Cellulolytic Ability of the Scab Fungus, Venturia inaequalis

Cellulase and β-D-glucosidase activity have been identified in Venturia inaequalis, the causal agent of apple scab. Most of the activity of the enzymes was associated with the mycelium after homogenization. The activities were relatively weak in vitro compared to those of other plant pathogens. With a new screening technique, β-D-glucosidase hyperproducing mutants were obtained.     

Venturia inaequalis Resistance in Apple

Apple scab caused by Venturia inaequalis has evoked the interest for quite different reasons of scientists, agronomists, producers and consumers since over a century. Consumers select spotless apples, producers want to avoid damage, agronomists are asked to develop and implement control measures mostly based on fungicides, scientists are challenged to find cheaper and less questioned control measures. Under these premises a high number of publications have appeared dealing with almost all aspects of the interaction V. inaequalis-Malus. This review considers the advances of the past 10 years due to new genetic tools. It tries to reevaluate and value earlier works. The complex genetic of scab resistance in Malus is viewed in the context of single resistance genes, QTLs and functional interactions at molecular level. Consequences for breeding and for the creation of genetically modified apples are discussed.     

Infection and Stroma Formation by Venturia inaequalis on Apple Leaves with Different Degrees of Susceptibility to Scab

Conidia of Venturia inaequalis germinated and formed appressoria on all leaves independently of age and origin within the first 15 hours after inoculation. On the youngest expanded leaves of the apple variety Golden Delicious 80% of the conidia developed stromata within the first 24 hours, sporulation occurred after 8 days. On the second leaf (counted from the top of the twig) stroma formation was slowed down compared to the first and sporulation occurred after 10 days only. On the third leaf the fungus rarely formed stromata and never sporulated. The fourth leaf already showed complete ontogenetic resistance. In the diploid variety Priscilla the gene Vf, which confers resistance against scab to apple leaves, was expressed as reduced stroma formation without sporulation, as in the case of ontogenetic resistance. In the triploid variety Sir Prize with the gene Vf, stroma formation was retarded and colonization and sporulation occurred later on the first leaf, similarly to the reaction of the second leaf of variety Golden Delicious.     

Effect of Post-infection Application of Fungicides on the Apple Scab Pathogen, Venturia inaequalis (Cke.) Wint.

The Ergosterol-biosynthesis inhibiting (EBI) fungicides had a prolonged inhibitory effect on the growth and sporulation of the fungus, Venturia inaequalis thereby preventing development of typical apple scab symptoms. Atypical lesions in the form of chlorotic or reddish brown flecks had reduced viable sporulation with deleterious effect on subcuticular hyphae when fungicide was applied beyond 144 h but before 170h after inoculation. Myclobutanil (45μg a.i./ml) had the maximum inhibitory effect on sporulation and colony growth while bitertanol (187.50 μg a.i./ml) showed only a temporary inhibition of fungal growth and sporulation. Ultra-structural studies also exhibited different levels of fungitoxicity by different fungicides when applied 144 h after inoculation.     

In vitro Degradation of Cell Walls of Apple Leaves by Pectinolytic Enzymes of the Scab Fungus, Venturia inaequalis, and by Commercial Pectinolytic and Cellulolytic Enzyme Preparations

A new method involving ¹⁴C-labelled cell walls of apple leaves was developed in order to study the process of cell wall degradation in vitro and its role in pathogenesis and host-resistance. ¹⁴C-labelled cell walls were efficiently digested by commercial enzyme preparations and less efficiently by the polygalacturonase of Venturia inaequalis, the causal agent of apple scab. Further, degradability of cell walls from a susceptible and a resistant variety were compared, but there was no evidence for a correlation of reduced degradability with resistance. The method presented here can be regarded as a useful tool for investigations where enzymatic processes of polymerization or depolymerization of plant material is involved.     

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.     

Interaction between Venturia inaequalis and Apple Callus Tissue Cultures: an Electron Microscopic Study

Untrastructural interactions between Venturia inaequalis and callus cultures from scab susceptible and resistant apple varieties, were similar. Host cell wall changes, appositions, and invagination of host plasmamembrane at sites of close contact with fungal hyphae were regularly observed. The ultrastructural observations are described and discussed. The host cell alterations as well as many fungal structures corresponded to those known in young leaves of susceptible apple varieties.     

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.     

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.     

Venturia inaequalis: the causal agent of apple scab

The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis. TAXONOMY: Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes.     

Immunodetection of Venturia inaequalis Ascospores with Phage Antibodies

We describe the bacterial expression of single chain variable fragment (scFv) antibodies that bind specifically to the ascospores of Venturia inaequalis (Cooke). A scFv phage display library was prepared from the expressed V‐gene repertoire of a mouse immunized with whole V. inaequalis ascospores. Affinity selection was then carried out using intact, non‐germinated ascospores. The binding of selected phage antibodies was monitored by enzyme‐linked immunosorbent assay, flow cytometry and fluorescence microscopy. Several scFv antibodies were found to bind specifically to V. inaequalis ascospores. No cross‐reactivity was detected with spores from other phytopathogenic fungi, such as Plectosphaerella cucumerina, Cladosporium spp. and Alternaria brassicicola. Moreover, the scFvs did not bind to V. inaequalis conidiospores or mycelia. This is the first report describing the immunodetection of V. inaequalis ascospores by phage‐derived scFv antibody fragments. The degree of specificity of the antibodies is sufficiently high to allow rapid detection of ascospores within environmental probes such as those from particle samplers.