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.     

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.     

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.     

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.     

Ganoderma – a basal stem rot disease of coconut palm in south Asia and Asia pacific regions

Basal stem rot (BSR) caused by the species of Ganoderma is one of the most devastating diseases of numerous perennial, coniferous and palmaceous hosts. In forest systems, Ganoderma has an ecological role in the breakdown or delignification of woody plants. Symptoms of BSR disease can take several years to develop, and the presence of the pathogen (such as indicated by fruiting bodies) is often only visible when the fungus is well established and more than half of the bole tissue has been decayed, leaving no chance for the grower to cure the infected palms. Soils with poor drainage and water stagnation during rainy seasons were found to favour the disease. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. Amendment of calcium nitrate in soil is one of the methods for the management of Ganoderma disease in palms. A combination of biological and systemic fungicides along with good cultural practices will effectively control the BSR disease in palms.     

Pathogenic Potential of Soil Fusaria from Malaysian Oil Palm Habitats

A total of 353 Fusarium isolates consisting of 8 species and 2 varieties obtained from the soils of 22 sampling sites in oil palm habitats in Peninsular Malaysia was tested on 2-leaf stage oil palm seedlings. None of the isolates tested produced any symptoms of vascular wilt or other diseases. However, some of the isolates did have an adverse effect on the growth and vigour of the palms. They were found to cause significant reduction in palm growth. Isolates causing a reduction in the growth of the oil palm seedlings were not confined to a certain type of soil or a particular location.     

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.     

Pathogenicity of Fusarium oxysporum Isolates from Malaysia and F. oxysporuin f. sp. elaeidis from Africa to Seedlings of Oil Palms (Elaeis guineensis)

Pathogenicity tests with Fusarium oxysporum isolated form Malaysian oil palm were made with oil palms seedlings raised form Malaysian seed as well s with wilt-susceptible seedlings gown from African seed. Oil palm seedlings grown form Malaysian seed were also inoculated with African isolates of F. oxysporum f. sp. elaeidis and F. oxysporum var. redolens. The experiments were made under normal soil moisture conditions and under water stress. F. oxysporum f. sp. elaeidis isolates form Africa were pathogenic to oil palm seedlings from Malaysian seeds but the Malaysian F oxysporum isolates were non-pathogenic to plams grown from Malaysian seed or the wilt-susceptible palms from African seed. Seedlings from Malaysian seed proved to be highly susceptible to the vascular wilt disease caused by F. oxysporum f. sp. elaeidis as 75–90% of the palms were infected. The susceptibility of the palms from Malaysian seed varied with different African isolates tested. The Yaligimba isolate from Zaire which was found to be F. oxysporum var. redolens was the most virulent. Disease was more severe when oil palm seedlings were subjected to a period of water stress. The incidence of death in the seedlings under stress conditions was 45% as compared with only 15% for palms grown under normal conditions.     

Growth of oil palm (Elaeis guineensis) seedlings on acid sulfate soils as affected by water regime and aluminium

In Malaysia, acid sulfate soils contain high amounts of aluminium and are usually utilized for oil palm cultivation. As these soils are frequently flooded during rainy periods, it is thought that this may affect the growth performance of the oil palm. A glasshouse experiment was, therefore, conducted to study the effects of water regime and aluminium on the growth of oil palm seedlings. Soils used in the experiment were Typic Sulfaquepts and Sulfic Tropaquepts from Pulau Lumut Island, Malaysia. Best growth was observed on a non-jarositic freely drained topsoil. Oil palm seedlings were found the be moderately tolerant to soil acidity. Growth was only affected if Al³⁺ and Al_sum activities in the soil solutions were above 100 and 700 M, respectively. Root length was found to be one of the better parameters to predict crop growth, while others included plant height, fron length and LAI. Soil solution attributes which could be used as indices of soil acidity for oil palm growth were pH and activities of Al³⁺, AlSO₄ ⁺ and Al_sum.     

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.     

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.     

Quantification and characterisation of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. from different ecosystems

Basal stem rot of oil palm caused by Ganoderma boninense is of major economic importance. Observations of the low incidence of disease due to Ganoderma species in natural stands, suggest that the disease is kept under control by some biological means. Trichoderma spp. are saprophytic fungi with high antagonistic activities against soil-borne pathogens. However, their abundance and distribution are soil and crop specific. Trichoderma species have been found to be concentrated in the A1 (0–30 cm) and Be soil horizons (30–60 cm), although the abundance of Trichoderma was not significantly different between the oil palm and non-oil palm ecosystems. Characterisation of Trichoderma isolates based on cultural, morphological and DNA polymorphism showed that T. harzianum, T. virens, T. koningii and T. longibrachiatum made up 72, 14, 10 and 4% of the total Trichoderma isolates isolated. As Trichoderma species are present in the oil palm ecosystem, but at lower numbers and in locations different from those desired, soil augmentation with antagonistic Trichoderma spp. can be developed as a strategy towards integrated management of basal stem rot of oil palm.     

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.     

Stem rots of oil palm caused by <Emphasis Type="Italic">Ganoderma boninense</Emphasis>: Pathogen biology and epidemiology

Oil palm (Elaeis guineensis Jacq.) has been grown in Papua New Guinea since the early 1960s. The most important disease of oil palm in PNG is a stem rot of the palm base. This is the same disease that constitutes a major threat to sustainable oil palm production in SE Asia. Investigations into the causal pathogen have revealed that the stem rots in PNG are caused predominantly by the basidiomycete Ganoderma boninense, with a minor pathogen identified as G. tornatum G. tornatum was found to have a broad host range whereas G. boninense appears to be restricted to palms. The population structure of G. boninense was investigated using inter-fertility studies between isolates collected from basal stem rots on oil palm. Although the G. boninense field populations are predominantly comprised of distinct individuals, a number of isolates were found that share single mating alleles. This indicates that out-crossing had occurred over several generations in the resident or wild population of G. boninense prior to colonization of oil palm. No direct hereditary relationship between isolates on neighbouring diseased palms was found, although an indirect link between isolates causing upper stem rot and basal stem rot was detected.     

Nematodes associated with date palm and their control measures

Date palm, Phoenix dactylifera L., is dioecious and can be artificially pollinated by man, and one-third of all the dates of the world are grown in Iraq. In Egypt, there are about 12?million date palm trees grown in 99,867?feddans (fed.?=?4200?m²). Productivity is 1352,954?million?tons with yield 111.7?kg/tree. Plant parasitic nematodes associated with date palm are Criconemoides spp., Helicotylenchus spp., Hemicriconemoides spp., Hemicycliophora spp., Hoplolaimus spp., Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica, Pratylenchus brachyurus, Pratylenchus jordanensis, Pratylenchus coffeae, Pratylenchus neglectus, Pratylenchus thornei, Trichodorus spp., Tylenchorhynchus goffarti, Tylenchorhynchus latus and Xiphinema spp.; Meloidogyne incognita-infected roots of susceptible cultivar favoured giant cell and galls formation. Date palm roots infected with Pratylenchus penetrans showed puncture of epidermal cells and disarrangement of cortical cells with large empty abnormal cavities. As control measures, it is advised to; 1 – plant immune or resistant cultivars against pathogenic nematodes, 2 – use oil cakes or poultry manure as organic amendments and a nematicide, carbofuran.These were tested and found effective in the control of Helicotylenchus multicinctus and P. penetrans, 3 – treat nematode-infested date palm seedlings with hot water at a suitable temperature for a given period before transplanting to open field, 4 – plant nematode -free date palm seedlings, 5 – soil solarisation and tillage before planting, 6 – weed control, 7 – intercrop with nematode-resistant horticultural crops and 8 – induce resistance in susceptible date palm cultivars against root knot nematode.     

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.     

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.     

Parrots consume sodium‐rich palms in the sodium‐deprived landscape of the Western Amazon Basin

Herbivorous animals face shortages of different minerals in different geographic areas. In the Amazon Basin, sodium is often limiting, driving herbivores to seek supplemental sources. In the lowlands of the western Amazon Basin, parrots commonly consume sodium‐rich soils at clay licks but lick use varies widely among species, and to date, parrots in the region have not been reported consuming other supplemental sodium sources. We document 11 species of psittacines consuming sodium‐rich leaves and trunks of Attalea butyracea palms growing on sodium‐rich soils in lowland Peru. Consumed palms had more sodium and less potassium than uneaten A. butyracea palms and other palm species in the area. Among A. butyracea palm parts, sodium and Na:K ratios were highest in trunks (consumed by parrots in 94% of the 387 foraging bouts recorded) and lowest in leaves (consumed in only 14% of foraging bouts). The low potassium and high Na:K ratio suggest that birds may be seeking not just any sodium sources, but those low in potassium, as potassium is known to exacerbate dietary sodium shortages. Use of the palms and species’ abundance in the study area were not correlated. Instead, parrot species that consumed palms the most were those that use relatively few traditional soil clay licks. This finding suggests that parrot species in the region have fundamental differences in preferred strategies for obtaining supplemental sodium and may help explain documented interspecific differences in geophagy.