agronomy Article In Vitro Pathogenesis Caused by Phytophthora cactorum and DNA Analysis of the Strawberry-Resistant Microplants with ISSR Markers Wojciech Marecki * and Jadwiga Zebrowska˙ Department of Genetics and Horticultural Plant Breeding, Institute of Plant Genetics, Breeding and Biotechnology, Faculty of Agrobioengineering, University of Life Sciences in Lublin, Akademicka 15 Street, 20-950 Lublin, Poland; [email protected] * Correspondence: [email protected] Abstract: The soil pathogenic fungus Phytophthora cactorum causes the most dangerous diseases occurring in strawberry plantations—strawberry crown rot and leather rot. Modern biotechnology methods, e.g., in vitro culture selection and molecular diagnostics can be utilized in the selection of cultivars that are less susceptible or resistant to Phytophthora diseases. In this study, in vitro selection of four strawberry microclones: ‘Elsanta’, ‘Feltar’, ‘Teresa’ and ‘Plena SVdT’ against Phytophthora cactorum (Lebert and Cohn) J. Schröt was carried out. Molecular analysis with inter simple sequence repeat (ISSR) markers was also used to evaluate genetic similarity of the selected resistant plants. None of the analyzed microclones showed complete resistance to the selection factor, but there were plants in all tested microclones that survived the pressure of the pathogen. Results showed that susceptibility to this pathogenic fungus was significantly differentiated and depended on the microclone. The ‘Feltar’ microclone had the significantly lowest susceptibility to Phytophthora disease, followed by the microclones ‘Elsanta’ and ‘Teresa’ with significantly higher susceptibility. The ‘Plena SVdT’ microclone showed the highest susceptibility to Phytophthora disease. This differentiation was linked to the genetic similarity observed at deoxyribonucleic acid (DNA) level between the Citation: Marecki, W.; Zebrowska,˙ J. resistant plants selected from microclones. Cluster analysis revealed that microclones with similar In Vitro Pathogenesis Caused by Phytophthora cactorum and DNA susceptibility to phytophthorosis, i.e., ‘Elsanta’, ‘Feltar’ and ‘Teresa’, appeared to be genetically Analysis of the Strawberry-Resistant similar. The microclone ‘Plena SVdT’ revealed a different course of phytophthorosis from the Microplants with ISSR Markers. aforementioned microclones, being the least genetically similar to them. Agronomy 2021, 11, 1279. https:// doi.org/10.3390/agronomy11071279 Keywords: Fragaria × ananassa; micropropagation; phytophthorosis; susceptibility; genetic similarity; selection Received: 25 May 2021 Accepted: 22 June 2021 Published: 24 June 2021 1. Introduction Publisher’s Note: MDPI stays neutral Breeding for conferring resistance is highly important in modern agriculture. The with regard to jurisdictional claims in cultivation of cultivars that are less susceptible or even resistant to pathogens allows the published maps and institutional affil- restriction of fungicides that are harmful to humans and the environment. Production iations. costs are thus reduced, and farmers obtain higher and more uniform yields of high-quality fruits [1]. The use of modern biotechnological methods, such as tissue cultures or molecular diagnostics can significantly accelerate the progress of breeding compared to conventional Copyright: © 2021 by the authors. selection. Currently, one of many useful techniques in modern plant breeding is in vitro Licensee MDPI, Basel, Switzerland. culture selection. Such selection is aimed at obtaining plants with new, valuable traits This article is an open access article that would also be present in plants transferred to field conditions, and these traits would distributed under the terms and be inherited by the next generations. The tested plants can be selected for both biotic conditions of the Creative Commons (resistance to fungal pathogens) and abiotic factors (drought, soil salinity, heavy metals, Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ low temperature). In vitro culture selection can only be carried out on traits expressed 4.0/). at the cell, callus or seedling level [2–4]. The plants obtained in this method can be Agronomy 2021, 11, 1279. https://doi.org/10.3390/agronomy11071279 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 1279 2 of 12 used for further breeding studies as a source of resistance genes. Sowik et al. [5] and Zebrowska˙ [6,7] investigated the resistance of strawberry cultivars and clones via the selection of the pathogenic fungus Verticillium dahliae in an in vitro culture. Zebrowska˙ [6] reported that the susceptibility of the tested strawberry cultivars in vitro was similar to the susceptibility of these cultivars under field conditions and that in vitro culture selection could be successfully applied in breeding programs. Two of the most dangerous diseases occurring in strawberry plantations are strawberry crown rot and leather rot [8]. Both of these diseases are caused by the fungus of the genus Phytophthora and more precisely by its specialized form—Phytophthora cactorum. Phytophthora cactorum is a common polyphagic organism in soils of the temperate climate zone. It causes diseases in 200 species of crops from 60 families [9]. This fungus mainly affects ornamental plants (such as rhododendron, pansy, pelargonium or begonia), fruit shrubs and trees (gooseberry, currant, peach, cherry, raspberry, apple tree and cherry) and forest trees (birch, black alder, Scots pine, common beech, common ash and spruce) causing a disease called phytophthorosis [8,10–14]. The source of P. cactorum on plantations may be the remains of infected plants left over from previous plantings, water drawn for irrigation from nearby ponds or rivers, irrigation systems, contaminated soil or infected seedlings [14]. Thick-walled spores of P. cactorum are called oospores and can survive in the soil for up to 6 years [14]. The first symptoms of strawberry crown rot are observed on the above-ground plant parts. Wilting of the youngest leaves in individual plants, which often have a blue-green color, is a characteristic symptom of the disease. The plant wilts and dies as a result of root infection. When removing infected strawberries from the ground, the upper part of the crown is often torn off, and the rest remains in the soil [15]. The newly attacked tissue is watery and light brown, and it turns an intensely brown-red color with disease development [15]. Tissue disintegration and necrosis are also observed. A day shorter than 13 h light per day is conducive to the disease. Plants are infected in nurseries (cryptic infection) and on production plantations [16]. Leather rot mainly occurs in years with high rainfall before and during harvest. Zoospores germinating on the surface of the fruit in the presence of water are responsible for the infection [15]. Fruits are infected at all stages of development, from fruit set to immature and fully developed ripe fruits. Light brown, later brown, dry, rotting spots develop on immature fruits that may cover the whole fruit. Light, grayish-yellow to pink- violet, discolored spots appear on ripening and ripe fruits [17]. The flesh of such fruits is firm, stringy, light brown in color with darker vascular bundles. Infected fruits are slightly softer than healthy ones, with a decidedly unpleasant smell and clearly bitter taste [18,19]. Ellis et al. [20] reported that the average losses due to this disease on strawberry plantations in the USA are 20–30%, and during a particularly severe epidemic in 1981 in Ohio, losses reached 50%. According to Bielenin [21], losses in strawberry seedlings caused by crown rot, originating from an infected plantation, reached 40% in 1994, while in the years 1995–1997, fruit losses ranged from 20% to 80% as a result of leather rot on the cultivars ‘Elsanta’, ‘Senga Sengana’ and ‘Sirius’. Selection in order to obtain new strawberry cultivars resistant or tolerant to Phytoph- thora cactorum is currently one of the most important directions of breeding this species in Poland and in the world. So far, research on the susceptibility of strawberry cultivars to P. cactorum has been conducted mainly in field conditions. There are few reports on the use of biotechnological methods for resistance breeding, i.e., tissue cultures in conjunction with the molecular analysis of selected resistant forms. Considering this breeding direction, the present study applied in vitro culture selection to assess the susceptibility of strawberry microclones to infection by the pathogen of the genus Phytophthora spp. in conjunction with molecular analysis of selected resistant forms. Agronomy 2021, 11, 1279 3 of 12 2. Materials and Methods The starting material for in vitro selection against Phytophthora cactorum (Lebert and Cohn) J. Schröt was obtained by in vitro propagating the ends of runners taken from the donor plants of strawberry cultivars: ‘Elsanta’, ‘Feltar’, ‘Plena’ and ‘Teresa’ derived from the collection of the Department of Genetics and Horticultural Plant Breeding of the University of Life Sciences in Lublin. The Dutch cultivar ‘Elsanta’ [‘Gorella’ × ‘Holiday’] is very susceptible to root system diseases (caused by Verticillium sp.). However, the susceptibility to soil pathogens, such as Phytophthora spp. of Polish cultivars ‘Feltar’ [(‘Senga Tigaiga’ × ‘Merton Dawn’) S1], ‘Plena’ [‘Senga Sengana’ × ‘Merton Dawn’] and ‘Teresa’ [‘Redgauntlet S1’ × ‘Senga Sengana S1’] selected at the University of Life Sciences in Lublin is not yet known.