Alternaria Black Spot of Crucifers: Symptoms, Importance of Disease, and Perspectives of Resistance Breeding: Alternaria black spot of cruciferous vegetables, incited by different species of Alternaria , remains an increasing threat to Brassicaceae crops throughout the world, including Poland. Brassica plants are attacked by conidia of A. brassicae (Berk.) Sacc., A. brassicicola (Schw.) Wiltsh., A. raphani Groves & Skolko, and A. alternata (Fr.) Kreissler. The pathogens have a wide spectrum of hosts, such as head cabbage, Chinese cabbage, cauliflower, broccoli, and other crucifers including cultivated and wild grown plants. Alternaria pathogens usually cause damping-off of seedlings, spotting of leaves of cabbages, blackleg of heads of cabbages, and spotting of cauliflower curds and broccoli florets. In oilseed rape, A. brassicae is the dominant invasive species, while in the cruciferous vegetables, both species, A. brassicae , and A. brassicicola are encountered. Infected seeds with spores on the seed coat or mycelium under the seed coat are the main means of distribution for these pathogens. The fungus can overwinter on susceptible weeds or crop debris and on seed plants, as well as on stecklings. Methods for disease prevention and control are based on combining agricultural management practices with chemical control. Using disease-free seeds or seeds treated with fungicides can greatly reduce disease incidence. After appearance of the first symptoms of disease, stringent fungicide spray program is an effective way to reduce losses. Many authors seem to agree, that the most economically feasible method of disease control is the development of resistant Brassicaceae crops varieties, as transgenic approach proved unsuccessful. Due to our increasing understanding of pathogen-host plant interactions, identification of resistance sources, and assessment of the resistance trait inheritance mode, breeding programs of Brassica crops for Alternaria resistance can be enhanced. This is of particular importance since recent years experience dynamic development of ecological and integrated plant production with an emphasis on plant biotic stress resistance. Highly resistant genetic resources have not been reported in Brassica cultivated species, although some varieties differ in their resistance/susceptibility level. Strong cross-incompatibility, polygenic background of the resistance (additive and dominant gene interactions), as well as the differences in ploidy between the Brassica species of interest, render the transfer of Alternaria resistance from the wild species into the cultivated forms difficult. Additionally, it is often connected with employment of in vitro hybridization techniques, including somatic hybridization, embryo and ovary rescue, or protoplast fusion.

Heavy losses incited yearly by Alternaria brassicicola on the vegetable Brassicaceae have prompted our search for sources of genetic resistance against the pathogen and the resultant disease, dark leaf spot. We optimized several parameters to test the performance of the plants under artificial inoculations with this pathogen, including leaf age and position, inoculum concentration, and incubation temperature. Using these optimized conditions, we screened a collection of 38 Brassicaceae cultigens with two methods (detached leaf and seedlings). Our results show that either method can be used for the A. brassicicola resistance breeding, and that the plant genotype was crucial in determining its response to the pathogen. The bio-assays for A. brassicicola resistance were run under more stringent lab conditions than the field tests (natural epidemics), resulting in identification of two interspecific hybrids that might be used in breeding programs. Based on the results of the biochemical analyses, reactive oxygen species and red-ox enzymes interplay has been suggested to determine the outcome of the plant-A. brassicicola interplay. Confocal microscopy analyses of the leaf samples provided data on the pathogen mode of infection: Direct epidermal infection or stomatal attack were related to plant resistance level against A. brassicicola among the cultigens tested. Further, the microscopic analyses suggested rapid actin network activation of the host cells around the papillas deposited under the pathogen appressorium.

Summary: Dramatic increase in confocal microscopy observation output has been gained by optimization of a simple trypan blue and aniline blue dual-stain and its application to two model pathosystems: Pseudoperonospora cubensiscucumber and Phytophthora infestans-tomato. Comparison of two dual-stain methods for confocal microscopy studies of P. cubensis-challenged cucumber leaves indicated the 'mild' approach most successful. This methodology provides simultaneous detection of different pathogen structures layered with the plant defense reactions. Moreover, ImageJ-assisted quantification of plant defense responses renders this method useful for addressing the host plant resistance reactions, as well as investigating the given isolate's pathogenicity. Application of this method for the P. infestans-challenged tomato leaf samples resulted in detection of several fungal infection structures, along with plant defense responses. The dual-stain also enabled detection of a peculiar aniline blue-sensitive material in the pathogen cell walls at the area of its hyphae emerging through the leaf stomata. Results presented herein indicate this method is applicable for detailed (possibly quantitative) investigations of multiple plant-fungal pathosystems.

Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part of the recovery plan for the restoration and protection of H. verticillatus, an efficient micropropagation protocol based on axillary shoot proliferation was developed. Various concentrations of 6-benzylaminopurine (BAP; 0 to 4.44 µM) were examined for their morphogenetic potential in the regeneration of six genotypes of H. verticillatus from the nodal explants derived from greenhouse-grown plants. Both the BAP concentration and genotype had significant effects on the regeneration capacity of H. verticillatus. Although the induced buds were observed on ½-strength Murashige and Skoog medium without plant growth regulators, a higher rate of induction and bud development were achieved on media with either 0.88 or 2.22 µM BAP, regardless of the genotype. Successful rooting of the induced shoots was achieved within four weeks after the transfer from the induction medium to the fresh ½-strength MS medium, but the rooting efficiency was dependent on the plant’s genetic background. Regenerated plantlets, with well-developed shoots and roots, were acclimatized successfully to greenhouse conditions with a 97% survival rate. Simple sequence repeats (SSRs) markers were employed to assess the genetic uniformity of the micropropagated plants of H. verticillatus. No extraneous bands were detected between regenerants and their respective donor plants, confirming the genetic fidelity and stability of regenerated plants. To our knowledge, the protocol developed in this study is the first such report for this endangered species.

Late blight (LB) caused by the oomycete Phytophthora infestans continues to thwart global tomato production, while only few resistant cultivars have been introduced locally. In order to gain from the released tomato germplasm with LB resistance, we compared the 5-year field performance of LB resistance in several tomato cultigens, with the results of controlled conditions testing (i.e., detached leaflet/leaf, whole plant). In case of these artificial screening techniques, the effects of plant age and inoculum concentration were additionally considered. In the field trials, LA 1033, L 3707, L 3708 displayed the highest LB resistance, and could be used for cultivar development under Polish conditions. Of the three methods using controlled conditions, the detached leaf and the whole plant tests had the highest correlation with thefield experiments. The plant age effect on LB resistance in tomato reported here, irrespective of the cultigen tested or inoculum concentration used, makes it important to standardize the test parameters when screening for resistance. Our results help show why other reports disagree on LB resistance in tomato.