Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro

Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.     

Gibberella ear rot of maize (Zea mays) in Nepal: distribution of the mycotoxins nivalenol and deoxynivalenol in naturally and experimentally infected maize

The fungus Fusarium graminearum (sexual stage Gibberella zeae) causes ear rot of maize (Zea mays) and contamination with the 8-ketotrichothecenes nivalenol (1) or 4-deoxynivalenol (2), depending on diversity of the fungal population for the 4-oxygenase gene (TRI13). To determine the importance of 1 and 2 in maize ear rot, a survey of naturally contaminated maize in Nepal was combined with experiments in the field and in a plant growth room. In the survey, 1 contamination was 4-fold more frequent than 2 contamination and 1-producers (TRI13) were isolated more than twice as frequently as 2-producers (Psi TRI13). In maize ear rot experiments, genetically diverse 1-producers and 2-producers caused ear rot and trichothecene contamination. Among strains with the same genetic background, however, 1-producers caused less ear rot and trichothecene contamination than did 2-producers. The high frequency of 1 contamination and the high virulence of many 1-producers are of concern because maize is a staple food of rural populations in Nepal and because 1 has proven to be more toxic than 2 to animals.     

Impact of competitive fungi on Trichothecene production by Fusarium graminearum

Bioassays were used to determine the production of the trichothecene mycotoxin, deoxynivalenol (DON), by two isolates of Fusarium graminearum when grown in association with potentially competitive fungi and an antifungal chemical, 6-pentyl-alpha-pyrone (6PAP). The presence of 6PAP in the culture medium reduced DON production by as much as 80%, but this effect was reduced for the F. graminearum isolate that most efficiently metabolized the added 6PAP. A 6PAP-producing Trichoderma isolate grown in a competition assay system with the F. graminearum isolates was also able to substantially reduce DON production. When Fusarium isolates (F. crookwellense, F. culmorum, F. subglutinans, F. poae, F. equiseti, F. avenaceum, and F. sambucinum), which co-occur with F. graminearum in New Zealand maize plants (Zea mays), were grown in competition assays, the effect on DON production was variable. However, all isolates of F. subglutinans tested were shown to cause reductions in DON production (by 13-76%, mean = 62%). F. subglutinans frequently co-occurs with F. graminearum, but its presence can vary with location and time of the season. When the competitive fungus tested was also a trichothecene producer (e.g., of nivalenol), both toxins were produced in the assay medium. The results indicate that mycotoxin production by F. graminearum can be affected by the presence of particular competitive fungi. These results have implications for an ecological understanding of pathogenicity and of mycotoxin accumulation in plants. Early establishment of F. subglutinans, for example, may act as a biological control mechanism providing a temporary protection against invasion by more commonly toxigenic fusaria such as F. graminearum.     

Characterization of a fusarium 2-gene cluster involved in trichothecene C-8 modification

The Fusarium trichothecenes T-2 toxin and deoxynivalenol (DON) are potent inhibitors of eukaryotic protein synthesis and are a significant agricultural problem. Three coregulated loci are required for T-2 toxin synthesis by Fusarium sporotrichioides. The core-trichothecene gene cluster consists of 12 genes (Tri3-Tri14) while the second locus consists of a single gene (Tri101). The third locus was recently partially described and encodes 1-2 biosynthetic enzymes and a putative regulatory gene. Here, we describe a detailed characterization of this locus. Located adjacent to Tri1 is Tri16, which is required for esterification of the C-8 hydroxyl. A putative regulatory gene, also adjacent to Tri1, is not required for T-2 toxin synthesis. The genomic sequence of Fusarium graminearum (a DON producer) contains a putative functional Tri1 and a nonfunctional Tri16. The presence of the Tri16 pseudogene is consistent with the chemical structure of DON, which has a C-8 keto group rather than the C-8 ester of T-2 toxin.     

Patterns of trichothecene production, genetic variability, and virulence to wheat of Fusarium graminearum from smallholder farms in Nepal

Fusarium graminearum causes wheat head blight and contaminates grain with the trichothecenes 4-deoxynivalenol and nivalenol. Sequence analysis of trichothecene genes indicates that nivalenol production is the ancestral trait; however, deoxynivalenol producers occur worldwide and predominate in North and South America and in Europe. Analysis of a large field population (>500 strains) from Nepal identified three groups that were both genetically distinct and polymorphic for trichothecene production: SCAR1 comprising 95% deoxynivalenol producers, SCAR2 comprising 94% nivalenol producers, and SCAR3/5 comprising 34% deoxynivalenol producers/63% nivalenol producers. The ability to cause wheat head blight differed between SCAR groups and trichothecene chemotypes: deoxynivalenol producers were more virulent than nivalenol producers across all three SCAR groups and within the SCAR3/5 genetic background. These data support the hypothesis that production of deoxynivalenol rather than nivalenol confers a selective advantage to this important wheat pathogen.     

Modified microwave-assisted extraction of ergosterol for measuring fungal biomass in grain cultures

Ergosterol is a measure for fungal biomass. The recovery rates using a previously described microwave-assisted-extraction (MAE) method for ergosterol analysis tended to be low for grain cultures (pure culture in sterilized 40% moisture content grain) inoculated with Fusarium graminearum . An improved MAE method for measuring ergosterol in grain cultures was developed and compared. Modification to the original MAE included alterations in duration of microwave exposure and extraction solvents. Four autoclaved grains (wheat, rice, barley, and corn) were inoculated with F. graminearum or spiked with ergosterol at concentrations from 0.88 to 100 microg/g and extracted with both methods. The ergosterol recovery rates were significantly different (p < 0.05) for the two methods in assaying both the spiked and grain culture samples. The modified method provided greater recovery rates than the previously reported MAE method for the spiked samples and F. graminearum grain cultures.     

浙贝母HMGR基因保守区序列的克隆及生物信息学分析

3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是甲羟戊酸途径中的第一个限速酶,在植物生物碱类物质代谢过程中发挥重要的作用。为探究HMGR酶在浙贝母甾体类生物碱合成代谢途径中的调控作用,以百合科浙贝母狭叶品种为材料,利用RT-PCR技术和Ta克隆技术首次从浙贝母中克隆出HMGR基因保守区序列,长度为390 bp。序列分析表明,浙贝母HMGR基因保守区序列与其他9种植物的HMGR基因有较高的相似性(84%~79%);蛋白质同源比对、特征区及系统进化树分析表明,浙贝母HMGR氨基酸片段与其他植物有较高的同源性(95%~88%),与球药隔重楼和海枣等单子叶植物的亲缘关系较近,据此,初步确定该基因为浙贝母HMGR基因,命名为Ft HMGR。Ft HMGR基因的克隆及分子鉴定为进一步解析该基因在浙贝母甾体类生物碱合成代谢途径中的分子调控作用奠定了基础,有助于对浙贝母活性物质的合成进行调控,从而提高产量。     

Structure-activity relationships of trichothecene toxins in an Arabidopsis thaliana leaf assay

Many Fusarium species produce trichothecenes, sesquiterpene epoxides that differ in patterns of oxygenation and esterification at carbon positions C-3, C-4, C-7, C-8, and C-15. For the first comprehensive and quantitative comparison of the effects of oxygenation and esterification on trichothecene phytotoxicity, we tested 24 precursors, intermediates, and end products of the trichothecene biosynthetic pathway in an Arabidopsis thaliana detached leaf assay. At 100 microM, the highest concentration tested, only the trichothecene precursor trichodiene was nontoxic. Among trichothecenes, toxicity varied more than 200-fold. Oxygenation at C-4, C-8, C-7/8, or C-15 was, on average, as likely to decrease as to increase toxicity. Esterification at C-4, C-8, or C-15 generally increased toxicity. Esterification at C-3 increased toxicity in one case and decreased toxicity in three of eight cases tested. Thus, the increase in structural complexity along the trichothecene biosynthetic pathway in Fusarium is not necessarily associated with an increase in phytotoxicity.     

Biosynthesis of trichothecenes and apotrichothecenes.

Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described.     

Interactions of arbuscular-mycorrhizal fungi and Bacillus strains and their effects on plant growth,microbial rhizosphere activity (thymidine and leucine incorporation) and fungal biomass (ergosterol and chitin)

The effects of two Bacillus strains (Bacillus pumillus and B. licheniformis) on Medicago sativa plants were determined in single or dual inoculation with three arbuscular-mycorrhizal (AM) fungi and compared to P-fertilization. Shoot and root plant biomass, values of thymidine and leucine incorporation as well as ergosterol and chitin in rhizosphere soil were evaluated to estimate metabolic activity and fungal biomass, respectively, according to inoculation treatments. For most of the plant parameters determined, the effectiveness of AM fungal species was influenced by the bacterial strain associated. Dual inoculation of Bacillus spp. and AM fungi did not always significantly increase shoot biomass compared to single AM-colonized plants. The most efficient treatment in terms of dry matter production was the dual Glomus deserticola plus B. pumillus inoculation, which produced similar shoot biomass and longer roots than P-fertilization and a 715% (shoot) and 190% (root length) increase over uninoculated control. The mycorrhizas were more important for N use-efficiency than for P use-efficiency, which suggests a direct mycorrhizal effect on N nutrition not mediated by P uptake. Both chemical and biological treatments affected thymidine and leucine incorporation in the rhizosphere soil differently. Thymidine was greater in inoculated than in control rhizospheres and B. licheniformis was more effective than B. pumillus in increasing thymidine. Non-inoculated rhizospheres showed the lowest thymidine and leucine values, which shows that indigenous rhizosphere bacteria increased with introduced inocula. The highest thymidine and leucine values found in P-fertilized soils indicate that AM plants are better adapted to compete with saprophytic soil bacteria for nutrients than P-amended plants. Chitin was only increased by coinoculation of B. licheniformis and G. intraradices. B. pumillus increased ergosterol (indicative of active saprophyte fungal populations) in the rhizosphere of AM plants and particularly when colonized by G. mosseae. The different AM fungi have different effects on bacterial and/or fungal saprophytic populations and for each AM fungus, this effect was specifically stimulated or reduced by the same bacterium. This is an indication of ecological compatibilities between microorganisms. Particular Glomus–bacterium interactions (in terms of effect on plant growth responses or rhizosphere population) do not seem to be related to the percentage of AM colonization. The effect on plant growth and stimulation of rhizosphere populations, as a consequence of selected microbial groups, may be decisive for the plant establishment under limiting soil conditions.     

Possible role of plant phenolics in the production of trichothecenes by Fusarium graminearum strains on different fractions of maize kernels

Four trichothecene-producing strains of Fusarium graminearum were grown on three maize grain fractions, whole grain, degermed grain, and the germ, to determine the effect of natural substrates on mycotoxin production. Monitoring the ergosterol content after 25 days of incubation indicated that fungal growth on all grain fractions was comparable. Trichothecene (TCT) production was highest on degermed grain, less on whole grain, and very low or nondetectable on the germ; similar results were found with four different strains. It was concluded that inhibitor(s) of TCT biosynthesis were present in maize germ. The presence of phenolic compounds was investigated in the different fractions. The hydroxamate 4-acetylbenzoxazolin-2-one (4-ABOA), a known inhibitor of mycotoxin production, was found in the degermed and whole grain fractions but not in the germ. Therefore, the TCT inhibition observed on the maize germ fraction used in our study is clearly not linked to 4-ABOA. Other soluble phenolic compounds were found at a much higher concentration in the germ than in the two other fractions. The inhibition property of the soluble ester-bound extracts was tested in liquid culture. A possible role for these compounds is discussed.     

Enzyme‐Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures,Extracts, and Plant Tissues

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme‐linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross‐reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L‐2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were detected in conidia and mycelia of MV, and in agar upon which wild‐type MV was grown, indicating secretion into the growth media. Two MV sectors (morphological variants of wild type) also tested positive for trichothecenes. Purified roridin A and concentrated extracts containing trichothecenes from MV spore cultures exhibited phytotoxicity (growth inhibition or necrosis) when applied to excised shoots of hemp sesbania seedlings and intact kudzu leaf tissues. Evidence of some translocation of trichothecenes from the application point in kudzu was found, but translocation to the upper shoot portion of hemp sesbania was not detected at the lowest limit of detection in this assay (0.14 ppb). This assay is also being employed to identify induced mutants and/or other naturally occurring sectors deficient in trichothecene mycotoxin production. Results indicated that ELISA is a sensitive and rapid assay method to quantify trichothecenes produced by this bioherbicidal fungus and in certain plant tissues treated with trichothecenes.     

Cobalt reduces nitrate inhibition of nodulation in mungbean (Vigna radiata)

 A cobalt-mediated decrease in ethylene production reduced the inhibition of nodulation by nitrate in Vigna radiata (mungbean). Nitrate increased the ethylene production in 5-day-old seedlings, while it caused a reduction in the nodulation status (nodule number and nodule weight) and nodule efficiency (acetylene reduction activity) in mungbean plants. The application of cobalt chloride inhibited nitrate-affected ethylene production and also decreased the inhibitory effect of nitrate on nodulation. The effect of cobalt was most marked on nodule number. Received: 6 August 1999     

紫外光与脉冲强光照射麦角甾醇转化VD2研究

常规紫外照射处理在生成天然VD₂的同时伴随着速甾醇等副产物的生成,为降低后期VD₂的提纯难度,本研究采用脉冲强光(IPL)与短波段紫外光(UVC)两种照射对麦角甾醇标准液与食用菌麦角甾醇提取液分别进行VD₂转化,分析照射剂量及后期反应对VD₂转化效率和副反应的影响。结果表明,双孢菇麦角甾醇的提取优化工艺条件为:采用提取时间60 min、乙醇浓度100%、料液比1∶20 g·mL^-1、提取温度80℃,此条件下双孢菇麦角甾醇提取率为3.59 mg·g^-1。麦角甾醇标准液经1.108 J·cm^-2 IPL照射处理并充分反应后,VD₂得率为7.34%;经0.656 J·cm^-2 UVC照射处理并充分反应的VD₂得率为9.68%;但相比UVC照射处理,IPL处理组的副产物量显著降低。麦角甾醇在经过光化反应后生成VD₂前体,在室温下转化VD₂速度缓慢,通过80℃、30 min加热,贮藏24 h后转化基本完全。食用菌麦角甾醇提取液经两种方式照射处理,其VD₂得率均低于同等浓度麦角甾醇标准溶液得率,可能是因为食用菌麦角甾醇提取液中成分较为复杂,且其他非麦角甾醇类物质对光照有一定的屏蔽作用。IPL照射处理能够有效减少麦角甾醇转化VD₂过程中副产物的生成,有助于以食用菌为原材料获得VD₂的规模化的生产要求。     

Cobalt chloride enhances crop duration,increases production,and productivity of chickpea

A field experiment was conducted to evaluate the response of chickpea, Cicer arietinum cv. GG 2 to cobalt sulfate and cobalt chloride at 0, 100, 200, 400, 800 and 1,600?g ha^?1. At three leaf stage chickpea seedlings were fertigated with both cobalt sources and levels. Both cobalt sources at the higher level (400 to 1,600?g ha^?1) were found injurious to chickpea. All growth, yield, and quality parameters were adversely affected by cobalt sulfate at every level; however, cobalt chloride has given appreciable result up to 100 and 200?g ha^?1 over no application of cobalt. Cobalt content in plant and soil increased linearly with increases in cobalt concentration, which reduced chickpea yield linearly. Cobalt sulfate was apparently more harmful than cobalt chloride. The study suggests cobalt chloride has not shown any toxicity up to 100?g ha^?1 and can be used for higher productivity of chickpea.     

FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis

Fumonisins are polyketide-derived mycotoxins produced by the filamentous fungus Gibberella moniliformis (anamorph Fusarium verticillioides). Wild-type strains of the fungus produce predominantly four B-series fumonisins, designated FB(1), FB(2), FB(3), and FB(4). Recently, a cluster of 15 putative fumonisin biosynthetic genes (FUM) was described in G. moniliformis. We have now conducted a functional analysis of FUM13, a gene in the cluster that is predicted by amino acid sequence similarity to encode a short chain dehydrogenase/reductase (SDR). Mass spectrometric analysis of metabolites from FUM13 deletion mutants revealed that they produce approximately 10% of wild-type levels of B-series fumonisins as well as two previously uncharacterized compounds. NMR analysis revealed that the new compounds are similar in structure to FB(3) and FB(4) but that they have a carbonyl function rather than a hydroxyl function at carbon atom 3 (C-3). These results indicate that the FUM13 protein catalyzes the reduction of the C-3 carbonyl to a hydroxyl group and are the first biochemical evidence directly linking a FUM gene to a specific reaction during fumonisin biosynthesis. The production of low levels of FB(1), FB(2), FB(3), and FB(4), which have a C-3 hydroxyl, by the FUM13 mutants suggests that G. moniliformis has an additional C-3 carbonyl reductase activity but that this enzyme functions less efficiently than the FUM13 protein.     

Growth rate of bacteria is affected by soil texture and extraction procedure

Effects of soil texture on the extraction efficiency of bacteria from soils and on biosynthetic activity of the extracted bacteria were studied. Bacterial extracts were prepared from three soils of different texture by homogenization (ultrasonication and mixing) or by homogenization-centrifugation at different speeds. Bacterial biosynthetic activity was estimated using thymidine and leucine incorporation techniques. In each step of the extraction procedure, a higher extractability of bacteria was obtained in finer soils than in coarse soil. Also cell-specific growth rates of bacteria were higher in the finer soils than in the coarse soil. However, in all soils, the extracted bacteria always had significantly lower cell-specific thymidine and leucine incorporation rates than the bacteria in soil slurries and thus did not represent so well the bacterial growth in the original soils. The total declines in cell-specific incorporation rates caused by the extraction were larger in fine soil (96-98%) than in coarse soil (90%), but bacteria in the coarse soil were more responsive to only minor intervention. The homogenization-centrifugation method eliminated the differences in bacterial biosynthesis found when working with soil slurries. Therefore, we recommend using of soil slurries or, optionally, soil suspensions to compare bacterial biosynthetic activity among soils of different textures.     

Influence of halogen salts on the production of the ochratoxins by Aspergillus ochraceus Wilh

The first report of the biological production of bromo ochratoxin B by Aspergillus ochraceus Wilh. is presented as well as a study of the influence of potassium bromide, potassium iodide, potassium fluoride, and potassium chloride on the production of ochratoxin A and ochratoxin B. Potassium fluoride and potassium iodide inhibited the growth of the fungus, whereas potassium chloride substantially stimulated the production of ochratoxin A in shaken solid substrate fermentation on whole wheat or shredded wheat, generally giving a high yield of ochratoxins. Increasing levels of potassium bromide led to a decline in ochratoxin A production and an increase in bromo-ochratoxin B, ochratoxin B, and 4-hydroxy ochratoxin B. Nevertheless, A. ochraceus was much less versatile in the bromo analogues than other fungi, which produce metabolites containing chlorine. Analysis included aminopropyl solid-phase extraction column cleanup followed by quantitative analysis on reversed-phase HPLC using fluorescence detection and employing N-(5-chloro-2-hydroxybenzoyl)phenylalanine as an internal standard.     

Rapid fluorescence polarization immunoassay for the mycotoxin deoxynivalenol in wheat

The fungus Fusarium graminearum, a pathogen of both wheat and maize, produces a toxin, deoxynivalenol (DON), that causes disease in livestock. A rapid test for DON in wheat was developed using the principle of fluorescence polarization (FP) immunoassay. The assay was based on the competition between DON and a novel DON-fluorescein tracer (DON-FL2) for a DON-specific monoclonal antibody in solution. The method, which is a substantial improvement over our previous DON FP immunoassay, combined a rapid (3 min) extraction step with a rapid (2 min) detection step. A series of naturally contaminated wheat and maize samples were analyzed by both FP immunoassay and liquid chromatography (HPLC-UV). For wheat the HPLC-UV and FP methods agreed well (linear regression r(2) = 0.936), but for maize the two methods did not (r (2) = 0.849). We conclude that the FP method is useful for screening wheat, but not maize, for DON.     

Concentrations of Some Metabolites Produced by Fungi of the Genus Fusarium and Selected Elements in Spring Spelt Grain

The aim of this study was to determine the content of selected elements and metabolites produced by fungi of the genus Fusarium in spelt (Triticum spelta L.) grain and husks and common wheat (T. aestivum L.) grain. Concentrations of trichothecenes, a volatile metabolite trichodiene (TRICH), as well as ergosterol (ERG) and adenosine 5′‐triphosphate (ATP) (a total microbial biomass indicator), were assessed. Toxin concentrations in spelt grain and husks harvested in 2003 and 2004 were comparable. Average deoxynivalenol concentrations reached 450 and 523 μg/kg in grain and 2,162 and 855 μg/kg in husks, respectively. Spelt grain, in comparison with common wheat grain, contained significantly higher concentrations of P, S, Mg, Zn, and Cu and a lower concentration of Al, whereas the concentrations of Ca, Fe, and Pb were significantly higher in the husks than in the grain of this cereal. A comparison of concentrations of Fusarium spp. metabolites in the grain of spelt and common wheat showed that the total concentration of mycotoxins and TRICH was slightly lower in T. spelta, whereas common wheat grain contained lower concentrations of ERG and ATP. The obtained results indicate that spelt husks contain considerable concentrations of trichothecenes.