Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.     

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.     

A rice GDSL esterase/lipase protein (GELP) is required for anther and pollen development

Molecular Breeding - Gibberella ear rot (GER) caused by Fusarium graminearum (teleomorph Gibberella zeae), is one of the most devastating diseases in maize that not only severely reduces grain...     

On the trail of a cereal killer: recent advances in Fusarium graminearum pathogenomics and host resistance

The ascomycete fungal pathogen Fusarium graminearum (sexual stage: Gibberella zeae) causes the devastating head blight or scab disease on wheat and barley, and cob or ear rot disease on maize. Fusarium graminearum infection causes significant crop and quality losses. In addition to roles as virulence factors during pathogenesis, trichothecene mycotoxins (e.g. deoxynivalenol) produced by this pathogen constitute a significant threat to human and animal health if consumed in respective food or feed products. In the last few years, significant progress has been made towards a better understanding of the processes involved in F. graminearum pathogenesis, toxin biosynthesis and host resistance mechanisms through the use of high-throughput genomic and phenomic technologies. In this article, we briefly review these new advances and also discuss how future research can contribute to the development of sustainable plant protection strategies against this important plant pathogen.     

Analysis of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides evidence of species-specific differences in host preference

Species identity and trichothecene toxin potential of 560 members of the Fusarium graminearum species complex (FGSC) collected from diseased wheat, barley and maize in South Africa was determined using a microsphere-based multilocus genotyping assay. Although three trichothecene types (3-ADON, 15-ADON and NIV) were represented among these isolates, strains with the 15-ADON type predominated on all three hosts. A significant difference, however, was identified in the composition of FGSC pathogens associated with Gibberella ear rot (GER) of maize as compared to Fusarium head blight (FHB) of wheat or barley (P<0.001). F. graminearum accounted for more than 85% of the FGSC isolates associated with FHB of wheat and barley (N=425), and was also the dominant species among isolates from maize roots (N=35). However, with the exception of a single isolate identified as an interspecific hybrid between Fusariumboothii and F. graminearum, GER of maize (N=100) was exclusively associated with F. boothii. The predominance of F. graminearum among FHB isolates, and the near exclusivity of F. boothii among GER isolates, was observed across all cultivars, collection dates, and provinces sampled. Because these results suggest a difference in host preference among species of the FGSC, we hypothesize that F. graminearum may be less well adapted to infect maize ears than other members of the FGSC.     

Tri1 in Fusarium graminearum encodes a P450 oxygenase

Gibberella zeae (asexual state Fusarium graminearum) is a major causal agent of wheat head blight and maize ear rot in North America and is responsible for contamination of grain with deoxynivalenol and related trichothecene mycotoxins. To identify additional trichothecene biosynthetic genes, cDNA libraries were prepared from fungal cultures under trichothecene-inducing conditions in culture and in planta. A gene designated LH1 that was highly expressed under these conditions exhibited only moderate (59%) similarity to known trichothecene biosynthetic cytochrome P450s. To determine the function of LH1, gene disruptants were produced and assessed for trichothecene production. Gene disruptants no longer produced 15-acetyldeoxynivalenol, which is oxygenated at carbon 7 (C-7) and C-8, but rather accumulated calonectrin and 3-deacetylcalonectrin, which are not oxygenated at either C-7 or C-8. These results indicate that gene LH1 encodes a cytochrome P450 responsible for oxygenation at one or both of these positions. Despite the relatively low level of DNA and amino acid sequence similarity between the two genes, LH1 from G. zeae is the probable homologue of Tri1, which encodes a cytochrome P450 required for C-8 oxygenation in F. sporotrichioides.     

FUM1--a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests

We have analyzed the role of fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for fumonisin biosynthesis. Maize ear rot, ear infection, and fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and fumonisin production. Two independent fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.     

玉米穗腐病抗性鉴定、遗传分析与分子机制*

玉米是我国主要农作物之一,其产量占全国谷物总产量三分之一左右,在国民生活中发挥了重要作用。玉米生长过程中受多种病害侵扰,其中穗腐病是一种由真菌导致的常见病害,目前已鉴定出40余种可诱发玉米穗腐病的病原菌。穗腐病不仅可造成玉米产量损失也导致籽粒品质严重下降,病原微生物所产生次生毒素更是危及人畜安全。目前穗腐病防治以化学方法为主,但是因此所造成的种植成本增加和环境污染问题日益突出,选育抗性品种成为防控玉米穗腐病最经济和安全有效的方法。玉米穗腐病抗性属于典型数量性状,国内外已有多个研究组通过建立玉米穗腐病抗性研究体系,开展玉米群体大规模穗腐病抗性鉴定工作,并筛选出一批抗病材料,这为玉米穗腐病抗性遗传改良奠定了材料基础。利用数量性状位点(quantitative trait locus, QTL)定位和全基因组关联分析(genome-wide association study, GWAS)等方法在玉米 1~10号染色体均检测到显著相关位点。尽管如此,玉米穗腐病抗性研究成果应用于生产的实例较少,生产上仍然缺乏综合性状优良且高抗穗腐病的玉米品种。这一方面是由于玉米穗腐病抗性遗传机制十分复杂、抗性基因克隆工作进展缓慢;另一方面为缺乏对玉米穗腐病抗性遗传研究进展进行系统总结,未有效开发分子标记用于分子育种所致。通过综述玉米穗腐病抗性遗传研究进展,汇总已定位QTL位点和显著关联SNP,构建一致性图谱和鉴定出定位热点区间,并进一步对比分析定位区间候选基因特征和转录组、代谢组研究进展,对促进玉米穗腐病抗性机制研究和玉米抗性育种均具有重要意义。     

A new QTL for resistance to Fusarium ear rot in maize

Understanding the inheritance of resistance to Fusarium ear rot is a basic prerequisite for an efficient resistance breeding in maize. In this study, 250 recombinant inbred lines (RILs) along with their resistant (BT-1) and susceptible (N6) parents were planted in Zhengzhou with three replications in 2007 and 2008. Each line was artificially inoculated using the nail-punch method. Significant genotypic variation in response to Fusarium ear rot was detected in both years. Based on a genetic map containing 207 polymorphic simple sequence repeat (SSR) markers with average genetic distances of 8.83?cM, the ear rot resistance quantitative trait loci (QTL) were analyzed by composite interval mapping with a mixed model (MCIM) across the environments. In total, four QTL were detected on chromosomes 3, 4, 5, and 6. The resistance allele at each of these four QTL was contributed by resistant parent BT-1, and accounted for 2.5-10.2% of the phenotypic variation. However, no significant epistasis interaction effect was detected after a two-dimensional genome scan. Among the four QTL, one QTL with the largest effect on chromosome 4 (bin 4.06) can be suggested to be a new locus for resistance to Fusarium ear rot, which broadens the genetic base for resistance to the disease and can be used for further genetic improvement in maize-breeding programs.     

Detection and verification of quantitative trait loci for resistance to Fusarium ear rot in maize

Fusarium ear rot caused by Fusarium verticillioides is a prevalent disease in maize which can severely reduce grain yields and quality. Identification of stable quantitative trait loci (QTL) for resistance to Fusarium ear rot is a basic prerequisite for understanding the genetic mechanism of resistance and for the use of marker-assisted selection. In this study, two hundred and ten F _2:3 families were developed from a cross between resistant inbred line BT-1 and susceptible inbred line Xi502, and were genotyped with 178 simple sequence repeat markers. The resistance of each line was evaluated in two environments by artificial inoculation using the nail-punch method. The resistance QTL were detected using the composite interval mapping method. Three QTL were detected on chromosomes 4, 5 and 10. Of them, the QTL on chromosome 4 (bin 4.05/06) had the largest resistance to Fusarium ear rot, and could explain 17.95?% of the phenotypic variation. For further verification of the QTL effect, we developed near-isogenic lines (NILs) carrying the QTL region on chromosome 4 using parental line Xi502 as the recurrent parent. In the NIL background, this QTL can increase the resistance by 33.7?C35.2?% if the resistance region is homozygous, and by 17.8?C26.5?% if the resistance region contains the heterozygous allele. The stable and significant resistance effect of the QTL on chromosome 4 lays the foundation for further marker-assisted selection and map-based cloning in maize.     

FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F. graminearum

Fusarium verticillioides (teleomorph Gibberella moniliformis) and F. graminearum (teleomorph G. zeae) are well known to cause devastating diseases on cereal crops. Despite their importance, our understanding of the molecular mechanisms involved in these host-pathogen interactions is limited. The FSR1 locus in F. verticillioides was identified by screening REMI mutants for loss of virulence in maize stalk rot inoculation studies. FSR1 encodes an 823-codon open reading frame interrupted by two introns. The Fsr1 protein shares 60% sequence identity with the Sordaria macrospora Pro11, a multimodular protein with four putative protein-protein binding domains (caveolin-binding domain, coiled-coil structure, calmodulin-binding motif, and seven-WD40 repeats), which plays a regulatory role in cell differentiation and ascocarp development. Our data demonstrate that FSR1 is essential for female fertility and virulence in F. verticillioides. Significantly, targeted disruption of the FSR1 ortholog in F. graminearum (FgFSR1) reduced virulence on barley and deterred perithecia formation. Cross-complementation experiments demonstrated that the gene function is conserved in the two Fusarium species. FSR1 is expressed constitutively, and we hypothesize that Fsr1 regulates virulence by acting as a scaffold for a signal transduction pathway. A survey of available genome databases indicates Fsr1 homologs are present in a number of filamentous fungi and animal systems but not in budding yeast or plants. A maximum likelihood analysis of this gene family reveals well-supported monophyletic clades associated with fungi and animals.     

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.     

玉米抗镰刀菌穗腐病接种方法及抗病资源筛选研究

对玉米抗穗腐病性鉴定中采用的牙签法与花丝通道注射法进行比较研究表明,牙签法接种果穗的发病程度重于花丝通道注射法接种。牙签法接种病菌于果穗子粒与穗轴之间,有利于病原菌的发育扩展,是一种比较理想的、容易操作的接种方法。采用牙签法接种串珠镰刀菌,对178份玉米自交系和15份杂交种进行抗穗腐病鉴定,筛选出高抗(HR)自交系1份、抗病(R)玉米自交系34份、抗病(R)玉米杂交种12份。     

Molecular mechanisms of deoxynivalenol resistance in the yeast<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The production of trichothecene toxins is a suspected virulence mechanism of several plant pathogenic fungi. This hypothesis has been confirmed forGibberella zeae (Fusarium graminearum) by gene disruption experiments, suggesting in turn, that resistance against the fungal toxin is a relevant component ofFusarium resistance of the host plant. Our goal is therefore to identify molecular mechanisms of trichothecene resistance. Using yeast as a model system we have found the following resistance mechanisms and genes: a) reduced uptake of deoxynivalenol (PDR5), b) toxin modification and reduction of toxicity (AYT1), and c) formation of a resistant toxin target (RPL3). Homologous plant genes exist and are attractive candidates forFusarium resistance genes.     

玉米茎基腐病生防菌的筛选及应用

【目的】拟针对禾谷镰孢进行生防菌的筛选和应用研究,以期为玉米茎基腐病生防菌剂的研制奠定基础。【方法】通过对峙培养法对玉米茎基腐病的主要致病菌禾谷镰孢进行玉米内生生防细菌的筛选,从玉米主栽品种九单48幼苗内部获得一株具有较强抑菌效果的内生生防细菌(简称48SJ7-1);基于传统鉴定、16S rRNA基因序列测定及系统发育分析,对48SJ7-1进行鉴定;并通过盆栽试验测定该生防菌株的防效。【结果】菌株48SJ7-1经鉴定为甲基营养型芽孢杆菌,GenBank登录号为KU377993,48SJ7-1盆栽防效为68.47%,与对照药剂2%戊唑醇悬浮种衣剂差异不显著。【结论】48SJ7-1对玉米茎基腐病的主要致病菌禾谷镰孢有较好防效,对玉米生长还有明显的促进作用,而且表观上无药害发生。     

Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors

The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.     

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.