Meteorus rubens—Steinernema carpocapsae interaction, field studies

The entomogenous nematode,Steinernema carpocapsae was applied in three forms in two fields cultivated with cotton and tomato, under each cotton seedling or tomato plant: a bait like form, a suspension and in irrigation water. High concentrations of 2000 and 1000 IJS/seedling induced 100% mortality (after 5 days), while lower concentrations showed the same result but after 15 days or later. Under all doses of nematode, bait form application was more effective than suspension or irrigated water.     

Meteorus rubens—Steinernema carpocapsae interaction, field studies

The entomogenous nematode,Steinernema carpocapsae was applied in three forms in two fields cultivated with cotton and tomato, under each cotton seedling or tomato plant: a bait like form, a suspension and in irrigation water. High concentrations of 2000 and 1000 IJS/seedling induced 100% mortality (after 5 days), while lower concentrations showed the same result but after 15 days or later. Under all doses of nematode, bait form application was more effective than suspension or irrigated water.     

高粱GRF基因家族鉴定及SbGRF4原核表达分析

为研究高粱生长调控因子(growth-regulating factor,GRF)在高粱生长过程中的功能,采用生物信息学分析方法对高粱GRF基因家族进行鉴定,并以高粱BTx623品种为材料,利用PCR技术扩增获得SbGRF4基因cDNA全长序列,构建pET-28a-SbGRF4重组质粒,采用原核表达技术分别对表达菌株、诱导温度以及异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导浓度进行优化,最后用Western blot技术对原核表达的SbGRF4蛋白进行验证。结果表明,在高粱中共鉴定到10个GRF基因SbGRF1～SbGRF10;亚细胞定位预测结果显示均定位于细胞核中;分布于高粱的6条染色体上,所有家族成员均含有QLQ和WRC保守结构域;系统进化结果显示,高粱GRF蛋白分为Class 2、Class 3、Class 4a、Class 4b四个亚家族;相同亚家族各个成员间外显子和内含子的数量差异较小;同一亚家族的高粱GRF蛋白保守基序具有相似性;克隆到的SbGRF4基因c DNA序列全长为1 221 bp,并成功构建pET-28a-SbGRF4融合表达载体;SbGRF4蛋白的最佳表达菌株为大肠杆菌Escherichia coli JM109(DE3)菌株,最佳诱导温度为25℃,最佳IPTG诱导浓度为0.6mmol/L;Western blot验证结果显示原核表达的蛋白为SbGRF4。表明SbGRF4蛋白可在大肠杆菌原核表达系统中表达。     

云南省元阳县籼/粳型稻瘟病菌无毒基因Avr-Pia、Avr-Pita1和Avr-Pii多样性分析

为明确云南省元阳县籼/粳型稻瘟病菌中无毒基因的多样性及差异,采用接种鉴别品种方法和PCR技术对9株粳型菌株和21株籼型菌株中无毒基因Avr-Pia、Avr-Pita1和Avr-Pii的多样性进行分析。结果显示,30株稻瘟病菌菌株中无毒基因Avr-Pia、Avr-Pita1和Avr-Pii的平均出现频率分别为33.3%、76.7%和0,Avr-Pia、Avr-Pita1在籼型菌株和粳型菌株中的出现频率分别为4.8%和100.0%、66.7%和100.0%。Avr-Pia主要为存在/缺失的多样性,籼/粳型菌株对水稻品种Achiasahi(Pia)的致病率分别为100.0%和11.1%;Avr-Pita1可划分为3个类群,除菌株CH1195属于PO类群外,籼型菌株均属于J1类群,粳型菌株则属于J2/J3类群;籼/粳型菌株对水稻品种K1(Pita)的致病率分别为100.0%和0,且籼/粳型菌株Avr-Pita1蛋白序列在第83位和第192位氨基酸差异明显。Avr-Pii在30株稻瘟病菌菌株中均未检出,籼/粳型菌株对水稻品种Fujisaka No. 5(Pii)的致病率分别为100.0%和0。表明Avr-Pia和Avr-Pita1在粳型菌株中的出现频率高于籼型菌株且变异程度低,粳型菌株中可能存在其它无毒基因使其不能侵染Fujisaka No. 5。     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)化学诱变hrp基因突变体及相关性状的研究

 硫酸二乙酯(diethyl sulfate,DES)化学诱变水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)日本系统III号小种(JXOIII),获得16株hrp-突变体,其在感病寄主水稻IR26、汕优63上失去致病性和在非寄主烟草上失去过敏反应。所得的hrp-突变体并无黄色素缺失现象。16株hrp-突变体的淀粉酶、果胶酶、蛋白酶、纤维素酶和脂酶等胞外酶活性基本与野生型一致。JCM2092、JCM2211、JCM2252和JCM2457这4株hrp-突变体为III型泌出通道突变体,JCM0066等另12株hrp-突变体的超声波破碎液、离心上清液中无激发HR的信号物质存在。细胞组分分析表明,激发烟草HR的信号物质不是LPS而是蛋白类物质。来自JXOIII基因文库hrp基因克隆pUHRX245中的1.1kb SacI-KpnI片段,能够互补hrp-突变体JCM0066和JCM2482,使其恢复在烟草上激发HR的能力,但不能恢复其致病性。     

Plum pox virus as a stress factor in the vegetative growth, fruit growth and yield of plum (Prunus domestica) cv. ‘Cacanska Rodna’

During 2005–2007, under the environmental conditions of Cacak (43°53’N; 20°21’E), western Serbia, the effect of Plum pox virus (PPV) on the phenology of vegetative shoot and fruit growth was investigated in a plum orchard of cv. ‘Cacanska Rodna’ including trees non-infected with PPV and those with infection and clearly visible symptoms, as verified by RT-PCR. The results showed that PPV did not affect the growth phenology of the vegetative shoot (length and thickness) and the fruit growth. However, it caused negative effects on the average length (22.31 ± 0.28 cm in non-infected trees, 18.35 ± 0.07 cm in infected trees) and thickness (4.21 ± 0.03 mm in non-infected trees, 4.01 ± 0.02 mm in infected trees) of vegetative shoots as well as on the average fruit weight (20.43 ± 0.16 g in non-infected fruits, 17.58 ± 0.18 g in infected fruits) and fruit dimensions. The greatest effect of PPV during the trial was the induction of a massive premature fruit drop and, consequently, a 52.94% decrease in total yields per tree (18.0 ± 1.10 kg) and unit area (12.0 ± 0.89 t ha^-1) in infected vs non-infected trees.     

番茄叶霉病高抗基因Cf-9、Cf-11和Cf-19的分子标记

 本研究以9个含不同叶霉病抗病基因的番茄品种为试材,通过接种鉴定表明,Cf-5、Cf-9、Cf-11和Cf-19基因对我国目前的2个叶霉菌优势生理小种均具有较强的抗性。根据Cf-9基因设计引物,扩增Cf-9基因的片段,含Cf-9、Cf-11和Cf-19基因的3种番茄均获得了2.7kb的扩增片段。但用限制性内切酶TaqⅠ对PCR产物酶切可以将3种材料明显区分开来,Cf-9的2个差异酶切片段为1170和460bp;Cf-11的2个差异酶切片段为1100和410bp;Cf-19的2个差异酶切片段为1210和300bp,从而建立了3个基因的分子标记。在F₂分离群体中验证表明,3个基因的分子标记鉴定结果与抗性接种鉴定结果是一致的,用这些标记可以进行分子标记辅助选择。     

转cry1Ab/1Ac基因水稻对野生褐家鼠Rattus norvegicus取食、体重及器官重量的影响

为评价转Bt基因水稻对野生啮齿类动物的影响,以野生褐家鼠Rattus norvegicus为试验对象,分别饲喂转cry1Ab/1Ac基因水稻华恢1号(HH1)及其亲本非转基因水稻明恢63(MH63),通过取食选择试验及110 d连续饲喂试验,测定转基因水稻对褐家鼠取食量、体重及器官的影响。结果显示,野生褐家鼠雌鼠日平均取食MH63和HH1分别为5.52 g和5.37 g,雄鼠日平均取食分别为7.00 g和6.52 g,在选择取食MH63和HH1稻谷上无显著差异;在连续饲喂试验的不同时期,褐家鼠体重及每日取食稻谷量在对照组与试验组间均无显著差异;饲喂试验结束后解剖观察,褐家鼠脾脏、肾脏、肾上腺,雌性卵巢和子宫以及雄性睾丸、附睾和储精囊的绝对重量及相对重量在对照组与试验组间均无显著差异。研究表明,与MH63相比,转cry1Ab/1Ac基因水稻对野生褐家鼠的取食、体重以及主要器官均无显著性影响。     

稻瘟病菌MoCOS1基因调控MoCMR1基因表达的研究

 以往研究证明MoCOS1可能是一个新型的转录因子,其突变体不产生分生孢子,黑色素合成明显减少。经过RNA-Seq分析,发现受MoCOS1调控的基因达到442个,其中MoCMR1的表达水平受到MoCOS1的下调。本研究中通过实时定量PCR进一步证实MoCOS1基因突变导致MoCMR1基因表达水平下降。基因MoCMR1突变后,黑色素产量略有减少,但分生孢子形态及产量和MoCOS1的表达量没有发生变化。     

小麦抗赤霉病3B-QTL和6B-QTL的遗传互作模式分析

为明确来自抗赤霉病的小麦品种望水白的2个主效抗扩展3B-QTL和6B-QTL的抗性遗传和互作模式,基于分子标记辅助选择方法,构建了回交分离群体,以病小穗数(NDS)和病轴长(LDR)为鉴定指标,采用单花滴注接种法对携带3B-QTL和6B-QTL的BC_3F_1、BC_3F_2、BC_3F_3世代以及抗感对照进行了抗赤霉病扩展的表型鉴定和评价。结果表明,3B-QTL和6B-QTL在烟农19和矮抗58不同的背景中,杂合基因型与纯合望水白基因型之间的NDS和LDR差异显著;携带3B-QTL和6B-QTL株系的抗扩展性与只含有单个3B-QTL的株系无显著差异,但显著高于只携带单个6B-QTL株系的抗性,抗感分离比经卡方检验符合4∶3∶9的分离比例,遵循2个独立孟德尔遗传因子控制的隐性遗传模式,且3B-QTL隐性上位于6B-QTL。研究表明,望水白的3B-QTL和6B-QTL抗扩展效应强且稳定,可以作为抗赤霉病基因资源在育种实践中充分利用。     

Antifeedant activity of a novel 6-(4,7-hydroxy-heptyl) quinone? from the leaves of the milkweed Pergularia daemia on the cotton bollworm Helicoverpa armigera (Hub.) and the tobacco armyworm Spodoptera litura (Fab.)

The effects of crude extracts and an isolated compound from the leaves of milkweed, Pergularia daemia (Forssk) Choiv., on the antifeedant activity against two important lepidopteran pests, Helicoverpa armigera (Hub.) and Spodoptera litura (F.), were studied. Maximum antifeedant activity was recorded in ethyl acetate crude extract against H. armigera (70.3%) and S. litura (71.82%) at 1% concentration. Ethyl acetate crude extract was further subjected to column chromatography, which was performed using hexane as initial solvent and then by increasing the polar strength using ethyl acetate. Fractions collected at hexane and ethyl acetate (80:20) yielded 6-(4,7-hydroxy-heptyl) quinone, a novel compound which showed significant antifeedant activity against H. armigera (80.22% at 2000 ppm) and S. litura (68.31% at 2000 ppm).     

水稻条斑病细菌hrp调节基因hrpGXooc和hrpXooc的克隆和序列分析

 Xanthomonas oryzae pv. oryzicola基因文库的hrp基因克隆pUHRS138携39.3-kb大小的hrp基因片段。经系列亚克隆和对hrp^-突变体的功能互补验证,4.5-kb BamHI-KpnI为最小功能片段,该片段可使X. o. pv. oryzicola的hrp^-突变体恢复在烟草上激发产生HR和在水稻上具致病性。序列测定和分析显示,4.5-kb hrp片段中含hrpXooc和hrpG_Xooc基因。单独的hrpG_Xooc和hrpXooc不能功能互补hrp^-突变体。hrpXooc与其它黄单胞菌中已克隆的hrpX的同一性达83%以上,推测的蛋白质水平上的差别主要在32、141、164、175、213、247和357位点上。HrpX序列中α-螺旋-转-α-螺旋结构在黄单胞菌中高度保守。hrpG_Xooc与水稻白叶枯病菌的hrpGXoo同一性达96%,与X. campestris pv. vesicatoria的hrpG_Xcv同一性达87%,与Ralstonia solanacearum的hrpG_Rs同源性较低,4种HrpG蛋白质水平上的差别主要集中在22、29、115和252位点上。HrpGXooc和HrpGXcv同列比较显示,3~9和216~220区域的氨基酸序列有所不同,可能反映了HrpG蛋白在感知环境信号和调节hrp基因表达方面的差别。     

cryIA(b)基因及其介导的抗性在转基因水稻中的遗传

研究了cryLA(b)基因及其介导的抗虫性在转基因水稻自交或回交后代中的遗传规律和遗传稳定性。结果表明：cryLA(b)基因及其连锁的bar基因作为一个显性遗传位点遵循孟德尔遗传分离规律；在T1代群体中遵循3：1的分离规律，T2代稳定纯合；在BC11～BC4代群体中遵循1：1的分离规律，BC群体自交的BCF9群体中遵循3：1分离规律。cryLA(b)基因及其介导抗性已稳定地遗传至T7代和BC4代。室内及田间抗虫性鉴定显示，不同世代的转基因植株均保持稳定的高度抗虫性。     

水稻纹枯病发病过程PR1和PBZ1的表达动态

 采用从福建省稻田分离纯化的纹枯病菌(Rhizoctonia solani)菌株FJ-15接种籼稻9311,分析了水稻在纹枯病菌侵染致病过程中,编码病程相关蛋白的基因表达动态,并观察了症状的变化。Northern blot分析表明:PR1在接种12 h后开始表达,在之后的4个时间段其表达量逐步增强;而PBZ1也在12 h开始表达,在48 h表达量激剧增强几乎与72 h表达量相当。组织和症状观察表明,接种12 h后叶鞘表面菌丝纵横分枝,接种36 h后出现零星病斑,接种48 h后表现典型的受害症状,接种72 h后病斑继续扩大,并可蔓延到非接种叶鞘。结果表明,PR1和PBZ1的表达与水稻和纹枯病菌亲和互作的过程存在对应关系。     

黑龙江省小苹果黑星病Venturia inaequalis(cooke)winter的初侵染来源

 1958-1963年调查研究证明黑龙江省小苹果黑星病病叶上的分生孢子全部不能越冬。在早春也没有发现带病的枝条,因此以菌丝在病枝上越冬、春天产生分生孢子的可能性极小。每年春天在去年病树的干枯落叶上能找到大量子囊壳及子囊孢子,经人工接种后侵染率都极高。空中孢子捕捉的结果是:子囊孢子出现在前,分生孢子的出现是在田间发病以后,两者相距半个月以上,说明所捕捉到的分生孢子是受子囊孢子侵染后产生的。可以得出结论:黑龙江省小苹果黑星病的初次侵染来源是子囊孢子。
早期调查发病情况说明离地面越近的叶片发病越早,越重。初步认为,清扫落叶是减轻病害的重要措施之一。     

Studies on the biology of Cryphalus piceae (Ratz.) (Coleoptera, Scolytidae) in the Bartin and Karabük regions of Turkey

The purpose of this study is to investigate the biology of Cryphalus piceae (Ratz.) (Col., Scolytidae) living in Abies bornmülleriana (Mattf.) in fir forests in the Western Black Sea region in Turkey. The dates of swarming, tree attack, egg-laying, and different stages of development are given. The species had two generations per year.     

Kombinierter Einsatz von synthetischen Insektiziden und Bacillus thuringiensis sowie Trichogramma evanescens gegen ausgewa¨hlte Scha¨dlinge an Vigna sesquipedalis unter tropischen Tieflandbedingungen

Field experiments with the synthetic insecticides, Methomyl and Carbaryl, alone and in combination with the biological control agents, Bacillus thuringiensis (Bt) and Trichogramma evanescens, were conducted to control pests in yardlong beans, Vigna sesquipedalis, during the dry season 1998/1999. The experimental area was located at the Central Luzon State University in the province of Nueva Ecija on the Philippine main island Luzon. Carbaryl was applied against bean flies 5 days after plant emergence. Methomyl was sprayed against pod sucking insects and the bean podborer, Maruca vitrata, 45 days after plant emergence. The biological control agents were applied with the beginning of the flowering stage of V. sesquipedalis, when plants were first attacked by M. vitrata.Major insect pests on V. sesquipedalis were cowpea aphid (Aphis craccivora), leafminer (Liriomyza sp.), and bean podborer (M. vitrata). Both, Bt and T. evanescens had no significant impact on yield. M. vitrata eggs, exposed for 48 hours in the fields to augmented T. evanescens, were used to measure field efficacy of the parasitoid. Highest parasitism rate was 13.3%, thus, generally low.Highest yield was obtained from plots treated with both synthetic insecticides. No significant difference in fresh pod yield was observed when, in addition to the synthetic insecticides, Bt and/or T. evanescens was applied. However, all treatments significantly out-yielded the untreated control.     

核盘菌自噬相关基因SsATG5和SsATG8的功能分析

为探究自噬在核盘菌Sclerotinia sclerotiorum致病过程中的作用,利用酵母Saccharomyces自噬相关基因（autophagy-related gene,ATG）编码的蛋白序列比对核盘菌基因组,获得核盘菌假定ATG,并以核盘菌1980菌株为出发菌株,基于同源重组的原理对假定ATG进行敲除和回补,并测定不同突变体的生长表型和致病能力。结果表明,从核盘菌基因组中比对到2个ATG,分别命名为SsATG5和SsATG8,两者在核盘菌致病过程中均上调表达。SsATG5和SsATG8敲除突变体在菌丝生长、产草酸和侵染垫形成方面与野生型菌株无明显差异,但SsATG5敲除突变体在离体拟南芥Arabidopsis thaliana叶片上的致病力显著下降了约40%,在活体拟南芥植株上的致病力显著下降了约80%,同时SsATG5回补突变体恢复了正常的致病力。表明SsATG5参与了核盘菌的致病过程,证实自噬在核盘菌致病过程中发挥着重要作用。     

Kombinierter Einsatz von synthetischen Insektiziden und Bacillus thuringiensis sowie Trichogramma evanescens gegen ausgewa¨hlte Scha¨dlinge an Vigna sesquipedalis unter tropischen Tieflandbedingungen

Field experiments with the synthetic insecticides, Methomyl and Carbaryl, alone and in combination with the biological control agents, Bacillus thuringiensis (Bt) and Trichogramma evanescens, were conducted to control pests in yardlong beans, Vigna sesquipedalis, during the dry season 1998/1999. The experimental area was located at the Central Luzon State University in the province of Nueva Ecija on the Philippine main island Luzon. Carbaryl was applied against bean flies 5 days after plant emergence. Methomyl was sprayed against pod sucking insects and the bean podborer, Maruca vitrata, 45 days after plant emergence. The biological control agents were applied with the beginning of the flowering stage of V. sesquipedalis, when plants were first attacked by M. vitrata.Major insect pests on V. sesquipedalis were cowpea aphid (Aphis craccivora), leafminer (Liriomyza sp.), and bean podborer (M. vitrata). Both, Bt and T. evanescens had no significant impact on yield. M. vitrata eggs, exposed for 48 hours in the fields to augmented T. evanescens, were used to measure field efficacy of the parasitoid. Highest parasitism rate was 13.3%, thus, generally low.