Alexander B. Filonow
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078
John M. Dole
Department of Horticulture and Landscape Architecture, Oklahoma State University, Stillwater, OK 74078
Pythium ultimum, which causes root rot and damping-off of many floricultural crops grown in Oklahoma greenhouses, produces oospores for survival and to initiate disease. Strains of Actinoplanes spp. that are hyperparasites of oospores were evaluated for their biological control of Pythium root rot of plants grown in a greenhouse. In soil-less potting mix infested with 103 oospores of P. ultimum /g mix, strains W57, W257, or 25844 of Actinoplanes spp. on clay granules applied at 5% or 0.5% w/w to the mix 5 d prior to replanting geranium or poinsettia seedlings reduced root rot severity and increased plant stand compared to nontreated plants after 6 wk. Granules of W257 applied at 5% w/w to mix 7 d prior to replanting poinsettia seedlings reduced root rot of the plants after 6 wk, whereas seedling dips in a suspension of macerated mycelium of W257 (109 colony-forming units/mL) had no effect. Strain W257 applied as granules (1% w/w) or as a root dip (8×108 colony-forming units/mL) 7 d after replanting poinsettia seedlings was as effective as a fungicide (metalaxyl) in reducing root rot after 9 weeks, but no treatment increased plant stand. Colonization of roots by P. ultimum was reduced by metalaxyl or W257 compared to roots from nontreated, pathogen-infested mix. Actinoplanes spp. show promise in the biological control of P. ultimum in greenhouse floricultural crops. ©Oklahoma Academy of Science.
Production of floriculture crops grown in greenhouses is frequently limited by Pythium spp. that cause seed damping-off and root rot (1-3). In Oklahoma, Pythium spp. attacked 10% of the flower and house plant samples submitted in 1992 to the Oklahoma State University Plant Disease Diagnostic Laboratory (Jacobs, unpublished data), causing an estimated $1 million annual loss to the Oklahoma ornamental and nursery industry. Genetic resistance to Pythium diseases is low in several important greenhouse crops, such as geraniums and poinsettias. Presently available fungicides are often costly to use and may become environmentally unacceptable in the future. In addition, fungicides have little effect against oospores, which are the small, hardy spores of Pythium spp. that may persist in dry soil or soilless media for years and still cause disease. Current microbial-based biological controls (1, 4-6) are an alternative to fungicides, but none destroys oospores.
Actinoplanes spp. are a novel group of filamentous bacteria that produce minute sporangia (7), which when hydrated release motile spores capable of parasitizing Pythium spp. or related fungi (8-10). A clay pellet formulation that was developed for applying sporangia to soil has been shown to reduce Pythium root rot of bush beans and table beets in field microplots (11). Little is known, however, about the potential of Actinoplanes spp. to control diseases of Pythium spp. in floricultural crops in greenhouses. Therefore, we evaluated the effectiveness of Actinoplanes spp. to biologically suppress the damping-off and root rot caused by P. ultimum Trow, which is a troublesome fungal pathogen in greenhouses, in geraniums (Pelargonium x hortorum Bailey) and poinsettias (Euphorbia pulcherrima Willd. ex Klotzsch).
Preparation of Pythium-Infested Potting Mix and Actinoplanes spp. Application: Oospores of P. ultimum were grown in dilute corn meal broth, harvested, (8) and mixed into potting mix (peat/ perlite/ vermiculite; v/v/v; limed to pH 6.8) at 103 oospores/g of mix. Strains A25844 (A. brasiliensis), W57, and W257 of Actinoplanes spp. were evaluated for biological control because of their high degree of parasitism of oospores of Pythium spp. (8). Actinoplanes spp. strains were applied as sporangia on clay granules (11) or as a root dip in a suspension of comminuted myce-
lia in 1% carboxymethylcellulose (CMC) as a sticker (12). All experiments were conducted in greenhouses under conditions similar to commercial greenhouse culture (2, 3). Plants were grown in peat /perlite /vermiculite potting mix, in 10-cm diameter pots, unless otherwise stated.
Effectiveness of Actinoplanes spp. Granules: Clay granules with Actinoplanes spp. were mixed into moist, P. ultimum-infested potting mix at 5% or 0.5% w/w in plastic bags. After 5 d incubation at 24°C to allow parasitism of oospores, the contents of the bags were transferred to pots. Red Orbit geraniums from seed and Guthier V-14 Glory poinsettias from cuttings in Oasis (Oasis, Smithers-Oasis, Kent, OH) blocks were replanted into the pots. There were 10 replicates per treatment per pathogen per host. Controls consisted of plants in nontreated, infested potting mix, and in infested mix treated with 5% granules without Actinoplanes sp. After 6 wk, plant stands were counted, and the roots harvested and washed in running tap water. Root rot severity in geraniums was rated using an index of 1-6, where 1= healthy root, 2 = 1-25% root rot, 3 = 26-50% rot , 4 = 51-75% rot, 5 = >75% root rot, and 6 = nonrecoverable root. Root rot severity in poinsettias was rated, using a 1-5 index, where 1 = healthy root, 2 = 1-25% root rot, 3 = 26-75% rot, 4 = >75% root rot, and 5 = nonrecoverable root.
Granules of W257 Compared to Root Dipping in a W257 Suspension: Potting mix infested with P. ultimum was amended with 5% w/w granules of W257 in plastic bags as described above and incubated for 7 d at 25°C prior to dispensing into pots. Poinsettia seedlings in Oasis blocks were then planted into the pots. Oasis blocks with poinsettia roots also were dipped into a W257 suspension (109 colony-forming units/mL) and immediately planted into pots with infested mix. There were 20 replicates per treatment. Controls were poinsettia roots dipped in CMC and poinsettia seedlings in nontreated, infested potting mix. Stand and root rot severity were determined 6 wk later.
Efficacy of W257 Applied as Granules or a Root Dip Compared to a Fungicide: Granules of W257 (1% w/w) and a root dip of W257 (8 × 108 colony-forming units /mL) were compared to a fungicide drench of metalaxyl (150 mL/pot of 0.2 cc Subdue /6L; Novartis, Inc., Greensboro, NC) for suppressing damping-off of poinsettia plants. Granules of W257 were added to infested potting mix in 15-cm diameter pots 7 d prior to planting. Root dips were planted immediately after treatment. Controls were 1% w/w granules without Actinoplanes sp.; root dips in CMC; and nontreated, pathogen-infested potting mix. There were 15 replicates per treatment. Root rot severity was measured 9 wk later. Pieces of roots from five randomly selected plants were planted on a Pythium selective medium (13) to assess the effect of treatments on root colonization by P. ultimum.
Data Analyses: Percentage plant emergence, root rot severity, and percentage recovery of P. ultimum from roots were arcsine transformed prior to ANOVA, and means were compared with the Student Newman Keul's test (P £0.05).
Effectiveness of Actinoplanes spp. Granules: Granules of strains A25844, W57, and W257 of Actinoplanes spp. applied at 5% or 0.5% (w/w) to root medium infested with P. ultimum oospores reduced root rot severity of geranium and poinsettia seedlings compared to untreated plants (Table 1). Suppression or root rot was greater with the 5% (w/w) treatment than with the 0.5% treatment. Clay granules without Actinoplanes spp. (5% w/w) did not reduce root rot severity, indicating that Actinoplanes spp. were the active agents of disease control.
Actinoplanes spp. also significantly increased stand after 6 wk. In control pots only 30-40% of plants survived, whereas 100% of geraniums survived in pots receiving granules with Actinoplanes spp. strains (5% w/w) (data not presented). At 0.5% w/w surviving geranium plants in pots treated with A25844, W57 or W257 were 80%, 100%, and 80%, respectively, of initial plantings. There was 100% survival of poinsettia plants in pots treated with either 0.5 or 5.0% (w/w) of Actinoplanes spp., but this survival was not significantly different (P = 0.05) than the 70% survival in the untreated pots.
Granules of W257 Compared to Root Dipping in a W257 Suspension: Poinsettias in root medium treated with strain W257 applied as 5% granules had reduced severity of root rot (Table 2) compared to the untreated control; however, root dips in a suspension of W257 did not decrease severity. The CMC treatment sticker did not reduce root rot symptoms compared to the untreated control, suggesting that disease suppression was due to Actinoplanes spp. No increase (P= 0.05) in poinsettia plant survival was found in Actinoplanes-treated pots (75-85%) compared to the controls (50%).
Efficacy of W257 Applied as Granules or a Root Dip Compared to a Fungicide: Poinsettias in pots that were treated with 1% w/w granules of W257, root dipping in a W257 suspension, or metalaxyl had less root rot than the control treatments (Table 3). Root rot control by W257 was
as effective as metalaxyl. None of the Actinoplanes or metalaxyl treatments increased plant survival after 9 wk compared to the control.
Recovery of P. ultimum from poinsettia roots in root medium treated with metalaxyl or with strain W257 was less frequent than that from roots in untreated, pathogen-infested medium (Table 3). Recovery from roots in medium treated with granules or CMC was not different from the untreated control. Less P. ultimum was recovered from root medium treated with granules of W257 than from root medium treated with unamended granules. Percentage recovery from roots in medium treated with W257 was not different from roots in medium treated with metalaxyl. These results indicate that the colonization of roots by P. ultimum was suppressed by strain W257.
Results from this study suggest that Actinoplanes spp. may have promise for reducing Pythium root rot in floricultural plants in the greenhouse. Actinoplanes strain W257 applied as granules or as a root dip was as effective as the fungicide metalaxyl in reducing root rot after 9 wk (Table 3). Colonization of poinsettia roots by P. ultimum was reduced by either metalaxyl or W257 compared to roots from untreated, pathogen-infested medium.
Realizing the potential of Actinoplanes depends on developing a practical application method and inoculation time. Application of Actinoplanes spp. on granules to root growing medium is a method compatible with greenhouse operation; however, the lowest granular rate (0.5% w/w) of Actinoplanes spp. effective in our study may not be an economical level to apply. A rate of 0.01-0.1% (w/w) would be more practical for growers. A lighter carrier than the currently used clay granules or an increase in sporulation density on the carrier could reduce the granular rate needed. Further study may produce an effective yet economical granular rate. In addition, microbial parasites need time to find their host and infect. In our experiments we al-
lowed 5-7 d of prior incubation of Pythium-infested medium with Actinoplanes spp. granules. We need to determine the minimum incubation time for control.
Our results with root dips as an application method for Actinoplanes were mixed; nevertheless, further study of this method of protecting roots against Pythium spp. is warranted. Root dips, especially roots encased in foam blocks which may absorb and retain biocontrol agents, are a method compatible with the greenhouse culture of several floricultural crops. The results with root dips also raises the question of mode of action, because roots are replanted immediately after dipping, and the incubation time needed for parasitism of oospores most likely would be limited. Actinoplanes spp. are known (14) to produce antibiotics, so antibiosis may play a role. However, little is known about the use of antibiotic-producing strains of Actinoplanes spp. in the biocontrol of Pythium spp., and further research is also warranted in this regard.
Other biocontrol agents, such as Trichoderma harzianum (4) or composted amendments (1) inhibit hyphal growth of P. ultimum in soil, but they have little effect against oospores of the pathogen. Biocontrol agents that effectively parasitize oospores in planting mix would reduce the survival of P. ultimum in soil and greenhouse root medium. Moreover, because the oospore is the sexual stage where genetic recombination occurs, reducing oospore populations may help maintain the efficacy of current anti-Pythium fungicides by reducing the frequency of new fungicide-resistant isolates (15). In addition, the combination of Actinoplanes spp. which are known parasites of oospores (8-10), with other biocontrol agents that inhibit hyphal growth may provide greater control than either agent used separately.
Approved for publication by the director of the Oklahoma Agricultural Experiment Station. This research was supported under project OKL 2215. The interest and generous financial support of the Fred C. Gloeckner Foundation for this work is greatly appreciated. We thank Randall Smith for technical assistance.
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Received: October 19,1998; Accepted: April 16,1999