Final Report for GS08-068
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.
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.
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.
An enzyme activity curve was generated to relate enzyme concentrations (units per milliliter) to activity levels (micromoles glucose equivalent, GE, per gram sample water). Mulch samples from a two-year established field site ranged in activity from 12.3 to 24.1 µmol GE /g aq, which corresponded to an enzyme concentration range of approximately 14-553 U/ml.
In pathogen trials with a single enzyme application, colony biomass did not decrease with increasing enzyme concentration in trials using a series of 0 to 100 U/ml, but did vary using a series of 0 to 1000 U/ml. With the higher concentration range (0-1000 U/ml), dry weights from the 1000 U/ml enzyme level were significantly different than all other treatments, and no other treatment levels differed from one another. P. cinnamomi colonies under sustained enzyme exposure at a range of 0-500 U/ml cellulase showed a pattern of decreasing dry weight with increasing enzyme concentration, and the 500 U/ml treatment caused significant reduction in dry weight relative to all other treatments in both trials (P<0.05).
In sporulation trials with a single enzyme application, sporangia production increased with enzyme concentration up to 100 U/ml, then decreased at 500 U/ml. Enzyme concentration significantly affected sporangia production (P<0.001), and the 500 U/ml enzyme level significantly reduced sporangia production relative to 10, 50, or 100 U/ml (P<0.05). In trials with sustained enzyme application, sporangia production decreased sharply with increasing enzyme concentration, and every treatment level was significantly different from each other level (P<0.01).
In soil application trials, cellulase activity varied across treatments and over time. Activity in cells treated with autoclaved enzyme solution was similar to that observed in control cells. In cells which received a single enzyme application, activity diminished significantly (P<0.001), to an average of 53% of original values over the course of two weeks. Cells which received three enzyme applications had activity which was intermediate between those receiving a single application at either one day or two weeks before planting. At time of planting, cellulase activity in the 100U/ml treatment was similar to that in an average two year field-applied mulch. Cellulase applications did not affect disease progress in lupine over a seven week study period. No significant differences were detected among treatments for percent mortality or for AUDPC.
In Fraser fir container trials with cellulytic-fungi enhanced mulches, there was no significant difference among treatments for AUDPC based on ratings at day 88 or day 144. At day 88, only 12% of the seedlings had died across all treatments, with a maximum mortality of 33% and no mortality at all in five of the eleven treatments. After the second inoculation, plants across all treatments declined rapidly, with an overall mortality rate of 78% by day 144. The highest final survival rate was 50% in the plants which received wood chip isolate number 775.3, followed by 43% survival in plants which received gypsum. A GLM analysis of cellulase activity in mulches at the end of the study was significant for treatment effects (P<0.001), with higher cellulase activity in mulch amended with wood chip isolate number 767.3 than in all other treatments. The lowest cellulase activity was observed in mulch amended with gypsum, but this difference was not significant.
Educational & Outreach Activities
A manuscript is in preparation for publication. Partial results have been presented at the annual American Phytopathological Society meeting and at a Christmas tree grower meeting.
Richter, B.S., Benson, D.M., and Ivors, K.L. (2009) Cellulase enzymes as a biocontrol mechanism for Phytophthora cinnamomi in mulching systems. American Phytopathological Society Annual Meeting. Portland, OR.
We were able to demonstrate that cellulase activity in mulches is a viable mechanism for suppression of Phytophthora cinnamomi. We found that sustained exposure of this pathogen to a cellulase compliment extracted from a single cellulytic fungus, T. reesei, at levels similar to those found in mulch was sufficient to disrupt sporulation and, in some cases, to damage hyphae. Activity in field applied mulches varied, and not all mulches could be expected to sustain cellulase levels sufficient to destroy the pathogen or entirely eliminate sporulation.
In container trials, cellulase applied directly to soil lost approximately 50% of its activity within two weeks; if the rate of this decrease is linear, all enhancement of cellulase activity due to the addition of the product would be lost within four weeks of application. Although cellulase activity at time of planting was sufficient in some treatments to cause pathogen suppression, based on our standard curve, root fragment inoculum remained infective, and there were no differences in lupine mortality among treatments. The enzyme levels tested in this study were insufficient to break down inoculum in the form of infected root fragments, and the duration of enzyme activity was insufficient to prevent the pathogen from emerging from infected root fragments to infect new host roots.
Trials with cellulytic fungi-enhanced mulches used a base mulch previously identified as having some suppressive ability against P. cinnamomi. The addition of individual fungal isolates did not provide additional suppression, although one isolate did provide additional cellulase enzyme activity beyond an already-high background level. These findings imply that there may be a threshold level of cellulase activity, beyond which additional increases do not further improve disease suppression. Once optimal cellulase levels are achieved, other mechanisms will likely have to be employed for further improvements of suppression within mulches.
Areas needing additional study
This study used an enzyme product extracted from a single fungus, T. reesei, to reproduce enzyme activity levels observed in field applied mulches. Mulches, however, may contain dozens or even hundreds of distinct cellulase enzymes, produced by the various fungi, bacteria, and actinobacteria that comprise the mulch microbial population. Different cellulase enzymes have different optimal conditions for their activity and vary in their efficacy on any given substrate. Standard assay methods are not always good predictors of enzyme activity on natural substrates, and the single enzyme selected for use in this study may or may not be the most effective against P. cinnamomi hyphae. This study has provided a first link between field enzyme activity and in vitro enzyme efficacy assays; in order to strengthen that link, additional studies should be conducted using multiple enzymes, and using adjusted procedures that include cellulase isolated from Phytophthora cell walls as the assay substrate. Once these multi-enzyme analyses have been conducted, we may better define the optimal cellulase levels for pathogen suppression within these systems.