Masters Degrees (Plant Pathology)

Singular and combined effect of postharvest treatments on viability and reproductive ability of phyllosticta citricarpa infections.

Wed, 01 Mar 2017 00:00:00 GMT

Singular and combined effect of postharvest treatments on viability and reproductive ability of phyllosticta citricarpa infections. Schreuder, Wouter ENGLISH ABSTRACT: Citrus is one of the most important fruit crops globally and is currently being produced in over 100 countries. South Africa is one of the biggest shipping exporters of fresh citrus, with approximately 40% exported to European markets. Packhouses have rigorous export quality control programmes to maintain quality and prevent postharvest decay during the shipping period. Citrus black spot (CBS) (caused by Phyllosticta citricarpa (McAlpine) van der Aa) is mostly a cosmetic disease that reduces the aesthetic quality of fruit and does not cause postharvest decay. However, P. citricarpa is regarded as a quarantine organism in certain countries, and despite scientific evidence to the contrary, trade restrictions are imposed, such as the zero tolerance for CBS lesions on fruit exported to European Union. Whilst fruit may be exported from areas where CBS occurs, very strict preharvest control programmes must be followed to ensure fruit production in orchards meet the zero tolerance requirements. The biggest danger surrounding CBS is the presence of latent, asymptomatic infections in harvested and packed fruit, which can sometimes manifest on the fruit long after packhouse treatment, cold storage and shipping. Previous studies have indicated that postharvest treatments delay symptom expression and control CBS by reducing lesion and pycnidiospore viability. The objective of this study was to evaluate the effect of more recent protocols and fungicides used in packhouses, as well as alternative fungicides, against latent CBS infections, including the reproductive potential of the lesions. Fruit with CBS lesions, as well as asymptomatic fruit with latent infections, were subjected to standard packhouse sanitation, fungicide treatment and cold storage (singularly and combined), and incubated at conditions that enable expression of latent infections. The full packhouse treatment along with storage period gave significantly control of latent infections. The over all reproductive ability of lesions were very low, with less than 2.1% of all lesion that formed on both Valencia’s and Eureka lemons developing pycnidia. Three alternative single treatments showed potential to control latent infections: FLU, potassium sorbate and Propirly 270 EC (PPZ + PYR). Treatment with (respectively) FLU and Propirly 270 EC resulted in moderate to significant control of latent infections on both Valencia oranges and Eureka lemons. Potassium sorbate moderately controlled latent CBS infections in both Valencia oranges and Eureka lemon trials. The combined epidemiological requirements for pycnidiospore release along with results from trials conducted in the current study indicate that harvested fruit is not an epidemiologically significant pathway for the spread of CBS.; AFRIKAANSE OPSOMMING: Sitrus is wêreldwyd een van die belangrikste vrugtegewasse en word tans geproduseer in meer as 100 lande. Suid-Afrika is een van die grootste uitvoerders van vars sitrus, met ongeveer 40% van die produksie uitgevoer na Europese markte. Pakhuise is onderworpe aan streng programme om uitvoergehalte te beheer deur die handhawing van hoë vrugkwaliteit en voorkoming van na-oes vrugbederf. Sitrus swartvlek (SSV) (veroorsaak deur Phyllosticta citricarpa (McAlpine) van der Aa) is hoofsaaklik 'n kosmetiese siekte wat die uitvoerkwaliteit van vrugte verswak. Alhoewel dit nie na-oes bederf veroorsaak nie, word dit egter in sommige lande beskou as ‘n kwarantyn organisme. Ten spyte van wetenskaplike bewyse tot die teendeel, word handelsbeperkinge teen SSV vrugte steeds opgelê, byvoorbeeld die nul toleransie van SSV letsels op vrugte uitgevoer na die Europese Unie. Produksie areas waar SSV voorkom volg streng vooroes programme om te verseker dat vrugte uit sulke boorde aan hierdie nul toleransie vereistes voldoen. Die grootste gevaar rondom swartvlek is die teenwoordigheid van latente, asimptomatiese infeksies op geoesde en verpakte vrugte Sulke infeksies word soms eers uitgedruk lank na na-oes behandeling, verkoeling en verskeping van die vrugte. Vorige studies het aangedui dat na-oes behandelings simptoomuitdrukking vertraag, met gevolglike swartvlekbeheer deur die vermindering van letseluitdrukking en die lewensvatbaarheid van piknidiospore. Die doel van hierdie studie is om die meer onlangse pakhuis protokolle en swamdoders, asook alternatiewe swamdoders, te evalueer vir hul uitwerking teen latente swartvlek infeksies en hul effek op die reproduktiewe potensiaal van letsels. Vrugte met swartvlekletsels, asook asimptomatiese vrugte met latente infeksies, was onderwerp aan standaard pakhuissanitasie, swamdoderbehandelinge en koelkameropberging (alleenstaande en gekombineer). Daarna was dit geïnkubeer onder omstandighede wat simptoomuitdrukking bevoordeel. Die volledige pakhuisbehandeling, tesame met ‘n kouestoor tydperk, het aansienlike beheer van latente infeksies gegee. Oor die algemeen was die voortplantingsvermoë van letsels baie swak, met minder as 2.1% van alle letsels op beide Valencia en Eureka suurlemoene wat uiteindelik piknidia ontwikkel het. Drie alternatiewe alleenstaande behandelings het potensiaal getoon vir die beheer van latente infeksies: FLU, kaliumsorbaat and Propirly 270 EC (PPZ + PYR). FLU en Propirly 270 EC het matige tot aansienlike beheer oor latente infeksies op beide Valencia lemoene en Eureka suurlemoene uitgeoefen. Kaliumsorbaat het matige beheer oor latente swartvlek infeksies op beide Valencia lemoene en Eureka suurlemoen uitgeoefen. Die kombinasie van epidemiologiese vereistes vir piknidiospoor vrystelling, tesame met die resultate van hierdie proewe, dui daarop dat ge-oesde vrugte is nie 'n noemenswaardige epidemiologiese weg bied vir die verspreiding van swartvlek nie. Thesis (MSc)--Stellenbosch University, 2017.

Further optimisation of in-line aqueous application of imazalil to control citrus green mould caused by Penicillium digitatum

Wed, 01 Mar 2017 00:00:00 GMT

Further optimisation of in-line aqueous application of imazalil to control citrus green mould caused by Penicillium digitatum Savage, Catherine ENGLISH ABSTRACT: South Africa has a successful citrus export industry. A threat to fresh citrus fruit is the fungal pathogen Penicillium digitatum causing green mould. Imazalil (IMZ) is the most important fungicide to combat green mould. Solution pH and temperature, and exposure time of the fruit to the solution, are important when using the sulphate form of IMZ. Research has increased our understanding of IMZ use, but further variables need to be investigated, along with an alternative application method. The control of green mould infection and sporulation by IMZ were tested using a heated flooder. Solution variables included the effects of pH (3; 4; 5; 6), temperature (45; 55; 65°C), and concentration (250 or 500 μg.mL-1) in a time of 8 s. Residues increased with increasing pH, temperature and concentration. The majority average residues loaded were between 0.4 and 3.0 μg.g-1. Treatments at pH 6 loaded higher residues at 55 and 65°C, where the maximum residue limit (MRL) of 5 μg.g-1 was almost always exceeded. The flooder loaded adequate residues, offering good curative and protective control. Sporulation inhibition of green mould was also linked to residues, and complete inhibition was achieved at the higher residue levels. The flooder was an effective applicator of IMZ. The fungicide bath is the most common IMZ application method in South Africa. The ability of IMZ to control green mould was investigated in a cold bath of 10°C and compared to ambient temperature and 35°C baths. Solution temperature had no significant effect on IMZ’s ability to cure 24 hr old green mould infections with all temperatures providing control above 80%. Sporulation inhibition and residue loading increased as solution pH, temperature, and exposure time increased. Sporulation inhibition was < 50% in pH 3 baths, irrespective of temperature, complete inhibition was obtained at 35°C and pH 6, but the IMZ MRL was exceeded at longer exposure times (> 45 s). The survival of Rhizopus stolonifer was studied in vitro at various water temperatures (10°C to 65°C) for exposure times of 1 or 60 min, and after a pasteurisation step. Sub-treatments included the addition of IMZ fungicide or green mould spores, with IMZ seemingly having a significant effect on Rhizopus spore survival. The same was not true for solutions at temperatures below 35°C, however, temperatures of 45, 55 and 65°C, particularly after a 60 min exposure, caused a significant reduction in Rhizopus spore viability. Complete Rhizopus eradication was achieved with 65°C and the pasteurisation step. In order to control fungal contaminants in the fungicide bath, packhouses need to apply IMZ in heated solutions (circa 45°C) and/or pasteurize fungicide baths overnight. Imazalil residue levels on citrus can be increased by increasing solution pH, temperature, concentration or exposure time. Most treatments gave excellent infection control and only a low residue is necessary to cure or prevent a green mould infection. Residue levels were closely linked to the level of sporulation inhibition achieved. Both the flooder and dip tank offered good green mould control. Contaminants that build up in solution can be eradicated at high temperatures.; AFRIKAANSE OPSOMMING: Suid Afrika het ‘n suksesvolle sitrusbedryf. Penicillium digitatum, die swampatogeen wat groenskimmel veroorsaak, is ‘n bedreiging vir vars sitrusvrugte. Imazalil (IMZ) is die belangrikste swamdoder in die bekamping van hierdie patogeen. Die pH en temperatuur van die oplossing, asook blootstellingstyd van die vrugte aan die oplossing is belangrik wanneer die sulfaat vorm van IMZ gebruik word. Navorsing het ons kennis van IMZ verbreed, maar verdere ondersoek van toepaslike veranderlikes is nodig, asook ‘n alternatiewe aanwendingsmetode. Die beheer van groenskimmelinfeksies en sporulasie deur IMZ na aanwending met ‘n verhitte vloedtoediener is ondersoek. Verskillende oplossingsveranderlikes het ingesluit pH (3; 4; 5; 6), temperatuur (45; 55; 65°C) en konsentrasie (250 of 500 μg.mL-1), na ‘n blootstellingstyd van 8 s. Residue het toegeneem met toenemende pH, temperatuur en konsentrasie. Die meeste residuwaardes was tussen 0.4 en 3.0 μg.g-1. Behandelings by pH 6 het hoër residue by 55 en 65°C gelaai, met die maksimum residulimiet (MRL) van 5 μg.g-1 omtrent deurgaans oorskry. Residulading deur die vloedtoediener was genoegsaam en het goeie genesende, sowel as beskermende beskerming verleen. Sporulasie inhibisie van groenskimmel was ook gekoppel aan residulading, met volledige inhibisie teen hoër residuladings. Die vloedtoediener gee effektiewe toediening van IMZ. Die swamdoderbad is die mees algemene IMZ toedieningsmetode in Suid Afrika. Die vermoë van IMZ om groenskimmel te beheer in ‘n koue bad teen 10°C is ondersoek en vergelyk met baddens teen omgewingstemperatuur en 35°C. Die oplossingstemperatuur het geen noemenswaardige effek gehad op die vermoë van IMZ om 24 uur-oue groenskimmel infeksies te beheer nie, met alle temperature wat tot meer as 80% beheer gelei het. Sporulasie inhibisie en residulading het toegeneem met toenemende pH en temperatuur van die oplossing, asook blootstellingstyd. Sporulasie inhibisie in pH 3 baddens was < 50%, ongeag die temperatuur, met volledig inhibisie behaal teen 35°C en pH 6, alhoewel die IMZ MRL oorskry is teen langer blootstellingstye (> 45 s). Die in vitro oorlewing van Rhizopus stolonifer is bestudeer teen verskeie watertemperature (10°C to 65°C) en vir blootstellingstye van 1 of 60 minute, asook nà ‘n pasteurisasie stap. Tussenbehandelings het die byvoeging van òf IMZ òf groenskimmelspore ingesluit, met IMZ wat oënskynlik ‘n noemenswaardige effek het op spooroorlewing gehad het. Dit het nie gegeld vir oplossings onder 35°C nie, maar temperature van 45, 55 en 65°C met veral 60 min blootstelling het ‘n noemenswaardige verlaging in Rhizopus spooroorlewing tot gevolg gehad het. Volledige uitwissing van Rhizopus is behaal met 65°C en die pasteurisasie stap. Ten einde swamkontaminasie in die swamdoderbad te beheer, behoort pakhuise IMZ in verhitte oplossings (circa 45°C) aan te wend en/of moet hulle oornag pasteuriseer. Imazalil residuvlakke op sitrus kan verhoog word met verhoging van die oplossing se pH, temperatuur, konsentrasie of verlenging van blootstellingstyd. Die meeste behandelings gee uitstekende infeksiebeheer en slegs ‘n lae residuwaarde is voldoende om ‘n groenskimmelinfeksie te genees of voorkom. Sporulasie inhibisie, daarteenoor, is nòù gekoppel aan die residuvlakke. Beide die verhitte vloedtoediener en die swamdoderbad het goeie groenskimmelbeheer gegee. Die opbou van kontaminante in die oplossing kan uitgewis word by hoë temperature. Thesis (MSc)--Stellenbosch University, 2017.

Optimisation of postharvest drench application of fungicides on citrus fruit

Tue, 01 Mar 2016 00:00:00 GMT

Optimisation of postharvest drench application of fungicides on citrus fruit Christie, Charmaine ENGLISH ABSTRACT: South Africa is the 2nd largest exporter of fresh citrus, after Spain, worldwide. Delays to the packline, i.e. degreening, can result in substantial postharvest decay such as green mould caused by Penicillium digitatum (PD). Pre-packline aqueous fungicide drench application is an important tool to minimize postharvest losses before degreening, which provides a favourable environment for infection. Sour rot, caused by Geotrichum citri-aurantii (GC), becomes an infection risk after rainfall and the availability of effective fungicides against this pathogen is limited. Thiabendazole (TBZ), pyrimethanil (PYR), guazatine (GZT) and 2,4-dichlorophenoxyacetic acid (2,4-D) are applied during drenching in South Africa for the control of postharvest diseases on citrus, although this application has not yet been standardized and guazatine use is restricted to certain export markets; GZT is the only fungicide in the drench mixture that is effective against sour rot. Therefore the aim of this study was to improve our understanding of drench application in terms of the influence of infection age, fruit orientation (pole), treatment exposure time and the addition of adjuvants and sanitisers on disease control. Lemon, Satsuma mandarin and navel orange fruit were drenched with TBZ and PYR (1000 μg.mL-1 each) at different exposure times (14 s, 28 s and 56 s) and inoculated with PD 0, 6, 12, 18, 24, 30, 42, 48 and 54 h before (curatively) and 24 h after (protectively) treatment. Sporulation inhibition and residue loading were evaluated. Lemon and Satsuma mandarin fruit were exposed to a lower drench volume compared to navel orange fruit (26.5 and 64.3 L.min⁻¹, respectively). Batch differences played a significant role in green mould control with lemon and Satsuma mandarin fruit requiring treatment by 33.1 to 44.5 h and 23.8 to 32.1 h infection age, respectively, to gain 90% control. Exposure time only became significant with ≥ 30 h old infections on navel orange fruit at the higher drench volume used, with control declining more rapidly for fruit drenched at shorter exposure times. Control on navel orange fruit differed as much as 30.2% between exposure times with 54 h old infections and > 90% control was achieved by drenching fruit before 27 h. Protective control was generally effective (> 90%). These results support the proposition to drench all citrus types ≤ 24 h in order to reduce the risk for green mould decay development as sporulation inhibition was poor (< 50%) and fruit batches differed as much as 8 to 12 h in infection age for similar control levels. Valencia orange fruit were drenched with TBZ, PYR and 2,4-D (1000, 1000 and 250 μg.mL-1, respectively; calyx-end facing upward, sideways and downwards) at 41.0 L.min⁻¹ for 18 s with different adjuvant concentrations (0.0, 0.025, 0.05, 0.1 and 0.2 μl.mL⁻¹). Almost no differences were evident between concentrations, other than a negative effect on residue loading, deposition quantity and green mould control at the highest adjuvant concentration tested. Fruit orientation was however significant, with fruit facing calyx-end upward resulting in higher residue levels, curative green mould control, deposition quantity and quality compared to the stylar-end. Since sour rot inoculum levels can accumulate in the drench solution with dirt from fruit during drenching, Chlorine (Cl; 80 μg.mL-1) and hydrogen peroxide/peracetic acid (HPPA; 0.6%) efficacy was compared for the control of GC spores (CFU.mL-1) in solution without reducing fungicide persistence and efficacy. Wounded navel orange fruit were drenched with TBZ, PYR, GZT and 2,4-D (1000, 1000, 500 and 250 μg.mL-1, respectively) during commercial packhouse trials with Cl or HPPA (80 μg.mL-1 and 0.6%, respectively) used as shock treatments at each bin stack (two bins) containing bin no. 1, 50, 100 and 150. Fungicide persistence and green mould infection (environmental inoculum) was similar regardless of whether sanitisers were present or not. Green mould infection increased by bin 150 (4.6 – 5.4% difference). Different sanitiser concentrations (0, 20, 40, 60 and 80 μg.mL-1 Cl or 0.00, 0.01, 0.10, 0.30 and 0.60% HPPA) were combined with a mixture of TBZ, PYR and 2,4-D (1000, 1000 and 250 μg.mL-1, respectively) and GC spores (≈ 3.175 × 104 spores.mL-1) for 1, 3 and 60 min exposure during in vitro trials. Fungicide concentration was generally not influenced by sanitisers although sanitisers, however, did not persist after 60 min in solution exposed to fungicides. Only HPPA could completely reduce sour rot inoculum (0.0 CFU.mL-1) after 1 – 3 min as Cl was not as effective at the high pH levels (> 10) of the solution. During in vivo trials, green mould inoculated (24 h before treatment) and wounded fruit were drenched with TBZ, PYR and 2,4-D (1000, 1000 and 250 μg.mL-1, respectively) and GC spores (similar to in vitro trials) containing either 80 μg.mL-1 Cl or 0.3% HPPA with the addition of 0, 500 or 1000 μg.mL-1 kaolin, used to simulate dust accumulation during drenching. Sanitiser addition mostly did not affect solution concentration and green mould control, although HPPA treatments improved sour rot control on Valencia and Nadorcott mandarin fruit and resulted in improved green mould control on Nadorcott mandarin fruit; the lower level of kaolin (500 μg.mL-1) tested in this study improved green mould and sour rot control in some cases. Timeous drench application (≤ 24 h) provides effective green mould control whereas exposure time and adjuvant concentration requires further investigation in order to improve fungicide retention and distribution throughout highly congested fruit bins. Since drench pH is not regulated, HPPA was superior to Cl at high pH levels (> 10) for reducing sour rot infection and inoculum levels in solution, although further research is required to determine shock treatment intervals (within 60 min) required and potential side effects.; AFRIKAANSE OPSOMMING: Suid-Afrika is wêreldwyd die tweede grootste uitvoerder van vars sitrus vrugte. Vertragings vanaf oes na die paklyn, vir onder andere ontgroening, kan lei tot aansienlike na-oes verliese, veral weens groenskimmel wat deur Penicillium digitatum (PD) veroorsaak word. ‘n Voor-paklyn stortstelsel is 'n belangrike instrument om na-oes verliese te beperk voor ontgroening, wat 'n gunstige omgewing vir infeksie ontwikkeling bied. Suurvrot, wat veroorsaak word deur Geotrichum citri-aurantii (GC), raak ‘n probleem in tye van hoë reënval en die beskikbaarheid van doeltreffende swamdoders teen hierdie patogeen is beperk. Thiabendazole (TBZ), pyrimethanil (PYR), guazatine (GZT) en 2,4-dichlorofenoksie-asynsuur (2,4-D) word aangewend in die voor-paklyn stortstelsels in Suid-Afrika vir die beheer van na-oes siektes op sitrus. Hierdie proses is nog nie gestandariseer nie en GZT gebruik is tot sekere uitvoer markte beperk; GZT is die enigste swamdoder wat effektief in die stortstelselmengsel teen suurvrot is. Die doel van hierdie studie was om die begrip van stortaanwending te verbeter in terme van die invloed van infeksie ouderdom, vrugoriëntasie, blootstellingstyd aan behandeling en die toevoeging van benatters en ontsmettingsmiddels om siektebeheer te verbeter. Suurlemoen, Satsuma manderyn en navel lemoen vrugte is gestort met TBZ en PYR (1000 μg.mL-1 elk) met verskillende blootstellingstye (14 s, 28 s en 56 s) en geïnokuleer met groenskimmel 0, 6, 12, 30, 42, 48 en 54 h voor (kuratief) en 24 uur na (beskermend) behandeling. Spoorvormingsinhibisie en residu-lading is ook geëvalueer. Suurlemoen en Satsuma manderyn vrugte is aan 'n laer stortingsvolume in vergelyking met navel lemoen vrugte blootgestel (26.5 en 64.3 L.min⁻¹, onderskeidelik). Vruglotverskille het ‘n beduidende rol in groenskimmel beheer met suurlemoen en Satsuma mandaryn vrugte gespeel, en behandelings van onderskeidelik 33.1 – 44.5 en 23.8 – 32.1 h oue infeksies was nodig om 90% beheer te kry, afhangende van die vruglot. Blootstellingstyd het eers beduidend geraak met ≥ 30 h ou infeksies in navel lemoen vrugte, met die hoër stortvolumes, met beheer wat vinniger afneem vir vrugte gestort met korter blootstellingstyd. Beheervlakke het verskil van 30,2% op 54 h oue infeksies en > 90% beheer is behaal op vrugte wat binne 27 h na infeksie behandel is. Beskermende beheer was oor die algemeen effektief (> 90%). Hierdie resultate ondersteun die aanbeveling om alle sitrus tipes ≤ 24 h na-oes te stort om so die risiko vir groenskimmel ontwikkeling te beperk. Spoorvorminginhibisie was in die algemeen swak (<50%). Om die effek van ‘n benatter te bepaal, is Valencia lemoen vrugte met TBZ, PYR en 2,4-D (1000, 1000 en 250 μg.mL-1, onderskeidelik) teen 41.0 L.min⁻¹ vir 18 s met verskillende benatter konsentrasies (0.0, 0.025, 0.05, 0.1 en 0.2 μl.mL⁻¹) gestort. Geen verskille is ondervind behalwe 'n negatiewe uitwerking op residu-lading, neerslag hoeveelheid en groenskimmel beheer teen die hoogste getoetste benatter konsentrasie. Vrug oriëntasie het egter ‘n beduidende rol gespeel, met hoër residu-vlakke, kuratiewe groenskimmel beheer, neerslag hoeveelheid en kwaliteit op vrugte wat kelk-end opwaarts gewys het, in vergelyking met die teenoorgestelde end van dieselfde vrug. Siende dat suurvrot inokulumvlakke in die stortstelsel oplossing saam met stof van vrugte tydens stortaanwending kan opbou, is chloor (Cl; 80 μg.mL-1) en waterstofperoksied / asynsuur (HPPA; 0,6%) se doeltreffendheid vir beheer van GC spore (CFU.mL-1) in oplossing vergelyk, sowel as om te toets dat die swamdoderkonsentrasie en doeltreffendheid daarvan nie verminder word nie. Gewonde navel lemoen vrugte is met TBZ, PYR, GZT en 2,4-D (1000, 1000, 500 en 250 μg.mL-1, onderskeidelik) gedurende kommersiële pakhuisproewe gestort, met Cl of HPPA (80 μg.mL-1 en 0.6%, onderskeidelik) wat toegedien is as skokbehandelings in die oplossing by elke vrugkratstapel (twee kratte) wat kratnommers 1, 50, 100 en 150 ingesluit het. Swamdoderbehoud en groenskimmel infeksie (vanweë omgewingsinokulum) was soortgelyk ongeag die eenwoordigheid van ontsmettingsmiddel. Groenskimmel infeksie het verhoog by krat 150 (4.6 – 5.4% verskil). Tydens in vitro proewe is verskillende ontsmettingsmiddel konsentrasies (0, 20, 40, 60 en 80 μg.mL-1 Cl of 0.00, 0.01, 0.10, 0.30 en 0.60% HPPA) met 'n mengsel van TBZ, PYR en 2,4-D (1000, 1000 en 250 μg.mL-1, onderskeidelik) en GC spore (≈ 3,175 × 104 spore.mL-1) gekombineer vir 1, 3 en 60 min blootstellingtyd. Swamdoderkonsentrasies is oor die algemeen nie beïnvloed deur ontsmettingsmiddels nie, maar ontsmettingsmiddels het egter nie in oplossing bly voortbestaan na 60 min blootstelling nie. HPPA kon suurvrot inokulum heeltemal uitwis (0,0 CFU.mL-1) na 1 – 3 min en Cl was nie so effektief in die hoë pH vlak (> 10) van die oplossing nie. Tydens in vivo proewe is groenskimmel geïnokuleerde (24 h voor behandeling) en gewonde vrugte gestort met ‘n mengsel van TBZ, PYR en 2,4-D (1000, 1000 en 250 μg.mL-1, onderskeidelik) en GC spore (soortgelyk aan in vitro proewe) wat 80 μg.mL-1 Cl of 0,3% HPPA bevat het, asook 0, 500 of 1000 μg.mL-1 kaolin. Die ontsmettingsmiddel het meestal geen negatiewe invloed op swamdoder konsentrasie en groenskimmel beheer gehad nie, alhoewel HPPA behandelings suurvrotbeheer op Valencia en Nadorcott manderyn vrugte verbeter het, asook verbeterde groenskimmelbeheer op Nadorcott manderyn vrugte. In sommige gevalle het die laer vlak van kaolin (500 μg.mL-1) gelei tot verbeterde groenskimmel en suurvrot beheer. Tydige stortbehandeling (≤ 24 h) lewer doeltreffende groenskimmel beheer, terwyl blootstellingstyd en benatter konsentrasie verder ondersoek moet word om swamdoderwerking en verspreiding deur dig-verpakte vrugkratte te verbeter. Met die wete dat die pH vlakke van stortstelseloplossings nie gereguleer word nie, is HPPA ‘n beter ontsmettingsopsie teen die hoë pH-vlakke (> 10) in stortoplossings. Verdere navorsing is nodig om skokbehandelingsintervalle en moontlike newe-effekte van ontsmettingsmiddels te bepaal. Behoorlike vermenging van oplossings is ook noodsaaklik vir verbeterde swamdodereenvormigheid in oplossing en die daaropvolgende residu-lading. Thesis (MSc)--Stellenbosch University, 2016.

A host-pathogen study of Fusarium Verticillioides in resistant and susceptible maize inbred lines

Sun, 01 Mar 2015 00:00:00 GMT

A host-pathogen study of Fusarium Verticillioides in resistant and susceptible maize inbred lines Vermeulen, Meagan ENGLISH ABSTRACT: Maize (Zea mays L.) is an important crop worldwide and forms the staple diet of many African countries including South Africa. Fusarium ear rot (FER) of maize is caused by a fungus, Fusarium verticillioides, which also produces the fumonisin mycotoxin group. The consumption of fumonisin contaminated maize grain has been associated with serious human and animal health complications. Several South African maize inbred lines exhibiting resistance to FER and fumonisin contamination have been identified. These locally adapted inbred lines could be used to generate mapping populations to identify QTLs associated with resistance to FER and fumonisin contamination. The corresponding markers could be utilised in breeding programmes through marker-assisted selection to ensure the development of commercial cultivars with resistance to FER and fumonisin contamination. In this study, resistant and susceptible maize inbred lines were utilised to commence the development of recombinant inbred line (RIL) populations for the mapping and validation of QTLs associated with FER and fumonisin resistance. One F2 population was phenotypically and genotypically analysed to produce a linkage map for the preliminary identification of QTLs associated with resistance to F. verticillioides infection and fumonisin deposition. A potential QTL for resistance to FER was detected and should be validated across several locations and years in the subsequent RIL population. Additionally, potential resistance barriers of maize to infection by F. verticillioides were investigated by histological studies. The importance of a closed stylar canal in determining resistance to FER was established for nine South African maize inbred lines by means of scanning electron microscopy (SEM). No significant association was observed between a closed stylar canal and the resistance/susceptible status of maize inbred lines before pollination, while the canals appeared closed in all inbred lines following pollination. The results suggest that the stylar canal architecture is not an essential factor determining resistance to F. verticillioides ingress in the maize inbred lines selected for this study. Furthermore, the possibility of resistance to FER and fumonisin contamination being initiated during the seedlings phase of a resistant and susceptible maize inbred line was investigated by means of confocal laser scanning microscopy (CLSM). Fusarium verticillioides growth originating from soil-borne or seed-borne contamination was monitored in various above and below soil plant tissues but no significant difference in the colonisation could be determined between resistant and susceptible maize seedlings. No fumonisin was produced regardless of the inoculation method or resistance status of the plant. These results suggests that the resistant and susceptible maize seedlings used in this study may not be resistant to systemic fungal ingress but may resist the deposition of fumonisins. The resistance associated with the resistant inbred line is not mediated during the seedling phase but potentially through structural and biochemical defence mechanisms during later plant developmental stages.; AFRIKAANSE OPSOMMING: Mielies (Zea mays L.) is ‘n belangrike graangewas wat wêreldwyd geproduseer word en dien as stapelvoedsel in talle Afrika-lande, insluitend Suid-Afrika. Fusarium kopvrot (FKV) in mielies word veroorsaak deur die swam, Fusarium verticillioides, wat ook die fumonisien mikotoksien groepe produseer. Die inname van fumonisien-geïnfekteerde mielies gaan gepaard met ernstige gesondheidsprobleme in mense en diere. Verskeie Suid-Afrikaanse ingeteelde mielielyne, wat weerstandbiedend is teen FKV en fumonisien kontaminasie, is voorheen identifiseer. Hierdie plaaslik-aangepaste teellyne kan gebruik word om kartering populasies te genereer om kwantitatiewe eienskap loci (KEL) te identifiseer wat verband hou met weerstandbiedenheid teen FKV en fumonisien kontaminasie. Die ooreenstemmende merkers kan gebruik word in teelprogramme deur gebruik te maak van merker-geassisteerde seleksie om kommersieële kultivars, wat weerstandbiedend is teenoor FKV en fumonisien kontaminasie, te ontwikkel. In hierdie studie is weerstandbiedende en vatbare mielie inteellyne gebruik om rekombinante inteellyn (RIL) populasies te begin ontwikkel vir die kartering en validasie van KEL’e geassosieer met FKV en fumonisien weerstandbiedenheid. Een F2 populasie was fenotipies en genotipies geanaliseer om ‘n koppeling-kaart te verwek vir die voorlopige identifikasie van KEL’e geassosieer met weerstandigheid tot F. verticillioides infeksie en fumonisein afsetting. ‘n Potensiële KEL vir weerstandbiedenheid is geïdentifiseer, wat verdere bevestiging in die daaropvolgende RIL populasie in verskeie geografiese areas en oor addisionele seisoene, benodig. Potensiële fisiese versperrings teen F. verticillioides tydens mieliesaad infeksie is ook ondersoek met behulp van histologiese studies. Die belangrikheid van ‘n geslote styl-kanaal vir weerstandbiedendheid teenoor FKV is bevestig in nege Suid-Afrikaanse inteellyne deur middel van skandeer elektron mikroskopie (SEM). Geen beduidende verwandskap tussen ‘n geslote styl-kanaal en die weerstandbiedenheid/vatbaarheid van die inteellyne voor bestuiwing is gevind nie, terwyl die kanaal in alle inteellyne gesluit was na bestuiwing. Die resultate dui daarop dat die styl-kanaal argitektuur nie ‘n noodsaaklike faktor is in die bepaling van weestand tot F. verticillioides besmetting in die suiwer mielielyne wat geselekteer was in hierdie studie nie. Verder is die moontlikheid dat weestand tot FKV en fumonisien kontaminasie geïnisieer kan word gedurende die saailing-fase ondersoek in beide ‘n weerstandbiedende en vatbare mielie inteellyn met behulp van konfokale laser skandering mikroskopie (CLSM). Die groei van F. verticillioides afkomstig vanuit die grond of saad is gemonitor in verskeie bo- en ondergrondse plantweefsels, maar geen beduidende verskille in kolonisasie kon opgespoor word tussen weerstandbiedende en vatbare mielie saailinge nie. Geen fumonisien produksie is waargeneem nie, ongeag die innokulasie metode of weerstand-status van die plant. Hierdie resultate dui daarop dat die weerstandbiedende en vatbare mielie saailinge wat in hierdie studie gebruik is moontlik nie weerstandbiedend is teen sistemiese swaminfeksie nie, maar wel weerstand kan bied tot afsetting van fumonisiene. Die weerstand geassosieër met die weerstandbiedende inteellyn word nie bemiddel gedurende die saailingfase nie maar waarskynlik deur strukturele en biochemiese verdedigingsmeganismes tydens latere plant ontwikkelings-stadia. Thesis (MSc)--Stellenbosch University, 2015

Fungicide resistance and control of citrus green mould

Tue, 01 Apr 2014 00:00:00 GMT

Fungicide resistance and control of citrus green mould Kellerman, Mareli ENGLISH ABSTRACT: Please refer to full text for abstract; AFRIKAANSE OPSOMMING: Sien volteks vir opsomming Thesis (MScAgric)--Stellenbosch University, 2014.

The role of sucker wounds as portals for grapevine trunk pathogen infections

Tue, 01 Apr 2014 00:00:00 GMT

The role of sucker wounds as portals for grapevine trunk pathogen infections Makatini, Gugulethu Joy ENGLISH ABSTRACT: Grapevine trunk diseases are responsible for reduced wine and table grape production world-wide. Trunk disease infections are caused by xylem-inhabiting pathogens which include species of Botryosphaeriaceae, Diatrypaceae, Hymenochaetales and Diaporthales, as well as Phaeomoniella chlamydospora and Phaeoacremonium spp. Winter pruning wounds are regarded as the main infection-sites for trunk disease pathogens. However, the role of sucker wounds as portals of trunk disease infections has been minimally investigated. Knowledge of the potential role of grapevine trunk pathogen infections that occur through sucker wounds is important for better wound protection strategies. The aim of this study was to determine the role of grapevine sucker wounds as portals of entry for trunk disease pathogens and to assess the use of Trichoderma spp. for sucker wound protection. The susceptibility of sucker wounds to different trunk disease pathogens was assessed from natural as well as artificial infections. In addition the duration of sucker wound susceptibility in the field was also ascertained. Sucker wounds were sampled from three wine and two table grape vineyards during 2011 and 2012 in the Western Cape province of South Africa. Thereafter, fungal isolations were made from 161 sucker wounds and the cultures were identified based on cultural and morphological characteristics as well as the internal transcribed spacer regions and 5.8S ribosomal RNA gene. Sixty-two percent of the wounds were naturally infected by at least one of the trunk pathogens. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) and Phaeomoniella (Ph.) chlamydospora (27%; 5%) were the most predominant trunk disease pathogens isolated from sucker wounds of field wine and table grape cultivars, respectively. Lower incidences of Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) and Neofusicoccum australe (1%) were obtained, however, only from wine grapes. Sucker wounds on 1-year-old potted grapevine plants of Chardonnay cultivar were inoculated with spore suspensions of Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora and Po. viticola in the glasshouse. After 4 months all the inoculated pathogens could be re-isolated at the following incidences: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) and E. lata (45%). Sucker wound susceptibility was further ascertained under field conditions on 12-year-old Cabernet Sauvignon vines by artificial inoculation of the same pathogen species. After 5 months three pathogens could be re-isolated at the following incidences: Po. viticola (65%), N. parvum (32.5%) and Ph. chlamydospora (7.5%). The duration of susceptibility of field sucker wounds to Ph. chlamydospora was assessed for a period of 4 weeks. The wounds remained susceptible for 4 weeks with a decline in susceptibility after one week. This study showed that sucker wounds are susceptible to the major trunk disease pathogens and thus could play an important role in grapevine trunk disease epidemiology. In the second part of this thesis a possible management strategy to prevent infections of sucker wounds was investigated. The use of Trichoderma (T.) harzianum against two trunk pathogens on sucker wounds was tested in the field. Additionally the sensitivity of T. harzianum and T. atroviride was tested in vitro against 16 fungicides that are used to control powdery mildew, downy mildew, Botrytis rot and Phomopsis cane and leaf spot. In October 2012, sucker wounds were made on 1-year-old wood of Cabernet Sauvignon and spray-treated with Eco-77® immediately after desuckering, and then inoculated with spore suspensions of either Ph. chlamydospora or Po. viticola after 24 hours. After 5 months, isolations were made from the sucker wounds to evaluate the efficacy of the Trichoderma treatment. Trichoderma harzianum reduced the incidence of Ph. chlamydospora by 66.65%. Although the incidence of Po. viticola was reduced by 15.37%, it was not significantly different from the control treatment. The inhibition of mycelial growth and conidial germination of T. harzianum and T. atroviride were screened against 16 fungicides. The fungicides were applied at 0, 0.25, 0.5, 1 and 2 times the recommended dosages. Systemic fungicides boscalid, metrafenone and trifloxystrobin, as well as contact fungicides quinoxyfen and meptyldinocap were least toxic to Trichoderma spp. isolates. For the conidial germination assay, boscalid, trifloxystrobin, penconazole and metrafenone (systemic) plus quinoxyfen and folpet (contact) were compatible with Trichoderma spp. These fungicides were regarded as being compatible with Trichoderma spp. isolates because they gave mean percentage inhibitions of less than 50% at all the tested dosages. Spiroxamine and pyrimethanil gave the highest mean percentage inhibitions for both mycelial inhibition and conidial germination. The findings of this study showed that T. harzianum can protect sucker wounds against Ph. chlamydospora in the field. Furthermore, some fungicides applied for the control of powdery mildew and Phomopsis cane and leaf spot can be alternatively or simultaneously applied with T. harzianum and T. atroviride, however, this will have to be verified with field trials.; AFRIKAANSE OPSOMMING: Wingerd stamsiektes is wêreldwyd verantwoordelik vir verminderde wyn- en tafeldruif produksie. Stamsiektes word veroorsaak deur patogene wat in die xileem voorkom, insluitend verskeie spesies in die Botryosphaeriaceae, Diatrypaceae, Hymenochaetales en Diaporthales, asook Phaeomoniella chlamydospora en Phaeoacremonium spp. Winter snoeiwonde word beskou as die hoof bron van infeksies vir stamsiekte patogene. Die rol van suierwonde as poorte van infeksie vir stamsiektes is nog nie goed bestudeer nie. Kennis van die potensiële rol van wingerd stamsiekte patogeen infeksies wat deur suierwonde plaasvind is belangrik vir die formulasie van beter wondbeskerming strategieë. Die mikpunt van hierdie studie was om die rol van suierwonde as ingangsportale vir wingerd stamsiekte patogene te bepaal en om die gebruik van Trichoderma spp. vir suierwond beskerming te evalueer. Die vatbaarheid van suierwonde vir verskillende stamsiekte patogene is geëvalueer vanuit natuurlike, sowel as kunsmatige infeksies. Die duur van suierwond vatbaarheid in die veld is ook bepaal. Suierwonde is versamel vanuit drie wyn- en twee tafeldruif wingerde gedurende 2011 en 2012 in die Wes Kaap provinsie van Suid Afrika. Hierna is swam isolasies gemaak vanuit 161 suierwonde en die kulture is geïdentifiseer volgens kultuur en morfologiese kenmerke, sowel as die interne transkribeerde spasieerders en 5.8S ribosomale RNA geen. Twee-en-sestig persent van die wonde was geïnfekteer deur ten minste een van die stamsiekte patogene. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) en Phaeomoniella (Ph.) chlamydospora (27%; 5%) was die mees algemene stamsiekte patogene wat, respektiewelik, vanuit die wyn- en tafeldruif kultivars verky is. Laer hoeveelhede Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) en Neofusicoccum australe (1%) is verkry, en slegs vanaf wyndruiwe. Suierwonde op 1-jaar oue Chardonnay wingerdplante in potte is in die glashuis geïnokuleer met spoorsuspensies van Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora en Po. viticola. Na 4 maande kon al die geïnokuleerde patogene her-isoleer word teen die volgende hoeveelhede: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) en E. lata (45%). Suierwond vatbaarheid is verder geëvalueer onder veld kondisies op 12-jaar oue Cabernet Sauvignon plante deur kunsmatige inokulasie van die selfde patogeen spesies. Na 5 maande kon drie patogene her-isoleer word teen die volgende hoeveelhede: Po. viticola (65%), N. parvum (32.5%) en Ph. chlamydospora (7.5%). Die duur van vatbaarheid van suierwonde teen Ph. chlamydospora in die veld is geevalueer oor ‘n periode van 4 weke. Die wonde het vatbaar gebly vir 4 weke met ‘n afname in vatbaarheid na ‘n week. Hierdie studie demonstreer dat suierwonde vatbaar is vir die hoof wingerd stamsiektes en dus ‘n belangrike rol in die epidemiologie van wingerd stamsiektes kan speel. In die tweede deel van hierdie tesis is ‘n moontlike bestuurs-strategie ondersoek om infeksie van suierwonde te verhoed. Die gebruik van Trichoderma (T.) harzianum teen twee stampatogene op suierwonde is getoets in die veld. Verder is die in vitro sensitiwiteit van T. harzianum en T. atroviride getoets teen 16 fungisiedes wat gebruik word in die beheer van poeieragtige meeldou, donsskimmel, Botrytis vrot en Phomopsis streepvlek. Gedurende Oktober 2012 is suierwonde gemaak op 1-jaar oue hout van Cabernet Sauvignon en onmiddelik behandel met Eco-77® na suiering. Wonde is dan geïnokuleer met spoorsuspensies van óf Ph. chlamydospora óf Po. viticola na 24 uur. Na 5 maande is isolasies gemaak vanaf suierwonde om die doeltreffendheid van van die Trichoderma behandeling te evalueer. Trichoderma harzianum het die voorkoms van Ph. chlamydospora met 66.65% verminder. Alhoewel die voorkoms van Po. viticola verminder is met 15.37%, was dit nie ‘n beduidende verskil in vergelyking met die kontrole behandeling nie. Die inhibisie van miselium groei en konidia ontkieming van T. harzianum en T. atroviride is getoets teen 16 fungisiedes. Die fungisiedes is aangewend teen 0, 0.25, 0.5, 1 en 2 keer die aanbevole dosisse. Sistemiese fungisiedes boscalid, metrafenone en trifloxystrobin, sowel as kontak fungisiedes quinoxyfen en meptyldinocap was die minste toksies teen Trichoderma spp. Gedurende die konidia ontkiemingstoets was boscalid, trifloxystrobin, penconazole en metrafenone (sistemies) en quinoxyfen en folpet (kontak) versoenbaar met Trichoderma spp. Die fungisiedes is beskou as bruikbaar met Trichoderma spp. isolate omdat hulle gemiddelde persentasie inhibisies van minder as 50% teen al die getoetste dosisse gelewer het. Spiroxamine en pyrimethanil het die hoogste gemiddelde persentasie inhibisie gelewer vir beide die miselium inhibisie en konidia ontkieming. Die bevindings van hierdie studie het gewys dat T. harzianum suierwonde kan beskerm teen Ph. chlamydospora in die veld. Verder sou sommige fungisiedes wat aangewend word vir die bestuur van poeieragtige meeldou en streepvlek moontlik alternatiewelik of gelyktydig met T. harzianum en T. atroviride aangewend word, alhowel dit met veldproewe bevestig moet word. Thesis (MScAgric)--Stellenbosch University, 2014.

Risk assessment of the Acacia cyclops dieback pathogen, Pseudolagarobasidium acaciicola, as a mycoherbicide in the South African strandveld and limestone fynbos

Tue, 01 Apr 2014 00:00:00 GMT

Risk assessment of the Acacia cyclops dieback pathogen, Pseudolagarobasidium acaciicola, as a mycoherbicide in the South African strandveld and limestone fynbos Kotze, Louis Jacobus Daniel ENGLISH ABSTRACT: Acacia cyclops, an invasive weed in South Africa, was initially imported to stabilize the sand dunes in the southern Cape. The spread of A. cyclops is a major threat to the fragile biodiversity of the strandveld and limestone fynbos vegetation. Acacia cyclops dieback has been observed for some time, although the causative agent, Pseudolagarobasidium acaciicola, has only recently been described. This fungus is nominated for development as a mycoherbicide to control A. cyclops. Although current biological and mechanical control efforts are proving to be partially effective, A. cyclops is still causing major damage to natural ecosystems. The introduction of a mycoherbicide would increase the cost effectiveness of controlling this weed in the long term. The majority of the literature that was reviewed supports the use of mycoherbicides as biocontrol agents, especially when taking into account the decrease in acceptance and availability of chemical control agents. Considering that the Pseudolagarobasidium genus consists of saprobes, opportunistic facultative pathogens and endophytes, P. acaciicola is predicted to have similar biological characteristics. The species is also highly likely to be indigenous, although with a wider distribution range than previously envisaged. Strict precautions should still however be taken to ensure that non-target species will not be threatened. This study consists of a unique risk assessment comprising different sections. A field survey was performed to record disease incidence among indigenous woody plant species around 100 diseased A. cyclops trees. Subsequently, DNA extractions were made from the roots of the diseased indigenous plants and A. cyclops trees to verify the presence of P. acaciicola. Of the 2432 indigenous woody plants observed, 22 (0.9%) were dead or dying, while P. acaciicola was detected in 10 of these (0.4%), representing six species. Pseudolagarobasidium acaciicola was detected in 47% of the A. cyclops trees. Although P. acaciicola could be a weak pathogen in a broad range of indigenous plant species, the extremely low disease incidence is an indication of a low level of risk associated with using P. acaciicola as a mycoherbicide. Additionally, pathogenicity trials on indigenous plant species were conducted to give an indication of host susceptibility. A total of 30 indigenous plant species were wound inoculated at two field sites, and potted plants representing 17 indigenous plant species were wound and soil inoculated in a nursery. The optimum growth temperature for P. acaciicola was determined in order to understand it’s seasonal and landscape preference. Mortality was recorded in five of nine indigenous Fabaceae species, while a single plant each of four other non-Fabaceae species died after inoculation. No plants outside the Fabaceae family died in the field. Only A. cyclops seedlings died following soil inoculation. Longitudinal sections of stem inoculated plants revealed no systemic infection in Fabaceae species that survived inoculation. Infection in susceptible Fabaceae species was generally more extensive than infection in susceptible non-Fabaceae species. The optimum growth rate for P. acaciicola was determined at 35°C, indicating an adaptation to summer conditions. Indigenous Fabaceae species do display greater susceptibility than species from other families, indicating some level of specificity, although susceptible species can not be phylogenetically circumscribed. Aside from being a facultative pathogen on A. cyclops, results from this study suggest that P. acaciicola is primarily a saprophyte and an occasional opportunistic pathogen on some indigenous Fabaceae, possibly only being a weak opportunistic pathogen on some non-Fabaceae species. However, the risk of not effectively managing A. cyclops populations in these threatened vegetation types outweighs the risk associated with using P. acaciicola as a mycoherbicide. Therefore the use of P. acaciicola as a mycoherbicide on A. cyclops would be recommended, provided that sufficient monitoring of treated sites is implemented that primarily focus on the indigenous Fabaceae species. The effective control of A. cyclops could be achieved when P. acaciicola is used to compliment current mechanical, biological and chemical control methods in an integrated management strategy.; AFRIKAANSE OPSOMMING: Acacia cyclops, ook bekend as rooikrans, is ‘n indringerplant in Suid-Afrika wat oorspronklik vanaf Australië ingevoer is om die sandduine in die Kaap te stabiliseer. Die verspreiding van rooikrans bedreig die sensitiewe biodiversiteit van die strandveld en kalksteen fynbos. Rooikrans terugsterwing is al vir ‘n geruime tyd opvallend in die grootste deel van die plant se verspreiding in Suid-Afrika, alhoewel die veroorsakende organisme, Pseudolagarobasidium acaciicola, eers onlangs beskryf is. Hierdie swam is as ‘n geskikte kandidaat vir die ontwikkeling van ‘n biologiese onkruiddoder om rooikrans te beheer, genomineer. Alhoewel die huidige biologiese- en meganiese beheer metodes vir rooikrans gedeeltelik suksesvol is, hou dié indringer steeds ‘n ernstige bedreiging vir die natuurlike ekosisteme in. Die gebruik van ‘n swam-gebaseerde onkruiddoder sal die beheer van rooikrans oor die langtermyn meer koste-effektief maak. Die oorgrote meerderheid van die literatuur wat hersien is, ondersteun die gebruik van swam-gebaseerde onkruiddoders as biologiese beheermiddels, veral as die afname in aanvaarbaarheid en beskikbaarheid van chemiese beheermiddels in ag geneem word. Aangesien die Pseudolagarobasidium genus uit saprofiete, opportunistiese fakultatiewe patogene en endofiete bestaan, word daar voorspel dat P. acaciicola ‘n soortgelyke biologiese karakter sal hê. Dit is hoogs waarskynlik dat hierdie swamspesie inheems is, alhoewel die verspreiding wyer mag wees as wat oorspronklik voorspel is. Streng maatreëls moet egter steeds in plek wees om te verseker dat nie-teiken plantspesies nie bedreig word nie. Hierdie studie bestaan uit ‘n unieke risiko-analise met verkeie onderafdelings. ‘n Veld-opname is uitgevoer om die siekte-voorkoms van die inheemse houtagtige plantspesies rondom ‘n 100 siek rooikrans plante te bepaal. DNA ekstraksies is vervolgens vanuit die wortels van siek inheemse plantspesies en -rooikrans uitgevoer, om uiteindelik die teenwoordigheid van P. acaciicola binne die hout te kon bevestig. Uit ‘n totaal van 2432 inheemse houtagtige plante wat aangeteken is, was 22 (0.9%) siek of dood, terwyl die teenwoordigheid van P. acaciicola in 10 van hierdie plante (0.4%), wat ses spesies teenwoordig, bevestig is. Die teewoordigheid van P. acaciicola is ook in 47% van die rooikrans bevestig. Alhoewel P. acaciicola moontlik ‘n swak opportunistiese patogeen op ‘n verskeidenheid inheemse plantspesies is, dui die lae verhouding van dooie inheemse plante teenoor gesonde plante in die veld op ‘n lae risiko vir die gebruik van P. acaciicola as ‘n biologiese onkruiddoder. Patogenisiteitstoetse is op inheemse plantspesies uitgevoer om ‘n aanduiding van gasheervatbaarheid te verkry. Wond-inokulasies is op ‘n totaal van 30 inheemse plantspesies by twee veldstudie-areas uitgevoer, terwyl wond- en grond-inokulasies op 17 inheemse spesies potplante in die kweekhuis uitgevoer is. Die optimale temperatuur waarby P. acaciicola groei, is bepaal om die swam se seisoenale- en habitatsvoorkeure beter te verstaan. Plante van vyf uit die nege inheemse Fabaceae spesies het doodgegaan, terwyl ‘n enkele plant van vier nie-Fabaceae spesies doodgegaan het. Alle plante buite die Fabaceae familie het oorleef in die veld na inokulasie. Slegs rooikranssaailinge het na grond inokulasie doodgegaan. Lengtedeursnee van die stam en wortels van elke geïnokuleerde plant het bevestig dat daar geen sistemiese infeksie in Fabaceae spesies wat inokulasie oorleef het, plaasgevind het nie. Infeksies in vatbare Fabaceae spesies was oor die algemeen meer ernstig as infeksies in vatbare nie-Fabaceae spesies. Die optimale groei van P. acaciicola het by 35°C plaasgevind, wat aandui op ‘n voorkeur vir somerstoestande. Inheemse Fabaceae spesies het meer vatbaar as vatbare plantspesies van ander families voorgekom. Hierdie verskynsel dui op ‘n sekere vlak van spesifisiteit, alhoewel daar geen duidelike filogenetiese grense vir vatbare spesies bepaal kon word nie. Behalwe vir die feit dat P. acaciicola ‘n fakultatiewe patogeen op rooikrans is, stel resultate van hierdie studie voor dat hierdie swam hoofsaaklik ‘n saprofiet is wat soms ook ‘n opportunisties patogeen op sekere inheemse Fabaceae is en moontlik slegs ‘n swak opportunistiese patogeen op plantspesies buite die Fabaceae familie is. Die swak en oneffektiewe bestuur van rooikrans in hierdie bedreigde plantegroeitipes hou egter ‘n groter bedreiging in as die gebruik van P. acaciicola as ‘n biologiese onkruiddoder. Pseudolagarobasidium acaciicola word daarom aanbeveel vir die beheer van rooikrans, mits voldoende monitering, wat fokus op inheemse Fabaceae spesies, gepaard gaan met die gebruik van hierdie biologiese onkruiddoder. Rooikrans kan effektief beheer word as P. acaciicola ingespan word om huidige meganiese-, biologiese- en chemiese beheermetodes in ‘n geïntegreerde bestuurstrategie te komplimenteer. Thesis (MSc)--Stellenbosch University, 2014.

The effect of garlic extracts on the control of postharvest pathogens and postharvest decay of apples

Tue, 01 Apr 2014 00:00:00 GMT

The effect of garlic extracts on the control of postharvest pathogens and postharvest decay of apples Daniel, Chanel Karousha ENGLISH ABSTRACT: Apples are an important export commodity for the South African market, and postharvest losses that occur as a result of decay due to infection with pathogenic fungi such as Botrytis cinerea Pers., Penicillium expansum (Link) Thom. and Neofabraea alba (E.J. Guthrie) are of major concern for all parties concerned with fruit production and distribution. Decay control of these fungi is primarily managed through the use of synthetic fungicides; however, pathogen development of resistance to these fungicides and recent worldwide concern over healthier living and a greener environment has called for the discriminate use of synthetic chemicals. This has opened up an avenue for the development of safer and more environmentally friendly alternatives to control postharvest decays. The use of plant extracts and essential oils are favoured as natural sources of antimicrobials whilst still being safe for human consumption and having no negative impact on the environment. Allium sativum (garlic) is one such plant species that is well documented for its value in improving human health and is readily available for consumption not just as a flavour component of food but also to be taken as a daily herbal diet supplement. Given the antimicrobial effectiveness of garlic against human pathogens and ailments, its value as an antifungal agent against postharvest pathogens causing grey mould, blue mould and bull’s eye rot of apples was investigated in vitro and in vivo within this study. Furthermore, an attempt was made to elucidate the chemical components of garlic extracts by gas chromatography-mass spectrometry (GC-MS). All experiments in this study were carried out with garlic extracts prepared from fresh garlic bulbs. For the in vitro experiments, two extract preparations of garlic, one containing ethanol (Extract 1) and one where ethanol had been removed by evaporation (Extract 2), was tested for antifungal action within an amended media experimental design. Both extract preparations were each subjected to two dilution series (0-80% garlic extract) with water and ethanol as diluents. Both extract preparations were successful at retarding pathogen mycelial growth and spore germination; however, concentrations of Extract 2 (ethanol evaporated) and diluted with distilled water provided markedly better inhibition of B. cinerea and P. expansum than the ethanolic dilutions of extract 2. Both extract preparations yielded similar inhibitory results when tested against N. alba. Due to the results achieved in the amended media experiments, the use of a crude garlic extract without ethanol and diluted in water was considered to be the best option for further tests throughout the remainder of the study. In vitro volatile effects of crude garlic extracts at concentrations between 0 and 40% garlic extract were subsequently tested. Garlic volatiles were effective in inhibiting pathogen mycelial growth and spore germination of all three pathogens, at lower concentrations compared to the amended media experiments. In vitro volatile exposure with garlic extracts was more effective at inhibiting N. alba than direct application of the extracts. Curative and protective application of garlic extracts and clove oil for increased fungal inhibition through synergism was tested by direct and volatile exposure to the pathogens in vivo on three economically important apple cultivars; ‘Granny Smith’, ‘Golden Delicious’, and ‘Pink Lady’. Direct exposure of artificially wounded and inoculated fruit to the garlic extract and clove oil revealed that garlic extracts applied curatively but not protectively effectively controlled decay caused by B. cinerea and P. expansum on all apple cultivars. Both curative and protective applications were ineffective in controlling N. alba. In vivo volatile exposure to the garlic extracts and clove oil did not inhibit decay on any of the cultivars and was not effective against any of the three pathogens investigated. A full chemical profile analysis was done by GC-MS analysis of garlic extract samples. The compounds diallyl disulphide, allyl methyl trisulphide, allyl methyl disulphide and dimethyl trisulphide were detected in relatively high amounts. This result suggests that the abundance of sulphur and sulphur related compounds detected may be responsible for the antifungal action noted in the experimental studies. In conclusion, garlic was shown to have antifungal activity against B. cinerea, P. expansum and N. alba. The pathogens used in this study were not compared with each other, but undoubtedly each pathogens reacts differently to exposure to the garlic extracts. It would therefore be advisable to investigate the effects of the extracts on each of the pathogens in a more in-depth study. More investigations into the application of the garlic extracts is required before it may be recommended for use; however, results for the use of garlic extracts against these postharvest pathogens and the postharvest decay they cause are promising.; AFRIKAANSE OPSOMMING: Appels is ‘n belangrike uitvoerproduk vir die Suid-Afrikaanse vrugtebedryf, maar noemenswaardige na-oes verliese word weens bederf deur patogeniese swamme soos Botrytis cinerea Pers., Penicillium expansum (Link) Thom. en Neofabraea alba (E.J. Guthrie) ervaar. Dit raak alle partye betrokke met die produksie en verspreiding van hierdie vrugsoort. Hierdie swamme word hoofsaaklik met behulp van kunsmatige swamdoders beheer, alhoewel weerstand-ontwikkeling en wêreldwye bewusmaking van ‘n gesonder leefstyl en omgewing die gebruik van kunsmatige middels streng aanspreek en die ontwikkeling van veiliger en meer omgewingsvriendelike alternatiewe middels verlang. Plant-ekstrakte en essensiële olies kan dien as sulke middels en is natuurlike bronne van anti-mikrobiese aktiwiteit, is veilig vir menslike verbruik en het ook geen negatiewe invloed op die omgewing nie. Allium sativum (knoffel) is so ‘n plantspesie wat as alternatiewe middel gebruik kan word. Dit is bekend vir sy waarde in die verbetering van menslike gesondheid, is maklik bekombaar en word nie net as ‘n geurmiddel vir voedsel gebruik nie, maar ook as ‘n daaglikse krui-aanvulling. Gegewe die anti-mikrobiese doeltreffendheid van knoffel teenoor menslike patogene en kwale, is die werking (in vitro en in vivo) teen na-oes patogene wat grys skimmel, blou skimmel en teikenvrot in appels veroorsaak, in hierdie studie ondersoek. Bepaling van die chemiese samestelling van die knoffel-ekstrak is ook met behulp van gaschromatografie massa spektrometrie (GK-MS) onderneem.Vars knoffelbolle is vir elke eksperiment in hierdie studie gebruik met die voorbereiding van die knoffel-ekstrak. Vir die in vitro eksperiment is twee knoffel-ekstrakte voorberei, naamlik: ‘n ekstrak wat etanol bevat (Ekstrak 1) en een waarvan die etanol verwyder is met verdamping (Ekstrak 2). Die ekstrakte is getoets vir werking teen fungi in kultuur-medium.. Albei ekstrakte is verdun tot twee konsentrasie reekse (0-80%) met water en etanol as verdunningsmiddels. Albei ekstrakte het suksesvolle werking getoon teenoor die patogene ten opsigte van vertraging van miseliumgroei en spoor-ontkieming, alhoewel konsentrasies van Ekstrak 2, verdun met gesuiwerede water, patogene B. cinerea en P. expansum beter onderdruk het as Ekstrak 2 verdunnings met etanol.. Beide ekstrakte en hul afsonderlike verdunnings met etanol en water het soortgelyke resultate gelewer met onderdrukking van N. alba. Volgens resultate wat verkry is van die kultuur-medium eksperimente, is Ekstrak 2 verdun met gesuiwerde water beskou as die geskikste vir verdere toetse in hierdie studie. Die vlugtige effek van Ekstrak 2 is in vitro getoets by konsentrasies tussen 0 tot 40%. Die vlugtige stowwe van knoffel het al drie patogene se groei en spoor-ontkieming effektief onderdrukby laer konsentrasies as wat gebruik is in die kultuur-medium eksperiment. Dus is in vitro blootstelling van N. alba aan die vlugtige stowwe meer effektief as direkte toediening van die ekstrakte. Die voorkomende en beskermende effek van die knoffel-ekstrak, asook naeltjie-olie, is in vivo ondersoek om te bepaal of die stowwe saam sterker onderdrukking van die patogene kon bewerkstellig. Direkte en vlugtige blootstelling is op drie ekonomies-belangrike appel-kultivars getoets, naamlik: ‘Granny Smith’, ‘Golden Delicious’ en ‘Pink Lady’. Direkte blootstelling met die knoffel-ekstrak en naeltjie-olie aan gewonde en ge-inokuleerde vrugte het aangedui dat B. cinerea- en P. Expansum-bederf net beheer kon word indien knoffel voorkomend toegedien is vir al die ondersoekte appel-variëteite. Voorkomende en beskermende toediening was onsuksesvolle om N. alba te beheer. In vivo blootstelling van die drie patogene aan die knoffel-ekstrak en naeltjie-olie se vlugtige stowwe kon nie enige van die patogene effektief onderdruk nie en was onsuksesvol in bederf-beheer. ‘n Volledige chemiese profiel is saamgestel deur GK-MS ontleding van die knoffelekstrakte. Hoë vlakke van verbindings dialliel disulfied, alliel-metiel-tri-sulfied, alliel-metieldisulfied en dimetiel-trisulfied is bespeur. Die aantal vrye sulfied en sulfied-verwante verbindings in die ekstrak kan moontlik ‘n verduideliking bied vir die anti-swam werking waargeneem gedurende hierdie studie. Ten slotte: knoffel toon ‘n anti-swam werking teenoor B. cinerea, P. expansum en N. alba. Die patogene in hierdie studie is nie met mekaar vergelyk nie, omdat elkeen uniek en uiteenlopend op knoffel reageer het. Alhoewel die huidige studie alreeds belowende resultate gelewer het, moet die ekstrak se effek op elke patogeen onderskeidelik nog in diepte ondersoek word, asook die wyse van die toediening in die na-oes praktyk voordat hierdie middel aanbeveel kan word vir gebruik. Thesis (MSc)--Stellenbosch University, 2014.

The role of arthropods in the dispersal of trunk disease pathogens associated with Petri disease and Esca

Fri, 01 Mar 2013 00:00:00 GMT

The role of arthropods in the dispersal of trunk disease pathogens associated with Petri disease and Esca Moyo, Providence ENGLISH ABSTRACT: Petri disease and esca are devastating grapevine trunk diseases and compromise the sustainability of viticulture world-wide. Despite being extensively studied, knowledge of inoculum sources and mechanisms of spread of the causal pathogens is limited. Arthropods have been suspected to play a role in the spread of Petri disease and esca pathogens. However, little information is known about the extent to which arthropods are associated with these pathogens. This study aimed to determine whether arthropods occurring within or on declining grapevines, are associated with trunk disease pathogens and to identify arthropods associated with pruning wounds. The potential of selected arthropods to act as vectors of trunk disease pathogens was also investigated. Two vineyards exhibiting grapevine trunk disease infections were sampled weekly for two years for collection of arthropods. Arthropods were collected using pruning wound traps, visual searches as well as trunk and cordon traps. Fungal spores from surfaces of arthropods were collected in water. Samples were subjected to nested PCR using primers Pm1/Pm2 and Pch1/Pch2 to verify the presence of Phaeoacremonium spp. and Phaeomoniella chlamydospora, respectively. Water samples were also cultured and grapevine trunk disease pathogens obtained were identified by sequencing the internal transcribed spacers 1 and 2 and the 5.8S rRNA gene or the partial beta-tubulin gene. A total of 10 875 arthropod individuals, belonging to more than 31 families, were collected from declining grapevines. The most abundant arthropods included millipedes, ants, spiders and beetles. Portuguese millipedes and cocktail ants were associated with fresh grapevine pruning wounds. Thirty-three percent of the 5677 water samples analysed, contained propagules of pathogens associated with Petri disease and esca. Of these, 37 % were recovered from millipedes, 22 % from cocktail ants, 15 % from spiders and 10 % from beetles. All the major groups of grapevine trunk diseases were detected on the arthropods. Phaeoacremonium species were detected in 1242 samples while Phaeomoniella chlamydospora was identified from 855 samples. Other fungi isolated included members of the Botryosphaeriaceae, Diatrypaceae and Diaporthales. The potential of grapevine sap as a food source for Portuguese millipedes and cocktail ants was investigated, in vitro. Millipede individuals were offered a choice between water and grapevine sap while ants in nests were presented with grapevine sap, tuna and water and monitored for ingestion of sap. Both taxa preferred grapevine sap over the other food items, indicating close association with pruning wounds. Subsequently, the ability of both taxa to transmit a DsRed-transformed Phaeomoniella chlamydospora isolate to fresh pruning wounds of canes in polystyrene strips, floating in water, and potted vines was tested. Arthropods were exposed to the fungus for 24 hours and transferred to the base of the plants and canes and were removed after three days. Isolations after a month revealed that millipedes and ants were capable of transmitting the fungus onto wounds and cause infection. Millipede faecal pellets were also evaluated as potential sources of inoculum. Millipedes were fed on Phaeomoniella chlamydospora for 24 hours, surface sterilised and allowed to defaecate in sterile Petri dishes overnight. Faecal material was collected, macerated in water and plated onto potato dextrose agar. Propagules of Phaeomoniella chlamydospora survived passage through the gut of millipedes and were passed out in a viable state to form colonies of Phaeomoniella chlamydospora. This study concludes that a wide variety of arthropods can be a source of inoculum of trunk diseases in vineyards. The results of the dissemination trial provides evidence that millipedes and ants are able to disseminate and infect vines with Phaeomoniella chlamydospora. It is therefore, highly likely that other grapevine trunk disease pathogens are transmitted in the same manner. This knowledge highlights the need for control of certain arthropods to be taken into consideration when managing grapevine trunk disease pathogens.; AFRIKAANSE OPSOMMING: Petri siekte en esca is verwoestende wingerd stamsiektes en verhinder die volhoubaarheid van wingerdproduksie wêreldwyd. Hierdie siektes is al intensief bestudeer, maar kennis rakende die inokulum bronne en meganismes van verspreiding van die veroorsakende patogene is beperk. Arthropoda is al vermoed om ‘n rol te speel in die verspreiding van Petri siekte en esca patogene, maar weinig informasie is bekend oor die mate waartoe arthropoda geassosieer is met die patogene. Hierdie studie het ten doel gestel om die arthropoda wat op of in wingerdstokke wat terugsterf voorkom te identifiseer en te bepaal watter van die arthropoda geassosieer is met stamsiekte patogene. Daar is ook ten doel gestel om die arthropoda wat geassosieer is met vars snoeiwonde te identifiseer en ook die moontlike vektor status van die stamsiekte patogene deur arthropoda. Arthropoda is weekliks vir twee jaar gekollekteer vanaf twee wingerde met stamsiekte infeksies. Snoeiwond lokvalle, visuele soektogte en stam- en kordon lokvalle was gebruik om arthropoda te vang. Swamspore van die oppervlak van die arthropoda is afgewas met water. Van hierdie water monsters is gebruik om dubbelvoudige polimerase ketting reaksies (PKR) te doen met die inleiers Pm1/Pm2 en Pch1/Pch2 om vir die teenwoordigheid van Phaeoacremonium spp. en Phaeomoniella chlamydospora onderskeidelik te toets. Die oorblywende water monster is gekweek op medium om die swamme teenwoordig te bepaal. Die wingerd stamsiekte patogene is verder geidentifiseer deur die DNS volgordes te bepaal van die interne getranskribeerde spasies 1 en 2 en die 5.8S rRNS geen of ‘n gedeelte van die beta-tubulien geen. In totaal is 10 875 arthropoda, wat behoort tot 31 families, gekollekteer vanaf wingerde wat terugsterf. Die mees algemene arthropoda was duisendpote, miere, spinnekoppe en kewers. Die Portugese duisendpote en die wipstert mier is geassosieer met vars wingerd snoeiwonde. Van die 5677 water monsters wat geanaliseer is, het 33% propagules van die Petri siekte of esca patogene gehad. Van hierdie was 37 % afkomstig vanaf duisendpote, 22 % van wipstert miere, 15 % van spinnekoppe en 10 % van kewers. Al die hoofgroepe van wingerd stampatogene is opgespoor op die arthropoda. Phaeoacremonium species is opgespoor in 1242 monsters en Phaeomoniella chlamydospora is gevind in 855 monsters. Ander swamme wat ook geisoleer is sluit lede van die Botryosphaeriaceae, Diatrypaceae en Diaporthales in. Die potensiaal van wingerdsap as ‘n bron van voedsel vir Portugese duisendpote en wipstert miere is in vitro ondersoek. Duisendpoot invidue is ‘n keuse gegee tussen water en wingerd sap terwyl mierneste ‘n keuse gehad het tussen water, wingerd sap en tuna. Die duisendpote en miere is gemonitor vir die inname van wingerdsap in die teenwoordigheid van die ander bronne. Beide die duisendpote en miere het wingerdsap verkies wat aandui dat hulle ‘n noue assosiasie met wingerd snoeiwonde het. Vervolgens is beide taksons getoets vir hul vermoë om ‘n DsRooi-getransformeerde Phaeomoniella chlamydospora isolaat te vektor na vars snoeiwonde op lote gemonteer op polistireen stroke wat in water dryf en op wingerd plante in potte. Die duisendpote en miere is blootgestel aan die swam vir 24 uur en oorgedra na die basis van die plante en lote en is weer verwyder na drie dae. Na ‘n maand is isolasies gedoen wat gewys het dat die duisendpote en miere die swam suksesvol kon oordra na die snoeiwonde en infeksie veroorsaak. Duisendpoot uitwerpsels is geëvalueer vir die potensiaal as inokulum bron. Duisendpote het gevoed op Phaeomoniella chlamydospora vir 24 uur, daarna oppervlakkig gesteriliseer en toegelaat om oornag uitwerpsels te maak in steriele Petri bakkies. Uitwerpsels was gekollekteer, fyngemaak in water en op aartappel dekstrose agar uitgeplaat. Propagules van Phaeomoniella chlamydospora het die verteringskanaal van die duisendpote oorleef en het tipiese kolonies op die agar gevorm. Hierdie studie het vasgestel dat ‘n verskeidenheid van arthropoda ‘n bron van inokulum van stamsiektes in wingerd kan wees. Die resultate van die vektor proewe het gewys dat duisendpote en miere die vermoë het om Phaeomoniella chlamydospora te versprei na snoeiwonde wat die swam dan suksesvol geinfekteer het. Dit is daarom hoogs waarskynlik dat van die ander wingerd stamsiekte patogene ook versprei kan word op dieselfde manier. Hierdie kennis demonstreer dat die beheer van spesifieke arthropoda in ag geneem moet word in die bestuur van wingerd stamsiektes. Thesis (MScAgric)--Stellenbosch University, 2013.

Simptomatologie en anatomie van gleufstam ('legno riccio') by die wingerdstok (Vitis)

Sat, 01 Dec 1973 00:00:00 GMT

Simptomatologie en anatomie van gleufstam ('legno riccio') by die wingerdstok (Vitis) Kriel, G. J. le R. (Gabriel Jacobus le Roux) ENGLISH ABSTRACT: no abstract available; AFRIKAANSE OPSOMMING: geen opsomming Thesis MSc(Agric)--Stellenbosch University, 1973.

Biochemiese veranderinge in druiwemos veroorsaak deur Botrytis cinerea en Rhizopus nigricans

Tue, 01 Dec 1964 00:00:00 GMT

Biochemiese veranderinge in druiwemos veroorsaak deur Botrytis cinerea en Rhizopus nigricans Hofmann, Gerhard ENGLISH ABSTRACT: no abstract available; AFRIKAANSE OPSOMMING: geen opsomming Thesis (MScAgric)--Stellenbosch University, 1964.

Bars van tafeldruiwe met spesiale verwysing na Queen of the Vineyard

Sat, 01 Dec 1956 00:00:00 GMT

Bars van tafeldruiwe met spesiale verwysing na Queen of the Vineyard Meynhardt, J. T. (Johann Theron) ENGLISH ABSTRACT: no abstract available; AFRIKAANSE OPSOMMING: geen opsomming Thesis (MScAgric)--Stellenbosch University, 1956.

n Studie van Botrytis cinerea met verwysing na die effek van swaeldioksiedtoedienings, verskillende tye na bespuiting van druiwekorrels met konidia, op die intensiteit van vaalvrot

Fri, 01 Dec 1972 00:00:00 GMT

n Studie van Botrytis cinerea met verwysing na die effek van swaeldioksiedtoedienings, verskillende tye na bespuiting van druiwekorrels met konidia, op die intensiteit van vaalvrot Combrink, J. C. (Jacob Coenraad), 1918- ENGLISH ABSTRACT: no abstract available Thesis (MScAgric)--Stellenbosch University, 1972.

Comparative studies on genetic variability and fungicide resistance in Tapesia yallundae

Tue, 01 Dec 1998 00:00:00 GMT

Comparative studies on genetic variability and fungicide resistance in Tapesia yallundae Ntushelo, Khayalethu ENGLISH ABSTRACT: Eyespot is an important disease of spring wheat (Triticum aestivum L.). Four species of Ramulispora are associated with this disease, of which Tapesia yallundae and T. acuformis. are common. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance in Tapesia yallundae. Each of the chapters treats specific but related topics. T. yallundae, which is the only species thus far reported from South Africa, has been associated with yield losses of up to 50%. To enable the implementation of more accurate and effective control measures, understanding the dynamics of reproduction and the genetics of the pathogen is of utmost importance. Of the many plant disease control measures such as cultural practices, sanitation, biological control, etc., fungicide application is the most commonly resorted to measure in eyespot control. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance of Tapesia yallzll7dae. Fungicide application, however, is not without problems. The pathogen can build up resistance to fungicides. The most commonly used fungicides in eyespot control include the benzimidazole carbendazim, triazoles such as flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol, fenbuconazole, triademinol, and the imidazole, prochloraz. Cases of resistance to the groups listed above have been reported. Frequent monitoring for resistance is thus crucial to prevent wastage of fungicide and unnecessary impregnantation of the environment with potentially ineffective chemicals. In chapter 2 of this thesis 300 isolates of T. yallundae from 15 fields were evaluated for resistance against carbendazim, flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol and fenbuconazole. These results indicated that to some triazoles, such as fenbuconazole, a high level of resistance was already present in field populations. In a sexually reproducing fungus such as T. yallundae, knowledge pertaining to its ability to pass resistance factors to offspring is equally important. Mating studies were, therefore, also conducted with parental strains that showed signs of triazole resistance. Three generations were subsequently tested for resistance to five triazoles, namely flusilazole, tebuconazole, propiconazole, bromuconazole and flutriafol. Results of this study showed variable sensitivity in progeny, which indicated quantitative inheritance of resistance to triazoles. Although the sexual stage has not yet been observed in the field in South Africa, this knowledge lays the foundation for the long-term understanding of the population dynamics of the fungus. The ability of a heterothallic ascomycete population to reproduce sexually is dependent on the availability of its two mating types, MATI-I and MATI-2, their distribution, and female fertility amongst other factors. In the UK. the teleomorph is commonly observed in the field, which is in contrast to the situation in South Africa, where it has only been induced in the laboratory. A comparative study between the South African and the UK. populations was therefore undertaken. Isolates representative of the two populations were mated with tester strains as both sperm recipients and as sperm donors. This allowed the percentage of hermaphrodites to be determined. No difference in terms of female fertility was observed between the South African and the UK. populations, with both populations showing low effective population numbers. These data suggested, therefore, that the teleomorph would also occur more frequently in South Africa if the climate was more indusive to its development. The overall results of this study indicated that eyes pot could still be controlled by means of fungicide application in South Africa. Although a shift in sensitivity was observed towards fenbuconazole and flusilazole, no resistance was detected towards carbendazim. The latter might be due to the absen<.:eof the sexual stage in the field, coupled by the monocyclic nature of the pathogen and sensible fungicide regimes. The absence of T. acujormis makes the disease situation less complicated in terms of fungicide application and management. Continuous surveys will have to be conducted, however, to monitor this situation in future.; AFRIKAANSE OPSOMMING: Hierdie studie ondersoek die genetiese variasie, reproduksie dinamika en fungisied weerstand in Tapesia yallundae. Elke hoofstuk handel oor spesifieke maar verwante onderwerpe. Oogvlek is 'n belangrike siekte van lentekoring (Triticum aestivum L.). Vier spesies van Ramulispora word geassosieer met die siekte, waarvan Tapesia yallundae en T. acuformis mees algemeen voorkom. T. yallundae, wat tans die enigste spesie is wat in Suid-Afrika aangeteken is, het al verliese van tot 50% veroorsaak. Om meer akkurate en effektiewe beheermaatreels te implementeer, is dit noodsaaklik om die oorlewingsdinamika van die patogeen te verstaan. Van al die siektebeheermaatreels soos kulturele praktyke, sanitasie, biologiese beheer ens., bly fungisiedbehandeling die mees algemene maatreel vir die beheer van oogvlek. Fungisiedtoediening het egter ook verskeie probleme. Die patogeen kan weerstand opbou teen die fungisied. Die mees algemene fungisiedes wat vir oogvlekbeheer aangewend word sluit onder meer die benzimidasool karbendazim in, triasole soos flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol, fenbukonasool, triadimenol, en die imidasool, prochloraz. Weerstand is egter reeds teen hierdie middels bekend. Gedurige monitering vir weerstand is dus krities om die vermorsing van fungisied en besoedeling van die omgewing met oneffektiewe middels te beperk. In hoofstuk 2 van hierdie manuskrip word 300 isolate van T. yallundae van 15 lande geevalueer vir weerstand teenoor karbendazim, flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol en fenbukonasool. Resultate dui daarop dat teen sommige van hierdie triasole, soos bv. fenbukonasool, daar reeds 'n hoe vlak van weerstand teenwoordig was in veldpopulasies. In 'n seksueel reproduserende fungus soos T. yalluJ1dae, is dit noodsaaklik om te bepaal wat sy vermoe is om weerstandbiedenheid aan die nageslag oor te dra. Om die rede is paringstudies ook op ouers wat tekens van weerstand teenoor triasole getoon het uitgevoer. Drie generasies was gevolglik getoets vir weerstand teenoor vyf triasole, naamlik flusilasool, tebuconasool, propikonasool, brumukonasool en flutriafol. Resultate van die studie het 'n variasie in sensitiwiteit van die nageslag getoon, wat op 'n kwantitatiewe oorerwing van weerstand teen £riasole dui. Alhoewel die teleomorf nog nie in lande in Suid-Afrika opgemerk is nie, Ie hierdie kennis die fondament vir die langtermyn vertolking van die populasie dinamika van hierdie fungus. Die vermoe van 'n heterotalliese askomiseet populasie om seksueel voort te plant is afhanklik van die beskikbaarheid van sy twee paringstipes, MATI-I en MATl-2, hul verpreiding, vroulike vrugbaarheid en ander faktore. Alhoewel die teleomorf algemeen in lande in die Verenigde Koninkryk opgemerk word, is dit in kontras met die situasie in Suid-Afrika, waar hierdie stadium nog slegs in die laboratorium gelnduseer kon word. 'n Studie is dus onderneem om die Suid-Afrikaanse en V.K. populasies met mekaar te vergelyk. Isolate van die twee populasies is dus gepaar met paringsisolate as beide sperm ontvangers en sperm donors. Hierdie prosedure het dit moontlik gemaak om die persentasie hermafrodiete te bepaal. Geen verskille in vroulike fertiliteit is tussen die Suid-Afrikaanse en V.K. populasies bespeur nie, en beide populasies het ook 'n lae effektiewe populasie getal getoon. Hierdie data het dus voorgestel dat die teleomorf ook meer algemeen in Suid-Afrika sou voorkom as die klimaat meer geskik was vir teleomorf vormmg. Die resultate van hierdie studie het tot die slotsom gelei dat oogvlek steeds deur fungisiedbehandeling in Suid-Afrika beheer kan word. Alhoewel daar 'n merkbare verskuiwing in sensitiwiteit teenoor fenbukonasool en flusilasool was, was geen weerstand teenoor karbendazim waargeneem nie. Laasgenoemde kan dalk toegeskryf word aan die afwesigheid van die teleomorf in die veld, gekombineer met die monosikliese natuur van die patogeen en gebruik van alternerende fungisiedes. Die afwesigheid van T. acuformis maak die plaaslike siektetoestand minder gekompliseerd in terme van fungisied aanwending en bestuur. Voortdurende opnames sal egter uitgevoer moet word om hierdie situasie ook in die toekoms te monitor. Thesis (MScAgric)--Stellenbosch University, 1998.

Colletotrichum diseases of Proteaceae

Mon, 01 Mar 2004 00:00:00 GMT

Colletotrichum diseases of Proteaceae Lubbe, Carolien M. ENGLISH ABSTRACT: This thesis consists of four chapters that present research findings on Colletotrichum diseases associated with Proteaceae worldwide. The first chapter is a review of literature regarding the taxonomy and histology of Colletotrichum species associated with Proteaceae. The literature is not restricted to Proteaceae hosts, as information regarding Colletotrichum on Proteaceae is very limited. In chapter two, Colletotrichum spp. associated with proteaceous hosts growing in various parts of the world were identified based on morphology, sequence data of the internal transcribed spacer region (ITS-I, ITS-2), the 5.8S gene, and partial sequences of the B-tubulin gene. Four species of Colletotrichum were associated with Proteaceae. Colletotrichum gloeosporioides was isolated from Protea cynaroides cultivated in South Africa and Zimbabwe and from a Leucospermum sp. in Portugal, but is known to occur worldwide on numerous hosts. A recently described species, C. boninense, was associated with Zimbabwean and Australian Proteaceae, but also occurred on a Eucalyptus sp. in South Africa. This represents a major geographical and host extension for the species, and a description of the African strains is provided. Colletotrichum crassipes was represented by a single isolate obtained from a Dryandra plant in Madeira. Colletotrichum acutatum was isolated from Protea and Leucadendron in South Africa as well as from other proteaceous hosts occurring elsewhere. Colletotrichum acutatum f. sp. hakea was isolated from Hakea in South Africa. In chapter three, pathogenicity of these Colletotrichum species to certain proteas was established, relative aggressiveness of the different species tested and host response to them were compared as well as the effect that wounding had on host response. From the results obtained it is concluded that C. acutatum and C. gloeosporioides are the primary pathogens associated with Colletotrichum leaf necrosis, and C. acutatum is the main cause of anthracnose and stem necrosis of Proteaceae in South Africa. A histological study was performed in chapter four in response to the findings from the previous chapter. The behaviour of two C. acutatum isolates (one originating from Protea and the other from Hakea, C. acuataum f.sp. hakea) was studied on inoculated Protea leaf surfaces using light and scanning electron microscopy. Colletotrichum acutatum from Protea formed melanised appressoria on the leaf surface, whereas C. acutatum from Hakea formed very low numbers of both melanised and unmelanised appressoria. Most of the appressoria formed by C. acutatum from Protea were formed on the cell junctions and on the periclinal walls of the epidermal cells. From this study it is clear that C. acutatum f. sp. hakea is not a pathogen of Protea. Consequently the current use of this isolate as a biological control agent of Hakea in South Africa poses no threat to indigenous Protea species. Colletotrichum acutatum from Protea (although closely related to C. acutatum f. sp. hakea), is a pathogen of Protea, which was confirmed by histological observations. In conclusion, the present study has shown that several species of Colletotrichum are associated with diseased Proteaceae. These species differed in their pathogenicity and aggressiveness when inoculated onto certain protea cultivars. These differences could be partially explained by examining the behaviour of C. acutatum on the leaf surface. It is clear, however, that the distribution of the different species, their aggressrveness on different Proteaceae and their modes of infection needs to be investigated further. This work provides a basis for future research on the long-term effective management of these pathogens in fynbos production.; AFRIKAANSE OPSOMMING: Hierdie tesis bestaan uit vier hoofstukke wat handeloor navorsing van Colletotrichum siektes van Proteaceae wêreldwyd. Die eerste hoofstuk is 'n oorsig van literatuur rakende die taksonomie en histologie van die Colletotrichum spesies wat met Proteaceae geassosieer word. Die literatuur oorsig is nie beperk tot die Proteaceae nie aangesien baie min inligting rakende Colletotrichum op Proteaceae bestaan. In die tweede hoofstuk word die Colletotrichum spesies wat met proteas in verskeie dele van die wêreld geassosieer word, op grond van morfologie, DNS volgorde data van die interne getranskribeerde spasieerder area ("ITS-I, ITS-2"), die 5.8S geen, en gedeeltelike DNS volgordes van die B-tubulin geen geïdentifiseer. Vier Colletotrichum spesies is met die Proteaceae geassosieer. Colletotrichum gloeosporioides is geïsoleer vanaf Protea cynaroides wat in Suid-Afrika en Zimbabwe gekweek is en vanaf 'n Leucospermum sp. in Portugal, maar is bekend op verskeie gashere wêreldwyd. 'n Spesie wat onlangs beskryf is, C. boninense, is met Zimbabwiese en Australiaanse Proteaceae geassosieer, maar kom ook op 'n Eucalyptus sp. in Suid-Afrika voor. Dit is 'n groot uitbreiding van die geografiese voorkoms en gasheerreeks van hierdie spesie en 'n beskrywing van die Afrikaanse rasse word gegee. Colletotrichum crassipes is verteenwoordig deur 'n enkele isolaat wat vanaf 'n Dryandra plant in Madeira verkry is. Colletotrichum acutatum is vanaf Protea en Leucadendron in Suid-Afrika asook vanaf ander proteas wat elders voorkom, geïsoleer. Colletotrichum acutatum f. sp. hakea is vanaf Hakea in Suid-Afrika geïsoleer. In hoofstuk drie is die patogenisiteit van hierdie Colletotrichum spesies teenoor sekere proteas getoets, die relatiewe aggressiwiteit van die verskillende spesies is vergelyk, asook die gasheer se reaksie teenoor die spesies en die effek wat verwonding op die gasheer gehad het. Daar kan afgelei word vanaf die resultate dat C. acutatum en C. gloeosporioides die primêre patogene is wat met Colletotrichum blaarnekrose geassosieer word, en dat C. acutatum die hoof oorsaak is van antraknose en lootnekrose van Proteaceae in Suid-Afrika. 'n Histologiese studie is in hoofstuk vier uitgevoer in reaksie op die bevindings van die vorige hoofstuk. Die gedrag van twee C. acutatum isolate (een vanaf Protea en die ander vanaf Hakea, C. acutatum f.sp. hakea) op die oppervlaktes van geïnokuleerde Protea blare is bestudeer deur gebruik te maak van lig- en skandeer-elektronmikroskopie. Colletotrichum acutatum vanaf Protea vorm gemelaniseerde appressoria op die blaaroppervlak, terwyl C. acutatum vanaf Hakea klein hoeveelhede van beide gemelaniseerde en ongemelaniseerde appressoria vorm. Meeste van die appressoria wat deur C. acutatum vanaf Protea gevorm word, vorm op die aanhegtingspunte tussen selle en op die periklinale wande van die epidermale selle. Vanuit hierdie studie is dit duidelik dat C. acutatum f. sp. hakea nie 'n patogeen van Protea is nie. Gevolglik hou die huidige gebruik van hierdie isolaat as biologiese beheer agent van Hakea in Suid-Afrika geen gevaar in vir inheemse Protea spesies nie. Colletotrichum acutatum vanaf Protea (alhoewel dit naverwant is aan C. acutatum f. sp. hakea) is 'n patogeen van Protea en hierdie stelling is ook bevestig deur histologiese waarnemmgs. Ter samevatting het hierdie studie getoon dat verskeie Colletotrichum spesies geassosieer word met siektes van Proteaceae. Hierdie spesies het van mekaar verskil rakende patogenisiteit en aggressiwiteit nadat hulle op sekere protea kultivars geïnokuleer is. Hierdie verskille kon gedeeltelik verklaar word deur die gedrag van C. acutatum op die blaaroppervlaktes van verskillende protea kultivars. Dit is duidelik dat die verspreiding van die verskillende spesies, hulle aggressiwiteit op verskillende Proteaceae en hul infeksie metodes verder ondersoek moet word. Hierdie studie verskaf 'n basis vir toekomstige navorsing rakende lang-termyn effektiewe bestuur van hierdie patogene in fynbos aanplantings. Thesis (MSc)--Stellenbosch University, 2004.

The expression of yeast antifungal genes in tobacco as possible pathogenesis-related proteins

Mon, 01 Dec 2003 00:00:00 GMT

The expression of yeast antifungal genes in tobacco as possible pathogenesis-related proteins Basson, Esmé Maree ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. Although integration of the MJ-EXG1 expression cassette was confirmed, no mRNA levels could be detected with northern blot or RT-PCR analysis of the MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops.; AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse. Thesis (MScAgric)--Stellenbosch University, 2003.

The detection of cherry leaf-roll nepovirus and the use of molecular markers for germplasm identification in walnuts (Juglans regia L.)

Mon, 01 Dec 2003 00:00:00 GMT

The detection of cherry leaf-roll nepovirus and the use of molecular markers for germplasm identification in walnuts (Juglans regia L.) Mkhize, Thokozani M ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed.; AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om twee algemene opsporingstegnieke te kombineer: serologiese toetsstelle en genetiese vingerafdrukke om cherry leaf-roll nepovirus (CLRV) te eien en om In merkersisteem te ontwikkel wat okkerneut kiemplasma kan karakteriseer. Die opsporing van plant virusse is baie moeilik. As gevolg van kwarantyn vereistes, word daar beperkinge geplaas word op die invoer van plant materiaal vanuit die buiteland. Moderne tegnieke soos hierdie een wat op PKR berus, word benodig om te verseker dat CLRV nie in plantmateriaal teenwoordig is nie. In Stel inleiers is ontwerp wat In 430 bp nie-koderende homoloë area amplifiseer. Hiervoor is konsensus volgordes bestudeer en slegs die volgordes wat 98,5% homologie getoon het, is gekies. In vergelyking met ELISA was die sensitiwiteit van hierdie deteksie metode 100 maal beter. DNA volgordebepaling is op die resulterende fragment gedoen om die PKR produk te verifieer. AFLP tegnologie is gebruik om polimorfiese fraqmente vir 6 okkerneut kultivars en 'n onderstok te identifiseer en SCARs is uit hierdie fragmente ontwikkel. Die AFLP tegniek kon tussen al die okkerneut kultivars en die onderstok onderskei. Die omskakeling van die AFLP fragmente in SCAR merkers om sodoende In eenvoudige kragtige tegniek vir kultivar onderskeiding te ontwikkel, was egter nie suksesvol nie. Met die gebruik van 27 AFLP inleier kombinasies, kon polimorfiese fragmente van so hoog as 47.8% verkry word. Die rede hoekom omskakeling onsuksesvol was lê by die aard van die AFLP tegniek. Die SCAR inleiers is ontwikkel uit volyordes intern tot die AFLP inleiers, maar die spesifisiteit van die merkers het juis in die AFLP inleiers gelê en nie in die interne volgordes nie. In hierdie studie, met die gebruik van AFLP, is gevind dat okkerneut kultivars baie naby verwant is. Die AFLP inleierstelle wat gebruik is, het polimorfiese fragmente gelewer. Uit hierdie fragmente is 7 SCAR merkers ontwikkel. Daar is verwag dat die SCARs wat uit die AFLP merkers ontwikkel is, klein verskille tussen kultivars sou opspoor. Dit was egter net die Paradox SCAR merker wat die kultivars in twee groepe kon verdeel. Restriksie ensiem vertering met Rsalop die Chandler SCAR produkte het dieselfde bandpatrone as die van die Paradox SCAR produkte gelewer. Thesis (MSc)--Stellenbosch University, 2003.

The role of the mediterranean fruit fly, Ceratitis capitata, in Botrytis bunch rot of grape

Fri, 01 Mar 2002 00:00:00 GMT

The role of the mediterranean fruit fly, Ceratitis capitata, in Botrytis bunch rot of grape Engelbrecht, Rene ENGLISH ABSTRACT: Botrytis bunch rot of grape is caused by Botrytis cinerea Pers. :Fr. Conidia of the pathogen, which is dispersed by wind, water droplets and by insects, can penetrate the intact grape berry cuticle, but disease expression occurs only under predisposing conditions. Since relatively high infection rates often occur in vineyards, predisposing factors must play a fundamental role in primary infection and subsequent disease occurrence. Insects can play a very important role in this regard by depositing inocula at wound sites during feeding and by providing fresh wounds during their oviposition and feeding activities. The aim of this study was (i) to determine the potential of the Mediterranean fruit fly to transfer B. cinerea and other bunch and fruit rot fungi in natura, (ii) to investigate the transport, deposition and subsequent disease expression on grape berries in vitro, and (iii) to investigate fruit fly activities and the nature of deposited conidia and mycelia of B. cinerea by aid of digital photography and epifluorescence microscopy, respectively. Two Sensus fruit fly traps containing the para-pheromone, Capilure, were installed in orchards and five neighboring vineyards on four farms in the Stellenbosch region. Ceratitis fruit flies were collected weekly, identified and counted to determine the fluctuations in fruit fly population. Following field collection, the fruit flies were plated on Kerssies' B. cinerea selective medium and the number of flies yielding the pathogen was recorded. Two fruit fly species, C. capitata and C. rosa, were captured during the study period. Ceratitis rosa numbers comprised only 1% of the total number of fruit flies captured. Ceratitis capitata numbers, and the percentage B. cinerea contaminated flies generally increased after harvest in the different orchards and vineyards. Following harvest, the percentage flies yielding B. cinerea was higher in vineyards compared to orchards. Furthermore, in each vineyard an increase in percentage B. cinerea contaminated fruit flies was preceded by a corresponding increase in its neighboring orchard. The levels of both Penicillium and Alternaria contaminated fruit flies stayed high throughout the investigation period, especially after harvest of the orchard cultivars. Low incidence of Aspergillus, Mucor and Rhizopus spp. were recorded on C. capitata. These findings suggest that the Mediterranean fruit fly may play an important role in the dispersal of inocula of fungi associated with postharvest decay from early-maturing stone fruit orchards to mid- and late-maturing wine grape vineyards, and in disease induction under conditions unfavourable for natural infection. Three experiments were conducted to determine the potential of fruit flies in provoking B. cinerea decay. In the first experiment, transport of conidia and disease expression were investigated on rachis segments bearing unwounded berries only. In the second experiment, the effect of wounding on disease expression was investigated. In the third experiment, the effect of inoculum type (mycelia and conidia) on transportation and disease expression was investigated on rachis segments bearing unwounded berries, and on segments with wounded berries. The table grape cultivar, Dauphine, and the wine grape cultivar, Shiraz, were used at véraison, two weeks before harvest and harvest, and the transport studies were conducted in ethanol-disinfected perspex cages. Disease expression was studied in dry (~56% RH), ethanol-disinfected perspex chambers incubated at 22°C. The isolations from berries revealed that the flies deposited, without preference, high amounts of B. cinerea at various positions on the grape berry's surface. The freezing studies showed that the deposited conidia germinated and penetrated the berry skin at various positions. However, B. cinerea developed more often at the pedicel end than on the cheek or style end, which indicated a peculiar interaction between B. cinerea, the fruit fly and host tissue at this part of the berry. This phenomenon was substantiated by the finding that B. cinerea also developed more often at the pedicel end of berries that were not frozen. Further evidence for this interaction was found on intact berries exposed to flies that carried mycelia after feeding on berries without sporulating colonies of the pathogen, but showing symptoms of slippery skin. Significantly more decay developed on wounded berries compared to the unwounded berries and more so at the wound site. In addition, female fruit flies were responsible for significantly more decay development than male fruit flies. The study thus proved that the Mediterranean fruit fly can promote B. cinerea disease development under conditions unfavorable to natural infection. The activities of the Mediterranean fruit fly, Ceratitis capitata, on grape berries were monitored by aid of digital photography. In addition, the deposition of conidia and mycelia of Botrytis cinerea at three sites (pedicel end, cheek and style end) on the grape berry, germination of the fungal structures after dry (±56% RH) and moist (±93% RH) incubation and wounds inflicted during ovipositioning were examined with an epifluorescence microscope. The observations revealed that the fruit fly's activities were generally restricted to the grape berry. They visited the grape berry cheek more often, but visitations to the pedicel end of berries increased substantially from véraison to harvest, indicating the possibility of nutrient leakages at this site. Microscopy revealed that the flies deposited conidia singular, in feeding packages and in faecal excrements on the berry surface. The conidia in feeding packages were ensheathed by salivical fluids and occurred in clusters of 10 to 50 conidia. An average of 60% of the conidia in feeding packages germinated under dry conditions (±56% RH). Conidia that passed through the intestinal tract of the fruit fly and that were deposited in faecal excrements were deformed and low in viability. These conidia did not occur in cluster format, but were proportionally spread with the faeces on the surface of the grape berry. Conidia that were deposited singular and in faecal excrements did not germinate unless incubated under moist conditions (± 93% RH). Wounds inflicted by female fruit flies during ovipositioning were most frequently observed on the cheek. This predisposition to B. cinerea infection of grape berries by the activities of fruit flies, suggested an important role for the flies in the initiation of Botrytis bunch rot epidemics in vineyards.; AFRIKAANSE OPSOMMING: DIE ROL VAN DIE MEDITERREENSE VRUGTEVLIEG, CERATITIS CAPITATA, IN BOTRYTIS CINEREA TROSVERROTTING VAN DRUIWE Botrytis-trosverrotting van druiwe word deur Botrytis cinerea Pers. :Fr. veroorsaak. Konidia van die patogeen wat deur wind, waterdruppels en insekte versprei word, kan die intakte druiweskil binnedring, maar siekte-uitdrukking vind slegs onder spesiale omstandighede plaas. Aangesien relatief hoë infeksie vlakke algemeen in wingerde voorkom, moet predisponerende faktore 'n fundamentele rol in die primêre infeksie, en die daaruit voortspruitende siektetoestand speel. Insekte kan 'n baie belangrike bydrae lewer deur inokuia tydens voeding by wonde te deponeer. Nuwe wonde kan ook tydens oviposisionering en voeding ontstaan. Die doel van hierdie studie was om (i) die potensiaal van die Mediterreense vrugtevlieg om B. cinerea en ander tros- en vrugverrottingswamme in natura oor te dra, te bepaal; om (ii) die verspreiding, deponering en daaropvolgende siekteuitdrukking op druiwekorrels in vitro te ondersoek; en om (iii) die aktiwiteite en aard van die gedeponeerde konidia en miselia met behulp van digitale fotografie sowel as epifluoressensiemikroskopie waar te neem. Twee Sensus-vrugtelokvalle met die paraferomoon, Capilure, IS In vrugteboorde en aangrensende wingerde in die Stellenbosch-omgewing aangebring. Ceratitis-vrugtevlieë is weekliks versamel, geïdentifiseer en getel om fluktuasies in die vrugtevliegpopulasie te bepaal. Na die veldversameling is die vrugtevlieë op Kerssies se B. cinerea-selektiewe medium uitgeplaat. Gedurende die studie is twee spesies vrugtevlieë, C. capitata en C. rosa, gevang. Na oesstyd het die aantal Ceratitis-vrugtevlieë en die persentasie vrugtevlieë, besmet met B. cinerea, in die verskillende boorde en wingerde toegeneem. Na oestyd was die persentasie vrugtevlieë wat B. cinerea gedra het, hoër in die wingerde as in die boorde. Elke toename in die persentasie B. cinerea-besmette vrugtevlieë in 'n wingerd is voorafgegaan deur 'n ooreenkomstige toename in die aangrensende vrugteboord. Die aantal vrugtevlieë besmet met Penicillium en Alternaria spp. het tydens die navorsingstydperk deurgaans hoog gebly, veral nadat die vrugteboord-kultivars geoes is. Die voorkoms van Aspergillus-, Mucor- en Rhizopus spp. op Ceratitis-vrugtevlieë was deurgaans laag. Hierdie bevinding wys daarop dat vrugtevlieë 'n belangrike rol speel in die verspreiding van swarninokula, wat met na-oes verrotting geassosieer word, van vroegrypwordende steenvrugteboorde na mid- en laatrypwordende wyndruifwingerde. Drie eksperimente is in vitro onderneem om vrugtevlieë se potensiaal om B. cinereaverrotting te veroorsaak te bepaal. In die eerste eksperiment is ragi met slegs ongewonde korrels gebruik om die oordrag van konidia en siekte-ontwikkeling te ondersoek. In die tweede eksperiment is die effek van verwonding op siekte-ontwikkeling ondersoek. In die derde eksperiment is die effek van inokulumtipe (miselia en konidia) op verspreiding en siekte-ontwikkeling ondersoek deur ragis-segmente met gewonde korrels sowel as ragissegmente met ongeskonde korrels te gebruik. Die tafeldruif-kultivar Dauphine en die wyndruif-kultivar Shiraz, by kleurbreuk, twee weke voor oes en by oestyd, is in die eksperimente gebruik. Die oordragstudies is in etanol-ontsmette perspex-hokke uitgevoer. Siekte-ontwikkeling is bestudeer in droeë (±56% RH), etanol-ontsmette perspex-kamers en geinkubeer by 22°C. By ondersoek is gevind dat vlieë, sonder voorkeur, groot hoeveelhede B. cinerea op verskeie dele op die druiwekorrel-oppervlak deponeer. Bevriesingstudies het aangetoon dat die gedeponeerde konidia op verskeie dele van die korrelontkiem en die skil binnedring. Botrytis cinerea het egter meer dikwels by die korrelsteelkant as by die stempelkant, of op die wang, ontwikkel. Hierdie bevinding het 'n eiesoortige interaksie tussen B. cinerea, die vrugtevlieg en gasheerweefsel by die korrelsteelkant van die korrel aangetoon. Die verskynsel is gestaaf deur die bevinding dat B. cinerea ook meer dikwels by die korrelsteelkant van die korrels wat nie gevries is nie, ontwikkel het. Verdere bewys van hierdie interaksie is gevind by ongeskonde korrels wat aan die vlieë wat miselia gedra het blootgestel is. Die siekte het beduidend meer dikwels op gewonde as ongewonde korrels en verder aansienlik meer dikwels op die wondoppervlakte ontwikkel. Dit was ook duidelik dat vroulike vrugtevlieë baie meer vir verrotting verantwoordelik was as manlike vrugtevlieë. Die studie bewys dus dat Mediterreense vrugtevlieë die ontwikkeling van B. cinerea kan bevorder in omstandighede wat ongunstig is vir natuurlike infeksie. Die aktiwiteite van die Mediterreense vrugtevlieg C. capitata op die druiwekorrels is met behulp van digitale fotografie waargeneem. Verder is die deponering van konidia en miselia van B. cinerea op die verskillende dele (korrelsteelkant, wang en stempelkant) van die korrel, ontkieming van die swamstrukture na droeë (±56% RH) en nat (±93% RH) inkubasie en wonde wat tydens oviposisionering veroorsaak is, met epifluoressensie-mikroskopie ondersoek. Die waarnemings het onthul dat die vrugtevlieg se aktiwiteite gewoonlik tot die druiwekorrel beperk is. Hulle het korrelwange meer dikwels besoek. Besoek aan die korrelsteelkant het aansienlik toegeneem van kleurbreuk tot oestyd, wat op die moontlikheid van voedingstof-lekkasie by die deel aandui. Mikroskoopstudies het aangedui dat vlieë konidia enkel, in voedingspakkies en in fekale uitskeidings op die korreloppervlakte deponeer. Die konidia in die voedingspakkies is deur speekselvloeistof omhul en het in groepe van 10 tot 50 konidia voorgekom. Gemiddeld 60% van die konidia in voedingspakkies het in droeë omstandighede (±56% RH) ontkiem. Konidia wat deur die spysverteringskanaal van die vrugtevlieg gegaan het en in die fekale ekskresie gedeponeer is, was misvorm en het lae lewensvatbaarheid gehad. Laasgenoemde konidia was nie in groepe gedeponeer nie, maar is proporsioneel met die feces op die oppervlak van die druiwekorrel versprei. Konidia wat enkel en in feces gedeponeer is, het nie ontkiem nie, tensy toestande vogtig (±56% RH) was. Wonde wat deur die vroulike vrugtevlieë tydens oviposisionering veroorsaak is, is meer dikwels op die wang van die korrelopgemerk. Hierdie predisposisie van druiwekorrels tot B. cinerea-infeksie, meegebring deur die aktiwiteit van die vrugtevlieg, dui daarop dat die rol wat die vrugtevlieg in die inisiëring van Botrytis trosverrottingepidemies in wingerde speel, van beduidende belang is. Thesis (MScAgric)--University of Stellenbosch, 2002.

Infection pathways of Botrytis cinerea on selected wine grape cultivars

Sun, 01 Dec 2002 00:00:00 GMT

Infection pathways of Botrytis cinerea on selected wine grape cultivars Du Preez, Izak Frederik 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 (MScAgric)--University of Stellenbosch

Infection by dry, airborne Botrytis cinerea conidia and fungicide efficacy on different parts of grape bunches and vinelets

Fri, 01 Mar 2002 00:00:00 GMT

Infection by dry, airborne Botrytis cinerea conidia and fungicide efficacy on different parts of grape bunches and vinelets Van Rooi, Cicelia ENGLISH ABSTRACT: The evaluation of fungicide efficacy in commercial vineyards can be influenced by the sporadic occurrence of Botrytis cinerea at various positions on vines, differences in bunch structure during bunch development and the phenomenon that symptom expression in shoots and bunches is governed by the resistance reaction of the various shoot and bunch parts. It has been postulated that, following air and water dispersal, infection by solitary conidia should playa prominent role in the epidemiology of B. cinerea on grapevine. The aim of this study was to determine (i) infection and (ii) fungicide efficacy at specific sites on shoots of vinelets and bunches (table grape cultivar Dauphine and the wine grape cultivar Merlot) inoculated with dry, airborne conidia of B. cinerea. Vinelets, prepared from cuttings, and bunches obtained from the vineyards at full bloom, pea size, bunch closure, véraison and harvest stages, were sprayed in a spray chamber at the recommended dosages with iprodione, pyrimethanil, cyprodinil/fludioxonil and fenhexamid or were left unsprayed. After 24 h the vinelets or bunches were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at a high relative humidity (±93%). Following incubation, both the vinelets or bunches were divided into three groups. Vinelets and bunches of the one group were surface-sterilised, the others were left unsterile. Vinelets and bunches of one unsterile group were placed in dry chambers, kept for 14 days at 22°C with a 12 h photoperiod daily and monitored for symptom expression and the development of B. cinerea. Vinelets and bunches of the sterile group, and from one unsterile group were used for isolation. From each of these vinelets leaf blades, leaf petioles, shoots and inflorescences were removed. Sites used for isolation in bunch parts were rachises, laterals and pedicels, and sites on berries were the pedicel-end, cheek and style-end. The different parts and segments were placed in Petri dishes on Kerssies' B. cinerea selective medium, or on water agar medium supplemented with paraquat and incubated for 14 days at 22°C with a 12 h photoperiod daily. Infection and fungicide efficacy was determined by observing intact vinelets and bunches for symptom expression, and by estimating the amount of B. cinerea at the various sites on the vinelets and bunches with isolation studies. No symptoms of B. cinerea decay developed on sprayed and unsprayed vinelets that were kept in dry chambers during the 2 wk observation period. The isolation and incubation studies showed that the different fungicides were highly and nearly equally efficient in reducing superficial B. cinerea inoculum and latent infection. .In the case of leaf blades, which showed a high amount of B. cinerea on unsprayed vinelets under the two sterility regimes, decay was significantly reduced by each fungicide on both cultivars. This was not the case for the other parts, which yielded B. cinerea at low incidences under the two sterility regimes. The study with bunches showed that dry, airborne conidia, and the fungicide sprays, penetrated loose and tight clustered bunches from bloom to harvest and evenly landed on the various bunch parts. At full bloom, the amount of B. cinerea in unsprayed bunches was high on the laterals and pedicels, but low on the embryos. Unsprayed intact bunches at full bloom were highly susceptible to B. cinerea and developed symptoms of grey mould. The fungicides inhibited symptom expression at full bloom, but could not prevent infection. Unsprayed bunches inoculated at the other stages remained asymptomatic. The amount of B. cinerea was generally high in the rachises and laterals at pea size and bunch closure stages, and in the pedicel end of berries at harvest. Infection was constantly low in the berry cheek. The fungicides had a differential effect on infection at the various sites. In the case of rachises, the amount of B. cinerea was at each growth stage drastically reduced by each fungicide. In laterals, it was effectively reduced at pea size and bunch closure. However, at these two sites, significant differences were found between the fungicides in efficacy at stages when the amount of B. cinerea was high. This study showed that if these fungicides are applied properly to vine in commercial vineyards between budding and prebloom, during flowering, and at bunch closure, they should effectively prevent infection and symptom expression and thus the development of B. cinerea epiphytotics.; AFRIKAANSE OPSOMMING: INFEKSIE DEUR DROË, LUGGEDRAAGDE BOTRYTIS CINEREA KONIDIA EN DIE EFFEK VAN FUNGISlEDE OP VERSKILLENDE SETELS BINNE WINGERDTROSSE EN OP LOTE: Evaluering van fungisieddoeltreffendheid in kommersiële wingerde word beïnvloed deur die sporadiese voorkoms van Botrytis cinerea op verskeie posisies van wingerddele, verskille in trosstruktuur tydens trosontwikkeling, en die feit dat simptoomuitdrukking in lote en trosse deur die weerstandsaksie van die verskillende morfologiese dele van lote en trosse beheer word. In die natuur speel infeksie deur enkel konidia 'n prominente rol in die epidemiologie van B. cinerea van wingerd. Die doel van hierdie studie was om (i) infeksie en (ii) die effek van fungisiede op verskillende posisies op lote en trosse (tafeldruif kultivar Dauphine, wyndruif kultivar Merlot), wat met droë, luggedraagde konidia van B. cinerea geïnokuleer is, te bepaal. Lote, verkry vanaf steggies, en trosse versamel vanuit die wingerde tydens blom-, ertjiekorrel-, trostoemaak-, deurslaan- en oesstadium, is teen aanbevole dosisse met iprodione, pyrimethanil, cyprodinillfludioxonil of fenhexamid in 'n spuitkas bespuit, of is onbehandeld gelaat. Na 24 h is die lote en trosse met droë konidia van B. cinerea in 'n inokulasietoring geïnokuleer en daarna vir 24 h onder hoë humiditeit [±93% RH] geïnkubeer. Na inkubasie is die lote en trosse in drie groepe verdeel. Die een groep lote en trosse is oppervlakkig gesteriliseer om die patogeen op die oppervlakte te elimineer, en die ander twee groepe is onbehandeld gelaat. Die lote en trosse van een nie-steriele groep is vir 14 dae in droë voghokke by 22°C met 'n 12 uur daaglikse fotoperiode geplaas, en daagliks vir siekteuitdrukking en die ontwikkeling van B. cinerea gemonitor. Lote en trosse van die ander twee groepe is vir isolasiestudies gebruik. Vanaf elke loot is blaarskywe, blaarstele, internodes en ongeopende blomtrossies verwyder. Vanaftrosse is ragisse, laterale en korreisteie verwyder, en vanaf korrels is skilsegmente aangrensend aan die korrelsteel, die stempel-end, en die wang verwyder. Die dele en segmente is op B. cinerea selektiewe medium, en op paraquat medium in Petri bakkies geplaas en vir 14 dae by 22°C met 'n 12 uur daaglikse fotoperiode geïnkubeer. Infeksie en die fungisiedeffek is bepaal deur die intakte lote en trosse vir siekte- uitdrukking te monitor, en deur die hoeveelheid B. cinerea op verskeie posisies op lote en trosse te bepaal. Geen simptome het op enige posisie op bespuite en onbespuite lote, wat in droë hokke gehou is, ontwikkel nie. Die isolasie- en inkubasiestudies het getoon dat die verskillende fungisiede hoogs effektief op lote was, en inokulumvlakke van die patogeen doeltreffend verlaag het. In die geval van blaarskywe, wat hoë vlakke van B. cinerea op onbespuite steggies onder die twee steriliteitskondisies getoon het, is verrotting op beide kultivars betekenisvol deur die fungisiedes verlaag. Dit het egter nie vir die ander dele, waarop daar 'n lae voorkoms van B. cinerea onder die twee steriliteitskondisies was, gegeld me. Die studie met trosse het getoon dat droë, luggedraagde konidia en fungisiednewels beide oop en kompakte trosse vanaf blomstadium tot oes penetreer en eweredig op die verskillende dele land. Met blomstadium was die hoeveelheid B. cinerea in onbespuite trosse hoog op laterale en korrelstele, maar laag op die embrios. Onbespuite, intakte trosse was hoogs vatbaar vir B. cinerea by blomstadium en het simptome van vaalvrot ontwikkel. Die fungisiede het siekte-uitdrukking by blomstadium voorkom, maar kon nie infeksie voorkom me. Onbespuite trosse wat op ander stadia geïnokuleer is, het geen siekte-uitdrukking getoon me. Die hoeveelheid B. cinerea was hoër in die ragi, asook in laterale by ertjiekorrel- en trostoemaak stadium, en hoër in korreisteie by oesstadium. Infeksie was konstant laag in die korrelskil. Die fungisiede het 'n differensiële effek op infeksie by die verskillende posisies gehad. In die geval van ragi was die hoeveelheid B. cinerea drasties deur elke fungisied by alle groeistadia verlaag. In laterale was dit effektief by ertjiekorrel- en trostoemaakstadium verminder. By hierdie twee posisies waar die hoeveelheid B. cinerea hoog was, is daar egter betekenisvolle verskille in die doeltreffendheid van fungisiedes gevind. Hierdie studie toon dat as fungisiede behoorlik in kommersiële wingerde tussen botvorming en blomstadium, en tydens blom- en trostoemaakstadium toegedien word, infeksie en siekte-uitdrukking, en dus ook die epifitotiese ontwikkeling van B. cinerea, voorkom behoort te word. Thesis (MScAgric)--University of Stellenbosch, 2002.