Defense response changes in roots of oil palm (Elaeis guineensis Jacq.) seedlings after internal symptoms of Ganoderma boninense Pat. infection.

BACKGROUND: The development of basal stem rot (BSR) disease in oil palm is associated with lignin during vegetative growth and salicylic acid (SA) biosynthesis. The increase in the lignin content, SA accumulation, growth, and root biomass could indicate the resistance of oil palm seedlings to BSR disease. Therefore, although there are many studies on the interactions between the Ganoderma boninense and oil palm, research on evaluation of physiological processes, biochemistry, and molecules occurring during early internal symptoms of BSR in roots of oil palm (Elaeis guineensis Jacq.) are essential. RESULTS: Ganoderma boninense inoculation indicated that C01, C02, and C05 seedlings were susceptible, while the other three seedlings, C03, C07, and C08, were resistant based on Ganoderma Disease Index (GDI). Infection by G. boninense in the most susceptible seedlings C05 reduced fresh weight of roots (FW) by 9.0%, and lignin content by 10.9%. The most resistant seedlings C08 were reduced by only 8.4%, and 0.2% regarding their fresh weight and lignin content, respectively. BSR disease induced SA accumulation in the most susceptible C08 and decreased peroxidase (PRX) enzyme (EC 1.11.1.7) activities in root tissues of oil palm seedlings except C07 and C08 where PRX activities remained high in the 4 months after planting. Infection with G. boninense also increased glutathione S-transferase U19-like (EgGSTU19) gene expression in the root tissues of susceptible seedlings, while laccase-24 (EgLCC24) gene expression was associated with resistance against BSR disease. Based on the relative expression of twelve genes, two genes are categorized as receptors (EgWAKL5, EgMIK1), two genes as biosynthesis signal transduction compound (EgOPR5, EgACO1), five genes as defense responses (EgROMT, EgSOT12, EgLCC24, EgGLT3, EgGSTU19), and one gene as trans-resveratrol di-O-methyltransferase-like (EgRNaseIII) predicted related to BSR infection. While two other genes remain unknown (EgUnk1, EgUnk2). CONCLUSIONS: Ganoderma infection-induced SA accumulation and lignification in resistant accessions promote the seedlings root biomass. Oil palm seedlings have a synergistic physical, biochemical, and molecular defense mechanism to the BSR disease. The utilization of nucleotide-based molecular markers using EgLCC24 gene is able to detect resistant oil palm seedlings to G. boninense.

Antifungal Peptides from a Burkholderia Strain Suppress Basal Stem Rot Disease of Oil Palm.

Basal stem rot (BSR) is the most common disease of oil palm (Elaeis guineensis) in Southeast Asia. BSR is caused by a white-rot fungus Ganoderma boninense. The disease is difficult to manage. Therefore, development of novel and environmentally safe approaches to control the disease is important. Species of Burkholderia are known to have diverse lifestyles, some of which can benefit plants by suppressing diseases or increasing plant growth. In the present study, antifungal peptides produced by a bacterial strain isolated from the rhizosphere of an oil palm tree, Burkholderia sp. strain CP01, exhibited strong growth inhibition on G. boninense. A loss-of-function mutant of CP01 was generated, and it has enabled the identification of a 1.2-kDa peptide and its variants as the active antifungal compounds. High-resolution mass spectrometry revealed six analogous compounds with monoisotopic masses similar to the previously reported cyclic lipopeptides occidiofungin and burkholdine. The antifungal compounds of CP01 were secreted into media, and we sought to use CP01 culture extract without living cells to control BSR disease. Glasshouse experiments showed that CP01 culture extract suppressed BSR disease in oil palm seedlings. The ability of CP01 to produce an antifungal substance and suppress plant disease suggests its potential applications as a biofungicide in agriculture.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum through protoplast fusion.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 107/mL in G. lingzhi and 5.57 ± 0.49 × 106/mL in G. applanatum. Osmotic stabilizer NaCl (0.4 M) at pH 5.8 and enzymolysis time 3.5 h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, ß and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.

EgJUB1 and EgERF113 transcription factors as potential master regulators of defense response in Elaeis guineensis against the hemibiotrophic Ganoderma boninense.

BACKGROUND: Hemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection. RESULTS: The present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during the fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells. CONCLUSION: Our findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection.

Biophysical characterization of antibacterial compounds derived from pathogenic fungi Ganoderma boninense.

There have been relatively few studies which support a link between Ganoderma boninense, a phytopathogenic fungus that is particularly cytotoxic and pathogenic to plant tissues and roots, and antimicrobial compounds. We previously observed that liquid-liquid extraction (LLE) using chloroformmethanol-water at a ratio (1:1:1) was superior at detecting antibacterial activities and significant quantities of antibacterial compounds. Herein, we demonstrate that antibacterial secondary metabolites are produced from G. boninense mycelia. Antibacterial compounds were monitored in concurrent biochemical and biophysical experiments. The combined methods included high performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. The antibacterial compounds derived from mycelia with chloroform-methanol extraction through LLE were isolated via a gradient solvent elution system using HPTLC. The antibacterial activity of the isolated compounds was observed to be the most potent against Staphylococcus aureus ATCC 25923 and multidrug-resistant S. aureus NCTC 11939. GC-MS, HPLC, and FTIR analysis confirmed two antibacterial compounds, which were identified as 4,4,14α-trimethylcholestane (m/z = 414.75; lanostane, C30H54) and ergosta-5,7,22-trien-3ß-ol (m/z = 396.65; ergosterol, C28H44O). With the aid of spectroscopic evaluations, ganoboninketal (m/z = 498.66, C30H42O6), which belongs to the 3,4-seco-27-norlanostane triterpene family, was additionally characterized by 2D-NMR analysis. Despite the lack of antibacterial potential exhibited by lanostane; both ergosterol and ganoboninketal displayed significant antibacterial activities against bacterial pathogens. Results provide evidence for the existence of bioactive compounds in the mycelia of the relatively unexplored phytopathogenic G. boninense, together with a robust method for estimating the corresponding potent antibacterial secondary metabolites.

Chitosan-Based Agronanofungicides as a Sustainable Alternative in the Basal Stem Rot Disease Management.

The rise of environmental and health concerns due to the excessive use of the conventional fungicide urges the search for sustainable alternatives of agronanofungicides where the latter is aimed to enhance plant uptake and minimize the volatilization, leaching, and runoff of fungicides. With this in mind, fungicides of hexaconazole and/or dazomet were encapsulated into chitosan nanoparticles for the formulation of chitosan-based agronanofungicides. In the present study, chitosan nanoparticles (2 nm), chitosan-hexaconazole nanoparticles (18 and 168 nm), chitosan-dazomet nanoparticles (7 and 32 nm), and chitosan-hexaconazole-dazomet nanoparticles (5 and 58 nm) were synthesized and used as potent antifungal agents in combating the basal stem rot (BSR) disease caused by Ganoderma boninense in which they were evaluated via an artificial inoculation of oil palm seedlings with the rubber woodblock, which was fully colonized with the fungal Ganoderma boninense mycelium. The results revealed that chitosan nanoparticles could act as dual modes of action, which are themselves as a biocide or as a nanocarrier for the existing fungicides. In addition, the particle size of the chitosan-based agronanofungicides plays a crucial role in suppressing and controlling the disease. The synergistic effect of the double-fungicide system of 5 nm chitosan-hexaconazole-dazomet nanoparticles can be observed as the system showed the highest disease reduction with 74.5%, compared to the untreated infected seedlings.

Classification, Biological Characteristics and Cultivations of Ganoderma.

Species of Ganoderma (Ling-zhi) have been widely researched and cultivated due to their highly prized medicinal value, which is famous as a traditional Chinese medicine. The aims of this chapter are to (1) review the historical taxonomy of the family Ganodermataceae, (2) provide an account of the genera and species of Ganoderma together with the distributions and habitats, (3) evaluate morphological features and phylogenetic methods to define the genera and species and (4) present two commonly used cultivated methods (wood-log cultivation and substitute cultivation) for Ganoderma.

Analysis of Changes in Oil Palm Canopy Architecture From Basal Stem Rot Using Terrestrial Laser Scanner.

Basal stem rot (BSR), caused by the Ganoderma fungus, is an infectious disease that affects oil palm (Elaeis guineensis) plantations. BSR leads to a significant economic loss and reductions in yields of up to Malaysian Ringgit (RM) 1.5 billion (US$400 million) yearly. By 2020, the disease may affect ∼1.7 million tonnes of fresh fruit bunches. The plants appear symptomless in the early stages of infection, although most plants die after they are infected. Thus, early, accurate, and nondestructive disease detection is crucial to control the impact of the disease on yields. Terrestrial laser scanning (TLS) is an active remote-sensing, noncontact, cost-effective, precise, and user-friendly method. Through high-resolution scanning of a tree's dimension and morphology, TLS offers an accurate indicator for health and development. This study proposes an efficient image processing technique using point clouds obtained from TLS ground input data. A total of 40 samples (10 samples for each severity level) of oil palm trees were collected from 9-year-old trees using a ground-based laser scanner. Each tree was scanned four times at a distance of 1.5 m. The recorded laser scans were synched and merged to create a cluster of point clouds. An overhead two-dimensional image of the oil palm tree canopy was used to analyze three canopy architectures in terms of the number of pixels inside the crown (crown pixel), the degree of angle between fronds (frond angle), and the number of fronds (frond number). The results show that the crown pixel, frond angle, and frond number are significantly related and that the BSR severity levels are highly correlated (R2 = 0.76, P < 0.0001; R2 = 0.96, P < 0.0001; and R2 = 0.97, P < 0.0001, respectively). Analysis of variance followed post hoc tests by Student-Newman-Keuls (Newman-Keuls) and Dunnett for frond number presented the best results and showed that all levels were significantly different at a 5% significance level. Therefore, the earliest stage that a Ganoderma infection could be detected was mildly infected (T1). For frond angle, all post hoc tests showed consistent results, and all levels were significantly separated except for T0 and T1. By using the crown pixel parameter, healthy trees (T0) were separated from unhealthy trees (moderate infection [T2] and severe infection [T3]), although there was still some overlap with T1. Thus, Ganoderma infection could be detected as early as the T2 level by using the crown pixel and the frond angle parameters. It is hard to differentiate between T0 and T1, because during mild infection, the symptoms are highly similar. Meanwhile, T2 and T3 were placed in the same group, because they showed the same trend. This study demonstrates that the TLS is useful for detecting low-level infection as early as T1 (mild severity). TLS proved beneficial in managing oil palm plantation disease.

Cultural characterization and chlamydospore function of the Ganodermataceae present in the eastern United States.

The cultural characteristics of fungi can provide useful information for studying the biology and ecology of a group of closely related species, but these features are often overlooked in the order Polyporales. Optimal temperature and growth rate data can also be of utility for strain selection of cultivated fungi such as reishi (i.e., laccate Ganoderma species) and potential novel management tactics (e.g., solarization) for butt rot diseases caused by Ganoderma species. Historically, the taxonomy of the laccate (shiny) Ganoderma species has been unresolved and many species have been treated together as G. lucidum. The cultural characteristics of Ganoderma species from the United States are needed to understand the biology of these unique species that have all been lumped under this name. Culture morphology, average growth rate, optimal temperatures, and resiliency to elevated temperature exposure were characterized for isolates of Ganodermataceae taxa from the eastern United States, including Ganoderma curtisii, G. martinicense, G. meredithiae, G. ravenelii, G. sessile, G. tsugae, G. tuberculosum, G. cf. weberianum, G. zonatum, and Tomophagus colossus. We documented differences in linear growth rates and optimal temperatures between taxa. Isolates of G. sessile and T. colossus grew the fastest, and isolates of G. meredithiae, G. ravenelii, and G. tsugae grew the slowest. Isolates of G. sessile, G. martinicense, G. cf. weberianum, and T. colossus constitutively produced chlamydospores on malt extract agar, and these species were the only species to survive long-term exposure (30 or 40 d) to 40 C. We hypothesize that chlamydospores function as survival structures that serve as propagules resilient to adverse temperature conditions, especially heat. Cultural characteristics of G. martinicense, G. ravenelii, G. tuberculosum, and G. cf. weberianum collected from the United States are described for the first time.

Transciptome profiling at early infection of Elaeis guineensis by Ganoderma boninense provides novel insights on fungal transition from biotrophic to necrotrophic phase.

BACKGROUND: Basal stem rot (BSR) caused by hemibiotroph Ganoderma boninense is a devastating disease resulting in a major loss to the oil palm industry. Since there is no physical symptom in oil palm at the early stage of G. boninense infection, characterisation of molecular defense responses in oil palm during early interaction with the fungus is of the utmost importance. Oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense inoculums and root samples were obtained following a time-course of 0, 3, 7, and 11 days-post-inoculation (d.p.i) for RNA sequencing (RNA-seq) and identification of differentially expressed genes (DEGs). RESULTS: The host counter-attack was evidenced based on fungal hyphae and Ganoderma DNA observed at 3 d.p.i which became significantly reduced at 7 and 11 d.p.i. DEGs revealed upregulation of multifaceted defense related genes such as PR-protein (EgPR-1), protease inhibitor (EgBGIA), PRR protein (EgLYK3) chitinase (EgCht) and expansin (EgEXPB18) at 3 d.p.i and 7 d.p.i which dropped at 11 d.p.i. Later stage involved highly expressed transcription factors EgERF113 and EgMYC2 as potential regulators of necrotrophic defense at 11 d.p.i. The reactive oxygen species (ROS) elicitor: peroxidase (EgPER) and NADPH oxidase (EgRBOH) were upregulated and maintained throughout the treatment period. Growth and nutrient distribution were probably compromised through suppression of auxin signalling and iron uptake genes. CONCLUSIONS: Based on the analysis of oil palm gene expression, it was deduced that the biotrophic phase of Ganoderma had possibly occurred at the early phase (3 until 7 d.p.i) before being challenged by the fungus via switching its lifestyle into the necrotrophic phase at later stage (11 d.p.i) and finally succumbed the host. Together, the findings suggest the dynamic defense process in oil palm and potential candidates that can serve as phase-specific biomarkers at the early stages of oil palm-G. boninense interaction.

Morphological and transcript changes in the biosynthesis of lignin in oil palm (Elaeis guineensis) during Ganoderma boninense infections in vitro.

Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4-hydroxylase (EC 1.14.13.11), caffeic acid O-methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ-hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.

Transcriptional profiling provides new insights into the role of nitric oxide in enhancing Ganoderma oregonense resistance to heat stress.

Ganoderma is well known for its use in traditional Chinese medicine and is widely cultivated in China, Korea, and Japan. Increased temperatures associated with global warming are negatively influencing the growth and development of Ganoderma. Nitric oxide is reported to play an important role in alleviating fungal heat stress (HS). However, the transcriptional profiling of Ganoderma oregonense in response to HS, as well as the transcriptional response regulated by NO to cope with HS has not been reported. We used RNA-Seq technology to generate large-scale transcriptome data from G. oregonense mycelia subjected to HS (32 °C) and exposed to concentrations of exogenous NO. The results showed that heat shock proteins (HSPs), "probable stress-induced proteins", and unigenes involved in "D-amino-acid oxidase activity" and "oxidoreductase activity" were significantly up-regulated in G. oregonense subjected to HS (P < 0.05). The significantly up-regulated HSPs, "monooxygenases", "alcohol dehydrogenase", and "FAD/NAD(P)-binding domain-containing proteins" (P < 0.05) regulated by exogenous NO may play important roles in the enhanced HS tolerance of G. oregonense. These results provide insights into the transcriptional response of G. oregonense to HS and the mechanism by which NO enhances the HS tolerance of fungi at the gene expression level.

Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 µm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Involvement of metabolites in early defense mechanism of oil palm (Elaeis guineensis Jacq.) against Ganoderma disease.

Understanding the mechanism of interaction between the oil palm and its key pathogen, Ganoderma spp. is crucial as the disease caused by this fungal pathogen leads to a major loss of revenue in leading palm oil producing countries in Southeast Asia. Here in this study, we assess the morphological and biochemical changes in Ganoderma disease infected oil palm seedling roots in both resistant and susceptible progenies. Rubber woodblocks fully colonized by G. boninense were applied as a source of inoculum to artificially infect the roots of resistant and susceptible oil palm progenies. Gas chromatography-mass spectrometry was used to measure an array of plant metabolites in 100 resistant and susceptible oil palm seedling roots treated with pathogenic Ganoderma boninense fungus. Statistical effects, univariate and multivariate analyses were used to identify key-Ganoderma disease associated metabolic agitations in both resistant and susceptible oil palm root tissues. Ganoderma disease related defense shifts were characterized based on (i) increased antifungal activity in crude extracts, (ii) increased lipid levels, beta- and gamma-sitosterol particularly in the resistant progeny, (iii) detection of heterocyclic aromatic organic compounds, benzo [h] quinoline, pyridine, pyrimidine (iv) elevation in antioxidants, alpha- and beta-tocopherol (iv) degraded cortical cell wall layers, possibly resulting from fungal hydrolytic enzyme activity needed for initial penetration. The present study suggested that plant metabolites mainly lipids and heterocyclic aromatic organic metabolites could be potentially involved in early oil palm defense mechanism against G. boninense infection, which may also highlight biomarkers for disease detection, treatment, development of resistant variety and monitoring.

Sporoderm-Broken Spores of Ganoderma lucidum Inhibit the Growth of Lung Cancer: Involvement of the Akt/mTOR Signaling Pathway.

The sporoderm-broken spores of Ganoderma lucidum (SBGS) and their extracts exhibited a wide range of biological activities. In the present study, we prepare ethanol/ethanol extract (E/E-SBGS) and ethanol/aqueous extract (E/A-SBGS) from SBGS and examine their antitumor activities against human lung cancer. Our results showed that E/E-SBGS, not E/A-SBGS, inhibited the survival and migration of lung cancer cells in a dose-dependent manner. E/E-SBGS arrested cell cycle at G2/M phase and triggered apoptosis by decreasing the expression and activity of cell cycle regulators, cyclin B1 and cdc2, as well as anti-apoptotic proteins, Bcl-2 and Bcl-xl. Consequently, colony formation of lung cancer cells was markedly blocked by E/E-SBGS at subtoxic concentrations. Oral administration of both E/E-SBGS and SBGS significantly suppressed tumor volume and tumor weight without gross toxicity in mice. Mechanism study showed that E/E-SBGS dose-dependently suppressed the activation of Akt, the mammalian target of rapamycin (mTOR) and their downstream molecules S6 kinase and 4E-BP1 in treated tumor cells. Taken together, these results indicate that the ethanol extract of sporoderm-broken spores of G. lucidum suppresses the growth of human lung cancer, at least in part, through inhibition of the Akt/mTOR signaling pathway, suggesting its potential role in cancer treatments.

Streptomyces sanglieri which colonised and enhanced the growth of Elaeis guineensis Jacq. seedlings was antagonistic to Ganoderma boninense 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 10(9) 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.

Basidiospore and Protoplast Regeneration from Raised Fruiting Bodies of Pathogenic Ganoderma boninense.

Ganoderma boninense, a phytopathogenic white rot fungus had sought minimal genetic characterizations despite huge biotechnological potentials. Thus, efficient collection of fruiting body, basidiospore and protoplast of G. boninense is described. Matured basidiocarp raised under the glasshouse conditions yielded a total of 8.3 × 104 basidiospores/ml using the low speed centrifugation technique. Mycelium aged 3-day-old treated under an incubation period of 3 h in lysing enzyme from Trichoderma harzianum (10 mg/ml) suspended in osmotic stabilizer (0.6 M potassium chloride and 20 mM dipotassium phosphate buffer) yielded the highest number of viable protoplasts (8.9 × 106 single colonies) among all possible combinations tested (regeneration media, age of mycelium, osmotic stabilizer, digestive enzyme and incubation period).

Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles.

Ganoderma is a fungal genus belonging to the Ganodermataceae family and Polyporales order. Plant-pathogenic species in this genus can cause severe diseases (stem, butt, and root rot) in economically important trees and perennial crops, especially in tropical countries. Ganoderma species are white rot fungi and have ecological importance in the breakdown of woody plants for nutrient mobilization. They possess effective machineries of lignocellulose-decomposing enzymes useful for bioenergy production and bioremediation. In addition, the genus contains many important species that produce pharmacologically active compounds used in health food and medicine. With the rapid adoption of next-generation DNA sequencing technologies, whole genome sequencing and systematic transcriptome analyses become affordable approaches to identify an organism's genes. In the last few years, numerous projects have been initiated to identify the genetic contents of several Ganoderma species, particularly in different strains of Ganoderma lucidum. In November 2013, eleven whole genome sequencing projects for Ganoderma species were registered in international databases, three of which were already completed with genomes being assembled to high quality. In addition to the nuclear genome, two mitochondrial genomes for Ganoderma species have also been reported. Complementing genome analysis, four transcriptome studies on various developmental stages of Ganoderma species have been performed. Information obtained from these studies has laid the foundation for the identification of genes involved in biological pathways that are critical for understanding the biology of Ganoderma, such as the mechanism of pathogenesis, the biosynthesis of active components, life cycle and cellular development, etc. With abundant genetic information becoming available, a few centralized resources have been established to disseminate the knowledge and integrate relevant data to support comparative genomic analyses of Ganoderma species. The current review carries out a detailed comparison of the nuclear genomes, mitochondrial genomes and transcriptomes from several Ganoderma species. Genes involved in biosynthetic pathways such as CYP450 genes and in cellular development such as matA and matB genes are characterized and compared in detail, as examples to demonstrate the usefulness of comparative genomic analyses for the identification of critical genes. Resources needed for future data integration and exploitation are also discussed.

Molecular defense response of oil palm to Ganoderma infection.

Basal stem rot (BSR) of oil palm roots is due to the invasion of fungal mycelia of Ganoderma species which spreads to the bole of the stem. In addition to root contact, BSR can also spread by airborne basidiospores. These fungi are able to break down cell wall components including lignin. BSR not only decreases oil yield, it also causes the stands to collapse thus causing severe economic loss to the oil palm industry. The transmission and mode of action of Ganoderma, its interactions with oil palm as a hemibiotroph, and the molecular defence responses of oil palm to the infection of Ganoderma boninense in BSR are reviewed, based on the transcript profiles of infected oil palms. The knowledge gaps that need to be filled in oil palm-Ganoderma molecular interactions i.e. the associations of hypersensitive reaction (HR)-induced cell death and reactive oxygen species (ROS) kinetics to the susceptibility of oil palm to Ganoderma spp., the interactions of phytohormones (salicylate, jasmonate and ethylene) at early and late stages of BSR, and cell wall strengthening through increased production of guaiacyl (G)-type lignin, are also discussed.

Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection.

Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection.