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Grapevine Trunk Diseases in German Viticulture. III. Biodiversity and Spatial Distribution of Fungal Pathogens in Rootstock Moth

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Grapevine Trunk Diseases in German Viticulture. III. Biodiversity and Spatial Distribution of Fungal Pathogens in Rootstock Moth Vitis 58, 141–149 (2019) DOI: 10.5073/vitis.2019.58.141-149 Grapevine trunk diseases in German viticulture. III. Biodiversity and spatial distribution of fungal pathogens in rootstock mother plants and possible relation to leaf symptoms M. FISCHER Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Plant Protection in Fruit Crops and Viticulture, Siebeldingen, Germany Summary Introduction Three rootstock mother blocks, planted with cul- Grapevine trunk diseases (GTDs) affecting young tivars SO4 (planted 2004), 125AA (2005) and 5BB vines and nursery processes include the Esca disease com- (2005), and located in southwestern Germany were plex (for definition of esca complex diseases see, among examined for the existence of grapevine trunk disease others, SURICO 2001, 2009), black-foot disease (AGUSTÍ-BRI- (GTD) pathogens and related internal and external SACH and ARMENGOL 2013, AGUSTÍ-BRISACH et al. 2013) and symptoms between 2011 and 2017. Frequency of leaf Botryosphaeria dieback (reviewed by BERTSCH et al. 2012). symptoms ranged from 0.2 % in six-year old blocks Eutypa dieback, due to several species of diatrypaceous to appr. 1.5 % in 12-year-old blocks. While the typical fungi, mostly Eutypa lata (Elata), is apparent in older vine- "tiger stripe pattern" was less common, the majority yards only (LECOMTE et al. 2000, ROLSHAUSEN et al. 2014). of affected leaves was characterized by irregularly ar- Between 25 and 50 millions of graftlings are annu- ranged necrotic spots spread over the leaf surface. Ir- ally produced by German nurseries (Verband Deutscher respective of leaf symptoms, in cross sections of 9-12 Rebenpflanzguterzeuger, pers. comm.). During the bench year old vines all sampled trunks (n ≥ 20 for each block) grafting process the scions, i.e. fruit cultivars, by means showed the typical GTD symptoms in the wood, with of matching cuts such as "omega" or "whip" are com- symptoms prevalent in the trunk head compared to the bined with suitable rootstocks. In viticulture worldwide, middle section and the basis of trunks. Pathogens were the grafting process is often discussed as one of the main isolated from all trunks, with Fomitiporia mediterranea reasons for Esca, particularly Petri disease providing en- (Fmed), Cadophora luteo-olivacea (Clo), Phaeomoniella try ports for the associated fungal pathogens by means of chlamydospora (Pch), Eutypa lata (Elata), and Phaeo- open wounds in the wood. For all these diseases, water and acremonium aleophilum (Pal) being the most common. air are considered as the main vectors for propagules, i.e. Other GTD species included Cadophora cf. novi-ebo- spores/conidia, of the pathogens, which in this way may raci (new for German viticulture), Diaporthe eres, D. spread readily within the nursery and between pre-infected nobilis (new for German viticulture), D. rudis (new for and non-infected wood. Rootstock mother plants, grafting German viticulture), Eutypa laevata (new for German tools, water baths, callusing media or soil all have been viticiculture), Ilyonectria europaea, I. liriodendri, and demonstrated as being potentially contaminated by infec- Pestalotiopsis sp. The significance of the once foundSa - tious spores in the past (REGO et al. 2001, WHITEMAN et al. rocladium strictum remains unclear. GTD species were 2007, AROCA et al. 2006, 2010). revealed from all sampled trunk parts, with maximum Out of reasons essentially unknown, during the propa- diversity and overall frequency in the trunk head. Fur- gation process rootstock wood has been found to be more ther species, not related to GTDs, existed in all parts visually affected than scions, and so emphasis of research of the trunk. GTD pathogens were also demonstrated always was biased towards the rootstock part. Several for all shoots (two shoots each of ten vines SO4, 125AA potential causal agents have been named in the past, the and 5BB, with five vines each externally symptomatic ones mostly cited the anamorphic fungus Phaeomoniel- and non-symptomatic), but mostly could be detected by la chlamydospora (Pch) as well as Phaeoacremonium molecular means only. Clo, Pch, and Pal were the pre- aleophilum (Pal). For the latter, Togninia minima has been dominant species in shoots; further GTD species were confirmed as the teleomorphic form (MOSTERT et al. 2003; Ilyonectria europaea, I. liriodendri, and Phaeoacremoni- ROONEY-LATHAM et al. 2005), and the designation P. min- um angustius. imum has been suggested for Pal (GRAMAJE et al. 2015). Additional pathogens are discussed in the meantime, with K e y w o r d s : biodiversity; grapevine trunk diseases; special emphasis on Cadophora spp. (GRAMAJE et al. 2009, rootstock mother plants; fungal pathogens. TRAVADON et al. 2015) or other, partly novel, species of Correspondence to: Prof. Dr. M. FISCHER, Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Plant Protection in Fruit Crops and Viticulture, Geilweilerhof, D-76833 Siebeldingen, Germany. E-mail: [email protected] © The author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative-commons.org/licenses/by-sa/4.0/). 142 M. FISCHER Phaeoacremonium (MOSTERT et al. 2006, GRAMAJE et al. FISCHER and KASSEMEYER 2003, CLOETE et al. 2016). Plates 2015, SPIES et al. 2018). For German viticulture, first re- were incubated under daylight conditions at appr. 23 °C ports on Petri disease as an emerging disease in young and were checked once a day for 4 weeks. Hyphal tips of vineyards date back to FISCHER and KASSEMEYER (2004); growing colonies were transferred to fresh PDA to obtain over the years, further studies have been conducted by the pure cultures. Fungal isolates are maintained in the culture first author (for instance, see FISCHER 2009), but until re- collection of the Institute for Plant Protection in Viticulture cently (FISCHER et al. 2016) results were presented in small at the Julius Kühn-Institut, Geilweilerhof, and are stored in scale journals on a regional basis only, mainly directed to tubes at +4 °C conditions. the practice side of viticulture. In this study, between 2011 Molecular studies on fungal pure and 2017 three rootstock mother gardens, SO4, 125AA and cultures (see also F ISCHER et al. 2016): 5BB, all located in the southwestern region of Germany Total DNA was extracted from pure cultures as described were monitored for the occurrence of leaf symptoms and by TILLETT and NEILAN (2000). Extracted fungal DNA was the existence of GTD-related fungi in the wood. Samples subjected to PCR amplification of the ITS region using were taken from i) different sections of trunks of mother primers ITS5 and ITS4 (for primer sequences, see WHITE plants, and ii) shoots still attached or freshly cut from the et al. 1990). PCR conditions were an initial denaturation mother vines. Examination and assessment of samples was for 5 min at 95 °C, followed by 25 cycles at 98 °C for 20 s, by visual, culturing and molecular means. As a result, new 57 °C for 15 s, 72 °C for 20 s and a final extension step for and more comprehensive data are presented with respect to 1 min at 72 °C. Amplification was carried out in a 20 µL the diversity, the spatial distribution and the frequency of volume with the KAPA HiFi™ HotStart PCR Kit (KAPA GTD organisms in rootstook mother vines. Besides, sever- Biosystems, Wilmington, USA). Reactions contained 1x al non-GTD fungi were recovered and are reported here for PCR buffer, 2,0 mM MgCl2, 300 µM of each dNTP, 0,3 the first time on grapevine. µM of each primer, 0,5 U of KAPA HiFi HotStart DNA Polymerase and 1 µL template (20 ng µL-1). After electro- phoretic examination PCR products were purified using Material and Methods the QIAquick PCR Purification Kit (Qiagen, Hilden, Ger- many) according to the manufacturer's recommendations. P l a n t a t i o n s : Mother gardens comprised the Purified PCR products were subsequently sequenced in rootstock varieties most common in German viticulture both directions with primers ITS5 and ITS4 in an Applied and included cultivars SO4 (planted 2004), 125AA (plant- Biosystems 3130XL DNA Analyzer using BigDye® Ter- ed 2005) and 5BB (planted 2005). Mother plants all are minator v3.1 Cycle Sequencing Kit (Applied Biosystems, raised, i.e. with a distinct trunk, and trellised. All blocks are Foster City, USA) at Seqlab (Sequence Laboratories Göt- located in the southwestern part of Germany. tingen, Göttingen, Germany). Assembling and examination Monitoring for leaf symptoms, wood of consensus sequences were performed using CLC Main samples from trunks and shoots, and iso- Workbench 7.8.1 (Qiagen Bioinformatics, Qiagen, Aarhus, lation technique (see also F ISCHER et al. Denmark). For identification of fungal isolates consensus 2 0 1 6 ) : During the period from 2011 to 2017, all blocks sequences were analysed with the Basic Local Alignment were monitored at least twice for leaf symptoms; monitor- Tool (BLAST) available from NCBI (National Center for ing was conducted between August and September. Biotechnology Information, Bethesda, USA), and Gen- Starting in 2013 mother gardens were repeatedly sam- Bank numbers were generated for representative isolates pled over the years; with this background, age of planta- of all GTD taxa. Supportive data for identification were tions during sampling ranged from 9 to 12 years. For sam- derived from microscopy and morphology of pure cultures. pling of the trunks (n ≥ 20 for each block, then between For this, fragments of pure cultures were mounted in water nine and twelve years old) wood pieces were taken from and Melzer's reagent and studied at 500x and 1000x under visually affected regions in three different cross sections phase contrast optics. made in: i) the trunk head, ii) the middle section and iii) the Molecular studies on wood samples basis. Sampled trunks were uprooted in September/Octo- ( f r o m s h o o t s o n l y ) : Wood samples of appr.
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