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.     

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.     

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.     

Biological conversion of olive pomace into compost by using Trichoderma harzianum and Phanerochaete chrysosporium

Olive pomace was composted by using a reactor for a period of 50 days in four bioreactors. Urea was added to adjust C/N ration between 25–30. At the end of 50 days of composting using Trichoderma harzianum and Phanerochaete chrysosporium, cellulose and lignin were highly degraded. It was found that after 30 days, P. chrysosporium and T. harzianum degraded approximately 71.9% of the lignin and 59.25% of the cellulose, respectively. The percent of ash content in the raw waste mixture was 13%. This percentage increased from 13% to 18.55% in treatment bioreactors and from 13% to 13.55% in control reactors during 50 day of composting process. The amount of CO₂ produced by the treated sample was 3 mg of CO₂/g organic carbon which is indicated that the treated sample was considered as stable compost. The results proved that the use of accelerating agents was found to be efficient in producing mature stable with nearly non-phytotoxicity compared to control sample in less than 50 days.     

Comparative study on the delignification of Azadirachta indica (L) Del., wood by Chrysosporium asperatum and Trichoderma harzianum

Structural alterations induced in response to degradation by two white rot Basidiomycetes on the secondary xylem of Azadirachta indica (L) Del., was compared. In vitro decay test was employed to investigate the pattern of delignification of Azadirachta wood by Trichoderma harzianum and Chrysosporium asperatum. Wood samples inoculated with both the strains were analyzed for different periods viz. 30, 60, 90 and 120 days after fungal inoculation. Initially there was no appreciable percent weight loss of the wood blocks but later on (after 60 days) it increased rapidly and was found similar for both the strains (43-46% of wood mass). Samples inoculated with both the strains showed dual pattern of degradation i.e. selective delignification in the initial stage followed by simultaneous rot during advance stage of decay. Separation of the cells due to dissolution of middle lamella was the characteristic feature of both strains but in the advanced stage of decay, formation of erosion troughs were conspicuous in all the cell types. Other features such as cell wall thinning, rounded pit erosion, formation of erosion channels and bore holes were also observed frequently. Initially, fungal invasion started through the vessel lumen, followed by all the cell types of the xylem. From the vessels, mycelia entered into the adjacent rays and parenchyma cells through the pits. In advanced stage, degradation was so pronounced that rays were partially or even completely destroyed while many cells including vessels were either deformed or destroyed due to loss of rigidity of their walls. Structural alterations induced in response to C. asperatum and T. harzianum attack is described in details.     

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.     

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.     

Expression of the HSP24 gene from Trichoderma harzianum in Saccharomyces cerevisiae

Hsp24 is a small heat-shock protein (sHSP). Such proteins are important endogenous cytoprotection factors involved in defense. A 1116-bp full-length cDNA of the Hsp24, with a 645-bp open reading frame nucleotide encoding a 24-kDa polypeptide consisting of 214 amino acid residues, was isolated from Trichoderma harzianum. Sequence analysis revealed that Hsp24 gene has more than 42–58% amino acid sequence homology with those of other fungi. The Hsp24 gene was integrated into pYES2 by inserting into a single site for recombination, yielding the recombinant of pYES2/Hsp24. Hsp24 expressed by pYES2/Hsp24 was induced by galactose. We tested whether Hsp24 could confer abiotic stress resistance when it was introduced into yeast cells. A transgenic yeast harboring T. harzianum Hsp24 was generated under the control of a constitutively expressed GAL promoter. The results indicated that Hsp24 yeast transformants had significantly higher resistance to salt, drought and heat stresses.     

Regeneration and molecular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion

The fungal strains Graphium putredinis and Trichoderma harzianum were selected as parents for fusant development. Protoplasts were isolated using the combination of lysing enzymes Novozym 234 and cellulase with 0.6 M KCl as osmotic stabilizer. The optimum conditions for release of viable protoplasts from the fungal mycelium viz. age of the mycelium, lytic enzymes, osmotic stabilizers, pH, incubation period and regeneration medium were determined. Intergeneric protoplast fusion was carried out using 50% polyethylene glycol with calcium chloride (CaCl₂) and glycine buffer and the conditions for effective protoplast fusion, viz. fusogen, osmotic stabilizer, pH, incubation period and regeneration medium were optimized. At optimum conditions, the regeneration frequency of the fused protoplasts on potato dextrose agar (PDA) medium and fusion frequency were calculated. The regeneration frequency on non-selective (PDA) and selective media (PDA amended with starch) was determined for the parental and fusant strains in which, fusant showed a higher rate of regeneration. Fusant formation was confirmed by morphological markers (colony morphology and spore size and shape) and genetical markers like, mycelial protein pattern, restriction digestion pattern and random amplified polymorphic DNA (RAPD) analysis. The efficiency of these parental strains and their intergeneric fusant in the production of hydrolytic enzymes – amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) – have probable applications in various industrial processes.     

Characterization of <Emphasis Type="Italic">Streptomyces</Emphasis> spp. isolated from the rhizosphere of oil palm and evaluation of their ability to suppress basal stem rot disease in oil palm seedlings when applied as powder formulations in a glasshouse trial

Ganoderma boninense, the main causal agent of oil palm (Elaeis guineensis) basal stem rot (BSR), severely reduces oil palm yields around the world. To reduce reliance on fungicide applications to control BSR, we are investigating the efficacy of alternative control methods, such as the application of biological control agents. In this study, we used four Streptomyces-like actinomycetes (isolates AGA43, AGA48, AGA347 and AGA506) that had been isolated from the oil palm rhizosphere and screened for antagonism towards G. boninense in a previous study. The aim of this study was to characterize these four isolates and then to assess their ability to suppress BSR in oil palm seedlings when applied individually to the soil in a vermiculite powder formulation. Analysis of partial 16S rRNA gene sequences (512 bp) revealed that the isolates exhibited a very high level of sequence similarity (>?98%) with GenBank reference sequences. Isolates AGA347 and AGA506 showed 99% similarity with Streptomyces hygroscopicus subsp. hygroscopicus and Streptomyces ahygroscopicus, respectively. Isolates AGA43 and AGA48 also belonged to the Streptomyces genus. The most effective formulation, AGA347, reduced BSR in seedlings by 73.1%. Formulations using the known antifungal producer Streptomyces noursei, AGA043, AGA048 or AGA506 reduced BSR by 47.4, 30.1, 54.8 and 44.1%, respectively. This glasshouse trial indicates that these Streptomyces spp. show promise as potential biological control agents against Ganoderma in oil palm. Further investigations are needed to determine the mechanism of antagonism and to increase the shelf life of Streptomyces formulations.     

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.     

Lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of nasopharyngeal carcinoma cell line C666-1 in vitro

Latent membrane protein 2A (LMP2A) is found to play a key role in the development of nasopharyngeal carcinoma (NPC). However, the role of LMP2A silencing in the inhibition of cell growth of NPC has not been clarified. In this study, we inhibited LMP2A gene expression by lentivirus-mediated RNAi, to explore the effects of LMP2A silencing on the growth of NPC cell line in vitro. A lentivirus-mediated RNAi technology was employed to specifically knock down the LMP2A gene in NPC cell line C666-1. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and colony formation assays were performed to evaluate the expression of LMP2A and biological behavior of cell line C666-1 in vitro. We successfully construct a highly efficient and stable lentivirus vector, which efficiently downregulate the expression of LMP2A gene in infected cell line C666-1. Down-regulation of the expression of LMP2A significantly inhibits the proliferation and colony formation of C666-1 cells. In addition, the specific down-regulation of LMP2A arrests cells in G0/G1 phase of cell cycle and increases apoptosis rate. Our findings suggest that lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of NPC cell line C666-1 in vitro, and LMP2A may be a potential target for gene therapy in treatment of NPC.