Eur J Plant Pathol https://doi.org/10.1007/s10658-018-1548-y Alternaria brassicicola – Brassicaceae pathosystem: insights into the infection process and resistance mechanisms under optimized artificial bio-assay Marzena Nowakowska & Małgorzata Wrzesińska & Piotr Kamiński & Wojciech Szczechura & Małgorzata Lichocka & Michał Tartanus & Elżbieta U. Kozik & Marcin Nowicki Accepted: 10 July 2018 # The Author(s) 2018 Abstract Heavy losses incited yearly by Alternaria that the plant genotype was crucial in determining its brassicicola on the vegetable Brassicaceae have response to the pathogen. The bio-assays for prompted our search for sources of genetic resistance A. brassicicola resistance were run under more stringent against the pathogen and the resultant disease, dark leaf lab conditions than the field tests (natural epidemics), spot. We optimized several parameters to test the perfor- resulting in identification of two interspecific hybrids mance of the plants under artificial inoculations with this that might be used in breeding programs. Based on the pathogen, including leaf age and position, inoculum results of the biochemical analyses, reactive oxygen concentration, and incubation temperature. Using these species and red-ox enzymes interplay has been suggested optimized conditions, we screened a collection of 38 to determine the outcome of the plant-A. brassicicola Brassicaceae cultigens with two methods (detached leaf interplay. Confocal microscopy analyses of the leaf sam- and seedlings). Our results show that either method can ples provided data on the pathogen mode of infection: be used for the A. brassicicola resistance breeding, and Direct epidermal infection or stomatal attack were relat- ed to plant resistance level against A. brassicicola among Electronic supplementary material The online version of this the cultigens tested. Further, the microscopic analyses article (https://doi.org/10.1007/s10658-018-1548-y) contains suggested rapid actin network activation of the host cells supplementary material, which is available to authorized users. around the papillas deposited under the pathogen M. Nowakowska : M. Wrzesińska : P. Kamiński (*) : appressorium. W. Szczechura : E. U. Kozik Department of Genetics, Breeding, and Biotechnology of Keywords Alternariaresistance .Bio-assays .Darkspot Vegetable Crops, Research Institute of Horticulture, Skierniewice, . Poland disease Resistance breeding Resistance mechanisms e-mail: [email protected] M. Lichocka Laboratory of Confocal and Fluorescence Microscopy, Institute of Introduction Biochemistry and Biophysics - Polish Academy of Sciences, Warszawa, Poland Cabbages and edible brassicas feed humans worldwide, M. Tartanus placing 5th in the global production as a major vegetable School of Informatics, State Higher Vocational School in crop (FAOSTAT data). Dark leaf spot of brassicas –also Skierniewice, Skierniewice, Poland referred to as black spot (Brazauskienė et al. 2011; Scholze and Ding 2005)orAlternaria blight (Kumar M. Nowicki (*) Department of Entomology and Plant Pathology, Institute of et al. 2014; Meena et al. 2004)– is caused by Alternaria Agriculture, University of Tennessee, Knoxville, TN, USA fungi (A. brassisicola [Schw.] Wiltsh, A. brassicae e-mail: [email protected] [Berk.] Sacc., A. raphani [Groves & Skolko], and Eur J Plant Pathol A. alternata [Fr.] Kreissler (Bock et al. 2002;Köhletal. breeding programs designed to develop pathogen- 2010; Kubota et al. 2006; Kumar et al. 2014)). The resistant cultivars, it is important to find sources of disease is the major bottleneck in the global production Alternaria resistance in the Brassica oleracea germ- of cultivated oilseed crops, Chinese cabbage, head cab- plasm, including white head cabbage or cauliflower. A bage, broccoli, cauliflower, and other important crops successful breeding program also relies on the methods from the Brassicaceae family. It leads to 15 to 70% for distinguishing the genetic differences in resistance losses, mainly by infection of seeds and seedlings, but early in the plant development. also of the edible produce (Kumar et al. 2014;Nowicki The main goal of our study was to search for sources et al. 2012b;Shresthaetal.2005). Production of the of A. brassicicola resistance in the Brassicaceae germ- vegetable brassicas is mainly affected by A. brassicicola plasm. To address this issue, we assessed techniques for and A. brassicae, whereas the oleiferous seed crops are rapid evaluation of A. brassicicola resistance in Brassi- mainly affected by A. brassicae (Kumar et al. 2014; ca oleraceaea under controlled conditions. This study Michereff et al. 2012; Nowicki et al. 2012b). The dark aimed to compare the detached leaf and seedlings bio- leaf spot disease is particularly common in tropical or assays regarding important variables such as age of leaf, subtropical regions, but also threatens the Brassicaceae leaf position, inoculum concentration, and incubation production in areas with high humidity and frequent temperature. Both testing methods were also compared rainfall (Humpherson-Jones and Phelps 1989). In Po- with the results of the field assessment of Alternaria land, this disease mainly impacts the mid- and late- resistance across a broad collection of germplasm. season cultivars of head cabbage, grown for storage or Moreover, confocal investigations were employed on sauerkraut processing (reviewed by Nowicki et al. the double-stained samples of inoculated leaves, to gain 2012b). Poland ranks 5th or 6th in global production insights into the subcellular processes accompanying of these crops (FAOSTAT), and thus, the yearly threats the A. brassicicola infection of plants differing in sus- of Alternaria spp. (Kasprzyk et al. 2013) causing yield ceptibility. Analyses of several biochemical markers in losses and necessitating heavy protective fungicide us- the course of infection helped conclude on the resistance age (Nowicki et al. 2012b) are economically important mechanisms in the plants of contrasting reactions to the issues. infection. Both main pathogens, A. brassicicola and A. brassicae, infect host plants at all developmental stages. Typical disease symptoms – dark brown spots/ Materials and methods lesions with characteristic concentric circumferences, often with a yellowish chlorotic halo – appear on leaves, Plant and pathogen material stems, and siliques. Under conducive conditions, the colored spots develop a layer of brown-black conidial Brassicaceae germplasm used in this study included spores. The enlarging necrotic lesions drastically reduce cultivars, breeding lines, interspecific hybrids, land- the photosynthetic efficiency, hasten the plant senes- races, and wild accessions from the germplasm collec- cence, and lead to collapse and death of plants under tion at the Research Institute of Horticulture (InHort; high pathogen pressure. Such symptoms generate sever- Skierniewice, Poland), and Institute of Natural Fibres al sources of Brassicaceae crops losses caused by black and Medicinal Plants (Sinapis alba and Camelina sativa spot: Damping-off of seedlings, spotting of leaves of cv Omega), collectively referred to as cultigens cabbages, blackleg of heads of cabbages (head cabbage (Table 1). Ten days after sowing, plants were and Chinese), and spotting/browning of cauliflower transplanted into ∅ 10-cm plastic pots containing a peat curds and broccoli florets (reviewed by Kumar et al. substrate Kronen-Klasmann. Seedlings were grown on 2014; Nowicki et al. 2012b). benches in a greenhouse at 21/16 °C (day/night) and In order to augment the integrated pathogen control, 10 h of light, fertilized and watered in accordance with resistant Brassicaceae crops are needed. Although sig- the accepted practice for the species. nificant efforts have been contributed, to date no Three A. brassicicola isolates (X2038, X2039, Alternaria-resistant cultivars exist, and the resistant wild X2040) used in this study were obtained from Geves – Brassicaceae plants do not cross well with the domesti- Snes National Seed Testing Station, France. Thirteen cated ones (Hansen and Earle 1997). For planning of the isolates were collected in recent years from Brassicaceae Eur J Plant Pathol crops symptomatic for dark leaf spot, grown in various concentration, then all six diluted isolate suspensions regions of Poland (2011 to 2014). Pathogen identity was were mixed in equal volumes, and the final inoculum confirmed by microscopic observations of morphologi- mix was recounted to ensure the appropriate conidial cal characteristics. Stock cultures of A. brassicicola concentration. were maintained on standard Potato Dextrose Agar (PDA) media at 4 °C. For the preparation of inoculum, each isolate was incubated on PDA at 24 ± 1 °C in the Experiments conditions dark. Conidia of 10-day old cultures were washed off the plate with sterile distilled water and filtered through Bio-assay optimization two layers of cheesecloth to remove the remaining my- celium. The conidial suspension was then shaken and Experiments were conducted under controlled environ- supplemented with 0.1% (w:v) agar solution. The inoc- ment conditions in the growth chambers of InHort. ulum concentration was determined by three-time mea- These assays included detached leaf and seedlings bio- surements with a hemocytometer using a stereoscopic assays, described below. We evaluated the influence of microscope Nikon Eclipse E200, and final inoculum several variables on the disease severity in cultigens concentrations were adjusted according to