Abstract:Mycoviruses (viruses of fungi) are one of the most important pathogens of edible fungi and cause significant losses in many mushroom production systems. Mycoviruses induce various response in mushrooms, often producing latent or no obvious symptoms that can lead to significant production losses. Research on viral diseases of edible fungi has increased significantly due to the economic importance as a result of production loses and potential application of mycovirus in plant disease management. Therefore, this paper aims to review the structure and classification of mycovirus, symptom expression and disease severity, mode of transmission, detection methods and techniques for eliminating and producing virus free spawns for mushroom cultivation, and suggest future research needs for sustainable management of mushroom viral diseases.

Abstract:Nowadays, global water quality has been improved significantly with the development of public health. However, the death rate caused by cholera epidemic is still high in developing countries. Vibrio cholerae, the exact pathogen for the cholera epidemic, has been categorized as a Class A infectious disease in China. At present, a series of effective detection techniques for Vibrio cholerae have been established worldwide. In this study, recent advances on detection technology of Vibrio cholerae, including microbiology immunology, molecular biology, biosensors and rapid detection technology methods, were summarized. In addition, the advantages and disadvantages of various technology were compared, and the market demand of vibrio cholerae detection in the future was addressed.

Abstract:Insect symbiotic microbes are a group of special microbial resources. The actinomycetes widely exist in the insect intestines, cuticle or the nest. Some of fungus-growing insects associated actinomycetes can produce secondary metabolites that inhabit pathogenic microorganisms of the insects, plants or human tumor cells. Thus, the research on insect symbiotic actinomycetes not only can facilitate understanding of the symbiotic mechanism between insects and symbionts, but also explore new bioactive compounds for the development of biological pesticides and medicines. This review summarizes the advances in the study on the secondary metabolites from fungus-growing insects associated actinomycetes.

Abstract:Griffithsin, originally isolated from Griffithsia spp. marine red algae., is a broad-spectrum antiviral lectin that inhibits viral entry through binding to viral glycoproteins. Drugs that inhibit virus's entry prevent infection of cells by cell-free virus particles and also prevent virus transmission between virus-infected and uninfected cells. Besides, griffithsin also has the advantages of good solubility, easy expression, strong stability, low immunogenicity and good safety. Thus, griffithsin, as a new potential class of antiviral drugs, has got the favor of scientists.

Abstract:Drosophila suzukii (Matsumura) (spotted wing Drosophila) is a notorious pest of small and stone fruits, and causes damage across Asian, American and European continents. Unlike most other Drosophila species, female D. suzukii flies lay their eggs in ripening and ripe fruits and the hatched larva eat inside, thereby causing substantial economic losses to the fruit industry. Insect-associated microbes are essential in manipulating insect physiology and ecology, such as insect development, fitness and reproduction. Here, we review recent researches on the diversity of microbes in D. suzukii and the functions of microbes in D. suzukii biology. That information may provide clues for searching strategies to control D. suzukii populations.

Abstract:Hyperthermophilic archaea are facing severe challenges due to their high temperature environment. Therefore, how to maintain genomic stability of hyperthermophilic archaea is one of the most important scientific questions in this field. Hyperthermophilic archaea have similar spontaneous mutation frequencies to mesophilic microorganisms, suggesting that they have a more efficient DNA repair system than mesophilic microorganisms to repair genomic DNA damage caused by high temperature. At present, the molecular mechanism of DNA repair of hyperthermophilic archaea is still unclear. Endonucleases play an important role in the DNA repair pathway. Genomic sequences show that hyperthermophilic archaea encode a few DNA repair endonucleases, however, the research on them is still in an early stage. In this paper, we reviewed the research progress of hyperthermophilic archaeal DNA repair endonucleases, including NucS, EndoV, EndoQ, XPF and Hjc. We also proposed future studies.

Abstract:[Objective] The dlt operon in Gram-positive bacteria encodes enzymes that are necessary for the modification of D-alanylation of teichoic acids in cell wall. D-alanylation generates net positive charge on cell surface and, as a consequence, repulses the positively charged molecules, such as cationic antimicrobial peptides, thereby confers resistance to host animal. Here, we investigated the impact of dlt operon on phenotypic traits of Bacillus thuringiensis and role in virulence to insect. [Methods] We constructed the loss-of-function mutant of dlt by homologous recombination technique, and performed its morphological observation, surface charge difference analysis, stress resistance analysis and cell experiment.[Results] The results revealed that inactivation of dltA significantly decreased net negative charge of cell wall, drastically impaired the resistance of Bacillus thuringiensis to cationic antimicrobial peptides (polymyxin B and lysozyme) and alkaline. ΔdltA_Bt mutant displayed an obviously altered profile of growth curve, irregular shape and rough surface of cell, decreased biofilm formation and increased swarming motility. Moreover, inactivation of dltA significantly decreased adhesion ability to mid-gut epithelial cell of insect, and greatly attenuated virulence to Bombyx mori. [Conclusion] These findings provide evidence that D-alanylation of TAs mediated by dlt operon is closely correlated to many phenotypic traits of Bt, and has putative roles in the pathogenicity of B. thuringiensis to insect and the protection of B. thuringiensis from insect humoral immunity.

Abstract:[Objective] To determine the effect of concentrations of P, S and CO₂ on the growth and total carbohydrate accumulation of Desmodesmus insignis, the algae were cultivated in modified BG-11 medium containing 8 different initial phosphorus and sulfur concentrations and 4 different carbon dioxide concentrations. [Methods] Biomass and total carbohydrate content were measured by dry weight and phenol-sulfuric acid method, respectively. [Results] The highest biomass concentration reached 6.37 g/L under 0.460 mmol/L K₂HPO₄ and the maximum content and productivity of carbohydrate was 45.40% of dry weight and 0.20 g/(L·d) under 0.230 mmol/L K₂HPO₄, respectively. High MgSO₄ concentration could promote the growth and carbohydrate accumulation of Desmodesmus insignis. The highest biomass concentration and the maximum content and productivity of carbohydrate were achieved at 1.217 mmol/L, 0.609 mmol/L and 1.824 mmol/L MgSO₄, which was 7.02 g/L, 51.60% of dry weight and 0.26 g/(L·d), respectively. In addition, the highest biomass concentration and the maximum content and productivity of carbohydrate were 6.81 g/L, 44.03% and 0.20 g/(L·d) when 3% (V/V) CO₂ was used. [Conclusion] Thus, the concentrations of P, S and CO₂ which were beneficial to the growth and carbohydrate accumulation were 0.230 mmol/L, 1.824 mmol/L and 3% (V/V), respectively.

Abstract:[Objective] Pseudomonas putida SJTE-1 can degrade 17β-estradiol (E2) efficiently, but the enzymes for the tranformation of E2 in this strain is still unclear. In this work, we identified and characterized a new 3-oxoacyl-acyl-carrier-protein reductase (3-oxoacyl-ACP reductase, ANI01589.1) involved in the E2 degradation. [Methods] We cloned the encoding gene of this 3-oxoacyl-ACP reductase and overexpressed it in Escherichia coli BL21(DE3) strain. We purified the recombinant protein by metal-ion affinity chromatography and characterized its enzymatic activity in vitro. Then we detected the product of this enzymatic reaction with High Performance Liquid Chromatography (HPLC). [Results] The transcription of the 3-oxoacyl-ACP reductase was induced by 17β-estradiol. Protein sequence alignment showed that it contained two consensus regions and the conserved residues of the short-chain dehydrogenase/reductase (SDR), and its structure was similar to that of the 3-oxoacyl-ACP reductase (4fw8.1.A). The recombinant protein was purified with the yield of 19.6 mg per liter culture. This 3-oxoacyl-ACP reductase could convert 17β-estradiol into estrone using NAD⁺ as the cofactor. Its K_m value was 0.071 mmol/L and its k_cat value was 2.4±0.06/s^-1; and the transformation efficiency of this enzyme to 17β-estradiol was over 95.8% in 5 min. Its optimal reaction temperature was 42℃ and the optimal pH was 8.0. Divalent ions had different effect on the enzymatic activity; Mg²⁺ and Mn²⁺ could enhance the enzymatic activity. [Conclusion] The 3-oxoacyl-ACP reductase (ANI01589.1) could catalyze the transformation of 17β-estraiol efficiently and was important for the estrogen metabolism of P. putida SJTE-1.

Abstract:[Objective] OmpA protein (VPA1186) of Vibrio parahaemolyticus SH112 stain plays an important role in pathogenesis and can be a potential vaccine candidate against V. parahaemolyticus infection. We expressed and to immunologically characterized OmpA protein from strain SH112. [Methods] The ompA gene from strain SH112 was amplified by PCR method and cloned into express vector. The coding protein was analyzed according to the sequencing analysis. The protein was expressed in Escherichia coli BL21(DE3). The mouse anti-OmpA antiserums were generated by immunization of ICR mice with the recombinant protein purified by Ni-NTA. The immunogenicity and the specificity of OmpA were detected by Western blotting analysis. The vaccine protective efficacy of OmpA was verified by animal challenge experiment. [Results] The 40 kDa recombinant protein His-OmpA was successfully expressed. ELISA result shows that the titer of antiserum was above 1:50000. Moreover, Western blotting results reveals the antiserum reacted not only specifically with the purified His-OmpA protein but also with outer membrane proteins and whole-cell proteins of V. parahaemolyticus, suggesting that the expressed protein remained the immunogenicity of original OmpA protein. In addition, Western blotting result reveals that the antiserum reacted specifically with about 36 kDa proteins from four other V. parahaemolyticus strains with major serotypes in domestic, but not reacted with other non-V. parahaemolyticus strains, suggesting the antiserums have a high specificity and the protein maybe a common protective antigen in different V. parahaemolyticus isolates. We further showed that OmpA conferred protective effect as about 35% of mice survived V. parahaemolyticus infection. [Conclusion] Our findings indicate that OmpA protein could play important roles in development of diagnostic test and may serve as candidate vaccine against V. parahaemolyticus infection.

Abstract:[Objective] Traditional fermented camel milk is a valuable source of food for people living in the arid areas, and it is often used to treat a variety of disease with good effects. Its nutritional and medical functions are closely related to its abundant lactic acid bacteria. However, the studies of bacterial diversity of fermented camel milk are scattered and the amount of available data are still limited. Therefore, it is necessary to explore the microbiota composition and diversity of the traditional fermented camel milk.[Methods] We sequenced the V3-V4 region of 16S rRNA genes in fermented camel milk of Alxa and Sonid Bactrian camel by Illumina Miseq platform, and the microbiota composition and diversity were compared and analyzed.[Results] The results of Alpha diversity showed that the fermented camel milk from Sonid has higher bacterial diversity and microbial community richness than those of Alxa. At the phylum level, Firmicutes and Proteobacteria were dominant in both of them. At the genus level, Lactobacillus and Lactococcus were dominant in Sonid samples, Lactobacillus and Acetobacter were dominant in Alxa samples. In addition, we also found some foodborne pathogens and environmentally contaminating bacteria, such as Enterobacter, Raoultella and Leuconostoc. There were significant differences in the types and dominant bacteria in fermented camel milk in different regions. [Conclusion] These results provide insights into the microbiota community and diversity of traditional fermented camel milk, and also lay the foundation for screening dominant bacteria and excavating new probiotics in the further studies.

Abstract:[Objective] Ralstonia solanacearum is the causative agent of a devastating bacterial wilt disease in solanaceous plants. The purpose of this work was to identify genes involved in the pathogenesis of R. solanacearum. [Methods] We used a Tn5-based mutagenesis strategy to generate random insertion mutants that were then assayed for biofilm formation, cell motility and pathogenicity. Thermal asymmetric interlaced PCR (TAIL-PCR) was performed to identify the Tn5 insertion site of mutants with altered phenotypes. [Results] A total of 400 mutants were generated in the model strain GMI1000. Two mutants were found to be unable to form biofilms and had reduced swimming and swarming motility on soft agar media. When inoculated into tomato plants, both mutants failed to cause wilting disease symptoms. Both mutants carried a Tn5 insertion within the NADH dehydrogenase subunit F gene (nuoF), and the insertion sites were at 103 bp and 225 bp from the translational start site. A complemented strain expressing nuoF under the control of the ripAY promoter fully restored the wild-type phenotype. [Conclusion] The NADH dehydrogenase complex is the first enzyme in the respiratory electron transport chain in microbes. Our data here indicated that NADH dehydrogenase plays a role in biofilm formation, cell motility and pathogenicity in R. solanacearum.

Abstract:[Objective] To obtain biocontrol bacteria to control bacterial leaf streak (BLS) of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc). [Methods] A bacterial strain 504 with antibacterial activity was screened from water spinach rhizosphere soil by agar disk dilution and inhibition zone method using an Xoc model strain RS105 as target. Strain 504 was identified through morphological, physiological and biochemical characteristics in combination with 16S rDNA and gyrA sequence analysis. Antagonistic activity of 504 against some plant pathogenic Xanthomonas spp. was measured and stability of its sterile fermentation broth against Xoc was analyzed by Oxford Cup method. We predicted the genes encoding the synthesis of multiple lipopeptide and polyketide compounds by PCR amplification. Antibacterial activities of 504 against Xoc in the leaves of rice seedlings were evaluated by injection inoculation method. [Results] Strain 504 was identified as Bacillus velezensis, named B. velezensis 504. B. velezensis 504 showed specific inhibitory effects on some plant pathogenic Xanthomonas, especially on X. oryzae pv. oryzae. The secondary metabolites prediction showed that B. velezensis 504 contains genes such as fenA, dhbA, sfrA, bmyA, beaS, dfnA and bacA encoding the biosynthesis of various peptidoglycans and polyketose antibacterial compounds. The cell-free supernatant showed stable inhibitory effects against Xoc under high temperature and by protease treatment, however, there is no inhibitory activities when the pH value of supernatant is beyond 5.5 to 8.9. B. velezensis 504 exhibits significantly inhibitory effect on water-soaked lesions caused by Xoc in the leaves of the high susceptible rice cultivar Yuanfengzao. [Conclusion] B. velezensis 504 exhibits the broad-spectrum antagonistic activity against Xanthomonas spp., and has the potential as an effective biocontrol agent on diseases caused by plant pathogenic Xanthomonas spp..

Abstract:[Objective] To understand the bacterial community structure and diversity of healthy and bacterial wilt tobacco plants in rhizosphere soil, diseased stem tissue, symptomatic-asymptomatic junction stem tissue, and non-infected stem tissue. [Methods] The V3-V4 region of the bacterial 16S rRNA gene in soil and stem tissue was amplified, and the amplified fragments were sequenced using Illumina MiSeq high-throughput sequencing technology. [Results] Proteobacteria were the dominant phylum in all samples. Bacteroidetes and Acidobacteria were the dominant phyla in all rhizosphere soil. Cyanobacteria was the dominant phylum in non-infected stem tissue; and Cyanobacteria and Firmicutes were dominant in diseased stem tissue and symptomatic-asymptomatic junction stemtissue. The dominant genera in all rhizosphere soil were Ralstonia, Pseudomonas and Sphingomonas. The dominant genera of tobacco stem were Ralstonia and Pseudomonas. [Conclusion] Compared with non-infected tobacco plants, the richness and diversity of bacterial community in rhizosphere soil and stem tissue of bacterial wilt tobacco plants were generally greater. Bacterial wilt is a typical soil-borne disease, although the pathogen Ralstonia solanacearum can spread and expand in the vascular bundles, it mainly distributes in the soil. Therefore, the control of bacterial wilt should not be limited to the tobacco plant itself, but targeting the pathogen in field soil may strengthen the control efforts.

Abstract:[Objective] c-di-GMP, an important second messenger regulating multiple functions of bacteria, is generally synthesized and hydrolysed by proteins containing GGDEF or EAL domain. In this study, we analyzed the genome-wide GGDEF/EAL domain-containing proteins of Azorhizobium caulinodans ORS571, and selected three GGDEF-EAL composite proteins (AZC_3085, AZC_3226 and AZC_4658) for functional analysis. [Methods] SMART and CLUSTALW were used for prediction and multi-alignment of GGDEF/EAL domain-containing proteins. Mutants were constructed by homologous recombination. Phenotypes including cell motility, exopolysaccharide (EPS) production, biofilm formation and nodulation with legume host were investigated. [Results] There were 37 GGDEF/EAL domain-containing proteins in A. caulinodans ORS571. Mutant △4658 showed deficiency in cell motility, while its EPS production and biofilm formation were higher than that of wild type. Mutant △4658 showed stronger competitiveness than wild type in competitive nodulation assay. The loss of AZC_4658 led to the increase of intracellular c-di-GMP level. Mutants △3085 and △3226 did not show obvious difference in comparison with wild type. [Conclusion] The vast number of GGDEF/EAL domain-containing proteins suggested that c-di-GMP may play an important role in signal transduction of ORS571. The GGDEF-EAL composite protein AZC_4658 was involved in cell motility, EPS production, biofilm formation and nodulation of A. caulinodans ORS571.

Abstract:[Objective] To construct a recombinant Escherichia coli BL21 (DE3)/pET-KT2440, a low-specificity L-threonine aldolase gene (ltaE) was cloned from the genome of Pseudomonas putida KT2440. The enzyme characterization and the effects of key amino acid mutations on the enzyme activity and thermo-stability were investigated.[Methods] The ltaE gene was amplified by PCR with the genome of P. putida KT2440 as template. The recombinant plasmid pET28a-KT2440 was constructed and transformed into E. coli BL21 (DE3). The low-specificity L-threonine aldolase (LTA) was purified by Ni²⁺ affinity chromatography and then was characterized. The key amino acids Thr206 and lys207 were mutated by site-directed mutagenesis. [Results] SDS-PAGE analysis showed that LTA were highly expressed in E. coli BL21 (DE3) with a molecular weight of about 40 kDa, consistent with the theoretical value. A single band was observed through Ni²⁺ affinity chromatography. LTA had a specific activity of 5577 U/mg by two coupled-enzyme assay method. The optimal temperature and pH were 50℃ and 8.0, respectively, and was stable below 40℃ and pH between 5.0 and 9.0. LTA exhibited K_m and k_cat values of 23.95 mmol/L and 19216.6 s^-1 under the optimal conditions. Mg²⁺ and Ca²⁺ obviously stimulated enzyme activity, whereas Ni²⁺, Cu²⁺, Zn²⁺ and Fe²⁺ obviously inhibited it. LTA enzyme presented good resistance in tert-butyl methyl ether. The residual activity was retained over 90% after pre-incubation of the enzyme in TBME for 1 h. Site-directed mutation indicated that Thr206Ser significantly increased the thermal stability. Lys207 is essential for enzymatic function. Any mutation of K207 was lethal for enzyme activity. [Conclusion] The thermostability was improved by site-directed mutation. The work provides a solid foundation for the efficient and stable biosynthesis of β-hydroxy-α-amino acids by LTA.

Abstract:[Objective] The aim of this study was to enrich species resources of actinobacteria colonized in plant roots and rhizosphere through isolating, screening and identifying and to provide resources for the development of biocontrol agents via examining the effects of these actinobacteria on antagonizing plant pathogens and promoting plant growth. [Methods] Actinobacteria were isolated from roots and rhizosphere of dominant herbaceous plants grown in three habitats (saline-sodic, wet, and polluted land) using the dilution plate method. The antibacterial strains were screened by plate confrontation test. Then Salkowski colorimetry, Chrome Azurol S plate assay and nitrogen free culture were used to further detect their effect on promoting plant growth. The isolated strains were examined by the combined morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis for taxonomical identification. [Results] A total of 283 isolates of actinobacteria colonized in plant roots and rhizosphere, belonging to genera Streptomyces, Nocardia and Micromonospora, were obtained. Among them, 77% belonged to 10 groups of the genus Streptomyces. Seven strains showed strong antagonizing activity and plant growth-promoting effects. Strain H6-1 gave the most significant antagonizing effect among the seven strains, and its fermentation broths inhibition ratio to pathogens Fusarium oxysporum, Fusarium, Botrytis cinerea, Rhizoctonia solani, Macrophoma kawatsukai, and Colletotrichum orbiculare were 32.3%, 42.6%, 48%, 72.2%, 58.1% and 60.5%, respectively. Strain D11-4 showed the strongest plant growth-promoting effect, which was found to be capable of producing IAA (22.3 mg/L), iron carrier (halo diameter 25.2 mm) and fixing nitrogen from air. The seven species of the actinobacteria were identified as Streptomyces angustmyceticus H4-6, Streptomyces rochei S2-2, Streptomyces globosus H6-1, Streptomyces iakyrus GD8-4, Streptomyces bottropensis GH8-6, Streptomyces paradoxus H8-2, and Streptomyces coralus D11-4. [Conclusion] The three habitats had abundant species of actinobacteria colonized in plant roots and rhizosphere, and the seven selected actinobacteria isolates were proven to possess biocontrol potential, which is worth of further research and development.

Abstract:[Objective] This study aimed to analyze the effect of Pennisetum sp. in different growth stage on soil physical-chemical properties and bacterial community structure, and provide a theoretical basis for better application of Pennisetum sp. to improve soil in Pisha sandstone area. [Methods] We used Illumina MiSeq sequencing to compare the effect of Pennisetum sp. in different growth stage on soil bacterial community structure. [Results] After planting Pennisetum sp., the water content of each plot was lowest in tillering stage and highest in maturity period. The soil organic content increased and soil pH did not change significantly. The dominant bacteria of every plot obtained at phylum level were Actinobacteria and Proteobacteria. At genus level, the highest abundance was unidentified. Bacterial diversity and species abundance in three sample plots were higher than those before planting Pennisetum sp. The soil bacterial diversity and species abundance of farmland were highest in jointing stage, the amount of bacteria was highest in tillering stage. The soil bacterial diversity and species abundance of hilltop were highest in tillering stage. The soil bacterial diversity of flood and was highest in seedling stage, the species abundance was highest in jointing stage. [Conclusion] The soil bacterial community structure in Pisha sandstone was similar in different growth stage of Pennisetum sp. Pennisetum sp. can obviously promote soil bacterial diversity and species abundance and improve soil organic matter content effectively. However, the soil bacterial diversity, amounts and organic matter content reached a maximum in different growth stage.