Mating Type Ratio Research Articles

Indirect evidence for sexual reproduction in Cercospora beticola populations from sugar beet

Cercospora beticola is the main causal agent of cercospora leaf spot on sugar beet and has a large negative impact on the yield and quality of sugar beet production worldwide. Previous studies have shown that both mating type idiomorphs of C. beticola are present in natural populations, suggesting that C. beticola is heterothallic and may be reproducing sexually. Cercospora beticola isolates are diverse in the morphology of their conidia, onset of disease symptoms and fungicide resistance. To find the source of this diversity and to determine if sexual reproduction occurs in this fungus, C. beticola populations were collected from Western Europe, Iran and New Zealand. The mating types of these isolates were determined and AFLP analyses were used to study the genetic diversity in these populations. The mating type ratios did not deviate significantly from a 1:1 ratio in most of the populations and AFLP analyses showed high levels of genetic variation within and between the populations, with 86·4% of the isolates having unique genotypes. All populations were in significant linkage disequilibrium but levels of disequilibrium were low, and loci from only one primer pair were in significant gametic equilibrium in populations from the Netherlands and Italy. From these results there is the possibility that C. beticola reproduces sexually. High levels of gene flow among the samples from Europe demonstrated a single panmictic European population. This study confirms C. beticola to be a genetically highly diverse species, supporting the assumption that some populations are reproducing sexually.

Distribution of Mating Types and Genetic Diversity of Ascochyta rabiei Populations in Tunisia Revealed by Mating-type-specific PCR and Random Amplified Polymorphic DNA Markers

The distribution of mating types of Ascochyta rabiei (teleomorph: Didymella rabiei) was determined in Tunisia using a MAT-specific PCR assay. Among 123 isolates tested, 80% were MAT1-1 and 20%MAT1-2. Only MAT1-1 isolates were present in the Beja and Bizerte regions of Tunisia, whereas both mating types were present in Nabeul, Kef and Jendouba. In the latter three regions, the hypothesis of random mating could not be rejected based on chi-squared tests of mating-type ratios (P > 0.05). The lower frequency of the MAT1-2 coupled with the restricted distribution of this mating type in Tunisia may indicate a recent introduction of MAT1-2 in Tunisia. This speculation is consistent with the recent (2001) observation of D. rabiei pseudothecia on chickpea debris in Tunisia. Forty isolates representative of the five regions were genetically analysed using 10 random amplified polymorphic DNA (RAPD) primers to provide a preliminary estimate of genetic diversity of the pathogen in Tunisia. Among 129 putative RAPD loci amplified, 81% were polymorphic and 32 unique RAPD fingerprints were detected. A high level of genetic differentiation was detected among subpopulations (GST = 0.33). Cluster analyses revealed that isolates from Bizerte, Beja and Jendouba were genetically similar and distinct from isolates sampled in Nabeul and Kef. MAT1-1 isolates were clustered separately from MAT1-2 isolates in Jendouba and Nabeul suggesting that recombination may not yet be occurring in these regions despite the occurrence of both mating types in equal frequency in these regions. This lack of recombination between MAT1-1 and MAT1-2 also supports the hypothesis of a recent introduction of MAT1-2 into Tunisia.

First Record of the Perfect Stage Calonectria pauciramosa on Mastic Tree in Italy.

Leaf spots, stem lesions, crown rot, and root rot, caused by heterothallic fungus Cylindrocladium pauciramosum C.L. Schoch & Crous,, on young plants in a nursery represent an important problem in many representative ornamental plant-growing areas of Italy since the disease was first reported in 1993 (2). Symptoms observed during surveys in Italy from 1993 to 2005 were attributed solely to the asexual stage, and the teleomorph (Calonectria pauciramosa C.L. Schoch & Crous) was never found. In October 2005, orange-to-red brown fungal fruiting bodies pertaining to the genus Calonectria were observed in belowground regions and at the soil line on collar and stem tissues of seedlings of the mastic tree (Pistacia lentiscus L., Anacardiaceae) affected by damping off in two nurseries of eastern Sicily. The sexual structures were solitary or in groups, orange-to-red brown, subglobose to ovoid. On the basis of 50 microscopic observations of leaf tissues, they ranged from 180 to 280 μm in diameter and from 270 to 400 μm high. In the presence of adequate moisture, ascospores were discharged from mature perithecia in visible, viscous, and white droplets. Asci containing eight ascospores were clavate and tapering to a long thin stalk. Fusoid ascospores were hyaline and guttulate with rounded ends, slightly curved, 1-septate, not or slightly constricted at the septum, and measured 33 to 39 μm long and 6 to 8 μm wide. However, measurements show that perithecia, asci, and ascospores also fall within the range described by Schoch et al. (3). Single ascospore cultures did not produce perithecia. On the basis of these descriptions and the ability of single-ascospore cultures to mate with two Italian (DISTEF-G87 and DISTEF-G128) and two South African (STE-U 971 and STE-U 1670) selected tester strains of Cylindrocladium pauciramosum (2), the perithecia were identified as C. pauciramosa. To our knowledge, this is the first natural occurrence of the perfect stage of Cylindrocladium pauciramosum in Italy. The presence of the teleomorph is very important because it represents a second means of spread after conidia. Furthermore, the occurrence of the teleomorph also could lead to a 1:1 mating type ratio. In this case, lower percentages of hermaphrodites should be expected (1,4).

Maintenance of Sex‐Linked Deleterious Alleles by Selfing and Group Selection in Metapopulations of the Phytopathogenic FungusMicrobotryum violaceum

Microbotryum violaceum is a fungus that causes the sterilizing anther smut disease in many Caryophyllaceae. Its diploid teliospores are heterozygous at the mating-type locus, normally producing equal proportions of haploid sporidia of the two mating types. However, natural populations contain high frequencies of individuals producing sporidia of only one mating type. This mating-type ratio bias is caused by the presence of deleterious alleles at haploid phase ("haplo-lethals") linked to the mating-type locus. These haplo-lethals can be transmitted if there is conjugation among the products of meiosis (intratetrad selfing). Haplo-lethals still suffer from selective disadvantages, through reducing the infection probability of strains that carry them, and thus cannot persist in a panmictic population. We develop a realistic model of a metapopulation of M. violaceum on its host Silene latifolia. Simulations show that if intratetrad selfing rate is high, haplo-lethals can be maintained under a metapopulation structure because of founder effects and selection at the population level. Populations founded only by strains carrying haplo-lethals experience a lower extinction rate precisely because of their lower infection ability; they spread more slowly and sterilize fewer plants, thereby allowing their host population to grow more rapidly and therefore to be less prone to extinction.

First Report of Solanum physalifolium as a Host Plant for Phytophthora infestans in Sweden.

In the early summer of 2003, lesions resembling those caused by Phytophthora infestans (Mont.) de Bary on potato were observed on Solanum physalifolium Rusby var. nitidibaccatum (Bitter) Edmonds (2) that was growing as a weed in a parsnip (Pastinaca sativa) field in southern Sweden. When infected leaves of S. physalifolium were observed under the microscope (×200 magnification), sporangia with the same shape and size as those of P. infestans were observed. Pieces of infected leaves of S. physalifolium were put under tuber slices of S. tuberosum (cv. Bintje) in petri dishes and kept at 20°C. After 4 days, mycelium grew through the slices and sporulated profusely. The sporangia on the slices were of the same shape and size as those observed on the infected S. physalifolium leaves. In Sweden, the ratio of A1 and A2 mating types of P. infestans is 50:50, and oospores are commonly found in infected potato crops (1), so isolates from S. physalifolium were tested for mating type by growing them together with reference isolates of a known mating type on agar plates. Nine of the tested isolates were A1 mating type and six were A2 mating type. One self-fertile isolate was found. Naturally infected leaves of S. physalifolium were incubated at 20°C at 100% relative humidity so the lesions could coalesce and to facilitate oospore formation. After 5 days, oospores identical to those of P. infestans were observed under the microscope (×200 magnification). Sporangia produced by isolates originating from S. physalifolium and S. tuberosum were harvested, and a suspension containing 104 sporangia per ml from each isolate was prepared. Five leaves each of S. nigrum, S. physalifolium, and S. tuberosum (cv. Bintje), were inoculated with 10 μl of each sporangial suspension. Inoculated leaves were incubated in sealed petri dishes at 15°C. After 4 days, all S. tuberosum leaves were infected. After 7 days, two of five leaves of S. physalifolium inoculated with the S. tuberosum isolate and two of five S. physalifolium leaves inoculated with the isolate from S. physalifolium were infected. All lesions produced sporangia similar to those formed by P. infestans. S. nigrum was not infected by any of the isolates. The ability of S. physalifolium to act as a host plant for P. infestans producing sporangia during the growing season and oospores for survival between growing seasons may increase the problems of controlling late blight in potato in Sweden.

Cloning and characterization of the mating type ( MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp.

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the MAT1-2 gene of Cochliobolus heterostrophus, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MAT1-2 sequence in both directions, then primers designed to MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomorph. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to MAT loci of other loculoascomycete fungi. MAT genes were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of MAT1-1 by MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A. rabiei on chickpea is phylogenetically distant from other legume-associated Ascochyta spp. and the specific status of A. rabiei, A. lentis, A. pisi, and A. fabae was confirmed by the HMG phylogeny

Female Fertility and Single Nucleotide Polymorphism Comparisons in Cylindrocladium pauciramosum.

Cylindrocladium pauciramosum is well established in South America, and has recently been collected from nurseries in South Africa, Italy, and the United States. Isolates were compared with respect to the percentages of hermaphrodites and the respective mating types in the different samples. Based on these data, the effective population size could be determined for the different areas studied. All nurseries had mating type ratios significantly different from an idealized 1:1 ratio. In the South African nursery, the MAT-1 mating type was dominant, while the MAT-2 mating type dominated in other samplings. This is consistent with an introduction of a small starter population. High percentages of hermaphrodites also agreed with recent introductions into nurseries in Italy and the United States. Variability of DNA sequences of the 5' end of the β-tubulin gene from a set of C. pauciramosum isolates from different geographic regions was low to high. Isolates from South Africa, the United States, and Australia had identical β-tubulin DNA sequences; this sequence was also found in the Italian sample, along with another unique group. Finally, a group of isolates obtained from South and Central America had the highest variation of all isolates investigated, and also included isolates that shared single nucleotide variations with another species, C. candelabrum. These findings suggest that C. pauciramosum most likely has a Central or South American center of origin.

Sporidial Mating-Type Ratios of Teliospores from Natural Populations of the Anther Smut Fungus Microbotryum (= Ustilago) violaceum

In the fungal plant pathogen Microbotryum violaceum, infection is achieved by the dikaryotic conjugation product between two haploid sporidia differing in mating type. Each diploid teliospore, the transmission unit, is heterozygous for the mating-type locus; therefore, germination and meiosis should produce haploid sporidia of both mating types. We investigated the mating-type ratios generated by growing sporidial colonies from single teliospores collected from six populations in two regions of northern Switzerland. One-third of the teliospores produced strongly or totally biased mating-type ratios. Usually, teliospores from a single fungal collection showed bias in both directions; however, in one population a consistent bias in one direction was found. Either more rapid replication of one mating type, perhaps on account of deficiency mutations, or a head start in replication can lead to the loss of one of the mating types after several cell cycles on laboratory medium. An experiment on replication rates of sporidia with the two different mating types confirmed that differential replication could lead to mating-type bias, although the observed difference in replication rates did not correlate with mating-type biases recorded from the teliospore from which these sporidial colonies originated. Teliospore collections from which we obtained only totally biased teliospore colonies in our survey formed conjugation hyphae when suspended on leaf tissue. Therefore, we suggest that, early after germination, meiotic products of both mating types must be present, and early self-conjugation is possible. A comparison of mating-type bias from colonies isolated 4 and 24 d after teliospore germination showed a significant change in the degree of bias, indicating that bias develops over time. Mating-type bias may influence the breeding system and the probability of self compared with outcross conjugations. However, infection initiation must take place quickly, before the flowers where spores arrive fall off the plant. Therefore, processes occurring early after spore deposition may be far more important for disease production than those that develop over several cycles of replication of the haploid sporidia.

Genetic Analysis of the Reproductive System of the True Slime MoldComatricha Lurida

An isolate of Comatricha lurida was analyzed genetically to determine the nature of its reproductive cycle. Ofthe 75 myxamoebal cultures isolated from a single mass-spore agarcultured sporophore, 33 formed plasmodia. Forty-two parental myxamoebal clones failed to produce plasmodia in serial subculture. However, plasmodia were produced when the clones were crossed in appropriate pairwise combinations, indicating this isolate possessed two mating alleles, A1 and A2, at one locus. The analysis of the Ft generation clones obtained from two parental crosses (A!l x A24, A40 x A219) showed them to be predominantly of the A1 mating-type. Further, F! generation analysis of one additional parental cross (A*6 x A22) produced similar results; however, nearly all of the F{ clones were of mating-type A2. The theoretical implications of these skewed F! mating-type ratios are discussed. A prelim? inary analysis ofthe F2 generation was begun by crossing 10 A1 Ft clones from crosses A11 x A24, A*10 x A219 and 5 A2 F, clones from cross A'6 x A22 in all possible pairwise com? binations. Plasmodia were produced only when clones of different mating-type were mixed, providing evidence that this isolate of C. lurida has a stable heterothallic life history.

Genetic Analysis of the Reproductive System of the True Slime Mold Comatricha lurida

An isolate of Comatricha lurida was analyzed genetically to determine the nature of its reproductive cycle. Of the 75 myxamoebal cultures isolated from a single mass-spore agarcultured sporophore, 33 formed Plasmodia. Forty-two parental myxamoebal clones failed to produce Plasmodia in serial subculture. However, Plasmodia were produced when the clones were crossed in appropriate pairwise combinations, indicating this isolate possessed two mating alleles, A1 and A2, at one locus. The analysis of the F1 generation clones obtained from two parental crosses (A11 × A24, A110 × A219) showed them to be predominantly of the A1 mating-type. Further, F1 generation analysis of one additional parental cross (A16 × A22) produced similar results; however, nearly all of the F1 clones were of mating-type A2. The theoretical implications of these skewed F1 mating-type ratios are discussed. A preliminary analysis of the F2 generation was begun by crossing 10 A1 F1 clones from crosses A11 × A24, A110 × A219 and 5 A2 F1 clones from cross A16 × A22 in all possible pairwise combinations. Plasmodia were produced only when clones of different mating-type were mixed, providing evidence that this isolate of C. lurida has a stable heterothallic life history.

Distribution of mating types among clinical isolates of the Microsporum gypseum complex.

Forty-five clinical isolates of Microsporum gypseum were crossed with the + and - tester strains of Nannizzia gypsea, N. incurvata and N. fulva to determine their mating type. Thirty-two produced fertile gymnothecia when crossed with N. gypsea, of which 13 reacted as + and 19 as -. Ten produced fertile gymnothecia when crossed with N. incurvata, of which 7 reacted as + and 3 as -. The remaining 3 were sterile in all crosses with N. gypsea. N. incurvata and N. fulva. Of these 3, 2 were fluffy, degenerated cultures of M. gypseum, and the other one appeared to bae identical with the description of M. gypseum var. vinosum in cultural and morphological characteristics. There was approximately a 1:1 overall ratio of mating types among a total of 90 clinical isolates of M. gypseum in Japan on the basis of the above results and the data recently reported in the literature by other Japanese workers. The present results suggest that the ability to cause skin infection of humans by the soil-inhabiting organisms is approximately equal in the mating types and probably independent of sexuality.

Mating types and reproductive isolation inClosterium ehrenbergii meneghini

Numerous clones ofClosterium ehrenbergii Meneghini were isolated from soil or liquid samples from various localities and clonal uni-algal or axenic cultures were established. Mating types of these clones were determined by crossing with tester clones. No homothallic clone has been encountered, even in such an extensively surveyed area as that in Japan. By some unknown factors, mating type ratios have been found to deviate largely from 1∶1 in some populations in liquid samples, but not in populations from soil samples. Representative clones from each locality were intercrossed with those from other localities in all possible combinations. Seven reproductively isolated groups were recognized on the basis of capability and frequency of zygospore formation. Cooccurrence of more than two groups in the same sample has been encountered in five cases out of 41 localities surveyed. In four cases the two groups found together have turned out to be completely isolated but in the last case two groups of incomplete isolation were found with the other group of complete isolation.

Mating Types and Convertibility among Soil Isolates of Histoplasma capsulatum

The complete life cycle of Histoplasma capsulatum Darling was revealed in 1972 when Kwon-Chung (1972a, b) discovered the sexual state, Emmonsiella capsulata, which has been recently transferred to the genus Ajellomyces by McGinnis and Katz (1979). The organism is heterothallic, having a (+) and (-) mating system (Kwon-Chung, 1973). In 1974 Kwon-Chung et al., reported a 1 (+): 1 (-) overall ratio of mating types among 939 soil isolates, and a 1 (+): 7 (-) ratio among 184 clinical isolates of H. capsulatum. They also reported that the (-) mating type converted to the yeast phase (tissue phase) more readily in vitro than the (+) type, and suggested that the higher conversion rate of the (-) strains might partly explain why the (-) type is more frequent among the clinical isolates. In this paper we report the results of our study of 338 soil isolates of H. capsulatum. The isolation procedures through mice and the origin of these isolates are reported elsewhere (Gaur and Lichtwardt, 1980). All isolates of H. capsulatum were paired with Dr. KwonChung's tester strains, T-4-2 (+) and T-3-1 (-) on yeast-extract-agar medium (Kwon-Chung, 1973) and incubated for 3 wk at room temperature. Production of cleistothecia was verified microscopically. Excluded from this study are 31 isolates which paired with both tester strains, possibly due to mixed cultures. In-vitro conversion from the filamentous phase to the yeast phase was done at 37 C on brain-heart-infusion agar (Difco) supplemented with 0.1% L-cystine and 1% glucose (BHI+). Up to five serial transfers of each isolate were made to fresh media every 4 to 5 da. Only those cultures which produced almost entirely unicellular growth were

The genetics of mating type in the suctorian Tokophrya lemnarum.

SUMMARYThe two clones produced by the two members of a conjugating pair are normally of the same mating type, about 50% of such synclones being mating type I, 50% type II. This ratio suggests (1) that mating type is determined by a pair of alleles at one locus or by a pair of mating type determining chromosomes, and (2) that one mating type is homozygous, the other heterozygous or hemizygous. Mating type ratios are unaffected by temperature or parental age. Exceptions are of three types: (1) mates that produce one clone of type I, the other of type II, (2) mates which fuse permanently and yield a clone pure for either mating I or mating type II, and (3) conjugation of three mates one of which is regularly non-viable, the two survivors producing either two clones of type I, two clones of type II or one of each type. The bases of these exceptional results remain obscure.

GENETIC EVIDENCE FOR ZYGOTIC MEIOSIS IN PHYTOPHTHORA CAPSICI

Variation of several traits among single‐oospore isolates of the heterothallic oomycete, Phytophthora capsici, was studied to determine genetically the place of meiosis in the life cycle. Low percentages of met– offspring were recovered from two crosses of met– X prototrophic isolates. Arg– cultures were also recovered from the crosses indicating the possible presence of a suppressed arg– gene in one of the parental cultures. Three arg– X met– crosses yielded all four of the possible classes of offspring. Thus meiosis was either zygotic or, if gametangial, the original parental cultures must have been heterozygous at both the arg and met loci. However, when these parental cultures were crossed, only prototrophic offspring were recovered. A ratio of 3 prototrophs: 1 met– was obtained from cross R in which two prototrophic offspring from a previous arg– X met– cross were mated. This indicated the presence of a suppressor of met– and may explain the low recovery of met– offspring from earlier crosses. From backcrosses of met– offspring from this cross to the A2 prototrophic parent, ratios close to the expected 1:1 were obtained. When the A1 prototrophic parent of cross R (K‐10) was crossed to an A2 prototrophic isolate, met– offspring were again recovered, which indicated that K‐10 carried a suppressed met deficiency. In two crosses of streptomycin‐resistant (Sr) X sensitive (Ss) isolates, ratio of 108 Ss: 14 Sr and 84 Ss:14 Sr were obtained. Although 1:1 ratios would have been expected if meiosis were zygotic, segregation of Ss and Sr in the F1 generation indicated that gametangial meiosis was unlikely. Segregation of the A1 and A2 mating types in all crosses also indicated that meiosis was zygotic in P. capsici. Possible control of mating type by a single pair of alleles, by plasmids, and by more complex mechanisms is discussed in relation to the mating type ratios obtained.

SEXUAL REPRODUCTION AND INTERCROSSING IN VOLVULINA STEINW(1).

Gametes of Volvulina steinii bear near-apical mating papillae. Zygospore germination yields a single biflagellate cell that develops into a colony xuhose asexual progeny are all of the same mating type. Backcrossing of clones of progeny indicated a 1 :1 ratio of mating types among the progeny. Of 20 clones from a number of localities, none was homothallic and 3 showed no matins: reaction. Mating reactions of clones crossed in all possible combinations indicated the presence of 2 sexually isolated groups of clones producing smooth-walled zygospores and 1 group that produced spiny-walled zygospores. In the latter group weak and nonreciprocal mating reactions occurred in some clone combinations. Failure of germination of spiny-walled zygospores from certain crosses suggests further subdivision into genetically isolated groups.