Physiological Effect of Laccase, Secreted by Rigidoporus microporus on the Host Pathogen Interactions

Abstract

Laccases (EC1.10.3.2) are multi-copper containing phenol oxidases that catalyse the
oxidation of phenolic compounds and aromatic amines. They are widely distributed in higher
plants and in many fungi. In fungi, they are involved in delignification, sporulation, pigment
production and plant pathogenesis and fruiting body formation. Rigidoporus microporus is a
white rot fungus efficient lignin degradation and causative agent of the most destructive
white root disease. In this study, the production of laccase was detected qualitatively.
Furthermore involvement of laccase in the host-pathogen interaction was also studied. Three
isolates of R. microporus were isolated from symptomatic Hevea brasiliensis (R1), Mucuna
bractiata (R2), and Artocarpus nobilis (R3). The level of laccase production was measured
qualitatively on solid media containing the colour indicator of guaiacol. 5 mm diameter of
mycelium discs from 7 day old cultures were innoculated into 2% PDA plates supplemented
with 0.2% glucose as a laccase degrading substrate with 0.02% Guaiacol (pH 5.5) and
incubated at RT (28±20 C) under continuous dark conditions. After, an incubation period of 4
days, the enzyme activity was examined by the decolorisation intensity. Morphology of the
three isolates was also evaluated on malt extract agar. Lignin degradation ability was studied
using artificial inocula prepared by fresh root pieces of H. brasiliensis and incubated for four
months; thereafter they were splited longitudinally to observe the lignin degradation ability of
the isolates. All the test isolates were laccase positive and R3 (A. nobilis) isolate produced
higher intensity showing comparatively larger diameter of the reddish brown halos indicting
higher level of laccase production. Moreover, the isolate R3 showed the formation of
rhizomorphs in vitro conditions while the other isolates did not show them. Formation of
rhizormorphs is associated with the laccase synthesis which has been reported to be
responsible for making polyphenolic glue that sticks the hyphae together. The degradation of
roots was observed to be comparatively faster by the isolate R3 leaving white pockets on
roots by degrading cell wall component due to the removal of lignin in localized areas of
wood and observation can be attributed with the higher laccase enzyme. The root pieces
inoculated with R1 and R2 did not show white pockets and a spongy nature until four month
incubation period. It has been revealed that the isolate R3 (A. nobilis) is a good laccase
producer among the other isolates under investigation.

Keywords: Laccase, Rhizormorphs, Rigidoporus microporus