High-throughput screening reveals small molecule modulators inhibitory to Acidovorax citrulli

Acidovorax citrulli is a seed-transmitted gram-negative bacterium that can cause substantial economic yield loss in watermelon and melon production worldwide. Four small-molecule libraries containing 4,952 compounds were selected for high-throughput screening against A. citrulli wild-type strain Xu3-14 by evaluation of growth inhibition. One hundred and twenty-seven molecules (2.5% hit rate) were identified as bactericidal or bacteriostatic against A. citrulli at 100 μM. Secondary screens indicated that 27 candidate compounds were more effective against A. citrulli Group II strains than Group I strains (classified using repetitive element PCR). Several compounds were inhibitory to other pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Xanthomonas campestris pv. campestris, but did not affect the growth of plant beneficial bacteria Pseudomonas fluorescens and Bacillus subtilis. More than half of the compounds did not inhibit germination of Arabidopsis or watermelon seeds. The effect of small molecules on A. citrulli seed-to-seedling transmission was evaluated by applying each compound to inoculated watermelon seeds and assessing seedling infection. Nine compounds were chosen for further investigation based on their reduction of percentage seedling infection and compiling scores on their specificity, sensitivity, and phytotoxicity obtained in the secondary screens. The five best compounds were selected (thiamphenicol, nadifloxacin, pipemidic acid, ciclopirox, and zinc pyrithione) for greenhouse tests and were found to effectively reduce the seed-to-seedling transmission of A. citrulli in both artificially and naturally infested seeds. These top five compounds provide a basis for future development of an A. citrulli-specific bactericide.     

pyrG基因对西瓜噬酸菌致病性和组氨酸利用的影响

为研究pyr G基因与西瓜噬酸菌(Acidovorax citrulli)致病相关性,利用同源重组的方法构建pyr G基因突变体(Δpyr G),对pyr G基因的功能进行分析。结果显示,pyr G基因影响病原菌的致病性、游动性、生物膜形成和定殖能力以及激发烟草过敏反应能力;pyr G基因影响his D基因的表达;此外,pyr G基因突变影响西瓜噬酸菌对组氨酸的利用。     

苋菜活性物对西瓜细菌性果斑病带菌种子萌发的影响

采用抑菌圈法测定苋菜活性物对西瓜细菌性果斑病菌的毒力,平皿培养法测定苋菜活性物对带菌西瓜种子萌发的影响。结果表明,苋菜活性物对西瓜细菌性果斑病菌的最小抑菌浓度为5 mg/m L,EC50为146.349 2 mg/m L;无菌种子、带菌种子、16.6%乙醇处理的带菌种子、苋菜活性物处理的带菌种子的发芽势分别为87.3%、42.7%、48.0%、60.0%,发芽率分别为93.3%、54.7%、58.7%、90.0%;经5 mg/m L的苋菜活性物处理的带菌西瓜种子的发芽势和发芽率分别为64.7%和90.7%,而经80 mg/m L的苋菜活性物处理的带菌种子的发芽势和发芽率分别为3.3%和4.7%。这表明苋菜活性物对西瓜细菌性果斑病菌有较强抑制作用,在低浓度时既有抑菌效果又可促进种子萌发,极具开发和应用前景。     

西瓜噬酸菌ftsH基因功能分析

 AAA ATPase (ATPase associated with diverse cellular activities) 在西瓜噬酸菌的多种生命过程中发挥重要作用。AAA家族中的FtsH蛋白(filamentation temperature-sensitive H)是由ftsH基因编码的,参与生长、致病、环境应激反应、膜内在蛋白质量控制等过程的蛋白,但其在西瓜噬酸菌中的作用尚未明确。本实验以西瓜噬酸菌Aac5菌株为研究对象,通过构建基因缺失突变体,测定致病力等各项表型以及突变株中致病相关基因和其他AAA ATPase基因的表达量,初步探索西瓜噬酸菌中ftsH的功能,为FtsH蛋白的功能研究奠定基础。结果表明,ftsH缺失显著降低菌株致病力、运动能力、生物膜形成能力、生长能力和环境胁迫耐受能力,不影响烟草过敏性反应,显著影响西瓜噬酸菌致病相关基因、AAA ATPase基因以及热激转录因子σ³²的表达量。这表明ftsH基因的功能与西瓜噬酸菌的致病性和环境耐受性密切相关。     

浙江省甘薯上发现瓜类细菌性果斑病菌

通过对浙江宁海县疑似甘薯茎腐病等检疫性病害的调查,在甘薯病株上分离纯化获得能够引起烟草过敏反应的4株分离物,通过菌落形态观察、革兰氏染色反应、16S rRNA序列分析以及致病性测定等细菌学测定,将这4株菌株都鉴定为西瓜噬酸菌(Acidovorax citrulli,原Acidovorax avenae subsp. citrulli),序列同源性高达99.00%~100.00%。瓜类细菌性果斑病菌为我国的检疫性有害生物,务必引起高度重视,以保障浙江重要经济作物西甜瓜产业的健康发展。     

Characterization of Acidovorax citrulli strains isolated from solanaceous plants

Acidovorax citrulli is the causal agent of bacterial fruit blotch disease of cucurbits. Strains of this pathogen are distributed into two major groups: Group I strains have been mainly isolated from melon and other non-watermelon cucurbits, while Group II strains have been mainly recovered from watermelon. Here we report the characterization of strains T1 and EP isolated from diseased tomato and eggplant plants, respectively, and further confirmed to belong to A. citrulli species. Based on PCR, PFGE, and rep-PCR, these strains showed high similarity to the Group II strain 7a1. Sequencing and comparative analyses revealed that the genomes of T1 and EP aligned with that of the Group II model strain AAC00-1, over 97.88% and 99.22%, respectively. The virulence of T1, EP, and 7a1 determined on tomato, eggplant, and watermelon was similar and significantly higher than that of Group I strain M6. In contrast, M6 was more virulent on melon. Expression levels of seven virulence genes measured 24 hr after inoculation of tomato, eggplant, watermelon, and melon showed that the expression pattern was generally similar in strains 7a1, T1, and EP, whereas for M6 the expression was high only on melon. Overall, our results indicate that the solanaceous strains belong to Group II. To the best of our knowledge, this is the first study that reports characterization of A. citrulli strains isolated from solanaceous species. The fact that A. citrulli is able to naturally colonize and cause disease in non-cucurbit crops poses additional challenges for management of this important pathogen.     

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds was investigated. Citron melon seed lots infected with A. citrulli were generated in the field by inoculating either the pistils (stigma) or pericarps (ovary wall) of the female blossoms. Seventeen A. citrulli isolates from 14 different haplotypes belonging to two different groups (group I and II) were used for inoculation. After confirming that 100% of seed lots were infected, they were stored at 4°C and 50% RH for 7 years. After storage, the viability of A. citrulli cells from individual lots was determined by plating macerated seeds on semiselective medium as well as growing seeds for 14 days and scoring for bacterial fruit blotch symptoms. The type of A. citrulli isolate (group I or group II) used did not significantly influence bacterial survival. However, A. citrulli survival was significantly greater in seed lots generated via pistil inoculation (52·9 and 29·4%) than via pericarp inoculation (23·5 and 17·6%). Repetitive extragenic palindrome (rep)‐PCR on A. citrulli isolated from citron melon seed lots after storage displayed similar fingerprinting patterns to those of the reference strains originally used for blossom inoculation, indicating that cross‐contamination did not occur. The results indicate that A. citrulli may survive/overwinter in citron melon seeds for at least 7 years and bacterial survival in seed was influenced more by method of blossom inoculation than by the type of bacterial isolate.     

甘蔗赤条病菌巢式PCR检测

甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。     

Bacterial pathogens of rice in the Kingdom of Cambodia and description of a new pathogen causing a serious sheath rot disease

A study of rice diseases in Cambodia from 2005 to 2007 showed widespread occurrence of diseases caused by Acidovorax avenae subsp. avenae, Burkholderia gladioli, B. cepacia and Pantoea ananatis. This is the first report of these pathogens in Cambodia. Additionally, a pseudomonad causing a widespread disease similar to sheath brown rot (caused by Pseudomonas fuscovaginae) was isolated. The studied strains were pathogenic to rice cvs Sen Pidau and IR 66, producing similar, though slightly less severe, symptoms to those observed in the field. Based on comparative 16S rDNA gene sequence analysis, combined with cell wall fatty acid analysis and metabolic profiles, the isolated strains were allocated to the genus Pseudomonas. The novel species were differentiated from Pseudomonas fuscovaginae and P. putida by their inability to metabolize d ‐fructose, d ‐galactose, d ‐galactonic acid lactone, d ‐galacturonic acid, d ‐glucosaminic acid, d ‐glucuronic acid, p‐hydroxy phenylacetic acid, d ‐saccharic acid and urocanic acid. The major fatty acids were C_16:0, summed feature 3 (C_16:1ω7c and C_16:1ω6c) and summed feature 8 (C_18:1ω7c), representing 80% of the total. Partial 16S rRNA gene sequences (1460 bp) were identical, except for two nucleotide changes amongst the six strains. Alignment of the causal strains within type‐culture databases revealed similarities of 99·7% with Pseudomonas parafulva AJ 2129^T, 99·2% with P. fulva IAM 1592^T, 98·9% with P. plecoglossicidia FPC 951^T, and 98·1% with P. fuscovaginae MAFF 301177^T. On the basis of data from this polyphasic study, it is proposed that the unknown strains isolated from rice represent a novel species of the genus Pseudomonas.     

西瓜嗜酸菌LuxR家族基因luxR_(4229)的功能研究

瓜类细菌性果斑病是葫芦科作物上的一种严重的世界性病害,其病原菌为西瓜嗜酸菌(Acidovorax citrulli)。本研究以野生型菌株Ac-5为研究对象,通过同源重组双交换的方法,构建了LuxR家族功能基因luxR_(4229)的全基因缺失突变株,并对突变株进行致病性、生物膜形成能力和运动能力等生物学特性测定。同时,qRT-PCR定量检测了鞭毛基因fliR和fliC以及III型分泌系统功能基因hrpE和hrcN的表达水平。结果显示:与野生型相比,突变株致病力明显降低,运动能力减弱,而生物膜形成能力增强。fliR、fliC、hrpE和hrcN基因在突变株中的表达量下调,luxR4229对fliR、fliC、hrpE和hrcN基因均为正调控。基因luxR_(4229)在对西瓜嗜酸菌致病力、运动能力及生物膜的形成具有重要调控作用。