Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides in Ontario isolates of Sclerotinia homoeocarpa

Four hundred and thirty-five isolates of Sclerotinia homoeocarpa from eight populations in southern Ontario were tested for sensitivity to the demethylation-inhibiting (DMI) fungicides, propiconazole, myclobutanil, fenarimol and tebuconazole. The isolates were collected in summer 1994 just prior to legal DMI fungicide use on turfgrass in Ontario. There were wide variations in sensitivities, and seven of the eight populations were very sensitive to the fungicides. Based on mean EC₅₀ and the distribution of DMI sensitivity, one population near the U.S. border was suspected of having been previously exposed to DMI fungicide. Pairwise comparisons of EC₅₀ values for the different fungicides showed low to moderate correlations between fungicides. EC₅₀ values of myclobutanil and propiconazole had the best correlation, followed by the pair of tebuconazole and fenarimol. Other pairwise comparisons were not statistically significant except for a barely significant relationship between EC₅₀ values of myclobutanil and tebuconazole. For field populations of plant pathogens, cross-resistance to different DMI fungicides may not be as strong as conventionally thought. The data collected here will allow comparison to subsequent years to look for detectable shifts in S. homoeocarpa sensitivity to DMI fungicides as they become more frequently used in Ontario.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

Analyses of RAPD data for detection of host specialization in Sclerotinia homoeocarpa

Upgma analysis, principal component analysis, genetic diversity analysis and genetic distance analysis of RAPD data were used to assess the extent of host specialization in 50 isolates of S. homoeocarpa from five turfgrass hosts. In upgma analysis and principal component analysis, the occurrence of host specialization was not readily apparent based on visual inspection. Genetic diversity analysis showed significant differentiation among isolates from different host species ( G _ST = 0.34, P  < 0.001). The strongest evidence for some degree of host specialization came from the statistical analysis of genetic distances among isolates. By grouping pairwise genetic distances between isolates based on their host species, and analysing for average distance within the same host species and among different host species, it was found that the average distance within species was less than among species ( P  < 0.0001). An analysis of molecular variance of the genetic distances among isolates found that 32.3% of the total variation was attributable to host species. It is concluded that these isolates of S. homoeocarpa showed a weak level of host specialization, which was not readily apparent by upgma or principal component analyses, but was revealed by genetic diversity analysis and statistical analysis of genetic distances among isolates. Inoculation tests on different host species and tests using a greater number of isolates are required to confirm the extent of specialization.     

Relative virulence of isolates of Sclerotinia homoeocarpa with varying sensitivity to propiconazole

Isolates of Sclerotinia homoeocarpa were collected from across southern Ontario from areas where demethylation inhibiting fungicides reportedly had never been used. Forty of these isolates with propiconazole EC₅₀ values ranging from 0.003 to 0.069 µg ml^-1 were inoculated onto creeping bentgrass (Agrostis palustris) in summer 1995 and summer 1996 to assess virulence. Each isolate was grown on mixed cereal grains, dried, ground up and applied separately at a rate of 10 g m^-2 to 0.25 m² plots with three replicates per isolate. For both years, no spots were visible on the plots at time of inoculation; however, by the end of each experiment, there were up to 180 spots per plot with significant differences between isolates. Growth rate in culture was not significantly related to fungicide sensitivity (log-EC₅₀ values). Statistically significant negative relationships were found in both years between AUDPC and log-EC₅₀ values. These significant relationships imply that in the absence of propiconazole use, isolates that are more sensitive to propiconazole may out-compete isolates that are less sensitive. However, further study is required to determine the time frame for this to occur, and whether DMI-resistance prevention strategies can feasibly be based on the existence of resistance-related fitness costs.     

Genetic variation among populations of Pythium irregulare in southern Australia

Isolates of Pythium irregulare were sampled from seven cereal crops throughout South Australia to determine the extent of genetic diversity within this pathogen and the scale of genetic differentiation among populations. Data derived from 29 individual restriction fragment length polymorphism (RFLP) loci differentiated 54 DNA fingerprints among the 92 isolates analysed. Some isolates had two alleles at several RFLP loci and were scored as heterozygous. One such isolate was selfed in vitro and segregation ratios in the progeny were not significantly different from those expected for allelic variation in a diploid. These data provided evidence that outcrossing occurs within P. irregulare and may contribute to the high level of genetic variation within the species ( D _T = 0·502). Allelic frequencies were significantly different among all seven populations and G _ST values showed significant genetic differentiation between populations. The average genetic identity among populations was low and hierarchical cluster analysis provided no clear evidence that populations formed geographically related groups. These analyses indicate low levels of interpopulation gene flow within P. irregulare and imply that population differentiation results from genetic drift.     

四川省不同寄主立枯丝核菌的遗传分化和致病力研究

 在四川省生态条件下,从不同水稻和玉米植株上分别分离到来源不同的立枯丝核致病菌15株和7株。致病力、菌丝融合实验结果表明,菌株均属于AG-11A群,各菌株间致病力差异显著。对分离菌株进行RAPD分析,结果显示,相似系数为0.92处菌株可聚合为5类,聚类分组和寄主来源有一定的相关性,来自相同寄主菌株的亲缘关系较近,不同寄主对立枯丝核菌的遗传分化有一定的影响,与病原菌的致病力差异没有直接的相关性。     

Microsatellite DNA variation within and among invasive populations of Ambrosia artemisiifolia from the southern Pannonian Plain

Although the Pannonian Plain presents one of the areas in Europe most highly infested by Ambrosia artemisiifolia, previously reported population genetic studies of the invasive species have included only a few populations from this region. Our goal was to determine the level of genetic diversity and genetic structure of A. artemisiifolia populations from the southern Pannonian Plain using microsatellite DNA markers. We documented a high level of genetic diversity within the Pannonian populations, as compared with the genetic diversity parameters previously published for the other invasive populations of A. artemisiifolia in Europe. The mean number of alleles per locus (N_A) ranged between 6.8 and 9.4, allelic richness (r) between 6.59 and 8.53 and mean number of rare alleles per locus (N_R) ranged between 3.4 and 6.4 per analysed population. A low level of among‐population differentiation was detected, with percentage of variation between populations of 3.3%, generally low pairwise F_ST values (ranged from ?0.007 to 0.152) and also according to principal coordinate analysis. A Bayesian approach revealed that most individuals did not strongly associate with any single genetic cluster, confirming a lack of genetic structuring in the analysed region. Together with the results of quantification of the level of gene flow (N_m = 5.671), these observations indicated a presence of extensive gene exchange and admixture between the populations. Therefore, A. artemisiifolia in the Pannonian Plain region has the potential for rapid expansion.     

Genetic differentiation between hosts and locations in populations of latent Botrytis cinerea in southern England

This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria×ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum officinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154‐bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9/166), and by the rarity (3/77) of a 112‐bp allele at locus 7 that was common (58/166) in isolates from other hosts. There was significant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.     

内蒙古和黑龙江的核盘菌菌丝融合群分化及致病性研究

为明确核盘菌的遗传多样性,对采自内蒙古和黑龙江不同地区的44株核盘菌进行了菌丝融合群确定,并比较了不同菌丝融合群间菌丝生长速度、致病力、草酸和总酸产量的差异。结果表明:供试44个菌株分为25个融合群,其中有14个融合群仅由单一菌株组成,所占比例为56.0%。菌丝融合群内和菌丝融合群间菌丝生长速度、致病力、草酸和总酸产量都表现出显著差异(P0.001),并与菌株的地理来源无关。相关分析表明核盘菌菌株的致病力与菌株草酸产量呈正相关(r=0.484,P≤0.01),与pH呈负相关(r=-0.580,P≤0.01),与菌株的生长速度无关;草酸产量与pH高低(表示总酸的分泌量)负相关(r=-0.392,P≤0.01),进一步表明核盘菌菌株产生的总酸中草酸量占了很大的比例。     

Genetic diversity within and between sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides in Japan

To reveal the effects of herbicide selection on genetic diversity in the outcrossing weed species Schoenoplectus juncoides, six sulfonylurea‐resistant (SU‐R) and eight sulfonylurea‐susceptible (SU‐S) populations were analysed using 40 polymorphic inter‐simple sequence repeat loci. The plants were collected from three widely separated regions: the Tohoku, Kanto and Kyushu districts of Japan. Genetic diversity values (Nei's gene diversity, h) within each SU‐S population ranged from h = 0.125 to h = 0.235. The average genetic diversity within the SU‐S populations was H_S = 0.161, and the total genetic diversity was H_T = 0.271. Although the H_S of the SU‐R populations (0.051) was lower than that of the SU‐S populations, the H_T of the SU‐R populations (0.202) was comparable with that of the SU‐S populations. Most of the genetic variation was found within the region for both the SU‐S and SU‐R populations (88% of the genetic variation respectively). Two of the SU‐R populations showed relatively high genetic diversity (h = 0.117 and 0.161), which were comparable with those of the SU‐S populations. In contrast, the genetic diversity within four SU‐R populations was much lower (from h = 0 to 0.018) than in the SU‐S populations. The results suggest that selection by sulfonylurea herbicides has decreased genetic diversity within some SU‐R populations of S. juncoides. The different level of genetic diversity in the SU‐R populations is most likely due to different levels of inbreeding in the populations.     

Antibiosis and Antagonism of Sclerotinia homoeocarpa and Drechslera poae by Pseudomonas fluorescens Pf-5 In Vitro and In Planta

ABSTRACT Pseudomonas fluorescens strain Pf-5, which produces several antifun-gal metabolites, including the antibiotics pyoluteorin, pyrrolnitrin, and 2,4-diacetylphloroglucinol, was tested for its ability to inhibit Sclerotinia homoeocarpa (causal agent of dollar spot) and Drechslerapoae (causal agent of 'melting-out') in vitro and in turfgrass; Tn5 mutants with altered antibiotic production also were tested. Inhibition in vitro differed with the medium used, but both fungi generally were inhibited by Pf-5. In most cases, a mutant deficient in pyoluteorin but not pyrrolnitrin or 2,4-di-acetylphloroglucinol was as inhibitory as Pf-5, whereas a pyrrolnitrin-deficient mutant was less inhibitory than Pf-5 in most fungus/medium combinations. High-performance liquid chromatography analysis of culture extracts showed that bacterial genotype and nutrition have an interactive effect on antibiotic production, such that conditions causing an increase in one antibiotic may increase or decrease another. The purported deficiencies for the pyrrolnitrin- and pyoluteorin-deficient mutants were confirmed. In S. homoeocarpa-infested grass clippings incubated in a moist chamber, Pf-5 reduced mycelial growth, whereas the pyrrolnitrin-deficient mutant did not and the pyoluteorin-deficient mutant was intermediate. In greenhouse experiments, Pf-5 reduced dollar spot disease incidence in bentgrass and bluegrass when sprayed over inoculated turf. In grass clippings infested with D. poae and incubated in a moist chamber under favorable conditions for spore production, Pf-5 did not reduce significantly the number of spores produced compared with the non-treated control. However, Pf-5 reduced melting-out disease incidence and severity in bluegrass inoculated with spores of D. poae under greenhouse conditions.     

Genetic variation and structure in native and invasive Solidago canadensis populations

Solidago canadensis is native to North America, but has become a noxious invasive plant in China. We know only a little about its invasion history and the effects of introductions on its genetic composition. Here, we investigated genetic variation and structure between 15 North American and 13 Chinese populations of S. canadensis using AFLP makers. Four AFLP loci suggested relatively high genetic diversity of this weed and similar genetic variation between the invasive range and the native range. Most genetic variation was within populations across two ranges, but the Chinese range had a higher degree of among‐population variation than the North American range. Multiple tests, including Bayesian assignment, UPGMA analysis, PCoA and analysis of ‘isolation by distance’, showed that the Chinese populations originated from at least two distinct native sources and that secondary introduction or dispersal should be common in China. Also, North American populations were possibly a single genetic group. Overall, S. canadensis in China was probably founded from multiple introductions and then spread through long‐distance dispersal associated with human activities. Genetic variability in the species in the invaded range appears to have favoured establishment and spread and may well provide a challenge to successful control.     

Attenuation of Virulence in Sclerotinia homoeocarpa during Storage is Associated with Latent Infection by Ophiostoma mitovirus 3a

The hypovirulence-associated mitovirus, Ophiostoma mitovirus 3a (OMV3a), has been shown to be widespread in eastern Canadian populations of Sclerotinia homoeocarpa in the form of latent infection. Latent infection by OMV3a was not associated with an apparent phenotype and did not significantly reduce the growth and virulence of the pathogen. In the present study, we found that isolates of S. homoeocarpa latently infected by OMV3a can change to hypovirulent isolates after storage at 4 °C, and that this attenuation of virulence was associated with increased concentration of the OMV3a virus. Recurrent observations revealed that up to 29.8% of latently infected isolates changed to hypovirulent isolates after 21 months of storage. Transmission of OMV3a dsRNA from latently infected isolates to virus-free isolates resulted in latent infection of the recipient isolates, indicating that latent infection by OMV3a was not associated with genetic differences in the fungal host. The RNA genomes of the OMV3a virus in an isogenic pair of latently infected and hypovirulent isolates were sequenced and compared. Each of the two RNAs contained an open reading frame of 726 amino acids with conserved motifs typical of RNA-dependent RNA polymerase (RdRp). The OMV3a RNA sequences in these two isolates share 95.1% nucleotide and 94.6% amino acid sequence identities. The development of hypovirulence from latent infection by OMV3a virus may provide new strategies to improve the biological control efficacy of hypovirulence in dollar spot management.     

Potential biocontrol of Sclerotinia homoeocarpa and Bipolaris sorokiniana on the phylloplane of Poa pratensis with strains of Pseudomonas spp.

Four Pseudomonas strains were evaluated for their ability to control Sclerotinia homoeocarpa and Bipolaris sorokiniana on the phylloplane of Poa pratensis (Kentucky bluegrass). The strains evaluated were Pseudomonas fluorescens (PSD-4, PSD-5, PSD-6) and Pseudomonas lindbergii (PSD-42). All evaluations were conducted on the upper epidermis of intact attached leaves of Poa pratensis and antagonism was measured as the ability of strains of Pseudomonas to prevent chlorophyll loss from leaves inoculated with either S. homoeocarpa or B. sorokiniana. All strains prevented infection and loss of chlorophyll following inoculation with S. homoeocarpa. Only P. lindbergii (PSD-42) was antagonistic to B. sorokiniana. None of the P. fluorescens strains controlled disease induced by B. sorokiniana.     

Genetic variation and variation in aggressiveness to native and exotic hosts among Brazilian populations of Ceratocystis fimbriata

Ceratocystis fimbriata is a complex of many species that cause wilt and cankers on woody plants and rot of storage roots or corms of many economically important crops worldwide. In Brazil, C. fimbriata infects different cultivated crop plants that are not native to Brazil, including Gmelina arborea, Eucalyptus spp., Mangifera indica (mango), Ficus carica (fig), and Colocasia esculenta (inhame). Phylogenetic analyses and inoculation studies were performed to test the hypothesis that there are host-specialized lineages of C. fimbriata in Brazil. The internal transcribed spacer region ribosomal DNA sequences varied greatly but there was little resolution of lineages based on these sequences. A portion of the MAT1-2 mating type gene showed less variation, and this variation corresponded more closely with host of origin. However, mango isolates were found scattered throughout the tree. Inoculation experiments on the five exotic hosts showed substantial variation in aggressiveness within and among pathogen populations. Native hosts from the same families as the exotic hosts tended to be less susceptible than the cultivated hosts, but there was little correlation between aggressiveness to the cultivated and native hosts of the same family. Cultivation and vegetative propagation of exotic crops may select for strains that are particularly aggressive on those crops.     

Genetic variation among asexual progeny of Phytophthora infestans detected with RAPD and AFLP markers

Genotypic variation among 32 single-zoospore isolates (SZI) of Phytophthora infestans , derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA (AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates. Polymorphism was detected with 51 RAPD primers and with all 18 AFLP primer pairs in PI-105 SZIs. In SZIs from PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six AFLP groups. No close correlation among RAPD, AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.     

Genetic variation and phylogenetic relationships of triazine-resistant and triazine-susceptible biotypes of Solanum nigrum– analysis using RAPD markers

The expansion of weed species is a major problem in agriculture, especially when the number of herbicide-resistant biotypes is rising continuously. The major ecological questions associated with the evolution of herbicide resistance involve an intricate understanding of the interplay between gene frequency, fitness, inheritance and gene flow. In this study, the RAPD (Random Amplified Polymorphic DNA) technique, which facilitates detection of variability at DNA level, was used to examine the spread of Solanum nigrum L. populations. Twenty-five populations, from Poland, France and the UK, were analysed. Six populations from Poland and one from France showed target site-based triazine resistance. The genetic relationship between individuals was studied using the RAPD technique. It was found that some resistant populations from the Gabin and Grojec areas show very high affinity levels compared with individuals from France. Three groups of populations in which resistance had developed independently were distinguished. The results of the present investigation suggest that migratory birds, such as Turdus pilaris L. and Sturnus vulgaris L., play an important in spreading S. nigrum seeds.     

Genetic diversity in populations of the fungi Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on grapevine in France

Isolates of Phaeomoniella chlamydospora ( Phc ) and Phaeoacremonium aleophilum ( Pha ), two haploid, deuteromycetous fungi, were obtained from vines showing symptoms of esca disease in different localities in two French regions, and within a single vineyard in one of these regions. The population genetic structure was determined in both fungi using random amplified polymorphic DNA (RAPD) analysis. Populations of Phc showed similar levels of diversity at local and regional levels. The most frequent Phc haplotypes were found in every population, and the frequencies of positive alleles of markers were similar across populations. The hypothesis that recombination had occurred was rejected for the full set of samples, but not for the samples reduced to haplotypes, indicating that Phc may be a recombining species. Different features were identified in Pha populations. First, the southern population of Pha appeared more diverse than the south-western populations. Second, genetic differentiation was identified between Pha populations from southern and south-western regions for several RAPDs. Finally, in the southern population of Pha no evidence for recombination was obtained, even by reducing the sample to haplotypes. Within the single vineyard surveyed, several haplotypes of both fungi were recovered and randomly distributed. Thus different infection events appeared to have occurred on a low spatial scale. Data from this study showed that haplotypes of both fungi were distributed over long distances geographically, and that most of the vineyards surveyed were infested by more than one haplotype of Phc and Pha .     

Genetic variation in populations of the cacao wilt pathogen, Ceratocystis cacaofunesta

Ceratocystis cacaofunesta (=  Ceratocystis fimbriata ) causes a lethal wilt disease of cacao ( Theobroma cacao ) in Latin America. Polymorphic microsatellite markers, (CAT)₅ nuclear DNA fingerprints and Hae III mitochondrial DNA fingerprints were used to compare genetic diversity among isolates of C. cacaofunesta collected from populations in western Ecuador, Costa Rica, Colombia, and Rondônia and Bahia in Brazil. Microsatellite markers and nuclear DNA fingerprints separated Ecuadorian isolates from isolates of the other four populations, and these two major groups correspond to genetic lineages already identified from ITS-rDNA sequences and intersterility groupings. Mitochondrial DNA fingerprints also demonstrated substantial diversity and split the Ecuadorian isolates into two groups. All marker types showed limited variation in the Colombian, Costa Rican and Bahian populations, as might be expected for introduced populations that have gone through recent genetic bottlenecks. In contrast, the Rondonian and western Ecuadorian populations showed gene diversity values similar to natural populations of other Ceratocystis species. The Rondonian population was the only sampled population in the native range of T. cacao (the Upper Amazon), and the putatively introduced populations were more closely related to the Rondonian population than to the western Ecuadorian population. The Ecuadorian population is in an area with other native Theobroma species, which may serve as natural hosts.     

Assessment of biological and molecular variability between and within field isolates of Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate biotroph that causes clubroot, one of the most damaging diseases of crucifers. Differential cultivars and random amplified polymorphic DNA markers were used to assess the extent of genetic diversity among nine single-gall populations of P. brassicae and 37 single-spore isolates (SSI) derived from four of those field samples. Isolates were classified into eight pathotypes, and each isolate was associated with a unique molecular genotype. Virulence and DNA polymorphisms were detected within and between field isolates, and among SSIs from different pathotypes, hosts and geographical origins. The relatively high level of genetic diversity among field isolates was similar to that among SSIs derived from a single-club field isolate. Molecular and pathogenicity-based classifications were not clearly correlated, but isolates belonging to pathotype P1 were clustered. Two RAPD markers were specific to pathotype P1. The finding that genetic differences can occur in P. brassicae field isolates will be an important consideration in resistance genetic studies and in choosing breeding strategies to develop durable clubroot resistance.