Perspective and challenges of mycorrhizal symbiosis in orchid medicinal plants.

The family Orchidaceae is of the most diverse taxon in the plant kingdom, and most of its members are highly valuable herbal medicines. Orchids have a unique mycorrhizal symbiotic relationship with specific fungi for carbohydrate and nutrient supplies in their whole lifecycle. The large-scale cultivation of the medicinal plant Gastodia elata is a successful example of using mycorrhizal symbiotic technology. In this review, we adopted G. elata and Dendrobium officinale as examples to describe the characteristics of orchid mycorrhiza and mycorrhizal benefits for host plants' growth and health (e.g. biotic and abiotic stress and secondary metabolite accumulation). The challenges in applying mycorrhizal technology to the cultivation of orchid medicinal plants in the future were also discussed. This review aims to serve as a theoretical guide for the cultivation of mycorrhizal technology in medicinal orchid plants.

Transcriptome and metabolome analysis reveals stage-specific metabolite accumulation during maturity of Chinese black truffle Tuber indicum.

Tuber indicum is the most economically important member of Tuber, with the highest production and widest distribution in China. However, the overexploitation of immature ascocarps not only has driven wild resources of the species toward extinction, but also has caused enconomic losses and a decline in the reputation of T.indicum quality. In this study, stage-specific metabolites of T. indicum in relation to nutritional quality and the mechanism of their accumulations were explored by transcriptome and metabolome analysis at five harvest times, representing four maturation stages. A total of 663 compounds were identified in T. indicum ascocarps by a widely targeted metabolomic approach. Lipid compounds are the most prominent metabolites (18%) in our samples and also are higher accumulation at the immature stage than at mature stage, representing 30.16% differential accumulated metabolites in this stage. Levels of some of the amino acids, such as S-(methyl) glutathione, S-adenosylmethionine, which are known truffle aroma precursors, were increased at the mature stage. The gene expression level related to the biosynthesis of volatile organic compounds were verified by qPCR. This study contributes to the preliminary understanding of metabolites variations in T. indicum ascocarps during maturity for quality evaluation and truffle biology.

Potential Specificity Between Mycorrhizal Fungi Isolated from Widespread Dendrobium spp. and Rare D. huoshanense Seeds.

In nature, orchid seed germination and seedling development depend on compatible mycorrhizal fungi. Mycorrhizal generalist and specificity affect the orchid distribution and rarity. Here, we investigated the specificity toward fungi in the rare D. huoshanense by mycorrhizal fungal isolation and symbiotic germination in vitro. Twenty mycorrhizal fungal strains were isolated from the roots of adult Dendrobium spp. (six and 12 strains from rare D. huoshanense and widespread D. officinale, respectively, and two strains from D. nobile and D. moniliforme, respectively) and 13 strains belong to Tulasnellaceae and seven strains belong to Serendipitaceae. Germination trials in vitro revealed that all 20 tested fungal strains can stimulate seed germination of D. huoshanense, but only nine strains (~ 50%) can support it up to the seedling stage. This finding indicates that generalistic fungi are important for early germination, but only a few can maintain a symbiosis with host in seedling stage. Thus, a shift of the microbial community from seedling to mature stage probably narrows the D. huoshanense distribution range. In addition, to further understand the relationship between the fungal capability to promote seed germination and fungal enzyme activity, we screened the laccase and pectase activity. The results showed that the two enzymes activities of fungi cannot be directly correlated with their germination-promoting activities. Understanding the host specificity degree toward fungi can help to better interpret the limited geographic distribution of D. huoshanense and provides opportunities for in situ and ex situ conservation and reintroduction programs.

Comparative Transcriptomics Analysis of the Symbiotic Germination of D. officinale (Orchidaceae) With Emphasis on Plant Cell Wall Modification and Cell Wall-Degrading Enzymes.

Orchid seed germination in nature is an extremely complex physiological and ecological process involving seed development and mutualistic interactions with a restricted range of compatible mycorrhizal fungi. The impact of the fungal species' partner on the orchids' transcriptomic and metabolic response is still unknown. In this study, we performed a comparative transcriptomic analysis between symbiotic and asymbiotic germination at three developmental stages based on two distinct fungi (Tulasnella sp. and Serendipita sp.) inoculated to the same host plant, Dendrobium officinale. Differentially expressed genes (DEGs) encoding important structural proteins of the host plant cell wall were identified, such as epidermis-specific secreted glycoprotein, proline-rich receptor-like protein, and leucine-rich repeat (LRR) extensin-like protein. These DEGs were significantly upregulated in the symbiotic germination stages and especially in the protocorm stage (stage 3) and seedling stage (stage 4). Differentially expressed carbohydrate-active enzymes (CAZymes) in symbiotic fungal mycelium were observed, they represented 66 out of the 266 and 99 out of the 270 CAZymes annotated in Tulasnella sp. and Serendipita sp., respectively. These genes were speculated to be involved in the reduction of plant immune response, successful colonization by fungi, or recognition of mycorrhizal fungi during symbiotic germination of orchid seed. Our study provides important data to further explore the molecular mechanism of symbiotic germination and orchid mycorrhiza and contribute to a better understanding of orchid seed biology.

Acetylome analysis of acetylation providing new insight into sclerotial generation in medicinal fungus Polyporus umbellatus.

Sclerotium-forming fungi are ecologically diverse and possess notable pathogenic or medicinal properties. The sclerotial generation mechanism is still elusive though Polyporus umbellatus sclerotia are typical Traditional Chinese Medicine with diuretic and antitumor effects. Protein acetylation displays a crucial role in several biological processes, but the functions of acetylation in this valuable fungus are unknown at present. In this study, acetylome of P. umbellatus was studied using nano LC-Triple TOF mass spectrometry system following immune-affinity-based enrichment. Totally, 648 acetylated sites in 342 proteins were identified and nine motifs were found to be conserved in P. umbellatus including KacY, KacA, KacL, KacG, MacS, MacA, RacA, RacL, and RacG. Acetylated proteins taken part in types of biological processes, particularly to those in biological processes associated with reactive oxygen species (ROS) metabolism. Inhibitors complement tests were carried out to verify the role of ROS in acetylation modification. It was concluded that oxidative stress regulated sclerotial generation via proteins acetylation in P. umbellatus. The present study presents new insight into the essential roles of acetylation in sclerotial formation, which may also be applicable for other sclerotium-forming fungi.

Phenolics, Flavonoids Content and Antioxidant Activities of Tuber indicum at Different Maturity Stages.

The Chinese black truffle Tuber indicum (Ascomycota, Pezizales) is an ectomycorrhizal fungus forming hypogeous edible ascocarps. As a famous wild edible mushroom in the world, this species also attracted an increasing interest in their chemical composition and pharmacological activity. In this study, the total phenolic content, total flavonoid content and antioxidant activities of Tuber indicum collected from July to November at different maturity stages in China were analyzed. Our results showed that T. indicum collected in July (immature stage) possessed the highest amount of flavonoids (3.89 mg/g dw) and the highest ABTS.+ scavenging activity (EC50 =3.73 mg/ml). In addition, those samples collected in August (moderate mature stage) contained the highest phenolics content (4.78 mg/g dw), the highest DPPH⋅ radical scavenging activity (EC50 =3.73 mg/ml) and ferric reducing activity power (243.63 µmol FeSO4 /g). The study reveals T. indicum in the early maturity stage yield significantly higher content of phenolics and flavonoids and possessed stronger antioxidant activity than those collected in other months. This study provided important data for understanding the relationship between maturity stages and truffle formation and evaluating the quality of Chinese black truffle at different maturity.

Use of transcriptomic profiling to identify candidate genes involved in Polyporus umbellatus sclerotial formation affected by oxalic acid.

Polyporus umbellatus is a precious medicinal fungus. Oxalic acid was observed to affect sclerotial formation and sclerotia possessed more medicinal compounds than mycelia. In this study, the transcriptome of P. umbellatus was analysed after the fungus was exposed to various concentrations of oxalic acid. The differentially expressed genes (DEGs) encoding a series of oxidases were upregulated, and reductases were downregulated, in the low-oxalic-acid (Low OA) group compared to the control (No OA) group, while the opposite phenomenon was observed in the high-oxalic-acid (High OA) group. The detection of reactive oxygen species (ROS) in P. umbellatus mycelia was performed visually, and Ca2+ and H2O2 fluxes were measured using non-invasive micro-test technology (NMT). The sclerotial biomass in the Low OA group increased by 66%, however, no sclerotia formed in the High OA group. The ROS fluorescence intensity increased significantly in the Low OA group but decreased considerably in the High OA group. Ca2+ and H2O2 influx significantly increased in the Low OA group, while H2O2 exhibited efflux in the High OA group. A higher level of oxidative stress formed in the Low OA group. Different concentrations of oxalic acid were determined to affect P. umbellatus sclerotial formation in different ways.

Armillaria mellea Symbiosis Drives Metabolomic and Transcriptomic Changes in Polyporus umbellatus Sclerotia.

Sclerotia, the medicinal part of Polyporus umbellatus, play important roles in diuresis and renal protection, with steroids and polysaccharides as the main active ingredients. The sclerotia grow and develop only after symbiosis with Armillaria sp. In this study, a systematic metabolomics based on non-targeted UPLC-MS method was carried out between the infected part of the separated cavity wall of the sclerotia (QR) and the uninfected part (the control group, CK) to find and identify differential metabolites. The biosynthetic pathway of characteristic steroids in sclerotia of P. umbellatus was deduced and the content of ergosterol, polyporusterone A and B in the QR and CK groups were detected with the High Performance Liquid Chromatography (HPLC). Furthermore, the expression patterns of putative genes associated with steroid biosynthesis pathway were also performed with quantitative real-time PCR. The results showed that a total of 258 metabolites originated from fungi with the fragmentation score more than 45 and high resolution mass were identified, based on UPLC-MS metabolomic analysis, and there were 118 differentially expressed metabolites (DEMs) between both groups. The metabolic pathways indicated that steroids, fatty acid and carbohydrate were active and enriched during P. umbellatus sclerotia infected by A. mellea. The content of ergosterol, polyporusterone A and B in the QR group increased by 32.2, 75.0, and 20.0%, in comparison to that of the control group. The qRT-PCR analysis showed that series of enzymes including C-8 sterol isomerase (ERG2), sterol C-24 methyltransferase (ERG6) and sterol 22-desaturase (ERG5), which played important roles in the final steps of ergosterol biosynthesis, all presented up-regulated patterns in the QR group in P. umbellatus. The comprehensive metabolomic and transcriptomic information will contribute to further study concerning the mechanisms of P. umbellatus sclerotial formation infected by A. mellea in the future.

Symbiotic and Asymbiotic Germination of Dendrobium officinale (Orchidaceae) Respond Differently to Exogenous Gibberellins.

Seeds of almost all orchids depend on mycorrhizal fungi to induce their germination in the wild. The regulation of this symbiotic germination of orchid seeds involves complex crosstalk interactions between mycorrhizal establishment and the germination process. The aim of this study was to investigate the effect of gibberellins (GAs) on the symbiotic germination of Dendrobium officinale seeds and its functioning in the mutualistic interaction between orchid species and their mycobionts. To do this, we used liquid chromatograph-mass spectrometer to quantify endogenous hormones across different development stages between symbiotic and asymbiotic germination of D. officinale, as well as real-time quantitative PCR to investigate gene expression levels during seed germination under the different treatment concentrations of exogenous gibberellic acids (GA3). Our results showed that the level of endogenous GA3 was not significantly different between the asymbiotic and symbiotic germination groups, but the ratio of GA3 and abscisic acids (ABA) was significantly higher during symbiotic germination than asymbiotic germination. Exogenous GA3 treatment showed that a high concentration of GA3 could inhibit fungal colonization in the embryo cell and decrease the seed germination rate, but did not significantly affect asymbiotic germination or the growth of the free-living fungal mycelium. The expression of genes involved in the common symbiotic pathway (e.g., calcium-binding protein and calcium-dependent protein kinase) responded to the changed concentrations of exogenous GA3. Taken together, our results demonstrate that GA3 is probably a key signal molecule for crosstalk between the seed germination pathway and mycorrhiza symbiosis during the orchid seed symbiotic germination.

Identification and expression of DoCCaMK during Sebacina sp. symbiosis of Dendrobium officinale.

Dendrobium officinale Kimura et Migo is a famous precious medicinal plant in China. Seed and seedling were cultivated with the mycorrhizal fungus Sebacina sp. CCaMK was initially cloned from D. officinale based on a SSH cDNA library of symbiotically germinated seeds with Sebacina sp. Phylogenetic analysis was performed among DoCCaMK and other CCaMKs. The particle bombardment technique was used to visualize DoCCaMK-GFP. qRT-PCR and western blot analysis were conducted to determine the tissue expression patterns of DoCCaMK with (SGS) and without (UGS) Sebacina sp. Furthermore, the effect of KN-93 on CCaMK expression was also examined. Using NMT the net Ca2+ fluxes and the CCaMK concentration were measured during D. officinale seed germination. DoCCaMK had the highest homology with Lilium longiflorum CCaMK. The DoCCaMK-GFP protein localized in the nucleus and cell membrane. CCaMK expression was significantly upregulated after symbiosis with Sebacina sp. KN-93 could be used as an inhibitor of CCaMK to inhibit D. officinale seed germination. Ca2+ influx and the concentration of the CCaMK in the SGS group was significantly more than that of the UGS group. The characterization of CCaMK provides certain genetic evidence for the involvement of this gene during seed germination and mycorrhizal cultivation in D. officinale.

Chinese Black Truffle-Associated Bacterial Communities of Tuber indicum From Different Geographical Regions With Nitrogen Fixing Bioactivity.

It is well known that the microbes associated with truffle fruiting bodies play a very important role during the truffle lifecycle. Tuber indicum, commonly called Chinese black truffle, is a species endemic to Eastern Asia and in the genus of Tuber. Here, we reported the bacterial communities of T. indicum from different geographical regions and described the bacterial diversity from three compartments (soil, ectomycorrhizae and ascocarps) of T. indicum using high-throughput sequencing combined tissue culture. The results revealed that Bradyrhizobium was the dominant genus in fruiting bodies of T. indicum from nine geographical sites in China, and the microbes in T. indicum ascocarps were influenced by geological locations and soil characteristics. More specific bacterial taxa were enriched in the fruiting bodies than in the ectomycorrhizae and soil. In addition, 60 cultural bacteria were isolated from T. indicum fruiting bodies (4 families, 24 genera), and Pseudomonas, Alcaligenes faecalis, Microbacterium, and Arthrobacter were dominant. One of 13 strains that have potential nitrogen-fixation activities was further verified by an acetylene reduction assay (ARA). Together, this research provides new and important data for better understanding of the interaction between truffle and associated microbe and the biology of truffle itself.

Cloning and expression of three thaumatin-like protein genes from Polyporus umbellatus.

Genes encoding thaumatin-like protein (TLPs) are frequently found in fungal genomes. However, information on TLP genes in Polyporus umbellatus is still limited. In this study, three TLP genes were cloned from P. umbellatus. The full-length coding sequence of PuTLP1, PuTLP2 and PuTLP3 were 768, 759 and 561 bp long, respectively, encoding for 256, 253 and 187 amino acids. Phylogenetic trees showed that P. umbellatus PuTLP1, PuTLP2 and PuTLP3 were clustered with sequences from Gloeophyllum trabeum, Trametes versicolor and Stereum hirsutum, respectively. The expression patterns of the three TLP genes were higher in P. umbellatus with Armillaria mellea infection than in the sclerotia without A. mellea. Furthermore, over-expression of three PuTLPs were carried out in Escherichia coli BL21 (DE3) strain, and high quality proteins were obtained using Ni-NTA resin that can be used for preparation of specific antibodies. These results suggest that PuTLP1, PuTLP2 and PuTLP3 in P. umbellatus may be involved in the defense response to A. mellea infections.

SWATH label-free proteomics analyses revealed the roles of oxidative stress and antioxidant defensing system in sclerotia formation of Polyporus umbellatus.

Understanding the initiation and maturing mechanisms is important for rational manipulating sclerotia differentiation and growth from hypha of Polyporus umbellatus. Proteomes in P. umbellatus sclerotia and hyphae at initial, developmental and mature phases were studied. 1391 proteins were identified by nano-liquid chromatograph-mass spectrometry (LC-MS) in Data Dependant Acquisition mode, and 1234 proteins were quantified successfully by Sequential Window Acquisition of all THeoretical fragment ion spectra-MS (SWATH-MS) technology. There were 347 differentially expressed proteins (DEPs) in sclerotia at initial phase compared with those in hypha, and the DEP profiles were dynamically changing with sclerotia growth. Oxidative stress (OS) in sclerotia at initial phase was indicated by the repressed proteins of respiratory chain, tricarboxylic acid cycle and the activation of glycolysis/gluconeogenesis pathways were determined based on DEPs. The impact of glycolysis/gluconeogenesis on sclerotium induction was further verified by glycerol addition assays, in which 5% glycerol significantly increased sclerotial differentiation rate and biomass. It can be speculated that OS played essential roles in triggering sclerotia differentiation from hypha of P. umbellatus, whereas antioxidant activity associated with glycolysis is critical for sclerotia growth. These findings reveal a mechanism for sclerotial differentiation in P. umbellatus, which may also be applicable for other fungi.

[Molecular cloning and prokaryotic expression of a type II ribosome inactivating protein from Polyporus umbellatus].

A type Ⅱ ribosome inactivating protein (RIP) gene was cloned from Polyporus umbellatus sclerotia by RT-PCR method. The full open reading frame cDNA sequence of this gene was 873 bp in length and encoded a 290-aa protein with a molecular weight of 32.33 kDa and an isoelectric point of 5.58. Multiple sequence alignment revealed that the deduced amino acids possessed conserved domains of RICIN superfamily protein. A neighbor joining phylogenetic analysis suggests that PuRIP was closely related to RIP in Marasmius oreades. Real time PCR results showed that this gene expressed in all tested tissues of P. umbellatus. Meanwhile, the expression of this gene was significantly up-regulated in the part infected by Armillaria mellea. This result suggested that this PuRIP might played important role with potential biotic stress tolerance of P. umbellatus. Otherwise, we successfully constructed the pET15b-PuRIP plasmid, produced and purified the His-PuRIP fusion protein, which would provide the basic material for polyclonal antibody preparation and gene function research.

[Molecular cloning and characterization of two kind of heat-shock protein gene from Polyporus umbellatus].

With RT-PCR approaches, the full-length cDNA of two heat shock protein genes were cloned from total RNA of the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of the Hsp90 was 2 091 bp, encoding 696 amino acid residues with a predicted molecular mass of 78.9 kDa. The full open reading frame cDNA sequence of the Hsp70 was 1 944 bp, encoding 647 amino acid residues with a predicted molecular mass of 70.5 kDa. The Hsp90 and Hsp70 protein contained the conservative structure domain, respectively. Phylogenetic analysis showed that Hsp90 and Hsp90 from Trametes versicolor were clustered into one group, Hsp70 and Hsp70 from Fistulina hepatica were clustered into one group. Real-time PCR analysis showed that, the expression of Hsp90 and Hsp70 in the infected part by Amillariella mellea was upregulated. The expression profiling of Hsp90 and Hsp70 showed same patterns underbiotic stress. The results indicate that these two genes may play an important role in response to Amillariella mellea infection.

[Habitat suitability assessment of medicinal Polyporus umbellatusin China based on Maxent modeling].

Geographic distribution of Polyporus umbellatus was predicted by using distribution records. Based on 42 distribution records from 12 provinces and bioclimatic data (1950-2000), georaphic distribution of P. umbellatus was modeled using Maxent. The results showed thatthe Receiver Operating Characteristic (ROC) curve analysis method was used to assess the accuracy of MAXENT model and the area under ROC curve (AUC) value of MAXENT was 0. 960 which suggested that the result of assessment was dependable. The geographic distribution pattern of were divided into three distribution block based on distribution values of 0.5-0.8: small area of Heilongjiang, Jilin, Liaoning and Hebei province, the board area of Yunnan, Guizhou and Sichuan, the southeast area of Tibet and the most area of Shanxi and Shannxi, the southeast board area of Shannxi, Gansu and Ningxia. Jackknife Test showed that average precipitation in warm seasons had the greatest contribution to the distribution gain of P. umbellatus, followed by mean temperature of driest quarter and annual mean temperature. The object suggests the potential distribution areasof P. umbellatus which is useful for the habitat conservation and introduction of P. umbellatus.

Transcriptome analysis of genes involved in defence response in Polyporus umbellatus with Armillaria mellea infection.

Polyporus umbellatus, a species symbiotic with Armillaria mellea and it also exhibits substantial defence response to Armillaria mellea infection. There are no genomics resources databases for understanding the molecular mechanism underlying the infection stress of P. umbellatus. Therefore, we performed a large-scale transcriptome sequencing of this fungus with A. mellea infection using Illumina sequencing technology. The assembly of the clean reads resulted in 120,576 transcripts, including 38,444 unigenes. Additionally, we performed a gene expression profiling analysis upon infection treatment. The results indicated significant differences in the gene expression profiles between the control and the infection group. In total, 10933 genes were identified between the two groups. Based on the differentially expressed genes, a Gene Ontology annotation analysis showed many defence-relevant categories. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes pathway analysis uncovered some important pathways. Furthermore, the expression patterns of 13 putative genes that are involved in defence response resulting from quantitative real-time PCR were consistent with their transcript abundance changes as identified by RNA-seq. The sequenced genes covered a considerable proportion of the P. umbellatus transcriptome, and the expression results may be useful to strengthen the knowledge on the defence response of this fungus defend against Armillaria mellea invasion.

Diversity Analysis of Polyporus umbellatus in China Using Inter-simple Sequence Repeat (ISSR) Markers.

Polyporus (P.) umbellatus, an endangered medicinal fungus in China, is distributed throughout most areas of the country. Thirty-seven natural P. umbellatus samples collected from 12 provinces in China were subjected to the inter-simple sequence repeat (ISSR) assay to investigate the genetic diversity within and among the 11 natural populations. Nine ISSR primers selected from 100 primers produced 88 discernible DNA bands, with 46 being polymorphic. The frequency of polymorphism varied from 19.57 to 93.48% with an average of 61.26% across all populations. At the population level, the within-population variance was much greater (92.04%) than the between-population variance (7.96%) as revealed by analysis of molecular variance. Eleven P. umbellatus populations were grouped into two major clusters, and the clustering pattern displayed four groups using the unweighted pair-group method with an arithmetic mean dendrogram. Principal coordinate analysis further indicated that the genetic diversity of P. umbellatus strains was unevenly distributed and displayed a clustered distribution pattern instead. Within these clusters, subgrouping (Henan and Hubei) and cluster II (Jilin and Heilongjiang) related to the geographic distribution were evident. The present study provides the first global overview of P. umbellatus diversity analysis in China, which may open up new opportunities in comparative genetic research on this medicinal fungus in other countries.

Oxalic acid and sclerotial differentiation of Polyporus umbellatus.

The present investigation aimed to uncover the effects of exogenous oxalic acid during the sclerotial formation of Polyporus umbellatus, with an emphasis on determining the content of the endogenic oxalic acid in the fungus. To this end, the oxalic acid content of the vegetative mycelia, sclerotia, culture mediums and sclerotial exudate were measured using High Performance Liquid Chromatography (HPLC). Furthermore, the lipid peroxidation was estimated by detecting thiobarbituric bituric acid reactive substances (TBARS). The results showed that the exogenous oxalic acid caused a delay in sclerotial differentiation (of up to 9 or more days), suppressed the sclerotial biomass and decreased the lipid peroxidation significantly in a concentration-dependent manner. Oxalic acid was found at very low levels in the mycelia and the maltose medium, whereas it was found at high levels in the mycelia and sucrose medium. After sclerotial differentiation, oxalic acid accumulated at high levels in both the sclerotia and the sclerotial exudate. Oxalic acid was therefore found to inhibit P. umbellatus sclerotial formation.

[Molecular cloning and characterization of two oxidative stress related genes from medicinal fungus Polyporus umbellatus].

OBJECTIVE: To clone the NADPH gene (PuNOX) and Glyoxal oxidase gene (PuGLOX) from a medicinal fungus Polyporus umbellatus, and to carry out the bioinformatic analysis. METHODS: We used the Rapid Amplification of cDNA ends (RACE) technique to obtain the full length cDNA of these two genes. We used a series of bioinformatic tools to characterize physiochemical properties of the two deduced protein. The analyses of multiple alignment and phylogenetic trees were performed using Bioeditor and MEGA 5.0 softwares. RESULTS: The entire cDNA of PuNOX and PuGLOX were 1674 bp, 1723 bp in length and encoded a 557-amino acid protein and 515-amino acid protein with a molecular weight of 63.845 kDa and 55.891 kDa and the isoelectric point of 5.58 and 4.82, respectively. PuNOX had high identities (74 to 80%) with NADPH peroxidase from other fungus. From the evolutionary tree, PuNOX was closely related to that of Pleurotus ostreatus. PuGLOX had high identities (> 50%) with Glyoxal oxidases from various fungus. Phylogenetic tree analysis suggested that PuGLOX was closely related to that of Phanerochaete chrysosporium. CONCLUSION: Molecular characterization of the two oxidative stress related genes will be useful for further functional determination of the genes involved in the sclerotium development of Polyporus umbellatus.