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Gibberella xylarioides sensu lato from Coffea canephora: a new mating population in the Gibberella fujikuroi species complex.

Gibberella xylarioides Heim & Saccas (presumed anamorph, Fusarium xylarioides Steyaert) is the causal agent of coffee wilt disease, an economically important tracheomycosis in Africa. In vitro crosses carried out with Congolese, Ugandan, and Tanzanian single-ascospore/conidial isolates originating from diseased Coffea canephora/excelsa demonstrated a heterothallic mating system, controlled by a single locus with two alleles, MAT-1 and MAT-2. Compatible isolates produced fertile perithecia within 2 to 8 weeks after mating. Mating type (MAT) was characterized by PCR with primer pairs previously developed for the Gibberella fujikuroi species complex (GFC) and for Fusarium oxysporum. All strains analyzed were morphologically identical and corresponded to Booth's description of the "female" F. xylarioides strain. Based on crossing results and MAT-2/translation elongation 1-alpha (tef) sequence data, G. xylarioides, as currently understood, is demonstrated to encompass at least three "groups": G. xylarioides sensu strictu Ia, defined hitherto by two "historical" West African strains originating from the severe 1930s to 1950s epidemic (CBS 25852 and CBS 74979); G. xylarioides sensu strictu Ib, defined by two "historical" Central African lowland strains (DSMZ 62457 and ATCC 15664); and G. xylarioides sensu lato II, containing Congolese, Ugandan, and Tanzanian C. canephora/excelsa isolates. Infertility of crosses between the coffee wilt pathogen and known GFC mating populations demonstrates that G. xylarioides sensu lato constitutes a new biological species within the G. fujikuroi complex. MUCL 44532/MUCL 43887 and MUCL 35223/MUCL 44549 are proposed as G. xylarioides sensu lato II MAT-1/MAT-2 reference mating type tester strains.

Citrus sinensis↗

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.

Chromosome Mapping↗

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.

Animals↗

Gibberella from A (venaceae) to Z (eae).

Gibberella species are destructive plant pathogens, although many are more familiar under their Fusarium anamorph names. The recent synthesis of phylogenetic, biological, and morphological species approaches has revitalized taxonomy of a genus that was first described almost 200 years ago. Twelve sexual species of Gibberella of agricultural importance were selected for this review to represent phylogenetic, biological, and chemical diversity of the genus. Even closely related Gibberella species can differ in reproductive mode, geographic and host distribution, plant pathogenesis, and production of toxins and other biologically active metabolites. Gibberella species have proven amenable to meiotic and molecular genetic analysis; A complete genome sequence of G. zeae should soon be available. Combining gene disruption strategies with new genomics technologies for expression profiling should help plant pathologists to understand the pathological and evolutionary significance of biological and chemical diversity in Gibberella and to identify novel strategies for disease control.

Gibberella↗

Phytotoxic effect of deoxynivalenol and gibberella ear rot resistance of corn.

The Fusarium graminearum mycotoxin deoxynivalenol was shown to be more phytotoxic to corn genotypes susceptible to gibberella ear rot than resistant genotypes. The toxin caused greater damage to the membranes of the susceptible genotypes, as evidenced by the release of Na and K ions into solution. Isolated protoplasts of ear rot-resistant genotypes were able to bind more radiolabelled deoxynivalenol than those of susceptible genotypes, providing a partial explanation for the membrane tolerance to the toxin. The results are discussed in relation to the role of resistance to this phytotoxin and tolerance to gibberella ear rot.

Binding Sites↗

Partial MAT-2 gene structure and the influence of temperature on mating success in Gibberella circinata.

Genetic analysis of Gibberella circinata, the causative agent of pitch canker disease of pines, historically has been thwarted by a low frequency of mating success in the laboratory. We describe two findings that should facilitate genetic analysis of this fungus and related species. First, we determined that previously described degenerate primers could be used to amplify a portion of the MAT-2 mating type gene from G. circinata. This led to the cloning and sequencing of a fragment of the MAT-2 gene, which in turn made it possible to distinguish between G. circinata isolates of opposite mating types. Second, we discovered that of the 18 G. circinata field isolates in our collection, the 1 female fertile isolate expressed its fertility at 15 and 20 degrees C but not at 25 degrees C, the temperature used for crossing many Gibberella species. It is evident, therefore, that when sexual reproduction in other closely related species is initially being investigated, the crosses should be established at a variety of temperatures. Once we learned that female fertility in this G. circinata isolate was expressed at 20 degrees C, a high frequency of mating success was achieved.

Base Sequence↗

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize.

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F1 and F2 populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F2 individuals. Analysis of resistance was performed on the F3 families obtained by selfing the F2 plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F2 population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.

Chromosome Mapping↗

Genetics and gibberellin production in Gibberella fujikuroi.

Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development. The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.

Crosses, Genetic↗

Separate compartments for the production of sterols, carotenoids and gibberellins in Gibberella fujikuroi.

Substrate flows in the sterol, carotenoid and gibberellin pathways of Gibberella fujikuroi were examined by isotope-dilution experiments. The wild type and two carotenoid mutants of this fungus were grown in minimal medium with abundant glucose, limiting ammonium nitrate and a radioactively labelled precursor (either acetate, mevalonate or leucine). The precursors did not affect growth or terpenoid production, with two exceptions; leucine allowed additional growth, as expected from the nitrogen limitation in the medium, and mevalonate inhibited the accumulation of gibberellins, but only if added before the onset of gibberellin production. The relative contributions of glucose, mevalonate, leucine and acetate as terpenoid precursors, calculated from the specific radioactivities of ergosterol, neurosporaxanthin and phytoene, were different for different products and different precursors. We conclude that the biosyntheses of sterols, gibberellins and carotenoids in Gibberella are physically separated in different subcellular compartments with independent substrate pools. The same results were obtained with the three strains, except for carotenoid production, indicating that this pathway is regulated independently from other terpenoid pathways.

Acetates↗

Carbon catabolite repression in plant pathogenic fungi: isolation and characterization of the Gibberella fujikuroi and Botrytis cinerea creA genes.

The creA genes of two plant pathogenic fungi, the gibberellin-producing rice pathogen Gibberella fujikuroi and the gray mold Botrytis cinerea, were isolated and characterized. The deduced amino acid sequences of both glucose repressors are 64% identical to each other and 59% (G. fujikuroi) and 61% (B. cinerea) identical to the CreA protein of Aspergillus nidulans. The zinc finger regions of the Gibberella and Botrytis CreA proteins shared 98% identity with the corresponding zinc finger region of the A. nidulans protein, and studies by complementation of a creA null mutant of A. nidulans showed that the proteins are functional homologues of A. nidulans CreA. Northern blot analysis revealed that creA transcript levels are independent of the carbon source in both fungi.

Amino Acid Sequence↗

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.

Alleles↗

Putative polyketide synthase and laccase genes for biosynthesis of aurofusarin in Gibberella zeae.

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.

Amino Acid Sequence↗

GIP2, a putative transcription factor that regulates the aurofusarin biosynthetic gene cluster in Gibberella zeae.

Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of maize, wheat, and rice. Colonies of G. zeae produce yellow-to-tan mycelia with the white-to-carmine red margins. In this study, we focused on nine putative open reading frames (ORFs) closely linked to PKS12 and GIP1, which are required for aurofusarin biosynthesis in G. zeae. Among them is an ORF designated GIP2 (for Gibberella zeae pigment gene 2), which encodes a putative protein of 398 amino acids that carries a Zn(II)2Cys6 binuclear cluster DNA-binding domain commonly found in transcription factors of yeasts and filamentous fungi. Targeted gene deletion and complementation analyses confirmed that GIP2 is required for aurofusarin biosynthesis. Expression of GIP2 in carrot medium correlated with aurofusarin production by G. zeae and was restricted to vegetative mycelia. Inactivation of the 10 contiguous genes in the DeltaGIP2 strain delineates an aurofusarin biosynthetic gene cluster. Overexpression of GIP2 in both the DeltaGIP2 and the wild-type strains increases aurofusarin production and reduces mycelial growth. Thus, GIP2 is a putative positive regulator of the aurofusarin biosynthetic gene cluster, and aurofusarin production is negatively correlated with vegetative growth by G. zeae.

Base Sequence↗

Gibberella xylarioides (anamorph: Fusarium xylarioides), a causative agent of coffee wilt disease in Africa, is a previously unrecognized member of the G. fujikuroi species complex.

Tracheomycosis or coffee wilt has emerged as a major disease of robusta coffee in Uganda in the past 10 years. Coffee wilt historically has been associated with Fusarium xylarioides Steyaert (teleomorph Gibberella xylarioides Heim and Sacc.), a species that has been classified as a member of Fusarium section Lateritium. We investigated the molecular phylogenetics of fusarial coffee wilt isolates by generating partial DNA sequences from two protein coding regions, translation elongation factor 1-alpha and beta-tubulin, in 36 isolates previously identified as F. xylarioides and related fusaria from coffee and other woody hosts, as well as from 12 isolates associated with a current coffee wilt outbreak in Uganda. These isolates fell into two morphologically and phylogenetically distinct groups. The first group was found to represent previously unidentified members of the Gibberella fujikuroi species complex (GFC), a clade that replaces the artificial Fusarium section Liseola. This group of isolates fit the original description of F. xylarioides, thus connecting it to the GFC. The second group, which was diverse in its morphology and DNA sequences, comprised four distinct lineages related to Fusarium lateritium. Our finding of unrelated species associated with coffee wilt disease has important implications regarding its epidemiology, etiology and control.

Coffee↗

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari.

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.

Crosses, Genetic↗

Purification and characterization of a novel lyase from Cellulomonas sp. that degrades Fusarium and Gibberella acidic polysaccharides.

A Cellulomonas sp. isolated from soil produced a novel lyase that degraded the acidic polysaccharide of Fusarium sp. M7-1 with the formation of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose. DEAE-Toyopearl 650M column chromatography showed three lyase activity peaks (fractions I, II, and III). The major fraction was purified to homogeneity by polyacrylamide gel electrophoresis analysis, and its molecular weight was 74,000. The optimum pH was 6.5 to 8.0 and the stable pH range was 6.0 to 8.0. The purified enzyme did not degrade glucuronic or galacturonic acid-containing polysaccharides such as chondroitin, hyaluronic acid, pectin, or pectic acid. However, the purified enzyme specifically degraded various Fusarium and Gibberella acidic polysaccharides, and unsaturated sugars were produced with the release of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose. These results suggest that the acidic polysaccharides derived from Fusarium and Gibberella have similar structures.

Bacterial Proteins↗

Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A).

Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes and therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, DeltaMAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down-regulated in the DeltaMAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the Gibberella zeae and Gibberella moniliformis genomes. Fifty-five percent of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 45% were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences down-regulated in the mutant.

DNA, Complementary↗

Monooxygenases involved in GA12 and GA14 synthesis in Gibberella fujikuroi.

A microsomal preparation from mycelia of the gibberellin (GA)-producing fungus Gibberella fujikuroi catalyzed the first two steps in the conversion of the biosynthetic intermediate GA12-aldehyde to gibberellic acid (GA3). [14C]GA12-Aldehyde was converted to radiolabelled GA14, the major product, together with smaller amounts of non-hydroxylated GA12. The microsomal activities required reduced pyridine nucleotides and molecular oxygen. However, GA12 and GA14 synthesis differed markedly in the preferred electron source. Formation of GA12 required NADH or NADPH, while GA14 synthesis from GA12-aldehyde occurred only with NADPH. Marked differences were also found in the activating effect of FAD. When NADPH was the reductant, the rate of GA14 synthesis was enhanced 3.5 times by 5 microM FAD while this flavin nucleotide did not alter the synthesis of GA12. In contrast, GA12 synthesis was activated 3.8 times by 50 microM FAD in the presence of NADH. Both activities were inhibited by carbon monoxide and cytochrome c. These properties suggest that the 3beta-hydroxylation of GA12-aldehyde and further oxidation of carbon 7 are catalyzed by cytochrome P-450 monooxygenases in Gibberella fujikuroi.

Ascomycota↗