First report of leaf blight on pumpkin caused by Nigrospora sphaerica in China.

Pumpkin (Cucurbita moschata), which belongs to the gourd family (Cucurbitaceae), is widely planted throughout the world. In June 2023, many pumpkin plants (cv. Miben) displayed leaf blight and chlorosis in fields located in Suizhou (31.99°N, 113.02°E), Hubei Province, China. The disease incidence ranged from 30 to 40% in nine fields, 6.3 ha in total. The symptoms were irregularly shaped lesions that expanded along the mid-vein until the leaf turned brown and wilted. Fungal isolations were performed as described previously (Liu et al. 2023). Twenty pumpkin leaf samples with typical symptoms were collected and cut into 1 cm×1 cm pieces. The diseased tissue was surface-sterilized in 75% ethanol for 30 sec, plated on potato dextrose agar (PDA) medium and incubated at 25℃ for 3 days. Then, the emerging single fungal hyphal tip was transferred onto PDA plates to obtain purified isolates. A total of eighteen isolates on PDA plates were initially white and then developed to dark gray. The 5-day-old cultures growing on mung bean medium produced conidia that were black, single-celled, smooth, spherical or oblate, and ranged in size from 14.5 to 20.8 µm×13.3 to 20.5 µm (n=50). Therefore, the isolates were morphologically identified as Nigrospora sphaerica. Moreover, the genomic DNA of the isolates (HB-P1,HB-P2, and HB-P3) was extracted for amplification and sequencing of the regions of internal transcribed spacer (ITS) (White et al. 1990), nuclear large subunit rRNA (nLSU) (O'Donnell 1992; Rehner and Samuels 1994), and ß-tubulin (TUB2) (Glass and Donaldson 1995), with primers ITS1/ITS4, LROR/LR3, and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank under accession numbers PP348112, PP348113, PP348114 (ITS), PP411414, PP411415, PP411416 (nLSU), and PP357438, PP357439, PP357440 (TUB2). BLASTn showed that the sequences ITS, nLSU, and TUB2 of HB-P1, HB-P2, and HB-P3 had >99% nucleotide identities ((ITS: 100%, 508/508 bp, MF996488.1; 99.8%, 506/507, ON326588.1; 100%, 500/500 ,MK748317.1), (nLSU: 99.83%, 573/574, KT462720.1; 99.83% , 574/575 bp, KT462720.1; 99.65%, 575/577, KT462720.1), and (TUB2: 100%, 388/388, MN719407.1; 99.74%, 387/388, MN719407.1; 100%, 387/387, MN719407.1)) with Nigrospora sphaerica, respectively. A multilocus (ITS, nLSU and TUB2) phylogenetic analysis indicated that the isolates were Nigrospora sphaerica. Pathogenicity of three isolates were tested on pumpkin plants (cv. Miben). Fifteen pumpkin plants were inoculated by spraying the leaves (1×106 spores/ml), respectively, and 10 pumpkin plants were treated with sterile water as a negative control. All plants were incubated in an artificial climate box (LongYue, ShangHai) at 25℃ for 12 days. The experiment was repeated three times. Twelve days later, the inoculated pumpkin plants developed symptoms of leaf blight, while the control plants remained healthy. Then, pathogens were re-isolated from the each leaf of inoculated pumpkin plants and not from the control plants. Nigrospora sphaerica has been previously reported to cause leaf spot on watermelon in Malaysia (Ismail and Abd Razak 2021). To our knowledge, this is the first report of N. sphaerica causing leaf blight on pumpkin in China. This new disease can cause leaf blight, which may affect pumpkin productivity.

Endophytic fungi of Tradescantia pallida mediated targeting of Multi-Drug resistant human pathogens.

Antimicrobial resistance (AMR) has emerged as one of the most serious worldwide public health issues of the twenty-first century. The expeditious rise of AMR has urged the development of new, natural effective therapeutic strategies against drug-resistant pathogens. Endophytic fungi, which inhabit distinctive environments like endosymbiotic relationships with plants, are gaining interest as alternative reservoirs for novel compounds that exhibit a broad range of chemical diversity and unique modes of action by releasing a variety of secondary metabolites with antimicrobial properties. The objective of the current research was to isolate and identify endophytic fungal species from leaves of Tradescantia pallida and to investigate their antagonistic effects on Multi-Drug-Resistant human pathogens. Endophytic fungus TPL11 and TPL14 showed maximum inhibition in agar plug and agar well diffusion assay. The ethyl acetate crude extract effectively suppressed growth of MRSA (Methicillin-resistant Staphylococcus aureus) ATCC 43300,700699 strains and VRE (Vancomycin-resistant Enterococcus) with the Inhibition zone of 22 ± 0.05, 23 ± 0.11 and 24 ± 0.11 mm respectively with minimum inhibitory concentration (MIC) of 3.125 µg/mL. Whereas TPL11 fungus revealed antibiosis of 22 ± 0.05 and 21 ± 0.15 mm against MRSA(ATCC 43300,700699) and 24 ± 0.05 mm for VRE with MIC of 6.25,3.125 and 1.56 µg/mL respectively. The MIC (Minimum inhibitory concentration) index further confirmed that both the extracts were bacteriostatic against MRSA and bactericidal against VRE. The isolates TPL11 and TPL14 were identified as Fusarium oxysporum and Nigrospora sphaerica by 18S rRNA internal transcribed spacer (ITS) sequencing. To our insight, it is the first report to reveal the presence of F.oxysporum and N.sphaerica in T.pallida and their antibacterial activity.

First Report of Nigrospora sphaerica causing Leaf Spot on Rhododendron simsii in China.

Rhododendron simsii (indoor azalea) is widely cultivated for its high ornamental value (Xu et al. 2021). In April to May 2023, a leaf spot disease occurred in a field study at the Baili Azalea Forest Area (27°12'N, 105°48'E), Guizhou Province, China. About 500 plants were investigated, and the results showed that the incidence of leaf spot was 20 ~ 30%. To study this disease, 10 plants showing severe symptoms were collected. Initially, the symptoms were round or irregularly shaped brown spots (1 to 10 mm). With time, the spots enlarged and merged. Symptomatic leaves were washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were removed. After surface sterilization (30 s with 75% ethanol, 2 min with 3% NaOCl, then washed three times with sterilized distilled water), the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Fungal colonies developed from leaf tissues, and the germinated spores were transferred onto PDA for further purification and morphological observation. Three isolates (GUBJ23, GUBJ24, and GUBJ12) with similar morphology were obtained from five affected leaves. The representative strain GUBJ23 was selected for further study. On PDA the mycelium was initially white but with sporulation turned gray and then black. Black, single-celled conidia, spherical to sub-spherical, from 11.80 to 21.39 × 13.38 to 21.83 µm (n = 50) in diameter were borne singly on hyaline vesicles at the tips of conidiophores. These morphological characteristics were similar to those of Nigrospora sphaerica (Wang et al. 2017). To confirm the identification, primer pairs for the internal transcribed spacer (ITS) region (ITS5/ITS4), ß-tubulin (TUB2) (Bt-2a/Bt-2b), and the translation elongation factor 1-alpha (TEF1-α) (EF1-728F/EF1-986R), were used for PCR amplification of DNA from strain GUBJ23 (Carbone and Kohn 1999; Glass et al. 1995; White et al. 1990). The resulting sequences were deposited in GenBank with accession numbers OR818025 (ITS), OR835150 (TUB2), and OR835147 (TEF1-α). BLAST searches of the sequences revealed 99.80% identity (503/504 bp) of the ITS sequence, 100.00% identity (395/395 bp) of the TUB2 sequence, and 100.00% identity of the TEF1-α sequence (241/241 bp) with N. sphaerica LC7294 (accessions KX985932, KY019602, and KY019397, respectively.) Based on a combined dataset of ITS, TEF1-α, and TUB2 sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that isolates GUBJ23, GUBJ24, and GUBJ12 were N. sphaerica (Wang et al. 2017). Leaves of three healthy R. simsii plants were spray-inoculated with a spore suspension (105 conidia/mL), and an additional three plants were sprayed with sterile water. These plants were incubated at 25℃ in 75% relative humidity. After 5 to 7 days of inoculation, 0.5 to 1.8 mm spots appeared on the leaves. At 10 to 14 days after inoculation, grayish brown, semicircular or irregular lesions appeared on the leaves, usually with a diameter of 0.8 to 3 mm. The symptoms were like symptoms seen on naturally infected leaves, while the control leaves remained asymptomatic. The pathogen was re-isolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, TUB and TEF1-α), and the reisolated pathogen was identical to N. sphaerica. Thus completing Koch's postulates. According to previous research, N. sphaerica is a widely distributed phytopathogenic fungus that has a wide host range (Wang et al. 2017). This study is the first to identify N. sphaerica as the cause of leaf spot disease in R. simsii. Given the popularity of R. simsii as a pot plant and landscape shrub in Asia and othr regions, the occurrence of leaf spot disease seriously affects its ornamental and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce impact of the disease.

First Report of Leaf Spot on Zanthoxylum bungeanum Caused by Nigrospora sphaerica in China.

Zanthoxylum bungeanum Maxim., a deciduous shrub in Zanthoxylum genus of the Rutaceae family, has not only highly economical values as condiment and medicine, but also significantly ecological values in soil and water conservation. In March 2023, a typical leaf spot disease on Z. bungeanum (Variety "Xiao Qingjiao") was observed in the field with an area of 26.68 ha with 35% incidence and 25.4% disease intensity in Zhenfeng County (25°38'57.60″ N, 105°64'98.64″ E, 1,156 m), Guizhou Province, China. The symptom leaves showed as irregularly shaped necrotic lesions, brown to dark brown with black margin. 30 samples with typical symptoms were collected and cut into 0.5 cm × 0.5 cm pieces. Their surfaces were disinfected with 1.5% NaClO for 2 min followed by 75% ethanol for 35 s, rinsed three times with sterile distilled water, finally incubated on PDA plates at 27°C. A total of 36 isolates were obtained through single-spore cultivation. The colonies on PDA were fluffy with abundant aerial mycelia and covered the whole plates (diameter 90 mm) in 7 days. Conidia were brown to black, single-celled, smooth, spherical or oblate, 12.0-17.0 × 12.5-18.5 µm (av. = 14.5 × 15.5 µm, n = 50) and grew on a colorless transparent vesicle at the apical cell of conidiophores. The morphological characteristics were similar with N. sphaerica (Wang et al. 2017). The 5.8S DNA (ITS), translation elongation factor 1-alpha (TEF1-α) and ß-tubulin (TUB2) genes were amplified with primers ITS4/ITS5, EF1-728F/EF2, and BT2A/BT2B, respectively (White et al. 1990; Carbone and Kohn 1999, O'Donnell et al. 1998; Glass and Donaldson 1995). The ITS, TEF1-α and TUB2 sequences of two randomly selected isolates, GUCC 21-187 and GUCC 21-235, had > 99% nucleotide identities (ITS: 99.60% (504/506 bp, OR646539) and 99.61% (506/508 bp, OR640300); TEF: 100% (470/470 bp, OR654285) and 100.00% (471/471 bp, OR654286); TUB: 100.00% (408/408 bp, OR661269) and 99.52% (411/413 bp, OR661270), respectively) with those sequences of N. sphaerica (LC 7294) in GenBank (KX985932, KY019397 and KY019602, respectively). The phylogenetic tree based on sequences of ITS, TEF1-α and TUB2 indicated that GUCC 21-187 and GUCC 21-235 were most closely related to N. sphaerica (LC 7294), supported with 100%/100%/1 bootstraps. Based on morphological characteristics and molecular datasets analyses, the isolates were identified as N. sphaerica. 10 healthy 2-years-old Z. bungeanum plants were sprayed with conidial suspensions (1 × 106 conidia/mL) of the isolates and the other 5ere sprayed with sterile water as the controls, all the treated plants were cultivated in a glasshouse at 25°C under 85% relative humidity. Typical leaf spot symptoms appeared on inoculated Z. bungeanum plants after 8 days, while the control plants remained asymptomatic. N. sphaerica was re-isolated from the lesions of inoculated plants and identified by morphological and molecular identification. Pathogenicity test was performed three times with analogous results, fulfilling Koch's postulates. N. sphaerica had been reported as a common pathogen on a variety of plants including sugarcane, kiwifruit and blueberry (Cui et al. 2018; Chen et al. 2016; Wright et al. 2008). To our knowledge, this is the first report of leaf spot disease caused by N. sphaerica on Z. bungeanum in China. Our report would be helpful to Z. bungeanum growers to recognize this leaf spot disease, and corresponding measures could be taken to minimize or avoid the economic losses caused by it.

First Report of Fungal Pathogens Causing Leaf Spot on Sorghum-Sudangrass Hybrids and Their Interactions with Plants.

The sorghum-sudangrass hybrid is the main high-quality forage grass in Southwest China, but, in recent years, it has suffered from leaf spot disease, with a prevalence of 88% in Bazhong, Sichuan, China, seriously affecting yield and quality. The causal agents were obtained from symptomatic leaves by tissue isolation and verified by pathogenicity assays. A combination of morphological characterization and sequence analysis revealed that strains SCBZSL1, SCBZSX5, and SCBZSW6 were Nigrospora sphaerica, Colletotrichum boninense, and Didymella corylicola, respectively, and the latter two were the first instance to be reported on sorghum-sudangrass hybrids in the world. SCBZSX5 significantly affected the growth of the plants, which can reduce plant height by 25%. The biological characteristics of SCBZSX5 were found to be less sensitive to the change in light and pH, and its most suitable culture medium was Potato Dextrose Agar (PDA), with the optimal temperature of 25 °C and lethal temperature of 35 °C. To clarify the interactions between the pathogen SCBZSX5 and plants, metabolomics analyses revealed that 211 differential metabolites were mainly enriched in amino acid metabolism and flavonoid metabolism. C. boninense disrupted the osmotic balance of the plant by decreasing the content of acetyl proline and caffeic acid in the plant, resulting in disease occurrence, whereas the sorghum-sudangrass hybrids improved tolerance and antioxidant properties through the accumulation of tyrosine, tryptophan, glutamic acid, leucine, glycitein, naringenin, and apigetrin to resist the damage caused by C. boninense. This study revealed the mutualistic relationship between sorghum-sudangrass hybrids and C. boninense, which provided a reference for the control of the disease.

Antioxidant and Antidiabetic Activities, and UHPLC-ESI-QTOF-MS-Based Metabolite Profiling of an Endophytic Fungus Nigrospora sphaerica BRN 01 Isolated from Bauhinia purpurea L.

Diabetes-associated postprandial hyperglycemia is a major risk factor in cardiovascular disease. Since enzyme α-glucosidase is primarily responsible for glucose release during digestion, inhibiting it mitigates post-meal spike in blood glucose level. Metabolites from endophytic fungi could be potential natural inhibitors of this enzyme. Endophytic fungi isolated from Bauhinia purpurea L. were screened for their potential antioxidant and antidiabetic activities. Ethyl acetate extract of Nigrospora sphaerica BRN 01 (NEE) displayed high antioxidant activity with an IC50 value of 9.72 ± 0.91 µg/ml for DPPH assay and ferric reducing antioxidant power (FRAP) of 1595 ± 0.23 µmol AAE g-1 DW. NEE also showed high degree of inhibition of α-glucosidase activity with an IC50 value of 0.020 ± 0.001 mg/ml, significantly greater than the standard drug acarbose (0.494 ± 0.009 mg/ml). Metabolite profiling of NEE was carried using ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) and 21 metabolites identified based on the MS/MS fragmentation patterns. Docking analysis of all 21 identified metabolites was carried out. Of these, 6 showed binding energies higher than acarbose (- 6.6 kcal/mol). Based on the analysis of interactions of feruloyl glucose with active site residues of the enzyme, it could be a potential α-glucosidase inhibitor. Metabolites of Nigrospora sphaerica BRN 01, therefore, could be potential lead molecules for design and development of antidiabetic drugs.

A novel steroid hydroxylase from Nigrospora sphaerica with various hydroxylation capabilities to different steroid substrates.

Fungal hydroxylation of steroids is a key step in the industrial production of various steroid drugs. The main enzymes that enable these reactions are Cytochrome P450s (CYP), though very few industrially important CYPs have been identified and characterized. In this study, we identified a CYP enzyme (CYP-N2) and a cytochrome P450 reductase (CPRns) from Nigrospora sphaerica 722 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris. Gene CYP-N2 co-expressed with CPRns in Pichia pastoris GS115 showed 6ß- and 15α-hydroxylation activities on progesterone. Different hydroxylation specificity of CYP-N2 was observed on different steroid substrates. CYP-N2 showed 1α-hydroxylation on cortisone and 1α-hydroxylation and 6ß-hydroxylation activities on androstenedione (AD). With dehydroepiandrosterone (DHEA) as a substrate, the hydroxylated products of CYP-N2 included 7α-hydroxy-DHEA and 7α,15α-dihydroxy-DHEA. In order to precisely elucidate CYP-N2 biological function and find out the key amino acids influencing its hydroxylation capabilities in the binding pocket, new generation artificial intelligence technology AlphaFold 2 was used to predict the function-structure of CYP-N2 with high reliability. Through molecular docking, it was concluded that the residues almost binding all substrates were located in the same substrate binding pocket and the various hydroxylation abilities might be due to the different binding conformations of different substrates in the binding pocket. Alanine scanning mutagenesis was used to verify key amino acids identified by the molecular docking with steroid substrates. The 128 THR mutation resulted in conversion rate increase for substrates AD and cortisone by 2.6-fold and 2.1-fold respectively. The information obtained in this study is beneficial to facilitating the engineering of more efficient steroid hydroxylases for industrial applications.

First Report of Stem brown spot on 'Thai Gold' Selenicereus megalanthus in Malaysia caused by Nigrospora sphaerica.

'Thai Gold' yellow pitahaya (family Cactaceae, Selenicereus megalanthus) is a new crop being planted commercially in Malaysia. In May 2021, reddish-brown necrotic lesions were observed on the stems of approximately 60% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). As the disease progressed, the smaller lesions merged into larger irregularly shaped areas that formed dark brown in color. Stems with reddish-brown spot symptoms from ten plants were collected from the field and brought to the laboratory in sterilized paper bags. The symptom margin was excised into small blocks (5 x 5 x 5 mm). The blocks were surface sterilized based on Khoo et al. (2022), and placed on potato dextrose agar (PDA). The pathogens were isolated (three isolates were obtained) and cultured on potato dextrose agar (PDA) at 25°C for 5 days in the dark. The isolates developed floccose, white colony that darkened with age in PDA. Conidia (n = 30) were single celled, black, smooth, globose to subglobose, 13.9 to 18.7 µm in diameter, and borne singly on a hyaline vesicle at the tip of each conidiophore. Genomic DNA was extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (tef1-a) region and ß-tubulin (tub2) genes were performed using ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T10/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) primer sets, respectively. The products were sent to Apical Scientific Sdn. Bhd. for purification and sequencing. BLASTn analysis of the newly generated ITS (OK448496, OM832586, OM832589) were 100% identical to Nigrospora sphaerica isolate 1SS (MN339998) (507/507 bp), tef1-a (OM223859, OM826971, OM826972) were 100% identical to Nigrospora sphaerica isolate F (MT708197) (497/497 bp) and tub2 (OL697400, OM826973, OM826974) were 100% identical to Nigrospora sphaerica isolate SN180517 (MN719407) (434/434 bp). The isolates established a supported clade to the related N. sphaerica type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, tef1-a and tub2 sequences. Morphological and molecular characterization matched the description of N. sphaerica (Kee et al. 2019). Koch's postulates were performed by spray inoculation (106 spores/ml) of isolate Keningau on the stem of three 'Thai Gold' yellow pitahaya plants in growth stage 4 (BBCH code: 419) (Kishore, 2016), while water was sprayed on three mock controls. The experiment was repeated using isolate Keningau02 and Keningau03 as inoculants. The inoculated stems on yellow pitahaya plants were covered with plastics for 48 h, and the plants were maintained in a greenhouse at room temperature 25 to 28°C with a relative humidity of 80 to 90%. All the inoculated stems developed symptoms 5 days post-inoculation, whereas no symptoms occurred on mock controls, thus fulfilling the Koch's postulates. No pathogen was isolated from the mock controls. The experiments were repeated two more times for each isolate. The reisolated fungi were identical to N. sphaerica morphologically and molecularly. Previously, N. sphaerica has been reported to cause stem brown spot disease on S. megalanthus in the Philippines (Taguiam et al. 2020). To our knowledge, this is the first report of N. sphaerica causing stem brown spot on 'Thai Gold' S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for the Malaysian yellow pitahaya production.

A Genome Sequence Resource of Nigrospora sphaerica Causing Fruit Dried-Shrink Disease in Akebia trifoliata.

Nigrospora sphaerica is a worldwide plant pathogen causing fruit or leaf diseases on a variety of plant hosts such as Citrullus lanatus, Vigna unguiculata, Hylocereus polyrhizus, and Akebia trifoliata and other potential hosts. Here we report the first genome resource with high-quality assembly of the N. sphaerica strain ZJJ-C1, which causes fruit dried-shrink disease in A. trifoliata in China. The genome sequence of ZJJ-C1 will be useful for studying the evolution, host adaptation, and pathogenicity of N. sphaerica, which will be beneficial for a better understanding of the mechanisms of host-pathogen interaction during the endophytic period.

Epigenetic Modifiers Revamp Secondary Metabolite Production in Endophytic Nigrospora sphaerica.

During plant interaction, endophytes provide benefits to the host plant. Endophytes also contribute a variety of structural attributes with biological potential. Nigrospora sphaerica, which produces phomalactone from Adiantum philippense L., was subjected to epigenetic modification. High-performance liquid chromatography (HPLC) and Gas chromatography-mass spectrometry (GCMS) analysis were used to determine secondary metabolite profiling. Epigenetic modifiers like DNA Methyltransferase (DNMT) and Histone deacetylase (HDAC) inhibitors increased the expression of biosynthetic pathways. The activation of new metabolites was observed as a result of the activation of cryptic biosynthetic gene clusters, as well as the silencing of phomalactone in some treatments. When compared to DNMT treatments, HDAC treatments showed a significant increase in cryptic metabolite induction. The induction of cryptic metabolites with biological significance by HDAC treatment is supported by our findings.

Multigene Phylogeny, Diversity and Antimicrobial Potential of Endophytic Sordariomycetes From Rosa roxburghii.

Rosa roxburghii Tratt. is widely applied in food, cosmetics, and traditional medicine, and has been demonstrated to possess diverse bioactivities. Plant endophytic fungi are important microbial resources with great potential for application in many fields. They not only establish mutualistic symbiosis with host plants but also produce a variety of bioactive compounds. Therefore, in the present study, endophytic fungi were isolated from R. roxburghii, the diversity and antimicrobial activities were evaluated. As a result, 242 strains of endophytic Sordariomycetes were successfully isolated. Multigene phylogenetic analyses showed that these isolates included eight orders, 19 families, 33 genera. The dominant genera were Diaporthe (31.4%), Fusarium (14.4%), Chaetomium (7.9%), Dactylonectria (7.0%), Graphium (4.5%), Colletotrichum (4.1%), and Clonostachys (4.1%). For different tissues of R. roxburghii, alpha diversity analysis revealed that the diversity of fungal communities decreased in the order of root, fruit, stem, flower, leaf, and seed, and Clonostachys and Dactylonectria exhibited obvious tissue specificity. Meanwhile, functional annotation of 33 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. Additionally, antimicrobial activities of endophytic Sordariomycetes against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Pyricularia oryzae, Rhizoctonia solani, Fusarium oxysporum, Pseudomonas syringae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa were screened. Dual culture test assays showed that there were 40 different endophytic species with strong inhibition of at least one or moderate inhibition of two or more against the 12 tested strains. The results from the filter paper diffusion method suggested that extracellular metabolites may be more advantageous than intracellular metabolites in the development of antimicrobial agents. Eleven isolates with good activities were screened. In particular, Hypomontagnella monticulosa HGUP194009 and Nigrospora sphaerica HGUP191020 have shown promise in both broad-spectrum and intensity. Finally, some fungi that commonly cause disease have been observed to have beneficial biological activities as endophytic fungi. In conclusion, this study showed the species composition, alpha diversity, and lifestyle diversity of endophytic Sordariomycetes from R. roxburghii and demonstrated these isolates are potential sources for exploring antimicrobial agents.

Identification of sustainable trypsin active-site inhibitors from Nigrospora sphaerica strain AVA-1.

Trypsin is a protein-digesting enzyme that is essential for the growth and regeneration of bone, muscle, cartilage, skin, and blood. The trypsin inhibitors have various role in diseases such as inflammation, Alzheimer's disease, pancreatitis, rheumatoid arthritis, cancer prognosis, metastasis and so forth. From 10 endophytic fungi isolated, we were able to screen only one strain with the required activity. The fungus with activity was obtained as an endophyte from Dendrophthoe falcata and was later identified as Nigrospora sphaerica. The activity was checked by enzyme assays using trypsin. The fungus was fermented and the metabolites were extracted and further purified by bioassay-guided chromatographic methods and the compound isolated was identified using gas chromatography-mass spectrometry. The compound was identified as quercetin. Docking studies were employed to study the interaction. The absorption, distribution, metabolism, and excretion analysis showed satisfactory results and the compound has no AMES and hepatotoxicity. This study reveals the ability of N. sphaerica to produce bioactive compound quercetin has been identified as a potential candidate for trypsin inhibition. The present communication describes the first report claiming that N. sphaerica strain AVA-1 can produce quercetin and it can be considered as a sustainable source of trypsin active-site inhibitors.

First report of passion fruit leaf blight caused by Nigrospora sphaerica in China.

Passion fruit (Passiflora edulis Sims) is a widely cultivated dicotyledonous perennial plant with woody vines (Asande et al. 2020). In November 2020, leaf blight was observed on leaves of P. edulis (cultivar: 'Panama Red') newly planted in Wangyou, Huishui county, Guizhou province, China (25°82'57" N, 106°50'49" E). The leaf blight occurred on both young and old leaves, starting from the margins, and then extended to the entire leaves. The color of the affected tissue was brown with a yellow hallo in the early period, and then gradually turned to grey. The disease incidence was 60%-70% on a 0.08-ha field. Following isolation of the potential pathogen from 12 diseased leaves, nine isolates were obtained. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after eight days on potato dextrose agar (PDA) medium, incubated at 25°C in the dark for 10 days. The single cell conidia were solitary, spherical or slightly ellipsoidal, black, shiny, smooth, aseptate, spherical, and 8.1-13.5 µm (n=50) in diameter. Conidiophores (5.2-9.9 × 4.4-7.2 µm) were mostly reduced to conidiogenous cells and aggregated in clusters on hyphae. Conidiogenous cells were hyaline to pale brown or black, globose to ampulliform or clavate. Morphological characteristics of the isolates matched the description of the genus Nigrospora Mei Wang & L. Cai (Wang et al. 2017). For molecular identification, DNA was extracted, and PCRs were performed with primers ITS1/ITS4 for the ITS region (White et al. 1990), primers Bt2a/Bt2b for the ß-tubulin gene (TUB) (Glass and Donaldson 1995), and primers EF1-728F/EF1-986R for the translation elongation factor 1-alpha gene (EF1-α) (Carbone and Kohn 1999). Representative sequences of the ITS region, EF1-α, and TUB sequences (from isolate WYR007) were deposited in GenBank (accession numbers: MW561355; MZ053463; MZ032030) and are included in the supplementary materials. BLAST analysis against sequences from previously published studies showed 99.58% (ITS region), 99.54% (EF1-α), and 99.45% (TUB) identity to Nigrospora sphaerica sequences (accession numbers: MN215808.1; MN864137.1; KY019606.1). In addition, homology was confirmed with a phylogenetic tree using concatenated sequences from ITS, EF1-α and TUB constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch's postulates, conidia suspensions of isolate WYR007 (prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 103 conidia/mL) were sprayed on 15 leaves (200 µL per leaf) of 5 one-year-old healthy P. edulis plants (cultivar: 'Panama Red'). The same number of leaves from control group plants was only treated with 0.05% Tween buffer. All plants were incubated at 26°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation. After 14 days, symptomatic blight appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control group. The disease assays were repeated three times. Pure cultures were re-isolated from diseased leaves and confirmed to be N. sphaerica based on the morphological and molecular methods mentioned above (ITS region, the TUB, and the EF1-α sequences). To our knowledge, this study is the first report of N. sphaerica as a pathogen on P. edulis causing leaf blight. The identification of the pathogen could provide relevant background for its future management.s Sims) is a widely cultivated dicotyledonous perennial plant with woody vines (Asande et al. 2020). In November 2020, leaf blight was observed on leaves of P. edulis (cultivar: 'Panama Red') newly planted in Wangyou, Huishui county, Guizhou province, China (25°82'57" N, 106°50'49" E). The leaf blight occurred on both young and old leaves, starting from the margins, and then extended to the entire leaves. The color of the affected tissue was brown with a yellow hallo in the early period, and then gradually turned to grey. The disease incidence was 60%-70% on a 0.08-ha field. Following isolation of the potential pathogen from 12 diseased leaves, nine isolates were obtained. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after eight days on potato dextrose agar (PDA) medium, incubated at 25°C in the dark for 10 days. The single cell conidia were solitary, spherical or slightly ellipsoidal, black, shiny, smooth, aseptate, spherical, and 8.1-13.5 µm (n=50) in diameter. Conidiophores (5.2-9.9 × 4.4-7.2 µm) were mostly reduced to conidiogenous cells and aggregated in clusters on hyphae. Conidiogenous cells were hyaline to pale brown or black, globose to ampulliform or clavate. Morphological characteristics of the isolates matched the description of the genus Nigrospora Mei Wang & L. Cai (Wang et al. 2017). For molecular identification, DNA was extracted, and PCRs were performed with primers ITS1/ITS4 for the ITS region (White et al. 1990), primers Bt2a/Bt2b for the ß-tubulin gene (TUB) (Glass and Donaldson 1995), and primers EF1-728F/EF1-986R for the translation elongation factor 1-alpha gene (EF1-α) (Carbone and Kohn 1999). Representative sequences of the ITS region, EF1-α, and TUB sequences (from isolate WYR007) were deposited in GenBank (accession numbers: MW561355; MZ053463; MZ032030) and are included in the supplementary materials. BLAST analysis against sequences from previously published studies showed 99.58% (ITS region), 99.54% (EF1-α), and 99.45% (TUB) identity to Nigrospora sphaerica sequences (accession numbers: MN215808.1; MN864137.1; KY019606.1). In addition, homology was confirmed with a phylogenetic tree using concatenated sequences from ITS, EF1-α and TUB constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch's postulates, conidia suspensions of isolate WYR007 (prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 103 conidia/mL) were sprayed on 15 leaves (200 µL per leaf) of 5 one-year-old healthy P. edulis plants (cultivar: 'Panama Red'). The same number of leaves from control group plants was only treated with 0.05% Tween buffer. All plants were incubated at 26°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation. After 14 days, symptomatic blight appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control group. The disease assays were repeated three times. Pure cultures were re-isolated from diseased leaves and confirmed to be N. sphaerica based on the morphological and molecular methods mentioned above (ITS region, the TUB, and the EF1-α sequences). To our knowledge, this study is the first report of N. sphaerica as a pathogen on P. edulis causing leaf blight. The identification of the pathogen could provide relevant background for its future management.

Optimal fermentation time for Nigrospora-fermented tea rich in bostrycin.

BACKGROUND: Bostrycin has many biological functions, such as anticancer activity, and is becoming increasingly popular. Nigrospora sphaerica HCH285, which has the ability to produce high levels of bostrycin, can be used to ferment sun-dried green tea of Camellia sinensis through acclimation, resulting in the development of a Nigrospora-fermented tea. The effects of fermentation time on the production of bostrycin by the HCH285 strain were investigated. RESULTS: After 45 days of fermentation, the bostrycin content reached 3.18 g kg-1 , which is the highest level during the whole fermentation. At 50 days, the tea liquor was red, had a strong mushroom odour and a sweet taste, and presented optimal quality. The contents of free amino acids, tea polyphenols and soluble sugars in the fermented tea decreased generally during the fermentation, although the content of water-soluble substances increased. Additionally, the results of a 14-day acute oral toxicity test showed that Nigrospora-fermented tea was nontoxic. CONCLUSION: The optimum fermentation time of Nigrospora-fermented tea was concluded to be 45-50 days. These results provide insights with respect to the development of tea biotechnology and new tea products with active ingredients. © 2020 Society of Chemical Industry.

Antimicrobial metabolite profiling of Nigrospora sphaerica from Adiantum philippense L.

BACKGROUND: Endophyte bestows beneficial aspects to its inhabiting host, along with a contribution to diverse structural attributes with biological potential. In this regard, antimicrobial profiling of fungal endophytes from medicinal plant Adiantum philippense revealed bioactive Nigrospora sphaerica from the leaf segment. Chemical and biological profiling through TLC-bioautography and hyphenated spectroscopic techniques confirmed the presence of phomalactone as an antimicrobial metabolite. RESULTS: The chemical investigation of the broth extract by bioassay-guided fractionation confirmed phomalactone as a bioactive antimicrobial secondary metabolite. The antimicrobial activity of phomalactone was found to be highest against Escherichia coli by disc diffusion assay. The MIC was found to be significant against both Escherichia coli and Xanthomonas campestris in the case of bacteria and dermatophyte Candida albicans at 150 µg/ml, respectively. CONCLUSIONS: Overall, the results highlighted the antimicrobial potential of phomalactone from the endophyte Nigrospora sphaerica exhibiting a broad spectrum of antimicrobial activity against human and phytopathogenic bacteria and fungi. This work is the first report regarding the antibacterial activity of phomalactone.

First Report of Nigrospora sphaerica causing leaf spot on watermelon (Citrullus lanatus L.) in Malaysia.

Watermelon (Citrullus lanatus L.) is widely cultivated and consumed in Malaysia for its nutritional value. In June 2018, nearly 40% of the 'Red Rocky' watermelon plants in experimental plots of the research farm at Faculty of Agriculture, UPM Serdang, Selangor, Malaysia had leaf spot symptoms. Leaf spots were small, ranging 5 to 30 mm, yellow to brown, and circular to irregular in shape. With ages, the leafspots gradually enlarged and coalesced. To investigate the disease, ten symptomatic leaves were collected from the experimental plots. Diseased tissues (5 x 5 mm) were excied and surface sterilized with 0.5% sodium hypochlorite (NaOCl) for 2 min, rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C for 5 days. A total of ten isolates with similar colony morphologies were obtained from tissue samples. A single representative isolate "F" was further characterized by molecular analysis. All colonies were initially white in color, but later turned gray to black upon sporulation after 7 days. Conidia were produced in culture and were single-celled, black, smooth-walled, spherical in shape measuring 11.4 to 14 µm x 13.8 to 19 µm in diameter (n=40). These were borne on hyaline vesicles at the tip of a conidiophore. For molecular identification, genomic DNA was extracted from fresh mycelium of isolate F using DNeasy Plant Mini kit (Qiagen, Germantown, MD, USA). The internal transcribed spacer (ITS) region of rDNA and the translation elongation factor 1-alpha (TEF1-α) gene were amplified using the ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. BLASTn analysis of the ITS sequence revealed 100% identity (526 bp out of 526 bp) to Nigrospora sphaerica (GenBank Accession no. HQ608063). TEF1-α sequence had 100% identity (494 bp out 494 bp) with N. sphaerica (GenBank Accession no. MN995332). The resulting sequences were deposited in GenBank (ITS: Accession no. MK544066; TEF1-α Accession no. MT708197). Based on morphological and molecular characteristics, isolate "F" was identified as Nigrospora sphaerica (Sacc.) Mason (Chen et al. 2018). A pathogenicity test was conducted on five healthy leaves of five one-month-old watermelon 'Red Rocky' plants grown in a greenhouse. Leaves were wounded using a 34-mm-diameter florist pin frog and spray-inoculated until runoff with a conidial suspension (1 × 106 conidia/ml) of a 7-day-old culture. Five leaves from additional 2 plants were sprayed with sterile distilled water to serve as controls. Inoculated plants were covered with polyethylene bags for 48 h to maintain high humidity. Ten days post-inoculation, symptoms on inoculated leaves developed brown-to-black lesions similar to those observed in the field, while control leaves remained asymptomatic. N. sphaerica was re-isolated from all symptomatic tissues confirming Koch's postulates. N. sphaerica is distributed on a wide range of hosts and has been reported from 40 different host genera including monocotyledonous and dicotyledonous hosts (Wang et al. 2017). N. sphaerica has been reported to cause leaf spot of date palm in Pakistan (Alam et al. 2020) and kiwifruit in China (Chen et al. 2016). To our knowledge, this is the first report of N. sphaerica causing leaf spot of watermelon in Malaysia. This new disease could reduce fruit quality since sweetness and ripening are dependent on healthy foliage. Additionally, this disease can cause premature defoliation which would also reduce watermelon productivity.

The study of antimicrobial, anti-cancer, anti-inflammatory and α-glucosidase inhibitory activities of Nigronapthaphenyl, isolated from an extract of Nigrospora sphaerica.

A new compound, nigronapthaphenyl, was extracted from the endophytic fungus Nigrospora sphaerica isolated from a mangrove plant Bruguiera gymnorrhyza. The structure of the compound was elucidated by analysis of 1D and 2D NMR spectra and mass spectrometric data. It was tested in vitro for its antimicrobial activity, cytotoxicity, anti-inflammatory activity and for its ability to inhibit α-glucosidase. Nigronapthaphenyl showed antibacterial activities against Bacillus subtilis TISTR 088 and Bacillus cereus TISTR 688 with MIC values of 4 and 2 µg/mL respectively. Cytotoxicity against colon cancer cell line HCT 116 was found to be an IC50 value of 9.62 ± 0.5 µM . This further showed potential anti-inflammatory activity amounting to an IC50 of 6.2 ± 0.5 µM and also α-glucosidase inhibitory activity, with an IC50 value of 6.9 ± 0.5 µM.

New Antimicrobial Cyclopentenones from Nigrospora sphaerica ZMT05, a Fungus Derived from Oxya chinensis Thunber.

Six new cyclopentenone derivatives (+)-nigrosporione A (+)-1, (-)-nigrosporione A (-)-1, nigrosporione B (2), nigrosporione C (3), (+)-nigrosporione D (+)-4, and (-)-nigrosporione D (-)-4 were isolated from an endophytic fungus Nigrospora sphaerica ZMT05, collected from the rice grasshopper ( Oxya chinensis Thunberg), which is an insect pest in rice and which is also used as a food for people in some countries. Their planar and spatial structures were determined by spectroscopic analyses and eletronic circular dichroism (ECD) calculations. Compounds (+)-1, (-)-1, and 2 inhibited the plant pathogens Fusarium oxysporum, Colletotrichum musae, Penicillium italicum, and Fusarium graminearum, compounds 3 and (-)-4 inhibited F. oxysporum, C. musae, and P. italicum, and compound (+)-4 inhibited F. oxysporum, C. musae, and F. graminearum, showing antifungal activities stronger than triadimefon. Additionally, compounds (+)-1, (-)-1, 2, and 3 displayed moderate antibacterial activities against Staphyloccocus aureus and Escherichia coli.

Two new compounds from Nigrospora sphaerica ZMT05, a fungus derivated from Oxya chinensis Thunber.

A new pyrrolidinone derivative named nigrosporamide A (1), and a new acetophenone derivative, 4-prenyloxyclavatol (2), were isolated from an endophytic fungus Nigrospora sphaerica (collection No. ZMT05) isolated from Oxya chinensis Thunberg. Their chemical structures were established on the basis of the interpretation of spectroscopic data. In primary in vitro bioassay, nigrosporamide A (1) exhibited strong antifungal activity against Colletotrichum gloeosporioides and high inhibitory activity towards α-glucosidase.

Antibacterial activity of endophytic fungi from leaves of Indigofera suffruticosa Miller (Fabaceae).

Endophytic fungi were isolated from healthy leaves of Indigofera suffruticosa Miller, a medicinal plant found in Brazil which is used in folk medicine to treat various diseases. Among 65 endophytic fungi isolated, 18 fungi showed activity against at least one tested microorganism in preliminary screening, and the best results were obtained with Nigrospora sphaerica (URM-6060) and Pestalotiopsis maculans (URM-6061). After fermentation in liquid media and in semisolid media, only N. sphaerica demonstrated antibacterial activity (in Potato Dextrose Broth-PDB and in semisolid rice culture medium). In the next step, a methanolic extract from rice culture medium (NsME) and an ethyl acetate extract (NsEAE) from the supernatant of PDB were prepared and both exhibited antimicrobial activity against Gram-negative and Gram-positive bacteria. The best result was observed against Staphylococcus aureus, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 1.56 mg/mL and 6.25 mg/mL, respectively, for NsME and MIC and MBC values of 0.39 mg/mL and 3.12 mg/mL, respectively, for NsEAE. This study is the first report about the antimicrobial activity of endophytic fungi residing in I. suffruticosa leaves, in which the fungus N. sphaerica demonstrated the ability to produce bioactive agents with pharmaceutical potential, and may provide a new lead in the pursuit of new biological sources of drug candidates.