Self-sterile Strains Research Articles

Evidence of Biparental Mitochondrial Inheritance from Self-Fertile Crosses between Closely Related Species of Ceratocystis.

Hybridization is recognized as a notable driver of evolution and adaptation, which closely related species may exploit in the form of incomplete reproductive barriers. Three closely related species of Ceratocystis (i.e., C. fimbriata, C. manginecans and C. eucalypticola) have previously been shown to hybridize. In such studies, naturally occurring self-sterile strains were mated with an unusual laboratory-generated sterile isolate type, which could have impacted conclusions regarding the prevalence of hybridization and inheritance of mitochondria. In the current study, we investigated whether interspecific crosses between fertile isolates of these three species are possible and, if so, how mitochondria are inherited by the progeny. For this purpose, a PCR-RFLP method and a mitochondrial DNA-specific PCR technique were custom-made. These were applied in a novel approach of typing complete ascospore drops collected from the fruiting bodies in each cross to distinguish between self-fertilizations and potential hybridization. These markers showed hybridization between C. fimbriata and C. eucalypticola and between C. fimbriata and C. manginecans, while no hybridization was detected in the crosses involving C. manginecans and C. eucalypticola. In both sets of hybrid progeny, we detected biparental inheritance of mitochondria. This study was the first to successfully produce hybrids from a cross involving self-fertile isolates of Ceratocystis and also provided the first direct evidence of biparental mitochondrial inheritance in the Ceratocystidaceae. This work lays the foundation for further research focused on investigating the role of hybridization in the speciation of Ceratocystis species and if mitochondrial conflict could have influenced the process.

Direct repeat-mediated DNA deletion of the mating type MAT1-2 genes results in unidirectional mating type switching in Sclerotinia trifoliorum.

The necrotrophic fungal pathogen Sclerotinia trifoliorum exhibits ascospore dimorphism and unidirectional mating type switching - self-fertile strains derived from large ascospores produce both self-fertile (large-spores) and self-sterile (small-spores) offsprings in a 4:4 ratio. The present study, comparing DNA sequences at MAT locus of both self-fertile and self-sterile strains, found four mating type genes (MAT1-1-1, MAT1-1-5, MAT1-2-1 and MAT1-2-4) in the self-fertile strain. However, a 2891-bp region including the entire MAT1-2-1 and MAT1-2-4 genes had been completely deleted from the MAT locus in the self-sterile strain. Meanwhile, two copies of a 146-bp direct repeat motif flanking the deleted region were found in the self-fertile strain, but only one copy of this 146-bp motif (a part of the MAT1-1-1 gene) was present in the self-sterile strain. The two direct repeats were believed to be responsible for the deletion through homologous intra-molecular recombination in meiosis. Tetrad analyses showed that all small ascospore-derived strains lacked the missing DNA between the two direct repeats that was found in all large ascospore-derived strains. In addition, heterokaryons at the MAT locus were observed in field isolates as well as in laboratory derived isolates.

A new conifer-inhabiting species of Ceratocystis from Norway

A new species, Ceratocystis norvegica J. Reid & Hausner sp. nov., is described from Norway. Based on morphological criteria and analyses of rDNA internal transcribed spacer and small subunit rDNA sequences, strains collected from galleries of the bark beetle Ips typographus on Picea abies (L.) H. Karst, were shown to be distinct both from members of the Ceratocystis coerulescens complex and from other species described previously from conifers. Ceratocystis norvegica has the following defining characteristics: convergent ostiolar hyphae; a sharply defined temperature optimum at 20 °C; an apparent lack of a conidial state; and ascospores that on germination produce either self-fertile or self-sterile strains.

Identification of differentially expressed proteins in a mat1-2-deleted strain of Gibberella zeae, using a comparative proteomics analysis

Gibberella zeae is a self-fertile ascomycetous fungus that causes important diseases of cereal crops. A comprehensive understanding of sexual reproduction in G. zeae is needed for disease control. To identify fungal proteins involved in this process, we compared the protein profiles of a wild-type strain and its self-sterile strain deleted for MAT1-2, a master regulator of sexual reproduction in G. zeae. Using 2-DE and either MALDI-TOF or ESI-Q-TOF MS, we identified 13 protein spots that showed statistically significant differences in expression levels between the two strains; 11 were reduced and two were increased in abundance in the DeltaMAT1-2 strain. Six of the 13 proteins were similar to those related to cell wall structure and the others were orthologs of proteins involved in metabolism or environmental stress responses. We confirmed that all the genes of the proteins examined were down-regulated during the sexual development stage in the DeltaMAT1-2, DeltaMAT1-1, and other strains deleted for a MAP kinase or a G-protein gene. These data suggest that differences in the protein expression levels are mostly affected by down-regulation of the corresponding genes in the DeltaMAT1-2 strain. To date, this is the first proteomics approach successfully identifying proteins differentially regulated by MAT1-2 in G. zeae.

Genetic analysis of cross fertility between two self-sterile strains of Glomerella graminicola

The ascomycete Glomerella cingulata has an unusual and complex mating system which is controlled by multiple, multiallelic loci. Cross fertility between different isolates occurs via complementation of mutated fertility genes, a process known as unbalanced heterothallism. We have examined the heritability of cross fertility among progeny of crosses between two self-sterile strains of G. graminicola to determine if unbalanced heterothallism also occurs in this species. Both random spore and tetrad progeny were analyzed. One-half of the progeny mated with one parent, one-fourth mated with the second parent, and one-fourth did not mate with either parent. None mated with both parents, and none were self fertile. The data support a model in which cross fertility between strains is regulated by two unlinked loci. One locus may be a component of the pathway for self fertility. The other locus appears to be involved in self/nonself recognition, because alternate functional alleles must be present in the parents. However, this second locus is unlikely to be a typical ascomycete MAT idiomorph, since the HMG box of a putative MAT2 idiomorph from G. graminicola was found in both parent strains at a locus which is not linked to the self/nonself recognition locus identified in this study. The data reported here suggest that cross fertility in G. graminicola is genetically complex, and that it functions at least in part via unbalanced heterothallism.

Genetic Analysis of Cross Fertility between Two Self-Sterile Strains of Glomerella graminicola

The ascomycete Glomerella cingulata has an unusual and complex mating system which is con- trolled by multiple, multiallelic loci. Cross fertility be- tween different isolates occurs via complementation of mutated fertility genes, a process known as unbal- anced heterothallism. We have examined the herita-

Sexuality of self-sterile strains of Glomerella graminicola

The mating system in the ascomycete Glomerella graminicola is unlike that in heterothallic ascomycetes where mating is controlled by a single locus with two alternate specificities. G. graminicola has multiple mating types that are determined by more than one genetic locus. Crosses between a melanin-deficient (Mel-) strain and a wild type (Mel+) strain of G. graminicola produced both darkly pigmented and lightly pigmented perithecia. In heterothallic ascomycetes, it is the ascogonial parent that produces the perithecial wall and the cytoplasm of the ascospores. Given the unusual nature of the mating system in G. graminicola, it was important to test the hypothesis that perithecium color is an indicator of parentage in matings of this fungus. Although Mel+ and Mel- progeny were recovered from both types of perithecia, mitochondrial DNA RFLPs were correlated with the color of the perithecium from which the progeny were derived. Crosses involving the Mel- strain and a second Mel+ strain produced Mel+ and Mel- progeny, but only one type of mitochondrial DNA which was like that of the Mel- parent, indicating that this Mel+ strain was female sterile. The putative female sterile strain, which displays normal sensitivity to potassium chlorate (Chls), was further examined by crossing it with a Mel+, female fertile, chlorate resistant (ChlR) strain. Among the progeny, the ChlR and Chls phenotypes segregated 1:1 but only the type of mitochondrial DNA found in the ChlR parent was detected. These results indicate that sexual differentiation and cytoplasmic inheritance in matings of G. graminicola are like those processes in heterothallic fungi, in spite of the fundamental differences between the genetic mechanisms that regulate mating specificity in these two groups.

Sexuality of Self-Sterile Strains of Glomerella graminicola

Sexuality of Self-Sterile Strains of Glomerella graminicola

Mating system of the filamentous ascomycete, Glomerella cingulata.

Mating in heterothallic filamentous ascomycetes is typically controlled by a single mating-type locus with two alternate alleles or idiomorphs. In this study, five self-sterile strains of Glomerella cingulata from pecan were crossed in all possible combinations. Four of the five strains could be placed into two mating-type groups, but the fifth strain was sexually compatible with all of the other strains. Single ascospore progeny were isolated from each of the successful crosses, tested for self-fertility, and backcrossed with both parents. In addition, subsets of F1 isolates were crossed with all five of the original strains from pecan and in all possible combinations with each other. Results from the crosses showed that the ascospore progeny had stably inherited the mating pattern of one of the parental strains and that the mating type had segregated 1:1 among the F1 isolates. Furthermore, the five strains from pecan were sexually compatible with five additional heterothallic strains in all but one combination. Data from these experiments are consistent with a mating system composed of a single mating-type locus with multiple alternate alleles. We believe that this is the first report of this type of mating system for an ascomycete species.

Pseudohomothallism and evolution of the mating-type chromosome in Neurospora tetrasperma.

Ascospores of Neurospora tetrasperma normally contain nuclei of both mating-type idiomorphs (a and A), resulting in self-fertile heterokaryons (a type of sexual reproduction termed pseudohomothallism). Occasional homokaryotic self-sterile strains (either a or A) behave as heterothallics and, in principle, provide N. tetrasperma with a means for facultative outcrossing. This study was conceived as an investigation of the population biology of N. tetrasperma to assess levels of intrastrain heterokaryosis (heterozygosity). The unexpected result was that the mating-type chromosome and autosomes exhibited very different patterns of evolution, apparently because of suppressed recombination between mating-type chromosomes. Analysis of sequences on the mating-type chromosomes of wild-collected self-fertile strains revealed high levels of genetic variability between sibling A and a nuclei. In contrast, sequences on autosomes of sibling A and a nuclei exhibited nearly complete homogeneity. Conservation of distinct haplotype combinations on A and a mating-type chromosomes in strains from diverse locations further suggested an absence of recombination over substantial periods of evolutionary time. The suppression of recombination on the N. tetrasperma mating-type chromosome, expected to ensure a high frequency of self fertility, presents an interesting parallel with, and possible model for studying aspects of, the evolution of mammalian sex chromosomes.

A Method for Genetic Analysis ofGlomerella graminicola(Colletotrichum graminicola) from Maize

Strains derived from nine different isolates of Colletotrichum graminicola from maize participated in the production of perithecia when incubated on pieces of autoclaved corn leaves in a humidity chamber. Matings occurred between self-fertile and self-sterile strains, and also between certain self-sterile strains. As many as 200 ascospore progeny were recovered easily from individual perithecia. Characterization of progeny showed that sexual recombination and Mendelian segregation of distinct traits could be detected. Segregation of markers for chlorate resistance (Chl R ), benomyl resistance (Bml R ), and melanin deficiency (Mel − ) approximated a 1:1 ratio and defined three separate linkage groups (...)

A genetic study of an extracellular elastin-hydrolysing protease in the ringworm fungus Arthroderma benhamiae.

Summary: Analysis of crosses between granular and downy self-sterile strains of Arthroderma benhamiae (= Trichophyton mentagrophytes var. granulosum) displaying high and low extracellular elastin-hydrolysing capabilities indicates that the locus for mating-type segregates independently of a colonial morphology locus and a locus determining elastin-hydrolysing capability. The colonial morphology locus consists of two alleles which govern the production of granular or downy phenotypes, and is closely linked to a locus consisting of two alleles which determine high or low extracellular elastolytic activity, showing a recombination frequency of approximately 5%.

The Origin of Self-Sterile, Cross-Fertile Strains and Culture Sterility in Ceratocystis Fimbriata

Although Ceratocystis fimbriata Ell. & Halst. is generally consid? ered to be homothallic (1, 2, 5, 8, 9), some reports have appeared concerning strains of this species that exhibit a form of heterothallic be? havior. Hansen and Snyder (6) reported, in an abstract, that C. fim? briata is a homothallic, hermaphroditic organism in which self-sterile male and female mutants occur. Olson (9), working with isolates from sweet potato, found that single ascospores obtained from self-fertile cultures gave rise to both the self-fertile parental strain and a selfsterile strain that was cross-fertile when mated with the parent. On the other hand, Andrus and Harter (1) reported that, large number of single ascospore isolates have failed to reveal any evidence of sexual strains,'' and that C. fimbriata is entirely homothallic. A similar conclusion was reached by Mittman (8). The present report confirms previous observations of self-sterile, cross-fertile strains in C. fimbriata and contains additional information regarding their origin, site of production and frequency of occurrence. The relationship of these observations to the maintenance of pure strains and fertile cultures is also considered.

A role for malic acid in tomato fruit-set

Studies with excised flowers of a self-sterile strain of John Baer tomatoes have demonstrated that: 1. 1. Malic acid, glutathione, and manganous sulfate each and collectively increase tomato fruit-set. 2. 2. Of all the compounds known to promote fruit-set, only malic acid will appreciably increase fruit-set in the presence of glutathione and manganous sulfate. These findings suggest that the promotion effect of the three compounds in combination may be due to the decarboxylation of malic acid by malic decarboxylase. 3. 3. The addition of two of the products of the malic decarboxylation reaction (carbon dioxide and pyruvic acid) each quantitatively inhibits fruit-set in the presence of malic acid, glutathione, and manganous sulfate. 4. 4. The addition of ascorbic acid with the three latter compounds further increases fruit-set. These promotive and inhibitory effects suggest that the decarboxylation of malic acid may be beneficial to fruit-set in the tomato. It is suggested that the beneficial effect of this system may be through the activation of a series of linked oxidation-reduction reactions.

PHYSIOLOGICAL FACTORS IN TOMATO FRUIT‐SET

THE CHANGE-OVER from the static condition of the flower ovary to the rapidly growing condition of a young fruit is a dramatic shift. This important phenomenon, commonly spoken of as fruit-set, is an essential step in the life cycle of all sexually reproducing higher plants. A major contribution to the understanding, of fruit-set was made by Gustafson (1936) who showed -that in some plants the simple application of auxins could brin, about fruit-set even without pollination. Subsequent work has demonstrated that fruit-set in general is at least in part a function of auxin in the ovary. Some genetic limitations to fruit-set?poken of as sterility and incompatability-are known (East, 1929), but the physiological basis for these limitations remains obscure. The requirement of moderately cool night temperatures for tomato fruit-set has been found by Went (1945). A decrease in fruit-set of tomatoes under low light invensiBies of winter has been noted by Hemphill and Murneek (1950). Other studies have indicated the existence of factors influencing fruit-set indirectly, such as flower abscission and pollen sterility (Howlett, 1936) associated with unbalanced carbohvdrate/nitrogen ratios in the plants. The present study was undertaken with the objective of identifying as far as possible the physiological factors involved in fruit-set of the tomato. METHODS.-The plants used in the present study were both fieldand greenhouse-grown, John Baer, self-sterile tomatoes. The stamens of the flowers of this self-sterile strain characteristically develop a brown rino of necrotic tissue at the anther tips which effectively prevents any pollen from reaching the stigma. By the use of these self-sterile tomato flowers complete control of fruit-set is made possible. In the experiments with excised flowers, the flower clusters were cut from the stems with a sharp blade and were immediately placed into separate vials of distilled water. They were then trimmed to 2-3 mature flowers per cluster and a fresh diagonal cut made at the base of the cluster. All stem material was cut away, leaving only the pedicels attached by approximately 0.5 cm. of peduncle. In order to force fruit-set, the flowers were sprayed individually with a 30 p.p.m. solution of PCA (parachlorophenoxyacetic acid). They were then placed in individual vials with 1 per cent agar media containing the materials to be tested. The flower cultures were maintained at a tempera-