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.     

土传黄瓜立枯病高效拮抗菌的筛选鉴定及其生物效应

采用平板对峙法从黄瓜根际土壤中分离出的400余株细菌菌株中筛选出16株对立枯丝核菌Rhizoctonia solani具有拮抗效果的菌株,抑菌带直径在0.81～1.93 cm之间。并从中选出3株抑菌带直径在1.6cm以上的菌株N33、N35和N43,结合形态、生理生化特性及16S rDNA序列比对分析,鉴定N33菌株为假单胞菌属Pseudomonas sp.,N35和N43菌株为芽胞杆菌属Bacillus sp.。通过在黄瓜育苗基质中添加选育的高效拮抗菌株,观测其对黄瓜苗生长的促进作用以及对立枯病的防治作用,3株菌株均具有促进黄瓜苗期生长和防治苗期立枯病的作用,其中N43菌株促生及防病效果均最显著,地上部鲜重比对照处理增加62.16%,防治效果达62%。     

和田鞘氨醇杆菌AMCC 100218菌株的筛选、鉴定及杀线虫特性

为筛选对南方根结线虫具有致死效果的生防细菌,从山东省10个县市蔬菜主产区番茄根际土壤中分离可培养细菌,采用离体杀线虫试验测定分离菌株对南方根结线虫Meloidogyne incognita的致死活性,结合生理生化特征及分子生物学方法对高效杀线虫菌株进行分类鉴定,同时对其杀线虫特性进行表征,并通过盆栽试验进一步验证其生防潜力。结果显示,从山东省蔬菜主产区番茄根际土中分离到1株高效杀线虫菌株AMCC 100218,结合生理生化试验与16S rRNA序列分析,鉴定此菌株为和田鞘氨醇杆菌Sphingobacterium hotanense;该菌株对南方根结线虫2龄幼虫的致死效果可达88.87%,其杀线虫活性物质具有较好的热稳定性和储存稳定性,且耐碱不耐酸;盆栽试验结果表明,该菌能够显著减少土壤中的虫口密度,降低番茄发病率。表明和田鞘氨醇杆菌AMCC 100218菌株是1株具有防治根结线虫病潜力的生防细菌。     

黄瓜幼苗内生真菌诱导植株对南方根结线虫的抗性

在2007-2008年的一项温室测验中,10株黄瓜幼苗内生真菌表现出了对南方根结线虫较好的防效,为了阐明其作用方式,用裂根试验设计研究了10个菌株诱导黄瓜产生根结线虫抗性的能力.同一植株的根系被分为彼此隔离的挑战根系和应答根系,镰刀菌Fusarium spp.菌株Fu234和Fu654、毛壳菌Chaetomium sp.菌株Ch1001、叶点霉Phyllosticta sp.菌株Ph511在挑战根系中接种后,应答根系中的线虫入侵总数显著降低,较对照分别降低了42.4％、35.7％、38.4％、23.6％(第1次测定)和63.6％、45.2％、51.0％、37.0％(重复测定),这4个菌株诱导了黄瓜对根结线虫入侵的抗性.同时,Fu234、Fu654、Ch1001和拟青霉Paecilomyces sp.菌株Pa972接种后应答根系中的卵与雌虫的比值显著减少,相比对照其比值分别降低了24.3％、37.7％、48.3％、21.2％(第1次测定)和21.6％、39.8％、30.0％、46.2％(重复测定),这4个菌株诱导了黄瓜对根结线虫繁殖的抗性.     

杧果炭疽菌拮抗细菌的筛选鉴定及其防病效果

为寻找对杧果炭疽菌Colletotrichum gloeosporioides有效的生防菌株,从瓜叶白粉病病斑上分离得到104株细菌,通过平板对峙培养筛选出16株拮抗细菌,分别测定其在8种不同培养基中的抑菌活性,筛选出有较强抑菌作用的9A、26A和4N菌株进行室内、果园防病试验,并对具有较好防治作用的9A、26A菌株进行了鉴定。结果显示,拮抗菌9A、26A和4N分别在南瓜汁、红薯汁和马铃薯汁培养基上的抑菌活性最高,对杧果炭疽菌菌丝生长抑制率分别为88.83%、87.83%和85.87%;室内9A、26A和4N菌株菌液处理离体果实,24 h后接种炭疽菌分生孢子液,对杧果炭疽病的防治效果均达100%,与咪鲜胺对照组相同;在果园9A和26A的防病效果分别为98.4%和90.5%,显著高于对照组的防效78.8%,而4N的防效只有58.2%。根据形态、生理生化特征及16S rDNA 序列分析结果,将菌株9A和26A鉴定为枯草芽胞杆菌Bacillus subtilis。研究表明,菌株9A和26A具有较好的生防利用价值。     

防治杨树溃疡病内生菌的分离筛选及鉴定

为筛选到对杨树溃疡病有良好生防效果的拮抗菌,采用平板对峙法初筛、发酵液“三明治”法和改良琼脂扩散法复筛,以及离体组织试验对杨树不同部位组织的内生菌进行了分离筛选,并对目的菌株进行了防治效果测定;采用形态学观察和ITS序列及其系统发育树分析对其进行初步鉴定。结果表明从115株杨树内生菌中筛选出有拮抗效果的菌株24株,并复筛出拮抗效果良好的菌株5株,最终从杨树内生菌中筛选出1株杨树溃疡病拮抗真菌,编号为YGF9,其对杨树离体组织溃疡病防治效果可达76.47%,初步鉴定该菌株为黄绿木霉Trichoderma aureoviride。表明菌株YGF9可有效抑制杨树溃疡病病原菌的生长,是防治杨树溃疡病的潜在生防菌株。     

土壤中南方根结线虫的实时荧光PCR检测和定量

为了准确测定土壤中南方根结线虫Meloidogyne incognita的群体密度,根据序列特异性扩增区标记设计了南方根结线虫特异性引物和TaqMan探针,分别建立了卵和2龄幼虫的实时荧光PCR定量检测体系,并将该检测体系与显微镜计数法进行了比较.结果表明,引物Mi-F/R及探针Mi-probe对南方根结线虫具有高度特异性,建立的标准曲线循环阈值与卵和2龄幼虫数量的对数值之间具有良好的线性关系,相关系数分别为0.9771和0.9853,扩增效率均为90％,检测灵敏度为10-1个卵及10-3条2龄幼虫;对10个田间土壤样本的检测显示,实时荧光PCR定量检测与显微镜计数法测定的南方根结线虫卵和2龄幼虫数量呈显著正相关,且两种方法对2龄幼虫的测定结果无显著差异.     

野生大豆内生细菌多样性及其杀线虫活性分析

为探明河北昌黎野生大豆内生细菌的多样性,寻找新的具有杀线虫活性的微生物资源,采用平板稀释法从野生大豆根、茎、叶等组织中分离内生细菌,结合形态特征以及16S rDNA基因序列进行比对分析,并对大豆孢囊线虫Heterodera glycines进行杀线虫活性比较.从野生大豆分离获得内生细菌180株,分属于7大类群:变形菌门α亚群、变形菌门β亚群、变形菌门γ亚群、厚壁菌门、放线菌门、酸杆菌门和疣微菌门,其中变形菌门α亚群和厚壁菌门为优势类群,分别占分离细菌总株数的48.9％和18.9％,野生大豆组织内生细菌Shannon-Wiener多样性指数为2.11 ～2.91.内生细菌Bacillussubtilis WSR93和B.megaterium WSR22菌株能明显地抑制大豆孢囊线虫卵的孵化,相对抑制率分别为97.9％和98.2％,对2龄幼虫也有较强的毒杀作用,校正死亡率分别为88.9％和90.9％.研究表明,野生大豆组织中内生细菌具丰富的多样性,其胞外分泌物中存在天然杀线活性物质.     

拮抗性链霉菌对大丽轮枝菌微菌核形成与萌发的影响

为探索拮抗性链霉菌对棉花黄萎病病原大丽轮枝菌Verticillium dahliae Kleb.的抑菌机制,采用菌丝生长速率法、微菌核萌发法研究了6株拮抗链霉菌无菌发酵滤液对大丽轮枝菌生长、微菌核形成与萌发的影响。链霉菌无菌发酵滤液对大丽轮枝菌菌落生长、菌核形成和微菌核萌发有明显抑制作用。其中菌株B49的抑菌效果最好,5倍稀释发酵液培养14天时对菌落生长的抑菌率达69.7%;菌株B49、D184和Act12的5倍稀释发酵液对微菌核形成的抑制率达100%;将经B49、D184和Act12发酵液处理后丧失形成微菌核能力的大丽轮枝菌菌株转接至不含发酵液的PDA培养基,连续传代至第5代,其仍然不能恢复形成微菌核的能力;微菌核在含有菌株D184 5倍稀释发酵液的培养基上培养168 h时,萌发率仅为38.3%。     

Methoxyfenozide and pyriproxifen alter the cellular immune reactions of Eurygaster integriceps Puton (Hemiptera: Scutelleridae) against Beauveria bassiana

Effects of the two insect growth regulators (IGRs) methoxyfenozide, 20-hydroxyecdysone (20E) agonist, and pyriproxifen, Juvenile hormone (JH) agonist, were examined on the cellular immune responses of the Sunn pest, Eurygaster integriceps versus the entomopathogenic fungus Beauveria bassiana. The simultaneous treatment with the IGRs and the fungal spores altered haemocyte count (total and differentiate), nodulation response and phenoloxidase (PO) activity in a dose- and time-dependent manner. It was observed that different concentrations of methoxyfenozide increased total and differentiate haemocyte numbers as well as B. bassiana-induced nodulation response. In contrast with the JH agonist, pyriproxifen significantly decreased total and differentiate haemocyte numbers and inhibited nodule formation in E. integriceps adults. The 20E agonist displayed major effects when injected at the doses 2.79 and 5.59 μg/mg adult. In contrast, injecting adults by pyriproxifen significantly impaired their ability to raise an efficacious response against the fungal spores. The ability of the two IGR analogues to interfere with activity of the PO system in haemolymph of E. integriceps adults was also investigated 6 h after injection by fungal spores. Methoxyfenozide had an excitatory effect on PO activity when the 5.59 μg/mg concentration was used against adults. Conversely, pyriproxifen had an inhibitory effect on PO activity when used at 1.49 μg/mg adult concentration. These findings demonstrate that pyriproxifen may interfere with cell-mediated immunity of E. integriceps. So, pyriproxifen could be a good candidate for the integrated control of the Sunn pest.     

Agrobacterium-mediated transformation efficiency is altered in a novel rice bacterial blight resistance cultivar and is influenced by environmental temperature

Y73 is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice (Oryza meyeriana), which shows high resistance to bacterial blight. It has a similar genetic background to its recurrent parent, Dalixiang, but Y73 has a high resistance to both bacterial blight and Agrobacterium. The transformation efficiency of Y73 was 35.7%, while Dalixiang had higher transformation efficiency (71.2%) under the same co-cultivation temperature (25 °C). These results indicate that the resistance to Agrobacterium tumefaciens in Y73 was linked with its characteristic of bacterial blight resistance, and was obtained from its donor parent, O. meyeriana. Further studies also showed that the resistance to A. tumefaciens was weakened at a lower temperature (20 °C). To study its molecular mechanism, the expression levels of genes associated with Agrobacterium-mediated transformation (OsMPKs, OsVIP1s and OsPR1s) in rice were investigated by Quantitative real-time PCR (qRT-PCR) analysis. The expression levels of OsMPK genes remained unchanged at different temperatures, while all OsVIP1s and almost all OsPR1s were up-regulated and transformation efficiency of Y73 was increased notably when infected by Agrobacterium at 20 °C compared with 25 °C. This study suggests that the bacterial blight resistance genes in Y73 also have an effect on Agrobacterium-mediated transformation and that the response to temperature is associated with the regulation of MAPK/VIP1 defense signaling pathway.     

三种化合物处理对南方根结线虫生长发育及致病能力的影响

为明确不同浓度NH4HCO3、Cu SO4·5H2O和Fe Cl3·6H2O对南方根结线虫Meloidogyne incognita 2龄幼虫活性的影响,在盆栽条件下将化合物处理后的2龄幼虫接种番茄,通过根染色和根系冰冻切片法观察2龄幼虫的发育状况和根系内巨细胞的形成,并测定了番茄根系各生理指标的变化。结果表明,50mmol/L NH4HCO3、1.4mmol/L Cu SO4·5H2O和1.4 mmol/L Fe Cl3·6H2O处理南方根结线虫后,番茄根系内2龄、3龄及4龄幼虫数量均显著小于对照;根系内丙二醛、可溶性糖和游离氨基酸的含量均显著低于对照,根系活力显著增强;经NH4HCO3处理后,根系内巨细胞形成缓慢,14 d时出现巨型细胞空洞现象。3种化合物处理对南方根结线虫生长发育的抑制作用显著,处理后的线虫对番茄植株致病力减弱,表现为NH4HCO3抑制作用最强,Cu SO4·5H2O次之,Fe Cl3·6H2O最弱。     

Influence of neem on susceptibility of Beauveria bassiana and investigation of their combined efficacy against sweetpotato whitefly, Bemisia tabaci on eggplant

A study on the compatibility of the entomopathogenic fungus Beauveria bassiana with neem was conducted against sweetpotato whitefly, Bemisia tabaci, on eggplant. Initially, three concentrations of neem (0.25%, 0.5% and 1.0%) were used to investigate the physiological responses of B. bassiana. Thereafter, above three concentrations of neem along with three concentrations of B. bassiana (10⁶, 10⁷ and 10⁸ conidia/ml) were used to investigate combined deterrence index, DI under two application methods (foliar and soil) of B. tabaci. Significant differences were observed among neem concentrations on all variables- germination percentage, vegetative growth, number of conidia, amount of biomass and proteolytic activity of B. bassiana. The reduction percentage of germination, vegetative growth, sporulation, biomass production and proteolytic activity of B. bassiana were as high as 12%, 13%, 35%, 38%, and 34%, respectively, to neem. Significant differences were also observed on deterrence index, DI (adult and oviposition) of B. tabaci. The current study investigated that the highest adult DI (80.15) and oviposition DI (88.25) occurred when 1.0% neem was combined with 10⁸ conidia/ml of B. bassiana. As the results show, neem is compatible with B. bassiana; and soil application of neem along with foliar application of B. bassiana might be useful for the control of B. tabaci.     

水稻纹枯病菌拮抗细菌的筛选及鉴定

为获得对水稻纹枯病有生防效果的拮抗细菌,从江苏南京、徐州和常州等地采集的土样中分离细菌分离物1914株,采用平板对峙法筛选获得70株对水稻纹枯病菌有较强抑菌活性的分离物,其中11株对5种水稻病害病原菌均有抑制作用;对11株拮抗菌进行田间防效和室内促生试验,测定菌株分泌的抑菌物质和促生物质,并进行种属鉴定.结果表明,拮抗菌对水稻纹枯病的盆栽和田间小区防效在48.41％和43.03％以上;均可产生蛋白酶与嗜铁素,而不产生几丁质酶,除XF-174外其余10个菌株均可产生纤维素酶;对水稻苗株高和鲜重具有促生作用,并均可产生赤霉素(GA3);除ZF-273和XF-174外的9个菌株可产生吲哚乙酸(IAA),且细菌发酵液中IAA和GA3含量与水稻株高和鲜重的增长率呈正相关.结合各菌株形态特征、生理生化特性和16S rDNA与gyr-B序列分析结果,鉴定SF-181为枯草芽胞杆菌Bacillus subtilis,XF-174为荧光假单胞菌Pseudomonas fluorescens,其余9个菌株为解淀粉芽胞杆菌B.amyloliquefaciens.     

抗不同生化型青枯菌的生防菌筛选鉴定及其活性分析

为更好地利用生防菌控制青枯病危害,从不同地区的土壤中分离到569株细菌菌株,筛选到3株对5种不同生化型青枯劳尔氏菌Ralstonia solanacearum具有较强拮抗活性的菌株,其中菌株BS2004的拮抗活性最强。以BS2004的菌悬液为对照,分别测定无菌滤液、蛋白酶K及高温热处理后拮抗物质抑菌活性的变化。结果显示,蛋白酶K及高温热处理后,该菌的抑菌活性显著降低,表明其主要抑菌成分为蛋白类物质。在设施栽培条件下用生防菌BS2004菌悬液处理番茄植株,能有效控制番茄青枯病的发生,防治效果达66.75%,同时还发现,重新分离得到的青枯菌菌体数明显受到生防菌的抑制。通过对BS2004的形态、生理生化特征、脂肪酸鉴定、16S rDNA序列等进行分析,该菌株被鉴定为解淀粉芽孢杆菌Bacillus amyloliquefaciens。     

昆虫病原线虫对小地老虎的致病力测定及防治效果

为探讨应用昆虫病原线虫防治小地老虎技术的可行性,采用培养皿滤纸法比较9个品系的昆虫病原线虫对小地老虎幼虫的致病力,同时通过盆栽试验验证了斯氏线虫属小卷蛾斯氏线虫Steinernema carpocapsae NC116品系、芫菁夜蛾斯氏线虫S.feltiae SF-SN品系和异小杆线虫属嗜菌异小杆线虫Heterorhabditis bacteriphora H06品系的保苗效果和防治效果,并测定了苦参碱与NC116品系混用对小地老虎3龄幼虫的联合作用效果。结果表明,NC116品系对小地老虎3龄幼虫致病力最高,其对3龄、4龄和5龄的LD50分别为2.4、7.5、和31.2条/头,表明随小地老虎幼虫龄期递增,其致病力下降。盆栽玉米苗上分别施用线虫100~200条/头时,对玉米保苗效果及对小地老虎3龄幼虫的控制效果依次为NC116H06SF-SN。0.8 mg/L苦参碱和NC116品系混用后,可使小地老虎3龄幼虫死亡率提高109.89%,二者表现增效作用。     

Activity and metabolism of cyano-oxime derivatives in various strains of Botrytis cinerea

The fungitoxicity of cymoxanil, 4 cyano-oxime analogs (in which the acetylurea group was replaced with various groups: amide, ester, propargyl, and cyanomethyl-amide), and 2 cymoxanil-metabolites was studied against various strains of Botrytis cinerea. The fungicidal effect was measured on germ-tube elongation and mycelial growth. Cymoxanil and the analogs bearing the amide and ester groups showed the best anti-botrytis activity. The strains studied can be classified into 3 groups according to the germ-tube sensitivity to cyano-oximes. These groups fitted well with the 3 phenotypes of sensitivity to cymoxanil previously characterized in our laboratory: CyaS₁ (highly sensitive phenotype); CyaS₂ (moderately sensitive phenotype); and CyaR (tolerant phenotype). The bio-transformations of all the cyano-oximes were monitored in the culture-medium of the different strains using HPLC- and IP-HPTLC-methods. HPLC-studies showed that cymoxanil and the analogs bearing the groups amide and ester were quickly metabolized in the culture-medium of the CyaS₁ strain. Moreover, these studies allowed us to correlate disappearance of these cyano-oximes, to their fungicidal activity towards the CyaS₁ strain. This suggests that cyano-oximes and particularly cymoxanil are probably activated in a fungitoxic compound. IP-HPTLC-studies strengthened the precedent results and allowed us to correlate disappearance of cyano-oximes studied with appearance of two acids metabolites of hydrolysis. These metabolites could be the actual active-principles.     

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。     

四种无机盐对南方根结线虫卵囊、卵孵化及其幼虫存活的影响

为了明确NH4HCO3、CuSO4.5H2O、CsCl、NH4NO34种无机盐对南方根结线虫卵囊、卵孵化及其幼虫存活的影响。在室内条件下,采用浸渍法测试了4种无机盐在不同浓度和时间处理的作用效果。结果显示：各种无机盐均对线虫卵、卵囊孵化有抑制作用,对2龄幼虫有致死作用。随着无机盐浓度的增加和处理时间的延长,线虫卵、卵囊孵化的抑制率和2龄幼虫的死亡率显著提高。其中0.05 mol/L NH4HCO3对线虫卵囊孵化的抑制效果最好,处理16 h达81.17%,24 h后达100%;对线虫卵孵化也有很强的抑制作用,处理16 h达93.33%,24 h后达100%。各种无机盐在不同浓度和处理时间内均对2龄幼虫有致死效果,且半致死中浓度LC50不同,其中CuSO4.5H2O的LC50最小,NH4HCO3和CsCl次之。表明CuSO4.5H2O对南方根结线虫2龄幼虫有较强的致死活性,南方根结线虫2龄幼虫对CsCl的敏感性较高。     

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.