Pancreatic triglyceride lipase is involved in the virulence of the brown planthopper to rice plants

The brown planthopper (BPH), Nilaparvata lugens, an important rice insect pest, can enhance its virulence to BPH-resistant rice within as short a span as several generations. Here, we cloned a pancreatic triglyceride lipase (PTL) gene (NlPTL) in N. lugens, and found that its mRNA level was higher in the high virulence population (fed on variety Rathu Heenati, P-RH) than in the low virulence population (fed on variety Taichung Native 1, P-TN1). Knocking down NlPTL caused BPH individuals to spend more time in non-penetration and the pathway phases and less time feeding on the phloem of rice plants; these changes consequently decreased food intake, lipid content, survival rate, and fecundity in the insects. These findings reveal for the first time that PTL in BPH is involved in its virulence to rice plants.     

Less hairy leaf 1, an RNaseH-like protein,regulates trichome formation in rice through auxin

The trichomes of rice leaves are formed by the differentiation and development of epidermal cells. Plant trichomes play an important role in stress resistance and protection against direct ultraviolet irradiation. However, the development of rice trichomes remains poorly understood. In this study, we conducted ethylmethane sulfonate (EMS)-mediated mutagenesis on the wild-type (WT) indica rice ‘Xida 1B’. Phenotypic analysis led to the screening of a mutant that is defective in trichome development, designated lhl1 (less hairy leaf 1). We performed map-based cloning and localized the mutated gene to the 70-kb interval between the molecular markers V-9 and V-10 on chromosome 2. The locus LOC_Os02g25230 was identified as the candidate gene by sequencing. We constructed RNA interference (LHL1-RNAi) and overexpression lines (LHL1-OE) to verity the candidate gene. The leaves of the LHL1-RNAi lines showed the same trichome developmental defects as the lhl1 mutant, whereas the trichome morphology on the leaf surface of the LHL1-OE lines was similar to that of the WT, although the number of trichomes was significantly higher. Quantitative real-time PCR (RT-qPCR) analysis revealed that the expression levels of auxin-related genes and positive regulators of trichome development in the lhl1 mutant were down-regulated compared with the WT. Hormone response analysis revealed that LHL1 expression was affected by auxin. The results indicate that the influence of LHL1 on trichome development in rice leaves may be associated with an auxin pathway.     

产后奶牛子宫内致病菌的分离、毒力基因检测及耐药性分析

为检测产后奶牛子宫内膜炎致病菌的毒力基因和耐药性,在生产3周后采用棉拭子法对65头确诊的临床型子宫内膜炎荷斯坦奶牛进行子宫黏液的采集;分别用阴门灌注大鼠子宫试验、PCR方法以及药敏纸片法来检验分离株的致病性。结果表明,灌注混合菌(1∶1,1.0×10~9 cfu/mL)72h后剖解大鼠子宫发现子宫充血,肿大,增厚,子宫内部有蓄脓现象。然而,Escherichia coli和Staphylococcus aureus从患病牛中最终检出率分别为58.69%、62.75%;并对27株致病性E.coli系统发育组ChuA、yjaA基因检出率达到(13/27,48.15%),且E.coli和S.aureus分离株中均含有毒力基因KpSMII和TSST-1;随机抽取10头患病牛且含毒力基因最多的分离株进行药敏试验且均耐受于青霉素、多西环素、多粘菌素B、阿莫西林和四环素等抗生素。由此可见,以上抗生素再继续应用将会导致E.coli和S.aureus耐药程度持续增加。     

枸杞根腐致病菌及拮抗菌的分离鉴定

旨在探索枸杞根腐病发生地根际土壤生防细菌资源,为该病的防治提供理论依据。采用组织分离法分离枸杞根腐致病菌,对其进行形态学、分子生物学鉴定和柯赫氏法则验证;再利用平板对峙法,以分离出的病原菌为靶标,筛选具有拮抗效果的菌株,对其进行鉴定。结果表明：从青海省海西蒙古族藏族自治州主要栽培枸杞林中分离鉴定出4种致病菌,分别为黄色镰刀菌（Fusarium culmorum）、三线镰刀菌（F.tricinctum）、尖孢镰刀菌（F.oxysporum）、木贼镰刀菌（F.equiseti）;经筛选,有8株生防细菌对4种病原菌具有不同程度的拮抗效果,其中,抑菌效果最好的是QH-588菌株,其抑制率在36.09%~51.79%;经形态学和16S rDNA/Gyr B基因分析鉴定,其中1株为萎缩芽孢杆菌（Bacillus atrophaeus）,5株为枯草芽孢杆菌（B.subtilis）,2株为短小芽孢杆菌（B.pumilus）。在国内首次报道了三线镰刀菌为枸杞根腐病病原菌,筛选出的生防细菌在室内对枸杞根腐病的4种病原均具有较好的拮抗效果,可作为防治青海省枸杞根腐病的候选菌株。     

蟹源弗氏柠檬酸杆菌拮抗菌PNB3的分离鉴定与安全性分析

摘 要：筛选和鉴定抗水产致病性弗氏柠檬酸杆菌的优良芽孢杆菌,并分析其安全性。以一株蟹源致病性弗氏柠檬酸杆C1作为筛选指示菌、抑菌圈直径和抑菌效果作为指标进行抗菌芽孢杆菌优良菌株的分离与筛选,综合生理生化鉴定与16S rRNA序列分析对抗菌芽孢杆菌优良菌株进行鉴定,并参照《HJ/T 415—2008 环保用微生物菌剂环境安全评价导则》、《NY/T1444-2007微生物饲料添加剂评价通则》等国家标准,从毒力基因、耐药性、对小球藻生长的影响以及对斑马鱼和中华绒螯蟹的致病性等方面分析抗菌芽孢杆菌优良菌株的安全性。结果显示,从养殖淤泥中分离并筛选出一株抗菌芽孢杆菌优良菌株PNB3,其在1.0×105 CFU/mL时与蟹源致病性弗氏柠檬酸杆菌C1共培养处理120 h的最高抑菌率达到了99.99%。结合生理生化鉴定以及16S rRNA序列分析,菌株PNB3被鉴定为地衣芽孢杆菌(Bacillus licheniformis),不含溶血素BL基因、肠毒素Nhe基因、肠毒素T基因、肠毒素FM 基因、细胞毒素 K 基因、呕吐基因、多效调控因子等毒力基因,对复方新诺明、氨苄西林、诺氟沙星、卡那霉素、多粘菌素B、氟苯尼考、庆大霉素、依诺沙星、头孢拉定、青霉素等10种抗生素高度敏感,对多西环素、四环素等2种抗生素中度敏感,对链霉素耐药,在终浓度为2.0×104~2.0×108 CFU/mL时对小球藻的生长有促进作用,对斑马鱼的半数致死浓度（LC50）大于2.0×108 CFU/mL,对中华绒螯蟹的半数致死浓度（LC50）大于2.0×108 CFU/g。地衣芽孢杆菌PNB3是一株具有潜在安全性的弗氏柠檬酸杆菌拮抗菌,具备应用于中华绒螯蟹弗氏柠檬酸杆菌病生物防控的安全基础。     

根癌农杆菌介导的稻瘟病菌近等菌系的构建与初步应用*

 利用根癌农杆菌C58C1介导转化1株致病力极弱的野生型稻瘟病菌菌株CY2,成功地获得3000多个转化子,混合接种于携带25个不同抗病单基因的水稻近等基因系上,获得4株单胞致病突变体;继代培养和PCR检测表明,这些突变体均为T-DNA插入CY2的后代,而非自发突变或外源菌株污染。将4株突变体与实验室已保存的20株致病突变体分别回接水稻近等基因系,推断其携带的致病基因类型,结果表明突变体分别针对13个抗性单基因的18个单基因系发生了突变,其中针对Pi-a,Pik-s,Pi-sh单基因系表现出较高的侵染频率。由于这些突变体均来自同一个菌株,仅致病性存在差异,属于近等菌系,利用其分析出102个水稻品种中的34个品种的抗病基因组成,其中,抗病基因Pik-s,Pi-a,Pi-sh,Pi-19(t)分布频率较高,分别达70.59%,61.76%,52.94%和44.12%,且带有这类基因的糯稻品种多于杂交水稻。     

鸽源大肠杆菌O157：非H7菌株的致病性和耐药性分析

为查明南京市某赛鸽公棚赛鸽出现腹泻、死亡的致病病原,本研究对采集的病料进行病原分离鉴定,分析致病菌的血清型、生物被膜表型、毒力基因、耐药菌谱,并对分离菌株进行了人工感染试验。研究表明:1)从患病鸽组织、拭子和粪便中分离出的细菌初步诊断为大肠杆菌O157;分离菌株在NA平板上长出白色、光滑的圆形菌落,在CT-SMAC平板上长出紫红色菌落。2)分离菌株的16S rRNA基因序列与大肠杆菌的相似性达98%~99%,命名为E.c.86246。3)血清学试验表明分离菌株为大肠杆菌O157:非H7菌株,动力试验进一步证实其为动力株。4)分离菌株携带tccp1014、eaeA和hly毒力基因,可导致雏鸡胃肠道出现不同程度的功能损伤,甚至导致死亡;其半数致死量为2.0×10⁵ CFU/mL,具有较强的致病性。5)分离菌株携带tetA、tetM、sul2、aadA1和bla_TEM 5种耐药基因,具有较弱的生物被膜形成能力,对四环素、氨基糖苷类等抗菌药物严重耐药,对阿莫西林、氧氟沙星和诺氟沙星敏感。综上,本研究分离的鸽源大肠杆菌O157:非H7菌株,具有动力性,有较强的致病性和一定的耐药性,为大肠杆菌O157的流行病学调查、致病机理分析和抗菌药物的精准施治提供理论依据和数据参考。     

Identification and Gene Mapping of a multi-floret spikelet 1 (mfs1) Mutant Associated with Spikelet Development in Rice

In this study, a rice spikelet mutant, multi-floret spikelet 1 (mfs1), which was derived from ethylmethane sulfonate (EMS)-treated Jinhui 10 (Oryza sativa L. ssp. indica) exhibited pleiotropic defects in spikelet development. The mfs1 spikelet displayed degenerated the empty glume, elongated the rachilla, the extra lemma-like organ and degraded the palea. Additionally, mfs1 flowers produced varied numbers of inner floral organs. The genetic analysis revealed that the mutational trait was controlled by a single recessive gene. With 401 recessive individuals from the F₂ segregation population, the MFS1 gene was finally mapped on chromosome 5, an approximate 350 kb region. The present study will be useful for cloning and functional analysis of MFS1, which would facilitate understanding of the molecular mechanism involved in spikelet development in rice.     

Genetic Diversity and Genetic Changes in the Introgression Lines Derived from Oryza sativa L. Mating with O. rufipogon Griff.

The objectives of the present study were to estimate genetic diversity and genetic changes of introgression lines (ILs) which derived from cultivated rice (Oryza sativa L. cv. Xieqingzao B, XB) mating with common wild rice (O. rufipogon Griff., CWR). The genetic data of 239 ILs were based on a total of 131 polymorphic microsatellite (SSR) markers distributed across the 12 chromosomes of rice. On average, these ILs possessed 77.1 and 14.31% homozygous bands from XB and CWR, respectively. Most of the ILs were clustered together with XB individual, which was revealed by principal coordinate analysis (PCA) and the program STRUCTURE analysis. The result from PCA demonstrated that some intermediate genotypes between XB and CWR were also found. Moreover, there were some genomic sequence changes including parental bands elimination and novel bands emergence in the ILs. The average Nei's gene diversity (He) was 0.296, which was higher than that of cultivated rice. It suggested that interspecific hybridization and gene introgression could broaden the base of genetic variation and lay an important foundation for rice genetic improvement. These different genotypic ILs would provide a better experimental system for understanding the evolution of rice species and the mechanism of alien gene introgression.     

‘秦美’猕猴桃贮藏期病原真菌的鉴定

以‘秦美’猕猴桃为试材,分离纯化在贮藏过程中引起腐烂的主要病原真菌。采用形态学观察及核糖体rDNA-ITS(Internal transcribed spacer)区序列分析法进行鉴定。结果表明:从腐烂猕猴桃中共分离出5株致病菌,对ITS区序列测序结果经GenBank数据库BLAST比对,A的ITS序列与Trichothecium roseum(EU552162.1)同一分支,支持率达100%;B的ITS序列与Fusarium tricinctum(AB587078.1)同一分支,支持率达99%;C的ITS序列与Penicillium expansum(AF330635.1)同一分支,支持率达99%;D的ITS序列与Colletotrichum boninense(KF819619.1)同一分支,支持率达99%;E的ITS序列与Botrytis elliptica(KJ638600.1)同一分支,支持率达100%。由形态学及ITS区序列分析,构建系统发育树,最终鉴定A为粉红聚端孢(Trichothecium roseum),B为镰刀菌属(Fusarium tricinctum),C为扩展青霉(Penicillium expansum),D为炭疽病菌(Colletotrichum boninense),E为灰葡萄孢(Botrytis cinerea)。5种病原真菌中灰葡萄孢为主要致病菌。     

产河鲀毒素细菌水平转移相关基因的鉴定

多种进化关系并不密切的细菌均能合成河鲀毒素,揭示了产河鲀毒素细菌间可能存在水平基因转移现象,但通过何种途径并不清楚。为调查产河鲀毒素的气单胞菌(Aeromonas sp.)质粒中是否存在与水平基因转移相关的元件,通过PCR扩增分别发现该质粒中存在tra,rum及vird4基因,扩增出的906 bp的tra,681bp的rum及1 319 bp的vird4,分别与多种菌株的转座酶、松弛酶和Ⅳ型分泌系统中的ATP酶基因具有高度的序列同源性,并具有这些酶的保守结构域,结果表明产河鲀毒素的气单胞菌具有通过接合转移和转座方式转移基因的能力,同时具有转移河鲀毒素至宿主的能力。对于进一步了解产河鲀毒素细菌之间以及产河鲀毒素细菌与河鲀之间的关系有重要的帮助。研究亮点:本研究首次在产河鲀毒素的气单胞菌质粒中明确鉴定出与水平转移相关的基因,表明该细菌不但具有通过接合转移和转座方式转移基因的能力,而且具有转移河鲀毒素至宿主的能力。     

重庆地区兔腹泻病原菌分离鉴定及检测方法的建立

对重庆地区兔细菌性腹泻病原菌进行分离鉴定并建立相应检测方法,为该病的检测及防控奠定基础。通过临床诊断与细菌分离培养,对引起兔腹泻的病原菌进行分离,利用细菌通用引物扩增分离菌16S rDNA基因序列,测序分析后构建系统进化发育树。同时,依据各病原菌特异性序列设计引物,建立PCR检测方法。结果表明,共分离到4种病原菌,分子生物学鉴定为:铜绿假单胞菌、产气荚膜梭菌、金黄色葡萄球菌和大肠杆菌;依据铜绿假单胞菌外毒素eta基因、产气荚膜梭菌a毒素基因、金黄色葡萄球菌耐热核酸酶nuc基因和大肠杆菌外膜蛋白eae基因设计特异性引物,成功建立多重PCR检测方法,可从4种病原菌基因组混合物中扩增出大小分别为1 043、324、200和609bp的目的条带,目的菌株最低检出浓度为103cfu·mL-1。对重庆地区200份临床样本进行检测,发现4种病原菌普遍存在,其中,金黄色葡萄球菌单独感染检出率高达37.6%,与大肠杆菌的混合感染检出率达28.0%。     

Identification and Analysis of Physiological Races of Magnaporthe oryzae in Heilongjiang Province

A totatl of 116 isolates of rice blast fungus, Magnaporthe oryzae, were collected from 45 samples in different counties of Heilongjiang Province, and 20 Chinese physiological races belonging to seven groups were identified by using seven standard Chinese rice blast identifying varieties. Results showed that the dominant groups could be ranked as ZA, ZD, and ZB, with the occurrence frequencies of 47.41%, 22.41% and 15.52%, respectively. The race ZA₄₉ was the dominant race with the occurrence frequency of 26.72%. The occurrence frequencies of the races ZD₅ and ZD₁ were 10.34% and 8.62%, respectively. The rising occurrence frequencies of these three dominant species were the most important reasons that causing Kongyu-131 more sensitive to rice blast. The results of virulence frequency indicated that the race harboring Pi-k, Pi-i, Pi-a gene were more susceptible to rice blast in Heilongjiang Province, and they should not be large-scale cultivated.     

Genetic diversity analysis and GWAS reveal the adaptive loci of milling and appearance quality of japonica rice (Oryza sativa L.) in Northeast China

Milling and appearance quality are important contributors to rice grain quality. Abundant genetic diversity and a suitable environment are crucial for rice improvement. In this study, we investigated the milling and appearance quality-related traits in a panel of 200 japonica rice cultivars selected from Liaoning, Jilin and Heilongjiang provinces in Northeast China. Pedigree assessment and genetic diversity analysis indicated that cultivars from Jilin harbored the highest genetic diversity among the three geographic regions. An evaluation of grain quality indicated that cultivars from Liaoning showed superior milling quality, whereas cultivars from Heilongjiang tended to exhibit superior appearance quality. Single- and multi-locus genome-wide association studies (GWAS) were conducted to identify loci associated with milling and appearance quality-related traits. Ninety-nine significant single-nucleotide polymorphisms (SNPs) were detected. Three common SNPs were detected using the mixed linear model (MLM), mrMLM, and FASTmrMLM methods. Linkage disequilibrium decay was estimated and indicated three candidate regions (qBRR-1, qBRR-9 and qDEC-3) for further candidate gene analysis. More than 300 genes were located in these candidate regions. Gene Ontology (GO) analysis was performed to discover the potential candidate genes. Genetic diversity analysis of the candidate regions revealed that qBRR-9 may have been subject to strong selection during breeding. These results provide information that will be valuable for the improvement of grain quality in rice breeding.     

稻瘟病菌二个无毒基因的初步分析

 分析了2个稻瘟病田间采集菌株Y95-223和Y94-64的杂交后代的54个菌株对合系35号、楚粳3号及日本鉴别品种新2号、梅雨明、PiNo.4和K60共11个水稻品种致病性测定结果。结果表明:陆稻菌株Y94-64对合系35号、楚粳3号、PiNo.4分别持有2个无毒基因,对新2号、K60分别持有一个无毒基因;菌株Y95-223对梅雨明持有一个无毒基因,因为Y95-223对梅雨明不致病,Y94-64对它致病,而杂交后代非致病菌株与致病菌株比为1∶1．