Antifungal activity of a Pinus monticola antimicrobial peptide 1 (Pm-AMP1) and its accumulation in western white pine infected with Cronartium ribicola

Abstract

Pinus monticola antimicrobial peptide 1 (Pm-AMP1) was expressed and purified from bacterial cell lysate and its identity and purity confirmed by Western blot analysis using the Pm-AMP1 antibody. Application of Pm-AMP1 resulted in visible hyphal growth inhibition of Cronartium ribicola , Phellinus sulphurascens , Ophiostoma montium , and Ophiostoma clavigerum 3-12 days post-treatment. Pm-AMP1 also inhibited spore germination of several other phytopathogenic fungi by 32%-84% 5days post-treatment. Microscopic examination of C. ribicola hyphae in contact with Pm-AMP1 showed distinct morphological changes. Seven western white pine ( Pinus monticola Douglas ex D. Don) families (Nos. 1, 2, 5, 6, 7, 8, 10) showing partial resistance to C.ribicola in the form of bark reaction (BR) were assessed by Western immunoblot for associations between Pm-AMP1 accumulation and family, phenotype, canker number, and virulence of C. ribicola. There was a significant difference (p< 0.001) in mean Pm-AMP1 protein accumulation between families, with higher levels detected in the full-sib BR families (Nos. 1, 2, 5) than the half-sib BR families (Nos. 6, 7). Family 8, previously described as a Mechanism 'X' BR family, had the highest number of BR seedlings and displayed high Pm-AMP1 levels, whereas the susceptible family (No. 10) showed the lowest levels (p< 0.05). Family 1 showed a significant association between Pm-AMP1 accumulation and overall seedling health (p< 0.01, R= 0.533), with higher protein levels observed in healthy versus severely infected seedlings. In general, low Pm-AMP1 levels were observed with an increase in the number of cankers per seedling (p< 0.05), and seedlings inoculated with the avirulent source of C. ribicola showed significantly higher Pm-AMP1 levels (p< 0.05) in the majority of BR families. Cis-acting regulatory elements, such as CCAAT binding factors, and an AG-motif binding protein were identified in the Pm-AMP1 promoter region. Multiple polymorphic sites were identified within the 5' untranslated region and promoter regions. Our results suggest that Pm-AMP1 is involved in the western white pine defense response to fungal infection, as observed by its antifungal activity on C. ribicola and a range of phytopathogens as well as through its association with different indicators of resistance to C.ribicola.