Biological Control of Damping - Off Caused by Pythium ultimum and Rhizoctonia solani with Gliocladium virens in Soilless Mix

Lumsden, R. D., and Locke, J. C. 1989. Biological control of damping-off caused by Pythium ultimum and Rhizoctonia solani with Gliocladium virens in soilless mix. Phytopathology 79:361-366. Gliocladium virens controlled damping-off of zinnia, cotton, and Disease control efficacy lasted for at least 2 mo when G. virens was cabbage caused by Pythium ultimum or Rhizoctonia solani in nonsterile introduced with the pathogen inoculum and the mix was planted with soilless mix. This antagonist most effectively controlled disease among 50 zinnia seeds at intervals. The number of colony-forming units of G. virens isolates of bacteria and fungi, including species of Pseudomonas, Bacillus, remained high during the testing period, but the number of pathogen Trichoderma, and Penicillium. Twenty isolates of G. virens varied in their propagules was greatly reduced. The efficacy of the isolates tested, efficacy in controlling P. ultimum and R. solani. Some isolates controlled however, was not correlated with the number of colony-forming units of P. ultimum but not R. solani, and vice versa. This range of activity suggests G. virens. Sodium alginate formulations of isolate G20 of G. virens, a complex mechanism of action that might apply to one pathogen but not selected for control of both pathogens, maintained a high population the other. Inoculant of G. virens routinely was preincubated in the soilless density in a dry formulation when stored for 2 mo at 4 and 20 C, but not at mix before contamination of the mix with pathogen inoculum. Control of 30 C. Storage of an alginate formulation at these same temperatures in P. ultimum was effective when sporangial inoculum of the pathogen was air-dried soilless mix was not successful. Alginate formulations of G. virens introduced at the time of planting the host seed; however, control of added to soilless mix before planting seed show promise as a control for R. solani required prior contact of G. virens with inoculum of R. solani. damping-off in the greenhouse production of bedding plants. Additional keywords: biocontrol, soilborne pathogen. Damping-off diseases in bedding plant production are MATERIALS AND METHODS commonly encountered in the greenhouse and are primarily caused by the ubiquitous pathogen Pythium ultimum Trow and Antagonist isolates and preparation of inoculant. Several Rhizoctonia solani KUhn (11,25-27). Among several Pythium spp. antagonists from the collection of the Biocontrol of Plant Diseases that cause damping-off, P. ultimum is the most consistently Laboratory and from C. Howell, College Station, TX, were tested virulent and the most frequently isolated (27). Additionally, for their ability to control Pythium and Rhizoctonia damping-off. R. solani (anastomosis group 4) is commonly isolated from These included Gliocladium virens Miller et al from several sources bedding plants in midwestern greenhouses (25). (Table 1), hereafter referred to as Gl through G20; G. catenulatum Control of damping-off traditionally has emphasized proper Gilm. & Abbott (POPPu2); Trichoderma harzianum Rifai sanitation and manipulation of the environment. Disease control (POPS2 and TH-15); T. viride Pers. ex Gray (TiR9); Fusarium has been improved by the recent introduction of soilless potting solani (Mart.) Sacc. (POPPa26); Farrowia longicollea (Krzem. & media (2,28). Despite the improved control of damping-off, losses Bodura) Hawks (POPPul 1); Penicillium sp. Link ex Fr. are significant, and reliance on chemical fungicides is an accepted (CHC4B); Humicola fuscoatra Traaen (TABPul1); and several practice (11,21,28). However, fungicides are not the most desirable isolates of Pseudomonas spp. and Bacillus spp. All were previously means of disease control, for several important reasons. reported to suppress Pythium damping-off (18,19). Fungicides are heavily regulated and vary from country to country Cultures were maintained on V-8 juice agar (200 ml of V-8 juice, in their use and registration (13). Additionally, they are expensive, 800 ml of water, 1 g of glucose, 20 g of agar, and 6.0 ml of 1.0 N can cause environmental pollution, and may induce pathogen NaOH). Antagonist inoculant usually was added to planting media resistance (11,13). Also, fungicides can cause stunting and as 3-day-old cultures on autoclaved wheat bran medium (a 1:1 chlorosis of young seedlings (11). The effectiveness of chemical mixture of bran and water) as previously described (14,16), at the fungicides may vary if they interact chemically with planting media rate of 1%, on a dry-weight basis. Other media used for mycelial or are adsorbed, inactivated, or decomposed by components of the cultures contained vermiculite (400 g), yeast (80 g), molasses (48 ml), media (28). and water (1,600 ml); and peat moss (720 g), yeast (80 g), molasses The objective of this study was to identify potentially useful (48 g), and water (800 ml). All media were sterilized before the antagonistic microorganisms that effectively control the two major addition of 1 X 107 conidia per 100 g of culture. Incubation was for causes of damping-off disease, P. ultimum and R. solani, in 3 days at room temperature under cool white fluorescent lights. greenhouse bedding plant production in nonsterile soilless growing Alginate pellets (prills) were prepared as previously described media. (15,17), but instead of fermenter biomass, 1 X 107 conidia of G. virens (G20) were added to 100 ml of a suspension containing 1% sodium alginate, 1% vermiculite, or 5% wheat bran in water. This article is in the public domain and not copyrightable. It may be freely Tesseso a rpe no02 aladtegle reprinted with customary crediting of the source. The American prills were rinsed in tap water and air-dried. Prills were added to Phytopathological Society, 1989. planting mix at the rate of 1%, on a dry-weight basis. Vol. 79, No. 3, 1989 361 Pathogen inoculum. Isolate PuZ3 of P. ultimum was from a mix and incubated for 1 wk at 20-30 C in plastic bags before damped-off zinnia seedling (Zinnia elegans L.) grown in a soil from planting, except when amended soilless mix was held for assessing Beltsville, M D. Other isolates used were PuCNJ from cabbage and storage ability or for determining the effect on disease after PuCTx from cotton. Cultures were maintained on cornmeal agar, extended incubation. and sporangial inoculum was prepared by a modification of a To simulate contamination of pathogen-free potting mix, we previously described method (3). Cultures (3 days old on cornmeal added inoculum of P. ultimum or R. solani at seeding, using agar) were flooded with 10% soil extract prepared as described inoculum prepared as described above. In later experiments, we previously (3). One-month-old cultures were harvested by scraping found that inoculum of R. solani could be added before seeding for sporangia from the surface of the plate and blending them for 30 enhanced biocontrol. Zinnia was selected as the host species sec in a Tissuemiser (Tekmar, Cincinnati, OH). Sporangia were because of its susceptibility to the damping-off pathogens and for counted with the aid of a hemacytometer and diluted in 100 ml of convenience in handling. The hybrid cultivars Gold Sun or State tap water to provide 300 sporangia per square centimeter when Fair (Park Seed Co., Greenville, SC) were used. After 40 seeds drenched onto the surface of the planting medium in a 16-X 12-cm were planted in each flat (four rows of 10 seeds each), flats (12 X flat. 16 cm) were drenched with sporangia of P. ultimum in 100 ml of The primary isolate of anastomosis group 4 of R. solani (R-85) tap water (300 sporangia per square centimeter). All flats were originally was from a Maryland-grown cucumber seedling; the watered thoroughly. Flats infested with Pythium were incubated in isolate was recultured from a damped-off zinnia seedling grown in a growth room at 15-20 C; flats infested with Rhizoctonia were greenhouse soil infested with this isolate. Other isolates used were incubated in a growth chamber at 25-30 C. Both were supplied R2 from cabbage in New Jersey and RDB1 from cotton in Texas. with supplemental fluorescent light, to provide about 60 and 50 Inoculum was grown in sterile cornmeal sand (240 g of clean quartz W/m 2 (400-850 nm), respectively. Seedling stand was determined sand, 6.0 g of yellow cornmeal, and 75 ml of water) for 2 wk at 25 C. for P. ultimum after 1 wk (primarily preemergence damping-off) In each of four replicate flats of planting mix, 1.25 g of inoculum and for R. solani after 1 and 2 wk (primarily postemergence was incorporated, damping-off). All tests were repeated at least twice and included at Inoculum of P. ultimum and R. solani was also prepared with least four replicate flats per treatment. soilless mix previously infested as described above and cropped Population density assays of G. virens and the pathogens. with zinnia seedlings to simulate inoculum from naturally infested Samples of soilless mix were taken at different intervals to soil (Fig. 1). The infested soilless mix was diluted with noninfested determine the population density of G. virens. Serial dilutions were mix to give about 90% disease. prepared after 10 min of vigorous stirring in distilled water, and Biological control bioassay. Biocontrol studies were performed 1.0and 0.1-ml samples were spread on the surface of TME in soilless potting mix (Redi-Earth, W. R. Grace & Co., semiselective medium (24). Fungal colonies on the agar plates were Cambridge, MA), pH 5.5-6.5, moistened with water to a moisture counted 5-7 days after incubation at 25-28 C under continuous level of approximately 60%, on a dry-weight basis. The ingredients fluorescent light, and colony-forming units (cfu) were calculated of the mix were as previously reported (2). Preparations of per gram (dry weight) of soilless mix. antagonists were incorporated into moistened, nonsterile soilless Statistical analyses. The experiments were arranged in a randomized complete block design and were repeated at least twice. Each treatment contained four replicates. Resultant data TABLE 1. Range of efficacy of Gliocladium virens isolates against from repeated experiments were combined, and statistics damping-off of Zinnia elegans caused by Pythium ultimum and performed on the combined data, except with repeated experiRhizoctonia solani ments in which differences in sampling times were not identical. Colony-forming Analyses of data were usually done by analysis of variance with unitsonfoG.ireins separation of means by Duncan's multiple range test. Data per gram of expressed in percentages were corrected for uneven distribution by Previous Percent plant standxy soilless mix arc sine transformation and back-transformed. Repeated measure Isolate designation P. ultimum R. solani (X 106)x.7 analyses with general linear model procedures were done on timeHealthy dependent experiments and on population density studies with HealthyP. ultimumn and R. solani. control 88.2 a 86.1 a <0.1 eP.utm adRsoni Pathogen Population densities of R. solani and P. ultimum were control 9.4 g 26.6 fgh <0.1 e determined with a pellet soil-sampling device (8). Fifteen pellets GI GVRJ-I 29.0 efg 23.6 gh 2.7 d (5 X 5 mm) were deposited on the surface of a medium selective for G2 GL2 77.7 abc 30.0 efgh 5.8 d P. ultimum (6) or water agar containing 50 ptg each of streptomycin G3 GL3 24.5 efg 23.8 gh 4.2 e sulfate and Aureomycin per milliliter for R. solani. After 24and G4 GL4A2 26.4 efg 27.4 fgh 15.4 b 48-hr incubations at 20 and 25 C, respectively, colonies of G5 MTD356-14 33.7 efg 41.2 cdefgh 8.4 c P. ultimum and R. solani growing from the pellets were counted. G6 GV6 74.5 abc 25.1 defgh 9.0 c Rgeso nlsso auscretdfrPisndsrbto G7 MTD29-l 86.8 a 43.9 cdefgh 5.3 d Rgeso nlsso auscretdfrPisndsrbto G8 G8 156 fg 22.0gh 92 ~was used to analyze relative numbers of propagules of the G9 GVP 40.6 cdefg 24.5 gh 6.8 c pathogens during the course of an experiment, as indicated GIO0 GVMT 89.5 a 25.7 gh 0.2 e elsewhere (8). GIl MTD510-1 51.1 bcde 65.5 be 8.8 c G12 MTD356-ll 34.9 defg 44.4 cdefg 11.3 b RESULTS G13 MTD29O-18 41.9 bcdefg 39.9 defgh l0.4 c G14 MTDI38-10 32.5 efg 25.1 gh 13.2 b Biocontrol microorganisms tested. In a preliminary survey, over G 15 MTDI189-10 79.4 ab 78.6 ab 5.5 d 50 fungal and bacterial isolates were tested for their efficacy against G16 MTD3I-10 33.7 cdefg 58.4 be 9.7 c damping-off pathogens of zinnia. Six isolates of Gliocladium spp., G17 GL17 20.9 efg 36.7 defgh 8.0 c 17 of Trichoderma spp., 18 of Talaromycesfiavus (KlbScker) Stolk G18 GV1828 19.7 efg 51.0edef 4.5 e G19 GVA2-3 13.3 g 20.1I h 37.6 a & Sampson, five of miscellaneous fungi, and 11 of bacteria were G20 GL2I 56.5 abede 54.1 ede 7.9 c tested against Pythium and Rhizoctonia damping-off in soilless 'Values in each column followed by the same letter are not significantly mix. The bacterial isolates and the isolates of T. flavus and different according to Duncan's multiple range test (P= 0.01). Trichoderma spp. were ineffective. Only isolates of G. virens YPlant stand was determined 2 wk after planting. Data were transformed to significantly improved seedling stand in the presence of P. ultinmum arc sines for statistical analyses and back-transformed. or R. solani. Isolate G20 (previously designated GL21) was 'Colony-forming units per gram of dry soilless mix I wk after the addition selected from this preliminary study because of its ability to control of 1.0% bran culture and incubation at 60% moisture content. diseases caused by P. ultimum and R. solani and because of past