Influence of bacteria isolated from rice plants and rhizospheres on antibiotic production by the antagonistic bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

Serratia marcescens strain B2 is an antagonistic bacterium that produces the red-pigmented antibiotic prodigiosin and suppresses rice sheath blight caused by Rhizoctonia solani AG-1 IA. Rice sheath blight disease was suppressed when plants were inoculated with this bacterium an hour before pathogen inoculation but not when plants were treated 4 weeks before pathogen inoculation. In both cases the bacteria were detected in the rice rhizosphere 4 weeks after inoculation. Bacteria isolated from the rice plant and rhizosphere inhibited biosynthesis of prodigiosin in S. marcescens strain B2. We suggest that bacteria isolated from rice plants and rhizospheres mediate the suppression of antibiotic production of biological control agents and that such suppression is common under field conditions.     

荧光素酶标记无致病力青枯雷尔氏菌的生物学和定殖特性

为示踪青枯雷尔氏菌Ralstonia solanacearum无致病力菌株FJAT1458在番茄植株及根际土壤中的定殖特性,采用电击法对菌株FJAT1458进行荧光素酶(luciferase,LUC)基因标记,并于室内采用生测法测定标记菌株的生物学特性、遗传稳定性、致病力及在番茄植株和根际土壤中的定殖能力。结果表明,成功将luc基因整合至无致病力菌株FJAT1458染色体上,标记菌株FJAT1458-LUC发出强烈的荧光,且PCR扩增出1 612 bp的luc基因片段;与野生型菌株FJAT1458相比,标记菌株FJAT1458-LUC的生长明显滞后,培养8h之后标记菌株FJAT1458-LUC的OD_600nm值均小于野生型菌株FJAT1458;且标记菌株FJAT1458-LUC连续传代20次后菌体浓度显著增加,发光菌体比例显著降低,LUC活性和luc基因表达量均随着传代数增加而显著降低;标记前、后菌株FJAT1458的弱化指数分别为0.90和0.89,且接种番茄植株30 d均未引起植株发病。标记菌株FJAT1458-LUC能在番茄根际土壤、根及茎中定殖,定殖数量呈...     

Quorum sensing as a target for developing control strategies for the plant pathogen <Emphasis Type="Italic">Pectobacterium</Emphasis>

Quorum sensing is a regulatory mechanism that connects gene expression to cell density in bacteria. Amongst proteobacteria, numerous functions are regulated in this way, including pathogenicity in the Enterobacteriaceae genus Pectobacterium. In Pectobacterium, the signalling molecules involved in this regulatory process belong to the N-acyl-homoserine lactone class. Over the last 6 years, various studies have shown that these signal molecules could be degraded by other bacteria or by plant and animal cells, opening the path to innovative biocontrol strategies. This review explores the various determinants of pathogenicity in Pectobacterium and describes approaches that have been developed to quench the quorum-sensing-dependent pathogenicity in Pectobacterium. These approaches range from signal degradation by physicochemical constraints to the identification of signal-sensing inhibitors and from the identification of enzymes degrading acyl-homoserine lactones to the construction of transgenic plants tolerant to Pectobacterium.     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Induction of systemic resistance to<Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis> in bean by a benzothiadiazole derivative and rhizobacteria

Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar-S-methyl), and the rhizobacteriaPseudomonas aeruginosa KMPCH andP. fluorescens WCS417 were tested for their ability to induce resistance toColletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10⁻³ to 10⁻⁷ M BTH 3 days before the challenge inoculation. At a high concentration (10⁻³ M), BTH induced resistance of the same order as resistance induced by the pathogenC. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg⁻¹ BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants.P. aeruginosa KMPCH induced resistance in bean againstC. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean—C. lindemuthianum system.P.fluorescens WCS417 could induce resistance toC. lindemuthianum in a susceptible and in moderately resistant interactions. http://www.phytoparasitica.org posting Jan. 16, 2002.     

苹果树腐烂病菌拮抗放线菌JPD-1的筛选及鉴定

为筛选对苹果树腐烂病菌Valsa mali具有拮抗作用的放线菌,从苹果树根际土壤中分离获得放线菌,利用平板对峙法和生长速率法筛选拮抗活性较高的菌株,通过菌株培养特征、形态特征、生理生化特性及16S rDNA序列分析确定其分类地位,并采用离体枝条烫伤接种法测定所筛选获得的拮抗菌株对苹果树腐烂病的防效。结果表明,从苹果树根际土壤中共分离获得28株放线菌,其中菌株JPD-1对苹果树腐烂病菌的拮抗作用最强,抑制率为72.50%,该菌在高氏合成1号培养基上生长最好,培养7 d时菌落生长旺盛,气生菌丝白色,基生菌丝深粉红色,无可溶性色素产生;经形态特征观察、生理生化试验及16S rDNA序列分析,将菌株JPD-1鉴定为公牛链霉菌Streptomyces tau‐ricus。菌株JPD-1发酵滤液对苹果树腐烂病菌的抑制率及对离体枝条上苹果树腐烂病的防效均随稀释倍数的增大而减弱,发酵滤液原液使苹果树腐烂病菌菌丝膨大、末端畸形,抑制率为78.14%,对离体枝条上苹果树腐烂病的防效为76.37%。表明分离到的放线菌菌株JPD-1可以用作苹果树腐烂病的生防材料,具有很好的开发及应用前景。     

Induced resistance against Fusarium diseases of <Emphasis Type="Italic">Cymbidium</Emphasis> species by weakly virulent strain HPF-1 (<Emphasis Type="Italic">Fusarium</Emphasis> sp.)

The mechanism by which Fusarium diseases of cymbidium plants are suppressed by a weakly virulent strain HPF-1 of Fusarium sp. was studied. Strain HPF-1 produced microscopic, necrotic local lesions on cymbidium leaves, causing minor damage to palisade tissues at the infection sites. This weakly virulent strain remained near the site of infection and did not develop further. It systemically and nonselectively suppressed some diseases of cymbidium such as yellow spot of leaves caused by Fusarium proliferatum and F. fractiflexum, bulb and root rot caused by F. oxysporum, and dry rot of bulbs and roots caused by F. solani. Because endogenous salicylic acid levels increased in cymbidium leaves inoculated with strain HPF-1, the mechanism of disease suppression is thought to be systemic acquired resistance.     

瓜类细菌性果斑病菌hrcN基因的生物学功能分析

以分离自西瓜上的Aac5菌株为例,通过同源重组的方法,构建了hrc N基因插入缺失突变体,通过PCR方法和Southern blot验证突变菌株,对突变体进行致病性、致敏性、生长曲线和运动性测定。为明确hrc N基因与其他基因的关系,通过实时荧光定量PCR法定量检测了hrp A、hrc V、hrc U、Lux I、LuxR 5个基因的表达量。结果显示:与野生型相比,突变体致病力和致敏性明显减弱,致病时间延迟,群体感应信号减弱,生长能力明显下降,运动性减弱,互补菌株只能恢复部分功能;5个基因在突变体中的表达量均上调,hrc N基因与这5个基因之间均为负调控关系。说明hrc N基因在果斑病菌致病能力上发挥重要作用。     

Effect of seed priming with <Emphasis Type="Italic">Serratia plymuthica</Emphasis> and <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> to control <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in different oilseed rape cultivars

The efficacy of a seed treatment of oilseed rape (OSR) (Brassica napus) with the rhizobacteria Serratia plymuthica (strain HRO-C48) and Pseudomonas chlororaphis (strain MA 342) applied alone or in combination against the blackleg disease caused by Leptosphaeria maculans was tested with different cultivars. Seeds were soaked in bacterial suspensions (bio-priming) to obtain log₁₀ 6–7 CFU seed⁻¹. Cotyledons were inoculated with a 10 ul droplet of L. maculans spore suspension of log₁₀ 7 spores ml⁻¹ and the disease index (size of lesions) was evaluated 14 days later. A mean disease reduction of 71.6% was recorded for S. plymuthica and of 54% for P. chlororaphis. The combined treatment was not superior to the treatment with S. plymuthica alone. The reduction of the disease caused by S. plymuthica was independent of the cultivar’s susceptibility, whereas the control effect recorded with P. chlororaphis increased with decreasing cultivar resistance to blackleg disease. The bacterial colonization of OSR was restricted to the roots and hypocotyl. No significant difference in bacterial colonization of the rhizosphere was observed between different cultivars, nor between single or combined bacterial seed treatments.     

Contribution of the type II secretion system in systemic infectivity of <Emphasis Type="Italic">Ralstonia </Emphasis><Emphasis Type="Italic">solanacearum</Emphasis> through xylem vessels

Ralstonia solanacearum strain OE1-1 (OE1-1) systemically invades tobacco plants and causes bacterial wilt. A type II secretion system (T2SS)-deficient mutant of OE1-1, derived from EZ::TN<KAN-2>transposon-insertion, retained the ability of the parent strain to produce exopolysaccharide in vitro and grow in intercellular spaces immediately after invasion of host plants, but lost the ability to systemically infect the host. With transmission electron microscopy, the mutant was not observed in xylem vessels. These findings suggest that the T2SS contributes to systemic infection by enabling the bacteria to invade xylem vessels.     

A new leaf blight disease of <Emphasis Type="Italic">Trifolium dasyurum</Emphasis> caused by <Emphasis Type="Italic">Botrytis fabae</Emphasis>

A new disease was observed on Trifolium dasyurum, with symptoms beginning as a halo spot and developing into a leaf blight. The causal organism was identified by microscopy and DNA sequence studies as Botrytis fabae. This strain of B. fabae was also demonstrated to cause disease on foliage of a range of pulse crops, including Vicia faba, Pisum sativum, and Lens culinaris. This study demonstrates the potential of this strain of B. fabae to not only pose a significant threat to T. dasyurum but also to pulses grown in rotation with T. dasyurum that are susceptible to this strain of B. fabae.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Tobacco cultivars vary in induction of systemic resistance against <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> and growth promotion by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> O6 and its <Emphasis Type="Italic">gacS</Emphasis> mutant

The colonization of plant roots with certain rhizosphere bacteria promotes plant growth and induces long lasting systemic protection against a broad spectrum of plant pathogens. The role of the global regulator, GacS, in the rhizosphere colonist Pseudomonas chlororaphis O6 in stimulating growth promotion and induced resistance against Cucumber mosaic virus was examined in tobacco. Responses were compared in tobacco cvs Samsun and GX3. Root colonization of Samsun with wild-type O6 and the gacS complemented mutant-elicited reduced viral symptoms and viral titre. On GX3, there was little affect on symptoms when roots were colonized by the wild-type, gacS mutant or complemented mutant but colonization by both the wild-type and the gacS mutant lowered viral titre. Wild-type O6 and the gacS mutant caused plant growth to be maintained in both tobacco cultivars after viral infection, although the affect was stronger with GX3 than Samsun. In contrast, although a chemical inducer, benzothiadiazole, reduced symptoms and viral titre in both cultivars, plant growth was suppressed. Our results indicate rhizobacteria-elicited induced viral resistance without a negative impact on growth but there was a differential response between cultivars. Detailed knowledge regarding the mechanisms inherent to these differences between cultivars requires further investigation.     

紫花苜蓿根际芽胞杆菌YB-2的鉴定及其生防和促生能力分析

为获得对紫花苜蓿Medicago sativa生长有益的生防菌,以尖孢镰刀菌Fusarium oxysporum为指示菌从紫花苜蓿根际土壤中分离筛选拮抗菌株,基于形态学特征、生理生化特性和16S rDNA与gyrB基因序列分析对其进行鉴定,并评价其对紫花苜蓿根腐病的生防效果以及提高寄主植物耐盐碱胁迫的能力。结果表明,从紫花苜蓿根际土壤中筛选到1株对尖孢镰刀菌有较强拮抗作用的菌株YB-2,该菌株对尖孢镰刀菌菌丝生长的抑制率为71.37%;结合形态学特征、生理生化特性和16S rDNA与gyrB基因序列分析结果将菌株YB-2鉴定为枯草芽胞杆菌Bacillus subtilis。菌株YB-2具有溶解无机磷的能力,且具有产氰化氢、羧甲基纤维素酶、1-氨基环丙烷-1-羧酸脱氨酶、吲哚乙酸和嗜铁素的能力,在NaCl含量为1%～9%、pH 7～9条件下均能生长。菌株YB-2对紫花苜蓿根腐病的相对防效达56.83%;在盐碱条件下,与对照相比,接种YB-2的紫花苜蓿株高极显著增加了17.82%,根长显著增加了28.22%,地上部鲜重和地上部干重分别显著增加了51.12%和48.57%。表明菌株YB-2兼...     

长刺蒺藜草根际土壤解磷细菌群落的多样性分析

为揭示外来入侵植物长刺蒺藜草 Cenchrus longispinus能够在贫瘠沙性土壤中快速生长、定殖的入侵机制,利用传统功能微生物筛选培养法、 16S rDNA高通量测序技术及细菌基因组重复序列PCR（genome repetitive sequence PCR, rep-PCR）基因指纹图谱技术分析其根际土壤中解磷细菌的丰富度及多样性,并与本地伴生植物冰草Agropyron cristatum和狗尾草Setaria viridis根际土壤中的解磷细菌群落进行比较。结果显示,长刺蒺藜草根际土壤中解磷细菌的数量分别达到8.61×10⁴ CFU/g和7.34×10⁴ CFU/g,显著高于其余土壤样品。相较于空白对照土壤,长刺蒺藜草根际土壤中的解有机磷细菌数量增加最多,增加了87.0%,冰草和狗尾草根际土壤中的解有机磷细菌数量则分别增加了28.3%和15.2%;而长刺蒺藜草、冰草和狗尾草根际土壤中的解无机磷细菌数量分别增加了36.7%、 89.8%和24.5%。聚类分析结果表明,不同根际土壤中解磷细菌之间没有共有的优势聚类群。长刺蒺藜草根际土壤中2种解磷细菌群落的物种均匀度指数显著高于空白对照土壤和冰草及狗尾草根际土壤,而物种丰富度指数和Shannon-Wiener多样性指数均显著低于冰草及狗尾草根际土壤;其根际土壤中解无机磷细菌优势菌属经鉴定为金黄杆菌属 Chryseobacterium。表明长刺蒺藜草在贫瘠沙土中对磷素的高效利用可能是其快速入侵并定殖的机制之一。     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.