Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp Histopathological evidence of concomitant sexual and asexual reproduction of Elsinoë ampelina in grapevine under subtropical climate Zélia Valente Braga a,1 , Ricardo Feliciano dos Santos b,1 , Lilian Amorim b , Beatriz Appezzato-da-Glória a,* a Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Universidade de São Paulo, 13418-900, Piracicaba, Brazil b Departamento de Fitopatologia e Nematologia, Escola Superior de Agricultura ‘Luiz de Queiroz’, Universidade de São Paulo, 13418-900, Piracicaba, Brazil ARTICLE INFO Keywords: Ascospore Histology Sexual structures Sphaceloma ampelinum Vitis labrusca ABSTRACT Anthracnose caused by Elsinoë ampelina is one of the most serious diseases affecting grapevine production worldwide. Previous studies have described many structural and histochemical changes in grapevine leaves associated with E. ampelina colonization and the presence of ascus-like structures in the lesions. In this paper, we characterized the colonization and reproduction of E. ampelina in grapevine tendrils, stems and berries using light, epifluorescence and scanning electron microscopy. In the early stages of colonization, shape changes and the collapse of epidermal and parenchyma cells were more pronounced in the center of lesions. At this stage, pectic substances accumulated inside the parenchyma cells and hyphae were observed colonizing tissues inter- and intracellularly, including tracheary elements. As colonization evolved, lesions enlarged and infected tissues became necrotic and collapsed. Tyloses and conidiogenous cells producing conidia were observed in the tracheary elements. Conidiogenous cells and conidia were also present on the lesion surface. On the same le- sions, bitunicate asci containing two-to three-celled ascospores were observed on the surface and throughout the stromatic tissue. Our analyses showed that both sexual and asexual stages of E. ampelina occur from the be- ginning of the epidemic, which may help to explain the explosive nature of the disease when early stages of grapevine development coincide with rainy seasons. 1. Introduction Anthracnose is an important fungal disease in both table and wine grape cultivars across many countries, especially during rainy seasons [1–5]. Although grapevine anthracnose is reported to be caused by Elsinoë ampelina and different Colletotrichum species in Asia [3,6,7], in Brazil, the disease is caused by E. ampelina [8]. During rainy seasons, E. ampelina can lead to significant economic losses by causing reductions in yield and fruit marketability in vineyards where the disease is well established [9,10]. E. ampelina infects young tissues, including berries, leaves, rachises, stems and tendrils. The first symptoms are characterized by isolated small reddish circular spots that enlarge up to 7 mm in length and become brownish sunken lesions with grey to dark centers [9,11]. As the disease progresses, coalescence of lesions is commonly observed under favorable conditions [8]. Humidity and temperature are the en- vironmental factors that most influence the infection of E. ampelina [12]. Minimum wetness periods necessary for infection range from 7 to 10 h at 12 °C and from 3 to 4 h at 21 °C [13], and the incubation period is 3 days on leaves and 5–6 days on stems and tendrils at 25 °C [5]. Abundant conidia are produced on lesions when they are wet from rain or dew for 24 h or more [9]. Conidia are dispersed by rain splash and anthracnose outbreaks have been reported in years with high rainfall [9]. Grapevine anthracnose was reported for the first time in São Paulo State in 1889 (Puttemans, 1932) and remained for a century as the most important grapevine disease in Southeast Brazil [14]. Nowadays, al- though downy mildew (Plasmopara viticola) is considered the main disease of the crop, anthracnose shows high severity when early stages of plant development coincide with prolonged periods of rain and high relative humidity. Despite the importance of grapevine anthracnose in Brazil, the disease cycle has never been completely clarified, particu- larly the presence and role of sexual reproduction in E. ampelina. The sexual stage of E. ampelina does not occur as regularly as the asexual stage and has not been found in several countries including Australia, Brazil, India and South Africa [10,15–17]. In the United States, where https://doi.org/10.1016/j.pmpp.2020.101517 Received 20 March 2020; Received in revised form 27 May 2020; Accepted 12 June 2020 * Corresponding author. E-mail address: bagloria@usp.br (B. Appezzato-da-Glória). 1 Authors contributed equally to this work. Physiological and Molecular Plant Pathology 111 (2020) 101517 Available online 02 July 2020 0885-5765/ © 2020 Elsevier Ltd. All rights reserved. T