The use of RAPD to characterize Bipolaris sorokiniana isolates

INTRODUCTION

Wheat is one of the most economically important crops world-wide including Brazil. Nevertheless, considerable economic losses happen chiefly due to diseases caused by fungi. The phytopathogen Bipolaris sorokiniana (Sacc. in Sorok.) Shoemaker, 1959 (teleomorph): Cochliobolus sativus (Ito & Kuribayashi) Drechsl. ex Dastur causes diseases in wheat and other cereal grasses, and occurs widely in wheat-producing regions in Brazil. The diseases caused by the fungus are common root rot, leaf spot disease, seedling blight, and black point of the grain. The main source of inoculum is considered to be soil-borne conidia, which, under favorable conditions, may result in severe damage by common root rot (Duczek, 1984).

Bipolaris sorokiniana shows high morphological and physiological variability. The mechanisms involved in the variability of B. sorokiniana include heterokaryosis, which occurs chiefly through the anastomosis between adjacent hyphae, enables the parasexual cycle, which is the main source of genetic diversity in fungi with asexual reproduction (Day, 1974). This diversity is one cause of the infective success of phytopathogenic fungi in overcoming host resistance (Guseva et al., 1979).

The morphological, physiological and biochemical characterizations of B. sorokiniana have been the aim of many studies (Christensen, 1925; Mitra, 1931; Hrushovetz, 1956; Wilson and Murphy, 1964; Tinline, 1986). However, knowledge about the genetic structure of this fungus is less available (Zhong and Steffenson, 2001a,b; Zhong et al., 2002).

Fungal species that are pathogenic on plants or animals exhibit variation in many important traits. Variability in morphology can make pathogen identification difficult while physiological variability can affect the evaluation of the damage potential (virulence) of a particular pathogen. The correct species identification and recognition of physiological strains (pathotypes) is critical information to understand the pathogen-host relationship (Strongman and MacKay, 1993).

Molecular markers have been increasingly used to examine the dynamics of phytopathogen populations. The development and refinement of molecular techniques have provided tools to advance the study of genetic diversity, of the intra- and inter-specific phylogenetic relationships, and also the identification of races and pathotypes (Mullins and Faloona, 1987; Bruns et al., 1991; Batista, 1993; Gallego and Martinez, 1997).

Since its development, the random-amplified polymorphic DNA (RAPD) protocol has acquired a diversity of uses, such as: the establishment of the genetic similarity degree between individuals within a population (Anderson and Fairbanks, 1990), the construction of genetic maps as well as the localization of economically interesting genes (Williams et al., 1990), the production of a genomic fingerprint (Welsh and McClelland, 1990), and the study of genetic diversity along with the identification of fungi (Fani et al., 1993; Fegan et al., 1993; Henson and French, 1993; Jungehülsing and Tudzynski, 1997; McEwen et al., 2000).

The aim of the present study was to assess the genetic variability of B. sorokiniana isolates from different wheat cultivars, by means of the RAPD technique. The detection of genetic diversity and, if possible, the establishment of races and/or formae specialis are two important pre-requisites for the development of resistant wheat varieties and establishment of molecular markers to ascertain pathogen identification.

MATERIAL AND METHODS

Bipolaris sorokiniana isolates

The B. sorokiniana isolates examined in this experiment were obtained from wheat seeds of different cultivars grown in different wheat-producing regions in Brazil (Table 1). The seeds were provided by the Centro Nacional de Pesquisas do Trigo (EMBRAPA/CNPT), Passo Fundo, RS, Brazil.

Isolation and maintenance of the polyconidial Bipolaris sorokiniana cultures

The wheat seeds were submersed in 2% sodium hypochlorite (w/v) for 2 min, rinsed three times in sterile distilled water, and subsequently transferred to Petri dishes containing potato dextrose agar. The samples were then incubated in a BOD at 24 ± 2°C for approximately 9 days with a 12-h photoperiod. After some replication of the isolates pure cultures were obtained. Inoculum from the samples was transferred to slant test tubes containing PDA medium and incubated as before. When colonies developed and were identified, the test tubes were stored at 4°C.

DNA extraction

Genomic DNA was obtained from each isolate grown in liquid culture (potato dextrose broth) following a method already developed by Ashktorab and Cohen (1992), with modifications to suit the phytopathogen under study. Mycelium of each isolate was retrieved through filtration, rinsed, and macerated in liquid nitrogen until a fine powder was obtained. Wet mycelium extraction-lysis buffer (1 mL/g), which consisted of 200 mM Tris-HCl, pH 8.0, 250 mM NaCl, 25 mM EDTA, pH 8.0, 1% sodium dodecyl sulfate (SDS), 10 µL/mL b-mercaptoethanol (Sigma), and 50 µg/mL proteinase K, was added to this powder. The tubes were incubated at 64°C for 60 min, shaken occasionally and centrifuged at 5000 rpm for 20 min. One volume of phenol (equilibrated with Tris-HCl, pH 8.0) was added to the supernatant recovered from the centrifugation. Homogenization was followed by centrifugation at 5000 rpm for 20 min. The aqueous phase was removed, transferred to new tubes and the extraction routine took place twice with phenol:chloroform, and once with 1X volume chloroform:isoamyl alcohol. Pancreatic RNAse A (50 µg/mL) was added to the supernatant and then incubated at 37°C for 20 min. DNA sample was precipitated with 0.1 volume 3 M sodium acetate and 2.5 volume isopropanol at -20°C. DNA was then rinsed with cooled 70% alcohol (v/v), centrifuged at 5000 rpm for 10 min, dried at room temperature, and finally resuspended in milliQ water and stored at -20°C.

RAPD analysis

Genomic DNA from the B. sorokiniana samples was amplified using the RAPD protocol (Williams et al., 1990). The primers tested comprised the series W (W01-W20), E (E01-E20), P (P01-P20), and O (O01-O10) (Operon Technologies, Alameda, CA, USA). Thirty primers were selected for the analysis of genetic diversity observed on the basis of the polymorphic DNA standard obtained after the RAPD assays. The primers chosen, OPW02-W10, W18-W20; OPP01-P09, P14; OPE03, E07, E11, E12, E14, and E16-E18 (Table 2), were retested in RAPD assays to assess the reproducibility of banding patterns. Only strong and reproducible polymorphic RAPD profiles were analyzed. The amplification was tested in a final volume of 25 µL for each reaction containing 30 ng genomic DNA, a dNTP mix (2.5 mM of each dATP, dCTP, dGTP, dTTP), 30 ng oligonucleotide primer, 100 ng bovine serum albumin (BSA), 1X reaction buffer (10X), 1 U Taq polymerase (Cenbiot, RS, Brazil), and 0.001% mineral oil. The reactions took place in a thermal cycler Techne-Progene with 46 cycles as follows: 1 initial cycle (1 min at 94°C/5 min at 42°C/2 min at 72°C), 45 cycles (30 s at 94°C/30 s at 42°C/1 min at 72°C). Fragments were resolved by electrophoresis in 1.4% agarose gel, at 75 V for 2.5 h.

Statistical analysis

The RAPD data were assessed using the Statistical Package for the Social Sciences (SPSS) software, 2nd edition, through which similarity coefficients were calculated and the dendrogram for genetic distances was constructed. The similarity was evaluated through simple association and the genetic distance as the Euclidean distance. The binary matrix was built pairwise, and the presence or absence of a determined RAPD band scored 1 and 0, respectively. The hierarchical groupings were based on the unweighted pair group using arithmetic averages - UPGMA (Sneath and Sokal, 1973).

RESULTS

A total of 282 fragments resulted from the primer amplifications with the DNA from the 20 isolates of B. sorokinana (Table 2). Fragments present in at least one and maximum 18 isolates (5-90% occurrence) were considered polymorphic, while fragments present in at least 19 isolates (95% occurrence) were monomorphic. This criterion established a total of 228 fragments (80.9%) as being polymorphic.

Figure 1A,B shows a monomorphic standard obtained with primer W06. The 1.10-, 0.94-, 0.84-, and 0.72-kb fragments were present in all samples, except in isolate I017, which did not amplify the 1.10- and 0.72-kb fragments. On the other hand, this was the only isolate to yield 0.41-, 0.47-, 0.55-, and 0.66-kb fragments (Table 2).

Figure 2A,B shows the amplification standard for primer P14. Fragments of 0.63 and 0.88 kb were monomorphic and did not amplify with isolate I017. In contrast isolate I017 was the only to amplify 0.56-, 0.65-, 0.79-, and 0.84-kb fragments. The 1.27-kb fragment was present in isolate I030 only. The 0.77-kb band amplified with isolates I032, I007, and I021 only, while the 0.80-kb band amplified with isolate I006. Isolates I004, I033, I014, I022, and I018 exhibited a 1.07-kb fragment. The 1.11-kb fragment was present in isolates I029 and I023 exclusively, and the 1.23-kb fragment was amplified with isolates I006, I033, and I022 (Table 2).

A dendrogram was constructed with 282 fragments generated from RAPD data as shown in Figure 3. The genetic similarity between the isolates assessed through simple association ranged between 0.3865 and 0.9681 (Table 3). The maximum coefficient (0.9681) was observed for pair I040-I029, while the lowest coefficient (0.3865) was observed for pair I006-I017. The remaining isolates had similarity coefficients between 0.9645 and 0.3936. The isolates were assigned to 3 groups: 2 closely related (groups I and II, with similarities ³78%), and one group (group III), related to the other groups with similarity coefficients between 0.3865 and 0.4681.

Group I was established between pairs I029-I040 and I021-I032 joined by isolates I031, I026, I007, I041, I043, and I023. Group II comprised pair I004-I014, and isolates I033, I018, I012, I003, I006, I022, I030. Isolate I017, which forms group III, showed a banding pattern significantly different from the other isolates.

DISCUSSION

The results suggest that, notwithstanding the isolates originated from different wheat cultivars, for the sampling carried out, the genetic variability of B. sorokiniana isolates was low, with 19 of the 20 isolates showing a similarity coefficient of ³78%. These results might be related to the geographic origin of the samples because all the isolates were obtained from wheat seeds from two states on the southern region of Brazil (Rio Grande do Sul and Paraná), the leading wheat-producing region in the country.

Zhong and Steffenson (2001b) assessed genetic diversity of Cochliobolus sativus based on virulence and AFLP markers. Ninety-five polymorphic AFLP markers were found with 8 primer pairs, and each isolate exhibited a unique AFLP marker. The cluster analysis did not reveal a close correlation between pathotypes and AFLP groups. In the same study, the authors also observed no close correlation between genetic similarity measured by AFLP markers and geographic origin, suggesting that these isolates were derived from the same founder source population and disseminated from one area to another in association with their hosts.

Chen et al. (1995) found a high degree of similarity between Puccinia striiformis individuals from the same formae specialis, and high degree of polymorphism was observed for individuals of different species. In the same study the RAPD groups formed generally were not associated with the geographic regions either. On the other hand, isolates of Fusarium oxysporum f. sp vasinfectum were differentiated into three main groups directly related to both virulence and geographic origin (Assigbetse et al., 1994).

The variation in intensity of some fragments in this study was observed, but this might be due to a more efficient amplification at some target sites, or to a greater complementarity between the primer used and the fungal DNA. Variation may appear in keeping with target sites present at highly repetitive sequences as well (Meunier and Grimont, 1993; Kelly et al., 1994; Tyler et al., 1997).

Nucleotide alterations, insertions and deletions at initiation sites may result in polymorphic DNA, which is detectable by the RAPD technique (Williams et al., 1990). In the present study, the multi-nucleated conditions of B. sorokiniana mycelial cells and conidia, with subsequent heterokaryosis - which in turn could lead to mitotic recombination and new haploidization arrangements - may account for a likely contribution to the DNA polymorphism detected for the phytopathogen. Even though, the amplification products of 33.3% of the primers W02 (1.54 kb), W05 (1.20 kb), W06 (0.84 and 0.94 kb), W07 (0.71 kb), W10 (0.93 kb), W19 (0.54 kb), P08 (1.05 kb), P09 (0.99 kb), P14 (0.63 and 0.88 kb), and E16 (0.66 kb) generated monomorphic fragments. These same primers are being used with monoconidial cultures of the isolates in order to verify the significance of the monomorphic fragments. If they repeat themselves it may be possible that these fragments can be used as specific molecular markers to identify B. sorokiniana.

Taxonomic identification of isolate I017 (Samambaia, PR, Brazil), was confirmed through conidial shape; hilum; germination; basal germ tube; direction of growth and position of emergence; septum ontogeny and conidiogenous nodes. This was the only isolate to show a very low similarity and the absence of significant monomorphic bands. Isolate I017 with all primers tested also showed a different banding pattern. This isolate had previously shown a differentiated pattern as to the degree of pathogenicity and established no infective interaction with wheat cultivars, and differed from other isolates analyzed (Santos AMPV, personal communication).The isolate I017 was the only isolate that could be well distinguished from all the others in this study. More studies must be done with this isolate in order to classify it.

RAPD assays were used to determine formae specialis and physiological races in pathogenic and antagonistic species of Fusarium oxysporum (Migheli and Cavallarin, 1994). RAPD has been successfully used to distinguish pathogenic and non-pathogenic isolates of Leptosphareia maculans (Plummer et al., 1994) and to differentiate pathotypes in Peronospora parasitica (Tham et al.,1994).

In a work done with Cochliobolus carbonum (Jones and Dunkle, 1993) RAPDs were used to distinguish races at molecular level. In their studies they found that all pathogenic races were substantially different from nonpathogenic races. From the 20 primers tested on all five races, the banding pattern was similar for all pathogenic races with 19 of the primers. Only one primer showed race 3 specific differences.

A compilation of Tudzynski and Tudzynski (1996) lists 22 species of phytopathogenic fungi, where RAPD has been used. In most of these cases RAPD could be used to discriminate between physiologically or genetically well-defined species subgroups, or even between species, using RAPD as a molecular confirmation for the subgrouping obtained by other methods, and as a diagnostic tool for the grouping of new isolates (Jungehülsing and Tudzynski, 1997).

RAPD results obtained in the present study enabled a fast and efficient variability analysis for B. sorokiniana. These results also led to the generation of electrophoretic profiles, which discriminated intra-specific polymorphism in the isolates studied. Also, in order to find more variability among the isolates of B. sorokiniana, and maybe to separate them in races, a study with a higher number of isolates from the same, and different wheat cultivars from Brazil and other countries and also isolates from other cereal grasses will be necessary.

ACKNOWLEDGMENTS

Research supported by CNPq (Conselho Nacional de Pesquisa). We thank Centro Nacional de Pesquisas de Trigo (EMBRAPA/CNPT) for providing the wheat seeds.

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