Production and characterization of single-spore isolates of Plasmodiophora brassicae

An improved procedure for inoculating Brassica seedlings with single resting spores of Plasmo-diophora brassicae is described. Using this procedure, eight out of a total of 600 seedlings inoculated with single spores developed severe symptoms of clubroot disease. Seven isolates of P. brassicae derived from single spores were characterized using the European Clubroot Differential (ECD) series. Four distinct pathotypes were obtained. The original populations of P. brassicae were shown t o be heterogeneous; this supports the findings of other workers. The isolates have been used to establish a reference collection of single-spore isolates of P. brassicae of known pathotype for use in Brassica breeding.     

Variation in Virulence of Plasmodiophora brassicae in Japan Tested with Clubroot-resistant Cultivars of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

The differential hosts of Williams (1966) and the European Clubroot Differential (ECD) (Buczacki et al., 1975) have been used commonly to identify populations of Plasmodiophora brassicae, which causes clubroot disease in Brassica crops. However, some of these hosts showed intermediate and fluctuating scores to most populations from Japan. Therefore, these hosts could not be used to provide a clear classification in Japan. We have tried to clarify the genetic diversity in pathogenicity of P. brassicae in Japan using Japanese clubroot-resistant (CR) F₁ hybrid (F₁) cultivars and lines of Brassica rapa. The responses of some CR F₁ cultivars were very clear. Four groups of field populations in Japan were recognized using the CR F₁ cultivars. The clear response obtained here may depend largely on the genetic purity of the F₁ cultivars. Moreover, it is possible to classify some of these Japanese populations into the same race using the Williams set and ECD 01 to ECD 05. The present differential hosts may be useful in the study of European populations of P. brassicae. The response of the differential hosts suggests that there are several major CR genes in B. rapa. It is suggested that pyramiding CR genes would be useful in breeding CR cultivars that can overcome the breakdown of the present CR cultivars of Chinese cabbage.     

The relationship between genotypes of three Brassica species and collections oi Plasmodiophora brassicae

Internationally collected data concerned with variation for pathogenicity in Plasmodiophora brassicae and for clubroot resistance in three Brassica species, together with additional experimental evidence, were used to elucidate the major genetic features of this host-parasite relationship. The major components of resistance in B. campestris and B. napus were differential and xobahly oligogenic. Good agreement was found between the data and simple gene-for-gene schemes. In contrast, the major component of resistance in most B. oleracea genotypes appeared to be non-differential and there was evidence of pathogen variability for non-differential pathogenicity. A strong association between pathogenicity on any resistant B. campestris genotype and on all resistant B. napus genotypes was apparent, suggesting the possibility of genes in common. There was evidence that P. brassicae collections carrying certain characters for differential pathogenicity on these two species were less pathogenic on B. oleracea. However, there was no ether evidence of pathogenic specialization to a particular host species. Several analytical procedures were employed which may prove useful in clarifying the important features of other host-parasite combinations.     

The effect of calcium cyanamide on the reaction of swede cultivars to populations of Plasmodiophora brassicae

The effect of using calcium cyanamide to control clubroot in four swede cultivars was examined in a field trial and two pot trials. Three populations of Plasmodiophora brassicae were used which differed in pathogenicity. The four swede cultivars showed a similar ranking for disease severity with all three pathogen populations; in intermediate environments which were neither very conducive nor unfavourable to disease development, resistance was expressed differentially.
Increasing the concentration of calcium cyanamide increased the yield of uninoculated plants, probably due to a response to nitrogen, but at the highest concentration (2.55 g/kg soil), yields were reduced due to phytotoxicity. In inoculated plants, disease severity was progressively reduced with increasing concentrations of calcium cyanamide. A higher inoculum density was required to initiate disease symptom development in the more resistant cultivars. Similarly, higher concentrations of inoculum were required to cause disease when increasing concentrations of calcium cyanamide were used. The effect of the chemical appeared to be to reduce the inoculum density. These data could be interpreted in terms of a disease threshold effect related to the number of viable pathogen propagules required to cause disease on particular host genotypes. The ways in which the breakdown products of calcium cyanamide may interact with host and pathogen are discussed in relation to disease development.     

Suppression of clubroot (Plasmodiophora brassicae) development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum

This study investigated the ability of an endophytic fungus Acremonium alternatum to reduce clubroot formation in the model plant Arabidopsis thaliana, which is highly susceptible to Plasmodiophora brassicae . Quantitative PCR demonstrated that A. alternatum colonized the P. brassicae -infected roots and shoots of the host plant. When Arabidopsis plants were co-inoculated with P. brassicae and A. alternatum , gall formation was reduced as shown by the reduction of the disease index (DI) by up to 50% compared to plants only infected with P. brassicae, whereas the infection rate was lowered by about 20% only in several, but not all, experiments. Clubroot was similarly suppressed when plants were inoculated with autoclaved A. alternatum spores or spore extracts, showing that viable spores were not needed. However, A. alternatum spores did not inhibit P. brassicae resting spore germination. Compared to the normal root galls, the smaller root galls on A. alternatum -inoculated plants contained fewer resting spores of the clubroot pathogen. It was thus hypothesized that inoculation with A. alternatum delayed the development of P. brassicae . Using quantitative RT-PCR to monitor the expression of P. brassicae genes differentially expressed during the development of the disease, a delayed pathogen development was corroborated. Furthermore, greenhouse experiments identified a time window in which the endophyte had to be administered, where the latest effective time point was 5 days before inoculation with P. brassicae and the optimum treatment was to administer A. alternatum and P. brassicae at the same time. These results indicate that A. alternatum and perhaps similar endophytes could be useful for the management of clubroot disease.     

广东稻瘟病菌的遗传宗谱与致病性的关系

不同病圃同一套寄虫主的田间表现及其穗颈瘟分离菌株的遗传宗谱和致病性分析试验结果表明,不同生态稻区稻瘟病菌的小种分布类型不同,对品种的致病性也存在差异。将已经过ＲＦＬＰ和ＰＣＲ技术进行ＤＮＡ指纹分析,并划分为不同遗传宗谱的稻瘟病菌株进行致病性测定。结果表明,菌株的致病性与其遗传宗谱类型、生理小种类型、寄主及采集地点存在密切的关系：（１）同一遗传宗谱、分离自相同寄主、鉴定出来的生理小种类型又较接近的菌     

甘蓝夜蛾赤眼蜂对几种重要农业害虫寄主卵的选择性

在室内采用选择性方法 ,研究了甘蓝夜蛾赤眼蜂G品系和F品系对我国几种重要农业害虫的寄主选择性。结果表明 ,G品系对亚洲玉米螟卵和麦蛾卵的选择性无明显差异 ;在对玉米螟卵的选择性上 ,G品系与玉米螟赤眼蜂的差异不明显 ,可作为防治亚洲玉米螟的候选蜂种。F品系对麦蛾卵的选择性强 ,已丧失对玉米螟卵的选择性。G和F品系以及螟黄赤眼蜂对棉铃虫卵的选择性较强 ,而对麦蛾卵的选择性较弱。在对棉铃虫卵的寄主选择性上 ,G和F品系同螟黄赤眼蜂无显著差异 ;但在寄生能力上 ,G和F品系均显著高于螟黄赤眼蜂 ,均可作为防治棉铃虫的候选蜂种。G和F品系以及广赤眼蜂对麦蛾卵的选择性较强 ,而对小菜蛾卵的选择性较弱。     

Characterization of isolates derived from single resting spores of Plasmodiophora brassicae and studies of their interaction

A technique for inoculation of Brassica seedlings with single resting spores of Plasmodiophora brassicae is described. Three isolates derived from single spores were produced from one population of P. brassicae. They gave different reactions with the European Clubroot Differential (ECD) series, and two gave different reactions from those of the spore suspensions from which they were derived. When two isolates were mixed together, spores of one isolate restricted infection by spores of the other isolate.     

土壤pH对芸薹根肿菌侵染及病害发生的影响

通过设置不同pH梯度,研究土壤pH对根肿菌侵染及病害发生的影响。结果表明:土壤酸性时病菌侵染速度快,碱性时慢,而强酸性和碱性土壤条件则抑制孢子萌发;pH为6.0时最有利于根肿菌休眠孢子萌发,萌发率最高,为53.96%;碱性条件可使初级原生质团变形凝结成球状,不能正常分裂或延迟形成游动孢子囊,从而不利于根肿菌侵染。白菜发病率与病情指数随pH升高,呈先上升后下降趋势。其中,pH为5.0时,发病率和病情指数最高,pH 7.0~8.0时发病轻。因此,适宜的偏酸性环境条件下,通过作用于病菌休眠孢子萌发和侵染,提高病害危害程度,而中性或碱性条件干扰该过程并降低病害发生。     

新疆北部棉区黄萎病菌种群致病性分化及变异

采用特异性引物PCR检测技术和鉴别寄主法对新疆北部棉区41个棉花黄萎病菌菌系进行检测,以期明确黄萎病菌种群致病性分化及变异。特异性引物(ND1/ND2和D1/D2)PCR检测结果显示,供试菌系中落叶型菌系16个,占39.0%;非落叶型菌系24个,占58.5%;1个菌系未能检测出致病类型。鉴别寄主法测定结果显示:41个供试菌株中致病性强、中、弱的菌系分别为21、12和8个,分别占51.2%、29.3%和19.5%。落叶型菌系的致病力明显高于非落叶型,平均病情指数分别为39.4和25.8。研究表明,无论落叶型菌系和非落叶型菌系,接种后都可产生落叶症状,但落叶的程度有明显差别,其落叶症状的轻重不仅与菌系的致病类型有关,还与品种的抗病性密切相关。     

Usefulness of nonhost plants in managing Plasmodiophora brassicae

Germination of resting spores of Plasmodiophora brassicae , causal agent of clubroot in crucifers, may be stimulated by certain nonhost plants. Without a host plant to infect, such germination would lead to a reduced persistence of resting spores in the soil. The effect of four nonhost plants on P. brassicae was investigated in a 3-year field experiment and a 14-month glasshouse experiment. Three of the plant species used, leek ( Allium porrum ), winter rye ( Secale cereale ) and perennial ryegrass ( Lolium perenne ), have been reported to stimulate resting spore germination, while the fourth, red clover ( Trifolium pratense ), does not. In the field experiment, none of the plant species reduced the concentration of P. brassicae in soils when tested with bioassay plants (Chinese cabbage, Brassica rapa var. pekinensis ). In the glasshouse experiment, there was a lower disease level in all plant treatments compared with the plant-free control following incorporation and decomposition of plant roots. At this time, pH in the soils with plant treatments was higher than that in the control soil. There were no indications of a species-specific interaction between any of the nonhost plants investigated and P. brassicae , and it cannot be concluded that any of them would be useful in the sanitation of P. brassicae -infested soils within short time periods.     

芦笋茎枯病病菌鉴别寄主的构建

<正>芦笋(Asparagus officinalis Linn)又称石刁柏,百合科天门冬属,是世界十大名菜之一,在国际市场上被称为"蔬菜之王"。由于其营养价值高,并能润肺、镇咳、祛痰,具有抑制肿瘤生长的药理功能,深受人们的喜爱[1]。近年来,随着芦笋市场与栽培面积的扩大,病害的发生也逐年加重,尤其茎枯病的严重发生已影响芦笋的产量与质量,轻者生长发育不良,降低产量与品质,重者病株提前枯死,全田毁灭,严重影响芦笋生产与出口创汇[2]。目前该     

棉花黄萎病菌致病力测定及评价方法研究

 棉花黄萎病菌致病力测定及评价是抗病育种及病害综合防治的基础。本文以我国三大棉区的167个黄萎菌菌株为对象,研究致病力测定及评价中不同批次之间病情指数的校准及致病力类型划分标准等关键技术环节。结果表明,以中等致病力类型菌株Vd076为校正菌株,以感病性稳定的冀棉11为校正鉴别寄主,以校正菌株在校正鉴别寄主上病情指数达到50.0±5.0时的调查结果进行各菌株的致病力评价,可获得较好的校正效果,使不同批次试验数据具有可比性;依据聚类分析结果,制定了致病力类型划分标准,强、中、弱3种致病力类型的平均校正病指分别为﹥40.0、20.1~40.0和20.0;通过对不同鉴别寄主组合的分析,推荐陆地棉中棉所41号、豫棉21、鲁棉研28、中棉所35号、中棉所8号、冀棉11作为鉴别寄主。该研究为棉花黄萎病菌致病力变异等相关研究工作提供了技术支撑。     

Induction of root-mat symptoms on cucumber plants by Rhizobium, but not by Ochrobactrum or Sinorhizobium, harbouring a cucumopine Ri plasmid

Ochrobactrum CSL 2573, Rhizobium CSL 2411 and Sinorhizobium CSL 2611 strains harbouring the Agrobacterium cucumopine Ri plasmid (pRi), previously were shown to induce root-mat symptoms in an in vitro cucumber cotyledon assay. In whole-plant, rockwool-grown cucumber host tests Rhizobium CSL 2411 was shown to be as efficient an inducer of root-mat symptoms as the virulent Agrobacterium radiobacter strain NCPPB 4042, which also harbours a cucumopine pRi. Conjugal transfer of pRi to ingressing, avirulent Agrobacterium isolates was observed within root tissues with symptoms. Ochrobactrum CSL 2573 and Sinorhizobium CSL 2611 were not able to induce root-mat symptoms on plants. Rhizobium CSL 2411 and Ochrobactrum CSL 2573 were reisolated from inoculated plants, but Sinorhizobium CSL 2611 was not detected or isolated from inoculated plants 68 days after inoculation. It was postulated that the differences in pathogenicity observed between the in vitro and in situ host tests were caused by a lack of proper attachment to inoculated root tissues by pRi-harbouring Ochrobactrum and Sinorhizobium in the whole-plant host tests.     

Pathotypic diversity of Hyaloperonospora brassicae collected from Brassica oleracea

Downy mildew caused by Hyaloperonospora brassicae is an economically destructive disease of brassica crops in many growing regions throughout the world. Specialised pathogenicity of downy mildews from different Brassica species and closely related ornamental or wild relatives has been described from host range studies. Pathotypic variation amongst Hyaloperonospora brassicae isolates from Brassica oleracea has also been described; however, a standard set of B. oleracea lines that could enable reproducible classification of H. brassicae pathotypes was poorly developed. For this purpose, we examined the use of eight genetically refined host lines derived from our previous collaborative work on downy mildew resistance as a differential set to characterise pathotypes in the European population of H. brassicae. Interaction phenotypes for each combination of isolate and host line were assessed following drop inoculation of cotyledons and a spectrum of seven phenotypes was observed based on the level of sporulation on cotyledons and visible host responses. Two host lines were resistant or moderately resistant to the entire collection of isolates, and another was universally susceptible. Five lines showed differential responses to the H. brassicae isolates. A minimum of six pathotypes and five major effect resistance genes are proposed to explain all of the observed interaction phenotypes. The B. oleracea lines from this study can be useful for monitoring pathotype frequencies in H. brassicae populations in the same or other vegetable growing regions, and to assess the potential durability of disease control from different combinations of the predicted downy mildew resistance genes.     

寄主诱导下的玉米弯孢叶斑病菌致病性分化相关蛋白的动态变化研究

 Reinoculation and two-dimensional electrophoresis were performed to analyze the pathogenicity differentiation of Curvularia lunata.Resistant host inbred lines Shen135,Mo17,and 78599-1 were reinoculated(six generations) with low virulent isolate WS18.Results showed that the disease index had no significant change for the first 2 generations of inoculation.At the third generation,the incidence of disease was increased and the number of differential expressed proteins of mycelia were more than that in the first 2 generations.More than 100 differential expressed proteins were found in the mycelia of fifth generation when compared with the original one.In the experiment,10 differentially expressed proteins were identified by MALDI-TOF-MS analysis.Three proteins were related directly to the differentiation of virulence,2 were related to allergen,4 were related to the metabolism of carbon or signaling pathway and 1 was unkonwn to Curvularia lunata.     

Phytopathogenic, morphological, genetic and molecular characterization of a Verticillium dahliae population from Crete, Greece

A population of 84?V. dahliae isolates mainly originating from Crete, Greece, was characterized in terms of pathogenicity and virulence on different hosts, in parallel with morphological/physiological characterization, vegetative compatibility grouping and mating type determination. Tomato race 2 was found to have supplanted race 1 and was more virulent on a tomato-susceptible cultivar than race 1. Using a differential host classification system which tests pathogenicity to tomato, eggplant, sweet pepper and turnip, 59 isolates were assigned to tomato, 19 to eggplant, one to sweet pepper and five to tomato-sweet pepper pathogenicity groups. All isolates from Crete fell into VCG subgroups 2A, 2B and 4B, while a remarkably high incidence of bridging isolates (compatible with two or more VCGs) was recorded. The tomato-sweet pepper pathogenicity group was morphologically quite distinct from the others, while conidial length and pigment intensity were discriminatory parameters among VCGs 2A, 2B and 4B. PCR-based molecular marker Tr1/Tr2 was reliable in race prediction among tomato-pathogenic isolates, except for members of VCG 4B, while the application of markers Tm5/Tm7 and 35-1/35-2 was highly successful for tomato-pathogenic isolates. E10 marker was related to VCG 2B, rather than to pathogenicity groups. A single nucleotide polymorphism in the ITS2 region, and two novel molecular markers, M1 and M2, proved useful for the fast and accurate determination of major VCGs 2A, 2B and 4B, and can be used for high-throughput population analyses in future studies. The mating type was unrelated to VCG classification and probably does not control heterokaryon incompatibility in V. dahliae.     

Discrimination of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> races among strains from northwestern Spain by <Emphasis Type="Italic">Brassica</Emphasis> spp. genotypes and rep-PCR

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a severe seedborne disease of Brassica crops around the world. Nine races are recognized, being races 1 and 4 the most aggressive and widespread. The identification of Xcc races affecting Brassica crops in a target area is necessary to establish adequate control measures and breeding strategies. The objectives of this study were to isolate and identify Xcc strains from northwestern Spain by using semi-selective medium and pathogenicity tests, determine the existing races of Xcc in this area by differential series of Brassica spp., and evaluate the use of repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) to differentiate among the nine existing Xcc races. Seventy five isolates recovered from infected fields were identified as Xcc. Race-typing tests determined the presence of the following seven pathogen races: 1, 4, 5, 6, 7, 8 and 9. Race 4 was the most frequent in Brassica oleracea and race 6 in Brassica rapa crops, therefore breeding should be focussed in obtaining resistant varieties to both races. Cluster analysis derived from the combined fingerprints showed four groups, but no clear relationship to race, crop or geographical origin was found. Rep-PCR analysis was found not to be a reliable method to discriminate among Xcc races, therefore race typing of Xcc isolates should be done by using the differential series of Brassica spp. genotypes or another alternative approach.     

APN1的原核表达及其与Cry1Ac蛋白体外结合

本研究分析了Cry1Ac蛋白与棉铃虫BBMVs的结合情况;并将棉铃虫apn1基因的全长和片段在大肠杆菌Rosetta(DE3)中进行表达,利用Ligand blotting技术分析原核表达的APN1全长及4个片段与Cry1Ac蛋白的结合情况。结果表明:Cry1Ac蛋白可以与棉铃虫的BBMVs结合;在大肠杆菌中表达的棉铃虫受体蛋白APN1的全长、片段H1(33~257)、H2(258~547)和H3(548~798)可以与Cry1Ac蛋白结合,其中片段H3是首次发现可以与Cry1Ac蛋白结合。本研究的结果也为其他Bt蛋白受体的体外功能研究提供新的借鉴。     

Diagnostics,genetic diversity and pathogenic variation of ascochyta blight of cool season food and feed legumes

Molecular diagnostic techniques have been developed to differentiate the Ascochyta pathogens that infect cool season food and feed legumes, as well as to improve the sensitivity of detecting latent infection in plant tissues. A seed sampling technique was developed to detect a 1% level of infection by Ascochyta rabiei in commercial chickpea seed. The Ascochyta pathogens were shown to be genetically diverse in countries where the pathogen and host have coexisted for a long time. However, where the pathogen was recently introduced, such as A. rabiei to Australia, the level of diversity remained relatively low, even as the pathogen spread to all chickpea-growing areas. Pathogenic variability of A. rabiei and Ascochyta pinodes pathogens in chickpea and field pea respectively, appears to be quantitative, where measures of disease severity were based on aggressiveness (quantitative level of infection) rather than on true qualitative virulence. In contrast, qualitative differences in pathogenicity in lentil and faba bean genotypes indicated the existence of pathotypes of Ascochyta lentis and Ascochyta fabae. Therefore, reports of pathotype discrimination based on quantitative differences in pathogenicity in a set of specific genotypes is questionable for several of the ascochyta-legume pathosystems such as A. rabiei and A. pinodes. This is not surprising since host resistance to these pathogens has been reported to be mainly quantitative, making it difficult for the pathogen to overcome specific resistance genes and form pathotypes. For robust pathogenicity assessment, there needs to be consistency in selection of differential host genotypes, screening conditions and disease evaluation techniques for each of the Ascochyta sp. in legume-growing countries throughout the world. Nevertheless, knowledge of pathotype diversity and aggressiveness within populations is important in the selection of resistant genotypes.