Genetic Variation among Pathogens Causing “helminthosporium” Diseases of Rice, Maize and Wheat

Vinte amostras de espécies de fungos, agentes da helmintosporiose em cereais, foram obtidas de diferentes regiões geográficas, sendo nove constituídas de Bipolaris oryzae, isoladas de cultura do arroz (Oryza sativa), sete de B. sorokiniana coletadas de trigo (Triticum aestivum), duas de B. maydis e duas de Exserohilum turcicum provenientes de milho (Zea mays). As amostras foram analisadas através das técnicas de PCR-RFLP e RAPD. O polimorfismo de tamanho observado entre as amostras na região ITS1-ITS2 e o espaço compreendido da região 5,8S do rDNA indicou diferenças genéticas entre as amostras, enquanto o fenograma construído através do método de UPGMA após a Twenty isolates of four fungal species, agents of “Helminthosporium” diseases in cereals , were collected from different regions: nine Bipolaris oryzae isolated from rice (Oryza sativa), seven B. sorokiniana from wheat (Triticum aestivum), two B. maydis, and two Exserohilum turcicum from maize (Zea mays). The strains were compared by PCR-RFLP and RAPD analysis. Size polymorphism among the isolates in the ITS region comprising the 5.8 S rDNA indicated genetic differences among the isolates, while a UPGMA phenogram constructed after the digestion of this region with restriction enzymes showed interand intra-specific polymorphism. The RAPD profiles indicated an expressive level of polymorphism among different species, compared with a low level of polymorphism among isolates of the same species. A UPGMA phenogram grouped the isolates according to the species and their host plant. RAPD profiles did not reveal polymorphism that directly correlated climatic factors with geographic source of the isolates of B. sorokiniana, and B. oryzae. Teleomorphic species revealed high similarity with their correspondent anamorphs. Additional key words: Bipolaris sorokiniana, Bipolaris oryzae, Bipolaris maydis, Exserohilum turcicum, PCR-RFLP, RAPD. RESUMO Variação genética entre patógenos agentes da doença de “Helminthosporium” de arroz, milho e trigo digestão com as enzimas de restrição, indicaram polimorfismo inter e intraespecífico. Os perfis de RAPD indicaram um expressivo grau de polimorfismo entre as diferentes espécies. Entre as amostras da mesma espécie ocorreu um baixo índice de polimorfismo. O fenograma, obtido pelo método de UPGMA, permitiu diferenciar as quatro espécies analisadas e agrupou as mesmas conforme a espécie hospedeira. Os perfis de RAPD obtidos revelaram ausência de correlação entre os fatores climáticos e a origem geográfica dos isolados de B. sorokiniana e B. oryzae. Espécies teleomórficas revelaram alto nível de similaridade com seus correspondentes anamorfos. Several species of Drechslera, Bipolaris, and Exserohilum are important plant pathogens and are associated with symptoms of dark spots on leaves, and root rot of seedlings. These fungi contain dark pigmentation due to the presence of melanin in their cell walls, which is considered an important factor of virulence. The “brown spot” agent in rice (Oryza sativa L.) is Bipolaris oryzae (Breda de Haan) Shoemaker, 1959 [syns. Drechslera oryzae (Breda de Haan) Subram & Jain, 1966 and Helminthosporium oryzae (Breda de Haan) Miybabe & Hori, 1901]. The teleomorph of this species is Cochliobolus miyabeanus (Ito & Kurib, 1942). The fungal species that attack wheat (Triticum aestivum L.) are Bipolaris sorokiniana [syns. Helminthosporium sativum Pamm., Kim & Bakkee, 1910 and Drechslera sorokiniana (Sacc.) Subram & Jain, 1966, teleomorph: Cochliobolus sativus (Ito & Kurib) Drechsl. & Dastur, 1942]. The two fungal species that affect corn (Zea mays L.) are Bipolaris maydis (Nisikado & Miyake) Shoem., 1959 [syns. Helminthosporium maydis Nisikado & Miyake, 1926 and Drechslera maydis Nisikado & Miyake, Subram & Jain, 1966], and Exserohilum turcicum (Pass.) Leonard & Suggs, 1974 [syns. Helminthosporium turcicum Pass, 1876, Bipolaris *CNPq fellowship 639 Fitopatol. bras. 27(6), nov dez 2002 640 Fitopatol. bras. 27(6), nov dez 2002 R.C.B. Weikert-Oliveira et al. turcica (Pass.) Subram & Jain, 1966, and Drechslera turcica (Pass.) Subram & Jain, 1966]. The teleomorph of these species belong to Cochliobolus heterostrophusm (Drechsler) Drechsler, 1934, and Setosphaeria turcica (Luttr.) Leonard & Suggs, 1974, respectively (Sivanesan, 1987). The generic name Helminthosporium is deeply entrenched in the phytopathological literature, and segregation of the graminicolous species into Drechslera, Bipolaris, and Exserohilum has not been accepted universally (Alcorn, 1988). It has been suggested that knowledge of the systematics of the two genera, Drechslera, and Bipolaris, is inadequate (Hawksworth, 1986). The classification of the causal agents of “Helminthosporium” diseases is based on morphological and, to a lesser degree, on biochemical and physiological characteristics (Matsumura et al., 1988). Some authors have used molecular markers to study the relationship among these species (Bakonyi et al., 1995; Abadi et al., 1996). Increasingly, molecular biology techniques have been used to explore genetic variability in fungi (Caligiorne et al., 1999a). However, there are still few published studies on the genetic relationships among species that cause “Helminthosporium” diseases (Bakonyi et al., 1995; Abadi et al., 1996). The RAPD technique (Welsh & McClelland, 1990; Williams et al., 1990) involve the use of arbitrary primers that anneal to genomic DNA at low temperature conditions. This technique detects genetic polymorphisms that can aid in understanding population structure, diversity and taxonomy, and does not depend on prior knowledge of species-specific sequences. Comparative studies of the nucleotide sequences of ribosomal RNA (rRNA) genes provide the means for analysing phylogenetic relationships over a wide range of taxonomic levels. The nuclear small-subunit rDNA sequences (16S-like) evolve relatively slowly and are useful for studying distantly related organisms, whereas the mitochondrial rRNA genes evolve more rapidly and can be useful at the ordinal or family level. The internal transcribed spacer (ITS) region and intergenic spacer (IGS) of the nuclear rRNA repeat units, evolve faster, and may vary among species within a genus or among populations. The ITS1 and ITS2 regions represent the most variable region of the rDNA. This study analyzed genetic variation among fungal agents of “Helminthosporium” diseases isolated from cultures of the cereals rice, wheat and maize from diverse geographic regions. In this study we report the use of PCR-RFLP and RAPD assays as a molecular tool to illustrate the relationship between genetic polymorphism, type of host plant and geographic distribution. Twenty fungal isolates causing “Helminthosporium” diseases from different regions of North and South America were analyzed, including nine isolated from rice, seven from wheat and four from maize (Table 1). Teleomorph isolates [C. miyabeanus (CM.DO.TEX2), C. sativus (CS.BS.A20), C. heterostrophus (CH.DM.C4), and S. turcica (ST.ET.NK2)] were obtained from international collections and were used as reference for both PCR-RFPL and RAPD tests. Isolates were maintained on potato-dextrose agar at 4 oC. According to protocol described by Caligiorne et al. (1999a) samples for DNA extraction were grown on potatodextrose broth for eight days at 28 °C. Two grams of mycelia were harvested, frozen in liquid nitrogen and then ground to disrupt cell walls. The pellet was washed three times with phenol-chloroform and precipitated with absolute ethanol and 0.3 M NaCl. After 15 min, the precipitate was centrifuged and washed twice with 70% ethanol, dried and resuspended in 100 μl of 10 mM Tris-HCl (pH 7.5). DNA aliquots were TABLE 1 Characteristics of the fungus species examined Species Host Source Origin Reference Bipolaris oryzae Rice Leaf GO-Brazil BO.CNPA1 B. oryzae Rice Grain GO-Brazil BO.CNPA2 B. oryzae Rice Leaf GO-Brazil BO.CNPA3 B. oryzae Rice Grain GO-Brazil BO.CNPA4 B. oryzae Rice Leaf SP-Brazil BO.ESALQ1 B. oryzae Rice Leaf GO-Brazil BO.ESALQ2 B. oryzae Rice Grain GO-Brazil BO.ESALQ3 B. oryzae Rice Grain GO-Brazil BO.ESALQ4 Cochliobolus miyabeanus Rice Seed USA CM.BO.TEX2 B. sorokiniana Wheat Grain GO-Brazil BS.CNPA5 B. sorokiniana Wheat Grain GO-Brazil BS.CNPA6 Wheat Leaf RS -Brazil BS.CNPT1 B. sorokiniana Wheat Leaf RS-Brazil BS.CNPT2 B. sorokiniana Wheat Grain RS-Brazil BS.UFRS1 B. sorokiniana Wheat Grain RS-Brazil BS.UFRS2 C. sativus Wheat Grain Canada CS.BS.A20 B. maydis Maize Seed MG-Brazil BM.CNPMS1 Exserohilum turcicum Maize Seed MG-Brazil ET.CNPMS2 C. heterostrophus Maize Leaf USA CH.BM.C4 Setosphaeria turcica Maize Leaf USA ST.ET.NK2 7 B. sorokiniana 1Goiás, 2São Paulo, 3Rio Grande do Sul, 4Minas Gerais States 5Embrapa Arroz e Feijão GO, 6Escola Superior de Agricultura Luíz de Queiroz – SP, 7Cornell University, 8Embrapa Trigo RS, 9Universidade Federal do Rio Grande do Sul RS, 10Embrapa Milho e Sorgo MG