Experiment 1: the effect of dry heat on colony growth and sporulation of P. ramorum
Results from experiment 1 showed that temperatures above 35°C merely slowed down the growth rate of P. ramorum, and even exposure to 40°C for 4 h did not affect pathogen survival. The ability to withstand exposure to such high temperature suggests this pathogen may be able to become established and survive in warmer regions. Heat tolerance of P. ramorum in planta has been shown by Harnik et al. (2004). For sanitation purposes, three treatments were found to kill the pathogen: 24 h at 40°C, 2 h at 45°C and 1 h at 55°C; 55°C is the minimum required temperature that needs to be obtained (and maintained for at least five pile turns) in commercial turned windrow composting facilities (Hay 1996). Based on these observations, we suggest that prolonged exposure to 45°C may be as effective at eliminating P. ramorum as 55°C. As temperature fluctuations are hard to avoid in most field situations, it is preferable to choose the highest temperature for the shortest period of time. However, flash-heat treatments of 55°C for 30 min are not likely to successfully eliminate the pathogen.
It should be noted that these results were obtained on cultures. Survival of the pathogen in infected plant tissue may vary significantly depending on plant species, type of substrate (e.g. wood, cambium, leaves; see below), and propagules produced by the pathogen on each host or host part.
Experiment 2: the effect of composting and heat treatment on infected plant material
All tested treatments reduced recovery of the pathogen to zero. Furthermore, pathogen recovery levels were significantly different when compared to those of untreated samples (P < 0·01). This was true regardless of type of treatment or substrate. At the end of the experiments, recovery of the pathogen in untreated controls was undistinguishable (P < 0·01) from recovery recorded before treatment. Direct plating results were used for the analysis, but pear baiting provided identical results.
Wood chips represented the most variable and least favourable substrate for recovery of P. ramorum. Baiting always failed from wood chips, suggesting that the pathogen does not sporulate on this substrate, or that leachates from the substrate inhibit sporulation. Isolations from cankers caused by the artificial inoculation of coast live oak saplings yielded the largest percentage of successful isolations, but only limited baiting success, suggesting that the stem sections supported mycelial growth beneath the bark, but provided only limited opportunities for sporangial formation. We hypothesize sporangia production on these stems occurred solely at the inoculation site, perhaps on the inoculum plug. Isolations from bay leaves were moderately successful, but baiting was significantly more successful than with the two other substrates. This is not surprising as bay leaves are known to support prolific sporulation of P. ramorum (Davidson et al. 2005).
Both turned windrow systems maintained temperatures above 55°C for the time mandated by EPA guidelines for commercial composting in California, as did the forced air static pile system. However, it should be noted that in these studies the infected plant material and temperature recorders were placed in comparatively ‘ideal’ locations within each pile, in an experimental design referred to as direct process evaluation (Christensen et al. 2002). While results from these experiments clearly indicate that the composting process is capable of sanitizing infected green waste under the conditions tested, they do not necessarily prove that composting as a whole achieves identical results. As Christensen points out, deviations from the conditions tested may affect the outcome and direct process evaluation is unreliable by itself as an evaluation tool for monitoring the overall sanitary process. The frequency and intensity of potential deviations from the conditions tested here will define the reliability of composting as a tool to eliminate P. ramorum from infected plant material. Results from experiment 2 suggest that, if conditions met in our sampling points are met throughout the pile, composting will be successful in eliminating P. ramorum from infected plant material.
Experiment 3: survival and spread of P. ramorum within a turned windrow compost pile characterized by high levels of pathogenic inoculum
Our last experiment was designed to test the validity of the composting process by monitoring viability and presence of the pathogen in a compost pile largely composed of infected plant material. In this instance, rather than use direct process evaluation as in experiment 2, we used spot test analysis, a method whereby many samples are taken throughout the pile, and tested for presence of P. ramorum. This is a much more accurate method for the analysis of the sanitary process (Christensen et al. 2002). Sampling in this experiment was either complete or very intensive, to maximize our chances of detecting even limited pathogen survival. A complex sampling strategy was adopted including baiting, direct plating, PCR-based detection and the use of sentinel plants. Each diagnostic test had individual strengths and weaknesses. The use of baiting techniques allowed us to sample large volumes of substrate, but relies on active infection of the bait by motile zoospores. Therefore this method was used to determine active sporangia production on the tested substrate. Zoospores are generally regarded as the primary infection propagules for Phytophthora species, including P. ramorum (Werres et al. 2001) and P. cinnamomi (Zentmeyer 1980). When using baiting as a diagnostic detection assay, negative results have to be interpreted with caution, as it has been shown that pear baiting may be insensitive enough to miss Phytophthora spore densities that are high enough to cause disease (Yamak et al. 2002). Composts can suppress sporangial production below detectable limits without eradicating P. cinnamomi (Hardy and Sivasithamparam 1991), and suppressed isolates may recover sufficiently to become a threat under more favourable conditions (Hardy and Sivasithamparam 1991; Sidhu et al. 1999).
The positive controls used for our baiting trials failed to produce any lesions on the pear bait, indicating that sporulation of P. ramorum was suppressed. In contrast to our inability to bait positive controls from solid compost samples, the flood baiting controls were successful, suggesting that the leachate itself is not suppressing pathogen activity, but that suppression requires either intimate contact with the compost substrate or that suppression of P. ramorum in compost is severe enough that sporangia are not formed on U. californica leaves once they have been subjected to the biotic or chemical environment of a compost pile. Some evidence for the latter comes from another study in which we were unable to retrieve P. ramorum from 24 infected U. californica leaves 6 h after placing them into a cool (approx. 15°C) pile of ground and wetted green waste, while we were able to retrieve P. ramorum from 10 of 12 leaves placed on the surface of the pile.
While compost dilution plate methods do not require pathogen sporulation, they can realistically be used to sample only a significantly smaller subset of substrate. Results should be interpreted with caution, as negative results may be the result of pathogen suppression rather than eradication. No P. ramorum colonies were formed on 160 PARP plates of a mixture of sieved inoculated compost and PARP, but positive controls simultaneously inoculated with P. ramorum failed to grow out as well. This result indicated that compost contains chemical or biological factors capable of suppressing P. ramorum growth. Suppression of Phytophthora species has occurred in compost preparations in the absence of high temperatures (Hardy and Sivasithamparam 1991) or in compost-amended soil (Hoitink and Fahy 1986; Hoitink and Boehm 1999, McKellar and Nelson, 2003), and has repeatedly been attributed to biological or chemical factors (Hoitink et al. 1976; Yuen and Raabe 1984). We believe that the antimicrobial properties of compost suppressed P. ramorum in this test, including the positive controls.
The use of highly susceptible sentinel plants at various distances downwind from the pile is a good indicator that infected green waste is non-contagious once in a compost pile. The closest plants were 1 m from the infested pile and were routinely showered with debris as the pile was turned. This occurred under environmental conditions conducive to infection (Davidson et al. 2001), particularly the turning conducted in the cold, wet, windy weather of 12–25, April 2003 (days 10–23). It is possible that foliar application of compost caused a suppression of infection due to induced resistance (Elad and Shtienberg 1994) during this phase of the experiment. However, the rhododendrons were later planted in this compost, prewatered with a fine mist and then heavily watered with large droplets allowing splash dispersal of any propagules in the compost. After a year of this treatment, not a single infected leaf was found, while control leaves retained 97% infection. In light of the recent nursery infections that have occurred on Rhododendron, Camellia spp. and other nursery stock, it seems clear that P. ramorum will readily infect such susceptible species given adequate conditions to do so (http://www.aphis.usda.gov/ppq/ispm/pramorum/regulations.html). The lack of infection strongly suggests that P. ramorum is heavily suppressed or eliminated under composting conditions.
Detection by PCR enabled detection of P. ramorum regardless of dormancy or reproductive status. As our above results seem to corroborate Malajczuk's (1983) statement that Phytophthora species are easily suppressed by competing fungi and bacteria (or perhaps are suppressed by chemical compounds), PCR as a detection tool compliments the use of viable culture methods. Positive PCR results may therefore be problematic from a regulatory perspective, because the technique may detect dead cells. Although PCR is performed on small volumes of substrate, our sampling assay was intensive enough (430) and sensitive enough (at 0·8 ng of DNA per sample) to provide >99% confidence that if P. ramorum DNA were still present at only one part in 100 000 of the introduced amount, it should have been detected. Unlike the other tests, PCR-based assays can detect dormant chlamydospores or any other resting structure and vegetative mycelium. We found no positive samples.
In experiment 3, the temperatures in the compost were initially lower than those required by US EPA 40 CFR Part 503. Limited heating was due to the excessive moisture level in the pile, due initially to a watering regime designed to produce collectable leachate and subsequently to the heavy rains occurring in that period. Turning of the pile caused a significant reduction in moisture level, but heating did not occur after this point (especially in the toe of the pile), until further nitrogen-rich material was incorporated in the pile. It is interesting to note that the toes of the pile heated up as quickly as the rest of the pile, but cooled down faster than the rest of the pile. Maximum exposure to high temperatures can thus be obtained by shifting composting material in different locations in the pile through turnings (EPA 1989). In spite of the fact that only moderate temperatures were reached in the first part of the experiment, no isolates of P. ramorum were obtained from the leachate. This result further indicates that the pathogen is at least effectively suppressed during the composting process due to biologically or chemically mediated processes. It should be noted that the compost piles used in this study were both quite large and carefully monitored, as should be typical of commercially run facilities. Smaller composting piles or those not turned and/or monitored on a rigorous schedule as might be found in ‘back yard’ composting, frequently do not meet the temperature requirements for effective sanitization (Yuen and Raabe 1984; Ryckeboer et al. 2002), and therefore should not be construed to meet phytosanitary conditions outlined in this report.
The combined results from the three experiments conclusively show that some heat and composting treatments suppress and eliminate P. ramorum from a variety of infected plant substrates. Even when the pathogen was present in the leaves of U. californica, its most resilient wild plant host, the host material was effectively sanitized by following the guidelines described by US EPA 40 CFR Part 503 for composting (Hay 1996). Our experiments also indicate that, when analysing compost for the presence of a pathogen like P. ramorum, it is necessary to employ an assay capable of directly assessing for pathogen presence. We believe the approach we describe here is extremely appropriate for this type of analyses. The approach included a very sensitive and specific nested Taqman PCR assay in which the detection threshold for the DNA of the target organism was checked for each sample. The ability to quantify the detection threshold combined with quantification of the DNA of the target organism prior to treatment allows to determine not only presence/absence of the pathogen but also to quantify the magnitude of detectable pathogen reduction.
Whether compost may serve as a route of spread for this aggressive plant pathogen will depend on other factors such as the ability of the pathogen to survive or colonize mature compost. Phytophthora ramorum has been recovered from dust samples immediately surrounding tub grinders used to process green waste (Shelly et al. 2006), and preliminary data suggest that P. ramorum is capable of surviving in finished compost if introduced, so although compost appears to eliminate the pathogen when processed correctly, this should not be construed to mean that all compost originating from infected green waste is safe by simple virtue of the fact that it has been processed in accordance with US EPA guidelines.