Progress 01/01/11 to 12/31/11
Target Audience:Target audiences included the Washington State Commission on Pesticide Registration, the United States Department of Agriculture Agricultural Research Service, the Weyerhaeuser Company, nursery growers, researchers, educators, students, and the general public. Changes/Problems:
What opportunities for training and professional development has the project provided?Two graduate students gained research skills through their involvement in this project. How have the results been disseminated to communities of interest?
What do you plan to do during the next reporting period to accomplish the goals?
What was accomplished under these goals?
Effect of Biological Control Agents and Fungicides The biological control agents Cease (Bacillus subtilis), Actinovate (Streptomyces lydicus) and Soil Guard (Gliocladium virens) were not very effective at inhibiting the growth of any of the Cylindrocarpon spp. in culture. Cease was better than Actinovate which was better than Soil Guard, but the highest inhibition was only 47.6%. Differences in bacterial and fungal antagonism methodologies made direct comparison of inhibition difficult between the fungal and bacterial tests. Overall, in all the tests we conducted all four biocontrol agents involved (B. subtilis, T. harzianum, G. virens, and S. lydicus) showed some degree of individual antagonism against isolates of Fusarium and Cylindrocarpon. Individual C. destructans and C. liriodendri isolates showed considerable variation when tested against four fungicides; FORE, Pantheon, 26GT, and Heritage. FORE, which had the highest active ingredient concentration (400 ppm) resulted in the highest growth rate reduction for C. destructans when compared to growth in control cultures. Some differences were seen in response to isolate tolerance to FORE and Pantheon, where the principle active ingredient was Mancozeb either formulated with Mn or Mn and Zn, respectively. It is surprising to observe tolerance to Heritage, which is a new class of fungicides (strobularins) that has only recently been added to fungicide protection programs in nurseries. C. destructans isolates were more tolerant of Chipco GT-26 than any other fungicide. Chipco (Iprolodiene) has been a primary fungicide for decades used to control top-blight (Phoma spp., Fusarium roseum, F. oxysporum) and foliar pathogens such as Botrytis cinerea. The observed Cylindrocarpon species and isolate tolerance to these four fungicides increases the likelihood of fungicide tolerance buildup with continued use. Previous work in theWeyerhaeuser Lab, Federal Way, Washignton) suggests Cylindrocarpon spp. is tolerant to most classes of fungicides, except those containing the active ingredient ethridizole (a component of Banrot fungicide). Unfortunately this fungicide is no longer registered for our nursery use. These results suggest a greater reliance on other Integrated Pest Management (IPM) strategies is needed to effectively control this pathogen group. Historically, biocontrol studies conducted in conifer nurseries have produced few positive results to reduce use of traditional fungicide control methods. Attempts to cultivate antagonistic populations of microbes using compost amendments, seed coatings, and fallow treatments have likewise failed to be successfully implemented. In this study, we attempted to establish and maintain levels of root colonization by biocontrol organisms using repeated drench applications. Biocontrol application strategy was timed to coincide with periods of active new root growth. We found that drench applications of various mixtures of biocontrol agents were successful at colonizing both greenhouse and nursery seedlings, but overall had little effect on reducing root pathogen colonization. In both the greenhouse and nursery, naturally occurring root bacteria identified as fluorescent pseudomonads (FP) populations were quantified on roots and observed to increase dramatically on Douglas-fir seedlings roots. FP isolates from Douglas-fir roots were shown to be more antagonistic of biocontrol agents than against either pathogen group. This observation may help explain the difficulty in the successful implementation of biocontrol treatments based on in-vitro findings into workable field disease control methods. Other researchers have found this same group of naturally occurring FP populations to suppress Cylindrocarpon spp. in diseases such as apple replant disease (GuMazzola 2003). We detected FP populations on roots of transplant seedlings, soil, and previously pasteurized potting mix used in this study, suggesting they are widely common. Further work is needed to understand the role of FP and other microbes play in root disease of conifer seedling nurseries. Frey et al. (1997) determined there was a lot of diversity within the FP population isolated from Douglas-fir roots. The discovery of a several Cylindrocarpon spp. in Douglas-fir seedling roots, coupled with isolate diversity with regards to root infection and tolerance to specific fungicides, has increased the difficulty of using fungicide management. Further complicating this task is the lack of fungicides which carry a "drench" label. In previous laboratory trials we have screened most available fungicides for efficacy against Cylindrocarpon (Weyerhaeuser, Unpublished Studies). Fungicides like Banrot and Dithane both showed the greatest inhibitory effect against Cylindrocarpon growth. Banner, Switch and Compass fungicides all showed moderate toxicity to Cylindrocarpon colony radial growth in culture. Daconil and Decree showed lower growth inhibition, while Captan and Chipco 20619 showed no inhibition at all. Unestum et al. (1989) found Benlate to be the best fungicide followed by Mancozeb and Maneb, then Daconil, and finally Captan and Curlan for control of C. destructans. Unfortunately, over the past few years Banrot and Benlate registrations have been removed from nursery use. Furthermore, Unestum et al. (1989) also observed that the very fungicide controls needed for control were inhibitory to biocontrol agents. The individual isolate fungicide tolerance measured in this study brings into question previous observations and knowledge concerning effective chemical treatments for disease control. Effect of Treatment on Seedling Growth None of the fungicide or biocontrol treatments negatively influenced seedling growth or root growth potential. Interestingly, seedlings receiving Cleary's drench showed significantly more height growth during the fall growth measurement period. In summary, F. commune, C. destructans and C. liriodendri remain two important root pathogens affecting both container and bare-root nursery culture in the Pacific Northwest. Both Cylindrocarpon spp. can produce chlamydospores, allowing the fungus to survive on root debris on discarded containers, fallow fields and equipment. Its presence on transplant seedlings, which are grown in fumigated sow fields, suggests Cylindrocarpon is somewhat tolerant of current fumigation practices as is F. commune. Molecular diagnostic tools (polymerase chain reaction -- PCR) have provided a means to increase our understanding of the complexity of root pathogens and their interactions with cultural and biological IPM. So far we have not found a successful method for controlling Cylindrocarpon or F. commune on Douglas-fir seedlings in containers or bare root nurseries. Specific Study Recommendations Use current PCR methodologies to screen a broader representation of the F. commune and C. destructans and C. liriodendri isolates commonly associated with conifer nursery root disease. Bridging the gap from laboratory studies to field biocontrol implementation may lie in using a genomics approach to solve this difficult problem. Refine current PCR analysis methodologies to allow a better understanding of the population genetics of these root pathogens as it relates to seedling pathology and fungicide resistance. Determine the variability of the response of different isolates to specific drench fungicide chemistries. Develop complimentary fungicide treatments which provide broad spectrum efficacy under greenhouse and bare-root growing conditions. This study suggests that a fungicide program based on a single effective fungicide will quickly fail. Increasing the number of available fungicides with a "drench" label are needed to build a robust IPM program to control this pathogen complex. Biocontrol agent applications as part of a nursery IPM management scheme will require a better understanding of interactions at the species level within the root rhizosphere.