620 Mycol. Res. 100 ( 5 ) : 6 2 M 2 4 (1996) Printed in Great Britain Genetic studies of the phytopathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) by analysis of ordered tetrads F. FARETRA A N D S. POLLASTRO Dipartimento di Protezione delle Piante dalle Malattie, University of Bari, Via G. Amendola 165/a, 70126 Bari, Italy Botryotinia fuckeliana (Botrytis cinerea) is a heterothallic ascomycete which produces linear 8-spored asci (tetrads)in apothecia. Ascospores were collected in their linear sequence from individual asci, and analysed to determine the segregation patterns of alleles at gene loci controlling mating type (MATI), resistance to dicarboximide fungicides (Dafl) and resistance to benzimidazole fungicides (Mbcr). The alleles were present in their expected linear sequence in 58% of the asci; most irregular sequences (32% of the asci) were caused by single exchanges between adjacent spores. The MAT1 locus was ca 12 map units from its centromere, and the Mbcl locus showed loose linkage with its centromere. The Mbcr and Dafl loci were ca 47 map units apart on the same chromosome and not linked with the MAT1 locus. Pairs of homothallic ascospores were found in seven out of 105 asci. Homothallism was not caused by inclusion of more than one nucleus in ascospores, and it occurred only in ascospores predicted to carry the MATI-2 allele after meiosis. Botyotinia fuckeliana (de Bary) Whetzel, teleomorph of Botytis cinerea Pers., is the pathogen which causes 'grey mould' disease on numerous economically important plant species. The fungus became amenable to genetic analysis when a reliable method for obtaining meiotic progeny under laboratory conditions was developed (Faretra & Antonacci, 1987; Faretra & Pollastro, 1988; Faretra, Antonacci & Pollastro, 1988a, b). Apothecia containing linear asci are obtained by cross-fertilization of sexually compatible isolates carrying different alleles of the mating type gene, MATI. Each ascus is derived from a single, heterozygous, diploid nucleus, and contains four pairs of ascospores; the nuclei in each pair of ascospores should be genetically identical, since they are the result of mitotic division of one of the four products of meiosis (Lorenz & Eichhom, 1983; Faretra & Antonacci, 1987; Faretra, Contesini & Pollastro, unpublished). Apothecia can be transferred to vials of sterile distilled water, and ruptured with tweezers to release thousands of ascospores for the analysis of random meiotic spores (Faretra & Grindle, 1992). This method has been used to determine the genetic basis of fungicide resistance (Faretra & Pollastro, 1991, 1993a, b; Faretra, Pollastro & Sansiviero, 1992; Pollastro & Faretra, 1992; Pollastro ef ai., 1995), morphological differences (Faretra & Mayer, 1992; Faretra & Pollastro, 1992), and auxotrophy ( ~ a r e t r a& Pollastro, unpublished). However, it cannot provide some kinds of genetic data for which tetrad analysis is essential; for example, the data needed to locate a gene in relation to its centromere (Burnett, 1975; Fincham ef al., 1979). This report deals with investigations of mating type and resistance to benzimidazole and dicarboximide fungicides by analysis of ascospores from individual asci of B. fuckeliana. MATERIALS A N D M E T H O D S Botryotinia fuckeliana isolates The isolates used in this study (Table 1) carried known alleles of the genes conferring differences in mating type (MATI), response to benzimidazole fungicides (Mbcl) and response to dicarboximide fungicides (Dafl), as determined by Faretra & Pollastro (1991, 1993a). Alleles of these three genes were used to detect segregation patterns in tetrads. Sexual crosses and derivation of ascospore progeny The following strains (sclerotial $? parent x spermatizing 6 parent) were crossed to obtain apothecia as described by Faretra ef al. (1988a): SAS405 x SAS56 (cross CRI), SAS405 x 2A/12 (CR2), SAS405 x C/4 (CR3), SAS405 x 2E/5 (CR4), SAS405 x G / I (CR5), SAS56 x SAS405 (CR6), SAS56 x 2B/1 (CR7), SASS6 x 2D/1 (CRS), SAS56 x F/1 (CR9), SAS56 x 2H/1 (CRIO). Intact asci were transferred individually to dishes of water agar (20 g Oxoid technical agar 1-1 d'istllled ' water), and the ascospores were dissected out in their linear sequence with a Leitz micromanipulator to obtain ordered 8-spored tetrads. Germinated ascospores were grown on slants of malt extract agar (MEA, 20 g Oxoid malt extract and 20 g Oxoid technical agar I-' distilled water) to obtain colonies. Genotypes of ascospore progeny Resistance or sensitivity of the ascospore progeny to fungicides was determined by transferring hyphal inocula to dishes of MEA containing benomyl or diethofencarb to detect F. Faretra and S. Poflastro 62 1 Table 1. Origins and genotypes of strains of Botryotinia fuckeliana used for tetrad analysis Genotypes Origin (9x parents in sexual crosses)* Straint - SAS56 2A/I2 G/1 c/4 2E/5 SAS405 2H/I 2D/I 28/1 F/I - MAT1 Mbcl Daf1 I I S S S LR HR" HR* S LR HR* HR'" S S HR S S LR HR S LR LR - WS55 x unknown SAS405 x SAS56 V29 x SAS56 SAS56 x 813 SAS405 x VB74 WS158 x unknown V29 x SAS56 SAS56 x 813 SAS56 x SAS405 SAS405 x VB74 1 1 I 2 2 2 2 2 t Each strain was a single ascospore isolate from the cross indicated. All parental strains excepting SAS were field-isolates. 5 S, sensitivity; LR, low resistance; HR, high resistance. + " Alleles also conferring hypersensitivity to diethofencarb. ** Alleles also conferring normal sensitivity to diethofencarb. alleles of the Mbc1 gene, and MEA containing vinclozolin to detect alleles of the Dafl gene, as described by Faretra & Pollastro (1991). The mating types of the progeny were determined by crossing each one to both the MATI-1 reference strain, SAS56, and the M A T I - 2 reference strain, SAS405 (Faretra et al., 1988b). Genetic symbols and terminology for B. fuckeliana isolates have been described by Faretra & Grindle (1992). Tetrad analysis Sequences of genotypes in individual asci were deduced from segregations of genes conferring differences in fungicide resistance. The sequences so obtained could often be corroborated by segregations of morphological traits of colonies; for instance, conidiation, number and size of sclerotia. Pairs of ascospore progeny with identical phenotypes were easily recognized in most tetrads. Ordered asci were analysed to determine whether the different alleles at a gene locus segregated from each other at the first meiotic division (FDS; asci with a 4:4 arrangement of alleles in the spores) or the second meiotic division (SDS; asci with a 2:2 :2: 2 arrangement of the alleles). Generally, in absence of particular modality of meiotic recombination, a proportion of SDS :FDS lower than 2 :1indicates that the gene locus is linked to its centromere, and 50% of the frequency of SDS asci is an estimate of their map distance (Fincham et al., 1979). Ordered and unordered asci were analysed for recombination between pairs of gene loci. Asci were classified as parental ditypes (PD) containing only parental genotypes; non-parental ditypes (NPD) containing only recombinant genotypes, and tetratypes (T) containing both parental and recombinant genotypes. The proportion of PD:NPD asci indicates whether the gene loci are on the same or different linkage groups (Fincham et al., 1979). Genes assigned to the same linkage group were mapped using the formula 50[(T 6NPD)/(PD NPD T)], including the correction for double crossovers (Fincharn et al., 1979). + + + Whether there was independent segregation of genecentromere and gene-gene loci was assessed by means of the X2 test incorporating the Yates correction term (Sachs, 1982). RESULTS A N D DISCUSSION Alleles of the Mbc1 and Dafl genes segregated in a normal 1: 1ratio in all asci from all crosses (167 asci were analysed for the Mbcl gene and 158 asci for the Daf1 gene). The M A T I - 1 and M A T I - 2 alleles of the mating type gene also segregated normally in a I :1ratio in most (98 out of 105) asci examined. The seven exceptional asci contained homothallic ascospores (see below). The sequences of genetic markers were determined in 190 asci (Table 2). Regular, normal, linear sequences (type I ) occurred in 110 asci (58%). Most of the irregular sequences (types 2 and 3), observed in 61 asci (32%), were due to single exchanges between two adjacent spores. Only 19 asci (10%) had irregular sequences which implied more complex spore displacements (types 4-7). The high frequency of irregular spore sequences in mature asci can be explained by cytological studies of meiosis in developing asci of B. fuckeliana (Faretra, Contesini & Pollastro, unpublished). Those showed that the nuclear spindles of the first post-meiotic mitosis often overlap to their oblique orientation in the ascus, and they may not be parallel. The overlapping spindles which could be responsible for irregular sequences in ascus types 2-7 are illustrated in Table 2. The frequencies of SDS tetrads in regard to the Mbcl, Dafl and M A T 1 gene loci were 58, 72 and 25 % respectively (Table 3). Results of x2tests (Table 3) show that only the M A T 1 gene is linked to its centromere and can be mapped (12.5 map units); the Mbc1 gene may also be loosely linked to its centromere. The number of PD, NPD and T tetrads relating to segregations of the three possible gene pairs (Table 4) show that the Mbcl and Dafl genes are probably linked, since the PD:NPD ratio differs significantly from 1:l. The calculated distance between the two genes is ca 47 map units. PD :NPD ~ the gene ratios were not significantly different from 1 : for pair M A T I - M b c l and MATT-Dafl (Table 4), and so these genes are probably unlinked. These conclusions are in agreement with those based on analysis of random ascospore progeny (Faretra & Pollastro, 1991, 1992). . Seven asci from four different crosses contained ascospores which were fertile with both M A T I - 1 and M A T I - 2 reference strains. Strains of this phenotype have been referred to as homothallic MATI-112 strains (Faretra & Grindle, 1992). In five of these asci, there were two pairs of MATI-1 ascospores and two pairs of MATI-112 ascospores; in the other two asci, there were two pairs of MATI-I, one pair of MATI-2 and one pair of MATI-112 ascospores. Thus, pairs of homothallic MATI-112 ascospores replaced pairs of MATI-2 ascospores. Since alleles of the Mbc1 gene and/or the Dafl gene segregated in these asci as ~redictedon the basis of parental genotypes, it seems very unlikely that the abnormal mating type ascospores were due to contamination or experimental errors during fertilization. Previous studies of field isolates and monoascospore isolates Genetics of Bofryofinia fuckeliana 622 Table 2. Numbers of ordered asci of Botryotinia fuckeliana in which the ascospores display regular and irregular sequences of marker genes Ascus type? Arrangement of ascospores in ascit crosses5 CRI CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CRlO Total asci 1 0-0 Q-Q 0-0@-@ 42 10 8 8 4 8 6 5 13 6 110 4 0Q-Q @ 0-0@-@ 5 I I o o o o o o o 7 82 18 16 9 8 13 II 7 18 8 190 Total asci t Type I shows the regular linear sequence of marker genes in the nuclei of eight spores. Types 2-7 are irregular sequences caused by overlapping nuclear spindles. Pairs of spores carrying identical genetical markers are coded with the same number. Ascus polarity is ignored. The markers were alleles of genes encoding differences in fungicide resistance and morphology. There should be four pairs of spores with identical genotypes since they are produced by post-meiotic mitosis of the four products of meiosis. Lines indicate overlapping nuclear spindles (during post-meiotic mitosis) which can account for each type of sequence irregularity. 6 Asci were obtained from apothecia of the ten CR crosses described in the text. Table 3. Numbers of ordered asci (tetrads) from crosses of Botyotinia fuckeliana strains showing particular types of segregation of alleles at three gene locit Gene locus* Mbcl Daf I Cross FDS SDS FDS CRI CRZ CR3 CR4 CR5 CR6 CR7 CR8 CR9 CRIO Total asci Frequency (%) value5 Probability level (P) 33 7 7 2 41 18 6 x2 8 5 7 4 4 6 8 3 6 3 5 5 13 na na 70 97 42 58 5.61 < 0.02 2 na 2 6 2 na 6 2 44 28 MAT1 SDS 55 9 10 na 6 8 8 na I2 6 114 72 1.65 > 0.19 FDS 32 7 3 5 3 9 4 4 9 3 79 75 SDS 8 2 5 0 3 I I 3 I 2 26 25 83.08 < 0.001 t Alleles of the gene segregated from each other at the first meiotic division (FDS) or second meiotic division (SDS). SDS tetrads are due to recombination between the gene locus and its centromere. Responsible for difference in resistance to benzirnidazole fungicides (Mbcl), resistance to dicarboximide fungicides (Dafl) and mating type (MATI). na, not applicable. 5 For independent gene-centromere segregation the null hypothesis tested is SDS:FDS = 2: 1. * of B. fuckeliana (Faretra ef al., 1988b) have identified functionally homothallic strains. Inclusion of two nuclei of opposite mating type in one ascospore is the rule in the fourspored asci of secondarily homothallic species such as Neurospora fefrasperma (Dodge, 1927) and Podospom anserina (Franke, 1957). A similar mechanism has been shown to operate in functionally homothallic field isolates of B. fuckeliana since they were shown to be heterokaryons containing both A4ATl-1 and M A T I - 2 alleles in separate nuclei (Faretra ef al., 19886). That is, they are secondarily (pseudo) homothallic. However, homothallism of monoascospore isolates cannot be due to heterokaryosis and has not been explained. Cytological observations have shown that ascospores incorporate a single post-meiotic nucleus (Lorenz & Eichhom, 1983; Faretra & Antonacci, 1987; Faretra, Contesini & Pollastro, unpublished); consequently, they are presumably not heterokaryotic. Also, homothallic ascospores have been detected in asci with eight viable ascospores and with the expected segregation of phenotypes. Irregular inclusion of more than one nucleus of opposite mating type in individual ascospores is then very unlikely since it would be expected to result in aborted (anucleate) spores in eight-spored asci. Molecular studies of mating type genes in Ascomycetes have shown that unique DNA sequences are associated with specific alleles (reviewed by Glass & Lorimer, 1991; Glass & Nelson, 1994). The sequence differences between alleles (idiomorphs, according to Metzenberg & Glass, 1990) are so extensive that changes from one allele to another do not occur; furthermore, no new alleles have been produced experimentally by mutation or intragenic recombination F. Faretra and S. Pollastro 623 Table 4. Numbers of ordered asci (tetrads) from crosses of Botyotinia fuckeliana strains showing particular types of segregation of gene pairst Gene-gene segregation Cross PD NPD CRI CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CRlO Total asci Frequency (%) X' value for independent segregation* Probability level (P) 28 4 4 na 7 0 0 na 0 0 0 na 0 na 7 4 25.41 1 3 1 na 3 na 44 26 T PD NPD T PD NPD T 50 14 12 na 7 I1 9 na 15 na 118 70 7 3 2 0 0 3 3 2 I na 21 21 13 20 5 6 5 6 6 2 4 8 na 62 62 5 4 0 na 3 I 3 na 2 32 1 0 0 0 I 0 1 1 na 17 17 0.24 1 3 2 na I 0 16 17 1 0 na 1 4 5 na 3 6 3 na 8 0 5 11 66 71 12 0.59 t PD, parental ditypes (only parental genotypes present); NPD, non-parental ditypes (only non-parental genotypes present); T, tetratypes (both parental and non-parental genotypes present). na, not applicable. $ The null hypothesis tested is PD:NPD = 1:I. (Newmeyer et al., 1973; Griffiths, 1982). Therefore, it is unlikely that homothallism of B. fuckeliana ascospores is due to simple mutation from the MATI-2 to the A4ATI-1 allele. Random ascospore progeny from crosses of two homo~ ~S A S ~with ~ , the reference SAS405 thallic isolates, S A S and (MATI-2), were back-crossed to both strains SASS6 (MATII) and SAS405 (MATI-2) in order to determine their mating types. Most of the 128 ascospores tested were heterothallic (59 were MATI-1 and 63 were MATI-2). Only six ascospores were homothallic MATI-112. Thus, homothallism of isolates SAS27 and SAS39 was not caused by a defective MAT1 gene nor by a stable 'homothallic' locus and they acted as MATI-1 partner in the cross with SAS405. Mating type instability has been documented in several species of fungi- In the case of the heterothallic yeasts, Saccharomyces cereuisiae and Schizosaccharomyces M om be, the molecular mechanism of mating type switching is known (Beach & Klar, 1984; Herskowitz, 1988), Perkins (1987) suggested that a similar mechanism might be responsible for unidirectional reversal of mating type observed in some filamentous Ascomycetes, including Chromocrea spinulosa (Mathieson, 1952), Fusarium subglufinans (Leslie ef al., 1986), Glomerella cingulafa (Wheeler, 1950) and Sclerotinia trifoliorum (Uhm & Fujii, 1983). Turgeon, Christiansen & Yoder (1993), however, observed that the phenomenon is uncommon and has not been found in any of the well-studied species of filamentous fungi. In these organisms, a unique copy of the mating-type genes is present in the genome so that homothallism through a switching mechanism as in yeasts is prevented. 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