Bosea beijingensis sp. nov., Telluria beijingensis sp. nov. and Agrococcus beijingensis sp. nov., isolated from baijiu mash.

The baijiu fermentation environment hosts a variety of micro-organisms, some of which still remain uncultured and uncharacterized. In this study, the isolation, cultivation and characterization of three novel aerobic bacterial strains are described. The cells of strain REN20T were Gram-negative, strictly aerobic, motile and grew at 26-37 °C, at pH 6.0-9.0 and in the presence of 0-5.0   % (w/v) NaCl. The cells of strain REN29T were Gram-negative, strictly aerobic, motile and grew at 15-30 °C, at pH 6.0-9.0 and in the presence of 0-10.0   % (w/v) NaCl. The cells of strain REN33T were Gram-positive, strictly aerobic, motile and grew at 15-37 °C, at pH 5.0-10.0 and in the presence of 0-7.0   % (w/v) NaCl. The digital DNA-DNA hybridization and average nucleotide identity by orthology values between type strains in related genera and REN20T (20.3-36.8 % and 79.8-89.9  %), REN29T (20.3-36.8  % and 74.5-88.5  %) and REN33T (22.6-48.6  % and 75.8-84.2  %) were below the standard cut-off criteria for the delineation of bacterial species, respectively. Based on polyphasic taxonomy analysis, we propose three new species, Bosea beijingensis sp. nov. (=REN20T=GDMCC 1.2894T=JCM 35118T), Telluria beijingensis sp. nov. (=REN29T=GDMCC 1.2896T=JCM 35119T) and Agrococcus beijingensis sp. nov. (=REN33T=GDMCC 1.2898T=JCM 35164T), which were recovered during cultivation and isolation from baijiu mash.

Transcriptomic Response of Clonostachys rosea Mycoparasitizing Rhizoctonia solani.

Clonostachys rosea is an important mycoparasitism biocontrol agent that exhibits excellent control efficacy against numerous fungal plant pathogens. Transcriptomic sequencing may be used to preliminarily screen mycoparasitism-related genes of C. rosea against fungal pathogens. The present study sequenced and analyzed the transcriptome of C. rosea mycoparasitizing a Basidiomycota (phylum) fungal pathogen, Rhizoctonia solani, under three touch stages: the pre-touch stage, touch stage and after-touch stage. The results showed that a number of genes were differentially expressed during C. rosea mycoparasitization of R. solani. At the pre-touch stage, 154 and 315 genes were up- and down-regulated, respectively. At the touch stage, the numbers of up- and down-regulated differentially expressed genes (DEGs) were 163 and 188, respectively. The after-touch stage obtained the highest number of DEGs, with 412 and 326 DEGs being up- and down-regulated, respectively. Among these DEGs, ABC transporter-, glucanase- and chitinase-encoding genes were selected as potential mycoparasitic genes according to a phylogenetic analysis. A comparative transcriptomic analysis between C. rosea mycoparasitizing R. solani and Sclerotinia sclerotiorum showed that several DEGs, including the tartrate transporter, SDR family oxidoreductase, metallophosphoesterase, gluconate 5-dehydrogenase and pyruvate carboxylase, were uniquely expressed in C. rosea mycoparasitizing R. solani. These results significantly expand our knowledge of mycoparasitism-related genes in C. rosea and elucidate the mycoparasitism mechanism of C. rosea.

The Adenylate Cyclase-Encoding Gene crac Is Involved in Clonostachys rosea Mycoparasitism.

Clonostachys rosea is an excellent biocontrol fungus against numerous fungal plant pathogens. The cAMP signaling pathway is a crucial signal transduction pathway in fungi. To date, the role of the cAMP signaling pathway in C. rosea mycoparasitism remains unknown. An adenylate cyclase-encoding gene, crac (an important component of the cAMP signaling pathway), was previously screened from C. rosea 67-1, and its expression level was dramatically upregulated during the C. rosea mycoparasitization of the sclerotia of Sclerotinia sclerotiorum. In this study, the function of crac in C. rosea mycoparasitism was explored through gene knockout and complementation. The obtained results show that the deletion of crac influenced the growth rate and colony morphology of C. rosea, as well as the tolerance to NaCl and H2O2 stress. The mycoparasitic effects on the sclerotia of S. sclerotiorum and the biocontrol capacity on soybean Sclerotinia stem rot in ∆crac-6 and ∆crac-13 were both attenuated compared with that of the wild-type strain and complementation transformants. To understand the regulatory mechanism of crac during C. rosea mycoparasitism, transcriptomic analysis was conducted between the wild-type strain and knockout mutant. A number of biocontrol-related genes, including genes encoding cell wall-degrading enzymes and transporters, were significantly differentially expressed during C. rosea mycoparasitism, suggesting that crac may be involved in C. rosea mycoparasitism by regulating the expression of these DEGs. These findings provide insight for further exploring the molecular mechanism of C. rosea mycoparasitism.

The Effect of Buckwheat Resistant Starch on Intestinal Physiological Function.

Resistant starch appears to have promising effects on hypertension, cardiovascular and enteric illness. The influence of resistant starch on intestinal physiological function has drawn great attention. In this study, we first analyzed the physicochemical characteristics, including the crystalline properties, amylose content, and anti-digestibility among different types of buckwheat-resistant starch. The influence of resistant starch on the physiological functions of the mouse intestinal system, contained defecation, and intestinal microbes were also evaluated. The results showed that the crystalline mold of buckwheat-resistant starch changed from A to B + V after acid hydrolysis treatment (AHT) and autoclaving enzymatic debranching treatment (AEDT). The amylose content in AEDT was higher than in AHT and raw buckwheat. Moreover, the anti-digestibility of AEDT was also stronger than that in AHT and raw buckwheat. The buckwheat-resistant starch can promote bowel intestinal tract movement. The quantity of intestinal microbe was regulated by buckwheat-resistant starch. Our research demonstrates an effective preparation method for improving the quality of buckwheat-resistant starch and found that buckwheat-resistant starch has the role of adjusting the distribution of the intestinal flora and maintaining the health of the body.

Cry1Ac Mixed with Gentamicin Influences the Intestinal Microbial Diversity and Community Composition of Pink Bollworms.

Pink bollworms severely affect the production of cotton. The method currently used for pink bollworm control is the planting of Bt (Bacillus thuringiensis) protein-expressing transgenic cotton. However, pink bollworms can develop strong resistance to Bt proteins in transgenic cotton because of the large planting area and long planting time of this crop, which severely affects the control of pink bollworms. Intestinal microorganisms play very important roles in insect growth, development and Bt resistance. However, the effect of intestinal microorganisms on pink bollworm Bt resistance is still unclear. The current study aimed to analyze the effect of intestinal microorganisms on the Bt resistance of pink bollworms. Intestinal microorganisms associated with Bt resistance were initially screened through Illumina MiSeq sequencing and analysis. The results showed that feeding with a mixture of gentamicin, Cry1Ac and an artificial diet could significantly increase the mortality of pink bollworm larvae compared with feeding with of a mixture of Cry1Ac and an artificial diet or an artificial diet alone. The microbial diversity, community structure and composition of the pink bollworm larval intestine were significantly influenced by feeding with a mixture of gentamicin, Cry1Ac and an artificial diet. Several intestinal bacteria with significantly altered abundances after treatment with gentamicin were preliminarily screened as potential resources for addressing Bt toxicity. This study provides useful strategies for addressing the Bt resistance of pink bollworms.

cAMP Signalling Pathway in Biocontrol Fungi.

Biocontrol is a complex process, in which a variety of physiological and biochemical characteristics are altered. The cAMP signalling pathway is an important signal transduction pathway in biocontrol fungi and consists of several key components. The G-protein system contains G-protein coupled receptors (GPCRs), heterotrimeric G-proteins, adenylate cyclase (AC), cAMP-dependent protein kinase (PKA), and downstream transcription factors (TFs). The cAMP signalling pathway can regulate fungal growth, development, differentiation, sporulation, morphology, secondary metabolite production, environmental stress tolerance, and the biocontrol of pathogens. However, few reviews of the cAMP signalling pathway in comprehensive biocontrol processes have been reported. This work reviews and discusses the functions and applications of genes encoding each component in the cAMP signalling pathway from biocontrol fungi, including the G-protein system components, AC, PKA, and TFs, in biocontrol behaviour. Finally, future suggestions are provided for constructing a complete cAMP signalling pathway in biocontrol fungi containing all the components and downstream effectors involved in biocontrol behavior. This review provides useful information for the understanding the biocontrol mechanism of biocontrol fungi by utilising the cAMP signalling pathway.

Long-term fertilization alters soil properties and fungal community composition in fluvo-aquic soil of the North China Plain.

Different fertilization regimes can substantially influence soil fungal community composition, yet fewer studies try to control for the effects of nitrogen input. Here, we investigated the impact of fertilization with equal nitrogen upon soil properties and soil fungal diversity and community composition in the North China Plain in a long-term field experiment. Long-term (32 years) fertilization regimes were applied with equal amounts of nitrogen: no chemical fertilizer or organic manure; chemical fertilization only; organic manure fertilization only, and; combination of 1/2 chemical fertilizer and 1/2 organic manure. Then we investigated the influence of these four fertilization regimes to soil properties, fungal diversity and community composition. The results showed that applying organic manure significantly influenced soil properties. Illumina MiSeq sequencing and its analysis revealed that organic manure fertilization significantly changed soil fungal alpha diversity, but chemical fertilization did not. Although soil fungal community composition did not differ significantly among all the fertilization regimes at the phylum and class levels, they did show differences in the abundance of dominant fungi. Yet at the genus level, soil fungal community composition, abundance, and beta diversity was affected by all fertilization regimes. Application of organic manure also reduced the abundance of soil-born fungal pathogens such as Fusarium. Our results suggest that long-term application of organic manure could markedly improve soil properties, altering soil fungal community composition and its diversity. Moreover, organic manure fertilization could limit soil-born fungal diseases, to further contribute to soil ecosystem sustainability.

Directed mutation of ß-glucanases from probiotics to enhance enzymatic activity, thermal and pH stability.

ß-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of ß-glucanases in probiotics. Here, ß-glucanase genes were cloned from Bacillus licheniformis, Lactobacillus fermentum and L. johnsonii. ß-glucanase genes, as blg, lfg and ljg isolated from B. licheniformis, L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that ß-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of α-helices and ß-strands in the three mutants were altered, which might result in ß-glucanase activity increasement. Analysis of ß-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30-50 â„ƒ than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about ß-glucanase-encoding genes in L. fermentum and L. johnsonii. These findings provide an efficient way to improve the activity of ß-glucanase.

The Mitogen-Activated Protein Kinase Gene Crmapk Is Involved in Clonostachys chloroleuca Mycoparasitism.

Clonostachys chloroleuca is a mycoparasite used for biocontrol of numerous fungal plant pathogens. Sequencing of the transcriptome of C. chloroleuca following mycoparasitization of the sclerotia of Sclerotinia sclerotiorum revealed significant upregulation of a mitogen-activated protein kinase (MAPK)-encoding gene, crmapk. Although MAPKs are known to regulate fungal growth and development, the function of crmapk in C. chloroleuca mycoparasitism is unclear. In this study, we investigated the role of crmapk in C. chloroleuca mycoparasitism through gene knockout and complementation. Deletion of crmapk had no influence on the C. chloroleuca morphological characteristics but could significantly reduce the mycoparasitic ability to sclerotia and biocontrol capacity to soybean Sclerotinia stem rot; crmapk complementation restored these abilities. Transcriptome analysis between Δcrmapk and the wild-type strain revealed numerous genes were significantly down-regulated after crmapk deletion, including cytochrome P450, transporters, and cell wall-degrading enzymes (CWDEs). Our findings indicate that crmapk influences C. chloroleuca mycoparasitism by regulation of genes controlling the activity of CWDEs or antibiotic production. This study provides a basis for further studies of the molecular mechanism of C. chloroleuca mycoparasitism.

The heat shock protein 70 gene is involved for colony morphology, sporulation and mycoparasitism of Clonostachys rosea.

Heat shock protein 70 (HSP70) is an evolutionarily conserved chaperone protein. However, the role of HSP70 in mycoparasitism is unclear. Clonostachys rosea shows great potential against plant fungal pathogens. An HSP70 encoding gene, crhsp, from C. rosea 67-1 was significantly upregulated during C. rosea parasitization of the sclerotia of Sclerotinia sclerotiorum. In the present study, we investigated the role of crhsp in mycoparasitism using gene knockout experiments. The results showed that disruption of crhsp had remarkabe effects on the morphological characteristics of C. rosea. In addition, the ability of C. rosea to parasitize sclerotia and control soybean Sclerotinia stem rot in the greenhouse was significantly reduced in the Δcrhsp mutant. The results indicated that crhsp is involved in C. rosea mycoparasitism and provide the basis for further study of the molecular mechanism of C. rosea mycoparasitism. This is the first report to demonstrate the involvement of the HSP70 gene in C. rosea mycoparasitism.

Identification of genes related to chlamydospore formation in Clonostachys rosea 67-1.

Chlamydospores are specific structures that are of great significance to the commercialization of fungal biopesticides. To explore the genes associated with chlamydospore formation, a biocontrol fungus Clonostachys rosea 67-1 that is capable of producing resistant spores under particular conditions was investigated by transcriptome sequencing and analysis. A total of 549,661,174 clean reads were obtained, and a series of differentially expressed genes potentially involved in fungal chlamydospore formation were identified. At 36 hr, 67 and 117 genes were up- and downregulated in C. rosea during chlamydospore production, compared with the control for conidiation, and 53 and 24 genes were up- and downregulated at 72 hr. GO classification suggested that the differentially expressed genes were related to cellular component, biological process, and molecular function categories. A total of 188 metabolism pathways were linked to chlamydospore production by KEGG analysis. Sixteen differentially expressed genes were verified by reverse transcription quantitative PCR, and the expression profiles were consistent with the transcriptome data. To the best of our knowledge, it is the first report on the genes associated with chlamydospore formation in C. rosea. The results provide insight into the molecular mechanisms underlying C. rosea sporulation, which will assist the development of fungal biocontrol agents.

The transcription factor-encoding gene crtf is involved in Clonostachys chloroleuca mycoparasitism on Sclerotinia sclerotiorum.

Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) is a potential biocontrol fungus active against various fungal plant pathogens. From transcriptome sequencing of 67-1 parasitizing sclerotia of Sclerotinia sclerotiorum, we identified the transcription factor-encoding gene crtf that is significantly up-regulated during mycoparasitism. Transcription factors are widely distributed in fungi and involved in multiple biological processes. However, their role and regulatory mechanisms in mycoparasitism remain poorly understood. In this study, the function of crtf during 67-1 mycoparasitism was verified through gene knockout and complementation. The results showed that deletion of crtf did not influence fungal morphological characteristics, but the ability of the Δcrtf mutant to parasitize sclerotia and suppress soybean Sclerotinia white mold in the greenhouse was markedly diminished compared with the wild type strain. The biocontrol activity of Δcrtf recovered wild type levels when complemented with a plasmid expressing the crtf gene. These findings suggest that crtf plays a crucial role in C. chloroleuca mycoparasitism and provide insight into the molecular mechanisms underlying C. chloroleuca mycoparasitism on plant pathogenic fungi.

Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum.

Clonostachys rosea is a promising biocontrol fungus active against various plant fungal pathogens. In this study, the endochitinase-encoding gene Chi67-1, the expression of which is sharply upregulated in C. rosea 67-1 when induced by sclerotia, was transformed into the original isolate by protoplast transformation, and transformants were screened against Sclerotinia rot of soybean. The transformation efficiency was approximately 50 transformants per 1 × 107 protoplasts, and 68 stably heritable recombinants were assayed. The parasitic rates of 32.4% of the tested strains increased by more than 50% compared to 43.3% of the wild type strain in 16 h, and the Rc4-4 transformant showed a parasitic rate of 100% in 16 h. The control efficiencies of the selected efficient transformants to soybean Sclerotinia stem rot were evaluated in pots in the greenhouse, and the results revealed that Rc4-4 achieved the highest efficiency of 81.4%, which was 31.7% and 28.7% higher than the control achieved by the wide type and the pesticide carbendazim, respectively. Furthermore, the expression level of Chi67-1 was 107-fold higher in Rc4-4 than in the wild type, and accordingly, the chitinase activity of the recombinant increased by 140%. The results lay a foundation for the development of efficient genetically engineered strains of C. rosea.

Identification of mycoparasitism-related genes in Clonostachys rosea 67-1 active against Sclerotinia sclerotiorum.

Clonostachys rosea is a mycoparasite that has shown great potential in controlling various plant fungal pathogens. In order to find mycoparasitism-related genes in C. rosea, the transcriptome of the efficient isolate 67-1 in association with sclerotia of Sclerotinia sclerotiorum was sequenced and analysed. The results identified 26,351 unigenes with a mean length of 1,102 nucleotides, among which 18,525 were annotated in one or more databases of NR, KEGG, Swiss-Prot, GO and COG. Differentially expressed genes at 8 h, 24 h and 48 h after sclerotial induction were analysed, and 6,890 unigenes were upregulated compared with the control without sclerotia. 713, 1,008 and 1,929 genes were specifically upregulated expressed, while 1,646, 283 and 529 genes were specifically downregulated, respectively. Gene ontology terms analysis indicated that these genes were mainly involved in metabolism of biological process, catalysis of molecular function and cellular component. The expression levels of 12 genes that were upregulated after encountering with S. sclerotiorum were monitored using real-time PCR. The results indicated that the quantitative detection was consistent with the transcriptome analysis. The study provides transcriptional gene expression information on C. rosea parasitizing S. sclerotiorum and forms the basis for further investigation of mycoparasitism-related genes of C. rosea.

Draft Genome Sequence of Mycoparasite Clonostachys rosea Strain 67-1.

Clonostachys rosea is a promising mycoparasite. In this study, we sequenced the draft genome of the highly effective strain 67-1 using the Illumina HiSeq 2500 sequencing platform. The genome is 55.4 Mb with a G+C content of 49.2% and provides a powerful resource for future studies on the molecular mechanisms underlying Clonostachys rosea's antagonism on fungal pathogens.

Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction.

Reference genes are important to precisely quantify gene expression by real-time PCR. In order to identify stable and reliable expressed genes in mycoparasite Clonostachys rosea in different modes of nutrition, seven commonly used housekeeping genes, 18S rRNA, actin, ß-tubulin, elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme and glyceraldehyde-3-phosphate dehydrogenase, from the effective biocontrol isolate C. rosea 67-1 were tested for their expression under sclerotial induction and during vegetative growth on PDA medium. Analysis by three software programs showed that differences existed among the candidates. Elongation factor 1 was most stable; the M value in geNorm, SD value in Bestkeeper and stability value in Normfinder analysis were 0.405, 0.450 and 0.442, respectively, indicating that the gene elongation factor 1 could be used to normalize gene expression in C. rosea in both vegetative growth and parasitic process. By using elongation factor 1, the expression of a serine protease gene, sep, in different conditions was assessed, which was consistent with the transcriptomic data. This research provides an effective method to quantitate expression changes of target genes in C. rosea, and will assist in further investigation of parasitism-related genes of this fungus.

A perilipin gene from Clonostachys rosea f. Catenulata HL-1-1 is related to sclerotial parasitism.

Clonostachys rosea f. catenulata is a promising biocontrol agent against many fungal plant pathogens. To identify mycoparasitism-related genes from C. rosea f. catenulata, a suppression subtractive hybridization (SSH) cDNA library of C. rosea f. catenulata HL-1-1 that parasitizes the sclerotia of S. sclerotiorum was constructed. 502 clones were sequenced randomly, and thereby 472 expressed sequence tags (ESTs) were identified. Forty-three unigenes were annotated and exhibited similarity to a wide diversity of genes. Quantitative real-time PCR showed that a perilipin-like protein encoding gene, Per3, was up-regulated by 6.6-fold over the control at 96 h under the induction of sclerotia. The full-length sequence of Per3 was obtained via 5' and 3' rapid identification of cDNA ends. Overexpression of Per3 in HL-1-1 significantly enhanced the parasitic ability on sclerotia. The results indicated that Per3 might be involved in the mycoparasitism of C. rosea f. catenulata HL-1-1. This is the first report of a perilipin as a potential biocontrol gene in mycoparasites. The study provides usefu l insights into the interaction between C. rosea f. catenulata and fungal plant pathogens.

Luteimonas cucumeris sp. nov., isolated a from cucumber leaf.

A Gram-negative, aerobic and non-motile rod, designated Y4(T), was isolated from a cucumber leaf from Pinggu District, east Beijing, PR China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Y4(T) was most closely related to Luteimonas aquatica RIB1-20(T) (96.7% 16S rRNA gene sequence similarity). DNA-DNA relatedness between strain Y4(T) and L. aquatica RIB1-20(T) was 42.5 ± 3.9%. The predominant fatty acids were iso-C(15:0), iso-C(17:1)ω9c, iso-C(16:0) and iso-C(17:0). The major ubiquinone was Q-8. The DNA G+C content of the type strain was 69.9 mol%. Based on the evidence above, strain Y4(T) represents a novel species of the genus Luteimonas, for which the name Luteimonas cucumeris sp. nov. is proposed. The type strain is Y4(T) ( = CGMCC 1.10821(T) = KCTC 23627(T)).