Biosynthesis ofFusarium mycotoxins and genomics ofFusarium verticillioides

Analyses of mycotoxin biosynthetic genes inFusarium indicate that interspecies variation in trichothecene structure can result from differences in gene function and interspecies variation in fumonisin production/non-production can result from differences in the presence/absence of genes. Such variation is not always correlated with phylogenetic relationships of species as determined by sequencing primary metabolic genes; distantly related species can share the same mycotoxin biosynthetic genotype and resulting phenotype, while more closely related species can differ. These findings provide further evidence that the evolution of mycotoxin biosynthesis inFusarium has not always been congruent with the evolution of species. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005.     

A survey of the natural occurrence ofFusarium toxins in wheat grown ina southwestern area of Germany

Wheat for feed use (84 samples) was collected after harvest from 79 farms in a southwestern part of Germany (Baden-Wuerttemberg). The 1987 crop year was characterized by heavy rainfall in the summer months. The internal mycoflora of wheat samples was primarily fusaria, and storage fungi were rarely present. TheFusarium toxins, zearalenone (ZON), - and -zearalenol (,-ZOL), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), T-2 Toxin (T-2), HT-2 toxin (HT-2) and diacetoxyscirpenol (DAS) were analysed by gas chromatography with mass selective detection (detection limit: 1–3 µg/kg). Each of the samples contained at least one of theFusarium toxins examined except DAS. DON, ZON, 3-AcDON, NIV, T-2, HT-2 and -ZOL were detected in 96%, 80%, 59%, 26%, 26%, 7% and 5% of samples, with an average of 1632, 178, 7, 9, 82, 10 and 23 µg/kg, and a maximum of 20538, 8036, 18, 32, 249, 20 and 71 µg/kg, respectively. -ZOL (12 µg/kg) was found in one sample with -ZOL (71 µg/kg). One, two, three, four, five and sixFusarium toxins were detected in 6%, 27%, 37%, 23%, 4%, and 4% of total samples, respectively. The most frequent combination was that of ZON with DON and 3-AcDON, followed by the combinations ZON/DON and ZON/DON/3-AcDON/NIV in 22, 20, and 11% of total samples, respectively.Abbreviations 3-AcDON 3-Acetyldeoxynivalenol - DAS Diacetoxyscirpenol - DON Deoxynivalenol - HT-2 HT-2 toxin - NIV Nivalenol - T-2 T-2 toxin - -ZOL -Zearalenol - -ZOL -Zearalenol - ZON Zearalenone     

Standardization of an indirect PTA-ELISA for detection ofFusarium spp. in infected grains

Plant pathogenic fungi of the genusFusarium cause agriculturally important diseases of small grain cereals and maize. Especially the contamination of grains with the mycotoxin deoxynivalenol (DON) is harmful for animals and humans. For quantification of the severity ofFusarium head blight (FHB) in resistance evaluation and selection of cereals a fast, economical and reliable method is essential. Immunological methods appear to be particularly suitable for such an assessment. The results demonstrate that a selected antiserum was appropriate for the detection ofFusarium-exoantigens (ExoAg) in cereal grains by an indirect ELISA-format, although a discrimination between variousFusarium-species was not possible. The polyclonal antibody detection system was optimized for different parameters and a standard test protocol was elaborated. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

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.     

The phytotoxicity ofFusarium metabolites: An update since 1989

The present article summarises the published phytotoxic effects of severalFusarium metabolites (mycotoxins, phytotoxins, antibiotics and pigments) since 1989. The phytotoxicity of many of the commonly isolated metabolites cannot be disputed, but their role in pathogenesis ofFusarium-induced plant diseases is uncertain. Plant species/varieties differ in their susceptibililty resistance to these toxinsin vitro, as well as toFusarium pathogens under field conditions. Such variations in plant response may reflect resistance mechanisms that operate at several levels, including an initial ability to prevent fungal invasion; prevention of fungal spread and toxin tolerance or degradation. Little is known about the mode of action of most of these metabolites on either animal or plant cells. Several novelFusarium metabolites have been isolated in the past few years. Many are toxic to animals and cell lines, but assessment of their phytotoxicity has largely been neglected. Since many plant pathogenic Fusaria produce a plethora of metabolites, the additive or synergistic actions of toxins in combination must be considered in plant pathology.     

Impact of tillage on the incidence ofFusarium spp. in soil

The influence of several long-term conventional and conservation tillage treatments on the incidence and the diversity ofFusarium spp. in soil was studied. Soil samples were randomly collected from naturally contaminated field trials and Fusarium species were isolated by using the dilution plate method. The identification of the species was done by direct microscopic observation onFusarium-specific media. The isolation frequency ofFusarium species and the total number of colony forming units was affected by the sampling year and the cultivated crop and showed significant differences between the tillage treatments. Moldboard plough-treatments resulted in a lower diversity of Fusarium species than the chisel plough and rotary tiller treatments. Besides the tillage system the tillage depth also appeared to affect theFusarium populations. The deeper the tillage the lower was the number of isolatedFusarium spp. TwentyFusarium species were identified over both years of investigation. In conservation tillage plots a higher diversity ofFusarium species was found than in the moldboard plough-based tillage plots. A correlation betweenFusarium species producing the mycotoxin deoxynivalenol (DON) isolated from soil and the DON-content of grain could not be observed. However, these investigations indicate that conservation soil tillage results in conditions which increase the incidence ofFusarium species in soil.     

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.     

The effect of increasing concentrations ofFusarium toxins in the diets for piglets on histological parameters of the uterus

The effects of feeding diets containing 0.01, 0.06, 0.15, 0.22 and 0.42 mg zearalenone and 0.2, 0.8, 1.0, 1.9 and 3.9 mg deoxynivalenol per kg, coming fromFusarium toxin contaminated maize, on the uterus of 50 prepubertal piglets were investigated. The mean weight of the uteri of the animals which received the most contaminated diet was significantly increased at the time of slaughtering. The histological investigation showed no marked differences between the feeding groups. Histometrical parameters of the surface epithelium of the uterus and of the uterine glands were not altered by the treatment. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Level ofFusarium infection in wheat spikelets related to location and number of inoculated spores

The flowering time is the most susceptible period for primary infection of wheat heads byFusarium spp. During this period spores can be deposited into the opened wheat florets where they may later cause infections. We quantitatively explored the relationship between variables related to the flowering process and the infection level byFusarium graminearum in single spikelets. We imitated open (chasmogamous) and closed (cleistogamous) flowering by injecting well-defined amounts of spores into and between wheat florets. Applying the spores between the florets resulted in weaker disease symptoms and significantly lower amounts ofFusarium mycotoxins. With larger numbers of spores, the disease symptoms became more pronounced and the mycotoxin amounts per spikelet increased significantly. Our results indicate that the probability of primary infection is approximately proportional to the number of spores reaching the open florets during the flowering process. The breeding of wheat lines which flower partially or completely cleistogamously might reduce theFusarium susceptibility in wheat.     

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.     

Grass-to-tree facilitation in an arid grazed environment (Aïr Mountains, Sahara)

The scarcity of tree regeneration is a major concern for the long-term conservation of tree cover in the Aïr-Ténéré Nature Reserve (Sahara, Niger), and the causes of this shortage are poorly understood. We raised the hypothesis that livestock pressure is a key-factor in the mortality of tree seedlings and that facilitation processes among plants may enhance tree regeneration. We tested whether the tussock grass Panicum turgidum could facilitate the regeneration of a keystone tree species, Acacia tortilis var. raddiana, under a grazing regime. We transplanted 3-month-old seedlings of Acacia to plots representing different neighbourhood conditions (in areas without vegetation, amongst intact tussocks, among cropped tussocks, and under branches), which were either fenced or exposed to grazing by large herbivores. Survival and height of 160 transplants were recorded during one year. Acacia performance was much lower in the grazed plots, indicating that large herbivores had a strong negative impact on tree regeneration. Presence of Panicum tussocks had a positive effect on the height of seedlings, likely because they improved soil properties and water availability. No aboveground positive effect on Acacia was detected though the grass did reduce atmospheric aridity. We detected an indirect positive effect of Panicum on Acacia survival in the presence of herbivores when aridity and grazing pressure reached their maximum levels. We obtained evidence that indirect facilitation may occur in arid grazed areas, although it has been predicted to mainly exist in mesic competitive environments. Additionally, our results support the stress-gradient hypothesis against assumptions that competitive effects may dominate in extremely arid environments. Since most of the positive effect on Acacia survival is indirect (biotic grazing refuge) due to the presence of herbivores, transplantation of tree seedlings amongst Panicum may be a simple and low-cost technique for the restoration of Saharan ecosystems degraded by herbivore grazing.     

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     

Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Grants from the National Technology Agency of Finland (No. 40168/03) and the Academy of Finland (No. 52104); travel grants from NorFA and the European Commission to the Laboratory of Dr. Ulf Thrane     

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the same mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.     

Edaphic factors and plant species distribution in a protected area in the desert of Bahrain Island

Effects of edaphic factors (salinity, pH, Na⁺, K⁺, Ca⁺⁺, CaCO₃, water holding capacity, and grain size) on the spatial distribution of plants were investigated. Soil was sampled at 22 stands. Sixteen plant species were recorded from these stands. Relation between edaphic factors and plant distribution was investigated using correlation statistical analysis. Distribution of some plants was found to be highly correlated with edaphic factor(s).     

Responses of four arid zone grass species from varying habitats to drought stress

The effects of 4 or 8 drought cycles on four grass species,Cenchrus pennisetiformis, Leptochloa fusca, Panicum turgidum, andPennisetum divisum were assessed in a pot experiment. There were significant differences between the species in biomass production under water stress.C. pennisetiformis andP. turgidum produced significantly greater fresh and dry matter thanP. divisum and especially thanL. fusca. L. fusca had the lowest andP. divisum highest osmotic potentials compared with the other species after the completion of 4 or 8 drought cycles. Osmotic adjustment (difference between osmotic potential of droughted/rehydrated plants and control plants) was highest inL. fusca. The stomatal conductance was significantly decreased with increased drought stress inC. pennisetiformis. The elasticity ofC. pennisetiformis, P. turgidum andP. divisum increased with increase in number of drought cycles, whereas that ofL. fusca remained unchanged.L. fusca andP. turgidum had the lowest leaf hydration of all species after 8 drought cycles. The chlorophyllsa andb in all species remained unaffected by drought treatments. The proline content ofC. pennisetiformis andL. fusca increased significantly with increased drought stress, whereas that ofP. turgidum remained unaffected after 4 or 8 drought cycles.L. fusca synthesized great amount of leaf soluble proteins during 8 drought cycles, whereasP. divisum had low protein content after 4 drought cycles. The protein contents ofC. pennisetiformis andP. turgidum remained unaffected after 8 drought cycles. The leaf epicuticular wax ofL. fusca increased consistently with increased drought stress, but leaf wax ofP. divisum increased only at the highest drought stress and that ofC. pennisetiformis andP. turgidum increased after 4 drought cycles. On the basis of these results it was established thatC. pennisetiformis andP. turgidum were the most tolerant,P. divisum intermediate, andL. fusca the most sensitive to drought stress. The osmotic adjustment did not positively correlate with the degree of drought resistance.     

Mycoparsitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui

Summary Mycoparasitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui was studiedin vitro. Growth of the hyphae ofF. oxysporum inside the hyphae and conidiophores ofA. luchuensis and formation of resting bodies ofF. oxysporum in conidiophores and hyphae ofA. luchuensis were observed. Bursting of the host hypha attacked by the parasitic hyphae was also noticed.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.     

Antibiotic inhibition of pectolytic and cellulolytic enzyme activity in twoFusarium species

Among all antibiotics tested, amoxycillin (500 ppm) completely inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activity inF. oxysporum; none of the antibiotics did so inF. moniliforme. No antibiotic completely inhibited the cellulase activity in both test organisms, however, amoxycillin was better than other antibiotics in inhibiting the cellulase activity in both the organisms.     

Toxicity ofFusarium sambucinum Fuckel sensu lato to brine shrimp

In a screening test sevenFusarium strains out of 17 proved to be toxic towards brine shrimp. Among these, fourF. sambucinum strains as well as threeF. venenatum isolates caused toxic effects. The chemical screening by TLC analysis revealed the presence of sambucinol and diacetoxyscirpenol in the extracts of the toxic isolates ofF. venenatum 64537.