Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum.

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.

Relatedness of Macrophomina phaseolina isolates from tallgrass prairie, maize, soybean and sorghum.

Agricultural and wild ecosystems may interact through shared pathogens such as Macrophomina phaseolina, a generalist clonal fungus with more than 284 plant hosts that is likely to become more important under climate change scenarios of increased heat and drought stress. To evaluate the degree of subdivision in populations of M. phaseolina in Kansas agriculture and wildlands, we compared 143 isolates from maize fields adjacent to tallgrass prairie, nearby sorghum fields, widely dispersed soybean fields and isolates from eight plant species in tallgrass prairie. Isolate growth phenotypes were evaluated on a medium containing chlorate. Genetic characteristics were analysed based on amplified fragment length polymorphisms and the sequence of the rDNA-internal transcribed spacer (ITS) region. The average genetic similarity was 58% among isolates in the tallgrass prairie, 71% in the maize fields, 75% in the sorghum fields and 80% in the dispersed soybean fields. The isolates were divided into four clusters: one containing most of the isolates from maize and soybean, two others containing isolates from wild plants and sorghum, and a fourth containing a single isolate recovered from Solidago canadensis in the tallgrass prairie. Most of the sorghum isolates had the dense phenotype on media containing chlorate, while those from other hosts had either feathery or restricted phenotypes. These results suggest that the tallgrass prairie supports a more diverse population of M. phaseolina per area than do any of the crop species. Subpopulations show incomplete specialization by host. These results also suggest that inoculum produced in agriculture may influence tallgrass prairie communities, and conversely that different pathogen subpopulations in tallgrass prairie can interact there to generate 'hybrids' with novel genetic profiles and pathogenic capabilities.

Population structure of Fusarium fujikuroi from California rice and water grass.

The recent observance of Fusarium fujikuroi, the causal agent of Bakanae disease of rice, in California provides a unique opportunity to assess the population diversity of an introduced pathogen in a new environment. We collected 172 isolates of this pathogen between 2000 and 2003 from California rice and two from water grass (Echinochloa spp.). Pathogenicity of F. fujikuroi was demonstrated on early water grass (E. oryzoides) and barnyard grass (E. crus-galli) indicating that weed control should be part of Bakanae management programs. Both mating types and six unique amplified fragment length polymorphism haplotypes corresponding to six identified vegetative compatibility groups were detected. The two most frequently isolated haplotypes encompassed 94% of the collected isolates, suggesting that clonal reproduction dominates. Coefficients of similarity between the unique haplotypes ranged from 0.94 to 0.98, and indicate that there is very little genotypic variation in the F. fujikuroi population in California. The near fixation of the MAT-1 idiomorph (observed ratio 170 MAT-1:4 MAT-2), is consistent with a hypothesis of predominant or exclusive asexual reproduction. The low level of introduced genotypic diversity, in conjunction with the asexual reproductive strategy of this population will slow evolutionary processes, including adaptation to the California environment.

Population genetic structure of Gibberella zeae isolated from wheat in Argentina.

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight of wheat. The authors used amplified fragment length polymorphisms (AFLPs) to characterize the genetic structure of two G. zeae populations from commercial wheat fields. The working hypothesis was that sufficient genetic exchange occurs between local populations to prevent significant partitioning of allelic variation. We analysed 216 AFLP loci for 113 isolates collected during the 2002 harvest season. All strains had AFLP profiles typical of G. zeae lineage 7. Both populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange probably occurring between the two subpopulations. Linkage disequilibrium was low, but higher than reported for many other populations of G. zeae, and about 20% of the alleles detected were specific to one of the two subpopulations - results consistent with limited gene exchange between the two subpopulations. This study extends previous work with populations of G. zeae to include those found in Argentina, one of the world's largest wheat growing countries.

A genetic map of Gibberella zeae (Fusarium graminearum).

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.

Genetic variation in Fusarium section Liseola from no-till maize in Argentina.

Strains of Fusarium species belonging to section Liseola cause stalk and ear rot of maize and produce important mycotoxins, such as fumonisins. We isolated two species, Fusarium verticillioides (Gibberella fujikuroi mating population A) and Fusarium proliferatum (G. fujikuroi mating population D) from maize cultivated under no-till conditions at five locations in the Córdoba province of Argentina. We determined the effective population number for mating population A (N(e)) and found that the N(e) for mating type was 89% of the count (total population) and that the N(e) for male or hermaphrodite status was 36%. Thus, the number of strains that can function as the female parent limits N(e), and sexual reproduction needs to occur only once every 54 to 220 asexual generations to maintain this level of sexual fertility. Our results indicate that the fungal populations isolated from no-till maize are similar to those recovered from maize managed with conventional tillage. We placed 36 strains from mating population A into 28 vegetative compatibility groups (VCGs). Of the 13 strains belonging to five multimember VCGs, only 2 isolates belonging to one VCG were clones based on amplified fragment length polymorphism (AFLP) fingerprints. Members of the other four multimember VCGs had an average similarity index of 0.89, and members of one VCG were no more closely related to other members of the same VCG than they were to other members of the population as a whole. This finding suggests that the common assumption that strains in the same VCG are either clonal or very closely related needs to be examined in more detail. The variability observed with AFLPs and VCGs suggests that sexual reproduction may occur more frequently than estimated by N(e).

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex.

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an approximately 800-bp fragment from MAT-2 or an approximately 200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.

Molecular standardization of mating type terminology in the Gibberella fujikuroi species complex.

Mating type in the Gibberella fujikuroi species complex is controlled by a single locus with two alleles and is usually identified following sexual crosses with standard, female-fertile tester isolates. The mating type alleles have been arbitrarily designated "+" and "-" within each biological species, and the nomenclature is tied to the standard tester strains. We developed a pair of PCR primers that can be used to amplify a unique fragment of one of the mating type alleles (MAT-2) from at least seven of the biological species in this species complex. Based on the amplification pattern, we propose a replacement for the existing, arbitrary +/- terminology that is presently in use. The new terminology is based on DNA sequence similarities between the mating type allele fragments from the biological species of the G. fujikuroi species complex and the corresponding fragments from other filamentous ascomycetes.

Sexual Recombination in Gibberella zeae.

ABSTRACT We developed a method for inducing sexual outcrosses in the homothallic Ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum). Strains were marked with different nitrate nonutilizing (nit) mutations, and vegetative compatibility groups served as additional markers in some crosses. Strains with complementary nit mutations were cocultured on carrot agar plates. Ascospores from individual perithecia were plated on a minimal medium (MM) containing nitrate as the sole nitrogen source. Crosses between different nit mutants segregated in expected ratios (3:1 nit(-):nit(+)) from heterozygous perithecia. Analysis of vegetative compatibility groups of progeny of two crosses indicated two and three vegetative incompatibility (vic) genes segregating, respectively. For rapid testing of sexual recombination between nit mutants, perithecia were inverted over MM to deposit actively discharged ascospores. Development of proto-trophic wild-type colonies was taken as evidence of sexual recombination. Strains of G. zeae group 2 from Japan, Nepal, and South Africa, and from Indiana, Kansas, and Ohio in the United States were sexually interfertile. Four group 1 strains were not interfertile among themselves or with seven group 2 strains. Attempts to cross G. zeae with representatives of F. acuminatum, F. avenaceum, F. culmorum, F. crookwellense, F. oxysporum, and three mating populations of G. fujikuroi were not successful.

Effects of the tol mutation on allelic interactions at het loci in Neurospora crassa.

A mutant at the tol locus of Neurospora crassa can suppress heterokaryon (vegetative) incompatibility associated with differences at the mating-type locus. We tested the ability of this allele to suppress the vegetative incompatibility reactions that can occur when strains differ at one of nine het loci (het-C, -D, -E, -5, -6, -7, -8, -9, and -10). We found no cases in which the tol mutant suppresses a heteroallelic het locus interaction. Thus, the interaction(s) that leads to vegetative incompatibility because of differences at the mating-type locus is distinct from the interaction(s) that leads to vegetative incompatibility because of differences at any of these nine het loci.

Production of fusaric acid by Fusarium species.

Fusaric acid is a mycotoxin with low to moderate toxicity, which is of concern since it might be synergistic with other cooccurring mycotoxins. Fusaric acid is widespread on corn and corn-based food and feeds and is frequently found in grain, where Fusarium spp. are also isolated. We surveyed 78 strains of Fusarium moniliforme, F. crookwellense, F. subglutinans, F. sambucinum, F. napiforme, F. heterosporum, F. oxysporum, F. solani, and F. proliferatum for their ability to produce fusaric acid. Strains in Fusarium section Liseola also were assigned to mating population of the Gibberella fujikuroi species complex. The fungi could be divided into three classes, low (< 100 micrograms/g), moderate (100 to 500 micrograms/g), and high (> 500 micrograms/g), based on the amounts of this mycotoxin produced in culture on autoclaved corn. Strains of mating populations C from rice consistently produced moderate to high concentrations of fusaric acid. Two isolates, one each from mating populations C and D, produced fusaric acid in excess of 1,000 micrograms/g of corn. No isolates of any of the Fusarium species examined were negative for the production of fusaric acid on autoclaved corn.

Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola).

Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven biological species. While emphasizing the discreteness of the mating populations as biological entities, our isozyme data also reaffirm the close genetic relationship among these groups.

Female fertility and mating type effects on effective population size and evolution in filamentous fungi.

The idealized individual in many fungal species is a haploid self-sterile hermaphrodite that may be propagated by asexually produced spores or that may reproduce sexually. In field populations, polymorphism occurs for female-sterile/hermaphrodite status, and female-sterile mutants, which function only as males during sexual reproduction, may comprise > 50% of the population. The effective population number may be based on the number of strains of different mating type or the relative frequency of hermaphrodites. The female-sterile mutants are at a selective disadvantage every time sexual reproduction occurs, and must have an advantage during vegetative propagation to persist at a significant frequency. When a high frequency of female-sterile strains is observed in field populations, it indicates that vegetative propagation is a significant component of the fungus' natural history. Depending on the mutation rate to female sterility and the selective advantage of the female-sterile strains during vegetative propagation, the ratio of sexual:asexual generations can range from 1:15 to 1:2300 for species in the Gibberella fujikuroi complex. The relative rarity of sexual reproduction may permit female-sterile strains to accumulate to a level such that local populations could completely lose sexuality and appear as asexual (imperfect) species.

A genetic map of Gibberella fujikuroi mating population A (Fusarium moniliforme).

We constructed a recombination-based map of the fungal plant pathogen Gibberella fujikuroi mating population A (asexual stage Fusarium moniliforme). The map is based on the segregation of 142 restriction fragment length polymorphism (RFLP) markers, two auxotrophic genes (arg1, nic1), mating type (matA+/matA-), female sterility (ste1), spore-killer (Sk), and a gene governing the production of the mycotoxin fumonisin B1 (fum1) among 121 random ascospore progeny from a single cross. We identified 12 linkage groups corresponding to the 12 chromosome-sized DNAs previously observed in contour-clamped homogeneous electric field (CHEF) gels. Linkage groups and chromosomes were correlated via Southern blots between appropriate RFLP markers and the CHEF gels. Eleven of the 12 chromosomes are meiotically stable, but the 12th (and smallest) is subject to deletions in 3% (4/121) of the progeny. Positive chiasma interference occurred on five of the 12 chromosomes, and nine of the 12 chromosomes averaged more than one crossover per chromosome. The average kb/cM ratio in this cross is approximately 32.

Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and E mating populations of Gibberella fujikuroi (Fusarium moniliforme).

Two biological species of Gibberella fujikuroi (A and F mating populations) share the Fusarium moniliforme anamorph. Twenty strains of each of these biological species were tested for the ability to produce fumonisins B1, B2, and B3 and moniliformin and for toxicity to 1-day-old ducklings. Most of the members of the A mating population (19 of 20 strains) produced more than 60 micrograms of total fumonisins per g, whereas only 3 of 20 members of the F mating population produced more than trace levels of these toxins and none produced more than 40 micrograms of total fumonisins per g. In addition, only 3 of 20 members of the A mating population produced more than 1 microgram of moniliformin per g (and none produced more than 175 micrograms/g), while all 20 strains of the F mating population produced more than 85 micrograms of this toxin per g and 1 strain produced 10,345 micrograms/g. The duckling toxicity profiles of the strains of the two mating populations were similar, however, and the level of either toxin by itself was not strongly correlated with duckling toxicity. On the basis of our data we think that it is likely that the members of both of these mating populations produce additional toxins that have yet to be chemically identified. These toxins may act singly or synergistically with other compounds to induce the observed duckling toxicity.

Introductory biology of Fusarium moniliforme.

Fusarium moniliforme is a name that has been applied to any of six biological species (or mating populations) that share the teleomorph (sexual stage) Gibberella fujikuroi. Two of these six biological species, termed "A" and "D", are known to produce fumonisin mycotoxins. Strains from the "A" biological species grow as endophytes on maize and often comprise 90+% of the Fusarium isolates recovered from healthy maize seed. It is possible to distinguish all six biological species using sexual fertility and isozymes. Other attributes, such as morphological characters and sequences from the ribosomal DNA internally transcribed spacer (rDNA-ITS) region, can be used to identify some, but not all, of the biological species. Within a biological species, genetic variability and population structure can be assessed with anonymous RFLPs and tests of vegetative compatibility. The "A" biological species is genetically diverse, and the sexual cycle appears to be important in the life cycle of field populations of this organism in the United States.

Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea mays) seedlings.

The phytopathogenic fungus Gibberella fujikuroi mating population A (anamorph, Fusarium moniliforme) produces fumonisins, which are toxic to a wide range of plant and animal species. Previous studies of field strains have identified a genetic locus, designated fum1, that can determine whether fumonisins are produced. To test the relationship between fumonisin production and virulence on maize seedlings, a cross between a fum1+ field strain that had a high degree of virulence and a fum1- field strain that had a low degree of virulence was made, and ascospore progeny were scored for these traits. Although a range of virulence levels was recovered among the progeny, high levels of virulence were associated with production of fumonisins, and highly virulent, fumonisin-nonproducing progeny were not obtained. A survey of field strains did identify a rare fumonisin-nonproducing strain that was quite high in virulence. Also, the addition of purified fumonisin B1 to virulence assays did not replicate all of the seedling blight symptoms obtained with autoclaved culture material containing fumonisin. These results support the hypothesis that fumonisin plays a role in virulence but also indicate that fumonisin production is not necessary or sufficient for virulence on maize seedlings.