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Fitopatologia Brasileira

Print version ISSN 0100-4158On-line version ISSN 1678-4677

Fitopatol. bras. vol.28 no.4 Brasília July/Aug. 2003

http://dx.doi.org/10.1590/S0100-41582003000400004 

ARTIGOS / ARTICLES

 

Cytogenetics of Colletotrichum lindemuthianum (Glomerella cingulata f. sp. phaseoli)*

 

Citogenética de Colletotrichum lindemuthianum (Glomerella cingulata f. sp. phaseoli)

 

 

M. Gabriela Roca M.; Lisete C. Davide*; Maria C. Mendes-Costa

Laboratorio de Citogenetica, Departamento de Biologia, Universidade Federal de Lavras, Cx. Postal 37, Lavras, MG, CEP 37200-000; E-mail: lcdavide@ufla.br

 

 


ABSTRACT

Cytogenetic and morphological studies were conducted with Colletotrichum lindemuthianum (Glomerella cingulata f. sp. phaseoli), the pathogen responsible for anthracnose of common bean (Phaseolus vulgaris). In this species, there is some evidence of genomic variation but it is unknown whether the process occurs in a manner similar to other fungal genetic models. Six isolates from bean plants were used and sexual reproduction was observed in vitro. Meiosis and ascospore formation were investigated by cytogenetical approaches and light microscopy. To study the nucleus and chromosome numbers, a mixture of carmine and orcein propionic dyes was used. Nucleus divisions as well as ascospore maturation were asynchronous. Meiosis was observed in three isolates. In the asexual form, chromosomal polymorphism in conidia was also observed microscopically and the mitosis process was described.

Additional keywords: conidia cytogenetics, chromosome polymorphism.


RESUMO

Um estudo citogenético e morfológico foi conduzido em Colletotrichum lindemuthianum (Glomerella cingulata f. sp. phaseoli), o patógeno responsável pela antracnose do feijoeiro (Phaseolus vulgaris). Nesta espécie há algumas evidências de variações genômicas, porém não se sabe se os processos envolvidos são análogos aos que ocorrem com fungos considerados modelos genéticos. Seis isolados de C. lindemuthianum do feijoeiro foram utilizados e a reprodução sexual foi observada in vitro. A meiose e a formação de ascósporos foi estudada utilizando-se técnicas de citogenética e microscopia ótica pela primeira vez. O estudo do núcleo e o número de cromossomos foram estudados utilizando-se uma mistura dos corantes carmin e orceína propiônica. A divisão nuclear e a maturação dos ascósporos foram assincrônicas. A meiose foi observada em três isolados desta espécie. Na fase assexual, polimorfismo cromossômico nos conídios foi observado microscopicamente e o processo de mitose foi descrito.


 

 

INTRODUCTION

Colletotrichum lindemuthianum (Sacc. & Magn.) Scribner is the fungus responsible for anthracnose, a serious disease of common bean (Phaseolus vulgaris L.) (Rava & Sartorato, 1994; Perfect et al., 1999). The sexual form (teleomorph), Glomerella cingulata (Stonem.) Spauld. & Schrenk f. sp. phaseoli, has never been found in nature. The genetics of the genus Glomerella were first studied by Edgerton (1912, 1914). Cytogenetic, morphogenetic and morphological studies have also been conducted with G. cingulata (Hüttig, 1935; Lucas, 1946; Wheeler et al., 1948; McGahen & Wheeler, 1951; Uecker, 1994), but isolates from common bean were not included until recently (Roca et al., 2000). The sexual form of C. lindemuthianum was not reported until 1970 when the formation of fertile perithecia through crosses in culture medium was observed (Kimati & Galli, 1970). Although genetic studies have been carried out with G. cingulata strains isolated from bean plants (Batista & Chaves, 1982; Bryson, 1990; Mendes-Costa, 1996, O'Sullivan et al., 1998), the only work with conventional cytogenetics in this genera was done in 1946 with strains isolated from Ipomea sp. (Lucas, 1946).

In C. lindemuthianum, chromosome polymorphism was found by pulsed-field gel electrophoresis (PFGE) (O'Sullivan et al., 1998), as in strains of Colletotrichum gloeosporioides (Penz.) Penz and Sacc. isolated from Stylosanthes spp. (Masel et al., 1990), and from Citrus spp. (Liyanage et al. 1992). O'Sullivan et al. (1998) reported that the study of genome structure with molecular cytogenetic analysis, which is normally conducted during meiosis, was not possible in C. lindemuthianum because of the absence of the sexual form. In C. lindemuthianum, there is still uncertainty about the number of chromosomes longer than 7 Mb (O'Sullivan et al., 1998). In some filamentous fungi karyotyping by conventional light microscopy is not consistent with the electrophoretic karyotypes generated by PFGE. In Nectria haematococca Berk and Br. visualization of asci through conventional light microscopy led to an underestimation of the chromosome number and PFGE was effective for analysing chromosomes smaller than ca. 6 Mb (Taga et al., 1998). Since no cytogenetic studies of the sexual stage of C. lindemuthianum (G. cingulata f. sp. phaseoli) have been made so far, and given the excellent material at our disposal to compare with the asexual form, the aim of this study was to describe and to characterise both meiosis and ascospore formation in G. cingulata f. sp. phaseoli and to compare this process with the mitosis and the conidial chromosome number in the asexual form C. lindemuthianum, using light microscopy. The understanding of the cytogenetics of this fungi during sexual and asexual reproduction could help to explain the great variation reported in this specie in the asexual form.

 

MATERIALS AND METHODS

Colletotrichum strains and media

The six Brazilian strains used in this study were originally isolated from bean plants and are now deposited in the culture collection of the Department of Biology, Federal University of Lavras, Minas Gerais, Brazil. Isolate numbers and their geographical origins are as follow: 531-Goiânia, GO; 1002-Lavras, MG; 1003-Viçosa, MG; 1007-Lavras, MG; 1010-Lambari, MG and 1013-Patos, MG. These strains were cultivated on PDA and M3 media (Junqueira et al., 1984).

Cytogenetics of ascospores and asci

Cytological preparations were made according to Robinow (1975), with modifications depending upon the strain. In order to stain asci, perithecia were fixed in 3:1 ethanol:propionic acid for 24 h, crushed, suspended in this solution, and dropped onto slides. After air drying and heating in an oven at 40 ºC, slides were stained for 24 h with a carmine and orcein dye mixture in 2% propionic acid one week later. Slides were then quickly washed in chloroform, followed by the fixative solution and dried at 40 ºC for microscopic observation. Ascospores were measured with a micrometer eyepiece and a 40X magnification objective.

Cytogenetics of conidia

Fresh, ungerminated conidia were used for size measurements. Culture age was up to seven days and the conidia were measured with a micrometer eyepiece and a 40X objective for both free conidia and conidia from acervuli. Staining was used to identify conidial nuclei after seven days and to count chromosomes after 15 days. Except for the carmine-orcein mixture, staining for cytogenetic study was made according to Robinow (1975), with some modifications, depending on the strain and nuclear phase (Table 1).

Data and image analyses

Photomicrography was carried out with a camera coupled to a Leica microscope. Black and white films, ISO 25 and 100, were used and pseudostaining with grey colours with Adobe Photoshop v 2.5 and Jandel SigmaScan® Pro v 2.0 software permitted better identification of small chromosomes as well as the measurement and definition of pairing chromosomes.

Sample size and statistics

Evaluation of ascospore size (length and width) and chromosome numbers were performed in a fully randomized design with 30 and 35 replications, respectively. Chromosome counts were made for isolates 531 and 1013. The genotypic and phenotypic variances of ascospore size were estimated from the expected mean square by inserting the mathematical model into the operational formulas for mean squares from the one-way analysis of variance (ANOVA) (Hicks, 1993).

Evaluation on cell division in asci and formation of ascospores were performed with a minimum of 100 perithecia and scored as the number of observation most frequently found.

For evaluation of conidial size, a sample consisting of 100 fresh conidia was randomly chosen from five replicates. To count the number of nuclei per conidia, 500 ungerminated conidia were randomly chosen from five replicates after 15 days of culture. For strains 531 and 1002, counts of chromosome were undertaken from 60 ungerminated conidia, from different replicates of different plates. At least two different treatments were employed to count chromosome numbers (Table 1). The chromosome counts for strain 1013 were made with approximately ten ungerminated conidia from each of five replicates of each of five different plates (total number of 250 cells). In the latter analysis, the culture age was up to 25 days and only one staining treatment was used (Table 1). The same sample size was used to describe the mitosis process.

A one-way analysis of variance was carried out to account for the variation among and within strains, in relation to length and width of conidia. The effect of strain was considered to be a random one; therefore ANOVA was used to estimate genetic variance among strains by the method of moments (Hicks, 1993). This allowed for the estimation of inheritability in the broad sense, at the level of strain means for these traits. For chromosome numbers with strain 1013, a nested design was used; taking into account potential differences in plates, replicate slides for individual plates, and different conidia within any one slide. The effects of all these factors were considered to be random for the estimation of the corresponding components of variance.

 

RESULTS

The exuberant proliferation of perithecia was shown in culture medium (Figure 1). Each dark point is a cluster of perithecia which, when crushed, liberates many asci and ascospores. Since the details of the nuclear divisions in all perithecia were observed to be strikingly similar, the following descriptions, unless otherwise stated, apply equally well to asci of the three isolates studied.

 

 

Ascus formation

Ascus formation began with a strongly stained, central-coiled cell that, in most cases, had a large nucleus, meaning that karyogamy had already occurred. The diameter of this fusion nucleus was of approximately 3.20 µm, contrasted to 1.76 µm for the nuclei in the surrounding cells. Two nuclei, assumed to be haploid, were less often observed, meaning that fusion occurred quickly. The ascus then rapidly elongated and fusion nuclei attained their greatest length, 5.17 µm. In some of the early fusion nuclei, each nucleolus was observed attached to a large chromosome, the nucleolus-organizing chromosome.

Chromosome number and meiotic divisions

It was not generally possible to distinguish with certainty all of the individual chromosomes during pachytene, particularly the accurate pairing of bivalents in all the cells examined. Unpaired chromosomes were occasionally observed.

In late diplotene, knots and knobs suggestive of chiasmata, appeared at various points on the chromosomes. At late prophase I, four morphologically distinct chromosomes, assumed to be bivalents were seen, with the nucleolus (Figure 2a-b). The longest of these (approximately 1.10 µm long) was bent near the centre in a manner characteristic of the nucleolus-organizing chromosome. At metaphase I, the nucleolus was still present as a reminiscent body, dissipating at the end of the metaphase.

 

 

The two shortest chromosomes were approximately 0.5 mm long. No nucleoli were visible after metaphase I and it seemed probable that chromosomes pass from telophase of division