Browsing by Subject "Phaeomoniella chlamydospora"

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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.
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    Grapevine Trunk Diseases : Epidemiologie und Molekulardiagnose wichtiger Esca-Erreger während der Pflanzguterzeugung
    (2018) Haag, Nicolai; Vögele, Ralf
    Esca is one of the most important grapevine trunk diseases (GTDs) worldwide. In Europe, the wood-inhabiting fungi Phaeomoniella chlamydospora (Pch), Phaeoacremonium aleophilum (Pch) and Fomitiporia mediterranea (Fmed) are considered the main causal agents of this disease. Even young vineyards and planting material can be affected by pathogens of Esca. So far, control possibilities were mainly limited to prophylactic measures, such as prevention of overly large pruning wounds, minimizing stress of affected grapevines and elimination of dead wood. By now, biological control is basically possible; its efficacy however, still remains to be fully proven under practical conditions. According to previous studies in Germany investigating the infection status of planting material, Pch is considered the most important causal agent for an early infection in grapevine nursery propagation. Based on this, the objective of the present study was to comprehensively assess the occurrence of Pch and other GTD-pathogens in grapevine wood and potential inoculum sources and to visually evaluate Esca-associated symptoms in rootstocks and scions during the propagation process including indoor working steps and the outdoor rooting phase. The results should then be used to point up possible control measures. For this purpose, from 2014 to 2016 visual ratings of Esca-associated wood symptoms were conducted on grapevine material collected from three different nurseries over the entire propagation process as well as from planting material ready for sale. Further assessments on pathogen occurrence by nested PCR and traditional culturing methods focused on rootstock material, which is more affected from experience, and were at first limited to the detection of Pch. For each year, a strong increase of wood symptoms, both in rootstocks and scions, was observed in the course of the production process. Compared to that, PCR- and culture-based detection rates of Pch in rootstock wood were considerably lower revealing a marked discrepancy between symptom incidence and the actual presence of the pathogen. Interestingly, a previously unconsidered fungus, Caophora luteo-olivacea (Clo), which is suspected to be a further GTD-pathogen, was frequently isolated from symptomatic rootstocks. Based on this observation, a multiplex nested PCR method was subsequently developed to specifically detect Pch, Clo as well as species of the Esca-relevant genus Phaeoacremonium (Pm). By using this method, Pch and Pm spp. were detected in ~9% and ~15% of tested rootstocks, respectively (average of all nurseries and years of observation), whereas detection rates of Clo were comparatively high at ~78%. This way, it was possible to harmonize previously observed discrepancies between symptom and pathogen incidence to a great extent. Regarding the occurrence of pathogens and expression of wood symptoms significant differences between nurseries were noticed at single sampling dates, in general however, infestation situations were quite similar. Further investigations were conducted to identify potential inoculum sources in the propagation process. Herefore, sampling was done from various hydration tanks, callusing media and nursery soil. In addition, spore traps were installed in nursery fields in order to monitor airborne inoculum. Samples were subsequently analysed by multiplex nested PCR and/or fungal isolation. PCR-based detections of Pch, Pm spp. and Clo were obtained from hydration tanks as well as from callusing media and air with Clo being the most common species in every respect. Furthermore, viable inoculum of Pch and Pm spp. were found in rootstock wood only, whereas Clo could be additionally isolated from hydration tanks, callusing media and nursery soil. However, isolation rates of Pch, Clo and Pm spp. respectively corresponded to only ~9%, ~9% and ~2% of the detection frequencies when using multiplex nested PCR. The present study allowed for a comprehensive and in part previously unknown insight into the infestation situations of planting material regarding Pch and further Esca-associated pathogens. It provides specific information on potential inoculum sources in grapevine nursery propagation and emphasizes the possible role of pre-infected planting in the development and spread of Esca. With respect to a targeted control of Esca and associated diseases, the obtained results provide an important basis to verify efficacy and practicabilty of existing or yet to be developed control measures.