Morphological differentiation ofFusarium sambucinum Fuckel sensu stricto,F. torulosum (Berk. & Curt.) Nirenberg comb. nov. andF. venenatum Nirenberg sp. nov.

WithinFusarium sambucinum Fuckel sensu lato three species were differentiated:F. sambucinum Fuckel s. str.,F. torulosum (Berk. et Curt.) Nirenberg comb. nov. andF. venenatum Nirenberg sp. nov. They are described and illustrated in detail.     

Analysis of esterase zymograms ofFusarium sambucinum and related species

Esterase zymograms were obtained following polyacrylamide slab gel electrophoresis of protein extractsFusarium sambucinum and related species originating from different geographic locations and different matrices. The sites of esterase activity were recorded, and the R_fs were calculated. The data were used for the construction of phenograms by cluster analysis and nonlinear mapping by computerized classification techniques. The fifteen isolates ofF. sambucinum, the eight isolates ofF. torulosum and the six isolates ofF. spec. nov. each had identical profiles, and are therefore electrophoretically distinct species. The isolates ofF. sarcochroum, one ofF. sambucinum sensu lato (BBA 64280) and fifteen isolates ofF. sambucinum were electrophoretically indistinguishable from each other. We assume they are synonymous. The isolate ofF. bactridioides, one ofF. sambucinum sensu lato (BBA 64993) and eight isolates ofF. torulosum had uniform EST patterns, therefore the two species are electrophoretically identical. We assume they are also synonymous. The remaining three isolates ofF. sambucinum sensu lato are somewhat closely related toF. sambucinum isolates on the basis of our investigations.     

Chemical and physiological characterization of taxa in theFusarium sambucinum complex

Forty-one isolates ofFusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains ofF. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remainingF. sambucinum strains produced T-2 toxin, TB1 and TB2.Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB.Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.     

Computer-aided morphometrical analysis of macroconidia inFusarium sambucinum Fuckel sensu lato strains

Forty-one strains ofFusarium sambucinum Fuckelsensu lato were classified on the basis of morphological data of the macroconidia obtained by automated microscopic image analysis. The classification factors were determined in sixFusarium strains of the sectionDiscolor. The results show the relative homogeneity within the groups proposed by Nirenberg. A remarkable difference could be detected especially between the groupsF. sambucinum, F. torulosum andF. sambucinum sensu lato on the one hand andF. spec nov. on the other.Abbreviations PDA potato dextrose agar - SNA synthetic nutriment agar     

<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.     

Phylogenetic relationship withinFusarium sambucinum Fuckel sensu lato,determined from ribosomal RNA sequences

Partial ribosomal RNA nucleotide sequences were determined for 11 strains ofFusarium sambucinum Fuckelsensu lato to assess by molecular genetic means, Nirenberg's recent morphotaxonomic interpretation which split the species into three distinct taxa:F. sambucinum sensu stricto, F. torulosum, and one other species, as yet unnamed (Fusarium species nova). Four sequence patterns were identified among the 11 strains. Two sequences that varied at one site were found among strains ofF. sambucinum, strains ofF. torulosum andFusarium sp. nov. showed no intraspecific variation. Interspecific comparisons revealed nucleotide sequence differences of 3–9 substitutions in the ca. 240 nucleotide rRNA segment examined. Although interspecific differences are not large in terms of percent nucleotide substitution, they are much larger than the observed intraspecific variation and support the morphological interpretation distinguishing three taxa. When the data were analysed using parsimony and bootstrapping, the three taxon tree was well supported. The phylogenetic arrangement of these strains is congruent with secondary metabolite profile similarities.     

Production of type A trichothecenes and enniatin B byFusarium sambucinum Fuckel sensu lato

Twenty-nineFusarium isolates, representing three new taxa originated by Nirenberg fromF. sambucinum Fuckel sensu lato, namely:F. sambucinum Fuckel sensu stricto,F. venenotum Nirenb., andF. torulosum (Berk. & Curt.) Nirenb., were tested for in vitro production of toxic secondary metabolites on autoclaved corn kernels.F. sambucinum sensu stricto was able to produce type A trichothecenes and enniatin B (EB). In particular, amongst the 14 isolates tested, 5 produced only diacetoxyscirpenol (DAS) (up to 700 µg/g); 1 produced only neosolaniol (NEOS) (250 µg/g); 2 produced T-2 toxin (T-2) + NEOS (up to 175 and 150 µg/g, respectively); 1 produced NEOS + DAS (300 and 100 µg/g, respectively); and 5 produced DAS + EB (up to 500 and 140 µg/g, respectively). All six isolates ofF. venenotum were able to produce only DAS (up to 100 µg/g).F. torulosum produced no trichothecenes, but four out of nine tested isolates were able to produce EB (up to 140 µg/g). Zearalenones and type B trichothecenes were not found. The toxicity of the culture extracts towardsArtemia salina L. was correlated in general with the occurrence of the above toxins, except for someF. torulosum strains. However, the lack of correlation between the amounts of toxins recovered and toxic activity observed in theGeotrichum candidum Link ex Pers. andA. salina assays suggested the presence of unknown toxic compounds.     

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.     

A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates

During the 1989 corn harvest season, numerous reports of equine leukoencephalomalacia (ELEM) outbreaks and a pulmonary edema (PPE) syndrome in swine from several regions of the United States were received by the National Veterinary Services Laboratories (NVSL), Ames, Iowa. Previous and concurrent research linked Fusarium moniliforme and fumonisin-contaminated feeds to both diseases. Chemical and mycological investigations revealed fumonisin B₁ (FB₁) concentrations of 20 to 360 ppm in suspect swine feeds and 8 to 117 ppm in suspect equine feeds. Nonproblem feeds contained concentrations below 8 ppm. Fusarium moniliforme and Fusarium proliferatum were isolated from both problem and nonproblem equine and swine feeds. When cultured on autoclaved corn, the F. moniliforme and F. proliferatum isolates produced respective FB₁ and fumonisin B₂ (FB₂) that range from less than 5 to more than 2450 ppm and less than 5 to more than 1000 ppm, respectively. Isolates from both problem and nonproblem feeds produced high levels (greater than 500 ppm) in culture. Reported here is a review of chemical and mycological data resulting from the study of several cases of PPE and ELEM.     

Diversity of the <Emphasis Type="Italic">Fusarium</Emphasis> species associated with head and seedling blight on wheat in Slovakia

Species associated with Fusarium head blight are depending on the production and edaphic conditions. The differences are found in the representation of various Fusarium spp. in the diseases, which sporadically occur all over the territory of Slovakia, in all agricultural production types. We identified fifteen Fusarium species during ten years of investigation. Most of the mentioned species F. culmorum (W.G. Smith) Sacc., F. graminearum Schwabe, recently F. cerealis (Cooke) Sacc. (crookwellense Burgess, Nelson & Tousson) and F. sambucinum Fuckel in diseased caryopsis are seed transmitted. The significant differences among species and intra species in cultural and pathogenicity assays in vitro and in vivo were correlated. Some of them are able to produce toxic metabolites — deoxynivalenone, which probably play a role in the aggressiveness of the pathogen and promote disease development and pathogen colonization.     

Untersuchungen zur pathogenität und mykotoxinbildung von<Emphasis Type="Italic">Fusarium sambucinum</Emphasis>, dem erreger der trockenfäule an kartoffeln

Investigation into virulence and mycotoxin formation of the dry rot causing pathogen Fusarium sambucinum on potatos 11 strains ofFusarium sambucinum were isolated from tubers with dry rot symptoms from three different depots in the Land Brandenburg and Saxony-Anhalt. All isolates produced diacetoxyscripenol in artificially infected potato tubers. Additionally, two isolates produced T-2 and HT-2 toxins as well. The virulence and mycotoxin formation of the isolates was dependent on the potato varieties ‘Sieglinde’ and ‘Berber’ used in the experiment. The amount of diacetoxyscripenol in diseased tissue was positively correlated with the virulence of theF. sambucinum isolate and the susceptibility of the potato variety as well.     

Toxicity of mycotoxins of Fusarium sambucinum for feeding in Galleria mellonella

Fusarium sambucinum Fuckel showed antifeedant activity towards larvae of Galleria mellonella L. when incorporated into insect diet. The activity appeared mostly due to the concentration of trichothecenes present in the fungal extracts. Diacetoxyscirpenol and neosolaniol showed similar levels of activity and were significant potent antifeedants against larvae at 50 and 100 ppm. On the contrary, enniatin B showed no activity up to 100 ppm.     

Biocontrol of Fusarium sambucinum,dry rot of potato,by Serratia plymuthica 5–6

Serratia grimesii 4–9 and Serratia plymuthica 5–6, isolated from the rhizosphere of pea, Pisum sativum (L), were evaluated for their potential to suppress growth of Fusarium sambucinum in vitro and to reduce Fusarium dry rot in stored potatoes (Solanum tuberosum L). In vitro studies indicated that these bacterial isolates suppressed growth of F. sambucinum by 60% or more at both 15 and 25°C. In a potato tuber slice assay the number of infection sites in potato slices exposed to F. sambucinum and treated with S. grimesii 4–9 and S. plymuthica 5–6 was reduced by 96 and 97%, respectively, at 15°C. The diameter (mm) of the infection sites was reduced 91 and 96%, respectively, when compared to slices treated with F. sambucinum alone. Studies with Fusarium-infected whole potato tubers also showed significant reduction in dry rot formation following treatment with the bacterial isolates or the fungicide thiabendazole. When applied simultaneously with the pathogen, S. grimesii 4–9 and S. plymuthica 5–6 suppressed development of Fusarium dry rot by 60 and 77%, respectively, at 15°C and by 63 and 84%, respectively, at 25°C compared to tubers inoculated with the pathogen alone. Thiabendazole suppressed development of Fusarium dry rot by 66 and 81% at 15 and 25°C, respectively, compared to tubers inoculated with the pathogen alone. These studies demonstrate the potential of soil bacteria as biofungicides for managing post-harvest crop diseases. Due to the potential risks to human health associated with S. grimesii 4–9, S. plymuthica 5–6 is recommended for further study for biofungicide development.     

A new fumonisin from solid cultures of Fusarium moniliforme

A new fumonisin has been isolated from Fusarium moniliforme isolate MRC826 grown on corn. It was shown by NMR and mass spectrometry to be an isomer of fumonisin B₂ that has free hydroxyl groups at C-3 and C-10 instead of the normal C-3 and C-5. This new fumonisin was detected in cultures of most isolates of F. moniliforme that were examined and was usually present at concentrations similar to those of fumonisin B₂. Two isolates of F. moniliforme that produce significantly higher levels of this new isomer were identified.Abbreviations ELEM equine leukoencephalomalacia Mention of companies or products by name does not imply their endorsement by the US Department of Agriculture over others not cited.     

Production of trichothecene mycotoxins byFusarium graminearum andFusarium culmorum on barley and wheat

Wheat cultivars (Stoa, MN87150, SuMai-3, YMI-6, Wheaton) and barley cultivars (Robust, Excel, Chevron, M69) were inoculated in the field with isolates ofFusarium graminearum andF. culmorum. The diseased (Fusarium head blight) kernels were analyzed for deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV).F. culmorum produced all three trichothecenes on all cultivars tested whereasF. graminearum only produced DON and 15-ADON. There was no well defined correlation between DON production in the host and resistance although the data tended to favor SuMai-3 as having definitive resistance to bothF. graminearum andF. culmorum.Minnesota Agricultural Experiment Station, Paper No. 20 279.     

Fusarium fractiflexum sp. nov. and two other species within theGibberella fujikuroi species complex recently discovered in Japan that form aerial conidia in false heads

Morphological and molecular phylogenetic analyses were conducted on 12 strains ofFusarium, deposited in MAFF asF. subglutinans (≡F. moniliforme var.subglutinans≡F. sacchari var.subglutinans) orFusarium sp. because they formed aerial conidia in false heads in the dark. These strains were resolved as three distinct species within theGibberella fujikuroi species complex. A new species,F. fractiflexum, and two species new to Japan,F. circinatum andF. concentricum, are described and illustrated and their morphological features are discussed.Fusarium fractiflexum, isolated from diseased yellow leaf spots ofCymbidium spp., is differentiated from other fusaria based on its yellowish colonies and aerial conidia formed in false heads in the dark and in zigzag-like conidial chains under black light. Japanese strains ofF. circinatum also formed elongate, coiled sterile hyphae. Phialidic aerial conidia with a pointed apex and a wedgeshaped base were found inF. concentricum cultured under black light and represent a new diagnostic character of the species, in addition to colonies with alternating concentric rings when cultured on PDA. Based on DNA sequences of the β-tubulin gene and two other loci, strains ofF. fractiflexum were resolved phylogenetically as members of the Asian clade of theG. fujikuroi species complex. In addition, Japanese strains ofF. circinatum andF. concentricum were phylogenetically identical to the ex-type strains.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Production of fumonisins byFusarium species of Taiwan

Twenty-nineFusarium species isolated from various sources in different districts of Taiwan were tested for their ability to produce fumonisins in corn cultures. OnlyFusarium moniliforme produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). The finding that the other 28Fusarium species produced neither FB₁ nor FB₂ is preliminary because only one strain per species was studied. The detection of FB₁ and FB₂ in cultures ofF. moniliforme was demonstrated by TLC and HPLC, and FB₁ was further confirmed by mass spectrometry. In a separate experiment, in which 38 strains ofF. moniliforme were tested for fumonisins, approximately 66% (25/38) produced FB₁ and/or FB₂. Of the 25 strains, 14 produced only FB₁ and 11 produced both FB₁ and FB₂, and the amounts of FB₁ and FB₂ produced by different strains varied greatly. This is the first report that fumonisins are found in corn cultures experimentally infected withF. moniliforme strains from Taiwan. It is safe to assume that fumonisin producing strains ofF. moniliforme are widely distributed among the economic crops such as corn, rice, sugarcane, and sorghum throughout the Island.Abbreviations FB₁ Fumonisin B₁ - FB₂ Fumonisin B₂ - OPA o-phthalidialdehyde     

Biosynthesis of labeled fumonisins in liquid cultures of Fusarium moniliforme

Fumonisins were readily produced in cultures of Fusarium moniliforme using a defined liquid medium. Addition of 200 mg of d₃-methyl L-methionine to 100-ml cultures of F. moniliforme gave increased overall yields and high levels of deuterium (²H) incorporation into fumonisin B₁. Approximately 90% of the resulting fumonisin B₁ contained 6 deuterium atoms, while 9% of the product contained 3 deuterium atoms. Deuterium was shown to be incorporated exclusively in the methyl groups of the fumonisin backbone. The addition of as little as 5 mg of labeled methionine stimulated fumonisin production, but only about 5% of the fumonisin produced contained 3 deuterium atoms.Abbreviations ELEM equine leukoencephalomalacia Mention of companies or products by name does not imply their endorsement by the US Department of Agriculture over others not cited.     

Species-specific PCR Assays for the Fungal Pathogens Fusarium moniliforme and Fusarium subglutinans and their Application to Diagnose Maize Ear Rot Disease

Fusarium moniliforme Sheldon (syn. F. verticillioides (Sacc.) Nirenberg) and F. subglutinans (Wollenweber & Reinking) Nelson Toussoun & Marasas comb. nov., two anamorphs of the so-called‘Gibberella fujikuroi species complex', are important maize pathogens. Together with F. proliferatum, F. culmorum, and F. graminearum (teleomorph: Gibberella zeae) they are involved in the stalk rot and ear rot disease of maize. All species produce secondary metabolites (mycotoxins) which are a potential health hazard for humans and animals that consume maize and maize products frequently. In this study the development of polymerase chain reaction (PCR) assays for an easy and sensitive identification of G. fujikuroi anamorphs in maize kernels are described. The primer pairs are based on sequences of randomly amplified polymorphic DNA (RAPD) fragments and are specific for F. moniliforme and F. subglutinans respectively. The PCR assays are independent of the high phenotypic variability of traits which may complicate classification by morphological characters. They detect approximately 100 to 200 fungal genomes in the presence of an excess of maize DNA. For the analysis of infected maize kernels a rapid and easy DNA extraction was used which does not introduce inhibitory substances into the PCR. Hence the assays enable an early identification and detection of the two pathogens in host tissue by plant breeders and plant health inspection services. The assays were successfully applied to identify field isolates from Poland and to detect the pathogens in maize ears of various hybrids in Germany.