Biological Control of Soft Rot of Chinese Cabbage Using Single and Mixed Treatments of Bacteriocin-producing Avirulent Mutants of Erwinia carotovora subsp. carotovora

Two pathogenic strains of E. carotovora subsp. carotovora 2T-2 and TT-4 with high bacteriocin activity but low sensitivity to the bacteriocins of other strains were treated with ethyl methane sulphonate (EMS). Two avirulent mutants A-f-39 and B-e-19 developed from 2T-2 and TT-4, respectively, by this treatment had the same bacteriocin activity as their respective parents and inhibited the in vitro growth of pathogenic strains of this species. The disease control of these two mutant strains were compared in the field in 1995 and 1997 to the control by CGE234M403 (M403) (a commercialized biocontrol agent), a mixture of A-f-39 and M403, and an agrochemical (basic dithianon-copper chloride). The protection obtained with A-f-39 was comparable to M403 and was better than that with the chemical. The mixture of A-f-39 and M403 consistently gave the best results in all the field trials. Received 17 September 1999/ Accepted in revised form 22 February 2000     

大白菜软腐病菌游动性突变体在大白菜体内增殖扩展研究

 用培养皿滤纸吸附测定法和不伤根土壤拌菌处理及针刺接种法,测定了大白菜软腐病菌游动性突变体进入大白菜体内、并在其中侵染定殖和扩展的特性。结果表明,游动性丧失和增强的突变体都可以通过种子萌发和主动接触进入大白菜体内、并可以在体内有短期的繁殖,但菌量远低于野生菌。大白菜叶片接种实验说明,这两种突变体也都可以进行短距离扩展,但扩展距离和菌的繁殖量低于野生菌。     

几种化学物质诱导彩色马蹄莲对软腐病抗性的研究

本试验测定了纳米硅、草酸、硅酸钠和硫酸亚铁4种化学物质在不同浓度下对彩色马蹄莲软腐病菌的室内抑菌活性和诱导抗病效果。结果表明,硫酸亚铁3种浓度对彩色马蹄莲软腐病菌均表现较强的抑制作用;1 g/L纳米硅、0.15 g/L草酸和0.2 g/L硅酸钠3种化学物质对软腐病菌无明显抑制作用且诱导抗病效果较好,分别达到62.28%、77.56%、88.46%;经3种化学物质诱导处理后再进行接种,诱导处理和接种期间彩色马蹄莲叶片组织内过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)酶活性均高于对照,其中硅酸钠处理后彩色马蹄莲叶片组织内POD、PAL活性高于纳米硅和草酸处理,草酸处理后叶片组织内PPO活性高于纳米硅和硅酸钠处理。     

Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora

 Erwinia carotovora subsp. carotovora (Ecc) is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated nonmotile mutants in Ecc by in vivo insertional mutagenesis using a transposon Tn5. The sequence disrupted by the Tn5 insertion in YMU1 and YMU5 mutants was highly homologous to that of flhC and flhD genes, respectively. They are involved in the initiation of the expression of flagellum-related genes in many gram-negative bacteria such as Escherichia coli and Salmonella. With electron microscopy, the flhC and the flhD homolog mutants were shown to be aflagellate. Furthermore, the virulence of these mutants was greatly reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that flagellar formation is required for the pathogenicity of Ecc. Received: November 5, 2002 / Accepted: December 2, 2002 Acknowledgments This research was supported in part by Grant-in-Aid (12052210) and by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13073).     

88％水合霉素可溶性粉剂防治大白菜软腐病的田间药效试验

田间药效试验表明,88％水合霉素可溶性粉剂对秋季大白菜软腐病有较好的防治效果,3000倍稀释液防效达91．63％,其防效明显优于77％可杀得。     

拮抗链霉菌26B的筛选及其在不同含水量土壤中对白菜软腐病的防病效果

为挖掘防治白菜软腐病的生防菌,本研究采用牛津杯法从38株白菜根际放线菌中筛选到一株拮抗放线菌26B,通过形态学观察和16S rRNA基因序列分析将菌株26B初步鉴定为链霉菌。该菌除菌发酵滤液对供试的白菜软腐病菌Pectobacterium carotovorum subsp. brasiliensis BC1及3种马铃薯黑胫病菌均表现较高的抑菌活性,其中对白菜软腐病菌抑菌直径达到23.97mm。萌发试验表明,白菜种子经26B除菌发酵滤液浸种后发芽势、发芽指数、根长及鲜重显著增加。采用浸根法测定菌株26B除菌发酵滤液对软腐病菌荧光标记菌株BC1-gfp在白菜根部定殖量的影响,浸根处理后48h,白菜根上BC1-gfp的数量达到2.3×109CFU/g;未从26B除菌发酵滤液与BC1-gfp菌悬液混和处理（体积比10%）的白菜根上检测到病原菌。盆栽防病试验结果表明,在含水量为14%和20%的土壤中,菌株26B除菌发酵滤液对白菜软腐病的防效分别达96.0%和89.8%。上述结果表明,链霉菌26B是一株具有潜在开发应用价值的生防菌株,将链霉菌26B应用与土壤水分管理相结合可进一步提高其防病效果。     

Pythium rot of Chinese cabbage (Brassica rapa L. subsp. pekinensis) caused by Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of Chinese cabbage (Brassica rapa L. subsp. pekinensis) in Ibaraki Prefecture every year in early September from 2002 through 2004. The causal fungus was identified as Pythium aphanidermatum (Edson) Fitzpatrick. This is the first report of P. aphanidermatum on Chinese cabbage. A similar disease of Chinese cabbage caused by P. ultimum Trow var. ultimum is known as Pythium rot. We propose adding P. aphanidermatum as a new pathogen of this disease.     

Evaluation Of Tests to Determine Resistance of Zantedeschia spp. (Araceae) to Soft Rot caused by Erwinia carotovora subsp. carotovora

Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in Zantedeschia spp., particularly in cultivars from the section Aestivae. The disease can be partly controlled by cultural measures, but by combining cultural methods with resistant plant material a promising strategy for control of soft rot can be developed. No tests are available for resistance testing in breeding Zantedeschia spp. Therefore, three tests developed for use in potato breeding were adapted for use on eight cultivars of Zantedeschia spp. Variation was found in all three tests. Resistant control cultivar Zantedeschia aethiopica Crowborough scored most resistant in all three tests. Within the section Aestivae, degrees of susceptibility were identified that were in agreement with each other and with field observations, indicating reliability of two of the methods in which tubers were used. The correlation coefficient of these two tests was high. A new non-destructive test method was developed for use on seedlings which involved immersion of leaf disks in a bacterial suspension. The percentage of decayed leaf area was a measure of resistance and results were in general agreement with the other tests. These methods will be useful for breeding for soft rot resistance and performing genetic analyses.     

Characterization of Erwinia sp. causing black rot of papaya (Carica papaya) first recorded in Okinawa Main Island,Japan

Since 2002, papaya black rot has been spreading over several islands of Okinawa Prefecture, Japan. To devise a prevention strategy for the disease, microbiological research on the pathogen was conducted. Twelve strains were isolated from papaya infected with black rot showing symptoms such as water-soaked lesions on stems and petioles, black spots on fruits, and rotted leaves turning yellow with necrotic spots. Through Koch's postulates, we confirmed that the isolated strains caused papaya black rot. Bacteriological assays showed that the strains have characteristics different from the type strains of Erwinia mallotivora, E. papayae, and E. psidii. Moreover, 16S rDNA sequence similarity searches showed that the isolated strains had less than 98.6% similarity with type strains. Additionally, phylogenetic analysis of 16S rDNA sequences suggested that the isolated strains were possibly a novel species belonging to the genus Erwinia, as the strains formed an independent cluster and had low sequence similarity with the type strains. Earlier studies indicated that papaya black rot is caused by E. cypripedii. Therefore, we propose to add the Erwinia sp. isolated in this study to the list of papaya black rot pathogens.     

大白菜软腐病新病原菌Pectobacterium aroidearum的鉴定及其生物学特性

细菌性软腐病是大白菜生产上主要发生病害之一,其病原菌通常为胡萝卜软腐果胶杆菌Pectobacterium carotovorum subsp.carotovorum(Pcc)。从北京房山区大白菜软腐病样中分离获得一株菌株KC20,其在LB固体培养基上的菌落呈圆形、乳白色、半透明、表面光滑且边缘整齐,在半选择性培养基CVP上产生典型的杯状凹陷。致病性测定结果表明,该菌株侵染引发的大白菜软腐症状与田间大白菜软腐自然发病症状相同。利用果胶杆菌属特异引物Y1/Y2可扩增出预期大小为434 bp的目的片段;ITS-PCR和ITS-PCR-RFLP结果发现,KC20与P.carotovorum菌株带型均不相同。KC20的16S rDNA基因完整序列与已报道的标准菌株P.aroidearum SCRI 109T(JN600323)的相应序列相似性高达99%以上;该序列系统发育关系分析表明,KC20与已报道的P.aroidearum菌株聚集,并形成了明显的P.aroidearum类群,基于pmrA基因序列和基于果胶杆菌8个看家基因(acnA、icd、gapA、mdh、mtlD、pgi、proA和rpoS)的MLSA-多位点序列分析进一步支持了这一结果。以上结果表明,引发北京地区大白菜软腐病的病原菌株KC20为果胶杆菌P.aroidearum。人工接种条件下,KC20还可侵染马蹄莲、虎眼万年青、马铃薯、鳄梨、西葫芦、胡萝卜、生菜和芹菜等植物;该菌株具备在37℃以及含有7%NaCl培养基中生长的能力,能液化明胶;可利用棉籽糖、纤维素二糖、蜜二糖,不能利用异麦芽酮糖、D-麦芽糖、D-阿拉伯糖、D-山梨醇、菊糖等。大白菜软腐病新病原菌Pectobacterium aroidearum的发现加深了我们对该病害的了解,为该病害的有效防治提供了参考。     

四川大白菜细菌性软腐病新病原菌Pectobacterium versatile的鉴定

为明确引起四川理县大白菜软腐病的病原菌种类, 从理县梭罗沟村田间采集具有典型软腐症状的大白菜病样, 采用组织分离、致病力测定、生理生化分析、形态学和分子生物学方法进行鉴定。结果表明, 分离获得的5株细菌在NA培养基上形成的菌落呈圆形、半透明、乳白色、中心突起、边缘平滑, 在半选择性培养基CVP上培养48 h后产生凹陷。5株菌均能使大白菜、胡萝卜、马铃薯和芹菜的离体组织腐烂, 接种于白菜苗后叶片呈湿腐萎蔫症状。通过大白菜叶柄接种部位形成的病斑长径来比较菌株致病力强弱, 各菌株的致病力有显著差异。基于看家基因pgi、rpoS、mdh、proA、mtlD 和icdA(NCBI登录号为OL963562~OL963571)的多基因联合系统发育树, 分离菌株与Pectobacterium versatile聚为一簇, dnaX-leuS-recA(NCBI登录号为OL963572~OL963576)的多位点序列分析(MLSA)进一步支持以上结果, 且准确性更高。分离菌株的生理生化特征与P.versatile模式菌株一致, 综上将其鉴定为P.versatile。这是我国首次报道P.versatile引起大白菜软腐病。     

Colonization by two isolates of Plasmodiophora brassicae with differing pathogenicity on a clubroot-resistant cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis)

A commercial clubroot-resistant F₁ cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis), Kukai 70, is resistant to an isolate of populations of Plasmodiophora brassicae from Hagi (HG) city and is susceptible to another from Yamaguchi (YMG) city. The degree and frequency of primary and secondary cycle colonization by the isolates in the root hairs and root tissues of cv. Kukai 70 were compared. Seedlings of cv. Kukai 70 were grown in soils amended with inoculum of either HG or YMG and harvested 10 days after inoculation to observe the primary cycle (number of root-hair infections) and 20, 30, and 40 days after inoculation to observe of the secondary cycle (frequency of infected cells and degree of plasmodial development based on the number of nuclei in infected cells). Although more root hairs were infected in HG than in YMG, fewer cells in root tissues including the cortex and medullary rays were infected in HG than in YMG. In addition, YMG developed plasmodia with many nuclei and formed resting spores, whereas plasmodia remained immature with a small number of nuclei in HG and did not form resting spores even by 40 days after inoculation. These results suggest that suppression of plasmodial development during secondary colonization is associated with resistance mechanisms to HG in cv. Kukai 70. Starch did not accumulate (i.e., development of amyloplasts) in HG-infected cells. This may be involved in the suppression of secondary colonization of P. brassicae in the cultivar.     

Genome-wide analysis and identification of TIR-NBS-LRR genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveal expression patterns to TuMV infection

TIR-NBS-LRR (TNL) genes greatly affect plant growth and development. Ninety TNL-type genes were identified and characterized in Chinese cabbage (Brassica rapa ssp. pekinensis). Tissue-expression profiling revealed different expression levels in different tissues. qRT-PCR analysis revealed the expression patterns of 69 genes challenged by Turnip mosaic virus (TuMV): 42 genes were up-regulated, and 11 genes down-regulated; genes were grouped according to their different expression patterns. Sixteen candidate genes were identified as responding to TuMV infection. This study supplies information on resistance genes involved in Chinese cabbage's response against TuMV, and furthers the understanding of resistance mechanisms in B. rapa crops.