The Chemical Constitution of Rubrofusarin

Further confirmation of 2-methyl-γ-pyrone structure in rubrofusarin through a color reaction with alkaline dinitrobenzene and through preparations of the styryl compound and isoxazole derivative is presented. Evidences which favor the linear naphthopyrone structure for rubrofusarin are also given.     

The Chemical Constitution of Rubrofusarin

Alkaline degradation of rubrofusarin and nor-rubrofusarin were studied; nor-rubrofusarin readily underwent hydrolysis to give a tetrahydroxynaphthalenc, acetone, and acetic acid; whereas, rubrofusarin, after prolonged time of hydrolysis, yielded a β-methoxytrihydroxynaphthalene instead of the naphthol. Physical and chemical studies revealed that the naphthol is 1,3,6,8-tetrahydroxynaphthalene and it has been confirmed by the synthesis from chromotropic acid (disodium salt). Thus, evidently, rubrofusarin has a naphthalene nucleus to which a methoxyl group is attached at β-position. The formation, on the hydrolysis, of acetone and acetic acid, along with the naphthol, indicates the presence of 2-methyl-γ-pyrone structure in rubrofusarin.     

The aerobiology of Fusarium graminearum

Current knowledge of the aerobiology of Fusarium graminearum sensu lato is based on decades of published research documenting the processes of spore discharge, atmospheric transport, and deposition in this important pathogen of cereal crops worldwide. Spores from both local and more distant sources have been shown to cause infection in susceptible cereal crops when environmental conditions are favorable. Susceptible crops may be exposed throughout a growing season to airborne spores deposited in rain events and in night-time hours through gravitational settling. Given that spores deposited on cereal florets originate from distant as well as local sources, disease risk forecasts, based currently on weather favoring local spore production during the days before peak infection (i.e., initiation of crop flowering), might be improved by placing greater emphasis on local weather directly favoring infection at and following the time of flowering. Also, considering the genetic diversity of fungal spores introduced to local agricultural fields following atmospheric transport, crop breeders should select resistant varieties based on screening against a set of fungal isolates that represent the range of virulence observed in fungal populations across a broader geographic region. An increased understanding of the aerobiology of F. graminearum contributes to the overall knowledge of plant pathogen transport in the atmosphere.     

Double-Stranded RNA Mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.     

Double-stranded RNA mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.     

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

The secreted glycoside hydrolase family 29 (GH29) α-l-fucosidase from plant pathogenic fungus Fusarium graminearum (FgFCO1) actively releases fucose from the xyloglucan fragment. We solved crystal structures of two active-site conformations, i.e. open and closed, of apoFgFCO1 and an open complex with product fucose at atomic resolution. The closed conformation supports catalysis by orienting the conserved general acid/base Glu-288 nearest the predicted glycosidic position, whereas the open conformation possibly represents an unreactive state with Glu-288 positioned away from the catalytic center. A flexible loop near the substrate binding site containing a non-conserved GGSFT sequence is ordered in the closed but not the open form. We also identified a novel C-terminal βγ-crystallin domain in FgFCO1 devoid of calcium binding motif whose homologous sequences are present in various glycoside hydrolase families. N-Glycosylated FgFCO1 adopts a monomeric state as verified by solution small angle x-ray scattering in contrast to reported multimeric fucosidases. Steady-state kinetics shows that FgFCO1 prefers α1,2 over α1,3/4 linkages and displays minimal activity with p-nitrophenyl fucoside with an acidic pH optimum of 4.6. Despite a retaining GH29 family fold, the overall specificity of FgFCO1 most closely resembles inverting GH95 α-fucosidase, which displays the highest specificity with two natural substrates harboring the Fucα1-2Gal glycosidic linkage, a xyloglucan-derived nonasaccharide, and 2′-fucosyllactose. Furthermore, FgFCO1 hydrolyzes H-disaccharide (lacking a +2 subsite sugar) at a rate 10³-fold slower than 2′-fucosyllactose. We demonstrated the structurally dynamic active site of FgFCO1 with flexible general acid/base Glu, a common feature shared by several bacterial GH29 fucosidases to various extents.     

Functional identification of high-affinity iron permeases from Fusarium graminearum

The ScFTR1 gene encodes an iron permease in Saccharomyces cerevisiae. Its homologues, FgFtr1 and FgFtr2, were identified from filamentous pathogenic plant fungus, Fusarium graminearum. Homologies between the deduced amino acid sequences of ScFtr1p and FgFtr1 and FgFtr2 were 56 and 54%, respectively, and both had REXXE sequences, which form the conserved amino acid sequence of ScFtr1p. FgFtr1 expression increased under iron depletion, and although FgFtr2 mRNA was not detected in the wild-type strain, it was detected in the deltafgftr1 strain in the iron-depleted condition. When the FgFtr1 and FgFtr2 were deleted, the amount of growth was found not to be different from the wild-type in iron-depleted media. However, the mRNA of FgSid, a homologue of the SIDA of Aspergillus fumigatus, was dramatically increased in the deltafgftr1/deltafgftr2 strain and in an iron-depleted condition. FgFtr1 and FgFtr2 genes act as functional complements when they are introduced into the S. cerevisiae deltaScftr1 strain. The deltaScftr1 strain, which contains either the FgFtr1 or FgFtr2, grew well in iron-depleted media. Moreover, specific alteration of the REXXE consensus sequence of FgFtr1 and FgFtr2 did not allow for sustained growth of the deltaScftr1 strain on iron-depleted medium. The iron uptake activity was recovered when FgFtr1 and FgFtr2 genes were introduced into the deltaScftr1 strain. Though the Fet3p in S. cerevisiae was found on the intracellular vesicle in the deltaScftr1 strain, Fet3p was found on the plasma membrane when FgFtr1 or FgFtr2 was introduced into the deltaftr1 strain. An infection test was carried out with deletion strains; however, no change in the ability of these strains to cause disease was observed. These results suggest that FgFtr1 and FgFtr2 may function as iron permeases in the reductive iron uptake pathway and that they do not play major roles in the pathogenicity of F. graminearum.     

Aggressiveness of different Fusarium graminearum chemotypes within a population from Northern-Central Italy

The aggressiveness of 32 Fusarium graminearum strains isolated from durum wheat cultivated in Northern-Central Italy was assessed. The chemotype (15-acetyldeoxynivalenol-, 3-acetyldeoxynivalenol- or nivalenol-producer) of each strain was characterized with multiplex PCR. Two aggressiveness assays (single floret inoculation and the Petri-dish test), which yielded three aggressiveness indices, were used. One strain from southern Italy (moderately aggressive) was added as the positive control to evaluate the accuracy of the aggressiveness quantification in both assays. The results showed that all 32 F. graminearum strains are pathogenic and a wide range of aggressiveness levels was observed. The positive control strain is pathogenic and showed moderately aggressive in both assays, thus indicated that our aggressiveness quantifications are valid. Six highly aggressive strains, representing all three chemotypes, were stable in the different aggressiveness assays. There was no significant difference in aggressiveness among the three different chemotypes (P = 0.909–0.972) determined by three aggressiveness indices. Highly significant correlations of AUDPC_standard to mean and terminal disease severity (r = 0.91, P < 0.001 in both cases) reveal the potential of using the Petri-dish test for selection of F. graminearum inocula with the proper and/or varying aggressiveness levels for breeding purposes.     

PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins which cause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a disease which produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial to prevent toxins entering the food chain and a useful tool in disease management practices. We describe the development of specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA from pure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol for DNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), a multicopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences.     

Production of Deepoxy-Diacetoxyscirpenol in a Culture of Fusarium graminearum

Deepoxy-diacetoxyscirpenol was isolated from a laboratory culture of Fusarium graminearum grown on a solid rice substrate. It was characterized as 3-hydroxy-4,15-diacetoxy-trichothec-9,12-diene by proton nuclear magnetic resonance and mass spectrometry. This is the first report of the occurrence of this metabolite in a fungus culture.     

Extracellular carboxylesterase activity of Fusarium graminearum

Summary The extracellular carboxylesterase produced by Fusarium graminearum was investigated. The esterase showed optimum activity at 37°C and at pH 8.7–9.0. Increased activity was observed when whey as a growth substrate was supplemented with either Tween 40 or olive oil. Good activity was also obtained when the fungus was grown on a semi-solid bran culture medium. The esterase showed increased activity towards esters containing short-chain fatty acids.     

青色荧光蛋白标记的禾谷镰孢转化子的构建

【背景】近年来玉米茎腐病在我国大部分玉米产区普遍重度发生,其中镰孢菌茎腐病不仅造成了重大的经济损失,而且镰孢菌产生的毒素给人体和动物的健康也带来严重威胁。【目的】玉米茎腐病的病原组成复杂,禾谷镰孢是其中的主要病原之一,该病原菌侵染寄主导致发病的机制急需深入研究。【方法】以pCAMBIA1300质粒为骨架,利用重叠PCR的方法构建表达青色荧光蛋白的质粒pCAMBIA1300-CFP-Kan,通过农杆菌介导的遗传转化技术,将青色荧光蛋白的编码基因整合到禾谷镰孢基因组中。【结果】经过PCR鉴定和荧光显微观察,确定获得了31株青色荧光标记的禾谷镰孢菌。【结论】侵染试验结果显示,激光共聚焦显微镜下禾谷镰孢在玉米茎秆组织中的定殖位置清晰可见,该结果为进一步研究不同镰孢菌在寄主中的定殖规律奠定了基础。     

Properties of Fusarium graminearum galactose oxidase

The kinetics and action mechanism of the galactose oxidase from Fusarium graminearum were studied. pH-optimum of the enzyme activity and stability was 7.0, the activity and stability of the galactose oxidase being decreased at any other values of pH. The enzyme is destabilized at acidic pH that is connected with protonization of its ionogenic group with pK 4.7. The temperature optimum of the galactose oxidase is 35 degrees C. When studying the enzyme thermoinactivation, it was found that at temperatures below 30 degrees C the energy of activation of denaturation was about 40 kcal/mole and at temperatures ranging from 30 to 70 degrees C - 13 kcal/mole. On the basis of the data obtained it was concluded that a low-temperature form of the galactose oxidase, possessing a higher energy of activation of denaturation, is more active than a high-temperature form. The value of Km for the enzyme in respect to galactose was 0.19 M, and the value of Vmax = 360 mumole/min per g of the preparation.     

The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the k(cat) decreased (8.3- and 3-fold respectively) and the K(m) increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (k(red)=750 s(-1)) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.     

The transcriptome of Fusarium graminearum during the infection of wheat

Fusarium graminearum causes head blight disease in wheat and barley. To help understand the infection process on wheat, we studied global gene expression of F. graminearum in a time series from 24 to 196 h after inoculation, compared with a noninoculated control. The infection was rapid and, after 48 h, over 4,000 fungal genes were expressed. The number of genes expressed increased over time up to 96 h (>8,000 genes), and then declined at the 144- and 192-h post-inoculation time points. After subtraction of genes found expressed on complete medium, during carbon or nitrogen starvation, and on barley, only 355 were found exclusively expressed in wheat, mostly genes with unknown function (72.6%). These genes were mainly found in single-nucleotide polymorphism-enriched islands on the chromosomes, suggesting a higher evolutionary selection pressure. The annotated genes were enriched in functional groups predicted to be involved in allantoin and allantoate transport, detoxification, nitrogen, sulfur and selenium metabolism, secondary metabolism, carbohydrate metabolism, and degradation of polysaccharides and ester compounds. Several putative secreted virulence factors were also found expressed in wheat.     

禾谷镰孢转化体系的优化

禾谷镰孢是小麦赤霉病和玉米茎腐病的主要病原真菌。试验优化了聚乙二醇介导的原生质体转化法,建立了禾谷镰孢高效转化的稳定体系,并总结了影响转化成功的主要因素。以此方法对禾谷镰孢进行活体荧光标记,并用转化菌株侵染小麦胚芽鞘,在荧光显微镜下追踪病原真菌在宿主植物体内的生长及扩展过程。     

Effects of Weather Variables on Ascospore Discharge from Fusarium graminearum Perithecia

Fusarium graminearum is a predominant component of the Fusarium head blight (FHB) complex of small grain cereals. Ascosporic infection plays a relevant role in the spread of the disease. A 3-year study was conducted on ascospore discharge. To separate the effect of weather on discharge from the effect of weather on the production and maturation of ascospores in perithecia, discharge was quantified with a volumetric spore sampler placed near maize stalk residues bearing perithecia with mature ascospores; the residues therefore served as a continuous source of ascospores. Ascospores were discharged from perithecia on 70% of 154 days. Rain (R) and vapor pressure deficit (VPD) were the variables that most affected ascospore discharge, with 84% of total discharges occurring on days with R≥0.2 mm or VPD≤11 hPa, and with 70% of total ascospore discharge peaks (≥ 30 ascospores/m³ air per day) occurring on days with R≥0.2 mm and VPD≤6.35 hPa. An ROC analysis using these criteria for R and VPD provided True Positive Proportion (TPP) = 0.84 and True Negative Proportion (TNP) = 0.63 for occurrence of ascospore discharge, and TPP = 0.70 and TNP = 0.89 for occurrence of peaks. Globally, 68 ascospores (2.5% of the total ascospores sampled) were trapped on the 17 days when no ascospores were erroneously predicted. When a discharge occurred, the numbers of F. graminearum ascospores sampled were predicted by a multiple regression model with R² = 0.68. This model, which includes average and maximum temperature and VPD as predicting variables, slightly underestimated the real data and especially ascospore peaks. Numbers of ascospores in peaks were best predicted by wetness duration of the previous day, minimum temperature, and VPD, with R² = 0.71. These results will help refine the epidemiological models used as decision aids in FHB management programs.     

纳米银对小麦赤霉病菌的抑制

采用化学还原法制备纳米银,以小麦赤霉病菌为受试菌株,研究纳米银对小麦赤霉病菌抗菌活性、对细胞内3种保护酶:超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性和对细胞渗透调节物质:丙二醛(MDA)、可溶性蛋白、可溶性糖含量的影响。结果表明:纳米银能显著抑制小麦赤霉病菌的生长,抑制作用随着浓度的增加而不断增大,10μg/mL的纳米银对病原菌的抑制率达90%以上,有效中浓度(EC_(50))为0.59μg/mL。随着纳米银处理时间(2、4、6、8和10 h)的增长,3种酶的活性均出现先增加后降低的变化。SOD、POD和CAT均在4 h出现最高值,10 h降至最低。纳米银使得菌体内丙二醛含量增加,可溶性蛋白和可溶性糖含量降低。纳米银破坏了病原真菌体内细胞的完整性,这可能是纳米银抑制病原菌生长的机理之一。     

The mitogenic properties of Fusarium graminearum and Rhodococcus erythropolis enzymes

Blast transformation of peripheral blood lymphocytes in healthy people is studied by enzymes of the microbic origin possessing the lectin activity. Galactose oxidase of Fusarium graminearum IMV-F-1060 is shown to be mitogenically active with respect to the lymphocytes in the culture in vitro and may be one of home sources of lymphocytic mitogens for the laboratory investigations.