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.     

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.     

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.     

Assessment of plant secondary metabolites in oil palm seedlings after being treated with calcium,copper ions and salicylic acid

The effect of calcium, copper ions and salicylic acid (SA) amendment on the incidence of basal stem rot and activity of secondary metabolites in oil palm seedlings were investigated in glasshouse study. Disease incidence (DI) in positive control (T8) was 75% at nine months after inoculation (9 MAI). However, weekly pre-immunisation with Ca^2+?+^?Cu^2+?+^?SA prior to inoculation significantly suppressed DI and delayed disease onset as noted in T7. In the present study, the lowest %DI was observed in T7 (15%) followed by T1, T5, T6, T3, T4 and T2. The Ca²⁺, Cu²⁺ and SA amendments were resulted in earlier and higher accumulation of plant secondary metabolites as noted in leaves, stems and root tissues in response to invasion by Ganoderma boninense. High total phenolic content concentration was detected in T7 (leaf: 233.38 ± 0.12 mg/g; stem: 132.78 ± 0.04 mg/g and root: 86.98 ± 0.28 mg/g). Similar trend was obtained in peroxidase activity, total lignin content and hydrogen peroxide scavenging activity. These results suggested that it could be due to the accumulation of phenolics, peroxidase activities, lignin content and hydrogen peroxide scavenging activities in oil palm seedling tissues which might have collectively contributed to induce resistance against G. boninense.     

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.     

Role of phenolic acids and enzymes of phenylpropanoid pathway in resistance of chayote fruit (Sechium edule) against infestation by melon fly,Bactrocera cucurbitae

Melon fly is a serious pest of cucurbits all over the world causing huge losses to yield. However, the only exception is the chayote fruit (Sechium edule) that shows resistance to melon fly infestation. Studies on culture of melon fly indicated the absence of plant traits resisting oviposition on chayote fruit. However, the melon fly was unable to complete its life cycle successfully on chayote showing that factors inhibiting larval development in melon fly could be attributed to biochemical constituents. Studies were, therefore, carried out to compare the biochemical responses of chayote, a melon fly resistant species and bitter gourd, a susceptible species to melon fly infestation with regard to the levels of phenolic acids and activities of the enzymes of phenylpropanoid pathway (PPP) leading to synthesis of lignin. The resistant chayote exhibited significantly higher accumulation of lignin associated with higher activities of phenylalanine ammonia‐lyase (PAL), tyrosine ammonia‐lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD). On the contrary, the susceptible bitter gourd recorded lower activities of PAL, CAD and POD and a decreasing trend of TAL during infestation associated with a lower lignin content. The monomer composition of lignin in the resistant chayote showed twofold higher level of guaiacyl (G) and syringyl (S) units compared to susceptible bitter gourd and the G/S ratio during infestation increased in chayote while decreasing in bitter gourd. The levels of PPP intermediates, p‐coumaric acid was higher in chayote while p‐hydroxy benzoic acid, a chemo‐attractant, was higher in bitter gourd. Incorporation of p‐coumaric acid in the larval diet strongly inhibited larval growth even as p‐hydroxy benzoic acid promoted growth confirming the direct role of p‐coumaric acid in conferring resistance to chayote. The level of salicylic acid, a signal molecule involved in induction of defence response, was higher in chayote compared to bitter gourd. Chayote also exhibited higher level of activity of POD in the phloem exudates compared to bitter gourd. The higher concentration of sugars in exudates of chayote might act like signalling molecules causing activation of plant genes, especially of the phenylpropanoid biosynthesis pathway and possibly produce an osmotic effect inducing resistance against the melon fly. Thus, the study revealed that the resistance in chayote to melon fly infestation is a complex, multi‐layered process in which the activities of PPP enzymes generating phenolic intermediates leading to lignin biosynthesis and the composition of exudates appear to play significant roles. Besides, the study also indicated that different forms of lignin might play a role in the resistance of chayote against melon fly infestation.     

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.     

An <Emphasis Type="Italic">in vitro</Emphasis> study of the antifungal activity of <Emphasis Type="Italic">Trichoderma virens</Emphasis> 7b and a profile of its non-polar antifungal components released against <Emphasis Type="Italic">Ganoderma boninense</Emphasis>

Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate^® indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense.     

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.     

<Emphasis Type="Italic">Streptomyces sanglieri</Emphasis> which colonised and enhanced the growth of <Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq. seedlings was antagonistic to <Emphasis Type="Italic">Ganoderma boninense</Emphasis> in in vitro studies

Actinomycete strain AUM 00500 was 99.5 % similar to Streptomyces sanglieri NBRC 100784^T and was evaluated for antagonistic activity towards Ganoderma boninense, the causative fungus of basal stem rot of oil palm. The strain showed strong antifungal activity towards G. boninense in in vitro and SEM analysis showed various modes of inhibition of the fungus. Ethyl acetate extracts of single culture and inhibition zone of cross-plug culture by HPLC indicated that strain AUM 00500 produced two different antibiotics of the glutarimide group namely cycloheximide and actiphenol. In greenhouse trials, oil palm seed treated with spores of S. sanglieri strain AUM 00500 at 10⁹ cfu/ml showed significant (P < 0.05) increase in oil palm seedlings growth when compared to the control. Streptomyces sanglieri strain AUM 00500 successfully colonised the epidermal surface of the roots of treated oil palm seedlings and it was recovered from root fragments plated on starch casein agar.     

Current strategies and perspectives in detection and control of basal stem rot of oil palm

The rapid expansion of oil palm (OP) has led to its emergence as a commodity of strategic global importance. Palm oil is used extensively in food and as a precursor for biodiesel. The oil generates export earnings and bolsters the economy of many countries, particularly Indonesia and Malaysia. However, oil palms are prone to basal stem rot (BSR) caused by Ganoderma boninense which is the most threatening disease of OP. The current control measures for BSR management including cultural practices, mechanical and chemical treatment have not proved satisfactory. Alternative control measures to overcome the G. boninense problem are focused on the use of biological control agents and many potential bioagents were identified with little proven practical application. Planting OP varieties resistant to G. boninense could provide the ideal long-term solution to basal stem rot. The total resistance of palms to G. boninense has not yet been reported, and few examples of partial resistances have been observed. Importantly, basidiospores are now recognized as the method by which the disease is spread, and control methods require to be revaluated because of this phenomenon. Many methods developed to prevent the spread of the disease effectively are only tested at nursery levels and are only reported in national journals inhibiting the development of useful techniques globally. The initial procedures employed by the fungus to infect the OP require consideration in terms of the physiology of the growth of the fungus and its possible control. This review assesses critically the progress that has been made in BSR development and management in OP.     

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.     

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.     

Overexpression of cinnamyl alcohol dehydrogenase gene from sweetpotato enhances oxidative stress tolerance in transgenic Arabidopsis

Cinnamyl alcohol dehydrogenase (CAD) is the enzyme in the last step of lignin biosynthetic pathway and is involved in the generation of lignin monomers. IbCAD1 gene in sweetpotato (Ipomoea batatas) was identified, and its expression was induced by abiotic stresses based on promoter analysis. In this study, transgenic Arabidopsis plants overexpressing IbCAD1 directed by CaMV 35S promoter were developed to determine the physiological function of IbCAD1. IbCAD1-overexpressing transgenic plants exhibited better plant growth and higher biomass compared to wild type (WT), under normal growth conditions. CAD activity was increased in leaves and roots of transgenic plants. Sinapyl alcohol dehydrogenase activity was induced to a high level in roots, which suggests that IbCAD1 may regulate biosynthesis of syringyl-type (S) lignin. Lignin content was increased in stems and roots of transgenic plants; this increase was in S lignin rather than guaiacyl (G) lignin. Overexpression of IbCAD1 in Arabidopsis resulted in enhanced seed germination rates and tolerance to reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Taken together, our results show that IbCAD1 controls lignin content by biosynthesizing S units and plays an important role in plant responses to oxidative stress.

     

Expression Analysis of Fatty Acid Biosynthetic Pathway Genes during Interactions of Oil Palm (Elaeis guineensis Jacq.) with the Pathogenic Ganoderma boninense and Symbiotic Trichoderma harzianum Fungal Organisms

The fatty acid (FA) signaling pathway is emerging as an important mechanism in plant responses during interactions with microbial organisms. For a comprehensive evaluation of key FA biosynthetic pathway genes during interactions of oil palm (Elaeis guineensis Jacq.) with the pathogenic Ganoderma boninense and symbiotic Trichoderma harzianum fungal organisms, a lane-based array analysis of gene expression in artificially inoculated oil palm seedlings was performed. The results obtained demonstrated that acetyl-CoA carboxylase (ACC), β-ketoacyl-ACP synthases (KAS) II and III, palmitoyl-ACP thioesterase (PTE), oleoyl-ACP thioesterase (OTE) and glycerol-3-phosphate acyltransferase (ACT) showed identical responses in root and leaf tissues for the same fungi. The expression of these genes was up-regulated in both root and leaf tissues at 21 days post-inoculation (dpi) during interaction of oil palm with G. boninense. Thereafter, production of physical symptoms occurred at 42 and 63 dpi concomitantly with suppression of expression of these genes. An increase in the expression level of these genes was observed in both tissues at 3–63 dpi, which correlated with the colonization of roots and promotion of plant growth by T. harzianum. These data suggest that FA biosynthetic pathway genes are involved in the defense response of oil palm to infection. Identical plant responses by FA biosynthetic pathway genes may lead to enhanced resistance against G. boninense and could be a useful marker to contribute towards early detection of infection. The distinct expression profile during symbiotic interaction demonstrated its role in plant resistance mechanisms and growth promotion by T. harzianum.