China General Microbiological Culture Collection Research Articles

First Report of Charcoal Rot on Tobacco Caused by Macrophomina phaseolina in Hunan Province, China.

Flue-cured tobacco (Nicotiana tabacum L.) is a significant cash crop globally. In August 2022, necrotic lesions on stem associated with root rot and wilting were observed on flue-cured tobacco (Cv. Yunyan 87) in fields located in Banxin village (27.95N,109.60E) of Fenghuang county in Xiangxi Autonomous Prefecture, Hunan Province, China. The affected and damaged area of tobacco is approximately 10 hectares, with adisease incidence of 60%. Lots of small black speckling within the lower stem of the affected plant, vascular tissue changed to black, dry rot, and looked like charcoal breezes. Small pieces were cut from healthy and diseased tissues, surface sterilized with 5% NaClO for 3 min and 75% ethanol for 1 min, rinsed with sterile distilled water and air-drying, incubated on oat medium incubated at 28℃ for five days. These isolates grew fast and produced typical black microsclerotia. The morphological were septate hyphae and microsclerotia. The microsclerotia were black and regularly round, with a 42.5 - 92.9 μm diameter. These morphological features were consistent with Macrophomina phaseolina (Smith and Wyllie 1999). The internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1-α) genes of three representative isolates were amplified and sequenced using the primers ITS1/ITS4 and EF1-728F/EF2R (Machado et al. 2019). Our resulting sequences (GenBank accessions OR435093, OR435101, OR435102 for ITS; OR891780, OR891781 and OR891782 for EF1-α) showed 99-100% similarity with M. phaseolina by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software with the NJ method. The combined sequences grouped with isolates to M. phaseolina with 100% bootstrap support. The strain XF22 has been sent to the China General Microbiological Culture Collection Center (CGMCC3.25349). Pathogenicity tests were conducted by inoculating potted plants (six plants per isolate, three times) from 45 day-old tobacco seedlings cv. Yunyan 87. Stems were randomly gently scratched with sterile needles, and a 5 mm agar disc with mycelium of the pathogen was attached to the surface of each wound, with a sterilized agar disc as control. Inoculated seedlings were incubated in growth chambers at 26℃ and 60% RH with a 12 h photoperiod/day. After ten days, symptoms that brown or black lesions on the inoculated lesions were dotted with numerous black, hard microsclerotia similar to those naturally occurring on the diseased plants, but not on the control plants. The same pathogen was re-isolated consistently, fulfilling Koch's postulates. Based on morphological, molecular, and pathogenicity test results, these isolates were identified as M. phaseolina. Charcoal rot of tobacco, caused by M. phaseolina was previously found in Guangxi in 1989 (Zhu et al. 2002), while this is the first report of M. phaseolina causing charcoal rot on flue-cured tobacco in Hunan, China. We speculate that the planting area is influenced by the preceding crop sesame. The soil carries M. phaseolina, which can cause stem rot of sesame, leading to the occurrence of tobacco charcoal rot. Our results indicated that charcoal rot caused by M. phaseolina is a new threat to flue-cured tobacco production and lue-cured tobacco might be acting as a reservoir and spreading this pathogen to other economically crops in China.

Cercospora polygonatum, a new species causing gray leaf spot disease in Polygonatum cyrtonema.

This study identified a new species (Cercospora Polygonatum) that causes gray leaf spot (GLS) disease in cultivated Polygonatum cyrtonema. This fungal species was isolated from the affected region of GLS on P. cyrtonema leaves. Pathogenicity bioassays were conducted based on Koch's postulates. Morphology was examined based on the features of conidiomata, conidiogenous loci, conidia/conidiophores, and conidiogenous cells. The rDNA internal transcribed spacer region, calmodulin, translation elongation factor 1-alpha, and histone genes were subjected to phylogenetic analysis using MrBayes tool via in Phylosuite. Bootstrap support analysis for phylogenetic placement confirmed the new species, which was significantly different from the closely related species C. senecionis-walkeri and C. zeae-maydis. The morphological characteristics also supported this finding, with the conidiogenous of C. polygonatum being considerably shorter than those of C. senecionis-walkeri or C. zeae-maydis. In addition, C. polygonatum was distinguished by its cultural characteristics. As this fungus was isolated from P. cyrtonema, it was named C. polygonatum F.Q. Yin, M. Liu&W. L. Ma, sp. nov. The type specimen (H8-2) was preserved at the China General Microbiological Culture Collection Center. This is the first report of GLS caused by C. polygonatum on P. cyrtonema leaves in China. The current study enriches the knowledge regarding Cercospora sp., contributes to the identification of a species causing GLS in P. cyrtonema, and provides useful information for the effective management of this disease.

Yueomyces silvicola sp. nov., a novel ascomycetous yeast species unable to utilize ammonium, glutamate, and glutamine as sole nitrogen sources.

Five yeast strains isolated from tree bark and rotten wood collected in central and southwestern China, together with four Brazilian strains (three from soil and rotting wood collected in an Amazonian rainforest biome and one from Bromeliad collected in Alagoas state) and one Costa Rican strain isolated from a flower beetle, represent a new species closely related with Yueomyces sinensis in Saccharomycetaceae, as revealed by the 26S ribosomal RNA gene D1/D2 domain and the internal transcribed spacer region sequence analysis. The name Yueomyces silvicola sp. nov. is proposed for this new species with the holotype China General Microbiological Culture Collection Center 2.6469 (= Japan Collection of Microorganisms 34885). The new species exhibits a whole-genome average nucleotide identity value of 77.8% with Y. sinensis. The two Yueomyces species shared unique physiological characteristics of being unable to utilize ammonium and the majority of the amino acids, including glutamate and glutamine, as sole nitrogen sources. Among the 20 amino acids tested, only leucine and tyrosine can be utilized by the Yueomyces species. Genome sequence comparison showed that GAT1, which encodes a GATA family protein participating in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae, is absent in the Yueomyces species. However, the failure of the Yueomyces species to utilize ammonium, glutamate, and glutamine, which are generally preferred nitrogen sources for microorganisms, implies that more complicated alterations in the central nitrogen metabolism pathway might occur in the genus Yueomyces.

Isolation and identification of a pathogenic strain of Bacillus cereus from bovine and establishment of a rapid detection method for RPA-LF

In order to identify the cause of death of a cattle that suddenly died on a cattle farm in Tongliao City, Inner Mongolia Autonomous Region, we isolated a strain of bacteria from the spleen and identified the pathogen. The study demonstrated that the isolated strain is Bacillus cereus. The isolates were highly pathogenic and contained toxin genes, such as hbl, nhe, and entFM, in addition to the groEL housekeeping gene. The isolated strain has been named TL strain (16S rDNA sequence number: OP028902, whole genome sequence number: CP129005). Isolates were deposited at China General Microbiological Culture Collection Center (CGMCC No.17626). In this study, we also developed a rapid recombinase polymerase amplification-based lateral flow (RPA-LF) for pathogenic Bacillus cereus. The virulence gene (nhe gene) was used as the target sequence to design RPA-LF assay primers and probes. The RPA reaction is fast (results can be observed in about 15 min), the results can be judged by the naked eye, and the specificity of the RPA-LF method is good. The RPA-LF method detects pathogenic Bacillus cereus in food, which reduces the risk of harm to human health. The RPA-LF assay provides a reference for the rapid detection of pathogenic Bacillus cereus.

The establishment of patent culture preservation system in China and the development of China Center for Type Culture Collection

<p indent="0mm">With the implementation of the Patent Law of the People’s Republic of China in 1985, the China Center for Type Culture Collection (CCTCC), which located at Wuhan University, and the China General Microbiological Culture Collection Center (CGMCC), which located in the Institute of Microbiology, Chinese Academy of Sciences, were designated by the State Patent Office as the official repositories for the preservation of cultures for patent procedures (“patent cultures” for short). In 1995, CCTCC and CGMCC joined the Budapest Treaty on behalf of China and formally became the International Depository Authority (IDA) recognized by the World Intellectual Property Organization (WIPO). Later in 2016, the Guangdong Microbiological Culture Collection Center (GDMCC), which located in Guangdong Institute of Microbiology, was also approved as IDA. After more than <sc>30 years</sc> of development, the total amount of patent culture collections in China is now the second largest in the world, second only to the United States, and the annual new increment of patent cultures ranks steadily number one in the world. CCTCC has also evolved from a specialized patent culture repository into a comprehensive organization and national sharing platform dedicated to the collection and preservation of diverse cultures and providing services, taking on a broader responsibility for the collection, preservation, and sharing of biological materials.

Genome Sequence Resource of Sarocladium terricola TR, an Endophytic Fungus as a Potential Biocontrol Agent Against Meloidogyne incognita.

Genome Sequence Resource of Sarocladium terricola TR, an Endophytic Fungus as a Potential Biocontrol Agent Against Meloidogyne incognita.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Characterization of an Aspergillus niger for Efficient Fatty Acid Ethyl Ester Synthesis in Aqueous Phase and the Molecular Mechanism.

Fatty acid ethyl esters are important flavor chemicals in strong-flavor baijiu. Microorganisms are the main contributors to ester synthesis during baijiu manufacture. However, the ester synthesis was unstable between batches. This was owing to a limited knowledge of the mechanisms for ester synthesis by microorganisms. In this work, a fatty acid ethyl ester synthesizing Aspergillus niger strain CGMCC (China General Microbiological Culture Collection) 3.4309 was identified. The conversion ratios of ethyl valerate, ethyl caproate, ethyl caprylate, and ethyl caprate were 7.87, 29.20, 94.80, and 85.20%, respectively, under the optimized conditions. A comparison of transcriptomes under the initial and optimized ester synthetic conditions indicated that 23 genes were upregulated in transcription level and encoded enzymes with potential abilities for ester synthesis. Eleven of the enzymes were expressed, and three of them, numbered An605, An1097, and An3131, showed the ability to catalyze fatty acid ethyl ester synthesis under aqueous phase, with capric acid as the preferred substrate. The possible enzymatic catalytic mechanism was proposed based on homology modeling and molecular docking. This study reported for the first time that A. niger showed the ability to efficiently catalyze the synthesis of short- and medium-chain fatty acid ethyl esters in aqueous phase, identified the key enzymes, and analyzed the basic enzymatic properties. This is helpful to promote the application of related microorganisms and enzyme resources in the baijiu industry.

First Report of Anthracnose Caused by Colletotrichum liaoningense on Trichosanthes kirilowii in China.

Trichosanthes kirilowii Maxim (T. kirilowii) is widely grown in central China for its medicinal and edible value. In August 2020, an anthracnose-like disease was observed on fruit of T. kirilowii (cv. Wanlou9) in four fields (0.9 ha) located in Taihu county, Anhui Province of China. Approximately 60% of the T. kirilowii plants were affected in the fields. The symptoms initially consisted of small off-white necrotic spots, and later became larger, irregular gray necrotic lesions on green fruit, causing fruit rot and sometimes fruit drop. More than 10 symptomatic fruits were sampled, and small pieces of diseased tissue were surface sterilized in 0.1% HgCl2 for 2 min, 75% ethanol for 45 s, rinsed three times with sterile distilled water, then placed on potato dextrose agar (PDA) and incubated at 25℃in the dark. A fungus was consistently (80%) isolated from symptomatic fruit samples.Aerial mycelia were light gray, and radially black with white in reverse medium. Conidia were rarely produced on PDA, but prolific on water agar. The conidia were cylindrical to clavate, both ends rounded, had obvious circular granule in the center, and ranged from 14.6 to 19.9 μm × 5.4 to 7.3 μm. The morphological characteristics were similar to the descriptions of C. liaoningense by Diao et al. (2017). For molecular identification, representative isolates LG5-4 and LG9-6 were selected. Genomic DNA was extracted, and the internal transcribed spacer (ITS) region, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUB2) genes were amplified by PCR (White et al. 1990; Duan et al. 2018), and sequenced bidirectionally. A BLAST search of GenBank revealed the ACT and TUB sequences had 95.83% (KP890097), 99.20%, 95.33% (KP890111) and 99.84%, respectively, to C. liaoningense CAUOS2. A phylogenetic analysis was conducted using MEGA7 based on concatenated sequences of the four genes, indicating that the isolates were closely clustered with reference strains of C. liaoningense (98% bootstrap value). The two strains were deposited in the China General Microbiological Culture Collection Center as CGMCC3.20344 and CGMCC3.20345, and their sequences deposited in GeneBank (Accession nos. MW082811-12, MW117926-31), respectively. Pathogenicity tests were conducted on healthy fruit of T. kirilowii (cv. Wanlou9) using the wound inoculation by pinpricking and droplet (106 conidia/mL) on fruit surface. The experiments were done with three fruit per isolate (LG5-4 and LG9-6), and replicated three times. The controls were inoculated with sterile water. The fruit were covered with plastic bags and kept in a chamber (>90% RH, 28 to 30°C) for 14 days. Typical symptoms of yellow-brown lesions appeared 14 days after inoculation. No symptoms were observed on the controls. The fungus was re-isolated from the diseased tissues and identified as C. liaoningense by sequencing of the four genes, confirming Koch's postulates. C. liaoningense has been reported to cause anthracnose of mango and chili in China (Diao et al. 2017; Li et al. 2019). To our knowledge, this is the first report of C. liaoningense causing anthracnose on T. kirilowii. Due to cultivation of T. kirilowii in the region, further studies are required to develop management strategy of this disease.

Complete Genome Sequence of Paradevosia shaoguanensis Type Strain J5-3, Obtained Using Nanopore and Illumina Sequencing Technologies.

ABSTRACTThe complete genome sequence of Paradevosia shaoguanensis J5-3T (China General Microbiological Culture Collection Center [CGMCC] 1.12430T) is presented here. The complete genome sequence of P. shaoguanensis J5-3T will provide valuable references for classification and comparative genome analysis.

First Report of Wheat Common Bunt Caused by Tilletia laevis in Henan Province, China.

Wheat common bunt is a serious disease that may lead to yield losses of 75-80% in many wheat regions of the world (Mathre 1996). The disease may reduce yield and flour quality by producing trimethylamine, a compound that smells like rotting fish (Castlebury et al. 2005; Hoffmann 1981; Mathre 1996). Two closely related basidiomycete species, Tilletia caries (DC.) Tul. & C. Tul. [syn. T. tritici (Bjerk.) Wint.] and T. laevis J. G. Kühn [syn. T. foetida (Wallr.) Liro], cause wheat common bunt. Teliospore morphology is used to differentiate the two species. Teliospores of T. caries have reticulates on the surface while teliospores of T. laevis have a smooth surface (Pieczul et al. 2018). T. laevis was reported in Liaoning, Shaanxi, Shandong, Beijing, Hebei, Shanxi, Jilin, Heilongjiang, Jiangsu, Gansu, Xinjiang, Sichuan, Yunnan, Inner Mongolia, and Tibet (Guo 2011; Wang 1963), but not in Henan, the biggest wheat production province in China, before the present study. In July 2019, we found wheat common bunt in three fields grown with cultivar Zhengmai 618 in Yugong Mountain, Henan province. The diseased wheat heads had bunt balls filled with black powder with fishy smell. The disease incidences in these fields were 20-50%, but no common bunt was found in other nearby fields. About 200 diseased heads were sampled from the three fields. Teliospores from each head were observed under a microscope, and they all had smooth surface. Observations using a scanning electron microscope also showed smooth-surfaced teliospores. Teliospores were measured 13.5 to 18.5 μm in diameter. After surface sterilization of diseased heads using 0.25% NaClO for 5 min, teliospore suspension (1×106/ml) was made using sterilized distilled water and spread on water agar (200 μl per plate), and the plates were kept at 15°C with 24 h light (Goates and Hoffman 1987). On the 6th days, teliospores were germinated. Based on the disease symptoms, teliospore morphology, and germination, the bunt fungus was identified as T. laevis. To fulfill Koch's postulates, 1 ml of germinating teliospore suspension at the concentration of 106 spores/ml was injected into the heads of susceptible wheat cultivar (Dongxuan 3) at the boot stage with a syringe, and the plants injected with sterile ddH2O were used as control. The inoculated plants were grown in a growth chamber at 17°C with 50% humidity and 24 h light (300 μmol/m2/s). After one month at the ripening stage, the kernels of the inoculated plants were filled with black teliospores releasing fishy smell, and the control plants did not have bunt heads. Under a scanning electron microscope, teliospores from the inoculated heads had smooth surface and were measured 13.5 to 18.5 μm in diameter, similar to the teliospores of bunt heads from the fields. The fungus was also confirmed through molecular characterization using sequence characterized amplification region (SCAR) markers specific for T. laevis, and the expected 660 bp (Yao et al., 2019) and 286 bp (Zhang et al. 2012) bands were obtained separately from the teliospore samples from both the fields and growth chamber. The collection named as CGMCC 3.20112 was deposited in China General Microbiological Culture Collection Center. To the best of our knowledge, this is the first report of T. laevis causing wheat common bunt in Henan Province of China. Because the pathogen is seedborne and soilborne, the disease may become a high risk to wheat production in Henan and other provinces of China.

First report of Colletotrichum chlorophyti causing peanut anthracnose in China.

Peanut (Arachis hypogaea L.) is an important oil crop - mainly in Shandong and Henan Provinces, in China. In 2018-2019, the occurrence of a black spot disease on the leaves and stems were found in two fields (a total of 10 ha) in Niulingguanzhuang village of Yishui county in Linyi, Shandong Province, China, and 20% to 40% of plants were infected, thereby reducing the amount of marketable product. Natural symptoms were circular, dark brown-to-black lesions (2-6 mm in diameter) and coalescent necrosis on leaves and black necrosis in stems. Six symptomatic leaves and stems collected from six plants of two fields, were used for isolation. Portions of infected tissue were surfaced-sterilized with 0.5% NaClO for two minutes, 70% alcohol for 30 seconds and washed twice with sterile water. The tissues were placed on potato dextrose agar (PDA) at 28℃ for 5 days. Felt or fleece forming colonies about 4 cm in diameter that were circular, flat, and with dark center and white narrow margins were formed. Three isolates (LSJF-4, HSGF, HSJF) were purified and one culture (LSJF-4) was deposited in the China General Microbiological Culture Collection Center (CGMCC, NO. 3.19617). The chlamydospore were dark brown, verruculose, organized in chains and clusters, yellow-brown to black-brown color, and mostly spherical, 10.3 - 18.4 μm × 8.3-11.4 μm (n = 50, av. 12.6 ± 2.1× 10.1 ± 1.1 µm). The conidia were colorless, crescent or sickle-shaped, with one acute end and one blunt end. They were 16.7 - 24.3 μm × 3.6 - 5.5 μm (n= 50, av. 21.2 ± 1.7× 4.5 ± 0.36 µm), with dark brown, straight, septate setae, 40.3 - 86.9 μm × 3.6-5.5 μm (n=50, av. 70.0 ± 19.1 × 4.5 ± 0.7 µm) in size. Setae were straight, dark brown. These morphological characteristics of our isolates were identical to Colletotrichum chlorophyti (Damm et al. 2009). The internal transcribed spacer (ITS, ITS1/ITS4) rDNA of three fungus (LSJF-4, HSGF, HSJF) were amplified using PCR and sequenced, as described by Damm et al (2009). The sequencing results (GenBank Accession No. MK796409, MN756650, MN756651) were submitted to the GenBankand blast analysis showed that it had 99.0% identity with those of Colletotrichum chlorophyti (No. GU227894). Actin (ACT, ACT-512F/ACT-783R), beta-tubulin (Tub2, T1/T2 ), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GDF1/GDR1), chitin synthase (CHS1, CHS-79f/CHS-345R), histone (H3, Cy1H3F/ Cy1H3R) of LSJF-4 (Nos. MN688800, MN688797, MN097811, MN688799 and MN688798, respectively) (Damm et al. 2009) were also sequenced and BLAST. The results showed high identity of all the five sequences to the CGMCC 3.19617 (Nos. GU228286/GU228287 in GAPDH = 99.6%, GU228384/GU22385 in CHS1 = 96.8%, GU228090/GU228091 in H3 = 99.0%, GU227992/GU227993 in ACT = 98.7%, and GU228188/GU228189 in Tub2 = 98.8%). Therefore, isolate CGMCC 3.19617 was identified as C. chlorophyti based on morphological and molecular characteristics. To determine pathogenicity, a conidial suspension containing 106 conidia/mL was sprayed on leaves and stems of six 40-day old peanut seedlings. Three control plants were similarly sprayed with sterile water. All treated plants were kept moist (>85% relative humidity) at 30℃ for 48 h in darkness, and then kept in 60% relative humidity and 28℃ conditions. Two weeks post-inoculation, small, dark, near elliptical lesions appeared on inoculated leaves and stems, which were similar to those naturally infected plants. While controls remained symptomless. C. chlorophyti was reisolated from infected tissues and identified based on the previous methods, fulfilling Koch's postulates. The test was repeated twice. C. chlorophyti has been reported as a pathogen associated with soybean anthracnose isolated from the Netherlands (Damm et al. 2009). C. chlorophyti was first detected on soybean plants imported from Uruguay (Li et al. 2017) in China. To our knowledge, this is the first report of C. chlorophyti causing peanut anthracnose in China. C. chlorophyti may be a new threat to Leguminous plant species, especially peanuts plants. We proposed paying close attention to taking necessary control meatures.

First Report of Colletotrichum karstii Causing Anthracnose on Cigar Tobacco in Hainan, China

Cigar tobacco (Nicotiana tabacum L.) is an important cash crop in China. Due to suitable ecological conditions, Hainan province is a major area for growing cigar tobacco. Cigar tobacco leaves are both an important source of fiscal taxation and also the main source of income for farmers in Hainan province. In 2018 and 2019, necrotic leaf spots were observed on 5 to 15% of cigar tobacco plants in Danzhou city (109.58°E, 19.53°N) and Wuzhishan city (109.52°E, 18.78°N) in Hainan province, China. Lesions on plants in the fields were small, nearly round spots (0.2 to 0.5 cm in diameter) with light gray centers and narrow dark brown margins, and sometimes there were tiny black dots composed of sporophores scattered in the lesion center. To determine the causal agent, more than 10 symptomatic leaves from Danzhou city and Wuzhishan city were used for isolation. Small pieces of tissue were cut from lesion borders, sterilized with 2% sodium hypochlorite (NaClO) for 4 min and 70% ethanol for 30 s, rinsed with sterilized water (SW), and dried on a sterile tissue paper. Clean and excised tissue pieces were placed on potato dextrose agar plates and incubated at 28°C for 5 days in the dark. Pure colonies of the fungus had grayish white aerial mycelia and orange conidial masses in the centers, and sometimes there were tiny black dots composed of clusters of conidiophores and conidia scattered in the centers. A pure culture was deposited in the China General Microbiological Culture Collection Center (CGMCC, no. 3.19502). Microscopic observations showed single, straight to slightly curved, hyaline, cylindrical conidia that were 12.1 to 14.7 μm long and 6.4 to 8.2 μm wide. To confirm the identities of the isolate, the internal transcribed spacer (ITS) rDNA of the fungus was amplified using PCR and sequenced, as described by Yang et al. (2011). The sequencing results were then compared with other DNA sequences in the GenBank database by BLAST. Analysis of the PCR product (GenBank accession no. MK050853) showed that the sequence had 99.5% identity with the nucleotide sequences of Colletotrichum karstii (HM585409). Glyceraldehyde-3-phosphate dehydrogenase (GPDH, GDF1/GDR1) (Yang et al. 2011) was also sequenced and BLAST searched. The PCR product sequence had 99.8% identity with the nucleotide sequences of C. karstii (HM585391). Based on the morphological and molecular characteristics, the isolate CGMCC 3.19502 was identified as C. karstii. Pathogenicity tests were conducted by artificially inoculating 20 cigar tobacco plants (40 days old, cv. H211). A spore suspension (10⁶ conidia/ml) was evenly sprayed on all leaves of the 20 plants. Ten control plants were treated similarly with SW. Twenty inoculated plants and 10 noninoculated plants were placed in a chamber maintained at 30°C and 90% relative humidity. After 7 days, the same symptoms observed from natural infections were on inoculated leaves, whereas the controls remained symptomless. This isolate also infected detached tobacco leaves. The pathogen was reisolated from inoculated leaves, and Koch’s postulates were completed by confirming the identity of the isolated fungus based on the morphology and ITS sequence. Anthracnose caused by C. karstii was previously detected on Schefflera octophylla in Sichuan province (Li et al. 2017). To our knowledge, this is the first report of C. karstii causing anthracnose on cigar tobacco in China. Damage caused by C. karstii could cause significant economic losses if not managed. More research is needed to better understand this disease and establish control strategies.

Isolation and Characterization of a Novel myovirus Infecting Shigella dysenteriae from the Aeration Tank Water.

The genome sequence, morphology, and genetic features of a novel phage, named SSE1, is reported here. Phage SSE1 that infects Shigella dysenteriae (China General Microbiological Culture Collection Center number: 1.1869) was isolated from the aeration tank water of a sewage treatment plant. SSE1 showed morphological features associated with those of phages in Myoviridae. The whole genome sequence of phage SSE1 is composed of 169,744bp with the GC content of 37.51%. The double-stranded DNA of SSE1 contains 270 open reading frameworks (ORFs). Phylogenetically, phage SSE1 showed a stronger homology (whole genome and terminase large subunit protein sequence) to Escherichia phages than other Shigella phages in the NCBI database, but SSE1 did not infect Escherichia stains. This indicates that phage SSE1 should be a novel phage infecting Shigella dysenteriae. Besides, the result of this study provided a new idea for phage therapy. SSE1 may become a candidate for potential therapy against Shigella dysenteriae infection in clinical applications.

Genomic characteristics of Dickeya fangzhongdai isolates from pear and the function of type IV pili in the chromosome

Dickeya fangzhongdai, the causal agent of bleeding canker of pear, is a new member of the Dickeya genus and the only one that infects woody plants. Recent studies have reclassified several Dickeya isolates as D. fangzhongdai, which were isolated from various environments, including water, Phalaenopsis sp. and Aglaonema sp. To provide genomic characterization of D. fangzhongdai isolates from pear, the genomes of D. fangzhongdai strain JS5 (=China General Microbiological Culture Collection Center, CGMCC 1.15464T=DSM 101947T), along with two other isolates, LN1 and QZH3, were sequenced and compared to those of other Dickeya spp. Homology greater than 99% was observed among three D. fangzhongdai strains. Plasmid, type IV secretion system (T4SS) and type IV pili (TFPs) were found in genomes of D. fangzhongdai isolates. Comparative analysis of the type III secretion systems (T3SS), type III secretion effectors (T3SE), plant cell wall degradation enzymes (PCWDE) and membrane transport proteins of Dickeya spp. showed some differences which might reflect the variations of virulence, phylogenetic and phenotypic characteristics of Dickeya spp. In addition, deletion mutant of TFP in D. fangzhongdai JS5 showed no twitching motility and reduced virulence and biofilm formation. The fingdings of the distinctive plasmid, T4SS and TFPs, as well as the differences of T3SE, PCWDE and membrane transport proteins make D. fangzhongdai isolates unique. These results also suggested that acquisition of virulence genes by horizontal gene transfer might play some role in the genetic variation of D. fangzhongdai.

Isolation and Characterization of a Novel Diethylstilbestrol-Degrading Bacillus subtilis JF and Biochemical Degradation Metabolite Analysis.

Diethylstilbestrol (DES) can adversely affect the immune system of developing fetuses or even elicit toxic responses such as nerve toxicity and genotoxicity in human beings, thereby warranting methods to remove DES from the environments. The present study characterized a novel DES-degrading Bacillus subtilis JF and analyzed the degradation metabolites. The strain was collected at the China General Microbiological Culture Collection Center (Collection number: CGMCC 7950). The environmental effects, such as DES concentrations, pH levels, and temperature, on the strain’s degradation ability were tested. Degradation metabolites of DES by strain JF were analyzed via high performance liquid chromatography (HPLC) and liquid-chromatography time of flight mass spectrometry (LC-TOF-MS). Results indicated that B. subtilis JF can effectively degrade DES within a concentration of 25–200 mg/L. Increasing pH levels (pH > 7) are reported to increase the degradation rate of DES by the strain. The optimal temperature for strain JF to degrade DES was identified as 45°C. In this study, 4, 4′-hexene estrogen quinones (DESQ) and DES-4-semiquinone were speculated as two degradation metabolites of DES, and both can be completely degraded by strain JF. A slight reduction of DES in the blank system [DES cultured in Luria-Bertani (LB) medium without strain JF] was observed in this study. The reduction trend in the blank system only occurred during the first few days (about 4 days) and was considerably lesser than the decomposition and transformation effect of DES via strain JF. Furthermore, the metabolite DESQ could not be further decomposed in blank LB medium without strain JF. All the results demonstrate that complete degradation of DES in the fermentation broth occurs due to the function of strain JF rather than organic decomposition. In conclusion, the high efficiency of degradation and the potential to degrade DES completely indicates that strain JF has potential for the bioremediation of DES-contaminated environments (soil, river, and so on) and fermented foods.

Complete Genome Sequence of Potential Deoxynivalenol-Degrading Bacterium Youhaiella tibetensis Type Strain F4.

Here, we report the first complete genome sequence of the potential deoxynivalenol-degrading strain Youhaiella tibetensis F4T (China General Microbiological Culture Collection Center [CGMCC] 1.12719T). To the best of our knowledge, this is also the first announcement of the complete genome sequence of a Youhaiella species.

Identification and clinical significance of Bordetella holmesii isolated from a rare case of bacteremia in Nantong China

Objective To analyze the identification, drug resistance and clinical significance of a rare bacterium of Bordetella holmesii (B.holmesii) to improve its detection and clinical diagnosis and treatment. Methods A strain isolated from a bacteremia case was identified by bacterial culture, biochemical tests and 16S rRNA gene sequencing. Mega 7.0 software was used to conduct a similarity analysis of 16S rRNA gene sequences between the type strains of Bordetella spp. and the isolate, and then a phylogenetic tree was constructed. Antibiotic resistance of the isolate was determined by E-test. Changes in bacterial growth were measured after adding different concentrations of riboflavin or its inhibitor lumiflavin to the culture medium. Results B. holmesii ABD2 was the pathogen causing bacteremia in the immunocompetent patient. It was deposited under the number of CGMCC 1.13721 in China General Microbiological Culture Collection Center (CGMCC), and the 16S rRNA gene sequences were deposited in National Center for Biotechnology Information (NCBI) with the accession number of KT828544.1. Unrooted tree showed that the B. holmesii strain was highly homologous with B. pertussis. Antibiotic susceptibility test showed that the minimum inhibitory concentrations (MIC) of piperacillin, ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, gentamicin, amikacin, erythromycin, tetracycline and polymyxin B against the isolate were low, while the MIC values of cefazolin, cefuroxime, cefoxitin, cefotaxime, aztreonam and trimethoprim-sulfamethoxazole were high. Riboflavin accelerated the growth of B. holmesii ABD2, while its inhibitor lumiflavin had an inhibitory effect. Conclusions As B. holmesii is hard to isolate and identify, limited clinical, microbiological and epidemiological data are available. It is an under-recognized pathogen with a considerable amount of information that remains to be studied. Key words: Bordetella holmesii; Bacteremia; Immunocompetence; 16S rRNA gene sequencing; Riboflavin; lumiflavin

Phylogeny of Aspergillus section Terrei with two new records from the China General Microbiological Culture Collection Centre

Aspergillus section Terrei isolates deposited in the China General Microbiological Culture Collection Centre (CGMCC), Institute of Microbiology, were evaluated using morphological and molecular approaches. Based on phylogenetic analyses of nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (BenA), and calmodulin (CaM) genes sequences, these isolates were identified as Aspergillus terreus, A. floccosus, A. niveus, A. pseudoterreus, A. allahabadii, and A. alabamensis. The last two were new records from China. The phylogenetic variation and morphological description of A. allahabadii and A. alabamensis are given. Morphologically, most section Terrei members are indistinguishable. The phylogenetic relationships of 16 species of section Terrei showed that the CaM and BenA loci could be used to distinguish section Terrei members. This is the first study to summarize section Terrei species with the new taxonomic criteria of Aspergillus in China.

Phylogenetic reassessment of the Chaetomium globosum species complex.

Chaetomium globosum, the type species of the genus, is ubiquitous, occurring on a wide variety of substrates, in air and in marine environments. This species is recognised as a cellulolytic and/or endophytic fungus. It is also known as a source of secondary metabolites with various biological activities, having great potential in the agricultural, medicinal and industrial fields. On the negative side, C. globosum has been reported as an air contaminant causing adverse health effects and as causal agent of human fungal infections. However, the taxonomic status of C. globosum is still poorly understood. The contemporary species concept for this fungus includes a broadly defined morphological diversity as well as a large number of synonymies with limited phylogenetic evidence. The aim of this study is, therefore, to resolve the phylogenetic limits of C. globosum s.str. and related species. Screening of isolates in the collections of the CBS-KNAW Fungal Biodiversity Centre (The Netherlands) and the China General Microbiological Culture Collection Centre (China) resulted in recognising 80 representative isolates of the C. globosum species complex. Thirty-six species are identified based on phylogenetic inference of six loci, supported by typical morphological characters, mainly ascospore shape. Of these, 12 species are newly described here. Additionally, C. cruentum, C. mollipilium, C. rectum, C. subterraneum and two varieties of C. globosum are synonymised under C. globosum s.str., and six species are resurrected, i.e. C. angustispirale, C. coarctatum, C. cochliodes, C. olivaceum, C. spiculipilium and C. subglobosum. Chaetomium ascotrichoides is segregated from C. madrasense and the genus name Chaetomidium is rejected. Five species, including C. globosum s.str., are typified here to stabilise their taxonomic status. A further evaluation of the six loci used in this study as potential barcodes indicated that the 28S large subunit (LSU) nrDNA and the internal transcribed spacer regions and intervening 5.8S nrRNA (ITS) gene regions were unreliable to resolve species, whereas β-tubulin (tub2) and RNA polymerase II second largest subunit (rpb2) showed the greatest promise as DNA barcodes for differentiating Chaetomium species. This study provides a starting point to establish a more robust classification system for Chaetomium and for the Chaetomiaceae.