Additionally, we wanted to test the contribution by each Phytophthora species to leaf decomposition and to determine if there was any difference depending on leaf senescence based on their differing trophic adaptations. While the inclusion of natural stream water in these experiments is an imperfect approximation of natural conditions, namely in excluding both shredder organisms and other microorganisms eliminated in the holding period, it had the potential to reflect the interaction of the inoculated Phytophthora species with bacteria, protozoa, fungal communities, and possibly micro-invertebrates that persisted in stream water. Though the effect of stream water treatment was not statistically significant in the models, a noticeably higher occurrence of P. ramorum on brown leaves in sterile stream water treatments compared with non-sterilized stream water additions and also higher detection of P. ramorum spores by baiting in sterile compared to non-sterile stream water treatments both indicated that there was some difference between the two treatments. Though a much greater diversity of organisms likely influences this system under natural conditions, our previous research exposing leaves in natural streams demonstrated both P. ramorum and clade 6 Phytophthora species effectively colonize California bay leaves under natural conditions. As expected, based on previous work, both Phytophthora species rapidly colonized more than 60% of the leaf area of green leaves in both experiments. That P. ramorum also persisted on green leaves at high levels for the entire 16 weeks despite the loss of approximately 40% of leaf biomass stands in contrast to our previous findings where its colonization of leaves peaked within a few weeks after exposure in natural streams,ebb and flow table but then rapidly dropped to very low levels as colonization by clade 6 Phytophthora species rose and persisted at high levels.

This is evidence that the reduced recovery of P. ramorum from green leaves in natural streams as decomposition progressed was due to displacement from saprotrophic organisms like clade 6 Phytophthora species. Unfortunately, P. ramorum was completely suppressed from colonizing leaves in combined inoculations with P. gonapodyides and it could not be determined if the pattern observed in field experiments would occur under these simulations when both species were present. The suppression of P. ramorum colonization of green leaves in combined Phytophthora inoculations—consistent across all three experiments—was surprising because both species were effective at colonizing leaves when inoculated alone. One explanation could be that sporulation of P. gonapodyides from mycelial mats occurred earlier than that of P. ramorum and that the latter was therefore precluded from leaves because in all experiments, full colonization of green leaves by P. gonapodyides occurred very rapidly. Indeed, in the first experiment, colonization of P. gonapodyides occurred more rapidly on green leaves than that of P. ramorum . However, baiting two days after inoculation in the second experiment showed that P. ramorum spores wereactive in the microcosms where it was inoculated alone, but almost absent in the combined inoculation microcosms. This suggests that the presence of P. gonapodyides itself may have suppressed sporulation by P. ramorum. The rapid leaf colonization by P. gonapodyides in these microcosms also contrasts with the slower colonization that was observed in natural streams and may be an artifact of high inoculum loads and the relative abundance of substrate. The aim of these experiments was to characterize the capacity of each organism for growing and reproducing from each type of leaf rather than estimating typical colonization and decomposition in streams. Though logistically more difficult to prepare and standardize for an experiment of this magnitude, using sporangia or zoospore inoculum rather than mycelial mats may overcome the problem of uneven inoculum activation, the success of which we have experienced in smaller scale experiments.

Alternatively, the use of colonized plant tissue instead of mycelial mats as a source of inoculum may also produce a different outcome from the suppression of P. ramorum that we found with this approach in mixed inoculations. Interestingly, the kind of succession observed in field experiments did occur in a few control microcosms into which both Phytophthora species were accidentally contaminated . However, the limited occurrence and unknown relative quantity of original inoculum precluded more substantial evaluation. In any case, the suppression of P. ramorum sporulation in treatments where P. gonapodyides was present raises the question of what mechanism was responsible for the effect. It also furthers the impression that P. gonapodyides and other clade 6 Phytophthora species may have a moderating effect on the presence of P. ramorum in streams. The green leaves that we used were of mature cuticle and collected in midwinter and late summer for the first and second experiments, respectively. While some seasonal variation in susceptibility to P. ramorum infection has been reported in California bay leaves, the physical and chemical properties of mature leaves have also been reported to be relatively consistent throughout the year. Our results were similar for both experiments, and therefore, any variation in the leaves was overcome by experiment factors. The extensive colonization of brown leaves by P. gonapodyides and their limited colonization by P. ramorum is consistent with previous work where we showed that the former is a competent saprotroph while the latter is relatively ineffective at colonizing dead tissue. A significant discovery in this work was that P. ramorum colonized yellow, senescent leaves that were still fresh and had an intact cuticle to nearly the same degree as it did green leaves. At this stage, though chloroplasts and most of the protein content are gone from leaves, the cells are expected to be still alive, while in brown leaves that have dried the cells are no longer biologically active.

In fact, colonization of the yellow leaves by P. ramorum was not quite as extensive as its colonization of green leaves in the second experiment, which ran more or less concurrently and in which green and brown leaves were maintained in separate microcosms , though the difference between the separate experiments was not analyzed statistically. Though green leaves are shed into streams as a relatively low proportion of total litter, yellow leaves, often shed directly into streams from trees, constitute a much greater proportion of leaf litter in streams . This indicates that a great proportion of leaf litter in the streams is suitable for colonization by P. ramorum, and conforms to the regular recovery of this pathogen from natural leaf litter. Furthermore, the degree of colonization of yellow leaves by both Phytophthora species remained persistent throughout the 16 weeks, as with green leaves in the other experiments,growing lettuce hydroponically suggesting that the same kind of succession may be expected in these leaves as seen with green leaves in natural streams. Also consistent with previous findings with leaves colonized in naturally infested streams, leaves colonized by both Phytophthora species were generally conducive to sporulation as detected by baiting from the microcosms. Phytophthora gonapodyides was consistently recovered from P. gonapodyides-only and combined Phytophthora species inoculation treatments where it had colonized all green and brown leaves at all sampling points. The results from baiting of P. ramorum spores from microcosms were less regular, but nonetheless, mostly successful from microcosms containing colonized green or yellow leaves and occurred minimally from microcosms containing brown leaves which were colonized at only very low levels. The relatively less frequent recovery of P. ramorum by baiting from microcosms with non-sterilized stream water, not observed for P. gonapodyides, may be the consequence of P. ramorum not being well adapted to sporulation in biologically active aquatic environments or relying on different environmental signals. Nevertheless, these results confirm that both of these Phytophthora species can sporulate from colonized, decomposing leaves, whether green, yellow or brown leaves. Furthermore, at least under these conditions, their spores persisted for weeks and even months after any visible substrate was available, though the effect occurred more definitively and for longer with P. gonapodyides. As P. gonapodyides is not known to produce long-term survival structures, the question arises of how P. gonapodyides persisted so long in the microcosms in the absence of leaves. This observation also stands in contrast to our successful elimination of Phytophthora spores from original stream water collections simply by holding the water at cool temperatures for approximately three weeks. The observed persistence of spores of both Phytophthora species may be the result of an abundance of zoospore cysts due to the compact nature of the microcosms, or perhaps because the spores originated from propagules that would not have been suspended in the water column of the flowing streams. While oomycetes have been acknowledged as decomposers in aquatic environments until recently they have primarily been regarded as acting on non-cellulosic detritus such as insect and animal tissue. As most Phytophthora species are known as plant pathogens, the recent evidence that they may also degrade plant tissue in detritus is not surprising. Parasitism is considered an early characteristic in the evolution of oomycetes, but the possible evolution of a saprotrophic lifestyle from parasitic precursors has been considered for fungi and oomycetes.

Clade 6 Phytophthora are known to be opportunistic pathogens. Stradling saprotrophic and parasitic lifestyles, stream-resident Phytophthora may play an important role in the early breakdown of leaves and vegetative matter that still contain living cells. As facultative pathogens,clade 6 Phytophthora species can enter living cells and open intact tissues to further colonization by other saprotrophic organisms with less ability to penetrate living tissue. This is analogous to the paradigm of ‘conditioning’ of vegetative litter by pioneer microbial species, though in this case with respect to secondary saprotrophic microorganisms that could not on their own overcome physical and chemical protections still present in senescent but still alive leaf tissue. Our results were consistent with this hypothesis, as green leaves decayed more slowly in the absence of Phytophthora. It is uncertain why in the first experiment green leaves in the treatments with no Phytophthora inoculation decomposed very little over the entire 16 weeks of the experiment. In this experiment, both green and brown leaves were maintained together in microcosms, and it is possible that leachate from the leaves, particularly the brown leaves, may have had an inhibitory effect on some microorganisms. In the second experiment, leaves were leached prior to being deployed in the experiment, and also green and brown leaves were kept in separate microcosms. Green leaves in non-inoculated controls in the second experiment lost biomass to a degree ultimately similar to that of inoculated treatments, albeit at a slower rate. This indicates that other organisms were present that could initiate the decomposition of green leaves through the presence of Phytophthora accelerated it. We attempted additional isolations from some samples of leaves on acidified potato dextrose agar medium and found that the leaves in both controls and inoculated treatments were generally well colonized by a multitude of fungi . The fact that similar fungi occurred on leaves from microcosms prepared with both sterile and non-sterilized stream water suggests that many of these fungi were present on the leaves before entering streams as leaf litter .Additionally, overall there were no differences in decomposition rates between treatments with sterile or non-sterilized stream water added. Decomposition was also similar for leaves colonized by either Phytophthora species, indicating that, though P. gonapodyides is a better adapted saprotroph, both species had a similar effect on the decomposition of live, green and yellow leaves. This would be consistent with Phytophthora having the effect of opening integral tissue to colonization by other saprotrophs that then push decomposition forward. Finally, it is interesting that the presence of fungi in these leaves did not affect the persistence of P. ramorum throughout the experiments, suggesting that they are using different resources and that the successive displacement of P. ramorum in previous work may be specific to competition with other Phytophthora species or similar organisms. Under natural conditions, leaves would be exposed to a greater diversity of organisms, including other oomycetes such as Phytopythium species. As P. gonapodyides can colonize dead leaf tissue, it could be expected that it would contribute to leaf decay in brown leaves as well. This was not observed, as loss of biomass in brown leaves was the same in all treatments unaffected by Phytophthora colonization. The fact that P. gonapodyides substantially colonized brown, senesced leaves, but did not increase the rate of biomass loss raises the question as to what resources the organism uses in this substrate.