柑桔溃疡病菌抗药突变体的生理特性研究

 经药剂驯化和紫外光诱导双重作用可获得柑桔溃疡病菌抗拌种灵突变体(XcR),病原菌抗药突变频率低于10-6。Xc R的生长速率与敏感菌株基本一致,对萎锈灵和叶枯唑具有潜在的正交互抗性,与浸种灵没有交互抗性。XcR胞外产物和胞外水解酶活性一般多于或高于敏感菌株,其中以淀粉酶活性变化最为明显。Xc R琥珀酸脱氢酶活性显著降低,并对拌种灵的敏感性亦降低。XcR能够引起烟草的过敏性反应,但失去对寄主的致病性。Xc R的性质表明,柑桔溃疡病菌对拌种灵产生抗药性的风险较低,病原菌获得抗药性状的同时可能导致其在田间的适合度下降。琥珀酸脱氢酶的活性可作为柑桔溃疡病菌抗药性的标记。     

柑桔溃疡病菌胞外产物在拌种灵与菌体互作中的作用

 柑桔溃疡病原细菌的胞外产物,在拌种灵与病原菌互作过程中具有一定的调控作用。胞外产物尤其是胞外粘多糖(EPS),对拌种灵具有很强的拮抗作用;经药剂处理的病原菌,其胞外产物如:电解质、EPS、胞外蛋白、以及胞外水解酶等,其产量和生物活性都呈现出一定规律的变化。研究表明,病原菌在药剂处理下,可通过胞外产物的变化产生一定的抗逆反应。     

拌种灵、叶枯唑和萎锈灵对病菌的毒力机制比较

苯氨基甲酰类杀菌剂是最早开发的一类内吸性杀菌剂 [1 ] ,能有效地防治一些真菌病害 ,持效性长 ,抗药性风险低 [2 ] 。对叶枯唑产生抗药性的水稻白叶枯病菌 ( Xanthomonas oryzae pv.oryzae)对拌种灵同样具有抗药性 [3] 。近年来 ,拌种灵被有效地用于防治果园中柑桔溃疡病 ( X.citri) [4]。目前对噻唑类杀菌剂防治细菌病害的作用机制尚不清楚 ,初步认为这类药剂最初的作用靶点可能与致病性相关因子有关 [3] 。我们对 3种具有相似结构的杀菌剂拌种灵、叶枯唑和萎锈灵在离体条件下对病原菌的毒力机制进行了初步研究 ,同时也对拌种灵防治柑桔…     

镧素对镰刀菌Fusarium solani及其致病酶的影响

采用琼脂平板生长速率法及液体培养基培养测定了La对镰刀菌Fusarium solani的抑制作用和毒力,并测定了其对病菌胞外的果胶酶、蛋白酶和纤维素酶等几种致病酶活性的影响。结果表明,随着La2O3浓度升高,对病菌菌丝生长的抑制作用增强,对病菌的EC50和EC95分别为278.2和552.0 mg/L;在一定浓度范围内,La提高了单位量菌丝所产生3种致病酶的活性,但由于菌丝生长受到抑制,除蛋白酶外,病菌胞外致病酶果胶酶和纤维素酶的总量或总活性受到了抑制,降低了病菌的致病力。     

3种杀螨剂对顶孢霉菌株胞外蛋白酶和几丁质酶活性的影响

研究了螺螨酯、噻嗪酮和吡螨胺在不同浓度下处理对顶孢霉胞外蛋白酶和几丁质酶活力的影响。结果表明,螺螨酯各浓度处理对顶孢霉胞外蛋白酶和几丁质酶活性均无影响,噻嗪酮高浓度处理顶孢霉在培养的前4 d对胞外蛋白酶活性有轻微的抑制作用,随培养时间延长,抑制作用不明显;而低浓度处理下,即在1.25 mg/L浓度下培养7 d,其酶活力是第1天的3.97倍,对顶孢霉胞外蛋白酶活性有刺激作用。吡螨胺对顶孢霉胞外蛋白酶和几丁质酶活性有抑制,且随浓度增高和培养时间延长抑制作用越明显。     

柑桔溃疡病菌的分子生物学研究进展

柑桔溃疡病菌 (Xanthomonasax onopodispv .citri)属重要的国内外检疫性有害生物 ,由其引起的柑桔溃疡病对柑橘产量和苗木及果品进出口影响极大。随着分子生物技术的研究进展 ,对柑桔溃疡病菌的菌系鉴别与系统分类 ,全基因组测序以及检测研究等方面都进入了分子水平。本文从柑桔溃疡病菌的分类地位演变、全基因组测序的完成及其在病害的分子快速鉴定技术上的应用作了详细的评述。1　柑桔溃疡病菌的分类研究随着黄单胞杆菌属 (XanthomonasDow son 1 939)属下分种或分型的演变 ,柑桔溃疡病菌的种名也发生了较大的变化 ,尤其是分子生物学的发…     

进境柠檬样品上柑桔溃疡病菌的检测

从进境柠檬样品上分离到一株疑似柑桔溃疡病菌的分离物X206,对分离物进行了菌落形态学特征观察、16S r RNA序列测定、PCR检测、Biolog测定及致病性测试。试验结果表明分离物X206在NA培养基上形成黄色,有光泽,圆形,全缘,微隆起,粘稠状的菌落。特异引物Xac01/Xac02扩增分离物X206的DNA得到582bp的预期产物,产物序列与柑桔溃疡病菌序列的相似性为100%。其16S r RNA序列与柑桔溃疡病菌的序列完全一致。Biolog测定将分离物X206和柑桔溃疡病菌阳性菌株FZ01均鉴定为Xanthomonas campestris pv.dieffenbachiae。致病性测试显示分离物X206能导致柑桔叶片产生明显的溃疡病斑。根据试验结果将分离物X206鉴定为柑桔溃疡病菌(Xanthomonas axonopodis pv.citri)。     

从水稻叶面分离的菌株E-14产生抗菌物质

为了获得生物防治植物病原细菌的有效菌株,供试了从25种植物叶面分离的146个菌株。利用重要的植物病原细菌作为指示菌并研究了抗菌物质的产生。结果,在分离的试菌株中,从水稻分离到的E—14菌株具有最广泛的抗谱,在YPDA培养基培养的E—14菌株用氟仿处种,利用指示菌显环,对西红柿溃疡病菌和马铃薯环腐病菌形成最大的抑制环,而对水稻白叶抗病菌和柑桔溃疡病菌形成较大的抑制环。确认在YPD液体培养基中对西红柿溃疡病菌和马铃薯环腐病菌有较强的活性。除此之外,在YPDA平板培养的冷冻后融化渗出液中,对水稻白叶枯病菌和柑桔溃疡病菌也有活性。     

苦皮藤内生真菌A10代谢产物的杀菌活性

从苦皮藤根皮中分离得到一株内生真菌,编号为A10.对其代谢产物进行了杀菌活性研究.生物测定结果表明,A10菌株发酵产物菌丝体的甲醇和乙酸乙酯提取物对多种病原真菌具有较强的杀菌活性.其中乙酸乙酯提取物的杀菌活性较甲醇好,在1000μg/ml浓度下对烟草赤星病菌、小麦根腐病菌、玉米大斑病菌、番茄灰霉病菌、苹果轮纹病菌的菌丝生长抑制率均大于90%.盆栽试验结果表明,A10菌株发酵产物菌丝体的乙酸乙酯提取物在2000μg/ml浓度下对小麦白粉病的治疗和保护作用均在70%以上.以生物活性追踪为指导,对活性较好的菌丝体乙酸乙酯提取物部分进行了杀菌活性成分的分离,得到化合物QY01,在浓度为50μg/ml时,QYO1对烟草赤星病菌孢子萌发的抑制率高达98.4%.     

在水稻悬浮细胞系中非亲和白叶枯病菌繁殖受到抑制的现象及其机理的研究

 研究了水稻白叶枯病菌在水稻悬浮细胞系中的繁殖动力学特征以及悬浮细胞胞外液中各类物质对白叶枯病菌繁殖的影响。结果表明,水稻-白叶枯病菌非亲和互作与亲和互作相比病原菌繁殖的数量明显减少。胞外液中粗提蛋白对白叶枯病菌繁殖不具有抑制作用。非亲和互作胞外液中二元酚含量显著增高,并且水稻细胞防卫反应基因的转录和翻译活性明显增强。表明非亲和互作中白叶枯病菌繁殖受到抑制与水稻细胞防卫反应的启动相关。     

A study on the molecular mechanism of resistance to amicarthiazol in Xanthomonas campestris pv. citri

Three amicarthiazol-resistant mutants (Xuv10, Xuv20 and Xuv40) were obtained by UV induction and used in this study. Minimal inhibition concentrations (MICs) of amicarthiazol against the growth of mutants and wild-type isolate were 400 and 100 microg ml(-1) respectively. Inhibition by amicarthiazol of succinate dehydrogenase (SDH) activities of Xanthomonas campestris pv. citri (Hasse) Dye wild-type isolate (Xcc) and three resistant mutants derived from this isolate were assayed using triphenyltetrazolium chloride (TTC). The SDH activities of these mutants were significantly lower than that of Xcc. The complete nucleotide sequences of four subunits (SdhA, SdhB, SdhC and SdhD) of succinate-ubiquinone oxidoreductase (SQR) were cloned by polymerase chain reaction (PCR) amplification. An amino acid mutation (His229--> Leu229) in sdhB was found to confer resistance of X. campestris pv. citri to amicarthiazol. It is suggested that this mutation alters the SDH complex in some way that prevents binding of amicarthiazol.     

Toxicity and biochemical action of amicarthiazol on citrus canker pathogen, Xanthomonas citri ex Hasse

Inhibitory effects of amicarthiazol, in vitro, against the growth of seven plant bacterial pathogens and its biochemical actions on Xanthomonas citri were investigated. The growth of Erwinia carotovora, Ralstonia solanacearum, and Pseudomonas syringe pv. syringae was not sensitive to amicarthiazol whilst Xanthomonads were all strongly inhibited in NB liquid medium; EC₅₀ of X. citri was 2.63 μg ml⁻¹ and the effectiveness of amicarthiazol depended on the timing of application. The inhibitions on the secretion of gross EPS and gross EPR as well as the activities of amylase, cellulase, and pectase of X. citri were increased with the concentrations of amicarthiazol, whereas the conductivities of the extracellular product suspensions and the protease activity were somewhat accelerated by the concentration less than 10 or 20 μg ml⁻¹ and inhibited only at higher concentration. EPS which was purified from X. citri showed obvious antagonistic effects on the inhibition of amicarthiazol, but not on the inhibition of Cu(OH)₂. The endogenous respirations and the activity of succinic dehydrogenase of X. citri were also inhibited and the inhibitions were increased with the toxic concentrations. Furthermore, the potential induced physiological adaptability of X. citri to amicarthiazol and the comparison with Cu(OH)₂ and carboxin were also discussed.     

A single amino acid substitution in the SdhB protein of succinate dehydrogenase determines resistance to amicarthiazol in Xanthomonas oryzae pv. oryzae

BACKGROUND: Xanthomonas oryzae pv. Oryzae Ishiyama, a causal agent of rice bacterial leaf blight, was found to be sensitive in vitro to the systemic fungicide amicarthiazol (2‐amino‐4‐methylthiazole ‐5‐carboxanilide), which is a potent inhibitor of succinate dehydrogenase (SDH, EC 1.3.99.1). This paper aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzae to amicarthiazol. RESULTS: UV‐induced resistant mutants of X. oryzae pv. oryzae to amicarthiazol were isolated. The activity of SDH in wild‐type X. oryzae pv. oryzae was strongly inhibited by amicarthiazol, while that in resistant mutants was insensitive, although their SDH activity was decreased compared with the wild‐type sensitive strain without amicarthiazol. A mutation of Histidine₂₂₉ (CAC) to Tyrosine₂₂₉ (TAC) was identified in sdhB, which encoded the iron–sulfur protein subunit of SDH. The sdhB from the mutant was ligated into a cosmid, pUFR034, to generate pUFR034RAX, which conferred resistance to amicarthiazol when transformed into the wild‐type sensitive strain. CONCLUSION: A mutation of His₂₂₉ (CAC) to Tyr₂₂₉ (TAC) in SdhB was responsible for determining amicarthiazol resistance. Copyright © 2010 Society of Chemical Industry     

Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from Southwest Asia

Strains of Xanthomonas axonopodis pv. citri were isolated from Mexican lime (Citrus aurantifolia) trees in several countries in southwest Asia. These strains produced typical erumpent bacterial canker lesions on Mexican lime but not on grapefruit (C. paradisi). Lesions on grapefruit were watersoaked and blister-like in contrast to the typical erumpent lesions seen after artificial inoculation with all described pathotypes of X. axonopodis pv. citri. This group of strains hydrolysed gelatin and casein and grew in the presence of 3% NaCl as is typical of X. axonopodis pv. citri pathotype A. RFLP analyses and DNA probe hybridization assays also gave results consistent with X. axonopodis pv. citri pathotype A. Metabolic fingerprints prepared with the Biolog® system showed similarities as well as differences to X. axonopodis pv. citri pathotype A. In spite of the physiological and genetic similarities to pathotype A of X. axonopodis pv. citri, these strains had no or very little affinity for polyclonal antiserum prepared against any of the reference strains of X. axonopodis pv. citri and also did not react with monoclonal antibody A1, an antibody that detects all strains of pathotype A of X. axonopodis pv. citri. These strains were also insensitive to bacteriophage Cp3 like X. axonopodis pv. citri pathotype A and unlike X. axonopodis pv. citri pathotype B. We conclude that these strains, designated Xcc-A*, represent a variant of X. axonopodis pv. citri pathotype-A with pathogenicity limited to C. aurantifolia. The existence of extensive genotypic and phenotypic variation within pathotype A of X. axonopodis pv. citri was unexpected and further complicates the systematics of this species.     

抑制植物病原细菌的植物筛选

以3种重要的植物病原细菌—辣椒青枯病菌(Ralstonia solanacearum)、柑橘溃疡病菌(Xanthomonas citri)和大白菜软腐病菌(Erwinia carotovorapv.carotovora)为供试菌,对43种植物甲醇提取物进行离体抑菌活性测定。研究结果表明,漆树对辣椒青枯病菌抑菌活性最强,其次是金秀清明茶;抑制柑橘溃疡病菌活性最高的植物有漆树和十大功劳;对大白菜软腐病菌没有筛选到抑菌活性较好的植物;垫状卷柏、七叶一枝花、少花龙葵、水半夏和豆瓣菜粗提物对3种植物病原细菌均无抑制作用。     

拌种灵在辣椒和土壤中的残留和消解动态研究

为明确拌种灵施用于辣椒后的残留消解情况,评价其使用后的生态环境安全性,指导它的科学合理施用,通过添加回收率,建立了拌种灵的残留分析方法,借助高效液相色谱检测技术,检测了田间试验中拌种灵在辣椒及其土壤中的残留动态和最终残留情况。拌种灵在辣椒及其土壤中较易消解,其半衰期分别为3.84、5.76d,按推荐使用剂量和使用方法按最后1次施药后5d采收辣椒,辣椒和土壤中的最终残留浓度均〈0.5mg/kg。     

广西地不容提取物及其化合物的抑菌活性

在室内用生长速率法测定了广西地不容块根提取物及其化合物对梨褐斑病菌、梨黑斑病菌、柑橘疮痂病菌和柑橘溃疡病菌的抑菌活性。结果表明,广西地不容块根提取物对上述4种病原菌均有明显的抑制作用,质量浓度为10 g/L时,72 h的抑菌率分别为100%、91.96%、84.76%和100%;在从广西地不容块根提取物分离出的7个化合物中,l-罗默碱对4种病菌均有很高的抑菌活性,质量浓度为1 g/L时,72 h的抑菌率分别为100%、100%、85.04%和100%。紫堇定对梨黑斑病菌抑菌活性高,72 h抑菌率为100%,对梨褐斑病菌和柑橘疮痂病菌也有较高的抑菌活性,而对柑橘溃疡病菌的抑菌活性低。广西地不容块根提取物对梨褐斑病菌、梨黑斑病菌、柑橘疮痂病菌和柑橘溃疡病菌的EC₅₀分别为1.252 5、2.379 3、1.758 2、1.510 0 g/L,l-罗默碱的EC₅₀分别为0.147 3、0.167 1、0.3464、0.118 2 g/L。