In vitro and in vivo selection of probiotic purple nonsulphur bacteria with an ability to inhibit shrimp pathogens: acute hepatopancreatic necrosis disease‐causing Vibrio parahaemolyticus and other vibrios

Shrimp cultivation has been faced with huge losses in productivity caused by infectious shrimp pathogenic vibrios, especially Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (AHPND). Hence, purple nonsulphur bacteria (PNSB) were isolated from shrimp ponds for investigating their abilities to control shrimp pathogenic Vibrio spp. and their use as probiotics for sustainable shrimp cultivation. Based on their probiotic properties, strains S3W10 and SS15 were selected because of their strong abilities to produce amylase, gelatinase and vitamin B12. However, only three PNSB strains (SS15, TKW17 and STW181) strongly inhibited V. harveyi_KSAAHRC and V. vulnificus_KSAAHRC including V. parahaemolyticusAHPND strains by secreting antivibrio compounds. Four selected PNSB also grew in the presence of pancreatic enzymes, and they were identified as Rhodobacter sphaeroides for strains S3W10, SS15 and TKW17 and Afifella marina for strain STW181. The effects of a mixed culture were also investigated as follows: T1 (S3W10 + SS15), T2 (S3W10 + TKW17) and T3 (S3W10 + STW181) on postlarval white shrimp (Litopenaeus vannamei) for 60 days by comparison with a control. All three probiotic PNSB sets significantly improved the digestive enzyme activities and shrimp growth with their proliferation in shrimp gastrointestinal tract although the shrimp survival was not significantly different. They also significantly reduced the cumulative mortality of shrimp exposed to a virulent AHPND strain (V. parahaemolyticusSR2). This is the first to conclude that selected probiotic PNSB strains have great potential to be used for shrimp cultivation to control vibrios including AHPND strains.     

Silage and total mixed ration hygienic quality on commercial farms: implications for animal production

Implications of silage hygienic quality for animal production were investigated on forty‐five dairy farms in South West England. Samples of grass and maize silages and of total mixed rations (TMR) were obtained together with information on silage technology, herd size and animal production. Samples were analysed for mycotoxins, bacteria, yeasts, moulds and chemical composition. Thirteen mycotoxins were assayed, but none were detected in the samples of grass silage. However, mycotoxins were found in 0·9 of all maize and other silage samples, with deoxynivalenol and zearalenone predominating. There was no relationship between total mycotoxin concentration and mean lactation milk yield per cow. Enterobacteria counts tended to be higher in maize silage than in grass silage and higher still in TMR – a cause for concern. There were no relationships between mould counts and mycotoxin concentrations in silages, implying that mycotoxins may have been produced in the field pre‐ensiling.     

Mycobacteriosis caused by Mycobacterium marinum in reared mullets: first evidence from Sardinia (Italy)

Mycobacterium marinum is a slow‐growing non‐tuberculous mycobacterium, and it is considered the most common aetiologic agent of mycobacteriosis in wild and cultured fish. The diagnosis is principally made by histology when positive Ziehl–Neelsen stain granulomas are detected. The aim of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae of two lagoons (Cabras and San Teodoro) from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Nine of 106 mullets examined were affected by mycobacteriosis, and the spleen was the most affected organ. The histology detected higher rate (100%) of infection in spleen than the culture and PCR (75% and 62.5%, respectively). The sequencing of hsp65 gene identified M. marinum as the primary cause of mycobacteriosis in the mullets examined. Mullets affected by mycobacteriosis were mainly fished in the San Teodoro lagoon characterized by critical environmental conditions. Histology remains the most common method in detecting fish affected by mycobacteriosis, and PCR‐based methods are essential for species identification. Our finding are worthy of attention because mycobacteriosis caused by M. marinum in reared mullets was evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species.     

具有III型分泌系统的Pseudomonas moraviensis FP1761植物益生功能及其全基因组分析

 本研究从节瓜根际分离到一株具有防病促生功能的荧光类假单胞菌FP1761。该菌株对部分植物病原真菌和细菌具有拮抗能力,能够解钾、解有机磷和无机磷,可产生氨、蛋白酶、嗜铁素、吲哚乙酸。生物测定表明菌株FP1761可显著促进小麦生长。生理生化、平均核苷酸相似度、16S rDNA和多基因分析将FP1761鉴定为摩拉维亚假单胞菌(Pseudomonas moraviensis)。菌株FP1761基因组草图全长6.12 Mb,(G+C)含量为59.9%,共编码5467个基因序列。将该菌株与种内3个代表性菌株进行泛基因组和核心基因组分析,共产生 4 357个共有基因,菌株FP1761特有基因327个。利用antiSMASH对菌株次生代谢基因簇进行预测,发现其含有8个潜在的次生代谢产物基因簇。其中两个基因簇与嗜铁素pyoverdine合成相关,未见聚酮类合成基因。基因组分析发现,该菌株具有与病原性假单胞菌相似的III型分泌系统,但丢失了效应蛋白调控因子hrpS和转运相关的hrpH和hrpK1基因。对全基因组扫描,菌株FP1761仅保留了病原性假单胞菌的保守效应蛋白AvrE和HopAA1-1。FP1761是目前已发现的唯一具有III型分泌系统的摩拉维亚假单胞菌。本研究表明摩拉维亚假单胞菌FP1761具有潜在的植物防病促生功能,但其III型分泌系统与植物益生互作机制有待进一步解析。     

连栽木麻黄根际微生物群落结构和功能特征

以不同栽培代数的木麻黄(第1代FCP、第2代SCP、第3代TCP)根际土壤为试验材料,运用BIOLOG微平板和磷脂脂肪酸(PLFA)技术分析根际土壤微生物群落结构和功能多样性对多代连栽响应。结果表明,不同代数的土壤微生物对各类碳源的利用程度存在显著差异,连栽后木麻黄根际土壤微生物对碳源利用率显著下降。在6类碳源中,除胺类外,其他5种碳源均呈现FCP>SCP>TCP。PLFA分析共检测到11种PLFA生物标记,FCP土壤微生物PLFA生物标记总量明显高于SCP和TCP,3个代数土壤中含量最高的PLFA生物标记是i16:0、a15:0和18:1ω9c。土壤中特征微生物含量差异明显,细菌分布量最大,其次是真菌和放线菌。随栽植代数增加,细菌含量减少,真菌含量增加。土壤微生物群落多样性指数均呈现FCP>TCP>SCP,与土壤理化性质变化密切相关。可见木麻黄连栽显著影响其根际土壤微生物群落结构与功能,因此根际土壤微生态失衡可能是导致木麻黄连栽障碍的重要因素。     

Bacterial communities associated with natural and commercially grown rooibos (Aspalathus linearis)

Aspalathus linearis is a commercially important plant species endemic to the Cape Floristic Region of South Africa and is used to produce a herbal tea known as rooibos tea. Symbiotic interactions between A. linearis and soil bacteria play an important role in the survival of Aspalathus plants in the highly nutrient-poor, acidic fynbos soil. The aim of this study was to characterize and compare rhizosphere and bulk soil bacterial communities associated with natural and commercially grown A. linearis, as well as the effect of seasonal changes on these communities. Bacterial communities were characterized using high throughput amplicon sequencing, and their correlations with soil chemical properties were investigated. The N-fixing bacterial community was characterized using terminal restriction fragment length polymorphism and real time quantitative polymerase chain reaction. Actinobacteria, Proteobacteria, and Acidobacteria were the most dominant bacterial phyla detected in this study. Highly similar bacterial communities were associated with natural and commercially grown plants. Significant differences in the bacterial community were observed between rhizosphere and bulk soils collected in the dry season, while no significant differences were detected in the wet season. This study provides insights into bacterial community structure and potential factors shaping bacterial community structure with commercially important A. linearis.     

马铃薯与玉米复合种植对土壤化感物质及土壤细菌群落结构的影响

为探究马铃薯与玉米复合种植对化感物质积累与细菌群落结构的影响,分析轮作、间作缓解连作障碍的机制,本研究以马铃薯连作、玉米连作、马铃薯||玉米间作、马铃薯-玉米轮作第8年的土壤为对象,利用GC-MS测定土壤中化感物质含量,并采用Illumina Miseq高通量测序技术对土壤细菌16Sr DNA V4-V5区域进行测序,分析土壤中细菌多样性和群落结构的变化,并对化感物质和优势菌属进行相关性分析。结果表明:玉米连作和马铃薯连作会导致化感物质的积累,玉米连作土壤积累了更多的油酸、亚油酸、花生酸、木焦油酸等脂肪酸,马铃薯连作土壤积累了更多的硬脂醇、二十烷醇等脂肪醇类物质。轮作降低了大部分化感物质的积累,间作降低的化感物质种类相对轮作较少。不同种植方式下土壤细菌群落结构发生了显著变化,相对于连作,间作和轮作Ace指数和Chao指数显著升高。在门水平上,轮作土壤放线菌丰度显著高于马铃薯连作土壤,间作土壤拟杆菌门丰度显著低于玉米连作土壤,两种连作土壤中酸杆菌门丰度都较轮作显著升高。在属水平上,一些有益细菌如节杆菌属、溶杆菌属等在复合种植土壤中相对丰度更高。通过相关性分析发现土微菌属、小梨形菌属与脂肪醇类物质呈显著正相关,黄杆菌属、溶杆菌属、微杆菌属等与脂肪酸类物质呈显著负相关。马铃薯与玉米复合种植降低了化感物质在土壤中的积累,从而抑制了土壤细菌丰度的降低,提高了有益菌属丰度,消减了连作障碍。     

一株盐芽孢杆菌的筛选及其粗酶的酶学特性研究

从浙江舟山市双峰盐场中筛选出一株分泌蛋白酶的嗜盐菌,结合16S rDNA基因序列鉴定、菌株形貌特征确定其为特氏盐芽孢杆菌(Halobacillustrueperi)命名为HalobacillustrueperiB1。经研究,该菌株最适生长NaCl浓度为5%,表明其为中度嗜盐菌。该蛋白酶在50℃和pH 7.5条件下具有最高的蛋白酶活力。对发酵培养时间的优化实验表明发酵8 h后B1分泌的蛋白酶活力最大。对发酵培养基的优化结果表明,脱脂奶粉为最适氮源和碳源;75 g·L~(-1)为最佳发酵盐浓度,对钙镁离子的依赖不明显。确定优化后的发酵培养基:Na HCO_30.06 g,KCl_2.0g,CaCl_2·2H_2O0.48 g,Mg SO_4·7H_2O 1.0 g,FeCl_3 0.001 g,NaCl 75.0 g,脱脂奶粉10.0 g。在初始pH 7.5,温度为40℃条件下发酵8 h。     

薄膜扩散梯度（DGT）技术在环境微界面物质运移过程研究中的应用

土壤、沉积物、水体和生物体之间的接触和作用形成了多种环境微界面。这些环境微界面是物质迁移转化的重要场所,而高度时空异质性的界面特征使得对其中化学反应信息的捕捉变得极其复杂且困难。薄膜梯度扩散(DGT)技术以其原位测量元素生物有效态和高空间分辨率等优势,适用于研究化学异质性的界面过程。本文系统总结了DGT技术在环境微界面的物质运移过程研究中的应用现状,包括以下3方面内容:一是一维物质浓度测定;二是二维化学分布成像;三是与薄膜扩散平衡技术(DET)、平衡式孔隙水采样器(Peeper)和平面光极(PO)等技术联用同步获取多种溶质分布信息。现有研究证据表明,DGT适合在亚毫米(几十至几百微米)至毫米尺度研究环境微界面营养盐和污染物运移的生物地球化学过程,并可与其他化学成像技术结合研究物质跨界面运移的驱动因子和动力学特征。最后,在DGT技术发展与应用场景扩展等方面提出了几点展望。