16S rDNA Sequence Analysis Research Articles

Seasonal dynamics, geographical range size, hosts, genetic diversity and phylogeography of Amblyomma sculptum in Argentina

The aim of this work was to generate knowledge on ecological aspects of Amblyomma sculptum in Argentina, such as seasonal dynamics, geographical range size, hosts, genetic diversity and phylogeography. Adult and immature A. sculptum ticks were collected in different localities of Argentina to know the geographical range size and hosts. The genetic diversity of this tick was studied through analyses of 16S rDNA sequences. To describe the seasonal dynamics, free-living ticks were monthly collected from October 2013 to October 2015. A. sculptum shows a marked ecological preference for Chaco Húmedo eco-region and “Albardones” forest of the great rivers in the wetlands in the Chaco Biogeographical Province, and for Selvas Pedemontanas and Selva Montana in the Yungas Biogeographical Province. This species has low host specificity, and it has large wild and domestic mammals as principal hosts to both immature and adult stages. Amblyomma sculptum is characterized by a one-year life cycle. Larvae peak in early winter, nymphs peaked during mid-spring, and adults during late summer and mid-summer. The genetic divergence was low and the total genetic variability was attributable to differences among populations. This fact could be associated to stochastics process linked to micro-habitat variations that could produce a partial restriction to gene flow among populations. The geographic regions do not contribute much to explain the A. sculptum population genetic structure, with an ancestral haplotype present in most populations, which gives rise to the rest of the haplotypes denoting a rapid population expansion.

Effects of Lactobacillus buchneri isolated from tropical maize silage on fermentation and aerobic stability of maize and sugarcane silages

AbstractThis study was aimed to perform a screening of Lactobacillus buchneri strains from maize silage and use them as inoculant in maize and sugarcane silages. In all, 151 lactic acid bacteria (LAB) strains were isolated from whole‐plant maize silage, and their identification was based on the sequence analysis of 16S rDNA. In total, 15 strains were categorized to the L. buchneri group and eight of these were selected based on growth rate and fermentation pattern. The selected strains were evaluated on fermentation and aerobic stability of maize and sugarcane silages. For maize, the inoculated silages had lower pH and higher LAB population, but lower acetic acid concentration in comparison with the untreated control silage. For sugarcane silage, the strains 56.1, 56.4 and 40788 resulted in highest dry‐matter (DM) content and lowest DM losses. However, only the strain 40788 showed lowest counts of yeasts and moulds. Sugarcane silages inoculated with the strains 56.9, 56.26 and the untreated control silage showed highest concentrations of lactic acid and ethanol, besides the great DM losses. Even so, for both crops, the aerobic stability was not affected by inoculation. After air exposure, all silages increased temperature and had high population of yeast and moulds. Nevertheless, the strains 56.1 and 56.4 are promising for use as a silage inoculant.

Characterization of Novel and Efficient Poly-3-hydroxybutyrate (PHB) Producing Bacteria Isolated from Rhizospheric Soils

Polyhydroxybutyrate (PHB) is an eco-friendly, biodegradable plastic which exhibits properties very similar to the conventional plastic, thus, having high potential for replacing non-degradable conventional polypropylene plastic that is responsible for several environmental problems at global level. The present study focuses on the isolation and characterization of potential novel PHB producers. A total of 194 PHB producing bacteria were isolated from rhizospheric soils of three different crops; and subjected to microscopic, biochemical and molecular characterization studies. Quantification of PHB production exhibited significant amount of PHB accumulation (120–132 mg/ml) by a few isolates (KW-4, MS-6, RoW-1, AW-1 and RoS-4). Fourier transform infrared spectroscopy (FTIR) analysis of PHB extracted from the isolates was found to be comparable to the spectra of standard PHB, thus, establishing the chemical nature of the extracted polymer. Detection of PHB granules by transmission electron microscopy (TEM) confirmed the isolates to be efficient PHB producers. For molecular characterization of the isolates, phbC gene amplification studies were undertaken, which resulted into an amplification product of 1089 bp, representing the complete PHB synthase gene. Using 16S rRNA gene amplification and sequencing, the selected isolates were identified as belonging to four major genera, viz., Bacillus, Lysinibacillus, Clostridium and Klebsiella; however, Bacillus being the predominant genera. Analysis of 16S rDNA sequences showed that a few isolates exhibited significant differences from their nearest neighbours (similarity level ranging between 93 and 99%), thus, strongly suggesting that they might represent novel strains. Some of these probable novel high PHB producers reported in the present study hold high potential to be exploited for further industrial-scale mass production of biodegradable polymer.

Identification and characterization of Candidatus Phytoplasma trifolii (16SrVI-D) inducing shoot proliferation disease of potato in India

Potato (Solanum tuberosum) is a widely cultivated and commercially important vegetable crop widely grown in India. Profuse shoot proliferation symptoms were observed in potato plants of two varieties, Kufri Khyati and Kufri Surya at the Research plots of All India Co-ordinated Research Project on Potato, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India during February, 2017. The symptomatic plants were tested for phytoplasma etiology. Nested and semi-nested PCR assays primed by primer pairs P1/P7 and 3Far/3Rev for 16S rRNA gene and secAF1/R3 for secA gene amplified DNA fragments of 1.3 kb (3Far/3Rev) and 840 bp (secAF1/R3), respectively from all the symptomatic potato samples but not from any asymptomatic plants. The 16S rDNA sequences of potato tuber (Acc. no. KY815100, Kufri Khyati; MG566064, Kufri Surya) and shoot phytoplasma strains (Acc. no. KY815099, Kufri Khyati; MG566063, Kufri Surya) shared 100% nucleotide sequence identity with 16S rDNA sequence of identified phytoplasma strains belonging to 16SrVI-D subgroup associated with Lens culinaris witches’ broom (Acc. no. KY439869), brinjal little leaf (Acc. no. KX284698) and Brassica oleracea var. capitata witches’ broom (Acc. no. KX671553) in pair wise sequence comparison. Sequence comparison of 840 bp of secA gene of potato phytoplasma strains from tuber of variety, Kufri Khyati (Acc. no. KY815101) and variety Kufri Surya (Acc. no. MG566065) showed 99% sequence identity to secA gene sequences of brinjal little leaf phytoplasma strains (Acc. no. KX610808, KX894792, KY064175), which belonged to 16SrVI-D subgroup. Phylogenetic analysis of 16S rRNA and secA gene sequences, and virtual RFLP analysis of 16S rDNA sequences of potato shoot proliferation phytoplasma strain confirmed their clustering and grouping with strains of clover proliferation subgroup D. Our results confirmed potato as a new host of 16SrVI-D subgroup of phytoplasmas in the world. The 16SrVI-D strain is wide spread in India and cause significant yield losses and hence poses serious socio-economic threat to commercially grown potato crops.

Ultrastructure of the marine benthic dinoflagellate Plagiodinium belizeanum (Dinophyceae) from the southeast Pacific island of Okinawa, Japan

:We isolated Plagiodinium belizeanum into clonal culture from the Pacific island of Okinawa (Japan) and characterized it using a combination of light microscopy, scanning electron microscopy, transmission electron microscopy and 18S/28S ribosomal (r) gene sequences. Although molecular phylogenetic analyses of 18S rDNA and 28S rDNA sequences were unable to resolve the phylogenetic position of P. belizeanum within dinoflagellates, the ultrastructural data provided some new traits for the species. For instance, double-membrane-bound vesicles, distinct from the mitochondria, were interpreted as autolysosomes containing electron-dense virus particles. The thecal plate pattern was Po 1′ 0a 5′′ 5(6)C 4S 5′′′ 0p 1′′′′, which is slightly different from the original description in having an additional epithecal plate and four sulcal plates. The laterally flattened cells were 22–34 μm long, 11–13 μm deep, and 15–18 μm wide and contained a peridinin-type plastid with lobes radiating from a central pyrenoid that lacked starch sheaths and was traversed by stacks of thylakoids. This isolate represents the first record of the species in Japan, and the new ultrastructural and DNA sequence data were used to emend the species description.

New records and host associations of the tick Ixodes apronophorus and the first detection of Ehrlichia sp. HF in Romania.

Ixodes (Ixodes) apronophorus is a neglected tick species and its geographical distribution, host associations, and role as a disease vector are not well known. We collected I. apronophorus from several locations in Romania. Morphological identification of ticks was confirmed by analysis of 16S rDNA and 12S rDNA gene sequences. We report new host associations of I. apronophorus, which was collected from dogs, foxes, and a hare-all new hosts for this tick species in Romania. Furthermore, we report for the first time occurrence of Ehrlichia sp. HF in I. apronophorus. Ehrlichia sp. HF was identified by sequencing a part of the 16S rDNA gene and was found in 16% (3/19) of the tested ticks. Ehrlichia sp. HF has not been previously reported in Eastern Europe and seems to have a much larger geographic distribution than previously known. Currently, it is unknown whether I. apronophorus is a competent vector for Ehrlichia sp. HF, or if the findings in this study represent infection in the hosts, namely dogs and fox.

Increasing the autotrophic growth of Chlorella USTB-01 via the control of bacterial contamination by Bdellovibrio USTB-06.

(i) To obtain and identify the predatory bacteria for the control of contaminated bacteria and to promote the autotrophic growth of Chlorella USTB-01. (ii) To identify and measure the different cell numbers in microalgal culture using flow cytometer. A predatory bacterial strain was isolated using Escherichia coli BL21 as a sole prey host, which was identified as Bdellovibrio USTB-06 by the analysis of 16S rDNA sequence. A flow cytometer was successfully used to identify and measure the cell numbers of Chlorella USTB-01, the contaminated bacteria and Bdellovibrio USTB-06 simultaneously in the autotrophic culture of Chlorella USTB-01 according to the identification of the different cell sizes. With the addition of Bdellovibrio USTB-06 at initial 104 plaque-forming units per ml, the contaminated bacteria severely decreased by about five counts (in log10 CFU per ml) and the growth of Chlorella USTB-01 was greatly increased by 37·0% compared with those of control respectively. Bdellovibrio USTB-06 could effectively promote the growth of Chlorella USTB-01 via the killing of the contaminated bacteria. Our study reveals a good biotechnology method to increase the growth of Chlorella USTB-01 which is very important in the industry of microalgal culture.

Molecular characterization of phytoplasmaassociated with four important ornamental plant species in India and identification of natural potential spread sources.

Phytoplasma suspected symptoms of phyllody, witches' broom, leaf yellowing, stunting and little leaf were observed in Chrysanthemum morifolium, Bougainvillea glabra, Jasminum sambac and Callistephus chinensis during survey of flower nurseries and experimental ornamental fields at Delhi, Maharashtra, Tamil Nadu and Karnataka from 2014 to 2016. Pleomorphic bodies typical to phytoplasma structures were observed in the phloem sieve elements of ultrathin sections of all the four symptomatic ornamental plants (stem tissue) in transmission electron microscope. Amplification of 1.8 and 1.2kb phytoplasma DNA products was observed in all the four test plants in PCR assays using universal primer pairs P1/P7 followed by nested primer pair R16F2n/R16R2, respectively. Pairwise sequence comparison, phylogeny and virtual RFLP analysis of 16S rDNA sequences confirmed the association of two phytoplasma subgroups (16SrI-B and 16SrII-D) in four ornamental plant species. 'Ca. P. aurantifolia' subgroup D (16SrII-D) was found associated with chrysanthemum phyllody and leaf yellowing at Delhi and Tamil Nadu, bougainvillea little leaf and yellowing at Delhi and Chinese aster phyllody at Bengaluru, Karnataka. However, jasmine little leaf and yellowing at Bengaluru, Karnataka and chrysanthemum stunting at Pune were found to be associated with 'Ca. P. asteris' subgroup B-related strains (16SrI-B). The identification of 16SrII-D subgroup phytoplasma infecting bougainvillea and 16SrI-B subgroup infecting jasmine are the new reports to the world. Besides weed species, Cannabis sativa showing witches' broom in jasmine fields at Bengaluru and Parthenium hysterophorus showing witches' broom symptoms in chrysanthemum fields at Delhi were identified to be caused by phytoplasma strains classified under subgroups 16SrI-B and 16SrII-D, respectively, by PCR assays and 16Sr DNA sequence comparison analysis. Among the three major leafhopper species identified, only Hishimonas phycitis was identified positive for 16SrI-B and 16SrII-D subgroups of phytoplasmas from chrysanthemum fields at Delhi and jasmine fields at Bengaluru, respectively. The identity of similar phytoplasma strains infecting ornamental species in leafhopper and the weed species in the present study suggested that H. phycitis and weeds may act as potential natural sources for secondary spread of the identified phytoplasma strains.

Molecular characterization of 'Clover proliferation' phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India.

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches' broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of~1.3kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit,bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of 'clover proliferation' (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetablesand one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of 'clover proliferation' subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.

Identification of bat trypanosomes from Minas Gerais state, Brazil, based on 18S rDNA and Cathepsin-L-like targets.

Several bat species can be infected by trypanosomes, but there is not much information about which of these parasites infect bats from Triângulo Mineiro and Alto Paranaíba, Minas Gerais state, Brazil, a formerly endemic region for Trypanosoma cruzi, the causative agent of Chagas disease. The aim of this study was to describe, characterize, and identify the presence of trypanosomes in bats. The captured bats (448) belong to four families and to 19 different species. Of those, 37 bats were found to be positive for trypanosomes by microhematocrit, (infection rate 8.3%) and 27 were positive after hemoculture analysis. Initially, the isolates were identified by PCR (18S rDNA, 24Sα rDNA, spliced leader, COII RFLP-PCR) using primers originally designed for T. cruzi. PCRs (18S rDNA, 24Sα rDNA) showed compatible bands for TcI, whereas COII RFLP-PCR showed a similar pattern associated to TcII. However, there was no DNA amplification using spliced leader as a target, revealing a discrepancy between the results. Phylogenetic analysis of Cathepsin L-like and 18S rDNA sequences proved that 15 of the isolates corresponded to Trypanosoma cruzi marinkellei and one to Trypanosoma dionisii. These results revealed that the diversity of trypanosome species in a region considered endemic for Chagas disease is greater than previous descriptions. All this can confirm the necessity of using DNA sequencing approaches in order to determinate trypanosomes species isolated from bats.

Association of ‘Ca. Phytoplasma asteris ’(16SrI group) with little leaf and witches ’broom symptoms of Catharanthus roseus

During survey of Gorakhpur area in August 2014, Cathranthus roseus plants showing little leaf and witches ’broom symptoms were analyzed for phytoplasma presence through nested PCR assays with universal phytoplasma primers P1/P6 and R16F2n/R16R2. 1.2kb amplicon was observed in symptomatic and positive control sample. The amplified products were eluted and directly sequenced. Aligned sequences were submitted in the GenBank with the accession number KX894734. BLASTn search analysis of 16S rDNA sequences of Catharanthus phytoplasma Gorakhpur isolate showed maximum identity with identified strains of 'Ca P asteris ’which was further supported by phylogenetic analysis.

Screening and characteristic analysis of Bacillus velezensis from sea cucumber ( Apostichopus japonicus ) ponds

PDF HTML阅读 XML下载 导出引用 引用提醒 刺参养殖池塘一株贝莱斯芽孢杆菌的分离及其生理特性 DOI: 作者: 作者单位: 1. 上海海洋大学 水产与生命学院, 上海 201306;2. 中国水产科学研究院黄海水产研究所, 青岛海洋科学与技术国家实验室, 海洋渔业科学与食物产出过程功能实验室, 山东 青岛 266071;3. 山东海跃水产科技有限公司, 山东 东营 257500 作者简介: 王成强(1988-),男,研究实习员,主要从事水产动物营养与饲料研究.E-mail:chengqiangwang@126.com 通讯作者: 中图分类号: S947 基金项目: 山东省农业良种工程重大课题；山东省自主创新与成果转化专项（2014ZZCX06205）；中央级公益性科研院所基本科研业务费专项资金项目（20603022016008）. Screening and characteristic analysis of Bacillus velezensis from sea cucumber (Apostichopus japonicus) ponds Author: Affiliation: 1. Shanghai Ocean University, Shanghai 201306, China;2. College of Fesheries and Life Science, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;Qingdao National Laboratory for Marine Science and Technology, Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao 266071, China;3. Shandong Haiyue Aquatic Science and Technology Co., Ltd, Dongying 257500, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:2014年5月，在东营的刺参（）养殖池塘环境中对水样和底泥样品进行了细菌分离和益生菌的筛选，共获得66株可培养细菌。以刺参“腐皮综合征”主要致病菌为指示菌进行拮抗作用实验，使用选择培养基对菌株的产淀粉酶和蛋白酶能力进行测定，筛选获得一株潜在益生菌株——DY-6，并进行了生理生化实验、16S rDNA基因序列分析、菌株生长特性及其对刺参的安全性研究。结果表明，DY-6对其指示菌假交替单胞菌（Vibrio parahaemolyticus）和溶藻弧菌（）均有良好的抑制作用，抑菌圈直径分别达到24 mm、22 mm、27 mm和37 mm，对淀粉和蛋白培养基的水解直径分别为28 mm和20 mm。利用该菌对刺参进行高浓度胁迫实验测试其安全性，实验期间浸浴组和投喂组刺参状态良好，没有发病和死亡现象。利用细菌生理生化和16S rDNA基因序列分析表明，DY-6与贝莱斯芽孢杆菌（ CP015911）的相似性为99%。因此，初步鉴定该菌株为贝莱斯芽孢杆菌。同时，将该菌株与商品化微生态制剂产品中分离的4株益生菌进行了比较，DY-6在温度20~35℃、盐度0~35范围内生长较快，优于上述4株菌，4 h进入对数生长期，10 h达到生长高峰。筛选的贝莱斯芽孢杆菌具有良好的抑菌能力，生长速度快，并且具有广温广盐的优点，不仅能够降低疾病发生率，还可更好地适应黄河口地区夏季水温偏高、盐度波动大的特点，由此具有良好的益生菌商业化开发潜力。 Abstract:To screen local probiotic strains, 66 cultivable bacterial strains were isolated from water and sediment samples from cultured ponds of sea cucumber () in Dongying in May 2014. Bacterial antagonistic experiments were conducted using V. parahaemolyticus and , which are main pathogenic bacteria of ‘skin ulcer syndrome’ for . In addition, selective medium was used to test the amylase production and protease activities of the isolated strains and to isolate a potential probiotic strain, DY-6. Then, its physiological-biochemical experiments and similarity analysis of 16s rDNA sequences as well as the growth characteristics of DY-6 experiments have been conducted. The safety experiment for the sea cucumber was also proceed. The DY-6 strain was successful in inhibiting V. alginolyticus, as indicated by inhibition zone diameters of 24, 22, 27, and 37 mm, respectively. Meanwhile, the hydrolysis zone diameters of amylase and protease were 28 and 20 mm, respectively. A safety experiment was designed to test the safety of DY-6 for sea cucumber. During the experiment, all sea cucumbers were in good conditions without falling ill. Based on physiological and biochemical experiments and 16s rDNA sequence analysis, strain DY-6 was determined to be 99% similar to B. velezensis. When compared to four bacterial strains isolated from commercial probiotic products, DY-6 exhibited superior growth at 20-35℃ and under salinities of 0 to 35. Moreover, DY-6 came into the logarithmic growth phase after 4 h of cultivation and reached growth peak after 10 h. In summary, strain DY-6 exhibited superb inhibition of sea cucumber pathogens, rapid growth, and both euryhalinous and euryhaline features. Therefore, DY-6 could be used to reduce the occurrence of sea cucumber disease and is also suitable for use at high temperatures during summer and in fluctuation salinity levels, as in the Yellow River Estuary. Thus, strain DY-6 has potential for commercial development. 参考文献 相似文献 引证文献

Diversity of phytoplasmas infecting different crops and weeds in Tripuraregion of India

Phytoplasma suspected symptoms of little leaf, chlorosis, yellowing, phyllody, witches’ broom, flat stem, flower virescence and stunting were observed in sixteen plant species during surveys in fields, gardens, fruit orchards and nurseries of six districts of Tripura, India in 2017–2018. Out of 15 symptomatic plant samples belonging to different species, 12 were positive through nested PCR assays for phytoplasma presence. Pair wise sequence comparison, phylogeny and virtual RFLP analysis of phytoplasma partial 16S rDNA sequences of these 12 samples indicated the presence of different phytoplasma groups and subgroups (16SrI-B, 16SrII-D, 16SrVI-D, 16SrXIV-A 16SrI, 16SrIX and 16SrXI). ‘Candidatus Phytoplasma asteris’-related phytoplasmas (16SrI-B) were identified in sesame phyllody affected plants at South Tripura and in crape jasmine, Celosia argentea and Zinnia elegans from Unakoti, Dhalai, North Tripura and West Tripura district. In West Tripura ‘Ca. P. australasiae'related phytoplasmas (16SrII-D) were identified with Jatropha hastata and bitter gourd; ‘Ca. P. trifolii’-related phytoplasmas (16SrVI-D) were identified in chili and brinjal and ‘Ca. P. cynodontis’-related phytoplasmas (16SrXIV-A) were identified in palm grass samples from Sipahijala district. ‘Ca. P. phoenicium’-related phytoplasmas (16SrIX) were identified in cowpea; ‘Ca. P. oryzae’-related phytoplasmas were identified in arecanut from North Tripura and ‘Ca. P. asteris’-related phytoplasmas were detected in Toona ciliata from Jampui Hills. This study suggests the presence of a wide genetic diversity of phytoplasmas infecting different crops and weeds in the Tripura state of India.

First Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Sweet Cherry Fasciation Disease in China

HomePlant DiseaseVol. 102, No. 1First Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Sweet Cherry Fasciation Disease in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Sweet Cherry Fasciation Disease in ChinaJ. Wang, C. X. Ai, X. M. Yu, K. P. Zhang, R. Gao, and X. D. LiJ. Wang†Corresponding author: J. Wang; E-mail: E-mail Address: jiewangsdau@163.comSearch for more papers by this author, C. X. AiSearch for more papers by this author, X. M. YuSearch for more papers by this author, K. P. ZhangSearch for more papers by this author, R. GaoSearch for more papers by this author, and X. D. LiSearch for more papers by this authorAffiliationsAuthors and Affiliations J. Wang † C. X. Ai X. M. Yu K. P. Zhang , Shandong Institute of Pomology, Taian, 271000, China R. Gao , Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, Shandong, 250100, China X. D. Li , Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Taian, Shangdong 271018, China. Published Online:24 Oct 2017https://doi.org/10.1094/PDIS-06-17-0816-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Sweet cherry (Prunus avium L.) is one of the most popular fruit crops grown worldwide as a nutritious horticultural fruit. It has high antioxidant potential because of the rich content of polyphenols, vitamin C, and other nutritious chemicals. Phytoplasmal diseases on cherry widely occur in China, Iran, Turkey, the Czech Republic, Lithuania, and Chile, with symptoms of branch proliferation, leaf yellowing, floral virescence, and so on (Wang et al. 2015). In June 2015, 2-year-old sweet cherry (Prunus avium L. cv. Brooks) trees with symptoms of typical stem fasciation and top sickle-like deformation, decline, delay in flowering, and stiff fruit were observed in a sweet cherry orchard in Zoucheng, Shandong, China (35°24′N, 116°58′E). Three diseased trees were found in 2 ha of cultivated area surveyed, indicating this disease may be sporadic and rare. To identify the causal agent, total genomic DNA was extracted from leaves and stems of three symptomatic trees and two asymptomatic trees using the SurePlant DNA Kit (CWBIO, Beijing, China). Phytoplasma specific 16SrRNA gene and tuf gene fragments were amplified using phytoplasma specific universal primers, P1/P7 and R16F2n/R16R2 (Lee et al. 1998), and fTuf1/rTuf1 and fTufu/rTufu (Schneider et al. 1997), respectively. Specific fragments, approximately 1.25 kb and 0.8 kb in length, were amplified from symptomatic plants, while no fragment was amplified from asymptomatic samples. After the fragments were cloned into the pMD18-T vector (Takara Bio Inc., Dalian, China), sequences of 1,244 bp and 841 bp were obtained (GenBank accession nos. KY828850 and MF174060). Phylogenetic analysis of phytoplasma 16S rDNA sequences or tuf gene sequences based on the maximum likelihood method using MEGA 6.0 placed the SCF phytoplasma within the 16SrI cluster. The iPhyClassifier (Zhao et al. 2009) analysis showed that the obtained 16S rDNA sequence shared 99.6% similarity with that of the ‘Candidatus Phytoplasma asteris’ reference strain (M30790), and sweet cherry fasciation (SCF) phytoplasma was almost identical (similarity coefficient of 0.98) to the 16SrI-B reference strain (NC005303). The profiles of in vitro RFLP analysis obtained by digestion of the nested-PCR products with TaqI and BfaI were similar to those of in silico RFLP analyses and confirmed that the SCF phytoplasma is a variant of 16SrI-B. Moreover, the SCF phytoplasma shared 99.1% similarity with cherry leaf bunchy phytoplasma ChBL strain (AY034089, Lithuania) and 99.0% similarity with sweet cherry virescence phytoplasmas (HQ148153, China), both of which belong to the 16SrI group. Furthermore, the SCF phytoplasma shared 90.0% similarity with sweet virescence phytoplasmas (KF148153, 16SrV-B), which have also been reported in China. As one of the most widespread phytoplasma groups, aster yellows group (16SrI) was divided into at least 20 subgroups (Fránová et al. 2016). To our knowledge, this is the first worldwide report on the 16SrI-B variant phytoplasma having a significant impact on sweet cherry production with the symptoms of stem fasciation, yellowing, and stunting.

Jenufa lobulosa sp. nov. (Chlorophyceae, Chlorophyta), a new epilithic, terrestrial species described from China

:Unicellular coccoid green algae are characterised by a high cryptic diversity, but such diversity is still poorly known, especially in terrestrial environments. The genus Jenufa, first described in 2011 from terrestrial habitats, currently includes three species: J. perforata, J. minuta and J. aeroterrestrica. We describe a new species, Jenufa lobulosa sp. nov. The type strain FACHB-2128 was isolated from sandstone surfaces in Nanning, Guangxi Province, a subtropical area in southwestern China, and the cultured material was examined using light and electron microscopy. It shared representative features of the genus: a parietal perforated chloroplast with several lobes, no pyrenoids, and asexual reproduction via autospores. However, J. lobulosa differed from congeners in the mean size of its vegetative cells and autosporangia, the number of autospores and cell wall thickness. Phylogenetic analyses of 18S rDNA and rbcL sequences showed that strain FACHB-2128 belonged to the Jenufa clade of the Chlorophyceae, forming an independent lineage and differing from any known species. The position of strain FACHB-2128 within Jenufa was also supported by ITS2 rDNA sequences.

Antibacterial activity and probiotic characterization of autochthonous Paenibacillus polymyxa isolated from Anabas testudineus (Bloch, 1792)

Microbial fish pathogens are prevalent in aquaculture. Control of bacterial fish pathogens is important and bio control of pathogenic bacteria is a novel field of study. The aim of this study was to evaluate the antagonistic activity of bacteria isolated from Anabas testudineus against potent fish pathogens. The cellular components/preparations and filtered cell free culture supernatants were effective against six fish pathogens. Altogether 110 strains were isolated from fish proximal and distal intestine, out of which 10 strains were selected through well diffusion method. From them a strain HGA4C having prominent antimicrobial activity was selected as candidate probiotic strain. The whole-cell product, heat-killed whole-cell product and the filtered broth were exhibited bactericidal activity against the tested pathogens. Among them cell free culture supernatant showed maximum inhibition. In addition, isolated candidate probiotic bacterium was capable of producing extracellular enzymes important for the digestion of food ingredients and was effectively grown in fish mucus obtained from Oreochromis niloticus. The strain tolerated gradient of bile juice secreted by the host and effectively produced biofilm. Analysis of 16S rDNA sequence revealed that isolated strain HGA4C was Paenibacillus polymyxa (MF457398.1). Furthermore intraperitoneal injection of the bacterium did not induce any pathological anomalies or mortalities in Oreochromis niloticus and disclosed the safety of this bacterium as a candidate probiotic in aquaculture.

Antibacterial activity of autochthonous bacteria isolated from Anabas testudineus (Bloch, 1792) and it's in vitro probiotic characterization

Antimicrobial potentials of bacteria isolated from Anabas testudineus have been evaluated through in vitro antagonistic activity against potent fish pathogens. The cellular components and filtered culture medium were effective against six fish pathogens. Altogether 110 strains were isolated from the fish gut, out of which 10 strains were selected through well diffusion method. From them, a strain HGA8B having cumulative maximum score was selected as candidate probiotic. The whole-cell product, heat-killed whole-cell product, Ethyl acetate extract, and the filtered broth were exhibited bactericidal activity against the tested pathogens. In addition, the isolated bacterium was capable of producing extracellular enzymes important for the digestion of food materials and was capable of growth in fish mucus from Oreochromis niloticus. The strain tolerated bile juice secreted by the host and effectively produced biofilm. Analysis of 16S rDNA sequence revealed that isolated strain HGA8B was Bacillus sp. (MF351637). Furthermore, intraperitoneal injection of the bacterium did not induce any pathological signs, symptoms or mortalities in Oreochromis niloticus and revealed the safety of this bacterium as a candidate probiotic in aquaculture.

Analysis of distribution characteristics of clinical isolates of nontuberculous mycobacteria in a general hospital

Objective To investigate the current prevalence of nontuberculous mycobacteria (NTM) in Shanghai, and to study the distribution characteristics of NTM clinical isolates, which may help to improve the diagnostic level of NTM and provide guidance for effective prevention and treatment of NTM infection. Methods Culture-positive isolates of clinical mycobacteria were collected from 2008 to 2013 in Huashan Hospital affiliated to Fudan University. All isolates were heat inactivated, and the genomic DNA was extracted and the species were identified by comprehensive comparative analysis of 16S rDNA, hsp65 and rpoB target genes sequencing. Results From January 2008 to December 2013, the overall mycobacterial culture-positive rate was 4.1% (411/10 015). After excluding the repeated isolates, a total of 253 culture-positive mycobacteria isolates were collected for the species identification. By genes sequencing analysis, 140 isolates were identified as mycobacterium tuberculosis complex (MTBc), 102 NTM and 11 Nocardias, accounting for 55.3%, 40.3% and 4.4%, respectively. Positive rate of NTM isolates had an increasing trend from 25.0% in 2008 to 42.7% in 2013, reaching a highest rate of 54.9% in 2012. In further analysis of 102 NTM isolates, 16 species were identified. Among them, 28 were M. abscesses, 18 strains of M. marinum, 17 strains of M. avium-intracellulare complex and 10 strains of M. fortuitum, accounted for 27.5%, 17.6%, 16.7% and 9.8%, respectively. Conclusions Both of the isolation number and isolation rate of NTM in the general hospital are increasing. NTM related cases are also increasing in recent years, which mainly caused by M. abscess, M. marinum, M. avium-intracellulare complex and M. fortuitum. Key words: Nontuberculous mycobacterium; Target gene sequencing; Species identification; Distribution type

Characterization of garlic endophytes isolated from the black garlic processing.

The objectives of this study were to isolate and identify garlic endophytes, and explore the characteristics of dominant strains. Garlic endophytes were studied through phenotypical characterization and comparative sequence analysis of 16S rDNA based on culture‐dependent approaches. Representative strains inferred from 16S rDNA sequencing were selected for further identification by gyrA and rpoB gene loci and phylogenetic analysis based on concateneted house‐keeping sequences. Seven kinds of Bacillus were found from garlic and black garlic, respectively. Further studies demonstrated that the total bacteria and endophytes showed a sharp decrease firstly, followed by a rapid rise, then maintained at a certain level, and finally slowed down during the black garlic processing. B. subtilis, B. methylotrophicus, and B. amyloliquefaciens were the dominant strains. The selected strains were capable of fermenting glucose, lactose, sucrose, and garlic polysaccharide to produce acid but no gas, with a strong ability of heat resistance. The results indicated that there were a certain number of garlic endophytes during the black garlic processing, and Bacillus was the dominant strains under the conventional culture‐dependent methods. This report provided useful information for the presence and type of garlic endophytes during the black garlic processing, which were of great significance to study the formation mechanism and quality improvement of black garlic in the future, as well as the security of garlic powder.

Reproductive isolation between sympatric Anguilla japonica and Anguilla marmorata.

Species-specific restriction fragment length polymorphism in the intron of the androgen receptor gene (ar5) was found in glass to silver-stage individuals of Anguilla japonica (n = 51) and A. marmorata (n = 21). The sequence analysis of 16S rDNA from 328 anguillid leptocephali collected in the North Equatorial Current of the western North Pacific Ocean revealed the specimens to be A. japonica (n = 194), A. marmorata (n = 128), A. bicolor pacifica (n = 5) and A. luzonensis (n = 1). All leptocephali of A. japonica and A. marmorata were monomorphic and did not share an allele at the ar5 locus, indicating that the two species are reproductively isolated.