Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa

Four recently described species, Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200–890 g/g), and F. napiforme (16–388 g/g), and by F. nygamai not obtained from millet in Africa (15–874 g/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300–18200g/g) and the 15 isolates from rice with Bakanae disease (2300–19300 g/g). The taxonomic position of these two uncertain taxa should be re-evaluated.     

Differentiation ofFusarium sambucinum Fuckel sensu lato and related species by RAPD PCR

DNA polymorphisms generated by the random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) were used to analyse 41 isolates investigated in the EuropeanFusarium sambucinum Project (EFSP). Employing ten arbitrary (10-mer) oligonucleotides and simple repeat sequences (M13, (GACA)₄) as single primers, informative banding patterns typical for identifying European populations ofFusarium sambucinum Fuckel s. str.,F. torulosum (Berk. & Curt.) Nirenberg andF. venenatum Nirenberg were obtained.     

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.     

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.     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

Protection of maize seedlings byFusarium moniliforme against infection byFusarium graminearum in the soil

The incidence ofFusarium moniliforme in surface-sterilized kernels in two commercial South African white maize cultivars was 64% and 6%, respectively. Heat treatment completely eliminated seedborneF. moniliforme from kernels of both cultivars. Heat treated, uncontaminated maize germlings were pre-inoculated with different isolates ofF. moniliforme and planted in steam-treated soil containing inoculum of different isolates ofF. graminearum Group 1 and Group 2. Seedling weights of germlings pre-inoculated with some isolates ofF. moniliforme were significantly higher than those of controls when exposed to some isolates ofF. graminearum in the soil. The protective effect of pre-inoculation withF. moniliforme was particularly evident in maize seedlings exposed to inoculum of an aggressive isolate ofF. graminearum Group 1. This is the first report of the protection of maize seedlings byF. moniliforme against infection byF. graminearum in the soil.     

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.     

Isolation and characterization of new chlamydosporol related metabolites ofFusarium chlamydosporum andFusarium tricinctum

Fusarium chlamydosporum strain T-826 isolated from corn in the USA produced chlamydosporol and two analogs which have been identified by various spectroscopic techniques as: 7,8-dihydro-5-hydroxy-4-methoxy-trans-7,8-dimethyl-2H,5H-pyrano(4,3-b)pyran-2-2-one (or isochlamydosporol) and 4-methoxy-5-hydroxymethyl-6-(3-butan-2-ol)-2H-pyran-2-one (or chlamydospordiol). Chlamydosporol (compounda+b) chlamydospordiol (compoundc) and isochlamydosporol (compoundd) were produced together (up to 6000 µg/g) by 3 out of 11 isolates ofF. chlamydosporum and by 3 out of 24 isolates ofF. tricinctum from various substrates and geographic origin. Three isolates ofF. chlamydosporum and one isolate ofF. tricinctum produced only chlamydospordiol and 2 isolates ofF. tricinctum produced chlamydosporol (a+b), and chlamydospordiol (c)PRC Publication, No. 1518     

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.     

Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A

Lipopeptides represent a unique class of bioactive microbial secondary metabolites, and iturin A shows attractive antibiotic properties among them. This study compares three methods, such as yeast/fungal growth inhibition assay, quantitative high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) for identifying a number of Bacillus species that produce iturin A. We examined the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD and lpa-14 amplification. Twenty standard strains and 120 field-collected Bacillus spp. isolates were tested in this study. Four B. subtilis and one B. circulan strains from ATCC, and B. amyloliquefaciens B128, a known iturin A producer, exhibited positive results. Of the 120 field-collected isolates, 42 strains were positive. The potential of producing iturin A by these PCR-positive strains were then confirmed by conventional methods such as fungal growth inhibition assay and HPLC analysis. The consistency between results of PCR, HPLC, and fungal growth inhibition assay suggests that the PCR method could be used as an alternative tool for fast screening of iturin A-producing Bacillus strains from the environment. This is the first report of detecting iturin A production from B. circulans.     

The distribution ofFusarium species in soils planted to millet and sorghum in Lesotho,Nigeria and Zimbabwe

Samples from soils planted to millet and sorghum from Lesotho, Nigeria, and Zimbabwe were processed and a total of 3,291Fusarium cultures were recovered. Of these 1,296 cultures were isolated from plant debris and 1,995 cultures were recovered from soil dilutions. The most prevalent species recovered wereF. oxysporum (37%),F. equiset (30%),F. solani (14%),F. moniliforme (6%),F. compactum (5%),F. nygamai (4%), andF. chlamydosporum (2%). OtherFusarium species isolated wereF. merismoides, F. polyphialidicum, F. graminearum, F. subglutinans, F. sambucinum, F. longipes, F. semitectum, F. dimerum, F. lateritium, and a group of cultures designated as population A which resembleF. camptoceras. Fusarium equiseti was the predominant species in soil samples from Nigeria and Zimbabwe, whileF. oxysporum was the predominant species recovered from soil from Lesotho.Contribution No. 1881, Fusarium Research Center, Department of Plant Pathology, The Pennsylvania State University.     

<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.     

Rapid screening of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mutants for overproduction of lovastatin

Summary A novel rapid screening method is demonstrated for isolating lovastatin-overproducing strains of Aspergillus terreus. The screening methodology, based on the activity of lovastatin against the yeast Candida albicans, is nearly three times as fast as the selection methods used earlier. The new 6-h assay shows a linear correlation between the quantity of lovastatin generated by A. terreus isolates and the inhibition zones obtained on plates of C. albicans. The new technique is less expensive and requires less labour.     

Natural variation of ascospore and conidial germination by Fusarium verticillioides and other Fusarium species

Fusarium verticillioides and other Fusarium species were examined for their spore germination phenotypes. In general, germinating spores of F. verticillioides formed germ tubes that immediately penetrated into agar. Such invasive germination was the predominant growth phenotype among 22 examined field isolates of F. verticillioides from a broad range hosts and locations. However, two of the field isolates were unique in that they formed conidial germ tubes and hyphae that grew along the surface of agar before penetration eventually occurred. Conidia of 22 other Fusarium species were assessed for their germination phenotypes, and only some strains of F. annulatum, F. fujikuroi, F. globosum, F. nygamai, and F. pseudoanthophilum had the surface germination phenotype (21 % of the strains assessed). Sexual crosses and segregation analyses involving one of the F. verticillioides surface germination strains, NRRL 25059, indicated a single locus, designated SIG1 (surface vs. invasive germination), controlled the germ tube growth phenotypes exhibited by both conidia and ascospores. Perfect correlation was observed between an ascospore germination phenotype and the germination phenotype of the conidia produced from the resulting ascospore-derived colony. Recombination data suggested SIG1 was linked (7 % recombination frequency) to FPH1, a recently described locus necessary for enteroblastic conidiogenesis. Corn seedling blight assays indicated surface germinating strains of F. verticillioides were less virulent than invasively germinating strains. Assays also indicated pathogenicity segregated independently of the FPH1 locus. Invasive germination is proposed as the dominant form of spore germination among Fusarium species. Furthermore, conidia were not necessary for corn seedling disease development, but invasive germination may have enhanced the virulence of conidiating strains.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Flavobacterium frigidimaris sp. nov., isolated from Antarctic seawater

We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flavobacterium hydatis, F. pectinovorum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA–DNA hybridization experiments showed that the DNA-similarities between strain KUC-1^T and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1^T can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1^T: JCM 12218^T and DSM 15937^T; mol% G+C of DNA of the type strain is 34.5 mol%). Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature, and cellular fatty acid composition, were also determined.     

Preservation of fungi in water (Castellani): 20 years

Sixty-two isolates of Fusarium were obtained from pasture grass and soil from various areas of New Zealand and identified as F. anthophilum [2], F. avenaceum [17], F. crookwellense [8], F. culmorum [4], F. graminearum [1], F. nivale [3], F. oxysporum [3], F. sambucinum [17], F. semitectum [1], F. tricinctum [1] and an unidentified Fusarium spp. [5]. These isolates were grown on autoclaved rice and tested for toxicity to rats in feeding tests. Eighty two percent of the isolates were toxic, of which twenty-four percent were severely toxic and caused hemorrhages of stomach and intestine, hematuria, and finally death. Cultures of the most toxic isolates contained 0.1 to 104 ppm of deoxynivalenol, 0.7 and 7 ppm of 15- and 3-acetyldeoxynivalenol respectively, 0.2 to 4 ppm of fusarenon- X, 11 to 1021 ppm zearalenone, 40 to 272 ppm of the hemorrhagic factor (wortmannin), 2,100 to 7,200 ppm of moniliformin, 565 ppm of the cytotoxic factor (HM-8) and enniatin in substantial concentrations. F. sambucinum is reported as a moniliformin producer for the first time.     

Biological Interactions to Select Biocontrol Agents Against Toxigenic Strains of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> from Maize

Biological control represent an alternative to the use of pesticides in crop protection. A key to progress in biological control to protect maize against Fusarium verticillioides and Aspergillus flavus maize pathogens are, to select in vitro, the best agent to be applied in the field. The aim of this study was to examine the antagonistic activity of bacterial and yeast isolates against F.verticillioides and A. flavus toxigenic strains. The first study showed the impact of Bacillus amyloliquefaciens BA-S13, Microbacterium oleovorans DMS 16091, Enterobacter hormomaechei EM-562T, and Kluyveromyces spp. L14 and L16 isolates on mycelial growth of two strains of A. flavus MPVPA 2092, 2094 and three strains of F. verticillioides MPVPA 285, 289, and 294 on 3% maize meal extract agar at different water activities (0.99, 0.97, 0.95, and 0.93). From this first assay antagonistics isolates M. oleovorans, B. amyloliquefaciens and Kluyveromyces sp. (L16) produced an increase of lag phase of growth and decreased a growth rate of all fungal strains. These isolates were selected for futher studies. In vitro non-rhizospheric maize soil (centrally and sprayed inoculated) and in vitro maize (ears apex and base inoculated) were treated with antagonistics and pathogenic strains alone in co-inoculated cultures. Bacillus amyloliquefaciens significantly reduced F. verticillioides and A. flavus count in maize soil inoculated centrally. Kluyveromyces sp. L16 reduced F. verticillioides and A. flavus count in maize soil inoculated by spray. Kluyveromyces sp. L16 was the most effective treatment limiting percent infections by F. verticillioides on the maize ears.     

Arcobacter ellisii sp. nov., isolated from mussels

As part of a study carried out for detecting Arcobacter spp. in shellfish, three mussel isolates that were Gram-negative slightly curved rods, non-spore forming, showed a new 16S rDNA-RFLP pattern with a specific identification method for the species of this genus. Sequences of the 16S rRNA gene and those of the housekeeping genes rpoB, gyrB and hsp60 provided evidence that these mussel strains belonged to an unknown genetic lineage within the genus Arcobacter. The similarity between the 16S rRNA gene sequence of the representative strain (F79-6^T) and type strains of the other Arcobacter species ranged between 94.1% with A. halophilus and 99.1% with the recently proposed species A. defluvii (CECT 7697^T). DDH results between strain F79-6^T and the type strain of the latter species were below 70% (53 ± 3.0%). Phenotypic characteristics together with MALDITOF mass spectra differentiated the new mussel strains from all other Arcobacter species. All the results indicate that these strains represent a new species, for which the name Arcobacter ellisii sp. nov. with the type strain F79-6^T (=CECT 7837^T = LMG 26155^T) is proposed.