Genetic Diversity of Alternaria alternata Causal Agent of Early Blight of Tomato in Khuzestan Province Using SSRs Marker

The early blight of tomato caused by Alternaria alternata is one of the most important and destructive diseases in Khuzestan province. Study genetic structure of A. alternata populations provides different levels of information in the management of early blight disease in tomato farms. Short sequence repeat (SSR) markers were used to determine the genetic structure and estimate genetic diversity in A. alternata isolates in Khuzestan province.
In this study to evaluate the genetic diversity and genetic populations of A. alternata pathogen, sampling was randomly carried out on aerial parts of tomato plants with leaf brown lesions in farms and glasshouses from five different regions in Khuzestan province such as: Shoshtar, Omidiyeh, Dashte azadagan, Ahvaz, and Dezful. Each sample was cut into 2–5-mm long pieces, were surface-sterilized with 1% sodium hypochlorite for 3 min and rinsed three times with sterile distilled water and air-dried with sterile filter paper. The sterilized samples were placed onto a general medium potato dextrose agar (PDA). A total of 64 A. alternata isolates were obtained from infected samples. Pathogenicity test was carried out on local susceptible cultivar under an artificial condition in the greenhouse. For obtaining the mycelia mass, liquid cultures were initiated by adding 2–4 mm 2 pieces of filter paper to 250-mL Erlenmeyer flasks containing 100 mL PDB medium (potato dextrose broth). Mycelium was collected by filtration through sterile filter paper with a vacuum funnel. Mycelia were harvested, frozen and stored at -20°C. DNA was extracted using a modified hexadecyl trimethyl-ammonium bromide (CTAB) procedure. A set of five paired sequence repeat primers (SSR) were used to determine the genetic diversity of A. alternata isolates. PCR amplification was performed in a 25 μl reaction volume. The bands generated by SSR primers that were repeatable and clearly visible with a high intensity were scored manually for the presence (1) or absence (0) of bands in each isolate. Data analyses for evaluating of genetic diversity of isolates were calculations using molecular software such as: NTysis, Gene Alex, and POP GENE.
A total of 21 alleles were produced by SSR primers with an average of 4.2 alleles in all populations. The highest and lowest amounts of alleles were related to locus AEM13 with eight alleles and loci of AEM6 and AEM9 with two alleles respectively. The average of allelic variability per locus was the highest in Shoshtar population and the lowest in Dezful population. Observed allele number and effective numbers of alleles were higher in Shoshtar in comparison of other populations. A Comparison of genetic diversity parameters in five population showed that Shoshtar population has the highest genetic diversity but lower values were estimated for Dashte azadagan. The highest and lowest genetic distance was detected between Ahvaz-Dezfol (0.066) and Shoshtar-Omidieh (0.005), respectively. Based on dendrogram of populations revealed two distinct groups, one group contained Dezful and the other Shoshtar, Omidiyeh, Dashte azadagan and Ahvaz. Analysis of molecular variance showed that 85 percent of the genetic diversity of all the isolates and 14% is allocated to different geographical areas. There was the high genetic similarity between isolates from different regions. High genetic similarity can be attributed to the migration of genes or genotypes of different factors. With according to of Cluster analysis based on UPGMA and Dice similarity coefficient at 62% level, eight groups were revealed. On the basis of microsatellite data indicated high genetic diversity within the isolates; this number of alleles could not lead in separation, on the basis of geographical locations between samples. In this study, the relationship detected between isolates within the six populations were probably due to exchange of tomato seeds between sampled regions and geographical closeness as well.
This study have been carried out for the first time in Iran, and in comparison of international populations, a different level of diversity was detected within and between populations of worldwide A. alternata isolates. In this study, the high genetic diversity of A. alternata detected in five populations exposed a potential risk to tomato farms. Genetic diversity of A. alternata in Khuzestan province as an air born pathogen is a warning for a breeder to apply the successful use of resistance genes in local disease management. This gene diversity helps breeders for screening potential resistant cultivars according to gene diversity of A. alternata population in order to develop of durable resistant. Quarantine regulations will need to prevent the introduction of more diverse isolates into these populations and prevent transmission any isolates from this area to other regions of the country. Understanding the genetic structure of pathogen populations in the present study may provide insights into the epidemiology and evolutionary potential of pathogens and could lead to improved strategies for managing the disease. The obtained results indicating the high genetic diversity due to mutation, recombinant and a sexual mating ability of the pathogen in the Khuzestan province. Results in this study will be useful in breeding for tomato early blight resistant cultivars and developing necessary control measures.