Isolation of a novel Carica papaya α-amylase inhibitor with deleterious activity toward Callosobruchus maculatus

Cowpea (Vigna unguiculata) is a subsistence crop for small and poor farmers from Latin America and Africa. This culture is commonly damaged by cowpea weevil (Callosobruchus maculatus), which burrow into stored seeds to fed on. Due to impact of larval predation, several plant defense studies have been developed, indicating that α-amylase inhibitors are able to impede and/or reduce bruchids digestive process. In this report, a novel α-amylase inhibitor from papaya seeds (Carica papaya) with activity against cowpea weevil enzymes was purified and biochemical characterized. Peeled seeds were macerated and extracted with a 0.6 M NaCl and 0.1% HCl solution. Crude extract was precipitated with ammonium sulphate (100%). After dialyses, this rich fraction was applied onto a CM-Cellulose column and retained peak was submitted to an analytic reversed-phase column HPLC (Vydac C-18TP) yielding several peaks. Only one fraction, with molecular mass of 4562 Da, showed significant inhibitory activity against C. maculatus α-amylases. Otherwise, no inhibitory activities against mammalian α-amylases were observed. Bioassays using artificial seeds containing C. papaya α-amylase inhibitor rich fraction (0.5% and 1.0%) were also conduced showing that α-amylase inhibitors were able to increase larval mortality (50%) and also decrease insect fecundity and adult longevity. These results showed the presence of an α-amylase inhibitor from C. papaya seeds with high specificity to insect enzymes, indicating that this inhibitor probably could be used, through genetic engineering, in the construction of transgenic plants with enhanced resistance toward cowpea weevil.     

异色瓢虫卵巢发育及卵子发生过程观察

为明确异色瓢虫卵巢发育和卵子发生过程,采用内部解剖和石蜡切片技术,分别对异色瓢虫卵巢形态和卵母细胞组织结构进行系统观察,并对其发育阶段进行了划分。结果表明:异色瓢虫雌性内生殖器官主要由1对卵巢、1对侧输卵管、1根中输卵管、受精囊、受精囊附腺、交配囊和黏腺组成;根据有无卵室和卵黄沉积情况将其卵巢发育划分为未分化期、生长发育初期、卵黄沉积期和成熟待产期4个等级;根据卵母细胞所在位置、体积和细胞核的变化,滋养细胞和滤泡细胞的形态变化以及卵黄沉积情况,将异色瓢虫卵子发生划分为卵母细胞分化期、卵母细胞营养期和卵黄形成期3个时期7个阶段。本研究首次对异色瓢虫的卵子发生进行了观察,并初步划分了异色瓢虫卵巢发育的分级标准。     

The effects of pyrethroids on the electrical activity of neurosecretory cells from the brain of Rhodnius prolixus

The effects of pyrethroids on the on-going electrical activity of the axons of neurosecretory cells from the brain of fifth instar Rhodnius prolixus have been studied using extracellular electrodes. Low concentrations of the pyrethroids decamethrin, bioresmethrin, permethrin, and bioallethrin all produce dramatic increases in the overall frequency and dramatic changes in the pattern of electrical activity when applied directly to the exposed brain and corpora cardiaca in an otherwise intact insect. This change in activity was brought about by a recruitment in active units and the production of phasic acivity. A doubling of frequency over that of controls was brought about by low doses of the pyrethroids, namely decamethrin, 1 × 10^?10M; bioresmethrin, 2 × 10^?10M; permethrin, 1 × 10^?9M; and bioallethrin, 2 × 10^?7M. Similar hyperactivity of this system occurred during intoxication of intact insects following topical application of LD₉₅ bioresmethrin. The enhanced sensitivity shown by neurosecretory cells over that of other cell types is discussed, as is the possibility that the increases in electrical activity of neurosecretory axons may result in massive neurohormonal release and thereby contribute to the eventual poisoning of the insect.     

长枝木霉对禾谷胞囊线虫卵的寄生和毒杀作用及其机制

为了明确长枝木霉Trichoderma longibrachiatum对小麦禾谷胞囊线虫Heterodera avenae卵的防治潜力和作用机制,在室内测定了长枝木霉分生孢子悬浮液对小麦禾谷胞囊线虫卵的寄生和毒杀作用,并通过温室盆栽试验测定了长枝木霉分生孢子悬浮液对小麦禾谷胞囊线虫卵的防治效果.室内试验中1.5×107 CFU/mL的长枝木霉分生孢子悬浮液处理12 d对小麦禾谷胞囊线虫卵的寄生率和卵孵化的相对抑制率分别为91.33％和88.35％.温室盆栽试验中1.5×107 CFU/mL处理对土壤中胞囊、线虫和根系中线虫的相对防治效果分别为89.84％、92.67％和88.30％;接种卵悬浮液和长枝木霉分生孢子悬浮液后,小麦幼苗株高、根系长度、地上部鲜重和根系鲜重的增长率分别为66.72％、159.31％、169.09％和170.19％;叶绿素总量和根系活力显著高于对照,分别为17.52 mg/g和356.25 μg·g-1·h-1.研究表明长枝木霉可作为小麦禾谷胞囊线虫的生防制剂.     

Developmental and tissue-specific expression of CHS1 from Plutella xylostella and its response to chlorfluazuron

The cDNA of chitin synthase-1 gene of Plutella xylostella (PxCHS1) was characterized and expression patterns of the two splice variants PxCHS1A and PxCHS1B were investigated in various developmental stages and in major body tissues by RT-PCR and real-time quantitative PCR (qPCR). The PxCHS1 cDNA was 5461 bp in length with an open reading frame of 4701 bp that encoded a putative protein of 1567 amino acids with predicted molecular mass of 179 kDa. The two splice variants were expressed from the mutually exclusive exons which were same in size (177 bp) but showed only 66% identity at the nucleotide level. Both splice variants were expressed in all developmental stages. The qPCR data suggested an uneven expression of the two variants in the body where expression of PxCHS1A was 3.7-fold higher than that of PxCHS1B. The expression of PxCHS1A was 1.5-fold higher in the head than the body whereas in case of PxCHS1B the difference between head and body was 6.3-fold. Chlorfluazuron did not change the expression of PxCHS1 in larvae.     

The effects of Artemisia annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera: Pieridae)

The biological effects of two important medicinal plants, Artemisia annua L. and Achillea millefolium (L.) (viz, mortality, growth, and feeding indices as well as enzyme and non-enzymatic activities) were studied on small white Pieris rapae L a deleterious pest of cruciferous plants under controlled conditions (16:8 h L:D at 25 ± 1 °C and 65 ± 5% RH). The LC₅₀ and LC₂₅ values were 9.387% and 3.645% for A. annua L. and 4.19% and 1.69% for A. millefolium (L.), respectively. At the lowest concentration (0.625%), the deterrency was 29.826% and 44.185% for A. annua L. and A. millefolium (L.), respectively. Feeding indices were variously affected with changes in a number of parameters and an increase in larval and pupal duration. The activity level of alkaline phosphatase increased sharply while alanin and aspartate aminotransferases showed a sharp decrease. For non-enzymatic compounds, the amount of glucose and uric acid increased, but total protein and cholesterol decreased. These results indicate that these two medicinal plants might possess potential secondary metabolites that may be useful for controlling potential insect pests.     

寄生Q型烟粉虱的丽蚜小蜂生长发育特性

为了阐明丽蚜小蜂Encarsia formosa对Q型烟粉虱的寄生特性及寄主植物携带病毒对其寄生的影响,采用微虫笼饲养法研究了寄生Q型烟粉虱的丽蚜小蜂生物学特性。结果显示,丽蚜小蜂寄生Q型烟粉虱3龄若虫时,其生长发育显著优于寄生Q型烟粉虱2龄若虫。丽蚜小蜂在感染黄化曲叶病毒（TYLCV）番茄和健康番茄上寄生Q型烟粉虱（分别为VW和NVW）3龄若虫时,其生长发育特性发生了明显变化。丽蚜小蜂以VW若虫为寄主时的寿命为10.25 d,显著长于寄生NVW的丽蚜小蜂寿命（5.90 d）;对VW和NVW后代3龄若虫的寄生率分别为23.81%和17.62%,致死率分别为38.95%和26.52%,差异均显著;而丽蚜小蜂的羽化率和发育历期不存在显著差异。说明丽蚜小蜂寄生Q型烟粉虱3龄若虫时更适宜其生长发育,寄主植物番茄携带TYLCV显著提高丽蚜小蜂对烟粉虱的寄生效果。     

木荷皂甙对稻瘟病菌细胞形态及生理生化指标的影响

为开发新型木荷皂甙杀稻瘟剂,分别采用菌丝生长速率法、显微观察、电导法和比色法研究木荷活性皂甙对稻瘟病菌Magnaporthe oryzae (Hebert) Barr.的活性,以及对病原细胞形态及其胞外生理生化成分含量的影响。结果显示:木荷皂甙M2IGR5抗稻瘟病菌的EC₅₀为16.41 μg/mL、EC₉₀为76.16 μg/mL;其EC₅₀和EC₉₀处理稻瘟病菌菌丝体24 h后,可导致菌丝断裂、细胞肿胀;处理去壁的原生质体2 h即可导致其变型、粘连、破裂,引起病原菌菌丝细胞内含物大量渗漏;处理2 h后的可溶性蛋白渗漏量和可溶性总糖分别较对照高10倍和30~60倍,且处理液的电导率升高、几丁质酶活性增强、胞壁几丁质组成成分N-乙酰葡萄糖胺脱落。研究表明,木荷皂甙抗稻瘟病菌不仅导致膜通透性增大、完整性丧失,而且还间接引起细胞壁降解,加速了稻瘟病菌细胞的凋亡。     

四种熏蒸剂对辣椒疫霉和南方根结线虫的毒力

为明确不同熏蒸剂对土传病原菌辣椒疫霉Phytophthora capsici和南方根结线虫Meloidogyne incognita的毒力,采用密闭熏蒸法测定了4种土壤熏蒸剂氯化苦、棉隆、威百亩、1,3-二氯丙烯的毒力及1,3-二氯丙烯和氯化苦复配的联合毒力。结果表明:氯化苦、棉隆、威百亩、1,3-二氯丙烯对辣椒疫霉的EC₅₀分别为0.12、2.44、8.30、8.45 mg/L; 对南方根结线虫的EC₅₀ 分别为1.23、0.22、0.30、0.18 mg/L。1,3-二氯丙烯和氯化苦以2:1、1:1、1:2比例复配时对辣椒疫霉的EC₅₀ 分别为1.13、0.24、0.14 mg/L;对南方根结线虫的EC₅₀分别为0.19、0.32、0.61 mg/L。结合经济效益和多种病害兼治的原则,推荐使用1,3-二氯丙烯与氯化苦1:1配比,可兼治辣椒疫病和根结线虫病。     

Flufenoxuron, an acylurea insect growth regulator, alters development of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) by modulating levels of chitin, soluble protein content, and HSP70 and p34 in the larval tissues

The effect of sublethal concentrations, 0.00141% (LC₂₀), 0.00251% (LC₃₀), and 0.0036% (LC₄₀) of a dispersible formulation of an acylurea insect growth regulator, flufenoxuron (Cascade) on certain biochemical parameters in the larvae of Tribolium castaneum was investigated. When neonates were fed on diet treated with sublethal concentrations for 24 h, it was observed that at all concentrations tested, there was a significant reduction in chitin content on the 15th day of development. Total soluble protein content at LC₂₀ and LC₃₀ decreased with increasing age of the larvae. At LC₂₀ and LC₄₀ concentrations there was a progressive increase in the protein: chitin ratio as a function of increase in age of the larvae. SDS-PAGE analysis of the larval tissue extracts indicated gross quantitative changes in some of the protein bands (MW 50-97 kDa). Western blot analysis revealed significant increase in the level of HSP70 in the extracts of larvae fed on LC₃₀ treated diet, on the 7th and 10th day of development in the decreasing order. Conversely, a significant decrease in the hyper-phosphorylated form of p34^cdc2 kinase due to flufenoxuron treatment indicating modulation of cell cycle regulation was observed. Thus, sublethal concentrations of flufenoxuron alter expression of developmentally regulated proteins, HSP70 and p34^cdc2 and chitin formation in a stage-specific manner thereby resulting developmental abnormalities in T. castaneum.     

小菜蛾感染玫烟色棒束孢后的病征及组织病理变化

为了明确玫烟色棒束孢对小菜蛾的致病作用,分别以1×10⁵孢子/mL和1×10⁸孢子/mL浓度的玫烟色棒束孢菌株EBCL03011孢子悬浮液感染小菜蛾幼虫,观察了感病幼虫体表和体内器官组织的病变情况。结果显示,感病虫体颜色由局部浅褐色逐渐变为大面积深褐色,病虫出现缩短、强直等外部形态的变化。组织切片观察显示,接种后4 h,附着于体壁的分生孢子开始萌发入侵;16 h后,小菜蛾内表皮被分解,菌丝段进入血腔。与此同时,被幼虫取食进入消化道的分生孢子也增殖产生菌丝段,以菌丝段突破肠壁细胞,向附近的脂肪体入侵;随着玫烟色棒束孢继续在虫体内增殖,24 h后各组织器官遭到不同程度破坏;48 h后,感病幼虫死亡,虫尸体内的菌丝突破体表,在虫体外形成菌丝层。表明玫烟色棒束孢入侵小菜蛾有两条途径,第1条为体表途径,第2条为消化道途径。     

哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达

采用室内筛选与田间防效相结合的方法,对哈茨木霉抑制黄瓜尖孢镰刀菌的拮抗机制进行了研究。对峙培养结果显示,木霉菌和病原菌间均形成了较明显的抑菌圈,对病原菌的抑菌率达66.7%~85.8%,其中菌株TG、TM对枯萎病菌抑制作用较强。木霉菌可有效提高根系对病原菌的抗性,黄瓜植株接种枯萎病菌后,根系细胞大量死亡,而先接种木霉菌再接种病原菌后则减小了对根系的伤害。田间防效试验结果表明,TG和TM孢子悬浮液浓度在108个/mL时防效最好,分别为54.9%和49.4%。接种木霉菌植株根系抗性基因的表达量均高于对照植株,呈双峰趋势。在第6天时抗性基因表达量最高,WRKY6、MYB、PR-1、PAL、GST和GLU的表达量分别为对照的5.15、5.22、6.07、6.00、3.16、和16.15倍。表明木霉菌通过激活与胁迫相关的基因表达提高了对病原菌的抗性。     

The effect of IKI-7899, a new chitin synthesis inhibitor,on larvae ofTribolium castaneum andSpodoptera littoralis

The effect of IKI-7899 and diflubenzuron on larval development and emergence ofTribolium castaneum and ofSpodoptera littoralis was determined. The LC₅₀ of IKI-7899 at adult emergence ofT. castaneum larvae fed the compound in their diet and the ED₅₀ of S.littoralis larvae treated topically were approximately one-sixth those of diflubenzuron.     

加州新小绥螨对土耳其斯坦叶螨的捕食作用

为明确加州新小绥螨Neoseiulus californicus （McGregor）对土耳其斯坦叶螨Tetranychus turkestani （Ugarov et Nikolski）的捕食潜力,采用捕食功能反应方法,研究了加州新小绥螨对土耳其斯坦叶螨各螨态的捕食作用。加州新小绥螨对雌成螨、若螨、卵的选择性捕食系数分别为0.44、1.25和1.35。在不同温度条件下,加州新小绥螨对土耳其斯坦叶螨的功能反应均属于Holling-Ⅱ型;28 ℃时捕食能力最强,对土耳其斯坦叶螨雌成螨、若螨和卵的攻击系数分别是0.6279、0.7203和0.7554,最大日捕食量分别为10.81头、28.49头和 40.82粒。在相同温度下,加州新小绥螨雌成螨寻找效应随着猎物密度的增加而降低;在相同密度下,寻找效应随温度的升高先增加后减小,在28 ℃时寻找效应最高,为0.535。加州新小绥螨的捕食作用存在较强的种内干扰作用（m=0.520）,随着捕食者密度的增大,平均捕食量逐渐减少,捕食作用率也相应降低。研究表明加州新小绥螨对土耳其斯坦叶螨具有很好的控制潜力。     

三种杀线虫剂对甘薯茎线虫扩散的Hormesis效应

在实验室条件下应用沙柱法及水琼脂平板法,测定了低剂量水平的涕灭威、甲氨基阿维菌素苯甲酸盐和丙溴磷对甘薯茎线虫Ditylenchus destructor扩散行为的影响。结果表明:在含5 μ g/mL 药剂的沙柱中,以及甘薯茎线虫经0.1 μ g/mL的药剂浸渍24 h后在不含药剂的沙柱中,其运动扩散能力均受到抑制;在含较低质量浓度(5×10－3 μ g/mL)药剂的沙柱中,以及线虫经较低质量浓度(10－2,10－3 μ g/mL)的涕灭威和(10－4,10－5 μ g/mL)甲氨基阿维菌素苯甲酸盐浸渍24 h后在不含药剂的沙柱中,其通过率均比空白对照高10%以上,差异显著,表现出Hormesis效应(兴奋效应);经较低质量浓度(10-1 μ g/mL)的丙溴磷浸渍后在不含药剂的沙柱中未表现出该效应。在水琼脂平板试验中,虽然在低剂量水平下药剂对线虫的扩散具有促进作用,但产生的Hormesis效应不如沙柱法的结果明显。     

甘蔗宿根矮化病菌巢式PCR检测

为了探索甘蔗宿根矮化病快速检测技术,以细菌16S-23S rDNA内转录间隔区(ITS)通用引物ITS1/ITS2为第1轮引物,以甘蔗宿根矮化病菌特异引物Lxx1/Lxx2为第2轮引物,建立甘蔗宿根矮化病菌巢式PCR检测技术。巢式PCR能扩增出438 bp的目的条带,其核苷酸序列与巴西、澳大利亚及美国甘蔗宿根矮化病菌分离物核苷酸序列同源率为100%或99.8%;其检测灵敏度为10fg甘蔗宿根矮化病菌基因组DNA,较常规PCR提高100倍;样品检测结果也表明巢式PCR检测灵敏度明显优于常规PCR,可用于+1片嫩叶和心叶等微量病菌样品的检测。     

渐狭蜡蚧菌Lecanicillium attenuatum产几丁质酶的活性及对南方根结线虫卵孵化的抑制作用

为探究卵寄生真菌产生的几丁质酶对南方根结线虫卵孵化的抑制作用,采用分离自黑龙江大豆孢囊线虫孢囊上的菌株CGMCC5328,利用NAG和pNP法测定其几丁质降解酶系和外切酶的活性,分析几丁质酶粗酶液对南方根结线虫卵的孵化抑制效果.结果表明,菌株CGMCC5328经形态学和ITS序列分析鉴定为渐狭蜡蚧菌Lecanicillium attenuatum;在几丁质酶诱导培养基中培养第4天时其分泌的几丁质降解酶系达酶活高峰,为16.90 U/mL;第6天外切酶达酶活高峰,为0.59 U/mL,之后酶活趋于稳定持续至第14天;几丁质酶分子量分别为79.6、65.6、54.1、42.1和32.9 kDa;其中培养第7天的几丁质酶粗酶原液对南方根结线虫卵孵化的抑制率为83.17%;第6天菌株CGMCC5328对卵的寄生率为85.10%.表明高效产几丁质酶的卵寄生真菌渐狭蜡蚧菌菌株CGMCC5328对南方根结线虫卵孵化具有明显的抑制效果.     

紫花苜蓿伴生菌对菌核病菌的抑制作用

为了从紫花苜蓿伴生菌中筛选生物防治菌种,采用对峙法和滤液培养法对紫花苜蓿伴生菌与菌核病菌的拮抗关系进行了研究,并采用茎秆接种法探讨了紫花苜蓿伴生菌对菌核病菌的控制作用。从紫花苜蓿植株中分离获得的22种真菌中,射丝孢、无孢菌丝体和丝葚霉对菌核病菌具有明显的拮抗作用;接种第5天,在含射丝孢、无孢菌丝体和丝葚霉3种真菌菌液的培养基上,三叶草核盘菌的菌落直径分别为4.8、26.8和28.9 mm,与对照相比,分别降低了75.2、53.2和51.1 mm。虽然射丝孢和无孢菌丝体都不能阻止菌核病菌侵入寄主组织,但对菌核病菌入侵后病斑的扩展表现出不同程度的抑制作用,其效果因品种而异,2种菌种对品种FGC901和Algonguin病斑扩展均表现出较好的抑制作用。     

Effect of Melia azedarach extract on the activity of NADPH-cytochrome c reductase and cholinesterase in insects

The effect of Melia azedarach extract on the activity of NADPH-cytochrome c reductase and cholinesterase was studied in Spodoptera frugiperda. Larvae were fed an artificial diet containing fruit extract and their midgut was used for enzyme determination. As compared to the control, consumption of the extract containing diet resulted in a 31% inhibition of the cholinesterase activity and a 34% activation of the NADPH-cytochrome c reductase activity. In the cockroach Leucophaea maderae, the effect on reductase activity was even more pronounced (43%).     

水稻条纹病毒胁迫对灰飞虱mucin基因表达量的影响

为分析灰飞虱mucin基因在水稻条纹病毒(Rice stripe virus, RSV)侵染过程中的作用,采用荧光定量PCR的方法分析了RSV 胁迫条件下灰飞虱mucin基因mRNA表达量的变化。饲喂RSV病叶1天和2天时灰飞虱mucin基因的表达量没有明显变化,而在3天和7天时灰飞虱mucin基因的表达量增加了3.60倍和1.97倍。饲喂健康水稻叶片1、2、3和7天后,灰飞虱mucin基因的表达量分别为饲喂前的1.07、1.12、0.78和0.34倍,而饲喂病叶1、2、3和7天后,mucin基因的表达量分别为饲喂前的1.15、1.19、3.19和1.01倍。结果表明,病毒胁迫使灰飞虱体内mucin基因的表达量增加,暗示mucin基因在RSV与灰飞虱互作过程中起着重要作用。