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Enhancement of potato defense mechanisms by BABA in multigenic resistance potato cultivars. Systemic response against Phytophthora and Fusarium. Olivieri FP, Lobato MC, Gonzalez Altamiranda E, Wolski E A, Guevara MG, Daleo GR and Andreu AB. IIB. FCEyN. Universidad Nacional de Mar del Plata (7600) Argentina. e- mail: [email protected] Plants have developed mechanisms to successfully co-exist with the presence of pathogenic organisms. Some interactions between plants and pathogens are based on recognition of specific elicitor molecules from avirulent pathogen races (avr gene products), which are described in the gene for gene resistance theory. Another type of resistance, multigenic (horizontal) resistance, is a less (well)-studied phenomenon that depends upon multiple genes in the plant host. All plants possess resistance mechanisms which can be induced upon pre-treatment of plants with a variety of compounds. This general phenomenon is known as systemic acquired resistance (SAR). At least in some plants species, SAR depends on the timely accumulation of multiple gene products, such as hydrolytic enzymes, peroxidases or other gene products related to plant defense. The pre-treatment of plants with an inducing organism or chemical compound appears to conduct the plant to mount an effective defense response upon subsequent encounter with pathogens, converting what would have been a compatible interaction to an incompatible one. Tuzum (2001) showed that in pathosystems (Lycopersicon spp.Alternaria solani) treated with BABA as elicitors, multigenic-resistant plants constituvely express specific isozymes of hydrolytic enzymes that release cell wall elicitors which, in turn, may activate other defense mechanisms. The aim of this work was to characterize, in systems of potato multigenic resistance, the components of biochemical mechanisms by which BABA (β-amino-butyric acid) increases resistance in potato against phytopathogens by inducing a mechanism SAR. Two potato cultivars with different level of horizontal resistance: the cultivar Bintje, susceptible a P. infestans (Wastie, 1991; Huarte, 2002) and the cultivar Pampeana (MP 159.789/12 x Huinkul MAG) with a high grade of horizontal resistance, free of known R genes Solanum demissum (Huarte et al., 1997) were analyzed in this work. BABA applications and defense response observations were carried out as follow: Three weeks after emergence, plants were sprayed with 2mM BABA, as chemical inducer or with water as control treatment. Three or twenty days after this treatment, leaves were cut and inoculated with P. infestans. At different times after inoculation, symptoms were registered and treated leaves were processed for biochemical analysis. Another set of treated plants were maintained in greenhouse until tuber maturation and during this period, BABA was applied three more times at 20 days intervals. Recently harvested tubers from treated or control plants were inoculated with P. infestans and, in order to evaluate the specificity of systemic acquired response, other tubers were inoculated with Fusarium solani f. sp. eumartii. Again, at different times after inoculations, symptoms were recorded and tuber tissue was processed for the analysis of defense components inductions. The highest level of protection in leaves against P. infestans was observed after 3 days of treatment with BABA, although this protection decreased in foliage at 20 days after treatment in both cultivars. At this time the protective effect was more evident in Pampeana than in Bintje. On the other hand, the levels of phytoalexins (antimicrobial compounds) and phenols were increased only after 20 days in treated leaves from plants of both cultivars. This increase was more than twice in each cultivar respect to the level detected in infected but untreated plants. Callose content was also quantified. Constitutively content was 40 % higher in Bintje than in Pampeana leaves but when foliage was inoculated with P. infestans, only Pampeana leaves presented an increase approximately of 25% in callose. The BABA treatment show a tendency to increase callose level in both cultivars and contrary the further infection suppress this response. The expression of two pathogenesis related proteins (PRPs), basic chitinases and aspartic proteases was analyzed in leaves at 24 and 48 hs after inoculation with P. infestans for both times of treatment with BABA (3 and 20 days). The accumulation of PRPs was estimated by western-blot analysis. In cv. Pampeana, the expression of these enzymes was increased in plants at 3 days of treatment; this induction decreased at 20 days. In cv. Bintje, the effect of BABA on the induction of these enzymes was less evident than in Pampeana at 3 days but increased at 20 days after BABA treatment. For the aspartic protease, the increase at this time was major than Pampeana cultivar. In summary, at least for these PRPs, BABA application induces an accumulation of these proteins and this response occurs later in the susceptible cultivar. The systemic acquired resistance (SAR) induced by BABA treatment was verified in harvested tubers coming from treated plants. BABA application in early stages of plant development did not affect tuber harvest yield. Moreover, in our greenhouse conditions, the number and weight of tuber recuperated from BABA treated plants increased respect to control plants. In these tubers the protection was observed for P. infestans and also for Fusarium eumartii. Tubers were inoculated with zoospores of P. infestans and maintained in moister chamber during 7 days. F .eumartii was inoculated as disks of mycelium introduced in tuber by wounding, and maintained for 10 days at 25 oC. At different times tuber tissue surrounding treated area was processed for protein analysis. For both cultivars, the expression level of aspartil proteases in tubers coming from treated plant was major than in control tubers. On the other hand, previous results demonstrated that the tissue coming from F. eumartii infected tubers present a high level of serine protease activity, called FESP (Fusarium Extracellular Serine Protease) and that the accumulation of this serine protease activity correlates with infection process (Olivieri et al, 2004 ). In our experiments, FESP activity decrease approximately 40% in infected tubers coming from BABA treated plants respect to infected tubers coming from non-treated plants. More than 3000 articles on induced pathogen resistance have been published since 1995. However, many questions are still unanswered and required further investigation. Stronger efforts are required to identify the compounds causing resistance, to quantify these compounds and to correlate with the biologically detectable resistance to characterize the induced stage in horizontal resistance. It is also necessary to evaluate the durability of this defense response in tuber during storage and to know the optimal time of BABA application to obtain a effective SAR. Literature cited 1. Huarte, M., 2002. Niveles Disponibles de Resistencia al Tizón Tardío en Latinoamérica. In: Centro Internacional de la Papa, 2002. E.N. Fernandez-Northcote (ed). Memorias del Taller Internacional Complementando la Resistencia al Tizón 2. 3. 4. 5. (Phytophthora infestans) en los Andes, 2002, febrero 13-16, 2001, Cochabamba, Bolivia, GILB, Taller Latinoamérica 1, Lima, Perú. p 59-66. Huarte, M., Butzonitch, M. Van Damme, M. Colavita, S. Capezio, S. Micheletto, E. Cacace y A. Clausen, 1997. Experiencias, dificultades y logros obtenidos en la búsqueda de resistencia durable a enfermedades. In: Primer Taller de PREDUZA en Resistencia Duradera en Cultivos Altos en la Zona Andina. Quito, Ecuador, 22-24 Setiembre 1997, Ed. D. Danial, pp: 154-163. Olivieri F, Maldonado S, Tonón C and C. Casalongué. 2004. Hydrolytic activities of Fusarium solani and Fusarium solani f. sp. eumartii associated with the infection process of potato tubers. Journal of Phytopathology 152: 337-344. Tuzum, S. 2001. The relationship between pathogen-induced systemic resistance (ISR) and multigenic (horizontal) resistance in plants. European Journal of Plant Pathology.1007: 85-93. Wastie R. 1991. Breeding for resistance In: Advances in Plant Pathology. Vol 7: Phytophthora infestans, the cause of Late blight of potato. Ingrams DS and Williams PH (eds) Academisc Press London pp 193-223.