Genetic Structure of Rhynchosporium secalis in Australia

ABSTRACT Restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Australian field populations of the barley scald pathogen Rhynchosporium secalis. Fungal isolates were collected by hierarchical sampling from five naturally infected barley fields in different geographic locations during a single growing season. Genetic variation was high in Australian R. secalis populations. Among the 265 fungal isolates analyzed, 214 distinct genotypes were identified. Average genotype diversity within a field population was 65% of its theoretical maximum. Nei's average gene diversity across seven RFLP loci was 0.54. The majority (76%) of gene diversity was distributed within sampling site areas measuring 1 m(2); 19% of gene diversity was distributed among sampling sites within fields; and 5% of gene diversity was distributed among fields. Fungal populations from different locations differed significantly both in allele frequencies and genotype diversities. The degree of genetic differentiation was significantly correlated with geographic distance between populations. Our results suggest that the R. secalis population in Western Australia has a different genetic structure than populations in Victoria and South Australia.     

Pathogenicity of Rhynchosporium secalis isolates from Norway on 30 cultivars of barley

The pathogenic variability of the barley scald fungus, Rhynchosporium secalis , in central Norway was examined in 1994. The climate in this region is usually cold and wet during the growing season of spring barley. Leaf blotch is prevalent and causes significant yield losses. Forty-two isolates of the fungus, from naturally infected spring barley in four counties, were differentiated into 32 pathotypes by the standard differential set for R. secalis . All pathotypes were complex and had virulence for nine to 22 differentials. The cultivar Osiris was resistant to all isolates tested. The cultivars C.I.8162, Hudson, Atlas 46 and C.I.3515 were resistant to the majority of the isolates. Several differentials with various resistance genes were susceptible to up to 100% of the isolates. Isolates were derived from local cultivars with no known resistance genes, suggesting that R. secalis populations in central Norway are characterized by a high degree of seemingly unnecessary pathogenicity. Because of the great variability and complexity of the pathotypes, traditional breeding methods using single major genes are not likely to be effective in central Norway.     

Asexual Genetic Exchange in the Barley Pathogen Rhynchosporium secalis

ABSTRACT The causal agent of barley scald, Rhynchosporium secalis, is a haploid anamorphic ascomycete with no known sexual stage. Nevertheless, a high degree of genetic variation has been observed in fungal populations on commercial barley cultivars and parasexuality has been suggested to contribute to this variation. In order to test whether asexual genetic exchange can occur, isolates of R. secalis were transformed to hygromycin B resistance or phleomycin resistance. Mixtures of transformants were co-inoculated either on agar or in planta and screened for the occurrence of dual-antibiotic-resistant colonies. No dual-antibiotic-resistant colonies resulted from mixing transformants of different fungal isolates. In contrast, with transformants originating from the same fungal isolate, asexual exchange of markers was demonstrated on agar plates and in planta. This is the first definitive evidence of asexual genetic exchange in R. secalis.     

Application of real-time and multiplex polymerase chain reaction assays to study leaf blotch epidemics in barley

ABSTRACT Leaf blotch, caused by Rhynchosporium secalis, was studied in a range of winter barley cultivars using a combination of traditional plant pathological techniques and newly developed multiplex and real-time polymerase chain reaction (PCR) assays. Using PCR, symptomless leaf blotch colonization was shown to occur throughout the growing season in the resistant winter barley cv. Leonie. The dynamics of colonization throughout the growing season were similar in both Leonie and Vertige, a susceptible cultivar. However, pathogen DNA levels were approximately 10-fold higher in the susceptible cultivar, which expressed symptoms throughout the growing season. Visual assessments and PCR also were used to determine levels of R. secalis colonization and infection in samples from a field experiment used to test a range of winter barley cultivars with different levels of leaf blotch resistance. The correlation between the PCR and visual assessment data was better at higher infection levels (R(2) = 0.81 for leaf samples with >0.3% disease). Although resistance ratings did not correlate well with levels of disease for all cultivars tested, low levels of infection were observed in the cultivar with the highest resistance rating and high levels of infection in the cultivar with the lowest resistance rating.     

Response of strawberry genotypes to inoculation with isolates of Verticillium dahliae differing in host origin

Eight strawberry genotypes known to differ in susceptibility to verticillium wilt were inoculated with eight isolates of Verticillium dahliae originally obtained from six different host crops: strawberry, potato, watermelon, mint, eggplant (aubergine) and cauliflower. Inoculation experiments were conducted in replicated field trials during two successive years. Known susceptible genotypes developed typical symptoms of verticillium wilt in both years. Although isolates manifested a wide range of aggressiveness, differences were significant only on the most susceptible strawberry genotype. Two isolates originating from strawberry were among the most aggressive of the eight tested, whereas the least aggressive isolate was obtained from cauliflower. Six strawberry genotypes that were regarded as resistant to verticillium wilt based on previous tests were also resistant in the present study, regardless of the isolate used. Overall, strawberry genotypes represented the largest source of variation in these experiments, with variance components approximately 10-fold greater than those associated with either isolate or the isolate × genotype interaction. The results suggest it should be possible to develop resistance to verticillium wilt in strawberry that is broadly effective against isolates of diverse host origin.     

Measuring Immigration and Sexual Reproduction in Field Populations of Mycosphaerella graminicola

ABSTRACT A field experiment was conducted to determine the relative contributions of immigration and sexual reproduction to the genetic structure of Mycosphaerella graminicola populations during the course of an epidemic. The genetic structure of M. graminicola populations sampled from wheat plots inoculated artificially with 10 isolates was compared with control plots infected naturally by airborne ascospores. Restriction fragment length polymorphisms (RFLPs) were used to test the randomness of associations among loci, and DNA fingerprints were used to identify clones. All isolates in the control plots had unique genotypes and RFLP loci were at gametic equilibrium, findings consistent with random mating. The proportion of isolates in the inoculated plots with DNA fingerprints that differed from the 10 inoculated isolates increased from 3% in the early to 39 and 34% in the mid- and late season, respectively. The degree of gametic disequilibrium was higher in the mid-season than in the late-season population. By the end of the growing season, we estimate that 66% of the isolates in the inoculated plots were asexual progeny of the 10 inoculated isolates, 10% were immigrants, and 24% were sexual recombinants. The proportion of infections caused by ascospores increased over the growing season.     

The Genetic Structure of Field Populations of Rhynchosporium secalis from Three Continents Suggests Moderate Gene Flow and Regular Recombination

ABSTRACT Restriction fragment length polymorphism (RFLP) markers were used to compare the genetic structure of field populations of Rhynchosporium secalis from barley. A total of 543 isolates representing 8 field populations were sampled from Australia, California, Finland, and Norway. Gene and genotype diversity were high in all populations. Nei's average gene diversity across seven RFLP loci was 0.513. Hierarchical gene diversity analysis showed that 9% of the total genetic variability was distributed among continents, 4% was distributed among fields within continents, and 13% was distributed among collection stations within a field. The majority (74%) of genetic variability was distributed within collection areas of approximately 1 m(2) within fields. Gene flow appears to be significant on a regional scale but more restricted among continents. Allele frequencies were significantly different at several RFLP loci. Genetic distances were small among populations within regions and large between regions. Pairwise comparisons of genotype diversity in the populations revealed significant differences among populations that were related mainly to differences in sampling strategies. Isolates from Norway and Finland showed a lower copy hybridization pattern with probe pRS26. This probe functioned as a fingerprint probe for the California and Australian isolates. Seven out of the eight populations studied were at gametic equilibrium for RFLP loci, suggesting that R. secalis populations in Norway, Finland, and Australia undergo regular recombination, although a teleomorph has not yet been recognized.     

Activity of carbendazim,dimethachlon, iprodione,procymidone and boscalid against Sclerotinia stem rot in Jiangsu Province of China

Carbendazim (MBC) was widely used to control Sclerotinia stem rot routinely during the 1980s in China, but development of MBC resistance in the causal agent Sclerotinia sclerotiorum led to control failures of this disease. In this study it was found that the MBC resistance in S. sclerotiorum populations was widespread throughout Jiangsu Province with a resistance frequency of 29.54% in the 1786 collected isolates during the growing seasons of 2006 to 2008. The resistance frequencies differed among sampled cities, ranging from 3.1% to 54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotia production and pathogenicity to the wild-type sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field MBC-sensitive isolates in the field. In the in vitro sensitivity test, boscalid showed greater activity against S. sclerotiorum than dicarboximide fungicides (dimethachlon, iprodione and procymidone). The treatment 50% boscalid (WG) 125 g a.i. ha⁻¹ was comparable in efficacy to the treatment 50% iprodione (WP) 600 g a.i. ha⁻¹, and better than other treatments of 6% dimethachlon (WP) 690 g a.i. ha⁻¹ and 50% procymidone (WP) 337.5 g a.i. ha⁻¹, whereas MBC failed to control Sclerotinia stem rot (control efficacy only 16.0%). The most active agent for controlling Sclerotinia stem rot was boscalid in our study.     

Genetic structure of a Pyrenophora teres f. teres population over time in an Australian barley field as revealed by Diversity Arrays Technology markers

Net form of net blotch caused by Pyrenophora teres f. teres (Ptt) is a major foliar disease of barley (Hordeum vulgare) worldwide. Knowledge of the evolution of Ptt pathogen populations is important for development of durable host-plant resistance. This study was conducted to investigate changes in genetic structure of a Ptt population within a barley field during three cropping years. The susceptible barley cultivar Henley was inoculated with Ptt isolate NB050. Leaf samples were collected during the years 2013–15 and 174 single spore Ptt isolates stored. Genotyping using Diversity Arrays Technology markers identified that 25% of isolates were clones of the inoculated isolate and 75% of isolates were multilocus genotypes (MLGs) differing from the original inoculated genotype. The novel genotypes probably originated from a combination of windborne spores from neighbouring fields, infected seed and sexual recombination in the field. The rapid change in the genotypic composition of the Ptt population in this study suggests adaptive potential of novel genotypes and demonstrates the need for barley breeders to use multiple sources of host-plant resistance to safeguard against resistance being overcome.     

Fitness effects of the triazine resistance mutation in Amaranthus hybridus: relative fitness in maize and soyabean crops

Fitness costs of the triazine resistance mutation were estimated in experimental populations of Amaranthus hybridus L. from Maryland and Virginia (USA). Estimated costs were attributed directly to effects of the resistance mutation, through comparisons of lines bearing similar nuclear genomes, but either resistant or susceptible cytoplasm. Experimental weed plants were established from seed in the field at a wide range of A. hybridus and crop (maize or soyabean) densities, and experiments were repeated over 3 years. Resistant cytoplasm caused large reductions in several components of fitness, but substantial differences were observed among years in these fitness costs of resistance. For the fitness component of total biomass above-ground biomass production, relative fitnesses of resistant genotypes (resistant/susceptible ratios) ranged between 0.42 and 0.70. Costs of resistance were similar in weeds growing with either maize or soyabean crops. Certain costs of resistance differed between populations: impairment of seedling establishment and early growth by the resistance mutation was evident in the Virginia population, but not in the Maryland population. This functional difference between populations was associated with greater costs of resistance in the Virginia population with respect to mid-season leaf number and total above-ground biomass production; however, only the former population difference in fitness cost was statistically significant.     

Vitis Resistance to Pierce's Disease Is Characterized by Differential Xylella fastidiosa Populations in Stems and Leaves

ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines representing a diverse selection of Vitis spp. was characterized through measurements of X. fastidiosa bacterial movement and accumulation in artificially inoculated greenhouse-grown grapevines. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was optimized for quantification of X. fastidiosa populations and tested on known amounts of X. fastidiosa added to grape tissue extracts. Predicted versus known X. fastidiosa concentrations proved to be highly correlated (R(2) = 0.99). Populations of X. fastidiosa in stem internode, stem node, petiole, and leaf blade samples from the genotypes in this study were measured at 12 weeks postinoculation using the optimized ELISA procedure. Samples from each plant part were taken at eight positions along the inoculated shoots. Systemic infection was detected in both susceptible and resistant genotypes. Resistant genotypes were characterized by significant differences in X. fastidiosa populations between stem internodes and leaves (1.0 x 10(6) and 1.1 x 10(7) cells/g of sample, respectively). In contrast, the susceptible genotypes were characterized by high mean X. fastidiosa populations in both stems and leaves (5.6 x 10(7) and 4.8 x 10(7) cells/g, respectively) the latter of which were not significantly different from the resistant genotypes. A high correlation (R(2) = 0.97) between stem X. fastidiosa numbers to previously characterized field Pierce's disease (PD) performance indicates that the quantitative ELISA measurements of X. fastidiosa in greenhouse-grown grapevines should be a useful tool for predicting PD resistance under field conditions.     

寄主品种-病原物小种寄生适合度中水平抗性与侵袭力关系的初步研究

 通过8个马铃薯品种与2个晚疫病菌生理小种组合寄生适合度的测定,对品种-小种寄生适合度中水平抗性与侵袭力之间的关系进行了研究。从初步研究结果可基本确定,在没有垂直抗性或垂直抗性被相应毒性基因克服了的组合中,品种水平抗性和小种侵袭力以乘积的方式构成寄生适合度。其关系可表示为:相对寄生适合度=品种水平抗性(以相对感病性表示)×小种侵袭力。     

Phenotype Instability in Botrytis cinerea in the Absence of Benzimidazole and Dicarboximide Fungicides

ABSTRACT Stability of phenotypes of isolates of Botrytis cinerea that were sensitive or resistant to benzimidazole and dicarboximide fungicides was examined in the absence of fungicides in laboratory and growth room experiments. Twelve greenhouse isolates of B. cinerea were subcultured on potato dextrose agar (PDA) for 20 generations and on geranium seedlings for 15 generations. Three isolates of each of the following four phenotypes were used: sensitive to the fungicides thiophanate-methy1 (a benzimidazole) and vinclozolin (a dicarboximide) (S(T)S(V)), resistant to both fungicides (R(T)R(V)), resistant to thiophanate-methy1 and sensitive to vinclozolin (R(T)S(V)), and sensitive to thiophanate-methy1 and resistant to vinclozolin (S(T)R(V)). In three trials on PDA, 36 populations were subcultured; 8 populations changed phenotypes by the end of 20 generations, as determined by conidium germination on fungicide-amended medium. Five of the eight initially were S(T)R(V); the resulting phenotypes were S(T)S(V), R(T)S(V), and R(T)R(V). Populations from eight other isolates exhibited temporary changes in phenotype during intermediate generations on PDA but reverted to initial phenotypes by the twentieth generation; five of these populations changed to phenotype R(T)R(V). In two geranium seedling trials, each of the 12 greenhouse isolates was inoculated onto a set of three seedlings for each generation, and diseased tissue that developed was used to initiate the next generation. Therefore, a total of 72 populations of B. cinerea were subcultured in the two trials; 5 of these populations changed phenotype at the end of 15 generations. Three of the five initially were S(T)R(V); these changed to phenotypes S(T)S(V) or R(T)R(V). In each of the two trials on geranium seedlings, a population subcultured from one S(T)S(V) isolate changed phenotype one to phenotype R(T)R(V) and one to phenotype R(T)S(V). In all trials, no population resistant to thiophanate-methy1 changed to a thiophanate-methy1-sensitive phenotype, and no population changed to phenotype S(T)R(V). Random amplified polymorphic DNA (RAPD) fingerprints were generated with the 12 initial isolates and 49 isolates subcultured on PDA or geranium seedlings. Cluster analyses of RAPD markers showed that subcultured isolates exhibiting the same phenotype clustered together and that subcultured isolates derived from a common greenhouse isolate but with different phenotypes were in different clusters. Some populations that did not change phenotype exhibited considerable differences in RAPD marker patterns. The results of this study indicate that, in the absence of fungicides, sensitive populations of B. cinerea can develop resistance to thiophanate-methy1 and vinclozolin, and this resistance can be maintained in populations through multiple generations. Populations resistant only to vinclozolin (S(T)R(V)) exhibited a high frequency of phenotype change, and populations resistant to both fungicides (R(T)R(V)) were stable.     

中国4省份葡萄霜霉病菌群体对烯酰吗啉的抗性及适合度

为明确我国山西、山东、河北和云南省葡萄霜霉病菌对烯酰吗啉的抗性以及田间病菌群体的适合度,本研究采用Taqman-MGB技术检测了2020年-2021年采自上述4省份292株葡萄霜霉病菌对烯酰吗啉的抗性频率;采用叶盘法测定了田间抗性菌株与敏感菌株的适合度。结果表明,上述4个地区的平均烯酰吗啉抗性频率及抗性等位基因频率分别为64.0%和74.7%,其中山东省蓬莱区最高(91.2%和96.3%),河北省廊坊市(73.8%和85.0%)和云南省宾川县(75.0%和82.6%)次之,山西省清徐县(16.7%和34.7%)最低。适合度分析发现,田间抗性菌株的产孢能力明显高于敏感菌株,但二者的复合适合度指数无显著差异。上述结果表明,4省份霜霉病菌群体均对烯酰吗啉产生了不同程度的抗性,田间抗性菌株与敏感菌株相比无明显适合度变化。     

Charcoal rot (Macrophomina phaseolina) resistance and the effects of water stress on disease development in sorghum

In three field experiments, plants subjected to post-flowering water stress and inoculated with Macrophomina phaseolina had greater development of charcoal rot symptoms than did inoculated plants not subjected to water stress. Two sorghum genotypes (B35-6 and SC265-14E) were found to be consistently more resistant to M. phaseolina , an assessment that was facilitated by the use of appropriate soil moisture conditions at the time of greatest plant susceptibility to charcoal rot.
In addition, an isolate of M. phaseolina originally isolated from a sorghum genotype with resistance to charcoal rot caused greater symptom development than did two other isolates originally obtained from sorghum genotypes susceptible to charcoal rot.     

Within-field variation of Fusarium graminearum isolates for aggressiveness and deoxynivalenol production in wheat head blight

Fusarium head blight (FHB), caused by Fusarium graminearum sensu stricto (s.s.), causes tremendous annual yield losses in wheat worldwide. Variation of aggressiveness of isolates from individual field populations in terms of FHB infection and deoxynivalenol (DON) concentration in the host are important population parameters reflecting parasitic ability. Our main objective was to estimate the variation of both traits within three populations of F. graminearum s.s., each consisting of 30 single-spore isolates collected from small wheat fields in Germany, and to compare it with 11 isolates of a collection (F. graminearum collection) from four countries. The same isolates were characterized using 19 single-sequence repeat markers. All isolates were spray inoculated on a moderately resistant spring wheat cultivar at two field locations over 2 years (i.e., in four environments). The genotypic proportion of phenotypic variance (σ(2)(G)) within populations was significant (P < 0.01) for both traits, and the σ(2)(G) × environment interaction was even more important for mean FHB severity. Ranges in mean FHB severity and DON concentration in the host were only slightly smaller for the field populations than for the F. graminearum collection. Both traits were significantly (P < 0.05) correlated within and across populations. A further partitioning of σ(2)(G) revealed 72% of σ(2)(G) within and 28% of σ(2)(G) across populations for both traits. Molecular variance of the three populations was similarly distributed (73.6% within versus 26.4% between populations). In view of this high within-field variation for traits of parasitic ability and selection, neutral molecular markers, multiple resistance genes of different origin should be employed in wheat breeding programs to obtain a long-term stable FHB resistance.     

Resistance to Scald (Rhynchosporium secalis) in Barley (Hordeum vulgare) Studied by Near-Isogenic Lines: I. Markers and Differential Isolates

ABSTRACT Near-isogenic lines (NILs) with resistance for scald in seventh generation backcross with 'Ingrid' as recurrent parent (RP) were tested with seven differential isolates of Rhynchosporium secalis in Norway and Canada. NILs of 'Turk', 'Brier', 'CI 8162', 'La Mesita', 'Hispont', 'Atlas 46', 'Modoc', 'Hudson', 'Abyssinian', 'Steudelli', and 'CI 2222' also were evaluated for field reactions. The genetic characterization of the NILs (degree of isogeneity with Ingrid) and with each other was carried out. The molecular marker pattern shows that the backcrossing program has resulted in from 86.3 to 100% RP genome in the NILs, depending on the marker system. On an average, 96% RP genome was found in the NILs. There were certain consistent (pairwise) differences between the NILs and RP on chromosomes 3H and 7H. Both chromosomes are known to contain loci conferring resistance to R. secalis, indicating successful introgression from the donors into the NILs. Approximately two-thirds of the observed RP-NIL polymorphisms were linked to the assumed resistance in the NIL. Based on the marker and phenotypic analyses of the NILs, suggestions for a more appropriate and updated terminology of genes for resistance to R. secalis in barley are made. The proposed changes in nomenclature also indicate the differentials that are available as NILs and those lacking.     

Aggressiveness of Mycosphaerella graminicola Isolates from Susceptible and Partially Resistant Wheat Cultivars

ABSTRACT The selective effect of quantitative host resistance on pathogen aggressiveness is poorly understood. Because two previous experiments with a small number of bread wheat cultivars and isolates of Mycosphaerella graminicola had indicated that more susceptible hosts selected for more aggressive isolates, we conducted a larger experiment to test that hypothesis. In each of 2 years, six cultivars differing in their levels of partial resistance were planted in field plots, and isolates were collected from each cultivar early and late in the growing season. The isolates were inoculated as populations bulked by cultivar of origin, field replicate, and collection date on seedlings of the same six cultivars in the greenhouse. The selective impact of a cultivar on aggressiveness was measured as the difference in aggressiveness between early and late isolates from that cultivar. Regression of those differences on disease severity in the field yielded significance values of 0.0531 and 0.0037 for the 2 years, with moderately resistant cultivars selecting for more aggressive isolates. In a related experiment, the protectant fungicide chlorothalonil was applied to plots of two susceptible cultivars to retard epidemic development. When tested in the greenhouse, isolates of M. graminicola from those plots were significantly more aggressive than isolates from the same cultivars unprotected by fungicide.     

Global Hierarchical Gene Diversity Analysis Suggests the Fertile Crescent Is Not the Center of Origin of the Barley Scald Pathogen Rhynchosporium secalis

A total of 1,366 Rhynchosporium secalis isolates causing scald on barley, rye, and wild barley (Hordeum spontaneum) were assayed for restriction fragment length polymorphism loci, DNA fingerprints, and mating type, to characterize global genetic structure. The isolates originated from 31 field populations on five continents. Hierarchical analysis revealed that more than 70% of the total genetic variation within regions was distributed within a barley field. At the global level, only 58% of the total genetic variation was distributed within fields, while 11% was distributed among fields within regions, and 31% was distributed among regions. A significant correlation was found between genetic and geographic distance. These findings suggest that gene flow is common at the local level while it is low between regions on the same continent, and rare between continents. Analyses of multilocus associations, genotype diversity, and mating type frequencies indicate that sexual recombination is occurring in most of the populations. We found the highest allele richness in Scandinavia followed by Switzerland. This suggests that R. secalis may not have originated at the center of origin of barley, the Fertile Crescent, nor in a secondary center of diversity of barley, Ethiopia.     

Development of two strains of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> with a different aggressiveness in the soil and on the roots of barley of two genotypes

Fusarium culmorum can affect plants including cereals or develop saprophytically in the soil. It is unknown whether its saprophytic ability is different in strains with a different aggressiveness. This knowledge could be used for effective breeding of resistant cultivars. Here, we aimed to study the development of two F. culmorum strains with a different aggressiveness in the soil under barley, to compare their saprophytic ability and to reveal the influence, if any, of the host plant on the development of strains in the soil and on the roots. Saprophytic development of the strains was studied on membranes inoculated with macroconidia and placed into non-sterile soil under barley of two genotypes. The fungus was identified on the membranes and on barley roots by indirect immunofluorescent method. Both strains could develop saprophytically. The more aggressive strain (Fc538) showed a lesser saprophytic fitness than the less aggressive strain (Fc885): its mycelial density was lower and the number of chlamydospores was greater. Barley genotypes influenced the development of the fungal strains. Interestingly, conditions for saprophytic development of both strains were more favourable in the soil under barley of the resistant genotype. The more aggressive strain colonized barley of the resistant genotype more actively. Rot symptoms appeared earlier in the barley of the resistant genotype, but the number of diseased plants was greater in the barley of the susceptible genotype. The presence of a saprophytic stage in life cycle should slow down the accumulation of aggressive races in field populations of F. culmorum. Possible interactions between F. culmorum strains and barley plants are discussed.