Mycelial compatibility reactions of Australian Fusarium graminearum and F. pseudograminearum isolates compared with AFLP groupings

Using the mycelial reactions of 435 combinations of 14 Fusarium pseudograminearum and 15 F. graminearum isolates, it was demonstrated for the first time that mycelial reactions/barrage formation cannot be clearly used to distinguish F. graminearum and F. pseudograminearum. Mutually compatible isolates produced very different patterns of compatibility with other isolates. However, about 60% of pairings between F. graminearum and F. pseudograminearum isolates were compatible, indicating common ancestry. The Mantel tests used to determine any possible associations between mycelial compatibility reactions and AFLP genotypic diversity data revealed no association between the two systems in either species. In addition, no association was found between mycelial compatibility reactions and sexual reproduction in the two species. Implications of the higher frequency of mycelial compatibility reactions observed in F. pseudograminearum than in F. graminearum are discussed.     

The Effects of Organomercury Seed Treatment on Barley Artificially Inoculated with Fusarium spp.1

Artificial field inoculation showed that in one year out of two, Fusarium roseum caused significant yield loss in barley grown from organomercury-treated seed. No significant yield loss occurred in either year as a result of inoculation with Fusarium nivale. Results from field, pot and blotter tests suggested that inoculation with F. nivale and sometimes F. roseum could at times be associated with increased vigour in barley. There was a fairly consistent trend particularly with F. nivale for this to occur to a greater extent in plants from untreated seed than in those from treated seed. Similarly, any reductions in vigour associated with inoculation with more pathogenic strains of either species occurred only, or to a greater extent, in plants from treated seed. There was some evidence of an increased capacity for regenerative root and shoot growth in barley plants damaged by Fusarium inoculation, and it is suggested that increased growth may be associated with increases in percentage dry weights of the shoot 8 but not 18 days after inoculation. In the absence of inoculation, the effect of seed treatment was to increase grain yields, though this was not clearly associated with the increases it sometimes caused in emergence and in 1000-grain weight.     

Natural Occurrence and Distribution of Fusarium Toxins in Contaminated Barley Cultivars

Grain samples of 15 naturally contaminated barley cultivars, collected after harvest in southeastern Poland, were analysed for occurrence of Fusarium trichothecenes and zearalenone (ZEA). Barley kernels were contaminated with the following toxic metabolites: deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), diacetoxyscirpenol (DAS), T-2 tetraol and ZEA. Significant correlations between concentrations of individual toxins and the dominant Fusarium species were found. Moreover, significant differences in toxin concentrations between cultivars were detected. Distribution of these mycotoxins was studied in two fractions of kernels (diameter > 2.5 mm and < 2.5 mm). A two-factor analysis of variance revealed significant differences between the two fractions, and between the analysed cultivars. Most of the interactions between fractions and cultivars were also significant. The highest concentration of the analysed toxins was in the fraction of small kernels. Kernel fraction <2.5 mm, although accounting for only 12.8% of sample weight, contained high proportions of the total toxin content: 80% of DON, 94% of NIV, 85% of ZEA, 83% of T-2 tetraol, 80% of DAS, 68% of HT-2 toxin and 81% of T-2 toxin. The results indicate that the level of contamination with Fusarium trichothecenes and ZEA, can be reduced by rejection of small kernels.     

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.     

Variation in Pathogenicity Associated with the Genetic Diversity of Fusarium graminearum

We screened 188 isolates of Fusarium graminearum, which originated from northwest Europe, the USA and Nepal, for genetic diversity using a sequence-characterised amplified region polymorphism (SCAR). On the basis of this analysis, 42 of the 118 isolates were selected for random amplified polymorphic DNA (RAPD) analysis. Three groups were identified, two of which, A and B, contained the isolates from Nepal, and a third, group C, contained the isolates from Europe and the USA. In pathogenicity tests on wheat and maize seedlings, group C isolates were more pathogenic than the group A and B isolates. The isolates were assigned chemotypes based on their ability to produce the trichothecene mycotoxins nivalenol (NIV) and deoxynivalenol (DON). Isolates from group A were equally likely to produce NIV or DON while group B isolates produced predominantly NIV, and group C isolates produced predominantly DON. Within group A, isolates of the two chemotypes were equally pathogenic to wheat but isolates with the NIV chemotype were significantly more pathogenic to maize. The results confirm that distinct genetic groups exist within F. graminearum and demonstrate that these groups have different biological properties, especially with respect to their pathogenicity to two of the most economically important hosts of this pathogen.     

Fusarium graminearum and Fusarium pseudograminearum caused the 2010 head blight epidemics in Australia

Wheat crops in southeast Queensland (Qld) and northern New South Wales (NSW) were infected with fusarium head blight (FHB)‐like symptoms during the 2010–11 wheat growing season. Wheat crops in this region were surveyed at soft dough or early maturity stage to determine the distribution, severity, aetiology and toxigenicity of FHB. FHB was widespread on bread wheat and durum, and Fusarium graminearum and/or F. pseudograminearum were diagnosed from 42 of the 44 sites using species‐specific PCR primers directly on spikelets or from monoconidial cultures obtained from spikelets. Stem base browning due to crown rot (CR) was also evident in some samples from both states. The overall FHB and CR severity was higher for NSW than Qld. Deoxynivalenol (DON) concentration of immature grains was more than 1 mg kg^?1 in samples from 11 Qld and 14 NSW sites, but only 13 of 498 mature grain samples sourced from the affected areas had more than 1 mg kg^?1 DON. DON concentration in straw also exceeded 1 mg kg^?1 in eight Qld and all but one NSW sites but this was not linked to DON concentration of immature grains. The proportion of spikelets with positive diagnosis for F. graminearum and/or F. pseudograminearum and weather‐related factors influenced DON levels in immature grains. The average monthly rainfall for August–November during crop anthesis and maturation exceeded the long‐term monthly average by 10–150%. Weather played a critical role in FHB epidemics for Qld sites but this was not apparent for the NSW sites, as weather was generally favourable at all sites.     

In vitro activity of isothiocyanates against Fusarium graminearum

Isothiocyanates are biotoxic degradation products formed as a result of enzymatic hydrolysis of glucosinolates present in Brassica species. The application of biofumigant Brassica crops, as an alternative crop protection method for soilborne pathogens and pests is increasingly gaining interest. However, little is known of the potential of biofumigation to reduce the inoculum of Fusarium species affecting cereals. The aim of this study was to evaluate the antifungal activity of five isothiocyanates, namely allyl, benzyl, ethyl, 2-phenylethyl and methyl isothiocyanates, against germination and growth of Fusarium graminearum under in vitro conditions. Aromatic isothiocyanates were more inhibitory than the aliphatic isothiocyanates against mycelial growth, whereas the reverse was observed for conidial germination. Among the tested isothiocyanates, allyl and methyl isothiocyanates were more efficient overall, showing lower ED₅₀ values (35–150 mg/L) for conidial germination and mycelial radial growth. The findings suggest that Brassica plants containing allyl and methyl glucosinolates could have a suppressive effect, reducing the inoculum of F. graminearum in soil prior to cereal production.     

禾谷镰孢菌γ-微管蛋白基因克隆及其与多菌灵抗药性关系分析

根据禾谷镰孢菌参考菌株NRRL31084(PH-1)的γ-微管蛋白基因核苷酸序列设计5对引物,采用PCR方法从禾谷镰孢菌(Fusarium graminearum)对多菌灵(MBC)的敏感菌株、室内诱导及田间多菌灵抗药性菌株中分段扩增测序,获得了γ-微管蛋白基因全序列.该基因全长1 868 bp,含有5个内元,编码一含493aa的多肽;与PH-1的γ-微管蛋白基因核苷酸序列同源性99%,存在10个差异核苷酸,与所编码的氨基酸序列同源性100%;与其它7种真菌的γ-微管蛋白基因所编码的氨基酸序列同源性为31%～72%.中国的2个敏感菌株和4个抗药菌株的γ-微管蛋白基因序列完全相同,认为多菌灵抗药性与该微管蛋白变异无关.     

Zearalenone production in Fusarium culmorum after transformation with DNA of F. graminearum

Total DNA from a cycloheximide-resistant mutant of Fusarium graminearum producing zearalenone was incubated with the protoplasts of a non-zearalenone-producing cycloheximide-susceptible isolate of F. culmorum. The regenerated protoplasts were inoculated on a 5-mM cycloheximide medium. The cultures which developed were examined for zearalenone production. Sixty-five cycloheximide-resistant isolates, including two isolates producing zearalenone, were obtained from approximately 3.6 × 10⁴ viable F. culmorum protoplasts. Similar incubations with homologous F. culmorum DNA and non-fungal DNA did not elicit zearalenone production. The results suggest the transformation of F. culmorum by the genetic factors responsible for cycloheximide resistance and zearalenone production from F. graminearum. The epidemiological and taxonomic significance of these observations is discussed.     

Trichothecene mycotoxin genotypes of Fusarium graminearum sensu stricto and Fusarium meridionale in wheat from southern Brazil

A total of 82 fungal isolates was obtained from wheat kernel samples affected by fusarium head blight collected from 20 locations in southern Brazil. Polymerase chain reaction (PCR) assays were used to characterize trichothecene mycotoxin genotypes [deoxynivalenol (DON), nivalenol (NIV) and two acetylated derivatives of DON]. To identify isolates that producing DON and NIV, portions of the Tri13 gene were amplified. To identify 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes, portions of Tri3 and Tri12 were amplified. Nearly all of the isolates studied (76/82) were of the DON/15-ADON genotype. Six of the isolates were of the NIV genotype. The DON/3-ADON genotype was not observed. Portions of three genes were sequenced from representative isolates of the NIV and DON/15-ADON genotypes and compared with sequences from curated reference isolates of Fusarium in GenBank. blast queries for individual gene sequences and pairwise comparisons of percentage identity and percentage divergence based on 1676 bp of concatenated DNA sequence suggested that the isolates representing the DON/15-ADON genotype were Fusarium graminearum sensu stricto and the isolates representing the NIV genotype were Fusarium meridionale . This is the first detailed report of trichothecene mycotoxin genotypes of F. graminearum and F. meridionale in Brazil.     

陕西省小麦赤霉病菌对多菌灵敏感性研究

 The mycelium growth rate method was used to test the sensitivity to carbendazim (MBC) at distinctive concentrations in 136 isolates of Fusarium graminearum from 19 counties of 6 districts in Shaanxi Province in 2008. The distinctive MBC concentration was 4 mg / L for testing of resistance and sensitivity. The results showed that average 50% effective concentration (EC50 ) of 136 tested sensitive isolates were (0. 908 6 ± 0. 062 3) mg / L. All the isolates were sensitive to MBC. The fungicide of MBC could be continually applied wheat production in Shaanxi.     

Molecular characterization of the Fusarium graminearum species complex in Japan

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.     

陕西省小麦禾谷镰刀菌的遗传多样性研究

为了明确陕西省小麦禾谷镰刀菌混合群(Fusarium graminearum species complex)的遗传多样性,利用4对E coRⅠ和M seⅠ引物对来自陕西省19个区县的162株小麦禾谷镰刀菌菌株进行了AFLP扩增。结果表明,4对引物均能扩增出数量不等的多态性条带,最少的6条,最多的20条,大部分扩增片段在100～750bp之间。利用NTSYS-2.1软件聚类分析表明,不同地区禾谷镰刀菌可分为两大类群,即类群A和类群B。这两大类群的分化和地理来源有明确相关性,类群A主要分布在关中地区,类群B主要分布在陕南地区。初步判断可能与两个地区生态环境和小麦主栽品种差异有关。各类群内的菌系与地理位置间的关系较为复杂,一些菌系与地理来源存在明确关系,而个别菌系与地理来源间的关系尚不能完全明确。还需进一步研究以明确各菌株与地理来源之间的关系。     

Genetic diversity of Australian Fusarium graminearum and F. pseudograminearum

Genotypic diversity in Fusarium pseudograminearum and F. graminearum from Australia and the relationship between diversity and pathogen aggressiveness for head blight and/or crown rot of wheat were examined. Amplified fragment length polymorphism (AFLP) analysis revealed a high level of genotypic diversity within each species. Sixty-three of the 149 AFLP loci were significantly different between the two species and 70 of 72 F. pseudograminearum and 56 of 59 F. graminearum isolates had distinct haplotypes. When head blight and crown rot severity data from a recently published work on isolates representing the entire range of aggressiveness were used, only the genotypic diversity of F. pseudograminearum was significantly associated with its aggressiveness for the two diseases. Cluster analyses clearly demonstrated the polyphyletic structures that exist in both pathogen populations. The spatial diversity within F. graminearum was high within a single field, while frequent gene flow ( N _m ∼ 14) and a low fixation index ( G _st = 0·03) were recorded among F. pseudograminearum isolates from the adjacent states of New South Wales and Queensland. The differences in population structure between the heterothallic F. pseudograminearum (teleomorph G. coronicola ) and the homothallic F. graminearum (teleomorph G. zeae ) were not as pronounced as expected given their contrasting mating systems. Neither species was panmictic or strictly clonal. This points to sexual recombination in F. pseudograminearum , suggesting that ascospores of G. coronicola may also play a role in its biology and epidemiology.     

PIRA-PCR for detection of Fusarium graminearum genotypes with moderate resistance to carbendazim

PIRA-PCR ( p rimer- i ntroduced r estriction a nalysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the β ₂-tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A Hin dIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and Taa I ( Tsp 4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both Hin dIII and Taa I. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7–8 h while the conventional method required 5–7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.     

Use of Fusarium graminearum transformed with gfp to follow infection patterns in barley and Arabidopsis

The fungal pathogen Fusarium graminearum attacks the seed spikes of barley and wheat, causing sterility, reduced seed weight and accumulation of mycotoxins. To explore infection patterns in barley and in the Arabidopsis model system, the green fluorescent protein gene (gfp) was used to transform F. graminearum. Inoculation of intact barley spikes resulted in rapid colonization of the brush hairs (ovary epithelial hairs) at the extruded seed tip within 7 h. Colonization followed a pattern of rapid basipetal growth along the pericarp epithelium (interior to the lemma and palea), accompanied by slower growth inward through the pericarp and testa. However, at 16 days after infection the aleurone and starchy endosperm remained uninfected, despite heavy colonization of the pericarp. Colonization of the outer lemma also occurred but was much slower. No increase in amylase enzyme activities was found, discounting the possibility that F. graminearum utilizes gibberellin-induced host enzymes to tap the endosperm for nutrients. The transformed Fusarium strain readily infected Arabidopsis thaliana leaves and produced copious spores within distant leaves. Results show the utility of gfp in tracing the growth of this pathogen, without misinterpretation due to contaminating fungi, and for resistance studies utilizing the Arabidopsis model system.     

Head infection and accumulation of Fusarium toxins in kernels of 12 barley genotypes inoculated with Fusarium graminearum isolates of two chemotypes

Twelve barley genotypes were inoculated with two F. graminearum isolates of different chemotype I₁ #148 (producing nivalenol/deoxynivalenol) and I₂ #108 (deoxynivalenol/acetyldeoxynivalenol). For both I₁ and I₂ isolates, respectively, reductions (%) in number of kernels head^-1 10.6 and 14.3; yield 39.6 and 35.7; weight of 1000 kernels 36.9 and 23.2 were observed in inoculated plants from control values. Chemical analysis revealed the presence (average concentration mg kg^-1) of deoxynivalenol (1.3) and nivalenol (3.2) in kernels of all genotypes inoculated with the I₁ isolate, and zearalenone (0.2) in three samples. After inoculation with the I₂ isolate, deoxynivalenol (37.8) and zearalenone (0.4) were found in kernels of all genotypes, while 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, respectively, were determined in five and four samples only. No significant correlation between examined characteristics was found for either the I₁ or I₂ isolate. The results obtained contribute information on the accumulation of toxins in cereal grain inoculated with F. graminearum isolates of different chemotypes.     

小麦赤霉病菌对多菌灵的抗药性研究

测定了1976 年从江苏南京采集的54 株小麦赤霉病菌菌株对多菌灵的敏感性,EC50值在0. 2617～ 0. 6544 μg. mL - 1 之间, 1983 年在同一地点采集的76 株, EC50 值相应为0. 2517～ 0. 7050 μg. mL- 1 , 而1998 年从浙江、湖北、上海、福建、安徽、江苏各地采集的104 株菌株EC50值为0. 2478～ 6. 4574 Lg. mL- 1 , 表明湖北、上海、浙江等地田间已检测到小麦赤霉病菌抗多菌灵菌株。紫外光诱导分生孢子也获得了该病菌抗多菌灵突变株, 其EC50 值为14. 1993 Lg. mL - 1 。抗药性突变体JPR 与敏感亲本菌株JPS 相比, 在菌丝生长、产孢量方面无明显差异, 但JPR 孢子萌发率为57. 1% , 而JPS 为100% , 而且50% 孢子萌发的时间较野生亲本菌株滞后12 h。JPR 产生脱氧雪腐镰刀菌烯醇(DON 毒素) 3. 90 μg mL - 1 , 而JPS 产生的DON 毒素为9. 28μg. mL - 1 。     

Scab Response and Moniliformin Accumulation in Kernels of Oat Genotypes Inoculated with Fusarium avenaceum in Poland

Inoculation experiments with 14 genotypes of oats (10 cultivars and 4 lines) were performed during 1996, 1997 and 1998 in Sitaniec, South-Eastern Poland. Panicles of oats were inoculated with a conidial suspension of Fusarium avenaceum, which caused a reduction in yield by 33% and in 1000 kernel weight (TKW) by 21%. During the period between inoculation and harvest, F. avenaceum was able to accumulate moniliformin (MON) in kernels at an average level of 0.13mgkg^–1 (gg^–1). The highest reduction of yield components caused by the F. avenaceum inoculation was found for cv. Santor, followed by lines CHD 1171, STH 2795 and cvs: Kwant and Farys, while cvs Slawko, Dukat, Borys and Komes exhibited the highest resistance to the disease in terms of TKW and yield reductions after inoculation.     

Role of Deoxynivalenol in Aggressiveness of Fusarium graminearum and F. culmorum and in Resistance to Fusarium Head Blight

The data available indicate that aggressiveness of Fusarium graminearum and F. culmorum depends on their deoxynivalenol (DON) and nivalenol-producing capacity: toxin-producing ability correlated closely with the level of aggressiveness measured. This agrees well with other literature findings. However, the resistance of a cultivar influenced DON production significantly. In the most resistant genotypes, toxin contamination remained near zero, whereas the same isolates and inoculum produced very high toxin levels in susceptible cultivars. As toxin levels were correlated with the ratio of Fusarium-damaged kernels (FDK) and this ratio is very low in highly resistant cultivars, the conclusion is that the level of resistance level is more important in governing DON accumulation in a given cultivar than is the aggressiveness of an isolate. In susceptible cultivars, DON producing ability is decisive, but in highly resistant cultivars resistance is the major factor in suppressing disease development and DON accumulation. In different years, the same FDK values were associated with different DON concentrations and this depended very much on the precipitation towards the end of May, the time of inoculation.