Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach

Highly specific rabbit polyclonal antibodies for the obligate sugar-beet root parasite, Polymyxa betae , were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro , and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta , and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar-beet roots and with the closely related Polymyxa graminis in barley roots, and to cross-react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross-reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea . In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar-beet, barley, cabbage and potato roots.     

Utilization of Asymmetric Somatic Hybridization for the Transfer of Disease Resistance from Brassica nigra to Brassica napus

Asymmetric somatic hybrid calli were produced between Brassica napus and a transgenic (Hyg ^R) line of B. nigra using a donor recipient fusion method for the production of cybrids. The transgenic line of B. nigra used as a donor also possessed genetic resistance to the pathogenic fungi Phoma lingam and Plasmodiophora brassicae. Using hygromycin for selection, 332 hybrid calli were obtained from which 30 produced shoots (1—-20 per callus) which were rooted on a hormone-free culture medium. The rooted shoots were transferred into soil and cultivated in a growth chamber where the plants were tested for resistance against the two pathogens. Out of 129 hybrid plants tested for resistance against P. brassicae, 30 (23.3 %) plants proved to be resistant and from 78 plants tested for resistance against P. lingam, 41 (52.6 %) plants remained disease-free after infection.     

Two-Dimensional Gel Electrophoresis of Proteins in Healthy Roots and Clubroots of Brassica oleracea

Abstract

Seedlings of Brassica oleracea were infected by the pathogenic fungus Plasmodiophora brassicae. causing clubroot formation. Proteins from healthy roots and clubroots were extracted by two different methods and analysed by two-dimensional gel electrophoresis. Host-proteins induced during clubroot infection were not analysed, since the fungus was always present in the infected roots. This work showed that several proteins present in non-infected roots were lost or strongly reduced in the infected roots.     

Clubroot resistance introgression in interspecific hybrids between Raphanus sativus and Brassica napus

Clubroot is a prevailing soil-borne disease affecting rapeseed production worldwide.However,few clubroot resistant rapeseed accessions were available for breeding.Identification and introgression of new clubroot resistant genes from closely related species by distant hybridization is an effective strategy.In the present study,9 radish(Raphanus sativus L.,2n=18,RR)lines resistant to Plasmodiophora brassicae pathotype 4 were used as donors to transfer clubroot resistance into a susceptible rapeseed(Brassica napus L.,2n=38,AACC)line by distant hybridization combined with embryo rescue.Nine intergeneric crosses were made but only 1(411×93039)produced F1 plants both from embryo rescue and natural seed-setting.Authenticity of triploid F1 hybrids(2n=28,ACR)were verified by flower color,cytological observation and molecular marker analysis,and 2 genuine F1 hybrids were identified.After chromosome doubling,these synthetic allohexaploid plants(2n=56,AACCRR)became partially fertile(pollen viability rate=35%)and were backcrossed with rapeseed parent to generate a BC1 population(2n=47,AACCR).Totally 178 BC1 plants were obtained,of which the majority(96.1%)were resistant to clubroot.These backcrossing progenies could be used for the breeding of new rapeseed varieties resistant to clubroot.     

Molecular Cloning and Sequence Analysis of Chitin Synthase cDNA from Mamestra brassicae （L.） （Lepidoptera： Noctuidae） Cuticle

Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761     

基于Dual RNA-seq分析的褪黑素影响萝卜与链格孢菌互作的作用研究

由芸薹链格孢菌(Alternaria brassicae)严重影响萝卜(Raphanus sativus)生产,外源喷施褪黑素可减轻病症,但褪黑素同时存在于植物和微生物中,外源喷施时可对感病植株上寄主和病原物的生命活动均有影响,其介导寄主-病原菌互作的作用模式。为解析褪黑素参与调控病叶上萝卜和链格孢菌互作的生理和基因转录机制,本研究通过对感黑斑病萝卜叶面外施0~1 500 μmol·L^-1浓度褪黑素,检测病情指数变化,同时利用互作转录组分析0、500和1 500 μmol·L^-1褪黑素处理下萝卜和链格孢菌转录水平变化。结果显示,50~500 μmol·L^-1褪黑素可分别显著提高萝卜和链格孢菌双方的生长势和抗逆性,且500 μmol·L^-1表现最优,1 000和1 500 μmol·L^-1与500 μmol·L^-1相比作用较小,呈明显剂量依赖型模式,但500 μmol·L^-1褪黑素处理下,萝卜对链格孢菌抗性明显提升。Dual RNA-seq检出萝卜基因组reads比对率>88%,链格孢菌基因reads比对率≤0.06%,萝卜差异表达基因数显著高于链格孢菌检出数。对萝卜及链格孢菌生长及抗逆性调控相关基因表达模式进行验证,基因表达模式基本与表型反应一致。综上所述,褪黑素介导萝卜与链格孢菌的作用存在低浓度促进、高浓度抑制的剂量依赖性效应,500 μmol·L^-1浓度下褪黑素对双方均具生长促进作用,但褪黑素对寄主的影响超过对链格孢菌的影响,最终表现为诱导萝卜对链格孢菌抗性增强。本研究为褪黑素在萝卜黑斑病抑制中的应用提供了数据支撑和理论基础,并在转录水平上初步解析了褪黑素参与萝卜对链格孢菌抗性变化的作用模式。     

Reduction of resting spore density of Plasmodiophora brassicae and clubroot disease severity by liming

Abstract

Relationships between the disease severity of clubroot caused by Plasmodiophora brassicae, the soil pH value and the concentration of exchangeable calcium associated with liming were investigated under controlled density of resting spores. Disease indices were lower in the plots treated with lime than in the control plots without lime application. The disease index was significantly lower when lime materials were mixed two weeks before sowing compared with four weeks before sowing. The reduction rate of the disease index was larger for a concentration of 2.0 g kg^?1 than 1.0 g kg^?1 of lime in soil. The density of the resting spores in soil at the time of sowing was significantly reduced by liming. The reduction rate was 17–31 % for calcium cyanamide, 12–29% for dolomite, and 20–39% for calcium carbonate compared with the control plot. It was suggested that the disease severity was influenced by both the soil pH and the content of exchangeable calcium in soil based on the analysis of covariance.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Peronospora parasitica and Plasmodiophora brassicae

Summary Forty-four landraces of Portuguese coles (Brassica oleracea L.), representing different morphotypes, were screened at the cotyledon stage for downy mildew resistance, caused by Peronospora parasitica. Two isolates of the pathogen, PHW 630 and PHW 828, were used in the experiment. The mean disease interaction phenotype varied among the landraces. Overall, the Portuguese coles have shown resistance to both isolates, particularly to isolate PHW 828. Some landraces were identified with differential reaction to the two P. parasitica isolates. Some of the Portuguese coles are, therefore, good sources of resistance genes to P. parasitica, and could be used in breeding programs or in the development of a downy mildew host differential set. Among the morphotypes tested, Couve Algarvia (ISA 207), Couve Galega (ISA 243 and 187) and Couve Glória de Portugal (ISA 84) have presented the highest resistance interaction. For the development of host differential sets, the landraces ISA 2 (Penca de Mirandela), ISA 35 (Couve de Valhascos) and ISA 600 (Penca Espanhola) could be further selected for the differentiation of P. parasitica isolates. They are resistant to PHW 828 and susceptible to PHW 643.The same morphotypes and landraces were also screened at the seedling stage for resistance to clubroot, caused by Plasmodiophora brassicae Woron.. Although some variation for resistance could be noticed in some the landraces tested, mainly Galega kale types, none of them presented a sufficient level of resistance as to be directly be useful in breeding for resistance to race 6 of P. brassicae.     

芸薹根肿菌生理小种鉴别方法研究进展

本文综述了芸薹根肿菌生理小种鉴别方法和国际通用的根肿病生理小种鉴别系统及鉴别寄主,详细介绍了“Williams”和“ECD”根肿病鉴别系统的优缺点及适用范围,分析了分子生物学鉴定根肿病生理小种的技术,以期为抗病品种选育和持续控制根肿病提供准确的抗病鉴定方法。     

Transfer of resistance to race 2 of Plasmodiophora brassicae from Brassica napus to cabbage (B. oleracea var. capitata). II. Meiosis in the interspecific hybrids between B. napus and 2x and 4x cabbage

Summary Meiosis in 14 interspecific F₁ hybrids with three chromosomal levels (triploid, tetraploid, hexaploid; 2n=28, 37 and 55) between Brassica napus L. and 2x and 4x cabbage (B. oleracea var. capitata L.) was studied. The oleracea genome from B. napus maintained close homology with the c genome of cabbage while the campestris genome of B. napus showed partial homology with the c genome contained in the hybrids. Genotypic influence on chromosome pairing was indicated. Structural chromosome differences and spontaneous chromosome breakage and reunion were suggested as causes for the abnormalities which related to the unbalance of the genotypes. The divergence of the genomes of B. napus and B. oleracea and the need for the qualification of the term secondary association were discussed.Contribution No. J. 673, Research Station, Agriculture Canada, St. Jean, Québec.