The Transaldolase,a Novel Allergen of Fusarium proliferatum,Demonstrates IgE Cross-Reactivity with Its Human Analogue

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.     

Restoration of Gibberellin Production in Fusarium proliferatum by Functional Complementation of Enzymatic Blocks

Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5′ noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.     

Discriminant analysis of volatile organic compounds of Fusarium oxysporum f. sp. cepae and Fusarium proliferatum isolates from onions as indicators of fungal growth

Basal rot is a common onion disease and is mainly caused by Fusarium oxysporum f. sp. cepae and Fusarium proliferatum. To study the possibility of using volatile organic compounds (VOCs) as biomarkers for these fungi, pathogenic isolates of F. oxysporum and F. proliferatum from onions were cultivated in onion medium and VOCs were measured by solid phase microextraction (SPME). Forty-two compounds were detected, and thirty of these compounds were highly related to fungal metabolic activity. Allyl mercaptan was specific to F. oxysporum isolate Fox006. Analysis of the VOCs showed significant differences between the two species and among different isolates within the same species. Sixteen of the VOCs showed were highly positively correlated with the fungal biomass estimated by real-time polymerase chain reaction (PCR). Ethanol, ethyl formate, ethyl acetate, 2-methyl-1-propanol, methyl thioacetate, n-propyl acetate and 3-methyl-1-butanol are volatile metabolites that were potential indicators of Fusarium growth on onions.     

Metabolism and Resistance of Fusarium spp. to the Manzamine Alkaloids via a Putative Retro Pictet-Spengler Reaction and Utility of the Rational Design of Antimalarial and Antifungal Agents

As a part of our continuing investigation of the manzamine alkaloids we studied the in vitro activity of the β-carboline containing manzamine alkaloids against Fusarium solani, Fusarium oxysporium, and Fusarium proliferatum by employing several bioassay techniques including one-dimensional direct bioautography, dilution, and plate susceptibility, and microtiter broth assays. In addition, we also studied the metabolism of the manzamine alkaloids by Fusarium spp. in order to facilitate the redesign of the compounds to prevent resistance of Fusarium spp. through metabolism. The present research reveals that the manzamine alkaloids are inactive against Fusarium spp. and the fungi transform manzamines via hydrolysis, reduction, and a retro Pictet-Spengler reaction. This is the first report to demonstrate an enzymatically retro Pictet-Spengler reaction. The results of this study reveal the utility of the rational design of metabolically stable antifungal agents from this class and the development of manzamine alkaloids as antimalarial drugs through the utilization of Fusarium’s metabolic products to reconstruct the molecule.     

Rhizobacterial Volatiles Affect the Growth of Fungi and Arabidopsis thaliana

Volatiles of Stenotrophomonas, Serratia, and Bacillus species inhibited mycelial growth of many fungi and Arabidopsis thaliana (40 to 98%), and volatiles of Pseudomonas species and Burkholderia cepacia retarded the growth to lesser extents. Aspergillus niger and Fusarium species were resistant, and B. cepacia and Staphylococcus epidermidis promoted the growth of Rhizoctonia solani and A. thaliana. Bacterial volatiles provide a new source of compounds with antibiotic and growth-promoting features.     

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler^® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.     

The Homologue of het-c of Neurospora crassa Lacks Vegetative Compatibility Function in Fusarium proliferatum

For two fungal strains to be vegetatively compatible and capable of forming a stable vegetative heterokaryon they must carry matching alleles at a series of loci variously termed het or vic genes. Cloned het/vic genes from Neurospora crassa and Podospora anserina have no obvious functional similarity and have various cellular functions. Our objective was to identify the homologue of the Neurospora het-c gene in Fusarium proliferatum and to determine if this gene has a vegetative compatibility function in this economically important and widely dispersed fungal pathogen. In F. proliferatum and five other closely related Fusarium species we found a few differences in the DNA sequence, but the changes were silent and did not alter the amino acid sequence of the resulting protein. Deleting the gene altered sexual fertility as the female parent, but it did not alter male fertility or existing vegetative compatibility interactions. Replacement of the allele-specific portion of the coding sequence with the sequence of an alternate allele in N. crassa did not result in a vegetative incompatibility response in transformed strains of F. proliferatum. Thus, the fphch gene in Fusarium appears unlikely to have the vegetative compatibility function associated with its homologue in N. crassa. These results suggest that the vegetative compatibility phenotype may result from convergent evolution. Thus, the genes involved in this process may need to be identified at the species level or at the level of a group of species and could prove to be attractive targets for the development of antifungal agents.     

Leaf axil sampling of midwest U.S. maize for mycotoxigenic Fusarium fungi using PCR analysis

PCR analysis was used to detect Fusarium species generically, as well as the mycotoxin-producing species F.␣subglutinans, F. proliferatum, and F. verticillioides in leaf axil and other maize tissues during ear fill in a multiyear study in central Illinois. The frequency of Fusarium detected varied from site to site and year to year. Fusarium was generically detected more frequently in leaf axil material than in leaf/husk lesions. In two growing seasons, the leaf axil samples were also tested for the presence of the mycotoxin producing species F. proliferatum, F. subglutinans, and F. verticillioides. Overall, F. proliferatum and F. verticillioides were detected less often than F. subglutinans. Fusarium was generically and specifically detected most commonly where visible fungal growth was present in leaf axil material. Disclaimer: The mention of firm names or trade products in this article does not imply that they are endorsed or recommended by the United States Department of Agriculture over other firms or similar products not mentioned.     

Beauvericin Production by Fusarium Species

Beauvericin is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and which can induce apoptosis in mammalian cells. Beauvericin is produced by some entomo- and phytopathogenic Fusarium species (Fusarium proliferatum, F. semitectum, and F. subglutinans) and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. We tested 94 Fusarium isolates belonging to 25 taxa, 21 in 6 of the 12 sections of the Fusarium genus and 4 that have been described recently, for the ability to produce beauvericin. Beauvericin was produced by the following species (with the number of toxigenic strains compared with the number of tested strains given in parentheses): Fusarium acuminatum var. acuminatum (1 of 4), Fusarium acuminatum var. armeniacum (1 of 3), F. anthophilum (1 of 2), F. avenaceum (1 of 6), F. beomiforme (1 of 1), F. dlamini (2 of 2), F. equiseti (2 of 3), F. longipes (1 of 2), F. nygamai (2 of 2), F. oxysporum (4 of 7), F. poae (4 of 4), F. sambucinum (12 of 14), and F. subglutinans (3 of 3). These results indicate that beauvericin is produced by many species in the genus Fusarium and that it may be a contaminant of cereals other than maize.     

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 μg/g by one strain), and variable but generally low to moderate amounts of beauvericin (4 to 320 μg/g) and fusaproliferin (50 to 540 μg/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.     

First report of molecular characterisation of Fusarium proliferatum associated with root rot disease of Indian mulberry (Morinda officinalis How.) in Viet Nam

Root rot is the most serious disease affecting Indian mulberry in Viet Nam. The objective of the present study was to confirm the identities and pathogenicity of species of Fusarium isolated from the roots of Indian mulberry. Based on pathogenicity assays, F. proliferatum was shown to be the pathogenic agent. The identity of F. proliferatum was confirmed by PCR and phylogenetic analyses based on the sequences of the ITS region and the TEF1-α gene. Of four semi-solid media (CA, PDA, CDA and YMA) tested, PDA was the most suitable medium for the growth of F. proliferatum. The optimal temperature range for the growth of F. proliferatum was 20–30?°C with a maximum at growth rate at 25?°C. The optimum pH for growth ranged from pH5.0–6.0 with maximum growth at 5.5. This is the first report of F. proliferatum causing root rot of Indian mulberry in Viet Nam.     

Biodiversity and Biogeography of Fusarium Species from Northeastern North American Asparagus Fields Based on Microbiological and Molecular Approaches

Sixteen Fusarium species were recovered from 52 asparagus commercial fields, representing all major ecological (edaphic and climatic) area of asparagus production in the province of Québec, eastern Canada. This study extends our understanding of the geographic range of these species. It also provides climatological and edaphic properties linked to community changes and adaptations. Fusarium oxysporum and F. proliferatum were omnipresent and abundant in all five ecological area under study, whereas F. redolens was less frequently found. Species of Fusarium that produce carmine red pigmentation on potato dextrose agar, i.e., F. acuminatum, F. avenaceum, etc., were common at the northern limit of asparagus production. Abundance of red Fusarium species corresponded with a low isolation frequency of F. proliferatum. Nevertheless, F. proliferatum had a high recovery rate throughout Québec asparagus growing areas, under climatic conditions as cold as those of northern Europe where this species is uncommon in asparagus fields. In the light of these results, redefinition of the geographical distribution of F. proliferatum in asparagus fields is proposed. Intraspecific molecular differences in F. proliferatum and F. oxysporum were detected in the EF-1 alpha sequences and compared with well-characterized strains of North America.     

Moniliformin production byFusarium species isolated from Argentinian corn

Forty-one isolates ofFusarium obtained from the main Argentinian corn production area were tested for their ability to produce moniliformin. One of 22 isolates ofF. moniliforme, 2/10 of F.proliferatum and 3/9 ofF. subglutinans, produced moniliformin in a range between 0,3 to 2,7 mg/g. These data represent the first report of the production of moniliformin byFusarium species from section Liseola in Argentina.     

Characterization of Fusarium spp. associated with pineapple fruit rot and leaf spot in Peninsular Malaysia

Pineapple (Ananas comosus) is one the important fruit crops planted in Malaysia, and this study was conducted to determine Fusarium spp. associated with diseases of the fruit crop as Fusarium is prevalent in tropical countries. Our objective was to identify and characterize Fusarium spp. associated with pineapple fruit rot and leaf spot mainly found on the fruits and leaves in Peninsular Malaysia. Fusarium isolates (n = 108) associated with pineapple fruit rot and leaf spot were characterized by morphological, molecular and phylogenetic analyses, a mating study and pathogenicity testing. TEF‐1α sequence analysis identified Fusarium proliferatum, Fusarium verticillioides, Fusarium sacchari and Fusarium sp. Mating was successful only between tester strains of F. proliferatum and F. verticillioides. Sexual crosses with standard tester strains showed that 82 isolates of F. proliferatum produced fertile crosses with mating population D (Gibberella intermedia) and three isolates of F. verticillioides were fertile with the tester strain of mating population A (Gibberella moniliformis). All isolates were pathogenic, causing pineapple fruit rot and leaf spot, thus fulfilling Koch's postulates.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.     

Fungal microbiota from rain water and pathogenicity of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.     

Volatile Compound-Mediated Interactions between Barley and Pathogenic Fungi in the Soil

Plants are able to interact with their environment by emitting volatile organic compounds. We investigated the volatile interactions that take place below ground between barley roots and two pathogenic fungi, Cochliobolus sativus and Fusarium culmorum. The volatile molecules emitted by each fungus, by non-infected barley roots and by barley roots infected with one of the fungi or the two of them were extracted by head-space solid phase micro extraction and analyzed by gas chromatography mass spectrometry. The effect of fungal volatiles on barley growth and the effect of barley root volatiles on fungal growth were assessed by cultivating both organisms in a shared atmosphere without any physical contact. The results show that volatile organic compounds, especially terpenes, are newly emitted during the interaction between fungi and barley roots. The volatile molecules released by non-infected barley roots did not significantly affect fungal growth, whereas the volatile molecules released by pathogenic fungi decreased the length of barley roots by 19 to 21.5% and the surface of aerial parts by 15%. The spectrum of the volatiles released by infected barley roots had no significant effect on F. culmorum growth, but decreased C. sativus growth by 13 to 17%. This paper identifies the volatile organic compounds emitted by two pathogenic fungi and shows that pathogenic fungi can modify volatile emission by infected plants. Our results open promising perspectives concerning the biological control of edaphic diseases.     

Dispersal of <Emphasis Type="Italic">Fusarium</Emphasis> spp. by rainwater and pathogenicity on four plant species

Research focused on the occurrence of Fusarium spp. in atmospheric dust or rainwater is not common. Preliminary studies with four sampling dates in 2007 revealed that several species of Fusarium may also be conveyed by rainwater. In order to determine the regular presence of Fusarium spp. in rainfall water, samples were systematically collected for a year (from October 2009 to October 2010) in three points on the Mediterranean coast of the province of Granada (Spain) 10-km distance between them. Throughout the year of sampling, a total of 179 rainwater samples were collected during every significant rainfall event. Eight different Fusarium species were isolated from the rainwater samples: F. oxysporum (32 %), F. proliferatum (26 %) and F. equiseti (20 %) coincide with previous studies, while F. dimerum (3 %), F. semitectum (4.7 %), F. solani (8 %), F. avenaceum (0.5 %) and F. chlamydosporum (3.7 %) were isolated for the first time from rainwater. Results were consistent with previous surveys conducted 100 km away from the sampling sites. Inoculation of 39 different isolates from five different Fusarium species showed pathogenicity on plants. Disease severity differed depending on the inoculated plant species, which means that rain water can be an effective vector to transport new pathogens into new cultivated areas. This work reveals some epidemiological aspects of Fusarium genus in natural environments. Some of the isolated Fusarium spp. are potential mycotoxin producers, such as zearalenone, fumonisin, moniliformin or nivalenol.     

Effects of Neem Leaf Volatiles on Submerged Cultures of Aflatoxigenic Aspergillus parasiticus

Microbe-free compressed air was passed continuously for a 3-day test period through an enclosed system containing fresh neem leaves; the resultant emitted volatiles were passed over the surface of submerged liquid cultures of a wild-type aflatoxigenic isolate of Aspergillus parasiticus. Aflatoxin determinations for the fungal culture that received neem-derived volatiles, after a 3-day incubation period, resulted in a 90% overall reduction in aflatoxin production and a 51% reduction in fungal biomass when compared with cultures that did not receive neem volatiles. In a separate experiment but in a similarly enclosed system, volatiles from fresh neem leaves were collected on a small Tenax column and were thermally desorbed and cryogenically focused on a capillary gas chromatography column. The neem volatiles were subsequently separated and identified by gas chromatography-mass spectrometry. Sixty-eight compounds were identified by comparison of retention times and mass spectra with either authentic compounds or spectra from a computer-assisted library database of mass spectra. It was found that 10% of the total headspace volatiles were composed of C₃ to C₉ alkenals, which are toxic to aflatoxigenic Aspergillus spp., which could explain the bioactivity that resulted in reduced biomass in the neem-treated cultures.     

Upgrading of an industrial lignin by using laccase produced by Fusarium proliferatum and different laccase-mediator systems

An industrial lignin recovered from a paper factory effluent was characterized and treated in buffered media under three different conditions. The first system contained only partially purified laccase produced by Fusarium proliferatum. The other two were supplemented with 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) or 1-hydroxylbenzotriazole (1-HBT) as mediators. During incubation the lignin remained solubilized and its spectral and chromatographic characteristics depended on time and conditions. UV–vis spectrum of lignin recovered from the first system showed a strong increase at the outset of incubation, reflecting the introduction of new functional groups in the polymer. No changes in its molecular mass distribution pattern were detected. Longer incubation produced minor alterations in its UV–vis spectrum, together with a polymerization in the recovered substrate, appearing as a new peak around 200 kDa. Lignin recovered from the laccase-ABTS system showed an intensive depolymerization together with a minor polymerization also in the high molecular mass fraction (200 kDa). That lignin sample was soluble under acid conditions. These sharp modifications contrasted with those detected in the lignin incubated with 1-HBT as mediator.Treating industrial lignin by these simple procedures rapidly enhances the technological potential of this paper-mill byproduct.