Effects of paclobutrazol on Botrytis cinerea isolates obtained from potted plants

The growth of different isolates of Botrytis cinerea, collected from potted plants affected by Botrytis blight in southern Spain during recent years, was studied. These isolates, which show wide phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid, and paclobutrazol--an efficient plant growth retardant used widely in nursery potted plants to reduce plant size, favouring compactness, a more intense green foliage and increased stress tolerance to maintain quality prior to sale. In addition, paclobutrazol may have a fungicidal effect since it belongs to the triazole chemical group. However, paclobutrazol is only used as a plant growth retardant in Spain. In this work, we evaluate the effect of paclobutrazol dose (0, 0.05, 0.25, 1.25, and 6.25 mg/plate) on the growth of a collection of different B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantana camara and Lonicera japonica. Mycelial growth curves and growth rates assessed from difference in colony areas during the linear phase, conidiation (measured as time of appearance), conidial length (microm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26 degrees C on Petri dishes containing potato dextrose agar for up to 36 days. Mycelial growth curves fitted a typical kinetic equation of fungus grown on solid media. The B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and paclobutrazol dose. This triazole delayed mycelial growth during the linear phase in an isolate-dependent manner, and isolates from C. persicum and L. japonica were more affected by paclobutrazol than H. macrophylla. On the other hand, 0.25 mg of paclobutrazol was the critical dose to significantly reduce the growth rate in all isolates. 6.25 mg paclobutrazol inhibited conidiation in isolates from C. persicum, and reduced the conidial length in isolates from H. macrophylla and L. camara. The sclerotia production process was blocked at paclobutrazol doses higher than 1.25 mg, while no sclerotia were produced in isolates from C. persicum and L. japonica with 0.25 mg. H. macrophylla was the isolate in which sclerotia production was most influenced by paclobutrazol. It was concluded that the exact effect of paclobutrazol on B. cinerea growth depends on the isolate, and new strategies should be considered for evaluating its use as retardant and fungicide.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Effects of indole-3-acetic acid on Botrytis cinerea isolates obtained from potted plants

We study the growth of different isolates of Botrytis cinerea collected from potted plants which were affected by Botrytis blight in southern Spain during recent years. These isolates, which show widely phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid applications and paclobutrazol, an efficient plant growth retardant and fungicide at the same time. In this work, we have evaluated the effect of the auxin indole-3-acetic acid (IAA) dose (0, 1, 10, and 100 mg/plate) on the growth of the collection of B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantona camara, and Lonicera japonica. B. cinerea produces indolacetic acid, but so far the precise biosynthetic pathway and some effects on this fungal species are still unclear, although recent studies have revealed an antifungal activity of IAA on several fungi, including B. cinerea isolated from harvested fruits. Mycelial growth curves and growth rates assessed from difference in colony areas during the both linear and deceleration phase, conidiation (measured as time of appearance), conidia length (microm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26 degrees C on Petri dishes containing potato dextrose agar for up to 35 days. Mycelial growth curves fitted a typical kinetic equation of fungi grown on solid media. B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and auxin dose. This plant growth substance delayed mycelial growth during the linear phase in an isolate-dependent manner, thus isolates from C. persicum, H. macrophylla and L. camara were more affected by IAA than L. japonica. On the other hand, 100 mg of IAA was the critical dose to significantly reduce the growth rate in all isolates and to promote brown-striped hyphae development, especially in isolate from C. persicum. 10 and 100 mg IAA delayed conidiation in isolates from H. macrophylla but scarcely effects were found in the conidia length. The sclerotia production process was blocked at IAA doses of 100 mg in isolates from L. camara and L. japonica, and was reduced in isolate from H. macrophylla. However, dose of 100 mg IAA had no effect on sclerotia production in isolate from C. persicum. It was concluded that the effect of IAA on B. cinerea growth depends on the isolate, thus isolates from H. macrophylla and L. camara were the most affected by IAA. B. cinerea reduced its development under IAA applications, depending on the isolate and dose. These results confirm those recently published on the inhibitory effect of IAA on Botrytris species growth.     

Genetic characterization of grapevine-infecting Botrytis cinerea isolates from Argentina

BackgroundBotrytis cinerea is an ascomycete with a high genetic diversity and complex population structure, as reported from several hosts and sites. However, nothing is known about its genetic diversity in Argentina.AimsThe aim of this work is to estimate the genetic diversity of a local population of B. cinerea isolates obtained from grapevine in Argentina.MethodsIn this work, 35 strains that had been isolated from grapevines were genotyped for the presence of transposable elements and PCR-based RFLP molecular markers. The obtained results were compared with those from a large French population of the fungus, and used to perform a population genetics analysis using the Genepop software.ResultsAll the analysed isolates were classified as Group II (according to the most recent proposed classification) and showed a high degree of genetic diversity, with 14 different haplotypes. A significant difference in allele frequency was recorded between the local and French populations.ConclusionsThese comparisons between fungal populations, led to the detection of a high level of diversity and the differentiation between local and French groups of isolates. This was confirmed by an Fst value of 0.3332, which was higher than that reported for other pairwise comparisons of populations. This work constitutes the first report on the genetic diversity of B. cinerea isolates and their population structure in Argentina.     

番茄灰霉病拮抗内生放线菌的筛选、鉴定及其活性评价

对安徽省淮北市番茄植株根、茎、叶中内生放线菌进行了分离、筛选,并测定了其抑菌活性。结果表明:番茄根、茎和叶中的内生放线菌的数量分别为5.66×104、0.67×104和0.39×104CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到93株内生放线菌,通过对峙实验,筛选到7株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生放线菌总数的7.5%。来自根组织中的菌株HNU-EA27的抑菌效果最佳,抑菌圈直径达28.3mm。根据形态特征、培养特征、生理生化特性、细胞壁组分和16SrDNA序列分析,将菌株HNU-EA27鉴定为毒三素链霉菌(Streptomyces toxytricini)。室内测定菌株HNU-EA27发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明:菌株HNU-EA27发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为30%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明:30%菌株HNU-EA27发酵滤液对番茄灰霉病的预防与治疗效果分别为80.6%和73.8%,均高于50%多菌灵可湿性粉剂600倍液。本研究表明,菌株HNU-EA27是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

Confocal microscopy study to understand Clonostachys rosea and Botrytis cinerea interactions in tomato plants

Clonostachys rosea, a biological control agent for plant diseases, is found in a variety of habitats and colonises and survives in different tissues. This antagonist is effective at controlling grey mould, which is caused by Botrytis cinerea, in different plant species. Despite the existing knowledge regarding the efficiency of C. rosea at biologically controlling grey mould, there are few studies concerning this interaction at the histological level. Therefore, we studied the antagonist–pathogen interactions using confocal microscopy. C. rosea survived in tomato tissues for at least 30 days between 18–30ºC. The antagonist colonised the wounded tomato stems faster and more efficiently than the pathogen. The colonisation of the leaf tissues by C. rosea was slow, and the spore concentration was poor in this experiment. Combined with the pathogen’s direct penetration into the leaves, this slow colonisation could cause the biological control to fail. C. rosea also preyed parasitically upon the pathogen’s hyphae, penetrated the tomato’s leaf tissue through the stomata and colonised the stem’s intercellular spaces. Root colonisation was abundant, with a dense hyphae network forming between epidermal cell junctions. This observation provided evidence that the fungus can penetrate via the roots. This paper will help to better define an application strategy for C. rosea in tomato propagation, with the goal of biological control or growth promotion, because to understand how the antagonist survives and interacts in its habitat will define how and when to apply it.     

Mutational tolerance to carbendazim in Botrytis cinerea

No spontaneous mutation for tolerance to the fungicide carbendazim was detected in C. 10⁸ conidia from each of eight carbendazim-sensitive field isolates of Botrytis cinerea. Conidia of B. cinerea were highly insensitive to u.v.-irradiation, although after severe irradiation treatments mutant strains showing the same levels of tolerance as two groups of carbendazim-tolerant field isolates were selected at frequencies of between 10^-9 and 10^-6 of survivors. Mutants with low levels of tolerance (ED₅₀ > 10 μg ml^-1 carbendazim; ‘partially-tolerant’) were selected from irradiated conidia obtained from sensitive field isolates and a further series of mutants capable of growth on 10 000 μg ml^-1 carbendazim (‘fully-tolerant’) were selected from irradiated conidia from either partially-tolerant mutants or from partially-tolerant field isolates. Both mutation steps were confirmed in similar experiments in which tolerance to an unrelated fungicide, 2, 6-dichloro-4-nitroaniline (DCNA), was incorporated as a genetic marker in the parent strains.     

Hormetic doses of UV-C light decrease the susceptibility of tomato plants to Botrytis cinerea infection

Preharvest hormetic doses of UV-C radiation can decrease the susceptibility of tomato leaves to Botrytis cinerea L. infection. UV-C light treatments have been shown to be very effective for reducing disease development in several species, including tomato (Solanum lycopersicum L.). Treating cultivated tomato plants with UV-C light is of interest not only because of the disinfecting effects of UV-C light but also because of its ability to stimulate plant defences (SDP) against diseases, provided that the applied doses are high enough to be effective while low enough to prevent deleterious effects. In the present study, the effects of UV-C light on plants were evaluated by biochemical analyses, including analyses of the activities of antioxidant enzymes and phenylalanine lyase (PAL); the concentration of malondialdehyde (MDA), an indicator of membrane integrity; and chlorophyll fluorescence parameters, such as the maximum quantum efficiency of photosystem II (F_V/F_M) and the Strasser performance index (PI). In this work, treatments with single doses of 0.85 kJ/m² of UV-C light were found to significantly increase plant defences against B. cinerea, reducing the affected leaf area by 51% compared to the affected area of control plants. This decrease in susceptibility was associated with increased PAL activity and the amount of bound phenolics compared to levels in control plants (not treated with UV-C).     

不同寄主来源的灰葡萄孢对番茄的致病力分化研究

从安徽合肥、蚌埠、长丰、和县等市、县的番茄、辣椒、草莓、葡萄等发病寄主上分离鉴定获得18个灰葡萄孢Botrytis cinerea菌株,采用菌丝块创伤接种法,分别测定了上述不同寄主来源的灰葡萄孢菌对番茄果实和叶片的致病力.结果表明,所有供试菌株接种番茄果实后均引起发病,但不同菌株所致病斑的平均直径有显著差异,显示灰葡萄孢菌株间对番茄果实的致病力存在明显分化.按照在番茄果实上所致病斑的平均直径大小可将供试菌株致病力划分为较强、中等和较弱3种类型.总体来说,来自番茄的菌株对番茄果实的致病力较强,来自草莓、葡萄和辣椒的菌株对番茄果实的致病力较弱,但来自相同寄主的菌株间致病力也存在差异,菌株致病力差异与菌株地域来源无明显相关.供试灰葡萄孢菌株接种番茄叶片后,除CF1外,均可引起番茄叶片发病,但不同菌株所致番茄叶片病斑的平均直径也有显著差异;供试菌株对番茄叶片的致病力差异与菌株的寄主和地域来源无显著相关.     

不同寄主来源的灰葡萄孢对番茄的致病力分化研究

Eighteen isolates of Botrytis cinerea were obtained from the diseased plant tissue collected in Hefei, Bengbu, Changfeng and Hexian in Anhui province, by means of tissue isolating method. The pathogenicity of the isolates of B. cinerea from different hosts to the fruits and leaves of tomato were investigated by applying wound inoculation with mycelial blocks. The results showed that all of the tested isolates caused grey mould on tomato fruits, but there was significant difference in the average diameters of the lesions caused by different isolates, suggesting that there was significant differentiation in pathogenicity of B. cinerea strains to tomato fruits among isolates. According to the average diameters of the lesions on tomato fruits, the pathogenicity of the all isolates was classified into three categories: strong, intermediate and weak. In general, the isolates from tomato were more strongly pathogenic to tomato fruits than the isolates from strawberry, grape and capsicum. However, there was difference in pathogenicity among the different isolates from the same host, and the pathogenicity difference was not obviously related to the localities of isolates. After inoculating of tomato leaves, all of the tested isolates except CF3 caused grey mould on tomato leaves, but there was significant difference in the average diameters of the lesions caused by different isolates; and the difference in pathogenicity to tomato leaves was not obviously related to the host and locality of isolates.     

源自不同寄主的灰葡萄孢生物学特性的比较研究

本研究以分离自番茄、辣椒、草莓、葡萄的灰葡萄孢Botrytis cinerea为供试菌株,从生长温度、pH适应性、碳源、氮源营养利用等方面对不同寄主来源的灰葡萄孢菌株的生物学性状进行了比较研究。结果表明,5个不同寄主来源的灰葡萄孢菌株的菌丝生长温度范围相同,均为0-35℃;但它们的最适生长温度和分生孢子致死温度存在差异,来自和县番茄菌株HX12最适生长温度为20℃,分生孢子致死温度为47℃ 10min,其余最适生长温度均为25℃,分生孢子致死温度均为48℃ 10min;不同菌株在相同温度下的生长速率有显著差异。pH对不同寄主来源灰葡萄孢菌株菌丝生长的影响存在差异,来自长丰辣椒的菌株LJ菌丝在pH2-9的范围内均能生长,以在pH3-6.5时生长较快,pH6时最快;其余4个菌株在pH2-12的范围内均能生长,以在pH3-9时生长较快,pH6左右最快。不同碳源、氮源营养对灰葡萄孢菌株菌丝生长和分生孢子产生均有显著影响,不同寄主来源的菌株间在碳源、氮源营养利用差异均极显著。在相同碳源、氮源营养条件下,不同寄主来源的菌株的线性生长、菌丝干重和分生孢子产量均有显著差异。     

Different products for biological control of Botrytis cinerea examined on wounded stem tissue of tomato plants

For the moment the agents that are used against Botrytis cinerea, in glasshouses were tomatoes are cultivated, are from chemical origin. For reducing the use of chemical agents in the future it is important to search for effective biological control agents against the fight of Botrytis cinerae. The following biological products Vital pasta, Vital gel and Elot-Vis were examined in there possibility to control Botrytis cinerea. Elot Vis was tested out in experiments that were carried out in climate chambers were leafs of 3 week old tomato plants were artificially infected with Botrytis cinerea spores. Also the biological products of Vital were first investigated in experiments that were carried out in climate chambers. In stead off leafs of tomato plants it were stem wounds of tomato plants who were treated with the pasta or the gel that was spread over the wounded surface after this has been inoculated with a suspension of conidia of Botrytis cinerae. The results of these first tests that were executed in the climate chambers were the circumstances for Botrytis cinerea were ideal seemed promising. In the next step these products were tested out on large scale in glasshouses. For each plant 5 wounds were created by removing the leafs, these wounds were or first treated with the Biological product and thereafter artificially infected with Botrytis cinerea spores to check out if these products can be used as a preventive agent or the wounds were first inoculated with a suspension of Botrytis cinerea spores and thereafter treated with the product. For the product Elot-Vis a few plants were totally sprayed with an Elot-Vis suspension before leafs were removed and the wounds were inoculated with conidia of Botrytis cinerea to check out if this product was able to activated the induced systemic resistance pathway. The experiments that were executed in glasshouses showed different percentages of succeeded Botrytis cinerea infections. This is probable due to the different weather conditions during both days that the experiments were executed. For the wounds that were treated with pasta it was difficult to distinguish wounds were Botrytis cinerea succeeded to infect the plant, because these wounds frequently didn't show any sign of infection on the surface but when the wounds with the pasta were cut open it was possible to see Botrytis cinerea infections inside the stem.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Hexanoic acid protects tomato plants against Botrytis cinerea by priming defence responses and reducing oxidative stress

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid‐induced resistance (Hx‐IR), we compared the expression profiles of three different conditions: Botrytis‐infected plants (Inf), Hx‐treated plants (Hx) and Hx‐treated + infected plants (Hx+Inf). The microarray analysis at 24 h post‐inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis‐induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up‐regulation of proteinase inhibitor genes, DNA‐binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis‐specific and non‐specific genes, preventing the harmful effects of oxidative stress produced by infection.     

Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.     

Nitrogen availability and susceptibility of tomato leaves to Botrytis cinerea

The aim of this study was to investigate the effect of nitrogen availability on susceptibility of tomato leaves to the fungal pathogen Botrytis cinerea. Plants with varying nitrogen availability were grown by adding N daily in exponentially increasing amounts to a nutrient solution at different rates. Leaves of plants grown at low nitrogen availability had a high leaf C/N ratio (21 g g^-1) and were about 2.5 times more susceptible to primary lesion formation by B. cinerea compared to plant grown at high nitrogen availability, which had a low leaf C/N ratio (11 g g^-1). Leaf C/N ratio accounted for 95% of variation in susceptibility. This relationship between C/N ratio and susceptibility persisted when plants were grown with exponential P addition and optimal N supply, and was thus independent of plant growth rate or related factors. We could not explain the effect of nitrogen availability by variation in the most obvious N-based resistance compound α-tomatine because more susceptible leaves with a high C/N ratio contained more α-tomatine. These leaves also contained more soluble carbohydrates. The level of soluble carbohydrates correlated positively with susceptibility, independent of the growth method. We therefore suggest that the effect of N availability on susceptibility must be explained by variation in levels of soluble carbohydrates and speculate about the role of these carbohydrates in the infection process. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of Botrytis cinerea populations isolated from two open-field cultivated host plants

The necrotrophic fungus Botrytis cinerea is reported to infect more than 220 host plants worldwide. In phylogenetical–taxonomical terms, the pathogen is considered a complex of two cryptic species, group I and group II. We sampled populations of B. cinerea on sympatric strawberry and raspberry cultivars in the North-East of Hungary for three years during flowering and the harvest period. Four hundred and ninety group II B. cinerea isolates were analyzed for the current study. Three different data sets were generated: (i) PCR-RFLP patterns of the ADP-ATP translocase and nitrate reductase genes, (ii) MSB1 minisatellite sequence data, and (iii) the fragment sizes of five microsatellite loci. The structures of the different populations were similar as indicated by Nei's gene diversity and haplotype diversity. The F statistics (F_st, G_st), and the gene flow indicated ongoing differentiation within sympatric populations. The population genetic parameters were influenced by polymorphisms within the three data sets as assessed using Bayesian algorithms. Data Mining analysis pointed towards the five microsatellite loci as the most defining markers to study differentiation in the 490 isolates. The results suggest the occurrence of host-specific, sympatric divergence of generalist phytoparasites in perennial hosts.     

Analyses of genetic and pathogenic variability among Botrytis cinerea isolates

Seventy nine isolates of Botrytis cinerea were collected from different host plants and different locations of India and Nepal. All the isolates were identified as B. cinerea based on morphological features and were confirmed using B. cinerea specific primers. Differentiation among the isolates was assessed using morphological, genetic and biochemical approaches. To analyze morphological variability, differences in conidial size, presence or absence of sclerotia and their arrangement were observed. Genetic variability was characterized using RAPD analysis, presence or absence of transposons and mating type genes. Cluster analysis based on RAPD markers was used for defining groups on the basis of geographical region and host. The biochemical approach included determining differences in concentration of oxalic acid and activity of lytic enzymes. All the isolates were categorized into different pathogenic groups on the basis their variable reaction towards chickpea plants. Isolates with higher concentration of oxalic acid and greater activity of lytic enzymes were generally more pathogenic. Pathogenicity was also correlated to transposons. Isolates containing transposa group showed some degree of correlation with pathogenic behavior. However, isolates could not be grouped on the basis of a single approach which provides evidence of their wide diversity and high evolution potential. Sensitivity of sampled isolates was also tested against five botryticides. Most of the isolates from same region were inhibited by a particular fungicide. This feature provided interesting cues and would assist in devising novel and more effective measures for managing the disease.