十字花科根肿病研究进展

总结了十字花科作物根肿病病原菌的分化、生物学特征、侵染方式、侵染症状、检测技术以及根肿病病害发生规律、抗病育种和防治方法,并展望了我国十字花科作物根肿病的研究方向。     

芸薹根肿菌活细胞PMAxx-qPCR快速定量检测方法的建立与应用

【目的】由芸薹根肿菌（Plasmodiophora brassicae）侵染引起的十字花科根肿病是一种世界性土传病害,病原菌长期存在于土壤中,对十字花科作物造成严重威胁。改良叠氮溴化丙锭（propidium monoazide xx,PMAxx）可选择性地穿透受损的死细胞膜,并抑制死细胞DNA的实时荧光定量PCR（qPCR）扩增。本文将PMAxx与qPCR技术相结合,建立一种快速检测芸薹根肿菌活菌的方法,为根肿病的早期诊断及制定科学的防控措施提供依据。【方法】配置浓度分别为0、5、10、20、40、60 µmol·L^-1的叠氮溴化丙锭PMA和改良叠氮溴化丙锭PMAxx,比较两种核酸染料对芸薹根肿菌死细胞DNA扩增的抑制效果,确定最佳核酸染料及工作浓度;设置光照时间分别为0、2、5、10、15和20 min,进行最佳光照时间的优化,建立芸薹根肿菌活细胞PMAxx-qPCR快速检测体系。设置芸薹根肿菌活孢子百分比为0、0.01%、0.1%、1%、10%、25%、50%、75%和100%的混合体系,验证PMAxx-qPCR体系的准确性,并应用于田间土壤样本中芸薹根肿菌活孢子的定量检测。【结果】PMAxx对芸薹根肿菌死细胞DNA的扩增抑制效果更好,当芸薹根肿菌浓度为1×10⁸个孢子/mL,PMAxx预处理的最适终浓度为4 µmol·L^-1,最佳光照时间为10 min时,可有效地抑制死孢子DNA的扩增,仅以有活力孢子DNA为靶标选择性地扩增。利用PMAxx-qPCR技术检测已知不同活孢子比例的菌悬液样品,各样品实测孢子存活率和理论存活率之间呈正相关（R²=0.992）。对田间采集的25份土壤样本,采用PMAxx-qPCR方法检测到11份样本中携带芸薹根肿菌,活细胞DNA浓度为32.35—6.97×10³ fg·g^-1。【结论】建立了基于PMAxx-qPCR的芸薹根肿菌活细胞定量检测技术,该技术具有快速、准确、灵敏的特点,解决了qPCR不能仅对活体病原菌进行准确鉴别和定量分析的问题,为制定有效的根肿病防控策略提供了依据。     

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.     

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).     

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.     

安徽油菜根肿病菌生理小种鉴定及品种抗病性评价

十字花科作物根肿病在安徽省黄山市油菜种植区发生日趋严重, 已成为限制油菜产量和品质的重要因素。为明确安徽黄山油菜产区根肿菌的生理小种及主栽油菜品种的抗病性,采用Williams法对安徽省黄山市部分地区的根肿病菌进行了生理小种鉴定,采用田间自然发病方法在根肿病发病严重地区对30个油菜品种进行抗性研究。结果表明,目前安徽省黄山油菜根肿病菌为生理小种4号和9号。荃银油5号、德油早1号等品种为抗病品种。这些品种可作为当地根肿病病区主栽品种及育种材料使用。     

Growth and movement of secondary plasmodia of Plasmodiophora brassicae in turnip suspension-culture cells

Growth of secondary plasmodia of the clubroot pathogen Plasmodiophora brassicae was studied in dual culture of P. brassicae and turnip suspension cells. Suspension culture of P. brassicae -infected turnip cells was achieved by using P. brassicae -infected callus in Murashige and Skoog medium supplemented with 0·1 mg 2,4-D L⁻¹ and 0·02 mg kinetin L⁻¹. The shape of secondary plasmodia in suspension cells was spherical-to-subspherical. A few young plasmodia divided and became numerous spherical, small plasmodia which eventually formed a plasmodial cluster. The plasmodia fused and became vegetative plasmodia. Infected cells were significantly larger than noninfected cells. Secondary plasmodia moved within transformed turnip suspension host cells by cytoplasmic streaming of the host cells. Secondary plasmodia divided in synchrony with the transformed turnip cells.     

Disease response of resynthesized Brassica napus L. lines carrying different combinations of resistance to Plasmodiophora brassicae Wor.

Resistance responses of resynthesized Brassica napus lines to infection with Plasmodiophora brassicae were investigated. Lines that were derived from interspecific crosses between clubroot-resistant B. rapa and resistant B. oleracea exhibited very broad and effective resistance in both greenhouse and field tests. When clubroot resistance was introduced into resynthesized lines from the B. oleracea parent only, the plants were mainly susceptible. Interspecific hybrids from the most resistant parental genotypes, i.e. B. campestris ECD-04 and the B. oleracea cultivars ECD-15 or ‘Bohmerwaldkohf’, were used to initiate a B. napus resistance-breeding programme. These artificial rapeseed lines were resistant to isolates that were virulent on all B. napus differential lines and/or parental lines. Preliminary segregation analysis suggests that their resistance is due to at least two dominant and unlinked genes. In some cases progenies from selfed resynthesized plants exhibited resistance reactions that differed from those of the parental hybrid plant; this may have been the result of cytological instability.     

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