The chitinase C gene PsChiC from Pseudomonas sp. and its synergistic effects on larvicidal activity

Pseudomonas sp. strain TXG6-1, a chitinolytic gram-negative bacterium, was isolated from a vegetable field in Taixing city, Jiangsu Province, China. In this study, a Pseudomonas chitinase C gene (PsChiC) was isolated from the chromosomal DNA of this bacterium using a pair of specific primers. The PsChiC gene consisted of an open reading frame of 1443 nucleotides and encoded 480 amino acid residues with a calculated molecular mass of 51.66 kDa. The deduced PsChiC amino acid sequence lacked a signal sequence and consisted of a glycoside hydrolase family 18 catalytic domain responsible for chitinase activity, a fibronectin type III-like domain (FLD) and a C-terminal chitin-binding domain (ChBD). The amino acid sequence of PsChiCshowed high sequence homology (> 95%) with chitinase C from Serratia marcescens. SDS-PAGE showed that the molecular mass of chitinase PsChiC was 52 kDa. Chitinase assays revealed that the chitobiosidase and endochitinase activities of PsChiCwere 51.6- and 84.1-fold higher than those of pET30a, respectively. Although PsChiC showed little insecticidal activity towards Spodoptera litura larvae, an insecticidal assay indicated that PsChiC increased the insecticidal toxicity of SpltNPV by 1.78-fold at 192 h and hastened death. These results suggest that PsChiC from Pseudomonas sp. could be useful in improving the pathogenicity of baculoviruses.     

Insect-resistant biotech crops and their impacts on beneficial arthropods

With a projected population of 10 billion by 2050, an immediate priority for agriculture is to achieve increased crop yields in a sustainable and cost-effective way. The concept of using a transgenic approach was realized in the mid-1990s with the commercial introduction of genetically modified (GM) crops. By 2010, the global value of the seed alone was US $11.2 billion, with commercial biotech maize, soya bean grain and cotton valued at approximately US $150 billion. In recent years, it has become evident that insect-resistant crops expressing δ-endotoxin genes from Bacillus thuringiensis have made a significant beneficial impact on global agriculture, not least in terms of pest reduction and improved quality. However, because of the potential for pest populations to evolve resistance, and owing to lack of effective control of homopteran pests, alternative strategies are being developed. Some of these are based on Bacillus spp. or other insect pathogens, while others are based on the use of plant- and animal-derived genes. However, if such approaches are to play a useful role in crop protection, it is desirable that they do not have a negative impact on beneficial organisms at higher trophic levels thus affecting the functioning of the agro-ecosystem. This widely held concern over the ecological impacts of GM crops has led to the extensive examination of the potential effects of a range of transgene proteins on non-target and beneficial insects. The findings to date with respect to both commercial and experimental GM crops expressing anti-insect genes are discussed here, with particular emphasis on insect predators and parasitoids.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

斑蝥素对粘虫几种代谢酶及多酚氧化酶的影响

为进一步探讨斑蝥素的杀虫作用机理, 本研究采用叶碟饲喂法处理粘虫Mythimna separata（Walker）5龄幼虫, 测定了饲喂处理后6, 12, 24, 36和48 h试虫体内羧酸酯酶(CarE)、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(ALP)、谷胱甘肽 S-转移酶(GST)和细胞色素P450 Ο-脱甲基酶及多酚氧化酶(PPO)的活性。结果表明: 斑蝥素可显著激活羧酸酯酶, 处理后48 h酶比活力最大, 为同期对照的1.68倍; 酸性磷酸酯酶在处理后6和12 h活性变化不明显, 处理24 h后逐渐被激活, 且与对照差异显著(P＜0.05); 明显抑制碱性磷酸酯酶, 且随着处理时间的延长, 抑制作用增强; 对Ο-脱甲基酶表现为先抑制后激活的趋势; 谷胱甘肽S-转移酶表现出先激活后抑制的影响; 离体活体条件下均可显著抑制PPO活性。可见, 斑蝥素可明显影响昆虫的代谢酶系, 且其对碱性磷酸酶和多酚氧化酶的抑制作用可能与其毒杀作用有关。     

18种中草药提取物对褐飞虱和甜菜夜蛾的杀虫活性分析

为了筛选对农产品和环境安全、对害虫高效的植物源杀虫剂,分别采用喷雾法和饲毒法,测定了18种中草药的3种溶剂（水、乙醇、石油醚）提取物对褐飞虱Nilaparvata lugens Stål的触杀活性和对甜菜夜蛾Spodoptera exigua Hübner的胃毒作用。结果表明,各种提取物（0.8 g/mL）对甜菜夜蛾的胃毒作用不明显,校正死亡率最高为33.33%;但对褐飞虱的杀虫活性较高,其中百部、黄柏、榧子、生山栀、鹤虱、木香的乙醇或石油醚提取物,对褐飞虱3龄若虫的24 h校正死亡率均在80%以上,其石油乙醚提取物的LC50值依次为：0.0826,0.0455,0.0145,0.0047,0.0038,0.0033 g/mL。采用系统溶剂法分析发现,木香和鹤虱的主要杀虫活性成分均在氯仿萃取物中,表明其杀虫成分易溶于较弱极性溶剂中。进一步通过活性追踪法,对鹤虱提取物的柱层析各流分的活性检测表明,其杀虫活性成分的Rf值为0.5556~0.5926。该研究为进一步纯化新型植物源杀虫物质并确定其分子结构,从而开发其应用前景及发现新的杀虫活性先导化合物奠定了基础。     

隆中风景区蕨类植物分布及其杀虫杀菌种筛选

初步调查隆中风景区蕨类植物的分布,发现该区共有蕨类植物91种,隶属于27科49属,以水龙骨科最多,有18种,其次为鳞毛蕨科,有10种。通过筛选,初步确定14个植物种具有杀虫杀菌价值和开发利用潜力。     

Bt基因甜菜叶绿体转化载体构建及毒蛋白表达

利用植物叶绿体基因组在进化中高度保守的特点,根据烟草、菠菜、水稻叶绿体基因组全序列资料设计合成引物,PCR扩增并克隆了甜菜叶绿体两个重要功能基因rbcL和atpB(GenBank登录号分别为DQ067450和DQ067451),并以其作为定点整合外源基因的同源重组片段,构建了Bt基因CryIAc甜菜叶绿体定点转化载体pSKARBt,酶切鉴定表明:所构建载体符合预期设计。对克隆菌菌体总蛋白进行了生物杀虫试验,结果表明:Bt基因CryIAc能够在叶绿体特异性启动子及终止子的调控下表达,并对二龄末甘蓝夜蛾有很强的毒杀作用。该载体构建对培育甜菜高抗虫品种具有重要应用价值。叶绿体转化及后续工作正在进行中。     

Insecticidal effects of some biological agents on Agelastica alni (Coleoptera: Chrysomelidae)

The alder leaf beetle (Agelastica alni L., Coleoptera: Chrysomelidae) causes approximately 10% of total economic damage to hazelnut product per year in Turkey. A. alni larvae are susceptible to several pathogens indigenous to the area in which these insects occur in Turkey. In the present study, in order to find a more effective and safer biological control agent against this common pest, we evaluated the various biological agents’ insecticidal activity during the four hazelnut seasons from 2002 to 2005 on the larvae of the alder leaf beetle collected from the vicinity of Trabzon, Turkey. The tested agents are 25 insect-originating bacteria, 2 bacterial toxins and 1 viral preparation. The results showed that the highest insecticidal activity was obtained by bacterial isolates at 1.8 × 10⁹ bacteria/mL dose, within ten days on the larvae of A. alni. These are 90% for Bacillus thuringiensis biovar tenebrionis (4AA1), Bacillus sphaericus (Ar4, isolated from Anoplus roboris L., Col.: Curculionidae), and Bacillus thuringiensis (Mm2, isolated from Melolontha melolontha L., Col.: Scarabaeidae). Our results indicate that these isolates may be valuable as biological control agent.     

Egg exposure to pyriproxyfen in the tomato leaf miner Tuta absoluta: ovicidal activity or behavioural‐modulated hatching mortality?

Insecticide susceptibility varies with the insect life stage and although the egg stage is sometimes perceived as the most vulnerable, it is a difficult target for insecticide application and little studied as such. Egg susceptibility to insecticides is generally considered important for insect growth regulators and insect pests of reduced mobility like leaf miners because of their placement on the host plant part targeted by the insecticidal application. The egg exposure to the juvenile hormone mimic pyriproxyfen was studied here in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae), a key tomato insect pest, aiming to assess if mortality is achieved due to insecticide ovicidal activity or due to behavioural impairment of the larvae preventing egg hatching. Survival analysis of treated tomato leaves and/or eggs indicated the importance of egg exposure to pyriproxyfen leading to a significant decrease in survival with a nearly 50% reduction in survival time, which peaked at the first instar. Ovicidal activity of pyriproxyfen in the tomato leaf miner was negligible, but image recording and behaviour analysis indicated behavioural impairment of larval activity compromising hatching and leaf mining by early instar larvae leading to high mortality at this stage (ca 50%). The likely causes and management implications of such findings are discussed.