Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts

To obtain a genetic marker to observe and study the interaction of Sclerotinia sclerotiorum with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs, both containing genes for the green fluorescent protein (GFP) and hygromycin B phosphotransferase. Putative transformants were obtained using polyethylene glycol-mediated transformation of protoplasts. Seven stable gfp transformants were identified and evaluated for fluorescence in vitro and in planta , pathogenicity and colonization of host tissues. Real-time quantitative polymerase chain reaction detected a single copy of the gfp gene in transformants. Fluorescence was quantified directly from mycelium and protein extracted from hyphae. The seven transformants (four from ND30 and three from ND21) were pathogenic on dry bean, canola, soybean and sunflower. However, depending on the host, three transformants differed significantly ( P  = 0·05) in the length of lesions formed compared to the wild-type. Hyphae fluoresced in plant tissue and could clearly be distinguished from plant cells. Infection and colonization of tissues were clearly visible with a fluorescent microscope. Transformants differed in the intensity of GFP expression both in vitro and in planta .     

Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

An optimized duplex real-time PCR tool for sensitive detection of the quarantine oomycete Plasmopara halstedii in sunflower seeds

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.     

Inter-isolate variation for virulence in Plasmopara halstedii (sunflower downy mildew) from Hungary

A total of 82 isolates of Plasmopara halstedii , collected from all production areas of Hungary between 1976 and 1993, were assessed for their virulence pattern on a standard set of sunflower differentials under glasshouse conditions. The isolates were classified into six pathogenic races each representing a particular virulence phenotype. From 1976 until 1988 all the isolates were found to be virulent only on sunflowers possessing no known resistance genes, thus classified as race 1. There was an apparent shift in the virulence of the P. halstedii population collected after 1988. Six races (1, 2, 3, 4, 8 and 9) were identified among the 45 samples collected between 1989 and 1993, with races 1 and 3 predominant, at a frequency of 35% each. While the increase in race virulence is undoubtedly due to selection imposed by resistant hybrids, the origin of the new races is unknown. Whether new races have arisen from the indigenous P. halstedii population, or whether they have been imported from abroad, can only be reliably determined by DNA techniques, such as fingerprinting.     

Isozyme analysis of Plasmopara halstedii using cellulose-acetate gel electrophoresis

Isozyme analysis by cellulose-acetate gel electrophoresis was used for the first time on Plasmopara halstedii , the causal agent of sunflower downy mildew. Forty-five isolates originating from sunflower, cocklebur and Helianthus  ×  laetiflorus were used, comprising 10 field isolates and 35 single-spore lines of an additional 30 field isolates representing 10 different virulence phenotypes. Sixteen isozyme systems were analysed, of which three, isocitrate dehydrogenase, malate dehydrogenase and phosphoglucomutase, resulted in clear, reproducible banding patterns and revealed some polymorphism among the isolates. Phosphoglucomutase differentiated two groups among the isolates collected from cultivated sunflower, while the other enzymes were polymorphic between isolates from the different hosts. Polymorphisms were not related to virulence phenotype.     

Variation in aggressiveness of Plasmopara halstedii (sunflower downy mildew)

Journal of Plant Diseases and Protection - Variability in aggressiveness was studied in seven Plasmopara halstedii (sunflower downy mildew) parental isolates of races 100, 300, 304, 314, 704, 710...     

Assessment of blast disease resistance in transgenic PRms rice using a gfp-expressing Magnaporthe oryzae strain

Rice blast caused by the fungus Magnaporthe oryzae (anamorph Pyricularia grisea ) is one of the most devastating diseases of cultivated rice worldwide. In this study, a green fluorescent protein ( gfp )-expressing M. oryzae strain was generated and used to investigate the infection process in a commercial rice cultivar. Expression of the gfp gene did not affect the pathogenicity of the M. oryzae transformants. Confocal microscopy allowed in vivo imaging of this pathogen during infection of rice tissues. Magnaporthe oryzae pathogenicity was examined on both leaf and root tissues. In roots of wild-type plants, the fungus penetrated into epidermal and cortical cells, and colonized the central cylinder and xylem vessels. However, the dimorphic growth pattern typically observed during the biotrophic and necrotrophic stages of leaf colonization was not observed during colonization of root tissues. Furthermore, events occurring during infection of rice plants constitutively expressing the maize pathogenesis-related PRms gene were characterized and compared with those occurring during the interaction of this pathogen with untransformed rice plants. Fungal penetration was drastically reduced and delayed in tissues of PRms plants compared to untransformed plants. These results indicated that the gfp -expressing M. oryzae represents a strategic tool for the assessment of blast disease resistance in transgenic rice which can be also applied to the analysis of the M. oryzae interaction with other cultivars or mutants of important crop species.     

Ultrastructural and Immunochemical Changes During the In Vitro Development of Plasmopara halstedii

ABSTRACT The asexual phase of the life cycle of Plasmopara halstedii, the causal agent of downy mildew of sunflower, plays a key role in the propagation of the disease. We investigated the morphological and ultrastructural changes that occur during the asexual development of the pathogen. Direct examination of infected cotyledons confirmed the presence of sporangiophores. In contact with water, important ultrastructural changes occurred, affecting the surface of zoosporangia, which became smoother, and their cytoplasm, which differentiated into flagellate zoospores. The subsequent encystment of zoospores was characterized by the synthesis of a cell wall and the loss of the flagella. In addition, two monoclonal antibodies (MAbs) specific for P. halstedii were used to analyze the immunochemical changes associated with these modifications. MAb 16A6, which bound to a 48-kDa glycoprotein, mainly labeled the surface of mobile or encysted zoospores and of mother cells of germ tubes. Conversely, MAb 2F9, which recognized highly glycosylated antigens, labeled the surface of zoosporangia and of flagellate zoospores, but not the encysted zoospores. These results provide new insights into the morphological and ultrastructural changes associated with the release and the encystment of zoospores which may be interesting targets for the development of new antimicrobial products.     

绿色荧光蛋白基因标记棉花黄萎病菌

以携带有潮霉素抗性筛选标记的pCTHyg载体为骨架,构建了含有增强型绿色荧光蛋白基因sGFP的载体pCH-sGFP,并通过农杆菌介导的遗传转化法导入引起棉花黄萎病的高致病力大丽轮枝菌Vd991,获得了sGFP整合到大丽轮枝菌基因组的转化株。通过转化子荧光信号、生长表型和致病力筛选鉴定,获得了1株与Vd991生长和致病力无显著差异且荧光信号强烈的转化株Vd gfp77。侵染棉花根部试验表明,Vd-gfp77侵染棉花后快速扩展繁殖,子代仍然能发出强烈的荧光信号。本试验绿色荧光蛋白标记大丽轮枝菌的成功构建,为后续大丽轮枝菌侵染棉花过程的组织学和致病机理研究提供了良好的研究材料。     

Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii

Plasmopara halstedii isolates showing an atypical reaction to metalaxyl were collected in France, in 1995 and 1996, and tested in the laboratory for their level of sensitivity to this fungicide. Primary and secondary infections caused by one of these isolates were not controlled by the metalaxyl concentration registered for seed treatment. The EC₅₀ of this isolate was 12 800 mg a.i. kg^-1 compared with 22 mg a.i. kg^-1 for sensitive isolate, indicating a 582-fold decrease in sensitivity to the compound. There was no reduction in the agressiveness of the resistant isolate. Using other anti-oomycete fungicides, it appeared that propamocarb, contact fungicides (fluazinam, folpet, mancozeb) and the mixed formulations dimethomorph + mancozeb, cymoxanil + mancozeb and ofurace + folpet were effective against primary infections made with metalaxyl resistant and sensitive isolates, but not against secondary infections. Metalaxyl mixed with fluazinam, folpet or mancozeb was more effective against primary infections with the resistant isolate than metalaxyl alone. The EC₅₀ of five other isolates ranged from 5 800 to 32 900 mg a.i. kg^-1, indicating a variability in metalaxyl sensitivity of resistant sunflower downy mildew isolates. This is the first report of physiological resistance to metalaxyl in Plasmopara halstedii.     

向日葵的检疫性有害生物

根据FAO“有害生物风险分析准则”评估，初步提出了12种向日葵的潜在检疫性有害生物。其中，我国尚未发生或尚未报道的有褐色茎腐病菌、茎点霉黑茎病菌、向日葵红色种子象、向日葵灰色种子象、向日葵叶甲和向日葵瘿蚊等6种，国内局部地区已有发生的有向日葵霜霉病菌和向日葵黑斑病菌等2种，已列入了我国各类检疫性有害生物名单的种类有黄萎轮枝孢、棉根腐病菌、列当属和菟丝子属寄生种子植物等。     

Dynamic changes in pod and fungal physiology associated with the shift from biotrophy to necrotrophy during the infection of Theobroma cacao by Moniliophthora roreri

Theobroma cacao pods were inoculated with meiospores of Moniliophthora roreri (Mr), a hemibiotrophic basidiomycete causing frosty pod rot. Pods were malformed 30 days after inoculation (DAI) and sporulation was observed 60 DAI. Glucose and asparagine concentrations decreased and mannitol and malonate increased in infected pods 30 DAI. By 60 DAI, most carbohydrates, amino acids, and organic acids were drastically reduced by infection. Mannitol and succinic acid levels increased 60 DAI and likely originated from Mr. RT-qPCR analysis of cacao ESTs indicated a strong response to infection 30 DAI in malformed pod. Evidence indicated that biotrophic hyphae colonized pods and a shift to necrotrophic growth occurred later (during the end stages of infection). Expression of cacao ESTs associated with plant hormone biosynthesis and action was altered. Changes in the expression of Mr ESTs in response to nutrient deficiency in pure culture were small. Changes in Mr gene expression patterns and levels of specific metabolites in necrotic sporulating pods 60 DAI compared to malformed pods 30 DAI indicated that the glyoxylate cycle of Mr was up regulated during the shift from biotrophic to necrotrophic phases of the disease cycle.     

草莓胶孢炭疽菌CFEM候选效应子的生物信息学鉴定及其侵染过程中的转录分析

病原真菌通常分泌效应子到寄主组织中调控寄主的生理过程,从而有利于其侵染。CFEM(common in several fungal extracellular membrane)蛋白是真菌所独有的,且与致病性密切相关。本研究利用Pfam数据库对草莓胶孢炭疽菌全基因组进行搜索,鉴定获得22个CFEM蛋白。对CFEM蛋白的信号肽、跨膜结构域和亚细胞定位进行分析,结果表明仅有8个CFEM蛋白为分泌蛋白。对CFEM分泌蛋白在不同侵染阶段的转录情况进行转录组学及RT-PCR分析,结果显示8个CFEM蛋白在侵染后不同时期均有表达。其中,1个CFEM分泌蛋白于附着胞形成期特异表达,2个于活体寄生阶段特异表达,2个于死体寄生阶段特异表达。综合上述分析结果,预测这8个分泌蛋白可能为草莓胶孢炭疽菌的效应子。本研究为深入解析植物病原真菌CFEM效应子提供了理论依据。     

GFP标记的多粘芽孢杆菌1114在番茄根际的定殖

多粘芽孢杆菌Paenibacillus polymyxa菌株1114对多种植物病原菌具有良好的防治效果。为研究其在作物根际土壤中的迁移和定殖规律,通过电击转化引入绿色荧光标记质粒pB-SVG101,得到稳定表达的荧光标记菌株,转化后的菌株对青枯菌Ralstonia solanacearum仍保持良好的抑制效果。通过多种接种方法,发现该菌株能通过浸根的方式侵入番茄的维管组织和周围细胞。     

Victoria Blight,defense turned upside down

Genes that confer disease resistance to biotrophic pathogens typically encode nucleotide-binding, leucine-rich-repeat proteins (NB-LRRs). These proteins confer resistance by detecting the presence of virulence effectors secreted by biotrophic pathogens. Recognition triggers NB-LRR activation and subsequently, the defense response which often includes localized host cell death. The fungus, Cochliobolus victoriae, is a necrotrophic pathogen that causes a disease called Victoria Blight. Virulence of this fungus is dependent on its production of a peptide called “victorin” that has been traditionally described as a toxin. Only plants that respond to victorin are susceptible to Cochliobolus victoriae whereas those that do not are resistant to the fungus. Genetic and molecular analyses have revealed that victorin functions like a biotrophic effector recognized by a NB-LRR resistance protein in Arabidopsis. Further, numerous plant species express victorin sensitivity suggesting there are numerous NB-LRRs that recognize victorin. Thus, through expression of victorin, C. victoriae is able to exploit plant defense to cause disease and is capable of evoking this response in an array of different plants.     

In vitro formation of infection structures of Phytophthora infestans is associated with synthesis of stage specific polypeptides

Efficient and synchronized production of infection structures of Phytophthora infestans, the causal agent of late blight of potato, was established on an artificial membrane without the host plant. Microscopic comparison of the in vitro and the in planta formed fungal structures revealed a high degree of similarity. In vitro development of infection structures enabled detailed cytological and biochemical investigations. By video microscopy the highly dynamic phenomenon of cytoplasmic migration was monitored within the living fungus. At four distinct developmental stages, hyphae, cysts, germinating cysts and appressoria, all grown in vitro, protein synthesis was analysed by comparative two-dimensional SDS-polyacrylamide gel electrophoresis. On two-dimensional gels of protein extracts of the four developmental stages a number of polypeptides were identified that showed stage-specific differences in their relative amounts. The de novo synthesis of proteins was investigated by in vivo labelling experiments. A number of polypeptides showed development-dependent expression. The majority of changes in protein synthesis occurred during germination of cysts and development of the germ tubes. In particular, at the stage of appressoria formation, the actual start of the infection process, several major polypeptides were newly synthesized.     

Comparative epidemiology of zoosporic plant pathogens

Loss of zoospores has happened independently several times in different phylogenic lines and has, it is claimed, no major phylogenetic significance. But whether or not, how, and under which conditions plant pathogens retain the ability to produce motile asexual spores has fundamental importance from an ecological and epidemiological perspective. Recent molecular investigations of the early evolution of fungi and oomycetes are shedding light on the issue of zoospore loss in organisms able to cause plant diseases. Zoospore loss may have accompanied the development of new forms of dispersal adapted to the terrestrial environment, or the simplification processes which often follow the shift to parasitic or biotrophic life-forms. In this review we consider hybridisation events between Phytophthora species, long distance dispersal of oomycetes, sporangia and zoospore survival, direct and indirect infection processes and newly observed sporulating structures. These aspects are all relevant features for an understanding of the epidemiology of zoosporic plant pathogens. Disease management should not be based on the presumption that the zoosporic stage is a weak link in the life cycle. Oomycete plant pathogens show remarkable flexibility in their life cycles and ability to adapt to changing environmental circumstances.

Allelopathic activity of Mexican sunflower [Tithonia diversifolia (Hemsl.) A. Gray] in soil under natural field conditions and different moisture conditions

The allelopathic activity of Mexican sunflower [ Tithonia diversifolia (Hemsl.) A. Gray] in soil under natural field conditions and the effect of water stress on the growth and allelopathic activity of this plant were investigated. Seed germination, shoot growth and root growth of tested plant species in soil collected from a field where Mexican sunflower had been grown for 5 years were less than those in soil from an area without the plant. Growth of young leaves, mature leaves, senescent leaves, stem and roots of Mexican sunflower was reduced with decrease in soil moisture level. The allelopathic activity of water extracts (per dry weight of starting material) from each part of the plants grown at low soil moisture levels was greater than that of the water extracts from the same part of the plants grown at high soil moisture levels. The allelopathic activity was found in the soil from the pots previously planted with Mexican sunflower to a similar extent (per plant) at different soil moisture levels. These results suggested that, in the field, under water stress conditions, the growth of Mexican sunflower was reduced but the plants contained a greater amount of allelopathic substance(s) per dry weight than in the absence of water stress. It was considered that allelopathic activity of Mexican sunflower in soil was maintained to a similar extent under various soil moisture conditions in natural fields.