Mycelial Samples Research Articles

TMT-Based Proteomic Analysis Reveals the Molecular Mechanisms of Sodium Pheophorbide A against Black Spot Needle Blight Caused by Pestalotiopsis neglecta in Pinus sylvestris var. mongolica.

Black spot needle blight is a minor disease in Mongolian Scots pine (Pinus sylvestris var. mongolica) caused by Pestalotiopsis neglecta, but it can cause economic losses in severe cases. Sodium pheophorbide a (SPA), an intermediate product of the chlorophyll metabolism pathway, is a compound with photoactivated antifungal activity, which has been previously shown to inhibit the growth of P. neglecta. In this study, SPA significantly reduced the incidence and disease index and enhanced the chlorophyll content and antioxidant enzyme activities of P. sylvestris var. mongolica. To further study the molecular mechanism of the inhibition, we conducted a comparative proteomic analysis of P. neglecta mycelia with and without SPA treatment. The cellular proteins were obtained from P. neglecta mycelial samples and subjected to a tandem mass tag (TMT)-labelling LC-MS/MS analysis. Based on the results of de novo transcriptome assembly, 613 differentially expressed proteins (DEPs) (p < 0.05) were identified, of which 360 were upregulated and 253 downregulated. The 527 annotated DEPs were classified into 50 functional groups according to Gene Ontology and linked to 256 different pathways using the Kyoto Encyclopedia of Genes and Genomes database as a reference. A joint analysis of the transcriptome and proteomics results showed that the top three pathways were Amino acid metabolism, Carbohydrate metabolism, and Lipid metabolism. These results provide new viewpoints into the molecular mechanism of the inhibition of P. neglecta by SPA at the protein level and a theoretical basis for evaluating SPA as an antifungal agent to protect forests.

The effect of casing and gypsum on the yield and psychoactive tryptamine content of Psilocybe cubensis (Earle) Singer

Psychedelic fungi have experienced a surge in interest in recent years. Most notably, the fungal secondary metabolite psilocybin has shown tremendous promise in the treatment of various psychiatric disorders. The mushroom species that produce this molecule are poorly understood. Here we sought to examine for the first time, the response of a psilocybin-producing species Psilocybe cubensis to casing (peat moss and vermiculite) and supplementation with gypsum (calcium sulfate dihydrate), two common practices in commercial mushroom cultivation. Mycelial samples of genetically authenticated P. cubensis were used to inoculate popcorn grain bags. The fully colonized bags of popcorn grain (0.15 kg) were transferred to bins of 0.85 kg pasteurized horse manure, with or without 1 cm thick layer of casing and/or 5 % gypsum. Our results indicate that the use of a casing layer significantly increases the biological efficiency (161.5 %), by approximately four fold, in comparison to control (40.5 %), albeit with a slight delay (∼2 days) for obtaining fruiting bodies and a somewhat reduced total tryptamine content (0.85 %) as gauged by High Performance Liquid Chromatography measurements. Supplementation with both casing and gypsum, however, appears to promote maximal yields (896.6 g/kg of dried substrate), with a biological efficiency of 89.6 %, while also maintaining high total tryptamine expressions (0.95 %). These findings, revealing methods for maximizing yield of harvest and expressions of psychoactive tryptamines, may prove useful for both home growers and commercial cultivators of this species, and ultimately support the growth of a robust industry with high quality natural products.

Comparative proteomics reveals the mechanism of cyclosporine production and mycelial growth in Tolypocladium inflatum affected by different carbon sources.

Cyclosporine A (CsA) is a secondary cyclopeptide metabolite produced by Tolypocladium inflatum that is widely used clinically as an immunosuppressant. CsA production and mycelial growth differed when T. inflatum was cultured in different carbon source media. During early fermentation, CsA was preferred to be produced in fructose medium, while the mycelium preferred to accumulate in sucrose medium. On the sixth day, the difference was most pronounced. In this study, high-throughput comparative proteomics methods were applied to analyze differences in protein expression of mycelial samples on day 6, revealing the proteins and mechanisms that positively regulate CsA production related to carbon metabolism. The differences included small molecule acid metabolism, lipid metabolism, organic catabolism, exocrine secretion, CsA substrate Bmt synthesis, and transcriptional regulation processes. The proteins involved in the regulation of mycelial growth related to carbon metabolism were also revealed and were associated with waste reoxidation processes or coenzyme metabolism, small molecule synthesis or metabolism, the stress response, genetic information or epigenetic changes, cell component assembly, cell wall integrity, membrane metabolism, vesicle transport, intramembrane localization, and the regulation of filamentous growth. This study provides a reliable reference for CsA production from high-efficiency fermentation. This study provides key information for obtaining more CsA high-yielding strains through metabolic engineering strategies.

Growth, laccase activity and role in 2,4-D degradation of Lentinus crinitus (L.) Fr. in a liquid medium

This study aimed to evaluate the role of L. crinitus laccase in the biotransformation of 2,4-D pesticide and the adsorption capacity and/or accumulation in the fungal biomass. Preliminary tests were carried out in liquid medium containing L. crinitus to detect the maximum concentration of 2,4-D in which the mycelium developed (5.025 g L−1). The growth and laccase activity for L. crinitus were analyzed using a complete factorial experimental design with variations in concentrations of glucose (16.65–33.35 g L−1), malt extract (0.825–9.175 g L−1), and 2,4-D (0.01–5 gL-1), and the mycelial samples were evaluated by SEM-EDS. The ability of L. crinitus to remove 2,4-D after 28 days was demonstrated by a decrease in concentrations of 2,4-D by up to 15.7% more than control treatments and occurred with both low (<2 g L−1) and high (>10 g L−1) glucose consumption conditions, reaching biomass concentrations below 2 g L−1, and presenting laccase activity between 3 and 6 g L−1. The laccase activity of the fungus depends on the initial amount of glucose and 2,4-D (up to 18 g L−1 and 4 g L−1). Lower concentrations of 2,4-DCP (0.01–2.70 mg L−1) may be an indicative of L. crinitus as its transformation agent. The presence of chlorine in the fungal mycelium (up to 4.0%) may represent its adsorption and/or accumulation by L. crinitus. L. crinitus can be described as a biotransformator of 2,4-D and 2,4-DCP through laccase activity, highlighting its potential for treatment of agricultural effluents.

Genome-Wide Gene Expression Analyses of the AtfA/AtfB-Mediated Menadione Stress Response in Aspergillus nidulans.

The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism, and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans. Comparative analyses of transcriptomes of control, ΔatfA, ΔatfB, and ΔatfAΔatfB mutant strains were performed. Dependence of a gene on AtfA (AtfB) was decided by its differential downregulation both between the reference and ΔatfA (ΔatfB) strains and between the ΔatfB (ΔatfA) and the ΔatfAΔatfB strains in vegetatively grown cells (mycelia) and asexual spores (conidia) of menadione sodium bisulfite (MSB)-treated or untreated cultures. As AtfA is the primary bZIP TF governing stress-response in A. nidulans, the number of differentially expressed genes for ΔatfA was significantly higher than for ΔatfB in both mycelial and conidial samples, and most of the AtfB-dependent genes showed AtfA dependence, too. Moreover, the low number of genes depending on AtfB but not on AtfA can be a consequence of ΔatfA leading to downregulation of atfB expression. Conidial samples showed much higher abundance of atfA and atfB mRNAs and more AtfA- and AtfB-affected genes than mycelial samples. In the presence of MSB, the number of AtfB- (but not of AtfA-) affected genes decreased markedly, which was accompanied with decreased mRNA levels of atfB in MSB-treated mycelial (reference strain) and conidial (ΔatfA mutant) samples. In mycelia, the overlap between the AtfA-dependent genes in MSB-treated and in untreated samples was low, demonstrating that distinct genes can be under AtfA control under different conditions. Carbohydrate metabolism genes were enriched in the set of AtfA-dependent genes. Among them, AtfA-dependence of glycolytic genes in conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, such as the Emericellamide cluster, also showed AtfA-dependent regulation. Genes encoding catalase and histidine-containing phosphotransfer proteins showed AtfA-dependence under all experimental conditions. There were 23 AtfB-dependent genes that did not depend on AtfA under any of our experimental conditions. These included a putative α-glucosidase (agdB), a putative α-amylase, calA, which is involved in early conidial germination, and an alternative oxidase. In summary, in A. nidulans there is a complex interaction between the two bZIP transcription factors, where AtfA plays the primary regulatory role.

MOLECULAR IDENTIFICATION AND GERMPLASM CONSERVATION OF WILD MUSHROOMS FROM MAYANTOC, TARLAC, PHILIPPINES

Tarlac is a landlocked province located in Central Luzon, Philippines. It has natural forests containing natural vegetation which offers favorable surroundings for the mushrooms to grow. In this study, a collection of macrofungi was conducted, followed by molecular identification. The genomic DNA was extracted through CTAB method, and amplified the ITS region using Internal Transcribe Spacer (ITS) gene marker. The amplified products were sequenced and identified using BLAST NCBI analysis. There are 25 mushroom samples that were successfully identified and with high similarity with those of other related species retrieved from GenBank. The samples were classified into 6 orders, 12 families, 15 genera, and 25 species from Basidiomycota and Ascomycota which separated into two branches in the phylogeny tree. The tree also shows that the most species collected were from the order Polyporales with 11 species grouped in the first clade followed by Agaricales with 8 mushroom species. A portion of the fruiting body with 15% glycerol and all the mycelial samples were transferred in cryovials and placed in a cryofreezer for preservation.

Gene expression of the white-rot fungus Lenzites gibbosa during wood degradation

ABSTRACT To determine the wood degradation mechanism and its key genes and biological processes of Lenzites gibbosa, we sequenced 15 transcriptomes of mycelial samples under woody environment at 3, 5, 7, and 11 d (D3, D5, D7, and D11) and nonwoody environment (control). All the transcripts were annotated as much as possible in eight databases to determine their function. The key genes and biological processes relating to wood degradation were predicted and screened. The expression of 11 key genes during wood degradation after 5 d of sawdust treatment was detected by quantitative polymerase chain reaction (PCR). A total of 2069 differentially expressed genes (DEGs) were obtained in 10 differential groups. Comparing wood with nonwood treatment condition, the key genes were those participating in oxidation-reduction process, they were oxidoreductase and peroxidase genes and their regulator genes; these genes mainly focused on the three biological processes of carbohydrate metabolism, lignin catabolism, and secondary metabolite biosynthesis, transport, and catabolism. The mostly enriched subcategories in molecular function were oxidoreductase activity, peroxidase activity, and heme binding in Gene Ontology (GO) annotation. One cellulose and hemicellulose degradation pathway and seven pathways related to lignin-derived aromatic compound degradation or the later degradation of lignin were found. In conclusion, during the process of L. gibbosa growing on wood, gene expression at the transcriptional level indicated that lignin catabolism and hyphal growth were promoted, but the metabolism of carbon and carbohydrates including cellulose in lignocellulose in overall trend was inhibited to some extent. The results have important reference value for the study of degradation mechanism of wood white rot.

An increase in the number of peroxisomes is coupled to the initial infection stage and stress response of Botrytis cinerea

Peroxisomes are very important organelles in eukaryotic cells and participate in various biological processes, including pathogen–host interactions. A variety of proteins involved in peroxisome proliferation and metabolic activity within peroxisomes have been shown to be essential for full virulence of several phytopathogenic fungi. However, the effects of changes in the number of peroxisomes and proteins involved in the peroxisome pathway on the pathogenicity of Botrytis cinerea have rarely been reported. In this study, by analysing transcriptome data and RT-qPCR validation, we found that more than half of the genes annotated to the peroxisome pathway in B. cinerea were upregulated more than twofold between mycelial samples cultured in medium with tomato leaves and without tomato leaves. A strain of B. cinerea with fluorescently labelled peroxisomes was obtained by overexpression of GFP fused to peroxisomal targeting signal 1 (the tripeptide ‘SKL’). The addition of tomato leaves to the liquid medium induced a significant increase in the number of peroxisomes, β-oxidation level, H2O2 content, and acetyl-CoA level in B. cinerea mycelia. When B. cinerea was cultured with oleic acid as the sole carbon source, the formation of infection-related structures and their penetration into plant cells were found to be associated with peroxisome pathway activity. Furthermore, peroxisome proliferation and lipid metabolism increased in response to different extracellular stresses in B. cinerea. Taken together, our results confirmed that activation of the peroxisome pathway in B. cinerea contributes to the initial infection and the ability to cope with environmental stress.

Development of PCR-Based Assays for Rapid and Reliable Detection and Identification of Canker-Causing Pathogens from Symptomatic Almond Trees.

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, β-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures.

Occurrence of similar mycoviruses in pathogenic, saprotrophic and mycorrhizal fungi inhabiting the same forest stand.

Fungal viruses (mycoviruses) are considered to be highly host specific, but phylogenetic analysis supports the occasional occurrence of horizontal transmission between species. We used an extensive sampling strategy to investigate whether similar viruses occur in more than one fungal species of the same forest habitat. Mycelial samples were collected from in-growth mesh bags (N = 259), fruiting bodies (N = 173) and cultured isolates (N = 68) at a forest site where the spatial distribution of viral infections in clonal individuals of the wood decay fungus Heterobasidion parviporum was mapped in detail earlier. The investigation revealed previously known Heterobasidion viruses in ∼2% of the single or pooled mycorrhizal samples from mesh bags, ∼3% of the fruiting body samples and none of the fungal cultures analyzed. Novel virus strains distinct from known Heterobasidion viruses were detected in cultures of ectomycorrhizal fungi (Lactarius tabidus, L. rufus) and saprotrophic fungi (Megacollybia platyphylla, Mucoraceae spp.). Overall, our results support the view that mycoviruses do not readily cross species borders. Regarding potential virocontrol applications, the introduction of Heterobasidion viruses into natural habitats is not expected to cause a major infection pressure towards the indigenous fungal community. However, the ecological consequences of the putative interspecies virus transmission events detected require further investigation.

Chemical Composition and Antioxidant and Antibacterial Activities of Cultured Mycelia of Four Clavicipitaceous Mushrooms (Ascomycetes) from the Indian Himalayas.

Cultured mycelia of 4 clavicipitaceous fungi belonging to 2 genera, Cordyceps (C. gracilis, C. cicadae, C. sinclairii) and Metacordyceps (M. dhauladharensis), were analyzed for their chemical composition and antioxidant and antibacterial activities. Experiments to determine these characteristics were performed following standard methods. The fatty acid profiles of mycelia of all the species were analysed using gas chromatography. Chemical composition analysis of mycelial samples revealed carbohydrates (46.72 ± 0.7% to 63.40 ± 0.3%), protein (14.19 ± 0.2% to 26.16 ± 0.1%), crude fibers (0.93 ± 0.0% to 1.22 ± 0.0%), and ash (0.77 ± 0.0% to 0.98 ± 0.0%). The amounts of calcium and iron in mycelia were significantly higher than amounts of magnesium, copper, and manganese. The amount of β-carotene ranged from 0.67 to 1.17 μg/100 g; lycopene, from 0.30 to 0.57 μg/100 g; phenolic compounds, from 29.16 to 43.12 mg/100 g; polysaccharides, from 104 to 126 mg/g; and flavonoids, from 2.15 to 4.25 mg/g. All tested species showed significant (P ≤ 0.05) antioxidant activities measured based on half-maximal effective concentrations. C. cicadae possessed the lowest half-maximal effective concentration compared with the other species. All 4 species showed a broad spectrum of antibacterial activity against 7 tested pathogenic bacterial strains.

Survival of Rhizoctonia solani AG‐1 IA, the Causal Agent of Rice Sheath Blight, under Different Environmental Conditions

AbstractThe ability of Rhizoctonia solani AG‐1 IA, the causal agent of rice sheath blight, to survive in diseased rice straw and as sclerotia and mycelia was investigated. After storage for 10 months at 4°C, 25°C and non‐air‐conditioned natural room temperature (NRT, temperature range from 6°C to 35°C), sclerotia placed inside a desiccator, soaked in sterile water or immersed in wet paddy soil were viable. In contrast, only 15% of sclerotia in dry paddy soil survived. Survival of mycelia was severely affected by temperature and humidity. After 10 months in a desiccator at 4°C, 55% of mycelia samples could survive, whereas at 25°C and NRT, mycelial samples survived for only 7 and 5 months, respectively. However, mycelia stored in sterile water at constant temperatures (4°C or 25°C) survived for 10 months. A certain amount of UV radiation had no obvious effect on the survival of sclerotia or mycelia. The survival rate of the fungus in diseased rice straw stored for 16 months could reach 100% at 4°C, 50% at 25°C and 35% at NRT. The survival rates of the pathogen in diseased rice straw buried in dry, wet and flooded paddy soils after 10‐month storage at NRT were 75, 100 and 100%, respectively, indicating that soil humidity is a crucial factor for the survival of this fungus.

Fatty Acid Composition of Different Collections of Coprinoid Mushrooms (Agaricomycetes) and Their Nutritional and Medicinal Values.

Qualitative and quantitative analyses of the free fatty acid (FA) composition of ethanol extracts obtained from mycelia of 30 genetically identified strains of 18 species of coprinoid mushrooms (CMs) or coprini belonging to clades Coprinellus (C. bisporus, C. curtus, C. disseminatus, C. domesticus, C. ellisii, C.flocculosus, C. micaceus, C. aff radians I, C. aff radians II, C. aff radians III, C. xanthothrix, Coprinellus sp. 4), Coprinopsis (C. cinerea, C. cothurnata, C. gonophylla, C. lagopides, C. maysoidispora, C. strossmayeri), and Coprinus (C. comatus) were evaluated using gas chromatography. Two unsaturated (lineolic, oleic) and 3 saturated (palmitic, stearic, myristic) FAs were detected in the tested mycelial samples. Amounts of unsaturated FAs prevailed over saturated FAs. Lineolic and palmitic acids were detected in all the species in high quantities (up to 64.73% and 53.82%, respectively). All 5 FAs were found in the Coprinus clade (family Agaricaceae) as opposed to the Coprinellus and Coprinopsis clades (family Psathyrellaceae). Based on the unsaturated and saturated FA content, the species from the clade Coprinellus were different from Coprinopsis species and C. comatus. The obtained data confirm that palmitic, linoleic, and stearic acids are predominant in mushrooms, including coprini. The presence of large amounts of unsaturated FAs in the tested coprini make them an option for further studies to formulate novel mushroom-based dietary supplements and nutriceuticals.

Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum

Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.

Expression of pathogenesis-related genes in Metarhizium anisopliae when infecting Spodoptera exigua

Characterization of pathogenesis genes of Metarhizium anisopliae, will provide better understanding of the role of these genes during pathogenesis. The expression profiles of pathogenesis-related genes encoding for a subtilisin-like protease (PR1), two types of chitinases (CHI2 and CHI3), and a peptide synthetase (PES) were studied during the different stages of M. anisopliae infection in Spodoptera exigua larvae using quantitative real-time RT-PCR. Sampling were at 0, 2, 12, and 24h after infection, when the infected larvae reached the moribund stage (36h), when mycelia emerged from the cadavers, when few conidia had formed on the mycelia, and when the cadavers were covered by conidia. For comparison, conidia and mycelial samples harvested from culture media were also included. Among the studied genes, PR1 expression was detected early at 2h after infection and increased as the infection progressed. CHI2 and CHI3 expressions were detected 12h after infection and when the mycelia emerged from cadavers, respectively. The expression levels of PR1, CHI2 and CHI3 genes increased significantly at the beginning of conidiogenesis on cadavers, but decreased at later stages. As expected, their expressions in pure fungal propagules were at very low levels. For PES gene, fold changes were not significant between different samples (less than onefold), indicating it might not have a major role in infecting stages. High expression levels of PR1, CHI2, and CHI3 genes during the post-mortem hyphal growth and conidiation stages of M. anisopliae clearly indicate the importance of these genes during the saprophytic phase of this fungus on host insect.

Altered proteomic polymorphisms in the caterpillar body and stroma of natural Cordyceps sinensis during maturation.

ObjectiveTo examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem.MethodsThe surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%–4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster.ConclusionProteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.

목질진흙버섯 Phellinus linteus HN1009K의 항산화 활성과 항염증효과

최근 농가에서 재배하고 있는 상황버섯 종에 비하여 균사생장이 빠르고 자실체 발생과 생육이 빠른 P. linteus HN1009K (HK1009)가 개발되었다. 본 연구는 HK1009의 균사체와 자실체 에탄올 추출물의 항산화 및 항염증효과를 기존에 재배되고 있는 P. linteus (PL, Korea Sangwhang)과 P. baumii (PB, Jangsu Sanghwang)를 대조구로 하여 비교분석 하였다. HK1009는 균사체와 자실체 추출물 모두 PL과 PB에 비하여 항산화 활성이 높았으며 xanthine oxidase분석에서는 PL, HK1009, PB순으로 항산화 활성이 높게 나타났다. RAW264.7세포를 이용한 상황버섯 균사체 추출물 PB(EX), PL(EX), HK1009(EX)시료의 NO assay 결과는 PB, PL, HK1009 모두 농도 의존적으로 원액 <TEX>$200{\mu}g/ml$</TEX> 에서는 <TEX>$7{\sim}8{\mu}M$</TEX> 정도로 염증 억제 효과가 뛰어났다. RAW 264.7 세포에 대한 MTT assay 결과에서 PB(EX), PL(EX), HK1009(EX) 추출물의 원액처리구 에서도 세포의 생존능이 70% 이상을 유지하여 세포 독성이 없는 것으로 판단되었다. Novel Phellinus linteus HN1009K (HK1009) has been characterized on speedy mycelial growth and yearly artificial cultivation. In this study, antioxidant and anti-inflammatory activities of ethanol extract from mycelium and fruiting body of P. linteus HN1009K (HK1009) were compared to those of P. linteus (PL, Korea Sangwhang) and P. baumii (PB, Jangsu Sanghwang) as controls. HK1009 extract showed higher DPPH free radical scavenging activity in both mycelial and fruiting body samples than those of PL and PB. However, in xanthine oxidase assay, antioxidant effect was highly measured on mycelial samples of PL and HK1009. Mycelial extract of HK1009 induced high inhibitory effect on LPS-induced nitric oxide (NO) production in RAW264.7 cells comparing to PL and PB without toxicity on the cells. The results suggest that P. linteus HN1009K possesses superior antioxidant activity and anti-inflammatory effects.

Rohitukine, a chromone alkaloid and a precursor of flavopiridol, is produced by endophytic fungi isolated from Dysoxylum binectariferum Hook.f and Amoora rohituka (Roxb).Wight & Arn

Rohitukine, a chromone alkaloid, has gained considerable international attention in recent years because of its novel semi-synthetic derivative, flavopiridol and P-276-00. Both these molecules are in advanced stages of clinical development and trial for cancer treatment. Recently, flavopiridol was approved as an orphan drug for treatment of chronic lymphocytic leukemia cancer. The natural occurrence of rohitukine is restricted to only four plant species, Amoora rohituka and Dysoxylum binectariferum (both from the Meliaceae family) and from Schumanniophyton magnificum and Schumanniophyton problematicum (both from the Rubiaceae family). Recently, an endophytic fungi isolated from D. binectariferum was reported to produce rohitukine in culture. In this study, we report the production of rohitukine and its subsequent attenuation by endophytic fungi, Fusarium oxysporum (MTCC-11383), Fusarium oxysporum (MTCC-11384) and Fusarium solani (MTCC-11385), all isolated from D. binectariferum and Gibberella fujikuroi (MTCC-11382) isolated from Amoora rohituka. The fungal rohitukine which was analyzed by HPLC, LC–MS and LC–MS/MS was identical to reference rohitukine and that produced by the plant. The rohitukine content in the mycelial samples ranged from 192.78μg to 359.55μg100g−1 of dry weight of and in broth it ranged from 14.10 to 71.90μg100ml−1. In all the fungal cultures, the production declined from first to fourth sub-culture. Studies are underway to unravel the mechanism by which the fungi produce the host metabolite in culture.

Antagonistic potentials of Trichoderma Spp. against fruit rot of custard apple caused by Phoma ligam

Context: Custard-apple (Annona squamosa Linn.) is a popular tropical fruit and fruit-rot disease caused by Phoma lingam leads to considerable qualitative and quantitative damages to the fruit in the area under study. Objective: Studies were conducted to investigate the antagonistic potential of five Trichoderma spp. namely, T. harzianum, T. hamatum, T. lignorum, T. reesei and T. viride against in vitro growth of the pathogen followed by field experiments. Materials and Methods: Dual culture plate, closed petriplate and food poisoning technique were followed in order to ascertain the antagonistic potential of the five species of Trichoderma. Hyphal interaction between the pathogen and T. viride was studied by collecting mycelial samples from the interaction zone of dual culture plate and was processed for scanning electron microscope. Plants with infected fruits were sprayed with spore suspension of T. viride and two commercial bioformulations of T. viride viz. Trichofix and Trichoguard for three times at a dose of 0.1, 0.5 and 1.0% prepared in distilled water. Results: All the Trichoderma species more or less effectively inhibited the growth of the pathogen through mycoparasitism, production of volatile and non-volatile metabolites. Scanning electron microscopic (SEM) micrographs revealed that hyphal interaction between P. lingam and T. viride leads to lysis of the pathogenic mycelium by the antagonist. Field experiments with spore suspension of T. viride and Trichofix and Trichoguard significantly reduced fruit rot incidence of custard-apple. Conclusion: The results of the current study indicate that adoption of biocontrol based disease management programmes can be effectively utilized against similar fruit diseases. DOI: http://dx.doi.org/10.3329/jbs.v19i0.12995 J. bio-sci. 19: 15-21, 2011

A Nested PCR Assay for Detecting Valsa mali var. mali in Different Tissues of Apple Trees.

A nested polymerase chain reaction (PCR) assay for detecting Valsa mali var. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. mali isolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsa spp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.