Content of phytoestrogen coumestrol in alfalfa genotypes infected with a viral disease

SILVA MP.; MANZUR ME; MAMANÍ E.; CHIACCHIERA S; BASIGALUP D.

Milazzo

Congreso; XXVII SILAE Congress; 2018

Società Italo-Latinoamericana di Etnomedicina-SILAE

IntroductionCrop plants are frequently exposed to different biotic and abiotic stress conditions1. In the last years, the production of alfalfa in Argentina has been affected by several viral diseases2, particularly a co-infection of rhabdovirus and alfalfa mosaic virus3. Consequently, when the plant defense machinery is activated, the synthesis of several secondary metabolites can increase1. In alfalfa, there are some metabolites with important nutraceutical properties which are mainly localized in leaves. Two of them, coumestrans and isoflavonoids4, rise dramatically when the plants are exposed to viral infection. The objective of this work was to compare coumestrol content in individual genotypes (clones) from two alfalfa varieties under two conditions: infected vs non-infected. Materials and MethodsSeven genotypes from cv Monarca SP INTA and six genotypes from cv Traful PV INTA were used in this study. Plants were cloned and divided into two groups: infected with viral complex (I) and free of infection (NI). Coumestrol content was measured in both conditions when plants reached 380° degres - days. For each genotype, plant homogenate was obtained adding 2 ml of 3.5M HCl and 8ml of ethanol to 0.5 grams of herbage dry sample. This mixture was heated at 83°C for 30 min. and cooled to room temperature. Then, the solution was centrifuged for 10 minutes at 10,000 rpm at 4°C. After filtration, 10 µl aliquot was injected into the HPLC (Agilent 1100 series) and Coumestrol was detected at 260 nm (Diode Array Detector, series G1315B) and identified using an external standard (Sigma-Aldrich®). All data were analyzed using the Linear Mix Model test, followed by a post-hoc comparison test DGC (Prueba de Di Rienzo, Guzmán y Casanoves, 2002).Results and DiscussionDifferences in coumestrol content among genotypes and cultivars were detected (Table 1). Overall, there was a general trend to increase coumestrol production in response to viral infection, but only three genotypes (M18, M29 and T124) showed significant increases when infected with virus over the virus-free treatment.Table 1. Content of coumestrol for 13genotypes from cultivars Monarca (M) and Traful (T). Genotypes were cloned and the clones were infected (I) or non-infected (NI) with a viral complex.MNIITNIIM10381,2± 39,6 F337,7± 41,3 FT112ndndM111107,9± 40,2 A1125,5± 40,2 AT124727,6± 40,2 D **856,4± 40,2 CM18382,1± 39,7 F*524,9± 39,8 ET129941,4± 39,7 B950,5± 39,8 BM23784,0± 39,7 C835,4± 41,0 CT140650,1± 39,6 D759,9± 39,8 DM29695,5± 39,7 D*795,1± 39,7 CT141728,6± 31,2 D741,3± 31,7 DM36444,3± 39,7 E521,0± 40,1 ET152ndndM37448,8± 40,2 E527,0± 39,8 EIndicated values are mean peak areas ± E.E. and are expressed in ppm/material dry weight. Values are mean of 18 plants ± EE. COU: coumestrol; NI: free of viral infection; I: infected with viral complex; Different letters indicate overall significant differences (P