Genotypic and Pathogenic Diversity Among Pea-Infecting Strains of Aphanomyces euteiches from the Central and Western United States

ABSTRACT Pathogenic and genotypic variability among four populations of Aphanomyces euteiches from individual fields in Minnesota, Wisconsin, and Oregon were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses. About 50 strains were isolated from each of two pea fields in Minnesota, and 11 and 6 strains from pea fields in Wisconsin and Oregon, respectively, using pea (Pisum sativum) as a baiting host. Pathogenic variability and host range were evaluated in greenhouse studies with five pea lines or cultivars having different levels of resistance to Aphanomyces root rot and one cultivar each of alfalfa and snap bean. All strains were pathogenic on one or more pea cultivars, and 18 and 14% were pathogenic on alfalfa and bean, respectively. Disease severity incited by different strains varied significantly on individual pea cultivars and on all hosts combined. The percentage of strains pathogenic on different hosts varied among locations. Genotypic variation among all 114 strains was evaluated with RAPD analysis. Ten decanucleotide primers detected 92 polymorphic bands. Cluster and principal coordinates analysis revealed one large group containing 102 of the 114 strains from all locations. Two closely related minor groups of strains (12 strains) were genotypically distinct, with about 55% similarity to the main group of 102 strains. The strains in the minor groups were all isolated from the Minnesota locations and were pathogenic on two disease-resistant pea breeding lines (MN313 and MN314). Estimates of genetic diversity based on RAPD analysis ranged from 0.24 to 0.33 within populations to 0.35 among all strains from all populations. A. euteiches populations were genotypically and phenotypically variable, but no distinct genotypic differences were identified among populations from the four isolated locations.     

Genetic variation among asexual progeny of Phytophthora infestans detected with RAPD and AFLP markers

Genotypic variation among 32 single-zoospore isolates (SZI) of Phytophthora infestans , derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA (AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates. Polymorphism was detected with 51 RAPD primers and with all 18 AFLP primer pairs in PI-105 SZIs. In SZIs from PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six AFLP groups. No close correlation among RAPD, AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.     

Contamination, error, and nonspecific molecular tools

ABSTRACT Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are widely used in studies of genetic variation. Although it is recognized that contamination should be avoided in DNA samples, little is known about the potential hazards of low level bacterial contamination of samples from which DNA is extracted for RAPD or AFLP analyses. We found that contamination of Aphanomyces cochlioides cultures with a prokaryote at visibly undetectable levels markedly altered the results of RAPD and AFLP analyses. The contamination resulted in seven contaminant-specific RAPD products and in the suppression of eight products characteristic of uncontaminated A. cochlioides cultures. Prokaryote contamination resulted in 39 contaminant-specific AFLP products, but did not cause suppression of AFLP products. Comparing A. cochlioides samples with outgroup A. euteiches did not clearly indicate the presence of contaminant DNA, because uneven product suppression in RAPD analysis increased the apparent similarity between contaminated samples and A. euteiches and because a high proportion of the contaminant-specific amplified products comigrated with products from A. euteiches in both RAPD and AFLP analyses. Work with organisms that are prone to contamination should employ techniques such as restriction fragment length polymorphism or DNA sequence comparisons rather than relying solely on RAPD or AFLP analyses.     

苎麻疫霉抗甲霜灵突变株对棉苗的致病力及其遗传

研究了苎麻疫霉抗甲霜灵(Mt^r)突变株对棉苗的致病力及其遗传,结果显示,抗性突变株对棉苗的致病力与其野生型亲本间无显著差异。而Mt^r突变株XC-6-2对棉苗的致病力在其无性第1代(ZG₁)单孢株间及其与亲本间均存在极显著差异,且XC-6-2对棉苗的致病力性状在单游动孢子第2代(ZG₂)持续发生分离;XC-6-2单卵孢第1代(OG₁)对棉苗的致病力间亦存在极显著差异。上述结果提示,抗甲霜灵突变株对棉苗的致病力在无性单孢和单卵孢子后代间均不能稳定遗传。对比研究发现,野生型亲本菌株XC-6对棉苗的致病力在单游动孢子和单卵孢后代有相似的遗传规律,且单游动孢子群体对棉苗致病力的变异系数较其单卵孢株间的变异系数大。     

Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses

Two molecular genetic screening techniques, RAPD (random amplified polymorphic DNAs) and ISSR (inter-simple sequence repeats), were applied to detect the level and pattern of genetic diversity of Monochoria vaginalis, a common weed of rice fields, in seven populations from southern China. Among these populations, 116 bands were amplified by 18 RAPD primers, of which 34 bands (29.31%) were polymorphic, and 14 ISSR primers produced 111 bands with 87 polymorphic bands (78.38%). Within each population, a relatively low level of genetic diversity was detected by both RAPD and ISSR analyses, with a mean genetic diversity (H) of 0.0348 and 0.0551 respectively. Analysis of molecular variance of the data from the RAPD and ISSR markers detected that the majority of total genetic variation existed among populations (73.50% and 76.70% respectively) and only minor genetic variation within populations (26.50% and 23.30% respectively). Cluster analysis divided the seven populations into two groups, indicating that the genetic relationships among populations have relatively low correlation with their geographical distribution (Mantel test; r = 0.45 and 0.48 respectively). Our results indicated that both RAPD and ISSR markers were effective and reliable for accurately assessing the degree of genetic variation of M. vaginalis. Comparing the two techniques, ISSR markers were more efficient than the RAPD assay. The Mantel test gave r = 0.16, suggesting no correlation between these two molecular markers.     

A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches

ABSTRACT Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.     

Pathogenic and Genetic Variation in the Japanese Strains of Fusarium oxysporum f. sp. melonis

ABSTRACT Pathogenic variation among 41 Japanese strains of Fusarium oxysporum f. sp. melonis was analyzed by pathogenicity tests with muskmelon, oriental melon, and oriental pickling melon cultivars. Based on pathogenicity to muskmelon cvs. Amus and Ohi and oriental melon cv. Ogon 9, 41 strains were divided into 3 groups that corresponded completely to Risser's races 0, 2, and 1,2y. To further characterize pathogenic variation within the forma specialis and races, strains were assayed for pathogenicity to 42 additional muskmelon, oriental melon, and oriental pickling melon cultivars. All strains of race 1,2y were pathogenic to all cultivars tested. Strains of race 0 were divided into six variants based on differences in pathogenicity to three muskmelon cultivars; strains of race 2 also were classified into six variants based on differences in pathogenicity to two muskmelon cultivars and one oriental melon cultivar. Genetic variation among strains was analyzed by DNA fingerprinting with four repetitive DNA sequences: FOLR1 to FOLR4. Thirty-six fingerprint types were detected among forty-one strains by pooling results of fingerprinting with four probes. Cluster analysis showed distinct genetic groups correlated with races: the fingerprint types detected in each of races 2 and 1,2y were grouped into a single cluster, and two distinct genetic groups were found in race 0. However, pathogenic variation detected within races 0 and 2 could not be differentiated based on the nuclear markers examined.     

Identification of Molecular Genetic Markers in Pyrenophora teres f. teres Associated with Low Virulence on 'Harbin' Barley

ABSTRACT Two isolates of the barley net blotch pathogen (Pyrenophora teres f. teres), one possessing high virulence (0-1) and the other possessing low virulence (15A) on the barley cultivar Harbin, were crossed and the progeny of the mating were isolated. Conidia from cultures of the parent and progeny isolates were used as inoculum to determine the inheritance of virulence in the pathogen. Of the 82 progeny tested, 42 exhibited high virulence and 40 exhibited low virulence on 'Harbin' barley. The data support a model in which a single, major gene controls virulence in P. teres f. teres on this barley cultivar (1:1 ratio; chi(2) = 0.05, P = 0.83). Preparations of DNA were made from parental and progeny isolates, and the DNA was subjected to the random amplified polymorphic DNA (RAPD) technique in a search for molecular genetic markers associated with the virulence phenotype. Five RAPD markers were obtained that were associated in coupling with low virulence. The data indicate that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres.     

Pathogenic characteristics of isolates of Aphanomyces euteiches from pea in France

Aphanomyces root rot ( Aphanomyces euteiches ) has become a very destructive disease in French pea crops since 1993. The host specificity of the French pea-infecting populations of this pathogen was investigated by inoculating pea, common vetch, alfalfa, broad bean and green bean with 91 pea-infecting A. euteiches isolates, originating from the main areas of infestation in France. These isolates were compared to 13 isolates from various countries and hosts (pea, green bean, alfalfa). Virulence phenotypes were defined according to the pathogenicity data on the different hosts: all isolates from France infected two to five legume species, with most infecting pea, vetch, alfalfa and broad bean. Four pathotypes were characterized within the French isolates: one type corresponded to broad host range isolates, the second was composed of isolates preferentially agressive on pea/vetch/alfalfa and weakly aggressive on broad bean, and two others corresponding to more specialized isolates that preferentially infected pea/vetch or pea/vetch/alfalfa. Most isolates from France were preferentially pathogenic on pea, like the pea-infecting isolates from other countries, but were less specialized than the alfalfa- and green bean-infecting isolates from other countries. These results suggest that A. euteiches isolates may be maintained on wild or cultivated legumes other than pea in France.     

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.     

Hierarchical Analysis of Diversity, Selfing, and Genetic Differentiation in Populations of the Oomycete Aphanomyces euteiches

ABSTRACT Relatively little is known about the population biology of the legume pathogen Aphanomyces euteiches. A. euteiches is a soilborne pathogen causing Aphanomyces root rot of several legumes, including alfalfa, bean, lentil, and pea. Our objectives were to assess the degree of diversity, selfing, and population differentiation in A. euteiches. We contrasted populations within and among two geographically separated fields with a history of pea production. Molecular genotyping relied on amplified fragment length polymorphism analysis. Samples of A. euteiches recovered from two fields in northeast Oregon and western Washington confirmed previous reports of moderately high genetic diversity in populations of A. euteiches at the regional scale, but revealed higher-than-expected genotypic diversity within individual soil samples. Populations of A. euteiches were significantly differentiated at the soil sample, field, and regional level. The population structure appears to be patterned by regular selfing via oospores, a mixed reproductive system including both asexual and sexual reproduction, with occasional migration of novel genotypes or outcrossing.     

中国不同地区致病疫霉遗传多样性的RAPD分析

 本文应用RAPD技术检测了我国主要马铃薯产区致病疫霉的遗传分化情况及不同地区菌株间的亲缘关系。用筛选出的10个随机引物对1997-2001年间采自我国9省市的82株及3株来自日本的致病疫霉DNA进行了PCR扩增,获得了79条谱带,其中多态性标记75条,占95%。根据扩增结果,运用UPGMA分析,获得了表现菌株间亲缘关系的树状图。菌株间的最大遗传距离为0.5,以距离0.3为阈值,可将供试菌株划分为10个组(RG1-10)。结果发现:A1交配型菌株群体内的差异大于A1和A2菌株群体之间的;RAPD分组与菌株的地理来源、交配型及对甲霜灵的敏感性无明显相关性。研究结果显示,来自中国北方甘肃、内蒙、吉林、黑龙江地区的菌株与一些来自云南、四川等西南地区的菌株亲缘关系相近。病原菌随种薯的迁移可能是导致这种现象的原因之一。     

香蕉枯萎病菌RAPD分析及4号生理小种的快速检测

 用随机扩增多态性DNA(RAPD)技术,对采自广东、广西的香蕉和粉蕉上的30个香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)菌株和3个其它尖孢镰刀菌专化型的菌株进行比较及聚类分析。在遗传相似系数0.67时,可将供试菌株划分为3个RAPD群(RGs),其中香蕉枯萎病菌4号生理小种(FOC4)共15个菌株属于RGⅠ,1号生理小种(FOC1)共15个菌株属于RGⅡ,供试的其它尖孢镰刀菌专化型的3个菌株则属于RGⅢ。这说明香蕉枯萎病菌和供试3个其它专化型菌株与致病性间存在明显的相关性。1号生理小种内菌株间的遗传分化大于4号生理小种内菌株间的遗传分化。从90条RAPD随机引物中筛选出2条引物可产生4号生理小种的RAPD标记2个。将这2个RAPD标记电泳切胶回收、克隆及测序,并根据这2个特异片段序列设计SCAR上下游特异引物,通过对30个菌株的PCR扩增检验,其中一个RAPD标记成功地转化为SCAR标记,初步建立了以此为基础的4号生理小种快速检测技术,其检测灵敏度为2 ng新鲜菌丝。对采自不同地区的显症样品、吸芽、室内接种未显症的香蕉苗以及发病的香蕉植株不同部位进行检测,能够准确灵敏地鉴定出4号生理小种,从而为香蕉枯萎病菌的快速检测及防治奠定了基础。同时,快速检测结果发现,田间发病植株果柄的各部位及果实内并没有枯萎病菌的存在。     

Assessment of Host-Induced Selection on Three Geographic Isolates of Heterodera schachtii Using RAPD and AFLP Markers

ABSTRACT The hypothesis that host plants exert selection pressure on Heterodera schachtii populations was tested. Host selection of genotypes from three genetically distinct isolates of H. schachtii was assessed using cabbage, sugar beet, oilseed radish (Raphanus sativus), and white mustard (Sinapis alba). The plants represent a range of susceptibility to H. schachtii and included R. sativus and S. alba, because cultivars of those species have been used as trap crops for H. schachtii in Europe. Genotypic differences in amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were detected among the isolates after they reproduced on the different hosts. The poorest host plant, R. sativus, resulted in the greatest number of changes in both AFLP and RAPD markers. Oilseed radish selected nematode genotypes in less than four nematode generations. The nematode population genotypes detected by RAPD analyses after selection on oilseed radish were observed even after nematode populations were transferred back to the other three hosts. The genetic markers that were detected after selection were influenced by the genotypes of the original nematode isolates. The results indicate the utility of RAPDs and AFLPs for identifying and monitoring intraspecific genetic variability in nematodes and for understanding nematode population responses to host plants. Nematode management practices such as using resistant cultivars may alter gene frequencies, thereby reducing the efficacy of the tactic and exacerbating the nematode's potential to damage subsequent crops.     

产纳他霉素生防菌A01和A02与已知产生菌的RAPD比较

为了明确自主分离的天然抗真菌活性产物——纳他霉素产生菌A01和A02与已知纳他霉素产生菌的遗传相似性,采用RAPD技术对这2株菌和3个产纳他霉素的标准菌株进行了比较分析。利用筛选出的7个随机引物对5个菌株的PCR扩增共得到154条清晰稳定的DNA条带,其中同源性条带86条,多态性条带68条,分别占总条带数的55.8%和44.2%,平均每个引物扩增出9.7个多态性条带,得到了丰富的DNA指纹图谱。所得数据经NTSYS pc 2.10e软件聚类分析,表明5个菌株间的遗传距离为0.0991~0.4738,其中A02与其它菌株间的遗传距离均较远。结合前期的分类研究结果,确证了菌株A02为新的纳他霉素产生菌。     

韩国栗疫病抗病品种的遗传变异分析

为了分析韩国栗疫病的抗病品种和感病品种的遗传变异和抗病性的筛选,利用抗病性的快速检测法和RAPD(random amplified polymorphic DNA)方法对13个栗树品种进行了抗病性检测和RAPD标记分析。抗病性的快速检测选出了5个抗病品种、5个感病品种和3个中度抗病(或中度感病)品种,并且这一结果与该品种的田间表现相一致。利用筛选的12个随机引物,扩增了100个多态性RAPD片段,但未发现与抗病性或感病性相关的特异RAPD片段。聚类分析结果表明,12个品种大致分为抗病、感病和中度抗病(或中度感病)等3个大组,并与抗病性的快速检测结果基本一致。抗病品种“MANSEKI”表现出了相对于12个品种较远的亲缘关系。     

Population Genetic Structure and Host Specificity of Alternaria spp. Causing Brown Spot of Minneola Tangelo and Rough Lemon in Florida

ABSTRACT Alternaria spp. were sampled from two rough lemon (RL) and two Minneola tangelo (MIN) groves in a limited geographic area in central Florida to test for host-specialized forms of the pathogen. Isolates of Alternaria spp. were scored for variation at 16 putative random amplified polymorphic DNA (RAPD) loci and for pathogenicity on both hosts. Subpopulations on each host were differentiated genetically and pathogenically, which was consistent with the hypothesis of host specialization. Highly significant genetic differentiation was detected among all four subpopulations (Nei's coefficient of gene differentiation [G(ST)] = 0.292, P = 0.000); most of the differentiation occurred between hosts (G(ST) = 0.278, P = 0.000). Phenograms of qualitative similarities among isolates within subpopulations revealed two or three distinct clusters of isolates within each subpopulation. The majority of isolates sampled from RL were pathogenic on RL and not on MIN, although a few RL isolates were able to induce disease on MIN, and 44% were nonpathogenic on either host. In contrast, isolates from MIN were pathogenic only on MIN, never on RL, and only 3% of the isolates were nonpathogenic. Overall, three genetically distinct clusters of isolates were detected on both hosts. One of the clusters (cluster A) sampled from RL was pathogenic on RL and not on MIN and consisted almost entirely of one RAPD genotype. This cluster also contained two isolates that were 93% similar to the majority genotype but were pathogenic on MIN and not RL. In isolates from MIN, two distinct clusters of isolates were found in one subpopulation (clusters B and C), and three distinct clusters were found in another subpopulation (clusters A, B, and C). Clusters A and B were found on both hosts, while cluster C was limited to MIN. Populations of Alternaria spp. sampled from RL and MIN showed a high degree of host specificity; however, the specificity obscured a high level of genetic variation within subpopulations.     

不同干扰生境中荒漠小灌木红砂种群遗传多样性研究

应用RAPD标记技术对荒漠小灌木红砂(Reaumuria soongorica)种群在不同扰动下的遗传多样性进行了分析。18条随机引物对红砂6个种群的120个个体进行扩增,共检测102个位点,其中多态位点99个。研究结果表明:红砂种群的多态位点比率(P)为96.86%,显示了不同生境中红砂种群内存在较高的遗传多样性。Shannon多样性指数(0.5007)、Nei基因多样性指数(0.3307)和基因分化系数(Gst=0.1952)揭示了红砂种群遗传变异多存在于种群内,种群间的遗传分化则较小。聚类分析表明:红砂种群遗传距离与地理距离之间存在一定相关性;遗传多样性水平与物种特性和不同干扰生境有关,与生态因子无相关性。     

Consistent Quantitative Trait Loci in Pea for Partial Resistance to Aphanomyces euteiches Isolates from the United States and France

ABSTRACT Development of pea cultivars resistant to Aphanomyces root rot, the most destructive root disease of pea worldwide, is a major disease management objective. In a previous study of a mapping population of 127 recombinant inbred lines (RILs) derived from the cross 'Puget' (susceptible) x '90-2079' (partially resistant), we identified seven genomic regions, including a major quantitative trait locus (QTL), Aph1, associated with partial resistance to Aphanomyces root rot in U.S. fields (21). The objective of the present study was to evaluate, in the same mapping population, the specificity versus consistency of Aphanomyces resistance QTL under two screening conditions (greenhouse and field, by comparison with the previous study) and with two isolates of Aphanomyces euteiches originating from the United States and France. The 127 RILs were evaluated in the greenhouse for resistance to pure culture isolates SP7 (United States) and Ae106 (France). Using the genetic map previously described, a total of 10 QTL were identified for resistance in greenhouse conditions to the two isolates. Among these were Aph1, Aph2, and Aph3, previously detected for partial field resistance in the United States. Aph1 and Aph3 were detected with both isolates and Aph2 with only the French isolate. Seven additional QTL were specifically detected with one of the two isolates and were not identified for partial field resistance in the United States. The consistency of the detected resistance QTL over two screening environments and isolates is discussed with regard to pathogen variability, and disease assessment and QTL detection methods. This study suggests the usefulness of three consistent QTL, Aph1, Aph2, and Aph3, for marker-assisted selection.     

Disease assessment of pea lines with resistance to foot rot pathogens: protocols for in vitro selection

Cultural conditions were optimized so that disease reactions of plantlets and callus tissue from partially resistant and susceptible genotypes of Pisum sativum could be differentiated when inoculated with Fusarium solani f.sp. pisi. Disease was assessed using both semi-quantitative (disease score/fungal diameter) and quantitative methods (ergosterol assay).
After optimization of inoculum concentration and incubation period it was possible to differentiate the disease reactions of plantlets using disease scores and amount of ergosterol in the tissues.
Incubation period, temperature and the auxin added to the media were important in allowing differentiation of callus tissue cultures. These differences were visible by measurement of the diameter of fungal growth on the callus and the amount of ergosterol in the tissues, but not with disease scores.
The plantlet test was also applicable to the other foot rot pathogens Phoma medicaginis var. pinodella and Aphanomyces euteiches , allowing differentiation of the disease reactions of a partially resistant and susceptible genotype using disease scores. The ergosterol assay could be used to quantify infection caused by P. medicaginis var. pinodella , but this sterol was not detectable in mycelium of A. euteiches. In this fungus the presence of cholesterol was detected, which may be used to quantify infection.