玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用

【目的】由大斑突脐蠕孢（Exserohilum turcicum）和玉蜀黍平脐蠕孢（Bipolaris maydis）引起的玉米大斑病和小斑病是玉米生产上重要的叶部真菌病害,本研究旨在建立玉米大斑病菌和小斑病菌交配型多重PCR检测方法,为玉米大斑病菌和小斑病菌的交配型田间分布和有性生殖研究提供技术方法。【方法】根据GenBank中已登录的玉米大斑病菌（登录号MAT1-1：GU997138和MAT1-2：GU997137）和小斑病菌（登录号MAT1-1：X68399和MAT1-2：X68398）交配型基因序列,利用Primer Premier 5.0软件设计2种病原菌交配型多重PCR检测特异性引物,采用单因素法对引物的退火温度以及扩增程序中延伸时间和循环数等重要参数进行优化,建立玉米大斑病菌和小斑病菌交配型多重PCR检测方法,并对2种病原菌的交配型多重PCR检测灵敏度和特异性进行检验。同时,对田间采集的129株玉米大斑病菌和194株玉米小斑病菌单孢菌株的交配型进行多重PCR检测,以明确建立的交配型多重PCR检测方法的适用性。【结果】建立的多重PCR方法和设计的交配型特异引物StMAT01-2F/R、StMAT02-3F/R、ChMAT01-3F/R和ChMAT02-2F/R可分别扩增出MAT1-1、MAT1-2型菌株大小为816、132 bp（大病斑菌）与490、136 bp（小病斑菌）的特异性目的条带。25 μL多重PCR扩增体系：2×Multiplex PCR Mix 12.5 μL,引物各10 pmol,DNA模板100 ng,退火温度为57.2℃（大病斑菌）和55.0℃（小斑病菌）,35个循环。该多重PCR对玉米大斑病菌MAT1-1、MAT1-2型单孢菌株的检测灵敏度分别为0.1、0.01 ng基因组DNA,而对玉米小斑病菌MAT1-1、MAT1-2交配型的检测灵敏度均为0.1 ng基因组DNA。该交配型多重PCR检测方法对玉米大斑病菌和小斑病菌特异性很强,能够很好地区分玉米大斑病菌和小斑病菌相应的近缘种和14株其他真菌菌株。不同地理来源的玉米大斑病菌和小斑病菌交配型检测结果表明,该多重PCR能够准确地检出129株玉米大斑病菌和194株玉米小斑病菌的交配型,且检测结果与随机抽取的菌株杂交验证结果完全吻合。【结论】构建的玉米大斑病菌和小斑病菌交配型多重PCR检测方法灵敏度高、特异性好、操作简便,能够准确、快速地检测出玉米大斑病菌和小斑病菌的交配型,为玉米大斑病菌和小斑病菌的交配型田间分布和监测及有性生殖研究提供了可靠的技术方法。     

Effect of Pheromone Blend Components,Sex Ratio,and Population Size on the Mating of Cadra cautella (Lepidoptera: Pyralidae)

The almond moth Cadra cautella (Walker), a key pest of storage facilities, is difficult to manage using synthetic chemicals. Pheromone-based management methods remain a high priority due to advantages over conventional management practices, which typically use insecticides. Cadra cautella females release a blend of pheromone including (Z, E)-9,12-tetradecadienyl acetate (ZETA) and (Z)-9-tetradecadien-1-yl acetate (ZTA). The effect of these components on mating of C. cautella and how response varies with the population density and sex ratio remain unknown. In this study, the mating status of C. cautella was studied inside mating cages under different ratios of ZETA and ZTA diluted in hexane and at different population sizes either with equal or unequal sex ratio. The lowest percentage of mated females (highest mating disruption [MD] effects), corresponding to roughly 12.5%, was produced by a 5:1 and 3.3:1 ratio of ZETA:ZTA. Populations with equal sex ratio showed the lowest percentage of mated females, at 20% and 12.5% under lower and higher density, respectively. The next lowest percentage of mated females was produced when the sex ratio was set to 1: 2 and 2:1 male:female, with just 25% and 22.5% of moths mated, respectively. This study shows that mating status of C. cautella is influenced by ZETA:ZTA ratio, sex ratio, and population size. This current knowledge would have useful implications for mating disruption programs.     

天山天池二倍体棱叶蒜的开花动态及交配系统

以分布于新疆天山天池的二倍体棱叶蒜(Allium caeruleum)为研究对象,观察记录了开花进程,测定其花粉活力和柱头可授性,并采用去雄、套袋、人工授粉等方法对其交配系统进行检测。结果表明:(1)伞形花序平均直径为(29.07±1.17)mm,花被片天蓝色,花期6—8月;两性花,平均小花数205.67±19.88个,密集,无株芽;小花开放历经弹头、张口、吐药、展开、抱合和萎蔫等过程,历时5~7 d;小花开放进程中花药和柱头间存在空间隔离,从第1枚花药伸出至6枚花药全部脱落以及柱头伸长至萎蔫各需要2~3 d;阴天比晴天分别延长散粉以及延迟柱头萎蔫0.5~1 d。棱叶蒜具有雄蕊先熟、雌雄异熟、雌雄异位以及吸引昆虫避免自交的花部特征。(2)花药开裂散粉4 h时花粉活力最强(79.23%±1.97%的花粉具有活力),36 h后降至很低,属于短寿命花粉。(3)联苯胺—过氧化氢和MTT法检测柱头可授性;花丝、花柱等长期部分柱头开始具有可授性,花丝、花柱合拢期至花丝萎蔫期柱头可授性最强。同一朵花雄蕊和雌蕊的可育期存在部分时间重叠,防止了杂交失败。(4)单花花粉量为31 275±3 651,胚珠数6,P/O值为5 212±608;杂交指数OCI为5;无自发的自花传粉现象,不存在风媒传粉和无融合生殖;倾向于异交为主、自交可育,具备兼性异交并需要传粉媒介的混合交配系统的特征。天山天池二倍体棱叶蒜还以种子休眠、鳞芽无性繁殖的生殖补偿方式保证特殊地理环境下的种群繁衍。     

Population structure,genetic diversity,and sexual state of the rice brown spot pathogen Bipolaris oryzae from three Asian countries

Bipolaris oryzae causes brown spot in rice (Oryza sativa) inflicting substantial grain yield losses worldwide. Knowledge of the population structure, genetic diversity and sexual recombination of the fungal pathogen can help to implement effective disease management strategies. In this study, B. oryzae isolates sampled from Iran, the Philippines and Japan were analysed with 12 simple‐sequence repeat (SSR) markers, newly developed from the genome sequence of the fungus. Among the 288 B. oryzae isolates genotyped, 278 unique haplotypes were identified. High genotype numbers (richness) with even distribution (evenness) were found within the collection sites. Both mating types, MAT1‐1 and MAT1‐2, were present in each collection area, and the sexual state was induced under controlled conditions with production of viable ascospores. However, the tests of linkage disequilibrium rejected of the hypothesis of random mating. Discriminant analysis of principal components (DAPC) revealed that the B. oryzae collection formed three clusters, each consisting of isolates from different collection sites. Analysis of molecular variance (amova ) showed that genetic variation among clusters was 18.7%, with the rest of the variation distributed within clusters (R_ST = 0.187, P < 0.001). Statistically significant pairwise genetic differentiation was found between the clusters. These results show that Asian B. oryzae isolates are genetically diverse, and, overall, distributed in three groups. These findings will be helpful in managing the disease and guide the use of representative isolates needed for selection of resistant rice varieties.     

Genetic differentiation in populations of Exserohilum turcicum from maize and sorghum in South Africa

Exserohilum turcicum is the causal agent of northern leaf blight, a devastating foliar disease of maize and sorghum. Specificity of E. turcicum to either maize or sorghum has been observed previously, but molecular evidence supporting host specialization is lacking. The aim of this study was to compare the genetic structure of E. turcicum isolates collected from adjacent maize and sorghum fields in Delmas and Greytown in South Africa. In addition, the mode of reproduction of this pathogen was investigated. Isolates from maize (N = 62) and sorghum (N = 64) were screened with 12 microsatellite markers as well as a multiplex mating type PCR assay. No shared haplotypes were observed between isolates from different hosts, although shared haplotypes were detected between isolates from maize from Delmas and Greytown. Population structure and principal coordinate analyses revealed genetic differentiation between E. turcicum isolates from maize and sorghum. Analysis of molecular variance indicated higher among‐population variation when comparing populations from different hosts, than comparing populations from different locations. Lack of shared haplotypes, high proportion of private alleles, greater among‐population variance between hosts than locations and significant pairwise population differentiation indicates genetic separation between isolates from maize and sorghum. The high haplotypic diversity in combination with unequal mating type ratios and significant linkage equilibrium indicates that both sexual and asexual reproduction contributes to the population genetic structure of E. turcicum in South Africa.     

灰树花交配型鉴定分析

[目的]鉴定灰树花交配型,为灰树花杂交育种提供理论依据.[方法]灰树花孢子萌发后挑取单孢进行镜检,收集孢子单核体;采用原生质体单核化获得原生质体单核体并通过互配确定标准测交菌株T1和T2;采用标准测交菌株与孢子单核体配对及孢子单核体互配后,进行孢子单核体交配型分类,并利用核迁移试验和OWE-SOJ技术鉴定孢子单核体交配型.[结果]分离鉴定获得7 1株灰树花孢子单核体菌株,原生质体单核化获得17株原生质体单核体菌株,其中标准测交菌株Tt(A1B1)10株、T2(A2B2)7株;鉴定71株孢子单核体分别为T1、T2、T3和T4型,其中T1型28株、T2型35株、T3型4株、T4型4株;核迁移试验鉴定T1、T2、T3和T4的交配型分别为A1B1、A2B2、A2B1和A1B2,OWE-SOJ鉴定结果分别为A1B1、A2B2、A1B2和A2B1.[结论]灰树花属于四极性交配型系统,其交配型分析应以核迁移试验为准,OWE-SOJ技术不适用于灰树花的交配型分析.     

绿盲蝽交配干扰剂丁酸己酯微囊的制备及田间防治效果

以乙基纤维素为壁材,丁酸己酯为芯材,采用相分离法制备了绿盲蝽Apolygus lucorum交配干扰剂丁酸己酯微囊。通过正交试验研究了乳化剂700#、聚乙烯醇、乙酸乙酯及控释剂正十二烷4个因素对丁酸己酯微囊形成的影响,得到最佳制备工艺条件。利用生物显微镜对微囊的形态进行了观察,同时测定了在最优条件下制备的丁酸己酯微囊的载药量、包封率和释放速率等指标,并进行了田间药效试验。结果表明:在乙基纤维素3.0 g、丁酸己酯3.0 g、乳化剂700#1.5 g、聚乙烯醇2.0 g、乙酸乙酯30 m L和正十二烷2.0 g,以及滴加速率为5 m L/min、转速为1 000 r/min条件下制备的丁酸己酯微囊的载药量和包封率分别为21.5%和91.9%,外观较完整,粒径分布较均匀,平均粒径约为301.85μm,缓释效果较好(能持续释放35 d以上)。田间试验结果显示,调查期间悬挂丁酸己酯微囊的样区诱捕到绿盲蝽224头,与对照区相比少442头,表明丁酸己酯微囊对绿盲蝽交配具有干扰作用,可明显减少绿盲蝽繁殖的数量。     

需引起警惕的棕榈科植物入侵害虫—椰子织蛾

椰子织蛾是为害棕榈科植物的重要食叶害虫之一,2013年8月在我国海南首次发现。本文报道了该害虫各发育阶段的外部形态、COⅠ/cox1测序和比对的分子鉴定结果、寄主及为害情况。序列比对显示,目标样本430bp的cox1序列与NCBI中已提交的Opisina arenosella的线粒体COⅠ条形码序列覆盖率为100%,一致性达99.8%,表明分子遗传数据支持形态鉴定结果。调查发现,椰子织蛾寄主主要为椰子、王棕和林刺葵,已在海南9个市县发生,局部地区为害严重。椰子织蛾已在海南定殖、扩散,并形成经济和景观危害,其入侵将对我国南方热带、亚热带地区棕榈科植物的安全生产和生态环境构成重大威胁,需提高警惕,加强检疫,防止其扩散蔓延,并开展防治研究。     

Variation in growth rates and aggressiveness of naturally occurring self‐fertile and self‐sterile isolates of the wilt pathogen Ceratocystis albifundus

Ceratocystis albifundus is the most important fungal pathogen of black wattle (Acacia mearnsii) grown in plantations in southern and eastern Africa. It is a homothallic fungus but also undergoes unidirectional mating type switching. As a result, the ascospore progeny can be either self‐fertile or self‐sterile. The only apparent difference between these mating types is the deletion of the MAT1‐2‐1 gene in self‐sterile isolates. There is some evidence suggesting that self‐sterile isolates grow more slowly than self‐fertile isolates, but this has not been tested rigorously. The aim of this study was to determine whether self‐sterile isolates are less fit by examining growth rate, relative germination rate and pathogenicity. Five self‐sterile isolates were generated from each of five self‐fertile isolates of C. albifundus and these 30 isolates were compared. The results showed that the self‐sterile isolates grew consistently slower and were less pathogenic than the self‐fertile isolates. The germination ratio of self‐fertile to self‐sterile isolates from single ascospores collected from the ascomata of five self‐fertile isolates was on average 7:3. This could be a consequence of the self‐sterile isolates having a lower germination rate. This observation, and the lower growth and pathogenicity levels, suggests that self‐sterile isolates are not likely to compete effectively in nature, raising intriguing questions regarding their role and value to C. albifundus and other fungi having a similar mating system.     

利用性信息素迷向技术防治桃园梨小食心虫的效果与分析

为了绿色防控桃园梨小食心虫的为害,进行了性信息素迷向技术防效实验。连片100×667m2以上的桃园,平均亩悬挂梨小食心虫迷向丝60枚,迷向丝对梨小食心虫各代成虫的迷向率可达96%以上,且持效期在4个月以上,对各代折梢防效在94%以上,蛀果防效在96%以上,全年用药次数减少4~7次。结果表明,该技术对桃树整个生育期梨小食心虫的为害具有良好的控制作用,可有效减少农药防治次数和用药量,值得大面积推广应用。