Suppression of take-all of wheat by seed treatments with fluorescent pseudomonads.

Weller, D. M., and Cook, R. J. 1983. Suppression of take-all of wheat by seed treatments with fluorescent pseudomonads. Phytopathology 73:463-469. Strains of fluorescent Pseudomonas spp. applied to wheat seeds from nontreated seed. Tests in field plots fumigated with methyl bromide, suppressed take-all in both greenhouseand field-grown winter and spring with and without the reintroduction of G. graminis var. tritici, established wheat. The effective strains were originally isolated from roots of wheat that the bacteria do not promote plant growth other than by controlling of grown in soil naturally suppressive to take-all and were selected on the basis take-all. The seed treatment resulted in increased yields of up to 147% in of in vitro antibiosis to Gaeumannomycesgraminis var. tritici. Isolate 2-79, fumigated soil and up to 27% in natural soil. An antibiotic-resistant strain alone or combined with isolate 13-79, suppressed take-all in five of six field of 2-79 was isolated from the roots of wheat in the field following tests conducted in nonfumigated soil infested with inoculum of G. graminis germination of bacteria-treated seed. The population of the introduced var. tritici. The combination treatment was more suppressive than 13-79 bacterium exceeded 106 colony-forming units per 0. 1 g of root tissue 3 wk alone in all field tests, and was slightly more suppressive than 2-79 alone in after planting. The populations of strains 2-79 and 13-79 applied on wheat three of six field tests. Suppression of take-all by the bacteria was expressed seeds with methylcellulose were stable for 21 days at 5 or 15 C, but declined in the field as fewer plants with foliage symptoms of take-all and taller rapidly at 25 C. plants, more heads, greater yield, and less root disease than those grown Additional key words: bacterization, biological control, soilborne pathogens, Triticum aestivum. Take-all, a disease that is caused in wheat (Triticum aestivum L.) will be necessary if the bacteria are to be used in practice. by Gaeumannomyces graminis (Sacc.) Oliver and Von Arx var. Pseudomonas putida Kl 1 applied to wheat seeds decreased tritici Walker, may be the most important root disease of wheat symptoms of take-all on the roots in greenhouse experiments (23). worldwide. The disease can be controlled by crop rotation. It can Treatment of planting material of potatoes, sugar beets, and also be controlled by wheat monoculture, which results in the soil radishes with fluorescent Pseudomonas spp. (plant growthbecoming suppressive to take-all (6,15). However, strict adherence promoting rhizobacteria [PGPR]), significantly increased yields to either of these cultural practices by growers is rare. In the Pacific (2,11,12,14,19,21), apparently by displacing deleterious Northwest, many growers would like the option of growing two, rhizosphere fungi and bacteria on the plants (14,20). Howell and three, or possibly more consecutive crops of wheat after alfalfa or Stipanovic (7,8) demonstrated that strains of Pseudomonas potatoes, to help eliminate soilborne pathogens of those crops. fluorescens Migula applied to cotton seed suppressed seedling Unfortunately, these crops cause the soil to again become damping-off caused by Rhizoctonia solani Kiihn and Pythium conducive to take-all (3), and G. graminis var. tritici rapidly ultimum Trow. recolonizes' the soil when wheat is again grown. In these cases, a This paper reports the results of experiments designed to test the method is needed to reintroduce the agent(s) responsible for takefeasibility and methods for using fluorescent pseudomonads as all suppression. seed treatments for control of take-all of wheat. A preliminary Cook and Rovira (5) suggested a role for fluorescent account of this work was given previously (25). pseudomonads in take-all suppression. Smiley (17) showed that the population of antibiotic-producing fluorescent pseudomonads in soil was higher when wheat only was grown than when wheat was MATERIALS AND METHODS rotated withl other crops. Weller and Cook (24) found that the Culture and preparation of inoculum of G. graminis var. tritici. population of antibiotic-producing fluorescent pseudomonads was All isolates of Gaeumannomyces graminis var. tritici were started higher on roots grown in either of two suppressive soils than in two from single ascospores and were highly virulent. The pathogen was conducive soils. These findings suggest that antibiotic-producing, grown on agar plates of dilute potato-dextrose agar (PDA) (40 g root-colonizing pseudomonads might be useful for controlling potatoes, 4 g dextrose, and 15 g agar in 1 L of water) and stored at 4 C (4). Fresh cultures were routinely isolated from diseased wheat Smiley (18) and Cook and Rovira (5) obtained suppression of seedlings to maintain virulence (4). take-all in the greenhouse by adding pseudomonads to infested soil. Soils for both greenhouse and field studies were infested with oat Sivasithamparam and Parker (16) suppressed take-all by dipping kernels colonized by G. graminis var. tritici to ensure adequate roots of wheat seedlings in a suspension containing a mixture of disease development. Whole oats (250 ml) in water (200 ml) were fluorescent pseudomonads prior to planting the wheat in soil autoclaved in l-L, wide-mouth flasks with the openings plugged infested with G. graminis var. tritici. However, application on seed with cotton wrapped in cheesecloth. On the next day, 100 ml of water was added and the oats were again autoclaved. Two petri dish The publication costs of this article were defrayed in part by page charge payment. This (100 w 1 d and the of G. ga in au to c i on di sh article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § (100 X 15 mm) cultures of G. graminis var. tritici on dilute PDA 1734 solely to indicate this fact. were chopped into small squares and mixed with the oats. The This article is in the public domain and not copyrightable. It may be freely cultures were incubated for 3 wk at room temperature and then reprinted with customary crediting of the source. The American tested for contamination by other fungi or bacteria by placing Phytopathological Society, 1983. several oat kernels on PDA and nutrient broth yeast (NBY) extract Vol. 73, No. 3, 1983 463 agar (22) prior to drying. Pure cultures then were dried and stored the seeds were dried (time 0) the number of CFU per seed was in paper bags at room temperature. For use in greenhouse studies, determined as described above. Coated seeds were stored in plastic oat kernels were fragmented in a Waring Blendor, and used either petri dishes at 5, 15, and 25 C and sampled periodically for 5 wk. as a mixture of particle sizes or were sieved into several uniform Tests in vitro. Candidate bacteria were initially selected for particle sizes: large > 1.0 mm, medium = 0.5-1.0 mm, and small = ability to inhibit G. graminis var. tritici in vitro. Four isolates were 0.25-0.50 mm (26). In a typical greenhouse experiment, the spotted on the edge of an agar plate and a 6-mm-diameter plug of inoculum was added to soil at 1, 0.5, or 0.1% (w/ w). In field studies, the fungus from dilute PDA was placed in the center. Zones of whole oat kernels were placed (5.0 g per 3-m row) in the seed furrow inhibition were measured 5 days later. Antibiotic and siderophore at planting. production by the bacteria were tested on full-strength PDA and Isolation, culture, and storage of bacteria. Soils used as sources KMB, respectively, at pH 5, 6, 7, and 8. The pH of the media was of bacteria were collected from fields (located near Quincy, Moses adjusted with lactic acid and 0.1 M sodium hydroxide after Lake, and Lind, WA) that had been cropped to wheat for 22, 22, autoclaving. In tests for siderophores, the bacterium was grown for and 14 consecutive years, respectively. Soils were tested for 2 days in the center of a plate of KMB with and without ferric suppressiveness by the pot bioassay method (5). A soil was diluted chloride (5-100 MM); spores from a 48-hr-old PDA culture of 1:10 with fumigated (methyl bromide) soil, amended with 1.0% Geotrichum candidum Lk. ex Pers. were suspended in sterile ground oat kernel inoculum, and planted to wheat in 500-ml pots. distilled water and then sprayed on the plates. A 1:10 dilution of suppressive soil (from a field with a history of Greenhouse pot tests. Bacteria were tested for ability to control wheat monoculture and take-all decline) suppresses take-all, but a take-all in pot tests as seed and soil treatments. Three 1:10 dilution of conducive soil (from a field with a history of nonfumigated soils were used: Palouse silt loam (PSL, pH 5.5) multiple cropping or virgin soil) allows as much disease from Pullman, Puget silt loam (PuSL, pH 5.1) from Mt. Vernon, development as does 100% fumigated soil (5). Bacteria were WA, and Ritzville silt loam (RSL, pH 7.5) from Lind, WA. For isolated from roots of wheat plants grown in suppressive soil, either tests of seed treatments, sieved soil was uniformly infested with undiluted or diluted 1:10 with fumigated soil. Roots of 4to 5-wkpulverized oat kernel inoculum of G. graminis var. tritici by mixing old seedlings with tightly adhering soil were macerated with 0.01 M in a twin-shell blender for 30 min. Infested soil (300 g) was placed in phosphate buffer (pH 7.2) in a mortar and pestle. Appropriate paper drinking cups (500 ml) and watered with 100 ml of diluted dilutions were plated (0.1 ml) on various media to obtain single (1:3, v/v) Hoagland's solution (macro-elements only) plus 0.0125 colonies. Fluorescent pseudomonads were isolated by plating ml of metalaxyl (Ciba-Geigy, Greensboro, NC 48898) at 2.5 mg/ ml samples on King's Medium B (KMB) (9) and on KMB active ingredient to control Pythium root rot. The pseudomonads supplemented with novobiocin, penicillin, and cycloheximide were not adversely affected by this fungicide in vitro. A 0.5-cm layer (NPC) (1