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.

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.

Memoria para optar al título profesional de Ingeniero Agrónomo / La sarna del manzano causada por Venturia inaequalis es la enfermedad más importante del manzano en Chile y a nivel mundial, y su control está basado principalmente en el uso de fungicidas. En los últimos años se ha observado en Chile una aparente disminución del nivel de control de la enfermedad con los fungicidas comúnmente utilizados con este propósito y se especula sobre una posible pérdida de sensibilidad de poblaciones del patógeno. Durante las temporadas 2006-2009, se obtuvo una colección de 128 cepas del patógeno que provino de hojas de manzanos infectados con sarna recolectadas entre las regiones VII y IX. Se realizaron pruebas de sensibilidad a los fungicidas mancozeb, difenoconazole, fenarimol y pyrimethanil en aislados monoconidiales de cepas silvestres del patógeno con el objetivo de elaborar curvas basales de sensibilidad y compararlas con la sensibilidad de aislados monoconidiales provenientes de seis huertos comerciales sin evidencias de resistencia práctica. La sensibilidad en las pruebas in vitro se midió como la inhibición de crecimiento miceliar (difenoconazole, fenarimol, y pyrimethanil) ó la inhibición de la germinación de conidias (mancozeb). A partir de los resultados obtenidos se propusieron dosis discrimininatorias de 0,04; 1,0; 0,6; y 0,2 μg mL-1 para difenoconazole, fenarimol, mancozeb y pyrimethanil, respectivamente. La población de huertos comerciales estudiada presentó una menor sensibilidad a los fungicidas difenoconazole, fenarimol y mancozeb con valores de factor de resistencia 4,7; 5,8 y 2,1, respectivamente. La mayor diferencia de sensibilidad entre la población silvestre y la de huerto se observó en fenarimol, fungicida para el cual la población de aislados de huerto presentó el mayor cambio hacia una menor sensibilidad. Por el contrario, para pyrimethanil, la población de huerto no evidenció pérdida significativa de sensibilidad en relación a la población basal. / Apple scab caused by Venturia Inaequalis is the most important disease of apples worldwide, and its control is primarily based on fungicides. In recent years an apparent disease control failure has been observed in Chile. A potential loss of fungicide sensitivity from pathogen populations is speculated. A collection of 128 strains were isolated from diseased leaves collected between VII and IX regions, during 2006 to 2009. Sensitivity tests to the fungicides mancozeb, difenoconazole, fenarimol, and pyrimethanil were conducted on wild isolates to build local baseline sensitivity distributions and to compare with the sensitivity of strains from six different commercial orchards without evidence of practical resistance. In vitro tests were measured as inhibition of mycelial growth or conidial germination (mancozeb). Discriminatory doses of 0.04; 1.0; 0.6; and 0.2 μg mL-1 were proposed according to the in vitro monitoring of sensitivity to difenoconazole, fenarimol, mancozeb and pyrimethanil, respectively. Departure from the baseline distribution towards less sensitivity was found for difenoconazole, fenarimol and mancozeb with resistance factors of 4.7; 5.8 and 2.1 respectively. The highest difference between baseline and orchard population was observed for fenarimol which showed the higher shift towards less sensitivity. In contrast, pyrimethanil did not show a not significant shift.

Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia. / Ph. D.