褐飞虱SRAP-PCR体系的优化与确立

采用L16（4 5）正交试验设计,对褐飞虱SRAP PCR反应体系中的Mg2+、dNTPs、TaqDNA聚合酶、引物浓度和DNA模板浓度5个因素进行优化,确立了褐飞虱SRAP PCR最佳的反应体系。结果表明：褐飞虱SRAP-PCR最佳反应体系为：总体积10 μL,Mg2+浓度3 mmol/L、dNTPs为150 μmol/L、TaqDNA聚合酶2 U、正反向引物各0.3 μmol/L、DNA用量25 ng以及1×PCR Buffer。使用生物型1雌虫与生物型2雄虫单对杂交F2群体对优化后的褐飞虱SRAP-PCR反应体系进行验证,获得了条带清晰、多态性丰富的图谱,并发现共显性条带,表明确定的反应体系稳定可靠。     

牛筋草ISSR-PCR反应体系的建立与优化

以牛筋草地上组织为材料,采用正交优化和单因子试验两种方法对影响牛筋草ISSR-PCR体系的Mg2+浓度、dNTP浓度、Taq DNA聚合酶浓度、引物浓度和DNA模板浓度5个因素进行优化试验,建立适合牛筋草ISSR-PCR的反应体系.结果表明,牛筋草ISSR-PCR反应体系的最佳条件为:Mg2+1.75 mmol/L,dNTP 0.4 mmol/L,Taq DNA聚合酶1 U/25 μL,引物0.3μmol/L,模板DNA 80 ng/25μL,10×PCR Buffer 2.5 μL/25 μL.利用优化体系进行牛筋草的ISSR-PCR反应,可获得稳定性高、重复性好、背景清晰的电泳结果.     

小菜蛾ISSR-PCR反应体系的建立与正交设计优化

以小菜蛾(Plutella xylostella)基因组DNA为模板,采用L16(45)正交试验设计方法,建立小菜蛾的ISSR最佳反应体系。通过梯度退火试验,确定不同引物的最适退火温度。优化得到的反应体系(20μL)为:Mg2+浓度1.5mmol/L、Taq DNA聚合酶2.5U、dNTPs浓度0.2mmol/L、引物浓度1.25μmol/L、模板DNA量20ng。反应程序为:94℃预变性5.0min;94℃变性45.0s,40～61℃(不同引物退火温度各异)退火1.0min,72℃延伸1.5min,40个循环;72℃延伸10.0min;10℃保存。利用所建立的ISSR-PCR反应体系,获得了清晰、重复性好的DNA谱带。     

白背飞虱ISSR-PCR反应体系构建的正交设计优化

白背飞虱是水稻重要害虫之一。研究采用L16(45)正交设计优化试验,构建了白背飞虱ISSR最优反应体系,并对引物退火温度和反应循环次数进行了优化。结果表明：白背飞虱ISSR PCR最优反应体系为2 μL 10×PCR Buffer（10 mmol/L Tris HCl;50 mmol/L KCl）,1.5 mmol/L Mg2+,0.25 mmol/L dNTPs,0.9 μmol/L引物,1U TaqPolymerase和50 ng 模板DNA,ddH2O补充至20 μL。本研究采用的10条引物的最佳退火温度在51.0~54.0 ℃间,以引物相应最优退火温度和40次反应循环可获得较好的白背飞虱ISSR-PCR结果。     

白背飞虱RAPD-PCR反应体系的正交设计优化

白背飞虱Sogatella furcifera(Horváth)是水稻重要害虫之一。研究采用L16(45)正交设计优化试验,构建了白背飞虱RAPD最优反应体系,并对引物退火温度和反应循环次数进行了优化。结果表明:白背飞虱RAPDPCR最优反应体系为2μL 10×PCR Buffer(10 mmol/L Tris-HCl;50 mmol/L KCl),2.4 mmol/L Mg2+,0.15 mmol/L dNTPs,0.7μmol/L引物,0.8U Taq DNA Polymerase和30 ng模板DNA,ddH2O补充至20μL。本研究采用的10条引物的最佳退火温度在35-39℃间,以引物相应最优退火温度和40次PCR反应循环可获得较好的白背飞虱RAPD-PCR结果。     

正交设计优化南方根结线虫RAPD-PCR体系

以南方根结线虫(Meloidogyne incognita)DNA为模板,对影响南方根结线虫RAPD-PCR扩增的重要参数进行了优化试验,以期建立南方根结线虫RAPD-PCR反应最佳体系。应用L16(45)正交设计研究了Taq酶、10×PCR buffer、dNTP、primer、模板DNA对扩增反应的影响。结果表明,南方根结线虫RAPD-PCR优化体系为20μL体系中含模板3μL、10μmol/L引物1.5μL、10×反应缓冲液2.5μL、2.5 mmol dNTP mixture 2.4μL、Taq DNA聚合酶0.4μL。在此基础上筛选出扩增稳定、多态性丰富的RAPD引物,并通过梯度PCR试验,确定了引物最佳退火温度。该优化RAPD-PCR反应体系具有良好的稳定性和重现性,可应用于南方根结线虫不同居群间亲缘关系和遗传多样性的分析。     

马铃薯早疫病病菌ISSR-PCR反应体系优化

为了应用ISSR分子标记技术分析马铃薯早疫病病菌遗传多样性,采用单因素水平对影响ISSR反应的Mg~(2+),d NTPs、Taq DNA聚合酶、引物浓度及引物退火温度等条件进行了优化,建立了适宜于早疫病病菌的ISSR最佳反应体系。25μL的反应体系中,Mg~(2+)2.0 mmo1/L,d NTPs 0.15 mmol/L,Taq DNA聚合酶0.75U,引物0.40μmol/L;从30条ISSR引物中筛选出10条多态性较好的ISSR引物,并确定了各条引物的退火温度。结果为应用ISSR分子标记技术研究马铃薯早疫病病菌遗传多样性奠定了基础。     

腐食酪螨ISSR最佳反应体系的设计

本文以腐食酪螨基因组DNA为模板,采用逐个参数优化法,探讨腐食酪螨ISSR实验的最佳反应体系。实验结果表明,腐食酪螨ISSR-PCR最佳反应体系:总体积25μL,其中Mg2+1.5 mM;10×Buffer 2.5 mM;dNTPs 0.2 mM;ISSR引物0.2μM;模板DNA 50 ng;Taq DNA聚合酶0.75 U。扩增程序为:94℃预变性5min,94℃变性1 min,(48℃、50℃、52℃)复性1 min,72℃延伸1.5 min,循环35次,结束后72℃延伸5 min,4℃保存。同时通过梯度退火实验,确定不同引物的最佳退火温度。     

美国白蛾基因组DNA提取及RAPD反应条件优化

用改进的SDS法从无水乙醇浸泡美国白蛾幼虫标本中提取到高分子量的可用于RAPD扩增的基因组DNA。用正交试验对影响RAPD扩增的条件进行优化得到最佳反应体系：20 μL反应体系中,模板浓度1.25 ng/μL、引物浓度1.2 ng/μL、dNTP浓度 0.2 mmol/L、Mg2+浓度 3.0 mmol/L、TaqDNA聚合酶用量2.5 U。确定最佳退火温度为38 ℃。PCR反应条件：94 ℃ 30 s、38 ℃ 60 s、72 ℃ 60 s 35个循环后,72 ℃延伸5 min。为在分子水平上讨论美国白蛾种群的遗传分化提供基础。     

生防棘孢木霉T31菌株的分离筛选及其生物学特性

为评价具有生防潜力的棘孢木霉,采用平板对峙法、对扣培养法、圆盘滤膜法从长白山土样中分离筛选出木霉菌株T31,结合形态学及分子生物学分析对其进行种类鉴定,并采用菌丝生长法研究了该菌株的生物学特性。所分离的菌株T31被鉴定为棘孢木霉Trichoderma asperellum,该菌株对8种靶标菌的抑菌率在44.49%~77.91%之间,其中,对玉米大斑病菌Exserohilum turcicum、核盘菌Sclerotinia sclerotiorum、腐霉菌Pythiumuhtimum sp.及镰刀菌Fusarium sp.均有较强生长竞争优势,对玉米灰斑病菌Magnaporthe oryzae具明显重寄生作用。菌株T31的最适培养基为PDA培养基,最适生长温度为30 ℃、最适产孢温度为25 ℃,最适pH值为6。持续光照可以促进木霉菌孢子产生,Zn²⁺、Mn²⁺、Ca²⁺和Mg²⁺利于菌丝生长,但是高浓度的Fe²⁺和Cu²⁺抑制菌丝生长,Mn²⁺和Mg²⁺可促进产孢。此外,与普通木霉菌相比,该菌株具有耐寒性及耐盐性。     

枯草芽胞杆菌对离体荔枝果实霜疫霉病、炭疽病的防治效果

为了进一步明确从土壤中筛选得到的拮抗细菌OR-1、OR-2、OR-3、ON-6的分类地位和生防效果,采用形态学观察、理化特性结合分子生物学方法,鉴定这4株细菌皆为枯草芽胞杆菌Bacillussubitilis.拮抗菌对荔枝霜疫霉菌、炭疽菌菌丝生长均具有明显抑制作用,ON-6菌株的发酵液对菌丝生长的抑制率最高,分别为92.34％和70.36％,其次是OR-1菌株.拮抗菌处理组褐变指数均小于对照组以及杀菌剂处理组,且差异达显著水平(P＜0.05),OR-1菌株防褐变效果最好.常温下,拮抗菌发酵液对离体鲜果病害防治效果优于对照组和杀菌剂处理组,其中ON-6菌株的防治效果最佳;4℃低温储藏处理40天后,拮抗菌发酵液处理组的防治效果高于对照和杀菌剂处理组,且差异显著(P＜0.05),以ON-6和ON-3菌株的防治效果最佳.表明拮抗菌发酵液对荔枝鲜果上的霜疫霉菌和炭疽病菌有一定的抑制作用.     

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Acacia farnesiana lectin-like protein (AFAL) showed bacterioestatic effects against Xanthomonas axonopodis pv. passiflorae (Gram-negative) and Clavibacter michiganensis michiganensis (Gram-positive), with the latter being more sensitive. This effect is probably due to the ability of AFAL to interact with the bacterial cell wall where we observed that AFAL induced macroscopic change. The maximum bacterial growth inhibition was approximately 78% when incubated with Gram-negative strains, and as high as 92% percent for the Gram-positive one. The antibacterial effect of flavonoids (rutin, quercetin and morin) was also observed using low concentrations against both bacterial strains. Prior incubation of both with AFAL at high concentrations increases the inhibitory effect of flavonoids on bacterial growth. The potential use of AFAL as a control agent against the root-knot nematode Meloidogyne incognita was investigated as well, showing anti-nematode properties involving both egg hatching and motility. In the juvenile second-stage, AFAL showed reduction in larval mobility when measured against a control group. The results suggest that AFAL is effective against M. incognita and could be used as a component of integrated pest management programs. These data also suggest that lectins probably play a role in plant defense not only against invertebrate phytopathogens, herbivores and fungi but also against bacteria.     

苏云金杆菌亚致死浓度对美国白蛾及其寄生蜂生长发育的影响

为阐明苏云金杆菌Bacillus thuringiensis(Bt)与白蛾周氏啮小蜂Chouioia cunea Yang的协同作用,分别用103~108孢子/m L的6个Bt浓度处理美国白蛾Hyphantria cunea 2龄幼虫后,通过室内试验测定了Bt对其致死、亚致死效应,及亚致死浓度Bt对寄生蜂子代出蜂的影响。结果表明,浓度106孢子/m L处理组美国白蛾均未能化蛹;而106孢子/m L处理组均能化蛹并羽化,且幼虫发育历期随Bt浓度的增加而延长,平均蛹重在74.92~92.18 mg之间,均高于对照的68.66 mg,但蛹重不呈线性规律变化。当Bt对美国白蛾平均校正死亡率低于17.17%时,幼虫发育历期延迟不明显,但平均蛹重为101.11~124.34 mg,均高于对照;接蜂后能够正常出蜂,出蜂率为33.13%~51.25%,每蛹出蜂量为117.43~143.42头,均显著高于对照。表明用平均校正死亡率低于20%的Bt处理对白蛾周氏啮小蜂的寄生有利,2种生防措施协同作用存在增效潜力。     

Analysis of two acetylcholinesterase genes in Bombyx mori

Two acetylcholinesterases (AChE, EC 3.1.1.7) cDNAs were identified and cloned from silkworm, Bombyx mori. One of those, BmAChE-o cDNA, is comprised of 3197 nucleotides which encode 638 amino acids, having an amino acid sequence homology of 72% with Drosophila melanogaster Ace-orthologous AChE (AO-AChE). In some species, another AChE group based on the sequence, Drosophila Ace-paralogous AChE (AP-AChE) has been recognized in relation to organophosphate- or carbamate-resistance, but there have been few reports of AP-AChE among lepidopteran species. However, we isolated the AP-AChE from lepidopteran silkworm, and cloned full ORF as BmAChE-p, which cDNA consisted of 2465 nucleotides that encode 683 amino acids. The homologies with other AP-AChEs were over 60% when compared. Although silkworm is not a target of pesticides, the genomic information obtained in this study will contribute to insecticide-resistance study on lepidopteran pest species.     

溴氰虫酰胺和氯虫苯甲酰胺对三种鳞翅目害虫的毒力作用比较

为比较溴氰虫酰胺和氯虫苯甲酰胺对甜菜夜蛾、玉米螟和小地老虎的作用差异,采用浸卵法、浸叶法、饲喂毒营养液法等测定了溴氰虫酰胺和氯虫苯甲酰胺对3种害虫的卵、3龄幼虫和成虫的毒力,以及对其3龄幼虫的拒食活性。结果表明,溴氰虫酰胺和氯虫苯甲酰胺均无杀卵作用,但能降低初孵幼虫存活率。溴氰虫酰胺对甜菜夜蛾、玉米螟和小地老虎3龄幼虫的LC₅₀值分别为0.11、0.05和0.13 mg/L,相对毒力分别是氯虫苯甲酰胺的2.8、2.7和5.1倍。溴氰虫酰胺对3种害虫成虫的LC₅₀值分别为0.11、0.09和0.22 mg/L,相对毒力分别是氯虫苯甲酰胺的3.9、3.8和2.7倍。溴氰虫酰胺在亚致死浓度0.10、0.05和0.14 mg/L的剂量下分别对甜菜夜蛾、玉米螟和小地老虎3龄幼虫表现出较强的拒食作用,处理48 h后达到最高,拒食率分别为85.58%、81.91%和86.11%。药剂处理试虫虫体皱缩,体节缩短,体重增加率明显低于对照处理。表明溴氰虫酰胺对3种鳞翅目害虫的毒力作用比氯虫苯甲酰胺好,可作为氯虫苯甲酰胺的替代药品。     

Biological and biochemical responses of Biomphalaria alexandrina to some extracts of the plants Solanum siniacum and Artemisia judaica L.

The molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of Solanum siniacum and Artemisia judaica L. plants against Biomphalaria alexandrina snails was carried out. The tests revealed plant’s ethanol extract was more toxic to the snails than the other tested extracts. Therefore, it was tested against snails’ fecundity (M_x), reproduction rate (R₀) and their infection with Schistosoma mansoni miracidia. In addition, biochemical parameters and the activities of some enzymes in tissues of snails treated with the two tested plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of B. alexandrina snails to plant’s ethanol extract led to a significant reduction in their survival and snails’ fecundity, reproduction rate. In addition, it caused a considerable reduction in the infectivity of S. mansoni miracidia to the snails. Also, it caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results revealed that the glucose concentration in hemolymph and Lactate level in soft tissues of treated snails were increased (P < 0.001) while glycogen, total protein, the lipid content and the pyruvate level in snail’s tissues decreased (P < 0.001). The activities of lactic dehydrogenase (LDH), pyruvatekinase (PK) and cytochrome oxidase (CY) enzymes in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with the two tested plants while protease (PR) activity increased (P < 0.001). It is concluded that the application of LC₂₅ of ethanol extract of S. siniacum and A. judaica L. may be helpful in snail control as it interferes with the snails’ biology and physiology.     

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.     

两株棒束孢菌的鉴定及其对茶卷叶蛾和茶小卷叶蛾的致病力

为明确分离自茶卷叶蛾和中华大刀螳僵虫的2株虫生真菌的归属及生防潜力,采用形态学特征和rDNA-ITS序列分析其分类地位,测定了其对茶卷叶蛾和茶小卷叶蛾幼虫的致病力,并初步筛选了其最适培养条件.结果表明:2株真菌均为环链棒束孢Isaria cateniannulata,分别命名为ICBS918和ICTL911.在1.0×10⁸孢子/mL浓度下,ICBS918和ICTL911对茶卷叶蛾幼虫的累计校正死亡率均达100%,LT₅₀分别为3.13 d和3.15 d;对茶小卷叶蛾幼虫的累计校正死亡率分别为100%和95%,LT₅₀分别为3.25 d和3.31 d.接菌后8 d,2菌株对茶卷叶蛾幼虫的LC₅₀分别为0.47×10⁵孢子/mL和1.01×10⁵孢子/mL;对茶小卷叶蛾幼虫的LC₅₀分别为2.20×10⁵孢子/mL和1.34×10⁵孢子/mL.2菌株23 ℃下最适生长培养基均为综合马铃薯培养基,最适产孢培养基均为蛋白胨马铃薯葡萄糖琼脂培养基;在萨氏培养基上,菌株ICBS918和ICTL911分别在24~30 ℃及24 ℃时生长速度最快,在27 ℃和21 ℃时产孢量最大.表明2株环链棒束孢对茶卷叶蛾和茶小卷叶蛾幼虫均具有较好的生防潜力,可作为生防菌进行开发和应用.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Assessment of Cyfluthrin commercial formulation on growth, photosynthesis and catalase activity of green algae

Aquatic environments of the pampasic region of Argentina are severely affected by agricultural contamination due to an increase in a glyphosate tolerant transgenic variety of soybean crops. The present study is aimed to determine the effects of a commonly used Cyfluthrin commercial formulation (CCF) on growth, some physiological and biochemical parameter of four species of green algae. Significant inhibition of algal growth was observed from 0.1 mg Cyf/l. 96 h IC₅₀ were between 0.92 and 4.85 mg Cyf/l. CCF caused algicidal effects. Photosynthesis was stimulated by 50% in Scenedesmus quadricauda cultures exposed to the lowest concentration (hormesis). Algal photosynthesis inhibition was observed at higher concentrations with IC₅₀ values between 1.7 and 8.9 mg Cyf/l. Similar toxicity endpoints were found as a consequence of applying the traditional methodology of short-term chronic toxicity test of 96 h of exposition and the methodology developed using the Clark type photosynthetic oxygen evolution method. CAT activity was significantly increased between 23% and 33% considering the four species, at a lower concentration than those affecting algal growth and photosynthesis, indicating a potential biomarker. Taking into account that the extent of the soybean crops in the region is about fourteen million hectares, the improvement and extension of environmental tools for early detection of the action of pesticides on this essential group of organisms are discussed.