Locally Isolated Trichoderma harzianum Species Have Broad Spectrum Biocontrol Activities against the Wood Rot Fungal Species through Both Volatile Inhibition and Mycoparasitism.

Pathogenic root/wood rot fungal species infect multiple urban tree species in Singapore. There is a need for sustainable and environmentally friendly mitigation. We report the local Trichoderma strains as potential biocontrol agents (BCAs) for pathogenic wood rot fungal species such as Phellinus noxius, Rigidoporus microporus, and Fulvifomes siamensis. Isolated Trichoderma strains were DNA-barcoded for their molecular identities and assessed for their potential as a BCA by their rate of growth in culture and effectiveness in inhibiting the pathogenic fungi in in vitro dual culture assays. Trichoderma harzianum strain CE92 was the most effective in inhibiting the growth of the pathogenic fungi tested. Preliminary results suggested both volatile organic compound (VOC) production and direct hyphal contact contributed to inhibition. SPME GC-MS identified known fungal inhibitory volatiles. Trichoderma harzianum strain CE92 hyphae were found to coil around Phellinus noxius and Lasiodiplodia theobromae upon contact in vitro and were possibly a part of the mycoparasitism. In summary, the work provides insight into Trichoderma inhibition of pathogenic fungi and identifies local strains with good potential for broad-spectrum BCAs against root/wood rot fungi in Singapore.

Live Biomass of Rigidoporus vinctus: A Sustainable Method for Decoloration and Detoxification of Dyes in Water.

In this study, white-rot fungus, Rigidoporus vinctus, collected from an unidentified fallen twig from Pathankot, Punjab, India, was used for biosorption of anionic Congo red and cationic Methylene blue dyes from an aqueous medium. The biosorption efficiency of the live biomass of Rigidoporus vinctus was investigated to optimize biosorbent dosage, process time, concentrations of dyes, and pH of solutions. The results indicated that Rigidoporus vinctus is more efficient than other reported bio-adsorbents for Congo red and Methylene blue dyes. The maximum biosorption activity of Rigidoporus vinctus for Congo red was found at pH 2, and that for Methylene blue was at pH 10, after 24 h of the reaction period. The process followed pseudo-second-order kinetics, which indicated that the interaction of both dyes to the adsorption sites on the surface of Rigidoporus vinctus was responsive to biosorption. The biosorption process could be well explained by the Langmuir isotherm for both dyes. The maximum monolayer biosorption capacity of Rigidoporus vinctus for Congo red and Methylene blue was observed to be 54.0 mg/g and 80.6 mg/g, respectively. The seed germination test was carried out, and it was assessed that the toxicity of dyes was reduced up to significant levels. Based on the present experimental findings, it can be concluded that biosorption using the live biomass of Rigidoporus vinctus can effectively decolorize dye-containing wastewater, thus reducing the hazardous effects of dyes on human beings.

Exploring the Biocontrol Efficacy of Trichoderma spp. against Rigidoporus microporus, the Causal Agent of White Root Rot Disease in Rubber Trees (Hevea brasiliensis).

Rigidoporus microporus, which causes white root rot disease (WRD) in Hevea brasiliensis, is a looming threat to rubber plantation in Malaysia. The current study was conducted to determine and evaluate the efficiency of fungal antagonists (Ascomycota) against R. microporus in rubber trees under laboratory and nursery conditions. A total of 35 fungal isolates established from the rubber tree rhizosphere soil were assessed for their antagonism against R. microporus by the dual culture technique. Trichoderma isolates can inhibit the radial growth of R. microporus by 75% or more in the dual culture test. Strains of T. asperellum, T. koningiopsis, T. spirale, and T. reesei were selected to assess the metabolites involved in their antifungal activity. Results indicated that T. asperellum exhibited an inhibitory effect against R. microporus in both volatile and non-volatile metabolite tests. All Trichoderma isolates were then tested for their ability in producing hydrolytic enzymes such as chitinase, cellulase and glucanase, indole acetic acid (IAA), siderophores production, and phosphate solubilization. From the positive results of the biochemical assays, T. asperellum and T. spirale were selected as the biocontrol candidates to be further tested in vivo against R. microporus. The nursery assessments revealed that rubber tree clone RRIM600 pretreated with only T. asperellum or with the combination of T. asperellum and T. spirale was able to reduce the disease severity index (DSI) and exert higher suppression of R. microporus compared to other pretreated samples, with the average DSI below 30%. Collectively, the present study demonstrates that T. asperellum represents a potential biocontrol agent that should be further explored to control R. microporus infection on rubber trees.

A novel rubber tree PR-10 protein involved in host-defense response against the white root rot fungus Rigidoporus microporus.

BACKGROUND: White root rot disease in rubber trees, caused by the pathogenic fungi Rigidoporus microporus, is currently considered a major problem in rubber tree plantations worldwide. Only a few reports have mentioned the response of rubber trees occurring at the non-infection sites, which is crucial for the disease understanding and protecting the yield losses. RESULTS: Through a comparative proteomic study using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique, the present study reveals some distal-responsive proteins in rubber tree leaves during the plant-fungal pathogen interaction. From a total of 12 selected differentially expressed protein spots, several defense-related proteins such as molecular chaperones and ROS-detoxifying enzymes were identified. The expression of 6 candidate proteins was investigated at the transcript level by Reverse Transcription Quantitative PCR (RT-qPCR). In silico, a highly-expressed uncharacterized protein LOC110648447 found in rubber trees was predicted to be a protein in the pathogenesis-related protein 10 (PR-10) class. In silico promoter analysis and structural-related characterization of this novel PR-10 protein suggest that it plays a potential role in defending rubber trees against R. microporus infection. The promoter contains WRKY-, MYB-, and other defense-related cis-acting elements. The structural model of the novel PR-10 protein predicted by I-TASSER showed a topology of the Bet v 1 protein family, including a conserved active site and a ligand-binding hydrophobic cavity. CONCLUSIONS: A novel protein in the PR-10 group increased sharply in rubber tree leaves during interaction with the white root rot pathogen, potentially contributing to host defense. The results of this study provide information useful for white root rot disease management of rubber trees in the future.

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by fungi originating from Vietnam.

Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

Rigidoporus corticola Colonization and Invasive Fungal Disease in Immunocompromised Patients, United States.

We report 2 cases of Rigidoporus corticola (Oxyporus corticola) infection in humans in the United States. Clinical manifestations consisted of angioinvasive fungal sinusitis in 1 patient and pulmonary intracavitary fungus ball in the other patient. These cases illustrate previously undescribed clinicopathologic manifestations of infection by this filamentous basidiomycete in humans.

Differential Analysis of Mycelial Proteins and Metabolites From Rigidoporus Microporus During In Vitro Interaction With Hevea Brasiliensis.

Rigidoporus microporus is the fungus accountable for the white root rot disease that is detrimental to the rubber tree, Hevea brasiliensis. The pathogenicity mechanism of R. microporus and the identity of the fungal proteins and metabolites involved during the infection process remain unclear. In this study, the protein and metabolite profiles of two R. microporus isolates, Segamat (SEG) and Ayer Molek (AM), were investigated during an in vitro interaction with H. brasiliensis. The isolates were used to inoculate H. brasiliensis clone RRIM 2025, and mycelia adhering to the roots of the plant were collected for analysis. Transmission electron microscope (TEM) images acquired confirms the hyphae attachment and colonization of the mycelia on the root of the H. brasiliensis clones after 4 days of inoculation. The protein samples were subjected to 2-DE analysis and analyzed using MALDI-ToF MS/MS, while the metabolites were extracted using methanol and analyzed using LC/MS-QTOF. Based on the differential analyses, upregulation of proteins that are essential for fungal evolution such as malate dehydrogenase, fructose 1,6-biphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase hints an indirect role in fungal pathogenicity, while metabolomic analysis suggests an increase in acidic compounds which may lead to increased cell wall degrading enzyme activity. Bioinformatics analyses revealed that the carbohydrate and amino acid metabolisms were prominently affected in response to the fungal pathogenicity. In addition to that, other pathways that were significantly affected include "Protein Ubiquitination Pathway," Unfolded Protein Response," "HIFα Signaling," and "Sirtuin Signaling Pathway." The identification of responsive proteins and metabolites from this study promotes a better understanding of mechanisms underlying R. microporus pathogenesis and provides a list of potential biological markers for early recognition of the white root rot disease.

Enzyme Activities of Five White-Rot Fungi in the Presence of Nanocellulose.

White-rot fungi can degrade all lignocellulose components due to their potent lignin and cellulose-degrading enzymes. In this study, five white-rot fungi, Trametes versicolor, Trametes pubescens, Ganoderma adspersum, Ganoderma lipsiense, and Rigidoporus vitreus were tested for endoglucanase, laccase, urease, and glucose-6-phosphate (G6P) production when grown with malt extract and nanocellulose in the form of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). Results show that temperature plays a key role in controlling the growth of all five fungi when cultured with malt extract alone. Endoglucanase activities were highest in cultures of G. adspersum and G. lipsiense and laccase activities were highest in cultures of T. versicolor and R. vitreus. Urease activities were highest in cultures of G. adspersum, G. lipsiense, and R. vitreus. Glucose-6-phosphate levels also indicate that cells were actively metabolizing glucose present in the cultures. These results show that TEMPO-oxidized CNF and CNC do not inhibit the production of specific lignocellulose enzymes by these white-rot fungi. The apparent lack of enzymatic inhibition makes TEMPO-oxidized CNF and CNC excellent candidates for future biotechnological applications in combination with the white-rot fungi studied here.

Lanostanoid triterpenes from the fungus Rigidoporus microporus.

Five new lanostanoid triterpenes were isolated from the extract of R. microporus. Three of the metabolites (1-3) present a Δ8,9 skeleton with an uncommon keto functionality at C-1. Another compound (4) has an unprecedented rearranged skeleton in which methyl-19 was transposed to C-1, with conjugated double bonds at Δ1-10 and Δ8-9. All of the compounds have hydroxylated or furane-cyclized side-chains. The structures were elucidated by spectroscopic methods, and the absolute configuration of the hydroxyl-bearing carbon in the side chain of compound 5 was established in silico. The metabolites were evaluated for their antifungal activity and the bioactivity as agonist/antagonists of the liver X receptors (LXRs). Compound 4 presents antifungal activity and compounds 3 and 5 are the agonists of LXRs.

Cloning and Expression Analysis of HbPR-1b and HbPR-3 in Hevea brasiliensis During Inoculation with Rigidoporus microporus.

BACKGROUND AND OBJECTIVE: Pathogenesis-related (PR) proteins are dramatically accumulated after pathogen infection. Innate defense response through increasing PR-proteins is important for rubber rootstock selection that is tolerant to the white root disease caused by Rigidoporus microporus. This study was aimed to investigate the expression levels of PR-1 and PR-3 genes in tolerant (PB5/51) and susceptible (BPM24 and RRIM600) rubber clones after R. microporus infection. MATERIALS AND METHODS: The mRNA of HbPR-1b and HbPR-3 was isolated and characterized from rubber leaves. Gene expression levels of HbPR-1b and HbPR-3 were compared among three rubber clones (PB5/51, BPM24 and RRIM600) after R. microporus infection at 0, 12, 24, 48, 72 and 96 h using quantitative real-time PCR. The relative transcript abundances between inoculated and control plants were compared using the means of gene expression between time points and by Tukey's HSD test. A probability value (p<0.05) was used to determine the significance of difference between time points. RESULTS: The open reading frame of HbPR-1b is 492 bp with deduced 163 amino acid residues and the phylogenetic analysis showed it shared significant evolutionary history and clustering into group I of PR-protein. Moreover, the partial HbPR-3 was isolated with 390 bp. Gene expression levels of HbPR-1b and HbPR-3 showed marked differences in both transcripts depending on the rubber clones. Two genes demonstrated up-regulation of both tolerance and susceptibility in response to attack by R. microporus. The highest expression levels were found in seedlings of PB5/51 after inoculation. In RRIM600, low expression levels of HbPR-1b and HbPR-3 were initially observed but gradually increased at 24 h post inoculation. The transcription profile of HbPR-1b was stable expression in BPM24. CONCLUSION: The results demonstrated that the level ofHbPR-1b and HbPR-3 transcription can distinguish between tolerant and susceptible clones. The candidate defense genes to the white root disease were observed in PB5/51 seedlings, particularly HbPR-1b.

De novo transcriptomic assembly and profiling of Rigidoporus microporus during saprotrophic growth on rubber wood.

BACKGROUND: The basidiomycete Rigidoporus microporus is a fungus that causes the white rot disease of the tropical rubber tree, Hevea brasiliensis, the major source of commercial natural rubber. Besides its lifestyle as a pathogen, the fungus is known to switch to saprotrophic growth on wood with the ability to degrade both lignin and cellulose. There is almost no genomic or transcriptomic information on the saprotrophic abilities of this fungus. In this study, we present the fungal transcriptomic profiles during saprotrophic growth on rubber wood. RESULTS: A total of 266.6 million RNA-Seq reads were generated from six libraries of the fungus growing either on rubber wood or without wood. De novo assembly produced 34, 518 unigenes with an average length of 2179 bp. Annotation of unigenes using public databases; GenBank, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups (COG) and Gene Ontology (GO) produced 25, 880 annotated unigenes. Transcriptomic profiling analysis revealed that the fungus expressed over 300 genes encoding lignocellulolytic enzymes. Among these, 175 genes were up-regulated in rubber wood. These include three members of the glycoside hydrolase family 43, as well as various glycosyl transferases, carbohydrate esterases and polysaccharide lyases. A large number of oxidoreductases which includes nine manganese peroxidases were also significantly up-regulated in rubber wood. Several genes involved in fatty acid metabolism and degradation as well as natural rubber degradation were expressed in the transcriptome. Four genes (acyl-CoA synthetase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA acetyltransferase) potentially involved in rubber latex degradation pathway were also induced. A number of ATP binding cassette (ABC) transporters and hydrophobin genes were significantly expressed in the transcriptome during saprotrophic growth. Some genes related to energy metabolism were also induced. CONCLUSIONS: The analysed data gives an insight into the activation of lignocellulose breakdown machinery of R. microporus. This study also revealed genes with relevance in antibiotic metabolism (e.g. cephalosporin esterase) as well as those with potential applications in fatty acid degradation. This is the first study on the transcriptomic analysis of R. microporus on rubber wood and should serve as a pioneering resource for future studies of the fungus at the genomic or transcriptomic level.