Aldsulfin, a novel unusual anti-mannheimiosis epithiodiketopiperazine antibiotic produced by Lasiodiplodia pseudotheobromae FKI-4499.

An anti-mannheimiosis agent, aldsulfin, was isolated from a culture broth of the fungus Lasiodiplodia pseudotheobromae FKI-4499, together with a known compound, lasiodipline C, using bioassay-guided fractionation. Spectroscopic analysis of aldsulfin, using NMR, mass spectrometry, and CD analyses revealed it to be an epithiodiketopiperazine with an unstable and unusual hemithioaminal moiety. Aldsulfin showed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida.

Genetic diversity studies based on morpho-pathological and molecular variability of the Sclerotinia sclerotiorum population infecting potato (Solanum tuberosum L.).

White mould or stem rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is a devastating fungal disease found in major potato cultivation areas worldwide. The aim of this study was to characterize genetic diversity in the S. sclerotiorum population from the main potato producing regions of India by means of morphological (mycelial growth, colony colour, number and distribution pattern of sclerotia) and molecular characteristics, as well as to evaluate the virulence of S. sclerotiorum isolates in potato for the first time. Among the S. sclerotiorum population analyzed, high phenotypic and genotypic diversity were observed. Using all the morphological characteristics, a dendrogram was constructed based on Gower's similarity coefficient that distributed all the isolates into three clusters at the 0.62 similarity coefficient. Carpogenic germination of apothecia revealed that larger sclerotia produced a greater number of apothecia while smaller sclerotia produced fewer apothecia. Pathogenicity test results revealed that out of 25 isolates, seven were highly aggressive, 14 were moderate and four had low aggressiveness, whilst isolates from Punjab were more pathogenic than those of Uttar Pradesh. Phylogenetic analysis of universal rice primer polymorphism showed high genetic variability within the isolates that grouped all the isolates in three evolutionary lineages in the resulting dendrogram and showed partial relationship with geographical locations of the isolates. Further, the findings suggest the occurrence of higher heterogeneity and genetic diversity among the S. sclerotiorum isolates that indicates the existence of both clonal and sexual reproduction in the pathogen population of potato producing areas in India.

Identification and characterization of fungi associated with blister blight lesions of tea (Camellia sinensis L. Kuntze) isolated from Meghalaya, India.

Diseases in plants are mostly caused by fungi. Fungal interactions with the host can be either biotrophic, necrotrophic or hemibiotrophic. Synergistic polymicrobial interactions have been recently recognized that can also attribute to the occurrence of complex plant diseases. Tea is one of the most widely consumed beverages worldwide, although tea plants are affected by many different diseases causing a significant reduction in global tea production. Blister blight is one such serious and damaging leaf disease of tea. An assessment of blister blight disease was carried out at the tea development center in Umsning, Meghalaya. A considerable number of tea varieties showed characteristic blister blight symptoms that ranged from preliminary yellow spots in the upper leaf surface, matured white sporulating blisters in the lower leaf surface, and delayed brown necrotic lesions throughout the surfaces of the leaves. A total of 42 isolates, 15 from initial, 15 from mature, and 12 from necrotic stages were isolated from the symptomatic leaf samples. Pestalotiopsis and Nigrospora were the two fungi incessantly isolated from the diseased leaves. Colony characteristics that included colony, hyphal, and spore morphologies were examined and mycelial accumulation, sporulation, and sporal germination were determined for all the isolates of Pestalotiopsis and Nigrospora. Molecular analysis based on ITS-RFLP was performed for identification and genetic variability. In vitro pathogenicity assay revealed that Pestalotiopsis spp. and Nigrospora sp. developed distinct characteristics symptoms on greenhouse acclimated TV17 tea clones. To our knowledge, this is the first report of the prevalence of tea blister blight disease in Meghalaya and it is an initial attempt to identify fungal pathogens during different stages of blister blight disease.

Species diversity of Pleosporalean taxa associated with Camellia sinensis (L.) Kuntze in Taiwan.

Pleosporales species are important plant pathogens, saprobes, and endophytes on a wide range of economically important plant hosts. The classification of Pleosporales has undergone various modifications in recent years due to the addition of many families described from multiple habitats with a high level of morphological deviation. Numerous asexual genera have been described in Pleosporales that can be either hyphomyceteous or coelomycetous. Phoma- or coniothyrium-like species are common and have been revealed as polyphyletic in the order Pleosporales and linked with several sexual genera. A total of 31 pleosporalean strains were isolated in different regions of Taiwan between 2017 and 2018 from the leaves of Camellia sinensis plants with symptoms of leaf spot disease. These strains were evaluated morphologically and genotypically using multi-locus sequence analyses of the ITS, LSU, SSU, rpb2, tef1 and tub2 genes. The results demonstrated the affiliation of these strains with the various families in Pleosporales and revealed the presence of one new genus (Neoshiraia) and eight new species (Alloconiothyrium camelliae, Amorocoelophoma camelliae, Leucaenicola camelliae, L. taiwanensis, Neoshiraia camelliae, N. taiwanensis, Paraconiothyrium camelliae and Paraphaeosphaeria camelliae). Furthermore, to the best of our understanding, Didymella segeticola, Ectophoma pomi and Roussoella mexican were reported for the first time from C. sinensis in Taiwan.

A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds.

Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.

[Isolation and identification of endophytic fungi from Huperzia serrata and their metabolites' inhibitory activities against acetylcholinesterase and anti-inflammatory activities].

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.

Characterization and Pathogenicity of Pestalotiopsis-Like Species Associated With Gray Blight Disease on Camellia sinensis in Anhui Province, China.

Gray blight disease, caused by Pestalotiopsis-like fungi, is one of the deadliest threats to tea (Camellia sinensis) production. However, little information is known about the traits and characteristics of this pathogen. Here, a systematic survey was performed, and a total of 20 representative isolates were obtained from the leaves of tea plants affected by gray blight in two main tea plantations located in Anhui Province, China. Further analyses showed that two isolates were identified as Neopestalotiopsis ellipsospora, three isolates were regarded as Pseudopestalotiopsis chinensis, one isolate was considered as Pseudopestalotiopsis camelliae-sinensis, and the remaining isolates belonged to Pseudopestalotiopsis spp., on the basis of morphological characteristics and multigene phylogenetic analyses of the internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α. Pathogenicity tests indicated that there were significant differences in virulence among the Neopestalotiopsis and Pseudopestalotiopsis isolates when inoculated on the leaves of the tea plant (C. sinensis 'Shuchazao'). Furthermore, varied pathogenicity was also observed for the same isolate when inoculated on different varieties of tea plants. To our knowledge, this is the first record of Neopestalotiopsis ellipsospora and Pseudopestalotiopsis chinensis causing gray blight disease of tea plants in China.

Diversity of Pestalotiopsis-Like Species Causing Gray Blight Disease of Tea Plants (Camellia sinensis) in China, Including two Novel Pestalotiopsis Species, and Analysis of Their Pathogenicity.

Several Pestalotiopsis-like species cause gray blight disease in tea plants, resulting in severe tea production losses. However, systematic and comprehensive research on the diversity, geographical distribution, and pathogenicity of pathogenic species associated with tea plants in China is limited. In this study, 168 Pestalotiopsis-like isolates were obtained from diseased tea plant leaves from 13 primary tea-producing provinces and cities in China. Based on a multilocus (internal transcribed spacer, translation elongation factor 1-α, and ß-tubulin gene region) phylogenetic analysis coupled with an assessment of conidial characteristics, 20 Neopestalotiopsis unclassified isolates, seven Pestalotiopsis species, including two novel (Pestalotiopsis menhaiensis and Pestalotiopsis sichuanensis), four known (Pestalotiopsis camelliae, Pestalotiopsis chamaeropis, Pestalotiopsis kenyana, and Pestalotiopsis rhodomyrtus) and one indistinguishable species, and three Pseudopestalotiopsis species, including two known (Pseudopestalotiopsis camelliae-sinensis and Pseudopestalotiopsis chinensis) and one indistinguishable species, were identified. This study is the first to evaluate Pestalotiopsis chamaeropis on tea plants in China. The geographical distribution and pathogenicity tests showed Pseudopestalotiopsis camelliae-sinensis to be the dominant cause of gray blight of tea plants in China. In vitro antifungal assays demonstrated that theobromine not only derepressed mycelial growth of the 29 representative isolates but also increased their growth. Correlation analysis revealed a linear positive relationship between the mycelial growth rate and pathogenicity (P = 0.0148).

Vochysiamides A and B: Two new bioactive carboxamides produced by the new species Diaporthe vochysiae.

Endophytic fungi have been considered a rich source for bioactive secondary metabolites with novel chemical structures. A high diverse group of endophytes, isolated from different medicinal plants, belongs to the genus Diaporthe. In a previously study performed by our group the crude extract of strain LGMF1583 showed considerable antibacterial activity mainly against Gram-negative bacteria. Based on ITS phylogeny analysis, strain LGMF1583 was identified as belonging to Diaporthe genus and may represent a new species. In the present study, we described the new species Diporthe vochysiae based on multilocus phylogeny analysis and morphological characteristics. The species name refers to the host, from which strain LGMF1583 was isolated, the medicinal plant Vochysia divergens. In view of the biotechnological potential of strain LGMF1583, we have also characterized the secondary metabolites produced by D. vochysiae. Chemical assessment of the D. vochysiae LGMF1583 revealed two new carboxamides, vochysiamides A (1) and B (2), in addition to the known metabolite, 2,5-dihydroxybenzyl alcohol (3). In the biological activity analysis, vochysiamide B (2) displayed considerable antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae (KPC), a producer of carbapenemases, MIC of 80 µg/mL. Carbapenemases are considered a major antimicrobial resistance threat, and infections caused by KPC have been considered a public health problem worldwide, and new compounds with activity against this bacterium are nowadays even more required.

Endophytic fungi from the branches of Camellia taliensis (W. W. Smith) Melchior, a widely distributed wild tea plant.

Camellia taliensis (W. W. Smith) Melchior is a wild tea plant endemic from the west and southwest of Yunnan province of China to the north of Myanmar and is used commonly to produce tea by the local people of its growing areas. Its chemical constituents are closely related to those of C. sinensis var. assamica, a widely cultivated tea plant. In this study, the α diversity and phylogeny of endophytic fungi in the branches of C. taliensis were explored for the first time. A total of 160 fungal strains were obtained and grouped into 42 species from 29 genera, which were identified based on rDNA internal transcribed spacer sequence analysis. Diversity analysis showed that the endophytic fungal community of the branches of C. taliensis had high species richness S (42), Margalef index D' (8.0785), Shannon-Wiener index H' (2.8494), Simpson diversity index DS (0.8891), PIE index (0.8947) and evenness Pielou index J (0.7623) but a low dominant index λ (0.1109). By contrast, that in the branches of C. taliensis had abundant species and high species evenness. Diaporthe tectonigena, Acrocalymma sp. and Colletotrichum magnisporum were the dominant endophytic fungi. The phylogenetic tree was established by maximum parsimony analysis, and the 11 orders observed for endophytic fungi belonging to Ascomycota and Basidiomycota were grouped into 4 classes.

Occurrence of oleaginous yeast from mangrove forest in Thailand.

A total of 191 yeasts were isolated from 197 samples collected from eight estuarine mangrove forests along four different coastlines of Thailand (Andaman Sea and the East, North and West coasts of the Gulf of Thailand). Of these, 178 isolates were identified as 32 species in 16 genera of Ascomycota, 12 species in nine genera of Basidiomycota, and 13 isolates as potential new species, respectively. Mangroves located along the Andaman Sea coastline had a higher yeast diversity at the species and genera levels than those along the Gulf of Thailand. Kluyveromyces siamensis was the most frequently isolated species, whilst Candida tropicalis was the only species isolated at all eight sites. Screening isolated yeast strains belonging to genera previously reported as oleaginous yeast plus the 13 potential new species, revealed two oleaginous strains, Rhodotorula sphaerocarpa 11-14.4 and Saitozyma podzolica 11-11.3.1. Both of these strains were isolated from the same mangrove forest on the Andaman Sea coastline. They could accumulate lipid when suspended in glucose solution without any supplementation, while the fatty acid composition and oil profile of Rh. sphaerocarpa 11-14.4 and Sait. podzolica 11-11.3.1 were similar to vegetable oil and cocoa butter, respectively.

Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Bioactive secondary metabolites from new endophytic fungus Curvularia. sp isolated from Rauwolfia macrophylla.

Over the last decades, endophytic fungi represent a new source of pharmacologically active secondary metabolites based on the underlying assumption that they live symbiotically within their plant host. In the present study, a new endophytic fungus was isolated from Rauwolfia macrophylla, a medicinal plant from Cameroon. The fungus showed a highest homology to Curvularia sp. based on complete nucleotide sequence data generated from the internal transcribed spacer (ITS) of ribosomal DNA region. Large scale fermentation, working-up and separation of the strain extract using different chromatographic techniques afforded three bioactive compounds: 2'-deoxyribolactone (1), hexylitaconic acid (2) and ergosterol (3). The chemical structures of compounds 1-3 were confirmed by 1 and 2D NMR spectroscopy and mass spectrometry, and comparison with corresponding literature data. Biologically, the antimicrobial, antioxidant activities and the acetylcholinesterase inhibitory of the isolated compounds were studied.

Alternative Hosts of Cercospora beticola in Field Surveys and Inoculation Trials.

Cercospora beticola, the cause of Cercospora leaf spot (CLS) of sugar beet and table beet, has a broad range of potential alternative hosts. The role of these hosts as inoculum sources in the field is unclear and has had limited investigation since the advent of DNA-based pathogen identification. The presence of C. beticola on alternative hosts associated with table beet fields of New York was assessed in field surveys during 2016. Lesions were collected, and 71 cercosporoid conidia were isolated for phylogenetic comparison. C. beticola was identified from Solanum ptycanthum (n = 4), Chenopodium album (n = 2), and Spinacia oleracea (n = 1), whereas C. chenopodii was identified on Chenopodium album (n = 51). Artificial inoculation of 21 plants species demonstrated that C. beticola was pathogenic to Brassica kaber, Chenopodium album, Carthamus tinctorius, Rumex obtusifolius, and Spinacia oleracea. These results indicate that although C. beticola may be pathogenic to a range of plant species, the role of symptomatic tissue for inoculum production on alternative hosts in the field appears limited. Observations of C. beticola on necrotic and naturally senescent tissue suggest saprophytic survival on plant debris of a range of species, which has implications for CLS epidemics and disease management.

Biotechnological application of endophytic filamentous bipolaris and curvularia: a review on bioeconomy impact.

The filamentous Bipolaris and Curvularia genera consist of species known to cause severe diseases in plants and animals amounting to an estimated annual loss of USD $10 billion worldwide. Despite the harmful effect of Bipolaris and Curvularia species, scarce attention is paid on beneficial areas where the fungi are used in industrial processes to generate biotechnological products. Catalytic potential of Bipolaris and Curvularia species in the production of biodiesel, bioflucculant, biosorbent, and mycoherbicide are promising for the bioeconomy. It is herein demonstrated that knowledge-based application of some endophytic Bipolaris and Curvularia species are indispensable vectors of sustainable economic development. In the twenty-first century, India, China, and the USA have taken progress in the biotechnological application of these fungi to generate wealth. As such, some Bipolaris and Curvularia species significantly impact on global crop improvement, act as catalyst in batch-reactors for biosynthesis of industrial enzymes and medicines, bioengineer of green-nanoparticle, agent of biofertilizer, bioremediation and bio-hydrometallurgy. For the first time, this study discusses the current advances in biotechnological application of Bipolaris and Curvularia species and provide new insights into the prospects of optimizing their bioengineering potential for developing bioeconomy.

Antiproliferative and antibiofilm potentials of endolichenic fungi associated with the lichen Nephroma laevigatum.

AIMS: The objective of this study was to explore the diversity of endolichenic fungi from Nephroma laevigatum and to investigate their antiproliferative and antibiofilm potential. METHODS AND RESULTS: Forty-six isolates were obtained and identified by DNA barcoding. They belonged to genera Nemania, Daldinia, Peziza and Coniochaeta. Six strains belonging to the most represented species were selected and tested for their antiproliferative and antibiofilm activities. Extracts were analysed by reversed-phase HPLC. Activities against fungal and bacterial biofilm were evaluated using tetrazolium salt (XTT) assay and crystal violet assay respectively. Antiproliferative responses of extracts were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction by two extracts was observed in two cell lines (HT-29 and PC-3) via morphological changes, pro-apoptotic and anti-apoptotic proteins analysis (Western blotting) and DNA fragmentation. Four extracts displayed activities against Candida albicans biofilm with IC50 values ranging from 25 to 200 µg ml-1 . All extracts were inactive against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The most active isolates against human colorectal (HT-29 and HCT116) and prostate (PC-3 and DU145) cancer cell lines were Nemania serpens (NL08) and Nemania aenea var. aureolatum (NL38) with IC50 values ranging from 13 to 39 µg ml-1 . These extracts induced an apoptotic process through activation of caspases 8 and 3, poly(ADP-ribose) polymerase cleavage and DNA fragmentation. CONCLUSIONS: Selected crude fungal extracts have antiproliferative and antibiofilm activities. Data suggest that this antipoliferative effect is due to apoptosis process. This is the first report showing the effects of endolichenic fungi from N. laevigatum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the therapeutic potential of endolichenic fungi metabolites as sources for drug discovery programmes.

Medicinal, Nutritional, and Cosmetic Values of Macrofungi Distributed in Mazandaran Province of Northern Iran (Review).

A total of 82 species and 2 variations of medicinal and edible macrofungi (Basidiomycota and Ascomy-cota) have been collected and identified in Mazandaran Province of Northern Iran. Among these species, 58 possess medicinal and culinary properties, whereas 21 possess only medicinal properties (antiinflammatory, antimicrobial, an-timutagenic, antioxidant, antitumor, antiplatelet, hypoglycemic, hypocholesterolemic, neurotropic, hypotensive, insec-ticidal, immunomodulating, mitogenic/regenerative, spasmolytic, etc.). No medicinal effect has yet been reported for 4 species (Cantharellus infundibuliformis, Mycena inclinata, Suillus collinitus, Xerocomus porosporus) and 2 variations (Pluteus cervinus var. albus, Russula cyanoxantha var. variata) of edible species. Among the listed species, 15 (such as Auricularia auricula-judae, Hericium erinaceus, Fomes fomentarius, Flammulina velutipes, Ganoderma lucidum, Lyophyllum decastes, Pleurotus ostreatus, Tremella mesenterica) are currently used for manufacturing organic cosmetic products. The well-known mushrooms with medicinal properties (e.g., Auricularia auricula-judae, Flammulina velutipes, Hericium erinaceus, Ganoderma lucidum, G. tsugae, G. adspersum, Trametes versicolor) and excellent edibility (e.g., Boletus edulis, Cantharellus cibarius, Morchella esculenta, Pleurotus ostreatus) are widely distributed in the studied area. The biological resources of macrofungi growing in Mazandaran Province of Northern Iran possess medicinal, nutritional, and cosmetic values and could be further used for biotechnological exploitation to develop mushroom-derived pharmaceuticals, nutriceuticals, nutraceuticals, and cosmeceuticals.

Diversity, specificity, co-occurrence and hub taxa of the bacterial-fungal pollen microbiome.

Flower pollen represents a unique microbial habitat, however the factors driving microbial assemblages and microbe-microbe interactions remain largely unexplored. Here we compared the structure and diversity of the bacterial-fungal microbiome between eight different pollen species (four wind-pollinated and four insect-pollinated) from close geographical locations, using high-throughput sequencing of the 16S the rRNA gene fragment (bacteria) and the internal transcribed spacer 2 (ITS2, fungi). Proteobacteria and Ascomycota were the most abundant bacterial and fungal phyla, respectively. Pseudomonas (bacterial) and Cladosporium (fungal) were the most abundant genera. Both bacterial and fungal microbiota were significantly influenced by plant species and pollination type, but showed a core microbiome consisting of 12 bacterial and 33 fungal genera. Co-occurrence analysis highlighted significant inter- and intra-kingdom interactions, and the interaction network was shaped by four bacterial hub taxa: Methylobacterium (two OTUs), Friedmanniella and Rosenbergiella. Rosenbergiella prevailed in insect-pollinated pollen and was negatively correlated with the other hubs, indicating habitat complementarity. Inter-kingdom co-occurrence showed a predominant effect of fungal on bacterial taxa. This study enhances our basic knowledge of pollen microbiota, and poses the basis for further inter- and intra-kingdom interaction studies in the plant reproductive organs.

[Isolation of endophytic fungi from Zanthoxylum simulans and screening of its active strain].

This study aims at making full use of microbial resources, and screening the active endophytic fungi of anti-rheumatoid arthritis from Zanthoxylum simulans. The endophytic fungi were cultured and isolated by tissue culture and scribing method, and the active strain of inhibiting the proliferation of human rheumatoid arthritis synovial fibroblasts (HFLS-RA) was screened by MTT method. Morphological characteristics and rDNA ITS1-5.8S-ITS2 sequences were applied for the taxonomy of endophytic fungi. Strains were isolated from Z. simulans. Among them, MK-05, MK-17, MK-19, MK-23 having inhibiting activity to HFLS-RA, the IC50 were 0.367, 0.775, 0.689, 0.757 g·L⁻¹, respectively. By classic morphologic classification and sequencing the PCR-amplified rDNA ITS1-5.8S-ITS2 regions, four effective strains were identified as Botryosphaeria dothidea, Phomopsis sp., P. liquidambari and Diaporthe perseae. The active endophyic fungi that inhibited the proliferation of HFLS-RA were screened from Z. simulans for the first time, and the results lay the foundation for the development and utilization of the Z. simulans resources.

In vitro susceptibilities of Neoscytalidium spp. sequence types to antifungal agents and antimicrobial photodynamic treatment with phenothiazinium photosensitizers.

Neoscytalidium spp. are ascomycetous fungi consisting of pigmented and hyaline varieties both able to cause skin and nail infection. Their color-based identification is inaccurate and may compromise the outcome of the studies with these fungi. The aim of this study was to genotype 32 isolates morphologically identified as Neoscytalidiumdimidiatum or N. dimidiatum var. hyalinum by multilocus sequence typing (MLST), differentiate the two varieties by their sequence types, evaluate their susceptibility to seven commercial antifungal drugs [amphotericin B (AMB), voriconazole (VOR), terbinafine (TER), 5-flucytosine (5FC), ketoconazole (KET), fluconazole (FLU), and caspofungin (CAS)], and also to the antimicrobial photodynamic treatment (APDT) with the phenothiazinium photosensitizers (PS) methylene blue (MB), new methylene blue (NMBN), toluidine blue O (TBO) and the pentacyclic derivative S137. The efficacy of each PS was determined, initially, based on its minimal inhibitory concentration (MIC). Additionally, the APDT effects with each PS on the survival of ungerminated and germinated arthroconidia of both varieties were evaluated. Seven loci of Neoscytalidium spp. were sequenced on MLST revealing eight polymorphic sites and six sequence types (ST). All N. dimidiatum var. hyalinum isolates were clustered in a single ST. AMB, VOR and TER were the most effective antifungal agents against both varieties. The hyaline variety isolates were much less tolerant to the azoles than the isolates of the pigmented variety. APDT with S137 showed the lowest MIC for all the isolates of both varieties. APDT with all the PS killed both ungerminated and germinated arthroconidia of both varieties reducing the survival up to 5 logs. Isolates of the hyaline variety were also less tolerant to APDT. APDT with the four PS also increased the plasma membrane permeability of arthroconidia of both varieties but only NMBN and S137 caused peroxidation of the membrane lipids.