间歇式流加补料对丁香假单胞菌大豆致病变种Z2-6冠菌素产量的影响

为提高具有诱导植物抵御逆境等多种生理功能的冠菌素的产量,基于传统分批发酵法建立丁香假单胞菌大豆致病变种Pseudomonas syringae pv. glycinea Z2-6间歇式流加补料发酵方式,并对补料方法中的起始补料时间、补料体积和补料中的合成前体L-异亮氨酸含量进行了优化。结果表明,利用间歇式流加补料发酵方式,于接种后第8天开始每隔24 h补加新鲜GC培养基(含0.75 g/L的L-异亮氨酸和0.3 g/L的丙酮酸钠)1次,每次补料体积占发酵液总体积的2.0%时,丁香假单胞菌大豆致病变种Z2-6的冠菌素产量最大,达241.5 mg/L,较传统分批发酵方式下的产量(152.5 mg/L)提高了58.4%,合成速率达到20.1 mg·L~(-1)·d~(-1)。表明此种补料发酵方式既可以使细菌高效、持续地利用营养物质,又可解除产物的反馈抑制,大幅提高冠菌素产量,最终达到高产目的。     

Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis

Rhizoctonia solani root-rot is a major soilborne disease causing growth and yield depression. The ability of Glomus mosseae BEG12 and Pseudomonas fluorescens A6RI to suppress this soilborne disease in tomato was assessed by comparing the shoot and root growth of plants infested with R. solani 1556 when protected or not by these beneficial strains. The epiphytic and parasitic growth of the pathogenic R. solani 1556 was compared in the presence and absence of the biocontrol agents by microscopical observations allowing the quantification of roots with hyphae appressed to epidermal cells (epiphytic growth) and of roots with intraradical infection (parasitic growth). The root architecture of the tomato plants under the different experimental conditions was further characterized by measuring total root length, mean root diameter, number of root tips and by calculating degree of root branching. G. mosseae BEG12 and P. fluorescens A6RI fully overcame the growth depression caused by R. solani 1556. This disease suppression was associated with a significant decrease of the epiphytic and parasitic growth of the pathogen together with an increase of root length and of the number of root tips of inoculated tomato plants. The combined effects of G. mosseae BEG12 and P. fluorescens A6RI on pathogen growth and on root morphogenesis are suggested to be involved in the efficient disease suppression.     

A new aggressive strain of Verticillium albo-atrum in Verticillium resistant cultivars of tomato in the Netherlands

In 1991 serious losses caused byVerticillium wilt were found on two holdings in the Westland glasshouse district in the Netherlands in which theVerticillium resistant tomato cultivars Calypso and Criterium were grown in soilless systems. Isolates from diseased plants were identified asVerticillium albo-atrum.In inoculation experimentsVerticillium resistant tomato cultivars were seriously affected by the new isolates but not by a control isolate. Moneydor, a susceptible cultivar without the Ve gene, was the most seriously diseased by all isolates. The isolates from theVerticillium resistant tomato cultivars were less virulent on the susceptible cultivar than the control isolate.     

Tolerance to a non-host isolate of Verticillium dahliae in tomato

Tolerance to Verticillium spp. is a condition in which a host plant develops few symptoms despite substantial colonization by the pathogen. In the present paper we have shown that Craigella tomatoes are tolerant to a non-host isolate of V. dahliae, Dvd E6. Symptom expression was used to quantify disease and quantitative PCR to assess the amount of fungus in the stems. The classical incompatible and compatible interactions between Craigella resistant or Craigella susceptible near isolines and V. dahliae, race 1 were used for comparative purposes. Additional experiments using cytological assessment and quantitative PCR showed that in the tolerant interactions one plant defence response, vascular coating, was deployed as effectively as in resistant plants, limiting pathogen distribution. However, a second defence response, which causes the cyclical elimination of fungus from the stem in the classical interactions either does not occur or is substantially delayed in tolerant plants. Thus, the Verticillium population remains stable and substantial throughout the studied time course.     

Subcellular localization of the protein encoded by virulence gene <Emphasis Type="Italic">psvA</Emphasis> of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">eriobotryae</Emphasis>

The plasmid-encoded virulence gene psvA was previously isolated from Pseudomonas syringae pv. eriobotryae and sequenced. The deduced protein of the psvA gene had no significant similarity to any other protein sequences in the database. To gain a better understanding of the function of the PsvA protein its subcellular localization was examined. To localize the PsvA protein within the bacteria, the cells were fractionated into cytoplasmic, inner membrane, and outer membrane components. The cell fractions and culture supernatant were analyzed by immunoblotting. The PsvA protein was predominantly detected in the outer membrane fraction. Immunoelectron microscopy also showed that the PsvA protein was located in the outer membrane.     

The hrpZ and hrpA genes are variable, and useful for grouping Pseudomonas syringae bacteria

The hrpS to hrpB regions from strains of Pseudomonas syringae were amplified by polymerase chain reaction (PCR) and the DNA sequence determined. The order of hrpS, hrpA, hrpZ, and hrpB was consistent among P. syringae strains. The sequence of hrpS was highly conserved. In a cluster analysis with the hrpS sequence, P. syringae strains were divided into four groups (I, II, III, and IV) and one undetermined strain, in agreement with previous studies. In contrast, the hrpZ sequences contained insertions, deletions, and base substitutions followed by changes in amino acids. Based on cluster analysis of hrpA, hrpZ, and hrpB, P. syringae strains could be divided into five groups. One of the four groups (group I) in the cluster analysis of hrpS could be further divided into two subgroups (groups IA and IB). Groups II, III, and IV were the same in the two analyses. Group-specific primers were designed, based on the DNA sequences of hrpZ, that could differentiate the groups of P. syringae strains. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB112552 to AB112581     

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZ_Pst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.     

Apoplastic redox metabolism: Synergistic phenolic oxidation and a novel oxidative burst

The plant apoplast is an important mediator of communication between the cell cytoplasm and its surroundings. Plant cell suspensions offer a convenient model system to gain insight into apoplastic physiology. Here, we describe a novel phenomenon that took place when two naturally occurring phenolics were added together to either soybean or tobacco cell suspensions. Acetosyringone (AS) and/or hydroxyacetophenone (HAP), phenolics found in the extracellular/apoplast of tobacco cells, were added to soybean or tobacco cell suspensions undergoing an oxidative burst. Individually, AS appeared to be utilized as a typical peroxidase substrate to scavenge hydrogen peroxide, while HAP was utilized at a much lower rate. However, when added together the rate of utilization of both phenolics increased and surprisingly resulted in the production of hydrogen peroxide. We have further characterized this novel phenomenon in suspension cells. This study demonstrates that certain phenolics in plants can cause co-oxidation which, as in animals, could alter the structure and bioactivity of surrounding phenolics.     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

Flagellin from Pseudomonas syringae pv. tabaci induced hrp-independent HR in tomato

We have previously shown that flagellin of Pseudomonas syringae pv. tabaci is an elicitor that induces a hypersensitive reaction (HR) in nonhost tomato cells. Flagellin is the major HR elicitor produced by this pathogen, as shown by the inability of a flagellin-defective mutant, ΔfliC, to induce HR. Also, a ΔfliD mutant that secretes large amounts of monomer flagellins induces a strong HR in tomato. In this study, the possible involvement of an Hrp type III secretion system (TTSS) in flagellin-induced HR was investigated using flagella-defective mutants or Hrp TTSS-defective mutants. The hrcC gene encodes HrcC protein, which is required for Hrp pilus formation in the outer membrane. An hrcC mutation, introduced into the wild-type, ΔfliC, and ΔfliD mutants of P. syringae pv. tabaci did not affect swimming motility or flagellin secretion, whereas all ΔhrcC, ΔfliC, and ΔfliD mutants lost the ability to cause disease on host tobacco leaves. However, the ΔhrcC mutant and the ΔfliD/ΔhrcC double mutant were still able to induce HR cell death, expression of one of the defense-related genes hsr203J, and the generation of hydrogen peroxide in nonhost tomato cells. Thus, flagellin is required for both pathogenicity in host tobacco and HR in nonhost tomato. On the other hand, hrp TTSS is necessary for pathogenicity on host tobacco but is not indispensable to induce HR in nonhost tomato. These results clearly show that flagellin-induced HR is hrp-independent in tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB049570     

Effect of phytotoxic compounds produced by Clavibacter michiganensis subsp. michiganensis on resistant and susceptible tomato plants

A phytotoxic fraction of high molecular weight was isolated from the culture filtrate ofClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker of tomato, and partly purified. This high molecular weight fraction consists of sugars and a minor protein moiety and is therefore probably of similar nature to that of the toxin fromC. michiganensis subsp.michiganensis reported earlier in literature.The high molecular weight fraction was albe to induce wilting, the predominant symptom of the disease, as shown in a bioassay with tomato cuttings. However, this wilting reaction turned out to be non-specific in the bioassay, since (partially) resistant and susceptible genotypes responded similarly. No correlation could be found between the degree of virulence of fiveC. michiganensis subsp.michiganensis strains and the amount of the phytotoxic high molecular weight fraction produced in vitro.As the isolated high molecular weight fraction showed a phytotoxic effect on tomato plants it is worthwhile to test its potential for use as a selective agent in in vitro selection.Samenvatting Een fytotoxische fractie werd geïsoleerd uit cultuurfiltraat vanClavibacter michiganensis subsp.michiganensis, de veroorzaker van de bacterieverwelkingsziekte bij tomaat. Een eerste karakterisering toonde aan dat deze toxische fractie hoog-moleculaire component(en) bevat, bestaande uit polysacchariden en een gering percentage eiwit. Dit is in overeenstemming met toxines vanC. michiganensis subsp.michiganensis die al eerder beschreven zijn.Deze hoogmoleculaire toxische fractie was in staat verwelking te induceren van stengeltoppen van verschillendeLycopersicon esculentum enL. peruvianum genotypen in een bioassay. Gewichtsverandering van de stengeltoppen, uitgedrukt als percentage ten opzichte van het begingewicht, werd gebruikt als parameter voor verwelking. De toxische fractie reageerde niet-specifiek in de bioassay, want er werd geen verschil gevonden in respons van (partieel) resistente en gevoelige genotypen. Er bleek geen correlatie te zijn tussen de mate van virulentie van verschillende isolaten vanC. michiganensis subsp.michiganensis en de hoeveelheid van de toxische fractie geproduceerd in vitro.Het mogelijke gebruik van deze hoogmoleculaire toxische fractie als selectief agens bij in vitro selectie zal nader onderzocht worden.     

Cowpea mild mottle virus could not be detected by ELISA in soybean and groundnut seeds in Indonesia

Using ELISA to determine whether cowpea mild mottle virus (CMMV) was present in soybean and groundnut seeds, the virus was not detected in 4144 seeds harvested from seven CMMV-infected soybean genotypes and in 214 seeds collected from CMMV-infected groundnut plants (cv. Gajah). These results, together with those of other researchers, suggest that, under the conditions tested, CMMV is not transmitted by seed. This in contrast to what is generally accepted and published in reviews.     

Effectiveness and profitability of the <Emphasis Type="Italic">Mi</Emphasis>-resistant tomatoes to control root-knot nematodes

Experiments were conducted to determine the effectiveness and profitability of the Mi-resistance gene in tomato in suppressing populations of Meloidogyne javanica in a plastic-house with a natural infestation of the nematode. Experiments were also conducted to test for virulence and durability of the resistance. Monika (Mi-gene resistant) and Durinta (susceptible) tomato cultivars were cropped for three consecutive seasons in non-fumigated or in soil fumigated with methyl bromide at 75 g m^–2 and at a cost of 2.44 euros m^–2. Nematode densities were determined at the beginning and end of each crop. Yield was assessed in eight plants per plot weekly for 6 weeks. The P_f/P_i values were 0.28 and 21.6 after three crops of resistant or susceptible cultivars, respectively. Growth of resistant as opposed to susceptible tomato cultivars in non-fumigated soil increased profits by 30,000 euros ha^–1. The resistant Monika in non-fumigated soil yielded similarly (P > 0.05) to the susceptible Durinta in methyl bromide fumigated soil but the resistant tomato provided a benefit of 8800 euros ha^–1 over the susceptible one because of the cost of fumigation. Selection for virulence did not occur, although the nematode population subjected to the resistant cultivar for three consecutive seasons produced four times more eggs than the population on the susceptible one. Such a difference was also shown when the resistant cultivar was subjected to high continuous inoculum pressure for 14 weeks. The Mi-resistance gene can be an effective and economic alternative to methyl bromide in plastic-houses infested with root-knot nematodes, but should be used in an integrated management context to preserve its durability and prevent the selection of virulent populations due to variability in isolate reproduction and environmental conditions.     

A New Cell Wall Located N-rich Protein is Strongly Induced During the Hypersensitive Response in Glycine Max L.

Soybean (Glycine max (L.) Merill, cv. Williams 82) plants and cell cultures respond to avirulent pathogens with a hypersensitive reaction. After inoculation of soybean with Pseudomonas syringae pv. glycinea, carrying the avirulence gene avrA, or zoospores from the fungus Phytophthora sojae Race 1, a resistance-gene-dependent cell death programme is activated. A new gene was identified by differential display of mRNAs that is specifically activated during the early phase of incompatible pathogen-soybean interactions but does not respond to compatible pathogens. The gene is strongly induced within 2h after addition of P. sojae zoospores. A similar kinetic pattern was observed for P. syringae (avrA) inoculated soybean cell cultures. The gene encodes a deduced protein of 368 amino acids with a very high content of asparagine and was therefore termed N-rich protein (NRP). The protein is composed of two distinct domains, of which only the C-terminal domain has striking homology to proteins of unknown function from other plants. An antibody raised against the recombinant NRP recognizes a protein of 42kDa. The protein is located in the cell wall as indicated by cell fractionation studies. Comparison of the genomic DNA-sequence with the cDNA, identified two introns within the open reading frame. The NRP-gene is not directly induced by salicylic acid or hydrogen peroxide, indicating a distinct and specific signal transduction pathway which is only activated during programmed cell death. The NRP-gene appears to be a new marker in soybean activated early in plant disease resistance.     

实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。     

Effects of fusaric acid on cells from tomato cultivars resistant or susceptible toFusarium oxysporum f. sp.Lycopersici

Cell suspension cultures were set up from two tomato cultivars, one resistant, (Rio grande) and one susceptible (63.5) toFusarium oxysporum f. sp.lycopersici. Growth rates of the two cell cultures were comparable. Toxicity of fusaric acid, expressed as the fresh weight loss, was analyzed: It was significant in both cases after 10 h, but toxicity was twice as high for 63.5 suspension cells. In the same way, electrolyte leakage caused by fusaric acid was three times more important for 63.5 suspension cells. Moreover, fusaric acid treatment resulted in an acidification of the extracellular medium for 63.5 suspension cells (0.4 pH unit), whereas an alkalization was observed for Rio grande suspension cells (0.2 pH unit). Preliminary experiments suggest that fusaric acid was partially metabolized by Rio grande suspension cells, however, no detoxified forms of fusaric acid were detected either in cells or in culture filtrates. For these two tomato cultivars, the differences in sensitivity to fusaric acid of cultivated cells correspond to the differences in plant susceptibility toFusarium oxysporum f. sp.lycopersici.Abbreviations BAP 6-benzylaminopurine - conductivity - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOAc ethyl acetate - FA fusaric acid - resistivity     

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.