Hot Water and Copper Coatings in Reused Containers Decrease Inoculum of Fusarium and Cylindrocarpon and Increase Douglas Fir Seedling Growth

Inoculum of Douglas fir root diseases caused by the fungi Fusarium and Cylindrocarpon is carried from crop to crop in reused containers. Soaking containers for 90 seconds in 80 °C water removed ≈99% of Fusarium and 100% of Cylindrocarpon inoculum between growing cycles. Overall seedling growth was also improved: seedlings grown in containers soaked between growing cycles were 10% taller and had 20% more biomass than seedlings grown in nonsoaked containers. We obtained a 13% increase in the number of deliverable seedlings from containers soaked in hot water between crops, from the use of copper coated containers, or from both practices combined. cals, problems of chemical disposal, and higher efficacy against pathogens (Dumroese et al., 1993; James and Sears, 1990; Peterson 1990, 1991). Another potential problem with containergrown seedlings is poor root egress from the upper portions of the root plug after planting (Burdett, 1978, 1979a). New roots often fail to grow in a natural pattern (Balisky et al., 1995) that can affect seedling stability (Burdett, 1978) and may limit seedling access to nutrients, water, and mycorrhizal inoculum (Dumroese, 2000). Researchers have found a coating of copper on interior surfaces of containers changes Douglas fir root morphology at the nursery and improves root egress and form after outplanting (Burdett, 1978; Burdett and Martin, 1982; Wenny et al., 1988; Wenny and Woollen, 1989). This technique is also effective on many other horticultural plants (Appleton, 1993; Arnold, 1996; Brass et al., 1996). In a pilot study, a copper-coating was also effective in reducing seedling infection and colonization by F. proliferatum (Dumroese et al., 1995). This was not surprising since copper is also one of the oldest and most effective fungicides (Pirone, 1978). Copper sulfate and lime (Bordeaux mixture) has widespread toxicity against many plant pathogenic fungi (Johnson, 1935), cuprous oxide effectively protects seeds against seed-borne pathogenic fungi (Horsfall, 1932), and several other insoluble copper compounds, applied as sprays or dusts, protect plants against pathogens (Walker, 1969). Although toxic at high concentrations, copper is an essential nutrient for Fusarium sp. at low concentrations (Steinberg, 1950; Woltz and Jones, 1981). Our objectives were to quantify the rate of fungal inoculum build-up on reused seedling containers, how that affected seedling growth, and compare the efficacy of hot water treatments with the copper-coated containers for controlling fungal inoculum in a commercial nursery setting. Materials and Methods Our experiment was a completely randomized two container type × two cleaning treatment factorial arrangement conducted for five growing cycles. The two container types were Styroblock and Copperblock containers that were identical (160 cavities per container aligned in 10 rows and 16 columns, 90-mL cavity volume, and 764 cavities per m; Beaver Plastics, Edmonton, Alberta, Canada) except that Copperblocks have a proprietary copper oxychloride coating on cavity walls. Cleaning treatments were either no container cleaning (control) or submerging (soaking) containers in hot water (77 to 82 °C) for 90 s. We assigned three containers to each container type–soaking combination to serve as replicates and these containers remained within that treatment for five growing cycles. For each 10-month growing cycle, the same northern Idaho source of Douglas fir was sown in March and seedlings were harvested in December. We randomly placed all conReceived for publication 13 Nov. 2001. Accepted for publication 18 Jan. 2002. We thank K.E. Quick and S.J. Morrison at the Research Nursery for their technical assistance; D. Gerdes and Silvaseed Co. for providing containers; the Idaho Dept. of Lands for financial assistance; K. Steinhorst for assistance with statistical analysis; and R.R. Tripepi, G. Newcombe, B. Maynard, and E. Hinesley, and the anonymous reviewers for their comments on earlier drafts. Plant Physiologist. Plant Pathologist. Professor/Director. Douglas fir [Pseudotsuga menziesii (Mirbel) Franco] is commonly grown for reforestation in the northern Rocky Mountains and Pacific Northwest. Seedlings are usually grown as a 1-year crop at high densities (up to 1076 seedlings/m) in styrofoam or hard plastic containers that are reused for several crops. Seedling production in containers is often hampered by Fusarium root disease (James et al., 1987), and losses vary with seedlot and nursery. Fusarium proliferatum (Matsushima) Nirenberg is the most common species, and often the most virulent pathogen, infesting containers and colonizing seedlings (Dumroese et al., 1993; James et al., 1997). Another root pathogen found in nursery environments is Cylindrocarpon (Beyer-Ericson et al., 1991), commonly found in the rhizosphere of container-grown conifer nursery stock (James et al., 1994; Kope et al., 1996). Many Cylindrocarpon sp. are readily isolated from roots of symptomatic and nonsymptomatic seedlings in inland Pacific Northwest nurseries (James et al., 1994). Often, infected plants lack aboveground symptoms (e.g., chlorotic or necrotic foliage) despite extensive root decay. Cylindrocarpon sp. may act in conjunction with other root pathogens to cause major disease problems (Bloomberg and Sutherland, 1971; Duda and Sierota, 1987). Generally, root disease is difficult to control because root systems usually are extensively colonized before shoot symptoms appear (James et al., 1987), and chemical fungicide applications are generally ineffective (James, 1986a; James et al., 1988b). Therefore, integrated pest management is the best alternative to control root disease (James et al., 1990). Reducing inoculum of pathogenic organisms is important for limiting seedling infection (James et al., 1990). Although seeds may be an important source of Fusarium (James, 1986b), inoculum may also be carried from crop to crop on interior walls of reused containers (James et al., 1988a; James and Gilligan, 1988a; Sturrock and Dennis, 1988), and it may be particularly concentrated at the bottom of containers (James, 1989; James and Gilligan, 1988b). Immersion in hot water is a common method to sterilize plastic pots (Hartmann et al., 1990) and works well for both styrofoam and plastic containers used in reforestation nurseries (Sturrock and Dennis, 1988; James, 1989; James and Woollen, 1989). In forest nursery situations, hot water is preferable to chemical treatments such as sodium metabisulfite or bleach solutions due to reduced potential for worker exposure to irritating or toxic chemi7109, p. 943-947 9/25/02, 10:56 AM 943 HORTSCIENCE, VOL. 37(6), OCTOBER 2002 944 PLANT PATHOLOGY tainers within a production crop of Douglas fir at the Univ. of Idaho Forest Research Nursery that were grown following our basic regime (Wenny and Dumroese, 1992) and without fungicides. Seedlings were grown the first two growing cycles in a 1 sphagnum peat moss : 1 vermiculite (v/v) growing medium in a fullyenclosed greenhouse. We grew seedlings the final three growing cycles in a 7 sphagnum peat moss : 3 Douglas fir sawdust (v/v) medium; seedlings were started inside the greenhouse and moved to an open-sided greenhouse when seedlings were at ≈75% of target height. After each growing cycle and from a randomly-selected starting point, we systematically extracted 20 seedlings from each container (60 seedlings per container type– soaking combination), avoiding seedlings in the outer two rows and two columns to minimize edge effects. Seedlings were classified as deliverable or cull based on nursery criteria: stem diameter >2.3 mm; height >15 cm, but <30 cm; and firm root plug. Roots were gently washed to remove adhering medium. We measured seedling root volume using Burdett’s (root collar to tip of terminal bud), and stem diameter at the root collar (RCD). From each seedling, 10 root tips, each ≈1 cm long, were randomly removed, surface sterilized in a 1 bleach (5.25% sodium hypochlorite) : 10 water (v/v) solution for 1 min, rinsed in sterile water, and aseptically placed on a Komada’s selective medium (Komada, 1975). Root tips were incubated under cool-white fluorescent, diurnal light (12-h photoperiod) at 22 to 24 °C for 7 d. Although isolations were made onto Komada’s medium which was initially designed to isolate Fusarium oxysporum Schlect. from natural soil (Komada, 1975), our subsequent work indicated that this medium is selective for other Fusarium sp. (James et al., 1989, 1997), Cylindrocarpon sp. (Dumroese et al., 2000; James et al., 1997), and Trichoderma sp. (Mousseaux et al., 1998) as well. Fungal identification involved obtaining single-spore isolates from colonies on Komada’s medium and culturing them on both carnation leaf agar (Fisher et al., 1982) and potato dextrose agar. Fusarium species were identified based on the presence or absence of chlamydospores, macroconidial morphology, and the production of microconidia on chains or in false heads borne on monoor polyphialides (Nelson et al., 1983). Cylindrocarpon sp. were identified using Booth’s (1966) taxonomy; our identifications were based on macroconidial morphology, length, and septations and presence or absence of microconidia and chlamydospores. Seedlings were classified as infected if one or more root tips were colonized. Percent colonization was calculated by dividing the number of root tips infected by the total number of root tips sampled for all seedlings within the treatment. Seedling biomass was determined after drying shoots and roots at 60 °C to constant weight. One month after seedlings were extracted from containers, we assayed containers for residual Fusarium inoculum. Two pieces of styrofoam, each ≈0.5 cm, were removed from the bottom drainage hole of 10 randomly selected cavities per container (30 cavities per container/soaking combination), placed on Komada’s selective medium and incubated as described above. Immediately after sampling, containers assigned to the soaking treatment were immersed, allowed to dry, and then the same cavities were resampled to assess the efficacy of the soak. This technique did not affect the ability of individual cavities to hold medium in subsequent cycles. Statistical analysis. Percentage data were transformed using the arcsin of the square root of the value. We used a general linear model (PROC GLM; SAS Institute, 1989). The error term for main effects (container type and soaking) was the residual variation of these variables averaged over time (cycles). Treatment means were separated using Tukey’s honestly significant different test (HSD) when P ≤ 0.05. Data were back transformed as necessary for tables.