The Morphological Identification And Genetic Diversity Of Fusarium In Shanxi Province

The genus Fusarium Link is one of the most important pathogens, which can infect many plant species, cause serious plant diseases and seriously affect agricultural production. It is one of the most difficult fungal groups to identify, as it has many characteristics such as variety, easy variation within species, obvious physiological differentiation. In this study, Fusarium strains were isolated and identified from samples of different plants with typical symptoms of Fusarium disease, which were collected from different regions from Shanxi, Hebei and Liaoning. At the same time, EST-SSR, SRAP and ISSR markers were used to analyse the genetic diversity of some representative Fusarium strains. This work could lay a theoretical foundation for the study of Fusarium genetic differentiation, taxonomy and phylogeny. The main results are as follows:1. From June2008to September2011, more than two hundred ninety samples of different plants with typical symptoms of Fusarium disease were collected from22cities of8regions from Shanxi and some from Hebei and Liaoning. A total of seven hundred and eight four Fusarium Single spores were acquired using tissue isolation method. According to Leslie, Nelson, Booth classification criteria, after the standardized cultivating, examination and comparison of the morphological characteristics, six hundred and twenty six Fusarium isolates belonging to28species of11sections were identified, including F. oxysporum, F. redolens, F. solani, F. coeruleum, F. verticillioides, F. proliferum, F. subglutinans, F. udumand, F. sporotrichioides, F. tricinctum, F. illudens, F. camptoceras, F. ventricosum, F. acuminatum, F. sambucinum var. coeruleum, F. sambucinum, F. culmorum, F. heterosporum, F. aquaeductuum, F. merismoides.F. avenaceum, F. chlamydosporum, F. equiseti, F. graminearum, F. lateritium, F. semitactum, F. sporotrichioides, F.trichothecioides, F.nivale. A key to the species and sections of the Fusarium strains were listed.2. The abundance and variations in simple sequence repeats (SSRs) are the valuable resources for studying genetic markers of eukaryotes. ESTs (Expressed sequence tags) databases of Fusarium oxsporum and Fusarium verticillium in NCB1were searched for SSRs using SSRhunter, and the organization and abundance of the SSRs were analyzed. The results showed that:(1) A total of92SSRs were searched in9304Fusarium oxysporum ESTs, which were distributed in90ESTs, with the frequency of8.27%. It was observed that dinucleotide repeats (59.78%) were most abundant followed by tri-nucleotide repeats (17.39%). AG/TC had the highest estimated frequency in the26types of repeat motifs.(2) A total of1147SSRs were searched in63875Fusarium Verticillium ESTs, which distributed in1112ESTs, with the frequency of1.8%. Tri-nucleotide repeats (44.55%) were the main types of repeats, followed by Tetra-nucleotide repeats (24.06%). AGA/TCT and CGA/CAA were the most frequent motifs in all163types of repeat motifs.3. Thirty and20pairs of primers were designed respectively according to the EST-SSR of Fusarium oxsporum and Fusarium verticillium with Primer premier5.0, which were screened against two seperate Fusarium species genomic DNA. Among them,20(80%) and22(88%) pairs of primers showed the amplification. Then all the available primers in two Fusarium species were subjected to detect polymorphism for DNAs extracted from38Fusarium oxsporum and16Fusarium verticillium strains. The result showed that19(63.3%) and13(52%) pairs of primers could reveal polymorphisms among two seperate Fusarium species. To examine the transferability of EST markers in other five Fusarium species, all effective primers were further used for PCR-mediated amplification of genomic DNA from five Fusarium species of strains. Seventeen out of20pair of Fusarium oxsporum primers were able to produce amplified products and14showed polymorphisms, accounting for85%and70%of total primers respectively. Among all Fusarium verticillium primers,22were able to produce amplified products and19showed polymorphisms, accounting for100%and86%of total primers respectively. Results obtained in the present paper proved that developing polymorphic markers based on Fusarium species EST could be feasible and this kind of markers would be transferable to closed related species.19EST-SSR primers with polymorphism among Fusarium strains were selected for genetic diversity analysis of117Fusarium strains belonging to9species of6sections and4unknown strains. A total of191bands were amplified, and all of them were polymorphic. Cluster analysis results based on the EST-SSR data indicated that at certain genetic similarity coefficient level, the same Fusarium species strains were preferentially classified into the same genetic similarity group or subgroup, and the strains isolated from the same species of plants were also preferentially grouped into the same branch. The genetic similarity ranged from0.607to0.990, with the average of0.782. Therefore Fusarium strains could be well separated using EST-SSR molecular marker into different sections, species and host specialization. The differentiation reflected by Fusarium microsatellite fragments amplified with EST-SSR matched with the morphological classification in a certain extent, and all the tested strains showed obvious genetic differentiation. All the results showed that the SSR primers developed on Fusarium oxsporum and Fusarium Verticillium EST sequences could be used to assess genetic diversity, identification, classification and system development of Fusarium strains.3. Sequence related amplified polymorphism (SRAP) analysis also was used to detect the genetic variation of Fusarium strains. Among25primer combinations screened,12pairs of primers had stable amplified polymorphic bands. They were further applied to analyse the genetic diversity of117Fusarium strains belonging to9species of6sections. In total,260SRAP markers were obtained and all the bands were polymorphic, with average of10.1polymorphic bands per primer. The result showed that SRAP can be used for genetic diversity analysis of Fusarium as a new molecular marker technique.Cluster analysis results based on the genetic similarity showed that all the strains tested were clustered into six groups at0.814, and each group was divided into several subgroups at certain genetic similarity value. The same Fusarium species strains were clustered into the same group or subgroup. Therefore the SRAP groups based on the genetic similarity were consistent with the morphological species. All the results demonstrated that genetic diversity among the Fusarium species was abundant using SRAP. Futhermore it is a very efficient technique to study the genetic diversity, taxonomy and phylogenesis of genus Fusarium.Genetic similarity analysis showed that there was obvious genetic differentiation between Fusarium sections and species. Phylogenetic relationship between species of the same section showed more closely than those of the different sections. Some genetic diversity was observed among the strains of the same species, although they were collected from the same region and their host plants were of the same species.4. To clarify genetic difference and phylogeny relationship among the strains of Fusarium oxysporum, the genetic diversity of33Fusarium oxysporum strains from different areas were examined by inter-simple sequence repeats (ISSR). The results indicated that a total of105bands were amplified with11selected ISSR primers,91(86.7%) of which were polymorphic. The cluster analysis based on the ISSR data showed that the genetic similarity ranged from0.606to0.962, with the average of0.756. All the strains tested could be distinguished at0.962. This indicated that the SSR loci in Fusarium oxysporum genomes were rich in polymorphism. In addition, there was some correlation between genetic similarity and geographic origin of strains isolated from the same host.5. EST-SSR, SRAP and ISSR all can be used in genetic diversity study of Fusarium strains, but there were some differences. SRAP primers had the largest number of polymorphic bands among all markers, ISSR provided rich genetic information, and the data provided by EST-SSR were between SRAP and ISSR. The grouping patterns generated with EST-SSR and SRAP were complied with that of the morphological sections and species in certain degree, but there were some differences between molecular marker technology and morphological taxonomy in genesis relationship of the sections. The genetic relationships among strains can be obtained more realistic and objective by SRAP markers combining with EST-SSR molecular markers. Meanwhile ISSR can reflect more genetic information and reveal more genetic differences among Fusarium oxysporum strains, but not as available as EST-SSR markers in detecting differences within species of Fusarium. Combining three markers, genetic and phylogenic relationship within and among Fusarium sections or species were clarified more comprehensively and more scientific conclusion can be obtained.