Sensitivity of Venturia inaequalis to trifloxystrobin

The sensitivity of Venturia inaequalis to trifloxystrobin was monitored by both in vitro (spore germination) and in vivo tests (on apple seedlings). There was good correlation between the in vitro and in vivo results. Baseline sensitivity was established with 95 bulk isolates from Europe between 1995 and 1998 which showed a narrow sensitivity range-factor of up to 17 between the least and most sensitive isolates. Monitoring of populations originating from trifloxystrobin-treated and untreated orchards between 1995 and 1999 showed only sensitive isolates and no performance deficiencies were reported. Data also show that trifloxystrobin is not cross-resistant to anilinopyrimidines and triazoles, but is cross-resistant to kresoxim-methyl, another strobilurin which also inhibits the cytochrome bc1 enzyme complex at the Qo-site in the respiration chain of fungal mitochondria (QoI inhibitors). Under experimental conditions from a specific trial site in Switzerland, where trifloxystrobin has been tested since 1994, isolates containing conidia resistant to Qo inhibitors were detected in 1997 and 1999. However no obvious performance deficiencies were reported. Nevertheless these results demonstrate a risk for the development of practical resistance in V inaequalis to Qo inhibitors and therefore a need to apply resistance management principles strictly.     

Characterization of Venturia inaequalis pathogenicity on leaf discs of apple trees

Characterization of pathogenicity on whole plants is required to study host-pathogen interactions between Malus × domestica and Venturia inaequalis. We studied the reliability of an in vitro test of pathogenicity on leaf discs. Three strains of V. inaequalis (races 1, 6 and an English race) were inoculated in vitro onto a range of 16 Malus sp. clones including susceptible and resistant clones. The results were compared to those previously obtained in vivo. Resistant clones contained the main major known genes, i.e. Va, Vb, Vbj, Vf, Vg, Vm and Vr. Scab severity and the sporulation of the fungus were assessed 21 days after the inoculation date. The results indicated that it was possible to reproduce incompatible and compatible situations in vitro. A null severity corresponded to the avirulence of the strain for the clone considered. The resistance given by the Vb, Vbj, Vf, Vg, Vm and Vr genes were expressed in vitro. Only the clone carrying the Va gene and inoculated with the race 6 strain presented a compatible situation which was inconsistent with the observations on the whole plant. Improving this test will facilitate studies on the pathogenicity of V. inaequalis populations in relation to resistance genes of the host expressed in vitro as well as its genetic determinism.     

Germinability of Venturia inaequalis Conidia Exposed to Sunlight

ABSTRACT Detached conidia of Venturia inaequalis were exposed simultaneously outdoors to direct sunlight or in an adjacent ventilated enclosure in darkness for periods ranging from 0.5 to 16.5 h. In addition, conidia were either exposed or not exposed to UV light (254 nm) from 1 to 64 min in the laboratory. After exposure, conidia were placed on water agar in closed petri dishes and allowed 24 h to germinate. Germinability of conidia in the laboratory was reduced up to 95% by doses of UV(254) of 8.1 to 10.8 kJ m(-2).Germinability of conidia in the field was reduced up to 95% by doses of UV-B of about 44 kJ m(-2). A significant percentage of conidia survived radiation doses equivalent to 12 h in full sunlight.     

Genetics of Host-Pathogen Relationships Between Venturia inaequalis Races 6 and 7 and Malus Species

ABSTRACT Resistance to scab originating from Malus floribunda clone 821 is the most widely form of resistance used in apple breeding programs. A dominant gene, named Vf, was introgressed from this clone into recent cultivars, although the genetic determinants of the resistance of M. floribunda 821 are apparently more complex than a single gene. The appearance of new races overcoming the resistance of cultivars with the Vf gene, the parental clone, or both made it possible to undertake a genetic analysis of host-pathogen interactions. The segregation of resistance in progenies of crosses from 'Golden Delicious' x M. floribunda 821 and 'Golden Delicious' x 'Idared' into five strains of Venturia inaequalis-races 1 (strains 104, 1093, and 301), 6 (strain 302), and 7 (strain 1066)-demonstrated the existence of a second dominant gene in M. floribunda 821. This gene, independent of Vf, was named Vfh because it seemed to induce a hypersensitive reaction. The results obtained with strain 1066, virulent to M. floribunda 821, allowed identification of another dominant gene, Vg, responsible for the resistance of 'Golden Delicious' to this strain. Vg is also carried by 'Florina', which was selected for its Vf resistance. The pathogenicity of a progeny originating from a cross between V. inaequalis strains 1066 and 301, characterized in vitro on leaf disks of differential genotypes, revealed two independent avirulence genes involved in the pathogenicity toward the Vg and Vf genes, respectively. These avirulence genes were named Avr Vg and Avr Vf. The host-pathogen interactions detected are consistent with a gene-for-gene relationship.     

Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl

A new class of agricultural fungicides derived from strobilurins act as respiration inhibitors by binding to mitochondrial cytochrome b. The effects of the strobilurin, kresoxim-methyl, on conidia germination, mycelial growth and the protection of apple leaves from scab development were investigated for two isolates of Venturia inaequalis randomly selected from a culture collection. Inhibition of mycelial growth required relatively high doses of kresoxim-methyl (ED₅₀=1 μg ml^-1) for both isolates. In comparison, germination of conidia was highly sensitive for one of the isolates (ED₅₀=0·005 μg ml^-1), while the level of inhibition achieved for the second isolate was 60-fold less (ED₅₀=0·3 μg ml^-1). As deduced from identical sequences of cytochrome b cDNAs prepared from both isolates, the different responses of germinating conidia to kresoxim-methyl were not caused by differences in the sequence of cytochrome b as the target site for strobilurin action. Strong synergistic effects of salicylhydroxamic acid on kresoxim-methyl inhibitory potency suggested that the reduced kresoxim-methyl sensitivity observed for germinating conidia was caused by interference of the alternative respiratory pathway with inhibitor action. However, this mechanism of target site circumvention in germinating conidia had no adverse effects on the protection of apple leaves from scab infection by kresoxim-methyl. © 1998 Society of Chemical Industry     

Evidence for the Predisposition of Fungicide-Resistant Isolates of Venturia inaequalis to a Preferential Selection for Resistance to Other Fungicides

In the United States, populations of the apple scab pathogen Venturia inaequalis have progressed through three consecutive rounds of fungicide resistance development, first to dodine, then to the benzimidazoles, and most recently to the sterol demethylation inhibitors (DMIs). Analysis of extensive monitoring data have to date provided no indication of detectable cross-resistance or partial cross-resistance of V. inaequalis populations to the three unrelated classes of fungicides prior to the selection of resistant subpopulations. However, in this study, resistance to both benomyl and DMIs developed to significantly higher frequencies within the previously established dodine-resistant population than in the population sensitive to dodine. Accelerated selection of phenotypes double resistant to dodine and the DMI fenarimol was apparent over the course of distinct seasons of apple scab management with either dodine or fenarimol. The data provide evidence for an accelerated speed of resistance development among phenotypes of V. inaequalis already resistant to an unrelated fungicide. This finding represents a departure from the previous model, which assumed entirely independent rounds of resistance developments. The data indicate that phenotypes of V. inaequalis might not only be selected for the trait of fungicide resistance but also for traits allowing a more flexible response to changes in the environment where they compete.     

Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis

The sensitivity of Venturia inaequalis field isolates to inhibitors of the cytochrome bc1 complex at the Qo site (QoIs) was characterised at the molecular, biochemical and physiological level, and compared to other respiration inhibitors. Comparison of a sensitive and a QoI-resistant isolate revealed very high resistance factors both in mycelium growth and spore germination assays. Cross-resistance was observed among QoIs such as trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. In the mycelium growth assay, antimycin A, an inhibitor of the cytochrome bc1 complex at the Qi site, was less active against the QoI-resistant than against the sensitive isolate. The mixture of QoIs with salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase, exerted synergistic effects in the spore germination but not in the mycelium growth assay. Thus, the cytochrome and the alternative respiration pathways are assumed to play different roles, depending on the developmental stage of the fungus. Induction of alternative oxidase (AOX) by trifloxystrobin was observed in mycelium cells at the molecular level for the sensitive but not the resistant isolate. Following QoI treatment, respiration parameters such as oxygen consumption, ATP level, membrane potential and succinate dehydrogenase activity were only slightly reduced in Qo-resistant mycelium cells, and remained at much higher levels than in sensitive cells. In contrast, no difference was observed between sensitive and resistant isolates when NADH consumption was measured. Comparison of the cytochrome b (cyt b) gene of the sensitive and resistant isolates did not reveal any point mutations as is known to occur in resistant isolates of other plant pathogens. It is assumed that QoI resistance in V inaequalis may be based on a compensation of the energy deficiency following QoI application upstream of the NADH dehydrogenase of the respiratory chain.     

Negative cross-resistance to N-phenylanilines in benzimidazole- resistant strains of Botrytis cinerea,Venturia nashicola and Venturia inaequalis

A new series of compounds with high contra-selective activity against benomyl-resistant fungal strains was found among ring-substituted N-phenyl-anilines. Hydrophobic substitution in one of the benzene rings, together with the secondary amine character of the molecule, are important factors for high fungitoxicity. The sensitivities of benomyl-resistant isolates to some representatives of the N-phenylanilines equal or even surpass their sensitivity to diethofencarb or MDPC. The negative cross-resistance with benzimdazole fungicides is valid for strains of Botrytis cinerea, Venturia nashicola and Venturia inaequalis. The strains of B. cinerea with double insensitivity to benzimidazoles and phenyl-carbamates are insensitive also to N-phenylanilines. However, the similarly double-insensitive strain of V. nashicola was found to be just as sensitive to the N-phenylanilines tested as the strain resistant only to benzimidazoles. The latter result revealed an important bonus compared with diethofencarb. Preventive application of one of the most active representatives of the N-phenylanilines to young cucumber plants was effective against infection with benomyl-resistant isolates of B. cinerea. Moderate apical translocation after root-dipping was also observed.     

A biochemical mechanism of natural resistance of apple to Venturia inaequalis

Samenvatting In cultuurfiltraten vanVenturia inaequalis, de verwekker van appelschurft, werden stoffen gevonden die toxisch zijn voor blad van appelrassen welke tegen dit physio resistent zijn. Blad van vatbare rassen is ongevoelig. De resistentie komt tot stand doordat deze toxische werking in het blad de vorming veroorzaakt van fungicide oxydatie-producten van phloridzine. Op grond van deze waarnemingen kan een mogelijke interpretatie van de gen-om-gen relatie bij deze plant-parasietverhouding worden opgesteld.     

Localized adhesion of nongerminated Venturia inaequalis conidia to leaves and artificial surfaces

Adhesion to the host surface is the first step for successful plant pathogen development and has been reported to be associated with both passive and active processes. For conidia of Venturia inaequalis, which depend on leaf wetness for germination, this process has not yet been described. Conidia of V. inaequalis adhered to wet hydrophobic surfaces immediately after contact to the surface, hours before initiation of germination. Attachment of nongerminated conidia was much better on hydrophobic surfaces, such as apple leaves and polystyrene, than on hydrophilic glass. Conidia released adhesive material localized in a droplet named spore tip glue (STG) at the spore apex which interacted with a contact surface only when water was present. Histochemical investigations indicated the presence of proteins and carbohydrates in STG, lectin labeling the presence of beta-galactose and N-acetylglucosaminyl residues. Transmission electron microscopy revealed two phases in the STG at the tip of dry mature conidia; as STG was present on the outer side of the intact fungal cell wall its formation should be associated with the secretion of glue through pores of the conidial wall. Surface-active substances affected the adhesion of conidia to hydrophobic surfaces stressing the importance of hydrophobic interactions. The use of protein biosynthesis inhibitors did not affect adhesion of conidia indicating that the adhesive material was preformed. It is concluded that the coincidence of STG, contact to a hydrophobic surface, and free water are essential for the adhesion of V. inaequalis conidia.     

Sensitivity to inhibitors of sterol biosynthesis in isolates of Venturia inaequalis from Italian and Dutch orchards

Samenvatting Een geringe, maar significante reductie in gevoeligheid vanVenturia inaequalis voor fungiciden die tot de sterol-biosyntheseremmers behoren werd vastgesteld bij isolaten uit proefboomgaar den in Italië, die gedurende enkele jaren met deze middelen behandeld waren. Bij isolaten uit enkele commerciële boomgaarden in Nederland werd nog geen significante verschuiving in gevoeligheid waargenomen.     

Detection and Quantification of Resistance of Venturia inaequalis Populations to Sterol Demethylation Inhibitors

ABSTRACT Monoconidial isolates of Venturia inaequalis were collected in 1990 and 1991 from orchards in New York, Michigan, and Nova Scotia that had never or only sporadically been treated with fungicides acting as sterol demethylation inhibitors (DMIs). Sensitivities of isolates to two representative DMIs (fenarimol and myclobutanil) were determined by a sensitivity test based on the relative growth (RG) of mycelial colonies at one discriminatory dose. Mean isolate sensitivities were not significantly different (P > 0.2) for the majority of the populations tested, and all sensitivity data obtained from these sites were combined to provide a baseline distribution of isolate sensitivities for both fenarimol and myclobutanil. The baseline distributions were compared with isolate sensitivities determined for an experimental orchard in Nova Scotia with a documented case of DMI resistance and for a commercial orchard in Michigan with a long history of DMI use and first evidence of practical DMI resistance. For both DMIs tested and in both treated orchards, frequencies of isolates with RG values <80 had decreased or only slightly increased compared to the baseline population. In contrast, frequencies of isolates with RG values >80 had increased more than 20-fold over baseline levels. Thus, isolates with RG values >80 were rated DMI resistant. The validity of a qualitative isolate classification was tested in controlled infection studies. At doses of fenarimol and myclobutanil recommended for commercial control of apple scab, reproduction of a typical sensitive isolate on treated apple leaves was suppressed completely. Lesions caused by a resistant isolate continued to expand and produced abundant conidia. Statistical analysis of orchard sensitivities revealed that the analysis of isolate counts grouped into the categories DMI sensitive or resistant was most indicative in comparisons of orchard sensitivities aimed at detection of practical DMI resistance. A high degree of cross-resistance between fenarimol and myclobutanil indicated that sensitivity tests with one of the DMIs employed as the diagnostic tool in this study can serve as a test for other DMIs.     

Pathogenicity and virulence of south African isolates of Venturia inaequalis

European Journal of Plant Pathology - Many abiotic and biotic factors can negatively affect the production and productivity of coconut cropping systems. The incidence of Lasiodiplodia species in...     

Identification of Microsatellite Markers and Their Application to Population Genetics of Venturia inaequalis

ABSTRACT Microsatellite markers of Venturia inaequalis were developed using genomic libraries of V. inaequalis enriched for the simple sequence repeats (TC)(n) and (AAC)(n). Seven markers, three with (TC)(n) repeats and four with (AAC)(n) repeats, were selected for the analyses of 350 isolates of V. inaequalis collected from 11 sites in Europe. Polymorphism in the (TC)(n) repeats was higher than in the (AAC)(n) repeats. Nei's expected genetic diversity (H(E)) varied between 0.52 and 0.96 for the microsatellites containing (TC)(n) stretches and between 0.09 and 0.36 for the microsatellites containing (AAC)(n) stretches. Within-population diversity (H(S)) was very high with values ranging from 0.28 to 0.49, whereas differentiation among all European populations (G(ST)) was low with an average of 0.07. In the population from Ahrensburg (northern Germany) where isolates were mainly collected from apple varieties carrying the Vf gene, usually resistant to V. inaequalis, we showed a bottleneck effect with reduced diversity and loss of alleles. The great advantages of microsatellite markers over random amplified polymorphic DNA and polymerase chain reaction-restriction fragment length polymorphism markers are their high specificity, high polymorphism, good reproducibility, and unambiguous scorability.     

Polyphenols and natural resistance of apple leaves against Venturia inaequalis

Phloridzin was oxidized via 3-hydroxyphloridzin to the correspondingo-quinone by a polyphenoloxidase present in homogenates and in acetone powders of apple leaves. Phloridzin could also be hydrolyzed by a β-glucosidase to phloretin, which was oxidized, although at a lower rate, to 3-hydroxyphloretin and its correspondingo-quinone. Phloretin and its oxidation products can couple with theo-quinone of 3-hydroxyphloridzin.Oxidation products of phloridzin inactivated pectinase. Moreover, the intermediate oxidation products showed fungicidal activity. Apparently, in this way apple leaves possess a defense mechanism against those organisms which are able to cause cell collapse resulting in phloridzin oxidation. Leaves of plants with a hypersensitive reaction to infection byVenturia inaequalis were also highly sensitive to toxic proteins secreted by this fungus in vitro.     

Acquisition of Resistance to Sterol Demethylation Inhibitors by Populations of Venturia inaequalis

ABSTRACT Acquisition of resistance to sterol demethylation inhibitors (DMIs) by populations of Venturia inaequalis was investigated using a microscopical method developed by C. Siebels and K. Mendgen. Microscopical analysis of conidiophore formation enabled the earlier detection of resistance and a clearer distinction between DMI-resistant and DMI-sensitive populations than other in vivo methods commonly used to analyze inhibitory effects of fungicides. In addition, because observations were made on the level of individuals, quantitative measures of the composition of conidial populations were obtained. The development of DMI sensitivity was followed over a period of 3 years in control apple orchards that had never been treated with fungicides and in orchards with DMI history. The 50% effective dose values determined by microscopical evaluation of conidio-phore development for untreated populations revealed the baseline sensitivities of 0.3, 0.96, 0.09, 1.22, and 1.92 mg/liter for flusilazole, fenarimol, difenoconazole, tebuconazole, and pyrifenox, respectively. As compared with the baseline sensitivity, all populations with DMI history showed significant resistance to flusilazole. A strong nonlinear correlation (R = 0.96) was found between the resistance factors and the sum of all DMI treatments of the 3 years before taking the sample. According to this correlation, resistance can be expected in all apple orchards of the fruit-growing area along Lake Constance, Germany, in which more than two DMI treatments per season have been applied. Due to cross-resistance, the recently introduced DMI fungicides difenoconazole, tebuconazole, and pyrifenox did not allow the control of V. inaequalis populations resistant to flusilazole.     

Genetic Characterization of Cercospora sorghi from Cultivated and Wild Sorghum and Its Relationship to Other Cercospora Fungi

ABSTRACT Genetic variability and population structure of Cercospora sorghi from wild and cultivated sorghum were investigated to gain insight into their potential impact on epidemics of gray leaf spot of sorghum in Africa. Population structure was examined using data derived from amplified fragment length polymorphism (AFLP) of C. sorghi by Nei's test for population differentiation, G(ST), and analysis of molecular variation (AMOVA). Two ecological populations of C. sorghi in Uganda were devoid of population structure (G(ST) = 0.03, small ef, CyrillicF(ST) = 0.01, P = 0.291). AMOVA revealed that genetic variability was due mainly to variations within (99%) rather than between (0.35%) populations, and Nei's genetic distance between the two populations was 0.014. Phenetic analysis based on AFLP data and polymerase chain reaction-restriction fragment length polymorphism analyses of the internal transcribed spacer regions of rDNA and mitochondrial small subunit rDNA separated Cercospora cereal pathogens from dicot pathogens but did not differentiate among C. sorghi isolates from wild and cultivated sorghum. Our results indicate that Ugandan populations of C. sorghi compose one epidemiological unit and suggest that wild sorghum, while not affecting genetic variability of the pathogen population, provides an alternative host for generating additional inoculum.     

新疆野扁桃与其近缘种的亲缘关系

野扁桃（Amygdalus ledebouriana Schlecht）为野生珍稀濒危植物资源,我国仅分布在新疆。为研究野扁桃与栽培扁桃及其近缘种的亲缘关系,对新疆野扁桃、栽培扁桃、栽培桃和桃属部分物种的叶绿体matK基因进行测序,基于Kimura 双参数模型计算种间遗传距离,并以邻接法（NJ）构建系统发育树。结果表明：野扁桃与蒙古扁桃和榆叶梅的遗传距离较小,与栽培扁桃和桃属物种（桃、山桃和蟠桃）的遗传距离较大;栽培扁桃与山桃、蟠桃和桃的亲缘关系较其与野扁桃、榆叶梅和蒙古扁桃的亲缘关系更近。     

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.