Polymerase chain reaction fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design of highly specific molecular markers

Erwinia amylovora, the causal agent of fire blight, is genetically very homogeneous, and current methodologies provide insufficient or contradictory information about the probable dispersal routes of the pathogen. With the final aim to obtain specific and reliable molecular markers for different lineages of the pathogen, we studied the molecular basis of rep-polymerase chain reaction (PCR) polymorphism using seven different arbitrary primers to fingerprint 93 E. amylovora strains from different countries, including Spain. Polymorphism was very low, and was displayed by only 11 E. amylovora strains, which produced 22 polymorphic bands. Five of 11 polymorphic bands cloned contained DNA that was present in more than 85% of the strains, whereas six bands were due to DNA present exclusively in the strains producing the rep-PCR polymorphism. Also, five of the polymorphic bands were due to the possession of either the ubiquitous plasmid pEA29, of plasmid pEU30, which was exclusively found in strains from North America, or of a 35-kb cryptic plasmid, present only in 28 strains from Northern Spain. We designed primer pairs from several cloned polymorphic bands that allowed the specific identification of the strains producing the polymorphism. Our results indicate that rep-PCR is not adequate for constructing genealogies of E. amylovora, although the strategy illustrated here, as well as the designed primers, can be used effectively in epidemiological studies with this pathogen.     

Genetic relationships between the dominant genotypes of Phytophthora infestans in Hokkaido, Japan

The genetic characteristics of the dominant genotypes of Phytophthora infestans in Japan (US-1, JP-1, Japanese A1-A, A1-B) were compared. Differences were evident in the peptidase genotype, amplified fragment length polymorphism, and RG57 DNA fingerprints. Almost all of the fingerprint bands for the Japanese genotype A1-B were also present in JP-1 and Japanese A1-A, and few bands were unique to Japanese A1-B. These results suggest that the Japanese A1-B genotype was generated from sexual reproduction involving Japanese A1-A and JP-1 or related genotypes.     

北方棉区棉花黄萎病菌落叶型菌系鉴定

 采用RAPD扩增与温室致病性测定2种方法,以黄萎病菌Verticillium dahliae的5个落叶型、7个非落叶型菌系和V.albo-atrum的2个菌系为对照,对采自北方棉区6省(自治区)的34个棉花黄萎病菌菌系进行致病型鉴定。94.1%的菌系在2种鉴定结果中表现一致,与对照菌系相比较,确定其中26个菌系为落叶型菌系,6个菌系为非落叶型菌系。从而证实了黄萎病菌落叶型菌系在北方棉区河北、河南、山东3省的存在,并发现所确定的26个北方落叶型菌系中的22个与来自美国的对照落叶型菌系T₉、V₄₄的关系比与来自江苏的对照落叶型菌系V_B、V₉₉₁的关系更近。本实验还初步筛选到2条用于鉴别V.dahliae落叶型与非落叶型菌系的RAPD特异条带OPB-19₉₆₆和OPM-20₁₆₉₁,将它们用于对34个北方菌系的RAPD扩增鉴定,则与温室致病性测定结果的一致性分别为88.2%和94.1%,证明这2条特异条带在鉴定棉花黄萎病菌落叶型菌系上具有一定的实用价值,并为进一步制作特异探针以形成一套简便、准确、规范化的鉴定技术奠定了基础。     

Electrophoretic karyotype variation among pathotypes of Fusarium oxysporum f.sp. dianthi

Karyotype analysis by pulsed-field gel electrophoresis was applied to characterize isolates of Fusarium oxysporum f.sp. dianthi , the causal agent of Fusarium wilt on carnation. Eleven distinct chromosomal DNA patterns were detected among 38 pathogenic isolates, and the total genome size was estimated to range from 23·7 to 36·4 Mb. Except for isolates belonging to pathotypes 2 and 4 , all members of the same pathotype shared overlapping electrophoretic karyotypes. Karyotypes of isolates assigned to pathotypes 1 and 8 showed a high degree of similarity, in accordance with VCG and RFLP analysis. The same electrophoretic karyotype was also shared by members of pathotypes 2 and 5, thus confirming results obtained by both VCG and RFLP grouping, A single representative of pathotype 6, previously confined to the same VCG and RFLP group as pathotypes 2 and 5, had a slightly different chromosomal pattern. Isolates assigned to pathotype 4 showed four related karyotypes which partially differed in both the number and size of chromosomal bands. However, all strains assigned to this pathotype shared a basic profile of nine chromosomal bands, while two low-molecular-weight bands were present or absent. The findings are discussed with regard both to the suitability of race distinction in the case of the special form dianthi of F. oxysporum and to the use of karyotype analysis by PFGE as a tool for the study of the population genetics of this fungus.     

DNA methylation changes in fusarium wilt resistant and sensitive chickpea genotypes (Cicer arietinum L.)

DNA methylation plays an important role in the regulation of gene expression in biotic and abiotic stresses. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in seven resistant and sensitive chickpea genotypes following inoculation with Fusarium oxysporum f. sp. ciceris. In all, 27468 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using nine selective primer pairs. DNA methylation was evaluated in leaves, stems and roots in control and inoculated plants. Extensive cytosine methylation alterations were found in the pathogen-treated genotypes compared with the corresponding control, including hypermethylation and demethylation as well as the potential conversion of methylation types. For all genotypes, the percentage of demethylated sites were more than methylated sites in infected plants compared with the corresponding control. No significant differences were observed for banding patterns in infected and control leaf tissues, while the differences between percentage of unchanged, methylated and demethylated sites were significant in stem and root tissues. The total numbers of methylated polymorphic bands ranged from 137 to 154 bands in Sel95th1716 and Arman, accounting for 36.81%–44.64% of all bands, respectively. Ten fragments that were differentially amplified between infected and control plants were isolated and sequenced in three tissues separately. Most of sequenced fragments showed homology with disease related genes in GenBank. The results suggest that significant differences in cytosine methylation exist between resistant and sensitive chickpea genotypes, and that hypermethylation or hypomethylation of specific genes may be involved in the chickpea resistance to Fusarium wilt.     

Germination induces accumulation of specific proteins and antifungal activities in corn kernels

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.     

捕食线虫真菌酯酶同工酶谱和可溶性蛋白质谱分析

 本文对5个属14个种共33株捕食线虫真菌采用聚丙烯酰胺凝胶电泳技术进行了酯酶同工酶谱和可溶性蛋白质谱分析。结果表明,酯酶同工酶谱带少,但各属的特征谱带是明显的,在同属不同种之间,多数种都有自已的特征谱带与其它种相区别,同种不同菌株间没有明显差异;蛋白质谱带较多,在每个属中均有1~2条特征谱带,属间的谱带差异明显,同属不同种和同种不同菌株间蛋白质谱带都表现出多型性。     

我国棉花枯萎病菌酯酶同工酶测定初报

 利用聚丙烯酰胺薄层凝胶电泳法测定了我国主产棉区棉花枯萎病菌的酯酶同工酶。三年试验结果表明,供试42个菌系的酯酶同工酶有三种类型主酶带,根据主酶带可以将供试的菌系区分为三个类型。类型一,有E₄、E₅、E₆、E₈四条主酶带,包括13省的菌系31个,类型二,有E₄、E₆、E₈三条主酶带,包括6省的菌系10个;类型三,有E₆、E₈两条主酶带,包括新疆吐鲁蕃的菌系一个。     

遥感多光谱数据在内蒙古西部湿地监测中最佳波段选取的应用研究——以乌梁素海域为例

湿地由于具有丰富资源、独特的生态结构和功能而享有“自然之肾”之称。为了更好地保护和开发利用湿地,近年来湿地研究成为国际上众多学科学者们关注的热点。本文尝试遥感技术在湿地监测中的应用,从多光谱遥感数据中选取最佳波段来突出湿地类型或湿地植被工作进行探讨。经研究表明最佳波段选取原则除主要考虑波段的信息量要大,而它们之间的相关性要小这两个因素外,还要使波段组合后地物之间的光谱特征差异尽量达到最大。以乌梁素海域湿地为研究对象,利用最佳指数OIF指数选取最佳波段,并结合研究对象的光谱特征、Landsat7卫星ETM各波段用途,最终选取TM453为最佳波段组合,这样的组合可以使不同地物的灰度值差异达到最大,有利于目视解译。     

Integration of cultural and mechanical practices for management of the mango mealybug <Emphasis Type="Italic">Drosicha mangiferae</Emphasis>

Mango (Mangifera indica L.) is one of the most economically important trees which has been cultivated for several centuries. The tree is susceptible to insect damage and is attacked by leaf eaters, termites, root grubs, sapsuckers, gall formers, stem borers, pod borers and fruit borers. The mango mealybug Drosicha mangiferae Green (Hemiptera: Monophlebidae) is one of the most destructive pests in the Indo-Pak subcontinent, sometimes causing premature fruit drop. A combination of several management strategies such as use of entomopathogens, chemical insecticides and burning of adult females has been used to control D. mangiferae. Overreliance on insecticides has eliminated natural enemies, and we were therefore interested in investigating the impact of alternative insect pest management strategies such as cultural and mechanical techniques on D. mangiferae. The results of our studies suggested that different bands significantly stopped the upward movement of overwintering and surviving populations of D. mangiferae. To augment the bands’ results, mounding the tree with debris on a plastic sheet once a year was the most effective technique and resulted in maximum reduction of the nymphal population of D. mangiferae. The newly developed Haider band—designed in our laboratory in the present studies—was very successful in the field in controlling the upward movement of newly hatched nymphs on trees: only 0.8% of the nymphs crossed the band. Only 7% to 10% of the nymphs crossed plastic sheets, polyethylene sheets and funnel bands. The cotton and gunny bag (jute) bands were the least effective: 42% of the nymphs managed to cross them. The results suggested that employing cultural practices and bands together could reduce the need for insecticide treatments and offer a sustainable method for D. mangiferae control.     

菜蛾盘绒茧蜂多分DNA病毒对寄主小菜蛾幼虫体内部分组织的影响

利用显微注射方法，观察了菜蛾盘绒茧蜂多分DNA病毒（CpBV）对其寄主小菜蛾幼虫部分组织的影响。结果表明，CpBV进入寄主体内后能够影响其精巢、前胸腺和中肠等的变化。当被注射CpBV后，寄主精巢体积的增长受到抑制，并且有被分解的现象；颜色也随之逐渐变淡．直至呈现透明。前胸腺大小在感染CpBV 12h时就已经变小。中肠发育受阻，被分解，分布的气管出现萎缩现象，颜色由正常发育时的绿色变为黄色，个别会呈现红色，约20％中肠出现红色斑点。凝胶电泳分析表明，小菜蛾幼虫注射过CpBV后，其精巢、中肠和前胸腺中蛋白种类均减少，特别是精巢，仅有一条蛋白条带（78．8kD）较为清晰，蛋白条带明显变淡，说明蛋白含量明显下降；但前胸腺81．2kD的蛋白条带在感染CpBV后要比对照更为清晰。中肠酯酶活性测定表明，感染CpBV后中肠酯酶的活力受到抑制，酯酶活性减弱。     

Elicitors of Plant Defense Responses from Biocontrol Strains of Trichoderma viren

ABSTRACT Effective biocontrol strains of Trichoderma virens can induce the production of defense-related compounds in the roots of cotton. Ineffective strains do not induce these compounds to significant levels. This elicittation was found to be heat stable, insoluble in chloroform, passed through a 5K molecular weight cut-off (MWCO) filter, but not a 3K MWCO filter, and was sensitive to treatment by proteinase K. When the active material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several bands were present in the material from biocontrol-active strains that were lacking in inactive strains. When eluted and tested for elicitation activity, with or without renaturation, four bands stimulated cotton terpenoid production. One band showed cross-reaction with an antibody to the ethylene-inducing xylanase from T. viride. Another band of approximately 18 kDa, gave significant stimulation of cotton terpenoid production and increased peroxidase activity in cotton radicles in all tests, with or without renaturation. The 18-kDa protein was subjected to amino-terminal sequence analysis, and the first 19 amino acids at the amino terminus were determined to be DTVSYDTGYDNGSRSLNDV. A database homology search using the BLASTp algorithm showed the highest similarity to a serine proteinase from Fusarium sporotrichioides.     

西藏高寒植物垫状雪灵芝酯酶同工酶变异研究

以色季拉山的高寒植物垫状雪灵芝为材料,选取分布在海拔4500~4650m范围内的垫状雪灵芝3个居群为研究对象,采用聚丙烯酰胺琼脂电泳技术,对垫状雪灵芝3个居群的48个个体功能叶片的酯酶同工酶谱带进行测定并编码,依据酶谱带测定结果进行聚类分析;同时结合酯酶同工酶的3个遗传指标进行相关分析。结果表明:来自同一居群的个体,同工酶谱带相似性较大,居群内的遗传杂合度较小(HeA=0.735,HeB=0.765,HeC=0.726),这也说明居群内个体存在一定程度的遗传分化;酯酶在不同居群间的相似性系数的平均值相对较小,遗传距离相对较大(IAB=0.392,IAC=0.542,IBC=0.610;DAB=0.937,DAC=0.613,DBC=0.495)。研究数据显示垫状雪灵芝酯酶同工酶在不同的居群内与居群间存在不同程度的遗传分化,缺少有效的基因交流。     

中国北方番茄细菌性溃疡病菌的rep-PCR基因指纹图谱分析

对来自全国若干省市的番茄细菌性溃疡病菌(Clavibacter michiganensis subsp.michiganensis)的33个菌株进行rep-PCR分析。结果表明,用引物BOX分别扩增出6～15条多态性条带,条带大多数集中在500～2800bp之间;用引物ERIC扩增的条带不清晰,可能是反应条件不适合,也可能是其不适合对番茄细菌性溃疡病菌进行多态性分析;BOX-PCR表明番茄细菌性溃疡病菌菌株具有丰富的遗传多态性和较大的遗传变异,对产生的指纹图谱进行分析:在遗传距离为0.18时,测试的33个菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ等7个遗传相似组群,其中Ⅵ组群包含的菌株最多。研究还表明番茄细菌性溃疡病菌的遗传分群与菌株的地理来源关系密切,但与该病的发病年份上没有必然联系,这也从另一侧面说明了该病害是土壤带菌引起的。     

Biological, biochemical, and molecular parameters of Helisoma duryi snails exposed to the pesticides Malathion and Deltamethrin

Aquatic environments get contaminated with pesticides residues that result from the application of pesticides in agricultural practices. The present study aimed to evaluate the pesticides Malathion and Deltamethrin on biological and biochemical parameters of Helisoma duryi snails. The results showed that LC₁₀ of the two pesticides caused considerable reduction in survival rates and egg production of treated snails. Glucose concentration in hemolymph of the exposed snails showed a significant increase. On the other hand, albumin in hemolymph and glycogen contents as well as the activities of enzymes in tissues of snails including lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), arginase, and ornithine aminotransferase (OAT) were significantly inhibited. Meanwhile, the activity of AMP deaminase of the exposed snails was significantly increased in the hemolymph in response to treatment. The electrophoretic pattern of total protein showed differences in number and molecular weights of protein bands. DNA concentration was investigated by measuring the intensity of the genomic bands and its showed its increase in the treated snails. It was concluded that the residues of Malathion and Deltamethrin pesticides in aquatic environments have toxic effects on non-target organism, e.g. H. duryi snails.     

Decrease of rice plant resistance and induction of hormesis and carboxylesterase titre in brown planthopper, Nilaparvata lugens (Stål) by xenobiotics

The brown planthopper (BPH), Nilaparvata lugens (Stål) is a serious threat to the rice production throughout Asia. The indiscriminate application of various xenobiotics in rice ecosystem is perceived as one of the factors for the frequent outbreak of BPH. The present study has critically analysed the secondary effects of some xenobiotics used in rice field on certain plant and insect parameters that subsequently favour BPH outbreak. Application of 2,4-D, carbendazim, deltamethrin and urea reduced the innate BPH resistance of PTB 33 rice variety due to favourable alterations in rice free amino acid and sucrose content. Similarly, these chemicals also induced hormesis and enhanced feeding in BPH. Alternatively, soil amendment with neem seed powder and Calotropis gigantea leaves improved plant innate resistance and showed no sign of hormesis or enhanced feeding in BPH. In addition, deltamethrin has the ability to stimulate BPH carboxylesterase titre. Native PAGE analysis of esterases from whole body homogenate of BPH revealed at least five esterase isozyme bands, prominent being E1 and E2. However, no difference in BPH esterase banding pattern was observed between different xenobiotic treatments. All these esterase bands are classified under carboxylesterase based on their inhibition by class specific esterase inhibitors.     

Dynamics of the IgG3 responses following immunisation of BALB/c mice with somatic and excretory/secretory antigens from various Trichinella species

Comparison of the dynamics and antigen recognition profiles of IgG3 following immunisation with larval crude extracts (LCE) and excretory-secretory (ES) products from muscle larvae of different species of Trichinella (T. spiralis, T. nativa, T. britovi, T. nelsoni and genotype T6) was made in BALB/c mice. High levels of gG3 response were obtained in ELISA following immunisation with LCE from all species with maximum levels achieved between days 59 and 64 post-immunisation (p.i.) and maintained up to the limit of the observation (day 164). Antigen recognition profiles as measured by western-blot showed dense and numerous bands in the range 45-64 kDa that were consistent from week 5th with variation in epitope recognition among the different species. Following immunisation with ES antigens a significant decrease in IgG3 response was observed for all species especially for T. nativa in comparison to LCE. Antigen recognition on ES antigens showed three main bands in the range of 45-60 kDa for all species excepting T. nelsoni and T. britovi where an additional band was also present. These results clearly show that IgG3 epitopes are more abundant in somatic (LCE) than in ES products of Trichinella muscle larvae and that quantitative as well as qualitative variations exist among different species.     

DON毒素和小麦互作对小麦幼穗细胞可溶性蛋白的影响

 本研究经小麦幼穗细胞和DON毒素混合处理,研究抗赤霉病程度不同的苏麦3号(R)、望水白(R)、延冈坊主(R)、Alondra's (S)、安徽11(S)和仪宁小麦(S)的可溶性蛋白谱带的变化。用3μg/ml的脱氧雪腐镰刀菌烯酸(DON)与小麦幼穗细胞混和处理2、8、24、30h,分别提取可溶性蛋白,进行PAGE测定。结果显示,感病品种有多条谱带消失,如仪宁小麦、安徽11、Alondra's在处理2h后Rf 0.13的条带消失,处理8h后Rf 0.02条带消失,感病品种Alondra's和安徽11在处理24h后Rf 0.05的条带消失。抗病品种的电泳谱带没有变化,如苏麦3号、望水白在毒素处理后蛋白条带没有发生变化,抗病品种延冈坊主在处理24h后RfO.OS、O.11的蛋白带仅有变浅的趋势。研究还表明抗病品种苏麦3号、望水白、延冈坊主具有感病品种没有的Rf 0.3的蛋白带,且在毒素处理前后保持一致。     

Effects of momilactone on the protein expression in Arabidopsis germination

Although momilactone is known to play an important role in the allelopathy of rice (Oryza sativa L.), there is no information available about the mode of action of momilactone on the allelopathy of Arabidopsis. The present research describes the allelopathic activity of momilactone A and B on the germination of Arabidopsis thaliana and the effects of momilactone A and B on protein expression during germination. Momilactone A and B inhibited the germination of Arabidopsis at concentrations >30 and 10 μmol L^?1, respectively. The protein bands of 18, 19 and 21 kDa on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) were abundant in the momilactone A‐ and B‐treated Arabidopsis compared with the control. The protein bands of 18, 19 and 21 kDa were identified by the N‐terminal amino acid sequencing as cruciferin 2, cruciferina and cruciferin 3, respectively. Cruciferins and cruciferina are important storage proteins and play an important role as the initial source of nitrogen for seed germination. These results suggest that momilactone A and B may inhibit the germination of Arabidopsis seeds by inhibiting the degradation process of cruciferin and cruciferina.