Detection of Latent Infection of Wheat Leaves Caused by Blumeria graminis f.sp. tritici Using Nested PCR

Wheat powdery mildew caused by Blumeria graminis f.sp. tritici (Bgt) is an important and destructive disease worldwide. Detection of latent infection of wheat seedlings is critical to estimate initial inoculum potential of epidemics in the fields. To improve the conventional method, a nested PCR approach had been established in this study to detect latent infections of wheat leaves caused by Bgt. The DNA primer sets including external and internal primer pairs for the nested PCR were designed followed by testing their specificities to Bgt by using Bgt and other fungal species of wheat. Sensitivity test demonstrated that the nested PCR could detect as low as 0.1 fg template DNA and about 10,000 times more sensitive than the standard PCR. Results of artificial inoculation experiments showed that the nested PCR assay can detect a low level of latent infection of wheat seedlings 2 days earlier than did standard PCR. The incidences of latent infection of wheat seedlings determined by the nested PCR linearly correlated with those by the conventional incubation method (r² = 0.66, P = 0.0023). The incidences of latent infection detected with nested PCR were higher than that with the conventional method. This study provides an accurate method to efficiently estimate the initial inoculum potential of wheat powdery mildew epidemics in the fields.     

Assimilatverteilung in Sommergerstensorten mit unterschiedlicher Resistenz gegenüber dem Echten Mehltau (Erysiphe graminis f. sp. hordei)

Distribution of assimilates in cultivars of spring barley with different resistance against powdery mildew (Erysiphe graminis f. sp. hordei) Transport and distribution of radioactive labelled assimilates in spring barley cultivars with different degrees of resistance to powdery mildew were studied after ¹⁴CO₂-treatment of single leaves. Plants of the cultivars ‘Amsel’ (susceptible), ‘Asse’ (adult plant resistant), and ‘Rupee’ (resistant) were analyzed at the vegetative growth stage (5. leaf unfolded) and the generative growth stage (anthesis). At the vegetative growth stage the assimilate export from the mildew inoculated 5. leaf of ‘Amsel’ and ‘Rupee’ is decreased; in ‘Asse’, there is no considerable change of assimilate distribution due to infection. At the generative growth stage the assimilate export from the infected flag leaf of ‘Amsel’ is reduced when the fungus, is sporulating. In the cultivar ‘Asse’ the assimilates are bound at the infection site until the seventh day after inoculation, then the transport of assimilates to the ear is increased. In ‘Rupee’ mildew inoculation causes an enhanced assimilate transport to the ear. The changes in assimilate distribution due to mildew inoculation are discussed with respect to the different types of host-parasite-interactions and the source-sink-activities in the different cultivars.     

Cell death in wheat roots induced by the powdery mildew fungus Blumeria graminis f. sp. tritici

Inoculation of wheat (Triticum aestivum L. cv. Huamai 8) leaves with wheat powderly mildew fungus (Blumeria graminis f. sp. tritici) induced cell death in wheat adventitious roots, where no fungal structures were observed. The cytological and molecular characterization of this cell death was shown as following: cell nuclei were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; chromatin condensed and formed peripheral conglomeration in nuclei; and perinuclear spaces partly dilated. These results suggested that, without pathogen spread, the infection could induce systemic PCD in adventitious roots. Comparison with a leaf-cutting experiment (LC)enabled us to speculate that lack of assimilates was not the only reason for the systemic PCD in wheat roots in powdery mildew experiment and that such systemic PCD might be mediated by long-distance signals. In addition, reactive oxygen species (ROS) and Ca²⁺ were related to the systemic PCD.     

Evidence for Resistance-inducing Compounds in Conidia of Erysiphe graminis f.sp. hordei

Preparations of Erysiphe graminis f.sp. hordei conidia were spray-applied to the first leaf of barley plants which were subsequently challenge inoculated with virulent powdery mildew. The powdery mildew reducing effect of the preparations was assessed by scoring the outcome of the challenge inoculation. Homogenates of ungerminated conidia, germinated conidia, and methanol-water extracts of germinated conidia reduced the number of powdery mildew colonies. Cell wall fragments from ungerminated conidia, germinated conidia, and conidial germination fluid obtained from conidia germinated in aqueous suspension did not reduce the number of powdery mildew colonies. Microsconical analysis of the infection course following challenge inoculation indicated that the powdery mildew reducing effect is due partly to induced resistance.     

Virulence analysis of wheat powdery mildew population (Erysiphe graminis f.sp.tritici) from the region of Slovakia and Hungary

In the year 1992 a total of 163 isolates of wheat powdery mildew were tested. The samples of mildew isolates were obtained by means of a mobile spore catching apparatus. The populations from 4 regions of Slovakia and 3 regions of Hungary were analyzed. The resistance due toPm5, Pm8 andMl-i genes at the observed locations has already been overcome. The resistance genesPm1, Pm2 and a gene combinationPm2+Pm6 ensure the protection only against a part of the patho-types of powdery mildew population. Virulence corresponding to thePm4b gene has been low so far. The regional patterns of pathogen virulence are in good agreement with the gene resistance spectrum by the cultivars grown regionally. Little differences in virulence among the populations from the regions of Slovakia and Hungary indicate that this part of Eastern Europe should be considered as an epidemiologic unit.     

Localisation of genes for resistance against Blumeria graminis f.sp. hordei and Puccinia graminis in a cross between a barley cultivar and a wild barley (Hordeum vulgare ssp. spontaneum) line

The aims of this investigation have been to map new (quantitative) resistance genes against powdery mildew, caused by Blumeria graminis f.sp. hordei L., and leaf rust, caused by Puccinia hordei L., in a cross between the barley ( Hordeum vulgare ssp. vulgare) cultivar "Vada" and the wild barley ( Hordeum vulgare ssp. spontaneum) line "1B-87" originating from Israel. The population consisted of 121 recombinant inbred lines. Resistance against leaf rust and powdery mildew was tested on detached leaves. The leaf rust isolate "I-80" and the powdery mildew isolate "Va-4", respectively, were used for the infection in this experiment. Moreover, powdery mildew disease severity was observed in the field at two different epidemic stages. In addition to other DNA markers, the map included 13 RGA (resistance gene analog) loci. The structure of the data demanded a non-parametric QTL-analysis. For each of the four observations, two QTLs with very high significance were localised. QTLs for resistance against powdery mildew were detected on chromosome 1H, 2H, 3H, 4H and 7H. QTLs for resistance against leaf rust were localised on 2H and 6H. Only one QTL was common for two of the powdery mildew related traits. Three of the seven QTLs were localised at the positions of the RGA-loci. Three of the five powdery mildew related QTLs are sharing their chromosomal position with known qualitative resistance genes. All detected QTLs behaved additively. Possible sources of the distorted segregation observed, the differences between the results for the different powdery mildew related traits and the relation between qualitative and quantitative resistance are discussed.     

Disease in wheat genotypes naturally infected with Gaeumannomyces graminis var. tritici

Wheat genotypes consisting of seven homozygous lines and 40 segregate families were studied at two sites naturally infested with the take-all pathogen, Gaeumannomyces graminis var. tritici. The numbers of seminal and coronal roots infected with G. graminis and other root pathogens were recorded. The genotypes differed in infection with G. graminis, with little evidence of genotype × environment interactions. The incidence of G. graminis and Rhizoctonia solani was negatively associated, but the association did not greatly influence differences between wheats in infection with G. graminis. The distribution of R. solani was negatively associated with the severity of take-all at only one site. Of symptoms of infection with G. graminis, wheat genotypes differed most in incidence of deadheads, but differences were not consistent over environments, and were associated with earliness of maturity. Wheats differed more in expression of disease than in infection with G. graminis. The course of disease was deduced from associations between the incidence of pathogens and components of plant growth and yield. G. graminis was the dominant pathogen at both sites, and caused a yield loss of 0–15% at one site, and an average 62% loss at the other. More components of yield were affected where disease was most severe.     

Effects of take-all (Gaeumannomyces graminis var. tritici) on crop N uptake and residual mineral N in soil at harvest of winter wheat

Background and aims

Take-all, caused by the fungus Gaeumannomyces graminis var. tritici, is the most damaging root disease of wheat. A severe attack often leads to premature ripening and death of the plant resulting in a reduction in grain yield and effects on grain quality (Gutteridge et al. in Pest Manag Sci 59:215–224, 2003). Premature death of the plant could also lead to inefficient use of applied nitrogen (Macdonald et al. in J Agric Sci 129(2):125–154, 1997). The aim of this study was to determine crop N uptake and the amount of residual mineral N in the soil after harvest where different severities of take-all had occurred.

Methods

Plant and soil samples were taken at anthesis and final harvest from areas showing good and poor growth (later confirmed to be caused by take-all disease) in three winter wheat crops grown on the same soil type on Rothamsted Farm in SE England in 1995, 2007 and 2008 (harvest sampling only). All crops received fertiliser N in spring at recomended rates (190–200?kg?N ha^?1). On each ocassion crops were assessed for severity of take-all infection (TAR) and crop N uptakes and soil nitrate plus ammonium (SMN) was determined. Grain yields were also measured.

Results

Grain yields (at 85% dry matter) of crops with moderate infection (good crops) ranged from 4.3 to 13.0?t ha^?1, compared with only 0.9–4.5?t ha^?1 for those with severe infection (poor crops). There were significant (P?<?0.05) negative relationships between crop N uptake and TAR at anthesis and final harvest. At harvest, good crops contained 129–245?kg?N ha^?1 in grain, straw and stubble, of which 85–200?kg?N ha^?1 was in the grain. In contrast, poor crops contained only 46–121?kg?N ha^?1, of which only 22–87?kg?N ha^?1 was in the grain. Positive relationships between SMN and TAR were found at anthesis and final harvest. The SMN in the 0–50?cm layer following harvest of poor crops was significantly (P?<?0.05) greater than that under good crops, and most (73–93%) was present as nitrate.

Conclusions

Localised patches of severe take-all infection decreased the efficiency with which hexaploid wheat plants recovered soil and fertiliser derived N, and increased the subsequent risk of nitrate leaching. The risk of gaseous N losses to the atmosphere from these areas may also have been enhanced.     

Expression of resistance to Blumeria graminis f.sp. tritici in 'Chinese Spring' wheat addition lines containing chromosomes from Hordeum vulgare and H. chilense

Blumeria graminis f.sp. tritici (syn. Erysiphe graminis f.sp. tritici) causes an important disease of wheat (powdery mildew) to which Hordeum vulgare and H. chilense are resistant. The study of chromosomal addition lines of H. vulgare and H. chilense in wheat showed that they possessed resistance to wheat powdery mildew. This was expressed as a reduction of disease severity but it was not associated with increased macroscopically visible necrosis. The resistance is of broad genetic basis, conferred by gene(s) present on different chromosomes of both H. vulgare and H. chilense. The feasibility of transferring this resistance to wheat is discussed.     

籽用美洲南瓜叶片对白粉病菌的抗逆生理响应

以籽用美洲南瓜(Cucurbita pepo L.)白粉病抗病品系F2和感病品系M3为试材,在人工气候箱内接种白粉病生理小种2US孢子悬浮液,考察在接种白粉病菌后南瓜幼苗植株与白粉病菌的互作、叶片活性氧代谢及保护酶活性的变化,探讨南瓜抵御白粉病的生理机制。结果表明：(1)与感病品系M3相比,接种白粉病菌后,抗病品系F2叶片上病原菌发育缓慢,较难侵染叶片。(2)抗病品系F2在感病初期叶片H₂O₂、O₂^-·含量迅速升高后逐渐下降,而感病品系在感病初期H₂O₂、O₂^-·含量上升缓慢,在达最大值后始终保持较高水平,且感病品系叶片MDA含量始终高于抗病品系;组织化学染色分析发现,抗病品系叶片着色比感病品系快,之后着色面积有所减少并趋于较低水平。(3)抗病品系F2和感病品系M3叶片抗氧化酶CAT、SOD、POD活性及PAL、PPO活性在接种白粉病菌后均显著增加,但抗病品系的活性及其增幅均高于感病品系。研究发现,籽用美洲南瓜抗病品系叶片上白粉病菌发育缓慢,较难受到侵染,生成菌丝体后叶片上粉状斑点较小;抗病品系在被白粉病菌侵染初期依靠活性氧的增加抵御病原菌的入侵,随着活性氧含量增加抗病品系通过迅速增加自身抗氧化酶活性来防止氧化胁迫;与感病品系相比,抗病品系在受病原菌侵染后能迅速增加PAL、PPO活性以抵御病原菌侵染。     

Evaluation of Certain Cowpea Genotypes for Partial Resistance to Powdery Mildew (Erysiphe polygoni)

Partial resistance to powdery mildew (Erysiphe polygoni) in twenty one cowpea cultivars was assessed over two to three seasons under field conditions. Mildew incidence was recorded as number of leaflets affected/plant and severity as AUDPC or infection rate using Gompertz transformation. Irrespective of the parameter used to measure mildew resistance or the season, mean response of cvs V-105, V-269, V-276, V-282 and V-385 showed clear slow-mildewing, while RC-48, S-488, TVX-944-02E, V-27, V-36 and V-118 were rated as susceptible. Mildew resistance in some cultivars was negatively correlated with that for the leaf rust (Uromyces vignae). AUDPC was a more robust disease parameter than infection rate and showed a good correlation (r²= 0.79) with incidence.     

Pathotypes of Blumeria graminis f. sp. tritici,the causal agent of wheat powdery mildew from some regions of Iran

Wheat powdery mildew is controlled mainly by race-specific resistance. To be effective, breeding wheat for resistance to powdery mildew requires knowledge of virulence diversity in local populations of the pathogen. Isolates of Blumeria graminis, collected in 2009 and 2010 from three areas of Iranian production, were analysed for virulence using a host differential series comprised of 16 known genes conferring resistance to powdery mildew. The results showed that high-virulence frequencies to genes Pm1, Pm2, Pm4a, Pm5, Pm6, Pm7, Pm8 and Pm9 were found over both years and across all three areas. Virulence frequencies for Pm3a and Pm3b were intermediate, while virulence frequencies for Pm3a, Pm3c, Pm4a and Pm2, 6 were low. Genes Pm1, 2, 9 and Pm2, 4b, 8 were highly resistant in all regions. Virulence to Pm8 increased to high levels, while virulence to Pm4a decreased across the area surveyed from 2009 to 2010.     

Use of seedlings for predicting field resistance of wheat toErysiphe graminis f. sp.tritici

Seedlings can be used to predict field resistance, if any, of wheat varieties and experimental lines to wheat powdery mildew fungus,Erysiphe graminis f. sp.tritici. First leaves of 8-day-old seedlings of a field-resistant wheat showed a greater frequency of primary infections in subsidiary cells of stomata than those in a susceptible wheat. This method of predicting field resistance of experimental lines from seedling studies should prove time-saving because crosses involving field-resistant experimental lines can be made in the same generation.     

CAPS and DHPLC Analysis of a Single Nucleotide Polymorphism in the Cytochrome b Gene Conferring Resistance to Strobilurins in Field Isolates of Blumeria graminis f. sp. hordei

A single nucleotide polymorphism in the wheat powdery mildew (Blumeria graminis f. sp. tritici) cytochrome b gene is responsible for resistance to inhibitors of the quinol outer binding site of the cytochrome bc₁ complex (QoI) fungicides. Analysis of a partial sequence of the cytochrome b gene from field isolates resistant and sensitive to QoI fungicides revealed the same point mutation in barley powdery mildew (B. graminis f. sp. hordei). Analysis of 118 and 40 barley powdery mildew isolates using a cleaved amplified polymorphic sequence assay and denaturing high performance liquid chromatography, respectively, confirmed that this single nucleotide polymorphism also confers resistance to QoI fungicides in barley powdery mildew.     

Effects of sowing date and vernalisation on the growth of winter barley and its resistance to powdery mildew (Erysiphe graminis f.sp. hordei)

Detailed studies on the production of individual leaves, and the development of powdery mildew on them, were made in field plots of winter barley sown on different dates. The greater severity of the disease on early-sown than on later-sown seedlings during the autumn and winter can probably be explained mainly by changes in the abundance of inoculum and the suitability of the weather for infection. Results from glasshouse experiments suggest that the differences may be reinforced by direct effects of vernalisation on the susceptibility of seedlings to the disease. Contrary effects of sowing date on mildew severity during summer are probably due to the progressively greater resistance to mildew of the later-formed than of seedling leaves, and the earlier appearance of corresponding leaves on early-sown than on later-sown plants. Early sowing can also increase the total number of leaves produced per stem. Therefore, because resistance of the leaves increases progressively, the maximum degree of resistance expressed by the later-formed (e.g. flag) leaves will often be greater on early-sown than on later-sown plants.     

The Effect of Soil Phosphate on Injury to Winter Barley Caused by Mildew Infection (Erysiphe graminis f.sp. Hordei)

Populations of winter barley were sown in autumn in large tubsof soil to half of which additional phosphate was supplied.Half the plants of each phosphate treatment were infected withpowdery mildew (Erysiphe graminis D.C. f. sp. hordei Marchal)and all plants were transferred to the-field. Infection inducedextensive leaf injury during mid-winter in plants at low phosphate,but injury was greatly reduced in the high soil phosphate treatment.The extent of winter injury had a marked effect on the increasein leaf area in spring, and the accumulation of plant d. wtwas positively correlated with the percentage of total leafarea remaining undamaged at the end of winter. This, in turn,was strongly influenced by the interaction between powdery mildewand soil phosphate. High soil phosphate may act as a ‘buffer’to the effects of infection, minimizing the combined effectsof infection and abiotic stresses suffered by plants in winter. Barley, powdery mildew (Erysiphe graminis hordei), overwinter, phosphate, temperature     

Virulence Structure of Blumeria graminis f. sp. tritici and Its Genetic Diversity by ISSR and SRAP Profiling Analyses

Blumeria graminis f. sp. tritici, which causes wheat powdery mildew, is an obligate biotrophic pathogen that can easily genetically adapt to its host plant. Understanding the virulence structure of and genetic variations in this pathogen is essential for disease control and for breeding resistance to wheat powdery mildew. This study investigated 17 pathogenic populations in Sichuan, China and classified 109 isolates into two distinct groups based on pathogenicity analysis: high virulence (HV, 92 isolates) and low virulence (LV, 17 isolates). Populations from Yibin (Southern region), Xichang (Western region), and Meishan (Middle region) showed lower virulence frequencies than populations from other regions. Many of the previously known resistance genes did not confer resistance in this study. The resistance gene Pm21 displayed an immune response to pathogenic challenge with all populations in Sichuan, and Pm13, Pm5b, Pm2+6, and PmXBD maintained resistance. AMOVA revealed significantly higher levels of variation within populations and lower levels of variation among populations within regions. High levels of gene flow were detected among populations in the four regions. Closely related populations within each region were distinguished by cluster analyses using ISSR and SRAP alleles. Both ISSR and SRAP allele profiling analyses revealed high levels of genetic diversity among pathogenic populations in Sichuan. Although ISSR and SRAP profiling analysis showed similar resolutions, the SRAP alleles appeared to be more informative. We did not detect any significant association between these alleles and the virulence or pathogenicity of the pathogen. Our results suggest that ISSR and SRAP alleles are more efficient for the characterization of small or closely related populations versus distantly related populations.     

Detection of Powdery Mildew in Two Winter Wheat Plant Densities and Prediction of Grain Yield Using Canopy Hyperspectral Reflectance

To determine the influence of plant density and powdery mildew infection of winter wheat and to predict grain yield, hyperspectral canopy reflectance of winter wheat was measured for two plant densities at Feekes growth stage (GS) 10.5.3, 10.5.4, and 11.1 in the 2009–2010 and 2010–2011 seasons. Reflectance in near infrared (NIR) regions was significantly correlated with disease index at GS 10.5.3, 10.5.4, and 11.1 at two plant densities in both seasons. For the two plant densities, the area of the red edge peak (Σdr _{680–760 nm}), difference vegetation index (DVI), and triangular vegetation index (TVI) were significantly correlated negatively with disease index at three GSs in two seasons. Compared with other parameters Σdr _{680–760 nm} was the most sensitive parameter for detecting powdery mildew. Linear regression models relating mildew severity to Σdr _{680–760 nm} were constructed at three GSs in two seasons for the two plant densities, demonstrating no significant difference in the slope estimates between the two plant densities at three GSs. Σdr _{680–760 nm} was correlated with grain yield at three GSs in two seasons. The accuracies of partial least square regression (PLSR) models were consistently higher than those of models based on Σdr _{680760 nm} for disease index and grain yield. PLSR can, therefore, provide more accurate estimation of disease index of wheat powdery mildew and grain yield using canopy reflectance.     

1种小麦白粉病菌DNA基因组的微量简捷提取方法

为了寻找适合小麦白粉菌基因组DNA微量提取的方法,分别采用改进破壁法,液氮研磨法和溶菌酶消化法进行破壁,提取专性寄生菌小麦白粉菌DNA。结果表明,用改进的破壁方法,仅用3~10 mg的分生孢子粉所获得DNA的收率为（12.23±3.46）~（40.32±5.67）ng/mg,且OD260/OD280比值为1.71~1.92之间,说明该破壁方法获得的DNA收率大且纯度高。通过PCR反应获得了良好的效果。同时该方法也适用于小麦条锈菌和大麦白粉菌专性寄生菌DNA的提取。