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Accession No.
Grant No.
Project No.
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Project Start Date
Jan 1, 2011
Project End Date
Dec 31, 2011
Grant Year
Project Director
Edmonds, R.
Recipient Organization
Performing Department
Forest Soils
Non Technical Summary
We wish to determine the efficacy of soil drenches of fungicides and biocontrol agents in protecting bareroot Douglas-fir seedlings from infection by Cylindrocarpon spp. isolates commonly found in conifer nurseries. We will also conduct phytotoxicity and Root Growth Potential tests, to determine if any of the fungicide/biocontrol treatments have any adverse effects of the root growth potential (RGP) of the seedlings after storage. Knowledge of the relative efficacy and compatibility of co-applied fungicide and biocontrol agents could have the potential to decrease fungicide use, increase the longevity of currently available fungicides and in so doing, reduce fears of fungicide tolerance buildup, while improving seedling yield and performance.
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Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
The objectives of this project are: 1. Determine the efficacy of soil drenches of fungicides and biocontrol agents in protecting bareroot Douglas-fir seedlings from infection by Cylindrocarpon spp. isolates commonly found in conifer nurseries. 2. Determine if any of the fungicide/biocontrol treatments have any adverse effects of the root growth potential (RGP) of the seedlings after storage.
Project Methods
1. Trials: Greenhouse experiments will be conducted at the University of Washington. Field trials, production of inoculum and isolations, and seedling production trials will be conducted at various Weyerhaeuser facilities. 2. Production of Plant Material: Container seedlings will be used to reduce the risk of introducing Cylindrocarpon infected plant material prior to the test. Prior to initiation of fungicide and biocontrol screenings, a representative sample of test seedlings will be removed and root plated to determine the incidence and severity of infection by Cylindrocarpon species. Root systems from twenty-five seedlings will be individually washed and surface sterilized before platin. Roots will be incubated to quantify levels of Cylindrocarpon infection. 3. Production of Fungal Inoculum: Isolates of Cylindrocarpon destructans and C. didymum previously isolated from Douglas-fir seedling roots will be used in the study. At least five distinct isolates of each fungus species will be combined to make the test "inoculum." Isolates will be transferred from previous stock cultures, plated, and grown to produce macroconidia. Macroconidial suspensions will be applied to seedling growing media. 4. Efficacy Tests: The efficacy of fungicide drench treatments and biocontrol agents in protecting seedling roots from Fusarium and Cylindrocarpon will be quantified. 5. Phytotoxicity Tests: To determine if any of the fungicides have any adverse effects on the seedlings, fungicides will be drenched on non-inoculated seedlings at the rates used in the efficacy tests. A root growth potential (RGP) test will be done to determine if any of the treatments have any adverse effects on growth of roots from the seedlings. 6. Root Growth Potential (RGP) Test: Seedlings from each treatment will be lifted, repotted, and grown in a temperature controlled stress room for 4 weeks. In the stress room, conditions will simulate spring outplant conditions. Seedlings will be grown with artificial light. At the conclusion of the tests the seedlings will be unpotted and the new roots greater than 1 cm counted. Morphological data will be taken on a representative sample. 7. Pathogen Testing- A: Experiment designs and data collection: The experiment designs for the efficacy and phytotoxicity test will be a randomized complete block design. The efficacy and phytotoxicity tests will be conducted in a greenhouse maintained at 15-20 degrees C. During this time, the seedling will be examined for any above ground symptoms. After 6 weeks, the efficacy test will assess the effects of the treatments on root health. To confirm that symptoms are associated with Cylindrocarpon infection, roots with disease symptoms will be surface sterilized and plated onto selective media. All data will be subjected to analysis of variance (ANOVA). The treatment means will be separated by Fisher's protected least significant difference (LSD) test at P = 0.05. All analyses will be conducted using SAS software.

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: Nothing Reported 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? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

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.