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

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

Summary

Phytophthora root rot (PRR), caused by P. cinnamomi and other soilborne Phytophthora species, impairs production in a wide variety of food and ornamental crops worldwide. In some cropping systems, organic mulches have proven to be a valuable tool in suppressing this disease. Suppression has been linked to biological activity within the mulch, and is correlated with total and cellulase enzyme activity. The production of cellulases by mulch microflora has been suggested as a primary mechanism of PRR suppression, but it has not been shown that the levels of cellulase produced in mulch are sufficient to inhibit Phytophthora growth or infectivity. The goal of this research is to elucidate the role of cellulases in PRR suppression within mulching systems, with the aim of improving current mulch applications to provide greater disease control. In vitro assays will allow us to identify concentrations of cellulase with activity similar to levels observed in mulches from ongoing field trials, and to determine the effects of those levels on Phytophthora growth and infection. Bench and container studies will be used to ascertain the effects of cellulases on the density of viable Phytophthora propagules in soil and mulch and on PRR development in Fraser fir seedlings, and to examine the potential use of several known cellulase-producing fungi as biocontrol agents in mulching systems. Results are expected to identify threshold levels of cellulase activity which should facilitate disease suppression, and may provide an organic, sustainable means of improving mulches for use in biological and cultural disease control efforts.

Objectives/Performance Targets

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 and mulch and on subsequent PRR development in Fraser fir seedlings;
(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.

Accomplishments/Milestones

Cellulase activity in a commercially available enzyme product has been characterized and correlated to activity levels in field-applied mulches. In laboratory assays, we have found that P. cinnamomi responds differently to single enzyme applications than to repeated applications, designed to provide sustained activity levels. Where enzymes were applied once, and activity was allowed to gradually diminish over time, sporangia production by P. cinnamomi was unaffected or increased within an enzyme concentration range of zero to 100 Units per milliliter of soil solution. When enzyme concentrations were maintained over time through repeated treatments, sporangia production was reduced at concentrations between ten and 100 Units per milliliter. Enzyme activity assays suggest that the levels of cellulase activity achieved in field-applied mulches are within the range that cause reduced sporulation in vitro, supporting the hypothesis that biological control of P. cinnamomi in mulches is attributable to cellulase production by mulch-inhabiting organisms.
In a container trial using isolates of cellulytic fungi added to a wood chip mulch (85% pine chips, 15% dairy compost) as potential biocontrol agents, only one of nine isolates resulted in a measurable increase in cellulase activity within the mulch, relative to the unamended control. The increase in activity was not associated with a decrease in root rot disease, relative to the other treatments. Earlier trials had suggested that the wood chip mulch alone has high cellulase activity and provides some disease suppression. It is possible that a threshold level of cellulase activity exists, beyond which no further improvements in cellulase-mediated disease control may be achieved.

Impacts and Contributions/Outcomes

Results of assays conducted thus far support the suggestion that microbial cellulase production by mulch-inhabiting organisms is sufficient to impact P. cinnamomi and provide disease suppression. The differences observed in single versus sustained enzyme applications also suggest that suppression may be achieved only with sustained enzyme activity, as would occur in a biological control system, and that a single, sub-lethal exposure to cellulase may even increase disease pressure. This implies that application of cellulase as a pre-plant soil treatment may not be a viable disease reduction strategy. Further studies are underway to determine the effects of single and repeated soil applications on Phytophthora root rot disease in infested soil.

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