Bibliography: leaves 82-111. This thesis analyses the molecular composition of the surface components of the Endoparasitoid "Venturia canescens" using serological methods and specific sugar-binding lectins as diagnostic tools. The data reveals that the protective layer consists of at least two parts: a mucin-like glycoprotein and additional components from the wasp calyx fluid and the host hemolymph. The study suggests that the wasp larval cuticle is protected in a similar fashion to the egg chorion, except that the calyx-specific VLPs are probably replaced on the larval cuticle by host hemolymph proteins. The findings suggest that the mechanism of passive immune evasion could emerge during the evolution of the wasp-host interactions. The implication is that structurally conserved components may have similar functions in the parasitic and non parasitic species and could constitute a useful pre-adaptations for an endoparasitoid lifestyle.

In 1992, a research program was initiated to select suitable antagonists against the saprophytic (or winter) phase of the apple scab pathogen, Venturia inaequalis. An improved method for the mass screening of a vast collection of fungi was developed for this purpose. Some of the previously reported criteria such as leaf rheology and overwintering structure production proved unreliable or fastidious and cannot be used for in vitro antagonist selection. The main antagonism selection criterium retained was the in vitro inhibition of ascospore formation. To measure ascospore production, a simplified method of in vitro pseudothecia production was devised. This new method eliminates the need for prior conidia production by using a mycelial suspension and greatly reduces the risks of sterile mating by using a cocktail of Venturia inaequalis strains of different origins. Finally, the production cycle duration was reduced by varying the incubation temperature during the simulated winter. Moreover, a quick and efficient method of ascospore collection was developed. Ascospores were forcibly ejected in a large scale bubbler type apparatus in only one hour. This bubbler can be used for other studies including axenically produced ascospores. This new method compared favourably to the previously reported method and was used to screen a collection of about forty-two fungi. Six proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of saprophytes can now be based on a reliable and simple in vitro screening methodology.

Venturia inaequalis, the causal agent of apple scab, overwinters in apple leaves on the orchard floor. To develop a control strategy based on the prevention of the maturation of overwintering pseudothecia, a sampling of fungi colonizing dead apple leaves was conducted from different orchard floors in Quebec during the spring and fall of 1993. A total of 345 different isolates were obtained, from which fifteen genera have never been previously recorded as colonizers of apple leaves in North America. Small differences were detected in genera richness among orchards but the fungal composition of each orchard was fairly unique. Different tests on growth on amended media and leaf decomposition demonstrated that leaf degradation is not a reliable parameter alone to screen antagonist against V. inaequalis. No significant relation between growth on amended media, leaf rheology and ascospore inhibition was detected. However, 40 fungi reduced significantly ascospore production more than 87% compared to the control (V. inaequalis only). Of these antagonists, 30% decomposed apple leaves, suggesting that competition for the substrate is involved in the mode of action of at least one third of the antagonits detected. Other possible modes of antagonism are discussed.

Thesis (PhD(Agric))--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The apple industry in South Africa currently relies entirely on chemical fungicides to control apple scab, caused by Venturia inaequalis. In this dissertation, alterative management strategies against V. inaequalis were tested for the first time in South Africa. New information on the behaviour of the sexual winter phase of V. inaequalis in different climatic conditions was found and sources of asexual inoculum overwintering in apple orchards were identified. The effect of leaf shredding on fruit and leaf scab incidence and severity was tested against a non-shredded, non-sprayed negative control, a positive control that followed a commercial fungicide programme and a combined treatment of a commercial fungicide programme with leaf shredding, from 2010 to 2013. Reductions in fruit and leaf scab incidence and severity in the leaf shredding treatment were significantly lower compared to the negative control. Quantitative real-time polymerase chain reaction (qPCR) of airborne ascospores trapped using volumetric spore traps was used to measure the reduction in airborne ascospores in the shredded plots, and confirmed the efficacy of shredding found by comparing scab incidence and severity on fruit and leaves. Shredding twice during leaf-drop increased the efficacy of the treatment. Results indicate that leaf shredding should be integrated into scab management strategies in future. However, practical considerations unique to South African orchards, e.g. timing of leaf shredding relative to leaf-drop and orchard layouts, need to be addressed. Pseudothecial densities (PD, number of pseudothecia per fertile lesion) and ascal densities (AD, number of asci per pseudothecium) were compared between in Koue Bokkeveld (KB), a cold winter region, and Elgin (EL), a warm winter region experiencing climate warming, in 2012 and 2013. Scabbed leaves were detached during leaf-drop and overwintered in their region of origin and in the other region. The PD in leaves collected in KB and overwintered in KB was significantly higher than for leaves collected in EL and overwintered in EL, and leaves collected in KB and overwintered in EL. These results agreed with what was expected, as temperature during pseudothecial formation (i.e. the first four weeks after leaf-drop) was significantly lower in KB than in EL. However, the PD for leaves collected in EL and overwintered in EL did not differ significantly from EL leaves overwintered in KB. AD values in all treatments did not differ significantly from one another. Results suggest that factors other than temperature may be involved in controlling PD, e.g. the EL population may include strains not present in the KB population, with higher optimal temperatures for pseudothecial formation. Apple buds and pygmy apples were collected and tested for presence, number and viability of conidia in 2010, 2011 and 2012. Pygmy apples are small, late season fruit that remain attached to the tree throughout winter, especially in regions with warmer winters where trees do not experience sufficient chilling to complete dormancy. High conidial numbers were found on outer bud tissue and low numbers on inner bud tissue, but viable conidia were only found on inner bud tissue, using microscopy, and generally in orchards with high scab levels in the previous season. Molecular methods using PCR-RFLP and qPCR confirmed the presence of high amounts of V. inaequalis DNA in outer bud tissues, although calculated conidial amounts were higher than data obtained when using microscopy, which could indicate presence of mycelia not detected during microscopic examination. Higher numbers of conidia with higher percentage viability were found on pygmy apples, which are a more likely source of asexual inoculum in South African apple orchards than the low number of viable conidia on inner bud tissue. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse appelbedryf is tans afhanklik van chemiese swamdoders vir die beheer van die appelskurf patogeen, Venturia inaequalis. In hierdie proefskrif is alternatiewe bestuurstrategiëe vir die eerste keer in Suid-Afrika ondersoek. Nuwe inligting te opsigte van die gedrag van die geslagtelike winterfase van V. inaequalis, is onder verskillende klimaatstoestande ingewin en bronne van die oorwinterende ongeslagtelike inokulum in appelboorde, is identifiseer. Die invloed van blaarversnippering op die voorkoms en erns van appelskurf op vrugte en blare, is vanaf 2010 tot 2013 ondersoek en met ʼn negatiewe kontrole (onversnipperde blare sonder spuitprogram), ʼn positiewe kontrole (ʼn kommersiële swamdoderspuitprogram is gevolg) en gekombineerde behandelings (kommersiële swamdoderspuitprogram en blaarversnippering) vergelyk. Daar was ʼn betekenisvolle verskil in die voorkoms en erns van skurf op vrugte en blare met blaarversnippering teenoor die negatiewe kontrole. Kwantitatiewe intydse polimerase kettingvermeerderingsreaksie (kPKR) van luggedraagde askospore, vasgevang in volumetriese lokvalle, is gebruik om die afname van luggedraagde askospore in versnipperde behandelings te meet. Die doeltreffendheid van versnippering as behandeling, is bevestig deur die voorkoms van appelskurf te vergelyk met die ernstigheidsgraad daarvan op vrugte en blare. Die uitvoer van blaarversnippering twee keer gedurende die blaarvalperiode het die effektiwiteit van hierdie behandeling verhoog. Hiervan kan dus afgelei word dat blaarversnippering voordelig sal wees vir die bestuur van appelskurf en in toekomstige bestuurspraktyke ingesluit moet word. Praktiese oorwegings, uniek aan Suid-Afrikaanse boorde, soos boorduitleg en die tydsberekening van blaarversnippering teenoor blaarval, moet egter in ag geneem word. Pseudothesiale digtheid (PD; die aantal pseudothesia per vrugbare letsel) en askale digtheid (AD; die aantal aski per pseudothesium) is gedurende 2012 en 2013 vir die Koue Bokkeveld (KB), 'n koue winterstreek, en warm winterstreek Elgin (EL), 'n winterstreek wat klimaatsverwarming ervaar, vergelyk. Blare, met skurf, is gedurende blaarval gepluk en oorwinter in hul gebied van oorsprong, asook in die ander klimaatstreek. Blare wat in KB versamel is en in KB oorwinter het, se PD was aansienlik hoër as dié wat in EL versamel is en in EL oorwinter het, sowel as dié wat in KB versamel is en in EL oorwinter het. Hierdie resultate stem ooreen met wat verwag is, om rede die temperatuur gedurende pseudothesiale vorming, d.w.s. die eerste vier weke na blaarval, aansienlik laer in KB as in EL was. Die PD van blare wat in EL versamel en daar oorwinter het, het egter nie betekenisvol verskil van blare wat in KB oorwinter het nie. Die AD-waardes tussen behandelings verskil nie noemenswaardig nie en word as onbeduidend beskou. Die verkrygde resultate dui aan dat daar ander faktore as temperatuur betrokke is by die beheer van PD, bv. die EL-skurfpopulasie, waar die warmer klimaat meer optimaal is vir pseudothesiale vorming, rasse wat nie in die KB-bevolking teenwoordig is nie, mag insluit. Appelknoppe en dwerg-appels is gedurende 2010, 2011 en 2012 versamel en vir die teenwoordigheid, aantal en lewensvatbaarheid van konidiospore getoets. Dwergappels is klein laatseisoen appeltjies wat reg deur die winter aan die boom bly hang; veral in die streke met warmer winters waar die bome nie die nodige koue ervaar om dormansie te voltooi nie. Met behulp van mikroskopie is ʼn hoë aantal spore op die buitenste knopweefsel en lae getalle in die binneweefsel bespeur; maar lewensvatbare spore is net in die binneweefsel van knoppe waargeneem, wat hoofsaaklik afkomstig is van boorde wat hoë vlakke van appelskurf in die vorige seisoen ervaar het. Molekulêre tegnieke, PKR-RFLP en kPKR, is gebruik vir bepaling van V. inequalis DNA hoeveelhede op die buitenste knopweefsel. Hoër getalle konidiospore is met die molekulêre analise gevind, as dié verkry met mikroskopiese ondersoek en dui op die moontlike teenwoordigheid van miselium wat nie met visuele waarneming sigbaar was nie. Meer konidiospore met 'n hoër vlak van lewensvatbaarheid is op dwerg-apples gevind en dit is moontlik 'n meer waarskynlike bron van ongeslagtelike inokulum in Suid-Afrikaanse appelboorde, as die lae getalle van lewensvatbare konidiospore op die binneweefsel van die appelknoppe.

Apple scab is one of the greatest apple management problems throughout the world Much work has been done on cultivars resistant to Venturia inaequalis (Cke.) Wint., but few have been a commercial success. This frequently leaves fungicides as the only control method used. As Quebec growers select new cultivars for planting, more information is needed on their relative susceptibility for efficient scab control. In this light, 21 cultivars common to central and eastern Canada, were examined for their relative susceptibility using several components of partial resistance; disease severity, incubation period, latent period, lesion size, and conidial production. The cultivars used were Cortland, Early Geneva, Empire, Golden Delicious, Golden Russet, Idared, Jersey Mac, Jonagold, Jonamac, Lobo, Lodi, Summerland McIntosh, Mutsu (Crispin), Northern Spy, Paulared, Red Cortland, Red Delicious, Royal Gala, Spartan, Sunrise, and Vista Bella. A final ranking of the cultivars and selection of partial resistance components was done using the principal components analysis. (Abstract shortened by UMI.)

The use of a biofungicide on the perfect stage of V. inaequalis on leaf litter is one potential way to reduce the number of fungicides used to control apple scab. The previous in vitro screenings of Quebec mycoflora have shown that several isolates are able to significantly reduce the primary inoculum of the pathogen. Among the screened fungi, P176A and P130A, reduced over 98% of the ascospore production and were as effective as Athelia bombacina. However, because in vitro tests are generally poor predictors of in vivo assays a re-evaluation of the antagonists was done under field conditions. Eight fungal isolates, leaf shredding, and two comparative treatments (A. bombacina, 5% urea) were applied to intact scabbed leaves in October 1994 and 1995. After the treatments, the leaves overwintered on the orchard ground until the next spring. In April, samples of treated leaves were randomly selected and placed in spore traps to collect the ejected ascospores during rainfall. Since the primary inoculum was ejected during a four-month period, antagonism was based upon ratings taken throughout the whole ejection season. To evaluate the effect of incubation conditions on the antagonistic performance we incubated separately, in vitro and in vivo, sterile leaf disks which were artificially inoculated with V. inaequalis and fungal isolates. (Abstract shortened by UMI.)

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.