots remaining in the ground Sanitized empty planting spot

7 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 255 An Attempt to Produce an Immunoassay-based Detection Kit Need for the development of detection tools From a practical standpoint, disease control in individual trees is hampered by our inability to detect symptoms and signs of infection at an early stage of disease development. Infected palms usually show symptoms only after a large portion of their base has been destroyed by the pathogen. Although soil drenching with fungicide may effectively kill the pathogen, large-scale application of this type is not economically feasible. The success of chemical treatments through trunk injection can be achieved only if they are applied at an early stage of disease development. Therefore an accurate, quick and cheap detection system needs to be developed. Although cultural studies and microscopic observation are highly accurate for diagnoses of the infection, these techniques are too slow and not amenable to large-scale application (Miller and Martin, 1988). Immunoassay and nucleic acid hybridization systems have been used for plant pathogen detection and disease diagnoses. These molecular probes are more specific, rapid and sensitive than conventional methods based on disease symptoms (Leach and White, 1990). Immunoassay techniques offer greater simplicity and need less equipment than those of DNAprobe analyses. Experiments on the development of polyclonal antibody (PAb) and monoclonal antibody (MAb) against Ganoderma sp. were initiated at the Biotechnology Research Unit for Estate Crops in 1993 (Darmono et al., 1993). The main objective of the experiment was to produce an immunoassay-based detection kit. Detection kit specification There are some requirements in order for new products or technology to be applicable and acceptable by the users. In the case of a detection kit based on immunoassay, these requirements are: It should be specific and sensitive. It should be able to detect antigenic material far from the infection site. It should be easily used for on-site application. It should be inexpensive. It should not be harmful. Because it is directed for field application, the antibody used in the kit should be specific enough so that it only recognizes Ganoderma associated with basal stem rot, regardless of strain dissimilarity and geographical origins. If it is too specific, the antibody will detect only a certain strain of the pathogen and, consequently, will be less useful for field application. There are at least two ways to overcome this problem. The first is by pooling several specific antibodies or monoclonal antibodies, but this will be hampered by limited

8 256 T.W. Darmono knowledge on the number of strains of Ganoderma found in oil palm and by the high cost of production of the antibody. The second, less expensive, way is the development and production of polyclonal antibody. The sensitivity of the antibody should be measured, based on laboratory and field exercises. In laboratory exercises the level of sensitivity is determined by the ability of the antibody (at certain levels of dilution) to detect the least amount of antigen. For field applications, the antibody should ideally be capable of detecting antigenic material at an early stage of disease infection. The root system of an individual mature oil palm occupies about 16 m 3 of soil, and Ganoderma infection could start at any point in that space. In that kind of situation, the use of a DNAhybridization technique to detect Ganoderma infection at an early stage of disease development may be unreliable as it would require DNAobtained from the infection point. Thus, the tool used should ideally be able to detect infection at a distance from the infection site. Signs of infection can be in the form of chemical compounds produced by either the pathogen or by the plant in response to infection. Acceptability of any new product known to be strongly dependent on its price and ease of use. It should be cheap and be of significant benefit to the growers. Ideally, it should be far less expensive than the cost of single nutrient content analyses, which is approximately US$2 per sample in Indonesia. For the detection of Ganoderma infection, it would be better if systematic sampling could be conducted in the field regularly during observation of disease incidence. Alternatively, spot-selected sampling can be practised for reducing the cost of use. Sending samples to a commercial institution for enzyme-linked immunosorbent assay (ELISA) will be costly so the tool should be suitable for on-site application by any person with no special skills. Sampling activities should not harm the palms. Special care should be taken if the sample has to be obtained from the trunk or root, since an open injury may function as the entry point for the pathogen. Development of PAb Mycelial wash as antigen In the first stage of antibody development, a mycelial wash was used as a source of antigen. An isolate of Ganoderma sp. (TK-1, obtained from an infected oil palm in Bogor Botanical Garden) was cultured in a chemically defined liquid medium (Leatham, 1983). The mycelium was harvested and washed three times with phosphate-buffered saline (PBS) by filtration through a single layer of Whatman No. 93 filter paper. The liquid fraction from the final wash was used as the antigen. To develop the polyclonal antibody, a hyperimmune Balb/c mouse was injected intraperitoneally four times, at 2-day intervals with 250 µl antigen. Two days before the blood was withdrawn, an intravenous booster injection was given. Blood serum was obtained and the optimum titre for the

9 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 257 antigen antibody reaction was determined, based on a conventional checker A board method (Moekti, 1991). Cross-reactivity tests of the PAb were conducted by indirect-elisa (I-ELISA), against: 1. A mycelial wash of five isolates of Ganoderma spp. associated with oil palm, and 12 isolates of non-oil-palm origin; 2. Solvent from a fruiting-body tissue wash of five isolates of Ganoderma spp. associated with oil palm (including isolate TK-1); and 3. Solvent from a spore wash of 10 isolates of Ganoderma spp. associated with oil palm (including isolate TK-1). The optical density (OD) value of I-ELISA was measured with an automatic EIA-Microplate Reader at wavelengths of 405 nm and 495 nm. The mycelial wash used as an immunogen in this study contained approximately mg protein ml 1, with a molecular weight of 70,000 Da. Even with this relatively low content of protein the mycelial wash was proven to be capable of inducing a high titre of antibody (Figs 19.2 and 19.3). This might indicate that it contained a high molecular weight antigenic material in the form of protein or other metabolites. Antigen that contains polypeptides or proteins with a molecular weight of more than 5000 Da possesses a high immunogenic reactivity (Smith, 1988). From this experiment it was found that with low PAb concentration, at a 100-fold dilution, the antibody was capable of detecting µg ml 1 antigenic material (Fig. 19.2). Undiluted antibody was capable of detecting µg ml 1 antigenic material (Fig. 19.3). This result showed that when antigenic materials are present at low concentration, an undiluted antibody should be used. Determination of the titres is necessary in the development of any new antibody. Fig Optical densities from enzyme-linked immunosorbent assay readings in titres between dilute antibody and concentrated antigen.

10 258 T.W. Darmono Fig Optical density from enzyme-linked immunosorbent assay readings in titres between concentrated antibody and dilute antigen. The successful use of the mycelial wash as a source of antigen in the development of molecular detection assays for plant pathogenic fungi has been reported (Brown, 1993), however in this project, we encountered several problems due to its high specificity. The antibody only recognized antigenic materials from the in vitro cultures and not from the in vivo sources from field fruiting bodies or spores. Furthermore, the antibody produced was not capable in distinguishing Ganoderma spp. from different host origins. To increase specificity and sensitivity, monoclonal antibody development and the use of an exudate of Ganoderma sp. were attempted. Exudate as antigen The brown aqueous exudate secreted on the surface of mycelium grown on rubber wood was used as an antigen to develop a PAb anti-exudate of Ganoderma (PAb-aeG). A6-month-old Red Island laying hen was intramuscularly immunized with 0.25 ml antigen five times at 2 3 day intervals. Fourteen days after the final immunization, antibodies developed in the egg yolk were isolated, as described by Darmono and Suharyanto (1995). The specificity and reactivity of PAb-aeG were evaluated against 10 isolates of Ganoderma sp., using I-ELISA. The antigen for the cross-reactivity test was prepared from air-dried mycelium of on-wood cultures of the reference isolate AD-2 and field fruiting bodies of Ganoderma spp. Two grams of mycelium or fruiting body were ground in liquid nitrogen and extracted with 15 ml Tris buffer. The homogenate was separated and used as the antigen in cross-reactivity tests. Two types of enzyme antibody conjugates, i.e. rabbit anti-chicken horseradish peroxidase conjugate and alkaline phosphatase

11 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 259 conjugate, were tested at a dilution of 1 : The OD value of I-ELISAwas measured with an automatic EIA-Microplate Reader at wavelengths of 405 nm and 495 nm. Accumulation of chicken antibody corresponded well with antigen injections, indicating that the antibody was produced specifically against the exudate of Ganoderma sp. The optimum level of antibody production was found in eggs collected on the thirteenth day after the final immunization or the twenty-third day after initial immunization (Fig. 19.4). One of the main advantages of using chicken antibody is the ease of handling of the animal and of obtaining the antibody. About 15 ml of antibody mixture was usually obtained from each egg in a relatively short period of time, compared to 70 days or longer in rabbits. This amount of yolk antibody is sufficient to run about 3000 reactions in microwells. PAb-aeG produced in this study was highly sensitive in recognizing all field fruiting bodies of Ganoderma spp. associated with oil palm, but not Ganoderma of non-oil-palm origins (Fig. 19.5). Asatisfactory result was obtained only with the use of horseradish peroxidase anti-chicken antibody conjugate but not with alkaline phosphatase anti-chicken antibody conjugate. Development of MAb Antibodies were developed in a hyperimmune Balb/c mouse. Immunization of the mouse was conducted using a mycelial wash of isolate TK-1 as an immunogen, through the same procedures as described above. Five days after the final injection, a blood sample was withdrawn and lymphocytes were Fig Development of antibody in egg yolk, induced after injection of the hen with exudate of Ganoderma.

12 260 T.W. Darmono Fig Cross-reactivity of Ganoderma isolates against PAb-aeG. harvested and fused with myeloma sp/2 cells. Cell fusion was performed by treating the mixed cell suspension with polyethylene glycol (PEG) 4000 at 37 C for 2 minutes. The treated cells were cultured on a selective medium, Dulbecco Modified Eagle Media (DMEM) supplemented with 15% fetal calf serum (FCS) and hypoxanthine aminopterin thymidine (HAT). Hybridoma cells were then cultured in the same media without HAT supplementation. Selection of antibodies produced by the hybridoma was conducted by crossreacting against antigen prepared from eight isolates of Ganoderma. Selected hybridoma cell lines were cloned using a limiting dilution method. Antibody secreted into the medium was purified by ammonium sulphate precipitation. Typing of the monoclonal antibody was conducted using antibody isotyping kits (Sigma Chemical Co.). From 21 hybridoma produced, three (H-7, B-8 and D8) were selected. The specificity of these three hybridomas against eight isolates of Ganoderma is shown in Table The hybridomas were highly specific. Hybridomas B-8 and D-8 recognized only the reference isolate TK-1 from Bogor, West Java, and MU-1 from North Sumatra, while H-7 recognized only TK-1, but not MU-1. Both isolates were collected from diseased oil palm. The three hybridomas were not capable of recognizing isolates of other oil-palm origins, SP-1 and AD-2, and isolates of non-oil-palm origins, GJ-4, CO-2, KR-11 and KR-15. The hybridomas have been cloned. The monoclonal antibodies produced were all IgM type.

13 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 261 Table Specificity of monoclonal antibodies produced by three selected hybridomas. Isolates of Ganoderma Hybridoma culture TK-1 SP-1 AD-2 MU-1 GJ-4 CO-2 KR-11 KR-15 H-7 B-8 D-8 () Control () Control,,, Weaker to stronger reaction., No reaction. Application of PAb and MAb The potential use of PAb-aeG for the detection of signs of infection was evaluated. Samples of oil-palm tissue were collected from severely infected trees planted in 1984and their neighbouring apparently healthy trees, as well as from a 2-year-old tree naturally infected by Ganoderma sp. Samples were obtained from Bekri Oil Palm Plantation of PT Perkebunan Nusantara VII in Central Lampung, Sumatra. The sample from each mature tree was a composite of two mm stem tissue samples collected from two opposing areas 100 cm above the soil. Samples from the young tree were obtained from various areas, including the infection site, infection zones, growing point and young leaves up to 100 cm from the infection site. One to two gram of sample was ground in liquid nitrogen in one volume of Tris HCl buffer ph 7.4. The extract from each sample was used as the antigen. Indirect ELISA was conducted according to Moekti (1991) with the use of peroxidase anti-chicken antibody conjugate. The dot immunobinding assay (DIBA) was also conducted on selected samples according to Robinson-Smith (1994). With samples obtained from the mature trees, the antigen was not detected in any of the severely infected trees but was detected in an average of three out four apparently healthy surrounding trees. Since the disease-spread to neighbouring trees occurs primarily through root contact, these apparently healthy trees may have been infected by the pathogen although no disease symptoms were visible. A similar result was obtained in the 2-year-old plants, where the antigen was not detected in the decomposed tissue but was detected in apparently healthy tissues, including leaf fronds and shoot tips (data not shown). The highest concentration of antigen was found in reaction zones, encountered as a brown discolouration at the base of leaf stalks near the diseased stem. The absence of antigenic material in the decomposed tissues of oil palm may be due to degradation of the product by the pathogen itself or through other mechanisms. The DIBA test, conducted with a limited number

14 262 T.W. Darmono of samples, produced the same result, showing that the antigenic materials could be detected with the simpler technique. It is interesting to note that low molecular weight proteins were highly expressed in apparently healthy tissues of an infected plant, but not in healthy tissues of a reference healthy plant. This indicates that the PAb-aeG produced in this study has the potential to be used in the detection of early stages of infection of oil palm by Ganoderma spp. In the second series of tests, the PAb-aeG was tested against antigens prepared from leaf samples obtained from mature trees. Leaf samples were taken from 200 palms in a block with high disease incidence (34% of palms showing symptoms or signs of infection) and from 200 palms in a block with low disease incidence (5% of palms showing symptoms or signs of infection). The ELISA readings of samples obtained from the block with low disease incidence ranged from to 2.110, while from the block with high disease incidence, readings ranged from to By assuming that palms with an OD value of more than 0.39 (the median) were categorized as infected by Ganoderma, it was found that in the block with high disease incidence 80% of palms were infected while in the block with low disease incidence, 58% of palms were infected by the pathogen (Fig. 19.6). Plants with high OD values but showing no visual disease symptoms were revealed to be infected by the pathogen after their bases were chopped and examined. This showed that the PAb-aeG developed has the potential for large-scale application with a high degree of sensitivity. This second series of experiments further confirmed that the antigenic materials could be detected in leaves of diseased palms, more than 3 m from the infection site at the stem base. This result was consistent with the previous finding that exudate or other substances secreted by Ganoderma might be transported to the leaves along with nutrient and water transport by the plant. Leaf sampling is desirable since it does not damage the tree. Large-scale experimentation needs to be conducted to verify the potential commercial application of this product. Detection of antigenic material from oven-dried leaf samples PT SMART Corporation, a large private company planting oil palms at Pakanbaru, Riau, Sumatra, provided three separate batches of leaf samples. They were obtained from mature trees in three separate localities. The first batch was from infected oil palms from a plantation with high disease incidence, while the second and the third batches were from healthy oil palms in plantations with no disease incidence. Leaf sampling was conducted using a technique recommended for nutrient content analyses. All leaf samples were oven-dried at 60 C before they were sent to Bogor for ELISA. Eight leaflets from each bulk were randomly selected and used for antigen preparation. They were individually ground into powder in liquid nitrogen. Extraction was with Tris HCl and the extract was then used as an antigen for the cross-reactivity test with PAb-aeG.

15 Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 263 Fig Histogram of frequency of oil palms with certain range of optical density (OD) value from blocks with high (top) and low (bottom) disease incidence. Cross-reactivity was 2 3 times higher in leaf samples from diseased trees than those from healthy trees (Table 19.3). From this result it can be concluded that antigenic material associated with Ganoderma infection can be detected in leaves of diseased trees even after oven drying. However, OD values from these samples were much lower than those from leaf samples preserved in liquid nitrogen directly in the field.

16 264 T.W. Darmono Table Optical density readings from enzyme-linked immunosorbent assay of leaf extract from oven-dried leaf samples tested against PAb-aeG. Leaf samples From diseased trees From healthy trees, Field Site 1 From healthy trees, Field Site 2 Average optical density readings at 405 and 492 nm* a b b *Values followed by the same letter are not different significantly at P = Concluding Remarks Research on BSR disease caused by Ganoderma in oil palm in Indonesia is progressing very well. Information on some biological and ecophysiological aspects of the pathogen, as well as information on the host pathogen relationship provides a better understanding of the natural occurrence of the disease. Some biological control agents and chemical fungicides have been shown to be effective in the laboratory, but successful disease management through chemical and biological control will be achieved only after generation of a better field application technique. Provision of an immunoassay-based detection kit will help in the detection of infection at the earliest stage of disease development and this may subsequently increase the efficiency of disease management. References Abadi, A.L., Tjitrosomo, S.S., Makmur, A., Sutakaria, J., Dharmaputra, O.S., Macmud, M. and Susilo, H. (1989) Biology of Ganoderma boninense on oil palm (Elaeis guineensis) and the effect of some soil micro-organisms on its growth. Forum Pascasarjana 12th year, No. 2, (in Indonesian). Bell, A.A. and Wheeler, M.H. (1986) Biosynthesis and functions of fungal melanins. Annual Review of Phytopathology 24, Brown, I. (1993) Molecular detection assays for plant pathogenic fungi. AgBiotech News and Information 5, 219N 222N. Chaidamsari, T., Tahardi, J.S. and Santoso, D. (1998) Agrobacterium-mediated transformation in leaf explant oil palm. In: Proceedings of the 1998 International Oil Palm Conference, Nusa Dua, Bali, September 1998, pp Darmono, T.W. (1996) Penampakan keunggulan bahan hayati dari bahan kimia untuk pengendalian patogen penyakit akar tanaman perkebunan. Seminar Nasional Mikrobiologi Lingkungan II, Bogor, 9 10 October Darmono, T.W. (1998) Development and survival of Ganoderma in oil palm tissue. In: Proceedings of the 1998 International Oil Palm Conference, Nusa Dua, Bali, September 1998, pp Darmono, T.W. (1998) Molecular approaches to the elucidation of basal stem rot disease of oil palm. Proceedings of the BTIG Workshop on Oil Palm Improvement through Biotechnology, Bogor, April 1997, pp

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