A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.

O objetivo deste estudo foi identificar, através da seqüência de nucleotídeos das regiões ITS-5.8S do rDNA, isolados de Rhizoctonia solani causadores de podridão de hipocótilos e de queima foliar em soja (Glycine max), no Brasil. A seqüência do gene 5.8S do rDNA (155 bp) foi altamente conservada entre todos os isolados, mas foram observadas diferenças no tamanho e na seqüência de nucleotídeos nas regiões ITS1 e ITS2 entre os isolados obtidos de soja e os padrões de grupos de anastomose (AGs). A similaridade na seqüência de nucleotídeos entre os isolados do AG-1 IA, causadores de queima foliar, foi 95,1-100% na região ITS1 e 98,5-100% na região ITS2. A similaridade na seqüência de nucleotídeos entre os subgrupos IA, IB e IC variaram de 84,3 a 89% no ITS1 e de 93,3 a 95,6% no ITS2. Entre os isolados obtidos de soja pertencentes ao AG-4 e o padrão AG-4 HGI foram observadas 99,1% e 99,3-100% de similaridades para ITS1 e ITS2, respectivamente. Foi possível confirmar através da análise das regiões ITS-5.8S do rDNA que os isolados de R. solani brasileiros, causadores de queima foliar são pertencentes ao AG-1 IA e que, os isolados causadores de podridão de hipocótilos pertencem ao AG-4 HGI. A análise das regiões ITS-5.8S do rDNA não foi determinante na identificação do isolado AG-2-2 IIIB obtido de soja.

The aim of this study was to identify isolates of Rhizoctonia solani causing hypocotyl rot and foliar blight in soybean (Glycine max) in Brazil by the nucleotide sequences of ITS-5.8S regions of rDNA. The 5.8S rDNA gene sequence (155 bp) was highly conserved among all isolates but differences in length and nucleotide sequence of the ITS1 and ITS2 regions were observed between soybean isolates and AG testers. The similarity of the nucleotide sequence among AG-1 IA isolates, causing foliar blight, was 95.1-100% and 98.5-100% in the ITS1 and ITS2 regions, respectively. The nucleotide sequence similarity among subgroups IA, IB and IC ranged from 84.3 to 89% in ITS1 and from 93.3 to 95.6% in ITS2. Nucleotide sequence similarity of 99.1% and 99.3-100% for ITS1 and ITS2, respectively, was observed between AG-4 soybean isolates causing hypocotyl rots and the AG-4 HGI tester. The similarity of the nucleotide sequence of the ITS-5.8S rDNA region confirmed that the R. solani Brazilian isolates causing foliar blight are AG-1 IA and isolates causing hypocotyl rot symptoms are AG-4 HGI. The ITS-5.8S rDNA sequence was not determinant for the identification of the AG-2-2 IIIB R. solani soybean isolate.