小豆锈病分子检测体系的构建及应用

由豇豆单胞锈菌（Uromyces vignae Barclay）引起的小豆锈病是小豆生产中危害最为严重的病害之一，建立早期分子检测技术体系对病害早期诊断和及时防控具有重要意义。本研究以豇豆单胞锈菌ITS序列为依据设计引物，结合已报道的单胞锈菌属特异检测引物，以豇豆单胞锈菌为目标菌，以茄链格孢、茄丝核菌、禾谷镰刀菌、稻瘟菌、苹果轮纹病菌、半裸镰刀菌、玉米链格孢菌、尖孢镰刀菌和禾顶囊壳等9种常见植物病原真菌为参照菌，采用CTAB法提取上述各菌株的基因组DNA，应用普通PCR技术筛选出豇豆单胞锈菌的特异性检测引物UV-ITS，在此基础上设计巢式PCR引物UV-MX，构建了基于巢式PCR的高灵敏度小豆锈病分子检测体系。该巢式PCR体系在基因组DNA浓度仅为4×10^-6 ng·μL^-1时仍可实现有效扩增，其灵敏度是普通PCR的10万倍。以感病小豆品种‘宝清红’接种锈菌后不同时间的叶片为材料检验检测体系的应用效果发现，接种后12 h的小豆叶片样本即可扩增出豇豆单胞锈菌的特异性条带。本研究建立的小豆锈病分子检测体系特异性强、灵敏度高，具备对潜伏期病害进行早期诊断的应用潜力，可为小豆锈病的早期快速诊断和及时防控提供重要的技术支撑。     

建兰胶孢炭疽菌ITS序列分析及其PCR快速检测

由胶孢炭疽菌Colletotrichum gloeosporioides引起的炭疽病是建兰的重要病害.为建立快速检测该病原菌的方法,以ITSl/ITS4为引物,对15个建兰胶孢炭疽菌的ITS进行PCR扩增及测序,将测定的序列与炭疽菌属其它种的ITS序列进行比对分析,设计特异性引物CFl/CR1,并通过常规和巢式PCR对建兰胶孢炭疽菌进行检测.结果显示,15个菌株中有13个菌株ITS序列与该菌的模式种序列相似性高达99％以上,而另外2个菌株相似性则为86％;供试菌株在系统发育树上聚为2个不同的分支;引物CFl/CR1通过常规PCR可从1 ng的建兰胶孢炭疽菌基因组DNA中扩增到目的条带,而利用引物ITSl/ITS4和CF1/CR1通过巢式PCR可从1 pg的基因组DNA中扩增到目的条带,即巢式PCR反应检测灵敏度较常规PCR至少高1 000倍.表明建立的巢式PCR法可从自然感病的建兰叶片组织中检测到胶孢炭疽菌.     

石斛内生细菌DEB-2对5种辣椒病原真菌的抑制作用

为了探明石斛内生细菌DEB-2菌株对贵州辣椒病原真菌的抑制作用,采用生长速率法测定了菌株DEB-2发酵液对辣椒黑斑病病原菌Alternaria alternate、辣椒疫病病原菌Phytophthora capsici、辣椒黑点炭疽病病原菌Colletotrichum capsici、辣椒红色炭疽病病原菌Colletotrichum gloesporioide和辣椒早疫病病原菌Alternaria solani的毒力。结果显示,内生菌DEB-2菌株能显著抑制5种辣椒病原真菌菌丝生长和孢子萌发,菌丝尖端均有不同程度变粗、膨大、崩解或者菌丝分枝增多的现象;分生孢子及芽管产生畸形。DEB-2菌株发酵液对5种病原菌菌丝生长的抑制中浓度EC₅₀值分别是28.5、76.2、80.7、79.9、72.0 μL/mL;对辣椒疫病病原菌和早疫病病原菌分生孢子萌发的EC₅₀分别为73.5 μL/mL和68.7 μL/mL。研究表明,DEB-2菌株能有效地同时抑制5种辣椒病原真菌菌丝生长和孢子萌发,对该5种辣椒真菌病原菌具有一定的生防潜力,可作为生防菌进行开发和应用。     

雪松疫霉(Phytophthora lateralis)的快速分子检测

由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。     

黄花菜叶斑病菌鉴定、生物学特性分析、防治药剂筛选及LAMP检测体系的建立

为明确浙江省黄花菜叶斑病病原菌的种类，探究其对杀菌剂的敏感性并建立快捷检测方法，采用组织分离法获得黄花菜叶斑病菌，通过形态学特征观察和分子生物学技术对病原菌进行鉴定，测定其致病力及生物学特性，分析6种杀菌剂对病原菌的生长抑制效果，并以ITS和EF-1α序列为靶标设计特异性引物建立环介导等温扩增（loop-mediated isothermal amplification，LAMP）检测方法。结果表明，根据形态学特征以及ITS与EF-1α序列分析结果确定黄花菜叶斑病菌为杏黄镰孢Fusarium armeniacum。该病原菌菌丝生长适宜温度为20~30℃，pH 7时菌丝生长最快，以葡萄糖为碳源、硝酸钠为氮源时利用率最高。96%己唑醇对菌丝生长的抑制作用最强，EC₅₀为0.119 μg/mL。本研究所建立的LAMP体系在60℃条件下反应60 min，琼脂糖凝胶电泳和SYBR Green I染料染色均可判断检测结果。该方法可特异性地检测出杏黄镰孢，最低检测灵敏度为 100 pg/μL，是常规PCR检测灵敏度的10倍。表明本研究所建立的LAMP检测体系可快速检测出人工接种和实际发病黄花菜叶片中的病原菌。     

基于ITS与factor1-alpha序列桉树焦枯病菌的双重PCR快速检测

桉树焦枯病是威胁桉树生长的首要病害,建立准确、有效的桉树焦枯病的PCR快速检测技术是桉树焦枯病前期诊断的必要手段。试验以桉树焦枯病原菌(Calonectria morganii)DNA为模板,分别以ITS和factor 1-alpha序列为靶区域,针对Calonectria属和C.morganii设计了CYS1/CYS2和EF-S-1/EF-A-1两对特异性引物,建立了基于属和种的双重PCR快速检测技术。利用引物CYS1/CYS2可以从全部Calonectria属的供试菌株中扩增出一条351bp大小的条带,单独用特异性引物EF-S-1/EF-A-1进行PCR扩增,仅病原菌扩增出197bp的条带,同时使用2对引物时,病原菌可扩增出两条明亮条带。当体系退火温度为53℃时,DNA灵敏度检测限度达到450fg/μL。野外田间时效检测结果显示,该体系能准确检测出不同发病程度桉树组织上的病原菌,完全符合田间检测的要求。这是关于桉树焦枯病快速检测的首次报道。     

核桃枝枯病小新壳梭孢巢式PCR检测方法的建立

近年来,小新壳梭孢Neofusicoccum parvum导致的核桃枝枯病屡有报道,该病防治困难,发病率高,是核桃的一种灾难性病害。为建立N.parvum的快速、灵敏的分子检测体系,根据N.parvum的几丁质合酶1基因(CHS1)及其前后100bp序列,分别设计了两对特异性引物CT-WK3-S/CT-WK3-A和CT-N-S/CT-N-A,建立了N.parvum的巢式PCR检测方法。结果表明,建立的体系可以从不同来源的5个N.parvum菌株中特异性地扩增出97bp的目的条带,灵敏度达30fg/μL,是常规PCR的10倍,而从其近缘种葡萄座腔菌属Botryosphaeriasp.的病原菌及其他供试菌株均未扩增出任何条带。以感染N.parvum的核桃组织总DNA为模板进行检测,可以在发病早期检测出N.parvum的存在。本研究建立的巢式PCR体系可以为核桃枝枯病的田间检测提供思路。     

浙麦冬黑斑病病原菌的分离鉴定及淀粉酶产色链霉菌对其生物防治效果

黑斑病是浙江省慈溪市浙麦冬种植基地常发病害,传染速度快且严重影响麦冬的产量与品质。为了明确引起黑斑病的病原菌以便后续有效防治该病害,本文通过对染病组织培养、病原菌分离纯化和柯赫法则验证、形态学观察与基因序列分析比对（ITS、EF-1α、RPB2、Alt a 1、His 3、ATP）,最终鉴定出引起浙麦冬黑斑病的病原菌是链格孢菌Alternaria alternata。研究淀粉酶产色链霉菌Streptomycesdiastatochromogenes 1628代谢产物对黑斑病的防效结果表明,1628代谢产物对链格孢菌菌丝生长具有较强的抑制作用,其EC₅₀=0.764%（体积浓度）。田间防效结果表明7和14 d校正防效分别可达74.08%和65.28%,均高于阳性对照多菌灵。本研究明确了浙麦冬黑斑病病原菌的分类地位,并筛选到能有效抑制该病原菌的生防菌株淀粉酶产色链霉菌,为后续浙麦冬黑斑病的防治提供依据。     

五味子茎基腐病发生初报

通过对辽宁省多个地区的调查,发现此病害发生普遍,尤其是二年生的五味子茎基腐病发生严重。五味子茎基腐病在5月上旬开始出现,6月初为发生盛期。本文对五味子茎基腐病进行了症状描述,并对不同地点采集的病样分别进行木质部和韧皮部病原物的分离、纯化。对分离得到的菌株按照柯赫氏法则进行致病性测定并对病原菌进行鉴定,结果表明,此病害可由木贼镰刀菌(Fusarium equiseti)、茄腐镰刀菌(F.solani)、尖孢镰刀菌(F.oxysporum)和半裸镰刀菌(F.semitectum)4种镰刀菌属真菌引起。针对此病害的发生特点,提出了相应的防治措施。     

基于多重PCR技术的西瓜噬酸菌分组检测方法及其应用

瓜类细菌性果斑病是瓜类作物上重要的种传细菌性病害,其病原菌西瓜噬酸菌Acidovorax citrulli为我国进境植物检疫性有害生物,种子种苗带菌是病害长距离传播的重要来源,种子种苗的快速检测对病害综合防控具有重要的意义。根据寄主的差异西瓜噬酸菌分为两个组,Ⅱ组菌株比Ⅰ组菌株具有更高的铜制剂敏感性,因此,西瓜噬酸菌的分组检测可为病害田间防治中铜制剂的精准使用提供科学依据,避免化学农药的过量施用。本研究筛选了现有的西瓜噬酸菌种间特异性引物和种内分组特异引物,建立并优化了一个多重PCR体系,实现了通过一步试验,就能够准确将西瓜噬酸菌与近缘种和其他植物病原细菌区分开来,并直接鉴定到西瓜噬酸菌不同组。该多重体系可以从带菌种子浸泡液和感病植物组织研磨液中直接检测到西瓜噬酸菌的不同组,且稳定性好,具有应用于生产实践的潜力。     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

浅黄恩蚜小蜂和丽蚜小蜂对温室白粉虱的寄生潜能分析

浅黄恩蚜小蜂Encarsia sophia GiraultDodd和丽蚜小蜂Encarsia formosa Gahan是防治粉虱类害虫的优势寄生蜂,通过生命表技术方法分析了2种寄生蜂对温室白粉虱Trialeurodes vaporariorum(Westwood)的防治潜能。结果表明,丽蚜小蜂在羽化第3天和第10天出现2次寄生高峰,占其总寄生量的13.7%和8.0%,在2次高峰之间逐日寄生粉虱数量比较平稳,单雌逐日平均产雌数保持在10.6~13.4头,10 d后寄生量呈明显的下降趋势;而浅黄恩蚜小蜂羽化10 d内逐日寄生粉虱量变化不大,单雌逐日产雌数稳定在4.2~5.4头,羽化14 d后寄生量呈明显下降趋势。丽蚜小蜂和浅黄恩蚜小蜂的R0、T、rm、λ值分别为171.5、18.0、0.2854、1.3303和61.6、16.2、0.2544、1.2897;粉虱若虫充足时,丽蚜小蜂平均单雌寄生若虫数是浅黄恩蚜小蜂的2.7倍,而后者平均单雌取食若虫数为60.6头,明显高于前者42.7头,总的来看,丽蚜小蜂通过寄生和取食杀死粉虱总量220.8头,明显高于浅黄恩蚜小蜂的127.9头。表明在应用寄生蜂防治温室白粉虱时,单独释放丽蚜小蜂比浅黄恩蚜小蜂显示出更好的防治潜能。     

桃蚜体内次生共生菌沙雷氏菌对宿主抵御寄生蜂和高温胁迫的影响

为明确桃蚜Myzus persicae体内次生共生菌沙雷氏菌Serratia symbiotica对宿主抵抗不良环境的影响,利用叶碟法测定短翅蚜小蜂Aphelinus asychi对自然感染沙雷氏菌桃蚜、自然未感染沙雷氏菌桃蚜和人工感染沙雷氏菌桃蚜的寄生特性和取食特性,并测定烟蚜茧蜂Aphidius gifuensis对这3种处理桃蚜的寄生特性及这3种处理桃蚜经高温胁迫后的生长繁殖特性。结果显示,短翅蚜小蜂在人工感染沙雷氏菌桃蚜上的产卵率比在自然未感染沙雷氏菌桃蚜上的下降近1/2,羽化率下降1/3左右,致死率、取食率、僵蚜率均无显著差异;烟蚜茧蜂对这3种处理桃蚜的致死率、过寄生率、僵蚜率及其产卵率和羽化率等均无显著差异;这3种处理桃蚜的2龄若蚜经高温胁迫后,发育时间和寿命均显著延长,开始产蚜时间明显推迟,繁殖力和日繁殖率显著降低,繁殖历期无明显变化;高温胁迫后,人工感染沙雷氏菌桃蚜比自然未感染沙雷氏菌桃蚜开始产蚜时间提前3.9 d,繁殖力增加7.0头。表明人工感染沙雷氏菌可以提高桃蚜对短翅蚜小蜂和高温胁迫的防御作用,对烟蚜茧蜂的寄生无明显效果。     

湖南桔园主要天敌对柑桔全爪螨的捕食作用及其综合评定

1984-1988年调查了湖南桔园主要天敌——尼氏钝绥螨、深点食螨瓢虫、塔六点蓟马、中华草蛉、龟纹瓢虫的田间消长动态,测定了它们对柑桔全爪螨的捕食功能反应,均属于Holling Ⅱ型。5种主要天敌中,尼氏钝绥螨发生量最多,与柑桔全爪螨数量相关极为显著:中华草蛉的日捕食量最大,对柑桔全爪螨处置时间最短;深点食螨瓢虫食性较专一,对柑桔全爪螨瞬时攻击率最高。根据每种天敌的发生量、发生期、捕食能力及抗药性等4个因素,应用模糊分级方法综合评定对柑桔全爪螨控制作用大小依次为:尼氏钝绥螨、深点食螨瓢虫、塔六点蓟马、中华草蛉、龟纹瓢虫。这些天敌的综合作用可以控制正常年份柑桔全爪螨的为害。     

两种蚜小蜂对烟粉虱MED隐种的田间笼罩控效评价

为明确烟粉虱Bemisia tabaci MED隐种优势寄生蜂海氏桨角蚜小蜂Eretmocerus hayati ZolnerowichRose与浅黄恩蚜小蜂Encarsia sophia GiraultDodd对其控制效果的影响,在棉田尼龙纱网笼罩中释放烟粉虱之后,再分别单独释放海氏桨角蚜小蜂、浅黄恩蚜小蜂以及二者以不同比例组合(1∶1、1∶3、3∶1)释放,定期调查统计2种蚜小蜂对烟粉虱的寄生量和烟粉虱的种群动态。结果表明,相对于不放蜂对照,自首次放蜂后40 d开始,所有放蜂处理均能显著降低烟粉虱若虫种群密度,每100 cm~2叶片上均少于1.00头,但各处理间的烟粉虱种群密度无显著差异;海氏桨角蚜小蜂和浅黄恩蚜小蜂以3∶1比例组合释放的处理中对烟粉虱的寄生量最高,每100 cm~2棉花叶片上能达到4.25头。表明在棉田中对烟粉虱进行生物防治时,以初级寄生蜂海氏桨角蚜小蜂与复寄生蜂浅黄恩蚜小蜂为3∶1的比例释放,可以到达较好的控制效果。     

Nematicidal Activity of Chrysanthemum coronarium

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.     

玫烟色虫草和球孢白僵菌对朱砂叶螨和二斑叶螨的致病力评价

为探寻具有杀螨潜力的生防真菌,采用喷雾法测定分析玫烟色虫草Cordyceps fumosorosea IF-1106菌株和球孢白僵菌Beauveria bassiana BB-1339菌株对朱砂叶螨Tetranychus cinnabarinus和二斑叶螨Tetranychus urticae卵、幼螨及雌成螨的致病力。结果表明,感染玫烟色虫草IF-1106菌株和球孢白僵菌BB-1339菌株后螨类的形态特征不一致,感染IF-1106菌株后形成棉絮状菌丝,而感染BB-1339菌株后则形成羊毛状菌丝。IF-1106菌株和BB-1339菌株对朱砂叶螨和二斑叶螨卵的LC₅₀分别为2.38×10⁷、8.26×10⁷CFU/mL和4.48×10⁷、1.21×10⁸CFU/mL,对朱砂叶螨和二斑叶螨幼螨的LC₅₀分别1.97×10⁷、8.26×10⁷CFU/mL和7.65×10⁶、8.99×10^5...     

Bioluminescence reporter assay system to monitor Arabidopsis MPK3 gene expression in response to infection by Botrytis cinerea

The Arabidopsis MPK3 gene product participates in disease resistance mediated by the MAP kinase cascade. The expression of the MPK3 gene is induced by pathogen inoculation and treatment with chemicals such as salicylic acid (SA) and methyl jasmonate (JA), but the detailed expression pattern of the MPK3 gene has been largely unknown. To investigate MPK3 gene expression in response to disease stress, we fused the MPK3 promoter to the firefly luciferase gene to create a real-time monitoring system for regulated gene expression in planta. The results of an in vivo reporter assay using transgenic Arabidopsis plants harboring MPK3::Fluc showed that the MPK3 promoter activity was induced by treatment with chemicals such as SA and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), that induce defense gene expression. Inoculation with the fungal pathogen Botrytis cinerea resulted in systemic induction of MPK3::Fluc.     

Detection of the phytotoxin coronatine by ELISA and localization in infected plant tissue

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR)in vitro, and the availability of purified toxin has facilitated development of immunological detection methods. A modified, indirect competitive ELISA using the COR-specific monoclonal antibody 11B8 was developed to detect COR in various host plants infected by P. syringae. The estimated detection limit for COR was 50 pg per well, and COR could be reliably quantified from 5 to 40 ng ml⁻¹. The subcellular localization of COR within infected tomato tissue was investigated using the COR-specific antibody MAb 8H3G2. Immunofluorescence microscopy and immunogold labelling showed that COR was present inside tomato cells and was associated with chloroplasts and particles of proteinase inhibitor I. Localization studies indicated that COR is mobile in infected plant tissue and can be detected in healthy tissue adjacent to the bacterial lesions.