Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

Les maladies du bois de la vigne, causées par des champignons nécrotrophes, ont un impact considérable sur l'économie viticole au niveau mondial. En effet, tous les cépages Vitis vinifera cultivés actuellement présentent une sensibilité plus ou moins forte à ces champignons.Dans le but d'établir un test rapide d'évaluation de la sensibilité de clones de vigne (nouvellement sélectionnés ou futures obtentions variétales) à Eutypa lata, le champignon responsable de l'Eutypiose, une recherche de marqueurs moléculaires de tolérance a été réalisée. Suite à l'infection in vivo et in vitro d'une douzaine de cépages de sensibilité différente par E. lata, plusieurs gènes candidats ont été identifiés comme marqueurs potentiels de la tolérance à la maladie à partir d'une étude transcriptomique.A l'aide d'un système innovant d'infection in vitro, le dialogue moléculaire sans contact physique entre des disques foliaires de V. vinifera et le mycélium d’E. lata a également été étudié chez les douze cépages. Cette étude a permis de mettre en évidence le rôle potentiel d'éliciteurs dans la mise en place des réponses de défenses. De plus, l'infection par E. lata régule différentiellement l'expression des gènes codant pour un transporteur d'hexoses et des invertases, ainsi que les activités invertasiques associées, chez les cépages de sensibilités variables.Les travaux de recherche présentées dans cette thèse ont ainsi permis l'identification des marqueurs de tolérance à l'Eutypiose et la mise au point d'un test d'infection in vitro efficace et fiable, permettant de diagnostiquer la sensibilité des futures créations variétales. De plus les résultats obtenus démontrent l'importance des éliciteurs dans la mise en place des défenses, de la régulation du transport et du métabolisme des sucres au cours de l'infection par E. lata. / Nowadays, grapevine wood decay diseases cause significant economic losses for the most sensitive varieties and represent a threat to the sustainability of the wine industry.This research focuses on the identification of molecular markers for sensitivity to Eutypa lata, responsible for Eutypiosis that could be used to diagnose the sensitivity of new grapevine clones or cultivars. Using an in vivo and in vitro infection assay, several potential markers genes for tolerance to Eutypiosis have been identified from a gene expression study on twelve different cultivars.Using an innovative in vitro infection system, the molecular dialogue (without physical contact) between Vitis vinfera foliars discs of and Eutypa lata was studied, leading to the identification of elicitors that could potentially play a role in the induction of defense responses in the cultivars. In addition, the expression of several sugar transport and invertase genes and their associated activities were demonstrated to be differentially regulated by the infection in tolerant and susceptible cultivars.Altogether, this research work led to the identification of several tolerance markers genes to Eutypiosis and to the development of a new, efficient and reliable in vitro infection system that could be used to diagnose new cultivar susceptibility. Furthermore, the results obtained demonstrated the importance of sugar transport and defense metabolism regulation during the infection of grapevine by Eutypa lata.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating impact on vineyards worldwide, reducing growth and yield, eventually killing the grapevine. The causal organism of Eutypa dieback was first described as Eutypa armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987 this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two species that are capable of infecting grapevines are responsible for Eutypa dieback. Consequently, the molecular identification and characterisation of Eutypa dieback was used to delineate the species occurring on infected grapevines in South Africa. This involved the molecular analyses of three molecular markers, namely, the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon, and the -tubulin gene. The results obtained revealed the presence of a second species, namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on infected grapevines. Also co-habiting with these pathogens were related fungi form the Diatrypaceae family, Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart. Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca, and E. vitis revealed that all were pathogenic to grapevine. Several species of Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also pathogenic to grapevine. The symptoms in grapevine commonly associated with Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback which prompted the need for the development of a detection method that can correctly identify the presence of multiple pathogens. A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify and label the regions of DNA that are used as pathogen specific probes. Consequently, membrane immobilised species-specific oligonucleotides synthesised from the ITS, - tubulin and LSU molecular data were evaluated during the application of this diagnostic method to detect Eutypa species. It was found that the species-specific oligonucleotides, designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca. The application of the RDBH method for the detection of these Eutypa species, based on -tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a RDBH method, utilising species-specific oligonucleotides designed from elongation factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the detection of Diplodia seriata De Not. In addition to the above-mentioned shortcomings, the RDBH was not amenable to the detection of pathogens directly from field or environmental samples, but required preparation of DNA from pure cultures. The method, however, allows for the identification of multiple pathogens in a single assay. As DNA extraction methods are amended, improved and honed to obtain DNA from environmental samples, so would it increase the usefulness of RDBH. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak. Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. & Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987 word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak. Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in geïnfekteerde plante voorkom. Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis (Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species. Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie. Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.

[EN] Almond is one of the main crops of Majorca Island. Since 2008, symptoms of severe decline of almond trees have been observed in several orchards from different areas of the Island. Disease symptoms are similar to those described by different authors in other parts of the world caused by fungal trunk pathogens. In order to study the etiology of this problem, surveys were conducted on almond orchards distributed throughout the main growing regions in Majorca for six consecutive years (2009-2014). Based on morphological and molecular identification, 14 fungal species were recovered from almond wood samples: Collophora hispanica, Diplodia olivarum, D. seriata, Eutypa lata, E. leptoplaca, Fomitiporia mediterranea, Neofusicoccum luteum, N. mediterraneum, N. parvum, Omphalotus olearius, Phaeoacremonium amygdalinum, Pm. iranianum, Phellinus pomaceus and Pleurostomophora richardsiae and two species were recovered from one apricots orchard near almonds orchards: : Pm. minimum and Pm. venezuelense. Based on the DNA sequence analyses and morphological features, C. hispanica and Pm. amygdalinum proved distinct from all known species, and have been described. The most common species recovered from almond samples were P. richardsiae and D. seriata, followed by other species belonging to the family Botryosphaeriaceae and C. hispanica. The most frequently species isolated were also widely distributed and present in more regions. Subsequently, two pathogenicity tests were carried on almond trees by using representative isolates of some of the most frequent species. The first one was held for two consecutive years (2013 and 2014) with five species of Botryosphaeriaceae (D. olivarum, D. seriata, N. luteum, N. mediterraneum and N. parvum) and two species of Diatrypaceae (E. lata and E. leptoplaca). Fungi were inoculated on 1-2 years old almond trees of four different cultivars ('Jordi', 'Ferragnes', 'Pons' and 'Vivot') under field conditions. Nine months after inoculation, the total length of internal necrosis was evaluated. All species were pathogenic on almond. Neofusicoccum luteum caused the longest average lesion during the first year, and N. mediterraneum and N. parvum caused the longest lesion during the second year. Eutypa leptoplaca caused the shortest lesion length in both years of study. In addition, fungal lesion length varied depending on the variety of almond evaluated. In the first year of study, the more tolerant variety was 'Jordi', while in the second year, 'Ferragnes' and 'Vivot' varieties showed the highest degree of tolerance to fungal infection. In the second trial, almond seedlings variety 'Ferragnes' were inoculated with C. hispanica, Pm. amygdalinum, Pm. iranianum and P. richardsiae. Six months after inoculation the lesion length was evaluated. All species inoculated were pathogenic on almond, being P. richardsiae the most virulent species. Finally, the ability of some commercial fungicides to protect pruning wounds from infection by four species of Botryosphaeriaceae (D. seriata, N. luteum, N. mediterraneum and N. parvum) was evaluated. This study was conducted in two phases, an initial in vitro evaluation (mycelial growth assay) with 10 fungicides, followed by an evaluation of five fungicides, which proved to be effective in the in vitro trial, applied on pruning wounds at 1 and 7 days after inoculation. Internal lesion length and the percentage of re-isolation of the pathogen were calculated. tebuconazole and pyraclostrobin were the most effective fungicides in the in vitro evaluation, followed by cyproconazole and thiophanate-methyl. Thiophanate-methyl was the most effective fungicide to protect pruning wounds from infections caused by species of Botryosphaeriaceae. / [ES] Desde el año 2008, en parcelas de diferentes zonas de la isla se ha constatado la presencia síntomas de decaimiento de ramas y muerte de almendros, que recuerdan a los descritos por diferentes autores en otras zonas del mundo causados por hongos patógenos de la madera en diversos cultivos. Para estudiar su etiología se realizaron prospecciones en parcelas de almendros de la isla durante seis años (2009-2014). Se caracterizaron los síntomas y se tomaron muestras que se analizaron en laboratorio. En los análisis se obtuvieron 14 especies fúngicas de muestras de almendro: Collophora hispanica, Diplodia olivarum, D. seriata, Eutypa lata, E. leptoplaca, Fomitiporia mediterranea, Neofusicoccum luteum, N. mediterraneum, N. parvum, Omphalotus olearius, Phaeoacremonium amygdalinum, Pm. iranianum, Phellinus pomaceus, Pleurostomophora richardsiae y dos especies encontradas sólo en muestras de una parcela de albaricoqueros junto a parcelas de almendros: Pm. minimum y Pm. venezuelense. Collophora hispanica y Pm. amygdalinum son dos nuevas especies fúngicas. Las especies más frecuentes en las parcelas de almendro estudiadas fueron P. richardsiae y D. seriata, seguidas por las otras especies pertenecientes a la familia Botryosphaeriaceae y por C. hispanica. Las especies que se aislaron con mayor frecuencia, fueron a su vez las que tuvieron una distribución más amplia; presentes en más comarcas. Posteriormente, se estudió la patogenicidad a almendro de algunas de las especies detectadas. Se realizaron dos ensayos de patogenicidad, el primero se realizó dos años (2013 y 2014) con las cinco especies de Botryosphaeriaceae (D. olivarum, D. seriata, N. luteum, N. mediterraneum y N. parvum) y las dos de Diatrypaceae (E. lata y E. leptoplaca) aisladas, que se inocularon en árboles de 1 a 2 años de cuatro variedades de almendro ('Jordi', 'Ferragnes', 'Pons' y 'Vivot') en una parcela experimental. El ensayo se evaluó a los nueve meses de la inoculación, midiendo la longitud de las necrosis internas producidas. En este ensayo se demostró la patogenicidad a almendro de las siete especies que se ensayaron. Las especies que causaron las lesiones de mayor longitud fueron N. luteum el primer año de ensayo, y N. parvum y N. mediterraneum el segundo año. Ambos años, la especie que causó las lesiones de menor longitud fue E. leptoplaca. La dimensión de la lesión producida por el hongo inoculado dependía de la variedad de almendro evaluada. La variedad 'Jordi' fue la menos sensible el primer año de estudio, y 'Vivot' y 'Ferragnes' lo fueron el segundo año. En el segundo ensayo se estudió la patogenicidad de las especies C. hispanica, Pm. amygdalinum, Pm. iranianum y P. richardsiae en plantones de almendro de la variedad 'Ferragnes' en invernadero. Nuevamente, la evaluación se realizó a los seis meses de la inoculación, midiendo la longitud de las lesiones internas. Todas las especies inoculadas resultaron patógenas a almendro, siendo P. richardsiae la especie que causó la mayor longitud de lesión. Finalmente, se evaluaron fungicidas para la protección de heridas de poda frente a la infección por cuatro especies de Botryosphaeriaceae (D. seriata, N. luteum, N. mediterraneum y N. parvum). Este estudio se llevó a cabo en dos fases; en primer lugar, una evaluación in vitro (reducción del crecimiento miceliar) con diez fungicidas y, posteriormente, una evaluación de cinco de estos fungicidas, elegidos entre los más efectivos in vitro, aplicándolos en heridas de poda uno o siete días tras el corte y la inoculación. Como en los casos anteriores, para evaluar este estudio se midió la longitud de la lesión, pero además también se calculó el porcentaje de reaislamiento del patógeno inoculado en cada caso. Los fungicidas tebuconazol y piraclostrobin, seguidos de ciproconazol y metil tiofanato, se mostraron como los más efectivos en la evaluación in vitro, mientras que el fungicida más efectivo para la protección de / [CAT] Des de l'any 2008, en parcel¿les de diferents zones de Mallorca s'han observat símptomes de decaïment de branques i mort d'ametllers, que recorden als que diferents autors han descrit en altres zones del món causats per fongs de fusta en diversos cultius. Per estudiar la seua etiologia es van realitzar prospeccions en parcel¿les d'ametllers de l'illa durant sis anys consecutius (2009-2014). En aquestes prospeccions es van caracteritzar símptomes i es van prendre mostres que es van analitzar al laboratori. En les anàlisis de laboratori es van obtenir 14 espècies fúngiques de mostres d'ametller: Collophora hispanica, Diplodia olivarum, D. seriata, Eutypa lata, E. leptoplaca, Fomitiporia mediterranea, Neofusicoccum luteum, N. mediterraneum, N. parvum, Omphalotus olearius, Phaeoacremonium amygdalinum, Pm. iranianum, Phellinus pomaceus i Pleurostomophora richardsiae i dues espècies trobades només a una parcel¿la d'albercoquers situada a prop de parcel¿les d'ametllers: Pm. minimum i Pm. venezuelense. Collophora hispanica i Pm. amygdalinum són dues espècies noves. Les espècies més freqüents en les parcel¿les d'ametller estudiades van ser P. richardsiae i D. seriata, seguides per les altres espècies pertanyents a la família Botryosphaeriaceae i per C. hispanica. Les espècies que es van aïllar amb més freqüència, van ser també les que van tenir una distribució més àmplia; presents en més comarques. Posteriorment, es va estudiar la patogenicitat d'algunes de les espècies detectades. Concretament, es van realitzar dos assajos de patogenicitat. El primer es va dur a terme dos anys (2013 i 2014), amb les cinc espècies de Botryosphaeriaceae (D. olivarum, D. seriata, N. luteum, N. mediterraneum i N. parvum) i les dues de Diatrypaceae (E. lata i E. leptoplaca) aïllades, que es van inocular en arbres d'1 a 2 anys de quatre varietats d'ametller ('Jordi', 'Ferragnes', 'Pons' i 'Vivot') en una parcel¿la experimental. L'assaig es va avaluar als nou mesos des de la inoculació, mesurant la longitud de les necrosis internes produïdes. En aquest assaig es va demostrar la patogenicitat respecte l'ametller de les set espècies que es van assajar. Les espècies que van causar les lesions de major longitud van ser N. luteum el primer any d'assaig, i N. parvum i N. mediterraneum el segon any. En els dos anys dels assajos l'espècie que va causar lesions de menor longitud va ser E. leptoplaca. Es va observar que la dimensió de la lesió causada pel fong inoculat depenia de la varietat d'ametller avaluada. La varietat 'Jordi' va ser la menys sensible el primer any d'estudi, i 'Vivot' i 'Ferragnes' ho van ser el segon any. En el segon assaig es va estudiar la patogenicitat de les espècies C. hispanica, Pm. amygdalinum, Pm. iranianum i P. richardsiae en plançons d'ametller de la varietat 'Ferragnes' en hivernacle. L'avaluació es va realitzar als 6 mesos de la inoculació, mesurant la longitud de les lesions necròtiques internes. Totes les espècies van resultar patògenes d'ametller, sent P. richardsiae l'espècie que va causar les lesions més extenses. Finalment, es va realitzar un estudi d'avaluació de fungicides per a la protecció de ferides de poda enfront de la infecció per quatre espècies de Botryosphaeriaceae (D. seriata, N. luteum, N. mediterraneum i N. parvum). Aquest estudi es va dur a terme en dues fases, una avaluació in vitro (reducció del creixement micelià) amb 10 fungicides i, posteriorment, una avaluació de cinc d'aquests fungicides, elegits entre els més efectius in vitro, aplicant-los en ferides de poda a 1 o 7 dies després del tall i la inoculació. Novament per avaluar aquest estudi es va mesurar la longitud de la lesió, però a més també es va calcular el percentatge de reaïllament del patogen inoculat en cada cas. Els fungicides tebuconazol i piraclostrobin, seguits de ciproconazol i metil tiofanat, es van mostrar com els més efectius in vitro, mentre que el fungicida més efect / Olmo García, D. (2016). Etiología y control de las enfermedades fúngicas de la madera del almendro en la isla de Mallorca [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/60158 / TESIS

L'agriculture a besoin de fongicides ayant la propriété d'être transportés par le phloème pour contrôler des pathogènes vasculaires et racinaires. Jusqu'à présent, les tentatives de modulation structurale de fongicides pour les rendre phloème mobiles ont généralement eu comme conséquence une perte d'activité biologique.<br />La première approche de notre travail reprend la stratégie utilisée avec succès pour le développement d'herbicides auxiniques. Nous avons choisi comme molécule modèle le fenpiclonil en raison de la possibilité d'ajouter un groupe acide carboxylique en divers sites de la molécule. L'un de ces dérivés, le N-(1-carboxyéthyl)-3-cyano-4-(2,3-dichlorophényl)pyrrole possède une mobilité phloémienne modérée et montre une activité fongicide contre une souche d'Eutypa lata comparable à celle du fenpiclonil.<br />La deuxième stratégie a consisté à synthétiser des propesticides mobiles dans le phloème en greffant un acide aminé à divers xénobiontes ou à des composés naturels impliqués dans la défense des plantes. Ces conjugués avec une fonction a-aminoacide sont manipulés par des perméases de la membrane plasmique et sont nettement phloème mobiles. Toutefois, les enzymes qui peuvent libérer le composé initial peuvent être plus spécifiques que le système de transport.<br />Enfin, la capacité du phloème de ricin à charger l'acide salicylique (AS) a été étudiée pour deux raisons : 1- l'AS est une molécule signal importante impliquée dans la résistance systémique acquise 2- dans les cellules animales, l'AS est manipulé par un système de transport qui manipule aussi des médicaments de taille importante. L'AS s'accumule fortement dans le phloème et est ambimobile. Quelques résultats conduisent à l'hypothèse de l'intervention d'un système de transporteurs dépendant du pH et contribuant au chargement phloémien de l'AS, outre le phénomène de piégeage d'acide.

L'esca est un syndrome cryptogamique vasculaire de la vigne (Vitis vinifera). Une phase pionnière, sous la dépendance de Phaeoacremonium aleophilum (PA), Phaeomoniella chlamydospora (PC) et éventuellement de Eutypa lata (EL) permet la mise en place d'autres espèces. Depuis l'interdiction de l'arsénite de sodium, cette maladie n'est plus contrôlée.<br /> Nous avons étudié les propriétés de PA, PC et EL, en particulier l'impact sur leur croissance de fongicides commerciaux et de fongicides systémiques synthétisés au laboratoire, ainsi que de molécules naturelles. Ces pathogènes ne présentent pas la même sensibilité à ces molécules et l'un d'eux (PA) est peu affecté par divers traitements. En parallèle, nous avons étudié les propriétés d'ambimobilité de l'acide salicylique (AS) et de quelques-uns de ses dérivés halogénés.<br /> Nous avons montré que F 30, un dérivé acide du fenpiclonil, est mobile dans les boutures de vigne après application foliaire. Il est en partie retenu dans le bois et libère la molécule parent dans les racines. L'acide 5-chlorosalicylique (5-ClAS), connu pour être plus actif que AS pour stimuler les défenses naturelles, présente une mobilité voisine de celle de AS. Sur ces bases, F 30 et 5-ClAS ont été retenus pour des tests préliminaires de traitement par voie foliaire de boutures de vigne infectées.<br /> Cette recherche exploratoire souligne la complexité de la problématique, une lutte chimique (fongicide), génératrice de contraintes, devant s'intégrer dans une stratégie globale de contrôle.