Auswahl der wissenschaftlichen Literatur zum Thema „Grapes Diseases and pests Australia“

Botrytis cinerea infection of wine grapes can result in a variety of symptoms. The most common symptom is botrytis bunch rot (BBR), where infected berries rot and shrivel, and eventually produce fungal sporulation. Another symptom is slip skin, where the skins of infected ripe berries slide easily from the pulp. It is hypothesised that a reduction in osmotic potential in grape berries due to late-season rainfall leads to slip skin symptom development. Hyphal growth of B. cinerea on osmotically adjusted agar was inhibited at osmotic potentials associated with near-ripe berries. Vine sheltering was used in a research vineyard to manipulate rainfall artificially and to alter berry sugar content in Vitis vinifera Sauvignon blanc vines, with the aim of increasing osmotic potential and altering symptom expression. Both BBR and slip skin symptoms were affected by the various sheltering conditions, with sheltered vines having lower BBR and higher slip skin at harvest. REFERENCES Becker T, Grimm E, Knoche M 2012. Substantial water uptake into detached grape berries occurs through the stem surface. Australian Journal of Grape and Wine Research 18: 109-114. https://doi.org/10.1111/j.1755-0238.2011.00177.x Beever RE, Laracy EP 1986. Osmotic adjustment in the filamentous fungus Aspergillus nidulans. Journal of Bacteriology 168: 1358-1365. https://doi.org/10.1128/jb.168.3.1358-1365.1986 Beresford RM, Hill GN 2008. Botrytis control without fungicide residues - is it just a load of rot? New Zealand Winegrower 12: 104-106. Beresford RM, Evans KJ, Wood PN, Mundy DC 2006. Disease assessment and epidemic monitoring methodology for bunch rot (Botrytis cinerea) in grapevines. New Zealand Plant Protection 59: 355-360. Bondada BR, Matthews MA, Shackel KA 2005. Functional xylem in the post-véraison grape berry. Journal of Experimental Botany 56: 2949-2957. https://doi.org/10.1093/jxb/eri291 Choat B, Gambetta GA, Shackel KA, Matthews MA 2009. Vascular function in grape berries across development and its relevance to apparent hydraulic isolation. Plant Physiology 151: 1677-1687. https://doi.org/10.1104/pp.109.143172 Clarke SJ, Hardie WJ, Rogiers SY 2010. Changes in susceptibility of grape berries to splitting are related to impaired osmotic water uptake associated with losses in cell vitality. Australian Journal of Grape and Wine Research 16: 469-476. https://doi.org/10.1111/j.1755-0238.2010.00108.x Diakou P, Moing A, Svanella L, Ollat N, Rolin DB, Gaudillere M, Gaudillere JP 1997. Biochemical comparison of two grape varieties differing in juice acidity. Australian Journal of Grape and Wine Research 3: 1-10. https://doi.org/10.1111/j.1755-0238.1997.tb00122.x Grolemund G, Wickham H 2011. Dates and times made easy with lubridate. 2011 40: 25. Harris RF 1981. Effect of water potential on microbial growth and activity. In: Parr JF, Gardner WR, Elliott LF eds. Water Potential Relations in Soil Microbiology. SSSA Special Publication. Soil Science Society of America. Pp. 23-95. Hill GN, Beresford RM, Evans KJ 2010. Tools for accurate assessment of botrytis bunch rot (Botrytis cinerea) on wine grapes. New Zealand Plant Protection 63: 174-181. Hill GN, Evans KJ, Beresford RM 2014a. Use of nitrate non-utilising (nit) mutants to determine phenological stages at which Botrytis cinerea infects wine grapes causing botrytis bunch rot. Plant Pathology 63: 1316-1325. https://doi.org/10.1111/ppa.12225 Hill GN, Evans KJ, Beresford RM, Dambergs RG 2014b. Comparison of methods for the quantification of botrytis bunch rot in white wine grapes. Australian Journal of Grape and Wine Research 20: 432—441. https://doi.org/10.1111/ajgw.12101 Keller M, Smith JP, Bondada BR 2006. Ripening grape berries remain hydraulically connected to the shoot. Journal of Experimental Botany 57: 2577-2587. https://doi.org/10.1093/jxb/erl020 Loschiavo A, Scholefield P, Morrison J, Ferris M 2010. The cost of pests and diseases to the Australian winegrape industry. Australian Viticulture 14: 15-19. McCarthy MG, Coombe BG 1999. Is weight loss in ripening grape berries cv. Shiraz caused by impeded phloem transport? Australian Journal of Grape and Wine Research 5: 17-21. https://doi.org/10.1111/j.1755-0238.1999.tb00146.x Mendiburu Fd 2016. agricolae: Statistical Procedures for Agricultural Research. https://CRAN.R-project.org/package=agricolae. Mundy DC, Beresford RM 2007. Susceptibility of grapes to Botrytis cinerea in relation to berry nitrogen and sugar concentration. New Zealand Plant Protection 60: 123-127. Nelson KE 1956. The effect of Botrytis infection on the tissue of Tokay grapes. Phytopathology 46: 223-229. NIWA 2017. Mean monthly rainfall (mm). https://www.niwa.co.nz/education-and-training/schools/resources/climate/meanrain (05-05-2017). Pezet R, Viret O, Perret C, Tabacchi R 2003. Latency of Botrytis cinerea Pers.: Fr. and biochemical studies during growth and ripening of two grape berry cultivars, respectively susceptible and resistant to grey mould. Journal of Phytopathology 151: 208-214. https://doi.org/10.1046/j.1439-0434.2003.00707.x R Core Team 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. R Studio Team 2016. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA. http://www.rstudio.com/. Rogiers SY, Smith JA, White R, Keller M, Holzapfel BP, Virgona JM 2001. Vascular function in berries of Vitis vinifera (L) cv. Shiraz. Australian Journal of Grape and Wine Research 7: 47-51. https://doi.org/10.1111/j.1755-0238.2001.tb00193.x Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A 2012. Fiji: an open-source platform for biological-image analysis. Nature Methods 9: 676-682. https://doi.org/10.1038/nmeth.2019 Smart R, Robinson M 1991. Sunlight into Wine. Winetitles, Adelaide, Australia. Taiz L, Zeiger E 1998. Plant Physiology. Sinauer Associates, Sunderland, MA, USA. Tyerman SD, Tilbrook J, Pardo C, Kotula L, Sullivan W, Steudle E 2004. Direct measurement of hydraulic properties in developing berries of Vitis vinifera L. cv Shiraz and Chardonnay. Australian Journal of Grape and Wine Research 10: 170-181. https://doi.org/10.1111/j.1755-0238.2004.tb00020.x Whiting EC, Rizzo DM 1999. Effect of water potential on radial colony growth of Armillaria mellea and A. gallica isolates in culture. Mycologia 91: 627-635. https://doi.org/10.2307/3761248 Wickham H 2009. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. Wickham H 2016. tidyverse: Easily Install and Load 'Tidyverse' Packages. https://CRAN.R-project.org/package=tidyverse. Wickham H, Bryan J 2017. readxl: Read Excel Files. https://CRAN.R-project.org/package=readxl. Wilcox WF, Gubler WD, Uyemoto JK 2015. Compendium of Grape Diseases, Disorders, and Pests: Second Edition. APS Press, St Paul, MN, USA.

Tomato is an important vegetable crop that contributes significantly to income security and healthy diets of people worldwide. Queensland produces the majority of tomatoes for fresh consumption accounting for 40 per cent of national supply in Australia. The purpose of this study is to provide an analytical summary of the Queensland tomato supply chain, by focusing on margins along the supply chain. For that, a representative tomato gross margin model in Southern Queensland was used to analyse the estimated income, grouped variable costs and the gross margin for four tomato varieties: gourmet, round, grape, and cherry. The mean yields of the sampled varieties varied considerably, depending on climatic conditions, pests and diseases, the season and whether tomatoes are grown on the ground or trellises. Driven by high revenues and relatively low freight costs, grape tomatoes have the highest gross margin ($73 thousand per hectare) as well as the highest market price at $4.64 per kilo compared to other varieties. The cost of growing the crop up to harvest can exceed $10,000 per hectare with high labour requirement for harvesting and packing. While costs for machinery, fertiliser, herbicide, weed control, insecticide, and fungicide largely remain constant across four tomato varieties, it is planting and irrigation that makes a difference. With average value of $9,303 per hectare, planting costs range from $5,134 for round tomatoes to $12,241 for cherry tomatoes. The results of this gross margin analysis can be helpful to explore profitability at the farm level, allowing regional and international comparisons

The Russian Federation imports large quantities of planting and grafting material of grapes, including from countries where dangerous harmful organisms are spread to vineyards. Plant protection specialists in this industry, as well as grape producers, must understand the possible risk associated with the danger of the entry of quarantine disease pathogens into the Russian Federation and the potential damage if infected plant material is imported. Studies have been carried out by the staff of All-Russian Plant Quarantine Center to assess the phytosanitary risk of pests associated with the import of seedlings, stocks and grape cuttings. Based on these studies, the list of pests for the vine that have quarantine status and potentially quarantine status for the Russian Federation, in particular bacterioses, phytoplasmas and viral diseases has been established. This article describes the main diseases that affect grapes and are included in the Unified List of Quarantine Pests of the Eurasian Economic Union, as well as potentially dangerous viral diseases that are not currently included, but planned for inclusion in the Unified List, which pose a serious threat if they penetrate to the territory of the Russian Federation. From quarantine pathogens, grapes are affected by grape bacteriosis (Pierce disease of grapevine) (Xylella fastidiosa Wells et al.), bacterial blight of grapevine (Xylophilus ampelinus (Panagopoulos) Willems et al.), Flavescence dorée of grapevine (Candidatus Phytoplasma vitis), from the planned inclusion grapes are affected by red spotting of grape leaves (Grapevine red blotch-associated virus), marbling of grape leaves (Grapevine vein clearing virus) and discoloration of the leaves of Roditis grape (Grapevine Roditis leaf discoloration-associated virus). The article considers the biological peculiarities of pathogens, ways of their possible penetration and spread on the territory of the cultivation of grape in the Russian Federation, host plants of quarantine and dangerous pests, the main symptoms of diseases on plants, possible vectors, damage caused by quarantine pests in the countries of spreading, diagnostic methods, as well as areas of possible harmfulness for the Russian Federation.

Turkey is one of main gene centers in the world for grapes. It is believed that cultivated grapes have their origins in Turkey and the surrounding countries. Vitis vinifera ssp sylvestris is the only wild grape species in this region. That is why Turkey has a very large amount of wild grapevine populations and grape cultivars which offer to grapevine breeders a valuable gene pool. Wild grapevines have significant characters for inducing the resistence to biotic and abiotic stress factors, such as resistance to lime, drought, pests and diseases. Turkey has over 1.600 local grape cultivars, among which the majority of them are conserved at the national grape collection vineyard in Tekirda?. They are mostly used as table grapes, dried grapes or for local consumptions. Wild grapes are distributed all over the country territory, mainly in the river basins and forests. Wild grape collection vineyards were established at some universities in Turkey. These grapevines will be screened for the resistance to biotic and abiotic stress factors.

In order to reduce the impact of pests and diseases on the yield and quality of Ningxia wine grapes and to improve the efficiency and intelligence of detection, this paper designs an intelligent detection platform for pests and diseases. The optimal underlying network is selected by comparing the recognition accuracy of both MobileNet V2 and YOLOX_s networks trained on the Public Dataset. Based on this network, the effect of adding attention mechanism and replacing loss function on recognition effect is investigated by permutation in the Custom Dataset, resulting in the improved network YOLOX_s + CBAM. The improved network was trained on the Overall Dataset, and finally a recognition model capable of identifying nine types of pests was obtained, with a recognition accuracy of 93.35% in the validation set, an improvement of 1.35% over the original network. The recognition model is deployed on the Web side and Raspberry Pi to achieve independent detection functions; the channel between the two platforms is built through Ngrok, and remote interconnection is achieved through VNC desktop. Users can choose to upload local images on the Web side for detection, handheld Raspberry Pi for field detection, or Raspberry Pi and Web interconnection for remote detection.

Obtaining resistant varieties to diseases without loss of organoleptic quality is a real challenge for oenology. Inter-specific hybridization of grapevines began in the 19th century and was initially aimed at introducing pest and disease resistance in offspring. Later, several breeding programmes implemented worldwide led to the development of varieties showing different characteristics such as cold-hardiness, short/long growing season, and pest resistance. Vitis vinifera grapes have preferred flavour characteristics for wine production, but they tend to be susceptible to pests, diseases, and extreme temperatures; species native to North America and East Asia are generally better adapted to these stressors. But these wild species tend to be low yielding and produce wines with undesirable sensory characteristics, including high acidity, low astringency, and excessive herbaceous or undesirable aromas. To be an innovative revolution during the 21st century, resistant varieties (actual and future) should be tested in different contexts for 3 major points: (i) vines can produce grapes without pesticides; (ii)

Non-native grape species such as V. vinifera and V. labrusca can not sustain the hot and humid environment of Florida due to their susceptibility to various diseases. Vitis rotundifolia (muscadine grapes) is native to Florida and the southeastern United States and adapted well to this climate condition. They are highly resistant to almost all grape foliage diseases and root pests such as nematode and phylloxera. Theoretically, muscadine grapes may become a valuable rootstock for bunch grapes. Unfortunately, most previous studies found that muscadine grapes were graft-incompatible with bunch grapes by normal grafting techniques. This study was to look for an alternative technique to graft V. vinifera onto muscadine rootstocks. A preliminary study indicated that bunch grape scions were successfully grafted on adult muscadine grapes. Two V. vinifera grape cultivars, `Thompson Seedless' and `Chardonnay', and two muscadine grape cultivars, `Carlos' and `Alachua', were used for this study. The muscadine grapes used as rootstocks are 6-year old field-grown vines and V. vinifera was used as scions. Using the common V-type grafting method was completely failed in more than 150 attempts. We then tried to insert the first-year buds of V. vinifera into 1- to 3-year-old muscadine canes. Surprisingly, the survival rate of the inserting buds was moderately high when `Carlos' was used as the rootstock. `Thompson Seedless'/`Carlos', `Chardonnay'/`Carlos' reached 53% and 33%, respectively. Successful grafting but lower survival rate was also obtained when `Alachua' was used as the rootstock (10% in `Thompson Seedless/`Alachua' and 3% in `Chardonny'/`Alachua'). The average survival rate of `Thompson Seedless' on the muscadine rootstocks was 36%, and `Chardonnay' was 12%. Regardless the cultivar of the scions, buds survived on `Carlos' and `Alachua' rootstocks were 44% and 5%, respectively. Results from this study indicated that V. vinifera grapes could be successfully grafted onto muscadine rootstocks. The survival rate varied depending on cultivars used for both scion and rootstock. It would be very interesting to see if resistance to certain diseases such as the Pierce's disease can be improved in those V. vinifera grape with muscadine roots and trunks derived from this preliminary study.

More than twenty exotic vertebrate species are now listed as pests in Australia. Collectively, these pests have a huge economic and environmental impact and pose a major threat to Australia’s ecosystems and unique biodiversity. Management of such pests on a continental scale is a major challenge. Recent advances in biotechnology suggest alternatives to the lethal diseases normally sought for use as biological control agents. One proposal, being investigated in the Pest Animal Control Cooperative Research Centre, Canberra, is the use of biotechnology to develop a new generation of agents that act through controlling reproduction to prevent the build up of pest populations. The core concept is fertility control through immunocontraceptive vaccines delivered by viruses that specifically infect the target pest population. Proof of this exciting concept has been obtained for the mouse and, very recently, the rabbit, and a candidate vaccine vector identified for the fox, portending better control of a trio of Australia’s most pervasive pests. Other advances in biotechnology suggest ways to negate the build up of both innate and acquired immune resistance in target pest populations that normally act to limit the efficacy and effective life of biocontrol agents in the field. Prospects for extending the use of virally vectored vaccines to the field management of wildlife diseases are also identified. Targets for such vaccines include a growing suite of emerging diseases, hosted by Australia’s wildlife, which pose a threat to human and livestock health. Numerous technical challenges remain to be addressed before any of these new agents are ready for use in the field. However, the major risk to their development is now no longer viewed as being technical, but the failure to gain public acceptance for their use in the field. This already significant risk is exasperated by the present heightened level of public concern about all introductions of genetically modified organisms.

Bibliography: leaves 148-166. This study identifies genetic variation in Australian Uncinula necator populations. Techniques were developed for molecular and phenotypic markers for U. necator. Mating types of Australian clonal lines were identified and viable cleistothecia and infective ascospores were produced in vitro. The study establishes the foundation for investigating the population biology of U. necator, by identifying two distinct genetic groups, A and B, and micro-geographical variation among 35 clonal lines from various Australian viticultural regions.

Downy mildew, caused by the biotrophic Oomycete Plasmopara viticola, is one of the most important diseases of grapevines world wide. It is particularly destructive in temperate viticultural regions that experience warm wet conditions during the vegetative growth of the vine (Wong et al., 2001). The disease is not normally a problem in mediterranean climates where the growing season tends to be hot and dry (Mullins et al., 1992; Sivasithamparam, 1993). Grape downy mildew is however a major disease in Australian viticulture (McLean et al., 1984; Magarey et al., 1991). Grape downy mildew was first reported in Europe in 1878 (Viennot-Bourgin, 1981). In Australia, it was recorded for the first time in 1917 at Rutherglen in Victoria (Vic) (de Castella, 1917). The first recorded outbreak of the disease in Western Australia (WA) occurred in 1997 in a small planting of vines in the far north of the state. In the subsequent year, it was detected in widespread commercial viticulture in the Swan Valley production area, near Perth (McKirdy et al., 1999). The pathogen has since been found in all grape growing regions of WA. Since its introduction into European vineyards in the 1880?s, P. viticola has become one of the world?s most investigated grapevine pathogens. Many aspects its basic biology however remain unknown (Wong et al., 2001). Due to the recent detection of P. viticola in WA, little is known of the nature of strains of the pathogen in the state and their response to local environmental conditions. Much of the research concerning the influence of environmental factors on the development of P. viticola has been conducted in Europe e.g. parts of France and Germany. Due to significant differences in climatic conditions and a shorter selection time on the pathogen in WA, much of the information described in European studies may not be directly applicable to the grape downy mildew disease situation in WA. The focus of this thesis was to examine epidemiological aspects of P. viticola in the mediterranean climate of WA. The environmental conditions that could favour the development of epidemics by strains of the pathogen that have been detected in the state were determined. The existence of P. viticola ecotypes and genetic variation among strains from WA and the Eastern states of Australia was also investigated.

Bibliography: leaves 81-100. Entry of inoculum into a crop and disease development in the crop cannot be prevented because spores are airborne and there is a lack of highly resistant varieties. This makes complete control of chocolate spot unlikely. It should however, be possible to improve current levels of disease control through the integration of the factors identified in the study.

Thesis (MScAgric.)--Stellenbosch University, 2000.
ENGLISH ABSTRACT: Phomopsis cane and leaf spot disease of grapevine is an economically important disease in many of the vine-growing areas of the world. Four different Phomopsis spp. have previously been associated with this disease. The present study investigates the taxonomic significance of the different taxa found on grapevines in South Africa, as well as the endophytic growth and fungicide sensitivity of Phomopsis viticola isolates. The thesis is compiled of several different parts, which deal with specific, but related topics, and hence some duplication has been unavoidable. Understanding the epidemiology of a disease is important for the correct timing of disease control. To investigate the endophytic growth of P. viticola, asymptomatic shoots were collected at eight different growth stages. Nodes, internodes, leaf petioles, leaves, tendrils and bunch peduncles were investigated. Two Phomopsis spp., taxon 1 and 2 were identified in this study. The Phomopsis viticola-complex had a relative importance of 9% and accounted for 3% of the isolations. P. viticola (taxon 2) is mainly isolated from the nodes and internodes. Inoculations of healthy, young vine tissue confirmed taxon 2 to be a virulent pathogen, suggesting that it is a latent pathogen rather than an endophyte. In contrast, taxon 1 appeared to be a true endophyte, and did not seem to be an important pathogen on vines. The true identity of the causal organism of Phomopsis cane and leaf spot disease was investigated by collecting samples from 58 different vineyards in the grapevine growing areas of the Western Cape. P. viiicola occurred in grapevine material collected from Lutzville to Swellendam, but was not found in the Oudtshoorn and Orange River grapevine areas. Diaporthe perjuncta (taxon 1), P. vutcola (taxon 2), taxon 3 and a Phomopsis species commonly associated with shoot blight of peaches in the U.S.A., P. amygdali, were identified among the South African grapevine isolates. Examination of the Australian culture designated as taxon 4 found it to be a species of Libertella, thus excluding it from the P. viticola-complex. An Italian isolate was found to represent a species of Phomopsis not previously known from grapevines, and this was subsequently described as taxon 5. Species delimitation was based on morphological and cultural characteristics, stem inoculations and the formation of the teleomorph in vitro. The identity of each morphological taxon was confirmed by means of phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacers (ITS 1 and ITS2) and the 5' end partial sequence of the mitochondrial small subunit (mtSSU). P. amygdali, associated with peach shoot blight in the U.S.A., was isolated once only and appeared to be of lesser importance in this disease complex. Furthermore, taxa 1 (Diaporthe perjuncta) and 3 were also rarely encountered and proved to be non-pathogenic, indicating their non-functional role in Phomopsis cane and leaf spot disease. Taxon 2 (Phomopsis viticolas was common and widely distributed in diseased vineyards. This taxon was associated with the typical disease symptoms and proved to be pathogenic. Morphologically taxon 2 corresponded best with P. viticola, which was also neotypified in this study. Taxon 2 was mostly isolated from buds and nodes, indicating that these are important sites in which the fungus survives during winter. Molecular data indicated that taxon 3 and P. amygdali were not host specific to grapevine. The currently used foliar fungicides were compared to the new strobilurin fungicides. The effects of nine fungicides (azoxystrobin, flusilazole, folpet, fosetyl- Al+mancozeb, kresoxim-methyl, mancozeb, penconazole, spiroxamine and trifloxystrobin) were tested in vitro on inhibition of mycelial growth. The following EC50 (ug/ml) values were obtained: azoxystrobin (0.350), flusilazole (0.007), folpet (4.489), fosetyl-Al+mancozeb (3.925), kresoxim-methyl (1.665), mancozeb (2.891), penconazole (0.023), spiroxamine (0.321) and trifloxystrobin (0.051). Additionally, azoxystrobin, folpet, kresoxim-methyl, mancozeb, propineb and trifloxystrobin were tested for their ability to inhibit spore germination in vitro. The subsequent EC50 (ug/ml) values were obtained: azoxystrobin 0.123), folpet (0.510), kresoxim-methyl (0.0037), mancozeb (0.250), propineb (0.156) and trifloxystrobin (0.003). The results reported in part 4 showed that the strobilurin fungicides inhibited the mycelial growth and spore germination of P. viticola. However, further trials need to be conducted to verify these findings under field conditions. In the present study taxa 1, 3 and P. amygdali were infrequently isolated, suggesting that they played a less prominent role in the P. viticolacomplex.
AFRIKAANSE OPSOMMING: Streepvleksiekte van wingerd is 'n ekonomies belangrike siekte wat in die meeste wingerdproduserende gebiede van die wêreld voorkom. Vier Phomopsis spesies is in die verlede met dié siekte geassosieer. Hierdie studie ondersoek die taksonomiese belangrikheid van die verskillende taksa wat op wingerd in Suid Afrika gevind word, asook die endofietiese groei en fungisiedsensitiwiteit van die Phomopsis vitico/a isolate. Hierdie tesis bestaan uit verskeie dele met spesifieke, maar verwante onderwerpe wat tot onafwendbare duplisering lei. Dit is belangrik om die epidemiologie van 'n siekte te verstaan sodat korrekte en tydsberekende siektebeheer toegepas kan word. Die endofietiese groei van P. vitico/a is ondersoek deur simptoomlose lote by agt verskillende groei stadiums te versamel. Nodusse, internodusse, blaarstele, blare, rankies en trosstele is ondersoek. Twee Phomopsis spp., takson 1 en 2 is geïdentifiseer. Die Phomopsis vitico/a-kompleks het 3% van die isolasies uitgemaak en 'n relatiewe belangrikheid van 9% getoon. P. vitico/a (takson 2) is meestal uit die nodus en internodus geïsoleer. lnokulasies van gesonde, jong wingerdweefsel het bevestig dat takson 2 'n virulente patogeen is en dat die takson eerder 'n latente patogeen as 'n endofiet is. In teenstelling hiermee is takson 1 'n ware endofiet en 'n onbelangrike patogeen op wingerd. Die ware identiteit van die veroorsakende organisme van streepvlek is ondersoek deur plantmateriaal vanaf 58 verskillende wingerde in die wingerproduserende gebiede van die Wes-Kaap te versamel. P. vitico/a is in wingerdmateriaal vanaf Lutzville tot Swellendam aangetref, maar nie in die Oudtshoorn en Oranjerivier wingerd produserende gebiede nie. Diaporthe perjuncta (takson 1), P. vitico/a (takson 2), takson 3 en P. amygdali is in die Suid Afrikaanse wingerdisolate geïdentifiseer. P. amygdali word met lootverskroeiing van perske bome in die V.S.A. geassosieer. Die Australiese isolaat wat benoem is as takson 4, is met die huidige ondersoek gevind om 'n spesie van Libertella te wees. Takson 4 is daarvolgens uit die P. vitico/a-kompleks gelaat. 'n Italiaanse isolaat het 'n nuwe spesie van Phomopsis op wingerd verteenwoordig en is vervolgens as takson 5 beskryf. Spesie-onderskeiding is op morfologiese en kulturele eienskappe, staminokulasies en die vorming van die teleomorf in vitro gebaseer. Die identiteit vanelke morfologiese takson is met behulp van filogenetiese analises van die nukleêre ribosomale DNS intern transkriberende spasieerders (ITS 1 en ITS2) en die 5' punt gedeeltelike nukleotied volgorde van die mitochondriale klein subeenheid (mtSSU) bevestig. P. amygdali is slegs een keer geïsoleer en blyk van minder belang in die siektekompleks te wees. Takson 1 (Diaporthe perjuneta) en takson 3 het ook min voorgekom en is nie-patogenies, wat hul nie-funksionele rol in streepvleksiekte aandui. Takson 2 (P. viticola) is algemeen geïsoleer en kom wyd verspreid voor. Hierdie takson is geassosieer met die tipiese siektesimptome en is ook patogenies. Morfologies stem takson 2 met P. viiicola ooreen en is ook geneotipifiseer in hierdie studie. Takson 2 is meestal vanaf die ogies en nodusse geïsoleer, wat daarop dui dat hierdie belangrike setels is waar die swam tydens die winter oorleef. Die molekulêre data toon aan dat takson 3 en P. amygdali nie gasheerspesifiek tot wingerd is nie. Die swamdoders wat tans teen streepvlek gebruik word, is met die nuwe strobilurin swamdoders vergelyk. Die effek van nege swamdoders (azoksistrobin, flusilasool, folpet, fosetyl-Al + mancozeb, kresoxirn-metiel, mankozeb, penconasool, spiroksamien en trifloksistrobin) is in vitro op die inhibisie van miseliumgroei getoets. Die volgende EKso-waardes (g/ml) is verkry: azoxystrobin (0.350), flusilasool (0.007), folpet (4.489), fosetiel-Al + mankozeb (3.925), kresoxirn-metiel (l.665), mankozeb (2.891), penkonasool (0.023), spiroksamien (0.321) en trifloxystrobin (0.051). Azoxystrobin, folpet, kresoxim-rnetiel, mankozeb, propineb en trifloksistrobin is ook in vitro getoets vir hul inhibisie op spoorontkieming. Die volgende EKso-waardes is verkry: azoxystrobin (0.123), folpet (0.510), kresoxim-metiel (0.0037), mankozeb (0.250), propineb (0.156) en trifloxystrobin (0.003). Die resultate vervat in deel 4 toon dat die strobilurin swamdoders die miseliumgroei en spoorontkieming van P. viticola inhibeer. Toetsing in die veld word egter benodig om die effektiwiteit van die middels te bevestig. In hierdie studie is taksa I, 3 en P. amygdali selde geïsoleer, wat aangedui het dat hierdie taksa 'n minder belangrike rol in die P. viticola-kompleks speel.

Thesis (MScAgric)--University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Knowledge of the presence of Botrytis cinerea in morphological parts of bunches and leaves of grapevine would help to find a reliable, sensitive, and specific assay to verify the actual occurrence of latent infection, and to plan strategies for the effective control of B. cinerea bunch rot. The aim of this study was (i) to determine natural B. cinerea infection at specific sites in leaves and bunches of grapevine at different phenological stages, and (ii) to determine resistance in the morphological parts to disease expression. Bunches and leaves of the wine grape cultivar Merlot and the table grape cultivar Dauphine, were collected at pea size, bunch closure and harvest from five vineyards in the Stellenbosch and De Dooms regions respectively. The material was divided into two groups and sealed in polythene bags. The bags were lined with wet paper towels to establish high relative humidity. Leaves and bunches incubated in one group of bags were first treated with paraquat in order to terminate active host responses. These treatments provided conditions that facilitated disease expression under two host resistance levels by different inocula during the period of moist incubation. Disease expression was positively identified by lesion development, and the formation of sporulating colonies of B. cinerea at a potential infection site. Sites in leaves were the blades and petioles. Sites in bunch parts were rachises, laterals and pedicels, and on berries sites were the pedicel-end, cheek and style-end. In Dauphine, the various sites were at all stages classified as resistant to moderately resistant. However, at pea size and bunch closure, in spite of their resistance, nearly all the sites carried high to very high inoculum levels. The only exception was the berry cheek, which carried intermediate inoculum levels at pea size, and low inoculum levels at bunch closure. In nearly all sites, inoculum levels were lower at harvest. The decrease was the most prominent in petioles, rachises, laterals, pedicels and the pedicel-end of the berry. All these sites carried intermediate to low inoculum levels at harvest. In Merlot, sites constantly exibited a resistant reaction, except for the pedicel and pedicel-end of the berry, which changed from resistant at the early developmental stages to susceptible at harvest. Inoculum levels decreased during the season in the rachises and laterals, but were constantly high during the season in the pedicel and pedicel-end of the berry. According to this pattern of natural occurrence, B. cinerea fruit rot in these vineyards was not caused by colonisation of the pistil, and subsequent latency in the style end of grape berries. However, fruit rot was primarily caused by colonisation of the pedicel, and subsequent latency in the pedicel or pedicel-end of the berry. These findings furthermore support the hypothesis of increased host resistance during development, but also indicate that in the Western Cape province, inoculum in vineyards is abundant during the early part of the season, and less abundant later in the season. More information is therefore needed on the behaviour of the different types of B. cinerea inocula on the different morphological parts of grapevine to validate the pathway described for natural B. cinerea infection in vineyards. The penetration and disease expression at the different morphological parts of bunches of two grape cultivars (Dauphine and Merlot) under conditions simulating natural infection by airborne conidia was therefore investigated. The two cultivars did not differ in resistance of the berry cheek, which was at all stages classified as resistant. However, in Dauphine, latent inoculum levels in berry cheeks declined from intermediate at pea size to low at the following stages, whereas in Merlot, levels were intermediate during pea size and at harvest. Some differences between cultivars were found in the resistance of the structural bunch parts, and of their latent inoculum levels. In Dauphine, the rachis reacted susceptible at pea size, and was classified moderately resistant later in the season. Laterals and pedicels were moderate resistant at pea size, and resistant at later stages. Inoculum levels in rachises, laterals and pedicels were high at pea size, but intermediate at bunch closure and at harvest. The finding that B. cinerea infected and naturally occurred more commonly in the tissues of immature than mature bunches, that the structural parts of the bunch carried more B. cinerea than the berry cheek, and that these infections may be more important in B. cinerea bunch rot than infection of the cheek or the style end, suggest that emphasis should be placed on the disease reaction of the pedicel and related parts of immature bunches rather than on the berry. The resistanc-e reaction of leaf blades, petioles, internodes and inflorescences on cuttings, compared to those on older shoots from the vineyard were therefore investigated. In the case of vinelets, leaf blades, petioles, internodes and inflorescences were all classified susceptible to highly susceptible. The different parts furthermore all carried very high latent inoculum levels. In vineyard shoots the petioles and inflorescences showed resistance, and carried intermediate to latent inoculum levels. This finding suggests that leaf blades are not appropriate parts for studying the behaviour of inoculum of B. cinerea and host responses in grape bunches. In stead, petioles and inflorescences of vineyard shoots should be used for this purpose.
AFRIKAANSE OPSOMMING: WEERSTAND TEEN BOTRYTIS CINEREA IN MORFOLOGIESE DELE VAN BLARE EN TROSSE VAN WINGERD Kennis oor die teenwoordigheid van Botrytis cinerea in morfologiese dele van wingerd word benodig vir die ontwerp van 'n betroubare, sensitiewe en spesifieke toets vir die bevestiging van latente infeksies, en vir die implementering van strategieë vir die effektiewe beheer van B. cinerea-vrot. Die doel van hierdie studie was om (i) natuurlike B. cinerea infeksie by spesifieke areas in blare en trosse van wingerd te bepaal, en (ii) om weerstand teen siekte-uitdrukking in hierdie morfologiese dele vas te stel. Trosse en blare van die wyndruif kultivar Merlot en die tafeldruif kultivar Dauphine, is by ertjiekorrel, tros-toemaak en oes in vyf wingerde in die Stellenbosch- en De Doomsomgewing, onderskeidelik, versamel. Die materiaal is in twee groepe verdeel en in polietileen sakkies verseël. Die sakkies is met klam papierdoekies uitgevoer om sodoende hoë relatiewe humiditeit te verseker. Blare en trosse wat in die een groep geïnkubeer is, is eers met paraquat behandel om aktiewe gasheerreaksies te beëindig. Hierdie behandelings het toestande geskep wat gedurende die periode van vogtige inkubasie gunstig was vir siekteontwikkeling deur verskillende inokula by twee gasheer-weerstandsvlakke. Siekteuitdrukking is positief geïdentifiseer deur letsel-ontwikkeling en die vorming van sporuierende kolonies van B. cinerea by 'n potensiële infeksie-area. Dele waarop in die blare gekonsentreer is, was die blaarskyf en -steel. In die trosse was die dele die rachis, lateraal en korrelsteel, en op korrels was dit die korrelsteel-end, wang en styl-end. In Dauphine is die verskillende dele tydens al die fenologiese stadia as weerstandbiedend tot matig weerstandbiedend geklassifiseer. Die verskillende dele her egter, ten spyte van hul weerstandbiedendheid, hoë tot baie hoë inokulumvlakke by ertjiekorrel- en tros-toemaakstadium gedra. Die enigste uitsondering was die korrelwang, wat 'n middelmatige inokulumvlak by ertjiekorrel, en 'n lae inokulumvlak by tros-toemaak, gedra het. Die inokulumvlakke was in byna al die dele laer by oes. Die afname in inokulumvlakke was die prominentste in die blaarstele, rachi, laterale, korreisteie en die korrelsteel-end van die korrel. Al hierdie dele het 'n middelmatige tot lae inokulumvlak by oes gehad. In Merlot was die dele konstant weerstandbiedend, behalwe vir die korrelsteel en die korrelsteel-end van die korrel, wat gewissel het van weerstandbiedend by die vroeë ontwikkelingstadia, tot vatbaar by oes. lnokulumvlakke in die rachis en lateraal het gedurende die seisoen afgeneem; maar was deur die seisoen konstant hoog in die korrelsteel en korrelsteel-end van die korrel. Volgens die patroon van natuurlike voorkoms, word B. cinerea-vrot in hierdie wingerde nie deur kolonisasie van die stamper, en die daaropvolgende latensie in die styl-end van die korrels, veroorsaak nie. Vrot word egter primêr deur kolonisasie van die korrelsteel, en die daaropvolgende latensie in die korrelsteel of korrelsteel-end van die korrel, veroorsaak. Hierdie bevindinge ondersteun die hipotese van toenemende gasheerweerstand gedurende ontwikkeling, en dui ook daarop dat inokulumvlakke in wingerde in die Wes-Kaap provinsie volop is gedurende die eerste deel van die seisoen, en minder volop is later in die seisoen. Meer inligting word dus benodig aangaande die gedrag van die verskillende inokulum tipes van B. cinerea op die verskillende morfologiese dele van wingerd, ten einde die infeksieweg vir natuurlike B. cinerea infeksie in wingerde te bevestig. Die vestiging van latente infeksies in die verskillende morfologiese dele van trosse van twee kultivars (Dauphine en Merlot), onder toestande wat natuurlike infeksie deur luggedraagde konidia simuleer, is dus ondersoek. Die twee kultivars se weerstand in die korrelwang het nie verskil nie en is by alle fenologiese stadia as weerstandbiedend geklassifiseer. Die latente inokulumvlakke in die korrelwang van Dauphine het egter van middelmatig by ertjiekorrel, tot laag in die daaropvolgende stadia afgeneem, terwyl die vlakke in Merlot middelmatig by ertjiekorrel en oes was. Verskille tussen die twee kultivars is gevind ten opsigte van die weerstand in die trosdele, asook hulle latente inokulumvlakke. Die rachis van Dauphine was by ertjiekorrel vatbaar, en matig weerstandbiedend later in die seisoen. Die lateraal en korrelsteel was matig weerstandbiedend by ertjiekorrel en weerstandbiedend by latere stadia. lnokulumvlakke in rachi, laterale en korreisteie was hoog by ertjiekorrel, maar middelmatig by tros-toemaak en oes. Die bevindinge dat B. cinerea natuurlik meer algemeen in die weefsel van onvolwasse trosse voorgekom en laasgenoemde meer algemeen geïnfekteer het, dat B. cinerea se voorkoms hoër was in die morfologiese dele van die tros as in die korrelwang, en dat hierdie infeksies van groter belang in B. cinerea-vrot mag wees as infeksie van die wang of styl-end, dui daarop dat klem gelê moet word op die siektereaksie van die strukturele dele van onvolwasse trosse, eerder as van die korrel. Die weerstand van blaarskywe, blaarstele, internodes en blomtrossies van steggies, in vergelyking met die op ouer lote in wingerde, is dus ondersoek. Blaarskywe, blaarstele, internodes en blomtrossies van steggies is almal as vatbaar tot hoogs vatbaar geklassifiseer. Die verskillende dele het verder ook almal baie hoë latente inokulumvlakke gedra. By die ouer lote van wingerde het die blaarstele en blomtrossies weerstandbiedend vertoon, en middelmatige latente inokulumvlakke gedra. Hierdie bevindinge dui daarop dat blaarskywe nie die ideale morfologiese deel is vir gedragstudies van B. cinerea in druiwetrosse nie. Blaarstele en blomtrossies van ouer lote moet eerder vir die doel gebruik word.

Thesis (MScAgric)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry.
AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.

Thesis (MScAgric)--University of Stellenbosch
ENGLISH ABSTRACT: An understanding of the infection pathways of Botrytis cinerea in grape bunches will help to combat this devastating pathogen of grape. Many studies have been done to determine the possible infection pathways of B. cinerea. Most of these studies made use of artificial inoculations that deposit groups of conidia on the plant surface. The deposition of clusters of conidia is not a common phenomenon in nature. The aim of this study was to investigate the infection pathways of (i) naturally- as well as (ii) artificially inoculated B. cinerea conidia during all the phenological stages of three wine grape cultivars, and to compare the (iii) pathogenicity and virulence, on grape and nectarine fruit, of isolates obtained from different host plants. In the natural infection study the occurrence of Botrytis cinerea and subsequent disease expression at different positions in bunches of wine grapes (cultivars Chenin Blanc, Shiraz and Chardonnay) was determined from 1999 to 2001. Different techniques were used to detect viable inoculum at different positions (rachises, laterals, pedicels, and the peicel end, cheek and style end of berries) in bunches. Isolations were made on Kerssies' B. cinerea selective medium, or bunches were used untreated, or treated with paraquat. Paraquat was used to terminate host resistance and to promote the development of the pathogen from the tissues. The material was used untreated to detect the pathogen on the surface, or were surface-sterilized to detect mycelia (latent infection) in the tissue. In the artificial inoculation study, bunches of wine grapes (cultivars Chenin Blanc, Chardonnay and Shiraz) at pea size, bunch closure, and harvest were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at high relative humidity (±93%). Following incubation, the bunches were divided in two groups. The one group was surface-sterilised in 70% ethanol for 5 s, the other group was left untreated. Bunches of the sterile group, and from the untreated group were used for isolation. From each bunch rachis segments, laterals, pedicels and berry skin segments (from the pedicel-end and cheek) were removed. The sections were placed in Petri dishes on Kerssies' B. cinerea selective medium and on a water agar medium supplemented with paraquat, and incubated at 22°C under diurnal light. Occupation by the pathogen was positively identified by the formation of sporulating colonies of B. cinerea on the different tissues. Lastly, in the virulence and pathogenicity experiment on grape and nectarine fruit Botrytis cinerea isolates, which were obtained from different host plants, were compared by simulating natural infection. Cold-stored fruit, considered highly susceptible to B. cinerea were therefore inoculated with single, airborne conidia of the pathogen. Different tests were conducted to assess surface penetration and lesion formation. Isolations were made from fruit skins on Kerssies' B. cinerea selective medium. Nectarine fruit were treated with paraquat, and grape berries were frozen for 1 h at -12°C. Paraquat and freezing were used to terminate host resistance and to promote the development of the pathogen from the tissues. In the natural infection studies B. cinerea occurred in a consistent pattern in bunches of the three cultivars. B. cinerea consistently developed from the tissue of the rachis, laterals, pedicel and pedicel-end, but not from the berry cheek. The rachis, lateral and pedicel contained much higher levels of B. cinerea than any position on the berry. Furthermore, the pathogen consistenly occurred at relatively high levels on the rachises throughout the season. Collectively, the data showed that in the Western Cape province, B. cinerea occured more regularly in wine grape bunches during the early part of the season, than later in the season. The data of the artificial studies confirmed the findings made with the natural infection studies. In these experiments the pathogen resided more often on the structural bunch parts than on the berries. Overall, the isolation studies revealed that conidia occurred predominantly on the rachis. The incidence of B. cinerea was furthermore constantly high in the inner bunch after each inoculation, and in bunches of different maturities. The data therefore indicated that, when available, conidia penetrated loose and tight clustered bunches in a similar way. Finally, in the virulence and pathogenicity experiments the results showed clearly that no host specialisation exists in the B. cinerea isolates used in this study. From these studies it is clear that in the Western Cape province B. cinerea occurs more readily in the inner structural parts of the bunches and more so during the earlier parts of the season. These findings should be considered when planning and implementing disease control programmes.
AFRIKAANSE OPSOMMING: INFEKSIEWEË VAN BOTRYTIS CINEREA OP GESELEKTEERDE WYNDRUIF KULTIVARS Indiepte kennis van die infeksieweë van Botrytis cinerea op druiwetrosse word benodig vir die beheer van dié vernietigende patogeen van druiwe. Vele studies is al gedoen om die moontlike infeksieweë van die swam op druiwe trosse te ondersoek. Die meeste van die studies het gebruik gemaak van kunsmatige inokulasie tegnieke waar die konidia van die swam in groepe op die korreloppervlak gedeponeer is. In die natuur is dit 'n rare verskynsel dat konidia in groepe op die korreloppervlak land. Die doel van die studie was om die infeksieweë van B. cinerea op drie wyndruif kultivars te ondersoek wat (i) natuurlik- en (ii) kunsmatig geïnokuleer is met konidia gedurende al die fenologiese stadia, en om die (iii) virulensie en patogenisisteit van isolate wat van verskillende gashere verkry is, op druiwe en nektariens te vergelyk. In die natuurlik-geïnokuleerde druiwe is die voorkoms van B. cinerea en die gevolglike siektevoorkoms op verkillende posisies in trosse van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz) gedurende 1999 tot 2001 bepaal. Verskillende tegnieke is gebruik om lewensvatbare inokulum by verskillende posisies (ragis, lateraal, pedisel en pedisel-end van die korrel) in die tros waar te neem. Isolasies is op Kerssies' B. cinerea selektiewe medium gemaak, of trosse is onbehandeld gebruik, of behandel met paraquat. Paraquat is gebruik om die gasheer se natuurlike weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. Die plantmateriaal is onbehandeld gelaat om die patogeen op die oppervlak waar te neem, of die oppervlak is gesteriliseer om die latente myselium in die weefsel waar te neem. In die kunsmatige inokulasiestudies is trosse, van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz), geïnokuleer met droë spore, van B. cinerea, in 'n inokulasietoring en die plantmateriaal is dan geinkubeer vir 24 h by 'n hoë relatiewe humiditeit (93%). Na die inkubasie proses is die trosse in twee groepe verdeel. Die een groep druiwe het oppervlak sterilisasie ondergaan in 70% etanol vir 5 s, en die ander groep was onbehandeld gelaat. Trosse van die onbehandelde en gesteriliseerde groep druiwe is gebruik vir isolasies. Vanuit elke tros is daar segmente van die ragis, laterale, pediselle en korrels (van die pedisel-end en wang gedeeltes) geïsoleer. Die segmente is in Petri bakkies met Kerssies' B. cinerea selektiewe medium en op water agar medium, wat paraquat bevat het, geïsoleer en geïnkubeer onder 'n 12 h dagligperiode teen 22°C. Die patogeen is positief geïdentifiseer deur sporuierende kolonies op die onderskeie weefseltipes. Laastens, in die virulensie- en patogenisiteitsproewe op druiwe en nektariens is verskillende isolate van B. cinerea, verkry vanaf verskillende gasheerplante, vergelyk deur natuurlike inokulasie toestande na te boots. Koue opgebergde vrugte, wat beskou word as hoogs vatbaar vir die infeksie van B. cinerea, is geïnokuleer met droë, enkel luggedraagde spore van die patogeen. Verskillende toetse is gedoen om die oppervlak penetrerende en letselvormende vermoëns van die onderskeie isolate te toets. Isolasies is van die skille van die vrugte gemaak en op Kerssies' B. cinerea selektiewe medium geplaas. Die nektarienvrugte is met paraquat behandel en die druifkorrels is gevries vir 1 h teen -12°C. Paraquat en bevriesing is gebruik om die gasheer se weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. In die natuurlik-geïnokuleerde studies het B. cinerea 'n konstante patroon getoon in die trosse van die drie verskillende wyndruif kultivars. B. cinerea het konstant ontwikkel uit die ragis, laterale, pedisel en pedisel-end, maar selde uit die korrelwang. Die ragis, lateral en pedisel dele het baie hoër vlakke van van die swam bevat as enige deel op die korrel. Die patogeen het ook konstant volop deur die hele seisoen op die ragis voorgekom. Gesamentlik wys die data dat, B. cinerea in wyndruiwe, in die Wes Kaap provinsie, meer geredelik vroeër in die seisoen voorkom, eerder as later. Data van die kunsmatige inokulasiestudies het die bevindinge van die natuurlike inokulasiestudies tot 'n groot mate bevestig. In dié studies het die patogeen meer geredelik die strukturele dele van die tros, eerder as op die korrels, bewoon. Oor die algemeen het die isolasieproewe gewys dat die konidia meer op die ragis voorkom as op enige ander deel. Die voorkoms van B. cinerea was ook oor die algemeen baie hoër in die strukturele dele van die tros, as op die korrel self. Die verskynsel het onder trosse van verskillende ontwikkelingsvlakke voorgekom. Die data het dus ook gewys dat konidia, wanner dit beskikbaar is, minder- sowel as meer kompakte trosse op 'n soortgelyke manier penetreer. Laastens, in die virulensie en patogenisiteitseksperimente het die resultate duidelik gewys dat daar geen gasheer spesifieke gedrag onder B. cinerea isolate is nie. In die studies het dit duidelik na vore gekom dat, B. cinerea meer geredelik in die strukturele binne dele van die wyndruif tros, in die Wes Kaap provinsie voorkom. En so ook eerder aan die begin van die seisoen, as later in die seisoen. Dié kennis moet in aanmerking geneem word by die beplanning en implementering van siektebeheerprogramme.

Thesis (MSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the pectin component of the cell wall, thus providing an entry route, as well as nutrients for the fungus. Plants have evolved mechanisms to counteract and suppress the action of the ePGs. This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays. Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that transgene integration was successful, the transgenes were being expressed and there were at least 29 transgenic lines with independent integration events. The transgenic lines were confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from 22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs). The plant lines with positive transgene integration, expression, independent integration events and exhibiting 100% transgene-derived PGIP activity were further selected for whole plant and detached leaf antifungal assays where they were challenged with B. cinerea. The whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be explained by a quicker and stronger recognition between the pathogen and the host and the stronger activation of defence responses in the host. A more active hypersensitive response in the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed of lesion development observed on the transgenics lines versus the wild type support this possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera PGIPs during infection of grapevine tissue; the potential changes that might be caused by expressing PGIPs in a grapevine host with a native PGIP with high homology to the transgenes (including potential gene silencing) and the potential impact on defence signalling and defence responses all provides further avenues of study to elucidate this very interesting phenotype further. Overall, this study provides a comprehensively characterised population of transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence, where the host plant harbours a native copy of the PGIP encoding gene.
AFRIKAANSE OPSOMMING: Plante word voortdurend blootgestel aan biotiese en abiotiese faktore, wat stres veroorsaak en hul bestaan bedreig. Biotiese faktore, soos patogene, veroorsaak groot verliese in wêreldwye gewasopbrengste, met swampatogene wat moontlik die grootste skade veroorsaak. Swampatogene, soos Botrytis cinerea, wat ‘n wye reeks gasheerplante kan infekteer, stel selwand-afbrekende ensieme tydens plantinfeksie vry, wat as endo-poligalakturonases (ePG’s). bekend staan. Hierdie ePG’s breek die pektienkomponent van die selwand af, wat gevolglik as ‘n ingangspunt dien,asook voedingstowwe vir die swam verskaf. Plante het meganismes ontwikkel om die aktiwiteit van hierdie ePG’s te bekamp en te onderdruk. Die aktiwiteit van die selwand-geassosieërde proteïene, genaamd poligalakturonase-inhiberende proteïene (PGIP’s), speel hier ‘n rol. PGIP’s inhibeer ePG’s direk en hul inhiberende aktiwiteit verleng ook die bestaan van langketting oligogalakturoniedresidu’s, wat blykbaar ‘n kaskade van weerstandsreaksies kan inisieer. ‘n PGIP-koderende geen, VvPGIP1, is voorheen uit wingerd geïsoleer en gekarakteriseer. VvPGIP1, asook nege nie-vinifera wingerd-PGIP’s is voorheen in tabak uitgedruk en bevestig as proteïene met sterk anti-swamaktiwiteit, soos bevestig deur die bevinding dat die transgeniese tabak ‘n weerstandsfenotipe teen Botrytis in heelplant-infeksietoetse het. Ná die tabakstudie is twee van die nie-vinifera PGIP’s uitgedruk in vatbare V. vinifera-kultivars. Karakterisering van die vermeende transgeniese bevolking het getoon dat die transgeen-integrasie suksesvol was, dat die transgeen uitgedruk word en dat daar ten minste 29 transgeniese lyne met onafhanklike integrasie gebeurtenisse geskep is. Daar is verder bevestig dat die transgeniese lyne aktiewe PGIP’s (transgeen-afkomstig) in hul blare het. Ongesuiwerde proteïenekstrakte van 22 lyne het 100% inhibisie teen ‘n mengsel van ongesuiwerde B. cinerea PGs (BcPGs) getoon. Die plantlyne met positiewe transgeenintegrasie en -uitdrukking, asook onafhanklike integrasiegebeure en wat 100% transgeen-afkomstige PGIP-aktiwiteit getoon het, is verder aan heel-plant en verwyderde blaarswaminfeksies met B cinerea onderwerp. Die heelplantinfeksietoetse het getoon dat uitdrukking van nie-vinifera PGIP’s in V. vinifera ‘n toename, in plaas van ‘n afname, in vatbaarheid teen B. cinerea veroorsaak. Hierdie verbasende resultaat kan moontlik toegeskryf word aan ‘n vinniger en sterker herkenningsreaksie tussen patogeen en gasheer en die moontlike sterker stimulering van weerstandsreaksies in die gasheer. ‘n Meer aktiewe hipersensitiewe reaksie in die gasheer sal tot die voordeel van Botrytis, wat ‘n nektrotroof is, wees. Die tipe letsel, asook die aanvang en spoed van letselontwikkeling wat waargeneem is in transgeniese lyne teenoor die wilde-tipe ondersteun hierdie moontlikheid. Gapings in kennis ten opsigte van die doeltreffendheid van die ePG-inhibisie deur die nievinifera PGIP’s tydens infeksie van wingerdweefsel, die moontlike veranderinge (insluitend ‘n moontlike geenuitdowingseffek) wat veroorsaak kan word deur die uitdrukking van PGIP-gene in ‘n kultivar met ‘n inheemse en baie homoloë PGIP-geen, kon ‘n invloed op weerstandseine en weerstandsreaksies gehad het. Hierdie aspekte lewer verdere studiemoontlikhede om hierdie interessante fenotipe verder te verklaar.Algeheel lewer hierdie studie ‘n breedvoeriggekarakteriseerde bevolking trangeniese plante, wat dien as nuttige hulpbronne vir in vivoanalise van PGIP se funksie in siekteweerstandbiedendheid, veral waar die gasheerplant ‘n inheemse kopie van die PGIP-koderende geen huisves.

The movement of humans around the earth has been associated with an amazing redistribution of a variety of organisms to new continents and exotic islands. The natural biodiversity of native communities is threatened by new invasive species, and many of the most serious insect and weed pests are exotics. Classical biological control is one approach to dealing with nonindigenous species. If introduced species that lack natural enemies are competitively superior in exotic habitats, introducing some of their predators (herbivores), diseases, or parasitoids may reduce their population densities. Thus, the introduction of more exotic species may be necessary to reduce the competitive superiority of nonindigenous pests. The intentional introduction of insects as biological control agents provides an experimental arena in which adaptations and interactions among species may be tested. We can use biological control programs to explore such evolutionary questions as: What characteristics make a natural enemy a successful biological control agent? Does coevolution of herbivores and hosts or predators (parasitoids) and prey result in few species of natural enemies having the potential to be successful biological control agents? Do introduced natural enemies make unexpected host range shifts in new environments? Do exotic species lose their defense against specialized natural enemies after living for many generations without them? If coevolution is a common force in nature, we expect biological control interactions to demonstrate a dynamic interplay between hosts and their natural enemies. In this chapter, I consider biological control introductions to be experiments that might yield evidence on how adaptation molds the interactions between species and their natural enemies. I argue that the best biological control agents will be those to which the target hosts have not evolved resistance. Classical biological control is the movement of natural enemies from a native habitat to an exotic habitat where their host has become a pest. This approach to exotic pests has been practiced since the late 1800s, when Albert Koebele explored the native habitat of the cottony cushion scale, Icrya purchasi, in Australia and introduced Vadalia cardinalis beetles (see below) to control the cottony cushion scale on citrus in California. This control has continued to be a success.