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.     

小麦种质对茎基腐病抗性评价及优异种质筛选

小麦茎基腐病是由镰孢菌侵染引起的一种世界性土传病害,近年来已严重威胁到我国小麦的安全生产。为筛选具有茎基腐病抗性的小麦种质资源,本研究采用孢子悬浮液浸种法,分别以国外抗病材料Sunco和中国品种陕253为抗病和感病对照,对670份我国小麦品种(系)进行了茎基腐病温室苗期抗性鉴定。结果发现,我国供试品种(系)感病材料(病情指数>30)所占比例达到84%,且包含多个近年来小麦生产中的主推品种,表明我国小麦品种总体抗性水平低是导致茎基腐病近年来发病频率与程度不断增加的重要原因之一。经多轮筛选,发掘获得15份抗病表现稳定、抗性水平与抗病对照Sunco相仿的材料。15份材料平均病情指数在10.9~19.4之间,其株高、抽穗期等农艺性状表现出较为丰富的变异,为我国小麦抗茎基腐病品种选育和抗性遗传研究提供了种质资源。     

Rapid Diagnosis of Soya Bean Root Rot Caused by Fusarium culmorum Using a Loop‐Mediated Isothermal Amplification Assay

We report a rapid diagnosis of soya bean (Glycine max L.) root rot caused by Fusarium culmorum, using a loop‐mediated isothermal amplification (LAMP) assay. We used the CYP51C gene sequence to design LAMP assay primers specific for F. culmorum. The LAMP assay amplified the target gene efficiently in 60 min at 63°C. The sensitivity of the assay was 100 pg/μl of genomic DNA. Among the tested soya bean pathogens, a positive colour (sky blue) was only observed in the presence of F. culmorum with the addition of hydroxynaphthol blue (HNB) dye prior to amplification, whereas other species isolates showed no colour change. Suspected diseased soya bean samples collected in the field from Jiangsu, Shandong and Anhui provinces and Beijing were diagnosed successfully using the LAMP assay reported here. This study provides a new and readily available method for rapid diagnosis of soya bean root rot caused by F. culmorum.     

Resistance Induction by Salicylic Acid Formulation in Cassava Plant against Fusarium solani

Fusarium root rot caused by the soil-borne fungus Fusarium solani is one of the most important fungal diseases of cassava in Thailand, resulting in high yield losses of more than 80%. This study aimed to investigate if the exogenous application of salicylic acid formulations (Zacha) can induce resistance in cassava against Fusarium root rot and observe the biochemical changes in induced cassava leaf tissues through synchrotron radiation based on Fourier-transform infrared (SR-FTIR) microspectroscopy. We demonstrated that the application of Zacha11 prototype formulations could induce resistance against Fusarium root rot in cassava. The in vitro experimental results showed that Zacha11 prototype formulations inhibited the growth of F. solani at approximately 34.83%. Furthermore, a significant reduction in the disease severity of Fusarium root rot disease at 60 days after challenge inoculation was observed in cassava plants treated with Zacha11 at a concentration of 500 ppm (9.0%). Population densities of F. solani were determined at 7 days after inoculation. Treatment of the Zacha11 at a concentration of 500 ppm resulted in reduced populations compared with the distilled water control and differences among treatment means at each assay date. Moreover, the SR-FTIR spectral changes of Zacha11-treated epidermal tissues of leaves had higher integral areas of lipids, lignins, and pectins (1,770–1,700/cm), amide I (1,700–1,600/cm), amide II (1,600–1,500/cm), hemicellulose, lignin (1,300–1,200/cm), and cellulose (1,155/cm). Therefore, alteration in defensive carbohydrates, lipids, and proteins contributed to generate barriers against Fusarium invasion in cassava roots, leading to lower the root rot disease severity.     

Quantification of Fusarium culmorum in Wheat and Barley Tissues Using Real-Time PCR in Comparison with DON Content

A real‐time polymerase chain reaction (PCR) assay was developed for the specific detection of Fusarium culmorum in infected seeds. Primers and TaqMan minor groove binder probe were derived from the sequences of a F. culmorum specific PCR product. The specificity of the assay was confirmed by test in seven Fusarium species and 21 non‐Fusarium fungal species. With serial dilutions of purified genomic DNA from F. culmorum isolate B as the template, the detection limit of the assay was found to be 0.9 pg of fungal genomic DNA per reaction. A significant correlation ( = 0.982) and collinearity was found between DNA concentration and Ct (cycle threshold) values of real‐time PCR assay with serial diluted DNAs extracted from three seed samples with different deoxynivalenol (DON) content. Eight barley and nine wheat varieties infected by F. culmorum isolate B were evaluated in 1 (barley samples) and in 4 years (wheat samples). The results of real‐time PCR analysis and enzyme‐linked immunosorbent assay testing for DON content were compared and a significant correlation was found for barley samples (r² = 0.935). Concerning wheat we found rather complicated relationship between Ct values and DON contents influenced by environmental conditions of field trials. The real‐time PCR assay was found to be highly specific and sensitive. It could be used in phytopathological studies and praxis.     

Evaluation of Macroscopic and Microscopic Components of Partial Resistance to Leaf Rust in Durum Wheat

Leaf rust, caused by the fungus Puccinia triticina, is considered one of the most important foliar diseases in durum wheat. Hypersensitive resistance (HR) may be rapidly overcome by the pathogen when resistant cultivars are grown on a large acreage or following changes in virulence in the pathogen population. Prolonging the durability of the resistance requires uses of other types of resistance such as partial resistance (PR). In this study, six durum wheat lines provided by the International Center for Corn and Wheat Improvement (CIMMYT) with a high level of PR to leaf rust were studied in monocyclic tests in a growth chamber. Inoculations were performed on both primary and fifth leaves using the Spanish race DGB/BN. UV fluorescence microscopy was employed to determine microscopic components of the resistance, such as the number of early aborted infection units not associated with plant cell necrosis (EA?) and relative colony size (RCS) of the established infection units. Macroscopic components of PR such as latency period, infection frequency and uredinium size were measured as well. All six resistant lines were characterized by a higher EA? and smaller RCS respect to the susceptible control ‘Don Rafael’. Line 3 showed the highest level of PR. It had 22% of EA? compared with 4% in the susceptible control, and the smallest RCS (17% respect to RCS of ‘Don Rafael’) at adult plant stage. Both EA? and RCS had a high heritability (more than 97%) and the correlation with macroscopic parameters (latency period and uredinium size) was also high (significant at 0.001 level). Hence, PR to leaf rust in these durum wheat genotypes has been revealed at microscopic level (higher EA? and smaller RCS).     

Population Genetics of Three Important Head Blight Pathogens Fusarium graminearum, F. pseudograminearum and F. culmorum

Homothallic Fusarium graminearum (teleomorph Gibberella zeae) and anamorphic F. culmorum are destructive pathogens causing Fusarium head blight (FHB) of small‐grain cereals worldwide, while heterothallic F. pseudograminearum (G. coronicola) seems to be restricted to Australia as a FHB pathogen. In a comprehensive treatise of pathogen population genetics, this review summarizes global knowledge of genetic diversity among isolates sampled at various spatial and temporal scales, examines the mechanisms that generate this diversity and explores the implications of pathogen diversity and plasticity to resistance breeding. Despite their different modes of reproduction, there is large variation among isolates of all three species originating from different countries and continents. With a few exceptions, haplotype diversity ranges from 60 to 100% even within populations from individual fields. In F. graminearum, over 90% of the variation is found within populations, even when samples are collected from areas as small as 0.25 m². Variation among populations is low (4–8%) with negligible population subdivision. This indicates a high level of gene flow (N_m = 8–71) with linkage equilibrium for the majority of selectively neutral molecular marker loci analysed. These findings for F. graminearum point to large random mating populations driven by occasional outcrossing, high gene flow across large geographical distances and a relatively low host‐mediated directional selection. Similar conclusions can be drawn for the Canadian population of F. pseudograminearum, but not for populations from Australia, where different pathogen ecology may have reduced the frequency of sexual recombination. Phylogenetic analyses indicate delineation of lineages in F. graminearum, often along geographically separated lines, while the related F. pseudograminearum is a single recombining species with limited or no lineage development. The anamorphic F. culmorum shows no obvious clonal structure in its population as might have been expected. High levels of diversity within fields may have been caused by balancing selection from frequent alternation between saprophytic and parasitical life cycle and/or a hidden or recently extinct teleomorph. Other mechanisms including parasexual cycles or active transposable elements may also be involved but these have not been investigated as yet. Crosses between and among F. graminearum lineages have shown a rather simple, additive inheritance of pathogenicity and aggressiveness with frequent transgressive segregation in crosses among isolates with moderate aggressiveness. This raises the spectre of highly aggressive and/or toxigenic isolates evolving if a limited range of quantitative trait locus for FHB resistance is deployed on a large scale. Combining more than one genetically distinct sources of resistance, possibly with different modes of action against the pathogen, will be necessary to avoid severe FHB outbreaks in the future.     

Copper and Manganese Increase Resistance of Clusterbean to Root Rot Caused by Rhizoctonia

Clusterbean seedlings growing in soil inoculated with Rhizoctonia were treated with either 5 or 10 ppm Cu²⁺ and Mn²⁺. Soil was inoculated by pretreatment with 250 mg (wet weight) of Rhizoctonia inoculum. A similar set of plants was maintained in uninoculated soil. Root rot incidence of plants treated with Cu²⁺ 5 ppm, Cu²⁺ 10 ppm, Mn²⁺ 5 ppm and Mn²⁺ 10 ppm was 26.6, 30.5, 11.8 and 29.2% less than the inoculated control, respectively. Inoculation with Rhizoctonia reduced chlorophyll, non‐structural carbohydrate and in vitro dry matter digestibility (IVDMD) content compared with uninoculated ones. Oxidative enzymes activities (polyphenol oxidase, peroxidase, phenylalanine ammonia lyase and tyrosine ammonia lyase), crude protein, phenolic content, structural components (acid detergent fibre, cellulose and lignin), silica, macronutrients and micronutrients increased in inoculated seedlings and this increase was further heightened by the Cu²⁺ 10 ppm treatment compared with the Cu²⁺ 5 ppm, Mn²⁺ 5 ppm and Mn²⁺ 10 ppm treatments in response to fungal invasion. It was concluded that the Cu²⁺ 10 ppm treatment may be an effective soil nutrient to provide enhanced resistance of clusterbean plants to root rot (fungal) diseases.     

小麦禾谷镰孢菌茎基腐病抗源的筛选与评价

为了发掘我国小麦种质资源中具有禾谷镰孢菌茎基腐病(crown rot)抗性的材料,采用禾谷镰孢菌苗期接种试验的方法,研究了82份小麦种质对禾谷镰孢菌茎基腐病的抗性.结果表明,在鉴定的材料中没有发现高抗材料;中抗材料13份,占总数的15.8%,包括CI12633、红蚰子、FHB143、Tiszataj和紫秆子等;大多数材料为感病材料.值得注意的是不同小麦材料对禾谷镰孢菌赤霉病和茎基腐病具有不同的抗性水平,两种病害没有正相关性,暗示小麦茎基腐病和赤霉病的抗性机制可能不同,因此,需要广泛挖掘具有茎基腐病抗性的小麦资源.     

柑橘与枳属间体细胞杂种再生及其对脚腐病抗性的评价

Page橘柚 (CitrusreticulataBlanco×C .paradisiMacf.)胚性细胞悬浮系原生质体与枳 (Poncirustrifoliata (L .)Raf.)叶肉原生质体经电场诱导融合 ,4～ 5个月再生 15 0余棵小植株。再生植株根系发达 ,叶片具三叶特征。随机检查 2 0余株再生苗根尖染色体数目 ,表明都为四倍体 (2n =4x =36 )。随机取 7株进行RAPD分析 ,表明被检测植株都为杂种。用引起脚腐病的寄生疫霉菌 (PhytophthoraparasiticaDastar)毒素接种体细胞杂种及双亲叶片 ,结果表明 ,Page橘柚中度感病 ,枳高抗 ,体细胞杂种为抗病类型。     

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root-tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.     

外引玉米种质对两种穗腐病原镰孢菌抗性鉴定

采用双牙签接种技术,2016-2017年连续2年对从美国、加拿大、罗马尼亚、德国、法国、荷兰等国家引进的177份玉米种质资源抗玉米穗腐病原拟轮枝镰孢(F.verticillioides)和禾谷镰孢(F.graminearum)进行了鉴定。结果表明,鉴定的177份玉米种质资源中,对拟轮枝镰孢表现高抗(HR)的资源2份,占总数的1.13%;抗病(R)38份,占总数的21.47%;中抗(MR)75份,占总数的42.37%;感病(S)48份,占总数的27.12%;高感(HS)14份,占总数的7.91%。177份玉米种质资源中,对禾谷镰孢(F.graminearum)表现抗病(R)12份,占鉴定资源总数的6.78%;中抗(MR)66份,占总数的37.29%;感病(S)64份,占总数的36.16%;高感(HS)35份,占总数的19.77%,未发现对禾谷镰孢菌表现高抗的材料。在177份玉米种质资源中,兼抗两种穗腐病原镰孢菌的种质资源有12份。     

Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples

AIMS: The aim of the study was to develop a sensitive detection method of Fusarium culmorum contamination in cereal samples. METHODS AND RESULTS: A nested-PCR method using a single closed tube was developed for the detection of F. culmorum in infected cereal samples. The concentrations of the first primer pair was diluted 10,000 times compared to the concentration used for the second primer pair. Differing annealing temperatures allowed both first and second polymerase chain reaction (PCR) reactions to be performed subsequently in the same closed tube. The detection limit was 5-50 fg of purified target DNA and allowed the detection of 1% infected seeds of wheat in a mixture with uninfected grains. CONCLUSIONS: F. culmorum can be specifically detected in cereal samples by the highly sensitive method of nested-PCR in a single closed tube. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the detection of F. culmorum in cereal samples that is approximately 100 times more sensitive than previous PCR methods, involves low risk of cross contaminations between samples, low costs and reduced hands-on time as compared to standard nested-PCR protocols.     

Evaluation of Barley and Wheat Germplasm for Resistance to Head Blight and Mycotoxin Production by Fusarium asiaticum and F. graminearum

Fusarium head blight (FHB) is one of the most serious diseases in barley and wheat, as it is usually accompanied by the production of harmful mycotoxins in the grains. To identify FHB-resistant breeding resources, we evaluated 60 elite germplasm accessions of barley (24) and wheat (36) for FHB and mycotoxin accumulation. Assessments were performed in a greenhouse and five heads per accession were inoculated with both Fusarium asiaticum (Fa73, nivalenol producer) and F. graminearum (Fg39, deoxynivalenol producer) strains. While the accessions varied in disease severity and mycotoxin production, four wheat and one barley showed <20% FHB severity repeatedly by both strains. Mycotoxin levels in these accessions ranged up to 3.9 mg/kg. FHB severity was generally higher in barley than in wheat, and Fa73 was more aggressive in both crops than Fg39. Fg39 itself, however, was more aggressive toward wheat and produced more mycotoxin in wheat than in barley. FHB severity by Fa73 and Fg39 were moderately correlated in both crops (r = 0.57/0.60 in barley and 0.42/0.58 in wheat). FHB severity and toxin production were also correlated in both crops, with a stronger correlation for Fa73 (r = 0.42/0.82 in barley, 0.70 in wheat) than for Fg39.     

Comparison of Screening Methods for Resistance to Fusarium Root Rot in Common Beans (Phaseolus vulgaris L.)

Root rot, caused by Fusarium solani f. sp. phaseoli, is one of the main root diseases impacting production of common beans throughout the world. Because resistance of common beans to root rot is a quantitative trait that is strongly influenced by environmental factors, reproducible methods to screen bean plants for resistance to root rot are critical to the selection process. In this study, we adapted the inoculum layer method (ILM) developed for screening soybeans for resistance to Phytophthora rot and compared it with the traditional liquid inoculum method (LIM) for screening common beans for resistance to Fusarium root rot. In addition, two methods of evaluating resistance using the ILM were compared. The most significant Pearson correlation coefficient between trials involving 80 recombinant inbred lines was achieved with the ILM and counting discoloured vascular bundles in the lower stem (r_p = 0.7113***) compared to rating the discoloration on root and hypocotyl (r_p = 0.5555***). The traditional (LIM) screening method and rating the discolouration on roots resulted in a non‐significant correlation between trials (r_p = 0.1084).     

Characterization and Pathogenicity of Lasiodiplodia theobromae Causing Black Root Rot and Identification of Novel Sources of Resistance in Mulberry Collections

Black root rot (BRR) caused by Lasiodiplodia theobromae is an alarming disease of mulberry that causes tremendous economic losses to sericulture farmers in India and China. Successful control of this disease can be attained by screening germplasm and identifying resistant sources. Seventy four diseased root samples were collected from farmer’s fields belonging to four major mulberry growing states of South India. Based on morpho-cultural and scanning electron microscopy studies, 57 fungal isolates were characterized and identified as L. theobromae. Phylogenetic analysis of concatenated internal transcribed spacer and β-tubulin sequences revealed variation of the representative 20 isolates of L. theobromae. Following the root dip method of inoculation, pathogenicity studies on susceptible mulberry genotypes (Victory-1 and Thailand male) recognized the virulent isolate MRR-142. Accordingly, MRR-142 isolate was used to evaluate resistance on a set of 45 diverse mulberry accessions. In the repeated experiments, the mulberry accession ME-0168 which is an Indonesian origin belonging to Morus latifolia was found to be highly resistant consistently against BRR. Eight accessions (G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-0489, and MI-0501) were found to be resistant. These promising resistant resources may be exploited in mulberry breeding for developing BRR resistant varieties and to develop mapping populations which successively helps in the identification of molecular markers associated with BRR.     

芝麻营养生长期枯萎病抗性鉴定技术研究

芝麻枯萎病是芝麻主要真菌病害之一,由尖孢镰刀菌芝麻专化型(FOS,Fusarium oxysporum f.sp.sesami)引起,主要在苗期和成株期发生。为精准评价营养生长时期(2对真叶~现蕾)芝麻种质对FOS菌株的抗性水平,试验分析了FOS菌液浓度、蘸根接菌时间、致病力等条件下芝麻种质枯萎病病症及病情指数变化规律,建立了营养生长期芝麻枯萎病抗性精准鉴定方法。结果表明,1×10^6 cfu/mL^5×10^6 cfu/mL浓度下,植株蘸根接菌处理1~2周即可发病;第4周样本枯萎病病情指数趋于稳定。上述方法反映不同芝麻种质营养生长期对FOS菌株的抗性水平以及不同FOS菌株的致病力。营养生长期芝麻枯萎病发生可分为0~4级共5个等级。采用上述鉴定方法对42份芝麻种质进行抗枯萎病鉴定结果显示,野生种Sesamum radiatum Thonn.ex Hornem.高抗枯萎病(DI=0),而S.angustifolium(Oliv.)Engl.高感枯萎病(DI=100)。40份栽培种资源中,高感(HS)种质比例极高(55%),抗病种质比例较低(27.5%)。研究结果为深入开展芝麻抗枯萎病遗传机理分析提供了技术支持。     

青霉TS67菌株对大豆根腐病和玉米小斑病的防治效果评价

采用盆栽试验的方法,通过病情指数和防治效率等指标研究青霉TS67菌株的发酵液、发酵上清液和菌体对大豆根腐病及玉米小斑病的影响,实验结果的SPSS统计分析表明,青霉TS67的所有处理均能显著(P<0.01)抑制大豆根腐病及玉米小斑病的发生,其中采用TS67菌株与大豆尖孢镰刀菌(Fusarium oxysporum)的混合物进行拌种,对大豆根腐病的防治效果最好,防治效率迭63.98%;在玉米小斑病发病前喷洒TS67菌株的发酵液,对玉米小斑病的防治效果最好,防治效率达到53.34%.此外实验结果还表明用青霉TS67进行大豆拌种能有效促进大豆幼苗生长.     

Competitive Aggressiveness in Binary Mixtures of Fusarium graminearum and F. culmorum Isolates Inoculated on Spring Wheat with Highly Effective Resistance QTL

Fusarium head blight (FHB) caused by Fusarium graminearum and F. culmorum is a devastating disease with high effects on grain yield and quality. We developed spring wheat lines incorporating the highly effective FHB resistance quantitative trait loci (QTL) Fhb1 and Qfhs.ifa‐5A. Whether these QTL lead to competition within Fusarium populations in the field resulting in isolates with higher aggressiveness has not been analysed. The aims of this study were to determine (i) the aggressiveness potential of F. graminearum and F. culmorum isolates, (ii) competition effects of these isolates in binary mixtures and (iii) the stability of resistant hosts. Six F. graminearum, two F. culmorum isolates and seven binary mixtures containing these isolates were tested for their aggressiveness and mycotoxin production at two locations in South Germany in 2007 and 2008. Host lines were four spring wheat lines containing the resistance QTL Fhb1 and/or Qfhs.ifa‐5A or none of them and one standard variety. Re‐isolates were sampled from plots inoculated with the binary mixtures to identify the percentage of each isolate in the mixture by simple sequence repeat markers. Resistant host lines reacted as expected and had a high stability to all isolates and mixtures. Only less important host × mixture interactions were detected. Aggressiveness among isolates and mixtures was significantly different. Type and amount of mycotoxin and high single isolate aggressiveness were not necessarily advantageous in the mixture. However, both F. culmorum isolates outcompeted F. graminearum isolates. Significant deviations from the inoculated 1 : 1 proportions occurred in 34 of 49 cases, illustrating that competition effects appeared in the mixtures. These differences depended mainly on the year and not on the level of host resistance. We conclude that resistance should not be affected by the Fusarium isolates and mixtures.