Resistance in apple to shoot infection by Venturia inaequalis

Eight apple genotypes, including cultivars and breeding selections resistant and susceptible to Venturia inaequalis on foliage, were screened for shoot infection and the development of wood pustules following inoculation of shoot tips of 1–year maiden trees in the greenhouse. Where genotypes were highly resistant in terms of foliar symptoms (cvs Prima and Gavin), no shoot infection was observed. Where genotypes were highly susceptible in terms of foliar symptoms, then shoot tissue was either resistant (cv. Shinko) or susceptible (cv. Starking). In one experiment, shoot tips were inoculated sequentially as shoots extended. No pustules developed where inoculations were made later than May. In a further experiment, two leaf internode positions were inoculated on one occasion. Pustules were only observed where inoculations were made above the youngest unrolled leaf. The results suggested that shoot tips were more susceptible during early extension growth of the shoot. Pustules were noted in abundance on petioles of susceptible cultivars, and these probably contributed to early leaf abscission.     

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.     

苹果黑星病菌SSR反应体系的优化

通过对苹果黑星病菌基因组DNA的SSR反应中一些重要参数进行优化,结果表明,最适反应体系为:2 5μL体系中,10×Buffer Mg Cl2 2 0 mm ol/ L 2 .5μL ,d NTP 10 0μmol·L- 1 、引物0 .5μmol·L- 1 、Taq DNA聚合酶1.5 U ,DNA模板2 m g·L- 1 ,dd H2 O 19.5μL .PCR扩增程序为93℃,2 min,5 7℃,30 s,1个循环;72℃,1m in,93℃,30 s,5 7℃,30 s,4 0个循环;72℃,10 min.     

Sensitivity of Venturia inaequalis to Trifloxystrobin and Difenoconazole in Uruguay

The sensitivity of Venturia inaequalis to trifloxystrobin and difenoconazole was studied in Uruguay. Populations of V. inaequalis were collected from apple orchards with different histories of trifloxystrobin use. Sensitivity of populations to trifloxystrobin was analysed using a method for testing spore germination published by FRAC, using a discriminatory concentration of 2.0 mg a.i./l. Resistance to trifloxystrobin was widespread in the region of commercial apple production with resistance detected in all orchards examined, the incidences ranging from 3% to 95%. The highest frequencies were found in orchards with the most intensive use of Quinone outside inhibitors (Q_oI) fungicides. Sensitivities of isolates of V. inaequalis to difenoconazole were assessed at five concentrations using a mycelial growth assay on isolates (33 isolates per orchard) from one non‐commercial (baseline orchard) and two commercial orchards having differing histories of difenoconazole use. Populations in both commercial orchards exhibited reduced sensitivities to difenoconazole compared to the baseline orchard. Resistance factor (RF) values of 6.6 and 11.74 were measured in the orchards with moderate (up to 4 sprays per season) and intensive use (more than 5 sprays per season) of difenoconazole, respectively. A single‐assessment concentration (SAC) was identified for assessing difenoconazole sensitivity of V. inaequalis isolates by fitting linear regressions between log₁₀ EC₅₀ and relative growth (RG) of the isolates at each fungicide concentration testing, and comparing the goodness‐of‐fit of the regression lines. Comparable results were obtained based on EC₅₀ values and RG values at a SAC of 0.05 mg of active ingredient per litre (a.i. per l). Populations from both commercial orchards differed from the baseline population, in that isolates with RG ≥70 were present at substantial levels in the former but absent from the latter.     

Localization of fungal fimbriae by immunocytochemistry in pathogenic and nonpathogenic isolates of Venturia inaequalis

Pathogenic and nonpathogenic isolates of Venturia inaequalis were grown in liquid culture. Hyphae were treated with two types of fimbrial antiserum (AU- and AV-1) and examined by immunofluorescent microscopy, in order to establish the distribution of fimbrial epitopes in whole cell mounts. The AV-1 antiserum was specific for the glycoprotein subunits while the AU-antiserum was specific for the protein moieties present on the fimbriae of Mycobotryum violaceum. The use of fimbrial antiserum with immunocytochemistry and transmission electron microscopy demonstrated a clear distinction between pathogenic and nonpathogenic isolates of V. inaequalis, based on the appearance of the fungal cell wall and the distribution of fimbrial epitopes labeled with AV-1 antiserum and immunogold complex. In actively growing hyphae of the pathogenic isolate, characterized by distinct cellular organelles, small vacuoles, and lipid bodies, fimbrial epitopes were concentrated in the fungal cell wall and were present minimally on the outer surface. In contrast, actively growing hyphae of the nonpathogenic isolate of V. inaequalis had extensive fine hair-like protrusions in the fungal cell wall which labeled with the AV-1 antiserum and immunogold. The distribution of fimbrial epitopes in V. inaequalis was highly dependent on the developmental growth stage of the fungal mycelium. Aging mycelia in both the pathogenic and nonpathogenic isolates of V. inaequalis were characterized by a large central vacuole and no label. In the pathogenic and nonpathogenic isolates of V. inaequalis grown in vitro, the distribution of fimbrial glycoprotein epitopes provided a more complex profile than that seen in M. violaceum.     

Two novel Venturia inaequalis genes induced upon morphogenetic differentiation during infection and in vitro growth on cellophane

Venturia inaequalis is a hemibiotrophic ascomycete that causes apple scab. Germ tubes, from conidia or ascospores, penetrate the leaf or fruit surface directly via appressoria-like swellings; subsequently the hyphae divide laterally to form a stroma between the cuticle and the outer wall of the epidermal cells. This morphological switch can be mimicked by growing the fungus in vitro on cellophane discs. The aim of this work was to identify genes upregulated in planta using growth on cellophane as a model. Four cDNA clones were found to be induced by growth on cellophane, and qRT-PCR showed two of these genes were up-regulated over a thousand fold in infected apple leaves compared to liquid culture. The predicted proteins for both genes possess putative signal peptides for secretion but have no similarity to sequences in publicly available databases. Both genes encode proteins with novel, imperfect repeat domain structures, the number of which vary in an isolate-specific fashion. Cin1 has seven or eight repeats of about 60 amino acids with four conserved cysteine residues per repeat, while Cin3 has four or five repeats of 32 amino acids with no cysteines. Both proteins appear to have evolved through internal duplication. Cin3, in particular, shows considerable between-strain variation in domain structure, indicating a high degree of recombination at this locus and revealing that the repeat structure has most likely arisen by unequal crossing-over. Results of this study support the hypothesis that cellophane-grown V. inaequalis mimics aspects of biotrophic infection and provide the first insights into novel fungal genes expressed during apple scab infection and their mechanisms of evolution.     

Virulence and Molecular Diversity of Venturia inaequalis in Commercial Apple Growing Regions in Kashmir

Seventy‐one isolates of Venturia inaequalis collected from commercial apple growing areas of Kashmir were characterized on international differential apple hosts and analyzed by Random Amplified Polymorphic Microsatellites (RAMS), PCR–RFLP and sequencing of rDNA for elucidation of variability. Virulence analysis on a differential set categorized them into four pathogenic races, viz. (0), (1), (2) and (1,2) in the first time comprehensive molecular analysis of this in India and especially from Jammu and Kashmir, a north‐western Himalayan state of India. Race groups (0), (1), (2) and (1,2) contained isolates from diverse areas without specificity to any geographical zone or region. Cluster analysis of the RAMS and PCR–RFLP revealed a high genotypic diversity within V. inaequalis isolates. Three major clusters were obtained and the isolates could not be categorized on the basis of either their geographical distribution or the cultivar from which they were isolated. amova analysis of pathogen populations at regional or race level revealed high diversity within the populations. Pairwise F_ST comparisons between the populations revealed less genetic differentiation, thereby indicating existence of frequent gene flow in Kashmir. The 24 rDNA sequences of V. inaequalis showed high haplotype diversity of 0.938 and 0.40 nucleotide diversity. Again clustering at regional or race level detected greater part of variability within groups than among groups, thereby indicating high diversity in V. inaequalis populations in Kashmir valley.     

Purification and partial characterization of an extracellular melanoprotein from the fungus Venturia inaequalis

The fungus Venturia inaequalis clone No. 36 isolated from Malus domestica cv. Gloster excretes a melanoprotein of 36 kDa in relatively high amounts during growth in liquid culture. The protein was isolated from the culture medium and purified to homogeneity. It was shown to contain melanin. After raising an antiserum against the isolated protein, the protein could be shown to be located in the apoplast fluid of the V. inaequalis infected Malus domestica cv. Elstar. Partial sequencing of the protein revealed no significant sequence homologies to so far sequenced proteins. The melanoprotein binds ferrous and ferric iron. Moreover, it could be shown that the binding of ferric iron (but not of ferrous iron) leads to a change in the absorbance of the protein suggesting a modification of the protein by ferric, but not by ferrous, iron. In addition to iron, the protein also binds copper, but does not bind manganese or nickel. A possible function of this protein in the recruiting and transport of iron and copper and/or in the protection of the fungus by metal-ion mediated oxidative stress is discussed.     

Isolation of 21 new polymorphic microsatellite loci in the phytopathogenic fungus Venturia inaequalis

Twenty‐one new polymorphic microsatellite markers were isolated in the phytopathogenic fungus Venturia inaequalis, the causal agent of apple scab. An enrichment protocol was used to isolate microsatellite loci and the level of polymorphism was assessed on 44 European isolates. All loci were polymorphic with an average of 9.1 alleles per locus (range 2–24). Tests of cross‐species amplifications suggest that at least some of these microsatellites could be used in different species, mainly Spilocaea pyracanthae and S. eriobotryae.     

Environmental Factors Influencing the Dispersal of Venturia inaequalis Ascospores in the Orchard Air

A 6-year study was carried out in an apple-growing region of North Italy by trapping airborne ascospores of Venturia inaequalis with a volumetric spore trap operated continuously during the ascospore season, with the aim of better defining the weather conditions that allow ascospores both to discharge and to disperse into the orchard air. A total of more than 60 ascospore trapping events occurred. Rain events were the only occurrences allowing ascospores to become airborne (a rain event is a period with measurable rainfall ≥0.2 mm/h – lasting one to several hours, uninterrupted or interrupted by a maximum of two dry hours); on the contrary, dew was always insufficient to allow ascospores to disperse into the air at a measurable rate, in the absence of rain. In some cases, rain events did not cause ascospore dispersal; this occurred when: (i) rain fell within 4–5 h after the beginning of a previous ascospore trapping; (ii) rain fell at night but the leaf litter dried rapidly; (iii) nightly rainfalls were followed by heavy dew deposition that persisted some hours after sunrise. Daytime rain events caused the instantaneous discharge and dispersal of mature ascospores so that they became airborne immediately; for night-time rainfall there was a delay, so that ascospores became airborne during the first 2 h after sunrise. This delay did not always occur, and consequently the ascospore trapping began in the dark, when: (i) the cumulative proportion of ascospores already trapped was greater than 80% of the total season's ascospores; (ii) more than one-third of the total season's ascospores was mature inside pseudothecia and ready to be discharged.     

Overwintering of Venturia inaequalis the causal agent of apple scab in relation to weather

Apple leaves collected at East Mailing and bearing colonies of Venturia inaequalis were exposed outdoors from November 1978 to May 1979 at 12 United Kingdom sites. Pseudothecial development and abundance were recorded from samples returned to East Mailing every 2 wk. Pseudothecia matured most rapidly with high rainfall in November and high temperatures in spring. Mature pseudothecia were most abundant with low temperatures in November and low rainfall in spring. In 1979-80 a cross-over experiment, in which scabbed leaves spent either November-January or February-April at a common site, and the other half of the winter at one of five sites, demonstrated the separate effects of weather following leaf-fall and in the spring. Equations for forecasting the time to pseudothecial maturity, but not the numbers of mature pseudothecia, were obtained. Experiments in controlled environments demonstrated the importance of low temperature and high moisture for initiation and early development of pseudothecia, but a controlled increase of temperature in the spring did not result in faster rates of maturation compared with lower temperatures outdoors. Ascospore production from leaves kept in controlled environments in spring was consistently lower than from leaves kept outside.