Browsing by Subject "Wound closure"

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Epidemiologie des Esca-Erregers Phaeomoniella chlamydospora und eine neue Bekämpfungsmöglichkeit mittels eines Wundverschlusses aus elektrogesponnenen Polymeren
(2018) Molnar, Melanie; Vögele, Ralf
Esca disease has become a global thread for viticulture over the last decades. It is caused by a complex of at least three different wood-inhabitating fungi Phaeomoniella chlamydospora (Pch), Phaeoacremonium aleophilum (Pal), und Fomitiporia mediterranea (Fmed). For all these fungi wounds in wood are seen as the main entrance way, especially pruning wounds, which are caused by winter pruning. Currently, there are no effective control mechanisms available Therefore, it was the main aim of this project to test a new wound closure made of electrospun fibers, which shall be applied on the pruning wounds to build a physical barrier against invading spores. For evaluating the effectiveness of this treatment the epidemiology of the fungus Pch was investigated, as this fungus is one of the first of this complex invading the vine. For this purpose new molecular techniques have been developed to identify and differentiate Pch reliably. For epidemiologic studies the focus was placed on the occurrence of spores of Pch in the vineyards during the year. Spore traps were placed in vineyards of the Julius Kühn-Intitute which were showing severe symptoms of Esca and in vineyards which were free of foliar symptoms. Traps were analyzed during the whole three year project period on a weekly basis. In this study a new method was developed using a Pch- specific Nested-PCR (polymerase chain reaction) to analyze the spore traps instead of the usual method, in which the trapped spores are spread on media and counting germinated spores. The new Nested-PCR turned out to be a very fast, reliable and sensitive method. It was the first time Pch could be detected in a German vineyard year round. Spores of this fungus could be detected over the whole trial period. Furthermore, a collection of 16 Pch strains with origins from Germany, Italy and South Africa was analyzed using a set of 17 RAMS (random amplified microsatellites) primer to link each one with a unique haplotype. By using this method some polymorphisms formed a pattern which could be interpreted as length polymorphisms. Further analyses of the sequences showed that there are in fact length polymorphisms which are based on repeats of the same sequence. The combination of RAMS primers and the sequence analysis resulted in a finer differentiation of isolates. Primers were developed flanking these repetitive sequences to detect polymorphisms directly without a previous RAMS-analysis. In the end ten haplotypes and two clusters each containing three isolates were found. However, it was not possible to link haplotypes to their geographical origin to gain knowledge of the spread of the fungus. Markers found using the RAMS-analysis were used to differentiate the fungal spores trapped in the spore traps. Furthermore, spores were also compared with strains found in vines of the same vineyard to verify, if the spores were possibly released by fungi in the plants or if they had a different source. This direct analysis was successful in first trials and proofed the existence of different strains in the traps. For an exact classification more markers and primer pairs have to be developed suitable for analyzing spore traps. The epidemiologic survey of this pathogen clearly shows the necessity of a good working wound closure which has to be applied directly after pruning, as the spores are present all year round and especially at the time of winter pruning. For this purpose, different electrospun fibermats made of different fibers of lactic acid were tested under laboratory conditions and in greenhouses for their impermeableness against spores of Pch. During these tightness tests in the greenhouse the new developed Nested-PCR was used to exclude, within the scope of its detection limits, an already exiting infection of the tested plants and to confirm that the a new infection was caused by the applied strain of Pch. Furthermore the degradation and aging of the materials was tested under laboratory and field conditions. Fiber mats with an added amount of glycolic acid turned out to be less stable. Among the different materials fiber mats based on pure lactic acid turned out to be principally suitable to build an applicable and efficient wound closure. First trials in the vineyard confirmed this positive result. However, the long term effect of this treatment on the incidence of Esca disease in vineyards has to be proven.