Elucidating the role of cellulases involved in biological control of Phytophthora root rot

• 2008 annual report

A standard curve was developed to compare cellulase enzyme activity in a cellulase product with activity in two-year-old mulch samples from Fraser fir production sites, where mulches are being evaluated for suppression of Phytophthora root rot. Cellulase activity in the commercial product was sufficient to impact pathogen growth and reproduction at concentrations similar to those in mulch samples, indicating that cellulase activity in mulches is sufficient to suppress Phytophthora cinnamomi. Cellulase activity in mulch could be increased by adding known cellulytic fungi, but no additional suppression was achieved beyond that provided by a wood chip and dairy compost mulch alone.

Introduction

The purpose of this project is to elucidate the role of cellulases produced in organic mulches implemented for biological control of Phytophthora root rot (PRR). Cellulose-rich mulches have been used successfully in Australia and California to reduce PRR, caused primarily by P. cinnamomi, in avocado orchards; similar systems are under evaluation for PRR suppression in North Carolina Fraser fir farms. Previous research has found that suppression of PRR with mulches is associated with microbial activity in the mulch, and it has been postulated that cellulases produced by mulch-inhabiting organisms degrade the cellulosic component of Phytophthora cell walls. Downer et al. (2001) determined that cellulase and laminarinase enzyme activities were elevated in mulch relative to underlying soil, and that enzyme activities were positively correlated with microbial activity and fungal counts but negatively correlated with Phytophthora recovery. They also found that addition of 10-25 U/ml cellulase to Phytophthora cultures in soil extract impaired spore development (zoospores and chlamydospores), and concentrations greater than 25 U/ml disrupted mycelium. Due to differences in units between the two studies, it is not possible to determine whether the cellulase levels in the mulch would be sufficient to achieve the effects observed in the in vitro assays. Soil assays for cellulase activity measure the mass of “glucose equivalents” released per unit mass of sample per unit time, while purified enzyme concentrations are expressed as enzyme units (U), where one unit is the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute, under defined conditions. The mass of cellulose is unknown for any given soil or mulch sample, as are the relative quantities and molecular weights of each sugar released in the hydrolysis. Therefore, data from in vivo studies cannot be translated for comparison with in vitro assays. While prior research has provided support for the microbial cellulase model of suppression in mulch, more work is needed to interpret the existing research and determine the effects of cellulase on Phytophthora propagules at concentrations achieved from mulch applications. Resolving the role of cellulases and their activity levels expected to be suppressive will provide the basis for improving mulch-based cultural systems. If disease suppression can be related to a threshold level of cellulase activity, mulch formulations and cellulolytic microbes utilized in these systems could be optimized to achieve target cellulase activity levels. Further, tracking of cellulase levels in mulch over time could be used to determine optimal mulch age, and re-application times and rates. Improved mulch systems offer a locally-available, sustainable and affordable means of managing PRR in a variety of crops, allowing continued productivity of land infested by soilborne Phytophthora spp.

The objectives of this research are to:

(1) Establish levels of cellulase enzyme products (Units/ml) in vitro which yield enzyme activities (µmol Glucose Equivalents/g·h) similar to those achieved in organic field mulches, and determine the impact of cellulase levels on P. cinnamomi;
(2) Ascertain the effects of exogenously applied cellulases on density of viable Phytophthora propagules in soil;
(3) Track the duration of cellulase activity associated with exogenous cellulase applications; and

(4) Determine the effects of several known cellulytic fungi, added to mulch as potential biocontrol agents, on both cellulase activity and disease progress.