Maximizing Blad-containing oligomer fungicidal activity in sweet cultivars of Lupinus albus seeds

Abstract

During seed germination and plantlet growth, an important aspect of Lupinus β-conglutin proteolysis is the accumulation of blad in the cotyledons. Blad, a 20.4 kDa, 173 residue polypeptide which inhibits fungal growth, is the main subunit of BCO (Blad-containing oligomer), a Lupinus bioactive polypeptide oligomer, which underwent a successful translational research during the last thirty years. The development of this recent broad-spectrum biological fungicide for plant disease control made evident the advantage of being non-toxic to the environment, plants, humans and other animals, an interesting characteristic given the increasing consumer’s concern about food safety. The industrial-scale production of the edible fungicide BCO for agricultural purposes involves germinating and growing for ca. 8 days massive amounts of Lupinus plantlets. Therefore, it becomes economically relevant to maximize/increment the amount of extractable BCO. Although BCO represents an alternative pathway for lupin production in Europe, there is no previous evidence on lupin cultivars concerning BCO activity or its cotyledonary concentration, and therefore on the most promising cultivars for BCO extraction and fungicide production. In this work, the amount of BCO and its level of fungicide activity was evaluated in seven sweet cultivars of L. albus (cvs. Amiga, Energy, Estoril, Ludic, Misak, Multitalia and Rumbo), along 20 days after the onset of germination. In addition, four distinct lots of cv. Energy and six lots of cv Misak, with different harvest years (1997, 2010, 2013 and 2014) and/or different sites of production in Portugal (Alto Alentejo, Baixo Alentejo and Beira Litoral) were also analysed. Quantitative assays demonstrated maximal accumulation of BCO in the cotyledons of 4-days-old L. albus plantlets, apart from significant differences between seeds’ harvest year or production site. The assays also showed that cv. Energy had significant higher accumulation of BCO (31.67 μg mg−1 wet weight) than Rumbo (25.67 μg mg−1) and Misak (22.12 μg mg−1), respectively second and third cultivars with highest accumulation of the oligomer. Additionally, very significant differences were also observed among seeds’ harvest year or production site. These observations reduced dramatically the original purpose of this work on the study of BCO variations at the level of sweet L. albus cultivars, since the ‘noise’ detected in BCO levels caused by the year and location of their production was far greater than its variation among cultivars. The observation that storing for several years the seeds at -20 °C did not seem to decrease BCO activity and suggests that the variation in cotyledonary BCO accumulation is essentially controlled by the prevailing edaphoclimatic conditions during seed formation. Furthermore, the activity tests exhibited significant antifungal activity between 3 and 5 days-old plantlets, with absence of antifungal activity on 8 days-old plantlets or older. Overall this study is of considerable importance to maximize BCO extraction from the cotyledons of sweet L. albus cultivars.