嗜线虫致病杆菌HB310菌株对苹掌舟蛾的生物活性

苹掌舟蛾[Phalera flavescens（BremeretGery）]又称为苹果舟形毛虫，属鳞翅目舟蛾科，是我国蔷薇科果树和花卉上的重要食叶害虫，分布广泛，大发生时可将全树叶片食光，影响产量，危及树势。因为苹掌舟蛾幼虫发生为害高峰在8—9月份，已经到达果实的成熟期，化学药剂防治不但污染环境，还会造成果实的农药残留超标。多年来，昆虫病原线虫一共生菌复合体作为生防制剂在生产中成功地用于防治地下害虫或钻蛀类害虫，但是该复合体对叶面害虫防治效果较差。自Bowen从昆虫病原线虫共生菌发光光杆状菌（Photorhabdus luminescens）中发现并分离纯化出具口服杀虫活性的蛋白后，已经陆续发现多种昆虫病原线虫共生菌具有口服杀虫活性，并从中克隆了多个杀虫基因。线虫共生菌的研究已经引起人们广泛关注，能否应用于植物叶面害虫的防治也正在成为人们关注和研究的重点。     

嗜线虫致病杆菌CB6菌株对小菜蛾幼虫生物活性的研究

用叶碟法测定了嗜线虫致病杆菌CB6菌株发酵液对小菜蛾幼虫的致死率、拒食活性以及对生长发育的影响。结果表明 ,其发酵液对幼虫有较高的生物活性 ,取食 3d后 ,1～3龄幼虫的校正死亡率分别为 84.75%、68.33%和 59.32%。 4龄幼虫取食量显著减少 ,选择性拒食率为 76.59% ,非选择性拒食率为 69.72%。 3龄幼虫取食发酵液处理的叶片后 ,生长极其缓慢 ,在 48h时体重抑制率达 100% ,化蛹率和羽化率均明显下降 ,其中化蛹率仅为 15.56% ,100%蛹不能羽化。     

嗜线虫致病杆菌北京变种CB6菌株杀虫蛋白对棉铃虫幼虫的生物活性

嗜线虫致病杆菌北京变种Xenorhabdus var.pekingensis CB6菌株是本研究室自主分离的新菌株,其代谢物对棉铃虫具有很强的拒食和抑制生长活性.为了进一步明确代谢物中杀虫蛋白的生物活性,作者用饲料染毒法和叶碟法测定了杀虫蛋白对棉铃虫不同龄期幼虫取食和生长发育的影响.结果表明,杀虫蛋白对棉铃虫幼虫有很强的抑制生长作用,用64μg/g含杀虫蛋白饲料饲喂初孵、1、2和3龄幼虫5天的生长抑制率分别达95.37%、92.73%、87.15%和88.64%,并明显延长幼虫发育历期,影响幼虫的化蛹及蛹的羽化.杀虫蛋白对5龄棉铃虫幼虫拒食效果明显,幼虫饲喂经1.6mg/mL杀虫蛋白处理的叶片24h,选择性拒食率和非选择性拒食率分别为76.22%和85.42%.当蛋白浓度为0.32mg/mL时,24h选择性拒食和非选择性拒食率分别为68.39%和74.75%.     

嗜线虫致病杆菌CB6菌株对棉铃虫的生物活性

致病杆菌属(Xenorhabdus)及近缘的发光杆菌属(Photorhabdu)是昆虫病原线虫肠道共生菌。共生菌被线虫释放到寄主血腔中后,能导致昆虫迅速死亡,且产生抗菌成分防止虫尸腐烂[1]。由于从土壤或水源中未能分离到自由存活的共生细菌,因此早期认为这类细菌只有在与线虫共生状态下才对昆虫有致死作用[2]。1990年以来,Ensign等[3]和潘映红等[4]研究发现这类细菌的胞外蛋白和脂多糖注射到大蜡螟(Galleriamellonella)体内后均有杀虫活性。1998年,Science报道了发光杆菌(P.luminescens)菌株W 14分泌的高分子量复合体蛋白对烟草天蛾(Manducasexta…     

嗜线虫致病杆菌对小菜蛾的致死和亚致死效应室内评价

嗜线虫致病杆菌是与昆虫病原线虫共生的细菌,对小菜蛾等多种害虫具有广谱的杀虫活性。为了深入了解该菌的杀虫活性,在室内测定了嗜线虫致病杆菌HB310 ( 简称 Xn HB310)菌株对小菜蛾幼虫的致死和亚致死效应以及持效期。结果表明, Xn HB310菌液对小菜蛾2龄和3龄幼虫72 h的LC50值分别是3.32×104 和8.56×104 CFU/mL,其活性组分主要存在于菌株细胞和经50 kDa超滤膜超滤后的上清液中。此外, Xn HB310菌液对小菜蛾幼虫还表现出明显的拒食活性,其2龄和3龄幼虫的拒食中浓度(AFC50)分别为4.03×105和8.48×105 CFU/mL。将2.24×106 CFU/mL菌液喷施到甘蓝苗上,其在室内的持效期为3~4 d。用亚致死剂量(LC30和LC50)的菌液饲喂小菜蛾2龄幼虫,存活幼虫的生长发育指标、蛹重、雌虫寿命以及成虫产卵量均明显下降,推测这可能导致小菜蛾下一代种群数量减少。研究结果表明,嗜线虫致病杆菌HB310菌株具有开发成为杀虫剂的潜力。     

嗜线虫致病杆菌高产菌株的选育

用 4种诱变方法 (紫外线、氯化锂、60 Co γ射线、自身代谢产物 )对嗜线虫致病杆菌进行了诱变处理。结果表明 ,菌株对各种诱变剂均比较敏感 ,低剂量诱变和复合诱变处理的菌株正变率较高 ,其中氯化锂加紫外线复合诱变的累加处理效果最好 ,效价提高了 6 0 %～ 70 % ;连续5代传代培养 ,其产素能力基本稳定。     

高毒力杀虫细菌嗜线虫致病杆菌CB6菌株的培养基优化

用正交试验法研究了嗜线虫致病杆菌北京变种菌株CB6的营养利用以及培养基成分的改变对产生杀虫活性物质的影响。通过筛选该菌对碳源、氮源以及无机盐的需求,确定了该菌的最佳发酵培养基为(g/L):豆粉40g,蔗糖15g,蛋白胨15g,酵母膏10g,玉米浆5g,NaH2PO41g,NaCl5g,谷氨酸5g。培养液上清液对玉米螟初孵幼虫的体重抑制率为90 81%,比基础培养基的杀虫活性提高了46.5%。     

嗜线虫致病杆菌对棉铃虫的生物活性研究

嗜线虫致病杆菌HB310(Xenorhabdus nematophila HB310)菌液中主要的杀虫活性物质是一种高分子量的复合蛋白—毒素Ⅱ。以该菌液和毒素Ⅱ分别饲喂棉铃虫Helicoverpa armigera幼虫,检测其对棉铃虫生长发育的影响,同时通过生化分析研究了该毒素对幼虫中肠内几种蛋白酶活力的影响。结果表明:菌液和毒素Ⅱ对棉铃虫幼虫的取食量和生长发育均有显著的影响,取食拌有菌液和毒素Ⅱ的人工饲料的棉铃虫食量明显减少,发育速度延缓,发育历期比对照明显推迟;尽管一直取食混合原菌液(6.5×108 cells/mL)人工饲料的棉铃虫2龄幼虫前期死亡率很低,但是其生长发育几乎完全被抑制,该处理组所有幼虫均不能化蛹;原菌液对4龄幼虫的食量、发育历期、蛹重及化蛹率均有显著影响。菌液对棉铃虫幼虫的影响与菌液的浓度和幼虫的龄期成反比,稀释50倍的菌液对2龄和4龄幼虫的生长发育仍有一定影响;毒素Ⅱ(51.9 μg/mL)对4龄棉铃虫的生长发育也有明显的抑制作用。短时间(2 d)饲喂原菌液后更换正常饲料,仅延缓了棉铃虫幼虫的发育历期,而对其化蛹率、蛹重及羽化率均无明显影响。饲喂毒素Ⅱ的棉铃虫幼虫中肠主要蛋白酶的活性受到明显的抑制。     

嗜线虫致病杆菌YL001菌株代谢产物的抑菌活性

从陕西杨凌土样中筛选得到一株昆虫病原线虫,从其体内分离到它的共生菌嗜线虫致病杆菌Xenorhabdusnematophilus,编号为YL001,为明确其代谢产物的抑菌活性,本试验采用抑制菌丝生长速率法、抑制孢子萌发法、组织测定法和盆栽试验法系统测定了其发酵液对植物病原真菌的抑制作用。结果表明,在离体条件下该菌株发酵液对供试的15种植物病原真菌菌丝生长均有一定的抑制作用,对番茄灰霉病菌和辣椒疫霉病菌的抑制作用最强,EC50分别为53.54和90.39mg·L-1;对供试的6种植物病原真菌孢子萌发均有一定的抑制作用,其中对番茄灰霉病菌和烟草赤星病菌孢子萌发有较强的抑制作用,EC50仅分别为16.97和21.87mg·L-1;组织法测定中,发酵液稀释10倍后,对番茄灰霉病的保护效果为74.98%,治疗效果为63.50%;对盆栽番茄灰霉病的保护效果为65.11%,治疗效果为54.64%。显微观察表明,YL001菌株的发酵液可使菌丝生长形态异常、变形萎缩,甚至断裂。     

嗜线虫致病杆菌HB310菌株对东亚飞蝗杀虫活性的研究

采用叶片浸渍法测定了嗜线虫致病杆菌(Xenorhabdus nematophila) HB310菌株对不同龄期东亚飞蝗蝗蝻(若虫)的口服胃毒活性。结果表明,菌液与细胞在相同浓度条件下对2龄和4龄蝗蝻杀虫活性无显著差异,但明显高于上清液活性(P0.05);在高浓度(2.24×106cfu/mL)条件下,菌液和细胞对东亚飞蝗成虫致病力无差异,但随着浓度降低,菌液的活性明显高于细胞和上清液,且东亚飞蝗龄期越小敏感性越强。该菌株不同分子量胞内蛋白粗提物对不同龄期东亚飞蝗蝗蝻均具有很好的胃毒活性。其中,30 kDa蛋白粗提物饲喂处理96 h后,不同龄期东亚飞蝗蝗蝻的死亡率均达到90%以上。     

深色和浅色两种表型东亚飞蝗对蝗虫微孢子的敏感性比较

为验证群居型飞蝗深浅2种表型对蝗虫微孢子Paranosema locustae的敏感性差异,本研究以东亚飞蝗Locusta migratoria manilensis为研究对象,测定并比较了深色型和浅色型2种表型的东亚飞蝗经蝗虫微孢子侵染后其发育历期、死亡率、免疫血淋巴细胞数量以及免疫基因表达量等变化情况。结果显示,浅色型东亚飞蝗的发育历期因蝗虫微孢子侵染而延长近1.0 d左右,比深色型东亚飞蝗多延长0.5 d;浅色型东亚飞蝗发死亡率明显高于深色型的;浅色型东亚飞蝗的免疫血淋巴细胞数量低于深色型的;蝗虫微孢子侵染后,深色型东亚飞蝗免疫基因toll基因和attacin基因的表达量上升,而浅色型东亚飞蝗的toll基因表达量无明显变化,attacin基因的表达量甚至出现下调。而且,蝗虫微孢子在浅色型飞蝗体内增殖更多。表明深色型东亚飞蝗在应对蝗虫微孢子侵染时较浅色型更有优势,浅色型东亚飞蝗较深色型对蝗虫微孢子更敏感。     

调控东亚飞蝗体温的主要环境因子分析

为明确东亚飞蝗Locusta migratoria manilensis体温在常年发生区与环境因子之间的关系,于2015—2016年采用直接抓取和网捕方法在天津市北大港蝗区捕捉东亚飞蝗的秋蝗和夏蝗,测量其瞬时体温以及周围4类8个环境指标,应用因子分析法评价东亚飞蝗体温与环境因子的关系。结果表明,东亚飞蝗白天体温比夜间高,平均体温高于26.5℃;且体温在白天出现2次高峰,分别是上午11点左右的49.0℃和下午14点左右的47.2℃。无论是夏蝗还是秋蝗,其体温均随着龄期增加呈现上升趋势,2～5龄蝗蝻、成虫的平均体温依次为36.9、37.0、37.6、37.2、38.0℃。因子分析结果表明,植被冠层温度是影响飞蝗体温的关键因子,飞蝗所处地面温度对其体温影响最大;风速和植株高度是影响飞蝗体温的重要分因子;植株高度与体温成负相关。表明植被冠层温度与东亚飞蝗的体温变化最为密切。     

东亚飞蝗FK506结合蛋白基因克隆及其免疫功能分析

为明确东亚飞蝗Locusta migratoria manilensis免疫相关FK506结合蛋白(FK506 binding protein,FKBP)的功能,了解FKBP对金龟子绿僵菌Metarhizium anisopliae侵染东亚飞蝗的影响,分离克隆获得FKBP46基因及其纯化蛋白,采用荧光定量PCR方法测定...     

不连续气体交换循环呼吸模式下西藏飞蝗和东亚飞蝗的呼吸特征参数

为解析西藏飞蝗对青藏高原干燥低氧环境的呼吸适应机制,本研究比较不连续气体交换循环(discontinuous gas exchange,DGC)呼吸模式下西藏飞蝗Locusta migratoria tibetensis与东亚飞蝗L.m.manilensis的呼吸特征参数.结果 表明:西藏飞蝗和东亚飞蝗4、5龄蝗蝻及雄...     

嗜线虫致病杆菌SN313的次生代谢产物及其抑制植物病原真菌活性研究

通过化学与生物活性筛选从土壤中分离得到一株菌株,利用16S rDNA方法将其鉴定为嗜线虫致病杆菌Xenorhabdus nematophila并命名为SN313。采用微生物发酵、液相萃取、柱层析及半制备高效液相色谱等技术,对SN313的发酵液进行分离纯化,得到3个化合物。利用质谱和核磁共振等波谱技术并依据文献数据确定了这3个化合物分别是N-(2-羟基苯基乙酰) 色胺 ( 1 )、吩嗪-1-羧酸 ( 2 ) 和环(脯氨酸-色氨酸) ( 3 ),其中化合物 1 是一个新的β-吲哚基乙胺类衍生物。通过微量稀释法测定了3个化合物对4种植物病原真菌的抑制活性。结果表明:化合物 1 对辣椒疫霉Phytophthoa capsici和番茄灰霉病菌Botrytis cinerea具有明显的抑制作用 (IC₅₀值分别为11.20 μg/mL和28.94 μg/mL);化合物 2 对番茄灰霉病菌、辣椒疫霉、水稻纹枯病菌Thanatephorus cucumeris和小麦根腐病菌Bipolaris sorokiniana有明显的抑制作用 (IC₅₀ < 40 μg/mL);化合物 3 对番茄灰霉病菌具有较好的抑制效果 (IC₅₀ = 41.58 μg/mL)。从土壤微生物中获取化合物 2 ,为生物农药申嗪霉素有效成分的天然获取提供了一种新的途径。     

西藏飞蝗研究进展

西藏飞蝗Locusta migratoria tibetensis是为害小麦Triticum aestivum、青稞Hordeum vulgare等禾本科作物及牧草的重要高原害虫,具有发生面积大、繁殖能力强和为害重等特点,在西藏自治区雅鲁藏布江沿岸、青海省玉树藏族自治州和四川省甘孜藏族自治州等地均有过多次暴发的历史记录,给当地农牧业造成了严重的经济损失。为全面了解西藏飞蝗的发生规律及其相关研究进展,本文主要综述了西藏飞蝗的地理分布、形态学特征、生物学特性、生态学特征和防治措施等内容,同时对西藏飞蝗未来的研究方向以及综合防控进行了展望。     

Increased activity and reduced sensitivity of acetylcholinesterase associated with malathion resistance in a field population of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibility to malathion, and the activity and sensitivity of acetylcholinesterase (AChE, EC 1.1.1.7) were compared between two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen) collected from Wudi County of Shandong Province in East China and Huangliu County of Hainan Province in South China. Huangliu population showed 8.5-fold resistance to malathion compared with Wudi population. AChE from Huangliu population showed 4.8-fold higher activity than that from Wudi population toward the model substrate acetylthiocholine (ATC). Kinetic studies indicated that AChE from Huangliu population had 2.6-fold lower affinity, but 5.0-fold higher catalytic activity toward ATC than AChE from Wudi population. Significantly increased activity of AChE in Huangliu population was also confirmed by non-denaturing polyacrylamide gel electrophoresis. Inhibition kinetics revealed that AChE from Huangliu population was 9.8-, 2.4-, 8.0- and 7.7-fold less sensitive to inhibition by paraoxon, malaoxon, chlopyrifos oxon, demeton-S-methyl, respectively, than that from Wudi population. Our studies revealed that a mild resistance to malathion in Huangliu population was associated with reduced sensitivity and increased catalytic activity of AChE. Our results suggest that alterations of AChE may play an important role conferring or contribute to malathion resistance in Huangliu population of the locust.     

Comparative studies of acetylcholinesterases purified from two field populations of the oriental migratory locust (Locusta migratoria manilensis): implications of insecticide resistance

Acetylcholinesterase (AChE) was purified by affinity chromatography from two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), collected from Huanghua and Pingshan Counties, Hebei Province of China. The purification factors and yields were 1661-fold and 19.3%, respectively, for the Huanghua population, and 3897-fold and 39.6% for the Pingshan population. Both the purification factor and yield were significantly lower in the Huanghua population than in the Pingshan population. AChE activity was almost completely inhibited by 10⁻⁶ M eserine and BW284C51, but ?5.8% of AChE activity was inhibited by ethopropazine at the same concentration, suggesting that purified AChE from either population was a typical insect AChE. However, AChE purified from the Huanghua population was 62-, 2.0-, and 1.6-fold less sensitive to inhibition by the three organophosphate compounds, chlorpyrifos oxon, demeton-S-methyl, and paraoxon, respectively, than that from the Pingshan population. Significantly lower purification factor and low yield associated with reduced sensitivity of AChE to inhibition by the organophosphates indicated that AChE purified from the Huanghua population was biochemically and pharmacologically different from that of the Pingshan population. Reduced sensitivity of AChE appeared to contribute to organophosphate resistance in the locust from Huanghua County, where insecticides have commonly been used to manage outbreaks of the locust.     

Characterizations of general esterases in relation to malathion susceptibility in two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

General esterases were characterized and compared from two populations of the oriental migratory locust, Locusta migratoria manilensis, collected from Huanghua and Pingshan Counties, Hebei Province, China. General esterases were most concentrated in the thorax and abdomen, which contained 46.1 and 36.1% of total esterase activity in females, and 42.7 and 36.0% in males, respectively, when α-naphthyl acetate was used as a substrate. There was no distinct difference in esterase banding patterns in different body regions for the substrates α-naphthyl acetate and β-naphthyl acetate on non-denaturing polyacrylamide gel electrophoresis (PAGE). However, the general esterase activities in the Huanghua population were 1.8-fold higher than those in the Pingshan population in both females and males. Increased esterase activity in the Huanghua population appeared to be mainly due to several additional esterase bands detected on non-denaturing PAGE. Inhibition studies of general esterases using four inhibitors, including paraoxon, malaoxon, eserine, and carbaryl, indicated that most general esterases in the two populations were B-type. The increased esterase activity in the Huanghua population appeared to be associated with a 1.8-fold decreased susceptibility to malathion. Such differences may attribute to the difference in control practices for the locust between Huanghua and Pingshan Counties.