Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

Ozone for control of post-harvest decay of table grapes caused byRhizopus stolonifer

The effect of ozone on post-harvest decay of table grapes was studied both with regard to its effectiveness and its possible mode of action. Ozone concentrations fell rapidly upon contact with organic matter and the amount which reacted with grape berries and the microflora on their surface was about 0.1mgg^-1, when supplied at a rate of 8mgmin^-1for 20min. The dose applied could be increased by longer periods of exposure, but symptoms of toxicity were observed on certain cultivars. The number of colony forming units (cfu) of fungi, yeasts and bacteria naturally present on the berry surface was considerably reduced by a 20min exposure to ozone. Ozone treatments significantly reduced the extent of berry decay caused by fungi following cold storage and increased shelf-life. A significant decrease in decay was observed in berries that were treated with ozone either before or after being inoculated withRhizopus stolonifer.This finding indicates that, in addition to its sterilizing effect, ozone also induced resistance to post-harvest decay development. The phytoalexins resveratrol and pterostilbene were elicited by ozone treatments, at levels similar to those produced by uv-c irradiation. Resveratrol accumulated in greater quantities than pterostilbene. Inoculation withR. stoloniferin addition to ozone treatment, raised the levels of both stilbenes even more. Exposing berries to ozone was almost as effective as SO₂fumigation for the control of storage decay caused byR. stoloniferand no deleterious effects were observed on the appearance of the grape cluster. Ozone treatments can therefore be considered as a possible substitute for SO₂fumigation for the control of post-harvest fungal decay.     

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.     

Elicitin诱导烟草微敏反应和防卫基因的表达

 研究了一种α型elicitin-parasiticein对烟草微敏反应(microscopic hypersensitive response, micro-HR)和防卫反应分子表征基因表达的诱导作用。用150 nmol/L的parasiticein喷洒烟草叶片12 h后, 经曲利本蓝(trypan blue)染色, 光镜下可观察到有细胞坏死, 说明parasiticein引起了micro-HR, 而水和低浓度的parasiticein不能引起micro-HR。用parasiticein注射叶片可引起肉眼可见的HR, 注射用parasiticein浓度远比喷洒引起micro-HR的浓度低。Parasiticein还可诱导对TMV的抗性, 浓度在30 nmol/L时比150 nmol/L的效果好。用parasiticein注射烟草30 min, HR表征基因hsr203J和hin1开始表达, 在9 h内逐渐降低, 12~16 h肉眼可观察到HR。Parasiticein喷洒烟草后, 病程相关蛋白(pathogenesis-related, PR)基因PR-1b在诱导的第1 d到第5 d都有一定量的表达。可见, parasiticein能同步诱导过敏反 应和抗病 性及其分 子表征基 因的表达 。     

aloxylon ammodendron (Amaranthaceae) fruit development delay caused by post-flowering non-inductive photoperiod

Haloxylon ammodendron (C. A. Mey.) is one of the economically and ecologically important desert trees used for sand fixation. The ovary of H. ammodendron is found not to swell after flowering in spring until at the end of August or early September in western China. However, what happens for ovary at anatomic level in that period and which crucial ecological factor regulates the phenomenon of H. ammodendron have not been fully understood. To characterize the phenomenon and explore the crucial environmental regulating factors, we carried out the morphological and anatomic observations at the different development stages of the fruits and three single-factor experiments (low air temperature, sufficient soil moisture, and short day length). Our results showed that under the natural conditions, the ovary of H. ammodendron after flowering developed slowly and the morphological changes of fruits were not significant for the period from May to August and after late August or early September; and then the ovary developed rapidly and matured in October. Cell division in embryo was observed to start approximately 25 days after flowering (DAF) and just developed to globular embryo stage at mid-August. Photoperiod was identified as the pivotal environmental factor regulating the fruit development of H. ammodendron. Moreover, the threshold value of day length for the fruit development was 14.0 h. A long day (>14.0 h) treatment began from 5 DAF could delay fruit development of H. ammodendron while a short day (<14.0 h) treatment could accelerate it. Moreover, a further longer day treatment (>15.0 h) could also delay fruit development even when they had developed for a long time (110 DAF). The present study indicated that H. ammodendron adopted a reproductive strategy of delayed fruit development and this strategy helps it survive and obtain offspring in harsh desert habitats.     

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。     

猕猴桃溃疡病菌不致病菌株G230的鉴定及形成原因分析

为探索田间猕猴桃溃疡病菌Pseudomonas syringae pv. actinidiae(Psa)致病力丧失的分子机制,针对从猕猴桃果园中分离获得的1株不致病菌株G230,通过特异性引物检测和多基因序列分析明确其分类地位,并设计引物检测其是否由已知遗传变异引起,通过比较基因组学、基因表达、超敏反应和荧光素酶报告菌株检测确定引起菌株G230致病力丧失的原因。结果表明,不致病菌株G230为Psa生物型3(Psa3),其致病缺陷不是由已报道的遗传变异引起;基于基因组比较分析发现菌株G230中的hrpS基因被转座子ISPsy36插入破坏,导致Ⅲ型分泌系统（type Ⅲ secretion system,T3SS）不能正常表达;而在不致病菌株G230中表达hrpS基因后能恢复其T3SS功能,使其具备致病能力及激发非寄主超敏反应的能力。表明转座子ISPsy36插入hrpS基因内部可以破坏Psa的T3SS功能进而使其丧失致病力,这是自然条件下Psa3丧失致病力的一种新型机制。     

A functional gene-for-gene interaction is required for the production of an oxidative burst in response to infection with avirulent Pseudomonas syringae pv. tomato in Arabidopsis thaliana

An early event correlated with the gene-for-gene hypersensitive response (HR) is the accumulation of active oxygen species (AOS), also known as the oxidative burst. We present data that genetically demonstrates that the oxidative burst is a downstream component of the RPS2- avrRpt2gene-for-gene signal cascade. An in planta AOS assay using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was modified for use with the Arabidopsis thaliana / Pseudomonas syringae pv.tomato (P. syringae pv. tomato) model system. An oxidative burst occurred between 8 and 15 hpi with avirulent P. syringae pv. tomato(avrRpt2), but not with virulent P. syringae pv. tomato. This burst preceded cell death and was not observed in the RPS2 Arabidopsis mutantsrps2-101C and rps2-201 inoculated with avirulent P. syringae pv. tomato. An HR-like response has been observed when plants undergoing a systemic acquired resistance (SAR) response are challenged with a normally virulent pathogen (manifestation stage of SAR), however an HR-like oxidative burst was not detected by the in planta AOS assay during this stage of SAR.     

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。     

Differential induction of pathogenesis-related proteins in tomato byAlternaria solaniand the association of a basic chitinase isozyme with resistance

Accumulation of pathogenesis-related proteins is thought to play a role in pathogen-induced plant defense responses. Although early accumulation of hydrolytic enzymes such as chitinase and β-1,3-glucanase has been associated previously with genetically-inherited and induced systemic resistance, their role in resistance in tomato(Lycopersicon esculentum)to the phytopathogenic fungusAlternaria solaniis not yet understood. Here we describe the accumulation patterns of specific isozymes of pathogenesis-related proteins in the resistant tomato genotypes 71B2, NC EBR-1, NC EBR-2 and the susceptible cultivar Piedmont. Western blot analysis demonstrated that four isozymes of chitinase (26, 27, 30, and 32kDa) were induced in all genotypes upon challenge withA. solani,but only resistant lines had significantly higher constitutive levels of the 30kDa isozyme as well as total chitinase activity. In addition, the 30kDa chitinase isozyme was found to accumulate to significantly higher levels in resistant lines during pathogenesis than the susceptible genotype. Two isozymes of β-1,3-glucanase (33 and 35kDa) were detected in all genotypes, but a slightly higher constitutive level was detectable in all resistant lines when compared to the susceptible. Similar accumulation patterns of these isozymes were observed in all genotypes during the course of pathogenesis. Purified preparations of acidic and basic tomato chitinase and β-1,3-glucanase isozymes were tested for their antifungal activity againstA. solani in vitro.Results presented in this study indicate that only basic isozymes of chitinase and β-1,3-glucanase were inhibitory toA. solaniwhereas, no inhibitory activity was observed with the acidic isozymes. The results of this study suggest that a higher constitutive level of chitinase and β-1,3-glucanase and the induction pattern of a 30kDa chitinase isozyme in early blight resistant breeding lines is related to genetically-inherited resistance of tomato toA. solani.     

The TIR–NBS but not LRR domains of two novel N-like proteins are functionally competent to induce the elicitor p50-dependent hypersensitive response

The tobacco N protein recognizes the helicase domain (p50) of the Tobacco mosaic virus (TMV) replicase as an elicitor and mediates hypersensitive response (HR). We obtained two cDNA clones encoding novel N-like (NL) proteins NL-C26 and NL-B69 from Nicotiana tabacum cv. Samsun NN. NL-C26 and NL-B69 had a Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) structure and showed 78% and 73% identities to N, respectively. The NL-C26 and NL-B69 genes were also expressed in N. tabacum cv. Samsun nn, which lacks the N gene. Unlike N, NL-C26 and NL-B69, when coexpressed with p50, failed to induce HR on the sites of agroinfiltration in Samsun nn leaves. However, the elicitor-dependent HR in Samsun nn was induced efficiently by chimeric N proteins with the continuous TIR–NBS domains of NL-C26 and NL-B69. On the other hand, the efficiency of HR induction varied significantly among chimeric N proteins with either of the TIR and NBS domains of the NL proteins. In contrast, chimeras carrying the LRR domains of the NL proteins did not induce HR. Thus, the TIR–NBS domains of NL-C26 and NL-B69 could functionally adapt to the LRR domain of N, which may determine the specificity for the elicitor. We speculate that the NL genes are potential HR-inducing resistance genes for undetermined pathogens other than TMV.     

Transcriptional regulation by iron and role during plant pathogenesis of genes encoding iron- and manganese-superoxide dismutases of Pseudomonas syringae pv. syringae B728a

The bean pathogen, Pseudomonas syringae B728a, may require iron-superoxide dismutase (FeSOD) and manganese–superoxide dismutase (MnSOD) activities in protection against reactive oxygen species (ROS) generated in planta. Genes encoding FeSOD or MnSOD of P. syringae B728a were cloned by hybridization with specific PCR probes amplified from P. syringae genomic DNA. The sodB gene was monocistronic whereas the sodA gene was transcribed as part of a 1.4 kb polycistronic operon, consisting of orfX-sodA. A putative Fur consensus sequence was located upstream of the orfX-sodA operon. The sodB (FeSOD) gene was expressed throughout growth, but was down regulated under iron deficient conditions. In contrast, the sodA (MnSOD) gene was expressed only under iron deficient conditions. Mutants defective in sodA and sodB genes were generated by marker exchange mutagenesis. Unlike other bacterial SOD deficient mutants, the P. syringae B728a sodAsodB mutant was not impaired in growth on rich or minimal medium, but it was more sensitive to paraquat than wild-type. The P. syringae B728a SOD deficient mutants caused bacterial brown spot disease on bean pods or leaves to the same extent as wild-type. Thus, these superoxide dismutases may not be key enzymes for aerobic metabolism and pathogenicity in P. syringae B728a.     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

PCR cloning, DNA sequencing and phylogenetic analysis of a xylanase gene from the phytopathogenic fungusAscochyta pisiLib.

A gene encoding a xylanase,xyl1, was isolated from the phytopathogenic fungusAscochyta pisiLib. by PCR cloning using degenerate primers. DNA sequence analysis revealed an open reading frame of 736 bp interrupted by an intron of 55 bp. The ORF encodes a predicted protein of 227 amino acids. The precise splicing site of the intron was identified from the sequence of a PCR product obtained using the same degenerated primers on a cDNA template. The cDNA product and a northern blot demonstrated that the gene is transcribed into mRNA when the fungus is cultured in media containing xylan as sole carbon source. The Neighbour-Joining method using the Clustal W(1.5) program demonstrated that theA. pisixylanase is a member of the family 11 glycosyl hydrolases, and that this family represents at least five phylogenetically consistent groups. The family 11 glycosyl hydrolases can be linked with family 10 glycosyl hydrolyses through bifunctional enzymes fromRuminococcus flavefaciensand, to a lesser extentNeocallimastix patriciarum.     

Factors impacting blackleg development caused by Dickeya spp. in the field

Stem rot symptoms caused by pectinolytic bacteria of Genus Pectobacterium and Genus Dickeya, which are commonly referred to as blackleg, strongly impact the quality of seed potato production in most European countries. Several biotic and abiotic factors, such as cultivar susceptibility, isolate aggressiveness, mother tuber infection density and a wide range of soil-related and climatic factors have been identified in the literature as having an effect on blackleg development. The aim of this study was to identify which biotic and/or abiotic factors are most critical to the development of blackleg in the field. In Switzerland, the predominant species have belonged to Genus Dickeya as far back as 1992, which is why this study only investigates blackleg symptoms induced by Dickeya isolates. Seven field trials, in which inoculated tubers were planted, were conducted during a 3-year period and the number of blackleg-diseased plants was counted. Multiple regression analysis was used in order to determine the factors that had the greatest impact on two different variables: (i) periods between emergence of the plant and disease outbreak and (ii) overall blackleg incidence throughout the growing season. The results of this analysis have revealed that environmental factors, such as evapotranspiration and soil moisture, explain about half of the variability in the number of days before disease outbreak, and the total number of diseased plants is widely dependent upon cultivar susceptibility and isolate aggressiveness.     

Pythium rot of chingensai ( Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of chingensai (Brassica campestris L. chinensis group) in Okayama Prefecture in 2000. The causal fungi were morphologically identified as Pythium ultimum Trow var. ultimum and P. aphanidermatum (Edson) Fitzpatrick. This is the first report of rot caused by Pythium species on chingensai. We named this disease Pythium rot of chingensai.     

玉米镰孢菌穗、茎腐病侵染循环的相互关系

玉米穗腐病和茎腐病是玉米主要病害。茎腐病多数病株明显发生根腐,茎基节间产生不规则状褐斑,随后很快变软,内部空松,导致果穗下垂、籽粒干瘪、整株青枯或黄枯,明显减产。玉米穗腐病的主要致病菌为串珠镰孢菌Fusarium moniliforme和禾谷镰孢菌Fusarium graminearum,茎腐病的主要致病菌除腐霉菌外,也有与穗腐病相同的禾谷镰孢菌     

四川省小麦条锈病流行区划及菌源传播路径分析

四川省是我国小麦条锈病菌Puccinia striiformis Westend f.sp.tritici Eriks的菌源地和常发区,近10年秋苗病害始见期提前、流行期加长、危害损失呈上升趋势.在实地调查基础上,利用GIS技术对全省132个小麦主产县(市、区)1987-2006年小麦条锈病发生情况进行系统分析.结果表明,四川省小麦条锈病流行区域可划分为川西北越夏区、川西南越夏及冬繁区、盆地冬繁区和川东南春季流行区.GIS软件分析发现病菌孢子在四川盆地内的传播路径为沿岷江、涪江、沱江和嘉陵江等4条河流自北向南、自西向东传播.