Virulence characteristics of apple scab (Venturia inaequalis) isolates from monoculture and mixed orchards

Isolates of Venturia inaequalis were sampled from monoculture and mixed orchards of three apple cultivars: Bramley, Cox and Worcester. In addition, single-ascospore progeny isolates were obtained from three crosses between pairs of isolates originating from the three cultivars in monoculture orchards. These field isolates and single-ascospore progenies were inoculated onto each of the three cultivars in a glasshouse. The patterns of infection showed that all three cultivars, commonly regarded as susceptible to scab, contained some specific resistance factors and that scab isolates from both mixed and monoculture orchards appeared to have co-evolved with cultivars. A much higher proportion of isolates from cv. Worcester in the mixed orchard were unable to infect any of the three cultivars than isolates from any other combination of cultivar and orchard type, but there was no other difference between isolates from mixed vs. monoculture orchards. Many isolates could infect both Bramley and Cox, or Cox and Worcester; but only a single isolate could infect both Bramley and Worcester. Results from the testing of 61 single-ascospore progeny isolates suggested that virulence towards Bramley, Cox and Worcester was controlled by at least one, two and three factors, respectively. Moreover, the exact nature of the interactions between these factors in determining virulence depended on the particular pair of isolates concerned.     

Differentiation in populations of the apple scab fungus Venturia inaequalis on cultivars in a mixed orchard remain over time

The ascomycete Venturia inaequalis causes annual epidemics of apple scab worldwide. Scab development is reduced in mixed cultivar orchards compared with monocultures. In order to use mixtures in commercial production, how the population of scab changes in a mixed orchard needs to be understood, together with how likely a super race, with virulence factors overcoming multiple resistance factors in the mixed orchard, is to emerge and become dominant. This study used simple sequence repeat (SSR) markers to investigate the temporal change of scab populations in two mixed cultivar orchards in the UK to infer the likelihood of emergence of a scab super race. There were no significant differences between the populations at the two sampling times (6 or 7 years apart) in either of the two mixed orchards. In one of the orchards, apple scab populations on different cultivars were significantly different and the differences did not diminish over time. These results suggest that it is not inevitable that a super race of V. inaequalis will become dominant during the lifetime of a commercial apple orchard.     

Within‐ and between‐orchard variability in the sensitivity of Venturia inaequalis to myclobutanil,a DMI fungicide,in the UK

BACKGROUND: Myclobutanil, a demethylation inhibitor (DMI) fungicide, is an important fungicide for controlling apple scab and powdery mildew. Overuse of this fungicide has led to establishment of scab isolates with reduced sensitivity to this fungicide in several countries. Experiments were conducted to determine the sensitivity of the causal agent of apple scab, Venturia inaequalis (Cooke) Winter, to myclobutanil in the UK, in order to assess whether there is a relationship between fungal insensitivity and the number of DMI applications, and establishing whether fungal sensitivity varied greatly within an orchard. RESULTS: Reduced sensitivity of V. inaequalis to myclobutanil was positively related linearly to the number of DMI applications. ED₅₀ values ranged from 0.028 to 1.017 mg L^?1 (average = 0.292) for the baseline population, whereas isolates from two other orchards had much greater ED₅₀ values, ranging from 0.085 to 5.213 mg L^?1 (average = 1.852). There was significant variation in fungal sensitivity to myclobutanil among fungal isolates from different locations within a single orchard. CONCLUSIONS: Spatial spread of insensitive isolates of V. inaequalis to myclobutanil is likely to be limited in distance. Conidia may be an important source of primary inoculum. Myclobutanil should still be effective for most field isolates, but its use should be strategically integrated with other groups of fungicides. Copyright © 2009 Society of Chemical Industry     

The threat of wild habitat to scab resistant apple cultivars

Evaluations of plant resistance to pathogens are rarely made using isolates from wild habitats, although the heterogeneity of such habitats may generate pathogen diversity which could be a source of new virulence in cultivated habitats. The aim of this study was to investigate whether scab resistance factors, identified and characterized in apples using isolates of Venturia inaequalis from a cultivated habitat, remained effective against isolates from a wild habitat. Three V. inaequalis core collections originating from the cultivated apple Malus × domestica and from two wild species, M. sieversii and M. sylvestris, were established to maximize pathogen diversity. For each core collection, 10 isolates were inoculated in mixtures onto 51 genotypes from an apple progeny segregating for two qualitative resistance genes and six quantitative resistance loci (QRL). On each apple genotype, isolates that contributed to the scab symptoms were identified within the mixture using microsatellite markers. The most frequently detected isolates were inoculated singly to compare their aggressiveness according to their host origin. The results showed that isolates from a wild habitat were able to infect the susceptible apple genotypes. However, these isolates were never more aggressive than isolates from the cultivated habitat on the resistance factors tested. It can therefore be concluded that the resistance factors used in this study, identified with V. inaequalis isolates from a cultivated habitat, remained effective against isolates from M. sylvestris and M. sieversii.     

The relative importance of conidia and ascospores as primary inoculum of Venturia inaequalis in a southeast England orchard

Apple scab, caused by Venturia inaequalis, can lead to large losses of marketable fruit if left uncontrolled. The disease appears in orchards during spring as lesions on leaves. These primary lesions are caused by spores released at bud burst from overwintering sources; these spores can be sexually produced ascospores from the leaf litter or asexual conidia from mycelium in wood scab or within buds. The relative importance of conidia and ascospores as primary inoculum were investigated in an orchard in southeast England, UK. Potted trees not previously exposed to apple scab were placed next to (c. 1 m) orchard trees to trap air‐dispersed ascospores. Number and position of scab lesions were assessed on the leaves of shoots from both the potted trees (infection by airborne ascospores) and neighbouring orchard trees (infection by both ascospores and splash‐dispersed, overwintered conidia). The distribution and population similarity of scab lesions were compared in the two tree types by molecular analysis and through modelling of scab incidence and count data. Molecular analysis was inconclusive. Statistical modelling of results suggested that conidia may have contributed approximately 20–50% of the primary inoculum in early spring within this orchard: incidence was estimated to be reduced by 20% on potted trees, and lesion number by 50%. These results indicate that, although conidia are still a minority contributor to primary inoculum, their contribution in this orchard is sufficient to require current management to be reviewed. This might also be true of other orchards with a similar climate.     

Negatively correlated cross-resistance to phenylcarbamate fungicides in benomyl-resistant Venturia inaequalis and V. pirina

Isolates ofVenturia inaequalis and ofV. pirina sensitive (S) or resistant (R) to benomyl were examined in vitro on media amended with two phenylcarbamate fungicides. There was a negatively correlated cross-resistance (NCCR) to both methyl N-(3,5-dichlorophenyl) carbamate (MDPC) and isopropyl N-(3,4-diethoxyphenyl) carbamate (NPC) in some benomylresistant isolates. InV. inaequalis, isolates with low benomyl resistance (LR) did not show NCCR to MDPC, whereas isolates with medium (MR), high (HR) and very high (VHR) resistance to benomyl were more sensitive to MDPC than were the benomyl-sensitive isolates. To NPC, MR and VHR isolates showed NCCR whereas LR and HR isolates reacted similarly as sensitive isolates. InV. pirina only HR and VHR isolates showed NCCR to MDPC. The VHR isolates were sensitive to NPC, whereas the reactions of S, LR, MR and HR to NPC were similar.Crosses between benomyl-sensitive and benomyl-resistantV. pirina as well as between different resistant isolates showed that NCCR is inheritable and controlled by a single Mendelian gene.Samenvatting Benomyl-gevoelige en-resistente isolaten vanVenturia inaequalis enV. pirina werden in vitro onderzocht op media met de fungiciden methyl N-(3,5-dichlorophenyl) carbamate (MDPC) en isopropyl N-(3,4-dichlorophenyl) carbamate (NPC). Een aantal benomyl-resistente isolaten van deze pathogeen bleken een negatief gecorreleerde kruisresistentie (NCCR) te vertonen ten opzichte van MDPC en NPC.Isolaten vanV. inaequalis met matige (MR), hoge (HR) en zeer hoge (VHR) benomyl-resistentie vertoonden NCCR. Ten opzichte van NPC vertoonden alleen MR en VHR isolaten NCCR, en niet de LR en HR isolaten. InV. pirina vertoonden HR en VHR isolaten NCCR ten opzichte van MDPC, maar alleen de VHR isolaten ten opzichte van NPC.Kruisingen tussen benomyl-gevoelige en-resistenteV. pririna, en tussen verschillende benomyl-resistente isolaten onderling, toonden aan dat NCCR erfelijk is en berust op een enkel gen.Contribution No. 1712-E, 1986 series, from the ARO.     

Aggressiveness of eight Venturia inaequalis isolates virulent or avirulent to the major resistance gene Rvi6 on a non‐Rvi6 apple cultivar

For sustainable management of scab‐resistant apple cultivars, it is necessary to understand the role of aggressiveness in the adaptation of Venturia inaequalis populations and particularly the costs to the organism of acquiring additional virulence. The aims of the present study were (i) to identify the quantitative variables that are most important in determining the differences in aggressiveness among groups of V. inaequalis isolates, and (ii) to ascertain whether virulent and avirulent isolates of V. inaequalis differ significantly in aggressiveness. The aggressiveness of eight isolates that differed in their virulence to the major resistance gene Rvi6 was compared on the non‐Rvi6 apple cv. Gala. Three components of aggressiveness, namely lesion density, the number of spores per square centimetre of leaf area, and the number of spores per lesion, were evaluated 21 days after inoculation, and the kinetics of lesion density over time were analysed in terms of maximum lesion density, length of latent period and rate of lesion appearance. On the second youngest but fully developed leaf at the time of inoculation, maximum lesion density in the virulent group was 20% lower and the latent period 7% longer, than in the avirulent group. However, the alternative hypothesis, namely that isolates had adapted to quantitative resistance present in cv. Gala depending on their cultivar of origin, could not be rejected. The analysis of the kinetics of lesion density by a non‐linear mixed‐effect model proved useful in the assessment of aggressiveness.     

Selection and orchard testing of antagonists suppressing conidial production by the apple scab pathogen <Emphasis Type="Italic">Venturia inaequalis</Emphasis>

Apple scab caused by Venturia inaequalis is a major disease in apple production. Epidemics in spring are initiated by ascospores produced on overwintering leaves whereas epidemics during summer are driven by conidia produced on apple leaves by biotrophic mycelium. Fungal colonisers of sporulating colonies of V. inaequalis were isolated and their potential to reduce the production of conidia of V. inaequalis was evaluated on apple seedlings under controlled conditions. The four most effective isolates of the 63 screened isolates were tested subsequently under Dutch orchard conditions in 2006. Repeated applications of conidial suspensions of Cladosporium cladosporioides H39 resulted in an average reduction of conidial production by V. inaequalis of approximately 40%. In 2007, applications of conidial suspensions of C. cladosporioides H39 reduced conidial production by V. inaequalis by 69% on August 6 and by 51% on August 16, but no effect was found on August 20. However, viability of available conidia of C. cladosporioides H39 was low at the end of the experiment. Epiphytic and endophytic colonisation by Cladosporium spp. of leaves treated during the experiment with C. cladosporioides H39 was significantly higher than on control leaves sampled 6 weeks after the last application. It is concluded that C. cladosporioides H39 has promising potential as a biological control agent for apple scab control. More information is needed on the effect of C. cladosporioides H39 on apple scab epidemics as well as on mass production, formulation and shelf life of conidia of the antagonist.     

Population variation of apple scab (Venturia inaequalis) within mixed orchards in the UK

Apple scab, caused by Venturia inaequalis, is one of the most important apple diseases worldwide. To investigate between- and within-orchard fungal variability, 212 isolates were sampled from two mixed orchards, one of 10?years of age and the other of 45?years of age, in the UK and genotyped with AFLP and SSR markers. Populations of isolates from the two orchards did not differ significantly in terms of allele frequencies at the screened AFLP and SSR loci. However, groups of isolates from individual cultivars differed significantly within each orchard and there were also significant differences between groups of isolates from individual trees of the same cultivar in the same orchard. These differences were less pronounced in the younger mixed orchard than in the older one. The existence of tree-to-tree fungal variability indicates a possible role for conidia as a source of primary inoculum. Non-random mating may be one of the factors causing the significant differences among fungal populations from different cultivars. These results suggest that apparently ??susceptible?? cultivars have different background genetic resistance factors, which can be exploited for disease management in mixtures.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia

Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens‐like isolates in clade 1, A. tenuissima/A. mali isolates in clade 2, A. alternata/A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes‐like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens‐like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata/A. tenuissima intermediate isolates (14%) and A. tenuissima/A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed.     

A method to monitor airborne Venturia inaequalis ascospores using volumetric spore traps and quantitative PCR

Apple scab caused by the fungus Venturia inaequalis can result in significant crop losses if not managed effectively. Sanitation as part of an integrated management strategy aims to significantly reduce primary inoculum to lower the disease pressure. This study evaluates the possibility of molecular detection and quantification of ascospore discharge and the use of this method to test for efficacy of orchard sanitation treatments. A method to detect and quantify airborne ascospores was developed using volumetric spore traps (VSTs). V. inaequalis specific primers were tested on daily VST samples from two orchard sections (leaf litter removed compared to leaf litter left) during spring. A molecular method to detect and quantify ascospores was tested by amplifying genomic regions of the mitochondrial CYP51A1 gene, and the ITS region using SYBR® green. Timing of ascospore discharge was compared to predicted infection risk and a degree day model using weather data. The average spore detection limit was estimated to be at levels of 1 pg μl^?1 DNA (approximately 37 ascospores) per daily spore trap reading using CYP51A1 primers. Using the CYP51A1 primer pair, primary inoculum was estimated to be 51 % lower in the orchard sections where leaves had been removed, indicating that this method could be used to evaluate the efficacy of alternative control strategies such as leaf removal to reduce potential ascospore dose. This is the first report of combining VSTs and quantitative PCR to monitor airborne V. inaequalis ascospores.     

Effect of six sanitation treatments on leaf litter density, ascospore production of Venturia inaequalis and scab incidence in integrated and organic apple orchards

A two-year study was conducted to determine the effect of six sanitation treatments on leaf litter density (LLD), relative ascospore production of Venturia inaequalis and scab incidence on spur-leaf clusters, leaves and harvested fruits, on two cultivars with low and high scab susceptibilities, in Hungarian integrated and organic apple orchards. The following sanitation treatments were used: sprays of lime sulphur in autumn, collecting fallen leaves in autumn, straw mulch cover in late winter, sprays of lime sulphur followed by mulch cover, collecting fallen leaves followed by mulch cover, collecting fallen leaves followed by covering the orchard floor with plastic foil, and non-sanitized control. LLD decreased continuously in all treatment plots by 0–23% by mid-May in both orchards and years; however, LLD reduction was 1.4–4.2 times higher in the organic orchard compared to the integrated one. All treatments, except for the lime sulphur treatment, resulted in significant (P < 0.05) reduction of LLD and ascospore production in both the integrated and organic apple orchards compared to non-sanitized plots. The most efficient treatment was leaf collection combined with plastic foil cover, followed by leaf collection combined with mulch cover, leaf collection alone, mulch cover alone, and lime sulphur spray combined with mulch cover, with a reduction in the ascospore production of >95, 72–92, 56–79, 24–38, and 27–46%, respectively, in the mean of both orchards and years. However, only treatments of leaf collection applied alone, or in combination with mulch or with plastic foil cover reduced significantly (P < 0.05) leaf and/or fruit scab incidence by 18–57% compared to non-sanitized plots. These three leaf collection treatments are recommended in both integrated and organic orchards and the possibilities of successfully incorporating these methods into orchard management practices are interpreted.     

Sensitivity to strobilurin fungicides of Italian Venturia inaequalis populations with different origin and scab control

BACKGROUND: Venturia inaequalis (Cooke) Winter with reduced sensitivity to strobilurins has been reported in several countries, including Italy. This study aimed to characterise the sensitivity to strobilurins of three different types of V. inaequalis population: (a) wild types; (b) from commercial orchards satisfactorily managed with strobilurins; (c) from an experimental orchard with control failures by trifloxystrobin and kresoxim‐methyl. In vitro sensitivity tests included antigerminative activity on population conidia and mycelial growth inhibition on monoconidial isolates. Cleaved amplified polymorphic sequence (CAPS) analysis was used for the detection of G143A substitution. RESULTS: Wild‐type populations showed EC₅₀ values lower than 0.031 mg L^?1, while those of orchards with good performance by strobilurins presented EC₅₀ values never higher than 0.063 mg L^?1. Samples with scab control failures showed a strongly reduced population sensitivity. Similar differences were confirmed in monoconidial isolates. The G143A substitution was always detected in low‐sensitivity populations, only sometimes in well‐controlled populations and generally not in wild types. CONCLUSIONS: In vitro sensitivity assays were able to discriminate the three population types with different scab management, while the qualitative PCR analysis (CAPS) was only partially reliable. High sensitivity differences among V. inaequalis populations with good and poor field control by strobilurins were observed. Copyright © 2011 Society of Chemical Industry     

Genetic diversity and pathogenicity of Sphaceloma rosarum (teleomorph Elsinoë rosarum) causing spot anthracnose on roses

Roses produced or grown in the field, as well as pot‐grown and cut roses, are attacked by different fungal pathogens causing leaf spot diseases. The incorrect identification and scoring of these pathogens and the lack of information about their genetic and pathotype diversity hamper resistance breeding. This is especially true for the hemibiotrophic ascomycete Sphaceloma rosarum, which is often confused with other fungi. Here for the first time, the genetic variability between isolates at both the molecular and morphological level is analysed. Eighty leaf spot samples were collected from different rose genotypes at five different locations, and 15 single conidial isolates established. All of the samples showed high morphological similarities to the reference isolate CBS 213.33 that was obtained from a public repository. By sequencing a part of the large subunit (LSU) of the 28S ribosomal RNA and phylogenetic analysis, high sequence similarities were shown to other Sphaceloma species for 13 of the isolates and the CBS reference. One of the isolates clustered with Septoria species and another clustered with Seimatosporium species. UPGMA clustering with 145 polymorphic AFLP markers resulted in five distinct groups in the majority rule consensus tree for the 14 S. rosarum isolates, including the CBS reference. Jaccard similarities ranged from 0·31 to 0·91. A detached leaf assay using a differential set of five rose genotypes led to the classification of the five tested isolates as five distinct pathotypes. Therefore, grouping depending on the avirulence gene diversity was clearly different from clustering using selectively neutral AFLP markers that were evenly distributed throughout the genome.     

Microsatellite primers indicate the presence of asexual populations of<Emphasis Type="Italic">Venturia inaequalis</Emphasis> in coastal Israeli apple orchards

This study was initiated to determine whether differences in genotypic diversity among populations ofVenturia inaequalis (Cke.) Wint., as detected using neutral genetic markers, were related to the ecological conditions in which apples are grown in Israel. Since sexual reproduction in this fungal pathogen has an obligate requirement for sustained low winter temperatures, and since these requirements in Israel are met only on the Golan Heights, we were interested in whether lower elevation populations of this pathogen might be comprised of asexual clonal lineages. Unlike temperate apple growing regions, where the primary spring inoculum is ascosporic derived from overwintered pseudothecia, Israeli apple orchards at lower elevations in the Hula Valley and along the coastal plain rarely if ever experience low winter temperatures and pseudothecia have never been recovered. Two orchards were sampled from the Golan Heights (El Rom and Ortal, n=38) and three orchards from the Hula Valley and coastal plain (Sede Eliezer, Ginaton and Be’er Tuvia, n=40). Microsatellite primers were used to analyze population structure and the resulting binary data analyzed by both cluster and parsimony analysis. Populations from the coastal plain were genetically uniform within each of the orchards sampled, whereas populations from the Golan Heights showed levels of genotypic diversity ten times as high. The data support field observations that this pathogen does not reproduce sexually in regions characterized by the absence of low winter temperatures and is instead composed of clonal lineages. This may have bearing on control strategies for the disease in Israel. http://www.phytoparasitica.org posting April 21, 2003.     

Pseudomonas syringae pv. syringae from cool climate Australian grapevine vineyards: new phylogroup PG02f associated with bacterial inflorescence rot

During the period 2006–2011, Pseudomonas syringae pv. syringae caused a bacterial inflorescence rot (BIR) epidemic in an Australian cool climate viticultural region. Molecular multilocus sequence typing of ‘housekeeping’ genes (MLST), biochemical testing and analysis of molecular variance (AMOVA) were used to characterize the genotypes and phenotypes of P. syringae pv. syringae grapevine isolates. Comparison of the MLST data with exemplars of phylogroups available at PAMDB demonstrated that the BIR isolates formed a new clade within P. syringae pv. syringae phylogroup 2 (PG02): putatively designated PG02f. Analysis of the MLST and phenotypic data by AMOVA demonstrated some genetic differences between the BIR isolates and the general vineyard P. syringae pv. syringae population. Isolates positive for syringopeptin, syringomycin and tyrosinase, tobacco leaf hypersensitivity reaction (HR), ampicillin resistance and grapevine leaf pathogenicity were genetically distinct from those negative for these factors. This study has shown that, generally, the core genome of P. syringae pv. syringae is only weakly associated with the virulence-associated traits. As the new phylogroup PG02f consists of the epidemic BIR isolates and nonpathogenic grapevine isolates, these genetically similar isolates differ greatly in pathogenicity and most of the other tested phenotypic traits. However, within the PG02f group, tobacco leaf HR and presence of sylC (the gene for phytotoxin syringolin A) are associated with the BIR and bacterial leaf spot (BLS) isolates, and negative for the nonpathogens, indicating that these two virulence factors may be associated with vineyard pathogenicity within the new Australian phylogroup.     

Development of a qPCR test to detect and quantify Elsinoë piri in unsprayed and organic apple orchards and assessment of apple cultivar susceptibility to the disease

Recently, Elsinoë piri has become an important pathogen in unsprayed and organic apple orchards. The increasing demand for organic apples as well as the limited use of fungicides imposed by European legislation has turned this pathogen, which has been known for over a century in Europe, into a resurgent phytosanitary threat. It also represents a new trait to be taken into account in apple breeding programmes. As E. piri is extremely difficult to isolate and causes symptoms that can be confused with those caused by other pathogens, a quantitative PCR (qPCR) test has been developed based on the internal transcribed spacer (ITS) region of the rDNA gene. This test, which displays a low limit of detection, allows the early detection of the fungus in apple leaves. As a quantitative method, it is a promising tool for breeding purposes as it can be used on leaves to assess the level of quantitative resistance of apple cultivars to the disease. The molecular test is also useful to gain knowledge on the epidemiology of this re-emerging fungal disease. Spore trapping conducted in 2015, 2016 and 2020 using Rotorod devices and the qPCR test showed that airborne inoculum is released at the end of the growing season. Tests were also carried out to demonstrate that the pathogen was detected in buds during winter. A comparison of 18 E. piri isolates based on the sequencing of five genome regions highlighted a high genotypic diversity. All these isolates were detected with the qPCR test developed in this study.     

Diversity of kresoxim-methyl sensitivities in baseline populations of Venturia inaequalis

Several strobilurin fungicides inhibiting fungal respiration by binding to cytochrome b have been introduced recently. A mechanism of strobilurin resistance identified as active in several plant pathogenic fungi is based on the activation of alternative respiration. Thus far, respective studies have been restricted to single isolates of respective pathogens. Here, we report a study on 250 Venturia inaequalis baseline isolates to the strobilurin kresoxim-methyl having a broad sensitivity distribution characterized by a 50-fold difference in sensitivity of the most- and least-sensitive isolates. For the majority (62%) of these isolates, differences in sensitivity were not caused by the interference of alternative respiration with the full inhibitory potency of kresoxim-methyl. Rather, variable dose-responses with largely different degrees of inhibition achieved at a low dose of kresoxim-methyl were found to be responsible. For 38% of the baseline isolates, alternative respiration was already active during the stage of conidia germination. Activation of this pathway was, again, dependent on the strobilurin dose. Selection of sub-populations of isolates resisting low doses of kresoxim-methyl by multiple mechanisms and the recombination among isolates expressing such mechanisms singly can be expected to be slowed by an anti-resistance strategy based on high strobilurin doses. © 1999 Society of Chemical Industry     

Development,characterization and application of genomic SSR markers for the oat stem rust pathogen Puccinia graminis f. sp. avenae

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. In this study, an Australian Pga isolate was sequenced, the abundance of simple sequence repeats (SSRs) was determined and PCR‐based polymorphic markers suitable for genetic diversity analysis were developed. The amplification of 194 primer pairs was initially assessed using a set of 12 isolates of different cereal rust species and their formae speciales. A high frequency of cross‐species amplification was observed for most markers; however, 36 SSRs were diagnostic for P. graminis only. A subset of 19 genome‐derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates, previously shown to undergo sexual recombination, were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two subpopulations. The genome‐derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species.