Tesis para optar al grado de Magíster en Ciencias Agropecuarias Mención Sanidad Vegetal / Botrytis cinerea produce importantes pérdidas en uva de mesa en Chile. En el control químico de este patógeno se utiliza entre otras moléculas a boscalid, carboxamida que actúa inhibiendo la enzima succinato deshidrogenasa. En aislados de Botrytis recolectados de cultivos sometidos a un uso intensivo del fungicida, se ha demostrado la asociación entre la pérdida de sensibilidad con mutaciones en el gen sdhB, en donde se han identificado las mutaciones P225F/L/T y N230I asociadas a aislados resistentes y en mayor frecuencia las mutaciones H272R/Y/L asociadas a aislados resistentes y moderadamente resistentes. Durante las últimas dos temporadas (2011-2012 y 2012- 2013), en la zona Central de Chile, se ha reportado una baja sensibilidad a boscalid. El objetivo de la presente investigación fue determinar la presencia de mutaciones en el gen sdhB en aislados chilenos de Botrytis cinerea de distinto nivel de sensibilidad a boscalid. Con este propósito, 50 aislados monoconidiales fueron caracterizados genética y fenotípicamente. Para la caracterización fenotípica se verificó la sensibilidad a boscalid mediante evaluación del comportamiento de germinación conidial, determinándose 4 categorías según valores EC50: Sensible (S) (>0,05-1,37μg.mL-1), Levemente Resistente (LR) (1,38-7,80μg.mL-1), Moderadamente Resistente (MR) (7,81-50μg.mL-1) y Resistente (R) (>50μg.mL-1). Aislados R y MR con mutaciones se compararon con sensibles, según parámetros como: crecimiento miceliar, esporulación, sensibilidad osmótica, capacidad formadora de esclerocios y virulencia. La detección de mutaciones se realizó mediante la técnica PCR-PIRA (Primer-Introduced Restriction Enzyme Analyses) y el uso de partidores específicos H272Y/R-fw y H272- rev, cuyos productos fueron digeridos con las enzimas de restricción EcoRV y HhaI respectivamente. Los aislados de Botrytis cinerea mostraron niveles de sensibilidad a boscalid variables entre 0,13 μg.mL-1 (S) y 1,1*109 μg.mL-1 (R). La utilización del PCR-PIRA, permitió identificar las mutaciones H272Y/R en el gen sdhB que resultaron ser inespecíficas de un determinado nivel de sensibilidad a boscalid. De acuerdo a los parámetros de adaptabilidad evaluados tales como crecimiento miceliar, capacidad formadora de esclerocios y esporulación de aislados resistentes con mutaciones H272Y/R respecto de los sensibles fueron significativamente diferentes (p<0,05), lo cual implicaría que las mutaciones detectadas y asociadas a la resistencia a boscalid generarían un costo metabólico en los aislados de Botrytis. / Botrytis cinerea produces serious losses in table grapes in Chile. In the chemical control of this pathogen is used among other molecules like boscalid, carboxamide that inhibits the succinate dehydrogenase enzyme. Botrytis isolates collected from different crops subjected to intensive fungicide application has shown an association between sensitivity loss and mutations in the sdhB gene, where P225F/L/T and N230I mutations have been identified in resistant isolates and most frequently mutations like H272R/Y/L on moderately resistant and resistant isolates. During the past two seasons (2011-2012 and 2012-2013) in the central Chile, has reported a low sensitivity to boscalid. The objective of this research was determine the mutations presence in the sdhB gene of Botrytis cinerea isolates with different sensitivity levels to boscalid. For this purpose, 50 monoconidial isolates were characterized genetically and phenotypically. For phenotypic characterization was verified the sensitivity to boscalid by conidial germination, were classified into four resistance phenotypes based on the EC50 values them: Sensitive (S) (>0.05-1.37 μg.mL-1), Low resistant (LR) (1.38-7.80 μg.mL-1), moderately resistant (MR) (7.81-50μg.mL-1) and resistant (R) (>50μg.mL-1). Isolate R and MR with mutations were compared with sensitive isolates, with parameters such as mycelial growth, sporulation, osmotic sensitivity, sclerotia production and virulence. Mutations detection was performed by PIRA-PCR (Primer- Introduced Restriction Enzyme Analyses) with specific primers H272-rev H272Y/R-fw whose products were digested with restriction enzymes EcoRV and HhaI respectively. Botrytis cinerea isolates showed different sensitivity levels between 0.13 μg.mL-1 boscalid (S) and 1.1*10-9 μg.mL-1 (R). The PIRA-PCR method has detected H272Y/R mutations, none were specific a sensitivity level to boscalid. According to adaptability parameters evaluated such as mycelial growth, sclerotia production and sporulation of resistant isolates with mutations H272Y/R compare with sensitive were significantly different (p <0.05), which imply that the mutations detected generate a metabolic cost in Botrytis cinerea isolates to boscalid.

Tesis para optar al título profesional de Ingeniera Agrónoma y al grado de Magíster en Ciencias Agropecuarias con mención Sanidad Vegetal / La base del control de Botrytis cinerea radica en el uso de fungicidas aplicados en los periodos críticos de infección (floración y envero a precosecha). Las estrobilurinas (QoIs) por tener efecto sobre Oídio y también sobre Botrytis son alternativas de control en pre y post-floración. Recientemente, en ciertas regiones de Europa, Norteamérica y Chile se ha detectado el inicio de pérdida de sensibilidad de botrytis a QoIs. La resistencia a este grupo de fungicidas en hongos fitopatógenos estaría asociada a tres tipos de mutaciones en el citocromo b, siendo la principal la mutación G143A (reemplazo de Glicina por Alanina en el codón 143). El objetivo del presente estudio fue determinar la posible relación entre pérdida de sensibilidad a QoIs, comportamiento del patógeno y presencia de algunas de las 3 mutaciones asociadas. Con este propósito 20 aislados de B. cinerea recuperados desde vid y seleccionados según antecedentes previos de sensibilidad a azoxystrobin, fueron caracterizados genéticamente, mediante PCR (Reacción en Cadena de la Polimerasa) alelo-específico, para detectar la presencia de las mutaciones asociadas a resistencia a QoIs y mediante PCR-duplex para detectar la presencia de los transposones Boty y Flipper. La caracterización fenotípica consideró los siguientes parámetros: aspecto miceliar, esporulación, capacidad formadora de esclerocios (medio Agar- Malta (AM) a 20ºC), crecimiento miceliar (medio AM a 15, 20 y 25ºC), y virulencia sobre bayas cv. Thompson Seedless, con y sin herida a 0 y 20ºC.

Memoria para optar al título profesional de Ingeniero Agrónomo / Con el fin de evaluar la virulencia y características genéticas de aislados chilenos de Botrytis cinerea con distinta sensibilidad a iprodione, se seleccionaron nueve aislados altamente resistentes (EC50: 1,35 – 2,47 μg·mL-1) y cuatro aislados sensibles (EC50: 0,26 – 0,31 μg·mL-1). La virulencia se evaluó en plántulas de pepino (Cucumis sativus L.) mediante inoculación de discos de agar con micelio del hongo de tres días de edad y en bayas de vid (Vitis vinífera L.) Thompson Seedless con distinto grado de madurez (7°, 14° y 17° Brix), mediante inoculación de una suspensión conidial (equivalente a 2·105 conidias·mL-1). Posterior a la inoculación las plántulas de pepino se mantuvieron a 20-25°C y 100% de humedad relativa y las bayas de vid en cámaras de crecimiento a 20°C. La evaluación se realizó mediante medición del diámetro de la lesión en la zona de inoculación luego de 96 y 72 horas en hojas de pepino y bayas de vid, respectivamente. Los resultados obtenidos fueron sometidos a un ANDEVA simple y en las dos pruebas de virulencia realizadas los aislados de Botrytis cinerea resistentes y sensibles presentaron niveles de agresividad similares. La caracterización genética de los aislados se realizó amplificando mediante PCR el gen bos1 asociado a resistencia de Botrytis cinerea a dicarboximidas, utilizando 5 pares de partidores específicos. Posteriormente los productos de PCR purificados fueron secuenciados por Macrogen USA Corp. El análisis de las secuencias nucleotídicas de los nueve aislados resistentes detectó un cambio aminoacídico en la posición T1259A de la proteína. En ocho de éstos fueron detectadas las mutaciones I365N y R104Q y en el aislado restante un cambio en la posición I365S y una sustitución en la posición L849V. Esta última sustitución no ha sido descrita previamente desconociéndose su implicancia en el nivel de resistencia. En ninguno de los cuatro aislados sensibles secuenciados, el gen bos1 presentó mutaciones asociadas a resistencia a dicarboximidas. / In order to evaluate the virulence and genetic characteristics to Chilean isolates Botrytis cinerea of with different sensitivity to iprodione, were selected nine highly resistant isolates (EC50: 1, 35 to 2.47 μg·mL-1) and four susceptible isolates (EC50: 0.26 to 0.31 μg·mL-1). The virulence was evaluated inoculating cucumber seedlings (Cucumis sativus L.) with three days old Botrytis cinerea mycelium and different ripe (7 °, 14 ° 17 ° Brix) Thompson Seedless grape berries (Vitis vinifera L.) with a conidial suspension (2·105 conidia·mL-1). Cucumber seedlings were kept at 20-25°C and relative humidity 100% and grape berries in growth chambers at 20°C. The evaluation was done measuring the lesion diameter after 96 and 72 hours in cucumber leaves and grape berries, respectively. The results were subjected to a simple ANOVA and in both virulence tests carried out, the sensitive and resistant Botrytis cinerea isolates showed similar levels of aggressiveness. The genetic characterization was performed by PCR amplification of the dicarboximide resistance associated Botrytis cinerea bos1 gene, by means of five specific primers. The purified PCR product was sequenced by Macrogen USA Corp. By means of sequences analysis the bos1 gene nucleotide, in all the nine resistant isolates was detected an amino acid change at the T1259A protein position. The I365N and R104Q mutations were detected in eight of the isolates and in the other isolate a change in the position I365S and a substitution at position L849V. The latter substitution has not been previously detected and it implications on resistance is unknown. The bos1 gene showed no mutations associated to the resistance to dicarboximides in any one of the four susceptible isolates sequenced.

Thesis (MScAgric)--University of Stellenbosch, 2002. / 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.

Memoria para optar al Título Profesional de Ingeniero Agrónomo / El control de enfermedades de poscosecha es una problemática constante en la producción de uva de mesa, siendo Botrytis cinerea y Penicillium expansum, dos de los patógenos más importantes. El cuestionamiento a la cantidad máxima de residuos de agroquímicos permitidos en la fruta en los mercados de destino y la acotada cantidad de fungicidas disponibles en el mercado, han llevado a la búsqueda de alternativas a los fungicidas sintéticos, la cual se ha enfocado a productos naturales, amigables con el medio ambiente y libres de residuos. El principal objetivo de este estudio fue evaluar la actividad de 15 extractos de macrohongos en el control de ambos patógenos. Los macrohongos fueron colectados desde su ambiente natural, posteriormente secados, triturados y macerados en alcohol al 95%, y los extractos resultantes fueron utilizados en bioensayos de inhibición in vitro e in vivo. Para el control in vitro se enmendó agar papa dextrosa con cada extracto en etanol al 1% donde se sembró micelio y conidias de los patógenos. El control in vivo se realizó sobre bayas de uva de mesa del cv. Red Globe sometidas o no a desinfección previa con NaClO (0,5%). A las bayas se les realizó una herida y luego se aplicaron los extractos 1 y 24 horas antes de la inoculación con conidias de los patógenos. Los extractos con mayor efectividad inhibitoria in vitro del crecimiento miceliar de B. cinerea fueron Suillus luteus, Agarical sp. y Amanita sp. los que inhibieron en un 92,9, 71,0 y 69,4%, respectivamente. Para el control in vitro de P. expansum los dos extractos que tuvieron un mayor efecto fueron los de Agarical sp. y Agaricus arvensis con un 35,3 y 34,6% de inhibición del crecimiento miceliar, respectivamente. Los extractos con mayor efectividad en la inhibición de la germinación de conidias fueron: Agarical sp. y Macrolepiota rhacodes, para B. cinerea, y Agaricus arvensis y Laetiporus sulphureus para P. expansum. En el control in vivo solo el extracto de Amanita sp. tuvo un efecto en la inhibición de la pudrición causada por B. cinerea sobre las bayas, aplicado 24 horas antes de la inoculación, e igualmente en bayas sin desinfectar inoculadas con P. expansum. / Postharvest rots of table grapes are a constant problem for the industry with Botrytis cinerea and Penicillium expansum as the most important pathogens. Restrictions on the maximum amount of pesticide residues allowed on the fruit, and the limited number of fungicides registered in the markets have prompted the look for alternatives to the traditional chemical fungicides, focusing mainly on natural products environmentally friendly and free from residue restrictions. This study was aimed to evaluate the activity of 15 extracts of mushrooms in the control of both pathogens. The mushrooms were collected from their natural sources, dried, grounded and macerated in ethanol 95%, and the extracts obtained were evaluated in vitro and in vivo. Potato dextrose agar media was amended with the ethanol extracts at a rate of 1%, for the in vitro tests with mycelia or conidia of the pathogens. In vivo assays were conducted on Red Globe berries that were surface disinfected with NaOCl (0,5 %) or not disinfected at all. The berries were wound inoculated with a spore suspension of each pathogen 1 or 24 hours after the extracts treatments. The extracts that gave the greatest inhibition of the mycelial growth of B. cinerea were those of Suillus luteus, Agarical sp. and Amanita sp, which gave a 92.9, 71.0 and 69.4 % inhibition, respectively. While the extracts with the higher inhibition of the mycelial growth of P. expansum were those of Agarical sp. and Agaricus arvensis with 35.3 and 34.6 % inhibition, respectively. The extracts with the higher inhibition of the conidia germination were those of Agarical sp. and Macrolepiota rhacodes, for B. cinerea, and Agaricus arvensis and Laetiporus sulphureus for P. expansum. Only the extract of Amanita sp. reduced the rot caused by B. cinerea when applied 24 hours before berry inoculation and the rot caused by P. expansum on berries that were not disinfected.

Memoria para optar al Título Profesional de: Ingeniero Agrónomo / El control químico para la pudrición gris es la principal herramienta para reducir su incidencia que puede ser lograda por medio de diversos compuestos fungicidas. El fludioxonil, ciprodinil y fenhexamid son moléculas con actividad botriticida y fueron desarrolladas a partir de los años 90. Sin embargo su eficacia se ha visto fuertemente afectada por la aparición de resistencia debido a su uso reiterado. Por otro lado algunos ingredientes activos utilizados en determinadas concentraciones suelen alterar el metabolismo de las plantas. El objetivo de este estudio fue evaluar el efecto de dos fungicidas aplicados al follaje de plantas jóvenes de Vitis vinifera, variedades Thompson Seedless y Chardonay sobre la actividad fotosintética. Las mediciones fueron realizadas 5 veces a lo largo del día durante 5 días. Se evaluaron los parámetros de fotoinhibición de la fotosíntesis, contenido de clorofilas, tasa de asimilación de CO2 y permeabilidad relativa de membranas. Para ello se realizaron dos ensayos consistentes en 4 tratamientos más un testigo (T1= ciprodinil (375g/kg) & fludioxonil (250g/kg), 60gr/hL; T2= ciprodinil (375g/kg) & fludioxonil (250g/kg), 120gr/hL agua; T3= fenhexamid (500g/L) 72,5cc/hL; T4= fenhexamid (500g/L) 145cc/hL agua). Los resultados no mostraron diferencias significativas entre tratamientos a lo largo del ensayo, indicando que los fungicidas no alteraron los parámetros fisiológicos registrados de las plantas. / Chemical control for gray mold is the principal tool to reduce its occurrence and it can be achieved with a number of different fungicide compounds. Fludioxonil, ciprodinil and fenhexamid are molecules with botriticide activity and were developed from the ’90s. However their efficacy has been seen to be markedly affected by the development of resistance due to repeated use. Moreover some active ingredients used in certain concentrations often alter plants’ metabolism. The aim of this study was to assess the effects of two fungicides applied to the leaves of young plants from Vitis vinifera varieties Thompson Seedless and Chardonnay on their photosynthetic activity. The measurements were carried out five times a day for five days. The variables that were tested were photoinhibition of photosynthesis; Chlorophylls content; Carbon dioxide assimilation and Membrane permeability. Two experiments were performed, consisting of four treatments and a control (T1= ciprodinil (375g/kg) & fludioxonil (250g/kg), 60gr/hL; T2= ciprodinil (375g/kg) & fludioxonil (250g/kg), 120gr/hL agua; T3= fenhexamid (500g/L) 72,5cc/hL; T4= fenhexamid (500g/L) 145cc/hL agua). The results did not show differences between plants subject to the different treatments over the course of the experiments, suggesting that fungicides do not affect the plants physiology.

Memoria para optar al título profesional de: Ingeniero Agrónomo Mención: Sanidad Vegetal / La pudrición gris, causada por el hongo Botrytis cinerea, corresponde al principal problema fitopatológico que enfrentan los productores de uva de mesa, puesto que limita su producción y exportación al desarrollarse pudriciones incluso en almacenaje. Para su control, se integran diferentes medidas entre las cuales el uso de fungicidas específicos es la base de los programas utilizados. Actualmente, la creciente preocupación por la presencia de residuos de fungicidas en la fruta y el riesgo ambiental asociado a su uso, además del desarrollo de resistencias en Botrytis a estas moléculas, han complicado su empleo como estrategia de control. Ante el cual se han aplicado métodos alternativos como la intensificación de prácticas culturales que disminuyan las condiciones predisponentes de la enfermedad, junto a herramientas naturales que incluyen el uso de extractos de cítricos y controladores biológicos como Trichoderma. Por otro lado, recientemente se ha elaborado un biofungicida con la bacteria antagonista Serratia plymuthica cepa CCGG2742, un controlador biológico de B. cinerea, que en este ensayo se ha probado su sensibilidad frente a fungicidas de uso común en vid, con el objeto de conocer su compatibilidad en un posible programa de control que integre estas estrategias. Se evaluaron los ingredientes activos boscalid, ciprodinil + fludioxonil, fenhexamida, iprodione, kresoxim methyl, pyrimethanil, tebuconazole y extracto de cítrico en concentraciones que fluctuaron entre 0 y 5.000 µg i.a. ∙ mL-1 y se calculó la EC50. Los valores de EC50 obtenidos fueron: 6,00∙104 ; 3,18∙107 ; 7,04∙1012 y 1,38∙1018 µg i.a. ∙ mL-1 , para fenhexamida, extracto de cítrico, tebuconazole y boscalid respectivamente, mientras que con iprodione, kresoxim methyl, pyrimethanil y la mezcla de c+f, no se obtuvieron valores de EC50 positivos, ni indicios de inhibición in vitro. De acuerdo a estos resultados, el uso de dosis comerciales de fungicidas no altera el desarrollo de la cepa CCGG2742. / Gray mold induced by Botrytis cinerea, is the most important disease of table grapes that causes significant losses to grape growers. Its ability to attack in the orchard and its ability to develop under conditions prevailing during storage, shipment and marketing make its control a challenge. Control programs have relied mainly on chemical strategies using specific fungicides. However, the growing public concern about fungicide residues in fruit, and the environmental risk associated to fungicide use, in addition to pathogen’s resistance development, have created a complicated situation for the continuous use of fungicides. Therefore, alternative methods have been developed for non-chemical control, such as cultural practices, and the use of natural products including citrus extracts and biological controls agents, as Trichoderma. Serratia plymuthica, antagonistic to B. cinerea, is a new biocontrol agent, and in this study the sensitivity of the strain CCGG2742 to fungicides commonly used in vineyards was tested, in order to know their compatibility for possible control programs that integrate these tools. Boscalid, ciprodinil + fludioxonil, fenhexamida, iprodione, kresoxim methyl, pyrimethanil, tebuconazole and citric extract were tested at concentrations ranging between 0 and 5,000 µg i.a. ∙ mL-1 . EC50 values obtained ranged between 5.48∙104 and 1.38∙1018 µg i.a. ∙ mL-1 . According to these results, commercial rates of the fungicides tested do not affect the development of S. plymuthica strain CCGG2742.

Ingeniera Civil en Biotecnología / Chile: Potencia Alimentaria y Forestal es el nuevo slogan adoptado por el país y simboliza el buen escenario del sector silvoagropecuario. Sin embargo, la planta y los frutos de la vid son atacados por el hongo patógeno Botrytis cinerea, produciendo grandes pérdidas productivas y económicas. Nuevas oportunidades de negocio se abren con el cultivo de flores de exportación. Para cuidar que las flores permanezcan en óptimas condiciones se debe combatir el principal patógeno que enferma a estas plantas, la bacteria Erwinia carotovora. Recientemente se ha demostrado que extractos de plantas, algas y esponjas marinas que contienen geranilfenoles y geranilhidroquinonas, poseen propiedades antifúngicas y antibacterianas. Debido a esto y los bajos rendimientos con que se obtienen desde la fuente natural es que se ha decidido sintetizar 6 de estos compuestos en el Laboratorio de Síntesis Orgánica de la UTFSM. Se estudió el efecto de los compuestos H1, M1, I1, I5, L1 y L2 sobre el crecimiento de Botrytis cinerea y Erwinia carotovora. Además, se planteó aumentar la solubilidad de los compuestos y su efectividad contra los patógenos, mediante un sistema de encapsulamiento. Del estudio de actividad antifúngica se obtuvo resultados prometedores para los compuestos, en particular el compuesto H1, que incluso logró porcentajes de inhibición mayores que los del producto comercial Captan, utilizando las mismas concentraciones. H1 a 250 ppm provocó una inhibición del crecimiento de B. cinerea del 86%. También exhibieron altos porcentajes de inhibición del crecimiento del hongo, H1 a 150 ppm con 82% e I1 a 250 ppm con 81%. Al probar la actividad antibacteriana de los compuestos también se obtuvo un buen candidato de estudio, L1. Aunque demostró tener poder bactericida contra Erwinia carotovora a 100 ppm y contra Bacillus subtilis a 50 ppm, esta actividad antibacteriana no fue estable en el tiempo. Sólo fue estable contra Bacillus en el caso en que la infección se realiza al mismo tiempo que la adición del compuesto. El sistema de encapsulamiento fue efectivo en la solubilización de los compuestos pero inefectivo en su uso en pruebas biológicas. Los altos porcentajes de inhibición del crecimiento de Botrytis obtenidos por los compuestos sin encapsular se vieron disminuidos al utilizar los compuestos en su forma encapsulada, llegando a bajar, en el peor de los casos, desde 81% a 26% para I1 a 250 ppm. Al utilizarse el compuesto L1 encapsulado en los ensayos de actividad antibacteriana, en todos los casos, se revirtió el poder bactericida del compuesto, provocando gran proliferación de ambos patógenos, E. carotovora y B. subtilis. Se postula que estos compuestos son muy estables dentro de la micela de encapsulamiento y su liberación es muy lenta como para ser registrada durante la duración de los experimentos. Entre las recomendaciones realizadas para estudios futuros, se hace énfasis en la necesidad de un estudio de cinética de liberación. Se propone el compuesto H1 como componente principal de un potencial producto comercial para combatir el hongo Botrytis cinerea. Para el caso de la actividad antibacteriana, se debe seguir investigando para aumentar la estabilidad del compuesto L1.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Various studies revealed that Botrytis cinerea, the causal pathogen of Botrytis bunch rot, is mostly associated with pedicels, rachises, laterals and berry bases, and not with berry skins as previously understood. Provided that sufficient coverage of inner bunch parts was achieved, laboratory studies have shown that fungicides can effectively reduce the amount of B. cinerea at the various positions in bunches, and prevent infection and symptom expression at all growth stages. The same efficacy was, however, not achieved with the same fungicides when using conventional spraying methods in vineyards. Poor disease control on fruit and leaves in vineyards is attributed to inappropriate timing of fungicide applications and/or insufficient coverage of susceptible tissue. Previously, spray coverage evaluations in South Africa were based on the use of water-sensitive cards. A variety of other methods have been used to assess spray coverage in vineyards, but none of these methods could assess spray deposits on a very small, three-dimensional area of interest such as the susceptible grape bunch parts. The methods were furthermore dependent on human objectivity, which lacks quantitative measuring and speed of measurement. Suitable technology to determine spray coverage on susceptible bunch parts is, therefore, not available. The aim of this study was to develop a protocol to visualise and quantify spray deposits in grape bunches, specifically on the inner bunch parts and to use the protocol to determine the effect of different levels of spray cover on artificially inoculated B. cinerea grape bunches, in order to facilitate future determination of minimum effective coverage levels for effective B. cinerea control. A spray coverage assessment protocol using fluorometry, photomicrography and digital image analyses was developed to measure spray coverage on susceptible grape bunch parts. Among several fluorescent pigments tested, a yellow fluorescent pigment (SARDI Fluorescent Pigment) from Australia was selected on the basis of its small particle size (2.45 - 4.90 μm). Bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and the yellow fluorescent pigment. Sprayed parts from bunches were illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope at 20 x magnification. Photos of the berry skin, pedicel and rachis were taken with a digital camera (Nikon DMX 1200). Image analysis of photos was done with Image- Pro Discovery version 4.5 for Windows (Media Cybernetics) software. The total area of deposited pigment in selected areas of interest (AOI) was calculated. The percentage area covered was subsequently calculated for each AOI. Good correlation was evident between the parameters, sum of objects and percentage area covered. Bunch parts at pea size generally had higher coverage values than at bunch closure. Spray applications earlier in the season would therefore result in higher and more effective spray coverage of the susceptible bunch parts. Similar deposition trends were observed on the inner bunch parts (pedicel and rachis). These were, however, significantly different from berry skins, which had significantly higher levels of spray deposits than the inner bunch parts. The variance component analysis indicated that the highest variance was observed for berries and bunches, and substantially less for image readings. For the same accuracy, means for percentage coverage values of at least 10 bunches per treatment (1 part per bunch and 3 readings per part) will be sufficient. In order to determine the biological efficacy of different levels of spray coverage on B. cinerea incidence on grape bunches, bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and a yellow fluorescent pigment and the percentage fluorescent pigment coverage on pedicels was determine. Bunches were subsequently dusted with dry airborne conidia of B. cinerea in a settling tower and incubated for 24 h at high relative humidity (98%). Infection was determined by estimating the amount of B. cinerea infections occurring on sprayed bunch parts with isolations on to paraquat and Kerssies mediums. Linear regressions for the part x stage combinations of percentage B. cinerea incidence on different bunch parts were fitted on mean coverage levels. An increase in spray cover caused linear reductions in levels of B. cinerea on susceptible bunch parts. Higher B. cinerea incidences were recorded at pea size. Furthermore, higher B. cinerea incidences were found on paraquat medium for both stages, than on Kerrsies medium. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches. In these validation experiments, the results clearly showed that the protocol can be used to determine the effect of different levels of spray coverage on B. cinerea incidence and that an increase in spray coverage will decrease B. cinerea incidence. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches and subsequently be used as benchmarks to evaluate spray application in vineyards. / AFRIKAANSE OPSOMMING: Vaalvrot by wingerde word veroorsaak deur Botrytis cinerea. Verskeie studies het getoon/gewys dat die oorsaaklike patogeen meestal geassosieer word met die pedisel, ragis, laterale en die korrelbasis, en nie met die korrelskil soos voorheen beweer nie. Laboratorium studies het getoon dat swamdoders wel effektief is om B. cinerea by alle trosdele te verminder en simptoomontwikkeling te voorkom tydens alle groeistadia, mits die binne-trosdele voldoende spuit bedekking ontvang het. Dieselfde effektiwiteit is egter nie gevind in wingerde met konvensionele spuittegnieke nie. Onvoldoende siektebeheer van vrugte en blare van wingerde kan toegeskryf word aan verkeerde spuit skedulering en/of swak spuitbedekking van vatbare gasheerweefsel. Evaluering van spuitbedekking is voorheen in Suid Afrika deur middel van water-sensitiewe papier gedoen. Verskeie ander metodes is al gebruik om spuitbedekking te evalueer in wingerde, maar nie een van hierdie metodes kan gebruik word om spuitbedekking op ’n baie klein, drie-dimensionele oppervlak, soos die vatbare trosdele, te evalueer nie. Verder was die tegnieke afhanklik van menslike objektiwiteit, en gevolglik ontbreek kwantitatiewe meting en metingspoed. Daar is dus nie geskikte tegnologie vir die evaluering van spuitbedekking op vatbare trosdele nie. Die doel van hierdie studie was die ontwikkeling van ‘n protokol vir die visualisering en kwantifisering van spuitbedekking op spesifiek die binne-tros dele en om die protokol dan te gebruik om die effek van verskillende vlakke van spuitbedekking op B. cinereageinokuleerde druiwetrosse te bepaal, Protokol vir evaluasie van spuitbedekking op vatbare druifdele is ontwikkel deur gebruik te maak van fluorometrie, fotomikrografie en digitale beeldanalise. Van die verskillende fluoresensie pigmente wat getoets is, is ‘n geel flouresensie pigment (SARDI Flourescent Pigment) van Australië gekies op grond van sy klein partikelgrootte (2.45 - 4.90 μm). Druiwetrosse is gespuit tydens ertjie- en trostoemaakstadia met verskillende volumes van ’n mengsel van fenheksamied en die geel fluorosensie pigment. Die gespuite druifdele is dan verlig onder swartlig buise (UV-A lig in die 365 nm spektrum) en gevisualiseer deur ’n stereo mikroskoop by 20x vergroting. Foto’s van die korrelskil, pedisel en ragis is met ‘n digitale kamera (Nikon DMX 1200) geneem. Beeldanalise is gedoen met ImagePro Discovery weergawe 4.5 vir Windows (Media Cybernetics) sagteware. Die totale area neerslag van die pigment is in geselekteerde areas bereken. Die presentasie area bedek is bereken vir elkeen van hierdie areas. Goeie korrelasie is gevind tussen die parameters aantal fluoresserende partikels en die persentasie bedekte area. Trosdele tydens ertjie-stadium het in die algemeen hoër waardes gehad as by trostoemaak. Dit blyk dus dat spuittoediening vroeg in die seisoen meer effektief sal wees vir die bedekking van vatbare trosdele. Soortgelyke bedekkings patrone is gevind by die binne trosdele (pedisel en ragis). Dit het egter betekenisvol verskil van die korrelskil, wat betekenisvol meer spuitbedekking as die binne trosdele gehad het. ’n Variasie komponent analise het getoon dat die meeste variasie gevind is tussen korrels en trosse, en heelwat minder vir die beeld analise lesings. Om dieselfde akkuraatheid te behou, is ten minste 10 trosse per behandeling (1 deel per tros en 3 lesings per deel) nodig. Vir die bepaling van biologiese effektiwiteit van verskillende vlakke van spuitbedekking op B. cinerea voorkoms op druiwe, is druiwe gespuit tydens ertjie- en trostoemaak-stadia met verskillende volumes van ’n mengsel van fenheksamied en die geel fluorosensie pigment. Die persentasie fluoresensie pigment is bepaal op die pedisels. Trosse is vervolgens geinokuleer met droë luggedraagde konidia van B. cinerea in ’n inokulasietoring en geïnkubeer vir 24 h by hoë relatiewe humiditeit (98%). Die voorkoms van B. cinerea infeksie op gespuite tros dele is bepaal deur middel van isolasies op paraquat en Kerssies medium. Liniêre regressies vir trosdeel x stadium kombinasies van persentasie B. cinerea voorkoms op verskillende trosdele is gepas vir gemiddelde bedekkings waardes. ’n Verhoging in spuit bedekking het ‘n liniêre vermindering van B. cinerea voorkoms op vatbare trosdele veroorsaak. Verder is hoër vlakke van B. cinerea op paraquat medium as op Kerssies medium vir beide die groeistadia gevind. Die kennis wat verkry is uit hierdie studie sal gebruik word om minimum effektiewe spuitbedekkingsvlakke vir die beheer van B. cinerea op druiwetrosse te bepaal.

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