Mutations affecting chemotaxis of Agrobacterium vitis strain E26 also alter attachment to grapevine roots and biocontrol of crown gall disease

Crown gall in grapevine, caused by tumorigenic Agrobacterium vitis strains, can cause severe losses in most viticulture regions worldwide. The only effective means of control is through cultivation practices. One non-tumorigenic A. vitis strain, E26, can prevent crown gall infection when applied to wounds on grapevine prior to or simultaneous with inoculation of tumorigenic strains. ME19, a Tn5 mutant of strain E26, was impaired in terms of its ability to be chemo-attracted by grapevine root tissue extracts and its attachment to grapevine roots, and had reduced biological control activity; it did not significantly differ from the wild-type strain of E26 in phenotypes of agrocin production, growth in minimum medium or swarming activity. Complementation of ME19 with the cosmid clone of CP1543 from an E26 DNA library restored the chemotaxis, attachment, and biocontrol phenotypes. A 7·3-kb Kpn I fragment from CP1543 was cloned and sequenced, and sequence analysis revealed that the Tn 5 insertion occurred in a region that shares a significant homology with genes coding for methyl-accepting chemotaxis proteins (MCPs) in many bacteria. Complementation of the mcp gene mutants restored the affected phenotypes to the level of wild-type E26. An in-frame deletion mutant of the mcp gene was generated and was determined to have the same phenotypes as the original Tn5 mutant.     

剪口龄期和水分胁迫对轮纹病菌从剪锯口侵染的影响

为了明确剪口龄期和水分胁迫对轮纹病菌从剪锯口侵染的影响,本研究采用脱脂棉蘸取轮纹病菌分生孢子悬浮液接种不同龄期和断水处理的‘富士’苹果枝条剪口,研究了剪口龄期和水分胁迫对轮纹病菌从剪口侵染的影响。结果表明,春季修剪形成的剪口在2个月内对轮纹病菌都较为敏感,接种发病率较高。夏秋季节形成的剪口在2周内对轮纹病菌非常敏感,2周后抗病性逐渐增强。苹果修剪前后,干旱能明显降低剪口对轮纹病菌的抗侵入和抗扩展能力。保护剪锯口不受侵染既能降低苹果苗木带菌率,又能有效控制轮纹病的危害。     

根癌病生防菌——葡萄土壤杆菌E26菌株在葡萄植株的定殖研究

 利用绿色荧光蛋白基因(gfp)标记示踪,研究了葡萄根癌病生防菌葡萄土壤杆菌E26菌株应用到田间后在玫瑰香葡萄(Vitis vinifera cv. Muscat Humbug)根表面和根际土壤中的群体数量变化,比较了E26菌株与葡萄根癌病原菌K308菌株室内人工接种后在玫瑰香葡萄苗茎和根外植体伤口部位的附着情况.在田间自然状况下,E26菌株可以在葡萄根表面和根际土壤中存活定殖.接种5个月后,E26在根表面的平均数量为10⁴cfu/g根(鲜重),在根际土壤中的平均数量为10⁴cfu/g土壤(干重).在室内,E26菌株和K308菌株分别单独接种时均能以相似水平附着在葡萄茎和根的伤口;E26和K308以相同数量同时接种时,附着在葡萄伤口细胞的K308的数量显著低于K308单独接种时所附着在葡萄伤口细胞的数量.扫描电镜显微观察证实E26菌株能够和病菌K308菌株一样附着于葡萄根部伤口处.     

接种大豆花叶病毒条件下大豆上位叶离体培养物的生长与形态发生

 本研究从接种大豆花叶病毒(SMV)江苏株系Sa、Sg的大豆植株上取上位叶为外植体,研究不同株系对愈伤组织产生及芽与根分化的影响。结果发现,愈伤组织的诱导率及其形态特点在接种与未接种间无明显差异;接种Sa的其芽分化率低于未接种的,但接种Sg的其芽分化率反略高于未接种Sg的;接种Sa、Sg后,根的发生能力减弱,但对抗病品种的影响小于感病品种。     

Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants

Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.     

Factors Affecting Pseudomonas savastanoi pv. savastanoi Plant Inoculations and Their Use for Evaluation of Olive Cultivar Susceptibility

ABSTRACT Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.     

Induced Resistance by Penicillium oxalicum Against Fusarium oxysporum f. sp. lycopersici: Histological Studies of Infected and Induced Tomato Stems

ABSTRACT Tomato (Lycopersicon esculentum) plants of 'Lorena' were induced with a conidial suspension (10(7) conidia per ml) of Penicillium oxalicum before inoculation with Fusarium oxysporum f. sp. lycopersici, the wilt pathogen. Histological changes occurred in plants under both growth chamber and glasshouse culture conditions and there was a reduction of disease severity. In noninduced plants, the pathogen produced almost a complete loss of cambium (75 to 100% reduction), an increase in the number of bundles, and a decrease in the number of xylem vessels (20% reduction), in which the diameter also was reduced by 20 to 30% in hypocotyls and epicotyls. The percentage of vessels colonized by F. oxysporum f. sp. lycopersici was positively correlated to the area under the disease progress curve (AUDPC). However, plants induced with P. oxalicum showed less disease, did not lose the cambium, had a lower number of bundles, and had less vascular colonization by F. oxysporum f. sp. lycopersici (35 to 99%). These effects also were observed in 'Precodor', which is susceptible to races 1 and 2 of F. oxysporum f. sp. lycopersici, and partially in 'Ramón', which is resistant to both races. Renewed or prolonged cambial activity that led to the formation of additional secondary xylem could be one of the reasons for disease reduction in P. oxalicum-induced tomato plants.     

Cytospora Canker and Dieback of Apricots

Cytosporal canker and dieback (Cytospora cincta Sacc.) is a widespread disease in apricot growing areas of Europe, excluding the Mediterranean. The pathogen attacks xylem and phloem. If the necrotic phloem completely girdles the branches or trunk, the section above the girdled region dies. When cambium necrosis does not girdle the branch or trunk, canker development ensues. Apricot trees are susceptible to Cytospora infection between July and the next bud opening.     

Pathogenicity of Hymenoscyphus fraxineus towards leaves of three European ash species: Fraxinus excelsior,F. angustifolia and F. ornus

This study aimed to demonstrate the association of the ash dieback pathogen Hymenoscyphus fraxineus with leaf symptoms on Fraxinus excelsior and to test its pathogenicity towards leaves of three European ash species, F. excelsior, F. angustifolia and F. ornus, in wound inoculation experiments. On F. excelsior, H. fraxineus was isolated from 94% of leaf rachises with necrotic lesions and from 74% of necrotic leaflet midribs. Following wound inoculation of leaf rachises, in two separate experiments performed in 2010 and 2011, the ash dieback pathogen caused symptoms (necrotic rachis lesions, leaf wilting and premature leaf shedding) on all three ash species, while control leaves remained symptomless. Hymenoscyphus fraxineus was consistently reisolated from fungus‐inoculated rachises. All 10 isolates tested were pathogenic to the three ash species and varied in virulence. Koch's postulates for H. fraxineus as causal agent of leaf symptoms on F. excelsior were fulfilled in this study. Complemented with the isolation of the fungus from naturally infected, symptomatic leaf rachises of F. angustifolia and F. ornus in previous investigations, H. fraxineus was confirmed to be a leaf pathogen of these ash species as well. The leaf inoculation experiments showed that F. excelsior was highly susceptible to H. fraxineus, F. angustifolia was equally or slightly less susceptible, whereas F. ornus was the least affected species; however, F. ornus should also be regarded as a host tree for the ash dieback pathogen. This susceptibility ranking corresponds well with field observations and previous stem inoculation experiments.     

The effects of inoculum dose, duration of wet period, temperature and wound age on infection by Nectria galligena of pruning wounds on apple

Four experiments were conducted with potted trees of several apple cultivars to study the effects of several factors on the incidence of canker and the length of the incubation period following the inoculation of pruning cuts with conidia of Nectria galligena. These factors included wound age (the interval between pruning and inoculation), inoculum dose and environment (wet-period temperature and duration). The most important factors affecting the incidence of canker and the incubation period were inoculum dose, cultivar and wound age. Low inoculum dose resulted in low incidence of canker. The incidence of canker decreased as the age of the pruning wound increased. The incubation period lengthened with low inoculum dose and increasing age of the wound. The degree of resistance related to the age of the wound varied with cultivar; likewise, it also varied with the time of year, but this was not due to temperature alone. On fresh wounds, the incidence of canker and the incubation period were not affected by temperature during the wet period. The effect of duration of wetness on canker incidence was significant in only one out of three experiments: the longer wet periods resulted in a slightly lower incidence on fresh wounds. In another experiment, wet periods longer than 2 h resulted in shorter incubation periods. The results are discussed in relation to wound healing.     

Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae

Several factors affecting the severity of bacterial canker of pear were studied. In the orchard, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 10⁶ colony-forming units/shoot. However, under favourable conditions in a growth chamber, cankers formed on detached shoots inoculated with 5 cfu/shoot. A second-order polynomial relationship was established between log₁₀ transformed canker length and log₁₀ transformed inoculum dose. In orchard and growth chamber experiments, shoots were susceptible from the time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at – 10°C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. The practical implications of these results are discussed as a basis for control of bacterial canker of pear.     

荧光假单胞杆菌的嗜铁素是控制桉树灰霉病的主要因子

 本文对3个假单胞杆菌菌株(Pseudomonas spp.)及其嗜铁素(pseudobactin siderophore)缺失突变体防治桉树灰霉病进行了研究.平板拮抗活性测定表明,荧光假单胞杆菌(P.fluorescens) WCS374r菌株和恶臭假单胞杆菌(P.putida) WCS358r菌株通过对铁离子的竞争抑制灰霉菌的生长.在接种灰霉病菌之前10 h将WCS358r、WCS374r和WCS417r施用于受伤的桉树叶片后,可分别降低发病率48.9%、58.3%和40.3%;当将3种生防菌分别与灰霉病菌混合后接种桉树叶片,WCS358r和WCS374r仍然能够显著地降低发病率;在接种灰霉病菌12 h后再施用生防菌,WCS358r和WCS374r对病菌仍具有一定的抑制作用,而在24 h后施用生防菌,3个菌株均未表现显著的防治效果.WCS358r和WCS417r的嗜铁素缺失突变体无防病作用,而WCS374r的嗜铁素缺失突变体虽然还能有效地防治灰霉病,但与WCS374r相比,防病效果减弱.本试验结果说明假单胞杆菌的嗜铁素是控制桉树灰霉病的重要因子.     

Simultaneous Infection by Pseudomonas syhngae van Hall and Cytospora cincta Sacc. on Apricots

Simultaneous inoculation with Pseudomonas syringae van Hall and Cytospora cincta Sacc. induced more extensive cankers than those caused by either pathogen alone. Freshly cut wounds remained susceptible to cytosporal infection for a longer period than to bacterial inoculation. Knowledge gained of the annual cycle of cytosporal and bacterial diseases offers possibilities for effective control against both pathogens.     

Biological control of crown gall of grapevine, rose, and tomato by nonpathogenic Agrobacterium vitis strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 10(6) colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens.     

一种镰刀菌对空心莲子草的致病力与寄主专一性测定

从罹病的空心莲子草上分离到一种镰刀菌,人工接种条件下,3叶期的空心莲子草幼苗最易发病,叶部次之.在供试的4个菌株中,以YZ-03菌株的致病力最强,该菌株接种空心莲子草幼苗5d后,接种的植株病情指数已达100,8d后幼苗全部枯死;人工接种叶片和茎秆9d后,叶片的病情指数达90.95,接种茎秆的病情指数达85.19.采用27科61种植物对该镰刀菌进行寄主专一性测定,结果表明,该镰刀菌能使莲子草严重发病,也能侵染藜的幼苗,对包括主要农作物在内的其他59种植物均无致病性.     

Mutations that Affect Agrobacterium vitis-Induced Grape Necrosis also Alter Its Ability to Cause a Hypersensitive Response on Tobacco

ABSTRACT Tn5-induced mutations in Agrobacterium vitis F2/5 resulted in both altered grape necrosis and tobacco leaf panel collapse phenotypes, suggesting that the underlying mechanisms of the reactions are related. The reaction on tobacco resembles the classical hypersensitive response (HR) caused by several plant pathogenic bacteria in that it is observable within 14 h, is inhibited by treatment of plants with metabolic inhibitors, and results in the inability to recover the pathogen from the necrotic zone. Strains of A. vitis differ with regard to their efficiency of causing the reaction on tobacco. An EcoRI fragment from one mutant, M6, which is necrosis-altered and HR-minus, was cloned and sequenced. Sequence analysis revealed that the Tn5 insertion occurred in a region that shares significant homology with genes involved in long chain fatty acid production by the marine bacteria Shewanella spp. and Moritella marina. Complementation of M6 with a cosmid clone from an F2/5 DNA library restored the tobacco HR and grape necrosis phenotypes.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

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.     

Differential Colonization of Tomato Roots by Nonpathogenic and Pathogenic Fusarium oxysporum Strains May Influence Fusarium Wilt Control

ABSTRACT Histochemical staining, beta-glucuronidase (GUS) activity, or placing roots on agar were methods used to characterize interactions between the pathogenic fungus, Fusarium oxysporum f. sp. lycopersici, and the nonpathogenic biocontrol F. oxysporum strain 70T01 with respect to colonization behaviors, interaction sites, and population densities on tomato roots. Mycelia of strain 70T01, a genetic transformant expressing stable GUS activity, hygromycin B resistance, and effective disease control, were localized in epidermal and cortex cell layers of tomato roots in a discontinuous and uneven pattern. In contrast, mycelia of F. oxysporum f. sp. lycopersici were found in the vascular bundles. Thus, direct interactions between the two fungi likely happen in the root surface cell layers. Colonization density of strain 70T01 was related to the inoculation density but decreased with distance from the inoculation site. Host defense reactions, including increased cell wall thickness or papilla deposits, were adjacent to 70T01 hyphae. Experiments done in soil showed that strain 70T01 densities in roots were highest at inoculation zones and barely detectable for root segments more than 2 cm away from the inoculation sites. F. oxysporum f. sp. lycopersici densities were lowest at 70T01 inoculation zones and highest (>10 times) where strain 70T01 was not directly applied. Newly elongating roots where strain 70T01 did not reach were available for infection by the pathogen. The higher strain 70T01 density was always found when the plants were simultaneously infected by F. oxysporum f. sp. lycopersici, suggesting that F. oxysporum f. sp. lycopersici has as much influence in predisposing the plant to colonization by strain 70T01 as strain 70T01 has on providing disease protection against the pathogen.     

Influence of rootstock on the susceptibility of sweet cherry scions to bacterial canker, caused by Pseudomonas syringae pvs morsprunorum and syringae

In a field trial to determine whether the rootstock influenced the susceptibility of cherry cultivars to bacterial canker three cultivars (Napoleon, Roundel and JI 14039), each grafted on two rootstocks (F 12/1 and Colt), were subjected to natural infectionand to inoculation with three bacterial canker pathogens (Pseudomonas syringae pv. morsprunorum races 1 and 2 and P. syringae pv. syringae). Inoculations were made through leaf scars and through wounds. The high susceptibility of Napoleon and high resistance of JI 14039 were confirmed. Napoleon was more susceptible to inoculation through branches when on F12/1 than when on Colt but the reverse was true for leaf scar inoculations. JI14039 was more susceptible to race 1 inoculated through leaf scars when grown on F12/1 than when on Colt. No rootstock/scion interaction was detected with Roundel.
The complexity of the relationships between the pseudomonad pathogens and their cherry hosts is briefly discussed.