Research on basal stem rot (BSR) of ornamental palms caused by basidiospores from <Emphasis Type="Italic">Ganoderma boninense</Emphasis>

Basidiospores were isolated from the fruiting bodies of Ganoderma infecting oil palms from an estate in Johor and from ornamental palms (including oil palms) from Singapore. The spores were then germinated to obtain homokaryotic mycelia. Based on clamp connection formation in paired hyphal fusions, tester strains were identified from the homokaryons isolated. Compatibility tests were then carried out using these testers to determine the relatedness of the homokaryotic Ganoderma isolates, both from Johor and from Singapore. Results from the compatibility tests showed that Ganoderma from both locations belong to the same species, while the Ganoderma isolates from Singapore share some common alleles. The pathogenicity tests carried out on Chrysalidocarpus lutescens seedlings using inoculum growing on rubber wood blocks showed that dikaryotic mycelia can cause basal stem rot infection.     

An insight into spore dispersal of <Emphasis Type="Italic">Ganoderma boninense</Emphasis> on oil palm

The disease of oil palm caused by Ganoderma boninense, although universally referred to as Ganoderma basal stem rot, occurs in three very distinct phases, with basal stem rot only part of the disease cycle. G. boninense also causes a seedling disease and an upper stem rot. An understanding of spore dispersal provides an insight into where spores of G. boninense have a role in the infection process. This role will be discussed in relation to each of these three infection phases. This understanding is a critical component of developing a successful disease control strategy.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

Pathogenicity Confirmation of Ganoderma Disease of Coconut Using Early Diagnosis Technique

The pathogenicity and diagnostic methods were standardized for Ganoderma disease of coconut. The pathogenicity of Ganoderma lucidum isolated from coconut was tested using six types of inoculation techniques. Two diagnostic methods, viz. indirect enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reactions (PCR) were applied for the confirmation of pathogenicity in coconut seedlings. Polyclonal antibodies (PAbs) were raised against mycelial, basidiocarp and specific proteins of Ganoderma and used for detection of Ganoderma in inoculated seedlings through indirect ELISA technique. All the three PAbs could detect Ganoderma in diseased coconut root tissues in early stage of the disease before symptom expression by indirect – ELISA at the antiserum dilution of 1 : 1000 for mycelial protein, 1 : 700 for Ganoderma specific protein and 1 : 3000 for basidiocarp protein. Low cross‐reactions were observed with saprophytic fungi occurring in coconut roots and also with other basidiomycetous fungi. In PCR, primers Gan1 and Gan2 generated from internal transcribed spacer region of rDNA were used the detection that produced a product of 167‐bp size in Ganoderma infected plants. In the present investigation, spawn inoculum responded earlier within 8 weeks compared with other methods of inoculation as expressed by OD value in ELISA test. This was also confirmed by PCR technique. The combination of these two diagnostic methods for detection of Ganoderma infection was highly reliable, rapid and sensitive.     

Enhancing biological control of basal stem rot disease (<Emphasis Type="Italic">Ganoderma boninense</Emphasis>) in oil palm plantations

Basal Stem Rot (BSR) disease caused by Ganoderma boninense is 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, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus 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.     

Association of Copper and Zinc Levels in Oil Palm (Elaeis guineensis) to the Spatial Distribution of Ganoderma Species in the Plantations on Peat

Nutrients are essential for normal physiological processes in plants, and they play important roles in defence mechanisms against pathogens. Oil palms cultivated on peat are more prone to nutrient deficiency, especially micronutrients, and this may affect their susceptibility to Ganoderma species, the major threat to the sustainability of oil palm throughout South‐East Asia. This study was conducted to investigate the association of copper (Cu) and zinc (Zn) in mature oil palm to the spatial distribution of Ganoderma species in the plantations on peat. Foliar samples (frond 17) of oil palm from two plantations (Betong and Miri) on peat in Sarawak, Malaysia, were collected based on the spatial distribution pattern of Ganoderma, and total Cu and Zn were quantified spectrometrically. The experiment was conducted twice at a 1‐year interval. The concentrations of Cu and Zn were significantly lower in oil palms from infected areas in contrast to those from uninfected areas. In addition, oil palms in infected areas in Miri suffered Cu and Zn deficiencies. Furthermore, Cu and Zn were significantly lower in the oil palms in Miri that had higher Ganoderma occurrence, as compared to those in Betong, which had significantly higher Cu and Zn but lower Ganoderma occurrence.     

Molecular biology of <Emphasis Type="Italic">Ganoderma</Emphasis> pathogenicity and diagnosis in coconut seedlings

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10–13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.     

The Effect of Post Inoculation Treatment with Mineral Oil on TMV Multiplication in Tobacco Leaves and Protoplasts

Half leaves of N. tabacum dipped into a 0.2 % emulsion of meneral oil 15min after inoculation with tabacco masaic virus (TMV) developed significantly fewer lesion than when not dipped Dipping reduced lesion numbers when applied up to 2½ h after inoculation and multiple dippings were more effective than one. The effect of oil was the same whether the inoculum was whole virus or RNA. Tobacco protoplasts treated with mineral oil contained less virus than untreated protoplasts. The oil probably acted by killing the protoplasts and was effective only when protoplasts were centrifuged through the oil emulsion, When water-treated leaves were dipped into a TMV solution there was an effect on TMV infection similar to that caused by dipping in oil after TMV inoculation.     

Life expectancy of oil palm (Elaeis guineensis) infected by Ganoderma boninense in coastal soils,Malaysia: a case study

Ganoderma boninense basal stem rot poses a serious threat to the oil palm industry. The effects of external disease symptoms and coastal soils (Briah – Typic Endoaquepts, Jawa – Typic Sulfaquepts, and Selangor – Typic Humaquepts) on the life expectancy of the infected palms, from disease detection to death, were studied. Six-monthly censuses on disease classes for each palm were recorded between 2004 and 2012. Survival curves of disease symptoms and soil types were compared using Kaplan–Meier and log-rank methods, respectively. Ganoderma-infected palms in acid-sulphate (AS) and potential AS soils recorded lower life expectancy. Survival duration of infected palms with foliar symptoms was 12-months shorter. External factors, such as soil type may influence the survival of infected palms and soil types may pre-dispose oil palm to higher risk of Ganoderma infection. More effective Ganoderma management for palms planted on Coastal soils (with and without AS layer) have been proposed.     

The role of wounds in the infection of oranges by Penicillium digitatum Sacc

Inocula of spores of Penicillium digitatum in water applied to apparently uninjured skin of oranges do not cause lesions to develop. Addition of citric acid, orange juice, or various extracts of rind had little effect on susceptibility to infection. When spores in water are applied to wounds made between oil vesicles, lesions develop only from wounds that penetrate deeply into the albedo. The flavedo of most oranges seems to be resistant to infection even when damaged, but in a few consignments it showed much less resistance. Increasing the number of conidia in the inoculum caused more lesions to develop but some fruits developed lesions from inocula containing very few spores. The method and timing of spore application to wounds had a considerable effect on the incidence of lesions; emanations from infected fruit had no effect. Lesions developed more rapidly and readily when suspensions of spores in water were applied to wounds in the skin that damaged oil vesicles; wounds as shallow as 0–25 mm allowed lesions to develop.     

Application of 15N natural abundance technique for evaluating biological nitrogen fixation in oil palm ecotypes at nursery stage in pot experiments and at mature plantation sites

A range of different species of diazotrophic bacteria has been found in tissues and the rhizosphere of oil palm plants, suggesting a potential to benefit from biological nitrogen fixation (BNF). A few studies have confirmed that plantlets at nursery stage can benefit significantly from BNF after inoculation with Azospirillum spp. but no data are available regarding the benefit from naturally-occurring diazotrophic bacteria in oil palm. The results described here were derived from two pot trials laid out under controlled conditions with plantlets from two important regions for palm oil production in Brazil, as well as from different field sites of mature oil palm plantations. The ¹⁵N natural abundance technique was employed to estimate plant dependence on BNF (%Ndfa) by the different ecotypes grown in soil and previously characterized as hosting diazotrophic bacteria. From both pot trials it was possible to identify some ecotypes of high potential for N₂-fixation that reached in some cases approximately 50%Ndfa. However, the accuracy of measurement still needs to be improved using more suitable reference plants for pot experiments. Values of δ ¹⁵N signals from oil palm and reference plants in the field were inconclusive concerning any benefit from BNF to oil palm, owing to apparently high temporal and spatial variability of δ ¹⁵N of the plant-available N in the heterogeneous soil matrix for the different palm and reference plant tested.     

Mycoparasitic Scytalidium parasiticum as a potential biocontrol agent against Ganoderma boninense basal stem rot in oil palm*

The objective of this study was to assess the interactions between Scytalidium parasiticum (Sp) and Ganoderma boninense, the causal agent of basal stem rot (BSR) in oil palm (Elaeis guineensis). When compared with Scytalidium ganodermophthorum and Scytalidium sphaerosporum, Sp showed greater inhibition towards all Ganoderma isolates during dual-culture assays. At the interaction zone, coiling of host hyphae, formation of short lateral enlarged contact structures, and production of appressorium-like organs organs were observed in Sp on G. boninense. These were followed by the degradation, shrinkage, and deformation of G. boninense mycelia. Sp reduced mycelial survival and fruiting body regeneration of G. boninense. Sp's non-volatile metabolites suppressed the growth of G. boninense. Our results show that Sp could be a necrotrophic mycoparasite of G. boninense. Nursery experiments revealed that Sp was non-pathogenic to oil palm seedlings, and it could suppress Ganoderma infection and reduce disease severity. Sp increased the height of palms in the positive control with non-Ganoderma-inoculated rubber wood block and Sp inoculum compared to similar control without Sp. Leaf area was greater in the G. boninense G8 inoculated palms when Sp was present compared to without Sp. These results show that Sp might be a potential biocontrol candidate against BSR.     

Studies on oil palm trunks as sources of infection in the field

Diseases of oil palm caused by Ganoderma boninense are of major economic importance in much of South-East Asia. This paper describes results from an ongoing field trial concerning the spread of the pathogen from artificially inoculated trunks used to simulate spread from windrowed trunks. Three planting distances for bait seedlings revealed that the closer the seedling was planted to the source of inoculum the sooner it succumbed to the disease. However, infection only occurred when the trunks were mounded (covered with soil), and seedlings planted around uncovered trunks (at any distance) have showed no symptoms of disease to date. Isolates are being collected from infected plants and molecular analysis is being undertaken to give more information on the spread of the pathogen.     

Following basal stem rot in young oil palm plantings

The PCR primer GanET has previously been shown to be suitable for the specific amplification of DNA from Ganoderma boninense. A DNA extraction and PCR method has been developed that allows for the amplification of the G. boninense DNA from environmental samples of oil palm tissue. The GanET primer reaction was used in conjunction with a palm-sampling programme to investigate the possible infection of young palms through cut frond base surfaces. Ganoderma DNA was detected in frond base material at a greater frequency than would be expected by comparison with current infection levels. Comparisons are made between the height of the frond base infected, the number of frond bases infected, and subsequent development of basal stem rot. The preliminary results suggest that the development of basal stem rot may be more likely to occur when young lower frond bases are infected.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Association of Phoma aquilina and Ascochyta pteridis with curl-tip disease of bracken

Many spores of Phoma aquilina and Ascochyta pteridis were rinsed from dry infected litter obtained from a site where curl tip was prevalent. More spores were released from litter which had been incubated under moist conditions for 24 h before rinsing. P. aquilina, known to cause curl-tip symptoms in bracken when inoculated via wounds (McElwee, 1983), did not damage healthy plants when spores were applied to intact tissues. A. pteridis appeared to infect undamaged plants under humid conditions causing the initial ‘fleck’ symptoms associated with the disease. Flecks formed on plants by A. pteridis may permit subsequent infection by P. aquilina. For bracken control, treating plants with fungal inoculum in disease-free areas may result in disease if inoculum supplements can be found which permit initial infection by A. pteridis or damage plants sufficiently to allow colonisation by P. aquilina directly.     

Multi-year study of Ganoderma aerobiology

Ganoderma basidiospores are dominant members ofthe airspora in many regions of the world and areconsidered important airborne allergens. Theaerobiology of Ganoderma spores in the Tulsa,Oklahoma area was examined using Burkard VolumetricSpore Traps from 1987–1996. Ganodermaspores were present in the atmosphere on more than95% of the days from June through October with peakconcentrations generally occurring from late August tomid-October. The data showed marked interannualvariation, with seasonal totals in 1994 and 1995significantly higher than other years. Stepwisebackward multiple regression showed that cumulativeseason total was significantly related to Junetemperature and May through August precipitation(R² = 0.97, p < 0.01).     

Early immunodiagnosis of Ganoderma infecting coconut seedlings and palms by using monospecific polyclonal antibodies

Among the various fungal diseases affecting plantation crops viz., coconut, aracanut, oil palm, etc. in India, basal stem rot (BSR) caused by species of Ganoderma is the most destructive. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study we generated two different types of antiserum for diagnosis of Ganoderma using the purified monospecific protein (62 kDa) (MS) and crude sporophore extract (SE). We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antiserum developed against the MS and SE showed 1:700 and 1:3000 titre values for the detection of Ganoderma. The MS antisera developed showed very low or almost no cross-reaction when compared to SE antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In DIBA test, both types of antisera were used separately for pathogenicity tests. MS antisera showed a positive reaction for purified protein, artificially infected roots and infected field palm. A mild reaction was observed against infected field trunk but a negative reaction was observed for lesions and leaf samples. In the case of SE antisera, a negative reaction was observed for all leaf samples, healthy roots and healthy trunk samples but positive reactions were observed for positive control, artificially inoculated roots, infected field roots, infected trunk and lesions samples. Therefore, both ELISA and DIBA tests may be useful in the detection of infection at the earliest stage of disease development and this will certainly help in the development of management strategies against Ganoderma disease in palm crops in advance.     

Unexpected progeny obtained from glasshouse study of soil pH influence onAcaulospora laevis inoculum

Summary Potential growth stimulation of two hosts by acid-tolerantAcaulospora laevis was tested in a soil adjusted to 5 pH levels from 5.0 to 7.7. By wet-sieving methods, the field-source inoculum was essentially a pure culture ofA. laevis spores. Upon harvest of sweetgum a completely different spore-type was found in large quantities;A. laevis spores were relatively few to non-existent. The results dramatically underscore the need for caution against relying solely on pre-trial identifications of inoculum spores or presuming that apparently single-species cultures/inoculum will remain pure throughout an experiment.Contribution of the Oregon State University Agricultural Experiment Station in cooperation with the US Department of Agriculture, Agricultural Research Service, technical paper #7366 of the former.