Variable levels of laccase are secreted by four species of Ilyonectria that infect grapevines

Abstract

Laccases are a family of enzymes (polyphenol oxidases; PP0-1 and PP0-2) implicated in pathogenesis and degradation of lignin by many phytopathogens, including those that infect grapevines. The aim of this study was to (i) confirm that Ilyonectria species pathogenic to grapevines secrete laccase, (ii) to determine whether isolates vary in laccase secretion and (iii) to determine whether the amino acid sequence of laccase (/eel) differs between species. Laccase activity was measured using ABTS (2, 2¹-azino-bis [3-ethyl-benzthiazoline-6-sulfonic acid]) and DMP (2,6-dimethoxy-phenol). Six isolates of I. liriodendri and five isolates of the I. macrodidyma complex, including, I. macrodidyma (n=3), I. torrensensis and I. novozelandica were inoculated as agar plugs into minimal liquid media and incubated at 20°C for 7 days. The mycelium free extracellular fluid was assayed for PP0-1 and PP0-2 activity by their oxidation of ABTS and DMP, respectively. The results showed that all isolates produced PP0-1 activity but only seven produced detectable PP0-2 activity. There was isolate variation in both PP0-1 and PP0-2 activity for all species for which >1 isolate was tested (P<0.000). Degenerate PCR was used to amplify the feel gene from I. macrodidyma, I. novozelandica, I. torrensensis and I. liriodendri. Six amino acid polymorphisms were identified within isolates of I. liriodendri and the I. macrodidyma complex. Amino acid polymorphism was not found between isolates of the same species. Thus, variable laccase activity is likely to result from variable amount of enzyme secretion rather than isolate differences in enzyme activity.