1. IN VITRO INFLUENCE OF FOLIAR FERTILIZERS ON ALTERNARIA DISEASE IN TOMATO Mali Sanda MANOLE 1, Beatrice Michaela IACOMI 1 1 University of Agricultural Sciences and Veterinary Medicine, Plant Protection Dept., Bucharest, Romania e-mail: malismanole@yahoo.com Early blight (also known as Alternaria leaf spot) is one of the most common disease in tomato worldwide. A complex of species is responsible for important yield losses: Alternaria porri f.sp. solani, A. alternata f.sp. lycopersici and A. tenuissima. These pathogens exhibit a necrotrophic lifestyle. Early blight can infect plants at any stage during the growing season. Apart from the leaf symptoms the pathogens can cause collar rot (basal stem lesions at the seedling stage), stem lesions on the adult plant and fruit rot. Epidemics can cause severe tomato crop defoliation in areas with high humidity and frequent nightly dew. Under field conditions, EB infection of tomato plants usually occurs after flowering; necrotic lesions may also appear in lower floral parts and fruits, both green and ripe. Control measures include resistant cultivars, a 3 to 5 year crop rotation, routine applications of fungicides. It is well known that the nutritional status of plants influences their response to pathogens. Accordingly, adjustment of fertilization rates and the composition of the nutrient solution are occasionally used as means for disease suppression. The interaction of fungal disease and fertilizers is of great practical interest and there have been studies into the interaction of (in particular) nitrogenous fertilizer and disease. More work is needed to establishe if is any direct effect of fertilizers on Alternaria pathogens in tomato. Our study investigated the effects of different tomato ferilizers on mycelial growth and sporulation of Alternaria species pathogenic in tomato (in vitro assay) as well as their influence on saprophytic microflora. Although variable responses towards each compound was observed, the results obtained showed an antifungal effect of some fertilizers, at different developmental stages of pathogens (inhibition of mycelial growth or sporulation). Megafol Miracolo verde Kendal Control Miracolo verde Kendal CropmaxControl Kendal Tomato Stim 2 Megafol Tomato Stim 3 Control Kelpak MATERIALS AND METHODS RESULTS REFERENCES CONCLUSIONS The possible fungitoxic activity of several fertilizers and plant growth stimulators against A. solani, A. lycopersici, A. tenuissima, Trichoderma viride and Trichothecium roseum was examined in vitro to identify whether or not they affect the pathogens or antagonists directly. The compounds tested were: Atonik (0.05%), Biofert Forte (0.5%), Cropmax (0.1%), Kelpak (0.2 and 1%), Kendal (0.2%), Megafol (0.3%), Miracolo Verde (0.5%) and Tomato-Stim (2 and 3%). Alternaria isolates were collected from tomatoeas leaves or fruits. T. viride and T. roseum were isolated from the tomatoes plylloplane. The effect on mycelial growth was tested by removing a 8mm diameter disk from the margins of a 7-day-old culture. Plugs were placed in the center of a 9 cm Petri dish containing PDA, amended with the tested compounds at recomanded concentrations to their application. Inoculated dishes were incubated at 240C, and the diameter of mycelial fungi growth was recorded after 7 days. Inhibition of mycelial growth was calculated. The effect of fertilizers was determined on spore germination also. Conidial suspensions (60µl) calibrated at 10 4 spores/ml was spread on 2% water agar. Germination was quantified after 18 hours at three sites by counting 100 conidia per site. A spore was considered germinated if the length of the germ tube was at least as long as the width of the spore. The inhibition of spore germination was calculated. Three replicates were made for each compound and concentration. The in vitro experiments were repeated twice. Kendal Megafol Tomato Stim 2% Tomato Stim 3% Control Kelpak Fig. 1 Effect of fertilizers on mycelial growth of Alternaria solani. FertilizerConcentration (%) inhibition of spores germination (%) A. solaniA. lycopersiciA. tenuissima Atonik0.05000 Biofert forte0,05028,50 Cropmax0.2028.50 Kelpak0,2000 1000 Kendal0.259.8100 Megafol0.369.230.60 Miracolo Verde0.303.122.5 Tomato Stim222.58.210.8 325.518.415.2 Fig. 2 Effect of fertilizers on mycelial growth of Alternaria lycopersici Fig. 5 Effect of fertilizers on mycelial growth of Trichoderma viride The interaction of fungal disease and fertilizers is of great practical interest and there have been studies into the interaction of (in particular) nitrogenous fertilizer and Alternaria or Botrytis diseases (Rotem, 1994; Blachinski et al., 1996; Dik and Wuben, 2004; Yermiyahu et al., 2006). More work is needed in this area, different fungi will respond in different ways to the application of fertilizers. In the present study the direct antifungal activities of different foliar fertilizers was assayed. Our in vitro experiments revealed variable effects at different development stages of pathogens’s life cycle (mycelial growth and spore germination). The most sensitive pathogen was A. lycopersici. The influence of foliar application of nutrients will be evaluated in vivo. Future studies will combine measurements of fungal growth and symptom production with plant biomass and in planta nutrient levels. The use of fungal strains carrying reporter gene constructs linked to nutrient-responsive promoters (Bowyer et al., 2000) is a major technical advance that is likely to elucidate this problem. Control Cropmax Tomato Stim 2 MegafolKendal Fig. 3 Effect of fertilizers on mycelial growth of Alternaria tenuissima. Fig. 6 Effect of fertilizers on mycelial growth of Trichothecium roseum Fig. 4 Effect of fertilizers on spore germination of Alternaria species In vitro experiments revealed that Tomato Stim, Megafol, Kendal, Biofert Forte and Atonik had an inhibitory effect on mycelial growth of both A. solani and A. lycopersici (figures 1 and 2). Cropmax and Miracolo Verde were effective in inhibition of mycelial growth of A. lycopersici. No effect was observed for Miracolo Verde, Kelpak and Cropmax on A. solani. Kelpak was the only fertilizers tested who had no inhibitory activity on A. lycopersici. Kelpak is commercially available seaweed extract and is marketed as a plant growth stimulator due to its content in natural cytokinins and auxins. Megafol and Kendal are plant growth stimulators, containing amino acids with vegetal origin. The reduction of growth rate varied with tested pathogens and treatment, depending by. Fungal pathogens can metabolize all the major substrates. Fungal demand for nutrient increases as fungal biomass accumulates. D. Blachinski, D. Shtienberg, A. Dinoor, U. Kafkafi,3 L.S. Sujkowski, T.A. Zitter4 and W.E. Fry. 1996. Influence of Foliar Application of Nitrogen and Potassium on Alternaria Diseases in Potato, Tomato and Cotton. Phytoparasitica 24(4):281-292 Bowyer, P., Mueller, E. and Lucas, J. (2000). Use of an isocitrate lyase promoter-GFP fusion to monitor carbon metabolism of the plant pathogen Tapesia yallundae during infection of wheat. Mol. Plant. Pathol. 1, 253–262. Dik A.J, Wubben J.P, 2004. Epidemiology of Botrytis cinerea diseases in greenhouses. In: Elad Y, Williamson B, Tudzynski P, Delen N, eds. Botrytis: Biology, Pathology and Control. Dordrecht, the Netherlands: Kluwer Academic,317–31. Rotem, J. (1994) The Genus Alternaria. Biology, Epidemiology and Pathogenicity. American Phytopathological Society Press, St. Paul, MN, USA. Yermiyahu, U., I. Shamai R. Peleg N. Dudai D. Shtienberg. 2006. Reduction of Botrytis cinerea sporulation in sweet basil by altering the concentrations of nitrogen and calcium in the irrigation solution. Plant Pathology 55, 544–552 Among the tested compounds, Kendal was the most effective on Alternaria tenuissima: it inhibited totally the radial mycelial growth. Miracolo Verde, Atonik, Biofert Forte, Kelpak and Tomato Stim inhibited the mycelial growth also. No effect on mycelial growth was observed in presence of Megafol and Cropmax (figure 3). Kendal was very effective in inhibiting spore germination of A. tenuissima and A. lycopersici (figure 4). The inhibition of germination was complete, with no evidence of germ tube emergence. In A. solani, Kendal also inhibited spore germination (59.8%). An inhibittion of spore germination was observed for Tomato Stim, for all theree isolates tested, Megafol (in A. solani and A. lycopersici), Biofert Forte and Cropmax in A. lycopesici. No effect was observed for Atonik and Kelpak for all Alternaria species tested. On both antagonists tested, there was generally no effect of fertilizers on mycelial growth. Kendal reduced the mycelial growth of T. roseum (65.2%). The mycelial growth of T. viride was inhibited only by Tomato Stim and Cropmax.