农杆菌介导的玉米大斑病菌的遗传转化

 玉米大斑病是玉米生产上的一种重要真菌病害,在世界主要玉米产区都曾造成过严重的经济损失。目前,有关该病原菌的生理分化、遗传变异及毒素产生等方面研究报道较多,而在玉米与大斑病菌互作的分子机理方面研究相对较少。     

农杆菌介导的西瓜枯萎病菌遗传转化

为获得带GFP标记的西瓜枯萎病菌转化株,用于后期观察病原菌侵染过程,采用农杆菌介导的方法,对西瓜枯萎病菌1号生理小种进行了遗传转化。结果表明:共培养时间为36h,枯萎病菌孢子和农杆菌AGL1比例为1∶1时该菌株的遗传转化效率最高,可以达到117.33个转化子/107个孢子。转化株的孢子、菌丝体及萌发的孢子均能发出稳定而强的绿色荧光。转化株的致病力检测显示其致病力与转化前的野生菌株致病力无明显差异。结果表明本研究获得的带GFP标记的西瓜枯萎病菌转化株可用于观察病菌在西瓜根系的侵染过程。     

Investigating phenotypic variability in Colletotrichum lindemuthianum populations

Colletotrichum lindemuthianum, causal agent of anthracnose in the common bean, has wide genetic variability. Differential bean cultivars and morphological and physiological characteristics were used to analyze 74 isolates of C. lindemuthianum collected in two counties in the state of Minas Gerais, Brazil. Six different races were found, with a predominance of race 65 at both locations. Isolates were classified according to their sensitivities to the fungicide thiophanate-methyl, normally used in the control of common bean anthracnose. In all, ≈10% of isolates were resistant to the fungicide in vitro. Characteristics such as indexes of mycelia growth rate, colony diameter, sporulation capacity, and percentage of germination demonstrated the high genetic variability of C. lindemuthianum. We also observed variation in conidial cytology. The conidia of most isolates showed septa formation after germination, in contrast to septa absence, previously reported in the literature. Sexual and asexual reproduction were evaluated for mechanisms that may contribute in the generation of variability in C. lindemuthianum. Conidial anastomosis tubes were commonly found, indicating that asexual reproduction can help increase variability in this species. Information from this study confirmed high variability in C. lindemuthianum and will guide future studies in basic knowledge and applied technologies.     

根癌农杆菌介导的香蕉枯萎病菌4号生理小种的转化

 本文针对香蕉枯萎病菌4号生理小种这一对我国香蕉生产构成了极大威胁的检疫性有害生物,建立了根癌农杆菌介导的该生理小种的转化体系,确定了影响转化效率主要因子的最佳条件分别是:共培养乙酰丁香酮(AS)浓度为200μmol/L、共培养时间为48 h、培养温度为25℃、诱导培养基pH值为5.5。此条件下,转化效率达到21~24个转化子/10⁴香蕉枯萎病菌孢子。PCR和Southern杂交分析表明外源的T-DNA已经成功随机地整合到该病原菌基因组中,且多为单拷贝。应用该转化体系已获得近25 000个转化子,为研究该生理小种致病机制奠定了基础。     

Metabolism of phaseollin by different races of Colletotrichum lindemuthianum

The ability of four races of the bean pathogenColletotrichum lindemuthianum to metabolize the phytoalexin phaseollin in shake cultures was compared. Apart from some differences in the rate of conversion, all races metabolized the phytoalexin in the same way. Phaseollin was first converted to 6a-hydroxyphaseollin, and this product was further metabolized to 6a, 7-dihydroxyphaseollin. No metabolites of the latter compound could be detected.6a, 7-Dihydroxyphaseollin was as inhibitory as phaseollin to race 11, but was only slightly inhibitory to races 1, 2 and 1.Samenvatting Een vergelijkend onderzoek werd verricht naar het vermogen van vier fysiologische rassen van het bonepathogeenColletotrichum lindemuthianum om het fytoalexine phaseolline om te zetten.In schudculturen waaraan 10 g phaseolline/ml was toegevoegd, werd dit door alle fysio's op gelijke wijze omgezet, hoewel met verschillende snelheid (Fig. 1). Steeds werd phaseolline eerst omgezet tot 6a-hydroxyphaseolline, en dit produkt vervolgens tot een verbinding die geïdentificeerd kon worden als 6a, 7-dihydroxyphaseolline. Hierna konden geen verdere produkten worden aangetoond.6a, 7-Dihydroxyphaseolline was even fungitoxisch als phaseolline voor fysio 11, maar was slechts weinig fungitoxisch voor de fysio's 1, 2 en 1 (Tabel 1).De verschillen in omzettingssnelheid van phaseolline en in gevoeligheid voor phaseolline ee zijn omzettingsprodukten die tussen de fysio's gevonden zijn, zijn onvoldoende om de fysiospecifieke interacties tussen de boon en de verschillende fysio's vanC. lindemuthianum te verklaren.     

Genetic variability of Colletotrichum lindemuthianum in wild populations of common bean

The genetic structure of wild populations of Colletotrichum lindemuthianum was evaluated for virulence and molecular markers. Forty-five isolates were collected from five wild common bean populations located in their South-Andean centre of origin. The five pathogen populations were monomorphic in their ITS regions, but 45 polymorphic markers were identified using RAPDs. Polymorphism for virulence was also observed; 15 pathotypes were characterized on an international set of 12 differentials. A molecular variance analysis ( AMOVA ) showed that a very large part of the total genetic variation was within populations. Statistical analysis showed that there was a weak though significant differentiation among the five populations for the RAPD and virulence markers. A positive and significant correlation was found between geographic distance and the distances from RAPD and virulence data, suggesting migration between adjacent populations along the Argentinian transect. Our results suggest that the Andean wild isolates of C. lindemuthianum do not reflect all the putative diversity found in the isolates from cultivated common bean.     

农杆菌介导的稻瘟病菌致病突变菌株的筛选

 利用1个致病能力非常弱的稻瘟病菌菌株CY2和农杆菌介导T-DNA方法,已成功地获得了5000多个转化子,并以多个转化子混合接种含25个不同抗病基因的近等基因系,获得了30个致病突变体。但突变体针对不同的无毒基因有很大差别,从含有Pi-ta²的F128-1上获得了14个突变体,从含有Pi-a的IRBL-2上获得了7个突变体,从含有Pik-s的IRBL-4上获得了2个突变体,而从其它抗病基因的品系上未发现任何突变体,故T-DNA似乎有插入热点。不同突变体在携带相同抗病基因的品系上的致病性存在很大差异,表明这些近等基因系可能还携带其它抗稻瘟病基因。CY2不能侵染普遍认为不含抗稻瘟病基因的丽江新团黑谷(LTH),而7个突变体则能成功地使其发病,表明LTH亦含有抗稻瘟病基因。     

Pathogenic and genetic variability among Colletotrichum lindemuthianum isolates of different geographic origins

The pathogenic and genetic diversity of Colletotrichum lindemuthianum isolates collected from a total of 10 Central and South American, European and African countries were characterized using common bean differential cultivar pathogenicity tests and amplified fragment length polymorphism (AFLP) analysis. On the basis of pathogenicity tests, 74 isolates were attributed to 30 different pathogenic races using the CIAT-defined binary race-classification system. Twenty-one races were restricted geographically, being exclusive to different countries. Race 9 was the most widespread, being detected in four different countries. Cluster analysis of consensus AFLP data generated using three selective AFLP primer combinations grouped 86 isolates (including the 74 subjected to pathogenicity tests) into three clusters (with one isolate as an outlier). This analysis showed that the majority of South and Central American isolates were divided among two clusters, and that the limited number of European and African isolates used in this study were genetically most similar to Central American isolates. Overall, the results of this research showed some associations between the genetic diversity of the isolates and their country of origin, but not between genetic diversity and race classification of isolates.     

Variation in genotype, pathotype and anastomosis groups of Colletotrichum lindemuthianum isolates from Mexico

Patterns of variation were determined in anastomosis, pathotype and genotype of Colletotrichum lindemuthianum samples collected from individual plants of common bean cultivars from four locations in the Mexican highlands. In Chihuahua, 22 polymorphic AFLP bands in isolates taken from a single plant identified five distinct genotypes. In Michoacán, nine genotypes were identified based on a total of only six polymorphic bands. Combined cluster analysis of all isolates from individual plants grouped them geographically. In Chihuahua, in isolates from 16 individual plants, only nine genotypes were identified and all samples were found to belong to the same anastomosis group. However, analysis of selected isolates revealed two new pathotypes not reported previously from Chihuahua (races 449 and 467). In contrast, in Michoacán all 13 isolates from individual plants were found to have distinct genotypes, and in Mexico State only two pairs of isolates among 20 samples had identical genotypes. Although no pathotype variation was determined, five anastomosis groups were identified in Michoacán and three in Mexico State. These results suggest that patterns of variation in genotype and anastomosis groups are complex in the different locations sampled, and that no strong relationship exists between genotype, pathotype or anastomosis group.     

A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.     

Surface characteristics of necrotrophic secondary hyphae produced by the bean anthracnose fungus, Colletotrichum lindemuthianum

During infection of bean (Phaseolus vulgaris), the hemibiotrophic anthracnose pathogen, Colletotrichum lindemuthianum, initially produces biotrophic primary hyphae that are large-diameter and entirely intracellular, followed by necrotrophic secondary hyphae that are narrower and either intercellular or intracellular. In the present study, transmission electron microscopy of infected tissues prepared by high-pressure freezing and freeze-substitution showed that secondary hyphae have much thinner cell walls (25–40 nm) than primary hyphae (100–130 nm) and are not surrounded by an extracellular matrix. Immunofluorescence labelling with a panel of monoclonal antibodies showed that glycoproteins which are present on conidia, germ-tubes, appressoria, primary hyphae and mycelium grown in vitro are absent from the surface of secondary hyphae. Chitin, detected with the lectin wheat germ agglutinin, was the only surface component shared by secondary hyphae and the other fungal cell types. The results suggest that the fungal cell surface becomes modified during necrotrophic growth, with none of the glycoproteins associated with earlier stages of the infection process being produced.     

根癌农杆菌介导的胶孢炭疽菌遗传转化体系的建立

本研究基于农杆菌介导的遗传转化方法,建立了芒果胶孢炭疽菌高效的遗传转化体系,获得一批炭疽菌的T-DNA插入突变体,其目的是为炭疽菌的功能基因组学研究和致病相关基因的克隆奠定基础。结果如下:通过摸索并优化了体系的各项因子,在潮霉素筛选浓度为200μg/mL,菌液浓度为OD₆₆₀=0.15条件下,AS为200μmol/L,选择pH5.5的IM共培养基中转化效果最好;进一步通过对转化子的继代稳定性和PCR检测,结果发现潮霉素抗性稳定遗传和假阳性率低;通过菌落形态观察和产孢能力的测定获得3个菌落形态异常突变体,6个产孢能力下降突变体。     

Characterization of Mexican Isolates of Colletotrichum lindemuthianum by Using Differential Cultivars and Molecular Markers

ABSTRACT Differential cultivars and molecular markers were used to analyze 59 isolates of the bean anthracnose pathogen, Colletotrichum lindemuthianum, from different regions of Mexico. Ten distinct races were determined, three of which had not been reported previously in Mexico. Isolates were found to infect only a narrow range of the differential cultivars used and were restricted to cultivars of Middle American origin. A comparison of random amplified polymorphic DNA and amplified fragment length polymorphism (AFLP) analyses was carried out on a subset of the fungal isolates. Determination of genetic distances based on AFLP data and production of a dendrogram demonstrated two levels of association: i) isolates classified into two major groups according to the type of cultivar or system of cultivation from which they originated, and ii) isolates could be classified into smaller subgroups generally associated with the geographic location from which they were obtained. Bootstrap analysis and determination of confidence intervals showed these geographic groupings to be extremely robust.     

Effect of culture medium on spore production and germination of races of Colletotrichum lindemuthianum

Spore production on the media neopeptone glucose agar (PGA), malt extract agar (MEA), Czapek Dox agar (CDA) and bean pod extract agar (BEA) was determined for 10 races ofColletotrichum lindemuthianum, as well as the aftereffects on spore germination on water agar.Clear race-medium interactions were found for spore production. PGA was a universal medium for races of this fungus, but five races gave a much higher production on MEA. CDA and BEA were less effective for spore production. There was no clear aftereffect of the four media on spore germination. The 10 races could be divided into good, moderate and bad germinators, independent of the medium on which they had been developed.Samenvatting Onderzoek in vitro werd gedaan naar de invloed van vier cultuurmedia op de sporenproduktie van 10 fysio's vanC. lindemuthianum. Bovendien werd nagegaan of de kieming van de sporen op water-agar afhankelijk was van het medium waarop de sporen waren gevormd.Er bleken voor sporenproduktie inderdaad aanwijsbare fysio/medium interacties te zijn. De beste algemene gechiktheid had neopepton-glucose-agar (PGA). Op moutextract-agar (MEA) werden echter met verschillende fysio's veel hogere sporenprodukties verkregen, terwijl de sporenproduktie van andere fysio's duidelijk lager lag dan op PGA. De resultaten met Czapek-Dox-agar (CDA) en bonepeul-extract-agar (BEA) waren minder goed. Voor de 10 fysio's kan derhalve met PGA en MEA worden volstaan. Media met natuurlijke extracten (mout, bonepeulen) of met pepton, waren beter voor sporenproduktie dan het synthetische medium Czapek-Dox-agar.De media hadden geen na-effect op de sporenkieming op water-agar. De fysio's konden echter worden ingedeeld in goede, matige en slechte kiemers.     

Virulence and Molecular Diversity in Colletotrichum lindemuthianum from South, Central, and North America

ABSTRACT Isolates of Colletotrichum lindemuthianum (138 total) from Argentina, Brazil, the Dominican Republic, Honduras, Mexico, and the United States were characterized into 41 races based on virulence to 12 differential cultivars of Phaseolus vulgaris. These 41 races were categorized into two groups: those found over a wide geographic area and those restricted to a single country. Races 7, 65, and 73 were widespread. Race 73 was the most common (28%). Race 7 was found once in Argentina and Mexico but at a higher frequency in the United States. Race 65 was found repeatedly in Brazil and the United States. Although 39% of the races were detected repeatedly and three races were widespread, no race was isolated from both P. vulgaris gene pools. Phenetic analyses showed no obvious patterns correlated with virulence clusters. No geographic pattern was evident. Molecular polymorphism generated by random amplified polymorphic DNA confirmed the extensive variability in virulence of C. lindemuthianum. Virulence phenotypes were grouped into 15 clusters. The two largest clusters contained isolates from all the geographic regions sampled. Molecular polymorphism was observed among isolates from races 65 and 73 within and among countries, except among Bra-zilian isolates of race 65. The genetic diversity of C. lindemuthianum was greatest in Mexico and Honduras. Our data suggest that C. lindemuthianum may not be highly structured to specific Phaseolus gene pools.     

Genetic variability of Colletotrichum lindemuthianum isolates from Slovenia and resistance of local Phaseolus vulgaris germplasm

Journal of Plant Diseases and Protection - Bean anthracnose caused by Colletotrichum lindemuthianum is one of the most destructive diseases in all bean growing areas. AFLP analysis was used to...     

Genetic diversity and gene flow estimates among five populations of Colletotrichum lindemuthianum across Himachal Pradesh

Five Colletotrichum lindemuthianum populations causing anthracnose in common bean, a premier legume crop of Himachal Pradesh were analyzed to know about genetic diversity, migration and the probable rate of spread of races capable of overcoming resistance present in elite cultivars. The genetic diversity was calculated on the basis of allele frequencies of 12 random amplified polymorphic DNA markers using Nei's genetic diversity formulae. Diversity within each population (H_S) was high with values ranging from 0.26 to 0.31. Average differentiation among populations (G_ST) was 0.12 and populations were isolated by distance between the two populations. This indicates that less short distance gene flow occurs in Himachal Pradesh. However, the topography of Himachal Pradesh could be a barrier to long distance dispersal. Normalized haplotype diversity based on RAPD data was very high with mean value of 0.91. Keeping this in view, new races would be expected soon outside the state of origin, as naturally occurring gene flow is likely to be increased by the human activity for comprehensive cultivation of beans in the near future. Therefore, it is difficult to predict durability of resistant sources in Himachal Pradesh, which in turn emphasizes identification and introgression of R genes into commercial cultivars.     

Genetic Diversity and Pathogenic Variation of Colletotrichum lindemuthianum in the Three Centers of Diversity of Its Host, Phaseolus vulgaris

ABSTRACT Population subdivision of Colletotrichum lindemuthianum, the causal agent of anthracnose, was studied in three regions located in three centers of diversity of its host, Phaseolus vulgaris. Random amplified polymorphic DNA (RAPD) markers, restriction endonuclease analysis of the amplified ribosomal internal transcribed spacer region, and virulence on a set of 12 cultivars were used to assess the genetic diversity of C. lindemuthianum strains isolated in Mexican, Ecuadorian, and Argentinean wild common bean populations. The three regions were significantly differentiated for molecular markers. For these markers, Mexico was the most polymorphic and the most distant from Ecuador and Argentina. The majority of the RAPD alleles present in Ecuador and Argentina were found in Mexico, suggesting that Andean populations have been derived from the Mesoamerican center. Pathogenicity tests on a set of 12 cultivars showed that all but one of the Mexican strains were virulent exclusively on Mesoamerican cultivars. Argentinean strains were virulent preferentially on southern Andes cultivars, and the Ecuadorian strains, except for one strain, were avirulent on all cultivars. These results suggest an adaptation of strains on cultivars of the same geographic origin. Thus, based on molecular and virulence markers, C. lindemuthianum strains isolated from wild common bean populations were divided into three groups corresponding to host gene pools.     

Identification of Random Amplified Polymorphic DNA Markers Linked to the Co-4 Resistance Gene to Colletotrichum lindemuthianum in Common Bean

ABSTRACT New cultivars of the common bean (Phaseolus vulgaris) with durable resistance to anthracnose can be developed by pyramiding major resistance genes using marker-assisted selection. To this end, it is necessary to identify sources of resistance and molecular markers tightly linked to the resistance genes. The objectives of this work were to study the inheritance of resistance to anthracnose in the cultivar TO (carrying the Co-4 gene), to identify random amplified polymorphic DNA (RAPD) markers linked to Co-4, and to introgress this gene in the cultivar Rudá. Populations F(1), F(2), F(2:3), BC(1)s, and BC(1)r from the cross Rudá x TO were inoculated with race 65 of Colletotrichum lindemuthianum, causal agent of bean anthracnose. The phenotypic ratios (resistant/susceptible) were 3:1 in the F(2) population, 1:1 in the BC(1)s, and 1:0 in the BC(1)r, confirming that resistance to anthracnose in the cultivar TO was monogenic and dominant. Six RAPD markers linked to the Co-4 gene were identified, four in the coupling phase: OPY20(830C) (0.0 centimorgan [cM]), OPC08(900C) (9.7 cM), OPI16(850C) (14.3 cM), and OPJ01(1,380C) (18.1 cM); and two in the repulsion phase: OPB03(1,800T) (3.7 cM) and OPA18(830T) (17.4 cM). OPY20(830C) and OPB03(1,800T), used in association as a codominant pair, allowed the identification of the three genotypic classes with a high degree of confidence. Marker OPY20(830C), which is tightly linked to Co-4, is being used to assist in breeding for resistance to anthracnose.