Fusarium Head Blight of Common Polish Winter Wheat Cultivars – Comparison of Effects of Fusarium avenaceum and Fusarium culmorum on Yield Components

The effects of Fusarium avenaceum and Fusarium culmorum on the reduction in yield components, after independent inoculation of 14 winter wheat cultivars, were investigated. Single isolates of F. avenaceum and F. culmorum were independently used in inoculations of winter wheat heads. Reductions in the following yield traits: 1000‐kernel weight (TKW), the weight (WKH) and number (NKH) of kernels per head after inoculation were analysed statistically. The results indicate differences between both pathogens in their effects on yield traits. The statistical calculations were performed using analysis of variance (a three‐factor experiment) for particular yield trait reductions and multivariate analysis of variance for the yield trait reductions jointly. Almost all of the univariate and multivariate hypotheses concerning no differences between pathogens (F. culmorum, F. avenaceum), climatic conditions (years) and cultivars as well as hypotheses concerning no interactions between factors (pathogens, years, cultivars) were rejected at least at P= 0.05 significance level. The reduction of yield traits indicated individual reactions of the tested winter wheat cultivars to different pathogens. Among the tested traits the highest influence on the rejection of the hypothesis concerning the equivalence of F. avenaceum and F. culmorum was observed for TKW and WKH. The effect of the pathogen on yield reduction was greater for F. avenaceum than for F. culmorum during 1996 and 1997. A comparison of the cultivars indicated that the Begra cultivar showed the highest tolerance to inoculation with both Fusarium pathogens. Moreover, this genotype as well as several others showed lower tolerance to F. avenaceum rather than to F. culmorum, whereas Elena was the only cultivar with the opposite tendency.     

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

Background

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.

Methods

The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1⁺NIL were also compared to identify pathogen-responsive proteins.

Results

Eight proteins were either induced or upregulated in inoculated Fhb1⁺NIL when compared with mock-inoculated Fhb1⁺NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1⁺NIL when compared with Fusarium-inoculated Fhb1⁻NIL. Proteins that were differentially expressed in the Fhb1⁺NIL, not in the Fhb1⁻NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.

Conclusions

Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1⁺NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.     

小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,7-1、s-3、s-4和w-2均能表达,说明这些片段可能是功能性抗病基因的部分序列;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。     

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.     

Metabolic Biomarker Panels of Response to Fusarium Head Blight Infection in Different Wheat Varieties

Metabolic changes in spikelets of wheat varieties FL62R1, Stettler, Muchmore and Sumai3 following Fusarium graminearum infection were explored using NMR analysis. Extensive 1D and 2D ¹H NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. In addition, metabolic changes that are observed in all studied varieties as well as wheat variety specific changes have been determined and discussed. A new method for metabolite quantification from NMR data that automatically aligns spectra of standards and samples prior to quantification using multivariate linear regression optimization of spectra of assigned metabolites to samples’ 1D spectra is described and utilized. Fusarium infection-induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance.     

Occurrence of beauvericin and enniatins in wheat affected by Fusarium avenaceum head blight.

We evaluated Fusarium contamination and the levels of hexadepsipeptide mycotoxins in 13 wheat samples affected by head blight in Finland. Fusarium avenaceum was the dominant species (91%) isolated from all samples, but isolates of F. culmorum (4%), F. tricinctum (3%), and F. poae (2%) also were recovered. Beauvericin (0.64 to 3.5 microg/g) was detected in all 13 samples. Enniatin B (trace to 4.8 microg/g) was detected in 12 samples, enniatin B(1) (trace to 1.9 microg/g) was detected in 8 samples, and enniatin A(1) (trace to 6.9 microg/g) was detected in 10 samples. Ten of 13 strains of F. avenaceum and 2 strains of F. poae and F. tricinctum produced beauvericin in culture on rice (trace to 70, 9.4, and 33 microg/g, respectively). All strains also produced enniatins (trace to 2,700 microg/g). This is the first report on the natural co-occurence of beauvericin and enniatins in wheat infected predominantly by F. avenaceum.     

Resistance against Fusarium Head Blight in Transgenic Wheat Plants Expressing the ScNPR1 gene

Fusarium head blight (FHB) is a severe global wheat disease that may cause severe yield losses, especially during epidemic years. Transforming the regulatory genes in the metabolic pathways of disease resistance into wheat via transgenic methods is one way to improve resistance to FHB. ScNPR1 (Secale cereale‐NPR1), a regulatory gene for systemic acquired resistance (SAR), was isolated from S. cereale cv Jingzhouheimai and transformed into the moderately FHB‐susceptible wheat variety Ningmai 13. RT‐PCR analysis indicated that the ScNPR1 gene was stably expressed in transgenic plants. An evaluation of the resistance to FHB revealed that six ScNPR1 transgenic lines (NP1, NP2, NP3, NP4, NP5 and NP6) exhibited significantly higher FHB resistance than the wild‐type wheat Ningmai 13 and the null‐segregated plants. The expression of pathogenesis‐related (PR) genes after Fusarium graminearum inoculation was earlier or higher than those in the wild‐type variety Ningmai 13. The high expression in the early stages of PR genes should account for the enhanced FHB resistance in the transgenic lines. Our results suggest that overexpression of ScNPR1 could be used to improve FHB resistance in wheat.     

The Effect of Nitrogen Fertilization on Fusarium Head Blight Development and Deoxynivalenol Contamination in Wheat

The impact of nitrogen (N) fertilization on the development of Fusarium head blight (FHB) in wheat and the resulting deoxynivalenol (DON) contamination in the kernels was studied. In a first experiment, the disease was assessed on two locations under natural infection pressure. Five different types of nitrogen fertilizer (both organic and mineral) were investigated, each applied at five input rates from 0 to 160 kg N/ha. With all fertilizers, a significant increase of disease intensity was observed with increasing N input, while the type of N fertilizer had poor or no effects on FHB. Depending on the fertilizer used, the percentage of diseased spikelets increased from 2.2% at zero N rate up to 6.6% at 160 kg N input per hectare. In a second series of trials, three spring wheat varieties including one Durum wheat line were artificially inoculated with a Fusarium graminearum and a F. culmorum strain, known producers of DON. A mineral N fertilizer was applied at five input levels from 0 to 160 kg N/ha. A significant increase in FHB intensity and DON contamination in the grain was observed with increasing N from 0 to 80 kg/ha. At higher input rates, relevant in contemporary crop husbandry, disease intensity and toxin contamination remained at constant levels. It is concluded that adaptation of N fertilization represents no relevant tool in managing FHB in practical wheat cultivation.     

Mutual Exclusion between Fungal Species of the Fusarium Head Blight Complex in a Wheat Spike

Fusarium head blight (FHB) is one of the most damaging diseases of wheat. FHB is caused by a species complex that includes two genera of Ascomycetes: Microdochium and Fusarium. Fusarium graminearum, Fusarium culmorum, Fusarium poae, and Microdochium nivale are among the most common FHB species in Europe and were chosen for these experiments. Field studies and surveys show that two or more species often coexist within the same field or grain sample. In this study, we investigated the competitiveness of isolates of different species against isolates of F. graminearum at the scale of a single spike. By performing point inoculations of a single floret, we ensured that each species was able to establish independent infections and competed for spike colonization only. The fungal colonization was assessed in each spike by quantitative PCR. After establishing that the spike colonization was mainly downwards, we compared the relative colonization of each species in coinoculations. Classical analysis of variance suggested a competitive interaction but remained partly inconclusive because of a large between-spike variance. Further data exploration revealed a clear exclusion of one of the competing species and the complete absence of coexistence at the spike level.     

Quantification of Trichothecene-Producing Fusarium Species in Harvested Grain by Competitive PCR To Determine Efficacies of Fungicides against Fusarium Head Blight of Winter Wheat

We developed a PCR-based assay to quantify trichothecene-producing Fusarium based on primers derived from the trichodiene synthase gene (Tri5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing Fusarium species. Amounts of the trichothecene-producing Fusarium and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing Fusarium and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing Fusarium compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing Fusarium in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing Fusarium within the FHB disease complex and not by altering the rate of DON production. The Tri5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.     

Ice Nucleation Activity in Fusarium acuminatum and Fusarium avenaceum

Twenty fungal genera, including 14 Fusarium species, were examined for ice nucleation activity at −5.0°C, and this activity was found only in Fusarium acuminatum and Fusarium avenaceum. This characteristic is unique to these two species. Ice nucleation activity of F. avenaceum was compared with ice nucleation activity of a Pseudomonas sp. strain. Cumulative nucleus spectra are similar for both microorganisms, while the maximum temperatures of ice nucleation were −2.5°C for F. avenaceum and −1.0°C for the bacteria. Ice nucleation activity of F. avenaceum was stable at pH levels from 1 to 13 and tolerated temperature treatments up to 60°C, suggesting that these ice nuclei are more similar to lichen ice nuclei than to bacterial ones. Ice nuclei of F. avenaceum, unlike bacterial ice nuclei, pass through a 0.22-μm-pore-size filter. Fusarial nuclei share some characteristics with the so-called leaf-derived nuclei with which they might be identified: they are cell free and stable up to 60°C, and they are found in the same kinds of environment. Highly stable ice nuclei produced by fast-growing microorganisms have potential applications in biotechnology. This is the first report of ice nucleation activity in free-living fungi.     

Whole Genome Association Mapping of Fusarium Head Blight Resistance in European Winter Wheat (Triticum aestivum L.)

A total of 358 recent European winter wheat varieties plus 14 spring wheat varieties were evaluated for resistance to Fusarium head blight (FHB) caused by Fusarium graminearum and Fusarium culmorum in four separate environments. The FHB scores based on FHB incidence (Type I resistance)×FHB severity (Type II resistance) indicated a wide phenotypic variation of the varieties with BLUE (best linear unbiased estimation) values ranging from 0.07 to 33.67. Genotyping with 732 microsatellite markers resulted in 782 loci of which 620 were placed on the ITMI map. The resulting average marker distance of 6.8 cM allowed genome wide association mapping employing a mixed model. Though no clear population structure was discovered, a kinship matrix was used for stratification. A total of 794 significant (−log₁₀(p)-value≥3.0) associations between SSR-loci and environment-specific FHB scores or BLUE values were detected, which included 323 SSR alleles. For FHB incidence and FHB severity a total of 861 and 877 individual marker-trait associations (MTA) were detected, respectively. Associations for both traits co-located with FHB score in most cases. Consistent associations detected in three or more environments were found on all chromosomes except chromosome 6B, and with the highest number of MTA on chromosome 5B. The dependence of the number of favourable and unfavourable alleles within a variety to the respective FHB scores indicated an additive effect of favourable and unfavourable alleles, i.e. genotypes with more favourable or less unfavourable alleles tended to show greater resistance to FHB. Assessment of a marker specific for the dwarfing gene Rht-D1 resulted in strong effects. The results provide a prerequisite for designing genome wide breeding strategies for FHB resistance.     

Susceptibility of Kenyan Wheat Varieties to Head Blight, Fungal Invasion and Deoxynivalenol Accumulation Inoculated with Fusarium graminearum

Fifteen wheat varieties commercially grown in Kenya were tested for their susceptibility to head blight and mycotoxin accumulation after inoculation with Fusarium graminearum in pot experiments. The strains of the pathogen used had been isolated from wheat collected in different growing areas of Kenya. Head blight susceptibility was assessed as the percentage of spikelets bleached and area under disease progress curve; kernel colonization by fungal mycelium was determined as ergosterol content. All varieties were found to be moderately to highly susceptible. However, the varieties differed in head blight susceptibility (29–68% of spikelets bleached; mean 54%), fungal colonization (67–187  μ g/g ergosterol content; mean 111  μ g/g) and the resulting mycotoxin contamination [deoxynivalenol (DON) 5–31  μ g/g; mean 13.5  μ g/g]. Grain weight reductions due to head blight ranged from 23 to 57% (mean 44%). The varieties could be therefore divided into partially resistant and highly susceptible genotypes. The kernels of highly susceptible varieties had higher mycotoxin and ergosterol contents. However, the kernels of some varieties contained more fungal mycelium (ergosterol) without the corresponding high amounts of DON, suggesting that they possess some resistance to DON accumulation. Less susceptible varieties showed resistance to fungal spread, as indicated by a slow disease development and lower content of fungal biomass.     

Fusarium avenaceum and Fusarium crookwellens cause onion basal rot in Iran

Abstract

Basal rot is the main and economically soil-borne disease of onion that caused by various Fusarium species worldwide. To identify the prevailing Fusarium species, 140 Fusarium isolates were obtained from red onion bulbs farms in 10 regions of East and West Azarbaijan provinces in 2015. By inoculating 80 selected isolates, 40 of them were pathogenic on onion. These 40 isolates were identified as F. oxysporum with 43.62%, F. subglutinans with 44%, F. culmorum with 50.66%, F. avenaceum with 51%, F. solani with 42.41%, F. crookwellens with 55%, F. proliferatum with 47.16% and F. redolens with 55.5% virulence. Their frequency were 20%, 2.5%, 7.5%, 5%, 42.5%, 2.5%, 15% and 5%, respectively. Forty studied isolates demonstrating that, 14.2% were highly virulent, 26.1% virulent, 40.3% moderately virulent and 19.4% weakly virulent. This is the first report of F. avenaceum and F. crookwellens as the causal agents of red onion basal rot in Iran.     

3B染色体短臂小麦赤霉病抗性主效QTL的分析

采用区间作图和复合区间作图方法对重组自交系群体宁894037／Alondram、望水白／Alondra和苏麦3号／A1ondra进行了抗赤霉病QTL分析,结果表明,用在田间和温室的赤霉病抗性鉴定资料,在3个赤霉病抗源宁894037、望水白和苏麦3号的3B染色体短臂上均检测到主效QTL的存在。宁894037主效QTL位于标记BARCl33与Xgwm493之间的5．0cM的区间内,最高可解释42．8％的赤霉病抗性;望水白的主效QTL位于标记BARCl47与Xgwm493之间11．5cM的区间内,最高可解释15．1％的赤霉病抗性;苏麦3号的主效QTL位于Xgwm533a与Xgwm493之间13．0cM的区间内,最高可解释10．6％的赤霉病抗性。与赤霉病抗性主效QTL紧密连锁的标记均为SSR标记,可直接用于分子辅助育种。     

Identification of Kernel Proteins Associated with the Resistance to Fusarium Head Blight in Winter Wheat (Triticum aestivum L.)

Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB.     

Cellulose and xylan degrading enzymes of Fusarium avenaceum

It was found that crude preparation obtained from the culture medium of Fusarium avenaceum degraded cellulose and xylan. After chromatography on CM-Sepharose CL-6B of this preparation six fraction were obtained. The eluted fractions II and V showed xylanase activity, fraction IV — cellulase activity and fraction III — xylanase and cellulase activity. The end products of xylan hydrolysis by all xylanase fractions (II, III, V) were xylobiose, xylose, xylotriose and xylotetrose. The end products of cellulose hydrolysis by fractions III and IV was cellobiose, glucose and cellotriose. The data from gel filtration on Sephacryl S-200 indicated a molecular weight of more than 250,000 for both cellulase IV and xylanase V. After gel filtration in the presence of urea disaggregation of those high molecular xylanase and cellulase particles was observed. Xylanase II in difference from the other fractions contained higher amount of sugar. Digestion of fraction II with cellulase-hemicellulase preparation from Phoma hibernica decreased the content of sugar from 17% to 8%, but did not change its enzymatic properties. Cellulase IV as well as xylanase V were inactivated by N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, hence it is suggested that tryptophan and tyrosine are the essential for the activity of these enzymes.     

Association Mapping and Haplotype Analysis of a 3.1-Mb Genomic Region Involved in Fusarium Head Blight Resistance on Wheat Chromosome 3BS

A previous study provided an in-depth understanding of molecular population genetics of European and Asian wheat gene pools using a sequenced 3.1-Mb contig (ctg954) on chromosome 3BS. This region is believed to carry the Fhb1 gene for response to Fusarium head blight. In this study, 266 wheat accessions were evaluated in three environments for Type II FHB response based on the single floret inoculation method. Hierarchical clustering (UPGMA) based on a Manhattan dissimilarity matrix divided the accessions into eight groups according to five FHB-related traits which have a high correlation between them; Group VIII comprised six accessions with FHB response levels similar to variety Sumai 3. Based on the compressed mixed linear model (MLM), association analysis between five FHB-related traits and 42 molecular markers along the 3.1-Mb region revealed 12 significant association signals at a threshold of P<0.05. The highest proportion of phenotypic variation (6.2%) in number of diseased spikelets (NDS) occurred at locus cfb6059, and the physical distance was about 2.9 Kb between umn10 and this marker. Haplotype block (HapB) analysis using a sliding window LD of 5 markers, detected six HapBs in the 3.1-Mb region at r²>0.1 and P<0.001 between random closely linked markers. F-tests among Haps with frequencies >0.05 within each HapB at r²>0.1 and P<0.001 showed significant differences between the Hap carried by FHB resistant resources, such as Sumai 3 and Wangshuibai, and susceptible genotypes in HapB3 and HapB6. These results suggest that Fhb1 is located within HapB6, with the possibility that another gene is located at or near HapB3. SSR markers and Haps detected in this study will be helpful in further understanding the genetic basis of FHB resistance, and provide useful information for marker-assisted selection of Fhb1 in wheat breeding.