Three new endophytic Apiospora species (Apiosporaceae, Amphisphaeriales) from China.

Apiospora species are widely distributed fungi with diverse lifestyles, primarily functioning as plant pathogens, as well as exhibiting saprophytic and endophytic behaviors. This study reports the discovery of three new species of Apiospora, namely A.gongcheniae, A.paragongcheniae, and A.neogongcheniae, isolated from healthy Poaceae plants in China. These novel species were identified through a multi-gene phylogenetic analysis. The phylogenetic analysis of the combined ITS, LSU, tef1, and tub2 sequence data revealed that the three new species formed a robustly supported clade with A.garethjonesii, A.neogarethjonesii, A.setostroma, A.subrosea, A.mytilomorpha, and A.neobambusae. Detailed descriptions of the newly discovered species are provided and compared with closely related species to enhance our understanding of the genus Apiospora.

Three new species of Trichoderma (Hypocreales, Hypocreaceae) from soils in China.

Trichoderma spp. are diverse fungi with wide distribution. In this study, we report on three new species of Trichoderma, namely T.nigricans, T.densissimum and T.paradensissimum, collected from soils in China. Their phylogenetic position of these novel species was determined by analyzing the concatenated sequences of the second largest nuclear RNA polymerase subunit encoding gene (rpb2) and the translation elongation factor 1- alpha encoding gene (tef1). The results of the phylogenetic analysis showed that each new species formed a distinct clade: T.nigricans is a new member of the Atroviride Clade, and T.densissimum and T.paradensissimum belong to the Harzianum Clade. A detailed description of the morphology and cultural characteristics of the newly discovered Trichoderma species is provided, and these characteristics were compared with those of closely related species to better understand the taxonomic relationships within the Trichoderma.

Biogeography and Ecology of Magnaporthales: A Case Study.

The order Magnaporthales belongs to Sordariomycetes, Ascomycota. Magnaporthales includes five families, namely Ceratosphaeriaceae, Pseudohalonectriaceae, Ophioceraceae, Pyriculariaceae, and Magnaporthaceae. Most Magnaporthales members are found in Poaceae plants and other monocotyledonous herbaceous plants ubiquitously as plant pathogens or endophytic fungi, and some members are found in decaying wood or dead grass as saprophytic fungi. Therefore, studying the biogeography and ecology of Magnaporthales is of great significance. Here, we described the biodiversity of endophytic Magnaporthales fungi from Poaceae at three latitudes in China and conducted a meta-analysis of the geography and ecology of Magnaporthales worldwide. We found that Magnaporthales is a dominant order in the endophytic fungi of Poaceae. More than half of the endophytic Magnaporthales fungi have a taxonomically uncertain placement. Notably, few endophytic fungi are grouped in the clusters with known saprophytic or pathogenic Magnaporthales fungi, indicating that they may have saprophytic and parasitic differentiation in nutritional modes and lifestyles. The meta-analysis revealed that most species of Magnaporthales have characteristic geographical, host, and tissue specificity. The geographical distribution of the three most studied genera, namely Gaeumannomyces, Magnaporthiopsis, and Pyricularia, in Magnaporthales may depend on the distribution of their hosts. Therefore, studies on the endophytic fungal Magnaporthales from monocotyledonous plants, including Poaceae, in middle and low latitudes will deepen our understanding of the biogeography and ecology of Magnaporthales.

A preliminary DNA barcode selection for the genus Russula (Russulales, Basidiomycota).

Russula is a worldwid genus which has a high species diversity . Aiming accurate and rapid species identification, candidate genes nLSU (28S), ITS, tef-1α, mtSSU, rpb1, and rpb2, were analysed as potential DNA barcodes. This analysis included 433 sequences from 38 well-circumscribed Russula species of eight subgenera. Two vital standards were analysed for success species identification using DNA barcodes, specifically inter- and intra-specific variations together with the success rates of PCR amplification and sequencing. Although the gap between inter- and intra-specific variations was narrow, ITS met the qualification standards for a target DNA barcode. Overlapping inter- and intra-specific pairwise distances were observed in nLSU, tef-1α, mtSSU, and rpb2. The success rates of PCR amplification and sequencing in mtSSU and rpb1 were lower than those of others. Gene combinations were also investigated for resolution of species recognition. ITS-rpb2 was suggested as the likely target DNA barcode for Russula, owing to the two viatal standards above. Since nLSU has the lowest minimum of inter-specific variation, and tef-1α has the highest overlap between intra- and inter-species variations among the candidate genes, they are disqualified from the selection for DNA barcode of Russula.

Identification and characterization of new Muscodor endophytes from gramineous plants in Xishuangbanna, China.

The endophytic fungi Muscodor spp. produce volatile organic compounds (VOCs) which can inhibit and even kill pathogenic fungi, bacteria, and nematodes. Nine endophytic fungal strains, isolated from the shoots of gramineous plants including Arthraxon hispidus, Eleusine indica, Oplismenus undulatifolius, and Oryza granulata, were identified as Muscodor through phylogenetic analysis of the internal transcribed spacer. Through an SPSS K-means cluster analysis, the nine Muscodor strains were divided into four groups based on the antifungal activities of the VOCs produced by these fungi determined by a two-section confrontation test. The first group contains the strains Y-L-54, W-S-41, Y-S-35, W-T-27, and Y-L-56, which showed the strongest activity. The second and third groups contain W-S-35 and Y-L-43, which showed stronger and moderate activity, respectively. The fourth group contains W-S-38 and N-L-7, which were the weakest in inhibiting the tested pathogens. Thirty-five compounds and the relative amounts of VOCs were determined by SPME-GC-MS and comparison with the NIST14 mass spectrometry database and Agilent MassHunter qualitative and quantitative analyses. These 35 compounds were classified into two different categories: (a) the product of fatty acid degradation, and (b) the intermediate and final metabolite of the metabolic pathway with the precursor of mevalonic acid. SPSS clustering analysis showed that the chemical components of VOCs might be correlated with their bioactivity rather than their phylogenetic assignment and some of the identified compounds might be responsible for antifungal activity. In conclusion, new Muscodor endophytes were recorded in tropical gramineous plants and a number of strains showed remarkable bioactive properties. Therefore, they have important potential applications in the fields of plant disease control.

A Novel Derivative of (-)mycousnine Produced by the Endophytic Fungus Mycosphaerella nawae, Exhibits High and Selective Immunosuppressive Activity on T Cells.

An endophytic fungus, Mycosphaerella nawae ZJLQ129, was isolated from the leaves of the traditional Chinese medicine Smilax china. From the fermentation broth and mycelium, a dibenzofurane compound (-)mycousnine (1) was isolated. Chemical modification of it to the amide derivative (-)mycousnine enamine (2), which is new to science, was found to have high and selective immunosuppressive activity: similar to cyclosporin A, (-)mycousnine enamine (2) selectively inhibited T cell proliferation, suppressed the expression of the surface activation antigens CD25 and CD69 and the formation and expression of the cytokines interleukin-2 as well as interferon γ in activated T cells, but did not show any effect on the proliferation of B cells and cancer cells (PANC-1 and A549) and the activation of macrophages. Furthermore, the cytotoxicity of (-)mycousnine enamine was lower than that of cyclosporin A, and its therapeutic index (TC50/EC50) was 4,463.5, which is five-fold higher than that of cyclosporin A. We conclude that (-)mycousnine enamine (2), the semi-synthestic product prepared from the native product (-)mycousnine (1) of the endophyte M. nawae is a novel effective immunosuppressant showing low toxicity and high selectivity.

An autophagy gene, HoATG5, is involved in sporulation, cell wall integrity and infection of wounded barley leaves.

The endophytic fungus Harpophora oryzae is a beneficial endosymbiont isolated from wild rice. H. oryzae can not only promote rice growth and biomass accumulation but also protect rice roots from invasion by its close relative Magnaporthe oryzae. Autophagy is a highly evolutionary conserved process from lower to higher eukaryotic organisms, and is involved in the maintenance of normal cell differentiation and development. In this study, we isolated a gene (HoATG5) which encodes an essential protein required for autophagy from the beneficial endophyte fungus H. oryzae. Using targeted gene replacement, a ΔHoATG5 mutant was generated and used to investigate the biological functions of autophagy in H. oryzae. We found that the autophagic process was blocked in the HoATG5 deletion mutant. The mutant showed increased vegetative growth and sporulation, and was sensitive to nutrient starvation. The ΔHoATG5 mutant lost its ability to penetrate and infect the wounded barley leaves. These results provide new knowledge to elaborate the molecular machinery of autophagy in endophytic fungi.

Trichoderma Biodiversity of Agricultural Fields in East China Reveals a Gradient Distribution of Species.

We surveyed the Trichoderma (Hypocreales, Ascomycota) biodiversity in agricultural fields in four major agricultural provinces of East China. Trichoderma strains were identified based on molecular approaches and morphological characteristics. In three sampled seasons (spring, summer and autumn), 2078 strains were isolated and identified to 17 known species: T. harzianum (429 isolates), T. asperellum (425), T. hamatum (397), T. virens (340), T. koningiopsis (248), T. brevicompactum (73), T. atroviride (73), T. fertile (26), T. longibrachiatum (22), T. pleuroticola (16), T. erinaceum (16), T. oblongisporum (2), T. polysporum (2), T. spirale (2), T. capillare (2), T. velutinum (2), and T. saturnisporum (1). T. harzianum, T. asperellum, T. hamatum, and T. virens were identified as the dominant species with dominance (Y) values of 0.057, 0.052, 0.048, and 0.039, respectively. The species amount, isolate numbers and the dominant species of Trichoderma varied between provinces. Zhejiang Province has shown the highest diversity, which was reflected in the highest species amount (14) and the highest Shannon-Wiener diversity index of Trichoderma haplotypes (1.46). We observed that relative frequencies of T. hamatum and T. koningiopsis under rice soil were higher than those under wheat and maize soil, indicating the preference of Trichoderma to different crops. Remarkable seasonal variation was shown, with summer exhibiting the highest biodiversity of the studied seasons. These results show that Trichoderma biodiversity in agricultural fields varies by region, crop, and season. Zhejiang Province (the southernmost province in the investigated area) had more T. hamatum than Shandong Province (the northernmost province), not only in isolate amounts but also in haplotype amounts. Furthermore, at haplotype level, only T. hamatum showed a gradient distribution from south to north in correspondence analysis among the four dominant species. The above results would contribute to the application of Trichoderma biocontrol strains.

Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 µM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.

Friend or foe: differential responses of rice to invasion by mutualistic or pathogenic fungi revealed by RNAseq and metabolite profiling.

The rice endophyte Harpophora oryzae shares a common pathogenic ancestor with the rice blast fungus Magnaporthe oryzae. Direct comparison of the interactions between a single plant species and two closely-related (1) pathogenic and (2) mutualistic fungi species can improve our understanding of the evolution of the interactions between plants and fungi that lead to either mutualistic or pathogenic interactions. Differences in the metabolome and transcriptome of rice in response to challenge by H. or M. oryzae were investigated with GC-MS, RNA-seq, and qRT-PCR. Levels of metabolites of the shikimate and lignin biosynthesis pathways increased continuously in the M. oryzae-challenged rice roots (Mo-roots); these pathways were initially induced, but then suppressed, in the H. oryzae-challenged rice roots (Ho-roots). Compared to control samples, concentrations of sucrose and maltose were reduced in the Ho-roots and Mo-roots. The expression of most genes encoding enzymes involved in glycolysis and the TCA cycle were suppressed in the Ho-roots, but enhanced in the Mo-roots. The suppressed glycolysis in Ho-roots would result in the accumulation of glucose and fructose which was not detected in the Mo-roots. A novel co-evolution pattern of fungi-host interaction is proposed which highlights the importance of plant host in the evolution of fungal symbioses.

The rice endophyte Harpophora oryzae genome reveals evolution from a pathogen to a mutualistic endophyte.

The fungus Harpophora oryzae is a close relative of the pathogen Magnaporthe oryzae and a beneficial endosymbiont of wild rice. Here, we show that H. oryzae evolved from a pathogenic ancestor. The overall genomic structures of H. and M. oryzae were found to be similar. However, during interactions with rice, the expression of 11.7% of all genes showed opposing trends in the two fungi, suggesting differences in gene regulation. Moreover, infection patterns, triggering of host defense responses, signal transduction and nutritional preferences exhibited remarkable differentiation between the two fungi. In addition, the H. oryzae genome was found to contain thousands of loci of transposon-like elements, which led to the disruption of 929 genes. Our results indicate that the gain or loss of orphan genes, DNA duplications, gene family expansions and the frequent translocation of transposon-like elements have been important factors in the evolution of this endosymbiont from a pathogenic ancestor.

Evidence for biotrophic lifestyle and biocontrol potential of dark septate endophyte Harpophora oryzae to rice blast disease.

The mutualism pattern of the dark septate endophyte (DSE) Harpophora oryzae in rice roots and its biocontrol potential in rice blast disease caused by Magnaporthe oryzae were investigated. Fluorescent protein-expressing H. oryzae was used to monitor the colonization pattern. Hyphae invaded from the epidermis to the inner cortex, but not into the root stele. Fungal colonization increased with root tissue maturation, showing no colonization in the meristematic zone, slight colonization in the elongation zone, and heavy colonization in the differentiation zone. H. oryzae adopted a biotrophic lifestyle in roots accompanied by programmed cell death. Real-time PCR facilitated the accurate quantification of fungal growth and the respective plant response. The biocontrol potential of H. oryzae was visualized by inoculation with eGFP-tagged M. oryzae in rice. H. oryzae protected rice from M. oryzae root invasion by the accumulation of H2O2 and elevated antioxidative capacity. H. oryzae also induced systemic resistance against rice blast. This systemic resistance was mediated by the OsWRKY45-dependent salicylic acid (SA) signaling pathway, as indicated by the strongly upregulated expression of OsWRKY45. The colonization pattern of H. oryzae was consistent with the typical characteristics of DSEs. H. oryzae enhanced local resistance by reactive oxygen species (ROS) and high antioxidative level and induced OsWRKY45-dependent SA-mediated systemic resistance against rice blast.

Bioactive metabolites from Phoma species, an endophytic fungus from the Chinese medicinal plant Arisaema erubescens.

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), ß-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC50 values of 413.22 and 48.5 µg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC50 values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 µM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.

An autophagy gene, TrATG5, affects conidiospore differentiation in Trichoderma reesei.

Autophagy is a highly conserved process in lower to higher eukaryotic organisms, and occurs in many types of cells as tissues are remodeled during development. In this study, we investigated the functional role of the Trichoderma reesei TrATG5 gene, which encodes an essential protein required for autophagy. TrATG5 is conserved in structure and function in the filamentous fungi and might clearly rescue the pathogenicity function of MgATG5 in Magnaporthe oryzae. Target gene disruption was used to study the functions of TrATG5. It was found that the autophagic process was blocked in the TrATG5 deletion mutant. The mutant was sensitive to nutrient starvation, with abnormal conidiophores and reduced production of conidia. This new evidence might help to elucidate the molecular machinery of autophagy in filamentous fungi.

Distinctive endophytic fungal assemblage in stems of wild rice (Oryza granulata) in China with special reference to two species of Muscodor (Xylariaceae).

Ecological niches in the rhizosphere and phyllosphere of grasses capable of sustaining endophytes have been extensively studied. In contrast, little information regarding the identity and functions of endophytic fungi in stems is available. In this study, we investigated the taxonomic affinities, diversity, and host specificities of culturable endophytes in stems of wild rice (Oryza granulata) in China. Seventy-four isolates were recovered. Low recovery rate (11.7%) indicated that there were relatively few sites for fungal infection. Identification using morphology, morphospecies sorting, and molecular techniques resulted in classification into 50 taxa, 36 of which were recovered only once. Nucleotide sequence similarity analysis indicated that 30% of the total taxa recovered were highly divergent from known species and thus may represent lineages new to science. Most of the taxa were classified as members of the classes Sordariomycetes or Dothideomycetes (mainly in Pleosporales). The presence of Arthrinium and Magnaporthaceae species, most often associated with poaceous plants, suggested a degree of host specificity. A polyphasic approach was employed to identify two Muscodor taxa based on (i) ITS and RPB2 phylogenies, (ii) volatile compounds produced, and (iii) an in vitro bioassay of antifungal activity. This to our knowledge is only the second report regarding the isolation of Muscodor spp. in China. Therefore, we hypothesize that wild plants represent a huge reservoir of unknown fungi. The prevalence, novelty, and species-specificity of unique isolates necessitate a reevaluation of their contribution to ecosystem function and fungal biodiversity.

From pattern to process: species and functional diversity in fungal endophytes of Abies beshanzuensis.

The biodiversity-functional relationship in fungal ecology was recently developed and debated, but has rarely been addressed in endophytes. In this study, an integrative culture system was designed to capture a rich fungal consortium from the conifer Abies beshanzuensis. Results indicate an impressive diversity of fungal lineages (a total of 84 taxa classified in Dikarya) and a relatively high proportion of hitherto unknown species (27.4%). The laccase gene was used as a functional marker due to its involvement in lignocellulose degradation. Remarkable diversity of laccase genes was found across a wide range of taxa, with at least 35 and 19 distinct sequences in ascomycetes and basidiomycetes respectively, were revealed. Many groups displayed variable ability to decompose needles. Furthermore, many ascomycetes, including three volatile-producing Muscodor species (Xylariaceae), showed the ability to inhibit pathogens. Notably, most laccase-producing species showed little or no antibiosis and vice versa. Clavicipitalean and ustilaginomycetous fungi, specifically toxic to insects, were inferred from taxonomic information. Intra-specific physiological variation in Pezicula sporulosa, a second dominant species, was clearly high. We conclude that a suite of defensive characteristics in endophytes contributes to improving host fitness under various stresses and that a diversity of laccase genes confers an ecological advantage in competition for nutrients. Intra-specific diversity may be of great ecological significance for ecotypic adaptation. These findings suggest a fair degree of functional complementarity rather than redundancy among endemic symbionts of natural plant populations.

[Effect of ischemia on the structure of bladder in rabbits with partial bladder outlet obstruction].

OBJECTIVE: To compare the structures and functions of rabbit bladder after partial bladder outlet obstruction versus without ischemia so as to explore the effects of ischemia on bladder pathogenesis in rabbits with partial bladder outlet obstruction. METHODS: A total of 64 mature male rabbits were divided into 4 groups (n = 16 each). Four of each group underwent operation to establish an ischemic animal model (ischemia group), another 4 underwent operation to establish a partial bladder outlet obstruction animal model (obstruct group), the other 4 underwent operation to establish an ischemic and partial bladder outlet obstruction animal model (combination group) and the remaining 4 underwent a sham operation as control. The rabbits in 4 groups were evaluated at Week 1, 2, 4 and 8 post-operation respectively. The weight of bladder, the thickness of mucosal, submucosa, muscular layer and placenta percreta and the activities of sarco/endoplasmic reticulum Ca(2+) ATPase citrate synthase of cystic smooth muscle were detected respectively. MASSON staining was used to observe the smooth muscle and collagen in stroma of bladder and S-100 staining for observing the neurons in bladder. RESULTS: In obstruct and combination groups, the weights of bladder at week 1 were (5.10 ± 0.29) g and (4.80 ± 0.37) g respectively. They were both significantly higher than control group [(1.93 ± 0.17) g, all P < 0.05]. The weights of bladder in obstruct and combination groups peaked at Week 4 and they were (18.48 ± 2.03) g and (12.35 ± 0.39) g respectively. The weight of bladder in obstruct group was significantly heavier than combination group in the same terms. And they were both significantly heavier than control and ischemia groups (all P < 0.05). Muscular tissue vicariously thickened during the first 4 weeks, and collagen and stroma increased at Week 4 in obstruct group. Muscular tissue, collagen and stroma all increased initially. But at Week 2 only collagen and stroma increased in combination group. Compare with control group, the other groups all have deletion of neurons, especially in combination group. The activities of sarco/endoplasmic reticulum Ca(2+) ATPase and citrate synthase of cystic smooth muscle of obstruct group peaked at Week 4. In combination group, the activities of sarco/endoplasmic reticulum Ca(2+) ATPase and citrate synthase of cystic smooth muscle were decreased over 2 - 8 weeks. In the same terms, the activities of sarco/endoplasmic reticulum Ca(2+) ATPase and citrate synthase of cystic smooth muscle in control group were significantly higher than those in obstruct and combination groups (all P < 0.05). CONCLUSION: Ischemia can reduce the tolerance of bladder and aggravate the impairment of bladder to partial outlet obstruction.

Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds.

The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.

A new species of Harpophora (Magnaporthaceae) recovered from healthy wild rice (Oryza granulata) roots, representing a novel member of a beneficial dark septate endophyte.

A survey of the endophytic fungal community of wild rice (Oryza granulata) in China was conducted. Two isolates recovered from healthy roots are assumed to be dark septate endophytes (DSEs). They are morphologically similar to species from the genus Harpophora and are identified as a new species, Harpophora oryzae, based on the molecular phylogeny and morphological characteristics. A neighbor-joining tree constructed from ITS-5.8S rRNA gene regions reveals that H. oryzae forms a distinctive subclade within the genus Harpophora, and is not genetically close to other species of Harpophora. Harpophora oryzae exhibits a moderate growth rate, with a frequent production of rope-like strands. It sporulates readily on artificial medium. Phialides are usually flask or bottle shaped and occur singly along hyphae or laterally and terminally on branched, hyaline to brown conidiophores, and also form whorls on metulae. Conidiophores are mostly branched with a slightly thickened wall, varying in dimensions. Conidia are one-celled and hyaline, most of them being falcate and strongly curved. The morphological differences between Harpophora spp. and Harpophora-like anamorphs representing different orders are also discussed. An in vitro inoculation test showed that H. oryzae may contribute towards improving rice (Oryza sativa L.) growth. Microscopic inspection of roots and phylogenetic placement of isolates further confirmed that H. oryzae represents a novel member of DSEs.

Identity, diversity, and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice (Oryza granulate) from a nature reserve in Yunnan, China.

Rice (Oryza sativa L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root endophytic mycobiota residing in roots of O. granulate by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia macrospinosa, and the Ceratobasidium/Rhizoctonia complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions.