Fusarium mirum sp. nov, intertwining Fusarium madaense and Fusarium andiyazi, pathogens of tropical grasses.

Many species in the Fusarium fujikuroi Species Complex (FFSC) have an affinity for grass species, with whom they live in an endophytic association or cause disease. We recovered isolates of Fusarium from agriculturally important grasses in Africa and Brazil, and characterized them with morphological markers, mating type, and Amplified Fragment Length Polymorphisms (AFLPs). We also conducted multi-locus phylogenetic analyses based on partial DNA sequences of translation elongation factor-1α (TEF1), ß-tubulin (TUB), and the second largest subunit of RNA polymerase (RPB2) gene regions. Sexual cross fertility was used to test the biological species concept and the sexual stage of F. madaense is described. A novel species within the FFSC, Fusarium mirum, that is different from the other known species in the complex, was formally described. Fusarium mirum, F. madaense, and Fusarium andiyazi are a tightly intertwined species trio that are morphologically identical, but phylogenetically distinguishable, and amongst whom interspecific genetic exchange may still occur. These three species are so close that they cannot be reliably distinguished if only sequences of the TEF1 gene are used. In pathogenicity tests, all tested isolates of F. madaense from sugarcane, sorghum, maize, millet and Brachiaria could induce stalk rot in sorghum, maize and millet, and pokkah boeng in sugarcane. This study increases our understanding of the diversity of species within the FFSC that cause disease in tropical grasses or act as endophytes, and their geographic distributions. The genetically close relationship between F. mirum, F. madaense, and F. andiyazi provides an opportunity to study and identify factors underlying their limited inter-specific cross-fertility and sympatric speciation.

Genome Resources of Four Distinct Pathogenic Races Within Fusarium oxysporum f. sp. vasinfectum that Cause Vascular Wilt Disease of Cotton.

Whole genome sequence (WGS) based identifications are being increasingly used by regulatory and public health agencies to facilitate the detection, investigation, and control of pathogens and pests. Fusarium oxysporum f. sp. vasinfectum is a significant vascular wilt pathogen of cultivated cotton and consists of several pathogenic races that are not each other's closest phylogenetic relatives. We have developed WGS assemblies for isolates of F. oxysporum f. sp. vasinfectum race 1 (FOV1), race 4 (FOV4), race 5 (FOV5), and race 8 (FOV8) using a combination of Nanopore (MinION) and Illumina sequencing technology (Mi-Seq). This resulted in assembled contigs with more than 100× coverage for each of the F. oxysporum f. sp. vasinfectum races and estimated genome sizes of FOV1 52 Mb, FOV4 68 Mb, FOV5 68 Mb, and FOV8 55 Mb. The AUGUSTUS gene prediction program predicted 16,263 genes in FOV1, 20,259 genes in FOV4, 20,375 genes in FOV5 and 16,615 genes in FOV8. We were able to identify 525 genes unique to FOV1, 570 unique to FOV4, 1,242 unique to FOV5, and 383 unique to FOV8. We expect that these findings will help in comparative genomics and in the identification of unique genes as candidate targets for diagnostic marker and methods development to permit rapid differentiation of F. oxysporum f. sp. vasinfectum subgroups.

Draft Genome Resource for the Ex-types of Phytophthora ramorum, P. kernoviae, and P. melonis, Species of Regulatory Concern, Using Ultra-Long Read MinION Nanopore Sequencing.

Phytophthora ramorum, P. kernoviae, and P. melonis are each species of current regulatory concern in the United States, the United Kingdom, and other areas of the world. Ex-type material are cultures and duplicates of the type that was used to describe each species and that are deposited in additional culture collections. Using these type specimens as references is essential to designing correct molecular identification and diagnostic systems. Here, we report a whole genome sequence for the Ex-type material of P. ramorum, P. kernoviae, and P. melonis generated using high-throughput sequencing via the MinION third generation platform from Oxford Nanopore Technology. We assembled the quality filtered reads into contigs for each species. We assembled the continuous contigs of P. ramorum, P. kernoviae, and P. melonis (1,322, 545, and 2,091 contigs, respectively). The ab initio prediction of genes from these species reveals that there are 16,838, 12,793, and 34,580 genes in P. ramorum, P. kernoviae, and P. melonis, respectively. Of the 34,580 P. melonis genes, 10,164 genes were conserved among all three of these Phytophthora species which may include pathogenicity genes. We compared the ex-type of P. ramorum EU1 lineage assembly with another selected isolate of EU1 available at the National Center for Biotechnology Information and found 251,859 single nucleotide polymorphisms (SNPs) genome-wide; the comparison with the EU2 lineage genome isolate revealed 441,859 SNPs genome-wide. This genome resource of the ex-types of P. ramorum, and P. kernoviae is a significant contribution as these species are among the most important pathogens of regulatory concern in different regions of the world.

Draft Genome Resources for the Phytopathogenic Fungi Monilinia fructicola, M. fructigena, M. polystroma, and M. laxa, the Causal Agents of Brown Rot.

Fungi in the genus Monilinia cause brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: M. fructicola, M. fructigena, M. polystroma, and M. laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.

Population structure of and mycotoxin production by Fusarium graminearum from maize in South Korea.

Fusarium graminearum (Gibberella zeae) is an important pathogen of wheat, maize, barley, and rice in South Korea, and harvested grain often is contaminated with trichothecenes such as deoxynivalenol and nivalenol. In this study, we examined 568 isolates of F. graminearum collected from maize at eight locations in South Korea. We used amplified fragment length polymorphisms (AFLPs) to identify four lineages (2, 3, 6, and 7); lineage 7 was the most common (75%), followed by lineage 6 (12%), lineage 3 (12%), and lineage 2 (1%). The genetic identity among populations was high (>0.98), and the effective migration rate between locations was higher than that between lineages. Female fertility varied by lineage: all lineage 7 isolates were fertile, while 70%, 26%, and 14% of the isolates in lineages 6, 3, and 2, respectively, were fertile. All lineage 3 and lineage 7 isolates produced deoxynivalenol, whereas most lineage 2 and 6 isolates produced nivalenol. Genotypic diversity in lineage 3 and lineage 6 populations is similar to that found in previously described Korean rice populations, but genotypic diversity in lineage 7 is much lower, even though similar levels of gene flow occur between lineage 7 populations. We conclude that lineage 7 was relatively recently introduced into South Korea, perhaps accompanying imported maize seeds.

Genetic Structure of Atmospheric Populations of Gibberella zeae.

ABSTRACT Gibberella zeae, causal agent of Fusarium head blight (FHB) of wheat and barley and Gibberella ear rot (GER) of corn, may be transported over long distances in the atmosphere. Epidemics of FHB and GER may be initiated by regional atmospheric sources of inoculum of G. zeae; however, little is known about the origin of inoculum for these epidemics. We tested the hypothesis that atmospheric populations of G. zeae are genetically diverse by determining the genetic structure of New York atmospheric populations (NYAPs) of G. zeae, and comparing them with populations of G. zeae collected from seven different states in the northern United States. Viable, airborne spores of G. zeae were collected in rotational (lacking any apparent within-field inoculum sources of G. zeae) wheat and corn fields in Aurora, NY in May through August over 3 years (2002 to 2004). We evaluated 23 amplified fragment length polymorphism (AFLP) loci in 780 isolates of G. zeae. Normalized genotypic diversity was high (ranging from 0.91 to 1.0) in NYAPs of G. zeae, and nearly all of the isolates in each of the populations represented unique AFLP haplotypes. Pairwise calculations of Nei's unbiased genetic identity were uniformly high (>0.99) for all of the possible NYAP comparisons. Although the NYAPs were genotypically diverse, they were genetically similar and potentially part of a large, interbreeding population of G. zeae in North America. Estimates of the fixation index (G(ST)) and the effective migration rate (Nm) for the NYAPs indicated significant genetic exchange among populations. Relatively low levels of linkage disequilibrium in the NYAPs suggest that outcrossing is common and that the populations are not a result of a recent bottleneck or invasion. When NYAPs were compared with those collected across the United States, the observed genetic identities between the populations ranged from 0.92 to 0.99. However, there was a significant negative correlation (R = -0.59, P < 0.001) between genetic identity and geographic distance, suggesting that some genetic isolation may occur on a continental scale. The contribution of long-distance transport of G. zeae to regional epidemics of FHB and GER remains unclear, but the diverse atmospheric populations of G. zeae suggest that inoculum may originate from multiple locations over large geographic distances. Practically, the long-distance transport of G. zeae suggests that management of inoculum sources on a local scale, unless performed over extensive production areas, will not be completely effective for the management of FHB and GER.

Toxicity, pathogenicity, and genetic differentiation of five species of fusarium from sorghum and millet.

ABSTRACT Fusarium isolates recovered from sorghum and millet are commonly identified as F. moniliforme, but with the recognition of new species in this group, the strains given this name are being re-evaluated. We analyzed five strains each from five Fusarium species (F. andiyazi, F. nygamai, F. pseudonygamai, F. thapsinum, and F. verticillioides) often associated with sorghum and millet for their ability to produce fumonisin and moniliformin, their toxicity to ducklings, and their ability to cause disease on sorghum seedlings in vitro. These species can be distinguished with isozymes (fumarase, NADP-dependent isocitrate dehydrogenase, and malate dehydrogenase) and with banding patterns resulting from amplified fragment length polymorphisms. Two species, F. pseudonygamai and F. thapsinum, produced high levels of moniliformin, but little or no fumonisins, and were consistently highly toxigenic in the duckling tests. Two species, F. verticillioides and F. nygamai, produced high levels of fumonisins, but little or no moniliformin, and also were toxigenic in the duckling tests. F. andiyazi produced little or no toxin and was the least toxigenic in the duckling tests. In sorghum seedling pathogenicity tests, F. thapsinum was the most virulent followed by F. andiyazi, then F. verticillioides, and finally F. nygamai and F. pseudonygamai, which were similar to each other. Thus, these five species, which would once have all been called F. moniliforme, differ sufficiently in terms of plant pathogenicity and toxin production profile, that their previous misidentification could explain inconsistencies in the literature and differences observed by researchers who thought they were all working with the same fungal species.

Species diversity of and toxin production by Gibberella fujikuroi species complex strains isolated from native prairie grasses in Kansas.

Fusarium species from agricultural crops have been well studied with respect to toxin production and genetic diversity, while similar studies of communities from nonagricultural plants are much more limited. We examined 72 Fusarium isolates from a native North American tallgrass prairie and found that Gibberella intermedia (Fusarium proliferatum), Gibberella moniliformis (Fusarium verticillioides), and Gibberella konza (Fusarium konzum) dominated. Gibberella thapsina (Fusarium thapsinum) and Gibberella subglutinans (Fusarium subglutinans) also were recovered, as were seven isolates that could not be assigned to any previously described species on the basis of either morphological or molecular characters. In general, isolates from the prairie grasses produced the same toxins in quantities similar to those produced by isolates of the same species recovered from agricultural hosts. The G. konza isolates produce little or no fumonisins (up to 120 micro g/g by one strain), and variable but generally low to moderate amounts of beauvericin (4 to 320 micro g/g) and fusaproliferin (50 to 540 micro g/g). Toxicity to Artemia salina larvae within most species was correlated with the concentration of either beauvericin or fusaproliferin produced. Organic isolates from some cultures of G. moniliformis were highly toxic towards A. salina even though they produced little, if any, beauvericin or fusaproliferin. Thus, additional potentially toxigenic compounds may be synthesized by G. moniliformis strains isolated from prairie grasses. The Fusarium community from these grasses appears to contain some species not found in surrounding agricultural communities, including some that probably are undescribed, and could be capable of serving as a reservoir for strains of potential agricultural importance.

Population differentiation and recombination in wheat scab populations of Gibberella zeae from the United States.

In limited previous studies of the Ascomycete fungus Gibberella zeae in North America, the populations examined were genetically and phenotypically diverse and could be viewed as subsamples of a larger population. Our objective in this study was to test the hypothesis that a homogeneous, randomly mating population of G. zeae is contiguous throughout the central and eastern United States across a span of several years. We analysed presence/absence alleles based on amplified fragment length polymorphisms (AFLPs) at 30 loci, 24 of which are defined genetically on a linkage map of G. zeae, from > 500 isolates in eight field populations from seven states collected during the 1998, 1999 and 2000 cropping seasons. All these strains had AFLP profiles similar to those of standard isolates of G. zeae phylogenetic lineage 7. All the populations are genetically similar, have high genotypic diversity and little or no detectable genetic disequilibrium, and show evidence of extensive interpopulation genetic exchange. Allele frequencies in some of the populations examined are not statistically different from one another, but others are. Thus, the populations examined are not mere subsamples from a single, large, randomly mating population. Geographic distance and genetic distance between populations are correlated significantly. The observed differences are relatively small, however, indicating that while genetic isolation by distance may occur, genetic exchange has occurred at a relatively high frequency among US populations of G. zeae. We think that these differences reflect the time required for the alleles to diffuse across the distances that separate them, because relatively little linkage disequilibrium is detected either in the population as a whole or in any of the individual subpopulations.

Amplified Fragment Length Polymorphism Diversity in Cephalosporium maydis from Egypt.

ABSTRACT Cephalosporium maydis, the causal agent of late wilt of maize, was first described in Egypt in the 1960s, where it can cause yield losses of up to 40% in susceptible plantings. We characterized 866 isolates of C. maydis collected from 14 governates in Egypt, 7 in the Nile River Delta and 7 in southern (Middle and Upper) Egypt, with amplified fragment length polymorphism (AFLP) markers. The four AFLP primer-pair combinations generated 68 bands, 25 of which were polymorphic, resulting in 52 clonal haplotypes that clustered the 866 isolates into four phylogenetic lineages. Three lineages were found in both the Nile River Delta and southern Egypt. Lineage IV, the most diverse group (20 haplotypes), was recovered only from governates in the Nile River Delta. In some locations, one lineage dominated (up to 98% of the isolates recovered) and, from some fields, only a single haplotype was recovered. Under field conditions in Egypt, there is no evidence that C. maydis reproduces sexually. The nonuniform geographic distribution of the pathogen lineages within the country could be due to differences in climate or in the farming system, because host material differs in susceptibility and C. maydis lineages differ in pathogenicity.

Diversity of Epidemic Populations of Gibberella zeae from Small Quadrats in Kansas and North Dakota.

ABSTRACT Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight (FHB) of wheat and barley and has been responsible for several billion dollars of losses in the United States since the early 1990s. We isolated G. zeae from the top, middle, and bottom positions of wheat spikes collected from 0.25-m(2) quadrats during severe FHB epidemics in a single Kansas (KS) field (1993) and in a single North Dakota (ND) field (1994). Three amplified fragment length polymorphism (AFLP) primer pairs were used to resolve 94 polymorphic loci from 253 isolates. Members of a subset of 26 isolates also were tested for vegetative compatibility groups (VCGs). Both methods indicated high levels of genotypic variability and identified the same sets of isolates as probable clones. The mean number of AFLP multilocus haplotypes per head was approximately 1.8 in each population, but this value probably underestimates the true mean due to the small number of samples taken from each head. Isolates with the same AFLP haplotype often were recovered from different positions in a single head, but only rarely were such apparently clonal isolates recovered from more than one head within a quadrat, a pattern that is consistent with a genetically diverse initial inoculum and limited secondary spread. The KS and ND samples had no common AFLP haplotypes. All G. zeae isolates had high AFLP fingerprint similarity (>70%, unweighted pair group method with arithmetic means similarity) to reference isolates of G. zeae lineage 7. The genetic identity between the KS and ND populations was >99% and the estimated effective migration rate was high (Nm approximately 70). Tests for linkage disequilibrium provide little evidence for nonrandom associations between loci. Our results suggest that these populations are parts of a single, panmictic population that experiences frequent recombination. Our results also suggest that a variety of population sampling designs may be satisfactory for assessing diversity in this fungus.

Gibberella konza (Fusarium konzum) sp. nov. from prairie grasses, a new species in the Gibberella fujikuroi species complex.

The Gibberella fujikuroi species complex (Fusarium section Liseola and allied taxa) is composed of an increasingly large number of morphological, biological and phylogenetic species. Most of the known species in this group have been isolated from agricultural ecosystems or have been described from a small number of isolates. We sampled Fusarium communities from native prairie grasses in Kansas and recovered a large number of isolates that superficially resemble F. anthophilum. We used a combination of morphological, biological and molecular characters to describe a new species, Gibberella konza (Gibberella fujikuroi mating population I [MP-I]), from native prairie grasses in Kansas. Although female fertility for field isolates of this species appears to be low, G. konza is heterothallic, and we developed reliably female fertile mating population tester strains for this species. The F. konzum anamorph is differentiated from F. anthophilum and from other Fusarium species in section Liseola by mating compatibility, morphology, AFLP fingerprint profile and differences in ß-tubulin DNA sequence.

Expanded genetic map of Gibberella moniliformis (Fusarium verticillioides).

Gibberella moniliformis (Fusarium verticillioides) is primarily a pathogen of maize, but it can also cause disease in other crop species. This pathogenicity, as well as the contamination of food- and feedstuffs with the fumonisin mycotoxins, results in economically significant losses to both farmers and food processors. The dissection of important biological characters in this fungus has been hampered by the lack of a uniformly dense genetic map. The existing restriction fragment length polymorphism-based map contains significant gaps, making it difficult to routinely locate biologically important genes, such as those involved in pathogenicity or mycotoxin production, with precision. We utilized amplified fragment length polymorphisms (AFLPs) to saturate the existing genetic map and added 486 AFLP markers to the approximately 150 markers on the existing map. The resulting map has an average marker interval of 3.9 map units and averages approximately 21 kb/map unit. The additional markers expanded the map from 1,452 to 2,188 map units distributed across 12 chromosomes. The maximum distance between adjacent markers is 29 map units. We identified AFLP markers less than 1 map unit from the mating type (MAT) locus and 2.5 map units from the spore killer (SK) locus; eight AFLP markers map within 8.5 units of the FUM1 (fumonisin biosynthetic) locus. The increased saturation of this map will facilitate further development of G. moniliformis as a model system for the genetic and population genetic studies of related, but less genetically tractable, plant pathogenic fungi.

Relative Competitiveness and Virulence of Four Clonal Lineages of Cephalosporium maydis from Egypt Toward Greenhouse-Grown Maize.

Four clonal lineages of Cephalosporium maydis, a soilborne vascular wilt pathogen that causes late wilt of maize, were differentiated previously with molecular markers. In Egypt, this fungus can cause significant losses in infected susceptible plants. In greenhouse tests of individual isolates we found that these lineages differ in their virulence toward a series of maize accessions commonly used in Egyptian maize breeding programs. We also determined the relative competitiveness of representatives of the four lineages when incorporated into the soil as a mixed inoculum. The lineage (IV) with greatest mean disease incidence (virulence), when tested alone, was the least competitive on susceptible maize accessions when coinoculated as a component of mixed inocula of all four lineages. In these coinoculation experiments, one of the less-virulent lineages (II) dominated (70% of infections) and appeared to be the most competitive. These results suggest that virulence and competitive ability are not the same in this host-pathogen system. These results also suggest that standard protocols that rely on mixed inocula for resistance screening need to be altered, and that the relative proportion of the different lineages of the pathogen recovered in a field may be influenced by the maize variety/hybrid planted.