Vegetative compatibility and heterokaryon formation between different isolates of Colletotrichum lindemuthianum by using the nit mutant system

Brazilian Journal of Microbiology (2011) 42: 346-353 ISSN 1517-8382 VEGETATIVE COMPATIBILITY AND HETEROKARYON FORMATION BETWEEN DIFFERENT ISOLATES OF Colletotrichum lindemuthianum BY USING THE nit MUTANT SYSTEM Camila Rodrigues de Carvalho, Maria Cristina Mendes-Costa* Laboratório de Pesquisa I, Centro Universitário de Lavras, Lavras, MG, Brasil. Submitted: May 23, 2010; Approved: June 21, 2010. ABSTRACT Colletotrichum lindemuthianum, the causative agent of bean anthracnose, is one of the most common pathogens leading to expressive damage to plants beyond presenting noticeable variability. The knowledge on vegetative compatibility groups (VCGs) is of particular interest in asexual fungi as they subdivide the population in groups that can exchange genetic information via heterokaryosis and the parasexual cycle. Among the techniques used in studies about vegetative compatibility groups, the obtainment of nit mutants is apparent. This paper is aimed at obtaining heterokaryons between different isolates of C. lindemuthianum, grouping them in VCGs and evaluating their genetic variability by using the nit mutants system. Nit mutants were obtained from 20 single spore isolates. The mutants were phenotypically classified and paired for complementation and formation of heterokaryons so as to group them in VCGs. Seventeen mutants from the different phenotypic-rates were recovered: nit1, nit2, nit3 and nitM. At the same time, 10 mutants were selected for pairing and division of the anastomosis groups. Nine heterokaryons were obtained and the isolates were divided into 9 vegetative compatibility groups. In the combinations for the formation of anastomosis, 31 compatible combinations and 24 incompatible combinations were observed. It was concluded that the methodology used to select nit mutants in C. lindemuthianum made it possible to determine the vegetative compatibility groups and that such a technique was adequate to prove genetic variability. Key words: Colletotrichum lindemuthianum; anthracnose; heterokaryosis; nit mutants; VCG. INTRODUCTION variability presented by this phytopathogen are not completely known. Colletotrichum lindemuthianum (Sacc. & Magn.) Scrib. (= The formation of heterokaryons between different strains is Glomerella cingulata (Stonem Spaulde & Schrenck) f. sp. an important and common component of the life cycle of many phaseoli) is the causal agent of bean anthracnose and stands out filamentous fungi. Lineages that are capable of fusing as one of the most common pathogens that provokes expressive (anastomosis) and forming stable and functional heterokaryons damage to these plants. The utilization of bean varieties are known as sexually or vegetatively compatible, the former resistant to anthracnose is one form of preventing the disease, being frequently described as members of the same group of but it is made difficult due to the great variability of fungi. vegetative compatibility or vegetative compatibility group — However, the genetic mechanisms responsible for the great VCG (13). Vegetative incompatibility or heterokaryon *Corresponding Author. Mailing address: Research Laboratory I (Fungi), Centro Universitário de Lavras, CEP 37200-000, Lavras, MG, Brazil.; E-mail: 346 Carvalho, C.R. et al. Isolates of C. lindemuthianum incompatibility is a genetic mechanism that restricts the verification. Fragments from these cultures were transferred to heterokaryosis between individuals who differ in one or more Petri dishes containing a minimal medium + NaNO3 (MM) (4). het or vic loci (10, 19). The isolates that presented poor growth colonies in this The knowledge of vegetative compatibility groups between medium and little mycelial production were considered to be different lineages is of particular interest in asexual fungi such nit mutants, while those presenting dense aerial mycelium as Colletotrichum spp. since the VCGs subdivide the growth, or wild-type, were discarded (14). population into groups that can exchange genetic information via heterokaryosis and the parasexual cycle (4). Studies on Phenotypic classification of the nit mutants VCGs involve obtaining mutants that are incapable of utilizing For the phenotypic classification of the nit mutants, nitrate as the only source of nitrogen (nit mutants) and are mycelial fragments from the same Petri dishes containing MM resistant to chlorate, which is a toxic analog of nitrate. were selected and transferred to the center of dishes containing Complementation among different nit mutants is indicated basal medium (BM) supplemented with sodium nitrite (0,5 by the development of dense mycelia in the zone of contact g/L), sodium nitrate (2,0 g/L), hypoxanthine (0,5 g/L), between the two mutant colonies (heterokaryon) so that the two ammonium tartrate (1,0 g/L) and uric acid (0,2 g/L) (4). Each isolates belong to the same vegetative compatibility group (13). nit mutant was transferred to three dishes (100 x 15 mm) with Thus, mutants can once again present wild-type growth since each of the aforementioned media; totaling 15 dishes for each complementation with another mutant isolate of the same VCG isolate. These dishes were maintained in a BOD incubator has occurred, and usage of nitrate is now possible (6). between 22/25ºC for a period of 14 to 21 days. Two Heterokaryosis can be a probable cause of the increased genetic variation in this species. The present study brought this evaluations were carried out: the former on the 14th and the latter on the 21st day. fact into focus to obtain heterokaryons between different The phenotypic classification was done according to the isolates of C. lindemuthianum, group them in VCGs and mycelial growth of the mutants in media supplemented with evaluate their genetic variability by using nit mutants system. different sources of nitrogen: BM + sodium nitrate (MM), BM + sodium nitrite (NM), BM + hypoxanthine (HM), BM + MATERIAL E METHODS ammonium tartrate (AM) and BM + uric acid (UAM). Media supplemented with sodium nitrate and ammonium tartrate were Origin of fungal isolates used as negative and positive controls, respectively. The isolates (Table 1) of C. lindemuthianum employed were kindly donated by Elaine A. Souza, PhD (Biology Department of the Universidade Federal de Lavras – MG, Brazil) and twenty single spore isolates were used. Anastomoses formation A number of 10 mutants were selected in the test for formation of anastomoses and a pairwise confrontation of all the isolates was performed using the methodology described by Recovering of nit mutants Rodriguez-Guerra et al. (17). The hyphae were stained with After cultures were grown in solid M3 culture medium aceto-orcein staining solution (2%). By means of an optical (11), a mycelial fragment was transferred from the isolates to microscope the origin of the anastomosed cells were traced, the center of the Petri dishes containing minimal me (...truncated)