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), β-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.     

Suppression of Fusarium oxysporum with recombinant polygalacturonase inhibiting proteins (BvPGIPs) extracted from sugar beet roots

Soil-borne fungus Fusarium oxysporum f. sp. betae (Fob) is the causative agent of Fusarium yellows in sugar beet. Leaf interveinal yellowing and root vascular discoloration significantly reduce root yield as well as sucrose content and juice purity. Fob, like other fungal pathogens, initiates disease development by secreting polygalacturonase (PG) enzymes to break down plant cell walls during early stages of infection. To protect themselves, plants produce polygalacturonase-inhibiting proteins (PGIPs). In our study of sugar beet root defense responses, several PGIP genes (BvPGIPs) were identified. To determine if BvPGIPs inhibit Fob PGs, genes BvPGIP1, BvPGIP2 and Bv(FC607)PGIP1 were fused with the CaMV 35S promoter and each was expressed individually in sugar beet hairy roots. We demonstrate that all three recombinant BvPGIP proteins inhibited Fob and F. oxysporum f. sp. gladioli (Fog) PGs. A comparable level of BvPGIP activity was observed against Fob PGs, while BvPGIP2 showed higher activity against Fog PGs. Similar results were obtained when recombinant PGIPs were used to bioassay effects on Fob and Fog spore germination and hyphal growth. This is a first report that documents F. oxysporum inhibition by overexpressing BvPGIPs that may lead to improved Fusarium yellows resistance in sugar beet.

     

Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.     

Regulation of a novel Fusarium cytokinin in Fusarium pseudograminearum

Fusarium pseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusarium graminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F. pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F. pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Distribution and incidence of yellowing viruses in sugar beet crops in Spain from 1990 to 1993

Surveys of the principal yellowing viruses of sugar beet, beet yellows virus (BYV) and beet mild yellowing virus (BMYV) in Spain were carried out from 1990–1993. Beet yellowing viruses were detected in all provinces, although the mean percentages of plants infected with BYV and BMYV were practically zero in the southern zone. Within the northern zone high variations from one province to another could be observed. The mean percentages of plants infected with BYV were higher in the Ebro Valley than in the Duero Valley. Areas infected with BYV were very restricted, while BMYV could be found to a variable extent all over Spain, although the infection levels were lower. The incidence and distribution of these viruses in the Spanish sugar beet crop makes the study and application of control measures for beet yellowing viruses necessary.     

Birth,death and horizontal transfer of the fumonisin biosynthetic gene cluster during the evolutionary diversification of Fusarium

Fumonisins are a family of carcinogenic secondary metabolites produced by members of the Fusarium fujikuroi species complex (FFSC) and rare strains of Fusarium oxysporum. In Fusarium, fumonisin biosynthetic genes (FUM) are clustered, and the cluster is uniform in gene organization. Here, sequence analyses indicated that the cluster exists in five different genomic contexts, defining five cluster types. In FUM gene genealogies, evolutionary relationships between fusaria with different cluster types were largely incongruent with species relationships inferred from primary‐metabolism (PM) gene genealogies, and FUM cluster types are not trans‐specific. In addition, synonymous site divergence analyses indicated that three FUM cluster types predate diversification of FFSC. The data are not consistent with balancing selection or interspecific hybridization, but they are consistent with two competing hypotheses: (i) multiple horizontal transfers of the cluster from unknown donors to FFSC recipients and (ii) cluster duplication and loss (birth and death). Furthermore, low levels of FUM gene divergence in F. bulbicola, an FFSC species, and F. oxysporum provide evidence for horizontal transfer of the cluster from the former, or a closely related species, to the latter. Thus, uniform gene organization within the FUM cluster belies a complex evolutionary history that has not always paralleled the evolution of Fusarium.     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

Variation in the spectrum of Fusarium species on winter wheat

In 1998–2000 a monitoring of the spectrum of Fusarium species on winter wheat was carried out in the Rhineland. The epidemic spread ofFusarium spp. on wheat plants during growing season was investigated as well as the grain contamination after harvest.F avenaceum was the Fusarium species isolated most frequently followed byF culmorum, F poae andF graminearum. Microdochium nivale occurred considerably only in 1998. Both, susceptibility and plant height of the cultivars were correlated to the incidence of Fusarium species /M nivale on harvested kernels; interactions with cropping intensities were detected. The incidence ofF poae seemed to be independent of the cultivar-specific Fusarium susceptibility. Despite the lack of disease symptoms, between growth stages 45–85 mycelium ofFusarium spp. was detectable in the leaves as well as conidia on the leaf surfaces.     

Early Events in the Fusarium verticillioides-Maize Interaction Characterized by Using a Green Fluorescent Protein-Expressing Transgenic Isolate

The infection of maize by Fusarium verticillioides can result in highly variable disease symptoms ranging from asymptomatic plants to severe rotting and wilting. We produced F. verticillioides green fluorescent protein-expressing transgenic isolates and used them to characterize early events in the F. verticillioides-maize interaction that may affect later symptom appearance. Plants grown in F. verticillioides-infested soil were smaller and chlorotic. The fungus colonized all of the underground parts of a plant but was found primarily in lateral roots and mesocotyl tissue. In some mesocotyl cells, conidia were produced within 14 to 21 days after infection. Intercellular mycelium was detected, but additional cells were not infected until 21 days after planting. At 25 to 30 days after planting, the mesocotyl and main roots were heavily infected, and rotting developed in these tissues. Other tissues, including the adventitious roots and the stem, appeared to be healthy and contained only a small number of hyphae. These results imply that asymptomatic systemic infection is characterized by a mode of fungal development that includes infection of certain tissues, intercellular growth of a limited number of fungal hyphae, and reproduction of the fungus in a few cells without invasion of other cells. Development of visibly rotted tissue is associated with massive production of fungal mycelium and much less organized growth.     

Phylogenetic Diversity and In Vitro Susceptibility Profiles of Human Pathogenic Members of the <Emphasis Type="Italic">Fusarium fujikuroi</Emphasis> Species Complex Isolated from South India

Availability of molecular methods, gene sequencing, and phylogenetic species recognition have led to rare fungi being recognized as opportunistic pathogens. Fungal keratitis and onychomycosis are fairly common mycoses in the tropics, especially among outdoor workers and enthusiasts. The frequently isolated etiological agents belong to genera Candida, Aspergillus, and Fusarium. Within the genus Fusarium, known to be recalcitrant to prolonged antifungal treatment and associated with poor outcome, members of the Fusarium solani species complex are reported to be most common, followed by members of the Fusarium oxysporum SC and the Fusarium fujikuroi SC (FFSC). Morphological differentiation among the various members is ineffective most times. In the present study, we describe different species of the FFSC isolated from clinical specimen in south India. All twelve isolates were characterized up to species level by nucleic acid sequencing and phylogenetic analysis. The molecular targets chosen were partial regions of the internal transcribed spacer rDNA region, the panfungal marker and translation elongation factor-1α gene, the marker of choice for Fusarium speciation. Phylogenetic analysis was executed using the Molecular Evolutionary Genetics Analysis software (MEGA7). In vitro susceptibility testing against amphotericin B, voriconazole, posaconazole, natamycin, and caspofungin diacetate was performed following the CLSI M38-A2 guidelines for broth microdilution method. The twelve isolates of the FFSC were F. verticillioides (n = 4), F. sacchari (n = 3), F. proliferatum (n = 2), F. thapsinum (n = 1), F. andiyazi (n = 1), and F. pseudocircinatum (n = 1). To the best of our knowledge, this is the first report of F. andiyazi from India and of F. pseudocircinatum as a human pathogen worldwide. Natamycin and voriconazole were found to be most active agents followed by amphotericin B. Elderly outdoor workers figured more among the patients and must be recommended protective eye wear.     

SUGAR BEET YELLOWS: A PRELIMINARY STUDY OF THE DISTRIBUTION AND INTERRELATIONSHIPS OF VIRUSES AND VIRUS STRAINS FOUND IN EAST ANGLIA, 1955-57

Aphid transmissions to sugar beet seedlings from yellowed sugar beet leaves collected from commercial fields in East Anglia during the summers of 1955, 1956 and 1957, showed the occurrence of two yellowing viruses. One was sugar beet yellows virus (SBYV) and produced vein-etch and yellowing symptoms on beet seedlings in the glasshouse; the other produced yellowing but no etch. These two viruses were apparently unrelated, so that sugar beet tolerant to one of them would not necessarily be tolerant to the other. The second virus, called 'sugar beet mild yellowing virus' (SBMYV), decreased the root yield of sugar beet plants grown under glass, by as much as did the milder SBYV strains, but less than did the severe SBYV strains. The proportions of the two viruses in the samples differed from year to year and from place to place.     

Mode of antagonism of a biocontrol bacterium Lysobacter sp. SB-K88 toward a damping-off pathogen Aphanomyces cochlioides

The biocontrol bacterium Lysobacter sp. SB-K88 suppresses damping-off disease in sugar beet and spinach caused by Aphanomyces cochlioides and Pythium sp. through characteristic plant colonization and antibiosis against the pathogens. This study aimed to unravel further details on mode of antagonism of SB-K88 against a damping-off pathogen A. cochlioides AC-5. The SB-K88 substantially inhibited growth and decomposed AC-5 mycelia and suppressed the release of zoospores from the hyphae. The excised root tips of sugar beet seedlings from seeds previously inoculated with SB-K88 were less attractive to AC-5 zoospores. Although aerial growth was not affected, however, root hairs of SB-K88 inoculated sugar beet seedlings were remarkably shorter and thicker than those of uninoculated control. When exposed to zoospores, the SB-K88 inhibited motility of zoospores and/or caused lysis, and then aggregated around the dead cystospores or lysed residues within 3–6 h likely to be micro-predatory behavior to a eukaryotic organism. Confocal laser scanning microscopic analysis revealed that number of lipid bodies and activities of mitochondria were markedly increased in the affected hyphae compared with control hyphae as visualized by established vital stains. Taken together, these results suggest that Lysobacter sp. SB-K88 suppresses damping-off diseases through exerting multifaceted antagonistic effects against the peronosporomycetes.     

Interrelationship of Heterodera glycines and Fusarium solani in Sudden Death Syndrome of Soybean

Experiments were established in field microplots to examine the association between Heterodera glycines and the blue form of Fusarium solani in sudden death syndrome of soybean (SDS). Foliar disease symptoms occurred on more plants per plot, appeared 3 to 7 days earlier, and were more severe on plants grown in plots infested with F. solani + H. glycines than on those inoculated with F. solani only. Yields were suppressed only in treatments that included the nematode. Numbers of H. glycines cysts and second-stage juveniles were significantly lower in plots infested with F. solani + H. glycines than with the nematode alone. Fusarium solani was able to infect cysts and eggs.     

Fusarium udum parasitic onAspergillus luchuensis andSyncephalastrum racemosum

Summary Mycoparasitic behaviour ofF. udum on two soil-inhabiting microfungi,Aspergillus luchuensis andSyncephalastrum racemosum, was studied in dual cultures.Fusarium udum coiled and penetrated the host fungi and formed chlamydospores inside their hyphae and reproductive structures. The vegetative hyphae ofA. luchuensis showed swellings due to diffusible toxic substances ofF. udum in the medium and formed vesicle-like structures. This is the first record of these fungi being hosts of the mycoparasiteF. udum.     

Antagonistic Interaction of Heterodera schachtii Schmidt and Fusarium oxysporum (Woll.) on Sugarbeets

In a field experiment, nematicides controlled the disease of sugarbeets caused by Heterodera schachtii and Fusarium oxysporum. Biocides that were both fungicidal and nematicidal also controlled the disease, but sugar yields were no higher than those obtained with the plain nematicides. In greenhouse experiments, the interaction between H. schachtii and F. oxysporam was disadvantageous to the nematode. Damage to sugarbeets was less when the fungus and the nematode were present than when only the nematode was present. The fungus inhibited nematode invasion and development in sugarbeet seedlings, thereby decreasing the number of nematodes that matured about 3-fold.     

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari,Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA₃ in the culture fluid (2.76–28.4 μg/mL), while two others (B-3828 and B-1725) were inactive. GA₃ levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA₁, at a low level (less than 0.1 μg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.     

Altered growth of Fusarium oxysporum f. sp. chrysanthemi in an in vitro dual culture system with the vesicular arbuscular mycorrhizal fungus Glomus intraradices growing on Daucus carota transformed roots

Vesicular arbuscular mycorrhizal fungi can reduce plant disease symptoms and populations of pathogens through mechanisms that are not well understood. Glomus intraradices was grown on Daucus carota transformed roots in a two-compartment in vitro system. One compartment contained mycorrhizal roots on a complete growth medium, while the other contained a medium lacking sugar on which only mycelial growth was allowed. The direct interaction between G. intraradices and Fusarium oxysporum f. sp. chrysanthemi was studied in the compartment lacking sugar during a 5-day period. G. intraradices hyphal density and spore number were estimated along with F. o. chrysanthemi conidial germination, mycelial growth and sporulation. Five hours after inoculation, germination of F. o. chrysanthemi conidia doubled in the presence of G. intraradices. Radial growth of F. o. chrysanthemi colonies was always slightly but significantly enhanced in the presence of G. intraradices. No correlation was obtained between G. intraradices hyphae or spore densities and F. o. chrysanthemi hyphal growth. Overall sporulation of the 5-day-old F. o. chrysanthemi colonies was not influenced by the presence of G. intraradices. However, significant negative correlations were found between F. o. chrysanthemi conidia production and G. intraradices hyphae or spore concentrations. G. intraradices increased F. o. chrysanthemi conidial germination and slightly stimulated its hyphal growth in dual culture without any root influences. No antibiosis was observed between the fungi. The significance of the results and their potential implication for rhizosphere biology are discussed.