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Characterization of Neopestalotiopsis, Pestalotiopsis and Truncatella Species Associated with Grapevine Trunk Diseases in France

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Characterization of Neopestalotiopsis, Pestalotiopsis and Truncatella Species Associated with Grapevine Trunk Diseases in France CORE Metadata, citation and similar papers at core.ac.uk Provided by Firenze University Press: E-Journals Phytopathologia Mediterranea (2016) 55, 3, 380−390 DOI: 10.14601/Phytopathol_Mediterr-18298 RESEARCH PAPERS Characterization of Neopestalotiopsis, Pestalotiopsis and Truncatella species associated with grapevine trunk diseases in France 1,2 3 4,5 2 SAJEEWA S. N. MAHARACHCHIKUMBURA , PHILIPPE LARIGNON , KEVIN D. HYDE , ABDULLAH M. AL-SADI and ZUO- 1, YI LIU * 1 Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Xiaohe District, Guiyang City, Guizhou Province, 550006 People’s Republic of China 2 Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Oman 3 Institut Français de la Vigne et du Vin, Pôle Rhône-Méditerranée, 7 avenue Cazeaux, 30230 Rodilhan, France 4 Institute of Excellence in Fungal Research, Mae Fah Luang University, Tasud, Muang, Chiang Rai, 57100 Thailand 5 School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai, 57100 Thailand Summary. Pestalotioid fungi associated with grapevine wood diseases in France are regularly found in vine grow- ing regions, and research was conducted to identify these fungi. Many of these taxa are morphologically indistin- guishable, but sequence data can resolve the cryptic species in the group. Thirty pestalotioid fungi were isolated from infected grapevines from seven field sites and seven diseased grapevine varieties in France. Analysis of internal transcribed spacer (ITS), partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) sequence data revealed several species of Neopestalotiopsis, Pestalotiopsis and Truncatella associated with the symp- toms. Three Neopestalotiopsis spp. and one Pestalotiopsis sp. are reported for the first time associated with wood diseases in grapevine in France and worldwide, and include Neopestalotiopsis asiatica, N. javaensis, Neopestalotiopsis sp. and Pestalotiopsis biciliata. The sequence data indicate that Truncatella angustata was also associated with wood grapevine diseases in France; this species was previously reported on grapevine in Iran. The importance of con- trolling Pestalotioid fungi associated with wood grapevine diseases is discussed, which was previously considered of minor importance. These fungi are isolated from plants in nurseries before marketing. Key words: cryptic species, pestalotioid fungi, trunk disease, Vitis vinifera. Introduction sidered to be the most destructive diseases of grape- vines (Hofstetter et al., 2012; Bruez et al., 2013; Grama- Vitis vinifera L. (grapevine) is native to the Medi- je et al., 2016; Larignon 2016). These diseases have terranean region, central Europe, and southwestern severe effects on the perennial plant parts, mainly Asia, and is widely grown for its fruits throughout the the trunks of vines. In France, three main diseases, world (Terral et al., 2010). The ancient Greeks intro- esca disease, Botryosphaeria dieback and eutypa die- duced grape cultivation to Europe in the Minoan age back, are the most destructive, and have long been (Phillips, 2000a). France is among the top five coun- known wherever grapes are grown (Larignon and tries in the world in grape production, with a total et al et al. production of 5.5 million tonnes in 2013 (FAO, 2016). Dubos, 1997; Mugnai ., 1999; Bertsch 2013). Grapevine trunk diseases impact the economic A complex of fungal species, especially Fomitiporia production and longevity of vineyards, and are con- mediterranea Fischer, Phaeomoniella chlamydospora (W. Gams et al.) Crous & W. Gams and Phaeoacremonium minimum (Durieu & Mont.) D. Gramaje et al., are as- Corresponding author: Z.-Y. Liu sociated with esca disease. Botryosphaeriaceae spp. E-mail: [email protected] cause Botryosphaeria dieback, Eutypa lata (Pers.) Tul. 380 ISSN (print): 0031-9465 www.fupress.com/pm ISSN (online): 1593-2095 © Firenze University Press © 2016 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pestalotioid trunk diseases on grapes & C. Tul. causes Eutypa dieback, and Diaporthe am- from various viticultural districts of France (Aqui- pelina (Berk. & M.A. Curtis) R.R. Gomes et al. caus- taine, Burgundy, Champagne, Languedoc-Roussil- es Phomopsis cane and leaf spot. (Phillips, 2000b; lon, Rhône-Alpes) between January 2010 to January Gramaje et al., 2016; Larignon 2016). All these diseas- 2012. Plant material was also obtained from nurs- es can be observed in the same plant and presented a eries in Midi-Pyrénées and PACA. Isolations were fungal and bacterial microflora accompanying these made from tissues according to Larignon and Du- pathogen agents (Bruez et al., 2014, 2015). Symptoms bos, (1997). Isolation plates were incubated at room of these diseases include stunted shoots with short- temperature for 3 weeks. Colonies grown on malt ened internodes, defoliated shoots, dieback of one agar (MA) plates were transferred to MA and stored or more shoots accompanied by leaf drop, leaf stripe at 4°C for further study. and apoplectic forms and woody necrosis (brown or These fungal colonies were then grown on potato grey wedge-shaped necrosis, white rot, brown stripe, dextrose agar (PDA) incubated at 25ºC for 7 to 10 d black dots) (Bertsch et al., 2013). These trunk diseases and were initially identified using morphological trigger gradual decline of grapevines due to inner al- traits following Maharachchikumbura et al. (2012; terations of the wood, and result in decreasing yield 2014). and quality of grapes (Moreno-Sanz et al., 2013). Many pathogens infect mature grapevines. However, there Molecular phylogenies are also many records of pathogens infecting young grapevines (Bertelli et al., 1998; Giménez-Jaime et al., DNA extraction, PCR amplification, and DNA sequencing 2006; Halleen et al., 2006; Dubrovsky and Fabritius, Total genomic DNA was extracted from fresh 2007; Schroers et al., 2008; Larignon et al., 2015). fungal mycelia (500 mg), scraped from the margin of Neopestalotiopsis, Pestalotiopsis and Truncatella be- a colony on a PDA plate incubated at 25°C for 7–10 long to the order Xylariales and are generally known d (Guo et al., 2000). The ITS, TUB and TEF regions as pestalotioid fungi (Maharachchikumbura et al., were amplified using primer pairs ITS4/ITS5 (White 2014). These taxa are significant phytopathogens et al., 1990), BT2A/BT2B (Glass and Donaldson, causing postharvest fruit rot and trunk diseases in 1995; O’Donnell and Cigelnik, 1997), and EF1- 526F et al grapevines in many countries (Arzanlou ., 2013; or EF728F/EF1-1567R or EF2 (Carbone and Kohn, et al Jayawardene ., 2015), and were the second most 1999; O’Donnell et al., 1998; Rehner, 2001). Polymer- common taxa isolated from grapevine cankers in ase chain reaction (PCR) was performed for each Texas, following Botryosphaeria spp. (Úrbez-Torres sample with the 25 μL reaction system consisting of et al., 2011). However, recognition of species, using 19.5 μL of double-distilled water, 2.5 μL of 10× Taq phenotypes, is difficult, as morphological characters buffer with MgCl2, 0.5 μL of dNTP (10 mM each), 0.5 used to differentiate species are limited, plastic and μL of each primer (10 μM), 0.25 μL of Taq DNA poly- vary between hosts and environments. In the pre- -1 merase (5 U μL ), and 1.0 μL of DNA template. The sent study, we surveyed diseases in vine growing re- thermal cycling programme followed that of Maha- gions in France constantly observing wood disease, rachchikumbura et al. (2012). and isolated the associated fungi. Based on an initial Three different datasets were used to estimate microscopic investigation of the conidia of isolates, three phylogenies: a Neopestalotiopsis tree, Pestaloti- the organisms were identified as pestalotioid fungi. opsis tree (ITS, TUB and TEF), and a Truncatella tree. We identified the pestalotioid fungi to species level The Neopestalotiopsis and Pestalotiopsis trees were based on ITS, TUB and TEF sequence data. Knowl- based on combined datasets, and the Truncatella tree edge about pestalotioid fungi associated with grape- was based on an ITS dataset. Sequences generated vines will help provide a basis for developing man- in this study (Table 1) were supplemented with ad- agement strategies of these pathogens. ditional sequences obtained from GenBank, based on blast searches and the literature. Multiple se- Materials and methods quence alignments were generated with MAFFT v. Sample collection, isolation and identification 7 (http://mafft.cbrc.jp/alignment/server/ index. html); the alignments were visually improved with Diseased grapevine samples characterized by Mesquite v. 2.75 (Maddison and Maddison, 2011) leaf stripes and defoliated shoots were obtained and MEGA v. 5.2.2 (Kumar et al., 2012) or BioEdit Vol. 55, No. 3, December, 2016 381 382 Table 1. Collection details and GenBank accession numbers of isolates includes in this study. S.S.N. Maharachchikumbura Culture accession No. Species Variety Location1 Symptom2 Collector3 ITS TUB TEF (MFLUCC) Phytopathologia Mediterranea Neopestalotiopsis asiatica 12-0573 Mourvèdre P (nursery) * L KX816909 KX816937 KX816878 N. asiatica 12-0575 Gamay R (vineyard) Leaf stripes L KX816901 KX816930 KX816871 N. asiatica 12-0585 Cabernet-Sauvignon A (vineyard) Leaf stripes L KX816923 KX816951
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