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During epidemiological surveillance of Feline calicivirus (FCV) isolates in Shanghai, China, a natural mutant of FCV, designated SH1909, was successfully isolated from a stray cat. The complete genome sequence of SH1909 was determined in this study. Sequence comparison and analysis showed that thirteen unique aa residues substitutions and single-aa insertion of N or Y were observed in SH1909, which indicated that SH1909 was a novel and natural mutant of FCV. Interestingly, phylogenetic analysis based on LC-VP1 showed SH1909 could be clustered into an independent evolutionary branch with some Chinese isolates and was more distantly related to vaccine strains, indicating its potential to escape from vaccine-elicited immunity. According to the predicted B-cell epitopes of LC-VP1, amino acid mutation sites and positive selective sites, peptide C (aa sites: 445–460) and, peptide D (aa sites: 425–440) located in the hypervariable regions of LC-VP1, may result in the decreased immunological protection. Moreover, amino acid sites 439 and 449 may be responsible for the potential immune escape of SH1909. This study provides an important insight into genetic variations of FCV and vaccine development.

Enterobacter cloacae is a symbiotic bacterium, which is one of the species in intestinal microbiota in many humans and animals. In some cases, it causes harmful diseases in humans. More and more studies showed that E. cloacae caused disease on plants, such as macadamia, ginger, mulberry, onion, chili pepper and rice. Garlic (Allium sativum L.) is one of crops with economic importance in the world. It is also widely grown in China. During 2018 to 2020, the naturally infected garlic bulbs from garlic fields in Kaifeng of Henan Province (34.55° N; 114.78° E) showed dry brown discoloration and rot symptoms. The diseased garlic seriously affected its edible value. Voucher specimens collected on June, 2019 were deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU190620. To identify the causal agent of this disease, the bulb tissues of infected garlic were surface-disinfested in 0.6% sodium hypochlorite, dipped in75% ethanol, and then dipped in sterile distilled water. These bulbs were plated on LB medium and incubated at 37℃. A number of white colonies grew on the medium after plating for 16 h. All colonies were round, white, opaque, smooth, and gram-negative, which is a typical characteristic of Enterobacter. To confirm the initial identification of the isolated bacterium, the fragments of 16S rRNA gene and gyrA gene of 6 colonies were amplified, respectively. The PCR products were purified and sequenced. All 16S rRNA and gyrA sequences were identical to each other. The sequences of 16S rRNA gene and gyrA gene were deposited in GenBank with accession numbers MW730711 and MW768876, respectively. BLAST searches were conducted using the sequences of 16S rRNA and gyrA. The results showed 99.72%, and 96.91% identity with the corresponding sequences of E. cloacae strain CBG15936 (CP046116.1), respectively. Phylogenetic trees were performed using the neighbor-joining (NJ) method of MAGA X based on the sequences of 16S rRNA gene and gyrA gene. Phylogenetic tree indicated that isolates are most likely E. cloacae. Pathogenicity tests were performed by puncturing garlic bulbs with a hypodermic needle, followed by dipping in bacterial suspension with the concentration of 2×108 CFU for 5 minutes. As control, the garlic bulbs were treated with sterile water. The inoculated and control were incubated at 30°C. 7 days after inoculation, brown discoloration and rot were developed on all inoculated garlic bulbs. No symptoms were observed in the control group.The symptoms were similar to that observed on the original diseased garlic bulbs. The garlic bulbs in inoculated and control were ten replicates in each independent biological experiments. The pathogenicity tests were conducted three times with similar results. The bacteria were re-isolated from the symptomatic diseased garlics and confirmed as E. cloacae by morphological and sequence analyses as above. The re-isolated bacteria were identified by biochemical and physiological characteristics using API 20E strips. The results of the identification were identical to those of the edible ginger strains and the chili pepper strains. As far as we know, this is the first report of bulb decay on garlic caused by E. cloacae. The results are of great significance not only for the management of garlic bulbs during postharvest handling and storage, but also for the further research of opportunistic human pathogens E. cloacae.

During the period of the epidemiological surveillance of Feline calicivirus (FCV) isolates, a natural mutant of FCV, designated SH1909, was successfully isolated from a stray cat in Shanghai, China. The complete genome sequence of SH1909 was also determined in this study. Sequence comparison and analysis show that thirteen unique aa residues substitutions and single-aa insertion of N or Y were observed in SH1909 when compared with other FCV isolates. Phylogenetic analysis showed that SH1909 was assigned into a major evolutionary branch and distantly related to vaccine strains. These results indicated that SH1909 was a natural mutant of FCV circulating in China, which has the potential to escape from immunity of FCV vaccine.

In the present study, a mild salt stress (1 M NaCl, for 30 min) pretreatment of Candida oleophila increased its subsequent tolerance to lethal levels of salt stress (6 M NaCl), and low pH (pH 3.4). The sublethal salt stress induced the activation of antioxidant enzymes in the antagonistic yeast, including catalase, thioredoxin reductase, glutathione peroxidase, and glutathione reductase, at both the transcript and enzyme activity level. Sublethal salt stress-adapted (SA) yeast cells exhibited higher survival, compared to non-stress-adapted (NSA) cells, when exposed to 6 M NaCl. SA cells exhibited increased efficacy in controlling infection of kiwifruit by Penicillium expansum and Botrytis cinerea, and a higher growth rate in fruit wounds, compared to the NSA yeast. Collectively, the study further supports the premise that stress adaptation of antagonistic yeasts can improve their biocontrol efficacy.

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 μL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

Biosynthesis of advanced biofuels by engineered non-natural microorganisms has been proposed to be the most promising approach for the replacement of dwindling fossil fuel resources. Brevibacterium flavum (Bf) is a model brevibacterium aerobe which lacks basic and applied research that could enable this species to produce biofuels. There are no reports regarding engineering this microorganism to produce advanced alcohols before. Here, for the first time, we developed the bacterium as a novel biosynthetic platform for advanced alcohols production via the mutagenesis and engineering to produce 2-ketoacids derived alcohols. In order to enhance the strain's capability of producing advanced alcohols, we preferentially improved intrinsic metabolism ability of the strain to obtain improved expression host (IEH) via generating mutagenesis libraries by whole cell mutagenesis (WCM). The IEH was determined via screening out the mutant strain with the highest production of branched-chain organic acids (BCOA) using high throughput screening method.. Subsequently, a novel vector system for Bf was established, and the corresponding biosynthetic pathway of directing carbon flux into the target advanced alcohols was recruited to make the bacterium possess the capability of producing advanced alcohols and further enhance the production using the IEH. Specifically, we generated bioengineered strains that were able to synthesize up to the highest 5362 and 4976 mg/L isobutanol, 1945 and 1747 mg/L 2-methyl-1-butanol (2 MB), and 785.34 and 781 mg/L 3-methyl-1-butanol (3 MB) from pure glucose and duckweed substrates, respectively. Our findings confirmed the feasibility and potential of using Bf as a novel biosynthetic platform to generate advanced biofuels with glucose and inexpensive renewable feedstock-duckweed as a fermentation substrate. Biotechnol. Bioeng. 2017;114: 1946-1958. © 2017 Wiley Periodicals, Inc.

Biological control utilizing antagonistic yeasts has been actively pursued as an alternative to synthetic fungicides for the management of postharvest diseases. Abiotic stress resistance is an important attribute for antagonistic yeasts, directly associated with their biocontrol efficacy. The MADS-box transcription factor, Rlm1, has been reported to regulate the response of model yeasts to cell wall stress. Rlm1 in the antagonistic yeast, Candida oleophila, was found to play a role in resistance to salt, heat, and oxidative stress. Two Rlm1 mutants (ΔRlm1-1 and ΔRlm1-2) were generated. Compared to the wild-type (WT), C. oleophila I-182, ΔRlm1-1, and ΔRlm1-2 were more sensitive to a variety of stresses, including heat, salt, and oxidative stress. The mutants also exhibited lower biocontrol efficacy against gray mold caused by Botrytis cinerea, and slower growth in kiwifruit wounds with respect to the WT. This study provided the information to understand the relationship between the Rlm1 transcription factor, stress resistance, and biocontrol efficacy of antagonistic yeasts used for the biocontrol of postharvest diseases.

Combing the one-dimensional infrared spectroscopy (FTIR) technology with two order derivative spectrum technology, the growth change rule of bacillus cereus, the common food borne pathogenic bacteria, are analyzed without destruction, It is found that capsule, spore and other structures of bacillus cereus can be identified, based on the two order derivative spectra characteristic absorption peak. Observing shows the symmetric & anti symmetric carboxyl group stretching vibration absorption peaks near 1604.48 cm−1 and 1396.21 cm−1 gradually weaken from lag phase to the stable phase. With protein amide absorption peak near 1654.63 cm−1 tending to be stable in the three stages, the structural changes of cell capsule can be acknowledged. The DPA absorption peak near 1617.98 cm−1, 1384.64 cm−1, and 1560.13 cm−1 indicates the presence of bacillus, changing in three stages from lag phase to stable phase. Experiments show that FTIR can distinguish cells’ material structure, which lays a theoretical foundation for the related devices of fast detection for bacillus cereus.

Bacillus cereus is a kind of common food-borne pathogen that can cause vomiting and diarrhea shortly after ingestion. The spectroscopy properties of Bacillus cereus were measured using the infrared reflectance spectroscopy, which was a nondestructive technology. The influence of Bacillus cereus concentration on the spectroscopy was explored based on the two-dimensional (2D) correlation spectroscopy method. The results showed that some functional groups of capsule in pure Bacillus cereus culture, such as carboxyl, protein amide I and amide II, C-H methyl and methylene could induced some self-correlation peaks near 1592 cm−1, 1652 cm−1, 1512 cm−1 and 1412 cm−1 respectively. Some functional groups of spore, such as COO- group and C-H bond could induced some self-correlation peaks near 1348 cm−1, 1616 cm−1 and 1592 cm−1 respectively. In milk sample, the functional groups of capsule and spore could induced some self-correlation peaks too. The infrared spectroscopy combined with 2D correlation spectroscopy analysis method could be a effective method for the Bacillus cereus detection.

Powdery mildew is one of the major diseases of facilities vegetables. In order to achieve early, fast, and accurate diagnosis of powdery mildew, with TCS3200 color sensor and infrared sensor as detecting port and 12864 dot matrix LCD as display, the system explores the external change such as the color change of the blade in health and disease stage and change of reflection spectra. Through tracking experiment of different stages of cucumber leaves infected, the results show that the system can identify change of optical frequency values and the RGB values in the health cucumber leaves and infected cucumber leaves and thus provides effective warning alarm for controlling early disease occurrence.

With succinic anhydride as acylating agent, three commercial lipases – Candida antarctica lipase B (CALB), Pseudomonas cepacia lipase and Pseudomonas fluorescens lipase – were employed in the kinetic resolution of a series of rac -alkyl alcohols: 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, 3-hexanol, 3-methyl-2-butanol, 6-methyl-5-heptene-2-ol, 3-methyl-2-cyclohexene-1-ol and 2-methyl-1-pentanol. The most effective tested enzyme, immobilized CALB, was able to resolve most of the alcohols with high enantioselectivity, even higher (with enantiomeric ratios up to 115 and 91, for 3-hexanol and 3-methyl-2-butanol, respectively) than when vinyl acetate was used as the acylating agent. More importantly, the unreacted alcohol and the monoester succinate produced could be easily separated by a simple aqueous base-organic solvent liquid–liquid extraction. Using succinic anhydride as acylating agent and CALB, enantiomerically pure ( S )-2-pentanol with 99% ee and ( R )-2-pentanol with 95% ee were prepared in gram-scale reactions.

Powdery mildew is a fungal disease found in a wide range of plants and can significantly reduce crop yields. Bacterial strain LJ02 is a biocontrol agent (BCA) isolated from a greenhouse in Tianjin, China. In combination of morphological, physiological, biochemical and phylogenetic analyses, strain LJ02 was classified as a new member of Bacillus amyloliquefaciens. Greenhouse trials showed that LJ02 fermentation broth (LJ02FB) can effectively diminish the occurrence of cucurbits powdery mildew. When treated with LJ02FB, cucumber seedlings produced significantly elevated production of superoxide dismutase, peroxidase, polyphenol oxidase and phenylalanine ammonia lyase as compared to that of the control. We further confirmed that the production of free salicylic acid (SA) and expression of one pathogenesis-related (PR) gene PR-1 in cucumber leaves were markedly elevated after treating with LJ02FB, suggesting that SA-mediated defense response was stimulated. Moreover, LJ02FB-treated cucumber leaves could secrete resistance-related substances into rhizosphere that inhibit the germination of fungi spores and the growth of pathogens. Finally, we separated bacterium and its fermented substances to test their respective effects and found that both components have SA-inducing activity and bacterium plays major roles. Altogether, we identified a BCA against powdery mildew and its mode of action by inducing systemic resistance such as SA signaling pathway.

To enhance the enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 (APE1547), a directed evolution approach is employed to generate mutant library from the native enzyme. A mutation (TBC26) is identified after one round of epPCR. The enantioselectivity of TBC26 is increased up to 2.6-fold compared to that of wild type enzyme. TBC26 contains five amino acid substitutions (R11G, L36P, V223A, I551L, A564T). The five mutation sites are spatially distant to the catalytic center. According to the published crystal structure of WT and considering the changes of secondary and tertiary structure, here we try to explain the change of enantioselectivity of the TBC26. The results suggest that the change of enantioselectivity of enzyme has a close relationship to the configuration of the enzyme.

The volatile oils of Arundo donax Linn (giant reed) were extracted by two methods, one was Water-steam distillation, the other was petroleum extraction and then fractionated with some chemical reagent. Components of the volatile oils were analyzed and identified by gas chromatograph-mass spectrometry (GC-MS), and the allelopathic effect of the volatile oils were also explored by bioassay of Microcystin aeruginosa. The results showed that the volatile oils had allelopathic activity. This study also compared components of the volatile oils from giant reed in different extract methods. The conclusion of the study provided important information for recovering eutrophic waters by aquatic macrophytes.