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

This research was conducted in three phases: enzyme characterization, in vitro pathogen studies, and container bioassays. Ecostone HPP5000, a powdered formulation of cellulases from Trichoderma reesei (AB Enzymes, Darmstadt, Germany) was selected for use in the laboratory trials. Two isolates of P. cinnamomi from North Carolina Fraser fir production sites were used for all in vitro assays; an additional avocado isolate from California was also used in some of the assays.
In order to develop a standard curve to relate product enzyme concentration (Units per milliliter) with enzyme activity (mass of glucose equivalents released per gram of sample water), a dilution series of the Ecostone cellulase product was processed for enzyme activity by a modified Prussian blue method commonly used for soil cellulase analysis (von Mersi and Schinner, 1996; Shi et al., 2006). For comparison with activity in field established mulch beds, six mulch samples were processed using the same concentration ratios (by weight) and incubation conditions used for the cellulase product. One of these was a pine bark mulch, and the remainder were pine wood chips blended with 15% dairy compost, by volume. Because in vitro assays are typically performed in liquid culture, and in vivo exposure to cellulase enzymes within mulches or soils would be most likely to occur within the aqueous fraction, rather than within solids, results were reported as glucose equivalents per gram aqueous fraction (µmol GE/g aq).
Assays for cellulase effects on biomass and sporangia production were conducted with both single enzyme applications and sustained enzyme exposure. Single applications were performed by replacing the culture medium at the end of an initial growth phase with solutions containing the cellulase enzyme product at various concentrations. Sustained applications were also initiated after an initial growth phase, with enzyme solutions replaced twice daily with fresh solution in order to maintain enzyme activity levels through the duration of the trial. Four sample replicates were used for each isolate at each enzyme level, and each trial was replicated twice.
For biomass assays, the culture medium was replaced with enzyme solutions diluted in the same medium used in the initial growth phase. For single-exposure trials, two enzyme concentration ranges were used. The first trials used a series of 0 to 100 U/ml, and the second trials used a log series from 1 to 1000 U/ml, also with a 0 U/ml control. Sustained exposure trials used a series of 0 to 500 U/ml. After enzyme treatments were applied, cultures were allowed to continue incubating until control colonies reached the dish edge. Colonies were washed onto pre-weighed filter papers in a Buchner funnel over vacuum, and dried at 70ºC for a minimum of 24 hours to obtain colony dry weights.
For sporangia assays, P. cinnamomi colonies were grown for three days, drained and washed in deionized water, and bathed in enzyme solutions diluted in a potting mix extract, with a dilution range of 0 to 500 U/ml. Treated dishes were incubated for two days at room temperature under fluorescent light. At the end of the sporulation period, colonies were blended in physiological saline solution (0.9% NaCl), and sporangia were counted in 0.5 ml samples drawn from the blended suspension.
A bioassay with lupine (Lupinus angustifolius) seedlings was conducted to determine the effects of exogenously applied cellulase on pathogen propagules in soil. Cellulase (Ecostone HPP5000) was applied to infested field soil in 150-cc nursery cells. Soil inoculum was artificially enhanced by blending infested root fragments into the soil mixture. Treatments included 100 U/ml and 1000 U/ml cellulase concentrations, applied once either one day or 15 days before planting, or applied three times at 7-day intervals from one to 15 days before planting, heat-treated enzyme (1000 U/ml, autoclaved 15 min at 121ºC) applied 15 days before planting, and a no-enzyme control. Cells were placed in racks in a greenhouse in a randomized complete block design, with eight cones per treatment, eight treatments per block (one rack), and five blocks. Two replicate trials were conducted. One day after the final treatments, pre-germinated lupine seedlings were transplanted into the soil cells. Mortality data were collected daily, and at each date new mortalities were removed and the roots plated on selective medium for confirmation of Phytophthora infection.
In order to track cellulase activity in soil over time, additional soil cells were set up as above for each of the treatments; these were randomly selected and processed for cellulase activity at 24 h after the first treatment and 24 h after the final treatment. Enzyme activity was measured with the same method (Schinner & Von Mersi) used earlier in standard curve development. Remaining soil in each cell was used to calculate moisture content. For comparisons of cellulase concentrations in soil over time, activity results were adjusted to a dry matter basis. Results were also calculated on a soil water basis for comparison with enzyme and mulch samples, based on the standard curve generated with the cellulase product.
An additional container study was conducted to address the use of several known cellulytic fungi as potential biocontrol agents for use in mulching systems. Eight cellulase-producing fungal isolates were selected for use in the study, including three from an established culture collection and five unidentified isolates from field sites where mulches are under evaluation for Phytophthora control. One commercially available biocontrol strain (Trichoderma hamatum T-382) was also included, as well as a gypsum treatment. Fungal isolates were grown on a vermiculite-rye base in culture bags, and then each treatment was individually blended into pine wood chip and diary compost (85:15) mulch at a rate of 5%, by volume. Fraser fir seedlings were potted into the treated mixtures and placed in a greenhouse in a complete randomized block design with seven inoculated and three uninoculated blocks. Inoculated pots received four P. cinnamomi infested rice grains, placed under the mulch surface at 14 days after planting. At eight weeks after planting, the containers were moved from the greenhouse to the Horticultural Field Lab facility, where they were maintained with daily irrigation under shade cloth for the duration of the study. At 100 days after planting, mortality was low across all treatments, and inoculation with P. cinnamomi was repeated, as above. Plants were monitored for an additional 60 days. At the end of the 160-day growth period, mulch was collected from a subset of the containers and analyzed for cellulase activity.
Analysis of variance was conducted on data from in vitro assays using the general linear model function of SAS software (SAS institute, Inc., Cary, NC), with Tukey’s HSD test applied for means separations. Where data were non-normal and transformations did not normalize them, non-parametric analyses were used. GLM analysis on ranked data was used to conduct post hoc analyses on non-normal data sets. For cellulase activity data from soil application trials, mixed model repeated measures analysis was conducted using the PROC MIXED function in SAS. Plant mortality data and area under the disease progress curve (AUDPC) from container trials were analyzed using GLM analysis or Friedman’s nonparametric analysis. An alpha level of 0.05 was used for all tests.