Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.     

Fusarium culmorum Infection of Barley Seedlings: Correlation between Aggressiveness and Deoxynivalenol Content

Fusarium culmorum is a serious plant pathogen, especially on cereals. The production of deoxynivalenol (DON) by F. culmorum is believed to play a role in pathogenesis. This relationship has been almost exclusively studied in connection with head blight. The present paper reports the first finding of DON in cereal seedlings infected with F. culmorum . A pathogenicity test was performed, including 70 isolates of this pathogen from different sites within northern and central Europe. All isolates caused disease on barley seedlings. For 15 isolates with varying aggressiveness, the DON content in the 19-day-old-barley seedlings was determined. There was a significant correlation between DON concentration and disease index. The aggressiveness of two outlying isolates with very low DON production is discussed. The results indicate that for F. culmorum isolates of the DON chemotype, production of this toxin influences the aggressiveness of the isolates towards barley seedlings.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

Chemotyping of Fusarium graminearum and F. culmorum Isolates from Turkey by PCR Assay

Fusarium graminearum and F. culmorum are the major causal agents of Fusarium head blight in Turkey. They produce trichothecenes such as deoxynivalenol (DON), nivalenol (NIV) and their several acetylated derivatives, 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON). In this study, a total of thirty-three isolates of F. graminearum and F. culmorum were collected from various regions and three different hosts. They were identified by amplification of tri5 gene cluster. Totally 32 isolates, 21 of F. culmorum and 11 of F. graminearum, were determined as DON chemotype, while only one F. graminearum isolate (1F) was detected as a NIV. A 282 base pair (bp) band for tri13 gene and also ranging from 458 to 535 bp bands for tri7 gene were amplified in all DON producers’ genomes. Further analysis of DON chemotype based on tri3 gene amplification showed that all isolates of F. graminearum displayed 15-ADON sub-chemotype. They yielded a 863 bp amplicon. Similarly, 3-ADON sub-chemotype was identified in F. culmorum’ isolates except F13. As a result of tri3 gene assay, it was produced a 583 bp fragment in these twenty isolates. It is the first report that a F. graminearum isolate depicts NIV chemotype in agricultural regions of Turkey. According to our findings, DON chemotype is predominating in our country. Also, it is presented that most of the F. graminearum isolates have 15-ADON sub-chemotype, while all F. culmorum’s belong to 3-ADON which possess full length amplicon of tri7 gene.     

噬菌体对国内谷氨酸生产菌的鉴别

利用噬菌体的宿主专一性,对收集的国内用于生产的谷氨酸生产菌进行鉴别,发现一株7338菌株对T6—13噬菌体敏感,而对7338的噬菌体不敏感。根据上述事实,工厂中为防止噬菌体侵袭,轮换使用不同菌株时,提出要了解菌株对不同噬菌体的敏感性,以免感染造成巨大经济损失。     

Production and secretion of 5-n-alkylresorcinols by Fusarium culmorum

Fusarium culmorum F1 was found to produce and secrete into the culture medium several of 5-n-alkylresorcinols. The amount of resorcinolic lipids was 5.3 microg/g and 0.9 microg/l in mycelium and in post-culture liquid, respectively. First of all F. culmorum F1 produces saturated homologues with C15 to C25 side chains. The extract from the medium contained only homologues with shorter carbon chains (C13 to C17).     

Comparison of Fusarium acuminatum and Fusarium culmorum isolates by means of tandem-crossed immunoelectrophoresis

F. acuminatum and F. culmorum strains were compared by means of tandemcrossed immunoelectrophoresis in order to estimate the possiblities of serological classification in Fusarium sections Gibbosum and Discolor. On the basis of qualitative similarity the two species could be distinguished well. By the use of anti-F. acuminatum serum a similarity of S_SM=0.52 was found between F. acuminatum and F. culmorum, but the S_SM coefficient reached a value of 0.67 when the anti-F. culmorum serum was tested. This asymmetric nature of the qualitative similarity is discussed. In the majority of cases the quantitative differences of the common antigens did not allow differentiation between the species.     

产菊粉酶酵母菌株的筛选及菌种鉴定

经过初筛、复筛,得到2株菊粉酶活力较高的酵母菌Y9和Y27,其发酵液酶活分别达到19.4 U/mL和14.1 U/mL,两者胞外菊粉酶分泌较少,主要分布在酵母菌菌体上。通过细胞形态、生理生化特征及Biolog微生物鉴定系统鉴定,将Y9确立为Cryptococcus albidus(浅白隐球酵母),Y27确立为Pichia guilliermondiiA(季也蒙毕赤氏酵母A)。     

Restriction analysis of PCR amplified nrDNA regions revealed intraspecific variation within populations of Fusarium culmorum

Seventy-five isolates of Fusarium culmorum with diverse geographical origin and host were analyzed using restriction digestion of polymerase chain reaction amplified nuclear ribosomal DNA intergenic spacer (IGS) and 28S gene regions. The 28S gene was conserved and has produced identical restriction patterns, however, the IGS region was substantially variable. The isolates were divided into 29 unique IGS haplotypes. There was limited resolution between clustering of isolates and their origin and/or host. The variability was distributed largely equally at both macro- and micro-geographical scale. The phylogeographic distribution pattern suggests a seed-borne dispersal of F. culmorum.     

Fine Structure and Spore Germination in Fusarium culmorum

The fine structure of the macroconidium of Fusarium culmorum(W. G. Smith) Sacc. is described. The presence of a mucilaginouscoat around the conidium and its expansion during germinationare confirmed. Studies of germinating conidia have shown changesin the numbers of organelles. The germ tube is shown to be formedfrom an entirely new wall laid down in the conidium, and theemergence of the germ tube is brought about by lysis of theoriginal condial wall.     

Identification of Toxigenic Fusarium Species using PCR Assays

Isolates of the toxigenic cereal pathogens Fusarium culmorum, Fusarium graminearum, Fusarium crookwellense and Fusarium avenaceum, from Poland (48 isolates) and 12 from England, New Zealand, Italy and Canada, were examined using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), sequence-characterized amplified regions (SCARs), morphology and mycotoxin production under laboratory conditions. Their DNA products were compared by RAPD-PCR, which showed species-specific bands and the greatest diversity among isolates of F. avenaceum. PCR using three 20-mer-primer-pairs that are reported to be useful for identification of F. culmorum and F. graminearum group 2 confirmed their species-specificity. The same species-specific PCR product was observed in isolates of both nivalenol and deoxynivalenol chemotypes of F. culmorum or F. graminearum. A clear relationship was found between morphological and species-specific PCR identification of F. culmorum and F. graminearum isolates. However, F. avenaceum can be confused when using primers FA-ITS F/R (SCAR 2-14) with Fusarium tricinctum because the same band 272 bp appears in the gel, in both species probes.     

Evaluation of Spring and Winter Wheat Reaction to Fusarium culmorum and Fusarium avenaceum

Winter (37), spring (8) wheat accessions and additionally, 7 double haploid (DH) lines were examined for susceptibility to Fusarium seedling blight after inoculation with F. culmorum and F. avenaceum. Winter accessions exhibited lower susceptibility of about 30% to both pathogens than spring cultivars. Susceptibility of winter cultivars varied from low (22%) to high (97%). Evaluation of the root was found to be more reliable than evaluation of coleoptile necrosis.
F. avenaceum infected mostly root and, to a lesser extent, coleoptile and leaves, with about a three times lower disease score of coleoptile against root. F. culmorum caused a 1.5 higher disease score on root than on coleoptile. Susceptibility of DH lines was different from susceptibility of parental forms. Reaction of individual accessions to F. culmorum and F. avenaceum was different.     

Wall Structure and spore Germination in Fusarium culmorum

The conidial and germ-tube walls of Fusarium culmorum (W. G.Smith) Sacc. have been examined by various chemical and electron-microscopetechniques. On the basis of these results and hypothesis isproposed for the organization of these walls. Microchemicaltests indicate the presence of chitin in the walls and suggestthat the mucilaginous layer around the conidium is mainly composedof xylan. Chemical analyses of isolated wall material confirmthe presence of chitin constituents in the wall, and a rylanlayer around the conidium. Furthermore, the wall contains apolypeptide moiety which has a different amino acid compositionfrom the rest of the protein of the cell. Electron microscopestudies of replicas and sections of conidia, germ tubes, andhyphae reveal a layered structure for the wall. The centrallayer is non-microfibrillar and is overlaid on both sides witha layer of randomly orientated microfibrils. The mucilaginouslayer of the conidium obscures the microfibrillar structurebeneath it unless the mucilage is removed by hydrolysis. Theproblem of hyphal growth is discussed on the basis of the structureof germ-tube tips and mature hyphae observed.     

甲醇利用型吡咯喹啉醌产生菌的筛选及鉴定

从虎杖内生细菌和黏细菌中筛选吡咯喹啉醌(PQQ)产生菌。采用3种以甲醇为唯一碳源的培养基对160株供试菌株进行摇瓶培养发酵,发酵产物采用光谱学分析法及HPLC法筛选。结果显示,通过初筛和复筛共得到甲醇利用型菌134株,PQQ产生菌4株,其中菌株083114的PQQ产量为64.34 mg/L。菌株083114的16S rRNA基因序列分析结果显示,其序列与酸快生芽孢杆菌(Bacillus acidiceler)的系统发育关系最近。虎杖内生细菌及黏细菌中存在PQQ产生菌。     

Transformations of 4- and 17alpha-substituted testosterone analogues by Fusarium culmorum

A series of 4- and/or 17alpha-substituted testosterone analogues has been incubated with the hydroxylating fungus Fusarium culmorum AM282. It was found that 19-norandrostenedione, 19-nortestosterone, 4-methoxytestosterone, 4-methyltestosterone, and 4-chloro-17alpha-methyltestosterone were hydroxylated exclusively or mainly at the 6beta-position. The mixtures of 6beta-, 15alpha-, and 12beta- or 11alpha-monohydroxy derivatives were obtained from 17alpha-methyltestosterone and 17alpha-ethyl-19-nortestosterone--the substrates with alkyl group at C-17alpha. 4-Chlorotestosterone was predominantly hydroxylated at 15alpha-position, but the reaction was accompanied by the reduction of 4-en-3-one system, which proceeded in the sequence: reduction of ketone to 3beta-alcohol and then reduction of the double 4,5 bond. The results obtained indicate an influence of stereoelectronic and steric effects of substitutes on regioselectivity of the hydroxylation of 4-en-3-one steroids by F. culmorum.     

Morphological and biochemical modifications induced by a static magnetic field on Fusarium culmorum

The effects of the exposure to a static magnetic field (sMF) of 0.3 +/- 0.03 T on the Fusarium culmorum were investigated in vitro. sMF inhibition of mycelia growth was accompanied by morphological and biochemical changes. Fungal conidia germination and cell viability were also reduced. We provide evidence of the influence of sMF on Ca(2+)-dependent signal transduction pathways involved in conidia germination. Perturbation of these pathways by adding different compounds (i.e. CaCl(2), phorbol 12-myristate 13-acetate, neomycin, EGTA, LiCl) to the medium, suggested that exposed conidia are unable to mobilise calcium from intracellular stores and that the hindered mechanism may be IP(3)-dependent.     

A serological investigation of hyphal growth in Fusarium culmorum

Summary Apical growth of hyphae of Fusarium culmorum was demonstrated using an immunofluorescent labelling technique. An antiserum prepared against hyphal tips contained a series of antibodies, detected by immunodiffusion, not present in antisera against mature hyphae or conidia. Absorption of the tip antiserum with hyphae allowed a specific immunofluorescence reaction with hyphal tips only. The antiserum against mature hyphae gave non-fluorescent tips to the hyphae.     

Purification and properties of an alkaline proteinase of Fusarium culmorum.

The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3-9.6 and 50 degrees C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman-Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.