重要产毒镰刀菌合成白僵菌素和恩镰孢菌素研究进展

白僵菌素(beauvericin,BEA)和恩镰孢菌素(enniatins,ENNs)是由镰刀菌属的多种真菌产生的六酯肽类真菌毒素,该类毒素对上皮细胞、免疫细胞、卵巢细胞等具有很强的毒性作用。本文主要针对能产生BEA和ENNs的重要产毒镰刀菌的形态学特征、分子遗传学特征以及影响BEA和ENNs产生的环境条件如温度、底物等因素进行概述。重点阐述了产毒镰刀菌在两类毒素合成酶的基因水平和氨基酸水平的差异及影响产毒等主要因素,为BEA和ENNs及其产毒镰刀菌的预防和控制提供理论依据。     

Potential use of antioxidants for control of growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on whole maize grain

The effect of interactions between two food grade antioxidants butylated hydroxyanisole (BHA) and propyl paraben (PP, 100, 200, 500 microg g(-1)) and water activity (a(w), 0.995, 0.98, 0.95) of irradiated maize on lag phase prior to growth, growth rate and fumonisin production by Fusarium verticillioides and Fusarium proliferatum was evaluated at 25 degrees C. Both antioxidants had an effect on growth characteristics, and fumonisin production. However, this was dependent on the dose used and the a(w) treatment. At 500 microg g(-1) BHA and PP increased the lag phase prior to growth, and reduced the growth rate of both Fusarium species significantly, especially at 0.95 a(w). Both antioxidants significantly reduced the production of fumonisin by both Fusarium species, especially at 0.98 and 0.95 a(w). These results suggest that these antioxidants have potential for treatment of maize grain for controlling growth of these mycotoxigenic species and prevent fumonisin accumulation.     

Influence of agronomic and climatic factors on Fusarium infestation and mycotoxin contamination of cereals in Norway

A total of 602 samples of organically and conventionally grown barley, oats and wheat was collected at grain harvest during 2002-2004 in Norway. Organic and conventional samples were comparable pairs regarding cereal species, growing site and harvest time, and were analysed for Fusarium mould and mycotoxins. Agronomic and climatic factors explained 10-30% of the variation in Fusarium species and mycotoxins. Significantly lower Fusarium infestation and concentrations of important mycotoxins were found in the organic cereals. The mycotoxins deoxynivalenol (DON) and HT-2 toxin (HT-2) constitute the main risk for human and animal health in Norwegian cereals. The impacts of various agronomic and climatic factors on DON and HT-2 as well as on their main producers F. graminearum and F. langsethiae and on total Fusarium were tested by multivariate statistics. Crop rotation with non-cereals was found to reduce all investigated characteristics significantly--mycotoxin concentrations as well as various Fusarium infestations. No use of mineral fertilisers and herbicides was also found to decrease F. graminearum, whereas lodged fields increased the occurrence of this species. No use of herbicides was also found to decrease F. langsethiae, but for this species the occurrence was lower in lodged fields. Total Fusarium infestation was decreased with no use of fungicides or mineral fertilisers, and with crop rotation, as well as by using herbicides and increased by lodged fields. Clay and to some extent silty soils seemed to reduce F. graminearum in comparison with sandy soils. Concerning climate factors, low temperature before grain harvest was found to increase DON; and high air humidity before harvest to increase HT-2. F. graminearum was negatively correlated with precipitation in July but correlated with air humidity before harvest. F. langsethiae was correlated with temperature in July. Total Fusarium increased with increasing precipitation in July. Organic cereal farmers have fewer cereal intense rotations than conventional farmers. Further, organic farmers do not apply mineral fertiliser or pesticides (fungicides, herbicides or insecticides), and have less problem with lodged fields. The study showed that these agronomic factors were related to the infestation of Fusarium species and the concentration of mycotoxins. Hence, it is reasonable to conclude that farming system (organic versus conventional) impacts Fusarium infestation, and that organic management tends to reduce Fusarium and mycotoxins. However, Fusarium infestation and mycotoxin concentrations may be influenced by a range of factors not studied here, such as local topography and more local climate, as well as cereal species and variety.     

Trichothecenes and zearalenone production by Fusarium equiseti and Fusarium semitectum species isolated from Argentinean soybean

Fusarium equiseti and Fusarium semitectum represent the most abundant species in the Fusarium complex isolated from flowers, soybean pods and seeds in Argentina. The aim of the present study was to assess the production of major type A and type B trichothecenes (diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin, nivalenol, deoxynivalenol) and zearalenone by 40?F. equiseti and 22 F. semitectum isolates on rice culture. Mycotoxins were determined by HPLC with fluorescence detection after derivatisation with 1-anthronylnitrile for type A trichothecenes (i.e. diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin), by HPLC with UV detection for type B trichothecenes (i.e. nivalenol and deoxynivalenol), and by TLC for zearalenone. A total of 22 of 40?F. equiseti isolates produced diacetoxyscirpenol, nivalenol and ZEA alone or in combination, whereas only two of 20?F. semitectum isolates were nivalenol and ZEA producers. Both Fusarium species did not produce any deoxynivalenol, neosolaniol, T-2 toxin and HT-2 toxin. The variable retention in toxigenicity displayed by both fungal species suggests that these species have a saprophytic lifestyle in the soybean agroecosystem in Argentina.     

Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops

Various species of Fusarium can produce trichothecene mycotoxins that contaminate food commodities and can represent a risk for human and animal health. In this paper, a full factorial design was applied to study the influence of incubation temperature, water activity (a(w)) and type of isolate on the production of deoxynivalenol (DON), nivalenol (NIV) and 3-acetyldeoxynivalenol (3-AcDON) in corn kernel cultures by three isolates of Fusarium graminearum and three isolates of Fusarium culmorum from crops grown in Spain. The tested temperatures were 15, 20, 28 and 32 degrees C. The a(w)-values were 0.960, 0.970 and 0.980. Moisture of cultures (within the studied range) did not affect significantly production of trichothecenes; however, the temperature affected significantly mycotoxin production and the optimal values were 28, 20 and 15 degrees C for DON, NIV and 3-AcDON, respectively. Four additional isolates of F. graminearum and two additional isolates of F. culmorum were examined for production of these mycotoxins at the optimal temperatures. Of the seven isolates of F. graminearum, four produced DON (0.88-3.97 microg/g), seven produced NIV (1.53-124 microg/g), and three produced 3-AcDON (0.65-10.6 microg/g). Of the five isolates of F. culmorum, four produced DON (1.20-4.93 microg/g), four produced NIV (6.94-701 microg/g), and four produced 3-AcDON (0.83-7.70 microg/g). Practically all isolates seem to belong to the NIV-chemotype. This is the first study done with regard to interaction between strain and ecological variables on type B trichothecene production by isolates of these two species from crops grown in Spain.     

Effect of water activity and temperature on growth and the relationship between fumonisin production and the radial growth of Fusarium verticillioides and Fusarium proliferatum on corn

The two major fumonisin-producing Fusarium species are Fusarium verticillioides and Fusarium proliferatum. The growth and fumonisin production of these two isolates on corn was studied at water activities (a(w)) between 0.860 and 0.975 and at temperatures between 15 and 30 degrees C. Growth rates (g, mm/day) were obtained by linear regression during the linear phase of growth. In general, growth rates for both isolates increased significantly (P < 0.05) with increases in a(w) and temperature. Both fumonisin production and radial growth (mycelial development) for both isolates increased with a(w) at all temperatures investigated, but the effect of temperature on this relationship was not obvious. The effect of temperature on fumonisin production at high a(w) values optimal for growth was only marginal, whereas at lower a(w) values the effect of temperature was more pronounced, with more fumonisin production occurring at temperatures not optimal for growth. The optimum temperature for fumonisin production was between 15 and 25 degrees C. For F. proliferatum, the optimum temperature for growth at all a(w) values, 30 degrees C, resulted in the poorest fumonisin production. For both isolates, the slowest initial rate of fumonisin production was at 15 degrees C, the temperature at which the slowest growth rates were obtained.     

In vitro antifungal activity of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens

The aim of this study was to examine the effect of five naturally occurring compounds from essential oils on 10 different species of mycotoxigenic fungi involved in several plant diseases. The antifungal activities of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol were observed in?vitro on Fusarium subglutinans, Fusarium cerealis, Fusarium verticillioides, Fusarium proliferatum, Fusarium oxysporum, Fusarium sporotrichioides, Aspergillus tubingensis, Aspergillus carbonarius, Alternaria alternata and Penicillium sp. The naturally occurring compounds tested showed toxic effects on in?vitro mycelium growth of all fungal species but with different level of potency. The results are encouraging for further investigations of in planta antifungal activities of these essential oils components.     

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.     

Natural occurrence of zearalenone and toxicogenic fungi in amaranth grain.

Zearalenone was detected as the natural contaminant in two samples of Amaranthus cruentus grains (1980 micrograms/kg and 420 micrograms/kg, respectively). Fungi isolated from these samples were screened for mycotoxin production. Two of eight isolates of Fusarium (F. equiseti and F. moniliforme) produced zearalenone. One of four isolates of Aspergillus flavus and all four isolates of A. parasiticus produced aflatoxins. Other species potentially toxicogenic such as Aspergillus versicolor, Penicillium viridicatum, P. puberulum, P. crustosum, P. citrinum, P. expansum and Fusarium solani were also found.     

In vitro antifungal activity of terpinen-4-ol,eugenol, carvone, 1,8-cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens

The aim of this study was to examine the effect of five naturally occurring compounds from essential oils on 10 different species of mycotoxigenic fungi involved in several plant diseases. The antifungal activities of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol were observed in vitro on Fusarium subglutinans, Fusarium cerealis, Fusarium verticillioides, Fusarium proliferatum, Fusarium oxysporum, Fusarium sporotrichioides, Aspergillus tubingensis, Aspergillus carbonarius, Alternaria alternata and Penicillium sp. The naturally occurring compounds tested showed toxic effects on in vitro mycelium growth of all fungal species but with different level of potency. The results are encouraging for further investigations of in planta antifungal activities of these essential oils components.     

Fusarium avenaceum -- the North European situation

The Fusarium species complex found on small-grain cereals in Northern Europe is largely dominated by F. avenaceum, while other important species include F. tricinctum, F. poae, F. culmorum and F. graminearum. The dominance of F. avenaceum has in recent years initiated extensive analytical activity in Norway and Finland in order to gain insight into the contamination of grain with secondary metabolites related to the fungus. Of these, moniliformin is the most studied compound with regard to toxicity. However, the data from analytical surveys indicate that field conditions in Northern Europe do not favour production of the metabolite. Instead, enniatins are regularly found in ppm-concentrations in grain, especially wheat and barley, while the bio-production of a range of other F. avenaceum related metabolites has so far barely been investigated. This paper summarises the results from mycological and chemical analyses of grain samples from Norway and Finland for major Fusarium species and F. avenaceum-related secondary metabolites.     

Immunological detection of Fusarium species in cornmeal

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect Fusarium species in foods. Antibodies to proteins extracted from the mycelia of Fusarium graminearum and Fusarium moniliforme (verticillioides) were produced in New Zealand white rabbits. These antibodies detected 13 Fusarium species in addition to the producer strains. Levels of Fusarium semitectum and Fusarium tricinctum strains were below the detection threshold. The specificity of the assay was tested against 70 molds and yeasts belonging to 23 genera. One strain of Monascus species and one strain of Phoma exigua were detected; however, these two molds are not common contaminants of cereal grains or foods and should not interfere with the assay. The indirect ELISA's detection limits for F. graminearum and F. moniliforme were 0.1 and 1 microg of mold mycelium per ml of a cornmeal mixture, respectively. When spores of each mold were added individually to cornmeal mixtures (at ca. 10 spores per g) and incubated at 25 degrees C, these spores were detected by the indirect ELISA when they reached levels of 10(2) to 10(3) CFU/ml after 24 to 36 h. The indirect ELISA developed here shows promise for the detection of Fusarium species in grains or foods.     

Production of trichothecene mycotoxins by Australian Fusarium species.

Australian isolates of Fusarium species were grown on potato dextrose agar. Trichothecenes produced by these species were extracted by ethyl acetate followed by methanol and a silica gel column was used to clean-up the extract. The extracted samples were derivatized by acetylation with trifluoroacetic anhydride and the derivatives analysed by gas chromatography/mass spectrometry (GC/MS). Multiple ion detection was used to trace ions characteristic of the trichothecenes expected to be present. Quantitation of those found was based on a known mass of pentabromophenol that was added as an internal standard. Eight species of Fusarium (nineteen strains) were surveyed, of which three species, F. acuminatum, F. equiseti and F. sporotrichioides, produced the trichothecenes scirpentriol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, T-2 tetraol and deoxynivalenol. Wheat samples were inoculated with four different species of Fusarium, F. acuminatum, F. equiseti, F. graminearum and F. sporotrichioides, and in these samples diacetoxyscirpenol, neosolaniol, HT-2 toxin and T-2 toxin were found.     

FUSARIUM MOLDS AND THEIR MYCOTOXINS

An overview is presented covering the occurrence of Fusarium species in raw food and feed materials and the mycotoxins produced by these molds. A study of the literature (reported from 1969 until now) revealed the occurrence of over 60 Fusarium species in these raw materials. It appears that the most reported species occur worldwide and in a large variety of raw food products, such as cereals, vegetables and fruits. These Fusarium species occurring in samples are producers of toxigenic secondary metabolites of which more than 100 have been described. Most of them are referred to as mycotoxins of which trichothecencs (e.g., deoxynivalenol, nivalenol, T-2 toxin, diacetoxyscirpenol), fumonisins and zearalenones are most frequently reported .     

Diversity in Beauvericin and Enniatins H, I, and MK1688 by Fusarium oxysporum isolated from potato

Beauvericins and enniatins are cyclohexadepsipeptide mycotoxins that exhibit phytotoxicity and insecticidal activities. In the present study, the production of beauvericin and newly found enniatins (H, I, and MK1688) was characterized in 28 Fusarium strains isolated from potato samples in Korea. The predominant Fusarium species in potato was F. oxysporum (53.6%). Fifteen strains of F. oxysporum and two strains of other Fusarium species produced beauvericin (at concentrations from 3.1 to 743.2 microg/g) in culture on rice. Enniatins H and I were produced by 3 and 11 strains at concentrations from 33.1 to 781.3 microg/g and from 6.5 to 730.3 microg/g, respectively. Five isolates produced enniatin MK1688 at concentrations from 4.6 to 432.6 microg/g. In particular, one isolate (No. 1501) identified as F. oxysporum and two other Fusarium strains (Nos. 804 and 910) produced all of the tested toxins. These results indicate that enniatins H, I, and MK1688 and beauvericin are produced by Fusarium isolates occurring on potato. We do not know if the toxins can accumulate in the environment since it was not demonstrated.     

Fusaria and fumonisins in maize from Ghana and their co-occurrence with aflatoxins

Fifteen maize samples from four markets and processing sites in Accra, Ghana were analysed for fumonisins B1, B2, and B3. All samples contained fumonisins. Total fumonisin levels for 14 samples ranged from 70 to 4222 microg kg(-1). One sample of visibly mouldy kernels contained 52 670 microg kg(-1) total fumonisins. Mycological examination of the samples showed Aspergillus spp. as the most dominant fungi (76.4%) followed by Penicillium spp. (19.9%). Fusarium formed 2.6% with Fusarium verticillioides as the predominant Fusarium species. Thirty-two Fusarium strains representing five species isolated from the maize samples were tested for the production of fumonisins in maize substrates. From 95% (21 of 22) of the F. verticillioides strains tested, all three types of fumonisins were produced. Total fumonisin levels ranged from 127 to 11 052 microg g(-1). Additional studies on maize samples from 15 processing sites in Accra revealed a co-occurrence of both fumonisins and aflatoxins in 53% (8 of 15) of the samples.     

Interaction of water activity and bicarbonate salts in the inhibition of growth and mycotoxin production by Fusarium and Aspergillus species of importance to corn

The combined effects of water activity (a(w)) and ammonium/sodium bicarbonate on growth and mycotoxin production in corn by Fusarium and Aspergillus species were investigated. Interaction was observed between the salts and a(w) on the colony growth rates and lag phase durations of all isolates. Growth stimulation at low salt levels was observed only for the Fusarium isolates as the fastest growth of F. verticillioides and F. proliferatum occurred at levels of 0.1-0.2 and 0.5% ammonium and sodium bicarbonate, respectively. Although the complete inhibition of the growth of the Fusarium and Aspergillus isolates investigated took place at a level of 1% ammonium bicarbonate as much as 4% sodium bicarbonate failed to completely inhibit the growth of the Aspergillus isolates. Increase in concentration of either salt generally resulted in large reductions of both fumonisin B(1) and aflatoxin B(1) production. According to the sensorial analysis performed, corn treated with up to 1% ammonium bicarbonate was still acceptable for consumption, whereas corn treated with at least 2% sodium bicarbonate was determined to be sensorially unsuitable. Ammonium bicarbonate can be concluded to be more suitable for protecting stored corn from fungal contamination as it was capable of completely inhibiting both growth and mycotoxin production of the Fusarium and Aspergillus isolates of most importance to corn at levels that were still sensorially acceptable. Therefore ammonium bicarbonate could possibly be applied as a cheap and easy to apply treatment for use in resource limited developing countries.     

进境加拿大大麦中真菌病害的分离与鉴定

目的分离与鉴定进境加拿大大麦中真菌病害。方法从进境加拿大大麦的可疑种子及病残体中分离获得真菌菌株,并对所分离的菌株进行培养性状及形态学观察,同时对核糖体DNA内转录间隔区(internal transcribed spacer,ITS)扩增和测序,进行鉴定。结果共分离出21个真菌菌株,鉴定出6个菌种,包括黑麦麦角菌(Claviceps purpurea)、梨孢镰刀菌(Fusarium poae)、燕麦镰刀菌(Fusarium avenaceum)、细极链格孢(Alternaria tenuissima)、大麦网纹病菌(Pyrenophora teres)、颖枯壳针孢(Phaeosphaeria nodorum)。结论做好进境大麦的检疫、监管工作,对保障啤酒工业安全生产、防止外来危险性病原菌在我国定殖、流行以致造成危害具有极其重要的意义。     

小豆内生真菌的筛选鉴定及其产玉米赤霉烯酮的可能性探究

玉米赤霉烯酮(zearalenone,ZEN)是目前全球范围污染谷物最广泛的霉菌毒素之一,对人体和动物健康都会产生极大威胁,目前从市售小豆检测到ZEN。【目的】筛选暴露ZEN的小豆内生真菌,结合经典方法和分子生物学方法鉴定所筛选内生真菌,并探索菌株潜在的ZEN产生风险。【方法】以暴露ZEN的小豆为样本,将分离纯化后的内生真菌进行经典形态观察;同时扩增真菌核糖体保守序列ITS1、ITS4,镰刀菌特异基因序列FU1、FU2确定小豆中镰刀菌种属,同时通过ZEN玉米赤霉烯酮产毒控制基因pks13进行PCR扩增,分析菌株ZEN合成的潜在风险。【结果】鉴别出3株镰刀菌,其中2株初步鉴定为砖红镰刀菌(Fusarium lateritium)、1株为花腐镰刀菌(Fusarium napiforme),未检测出ZEN合成关键基因;1株玛利节菱孢霉(Arthrinium marii),分子检测具有产ZEN玉米赤霉烯酮毒素的可能性。将以上4株菌分别回接至ZEN的产毒培养基,LC-MS/MS法未检测出ZEN毒素。【结论】暴露ZEN的小豆具有内生镰刀菌,存在可能产ZEN的内生节菱孢霉;回接培养基未检测到ZEN,但不排除小豆产生ZEN的风险。     

Specific detection of Fusarium langsethiae and related species by DGGE and ARMS-PCR of a beta-tubulin (tub1) gene fragment

Fusarium langsethiae was recently described as a new toxigenic Fusarium species, which morphologically resembles Fusarium poae, but exhibits a mycotoxin pattern related to Fusarium sporotrichioides. To develop tools for early and specific detection of F. langsethiae and distinguishing it from related species of section Sporotrichiella and Discolor (F. poae, F. sporotrichioides, Fusarium kyushuense, Fusarium robustum, Fusarium sambucinum and Fusarium tumidum) sequence variations in their beta-tubulin-encoding (tub1) gene were employed to design two PCR-based methods, denaturing gradient gel electrophoresis (DGGE) and amplification refractory mutation system (ARMS)-PCR. DGGE reliably separated all these strains, even from mixtures and in the presence of DNA from their natural hosts Zea mais, Triticum aestivum and Avena sativa. In addition, a tetraprimer ARMS-PCR, which employs two primer pairs to amplify, respectively, two different fragments of tub1 in a single PCR reaction resulted in rapid differentiation between F. langsethiae, F. sporotrichioides and F. poae according to the number of amplicons (four, two and one, respectively). These two methods will thus be worthwhile tools in the specific detection of F. langsethiae in infected crops.