Bursaphelenchus xylophilus detection and analysis system based on CRISPR - Cas12.

Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.

Brenneria corticis sp. nov., isolated from symptomatic bark of Populus×euramericana canker.

A Gram-stain-negative, facultatively anaerobic, motile bacterial strain, designated gBX10-1-2T, was isolated from symptomatic bark of Populus×euramericana canker in China. Phylogenetic analysis based on its 16S rRNA gene sequence showed that the novel isolate belonged to the genus Brenneria, and shared the highest sequence similarity to Brenneria nigrifluens LMG 2694T (98.3 %). In the phylogenetic trees based on the four housekeeping genes sequences, the novel strain formed a separate branch different from B. nigrifluens LMG 2694T, indicating that the novel strain should be classified as a novel species. The genome sequence-derived average nucleotide identity (ANI) values between the novel isolate and B. nigrifluens LMG 2694T, Brenneria roseaesubsp. roseae FRB 222T and Brenneria roseaesubsp. americana FRB 223T were less than 85 %, lower than the proposed species boundary ANI cut-off value (95-96 %). The DNA G+C content was 56.2 mol%, and the main fatty acids were C16 : 0, C16 : 1ω7c, C18 : 1ω7c and C17 : 0cyclo. Based on the phenotypic and genotypic characteristics, strain gBX10-1-2T represents a novel species of genus Brenneria, for which the name Brenneria corticis sp. nov. is proposed. The type strain is gBX10-1-2T (=CFCC 11842T=KCTC 42840T).

Sphingomonas aeria sp. nov., isolated from air.

A Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented and rod-shaped bacterial strain, designated B093034T, was isolated from air at the foot of Xiangshan mountain, located in Beijing, China. Cells of strain B093034T were oxidase-negative and catalase-positive. Growth was observed at 4-41 °C, at pH 4.5-10.0 and at 0-7 % (w/v) NaCl. The isolate contained Q-10 as the predominant isoprenoid quinone, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0 and C14 : 02-OH as the major fatty acids, sym-homospermidine as the major polyamine, and sphingoglycolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, aminolipid, two unidentified phospholipids and three unidentified polar lipids as the polar lipids. The DNA G+C content was 67.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain B093034T grouped with members of the genus Sphingomonas and was closely related to Sphingomonas sanguinis IFO 13937T (96.49 % similarity), Sphingomonas pseudosanguinis G1-2T (96.37 %), Sphingomonas ginsenosidimutansGsoil 1429T (95.99 %) and Sphingomonas endophytica YIM 65583T (95.78 %). On the basis of the polyphasic evidence presented here, strain B093034T represents a novel species of the genus Sphingomonas, for which the name Sphingomonasaeria sp. nov. is proposed. The type strain is B093034T (=CFCC 13949T=LMG 30133T).

Phylogenetic analysis of family Neisseriaceae based on genome sequences and description of Populibacter corticis gen. nov., sp. nov., a member of the family Neisseriaceae, isolated from symptomatic bark of Populus × euramericana canker.

Two Gram-stain negative aerobic bacterial strains were isolated from the bark tissue of Populus × euramericana. The novel isolates were investigated using a polyphasic approach including 16S rRNA gene sequencing, genome sequencing, average nucleotide identity (ANI) and both phenotypic and chemotaxonomic assays. The genome core gene sequence and 16S rRNA gene phylogenies suggest that the novel isolates are different from the genera Snodgrassella and Stenoxybacter. Additionally, the ANI, G+C content, main fatty acids and phospholipid profile data supported the distinctiveness of the novel strain from genus Snodgrassella. Therefore, based on the data presented, the strains constitute a novel species of a novel genus within the family Neisseriaceae, for which the name Populibacter corticis gen. nov., sp. nov. is proposed. The type strain is 15-3-5T (= CFCC 13594T = KCTC 42251T).

Populibacterium corticicola gen. nov., sp. nov., a member of the family Jonesiaceae, isolated from symptomatic bark of Populus × euramericana canker.

Four Gram-stain-positive, aerobic, motile bacterial strains were isolated from the bark tissue of Populus × euramericana canker. Growth occurred between 10 and 37 °C and at pH 6-10, with optimal growth at 28-30 °C and pH 7.0-8.0. Growth occurred at 0-3 % (w/v) salinity. The strains were positive for oxidase and catalase activity. The major fatty acids were anteiso-C15 : 0 and C16 : 0. The phospholipid profiles contained diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, two phospholipids and five glycolipids. The peptidoglycan type was A4α, which is based on l-Lys-d-Ser-d-Asp. The DNA G+C content was 58.5 mol%. Based on 16S rRNA gene sequence analysis, as well as physiological and biochemical characteristics, the strains are considered to represent a novel species of a new genus in the family Jonesiaceae. The name proposed is Populibacterium corticicola gen. nov., sp. nov. The type strain of Populibacterium corticicola is 2D-4T (=CFCC 11886T=KCTC 33576T).

Brenneria populi sp. nov., isolated from symptomatic bark of Populus×euramericana canker.

Five Gran-stain-negative, facultatively anaerobic, motile, bacterial strains were isolated from symptomatic bark tissue of Populus×euramericana canker. Strains grew at 4-41 °C, pH 4-10 and 0-6 % (w/v) salinity. They were positive with respect to catalase activity and negative for oxidase activity, nitrate reduction and the Voges-Proskauer reaction. Analysis of 16S rRNA gene sequences indicated that these five poplar isolates belong to the genus Brenneria, having highest sequence similarity of 95.98 % with Brenneria goodwinii LMG 26270(T). These five isolates formed a single cluster based on multilocus sequence analysis, indicating that they all belong to a single taxon within the genus Brenneria, which was confirmed by DNA-DNA hybridization. The DNA G+C content was 54.9-55.7 mol%, and the main fatty acids were C16 : 0, C18 : 1ω7c, C17 : 0 cyclo and C16 : 1ω7c/iso-C15 : 0 2-OH. Based on these results, we describe a novel species of the genus Brenneria with the proposed name Brenneria populi sp. nov. The type strain is D9-5(T) ( = CFCC 11963(T) = KCTC 42088(T)).

Effects of application of corn straw on soil microbial community structure during the maize growing season.

This study investigated the influence of corn straw application on soil microbial communities and the relationship between such communities and soil properties in black soil. The crop used in this study was maize (Zea mays L.). The five treatments consisted of applying a gradient (50, 100, 150, and 200%) of shattered corn straw residue to the soil. Soil samples were taken from May through September during the 2012 maize growing season. The microbial community structure was determined using phospholipid fatty acid (PLFA) analysis. Our results revealed that the application of corn straw influenced the soil properties and increased the soil organic carbon and total nitrogen. Applying corn straw to fields also influenced the variation in soil microbial biomass and community composition, which is consistent with the variations found in soil total nitrogen (TN) and soil respiration (SR). However, the soil carbon-to-nitrogen ratio had no effect on soil microbial communities. The abundance of PLFAs, TN, and SR was higher in C1.5 than those in other treatments, suggesting that the soil properties and soil microbial community composition were affected positively by the application of corn straw to black soil. A Principal Component Analysis indicated that soil microbial communities were different in the straw decomposition processes. Moreover, the soil microbial communities from C1.5 were significantly different from those of CK (p < 0.05). We also found a high ratio of fungal-to-bacterial PLFAs in black soil and significant variations in the ratio of monounsaturated-to-branched fatty acids with different straw treatments that correlated with SR (p < 0.05). These results indicated that the application of corn straw positively influences soil properties and soil microbial communities and that these properties affect these communities. The individual PLFA signatures were sensitive indicators that reflected the changes in the soil environment condition.

Multilocus sequences confirm the close genetic relationship of four phytoplasmas of peanut witches'-broom group 16SrII-A.

Four witches'-broom diseases associated with Arachis hypogaea (peanut), Crotalaria pallida, Tephrosia purpurea, and Cleome viscosa were observed in Hainan Province, China during field surveys in 2004, 2005, and 2007. In previously reported studies, we identified these four phytoplasmas as members of subgroup 16SrII-A, and discovered that their 16S rRNA gene sequences were 99.9-100% identical to one another. In this study, we performed extensive phylogenetic analyses to elucidate relationships among them. We analyzed sequences of the 16S rRNA gene and rplV-rpsC, rpoB, gyrB, dnaK, dnaJ, recA, and secY combined sequence data from two strains each of the four phytoplasmas from Hainan province, as well as strains of peanut witches'-broom from Taiwan (PnWB-TW), "Candidatus Phytoplasma australiense", "Ca. Phytoplasma mali AT", aster yellows witches'-broom phytoplasma AYWB, and onion yellows phytoplasma OY-M. In the 16S rRNA phylogenetic tree, the eight Hainan strains form a clade with PnWB-TW. Analysis of the seven concatenated gene regions indicated that the four phytoplasmas collected from Hainan province cluster most closely with one another, but are closely related to PnWB-TW. The results of field survey and phylogenetic analysis indicated that Cr. pallida, T. purpurea, and Cl. viscosa may be natural plant hosts of peanut witches'-broom phytoplasma.

Acinetobacter puyangensis sp. nov., isolated from the healthy and diseased part of Populus xeuramericana canker bark.

Two Gram-negative, non-motile, rod-shaped strains, BQ4-1(T) and NHI3-2, isolated respectively from the healthy and diseased part of Populus ×euramericana canker bark, were characterized using a polyphasic approach. Chemotaxonomic characterization supported the inclusion of the two strains in the genus Acinetobacter, with genomic DNA G+C contents (42.5-43 mol%) within the range observed for this genus (38-47 mol%) and 9-octadecenoic acid (C18 : 1ω9c, 39.87 %), hexadecanoic acid (C16 : 0, 11.26 %) and summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c, 18.90 %) as major fatty acids. Phylogenetic analysis based on 16S rRNA, rpoB and gyrB gene sequences revealed that strains BQ4-1(T) and NHI3 did not cluster with any species with validly published names, and formed a distinct cluster with 99-100 % bootstrap support on three phylogenetic trees within the genus Acinetobacter. Acid was not produced from d-glucose, and haemolysis was not observed on agar media supplemented with sheep erythrocytes. On the basis of phenotypic, genotypic and phylogenetic characteristics, the two strains are considered to represent a novel species of the genus Acinetobacter, for which the name Acinetobacter puyangensis sp. nov. is proposed. The type strain is BQ4-1(T) (= CFCC 10780(T) = JCM 18011(T)).