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.     

Nucleic acid based detection technique for Ganoderma lucidum in coconut

Basal stem rot caused by Ganoderma lucidum is the most serious disease in coconut and arecanut gardens. Twenty-five Ganoderma isolates were collected from different parts of India and the pathogenicity of Ganoderma was proved on coconut seedlings. Mature sporophores developed within 10–13?weeks after inoculation of pathogen under in vivo. To detect the pathogen at early stage, DNA-based technology, polymerase chain reaction was used. In this, the primers Gan1 and Gan2 produced a product of 167?bp in size for all the Ganoderma isolates tested. Simultaneously, ITS 1 and ITS 4 primers amplified a fragment of 680?bp in the Ganoderma isolates. In addition, Ganoderma isolates showed polymorphism in the random amplified polymorphic DNA analysis.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA) for the detection of Ganoderma infecting palms

The genus Ganoderma has a worldwide distribution causing root and stem rot of many plantation crops. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study, we developed polyclonal antiserum for Ganoderma mycelial and extracellular protein, and evaluated its efficacy with different plant samples collected from artificially inoculated coconut seedlings and Ganoderma infected field palms. We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antisera developed against the crude mycelial protein (CMP) and extracellular protein (ECP) showed a 1:1000 titre value for the detection of Ganoderma. The CMP antisera developed showed more cross-reaction when compared to ECP 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 the DIBA test, ECP antisera detected positive control (ECP of Ganoderma MTP and CRS-1 isolate), artificially inoculated roots, infected field roots, infected basal trunk and additionally lesions gave positive reactions which were not found in the CMP antisera tested. Therefore, both ELISA and DIBA tests may be useful for screening a large number of samples and help in the detection of infection at the earliest stage of disease development and this will certainly help to adopt suitable management strategies against Ganoderma disease in palm crops in advance.     

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.     

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.     

Development and comparison of ELISA and PCR methods for the early detection of Ganoderma disease of coconut

Abstract

Molecular diagnosis, chemo-diagnosis and physiological parameter have been applied for detecting the Ganoderma disease of coconut. Polyclonal antibodies (PAbs) raised against mycelial protein of Ganoderma, specific mycelial protein (62 kDa) of Ganoderma isolates and basidiocarp protein of Ganoderma were used for detection. All the 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 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. For polymerase chain reaction tests, the primer was generated from the internal transcribed spacer region one (ITS 1) of rDNA of Ganoderma, which produced a PCR product of 167 bp in size. Utility of this method was confirmed at the field level.     

Comparing interfertility data with random amplified microsatellites DNA (RAMS) studies in <Emphasis Type="Italic">Ganoderma</Emphasis> Karst. Taxonomy

The taxonomy of the causal pathogen of basal stem rot of oil palms, Ganoderma is somewhat problematic at present. In order to determine the genetic distance relationship between G. boninense isolates and non-boninense isolates, a random amplified microsatellites DNA (RAMS) technique was carried out. The result was then compared with interfertility data of G. boninense that had been determined in previous mating studies to confirm the species of G. boninense. Dendrogram from cluster analysis based on UPGMA of RAMS data showed that two major clusters, I and II which separated at a genetic distance of 0.7935 were generated. Cluster I consisted of all the biological species G. boninense isolates namely CNLB, GSDK 3, PER 71, WD 814, GBL 3, GBL 6, OC, GH 02, 170 SL and 348781 while all non-boninense isolates namely G. ASAM, WRR, TFRI 129, G. RES, GJ, and CNLM were grouped together in cluster II. Although the RAMS markers showed polymorphisms in all the isolates tested, the results obtained were in agreement with the interfertility data. Therefore, the RAMS data could support the interfertility data for the identification of Ganoderma isolates.     

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.     

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.     

Lethal bole rot disease of coconut in East Africa

A lethal bole, rot disease of coconut, caused by Marasmiellus cocophilus Pegler, sp. nov., reaches epidemic proportions in several areas along the coasts of Kenya and Tanzania. The first symptoms noticed on palms 8 or more years old are a frond wilt and a crown rot, but these follow a primary bole rot. Highest mortality is among seedlings and young palms up to 8 years old. Where the disease occurs sporophores are sometimes common on exposed roots, dead seedlings and the soil surface where diseased palms have been dug out. The fungus appears to be a persistent colonizer of coconut debris in the soil. Mycelial cultures from infected bole tissues and from sporophores were highly pathogenic to seedlings, and slowly invaded older tissue. M. cocophilus reaches the inner bole tissues only through the roots, and wounding of roots of their aerenchymatous protuberances is important in infection. Seedlings may become infected through roots damaged during transplanting from nurseries to the field; movement of seedlings from affected areas is one certain method of dispersal. Suggested control measures are: (1) selection of seedlings in nurseries and subsequent transplanting should be as early as possible; (2) seedlings should be transplanted very carefully, and damaged roots pruned and disinfected; (3) seedlings should never be taken from affected areas; (4) periodic soil sterilization of nurseries; and (5) no cultivation should be done between palms, especially where disease is present.     

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.     

In vitro Antagonistic Interactions Between Endophytic Basidiomycetes of Oil Palm (Elaeis guineensis) and Ganoderma boninense

Ganoderma boninense is a white rot basidiomycete that causes basal stem rot disease of oil palm (Elaeis guineensis). The aims of this study were to identify endophytic basidiomycetes occurring naturally within oil palm and to assess their potential as biocontrol agents against G. boninense strain PER71 in vitro. In total, 376 isolates were recovered from samples collected from the root, stem and leaves of oil palm using Ganoderma‐selective medium. Ten of these isolates (2.7% of the total 376 isolates) were identified as basidiomycetes on the basis of clamp connections and the production of poroid basidiomes after incubation in glass jars containing PDA medium for 7–12 days. The isolates were identified using ITS rDNA sequencing as Neonothopanus nambi (five isolates), Schizophyllum commune (four isolates) and Ganoderma orbiforme (one isolate). The N. nambi isolates showed the greatest antagonistic activity against G. boninense, based on 73–85% inhibition of the radial growth measurements of G. boninense in dual culture and 76–100% inhibition of G. boninense growth in a culture filtrate assay. Possible modes of action for the antagonism shown by N. nambi against G. boninense in vitro include competition for substrate availability, space and the production of non‐volatile metabolites or antibiotics that inhibited the growth of G. boninense. Further in vivo investigations are required to determine the ability of N. nambi isolates to colonize oil palm seedlings and to protect oil palm from infection when challenged with G. boninense.     

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.     

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.     

Influence of microclimate on Drechslera leaf spot of young coconuts: effect of desiccation on spore survival and of moisture and shade on infection and disease development

Culture-produced conidia of Drechslera incurvata from coconut failed to germinate on the leaves of coconut seedlings incubating under the dry conditions of a greenhouse. Viability and rate of appressorium formation of artificially-dispersed, culture-produced conidia fell significantly during extended incubation of inoculated seedlings in the greenhouse, when 43% of conidia germinated after 90 days incubation compared with 62% at 59 days and 90% at 24 days. Field-produced conidia on excised leaves also lost viability upon storage in situ on the laboratory bench; germination fell from 60% at 3 months storage to 0×5% at 5 months and no germination at 6 months. Shading of seedlings in the field with saran cloth producing 30% shade or 50% shade depressed the amount of dew forming on leaves of young coconuts and significantly reduced both the number of infections from artificial inoculations and the severity of leaf spot disease developing subsequently.     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Characterization and Identification of an Epidemic Strain of Ascochyta rabiei on Chickpeas in Northwest China

The chickpea (Cicer arietinum L.) is the primary cultivated rain fed crop in the Xinjiang region of Northwest China. In 2007, chickpeas suffered an epidemic disease that caused significant economic losses. To identify the causative agent, debris and seeds of infected chickpea plants were collected from cultivated fields. We isolated and purified the responsible pathogen and tested its pathogenicity both in greenhouse and in the field. Morphological analysis using tissue culture as well as molecular techniques confirmed that the pathogen was the blight fungus Ascochyta rabiei.     

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.