Role of hydroxycinnamic acids in the infection of maize silks by Fusarium graminearum Schwabe

In the current study, the hydroxycinnamic acids in silks of diverse maize inbred lines differing in Fusarium resistance were determined at several times after inoculation with Fusarium graminearum or sterile water as control. The main objective was to determine the possible relationship between the hydroxycinnamic acid changes in silks and ear rot resistance. Several changes in the cell-wall-bound hydroxycinnamic acid concentrations were observed after inoculation with F. graminearum, although these changes were not directly correlated with genotypic resistance to this fungus. Ester-bound ferulic acid decreased, probably due to degradation of hemicellulose by hydrolytic enzymes produced by Fusarium spp., while p-coumaric acid and diferulates showed slight increases that, in conjunction, did not result in delayed F. graminearum progression through the silks. It is important to note that the decrease of ferulic acid in the F. graminearum treatment was faster in susceptible than in resistant genotypes, suggesting a differential hemicellulose degradation in silk tissues. Therefore, the ability of the maize genotypes to slow down that process through hemicellulose structural features or xylanase inhibitors needs to be addressed in future studies.     

Molecular Genetic Diversity and Variation for Aggressiveness in Populations of Fusarium graminearum and Fusarium culmorum Sampled from Wheat Fields in Different Countries

Fusarium graminearum and Fusarium culmorum are the major pathogenic organisms causing head blight in small-grain cereals. Natural epidemics may result in severe yield losses, reduction in quality, and contamination of the grain by mycotoxins. The genetic diversity of four field populations of F. graminearum from Germany, Hungary, and Canada, and one population of F. culmorum from Russia was investigated by polymerase chain reaction (PCR)-based fingerprinting. Additionally, a world-wide collection and two of the F. graminearum populations were analysed for their aggressiveness on young plants of winter rye in the greenhouse. The number of isolates analysed per population varied from 25 to 70. Significant quantitative variation for aggressiveness was observed within each of the individual field populations amounting to the same range as the world-wide collection. Abundant variation within populations was also revealed by DNA markers. The F. graminearum populations from Hungary and Winnipeg displayed the least genotypic diversity, the two German F. graminearum populations and the Russian F. culmorum population were highly diverse. Population diversity, however, followed no spatial pattern among samples within a German field for aggressiveness or molecular markers. For F. graminearum , sexual recombination is the most likely explanation for the large genetic diversity within field populations. Asexual and/or parasexual recombination, and balancing selection caused by the periodic alternation between the saprophytic and parasitic phase might play an additional role and account for the variation within the F. culmorum population. For improving Fusarium resistance, several resistance genes of different sources should be combined to avoid an unspecific adaptation of the genetically variable pathogen to an increased resistance level.     

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F.?graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F.?graminearum. Our data suggest that PGs likely play a role in F.?graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.     

Sphaerodes mycoparasitica biotrophic mycoparasite of 3-acetyldeoxynivalenol- and 15-acetyldeoxynivalenol-producing toxigenic Fusarium graminearum chemotypes

Fusarium spp. are economically important crop pathogens and causal agents of Fusarium head blight (FHB) of cereals worldwide. Of the FHB pathogens, Fusarium graminearum 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) are the most aggressive mycotoxigenic chemotypes, threatening food and feed quality as well as animal and human health. The objective of the study was to evaluate host specificity and fungal-fungal interactions of Sphaerodes mycoparasitica- a recently described mycoparasite - with F. graminearum 3- and 15-ADON strains by employing in vitro, microscopic and PCR techniques. Results obtained in this study show that the germination of mycoparasite ascospore in the presence of F. graminearum 3- and 15-ADON filtrates was greatly improved compared with Fusarium proliferatum and Fusarium sporotrichioides filtrates, suggesting a compatible interaction. Using quantitative real-time PCR with Fusarium-specific (Fg16N) and trichothecene Tri5 (Tox5-1/2)-specific primer sets, S. mycoparasitica was found to reduce the amount of F. graminearum 3-ADON and 15-ADON DNAs under separate coinoculation assays. Sphaerodes mycoparasitica was not only able to germinate in the presence of F. graminearum filtrates, but also established biotrophic mycoparasitic relations with two F. graminearum chemotypes and suppressed Fusarium growth.     

Salicylic acid regulates basal resistance to Fusarium head blight in wheat

Fusarium head blight (FHB) is a destructive disease of cereal crops such as wheat and barley. Previously, expression in wheat of the Arabidopsis NPR1 gene (AtNPR1), which encodes a key regulator of salicylic acid (SA) signaling, was shown to reduce severity of FHB caused by Fusarium graminearum. It was hypothesized that SA signaling contributes to wheat defense against F. graminearum. Here, we show that increased accumulation of SA in fungus-infected spikes correlated with elevated expression of the SA-inducible pathogenesis-related 1 (PR1) gene and FHB resistance. In addition, FHB severity and mycotoxin accumulation were curtailed in wheat plants treated with SA and in AtNPR1 wheat, which is hyper-responsive to SA. In support of a critical role for SA in basal resistance to FHB, disease severity was higher in wheat expressing the NahG-encoded salicylate hydroxylase, which metabolizes SA. The FHB-promoting effect of NahG was overcome by application of benzo (1,2,3), thiadiazole-7 carbothioic acid S-methyl ester, a synthetic functional analog of SA, thus confirming an important role for SA signaling in basal resistance to FHB. We further demonstrate that jasmonate signaling has a dichotomous role in wheat interaction with F. graminearum, constraining activation of SA signaling during early stages of infection and promoting resistance during the later stages of infection.     

Effect of salicylic acid on Fusarium graminearum, the major causal agent of fusarium head blight in wheat

Salicylic acid (SA) is one of the key signal molecules in regulating plant resistance to diverse pathogens. In Arabidopsis thaliana, it is predominantly associated with resistance against biotrophic and hemibiotrophic pathogens, and triggering systemic acquired resistance. In contrast, the effect of SA on the defence efficiency of wheat against fusarium head blight (FHB) and its causal agent, Fusarium graminearum, is still poorly understood. Here we show that the F. graminearum mycelial growth and conidia germination were significantly inhibited, and eventually halted in the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol (DON). However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a carbon source. High performance liquid chromatography (HPLC) analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom-commercial microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum conidia in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cv. Roblin. In contrast, no inhibition was observed when SA and conidia were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdf1.2, and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrate that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.     

Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from maize in Nepal and identification of a putative new lineage

On smallholder farms in the foothills of the Himalayan Mountains in Nepal, fungi of the Fusarium graminearum clade cause Gibberella ear rot of maize and contamination with the 8-ketotrichothecenes nivalenol and deoxynivalenol. Previous DNA marker analyses of the F.?graminearum clade from maize in Nepal found a high level of genetic diversity but were limited in detail or scope. The present study incorporated a collection of 251 field strains from a wide geographic distribution in Nepal and utilized sequencing of the MAT1-1-3 gene of the mating type locus to determine the number and frequency of lineages and species of the F. graminearum clade. The frequency of nivalenol and deoxynivalenol chemotypes was determined by chemical analysis and by TRI13 deletion-marker analysis. We found that Gibberella ear rot of maize in Nepal is associated with a complex of species of the F. graminearum clade - mainly Fusarium asiaticum and Fusarium meridionale, but also Fusarium boothii and a putative new lineage, which we have designated the 'Nepal lineage'. Fusarium graminearum sensu stricto, which dominates in maize elsewhere in Asia and worldwide, was not detected in Nepal. Although nivalenol production has been associated experimentally with lower virulence in maize ear rot and wheat head blight, this collection of the F. graminearum clade from maize in Nepal is dominated (4:1) by nivalenol producers, suggesting that traits other than crop plant pathogenesis affect population structure in this complex agroecosystem.     

The Type 2C protein phosphatase FgPtc1p of the plant fungal pathogen Fusarium graminearum is involved in lithium toxicity and virulence

Type 2C protein phosphatases (PP2Cs) are monomeric protein serine/threonine phosphatases that play various roles in eukaryotic organisms. In this study, we characterized the PP2C encoded by FgPTC1 in Fusarium graminearum , the major causal agent of Fusarium head blight on wheat and barley. We found that deletion of FgPTC1 delays the mycelium growth of F. graminearum in response to lithium. Consistently, FgPTC1 complemented the function of ScPTC1 in lithium toxicity in Saccharomyces cerevisiae . Furthermore, we showed that deletion of FgPTC1 attenuated the virulence of F. graminearum on wheat. Therefore, FgPTC1 plays an important role in regulating the hyphal growth and virulence of F. graminearum .     

以nit为遗传标记研究禾谷镰刀菌对氰烯菌酯的抗药性在菌丝融合过程中的遗传

分别从对氰烯菌酯敏感和抗性禾谷镰刀菌菌株中诱导了22个和50个nit突变体。通过比较它们的生物学特性表明,nit突变体在菌丝生长速率、培养形状以及致病性方面与其亲本没有显著差异,但是无性繁殖和有性繁殖能力有所改变。同时实验还表明,禾谷镰刀菌对氰烯菌酯和氯酸盐不存在交互抗性,且对氰烯菌酯和氯酸盐的双重抗性能够稳定地遗传。因此,可以将nit作为一个优良的遗传标记研究禾谷镰刀菌对氰烯菌酯的抗药性遗传。另外成功运用nit作为分子标记研究了禾谷镰刀菌对氰烯菌酯的抗药性在菌丝融合过程中的遗传和变异。研究结果表明,抗药性基因不能通过菌丝融合传递给另一个菌株或发生的概率极低,这将不利于对氰烯菌酯的抗性群体的发展。因此,菌丝融合在禾谷镰孢菌对氰烯菌酯的抗药性群体发展中的作用较小。     

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F.?poae isolates. A total of 125 F.?poae, four F.?cerealis, two F.?culmorum, one F.?langsethiae, one F.?sporotrichioides and seven F.?graminearum, plus F.?austroamericanum, F.?meridionale, F.?graminearum sensu stricto and F.?cortaderiae from the NRRL collection were analysed, and only F.?poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F.?poae and other Fusarium species with a negative result for the specific reaction ( F.?graminearum, F.?oxysporum, F.?chlamydosporum, F.?sporotrichioides, F.?equiseti and F.?acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F.?poae isolates.     

A molecular based strategy for rapid diagnosis of toxigenic Fusarium species associated to cereal grains from Argentina

Fusarium species are worldwide causal agents of ear rot in cereals. Their toxigenic potential is a health risk for both humans and animals. In Argentina, most identification of these fungi has been based on morphological and cross-fertility criteria which are time consuming and require considerable expertise in Fusarium taxonomy and physiology. DNA based approaches have been reported as rapid, sensitive and specific alternatives to identify the main fumonisin and trichothecene-producing Fusarium species. In this work, we used PCR assays and the partial sequence of TEF1-alpha gene (Translation Elongation Factor-1 alpha) to identify the fumonisin and trichothecene-producing species in Fusarium isolates from diverse regions of Argentina. The relative efficiency and reliability of those methods to improve mycotoxin risk prediction in this country were also assessed. Species-specific PCR assays were targeted toward multicopy IGS (Intergenic Spacer of rDNA units) and on the toxin biosynthetic genes FUM1 (fumonisins) and TRI13 and TRI7 genes (trichothecenes). PCR assays based on FUM1 gene and IGS sequences allowed detection and discrimination of the fumonisin producers Fusarium proliferatum and Fusarium verticillioides. Molecular identification of nonfumonisin producers from Gibberella fujikuroi species complex was possible after determination of TEF1-alplha gene sequences, which indicated the presence of Fusarium subglutinans, Fusarium andiyazi and Fusarium thapsinum. TEF-1 alpha gene sequences also allowed discrimination of the different species of the Fusarium graminearum complex (F. graminearum sensu lato) as F. graminearum sensu stricto, Fusarium meridionale and Fusarium boothii. The last two species belonged to NIV chemotype and were detected for the first time in the subtropical region of Argentina while F. graminearum sensu stricto was DON producer only, which was also confirmed by specific PCR assays based on TRI137/TRI7 genes. Our results indicated that the PCR assays evaluated in this work are reliable diagnostic tools to detect the main toxigenic Fusarium species associated to cereal grains in Argentina. An extensive epidemiological survey based on the approach presented in this work is currently in progress to know the mycotoxigenic hazard of Fusarium species in cereal grains from the subtropical region of Argentina.     

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。     

Identification of 11 polymorphic simple sequence repeat loci in the phytopathogenic fungus Fusarium pseudograminearum as a tool for genetic studies

Simple sequence repeat (SSR) markers for Fusarium pseudograminearum with 2 to 3 bp repeat motifs were identified by screening the genome database of the related species Fusarium graminearum. Twelve SSRs amplified single loci in both F. graminearum and F. pseudograminearum. Forty F. pseudograminearum and six F. graminearum individual isolates were screened to determine levels of polymorphism, with all SSRs displaying three to 14 alleles across all isolates. Eleven SSRs were polymorphic across F. pseudograminearum isolates tested proving the usefulness of genome databases of closely related species in identifying genetic markers.     

Toxin production by Fusarium culmorum IMI 309344 and F. graminearum NRRL 5883 on grain substrates

The production of deoxynivalenol, acetyl deoxynivalenol and zearalenone by Fusarium culmorum and F. graminearum on autoclave-sterilized grain (maize, rice, wheat and barley) was investigated. Fusarium culmorum produced significantly greater levels of toxins than F. graminearum. The four substrates examined differed in their ability to support toxin production. Toxin production on maize and rice was significantly greater than toxin production on barley or wheat.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.     

小麦抗茎腐病种质筛选及鉴定新方法的建立

为筛选针对我国黄淮麦区小麦茎腐病抗病新种质,建立可区分小麦其他茎基部病害的茎腐病抗性鉴定方法,本文采用室内苗期鉴定方法对主要来自黄淮麦区的108份小麦品种和高代品系进行茎腐病抗性评价,对其中45份小麦材料同时采用荧光定量PCR方法测定基部茎秆的禾谷镰刀菌DNA含量并与其茎腐病平均病级进行相关分析。共筛选到中抗茎腐病材料22份,未发现高抗和免疫品种(系);相关分析结果表明,小麦基部茎秆禾谷镰刀菌DNA含量与其茎腐病平均病级呈极显著正相关(r=0.73**),小麦基部茎秆禾谷镰刀菌DNA含量可以作为小麦茎腐病抗性的重要参考。抗病新种质的筛选和荧光定量PCR抗性评价方法的建立将为今后黄淮麦区小麦抗茎腐病品种的培育提供帮助。