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.     

Nivalenol‐producing Fusarium cerealis associated with fusarium head blight in winter wheat in Manitoba,Canada

Fusarium head blight (FHB) in small grain cereals is primarily caused by the members of the Fusarium graminearum species complex. These produce mycotoxins in infected grains, primarily deoxynivalenol (DON); acetylated derivatives of DON, 3‐acetyl‐DON (3‐ADON) and 15‐acetyl‐DON (15‐ADON); and nivalenol (NIV). This study reports the isolation of Fusarium cerealis in infected winter wheat heads for the first time in Canada. A phylogenetic analysis based on the TRI101 gene and F. graminearum species‐specific primers revealed two species of Fusarium: F. graminearum sensu stricto (127 isolates) and F. cerealis (five isolates). Chemotype determination based on the TRI3 gene revealed that 65% of the isolates were 3‐ADON, 31% were 15‐ADON and 4% were NIV producers. All the F. cerealis isolates were of NIV chemotype. Fusarium cerealis isolates can often be misidentified as F. graminearum as the morphological characteristics are similar. Although the cultural and macroconidial characteristics of F. graminearum and F. cerealis isolates were similar, the aggressiveness of these isolates on susceptible wheat cultivar Roblin and moderately resistant cultivar Carberry differed significantly. The F. graminearum 3‐ADON isolates were most aggressive, followed by F. graminearum 15‐ADON and F. cerealis NIV isolates. The findings from this study confirm the continuous shift of chemotypes from 15‐ADON to 3‐ADON in North America. In Canada, the presence of NIV is limited to barley samples and the discovery of NIV‐producing F. cerealis species in Canadian wheat fields may pose a serious concern to the Canadian wheat industry in the future.     

Transfer of the β‐tubulin gene of Botrytis cinerea with resistance to carbendazim into Fusarium graminearum

BACKGROUND: Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea (Pers. ex Fr.) and most other fungi is usually conferred by mutation(s) in a single chromosomal β‐tubulin gene, often with several allelic mutations. In Fusarium graminearum Schwade, however, carbendazim resistance is not associated with a mutation in the corresponding β‐tubulin gene. RESULTS: The β‐tubulin gene conferring carbendazim resistance in B. cinerea was cloned and connected with two homologous arms of the β‐tubulin gene of F. graminearum by using a double‐joint polymerase chain reaction (PCR). This fragment was transferred into F. graminearum via homologous double crossover at the site where the β‐tubulin gene of F. graminearum is normally located (the β‐tubulin gene of F. graminearum had been deleted). The transformants were confirmed and tested for their sensitivity to carbendazim. CONCLUSION: The β‐tubulin gene conferring carbendazim resistance in B. cinerea could not express this resistance in F. graminearum, as transformants were still very sensitive to carbendazim. Copyright © 2010 Society of Chemical Industry     

Natural occurrence of fusarium head blight,mycotoxins and mycotoxin‐producing isolates of Fusarium in commercial fields of wheat in Hubei

Combined analyses of the natural occurrence of fusarium head blight (FHB), mycotoxins and mycotoxin‐producing isolates of Fusarium spp. in fields of wheat revealed FHB epidemics in 12 of 14 regions in Hubei in 2009. Mycotoxin contamination ranged from 0·59 to 15·28 μg g^?1 in grains. Of the causal agents associated with symptoms of FHB, 84% were Fusarium asiaticum and 9·5% were Fusarium graminearum, while the remaining 6·5% were other Fusarium species. Genetic chemotyping demonstrated that F. asiaticum comprised deoxynivalenol (DON), 3‐acetyldeoxynivalenol (3‐AcDON), 15‐acetyldeoxynivalenol (15‐AcDON) and nivalenol (NIV) producers, whereas F. graminearum only included DON and 15‐AcDON producers. Compared with the chemotype patterns in 1999, there appeared to be a modest shift towards 3‐AcDON chemotypes in field populations during the following decade. However, isolates genetically chemotyped as 3‐AcDON were present in all regions, whereas the chemical 3‐AcDON was only detected in three of the 14 regions where 3‐AcDON accounted for 15–20% of the DON and acetylated forms. NIV mycotoxins were detected in seven regions, six of which also yielded NIV chemotypes. The number of genetic 3‐AcDON producers was positively correlated with amounts of total mycotoxins (DON, NIV and acetylated forms) or DON in wheat grains. Chemical analyses of wheat grains and rice cultures inoculated with different isolates from the fields confirmed their genetic chemotypes and revealed a preferential biosynthesis of 3‐AcDON and 4‐AcNIV in rice. These findings suggest the importance of chemotyping coupled with species identification for improved prediction of mycotoxin contamination in wheat.     

Virulence and toxin synthesis of an azole insensitive Fusarium culmorum strain in wheat cultivars with different levels of resistance to fusarium head blight

Fusarium culmorum causes head blight, produces toxins and reduces yield and quality of cereals. To prevent damage caused by fusarium head blight (FHB), azole fungicides are mainly applied. The occurrence of insensitivity to azoles is a major problem in agriculture. The present study shows that a tebuconazole insensitive strain of F. culmorum can be readily produced in the laboratory, but that the resulting strain of the fungus is of lower fitness in vitro. Insensitivity was confirmed microscopically and by cell viability and metabolic activity. The tebuconazole insensitive strain shows cross insensitivity to nine important azoles. In addition, plants inoculated with the insensitive F. culmorum strain showed no reduction of FHB symptoms and deoxynivalenol (DON) content after tebuconazole treatment, compared to an inoculation with the sensitive strain. Use of wheat cultivars carrying a high resistance level (i.e. cv. Toras) was the most effective method for reducing symptoms and decreasing DON content, independent from the level of fungicide insensitivity of the F. culmorum strain. In conclusion, resistant cultivars and a fungicide mixture which combines different mechanisms of action in fungal metabolism should be applied to avoid fungicide insensitivity of Fusarium spp. in future.     

Application of cycleave PCR to the detection of a point mutation (F167Y) in the β2‐tubulin gene of Fusarium graminearum

BACKGROUND: Resistance of Fusarium graminearum to the benzimidazole fungicide carbendazim is caused by point mutations in the β₂‐tubulin gene (FGSG_06611.3). The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field isolates in China. It is important to find a suitable method for rapid detection and quantification of this point mutation in the F. graminearum populations. RESULTS: A pair of primers, Codon167F/Codon167R, were designed to amplify a fragment containing the mutation site, and two cycling probes labelled with different fluorescent reporters were used to detect whether the mutation was present. A cycleave real‐time PCR method was developed for rapid determination of the frequency of this point mutation in 282 F. graminearum perithecia collected from different fields in Jiangsu Province, China. The mutation frequency in ascospores from the perithecia to carbendazim by a spore germination assay was 6.0%, while the frequency of the point mutation at codon 167 by the cycleave real‐time PCR assay was 3.9%. CONCLUSION: The cycleave real‐time PCR method is suitable for accurate detection of the codon 167 point mutation. The frequency of this mutation in the β₂‐tubulin gene represents the resistance frequency in F. graminearum populations to carbendazim. Copyright © 2011 Society of Chemical Industry     

茉莉酸对PR-1、PR-2、PR-5和Ta-JA2基因 表达以及小麦白粉病抗性的诱导

 为了探索小麦抗白粉病分子机理,明确茉莉酸对小麦白粉病抗性的诱导作用、对植物抗病性标志基因PR-1、PR-2、PR-5和本实验室克隆的1个新基因Ta-JA2的激活作用,以及抗病性变化与基因表达变化之间的相关性,本研究以感白粉病的小麦品种“中国春”、“濮麦9号” 和“周麦18”为材料,用茉莉酸甲酯（methyl jasmonate,MeJA）喷洒小麦幼苗叶片进行诱导,通过离体叶段培养法接种白粉菌（Blumeria graminis f. sp. tritici,Bgt）进行抗性鉴定;用实时定量PCR技术检测小麦叶片中PR-1、PR-2、PR-5和Ta-JA2基因的表达变化。结果表明MeJA处理可以显著提高“中国春”、“濮麦9号”和“周麦18”对白粉菌的抗病水平。茉莉酸处理显著激活了PR-1、PR-2、PR-5和Ta-JA2的转录。茉莉酸诱导的抗病性提高与抗病标志基因PR-1、PR-2、PR-5及Ta-JA2的表达增强呈正相关。植物激素茉莉酸是小麦抗白粉病反应的信号分子。     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

Biocontrol traits of two Paenibacillus polymyxa strains SQR‐21 and WR‐2 in response to fusaric acid,a phytotoxin produced by Fusarium species

The aim of the present study was to investigate the effects of the phytotoxin fusaric acid (FA) on the biocontrol traits of two biocontrol strains Paenibacillus polymyxa WR‐2 and SQR‐21. The results showed that the growth of both WR‐2 and SQR‐21 decreased with increasing FA concentration, and at 70 and 80 μg mL^?1 FA, respectively, the strains were unable to grow. The biocontrol traits of both strains were negatively affected by FA concentration higher than 2·5 μg mL^?1. However, at 2·5 μg mL^?1 FA, biofilm formation and root colonization were not affected and there was even a positive effect on the production of spores and hydrolytic enzymes (protease and β‐l,3‐glucanase). The production of fusaricidin‐type antifungal compounds was increased with an increase in FA concentration up to 50 and 60 μg mL^?1 for WR‐2 and SQR‐21, respectively. The production of antifungal volatile organic compounds by WR‐2 and SQR‐21 was increased only at 2·5 μg mL^?1 FA. The effect of FA on the overall metabolic activity of WR‐2 and SQR‐21 was also determined. This study will help to understand the response of P. polymyxa strains to FA and will help to improve their biocontrol efficiency.     

Real‐time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long‐distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real‐time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop‐infected seeds with Fop‐free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.     

Population genetics of Phytophthora infestans in Denmark reveals dominantly clonal populations and specific alleles linked to metalaxyl‐M resistance

Control of the potato late blight pathogen Phytophthora infestans relies heavily on chemicals. The fungicide metalaxyl‐M (Mefenoxam) has played an important role in controlling the disease, but insensitivity to the fungicide in certain isolates is now of major concern. A genetic basis for resistance to metalaxyl suggests the possibility for linking resistance phenotypes to specific population genetic markers, but in order to do this, the population genetic structure and mode of reproduction in a population must first be well described. The dynamics of metalaxyl‐M resistance in the Danish population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)‐based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating the potential for a more precise SNP‐based marker system for predicting resistance to metalaxyl‐M.     

Survey of the response of 82 domestic landraces of Zea mays to cucumber mosaic virus (CMV) reveals geographical region‐related resistance to CMV in Japan

Zea mays has been historically imported to Japan via two independent geographical routes: into southern Japan by trading with Europe in the 16th century and into northern Japan by import from North America in the 19th century. Breeding to genetically improve on quality traits and high yields has led to the current domestic landraces in each region. In a survey of 82 domestic landraces, nine out of 38 landraces originating from southern Japan showed complete immunity to cucumber mosaic virus yellow strain (CMV(Y)) without the formation of necrotic local lesions (NLLs). In contrast, three out of 44 landraces originating from northern Japan developed NLLs, but revealed no systemic spread of the virus. Due to the absence of good documentation on NLL formation in Z. mays, the response of domestic landraces Aso‐1 and Aso‐3, originating from Ibaraki in northern Japan, to a challenge with CMV(Y) and CMV(Ma‐1) was further analysed. Aso‐3 only formed NLL in response to CMV(Y) but not to CMV(Ma‐1). Moreover, in CMV(Y)‐inoculated Aso‐3, virus spread was restricted to the primary infection site and the expression of defence‐related genes was up‐regulated, whereas Aso‐1 became systemically infected with either CMV(Y) or CMV(Ma‐1). The response of Aso‐3 to CMV(Y) was inherited as a single dominant trait. Together, these results pointed towards the induction of hypersensitive response (HR)‐mediated resistance to CMV(Y) in Aso‐3. Although HR‐mediated resistance to viruses has been studied mainly in dicots, the pathosystem CMV–Z. mays may provide a model to investigate HR‐mediated resistance to viruses in monocot plants.     

The importance of non‐penetrated papillae formation in the resistance response of triticale to powdery mildew (Blumeria graminis)

Triticale is the intergeneric hybrid between wheat and rye. With the expansion of the triticale growing area, powdery mildew has emerged and become a significant disease on this new host. Recent research demonstrated that this ‘new’ powdery mildew on triticale has emerged through a host range expansion of powdery mildew of wheat. Moreover, isolates sampled from triticale still infect their previous host, wheat, but isolates sampled from wheat hardly infect triticale. Race‐specific and adult‐plant resistance have been identified in triticale cultivars. The main objective of this study was to characterize the cellular basis of powdery mildew resistance in triticale. Commonalities with resistance responses in other cereals such as wheat, barley and oat are discussed. A detailed comparative histological study of various resistance responses during cross‐inoculation of either virulent or avirulent wheat and triticale isolates on both hosts was carried out. The present data provide evidence that for incompatible interactions, the formation of non‐penetrated papillae is the predominant resistance response, while the hypersensitive response (HR) acts as a second line of defence, to cut the fungus off from nutrients, if penetration resistance fails. It is not clear yet what causes the slower growth and reduced colony size of triticale isolates when inoculated on wheat. Possibly, post‐penetration resistance mechanisms, other than HR, are switched on during these (semi‐) compatible interactions. Molecular studies on gene expression and gene function of defence‐related genes might reveal further insights into the genetic basis of these resistance responses.     

Multiple resistance of Papaver rhoeas L. to 2,4‐D and acetolactate synthase inhibitors in four European countries

The issue of cross‐ or multiple resistance to acetolactate synthase (ALS) inhibitors and the auxinic herbicide 2,4‐D was investigated in Papaver rhoeas L., a common and troublesome weed in winter cereals, in a broad‐scale study across four European countries. A combination of herbicide sensitivity bioassays and molecular assays targeting mutations involved in resistance was conducted on 27 populations of P. rhoeas originating from Greece (9), Italy (5), France (10) and Spain (3). Plants resistant to the field rate of 2,4‐D were observed in 25 of the 27 populations assayed, in frequencies ranging from 5% to 85%. Plants resistant to ALS‐inhibiting herbicides (sulfonylureas) were present in 24 of the 27 populations, in frequencies ranging from 4% to 100%. Plants resistant to 2,4‐D co‐occurred with plants resistant to sulfonylureas in 23 populations. In four of these, the probability of presence of plants with cross‐ or multiple resistance to 2,4‐D and sulfonylureas was higher than 0.5. ALS genotyping of plants from the field populations or of their progenies, identified ALS alleles carrying a mutation at codon Pro197 or Trp574 in 2,4‐D‐sensitive and in 2,4‐D‐resistant plants. The latter case confirmed multiple resistance to 2,4‐D and ALS inhibitors at the level of individual plants in all four countries investigated. This study is the first to identify individual plants with multiple resistance in P. rhoeas, an attribute rarely assessed in other weed species, but one with significant implications in designing chemical control strategies.     

Evaluation of Malaysian oil palm progenies for susceptibility,resistance or tolerance to Fusarium oxysporum f. sp. elaeidis and defence‐related gene expression in roots

Vascular wilt of oil palm caused by Fusarium oxysporum f. sp. elaeidis (Foe) is a devastating disease in West and Central Africa. As the oil palm industry in southeast Asia is still expanding, so is the oil palm germplasm collection through the importation of seed and pollen from Africa, the centre of diversity for Elaeis guineensis. There is a risk of inadvertent spread of the disease on contaminated seed or pollen. Regular re‐evaluation of the reaction of currently grown palm genotypes towards Foe is clearly required for biosecurity. This study has demonstrated that four Malaysian oil palm progenies, three in current or recent commercial use, are highly susceptible to infection by at least one of two African isolates of Foe, representing different countries, aggressiveness and vegetative compatibility groups. Symptoms and reduction of palm growth generally reflected the extent and intensity of systemic colonization by Foe. Progeny PK 5463 expressed partial resistance to Foe isolate F3, but not to isolate 16F, displaying significantly milder symptoms and supporting less widespread vascular colonization. This relatively incompatible interaction was used to study expression of potential defence‐related genes during root infection when compared to a susceptible palm–isolate combination. The only significant response was an early up‐regulation of chitinase in resistant palms. The research revealed at least one progeny–isolate differential interaction, and the associated resistance expression suggests a component of tolerance, because colonization by Foe was systemic in both compatible and incompatible combinations.