Plant Pathology (2018) 67, 1088–1096 Doi: 10.1111/ppa.12828 Infection cycle of Alternaria brassicicola on Brassica oleracea leaves under growth room conditions V. K. Macioszeka, C. B. Lawrenceb and A. K. Kononowicza* aDepartment of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, 90-237 Lodz, Poland; and bDepartment of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA Development of the necrotrophic fungus Alternaria brassicicola was evaluated during infection of three cabbage vari- eties: Brassica oleracea var. capitata f. alba ‘Stone Head’ (white cabbage), B. oleracea var. capitata f. rubra ‘Langedi- jker Dauer’ (red cabbage) and B. oleracea var. capitata f. sabauda ‘Langedijker Dauerwirsing’ (Savoy cabbage). Following inoculation of cabbage leaves, conidial germination, germ tube growth, and appressorium formation were analysed during the first 24 h of infection. Differences in the dynamics of fungal development on leaves were observed, e.g. approximately 40% of conidia germinated on Savoy cabbage leaves at 4 h post-inoculation (hpi) while only 20% germinated on red and white cabbage leaves. Leaf penetration on the three cabbage varieties mainly occurred through appressoria, rarely through stomata. Formation of infection cushions was found exclusively on red cabbage. Appresso- ria were first observed on red cabbage leaves at 6 hpi, and on white and Savoy cabbage leaves at 8 hpi. Conidiogenesis occurred directly from mature conidia at an early stage of fungal development (10 hpi), but later (48 hpi) it occurred through conidiophores. Disease progress and changes in the morphology of leaf surfaces were also observed. At the final 120 hpi measurement point, necroses on all investigated varieties were approximately the same size. Based on detailed light and electron microscopic analyses, an outline of the A. brassicicola infection cycle on B. oleracea leaves under growth room conditions has been proposed. According to the authors’ knowledge, this report represents the most detailed up-to-date description of infection of B. oleracea varieties by A. brassicicola. Keywords: Alternaria brassicicola, black spot disease, infection stages, susceptibility Introduction plant remains or necrotrophic on a living host. Although mating genes have been found in the A. brassicicola gen- The plant pathogenic fungus Alternaria brassicicola ome, there is no known sexual stage of this fungus (Bock causes one of the most destructive leaf and stem spot dis- et al., 2005; Linde et al., 2010; Dang et al., 2015). eases in cultivated Brassica species worldwide. Spreading Most of the necrotrophic phytopathogenic fungi such necrotic lesions lead to the death of infected seedlings or as A. alternata, A. brassicae, A. brassicicola, Phoma lin- foliar tissues of mature plants, consequently leading to gam, Sclerotinia spp. or Botrytis cinerea share similarities the host plant’s decay (Thomma, 2003; Lawrence et al., during infection; however, their life cycle, host range and 2008; Nowicki et al., 2012). Although A. brassicicola is environmental conditions necessary for successful infec- widely used as a model pathogen on Arabidopsis thali- tion constitute the difference in the behaviour of fungal ana for research into plant disease resistance, early stages species during host colonization. Generally, fungal infec- of A. brassicicola development during infection of tion cycles on host plants have been divided into three susceptible hosts have yet to be studied in great detail major stages: (i) pre-penetration (adhesion and germina- (Łaźniewska et al., 2010). It has to be emphasized that tion), (ii) penetration, and (iii) colonization stage (Guest most cultivars of cultivated brassicas show varying & Brown, 1997). It is known that A. brassicicola conidia degrees of susceptibility to A. brassicicola, with develop- release several cell wall-degrading enzymes during con- ment of necrosis observed within 48 h of infection under tact with a compatible host, which enable their adhesion growth room conditions, depending on temperature and to the host surface and initial tissue destruction (Cho humidity as well as conidial concentration (Rotem, et al., 2007). A microscopic study of A. brassicicola 1994). conidial germination in vitro revealed that initiation of The life cycle of A. brassicicola is that of a typical germ tube formation started after approximately 3 h via necrotrophic pathogen: it can be saprophytic on dead mechanical breaking of the outer layer of the conidial cell wall (Campbell, 1969). Moreover, the rate of coni- dial germination and appressorium formation on differ- *E-mail: [email protected] ent species of Brassica leaves was also investigated to a limited degree (McRoberts & Lennard, 1996). Alternaria brassicicola mainly penetrates host tissues through Published online 30 January 2018 1088 ª 2018 British Society for Plant Pathology A. brassicicola infection cycle 1089 appressoria, rarely through stomata (Dixon, 1981). Pene- To analyse the distribution of hyphae in plant tissue, tration by a necrotroph such as A. brassicicola has been infected Brassica leaves were paraffin-embedded at 24, 48 and strictly correlated with production of phytotoxins (Otani 72 hpi. Sections were cut and dehydrated in 92% ethanol et al., 1998; Oka et al., 2005) and subsequent killing of before immersing in Pianese’s stain (naphthol yellow 0.01 g, host cells (Cho, 2015). Necrotic lesions observed on malachite green 0.5 g, acid fuchsin 0.5 g in 150 mL distilled water and 50 mL of 92% ethanol) for 45 min. They were sub- leaves and stems as a consequence of A. brassicicola – sequently rinsed in 92% ethanol, differentiated in a mixture of infection were found to measure 0.5 2.5 cm in diameter 99 mL of 92% ethanol and 1 mL of acetic acid for 1 min, and were often surrounded by a chlorotic ring (Dixon, dehydrated in isopropanol, and then mounted in Histopaque 1981). (Difco). Sections were examined under a light microscope Here, details of the infection stages of A. brassicicola Olympus BX-60. All images were subjected to alterations to a and their impact on host tissue colonization during infec- greyscale using PHOTOSHOP v. 6.0 (Adobe) or CORELDRAW X4 tion of three B. oleracea varieties under growth room (Corel) software. conditions are presented. Table-top electron microscopy Materials and methods To investigate stages of A. brassicicola development on leaf sur- faces, 1.5 cm diameter leaf discs were cut from the second Plant material, fungal strain and inoculation mature leaves, which had been sprayed with the conidial suspen- sion. Discs were examined without any pretreatment at 15 kV In all experiments, three varieties of Brassica oleracea were with a table-top electron microscope TM-1000 (Hitachi), used: B. oleracea var. capitata f. alba ‘Stone Head’ (white cab- according to the manufacturer’s protocol. Samples were har- bage), Brassica oleracea var. capitata f. rubra ‘Langedijker vested from three inoculated plants per time point at 12, 24, 48 Dauer’ (red cabbage) and B. oleracea var. capitata f. sabauda and 72 hpi. ‘Langedijker Dauerwirsing’ (Savoy cabbage). Seeds were germi- The figures were composed using PHOTOSHOP v. 6.0 or COREL- nated under in vitro conditions on 1/29 MS solid medium DRAW X4 software. (Sigma-Aldrich) with the addition of 1% sucrose. After 7– 10 days, seedlings were transferred to soil containing 1/15 by volume of perlite and grown under controlled conditions: 16 h Disease development day/8 h night photoperiod under fluorescent light (Super TLD À À Philips 865) at a minimum of 100 lmol m 2 s 1,at22Æ 2 °C Disease progress on plants’ second leaves was determined and relative humidity of approximately 70%. Plants with four through measurement of spreading necrosis with a calliper every mature leaves were used for infection. 24 h for 5 days. Six plants were used per treatment and the The wildtype strain of A. brassicicola (ATCC 96836), with experiment was repeated three times. Statistical analysis was sequenced genome (Genome Sequencing Center at Washington performed with one-way ANOVA and Duncan’s test using University School of Medicine, USA, http://genome.jgi.doe.gov/ STATISTICA v. 12.5. Altbr1/Altbr1.home.html, http://alternaria.vbi.vt.edu/index.html), was cultured on potato dextrose agar (PDA; Difco) plates Results and grown in the dark at 22 Æ 2 °C for 7–10 days. Conidia were harvested by shaking Petri dishes flooded with sterile distilled water and diluted to a final concentration of Quantitative fungal development 9 5 À1 5 10 conidia mL . The second leaf of each plant was inoc- At 4 hpi, the largest number of germinating conidia ulated with one or two 10 lL drops of conidial suspension (38.33 Æ 3.18) was observed on leaves of Savoy cabbage between the second and fourth subsidiary leaf vein, and the ″ plants were incubated in plastic translucent boxes to maintain (Figs 1c & S1a ). At subsequent time points, a similar high humidity under the same light and temperature conditions level of conidial germination on Savoy cabbage was for plant growth described above. observed with significant increases at 16 (77.0 Æ 4.7) and 20 hpi (91.33 Æ 4.18). The number of germinating conidia on Savoy cabbage at 24 hpi (92.67 Æ 4.33) was Light microscopy similar to that observed at 20 hpi (Figs 1c & S1). Infected leaves of three plants were stained with aniline blue The number of germinating conidia on white and red (0.05%, VWR International)-lactophenol and conidial germina- cabbage leaves at 4 hpi (27.67 Æ 2.60 and 22.33 Æ 7.54, tion was analysed at 2, 4, 6, 8, 10, 12, 16, 20, 24 and 48 hpi. respectively) was significantly lower than on Savoy cab- For each leaf, the number of germinating conidia, germ tubes bage (Figs 1a,b & S1a,a0). However, their number on and appressorium formation per 100 conidia was determined. white cabbage leaves increased at 6 hpi (45.67 Æ 0.88) Germ tubes were counted when their length was at least half the and remained at the same level until 12 hpi (50.33 Æ 0.67; length of the conidium.