Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the field of agriculture, plant pathogens are a major concern because of the severe damage these organisms cause to crops yearly. Fundamental studies regarding plant pathogens and their modes of action made it possible for researchers in the field of molecular biology to investigate pathogens further on a molecular level. Botrytis cinerea, has been used to great effect as a model system to investigate various aspects regarding pathogenesis, also on a molecular level. Molecular research done on B. cinerea over the last few years has shown that the endopolygalacturonases (EPGs) of this fungus are key role players in pathogenesis. This hydrolytic enzyme family of six members, encoded by the Bcpg1-6 genes, are important in breaking down the complex cell wall polymers of host plants, enabling the fungus to penetrate its host sufficiently. It has been shown that both BcPG1 and 2 are crucial for virulence of B. cinerea. A leucine-rich repeat inhibitor protein situated in the cell wall of various plant species, the polygalacturonase-inhibiting protein (PGIP), has been proven to interact with and inhibit EPGs, and thus the necrotic actions of B. cinerea. From literature it was clear that specific data regarding individual interactions of fungal EPGs with PGIPs are lacking currently. Furthermore, most experiments regarding the effects of EPG as well as interaction and inhibition studies of EPGs and PGIPs, rely on in vitro methods, without the possibility to contextualize the results on an in vivo or in planta level. The scope of this study was to specifically address the issues of individual EPG:PGIP interactions and the use of possible in vivo methodology by using EPGs from a highly virulent South African strain of B. cinerea and the grapevine VvPGIP1 that has been previously isolated in our laboratory. This PGIP, originally isolated from Vitis vinifera cv Pinotage, has been shown to inhibit a crude EPG extract from this strain with great efficiency. The approach taken relied on heterologous over-expression of the individual Bcpg genes and the isolation of pure and active enzymes to evaluate the inhibition of the EPGs with VvPGIP1. The genes were all successfully over-expressed in Saccharomyces cerevisiae with a strong and inducible promoter, but active enzyme preparations have been obtained only for the encoding Bcpg2 gene, as measured with an agarose diffusion assay. The in vitro PGIP inhibition assay is also based on the agarose diffusion assay and relies on activity of the EPGs to visualize the inhibiting effect of the PGIP being tested. The active EPG2, however, was not inhibited by VvPGIP1 when tested with this assay. The EPG encoding genes from B. cinerea were transiently over-expressed also in Nicotiana benthamiana by using the Agrobacterium-infiltration technique. Transgene expression was confirmed by Northern blot analysis and EPG-related symptoms were observed five to eight days post-infiltration. Differential symptoms appeared with the various EPGs, providing some evidence that the symptoms were not random events due to the infiltration or a hypersensitive response. Moreover, the symptoms observed for EPG2 was similar to those that were reported recently by another group on the same host. In spite of the expression data and the clear symptoms that developed, active preparations, as measured with the agarose diffusion plate asay, could only be obtained for EPG2 again. In our search for a possible in vivo method to detect and quantify EPG activity and inhibition by PGIPs, we tested and evaluated a technique based on chlorophyll fluorescence to detect the effect of EPGs on the rate of photosynthesis. Our results showed that the over-expression of these genes reduced the rate of electrons flowing through photosystem II, indicating metabolic stress occurring in the plant. We used the same technique to evaluate possible interaction between VvPGIP1 respectively with BcPG1 and 2 and found that the co-expressing of the Vvpgip1 gene caused protection of the infiltrated tissue, indicating inhibition of EPG1 and 2 by VvPGIP1. For EPG2, the observed interaction and possible inhibition by VvPGIP1 is the first report to our knowledge of an interaction between this specific EPG2 and a PGIP. Moreover, to further elucidate the in planta interaction between VvPGIP1 and the EPGs from the South African B. cinerea strain, we tested for possible interactions by making use of a plant two-hybrid fusion assay, but the results are inconclusive at this stage. Previous studies in our laboratory have shown that several natural mutations exist between PGIP encoding genes from different V. vinifera cultivars. Based on this finding and the fact that these natural mutations could result in changes with regard to EPG inhibition and ultimately disease susceptibility, we isolated an additional 37 PGIP encoding genes from various grapevine genotypes, some of which are known for their resistance to pathogens. Combined, these results make a valuable contribution to understand plant pathogen interactions, specifically in this case by modeling the interactions of pathogen and plant derived proteins. The possibility to use in vivo methods such as chlorophyll fluorescence to follow these interactions on an in planta level, provides exciting possibilities to strenghten and contextualize in vitro results. / AFRIKAANSE OPSOMMING: Plantpatogene organismes veroorsaak jaarliks erge skade aan landbougewasse en word dus as ’n ernstige probleem in die landbousektor beskou. Diepgaande studies wat handel oor plantpatogene en hul metodes van infeksie het dit vir molekulêre bioloë moontlik gemaak om patogene nou ook op molekulêre vlak verder te bestudeer. Botrytis cinerea is baie effektief as modelsisteem gebruik om verskeie aspekte van patogenese verder te bestudeer, ook op ‘n molekulêre vlak. Molekulêre navorsing op B. cinerea, het getoon dat die endopoligalakturonases (EPGs) van dié swam kernrolbelangrik in patogenese is. Hierdie sesledige hidrolitiese ensiemfamilie word gekodeer deur die Bcpg1-6 gene en is belangrik vir die afbraak van die komplekse selwandpolimere van plantgashere, om suksesvolle gasheerpenetrasie te veroorsaak. Daar is aangetoon dat beide BcPG1 en 2 essensieël vir virulensie van die patogeen is. ’n Leusienryke-herhalings inhibitorproteïen wat in die selwand van verskeie plantspesies voorkom, die poligalakturonase-inhiberende proteïen (PGIP), het interaksie met en inhibeer EPGs en gevolglik ook die nekrotiserende aksies van B. cinerea. Uit die literatuur is dit duidelik dat spesifieke inligting aangaande individuele interaksies van fungiese EPGs met PGIPs tans nog ontbreek. Verder word daar op in vitro metodologie staatgemaak wannneer die effekte van EPGs asook die interaksie en inhibisie met PGIPs bestudeer word, sonder om die konteks van die in vivo- of in planta-omgewing in ag te neem. Die fokus van hierdie studie was om aspekte van individuele EPG:PGIP interaksies, asook die moontlike gebruik van in vivo metodologie te bestudeer deur EPGs, afkomstig van ’n hoogs virulente Suid-Afrikaanse ras van B. cinerea en die wingerd VvPGIP1, wat vroeër in ons laboratorium geïsoleer is, te gebrruik. Hierdie PGIP wat uit Vitis vinifera cv Pinotage geïsoleer is, inhibeer ’n kru EPG-ekstrak van bogenoemde ras baie effektief. Die benadering wat gevolg is het op die ooruitdrukking van die individuele Bcpg-gene in heteroloë sisteme staatgemaak en die gevolglike isolering van suiwer en aktiewe ensieme om EPG-inhibisie deur VvPGIP1 te beoordeel. Al die gene is suksesvol in Saccharomyces cerevisiae ooruitgedruk onder ’n sterk induseerbare promotor, maar volgens ’n agarose-diffundeerbare toets kon aktiewe ensiempreparate slegs vir die enkoderende Bcpg2 verkry word. Die in vitro PGIP-inhibisie toets is ook op die gemelde toets gebasseer en vereis EPG-aktiwiteit om die inhiberende effek van die PGIP, te visualiseer. Die aktiewe EPG2 is egter nie deur VvPGIP1 geïnhibeer met die aanleg van hierdie toets nie. Die EPG-enkoderende gene van B. cinerea is ook tydelik in Nicotiana benthamiana ooruitgedruk deur gebruik te maak van ’n Agrobacterium-infiltrasietegniek. Transgeenuitdrukking kon met die Noordelike kladtegniek bevestig word en EPG-verwante simptome is vyf tot agt dae na infiltrasie waargeneem. Verskillende simptome vir die verskillende EPGs is waargeneem, wat aanduidend is dat die simptome nie lukrake gevolge van die infiltrasies, of ’n hipersensitiewe respons is nie. Verder kon die simptome wat EPG2 vertoon het, gekorreleer word met dié wat onlangs deur ’n ander groep op dieselfde gasheer waargeneem is. Ten spyte van die ekspressiedata en die waargenome simptome, kon aktiewe ensiempreparate op die agarose-diffundeerbare toets, weereens slegs vir EPG2 waargeneem word. ’n Metode wat gebasseer is op chlorofilfluoressensie is getoets en geëvalueer as ’n moontlike in vivo metode om EPG aktiwiteit en inhibisie deur PGIPs waar te neem en te kwantifiseer. Die resultate het bevestig dat die ooruitdrukking van hierdie gene die elektronvloeitempo deur fotosisteem II verminder het wat ’n aanduiding is dat metaboliese stres in die plant heers. Dieselfde tegniek is gebruik om die moontlike interaksies tussen BcPG1 en 2 en VvPGIP1 te bestudeer en het aangetoon dat die mede-uitdrukking van die Vvpgip1-geen aanleiding gee tot ’n beskermende effek van die geinfiltreerde weefsel, wat aanduidend is van inhibisie van EPG1 en 2 deur VvPGIP1. In die geval van EPG2 is hierdie interaksie en moontlike inhibisie met ’n PGIP die eerste waarneming in die verband. In ’n verdere poging om die in planta-interaksie tussen VvPGIP1 en die EPGs van die Suid-Afrikaanse B. cinerea ras uit te klaar, is ’n plantgebasseerde twee-hibriede toets aangelê, maar geen klinkklare resultate kon verkry word nie. Vorige werk het bevestig dat verskeie natuurlike mutasies in PGIP-enkoderende gene, afkomstig van verskillende V. vinifera kultivars, voorkom. Hierdie resultaat en die feit dat hierdie mutasies verskille in EPG inhibisie en uiteindelik vatbaarheid vir siektes kan beïnvloed, het aanleiding gegee tot die isolering van ’n verdere 37 PGIP-enkoderende gene uit ‘n verskeidenheid druifplantgenotipes, sommige waarvan juis bekend vir hul weerstand teen patogene is. Die gekombineerde resultate wat in dié studie verkry is, maak ’n waardevolle bydrae tot die verstaan van plant-patogeeninteraksies, spesifiek met die modelering van interaksies van patogeen- en plantgebasseerde proteïene. Die moontlikheid om in vivo-metodes soos chlorofilfluoressensie te gebruik in in planta-analises, is besonder bemoedigend om in vitro-resultate te versterk en ook in konteks te plaas.

Thesis (PhD(Agric))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Despite adherence to fungicide spray schedules and label recommendations, table and wine grape producers invariably suffer crop losses when environmental conditions are conducive to fruit and foliar pathogens. Registered fungicides are effective and poor control is often attributed to: 1) improper spray timing, 2) reduced sensitivity to fungicides in the pathogen populations, and 3) poor spray deposition. Spray timing, management of fungicide resistance and the epidemiology of Botrytis cinerea have been thoroughly researched under South African conditions on grape crops. However, limited research regarding spray deposition exists in South Africa, probably due to a lack of proper spray deposition assessment protocols. To determine minimum spray deposition quantity and quality levels needed for effective B. cinerea control, bunches and leaves of table (Waltham Cross) and wine grapes (Chenin blanc) were sprayed at various stages using different volumes with a precision spray gun. A deposition assessment protocol using fluorometry, photomicrography and digital image analyses was improved. Deposition values correlated favourably with Botrytis infection. Increasing spray volume increased spray deposition; however, at a certain point, deposition quality remained constant and B. cinerea infections did not decrease significantly with increasing spray volume, indicating the importance of both spray deposition quantity and quality. Fluorescent pigment area that effected 75% control of B. cinerea infection (FPC75 values) was calculated for leaves, pedicels and receptacles at different growth stages. The FPC75 values obtained in this study can be used as benchmarks to evaluate future spray application. In order to study the optimisation of spray deposition with existing application technology (air blast and air shear sprayers) in commercial vineyards, spray deposition quantity and quality values were assessed from leaves and structural bunch parts of wine (Chenin blanc) and table grapes (Waltham Cross) and compared with FPC75 values. Spray trials were conducted at different growth stages at current best-practice recommendations, and with a range of spray volumes but with spray mixture concentration amended accordingly (i.e. fixed dosage per hectare). Spray trails indicated that deposition levels following current best-practice spray application were sub-optimal to control B. cinerea infections on bunches and leaves. Deposition values between air blast and air shear sprayers were generally similar. The air blast sprayer resulted in higher deposition levels with diluted spraying and increased spray volume; however, when dosage per hectare was kept constant, no significant differences were calculated between spray volumes (250-1000 L/ha), indicating that this sprayer can as effectively but more efficiently be used at lower spray volume. The air shear were not as efficient at higher spray volumes (>500 L/ha), but was superior at low volume concentrate application (≈250 L/ha at 4× concentration). This study clearly demonstrated the efficacy and cost-saving potential in optimising spray application with respect to application technology. / AFRIKAANSE OPSOMMING: Wingerdprodusente kan oesverliese ondervind indien omgewingstoestande bevorderlik is vir swampatogene. Siektes word onvoldoende beheer ten spyte van die nakoming van korrekte swamdoder aanbevelings. Geregistreerde swamdoders is effektief, mits die vatbare plantdele voldoende spuitbedekking ontvang. Onvoldoende siekte beheer kan gewoonlik toegeskryf word aan: 1) verkeerde spuit tydsberekening, 2) vermindere sensitiwiteit in patogeen-populasies teen swamdoders, en 3) swak spuitbedekking. Spuit tydsberekening, die bestuur van weerstand teen swamdoders en die epidemiologie van Botrytis cinerea is deeglik onder Suid-Afrikaanse toestande nagevors. Nietemin is daar beperkte navorsing oor spuitbedekking, waarskynlik weens 'n gebrek aan behoorlike spuitbedekking assesseringsprotokol. Om te bepaal hoeveel spuitbedekking (% area bedek deur fluoresserende pigment) nodig is om 75% van B. cinerea infeksies (FPC75 waardes) op vatbare wingerddele te beheer, is druiwetrosse en blare van tafel- en wyndruiwe (Waltham Cross en Chenin blanc, onderskeidelik) op verskillende groei stadiums en spuitvolumes in die laboratorium gespuit. ‘n Assesseringsprotokol van spuitbedekking op vatbare druifdele en blare is ontwikkel deur gebruik te maak van fluorometrie, fotomikrografie en digitale beeldanalise. Spuitbedekking het goed met Botrytis infeksies gekorreleer. Toenemende spuitvolume het bedekking laat toeneem, maar egter net tot 'n sekere punt, waar die kwantiteit van die bedekking nog toegeneem het, maar die kwaliteit van bedekking en B. cinerea infeksies nie beduidend toegeneem het nie. Dit is ‘n aanduiding van die belangrikheid van beide die kwantiteit en kwaliteit van spuitbedekking. Die FPC75 waardes wat in hierdie studie verkry is, kan as drempelwaardes om toekomstige spuittoediening te evalueer, gebruik word. Ten einde spuitbedekking met bestaande tegnologie (druk en waaierpomp spuitmasjiene) te optimiseer, is kommersiële wyn- en tafeldruiwe (Chenin blanc en Waltham Cross, onderskeidelik), volgens huidige spuit aanbevelings vir wingerde tydens verskillende groeistadiums en met ‘n reeks van verskillende spuitvolumes gespuit. Die konsentrasie van die spuitmengsel is dienooreenkomstig gewysig, i.t.v. ‘n vaste dosis per hektaar ongeag die spuitvolume. Bedekkingswaardes is met FPC75 waardes vergelyk en het aangedui dat kommersiële spuit aanbevelings aan produsente sal lei tot sub-optimale beheer van B. cinerea op beide blare en druiwetrosse. In die algemeen was bedekkingswaardes vir beide druk- en waaierpomp spuitmasjiene soortgelyk. Vir die waaierpomp teen verskillende spuitvolumes en aanbevole konsentrasie het ‘n toename in spuitvolumes tot hoër beddekingswaardes gelei, maar indien die dosis per hektaar van die spuitmengsel konstant behou is, is geen betekenisvolle verskille tussen spuitvolumes (250-1000 L/ha) voorspel nie. Hierdie dui aan dat die waaierpomp net so doeltreffend, maar meer effektief teen laer spuitvolumes gebruik kan word. Die drukpomp was nie so doeltreffend teen hoër spuitvolumes (> 500 L/ha) nie, maar was aansienlik beter by lae volume konsentraat toediening (≈ 250 L/ha op 4 × konsentrasie). Die studie toon duidelik die doeltreffendheid en moontlike kostebesparing moontlikhede deur bespuiting relatief tot bespuitingstegnologie te optimiseer. / Department of Plant Pathology, National Research Foundation, THRIP, Deciduous Fruit Producers’ Trust, Winetech, Bayer, BASF, Dow Agrosciences, DuPont, Syngenta, Nexus, Terason, UAP and Wenkem for financial assistance

Greenhouse and laboratory experiments in 2011 determined that Clonostachys rosea can effectively prevent Botrytis cinerea infection in Vaccinium angustifolium blossoms. In vitro testing demonstrated that C. rosea germination was not significantly affected by the presence of Switch®, but was by either Pristine® or Maestro®. Field experiments completed during the summer of 2010 and 2011 indicated that the dispenser designs tested had no significant effects on Bombus impatiens foraging behaviours, aside from hive-activity. There was also no difference in the quantity of C. rosea applied by each to bees, the distribution of product in the field, or for blossoms exposed to bees from each dispenser to resist infection by B. cinerea. However, B. cinerea prevalence in blossoms from both treatments was significantly different from the control, with infection reduced by 10-20%. Technical issues with dispensers currently appear to be the limiting factor for application within commercial wild blueberry production.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 23, 2010). Thesis advisor: Dr. Mengshi Lin. Includes bibliographical references.

O eucalipto participa de modo importante na silvicultura brasileira pela sua adaptabilidade, rápido crescimento e produtividade. Além disso, possui outras características como qualidade, diversidade e adequação de sua madeira para a indústria. A continuidade dos reflorestamentos com eucalipto demanda uma produção contínua de mudas. Entretanto, nos viveiros estas podem ser atacadas por doenças, como o mofo-cinzento e o oídio, causados por Botrytis cinerea e Oidium sp., respectivamente, principais doenças fúngicas que ocorrem na região Sul. O controle destas doenças é feito com fungicidas em outras culturas e em eucalipto o seu uso não é recomendado pela falta de produtos registrados. Além disso, podem surgir efeitos indesejáveis, como a poluição ambiental e a intoxicação do homem e de animais. O objetivo deste trabalho foi o estudo e a seleção de produtos eficientes para o controle alternativo dessas doenças. Para a execução dos experimentos foram utilizadas mudas de Eucalyptus dunnii nos experimentos com B. cinerea e mudas de E. benthamii com Oidium sp. Estas foram pulverizadas com fungicidas (parâmetro de controle), produtos químicos não fungicidas, óleos essenciais, extratos de plantas, leite e derivados e microrganismos. De cada um destes grupos foi selecionado o tratamento com menor valor de severidade, que foram testados entre si. A avaliação consistiu da medição da severidade das doenças, que foi determinada por meio de escalas descritivas de 0 (ausência de sintomas) a 4 (sintomas muito severos), específicas para cada patógeno. Verificou-se, em testes preliminares, os menores valores de severidade das doenças para o tanino e Mentha x villosa no controle do mofo -cinzento e para o leite de vaca e Lecanicillium sp. no controle do oídio. No experimento final, os produtos alternativos que apresentaram os menores valores de severidade foram: tanino controlando o mofo-cinzento e leite de vaca e Lecanicillium sp. controlando o oídio

Les vignerons du Val de Loire souhaitent faire évoluer les différents types de vins liquoreux produits actuellement dans leurs appellations en lien avec l’élaboration d’une qualité de raisin correspondant à ces types. Pour cela, il est nécessaire de caractériser objectivement les baies de raisin et leur qualité.Notre étude porte sur le développement d’une méthode multicritères combinant des mesures physiques et biochimiques des raisins, permettant d’identifier et de caractériser différents niveaux de développement de la pourriture noble sur raisins de Chenin blanc dans un objectif de production de vins liquoreux.Pour cela, des raisins correspondant à trois niveaux de pourriture noble ont été sélectionnés, lors de deux millésimes, 2012 et 2013. Des analyses classiques de qualité du raisin ont été réalisées et complétées par des analyses de couleur, de texture et de composés phénoliques.Outre la caractérisation phénolique du Chenin (sain et botrytisé), cette étude met en évidence la présence de myricétine, que l’on retrouve traditionnellement dans les variétés de raisin rouge, et qui semblerait être aussi synthétisée dans les baies de raisins blancs fortement atteintes par la pourriture noble.Nous avons également montré qu’une analyse multicritères permet une très bonne différenciation des baies plus ou moins botrytisées. Une combinaison des teneurs en myricétine, astilbine et glycérol permet notamment une très bonne discrimination des baies en fonction de leur niveau de botrytisation. Enfin, nous avons détectés de nouveaux composés dans les baies suite à l’attaque fongique, qui pourraient provenir de réactions d’oxydation. Ces résultats restent toutefois à confirmer. / Winemakers from the Loire valley are willing to improve the various types of sweet wines currently produced in their designation of origin altogether with the elaboration of grape quality that corresponds to these wines. For that purpose, it is necessary to objectively characterize the grapes and their quality. Our study implies the development of a multcriteria method that combines physical and biochemical measures on the grape, allowing the identification and characterization of the different levels of noble rot development on Chenin blanc grapes for the production of sweet wines. To do so, grapes corresponding to three noble rot levels were selected during two vintages, 2012 and 2013. Classical analysis of grape quality were carried out and complemented with color, texture and phenolic composition analysis. Besides the phenolic characterization of Chenin (healthy and botrytised), this study revealed the presence of myricetin, that is traditionally detected on red grape varieties, and that seems to be also synthetized on white grapes strongly affected by noble rot. We have also showed that a multicriteria analysis brings a very good differentiation of grapes more or less botrytised. A combination of myricetin, astilbin and glycerol concentrations showed a significantly good discrimination of grapes according to botrytisation level. Finally, we have detected new compounds on the grapes after the fungal attack that could be the result of oxidative reactions. These results are to be confirmed

Botrytis bunch rot, caused by Botrytis cinerea, is a fungal disease that primarily affects the fruit of wine grapes. Infection of fruit consequently results in reduced yields and wine quality. These factors lead to significant economic losses for growers which prompts the implementation of management practices to control the disease. One objective of this study was to evaluate the level of resistance that populations of B. cinerea in the Central Coast region showed to various chemicals. A fungicide assay was conducted to determine resistant phenotypes to six fungicide active ingredients (pyrimethanil, iprodione, fenhexamid, fludioxonil, trifloxystrobin, boscalid). Thirty-five (2020) and 88 (2021) B. cinerea isolates were collected from Santa Maria, Cambria, Paso Robles, and Edna Valley in California and screened for resistance. The frequencies of populations (2020, 2021) showing resistance to each active ingredient were: pyrimethanil (94.3%, 81.8%), trifloxystrobin (97.1%, 100%), boscalid (77.1%, 77.3%), fenhexamid (8.6%, 25%). The majority of isolates were sensitive to iprodione (100%, 100%), fludioxonil (100%, 100%), fenhexamid (88.6%, 75%), and boscalid (22.9%, 22.7%). These results documented the accumulation of resistance in B. cinerea to various fungicides commonly used for Botrytis bunch rot management in California’s Central Coast. Another objective of this study was to determine the effective concentration of the six fungicides that reduces mycelial growth of the fungus by 50% (EC50). Seven B. cinerea isolates in 2020 and ten isolates in 2021 were selected and subjected to a sensitivity screening with serial dilutions of the different fungicide active ingredients. The fungicides found to have the highest EC50 values indicating reduced efficacy for inhibiting B. cinerea growth were Scala® (FRAC 9), Flint® (FRAC 11), and Endura® (FRAC 7). The fungicides found to have the lowest EC50 values indicating higher efficacy for inhibiting B. cinerea growth were Scholar® (FRAC 12) and Rovral® (FRAC 2). The results from this study provided information regarding the accumulated resistance of B. cinerea populations to certain chemical groups and therefore the efficacy of different fungicide active ingredients. This information can be utilized by growers as a tool to enhance and develop fungicide spray programs that effectively manage Botrytis bunch rot in Central Coast vineyards.