Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.     

晋中地区甘蓝蚜对杀虫剂的敏感性变化及药剂混配的增效作用

为了了解甘蓝蚜对药剂的敏感性变化及药剂混配的增效作用,采用FAO推荐的微量点滴法,测定了山西省晋中地区田间甘蓝蚜种群对药剂的敏感性、相对敏感基线,及不同类型杀虫剂混配的增效作用。结果表明:甘蓝蚜对杀虫剂的敏感性降低,对啶虫脒、高效氯氰菊酯和齐螨素抗药性为原来的11.10倍、14.34倍和16.13倍,对氰戊菊酯、辛硫磷和吡虫啉的抗药性为原来的83.33倍、101.50倍和143.38倍,对灭多威的抗药性为原来的161.75倍;混配增效试验结果表明,辛硫磷和灭多威以有效成分1∶0.5,高效氯氰菊酯和齐螨素以1∶3、1∶6和1∶9混配后都具有明显的增效作用。     

Comparison of techniques for estimation of resting spores of Plasmodiophora brassicae in soil

Clubroot (Plasmodiophora brassicae) is an important disease of canola (Brassica napus) and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating P. brassicae resting spores in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC-PCR), propidium monoazide PCR (PMA-PCR), droplet digital PCR (ddPCR) and loop-mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long-term rotation study at Normandin, Québec, with replicated plots representing 0-, 1-, 2-, 3-, 5- and 6-year breaks following susceptible canola infested with clubroot. CPIC-PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC-PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA-PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly subsequently.     

RAPD markers linked to a clubroot-resistance locus in Brassica rapa L.

Linkage of random amplified polymorphic DNA (RAPD) markers with resistance genes to clubroot (Plasmodiophora brassicae Wor.) in Brassica rapa L. was studied in a doubled haploid (DH population obtained by microspore culture. Thirty-six DH lines were obtained from F₁ plants from a cross between susceptible ‘Homei P09’ and resistant ‘Siloga S2’ plants. ‘Homei P09’ was a DH line obtained by microspore culture of the Chinese cabbage variety ‘Homei’, which is highly responsive in microspore culture. The resistant line ‘Siloga S2’ was obtained by two rounds of selfing of the fodder turnip ‘Siloga’. Three RAPD markers, RA12-75A, WE22B and WE49B, were found to be linked to a clubroot-resistance locus. These three markers were linked in the DH lines and an F₂ population and should be useful for marker-assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

云南白菜根肿病有机防治技术的研究

选择高锰酸钾、石灰或草木灰、腐熟农家肥等有机投入品对根肿病的控制效果和施用方法进行了初步尝试。实验结果表明,有机肥、石灰、石灰水、高锰酸钾可以作为控制根肿病防治的物质,随着使用时间和使用方式的不同,对根肿病和蔬菜的产量的影响不同。石灰或草木灰塘施或对水浇施,腐熟农家肥和石灰或高锰酸钾配合使用对根肿病的发生均有较好的抑制作用;单施农家肥和高锰酸钾对根肿病控制效果不理想,用腐熟农家肥作底肥＋1%石灰水播种后浇塘＋1%石灰水三叶期浇根＋1%石灰水六叶期再浇根的组合模式可以有效地控制对根肿病的发生与危害。     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

甘蓝夜蛾研究进展

为给甜菜田甘蓝夜蛾的防治提供参考依据,笔者综述了甘蓝夜蛾生物学特性、生理生化特性和防治策略三个方面的研究进展。在生物学特性方面总结了虫态和发育历期、趋光性、滞育、人工饲养饲料配方、取食规律、迁移规律、种群消长规律、卵空间分布型和天敌种类9部分内容。在生理生化特性方面归纳了热休克、性信息素、抗药性基因P450、几丁质酶基因、消化酶5部分内容。在防治策略方面,简述了化学防治和生物防治施用的药剂种类、剂量、时期,提出了甜菜田甘蓝夜蛾的治理应遵循“以种群发生动态监测和抗药性监测为指导,以化学农药减量增效为核心,化学防治、生物防治并行”的治理策略。     

The exploration of genetic resources of Portuguese cabbage and kale for resistance to several Brassica diseases

Summary Twenty three accessions of nine Portuguese cabbage and kale land races from different geographic origins were tested at the seedling stage for resistance to several important brassica diseases. Resistance to downy mildew (Peronospora parasitica), expressed as necrosis of the cotyledon mesophyll, was found in all the accessions. Type A resistance to cabbage yellows (Fusarium oxysporum f. sp. conglutinans race 1) was present in most of the landraces. Resistance to clubroot (Plasmodiophora brassicae race 6) was found in one accession of the Portuguese tree kale. High resistance to blackleg (Leptosphaeria maculans) and white rust (Albuco candida) was not detected, although several accessions showed 20 to 30% of plants with intermediate expression of resistance. All Portuguese cole accessions were susceptible to blackrot (Xanthomonas campestris pv. campestris).     

芸薹根肿菌致病机理的研究进展

十字花科植物根肿病是由芸薹根肿菌的侵染而引起,由于该病原菌存活时间长,传染性强,传播速度快,传播途径多样,以及对根肿菌的生活史和致病成灾机制认识不足,使得近期根肿病在中国的发生越来越严重。笔者从近年来芸薹根肿菌致病机理的相关研究进展入手,综述了生长素、细胞分裂素、水杨酸、茉莉酸和脱落酸等植物激素,氧化酶、芥子油苷和类黄酮、多胺等次生代谢物质的变化与芸薹根肿菌致病的关系,分析了芸薹根肿菌致病过程和十字花科植物在应对根肿菌侵入过程中的主要生理生化反应、代谢改变过程,以期对进一步揭示芸薹根肿菌致病机理研究及根肿病有效防控起到一定的参考和借鉴作用。     

Plasmodiophora brassicae: aspects of pathogenesis and resistance inBrassica oleracea

Summary Clubroot is one of the most damaging diseases inBrassica oleracea crops world-wide. The pathogenicity ofPlasmodiophora brassicae is highly variable between as well as within field populations. Several sources of resistance to clubroot have been identified inB. oleracea. Generally, resistance tends to inherit partly as a recessive, partly as an additive trait, and appears to be controlled by few major genes. Progress in the understanding of the inheritance of resistance is being made through the use of single-spore isolates of the pathogen, and the use of molecular markers for resistance genes.Abbreviations cv cultivar - DH doubled haploid - ECD European Clubroot Differential set - RFLP Restriction Fragment Length Polymorphism