Implication of C-terminal mutation of PopA of Ralstonia solanacearum strain OE1-1 in suppression of bacterial wilt

Ralstonia solanacearum strain OE1-1 causes bacterial wilt on tobacco plants. The popA -mutant 31b, derived from OE1-1 by insertion of transposon Tn 4431 , did not cause wilt on tobacco plants inoculated through the roots. However, when 31b was directly inoculated into xylem vessels, the tobacco plants wilted, similarly to those inoculated with OE1-1. 31b retained its exopolysaccharide productivity and its type-III secretion function. Furthermore, 31b grew in intercellular spaces and systemically infected tobacco plants, similarly to OE1-1. popA consists of an operon with popB and popC , and suppression of popB and popC expression resulting from polar mutation by transposon insertion did not affect the virulence of 31b. The mutated popA ( popA31b ) was composed of 960 nucleotides, including 39 derived from Tn 4431. A recombinant mutant from OE1-1, where popA31b was introduced by marker exchange, showed the same phenotype as 31b. PopA31b protein was extracellularly secreted by 31b co-cultured with Arabidopsis thaliana . These results suggest that PopA31b extracellularly secreted by 31b in intercellular spaces may be implicated in suppression of disease development, leading to inability of the bacteria to induce wilt on plants. Taken together, interactions between host plants and R. solanacearum existing in intercellular spaces immediately after invasion may be involved in disease development.     

青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析

 利用青枯雷尔氏菌(Ralstonia solanacearum)无致病力菌株防治番茄青枯病具有很好的应用潜力。作者通过分离筛选自然弱毒株、⁶⁰Co辐射诱变和EZ-Tn5插入诱变,分别获得3、12和40株青枯雷尔氏菌无致病力突变菌株。经盆栽番茄苗致病性检测,15 d后均未发病,证实均为无致病力青枯雷尔氏菌。进一步对番茄青枯病的防治试验表明,从番茄青枯病发病田块分离的无致病力突变菌株FJAT1458的防治效果最好,防效达100%。该菌株能定殖番茄植株根系土壤、根部和茎部,定殖数量均表现为“先增后减”的趋势,并且接种浓度越大、苗龄越小,定殖数量越大。从构建的防效模型可以看出,不同接种浓度条件下,植株发病率随时间变化符合的回归方程不同,相关系数R值也不同,接种浓度越大,R值越小。本研究获得的青枯雷尔氏菌无致病力突变菌株FJAT1458对番茄青枯病具有很好的防病效果。     

烟草青枯病菌拮抗细菌的筛选、鉴定和抑菌机制研究

青枯雷尔氏菌Ralstonia solanacearum造成的烟草青枯病是烟草主要毁灭性病害之一.本研究采用牛津杯法从运城盐湖湖岸土壤中筛选获得一株对烟草青枯病菌具有较好拮抗效果的菌株FY-C;并进一步分析了菌株FY-C的抑菌谱、对青枯病菌的潜在生防效果.结果 显示,经菌株FY-C无菌滤液处理24 h后的青枯病菌细胞壁...     

甘薯瘟菌系致病型与寄主品种的互作关系

阐述了甘薯瘟菌系致病类型、品种抗性分类、抗性遗传趋势和病菌潜伏侵染特征;介绍了近年来我国南方甘薯瘟病区菌系分布状况,抗病品种利用与抗病育种进展;讨论了病原菌与寄主相互作用中的遗传关系。     

紫油厚朴叶提取物对马铃薯青枯病菌的抑制作用及防病效果

采用快速萃取法提取了紫油厚朴叶中的有效成分,用其提取物对马铃薯青枯病菌进行了抑菌作用及马铃薯青枯病的盆栽防治试验研究。结果表明:紫油厚朴叶提取物在浓度为含厚朴总酚0.6、0.3、0.2、0.1 mg/mL时对马铃薯青枯病菌均表现较强的抑菌活性;厚朴叶提取物对受试的马铃薯青枯病菌的最低抑菌浓度(MIC)为厚朴总酚0.1 mg/mL;盆栽防治试验效果亦很好。     

番茄青枯病植物疫苗胶悬菌剂的制备及其对病害的防治效果

研究了植物疫苗胶悬剂的制备工艺,确定胶悬剂制备的适宜参数为:琼脂终浓度为2.0‰、氯化钠终浓度为2.0%和pH为7.0。在此条件下,胶悬效果最好,质地均匀,无上下分层。试验了植物疫苗胶悬剂对番茄青枯病的防治效果,结果表明,该胶悬剂能较好地抑制番茄青枯病的发生,其防效(77.45%)与化学农药72%农用硫酸链霉素SP(77.23%)相当,配合生防菌剂20亿活芽孢/g蜡质芽孢杆菌WP施用,效果更佳,防效达81.81%。     

空心菜青枯病病原菌的鉴定

 空心菜青枯病是近年来在广东空心菜产区新发生的病害,病株率一般为5%~20%,严重达50%以上。症状初始表现为植株上部1~2片叶萎蔫、下垂,病叶失去光泽呈灰绿色;随着病情的发展,病叶不断增多,最终整株萎蔫,甚至倒伏。经细菌学鉴定、室内致病性测定、16S rDNA序列比较及系统进化分析,结果表明引起该病害的病原是茄科雷尔氏菌(Ralstonia solanacearum);致病性和碳水化合物利用试验结果表明,该病原菌属茄科雷尔氏菌1号生理小种,生化变种4。     

壳聚糖对青枯劳尔氏菌生长及其生物膜形成的影响

为建立快速检测壳聚糖抑菌效果的方法,研究其对青枯菌生物膜形成的影响并探索其抑菌机理,利用酶标仪检测壳聚糖与菌悬液混合培养物的吸光值,计算抑菌率,同时利用结晶紫染色法对壳聚糖处理后青枯菌生物膜形成能力进行评价,并进行透射电镜观察.结果表明,在壳聚糖浓度低于0.15 mg/mL时,随浓度增加其抑菌效果亦显著增加,其浓度超过0.15 mg/mL之后,其抑菌效果达到同一水平,最高抑菌率为74.3％;而壳聚糖浓度对青枯菌生物膜形成能力未产生显著影响;透射电镜观察结果显示:壳聚糖通过破坏青枯菌细胞膜抑制细菌生长.     

青枯菌无致病力菌株对烟草青枯病的控病作用初步研究

从茄子、番茄、辣椒、烟草青枯病株中分离出116株无致病力青枯菌,室内平板喷雾法拈抗试验结果表明,有21株菌在NA培养基上可明显抑制青枯菌TbRs的生长;烟草MSK326品种温室盆栽控病试验表明,Tnljdl-3和Aujd8—2—1两株菌具有较好控病效果,20d后的相对防效分别为58．4％和97％。     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.     

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

In the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex-PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.     

Control of soil rot of sweet potato by the incorporation of the fresh aerial tissue of Geranium carolinianum L. into the soil

In a previous study, we found that a 70% aqueous ethanol extract of the fresh aerial tissue of Geranium carolinianum L. showed antimicrobial activity against the pathogen causing soil rot of sweet potato. As the appropriate time for cultivation of sweet potato and the growing period of G. carolinianum do not overlap in Okinawa Prefecture, Japan, the fresh aerial tissue is available in order to control soil rot of sweet potato. Thus, we examined the control effect of fresh aerial tissue against soil rot of sweet potato. The various trials (a single repetition of 20 m²) were performed in fields that had undergone 8 years of continuous cropping of sweet potato at Okinawa Prefectural Agricultural Experiment Station, Horticultural branch. After harvest, when the disease severity was evaluated by determining the necrotic area of the storage root, the incorporation of fresh aerial tissue (5000 kg 1000 m^-2) into the soil was considered to be highly effective, with a protective value of 75.4. This result shows that G. carolinianum could be used as a biological agent for the control of soil rot of sweet potato.     

罗汉果青枯病菌生物学特性初步研究

对罗汉果青枯病菌的生长温度、pH、碳氮营养等生物学特性进行了测定。试验结果表明,罗汉果青枯病菌Lu L3菌株能在pH5.0～8.5和12～40 ℃的范围内生长,最适生长的pH范围为6.0～7.5,最适生长的温度范围为28～35 ℃。测试的6株病原菌株,其生长均能利用蔗糖、葡萄糖、乳糖、山梨醇、麦芽糖、甜醇和甘露醇作为碳源,利用蛋白胨、牛肉浸膏、酵母浸膏、硝酸铵、硝酸钾和脲作为氮源。气温、土壤种类和罗汉果的生育期对病原菌入侵寄主有影响。在20～35 ℃范围内,气温升高利于病原菌的入侵,病害发生严重;病原菌在黏土中入侵寄主容易,在壤土中入侵寄主困难;罗汉果植株越小越利于病原菌的入侵,花蕾期后,未观察到植株被病原菌侵染。接种病原菌Lu L3菌株后,12 h时植株的根表分离到接种的病原菌株;24 h时根内分离到接种的病原菌株;36 h时茎内分离到接种的病原菌株。     

青枯病的化学与生物防治研究进展

本文阐述了近年来在植物细菌性青枯病化学和生物防治研究方面所取得的进展。首先介绍了普通化学药剂和抗生素及植物生长调节剂等防治青枯病的新近应用，作用效果和防治特点；然后回顾了应用无致病力青枯菌菌株，拮抗细菌，菌根和基因工程技术进行生物防治的研究情况；最后对化学和生物防治的有关问题及应用前景进行了讨论。     

Identification and activity of ethyl gallate as an antimicrobial compound produced by Geranium carolinianum

We isolated an antimicrobial compound from the aerial tissue of Geranium carolinianum and identified it as ethyl 3, 4, 5-trihydroxy benzoate (ethyl gallate) by ¹H-NMR and ¹³C-NMR and gas chromatography–mass spectrometry. The antimicrobial activity of ethyl gallate against three potato pathogens was assayed by the paper disk method. The activity against Ralstonia solanacerum , Streptomyces scabies , and Streptomyces acidiscabies was observed at concentrations >200, >300, and >300 µg disk⁻¹, respectively. These results suggest that the antimicrobial activity of Geranium carolinianum against soil-borne plant disease pathogens is partly related to ethyl gallate.     

烟草青枯病内生拮抗菌的筛选、鉴定及其防效测定

 植物体内的大量微生物,在进化的过程中,由于长期生活在植物特定部位,因而与植物形成了一种特殊的关系,这些微生物在植物体内大量繁殖和扩散,有望成为生物防治有潜力的微生物。内生细菌是植物体内大量存在的微生物之一,具有在植株体内分布广、定殖能力强、防效好以及增殖和扩散快等优点,因而成为发展前景很好的植物病害生防菌。     

番茄枯萎病和青枯病拮抗细菌的筛选、评价与鉴定

从宁夏银川、江苏沭阳和福建厦门的番茄、辣椒、西瓜等作物根际土壤中,分离纯化获得367株细菌菌株。以番茄枯萎病菌Fusarium oxysporum f.sp.lycopersici和番茄青枯病菌Ralstonia solanacearum为靶标菌,从367株菌株中筛选出对两种病菌皆具有很强拮抗作用的菌株22株。拮抗细菌抑菌物质的研究结果表明：22株拮抗细菌均能分泌蛋白酶;不能分泌几丁质酶;3株细菌能分泌纤维素酶;3株细菌能分泌嗜铁素。盆栽试验结果表明：拮抗细菌PTS-394对番茄枯萎病和青枯病的防效最高,分别为77.4%和80%;菌株H-70、L-1和SJ-280对番茄枯萎病和青枯病的防效均大于60%。对上述4株拮抗细菌进行16S rRNA种属鉴定,均为枯草芽孢杆菌Bacillus subtilis。     

抗姜青枯菌银杏内生真菌的分离及抑菌特性

为了筛选新的生防菌种资源,从银杏根、叶中分离出对青枯劳尔氏菌Ralstonia solanacearum有拮抗作用的内生真菌,结合形态特征和ITS序列对该菌株进行了鉴定,分析了菌株发酵液的抑菌特性。结果表明,分离获得的80株内生真菌中有5株对青枯菌有拮抗作用,其中Gbh45具有良好的抑菌效果和遗传稳定性,根据形态特征和ITS序列比对,该菌株被鉴定为球黑孢菌Nigrospora sphaerica。该菌株的发酵产物经紫外照射、不同温度处理后其抑菌活性不发生变化,在碱性条件的耐受能力高于酸性,表明菌株Gbh45的发酵产物在自然条件下稳定性高,该菌对姜青枯病的生物防治具有巨大的应用潜力。     

生姜青枯病防治药剂室内筛选及评价

采用比浊法测定了10种药剂对生姜青枯菌的毒力,结果显示乳酸环丙沙星、盐酸环丙沙星、土霉素的毒力最强,其EC50分别为1.75、1.81和2.21μg/L;而硫酸庆大霉素、硫酸链霉素、头孢曲松钠、中生菌素次之,EC_(50)分别为11.17、11.23、19.23和24.20μg/L;姜瘟净、硫酸多黏菌素B和大蒜油的EC_(50)最高,分别为549.61,1 223.22和2 064.33μg/L。通过盆栽试验,采用药剂浸种、先灌药后接种病原、以及先接种病原后灌药等处理方式评价了这10种药剂对生姜青枯病预防和治疗的潜能,结果显示,硫酸链霉素和中生菌素浸种处理对生姜青枯病防效最好,分别为93.0%和75.9%;硫酸链霉素、硫酸多黏菌素B、姜瘟净、大蒜油表现出较好的预防作用;除硫酸链霉素外,其他药剂对青枯病治疗作用差。     

橘-芝麻套种模式下芝麻青枯病初侵染源分析

净作11年的橘园首次套种芝麻,发现园内芝麻青枯病发生严重。为探明芝麻青枯病初侵染源,本研究从芝麻青枯病株上分离到菌株JXRs02,通过形态学、分子生物学以及聚类分析,鉴定其为青枯雷尔氏菌演化型I。利用针刺接种法,将JXRs02菌株接种至柑橘园中的4种杂草凹头苋、青葙、空心莲子草以及加拿大蓬,发现JXRs02能够侵染凹头苋杂草,造成枯萎症状;LAMP检测的结果显示,在凹头苋病株组织内能够检测到青枯雷尔氏菌。依据柯赫氏法则,利用分子生物学方法证明了对凹头苋杂草具有致病性的菌株和造成芝麻青枯症状的病原菌同为青枯雷尔氏菌演化型I。同时发现,在凹头苋、青葙、空心莲子草和加拿大蓬4种杂草的根际土壤中芝麻青枯病菌均能够定殖繁衍。推测凹头苋杂草可能为芝麻青枯病菌的中间寄主,凹头苋、青葙、空心莲子草和加拿大蓬等杂草根际土壤积累的菌源可能为橘园套种芝麻后青枯病的初侵染来源。本研究对制定作物青枯病的防治策略具有一定科学意义。