草莓灰霉病菌的培养、毒素提取及生物测定

 草莓灰霉病菌(Botrytis cinerea Persoon)在25℃、pH3~4的Peberdy培养基中生长良好,并可产生引起草莓愈伤组织细胞死亡的毒素。产生毒素的高峰期为静置培养20天。灰霉菌发酵液中的毒素可用氯仿进行提取,经提取后的毒素液在269nm处有吸收峰。本研究建立了利用草莓愈伤组织细胞的荧光活性测定毒力的方法。     

灰葡萄孢毒素的组分分析和生物测定

 用PD培养液培养灰葡萄孢菌,过滤液中含活性成分,经氯仿提取后TLC分离获6种组分,Rf值分别为Ⅰ(0.36)、Ⅱ(0.48)、Ⅲ(0.54)、Ⅳ(0.65)、Ⅴ(0.11)、Ⅵ(0.77),经针刺果实生物测定发现,Ⅲ、Ⅴ、Ⅵ具有毒性。BC-毒素具有一定的广谱性,对不同科属的番茄、茄子、黄瓜、白菜、油菜、小麦、玉米、棉花等都具有不同程度的毒害作用。     

香豆素对灰葡萄孢霉菌抑菌作用机理初探

为探究香豆素对灰葡萄孢霉菌Botrytis cinerea作用机理的影响，通过抑菌试验、酶活试验及电导率试验，分析香豆素对灰葡萄孢霉菌的抑菌活性，以及经香豆素处理后灰葡萄孢霉菌胞内外蛋白质含量、酶活性以及细胞膜透性的变化规律，揭示香豆素对灰葡萄孢霉菌的抑菌机理。结果表明:在PD液体培养基上香豆素对灰葡萄孢霉菌的EC₅₀值为101 mg/L；处理组胞内及胞外蛋白浓度均低于对照组蛋白浓度；香豆素浓度为101 mg/L时对灰葡萄孢霉菌三磷酸腺苷酶 (ATPase) 和过氧化氢酶 (CAT) 的抑制率分别为62.60%和76.5%，对纤维素酶 (CL)、多聚半乳糖醛酸酶 (PG) 和果胶酶 (pectinase) 的抑制率分别为57.63%、64.51%和61.13%；香豆素处理可显著增强灰葡萄孢霉菌的细胞膜透性，经香豆素处理48 h，灰葡萄孢霉菌的电导率为17568.79 μS/cm，为对照组电导率的1.9倍。可见，香豆素可以抑制灰葡萄孢霉菌的生长，使其呼吸代谢受阻，干扰灰葡萄孢霉菌细胞膜的渗透，抑制灰葡萄孢霉菌细胞壁酶的活性。该结果可为以香豆素为主的新型植物源农药开发以及灰霉病的防治提供理论支撑。     

玉米大斑菌Ht-毒素组分分析

 对分别属于玉米大斑病菌(Helminthosporium turcicum)1号和2号生理小种的致病毒素进行硅胶TL层析、薄层扫描和生物监测的结果发现,两个小种的毒性组分不同。2号小种可分离到I(Rf0.50)、Ⅱ(Rf0.36)、Ⅲ(Rf0.67)、Ⅳ(Rf0.80)、Ⅴ(Rf0.13)、Ⅵ(Rf0.25)6种组分,而1号小种最多可分离到除Ⅳ以外的其它5种组分,这些组分均有不同程度的紫外吸收。对在200~360nm有明显紫外吸收的4种组分(Ⅰ、Ⅱ、Ⅲ、Ⅳ)的生物监测结果表明,只有组分Ⅲ对带Ht₁基因和不带Ht₁基因的Oh43和B372对玉米自交系不具任何致病活性,组分Ⅰ、Ⅱ对不带Ht₁基因的玉米自交系具有致病活性,而对带Ht₁基因的玉米自交系却无毒性。值得重视的是组分Ⅳ的致病特性与组分Ⅰ、Ⅱ正好相反,即对带Ht₁基因的玉米自交系具有致病活性,却对不带Ht₁基因的玉米自交系没有致病作用。结论指出,Ht-毒素的TLC组分Ⅰ、Ⅱ、Ⅳ玉米斑菌诱致玉米发病的主要活性成份,其中组分Ⅳ是2号小种与Ht₁基因玉米互作的特异性因子。     

黄瓜黑星病菌致病机理的研究Ⅰ 黄瓜黑星病菌致病毒素的分离和纯化

本文明确了在接种后的黄瓜黄化子叶中能够提取到黄瓜黑星病菌(Cladosporium cucum eri-num ) 的粗毒素,最佳培养时间为接种后4 d。该毒素具有明显的生物活性,能够抑制黄瓜胚根与胚芽的生长,能使扦插的黄瓜水培幼苗致萎。通过硅胶柱层析纯化黄瓜黑星病菌的粗毒素,根据洗脱顺序和洗脱液的种类将其分为4 个洗脱组分。根据硅胶薄层层析紫外显色及生物活性分析可确定,活性物质主要集中在无色的洗脱组分Ⅱ中,该洗脱组分中有 3 组活性物质:CCⅠ:Rf= 0.51; CCⅡ:Rf=0.25; CCⅢ:Rf= 0.06。     

黄瓜黑星病菌致病机理的研究Ⅰ黄瓜黑星病菌致病毒素的分离和纯化

 本文明确了在接种后的黄瓜黄化子叶中能够提取到黄瓜黑星病菌(Cladosporium cucumerinum)的粗毒素,最佳培养时间为接种后4 d。该毒素具有明显的生物活性,能够抑制黄瓜胚根与胚芽的生长,能使扦插的黄瓜水培幼苗致萎。通过硅胶柱层析纯化黄瓜黑星病菌的粗毒素,根据洗脱顺序和洗脱液的种类将其分为4个洗脱组分。根据硅胶薄层层析紫外显色及生物活性分析可确定,活性物质主要集中在无色的洗脱组分Ⅱ中,该洗脱组分中有3组活性物质:CCⅠ:Rf=0.51;CCⅡ:Rf=0.25;CCⅢ:Rf=0.06。     

葱叶枯病菌 Stemphylium botryosum毒素的分离与除草活性研究

葱叶枯病菌在活体外可以产生致病毒素, 用改良PS 培养基培养所得滤液经TLC 分离获得Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ 5 种组分, 其Rf 值分别为0.31、0.47、0.74、0.80 和0.90, 在乙醇中最大紫外吸收峰分别为240、247、223、236 和258 nm。生物测定结果表明, 组分Ⅱ、Ⅲ、Ⅳ对马唐的生长有明显的抑制作用, 其中以组分Ⅳ 生物活性最高。粗毒素对禾本科杂草种子萌发抑制效果高于对阔叶杂草, 而对玉米、水稻、油菜等种子萌发影响很小。试验还发现, 毒素对马唐的防效最高, 与百草枯药效相当。毒素对马唐叶绿素a 的含量影响不大, 但对叶绿素b 和叶绿素总含量影响较大。     

淡紫灰链霉菌除草活性物质对苘麻叶片生理生化的影响

以苘麻成株叶片为材料,采用植物抗性生理分析测定技术研究了淡紫灰链霉菌除草活性物质D-13组分对苘麻叶片细胞膜透性、叶片内MAD含量、SOD、CAT和APX酶活性变化的影响,以及对膜系统的影响。结果表明,苘麻叶片经D-13组分处理后,细胞膜透性随着D-13组分浓度的增加而增加,MDA含量在D-13组分处理后,随着D-13组分浓度的增加而升高。在电镜观察下,叶片细胞的膜系统随着D-13组分浓度的升高呈现出不同的受害情况。随着D-13组分处理时间的延长,SOD、CAT、APX活性均出现了不同程度的下降,与对照相比,SOD、CAT、APX在40h时活性达到最低,分别比对照活性降低超过了60%、30%和50%。可见,淡紫灰链霉菌除草活性物质可严重降低苘麻叶片的生理生化活性。     

玉米大斑菌Ht－毒素组分分析

对分别属于玉米大斑病菌（Ｈｅｌｍｉｎｔｈｏｓｐｏｒｉｕｍｔｕｒｃｉｃｕｍ）１号和２号生理小种的致病毒素进行硅胶ＴＬ层析、薄层扫描和生物监测的结果发现，两个小种的毒性组分不同。２号小种可分离到Ｉ（Ｒｆ０．５０）、Ⅱ（Ｒｆ０．３６）、Ⅲ（Ｒｆ０．６７）、Ⅳ（Ｒｆ０．８０）、Ⅴ（Ｒｆ０．１３）、Ⅵ（Ｒｆ０．２５）６种组分，而１号小种最多可分离到除Ⅳ以外的其它５种组分，这些组分均有不同程度的紫外吸收。对在２００～３６０ｎｍ有明显紫外吸收的４种组分（Ⅰ、Ⅱ、Ⅲ、Ⅳ）的生物监测结果表明，只有组分Ⅲ对带Ｈｔ１基因和不带Ｈｔ１基因的Ｏｈ４３和Ｂ３７２对玉米自交系不具任何致病活性，组分Ⅰ、Ⅱ对不带Ｈｔ１基因的玉米自交系具有致病活性，而对带Ｈｔ１基因的玉米自交系却无毒性。值得重视的是组分Ⅳ的致病特性与组分Ⅰ、Ⅱ正好相反，即对带Ｈｔ１基因的玉米自交系具有致病活性，却对不带Ｈｔ１基因的玉米自交系没有致病作用。结论指出，Ｈｔ－毒素的ＴＬＣ组分Ⅰ、Ⅱ、Ⅳ玉米斑菌诱致玉米发病的主要活性成份，其中组分Ⅳ是２号小种与Ｈｔ１基因玉米互作的特异性因子     

草莓根腐病防控生物药剂筛选与田间防治效果验证

草莓根腐病是陕西关中地区草莓生产中的主要病害之一。采用组织分离法对引起草莓根腐病的病原菌进行分离,通过形态学和分子生物学的方法鉴定其为茄腐镰刀菌Fusarium solani和尖孢镰刀菌Fusarium oxysporum。采用菌丝生长速率法测定了4种植物源农药对上述两种镰刀菌的抑菌活性。结果表明,1.5%苦参·蛇床子素水剂(AS)对尖孢镰刀菌的抑菌活性最强,EC50值为2.7μg/mL,0.3%苦参碱乳油(EC)对茄腐镰刀菌的抑菌活性最强,EC₅₀值为4.5μg/mL。选择室内抑菌活性较高的3种植物源杀菌剂和3种微生物菌剂(枯草芽孢杆菌、多粘类芽孢杆菌和哈茨木霉菌)为供试药剂进行田间试验,发现1 000亿芽孢/g枯草芽孢杆菌可湿性粉剂(WP) 600倍液对草莓根腐病的防治效果最好,达69.25%,10亿芽孢/g多粘类芽孢杆菌WP 800倍液、1.5%苦参·蛇床子素AS 700倍液、2亿芽孢/g哈茨木霉菌WP 200倍液防治效果分别为63.14%、62.84%和59.74%。因此推荐使用枯草芽孢杆菌WP、多粘类芽孢杆菌WP、苦参·蛇床子素AS和哈茨木霉菌WP作为草...     

Synergistic Antifungal Activity of Chitinolytic Enzymes and Prodigiosin Produced by Biocontrol Bacterium, Serratia marcescens Strain B2 against Gray Mold Pathogen, Botrytis cinerea

Serratia marcescens strain B2 is an effective biocontrol agent against gray mold of cyclamen, caused by Botrytis cinerea Persoon. Strain B2 has strong antifungal activity against B. cinerea in vitro. The culture filtrate was found to contain different types of chitinolytic enzyme activity, including endochitinase and chitobiase activity. Four chitinolytic enzymes were detected among the extracellular proteins of strain B2. Two major enzymes, a 58-kDa endochitinase and a 98-kDa chitobiase, were purified by chromatography and electrofocusing, respectively. Both enzymes inhibited spore germination of B. cinerea. And a red pigment, prodigiosin, extracted and purified from the bacterial cells, also inhibited spore germination. When prodigiosin and the chitinolytic enzymes were applied in concert, a synergistic inhibitory effect was observed. S. marcescens strain B2 has multiple modes of action against the pathogen. Received 14 February 2001/ Accepted in revised form 31 May 2001     

水稻白叶枯病菌毒素对水稻不育系珍汕97A的专化毒性

 采用乙酸乙酯法提取对珍汕97不育胞质具有专化毒性的白叶枯病菌AH28、GX50和非专化毒性的OS14菌株的毒素。以不同浓度的毒素处理珍汕97不育系和保持系的种子或幼苗,可引起水稻叶片褪绿和细胞坏死,产生与接种病菌相似的症状,在蚕豆叶片上形成过敏性坏死斑;毒素可导致水稻幼苗萎蔫、胚根胚芽生长受抑和根冠细胞死亡;毒素的毒性与毒素浓度和产毒素菌株的致病力成正比;专化性毒素AT对珍汕97不育系的毒害程度显著高于对保持系的毒害。     

水稻白叶枯病菌毒素对水稻不育系珍汕97A的专化毒性

采用乙酸乙酯法提取对珍汕９７不育胞质具有专化毒性的白叶枯病菌ＡＨ２８、ＧＸ５０和非专化毒性的ＯＳ１４菌株的毒素。以不同浓度的毒素处理珍汕９７不育系和保持系的种子或幼苗,可引起水稻叶片褪绿和细胞坏死,产生与接种病菌相似的症状,在蚕豆叶片上形成过敏性坏死斑;毒素可导致水稻幼苗萎蔫、胚根胚芽生长受抑和根冠细胞死亡;毒素的毒性与毒素浓度和产毒素菌株的致病力成正比;专化性毒素ＡＴ对珍汕９７不育系的毒害程度显著高于对保持系的毒害。     

Purification and Biological Characterization of Host-Specific SV-Toxins from Stemphylium vesicarium Causing Brown Spot of European Pear

ABSTRACT Culture filtrates of a pathogenic isolate (IT37) of Stemphylium vesicarium, causing brown spot of European pear, induced veinal necrosis only on pear leaves susceptible to the pathogen. Two host-specific toxins, SV-toxins I and II, were purified from culture filtrates of IT37 by successively using Amberlite XAD-2 resin adsorption, cellulose thin-layer chromatography, and high-performance liquid chromatography under three different sets of conditions. Susceptible cultivars showed veinal necrosis at a SV-toxin I concentration of 0.01 to 0.1 mug/ml, whereas resistant cultivars were insensitive to the toxin at 1,000 mug/ml. SV-toxins I and II caused a dose-dependent increase in electrolyte loss from susceptible leaf tissues. No increase in electrolyte loss was detected in leaf tissues from resistant cultivars. The results of physiological studies indicated that SV-toxins appear to have an early effect on plasma membranes of susceptible leaves. Spores of a nonpathogenic isolate induced necrotic lesions on susceptible leaves in the presence of a small amount of toxin. SV-toxins were detected in intercellular fluids obtained from diseased leaves after inoculation with the pathogen. The results indicate that SV-toxins I and II produced by S. vesicarium can be characterized as host-specific toxins.     

四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化

为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。     

Molecular characterization of pyraclostrobin resistance and structural diversity of the cytochrome b gene in Botrytis cinerea from apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.     

灰葡萄孢对腐霉利的抗性分子机制及快速检测技术

为明确灰葡萄孢Botrytis cinerea对腐霉利的抗性现状,于2017—2018年采用单孢分离法从浙江省5个地区的草莓大棚共分离获得200个菌株.通过区分剂量法测定了其对腐霉利的抗性,对抗药性菌株的分子机制进行了分析,并根据抗药性分子机制,建立了B.cinerea腐霉利高抗基因型的环介导等温扩增(loop-med...     

香蕉假茎细胞对枯萎病菌不同小种及其粗毒素的病理反应

 以香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)1号小种和4号小种及其粗毒素分别接种香牙蕉和粉蕉的组培苗及离体假茎后,用组织切片法观察香蕉假茎细胞的病理反应,以探明香蕉枯萎病菌不同小种及其粗毒素的致病作用。结果表明,枯萎病菌不同小种人工接种仅能感染相应的香蕉种类,但不同香蕉种类的离体假茎细胞用不同小种接种及其粗毒素处理,均产生褐变等病理反应,且病变程度不存在小种间的差异。表明枯萎病菌不同小种对香蕉不同种类的致病力差异可能与存在其它致病因子或专化性识别的因子有关。同时证实了病菌不同小种的毒素对蕉类不存在着选择毒性