Fusarium fujikuroi associated with stem rot of red‐fleshed dragon fruit (Hylocereus polyrhizus) in Malaysia

Stem rot was recorded as one of serious diseases of red‐fleshed dragon fruit, (Hylocereus polyrhizus), in Malaysia. Fusarium fujikuroi was recovered from stem rot lesion of H. polyrhizus and the species was identified using TEF1‐α sequence and mating study. From maximum likelihood phylogenetic tree using combined TEF1‐α and β‐tubulin sequences, the F. fujikuroi isolates from stem rot were grouped according to three geographical locations, namely Peninsular Malaysia, Sabah and Sarawak. Phylogenetic analysis indicated that F. fujikuroi isolates from stem rot of H. polyrhizus were clustered separately from F. fujikuroi isolates from rice because of intraspecific variation. From amplification of MAT allele‐specific primers, 20% of the isolates carried MAT‐1 allele while 80% carried MAT‐2 allele. From isolates that carried MAT‐1 allele, 65% crossed‐fertile with MP‐C (mating population of F. fujikuroi) tester strain while for MAT‐2 allele, 56% crossed‐fertile with MP‐C. None of the isolates were identified as MP‐D (mating population of F. proliferatum). Pathogenicity test conducted on 40 representative isolates showed that the stem rot symptoms were similar with the symptoms observed in the field, and can be categorized as low, moderate and high aggressiveness, which indicated variation in pathogenicity and virulence among the isolates. This study provides novel findings regarding Fusarium species associated with stem rot of H. polyrhizus and indicated that F. fujikuroi as a new causal pathogen of the disease.     

Characterization and pathogenicity of Fusarium proliferatum causing stem rot of Hylocereus polyrhizus in Malaysia

During a series of sampling in 2008 and 2009, stem rot disease was detected in Hylocereus polyrhizus plantations in Malaysia, with symptom appeared as circular, brown sunken lesion with orange sporodochia and white mycelium formation on the lesion surface. Eighty‐three isolates of Fusarium were isolated from 20 plantations and were morphologically identified as F. proliferatum based on the variability of colony appearance, pigmentation, growth rate, length of chains, production of bluish sclerotia, concentric ring aerial mycelium and sporodochia. Three species‐specific primers, namely ITS1/proITS‐R, PRO1/2 and Fp3‐F/4‐R successfully produced PCR products and confirmed that the isolates from stem rot of H. polyrhizus were F. proliferatum isolates. From BLAST search of translation elongation factor 1‐alpha (TEF1‐α) sequences, the isolates showed 99–100% similarity with F. proliferatum deposited in GenBank which further confirmed that the isolates were F. proliferatum. The results from amplification of MAT‐allele specific primers indicated that 14.5% of F. proliferatum isolates carried MAT‐1 allele and 85.5% carried MAT‐2. Crossing results showed that all 83 F. proliferatum isolates were male fertile showing positive crosses with the tester strains of MATD‐1 and MATD‐2. Perithecia oozing ascospore were produced. Forty isolates as representative were evaluated for pathogenicity test, produced rot symptoms similar to those observed in the fields which confirmed the isolates as the causal agent of stem rot of H. polyrhizus. To our knowledge, this is the first report of stem rot of H. polyrhizus caused by F. proliferatum in Malaysia.     

<Emphasis Type="Italic">Fusarium</Emphasis> species of the <Emphasis Type="Italic">Gibberella fujikuroi</Emphasis> complex and fumonisin contamination of pearl millet and corn in Georgia,USA

This study was designed to identify and compare the Fusarium species of the Gibberella fujikuroi complex on pearl millet (Pennisetum glaucum (L.) R. Br) and corn (Zea mays L.) crops grown in southern Georgia, and to determine their influence on potential fumonisin production. Pearl millet and corn samples were collected in Georgia in 1996, 1997 and 1998. Three percent of the pearl millet seeds had fungi similar to the Fusarium species of the G. fujikuroi species complex. One hundred and nineteen representative isolates visually similar to the G. fujikuroi species complex from pearl millet were paired with mating population A (Fusarium verticillioides (Sacc.) Nirenberg), mating population D (F. proliferatum (Matsushima) Nirenberg) and mating population F (F. thapsinum (Klittich, Leslie, Nelson and Marasas) tester strains. Successful crosses were obtained with 50.4%, 10.1% and 0.0% of these isolates with the A, D and F tester strains, while 39.5 of the isolates did not form perithecia with any tester strains. Two of the typical infertile isolates were characterized by DNA sequence comparisons and were identified as Fusarium pseudonygamai (Nirenberg and ODonnell), which is the first known isolation of this species in the United States. Based on the pattern of cross-compatibility, conidiogenesis, colony characteristics and media pigmentation, a majority of the infertile isolates belong to this species. Fumonisins FB₁ and FB₂ were not detected in any of the 81 pearl millet samples analyzed. The species of the G. fujikuroi species complex were dominant in corn and were isolated from 84%, 74% and 65% of the seed in 1996, 1997 and 1998, respectively. Representative species of the G. fujikuroi species complex were isolated from 1996 to 1998 Georgia corn survey (162, 104 and 111 isolates, respectively) and tested for mating compatibility. The incidence of isolates belonging to mating population A (F. verticillioides) ranged from 70.2% to 89.5%. Corn survey samples were assayed for fumonisins, and 63% to 91% of the 1996, 1997 and 1998 samples were contaminated. The total amount of fumonisins in the corn samples ranged from 0.6 to 33.3 g/g.     

Fusarium subglutinans f. sp. Pini represents a distinct mating population in the gibberella fujikuroi species complex

Fusarium strains in the Gibberella fujikuroi species complex cause diseases on a variety of economically important plants. One of these diseases, pitch canker of Pinus spp., is caused by strains identified as Fusarium subglutinans f. sp. pini. Fertile crosses were detected between F. subglutinans f. sp. pini strains from South Africa, California, and Florida. F. subglutinans f. sp. pini strains were not cross-fertile with the standard tester strains of six of the seven other mating populations of G. fujikuroi. Sporadic perithecia with ascospores were obtained in two crosses with the mating population B tester strains. These perithecia were homothallic, and the ascospores derived from these perithecia were vegetatively compatible with the mating population B tester strain parent. We concluded that fertile F. subglutinans f. sp. pini isolates represent a new mating population (mating population H) of G. fujikuroi and that they belong to a unique biological species in a distinct taxon.     

Sexual fertility of fortyFusarium strains from the EuropeanFusarium sambucinum project

Fungus strains designated asFusarium sambucinum, F. torulosum, orFusarium sp. nov. were crossed withMAT1-1 andMAT1–2 tester strains ofGibberella pulicaris. Of the 40 field strains that were crossed with the tester strains, 13 strains produced fertile crosses and 27 strains did not produce fertile crosses. One strain designated asF. torulosum was fertile with a tester strain ofG. pulicaris, suggesting that this is an intraspecies cross and that the strain isG. pulicaris, and, consequently,F. sambucinum rather thanF. torulosum. The lack of fertile crosses between tester strains and 27 of the 40 field strains suggests that these strains are notG. pulicaris. Although the ability to form a fully fertile cross with a tester strain can determine the species of a fertile strain, it is more problematic to exclude a strain only because it is infertile.     

Diversity of Fusarium species and mycotoxins contaminating pineapple

Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B₁–B₃, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB₁ content was 250 μg g^?1 in pineapple skin and 20 μg ml^?1 in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.     

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran

Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20–35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.     

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.     

Differentiation of Fusarium subglutinans f. sp. pini by Histone Gene Sequence Data

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.     

Biodiversity of Fusarium species in Mexico associated with ear rot in maize, and their identification using a phylogenetic approach

Fusarium proliferatum, F. subglutinans, and F. verticillioides are known causes of ear and kernel rot in maize worldwide. In Mexico, only F. verticillioides and F. subglutinans, have been reported previously as causal agents of this disease. However, Fusarium isolates with different morphological characteristics to the species that are known to cause this disease were obtained in the Highland-Valley region of this country from symptomatic and symptomless ears of native and commercial maize genotypes. Moreover, while the morphological studies were not sufficient to identify the correct taxonomic position at the species level, analyses based in the Internal Transcribed Spacer region and the Nuclear Large Subunit Ribosomal partial sequences allowed for the identification of F. subglutinans, F. solani, and F. verticillioides, as well as four species (F. chlamydosporum, F. napiforme, F. poae, and F. pseudonygamai) that had not previously been reported to be associated with ear rot. In addition, F. napiforme and F. solani were absent from symptomless kernels. Phylogenetic analysis showed genetic changes in F. napiforme, and F. pseudonygamai isolates because they were not true clones, and probably constitute separate sibling species. The results of this study suggest that the biodiversity of Fusarium species involved in ear rot in Mexico is greater than that reported previously in other places in the world. This new knowledge will permit a better understanding of the relationship between all the species involved in ear rot disease and their relationship with maize.     

Fusarium temperatum sp. nov. from maize, an emergent species closely related to Fusarium subglutinans

A large number of Fusarium isolates closely related to F. subglutinans were collected from maize in Belgium. We used a robust polyphasic approach to describe a new biological species, Fusarium temperatum, within the Gibberella fujikuroi species complex. F. temperatum can be distinguished from F. subglutinans and from other Fusarium species within the Gibberella fujikuroi species complex with AFLP fingerprint profile, differences in the translation elongation factor 1-α and β-tubulin DNA sequence and interspecies mating compatibility analyses. Intraspecies mating compatibility suggests that sexual reproduction might be common for field isolates of F. temperatum, and reliable female fertile mating population tester strains were proposed for this heterothallic species.     

Mating-type Distribution and Fertility Status in Magnaporthe grisea Populations from Argentina

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.     

Fusarium Species from Nepalese Rice and Production of Mycotoxins and Gibberellic Acid by Selected Species

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.     

Fusarium Species Associated with Mango Malformation in Peninsular Malaysia

Mango malformation has become the most important global disease on mango. Fusarium species previously associated with this disease include F. mangiferae, F. mexicanum, F. sterilihyphosum, F. proliferatum, F. subglutinans and F. tupiense. A few strains of F. proliferatum have been reported from Malaysia, but in this study, we report the results of more extensive sampling. The recovered strains were evaluated with morphology, mating tester strain cross‐fertility, amplified fragment length polymorphisms (AFLPs), and partial DNA sequences of the genes encoding translation elongation factor 1‐α (tef‐1α) and β‐tubulin (tub‐2). Amongst the 43 strains evaluated, three species were identified – F. proliferatum, F. mangiferae and F. subglutinans – with F. proliferatum being the most frequent (69%). None of the Fusarium species that appear to originate in the Americas were recovered in Malaysia, which suggests special measures may be warranted to keep these species from entering the country.     

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.     

Fusarium spp. associated with rice Bakanae: ecology,genetic diversity,pathogenicity and toxigenicity

African and Asian populations of Fusarium spp. (Gibberella fujikuroi species complex) associated with Bakanae of rice (Oryzae sativa L.) were isolated from seeds and characterized with respect to ecology, phylogenetics, pathogenicity and mycotoxin production. Independent of the origin, Fusarium spp. were detected in the different rice seed samples with infection rate ranges that varied from 0.25% to 9%. Four Fusaria (F. andiyazi, F. fujikuroi, F. proliferatum and F. verticillioides) were found associated with Bakanae of rice. While three of the Fusaria were found in both African and Asian seed samples, F. fujikuroi was only detected in seed samples from Asia. Phylogenetic studies showed a broad genetic variation among the strains that were distributed into four different genetic clades. Pathogenicity tests showed that all strains reduced seed germination and possessed varying ability to cause symptoms of Bakanae on rice, some species (i.e. F. fujikuroi) being more pathogenic than others. The ability to produce fumonisins (FB₁ and FB₂) and gibberellin A3 in vitro also differed according to the Fusarium species. While fumonisins were produced by most of the strains of F. verticillioides and F. proliferatum, gibberellin A3 was only produced by F. fujikuroi. Neither fumonisin nor gibberellin was synthesized by most of the strains of F. andiyazi. These findings provide new information on the variation within the G. fujikuroi species complex associated with rice seed and Bakanae disease.     

Identification and Characterization of Toxigenic Fusaria Associated with Sorghum Grain Mold Complex in India

Fusarium species are dominant within the sorghum grain mold complex. Some species of Fusarium involved in grain mold complex produce mycotoxins, such as fumonisins. An attempt was made to identify Fusarium spp. associated with grain mold complex in major sorghum-growing areas in India through AFLP-based grouping of the isolates and to further confirm the species by sequencing part of α-Elongation factor gene and comparing the sequences with that available in the NCBI database. The dendrogram generated from the AFLP data clustered the isolates into 5 groups. Five species of Fusarium—F. proliferatum, F. thapsinum, F. equiseti, F. andiyazi and F. sacchari were identified based on sequence similarity of α-Elongation factor gene of the test isolates with those in the NCBI database. Fusarium thapsinum was identified as predominant species in Fusarium—grain mold complex in India and F. proliferatum as highly toxigenic for fumonisins production. Analysis of molecular variance (AMOVA) revealed 54% of the variation in the AFLP patterns of 63 isolates was due to the differences between Fusarium species, and 46% was due to differences between the strains within a species.     

Identification of Fusarium proliferatum causing leaf spots on Cymbidium orchids in Taiwan

Previous research indicated that black and yellow leaf spots on Cymbidium, Ondontioda, Dendrobium and Cattleya could be caused by Fusarium proliferatum worldwide. However, the agent causing leaf spot on Cymbidium spp. plants is still obscure in Taiwan. Thirty‐five F. fujikuroi species complex (FFSC)‐like isolates were collected from Cymbidium leaf spot from different greenhouses in Taiwan. All isolates were identified as F. proliferatum based on morphological characteristics and molecular analysis. Sequence of translation elongation factor 1‐alpha gene showed 99%–100% homology with F. proliferatum. In addition, two assay techniques using either detached leaves or seedlings were used to evaluate the pathogenicity and host range of the isolates and consequently their effects on Cymbidium and other orchid plants. Pathogenicity assays revealed that all isolates induced black and necrotic spots on detached leaves of Cymbidium, showing 9.4%–29.5% severity on seedlings of Cymbidium. Results of host specificity tests on detached leaves of different plants indicated that the F. proliferatum isolates collected from Cymbidium plants caused severe black spots on Oncidium, Cymbidium, Dendrobium and Cattleya plants. The symptoms on Phalaenopsis plants were relatively mild. Results of host specificity tests on plant seedlings indicated that the F. proliferatum isolates of Cymbidium origin were also pathogenic to Oncidium, Cymbidium and Dendrobium, but not to Cattleya and Phalaenopsis. Phylogenetic analysis of the translation elongation factor (TEF) gene among all fungal isolates using maximum parsimony (MP) and Bayesian inference (BI) methods revealed that the isolates of F. proliferatum from Cymbidium spp. could be separated from other FFSC‐like species with high phylogenetic support.     

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.     

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.