The Spread of Ganoderma from Infective Sources in the Field and its Implications for Management of the Disease in Oil Palm

Transcription

1 J. Spread 8 Floodof et Ganoderma al. from Infective Sources in the Field The Spread of Ganoderma from Infective Sources in the Field and its Implications for Management of the Disease in Oil Palm J. Flood, Y. Hasan 2, P.D. Turner 3 and E.B. O Grady 8 CABI Bioscience, Egham, UK; 2 Bah Lias Research Station, P.T.P.P. London, Medan, North Sumatra, Indonesia; 3 PO Box 5, Quilpie, Queensland, Australia Introduction Basal stem rot (), caused by species of Ganoderma, has been recognized as a serious disease of oil palm (Elaeis guineensis) for over 8 years and has caused severe economic loss in Malaysia (Turner, 98; Singh, 99; Ariffin et al., 996) and North Sumatra, especially during the past 3 years (Hasan and Turner, 998). Initially, the disease was considered to affect only old palms (at least 25 years old) but, with successive palm generations on the same land, a higher disease incidence has been observed and the symptoms occur earlier with each replanting. For example, in Sumatra, where replanting was conducted by pushing over the old stand, with no attempt to remove -infected tissues, young replanted palms have died from the second year onwards after planting. Economic loss may begin to occur within years and severe loss after 5 years; the normal life span of a planting of oil palm would be 25 3 years. Where this phenomenon has occurred, it has generally been accepted that there has been an increase in available inoculum from the previous palm planting (Turner, 98). Thus, efforts to manage the disease have been directed largely towards disease avoidance through reducing the amounts of potential infection sources for the replanting at the time of clearing the old stand (Singh, 99). In Malaysia, the benefit of this clean-clearing approach over no disease avoidance measures has been demonstrated (Hashim, 99). However, the total removal of all infective tissues from an old stand with a high CAB International 2. Ganoderma Diseases of Perennial Crops (eds J. Flood, P.D. Bridge and M. Holderness)

2 2 J. Flood et al. disease incidence is a practical impossibility, and so the aim has been to concentrate on removal of as many of the larger tissue sections as economically feasible. To investigate the efficacy of sanitation in management, a series of trials was undertaken at Bah Lias Research Station (BLRS) of P.T.P.P. London in North Sumatra, Indonesia over a period of several years. The trials were designed to assess the relative importance of various tissue remnants from the old palm stand as potential sources of inoculum at replanting (Hasan and Turner, 998) so as to make practical recommendations for management of the disease in Sumatra. The trials were set up as to be sufficiently large to overcome any variations in inoculum, with each treatment being replicated at different sites in the plantation. Six-month-old seedlings were used to bait the Ganoderma-infected material. External leaf symptoms developing on these bait seedlings were recorded for the duration of each trial, while at the end of each trial, all seedlings were examined internally for Ganoderma infection by destructive sampling and plating to Ganoderma-selective medium (GSM) (Ariffin and Idris, 99). Each experimental plot was isolated by a deep trench to increase the likelihood that any infection recorded was derived from the tissue being tested and not from an outside source, but more recently, molecular fingerprinting techniques (Miller et al., this volume; Bridge et al., this volume; Rolph et al., this volume) became available which allowed confirmation of the origin of the pathogen in infected seedlings. Stump Tissues Astump comprises the base of the palm, or bole, and the thick crust of roots immediately surrounding it. Stumps are usually recognized as major sources of. The first trial compared stumps, prepared by felling diseased palms about 2 cm above the ground (standard practice), as an inoculum source with stumps derived from healthy palms. Around each stump eight bait seedlings were planted. An additional treatment, planting additional seedlings immediately outside the plot isolation trench and isolating these by a further trench m from the inner trench, in order to emphasize disease origin, was added. Each treatment was replicated eight times at different sites. Six months after planting, a small number of seedlings began to exhibit disease symptoms and by the end of the 28-month trial period, 76% of all bait seedlings showed symptoms; Ganoderma was isolated from these seedlings. In comparison, seedlings planted outside the first trench and within the second isolation trench perimeter showed very little infection only.6% of these seedlings were diseased and, at 8% of the replicate sites, these seedlings exhibited no symptoms at all. No disease was recorded in bait seedlings planted around healthy palm stumps within the period. Another trial aimed to assess the effect of stump size on disease incidence. Additional treatments in this trial were comparisons with stumps derived from

3 Spread of Ganoderma from Infective Sources in the Field 3 healthy palms and the effects of pre-felling poisoning by paraquat, using 6 ml per palm Gramoxone which was injected into the trunk. Stump size was found to exert a marked influence on disease occurrence, with more bait seedlings around smaller, lower stumps (2 cm high) exhibiting disease symptoms after 2 years than those around larger, higher stumps (5 cm high). Rate of decomposition and bait seedling root ingress into Ganoderma-colonized tissues would appear to be the most likely explanations for the difference. The effects of poisoning, which had accelerated tissue breakdown, supported this, with more seedling infection recorded around larger stumps where poisoning treatment had been carried out. The importance of inoculum sources at different soil depths adjacent to -infected stumps, which is of considerable relevance to sanitation practices, was also investigated. Thus, soil and palm tissue adjacent to infected stumps were removed to one of the following depths: 2, 4, 6, 8 and cm. Eight replicate bait seedlings were planted at each depth and these treatments were compared with diseased stumps that were undisturbed after felling (no soil or tissues removed) and sites around healthy palms excavated to a depth of 6 cm. In the absence of any sanitation, 75% of seedlings had become infected and 97% of replicate sites had infected plants within 2 years of planting (Table 8.). In comparison, disease incidence in the baited seedlings decreased to 2% where soil and debris had been removed to a depth of 6 cm, and no disease was recorded where soil and debris had been removed to 8 or cm (Table 8.). In an extension of this trial, the same sites were replanted with bait seedlings after 2 years and no disease was recorded at any depth 2 years later. Similarly, when new bait seedlings were planted around previously highly infective diseased stumps after 2 years, none of these bait seedlings developed symptoms. Even after 2 further years of recording, these seedlings remained symptomless, which would suggest that the potential of these stumps to act as sources of inoculum had declined after 2 years. Data of percentage infection over time at two sites (Table 8.2) further supported the view that fewer seedling infections occurred after 2 24 months. Some variation between sites is Table 8.. Effects of the removal of soil and palm tissues from around healthy basal stem rot ()-infected stumps on disease incidence after 24 months. Disease status Depth of bole removed (cm) % Replicate sites with infected seedlings % Seedlings infected

4 4 J. Flood et al. to be expected since the amount of infective tissue within stumps and its location in relation to seedling root contact will differ considerably, as will the rates of subsequent decay. During the course of these trials, molecular fingerprinting techniques became available for Ganoderma and were used to confirm the origin of the Ganoderma from infected bait seedlings. Material was collected from diseased seedlings, stump tissues and sporophores growing on the stumps and isolations made on GSM. DNAwas extracted from pure cultures (Miller et al., 999) and purified DNAsamples tested with the ITS3/GanET primer (Bridge et al., this volume) to check their identity. All isolates were positive with the ITS3/GanET primer, confirming that the pathogen had been isolated from the various tissues (Fig. 8.). Mitochondrial profiles were generated using the enzyme HaeIII as the restriction enzyme (Miller et al., 999; Rolph et al., this volume) and revealed that identical profiles were present in the stumps and the infected bait seedling material (Figure 8.2). Table 8.2. Percentage of total bait seedling infection appearing around basal stem rot stumps over time. Months after planting % Seedling infection Site A Site B Fig. 8.. Confirmation of the presence of the pathogen from stump tissues and infected seedlings.

5 Spread of Ganoderma from Infective Sources in the Field 5 Fig Mitochondrial DNA restriction fragment length polymorphisms of Ganoderma isolates from an infected basal stem rot stump and from a baited infected seedling planted near the infected stump. As mitochondrial (mtdna) inheritance is believed to be unilinear (Forster and Coffey, 99), isolates from the same sibling family would therefore have the same profile. However, generally, mtdnaprofiles are highly variable in Ganoderma isolates, even from the same and adjacent oil palms (Miller et al., 999). Thus, identical mtdnaprofiles from -infected stumps and from infected bait seedlings may indicate that mycelial spread or root-to-root contact has occurred, but, equally, the role of basidiospores cannot be ruled out (Miller et al., 999). To clarify this point, a third molecular profiling technique was used, namely amplification fragment length polymorphisms (AFLPs), as described by Vos et al. (995). This technique assesses the total cellular DNA profile (nuclear and mitochondrial DNA) and is a more stable and reliable method of studying variation (Rolph et al., this volume). Identical AFLP profiles were produced using several primers, including primer E (Rolph et al., this volume) (Fig. 8.3) confirming that the baited seedlings were infected with the same genotype as that in the -infected stump. Trunk Tissues Unless trunks of the old palm stand are destroyed at the time of replanting, they are usually windrowed, i.e. placed in rows. Such trunks are colonized by many species of fungi, including Ganoderma. Trunks will also remain following a number of estate practices, e.g. following underplanting, those excavated as low-yielding, palms removed for thinning or road construction and excavated diseased palms, and palms affected by upper stem rot (USR) often remain standing for long periods, as do palms killed by lightning. The trials summarized below assessed the significance of trunk sections as sources of

6 6 J. Flood et al. Fig Amplification fragment length polymorphisms from basal stem rot stump, baited seedling and Ganoderma sporophore (fruit body) growing on the infected stump. following various treatments and compared these sources with -infected stumps. Palms were felled as close as possible to the ground and the trunk then cut at m and 4.75 m from the base, with the remainder being discarded. The stump and each trunk section were isolated by trenches and bait seedlings were planted close to the sections. Apparently healthy palms were also included. Stump tissues remained the most important source of, with 27 38% seedling infection occurring, and although the incidence of disease arising from trunk sections was much lower (Table 8.3), this would remain of considerable practical significance. There was a marked increase over the 2-year period in the number of infection foci on what had previously been considered as healthy stumps, with the highest disease incidence (2%) being recorded where palms had been poisoned before felling and where legume overgrowth had been successful. The presence of diseased seedlings around what had previously been considered to be healthy palms would indicate that the pathogen is present in the palm for what maybe a considerable time before symptoms are seen. Infection rates of bait seedlings when planted around standing diseased and apparently healthy palms were compared with that from stumps; the infection rate of bait seedlings around standing palms was much lower (Table 8.4). However, the period of infectivity of standing palms is likely to be much longer, demonstrating the need to remove such palms in management of the disease. Also, while diseased tissues appear to lose much of their infective ability from about 8 2 months after felling, the majority of apparently healthy stumps and trunks had yet to show the extent to which they would

7 Spread of Ganoderma from Infective Sources in the Field 7 Table 8.3. Disease status Basal stem rot incidence in bait seedlings around oil-palm residues. % Seedling infection around stumps % Seedling infection around proximal trunks (stem) % Seedling infection around distal trunks (stem) Treatment Yr Yr2 Yr Yr2 Yr Yr2 Diseased Diseased Diseased Diseased Nil P C PC Nil P C PC P, Poisoned before felling; C, legume cover. become sources of disease at the end of 2 years (Table 8.4). However, from the 2-year data alone, it is clear that under field conditions they will certainly present a significant disease risk. In another trunk treatment, pieces were cut to simulate shredding as a clearing method, with and without poisoning prior to preparation. These were either placed on the soil surface or buried at 2 cm deep. Both infected and healthy trunk tissues were examined, with seedling baits used to detect in plots isolated by trenches. Both diseased and healthy shredded tissues can give rise to disease after burial. Except in a single instance, superficially placed tissues were not a disease hazard. In plots with buried tissues where disease was recorded, sporophores of Ganoderma were produced on the soil surface. Roots The current recommendation for sanitation procedure concentrates on a.5 m square centred on the point where the palm is planted. The assumption has been that the remaining inter-space presents no serious disease hazard. In a trial to examine this, areas between neighbouring diseased palms were each divided into three equal parts and isolated by deep trenches. Bait seedlings were then planted in each sector, as well as around the bases of the affected palms. Similar sectors between apparently healthy palms were also baited. In the plots, Ganoderma fructifications developed on cut root ends, signifying the presence of infected roots. The overall incidence of seedling infection was low (4%) and was confined to the sectors closest to the diseased palms, whereas 69% of bait seedlings planted around the main disease sources became infected. No disease was recorded between healthy palms.

8 8 J. Flood et al. Table 8.4. Comparison of basal stem rot () in bait seedlings around standing and healthy palms compared with stumps, 2 years after treatment. Standing palms Low stumps Disease status Treatment % Infective sites % Seedling infection % Infective sites % Seedling infection Nil P Nil P P, Poisoned. Also, records of the production of Ganoderma sporophores on cut ends of roots on the inside of isolation trenches from the depth trial (Table 8.) revealed that where no soil or palm tissues had been removed, 67% of all replicate sites had Ganoderma sporophores, while where soil had been removed to a depth of 6 cm, this had decreased to only %. Thus, diseased roots can comprise a small, but still significant, source of in a replant, although this probably requires dense root aggregations. Discussion It is apparent from these results that, when suitable disease sources are present, oil-palm seedlings can be attacked by Ganoderma soon after planting. Disease development and overt symptom appearance will depend on the size of the palm when it becomes diseased, its continued growth vigour and the size of the inoculum. Small seedlings close to large disease sources are killed rapidly. Larger, rapidly growing plants are also affected, but frequently do not die quickly. Numerous investigations have reported that many infected palms continue to grow well, often for very long periods, before the internal lesion becomes so extensive that visible external symptoms develop. This explains why so many cases of occur long after planting and also after obvious sources of primary infection have disappeared. Once a few palms in a field are infected it has been considered that further colonization of palms in the field is due to root-to-root contact by the palms or mycelial spread. Both Singh (99) and Hashim (994) reported the disease as occurring in patches or groups, which would support palm-to-palm infection, but this view has been challenged recently by Miller et al. (999). Studies of somatic incompatibility and mtdnaprofiling of isolates taken from many adult palms within two oil-palm blocks (Miller et al., this volume) revealed considerable variation between isolates, and led to the conclusion that isolates occurred as numerous distinct genotypes, even within the same palm. Thus,

9 Spread of Ganoderma from Infective Sources in the Field 9 mycelial spread to adjacent palms or root-to-root contact was very unlikely. Ariffin et al. (996) similarly reported a high degree of heterogeneity between isolates taken from adjacent infected adult palms. This contrasts with other wood-rotting fungi, such as Heterobasidion annosum (Stenlid, 985) or Phellinus noxius (Hattori et al., 996), where one clone of the pathogen can extend over several metres. However, the preliminary mtdnaand AFLP profiling described here has demonstrated that the same genotype is present in the diseased stump and in baited seedlings. Thus, the experimental assumption that the infected stump acts as a direct source of infection to the young seedlings was validated. Infection probably occurred due to the growth of seedling roots towards the decaying stump which is a rich source of nutrients. However, molecular analysis has only been conducted on a small number of stumps, and other sources of infection for young seedlings in the field cannot be ruled out. To date, the role of basidiospores has never been fully explained in this disease. Thompson (93) suggested that they were responsible for USR, usually in association with Phellinus spp., but Turner (965) failed to infect oil palm following direct spore inoculation of cut frond bases, and Yeong (972) reported no infection following direct inoculation of oil-palm seedlings. However, it is possible that basidiospores could infect palms indirectly, i.e. are able to colonize debris which subsequently becomes the source of infection for living palms (Miller, 995). This would account for the heterogeneity determined using molecular markers (Miller et al., 999). Thus, much more molecular analysis remains to be conducted so far only diseased stumps have been studied, but trunks and even roots can act as significant sources of infection. The investigations reported here have confirmed that the times of greatest practical significance for the control of Ganoderma in oil palm are: (i) soon after planting, when suitable inocula remain in the ground from the previous planting (oil-palm stumps or root debris); and (ii) later in the planting cycle, when root contact is made with Ganoderma-colonized sections of palm trunks resting on the ground in rows (windrows). Results of this study would seem to suggest that this danger extends over a much longer period when windrowed palms are not poisoned prior to felling and are not covered by legumes to accelerate decomposition. Fungi that cause root disease frequently require substantial inoculum potential before they are able to initiate infection and subsequently become established within the host plant. Thus, infection must require either a block of Ganoderma-colonized tissue of adequate size or a conglomerate of tissues, e.g. a mass of infected roots, which collectively become an infection source. In the trials summarized here, the importance of large blocks of inoculum is evident. Bait seedling infection was very rapid when planted close to -infected stumps. Gradual removal of this source with increasing depth showed a clear relationship between availability of infective material and both the occurrence and incidence of. This was not confined to the stump tissues. At a depth of 6 cm there was no mass of stump tissue, only a few infected roots, but these

10 J. Flood et al. root masses can become significant sources of infection. Even where a field that has been carefully cleaned of debris at replanting, as the new seedlings grow, more and more root debris is produced. This will include a large amount of root material from self-pruning (Hartley, 988; Jourdan and Rey, 996) and large numbers of fine quaternary roots are present in the upper layers of the soil. The hypothesis that this material could become the substrate for basidiospore colonization requires further study. The depth factor poses considerable problems from the practical viewpoint of sanitation at the time of clearing for replanting. Breaking up deeply located root masses requires deep tining, for which equipment is not always available. If seedlings are planted at the same points of former palms, there is a distinct possibility that their roots will soon encounter infective sources of Ganoderma, and thus as much of the diseased stump tissue as possible should be removed. However, further baiting using seedlings showed that these potential sources were less of a disease hazard after 2 years. This means that their importance could be expected to be very much reduced, or even negligible, if new palms are planted as far as possible from the old planting points. Their disease potential would have greatly diminished by the time the roots of the new planting reach the hazard sources, provided the old stand had been poisoned before felling. Alternatively, delayed planting could be a useful method of disease avoidance. Windrowed palm trunks represent another significant problem, and the same considerations apply to the necessity for planting as far away as possible from windrows. The lateral extent of root development during immaturity reaches roughly the edge of the canopy, meaning that it should take 2 3 years before reaching this particular disease source if planted at the furthest possible distance. An important observation is that the period over which windrows remain a disease hazard is greatly reduced when palms of the old stand are poisoned by paraquat prior to felling, and this effect is further enhanced when they are cut into sections and with a thick overgrowth of legume cover. Where there has been no poisoning, the tissues remain a disease hazard for years. In such situations older palms of the replant become infected, with overt disease symptoms only appearing long after the original infection sources have disappeared. One solution is to shred palm tissues so that they do not become sources over long periods, which is already a common practice in Malaysia but not in Sumatra. However, even this does not provide a total answer to the problem. Occurrence of in bait seedlings, arising from buried, shredded diseased and healthy trunk segments, was limited, but illustrated that the technique still contains a degree of disease risk. Disease arising from superficially placed segments was very slight and unexpected. It was in some ways remarkable that in such segments, buried or superficially placed, disease occurred at all, since many attempts at artificial inoculation of seedlings in polybags using such tissues have failed. The appearance of Ganoderma sporophores on the soil surface above buried sections indicated that a sufficient

11 Spread of Ganoderma from Infective Sources in the Field mass of Ganoderma-colonized tissue can overcome the inhibitory effects in soil which normally prevent its development there. Another possible control method for the future lies in the fact that trunk tissues, in particular, support the rapid development of many fungi other than Ganoderma, and this points to a possible biological control approach to the windrow disease hazard problem. Rapid degradation of the windrowed tissues, especially by fungi antagonistic to Ganoderma, would have obvious advantages for and Oryctes control. However, this approach needs more investigation, not least because woody tissues contain very little nitrogen, this influencing the extent of colonization by certain rotting microorganisms, so that manipulation of the nitrogen status of the debris will need to be conducted (Paterson et al., this volume). Acknowledgements This chapter is published with the permission of P.T.P.P. London, Sumatra, Indonesia. The considerable assistance of field staff in the execution of trials is gratefully acknowledged. The authors would like to thank the Crop Protection Programme (CPP) of the Department for International Development (DFID) for funding some of the research reported here, which was administered through NRI (RNRRS Project 6628). References Ariffin, D. and Idris, A.S. (99) A selective medium for the isolation of Ganoderma from diseases tissues. In: Basiron et al. (eds) Proceedings of the 99 International Palm Oil Conference, Progress, Prospects and Challenges Towards the 2st Century, September 99. PORIM, Selangor, Malaysia, pp Ariffin, D., Idris, A.S. and Azahari, M. (996) Spread of Ganoderma boniense and vegetative compatibility studies of palm isolates in a single field. In: Darus et al. (eds) Proceedings of the 996 PORIM International Palm Oil Congress Competitiveness for the 2st Century. PORIM, Malaysia, pp Forster, H. and Coffey, M.D. (99) Mating behaviour of Phytophthora parasitica: evidence for sexual recombination in oospores using DNArestriction fragment length polymorphisms as genetic markers. Experimental Mycology 4, Hartley, C.W.S. (988) The Oil Palm. Longman Scientific and Technical Press, UK. Hasan, Y. and Turner, P.D. (998) The comparative importance of different oil palm tissues as infection sources for basal stem rot in replantings. Planter 74, Hashim, K.B. (99) Results of four trials on Ganoderma basal stem rot of oil palm in Golden Hope Estates. In: Proceedings of the Ganoderma Workshop organised by PORIM, Selangor, Malaysia, September 99. Hashim, K.B. (994) Basal stem rot of oil palm caused by Ganoderma boninense an update. In: Sukaimi et al. (eds) Proceedings of the PORIM International Palm Oil Congress Update and Revision (Agriculture) 993. PORIM, Malaysia.

12 2 J. Flood et al. Hattori, T., Abe, Y. and Usugi, T. (996) Distribution of clones of Phellinus noxius in a windbreak on Ishigaki Island. European Journal of Forest Pathology 26, Jourdan, C. and Rey, H. (996) Modelling and simulation of the architecture and development of the oil palm (Elaeis guineensis) root system with special attention to practical application. In: Darus et al. (eds) Proceedings of the PORIM International Palm Oil Conference Competitiveness for the 2st Century. PORIM, Malaysia, pp. 97. Miller, R.N.G. (995) The characterization of Ganoderma populations in oil palm cropping systems. PhD thesis, University of Reading, UK. Miller, R.N.G., Holderness, M., Bridge, P.D., Chung, G.F. and Zakaria, M.H. (999) Genetic diversity of Ganoderma in oil palm plantings. Plant Pathology 48, Singh, G. (99) Ganoderma the scourge of oil palm in the coastal areas. Planter 67, Stenlid, J. (985) Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility and isozyme patterns. Canadian Journal of Botany 63, Thompson, A. (93) Stem rot of oil palm in Malaysia. Bulletin of the Department of Agriculture of the Straits Settlements and F.M.S. Science Series, Serdang 6. Turner, P.D. (965) Infection of oil palms by Ganoderma. Phytopathology 55, 937. Turner, P.D. (98) Oil Palm Diseases and Disorders. Oxford University Press, Oxford, pp. 88. Vos, P., Hogers, R., Bleeker, M., Reijans, H., Vandelee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (995) AFLP a new technique for DNAfingerprinting. Nucleic Acids Research 23(2), Yeong, W.L. (972) Studies into certain aspects of the biology of wood decay pathogens of Hevea rubber and oil palm (Elaeis guineensis). Bulletin of the Agricultural Science Project Report, University of Malaya.