Thesis (Ph. D.)--University of Wisconsin--Madison, 1960. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1935. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Antagonistic effects of Epicoccum purpurascens, Aureobasidium pullulans, Trichoderma spp., and Botrytis cinerea on establishment of Monilinia fructicola infections in cherry and peach blossoms were assessed in field and mist chamber studies. Conidia of each fungus were applied to blossoms that were subsequently inoculated with conidia of M fructicola. Mist chamber experiments on forced cherry blossoms demonstrated that recovery of M. fructicola was significantly reduced (P=.05) when spores of E. purpurascens and B.cinerea had been applied 24 hr prior to inoculation with M. fructicola. Reduction in recovery of M. fructicola was comparable to that obtained with the fungicide benomyl. In field trials done in 1990 and 1991, applications of E. purpurascens and A. pullulans reduced cherry blossom blight relative to nontreated blossoms by 47 to 65 and 54 to 58%, respectively, compared to reductions of 80 to 96 and 84 to 97% with the fungicides benomyl and iprodione, respectively. Twig blight in peach, an indicator of blossom blight infection, was reduced by 37% relative to nontreated blossoms with applications of E. purpurascens, compared to 54 and 51% reductions with benomyl and iprodione, respectively. Analysis of the influence of antagonistic fungi sprayed onto blossoms on fruit set indicated that B.cinerea was a weak pathogen of stone fruit blossoms. Significant reductions (P=.05 and P=.10) were obtained in fruit set compared with the nontreated control when conidia of B. cinerea were applied to both cherry and peach blossoms in 1991. Latent Monilinia infections were evaluated by dipping green cherries in the herbicide paraquat. Applications of E. purpurascens and A. pullulans to blossoms caused reductions in the number of latent Monilinia infections in green cherries by 18 and 49%, respectively in 1990, and 61 and 66% respectively in 1991. This compares with reductions of 98 and 92% in 1990 and 1991, respectively, with the fungicide iprodione. It was observed that the antagonists E. purpurascens and B. cinerea also became established as latent infections. These fungi were recovered at a significantly (P=.05) higher percentage on green cherries where they had been applied as antagonists to blossoms. No meaningful differences were detected in the amount of brown rot that developed on fruit due to the influence of fungal treatments on blossoms. / Graduation date: 1992

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 45-48).

Monilinia fructicola (Wint.) Honey, and Rhizopus nigricans Erh., the causal organisms of brown rot and Rhizopus rot, respectively, have long been recognized as the two major causes of post-harvest rots on peach and nectarine fruits. Fruit losses of as much as 50% may occur during storage, transport, and marketing from these two diseases if effective fungicide treatments are not used. Application of fungicide suspensions as post-harvest dips have been reported to reduce losses from post-harvest rots considerably, however, more effective treatments are needed. Tests were made of three fungicides: benomyl [methyl-l-(butylcarbamoyl)-2-benzimidazole carbamate], Topsin-M [dimethyl-4,4'-ophenelenebis (3-thioallophanate) , and Botran (2,6-dichloro-4-nitroanaline) against the two post-harvest rot fungi, M. fructicola and R. nigricans, on peach and nectarine fruits and in vitro. Fruits were inoculated with an equal concentration of spores of both fungi, then treated, by dipping in the different fungicide suspensions at 0, 4, 8, and 12 hour intervals after inoculation. Three concentrations of benomyl or Topsin-M (300, 450, and 600 ~g/ml) were used in combination with corresponding concentrations of Botran (600" 900, and 1200 Ug/ml). Number of decayed fruits increased with time of treatment after inoculation up to 8 hours then leveled off. Lesion progression, however, was slower with all fungicide treatments as compared to those of the untreated check fruit. Percent of decayed fruit remained lower when treatments were applied within 4 hours after inoculation. Later application times were ineffective and fruits showed approximately the same level of infection as controls. Treated nectarines appeared to be more susceptible to the post harvest rots than peaches. This may be due to lower residues of the chemicals remaining on the smooth skinned fruit. Benomyl and Topsin-M were fungitoxic to M. fructicola in vitro but neither was effective against R. nigricans. Botran alone was fungistatic to both R. nigricans and M. fructicola, but often lost effectiveness after 96 hours. Combinations of benomyl plus Botran, or Topsin-M plus Botran, were fungitoxic to M. fructicola and fungistatic to R. nigricans. Botran alone and Botran in combination with benomyl or Topsin-M inhibited germination of M. fructicola and R. nigricans spores more effectively than benomyl or Topsin-M alone. There were no major significant differences in control provided by treatments containing benomyl or Topsin-M. Treatments using Botran with benomyl or Topsin-M did not effectively control post harvest rot when applied later than 4 hours after inoculation. In vitro tests, however, indicated either a synergistic or additive effect of the fungicide combinations against R. nigricans. / Master of Science

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The aim of the present work was to evaluate the efficiency of pre-harvest application of fungicides on control of brown rot on postharvest and verify their effect when associated with fungal antagonists, ensuring efficiency in the control of disease and quality of fruit. We evaluated the effect of application of fungicides on pre-harvest in the control of brown rot after 15 and 40 days of refrigerated storage of fruits, in addition to the association of fungicides with postharvest treatment with the fungus antagonist, Trichoderma harzianum. To do this, were established two experiments. At the first, phytosanitary programs (active ingredients) were evaluated: [1] control (application of water); [2] captan; [3] iprodione; [4] iminoctadine; [5] tebuconazole; [6] procymidone; [7] azoxystrobin; [8] difenoconazole; [9] fungicide (azoxystrobin/difenoconazole); [10] fungicide (trifloxystrobin/tebuconazole); [11] sequence iminoctadine + captana; [12] sequence of iprodione + iminoctadina; [13] sequence of tebuconazole + captan; [14] sequence of tebuconazole + iprodione. All treatments were applied in the pre-harvest phase, in recommended doses for culture. After harvesting the fruits were stored at -0.5 °C for 15 and 40 days. In the second experiment were evaluated the pre-harvest application of following products: [1] control (application of water); [2] captan; [3] iprodione; [4] iminoctadine; [5] tebuconazole. After harvesting the fruits were stored at -0.5 °C for 40 days and following, in half of the fruits of each treatment was applied the antagonist Trichoderma harzianum, through immersion. Both experiments were evaluated after exposure of fruit to 20 °C for a period that varied from four to 12 days, depending on the storage time of each experiment. The parameters evaluated were: incidence of brown rot, percentage of healthy fruits, respiratory rate, ethylene production and skin browning. Until the time of harvesting the fungicide which best controlled the brown rot was the difenoconazole, whereas, the fungicides tebuconazole and iminoctadina have satisfactory results in control of brown rot, after 15 and 40 days of refrigerated storage. The use of Trichoderma harzianum has no effect on postharvest control of brown rot, however, when associated with fungicides is effective in the control of Rhizopus stolonifer. Pre-harvest application of fungicide captan and postharvest of Trichoderma harzianum affect postharvest quality of fruits, causing skin browning. / O objetivo do presente trabalho foi avaliar a eficiência da aplicação pré-colheita de fungicidas no controle da podridão parda em pós-colheita e verificar o seu efeito quando associado a fungos antagonistas, assegurando eficiência no controle da doença e qualidade dos frutos. Para tanto, avaliou-se o efeito da aplicação de fungicidas em pré-colheita no controle da podridão parda do pessegueiro após 15 e 40 dias de armazenamento refrigerado dos frutos, além da associação de fungicidas com o tratamento pós-colheita com o fungo antagonista, Trichoderma harzianum. Para isso, foram instalados dois experimentos. No primeiro foram avaliados programas fitossanitários com os seguintes produtos (ingredientes ativos): [1] testemunha (aplicação de água); [2] captana; [3] iprodiona; [4] iminoctadina; [5] tebuconazol; [6] procimidona; [7] azoxistrobina; [8] difenoconazol; [9] fungicida (azoxistrobina/difenoconazol); [10] fungicida (trifloxistrobina/tebuconazol); [11] sequência de iminoctadina + captana; [12] sequência de iminoctadina + iprodiona; [13] sequência de tebuconazol + captana; [14] sequência de tebuconazol + iprodiona. Todos os tratamentos foram aplicados na fase pré-colheita, nas doses recomendadas para a cultura. Após a colheita os frutos foram armazenados a -0,5 ºC por 15 e 40 dias. O segundo experimento constituiu-se da aplicação pré-colheita dos seguintes produtos: [1] testemunha (aplicação de água); [2] captana; [3] iprodiona; [4] iminoctadina; [5] tebuconazol. Após a colheita os frutos foram armazenados a -0,5 ºC por 40 dias e na sequência, na metade dos frutos de cada tratamento foi aplicado o fungo antagonista Trichoderma harzianum, por meio de imersão. Ambos os experimento foram avaliados após a exposição dos frutos à temperatura de 20 ºC, por um período que variou de quatro a 12 dias, dependendo do tempo de armazenamento de cada experimento. Os parâmetros avaliados foram: incidência de podridão parda, porcentagem de frutos sadios, taxa respiratória, produção de etileno e escurecimento da epiderme. Até o momento da colheita, o fungicida que melhor controlou a podridão parda foi o difenoconazol, enquanto que, os fungicidas iminoctadina e tebuconazol apresentam resultados satisfatórios no controle da podridão parda, após 15 e 40 dias de armazenamento refrigerado. A utilização do Trichoderma harzianum em pós-colheita não apresenta efeito no controle da podridão parda do pessegueiro, entretanto, quando associado a fungicidas é eficiente no controle de Rhizopus stolonifer. A aplicação pré-colheita do fungicida captana e a utilização pós-colheita de Trichoderma harzianum afetam a qualidade pós-colheita dos frutos, causando escurecimento da epiderme.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Brown rot is the main diseases of peaches, with pre and postharvest losses. Control it is difficult because the fungus infect fruit and remain latent until conditions are favorable to its manifestation. Therefore, to understand the role of latent infection of the disease incidence and find strategies to minimize losses, with low environmental impact, is essential for sustainable crop management. This thesis consists of four papers that resulted from work with the following objectives: to study the relationship between latent infection and the occurrence of postharvest brown rot; to evaluate the effect of preharvest fungicide application on the Monilinia fructicola control during storage and marketing of peaches; to investigate the pre and postharvest applications of silicon on the brown rot control in peach. They were used peaches of Eldorado and Pepita cultivars, in field experiments involving preharvest spraying of fungicides and pre and postharvest applications of silicon and experiments in refrigerated storage conditions at -0.5 ° C. Iprodione, tebuconazole and difenoconazole are effective in brown rot control in the field conditions, while in postharvest, the incidence was controlled in 96.2% with iminoctadine. These products decreased the incidence of injuries resulting from latent infections. There is a high correlation between the presences of latent infections in preharvest with the incidence of the disease in postharvest, with most of the damage after cold storage resulting from the latent infections of Monilinia fructicola. Foliar application of 6 g L-1 sodium silicate or sodium metasilicate is effective in controlling the disease without causing phytotoxicity to the plants. Six sodium silicate applications had the same effect as six fungicides applications to brown rot control in peaches Eldorado . Postharvest sodium metasilicate application, in 6 g L-1, reduced the diseases incidence but caused fruit skin darkening. Physical defense mechanisms of the plants are affected by silicon application, because it increased the element content in the leaves and fruits, and increased skin texture. However, the effect of silicon on the biochemical defense mechanisms may involve the synthesis of polyphenols, because in the one year of search we found a higher content of these compounds in fruit treated with silicon. In general, products based on silicon, applied alone or intercalated with fungicides, control the disease in pre and postharvest, allowing a decrease in fungicide use and production cost. / A podridão parda do pessegueiro é a doença de maior importância para a cultura, com danos em pré e pós-colheita. O controle é dificultado devido à habilidade do fungo de infectar os frutos e permanecer latente até que as condições tonarem-se favoráveis à sua manifestação. Portanto, compreender o papel da infecção latente na incidência da doença, bem como encontrar estratégias que minimizem as perdas, com baixo impacto ambiental, é fundamental para o manejo sustentável da cultura. Esta tese é composta por quatro artigos científicos que resultaram de trabalhos com os seguintes objetivos: estudar a relação da infecção latente com a ocorrência da podridão parda em pós-colheita; avaliar o efeito da aplicação pré-colheita de fungicidas no controle do fungo Monilinia fructicola durante o armazenamento e comercialização de pêssegos; investigar o potencial do silício em pré e pós-colheita no controle da podridão parda do pessegueiro. Foram utilizados pêssegos das cultivares Eldorado e Pepita em experimentos a campo envolvendo pulverizações pré-colheita de fungicidas e aplicações pré e pós-colheita de produtos à base de silício e experimentos em condições de armazenamento refrigerado a 0,5 ºC. Os fungicidas iprodione, tebuconazole e difenoconazole foram eficazes no controle da podridão parda a campo, enquanto que em pós-colheita, a incidência foi controlada em 96,2% com iminoctadina. Esses produtos também reduziram a incidência de lesões decorrentes de infecções latentes. Há uma alta correlação entre a presença de infecções latentes em pré-colheita com a incidência da doença em pós-colheita, sendo a maior parte das lesões após o armazenamento refrigerado decorrentes das infecções latentes do fungo Monilinia fructicola. Aplicação foliar de 6 g L-1 de metassilicato de sódio ou de silicato de sódio foram eficazes no controle da doença sem causar fitotoxidez às plantas. Seis aplicações de silicato de sódio tiveram o mesmo efeito que seis aplicações de fungicidas no controle da podridão parda em pêssegos cv. Eldorado. A aplicação de metassilicato de sódio em pós-colheita, na dose de 6 g L-1, reduziu a incidência da doença, mas causou escurecimento da epiderme dos frutos. Os mecanismos físicos de defesa das plantas são afetados pelo Si, pois houve aumento do conteúdo do elemento nas folhas e frutos e aumento da textura da epiderme, entretanto, o efeito do silício sobre os mecanismos bioquímicos de defesa pode envolver a síntese de polifenóis, pois em um dos anos da pesquisa verificou-se maior conteúdo destes compostos em frutos tratados com silício. Em geral, produtos à base de silício, aplicados isoladamente ou intercalados com fungicidas, controlam a doença em pré e pós-colheita proporcionando redução no uso de fungicidas e no custo de produção.

La limitació en l’ús de fungicides per al control de malalties en postcollita de fruita és una problemàtica d’elevada magnitud en el sector fructícola actual. Degut a això l’ús d’estratègies alternatives com el control biològic microbià són fonamentals per a la producció de fruita de qualitat. Malgrat tot, el desenvolupament de programes de biocontrol eficaços requereix d’un coneixement profund de la capacitat de control i els mecanismes d’acció utilitzats per l’agent microbià que es pretén emprar, així com de la possibilitat per a què aquest pugui ésser produït i formulat a nivell comercial. En aquest context, la bactèria Bacillus subtilis soca CPA–8 aïllada de la superfície de nectarines al Laboratori de Patologia de Postcollita del Centre IRTA (Lleida) ha demostrat tenir una bona capacitat de biocontrol de malalties de postcollita de fruita de pinyol. El seu futur ús a nivell comercial depèn de l’estudi del seu potencial de biocontrol de malalties de postcollita de fruita; així com del desenvolupament de processos per a l’optimització de la producció i formulació. La present tesi tenia com a objectiu principal avaluar aspectes clau per al desenvolupament de B. subtilis CPA–8 com agent efectiu per al control biològic de malalties de postcollita de fruita. Per a complir amb aquest objectiu, en primer lloc, es va dur a terme una anàlisi detallada de les característiques biològiques de la soca CPA–8 com ara el seu creixement en medi de cultiu, producció d’endòspores i substàncies antifúngiques; així com el seu potencial per al control de malalties importants en postcollita de taronja, poma i fruita de pinyol (Capítol 1). Amb aquests resultats, el següent pas fou estudiar el mode d’acció utilitzat per B. subtilis CPA–8 per a la supressió de patògens de postcollita, concretament contra Monilinia spp. causant de la podridura marró en fruita de pinyol (Capítol 2). Mitjançant la combinació d’eines d‘anàlisi químic, moleculars i biològiques es van determinar els principals factors implicats en la capacitat antagònica de la soca CPA–8 contra Monilinia spp. El pas següent fou optimitzar la producció de B. subtilis CPA–8, en aquest sentit es va desenvolupar un medi de cultiu de baix cost que proporcionés un creixement alt i mantingués l’eficàcia de biocontrol (Capítol 3). Primer es van buscar fonts de nitrogen i de carboni econòmiques entre productes comercials i subproductes agroalimentaris. Posteriorment es va dur a terme l’escalat de la producció de la bactèria en un bioreactor de 5 L i es va comprovar l’efectivitat per al control de Monilinia spp. en préssecs. El pas final fou la formulació de B. subtilis CPA–8 mitjançant l’assecat per atomització (Capítols 4 i 5). Aquest mètode fou seleccionat per dos motius, d’una banda perquè aquesta és una tècnica d’assecat viable per a formulació de bactèries a baix cost i per l’altra banda en base als resultats obtinguts en el capítol 1 sobre la capacitat de la bactèria per produir endòspores resistents al calor i efectives contra patògens de postcollita de fruita. Primer es va dur a terme un estudi comparatiu de l’efecte de l’atomització sobre la supervivència de B. subtilis CPA–8 i de l’agent de biocontrol Pantoea agglomerans CPA–2 (Capítol 4). Posteriorment es va realitzar una avaluació de les substàncies protectores/material de suport més adequades per a l’atomització de la soca CPA–8, i amb els millors productes atomitzats es van avaluar diferents medis de rehidratació i l’activitat antifúngica; així com la vida útil y l’efectivitat durant l’emmagatzemament (Capítol 5) Els resultats obtinguts en el capítol 1 demostraren que B. subtilis CPA–8 produeix cèl lules, endòspores resistents al calor i compostos que tenen una alta activitat antagònica in vitro contra els principals patògens de postcollita de fruita Botrytis cinerea, Monilinia laxa, Monilinia fructicola, Penicillium digitatum, Penicillium italicum i Penicillium expansum. Els tractaments de cèl lules, endòspores y sobrenedants lliures de cèl lules de la soca CPA–8 mostraren diferents nivells d’efectivitat per al control de podridures en taronja, poma i fruita de pinyol, presentant els millors resultats contra Monilinia spp. en préssecs i nectarines amb reduccions de la incidència de la malaltia fins al 100 %. L’assaig de dosis de diferents tractaments de la soca CPA–8 a 108, 107 i 106 UFC mL–1 foren efectius contra Monilinia spp. de forma semblant o millor al Serenade Max®. L’estudi del mode d’acció de B. subtilis CPA–8 (Capítol 2) va demostrar que els sobrenedants lliures de cèl lules provinents de cultius líquids tenen una alta activitat antifúngica in vitro contra Monilinia spp. similar a l’observada amb suspensions cel lulars. Les anàlisis mitjançant PCR i bioautografia en TLC d’extractes butanòlics d’aquests sobrenedants en comparació amb els de les soques de referència de B. subtilis UMAF6614 i UMAF6639, van revelar la producció de les principals famílies de lipopèptids antifúngics coneguts en Bacillus (fengicines, iturines i surfactines), fet que apuntava l’antibiosi com a principal mecanisme d’acció implicat en la capacitat de biocontrol de B. subtilis CPA–8. Les fraccions corresponents a les fengicines foren les responsables de l’activitat inhibitòria de la soca CPA–8 enfront de Monilinia spp. Aquests resultats van ser corroborats definitivament mitjançant la construcció de mutants de B. subtilis CPA–8 defectius per a la producció de fengicina interrompent l’expressió del gen fenB. Les anàlisis per PCR i bioautografia en TLC revelaren que els mutants defectius de la soca CPA–8 perdien la capacitat d’inhibir a Monilinia spp. per la seva incapacitat per a produir fengicines. Els assaigs d’efectivitat en fruita utilitzant tractaments provinents dels mutants de la soca CPA–8 van demostrar que aquests havien perdut la capacitat de controlar la podridura causada per Monilinia spp. amb percentatges d’incidència similars als observats en el control sense tractar, mentre que els tractaments amb la soca parental o Serenade Max® presentaven reduccions de la malaltia de fins el 100 %. Tots aquests resultats demostraren que la producció de fengicines juga un paper molt important en l’efectivitat de B. subtilis CPA–8 per a controlar la podridura marró del préssec; i que aquest resulta ser el principal mode d’acció implicat en la seva capacitat de biocontrol. En l’optimització de la producció de B. subtilis CPA–8 (Capítol 3) els resultats obtinguts van demostrar que és possible aconseguir alts nivells de biomassa (superiors a 3×109 UFC mL–1) emprant dos medis de baix cost composats per farina de soja desengreixada 44 % a 40 g L–1 com a font de nitrogen en combinació amb sacarosa a 20 g L–1 o melassa a 5 g L–1 com a fonts de carboni. A més a més, la producció de la soca CPA–8 en aquest medi de baix cost va poder ser escalada a nivell de laboratori en un bioreactor de 5 L de capacitat a 30 °C, amb agitació de 200 rpm i flux d’aire de 100 L h–1, mantenint les concentracions de la bactèria a 3×109 UFC mL–1. Els assaigs en fruita amb tractaments de la soca CPA–8 crescuda en el medi optimitzat de baix cost van demostrar que aquesta mantenia la seva eficàcia de biocontrol contra M. fructicola en préssecs amb reduccions de la malaltia de fins al 95 %, similar als tractaments de la bactèria crescuts en medis de laboratori. Les poblacions de CPA–8 van sobreviure en ferides de préssecs inoculats, independentment dels medis de cultiu utilitzats. Aquests resultats proporcionen una base fiable per a la producció de la soca CPA–8 a nivell industrial. En la formulació de B. subtilis CPA–8 mitjançant assecat per atomització (Capítols 4 i 5) els resultats demostraren que aquesta bactèria és capaç de sobreviure a les altes temperatures del procés (32.3 % de viabilitat i 3.3×109 UFC g–1 de concentració final de producte), comparada amb P. agglomerans CPA–2 utilitzada com a model de bactèria sensible a la calor i no formadora d’endòspores que no fou resistent (menys del 2 % de viabilitat). La supervivència de la soca CPA–8 a l’atomització va estar directament relacionada amb la seva capacitat per a produir endòspores resistents a les altes temperatures. La resistència a la calor de la soca CPA–8 a més a més va dependre de la fase de creixement, essent el cultiu de 72 h, més resistent que el de 24 h, probablement pel seu major contingut d’endòspores. Els resultats obtinguts en l’estudi de la substància protectora/material de suport més adequada per a l’atomització de B. subtilis CPA–8 van demostrar que quatre diferents combinacions de llet desnatada en pols i MgSO4 proveïen una bona recuperació de pols (28–38 %) i continguts d’humitat del 7–13 %. La supervivència de la soca CPA–8 fou diferent en els atomitzats dels cultius de 24 i 72 h. Les formulacions de 72 h mostraven una major supervivència (28–32 %) y amb concentracions finals al voltant d’1.6–3.3×109 UFC g–1, mentre que la viabilitat dels atomitzats de 24 h fou inferior a l’1 %, fet pel qual les primeres van seleccionar–se per a la seva posterior avaluació. Diferents rehidratants como l’aigua o el tampó fosfat van proporcionar una bona recuperació de cèl lules viables en les formulacions de CPA–8 similars a les obtingudes amb llet desnatada en pols o sacarosa al 10 % amb el que es dedueix que l’aigua pot utilizar–se com a rehidratant amb l’avantatge a nivell pràctic que això suposa. L’estudi de la vida útil de les formulacions de la soca CPA–8 emmagatzemades a 4 °C (fred) i a 20 °C (temperatura ambient) va demostrar que la viabilitat es mantenia o disminuïa lleugerament (0.2–0.3 log) durant 6 mesos d’emmagatzematge. A més a més, després de 4 i 6 mesos de conservació aquestes formulacions controlaven la podridura marró causada per Monilinia spp. en nectarines i préssecs mostrant reduccions de la incidència de la malaltia entre el 90 i 100 %. Els resultats obtinguts van demostrar que l’atomització podria ser un mètode d’assecat adequat per a obtenir formulacions estables i efectives de B. subtilis CPA–8.En conclusió els estudis realitzats en aquesta tesi demostren el potencial de l’antagonista B. subtilis CPA–8 per al control de malalties de postcollita de fruita i estableixen les bases per a la seva posterior implementació de la producció i formulació a nivell comercial. / La limitación en el uso de fungicidas para el control de enfermedades en postcosecha de fruta es un grave problema en el sector frutícola actual. Debido a esto, el uso de estrategias alternativas como el control biológico microbiano son fundamentales para la producción de fruta de calidad. Sin embargo, el desarrollo de programas de biocontrol eficaces requiere de un fuerte conocimiento de la capacidad de control y los mecanismos de acción usados por el agente microbiano que se pretende emplear, así como de la posibilidad para que éste pueda ser producido y formulado a nivel comercial. En este contexto, la bacteria Bacillus subtilis cepa CPA–8 aislada de la superficie de nectarinas en el Laboratorio de Patología de Postcosecha del Centro IRTA (Lleida) ha demostrado tener una importante capacidad de biocontrol de enfermedades de postcosecha de fruta de hueso. Su futuro uso a nivel comercial depende del estudio de su potencial de biocontrol de enfermedades de postcosecha de fruta; así como del desarrollo de procesos para su óptima producción y formulación. La presente tesis tiene como objetivo fundamental evaluar aspectos clave para el desarrollo de B. subtilis CPA–8 como agente eficaz de control biológico de enfermedades de postcosecha de fruta. Para cumplir con este objetivo, en primer lugar, se realizó un análisis detallado de las características biológicas de la cepa CPA–8 como su crecimiento en medio de cultivo, producción de endosporas y sustancias antifúngicas; así como su potencial para el control de importantes podredumbres de postcosecha de naranja, manzana y fruta de hueso (Capítulo 1). Con estos resultados el siguiente paso fue estudiar el modo de acción usado por B. subtilis CPA–8 para la supresión de patógenos de postcosecha, concretamente contra Monilinia spp. causante de la podredumbre marrón en fruta de hueso (Capítulo 2). Mediante la combinación de herramientas de análisis químico, molecular y biológico se determinaron los principales factores implicados en la capacidad antagónica de la cepa CPA–8 contra Monilinia spp. El siguiente paso fue optimizar la producción de B. subtilis CPA–8, para lo cual se desarrolló un medio de cultivo de bajo coste que proporcionase un crecimiento alto y mantuviese la eficacia de biocontrol (Capítulo 3). Primero se buscaron fuentes de nitrógeno y carbono económicas entre productos comerciales y subproductos agroalimentarios. Posteriormente se realizó el escalado de producción de la bacteria en un bioreactor de 5 L y se comprobó la efectividad para el control de Monilinia spp. en melocotones. El paso final fue la formulación de B. subtilis CPA–8 mediante secado por atomización (Capítulos 4 y 5). Este método fue seleccionado por dos motivos, por un lado porque ésta es una técnica de secado viable para formulación de bacterias a bajo coste y en base a los resultados obtenidos en el capítulo 1 sobre la capacidad de la bacteria para producir endosporas resistentes al calor y eficaces contra patógenos de postcosecha de fruta. Primero se realizó un estudio comparativo del efecto de la atomización sobre la supervivencia de B. subtilis CPA–8 y del agente de biocontrol Pantoea agglomerans CPA–2 (Capítulo 4). Posteriormente se realizó una evaluación de las sustancias protectoras/material de soporte más adecuadas para la atomización de la cepa CPA–8, y de los mejores productos atomizados se evaluaron los distintos medios de rehidratación y la actividad antifúngica; así como su vida útil y efectividad durante conservación (Capítulo 5).Los resultados obtenidos en el capítulo 1 indicaron que B. subtilis CPA–8 produce células, endosporas resistentes al calor y compuestos que tienen una alta actividad antagónica in vitro contra los principales patógenos de postcosecha de fruta Botrytis cinerea, Monilinia laxa, Monilinia fructicola, Penicillium digitatum, Penicillium italicum y Penicillium expansum. Los tratamientos de células, endosporas y sobrenadantes libres de células de la cepa CPA–8 mostraron diferentes niveles de efectividad para el control de podredumbres en naranja, manzana y fruta de hueso, presentando los mejores resultados contra Monilinia spp. en fruta de hueso con reducciones de la incidencia de la enfermedad hasta del 100 %. El ensayo de dosis de diferentes tratamientos de la cepa CPA–8 a 108, 107 y 106 UFC mL–1 fueron efectivos contra Monilinia spp., de forma similar o mejor que Serenade Max®. El estudio del modo de acción de B. subtilis CPA–8 (Capítulo 2) demostró que los sobrenadantes libres de células provenientes de cultivos líquidos tenían una alta actividad antifúngica in vitro contra Monilinia spp. similar a la observada con suspensiones celulares. Los análisis mediante PCR y bioautografía en TLC de extractos butanólicos de estos sobrenadantes en comparación con los de las cepas de referencia de B. subtilis UMAF6614 y UMAF6639, revelaron la producción de las principales familias de lipopéptidos antifúngicos conocidos en Bacillus (fengicinas, iturinas y surfactinas), lo que apuntó a la antibiosis como el principal mecanismo de acción implicado en la capacidad de biocontrol de B. subtilis CPA–8. Las fracciones correspondientes a las fengicinas fueron las responsables de la actividad inhibitoria de la cepa CPA–8 frente a Monilinia spp. Estos resultados fueron definitivamente corroborados mediante la construcción de mutantes de B. subtilis CPA–8 defectivos para la producción de fengicina interrumpiendo la expresión del gen fenB. Los análisis por PCR y bioautografía en TLC revelaron que los mutantes defectivos de la cepa CPA–8 perdieron la capacidad de inhibir a Monilinia spp. por su incapacidad para producir fengicinas. Los ensayos de efectividad en fruta utilizando tratamientos provenientes de los mutantes de la cepa CPA–8 demostraron que éstos habían perdido su capacidad para controlar la podredumbre causada por Monilinia spp. con porcentajes de incidencia similares a los observados en el control sin tratar, mientras que los tratamientos con la cepa parental o Serenade Max® presentaron reducciones de enfermedad de hasta el 100 %. Todos estos resultados demostraron que la producción de fengicinas juega un papel muy importante en la efectividad de B. subtilis CPA–8 para controlar la podredumbre marrón del melocotón; y que éste resulta ser el principal mecanismo de acción implicado en su capacidad de biocontrol. En la optimización de la producción de B. subtilis CPA–8 (Capítulo 3) los resultados obtenidos demostraron que se pueden conseguir altos niveles de biomasa (superiores a 3×109 UFC mL–1) usando dos medios de bajo coste compuestos por harina de soja desengrasada 44 % a 40 g L–1 como fuente de nitrógeno en combinación con sacarosa a 20 g L–1 o melaza a 5 g L–1 como fuentes de carbono. Además la producción de la cepa CPA–8 en este medio de bajo coste pudo ser escalada a nivel de laboratorio en un bioreactor de 5 L de capacidad a 30 °C, con agitación de 200 rpm y flujo de aire de 100 L h–1, manteniendo las concentraciones de la bacteria a 3×109 UFC mL–1. Los ensayos en fruta con tratamientos de la cepa CPA–8 crecida en el medio optimizado de bajo coste demostraron que ésta mantenía su eficacia de biocontrol contra M. fructicola en melocotones con reducciones de enfermedad de hasta el 95 %, similar a los tratamientos de la bacteria crecidos en medios de laboratorio. Las poblaciones de CPA–8 sobrevivieron en heridas de melocotones inoculados, independientemente de los medios de cultivo utilizados. Estos resultados proporcionan una base fiable para la producción de la cepa CPA–8 a nivel industrial. En la formulación de B. subtilis CPA–8 mediante secado por atomización (Capítulos 4 y 5) los resultados demostraron que esta bacteria es capaz de sobrevivir a las altas temperaturas del proceso (32.3 % de viabilidad y 3.3×109 UFC g–1 de concentración final de producto), comparado con P. agglomerans CPA–2 utilizada como modelo de bacteria sensible al calor y no formadora de endosporas que no fue resistente (menos del 2 % de viabilidad). La supervivencia de la cepa CPA–8 a la atomización estuvo directamente relacionada con su capacidad para producir endosporas resistentes a las altas temperaturas. La resistencia al calor de la cepa CPA–8 además dependió de la fase de crecimiento, siendo el cultivo de 72 h, más resistente que el de 24 h, probablemente por su mayor contenido de endosporas. Los resultados obtenidos en el estudio de la sustancia protectora/material de soporte más adecuada para la atomización de B. subtilis CPA–8 demostraron que cuatro diferentes combinaciones de leche desnatada en polvo y MgSO4 proveían una buena recuperación de polvo (28–38 %) y contenidos de humedad del 7–13 %. La supervivencia de la cepa CPA–8 varió considerablemente en los atomizados con los cultivos de 24 h y los de 72 h. Las formulaciones de 72 h mostraron una mayor supervivencia (28–32 %) y con concentraciones finales entorno a 1.6–3.3×109 UFC g–1, mientras que la viabilidad de los atomizados de 24 h fue inferior al 1 %, por lo cual se seleccionaron para su posterior evaluación. Diferentes rehidratantes como el agua o tampón fosfato proporcionaron una buena recuperación de células viables en las formulaciones de CPA–8 similares a las obtenidas en leche desnatada en polvo o sacarosa al 10 % por lo que el agua puede utilizarse como rehidratante con la ventaja a nivel práctico que supone. El estudio de la vida útil de las formulaciones de la cepa CPA–8 almacenadas a 4 °C (frío) y a 20 °C (temperatura ambiente) demostró que la viabilidad se mantuvo o disminuyó ligeramente entorno a 0.2–0.3 log durante 6 meses de almacenamiento. Además después de 4 y 6 meses de almacenamiento estas formulaciones controlaron la podredumbre marrón causada por Monilinia spp. en nectarinas y melocotones mostrando reducciones de la incidencia de la enfermedad entre el 90 y 100 %. Los resultados obtenidos demostraron que la atomización podría ser un método de secado adecuado para obtener formulaciones estables y eficaces de B. subtilis CPA–8. En conclusión los estudios realizados en esta tesis demuestran el potencial del agente de biocontrol B. subtilis CPA–8 para el control de enfermedades de postcosecha de fruta y sientan las bases para su posterior implementación de la producción y formulación a nivel comercial / Synthetic fungicides are the primary means to reduce losses caused by postharvest diseases. However, public concern for their negative impact on human health and environment associated with undesirable chemical residues on fruit and proliferation of fungicide–resistant isolates have impelled the search for alternative methods. Biological control using microorganisms has emerged as an effective alternative to control postharvest diseases and to produce quality fruit free of fungicide residues. However, the development of a successful biocontrol product requires a strong knowledge about the antagonistic ability and mechanisms of action used by the microbial agent to disease suppression and the possibility for its production and formulation to commercial application. In this context, Bacillus subtilis strain CPA–8 isolated from nectarines surface in the Postharvest Pathology Laboratory from IRTA centre (Lleida) has shown a significant capacity for biocontrol of postharvest diseases of stone fruit. Its future use on a commercial level depends on its antagonistic potential to control fruit postharvest diseases as well as an optimum production and formulation systems. This thesis has the main objective to evaluate key aspects involved in development of B. subtilis CPA–8 as an effective biocontrol agent of postharvest diseases on fruit. To achieve this objective, first, the CPA–8 growth and production of endospores and antifungal substances were characterized. Then, biocontrol potential of B. subtilis CPA–8 was tested against the main postharvest decay on oranges, apples and stone fruit (Chapter 1). Based on these results, the mechanism of action used by CPA–8 to suppress postharvest pathogens, particularly against Monilinia spp. causing brown rot in stone fruit was studied (Chapter 2). Based on chemical, molecular and biological analysis the key factors involved in the biocontrol activity of CPA–8 against Monilinia spp. were identified. Next step was optimizing B. subtilis CPA–8 production by developing a low cost medium that provide maximum bacterium growth and maintain its biocontrol efficacy (Chapter 3). First, different media combining economical nitrogen and carbon sources from commercial products and by–products were evaluated. Second, CPA–8 production was scaled up in a 5–liter bioreactor and the efficacy to control of Monilinia spp. in peaches was evaluated. The final step was B. subtilis CPA–8 formulation by spray drying (Chapters 4 and 5). This method was selected because it is a cost effective technique for bacteria preservation and results of Chapter 1 indicated that CPA–8 was heat resistant by endospores production capacity. These endospores had also demonstrated good antifungal activity against fruit postharvest pathogens. First, the role of endospore production by B. subtilis CPA–8 on its survival to spray–drying process was investigated by comparing CPA–8 with the biocontrol agent Pantoea agglomerans CPA–2 as model of heat–sensitive and non–spore forming bacterium (Chapter 4). Finally, carriers/protectants were evaluated to prepare CPA–8 formulations by spray drying; and with the best CPA–8 formulations rehydration media, shelf life stability and biocontrol efficacy during storage were evaluated (Chapter 5). The results obtained in Chapter 1 indicated that CPA–8 produces cells, heat–resistant endospores and compounds with high antifungal activity in vitro against the major fruit postharvest pathogens Botrytis cinerea, Monilinia laxa, Monilinia fructicola, Penicillium digitatum, Penicillium italicum and Penicillium expansum. Treatment of cells, endospores and cell–free supernatants of CPA–8 showed different efficacy levels to control fugal decay on oranges, apples and stone fruit, obtaining the best results against Monilinia spp. on stone fruit with disease reductions up to 100%. Dose tests demonstrated that different CPA–8 treatments at 108, 107 and 106 CFU mL–1 were effective against Monilinia spp., similar or better than Serenade Max®, a commercial biocontrol product based in a B. subtilis strain. Experimental evidence suggested that B. subtilis CPA–8 has biocontrol potential to control postharvest diseases on several fruit types, particularly against peach brown rot. The mode of action study (Chapter 2) showed that cell free supernatants and butanolic extracts from liquid cultures of B. subtilis CPA–8 had a strong antifungal activity in vitro against Monilinia spp. similar to that observed with cell suspensions. Fengycin, iturin and surfactin lipopeptides were identified by TLC in butanolic extracts from cell free supernatants of CPA–a by comparison with the B. subtilis reference strains UMAF6614 and UMAF6639, indicating that antibiosis could be the major factor involved in the CPA–8 biological control ability. TLC–bioautography analysis showed that CPA–8 antifungal activity was associated only with fengycin lipopeptide. These results were definitively supported by mutagenesis analysis targeted to suppress fengycin biosynthesis by disruption of the fenB gene. PCR and TLC–bioautography analysis allowed to identify CPA–8 transformants with reduced or suppressed antifungal activity and to select the defective phenotype associated with the lack of fengycin bands. Fruit trials confirmed that fengycin–defective mutants lost their ability to control peach brown rot disease in comparison with CPA–8 wild type strain or Serenade Max®. Taken together our data indicate that fengycin–like lipopeptides play a major role in the biological control potential of B. subtilis CPA–8 against peach brown rot. The results obtained in Chapter 3 indicate that high production levels of B. subtilis CPA–8 (>3×109 CFU mL–1) could be achieved in a low cost medium based on defatted soy flour 44 % (40 g L–1) with sucrose (20 g L–1) or molasses (5 g L–1). CPA–8 production in the optimized low cost medium was scaled–up in a 5–L bioreactor at 30 °C under shaking at 200 rpm and with air flow of 100 L h–1 and production was maintained around 3×109 CFU mL–1. Fruit trials with cells and cell free supernatants obtained from CPA–8 grown in optimized medium maintained biocontrol efficacy against M. fructicola in peaches showing disease reduction up to 95 %, similar to the treatment from bacterium grown in expensive laboratory media. CPA–8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results could be used to provide a reliable basis for the fermentation scaling–up process to an industrial level. The results obtained in Chapters 4 and 5 indicate that B. subtilis CPA–8 is heat resistant to high temperatures involved in spray–drying process by endospore production comparing with the heat sensitive and non–spore forming P. agglomerans CPA–2. The 72–h–old CPA–8 cultures spray–dried showed the best survival with 32.3 % viable cells recovery and a final concentration product of 3.3×109 CFU g–1, while CPA–2 viability was lower than 2 %. Heat resistance of CPA–8 was also depending on growth time, being 72–h–old culture more resistant than 24–h–old culture, probably due to the higher content of endospores. These results suggest that endospore production improves CPA–8 resistance to spray–drying formulation system. The study of carriers/protectants addition in spray–drying CPA–8 formulations showed that four different combinations of skim milk and MgSO4 provided reasonable recovery powder (28–38 %) and moisture content (7–13 %). CPA–8 survival varied considerably among spray–dried 24– and 72–h–old formulations. The 72 h–old CPA–8 culture spray dried showed the highest survival (28–32 %) and a final concentration product of 1.6–3.3×109 CFU g–1, while viability of 24 h–old culture formulations were lower than 1 %. Rehydration media as water or phosphate buffer provided good recovery of dried cells from CPA–8 formulations as well as skim milk or sucrose both at 10%. This result is a practical advantage. CPA–8 formulations after 4 and 6 month of storage at 4 ºC (cold) or at 20 ºC (room temperature) maintained survival and efficacy to control brown rot caused by Monilinia spp. on nectarines and peaches achieving disease incidence reductions among 90 and 100 %. Spray drying could be considered a suitable method to obtain stable and effective formulations of B. subtilis CPA–8. In conclusion, the studies in this thesis demonstrated the biocontrol potential of B. subtilis CPA–8 to control postharvest diseases of fruit and established the bases for subsequent implementation of production and formulation at commercial level.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2016-10-13T15:32:28Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação completa Maximiliano Dini.pdf: 5135504 bytes, checksum: 0af878399c0b0f849228a9eae5e33c8d (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-01-04T14:56:39Z (GMT) No. of bitstreams: 2 Dissertação completa Maximiliano Dini.pdf: 5135504 bytes, checksum: 0af878399c0b0f849228a9eae5e33c8d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-04T14:56:39Z (GMT). No. of bitstreams: 2 Dissertação completa Maximiliano Dini.pdf: 5135504 bytes, checksum: 0af878399c0b0f849228a9eae5e33c8d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-29 / A cultura do pessegueiro [Prunus persica (L.) Batsch] vem crescendo em todo o mundo, devido ao aumento no consumo de frutas in natura e de seus produtos industrializados. No Brasil, em parte, o crescimento deve-se ao melhoramento genético. Essa área de pesquisa, visando a obtenção de novas cultivares que produzam frutos de qualidade, tenham alta produção, adaptação climática, e apresentem resistência às principais doenças é de muita importância para a expansão da cultura. O conhecimento dos parâmetros genéticos, fenotípicos e ambientais que influenciam direta ou indiretamente nos caracteres de importância econômica em pessegueiro são de fundamental importância para o delineamento dos programas de melhoramento desta frutífera, permitindo antever a possibilidade de sucesso com a seleção de diferentes genótipos em diferentes ambientes. O objetivo central deste trabalho foi buscar fontes de resistência à podridão-parda; estudar a segregação; estimar a herdabilidade e verificar a possível existência de efeito materno em alguns caracteres de importância econômica no pessegueiro. Foi constatada variabilidade fenotípica e segregação transgressiva, para os caracteres fenológicos estudados nas populações avaliadas. A herdabilidade para o período de desenvolvimento do fruto, data de plena floração e data de maturação, é alta a muito alta, permitindo um rápido ganho genético para estes caracteres fenológicos. A herança desses caracteres fenológicos estudados é predominantemente aditiva, e os desvios podem ser atribuídos a um possível efeito materno ou a genes de efeito maior. Foi observada a presença de segregação transgressiva para os parâmetros referentes à tonalidade da cor da polpa (ângulo Hue, croma e luminosidade), e a herdabilidade estimada foi alta, possibilitando rápido avanço genético também para este caráter. A herança da tonalidade da cor da polpa é predominantemente aditiva, e os desvios podem ser atribuídos a genes de efeito menor, sem constatar-se indícios de efeitos maternos. O ângulo Hue é o parâmetro correto para classificar e estudar a tonalidade de pêssegos e nectarinas de polpa amarela, e o parâmetro luminosidade pode ser uma opção para o estudo dos frutos de polpa branca. O uso dos três parâmetros em conjunto possibilita melhor representação deste caráter. Para os caracteres de resistência à podridãoparda em flores e frutos, nas populações estudadas, foi verificada alta variabilidade genética. As cultivares Maciel e Cerrito foram os genitores de maior resistência à podridão-parda nas flores, e transmitiram essa característica a suas progênies. As seleções Conserva 947 e Conserva 1600 foram os genitores de maior resistência à podridão-parda nos frutos, com um nível de resistência similar a ‘Bolinha’, transmitindo essa característica a suas progênies. A herdabilidade da resistência à podridão-parda nas flores (incidência e severidade) e nos frutos (diâmetro da lesão e esporulação), no pessegueiro, é média. A seleção dos genitores, baseada no fenótipo, possibilita um médio avanço genético para esta característica. / The peach culture [Prunus persica (L.) Batsch] has been growing throughout the world, due to an increase in consumption of fresh fruits and processed products. Aiming the development of new productive and adapted cultivars, with good quality fruits and disease resistance, breeding programs are partially responsible for the expansion of the culture, in Brazil. The knowledge of the genetic, phenotypic and environmental parameters that influence, directly or indirectly, on the economically important characters in peach is of fundamental importance for the fruit breeding programs, allowing to anticipate the possibility of success with the selection of genotypes under different environments. The main objective of this work was to seek sources of brown rot resistant genotypes, as well as study the segregation, estimate the heritability and verify the possible existence of maternal effect on some characters of economic importance in peach. Phenotypic variability and transgressive segregation were observed for the phenological characters studied on the evaluated populations. The heritability of the fruit development period, date of full blooming and maturity date, is high to very high, allowing a rapid genetic gain for these phenological characters. The inheritance of the studied phenological characters is predominantly additive, and the deviations can be attributed to a possible maternal effect or major genes. It was observed the presence of transgressive segregation for the parameters relating to the tonality of flesh color (Hue angle, chroma and luminance), and the estimated heritability was high, enabling fast genetic advance for this character. The heritability of this character is predominantly additive, and the deviations can be attributed to the minor genes, without observing signs of maternal effects. The Hue angle is the correct parameter to classify and study the flesh color tonality of yellow peaches and nectarines. The luminance parameter may be an option for the study of white flesh. The use of the three parameters together, enables better representation of this character. For the character brown rot resistance on flowers and fruits, a high genetic variability was verified. The cultivars Maciel and Cerrito were the parentals with higher resistance to flower blight, and they transmitted this trait to their offspring. The selection Conserva 947 and selection Conserva 1600 were the parentals with higher brown rot resistance to infection on fruit, similar to ‘Bolinha’, passing this trait to their offspring. The heritability of brown rot resistance on flowers (incidence and severity) and fruits (diameter of the lesion and sporulation), in peach, is medium. Parental selection based on phenotype, enables a genetic breakthrough for medium resistance to brown rot.

Isolates of Monilinia fructicola (Wint.) Honey obtained from stone fruit orchards in Hawkes Bay, North Island and from Californian fruit exported to New Zealand, were tested for resistance to methyl benzimidazole carbamate (MBC). Resistant isolates from the North Island had EC₅₀ values of >30,000, and most isolates from the imported fruit had of values approximately 1.5 mg a.i./l carbendazim. Sensitive isolates failed to grow on 1 mg a.i./l carbendazim. A detached peach shoot system was used in controlled conditions for estimation of values for incubation period, latent period and rate of spore production on flowers (cv Glohaven). The same variables and the rate of colonisation of host tissue were measured on fruit (cv Fantasia) in controlled conditions. An inoculum density of 1x10⁴ spore/flower or fruit greatly increased fitness in vivo compared to an inoculum density of 1x10² spore/flower (fruit). Isolates varied considerably, but there was no consistent relationship between the degrees of resistance and fitness. This was in contrast to earlier studies with dicarboximide resistant strains of M. fructicola. The survival in the field of 10 isolates resistant or sensitive to MBC or dicarboximide fungicides on twig cankers and mummified fruit was compared. The ability to produce conidia on twig cankers inoculated in late spring 1989 was maintained by all sensitive and MBC resistant isolates for at least 1 year. The production of conidia on mummified fruit inoculated in February 1990 decreased after 2-3 months in the field but some conidia were still produced on all fruit in the following spring. Dicarboximide resistant isolates produced less conidia than either the MBC resistant and the sensitive isolates. The pathogenicity and fitness of all isolates were similar to the original values after survival for 1 year. A technique was developed to produce apothecia reliably from inoculated peach (cv Black Boy) and nectarine (cv Fantasia) fruit in controlled conditions in the laboratory. The fruit were inoculated with resistant or sensitive isolates, or combinations, and were incubated for 8 weeks at 25°C (±1°C) with 12 hours photoperiod of fluorescent light (Sylvania 2x65 W, daylight) to produce mummified fruit. The fruit were then buried in moist autoclaved peat moss for 10 weeks at 25°C (±1°C) in the dark to form stromata. These fruit were then hydrated with running tap-water (total hardness (CaCO₃) = 47 g/m³ and conductivity at 20°C = 12.7 mS/m) for 72 hours. The hydrated mummified fruit were placed in moist peat moss and were incubated for 13-14 weeks at 8°C (±0.5°C) in the dark. At the end of this period, stipe initials were visible. Differentiation of stipe initials into mature apothecia occurred within 15-20 days after transfer to 12°C (±2 °C) with a 12 hour photoperiod of fluorescent and incandescent light. All isolates produced apothecia when treated in this way. A technique for isolation of ascospore sets in linear arrangement was developed for tetrad analysis of the inheritance of resistance. At least 3 hours of fluorescent and incandescent light at 12°C (±2°C) was essential to allow ascospore ejection from individual asci taken from apothecia previously maintained in a 12 hour photoperiod at 12°C (±1°C). A water film on the surface of water agar was necessary to hold a set of ejected ascospores in linear sequence. Single ascospores were obtained in sequence with the aid of a micromanipulator. Genetic analysis of MBC resistant isolates was carried out on ascospores derived from apothecia produced in the laboratory. Analysis of ascospore sets in linear arrangement and ascospore populations indicated that resistance to >30,000 mg a.i./l carbendazim (high-resistant) is governed by a single major gene and is affected by gene conversion mechanisms. Crossing over was frequent, suggesting that recombination of resistance with other characters, such as pathogenicity and fitness, may occur readily. The segregation ratio (1:1) from most resistant isolates revealed that heterokaryons containing both resistant and sensitive alleles were common in resistant populations and that resistance is dominant. Allozyme analysis of ascospore progeny through electrophoresis revealed a narrow genetic base of M. fructicola in New Zealand. The technique for reliable apothecial production in controlled conditions developed in this study provided an important step for the determination of the biology of M. fructicola strains resistant to MBC fungicides, and the complexity of its life cycle. Genetic heterogeneity in field populations can be conserved in one isolate through heterokaryosis, thus providing for adaptability of the pathogen to the changing environmental conditions. Knowledge on genetic variability, overwintering ability, pathogenicity and fitness factors may be useful for future management strategies of stone fruit brown rot. Special emphasis should be made in particular to prevent primary infection on blossoms, which would delay the establishment of recombinant strains of M. fructicola and the onset of brown rot epidemics.