Paecilomyces lilacinus on Colonization of Polyfoam

Transcription

1 APPLED AND ENVRONMENTAL MCROBOLOGY, Aug. 1994, p Vol. 6, No /94/$4.+ Copyright D 1994, American Society for Microbiology Effect of Population Dynamics of Pseudomonas cepacia and Paecilomyces lilacinus on Colonization of Polyfoam Rooting Cubes by Rhizoctonia solani D. KELLY CARTWRGHT* AND D. M. BENSON Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina Received 18 January 1994/Accepted 6 June 1994 Suspensions of Pseudomonas cepacia (strain 5.5B) and Paecilomyces lilacinus (isolate 6.2F) were applied to polyfoam rooting cubes for control of stem rot of poinsettia caused by Rhizoctonia solani. The populations of antagonists and colonization of rooting cubes by R. solani were monitored during a 3-week period. Colonization of cubes by R. solani was reduced in cubes treated with P. cepacia, but the population of P. cepacia decreased by as much as 97% during the test period. ncreased colonization by R. solani was correlated with a decline in population of P. cepacia. P. lilacinus was more persistent than P. cepacia in cubes, with only a 21% reduction observed during the 3-week period. Colonization of the P. lilacinus-treated cubes by R. solani was significantly less than colonization of infested controls. No correlation existed between population of P. lilacinus and colonization of cubes by R. solani. ssues about groundwater contamination, worker exposure, pest resistance, and other problems associated with chemical herbicides, insecticides, and fungicides have raised concerns about their safety and use (2, 29, 32, 33). The use of biological control agents to manage plant diseases is considered a promising alternative to chemical fungicide use (2, 1). Commercially acceptable biocontrol products using antagonistic microorganisms have been developed (19, 21, 31). However, unpredictable and varied results often lead to abandonment of promising organisms under development (13, 25). Most efforts in biological control research have emphasized methodology to elucidate desirable characteristics of candidate microorganisms (3, 6, 14, 2, 26, 28). These characteristics coupled with the determination of optimal environmental and edaphic conditions necessary for disease control are prerequisite in development (4, 15, 18, 22, 28). However, there is comparatively limited knowledge on the ecological interactions between antagonist(s), indigenous microflora, and target pathogen(s) and the relation of these factors in disease suppression. A thorough understanding of these interactions could assist in evaluating and manipulating important developmental components, such as delivery systems, determination of optimal target area(s) and stages of plant development for application, the optimal mechanism(s) of action for a specific system, and predictability of biocontrol agents. Poinsettias (Euphorbia pulcherrima Willd. ex Klotzsch) are susceptible to stem rot caused by Rhizoctonia solani Kuhn (teleomorph = Thanatephorus cucumeris (Frank) Donk) (16, 3). During the late summer months when poinsettia propagation begins, many growers root poinsettia cuttings in soilless, polyfoam rooting cubes that provide easy handling and uniform root development (12). However, the warm, moist conditions of the cube environment can be conducive to diseases such as stem rot. n addition, rooting cubes are placed on greenhouse benches in joined strips for easy separation and the close proximity of individual cubes allows R. solani to easily * Corresponding author. Mailing address: Crops Research Laboratory, USDA/ARS/SAA, Oxford, NC Phone: (919) , ext Fax: (919) Electronic mail address: Kelly Cartwright@ncsu.edu grow from one cube to the next. nfected cuttings then serve as inoculum sources for other cuttings within the cube strip. Thus, R. solani spreads rapidly in cubes under greenhouse conditions. The most effective way to control stem rot in rooting cubes is through the use of cultural practices and chemical fungicides (1, 24, 27, 3). Effective fungicides are applied to cubes in a preventative manner to restrict colonization and subsequent spread of R solani (5). A successful biocontrol agent would be most effective if characteristics of the agent allowed use in a similar manner to prevent colonization of rooting cubes by R. solani. A strain of Pseudomonas cepacia and an isolate of Paecilomyces lilacinus are effective biocontrol agents of stem rot of poinsettia in polyfoam rooting cubes (8, 9). However, little is known about the survival, distribution, and population fluctuation of these antagonists after application to rooting cubes and the effect of these factors on colonization of cubes by R. solani and stem rot suppression. The purpose of these experiments was to investigate the relationship between population dynamics of P. cepacia (strain 5.5B) and P. lilacinus (isolate 6.2F) in polyfoam rooting cubes and colonization of rooting cubes by R. solani. MATERLALS AND METHODS Laboratory procedures. Both antagonists were isolated from native, North Carolina soils near Raleigh, N.C. Strain 5.5B of P. cepacia (ATCC 55344) was isolated from the rhizosphere of a grass species collected from a low-lying noncultivated site and identified by Harvey Spurr at the Oxford Tobacco Research Laboratory, USDA Agricultural Research Service, Oxford, N.C., with the HP 5898A Microbial dentification System (Hewlett-Packard Company, Avondale, Pa.). solate 6.2F of P. lilacinus (NRRL 22772) was recovered from soil sampled in a low-lying, noncultivated grassy area and identified by D. T. Wicklow at the Northern Regional Research Center, USDA Agricultural Research Service, Peoria, ll. For these tests, strain 5.5B and isolate 6.2F were retrieved from cultures stored in sterile water at 2 C (in the dark). On the basis of preliminary experiments, the most effective control of stem rot in rooting cubes was achieved with an

2 VOL. 6, 1994 EFFECT OF POPULATON DYNAMCS 2853 L F Entire sampled cube Cube-half used for colonization assay Sections A-D J Cube-half used for population assay 55mm ~~~~1 1l r ~ t 6 9mnn7m A B C D FG. 1. Four cross-sections (each section has nine subcube sections) of half cube from the outside (A) to the middle (D) of rooting cube showing top (sections 1 to 3, 1 to 12, 19 to 21, and 28 to 3), middle (sections 4 to 6, 13 to 15, 22 to 24, and 31 to 33), and bottom (sections 7 to 9, 16 to 18, 25 to 27, and 34 to 36) of cube and divisions A (sections 1 to 9), B (sections 1 to 18), C (sections 19 to 27), and D (sections 28 to 36). Each plated section measures 5 by 5 by 9 mm. inoculum of strain 5.5B or isolate 6.2F cultured for 1 to 14 days on potato dextrose agar (PDA; Difco Laboratories, Detroit, Mich.). For these experiments, both antagonists were cultured on PDA at ambient temperature on a laboratory bench for 13 to 15 days. Several methods of inoculum preparation were previously attempted, and the following method was chosen: preparations of antagonists were made in about a.1% PDA solution by blending the contents (colony plus agar medium) of a single petri dish with colonies only (colonies removed from the agar surface) from two to three additional cultures in 6 ml of sterile, deionized water in a Waring blender at high speed for 45 to 6 s. The suspension was strained through one layer of cheesecloth. Nutrient-based control suspensions were prepared by blending the contents of a single petri dish containing PDA in a Waring blender before straining the suspension through cheesecloth. For the inoculum of R. solani, rice grains were colonized with R. solani (isolate RS-3 from poinsettia; anastomosis group (AG) 4, NRRL 2285) by seeding twice-autoclaved rice (25 g of rice per 18 ml of sterile, deionized water) contained in 25-ml flasks with mycelial disks from a colony of R. solani and incubating the flasks at ambient temperature for 7 to 9 days. Greenhouse procedures. Poinsettia stock plants [Gutbier V-14 Glory (red)] were maintained in 22 Metro mix (W.R. Grace Co., Cambridge, Mass.) contained in 6-liter plastic pots on greenhouse benches. Plants were fertilized weekly by drenching the potting medium with a commercial (N-P-K) fertilizer (2.7 g/liter). Magnesium sulfate (2.4 g/liter) was applied as a drench each month. Every 3 months, soil drenches of potassium nitrate (.45 g/liter), calcium nitrate (1.6 g/liter), and a foliar spray of 1% molybdenum were applied. Stock plants were occasionally pruned to facilitate growth. Antagonist preparations were applied to polyfoam rooting cubes (Smithers-Oasis U.S.A., Kent, Ohio) in a manner consistent with that previously described (8, 9). The average concentrations (CFU per milliliter) for all experiments were log 9.7 for strain 5.5B and log 7.8 for isolate 6.2F. Cube strips (five individual cubes per strip) were soaked (until saturation) in antagonist preparations in plastic containers and wrapped in a styrofoam sleeve, and the sleeve was secured with rubber bands. ndividual cubes were 25 mm wide by 51 mm long by 37 mm high (47 cm3) and held 4 ml of suspension (per cube). After soaking, cube strips were placed on greenhouse benches and poinsettia cuttings were taken from stock plants, placed in cubes (one cutting per cube, five cuttings per strip), and misted. After 18 to 24 h, rice grains colonized by R. solani were placed on top (in the center) between each cube and at both ends of each cube strip (about 2 cm from the stem of each cutting). A watering regime of 1 min of mist per h, 14 times per day was used. Cubes soaked in sterile, deionized water or nutrient solution (.1% PDA) were used as water- and nutrient-based controls (infested and noninfested). Population assay for antagonists. Populations of P. cepacia and P. lilacinus were determined as follows. (i) Each sampled cube was aseptically dissected into equal halves for processing. (ii) For population studies, each half cube was weighed (1 g wet weight of cube contains 1 ml of water) and enough sterile, deionized water was added to make 2 ml of solution (i.e., 178 ml of water was added to a 22-g half cube). (iii) The cube and water were then blended in a Waring blender at high speed for 3 s (enough time to thoroughly pulverize the cube). (iv) From the resultant suspensions, serial dilutions (1:1) were made and.1-ml aliquots from appropriate dilutions were plated onto Kings medium B (11) (P. cepacia), or acidified, one-half strength PDA (P. lilacinus). All plates were inverted and placed in the dark for 48 to 72 h. Colonies of P. cepacia 5.5B were recognizable by distinctive color, shape, growth rate, and pattern, and colonies were occasionally transferred to Kings medium B or PDA to confirm identification. n some experiments, populations of bacteria indigenous to the cubes were assayed by counting all colonies that were not characteristic of strain 5.5B of P. cepacia. Colonies of P. lilacinus were formed from mycelial fragments or conidia, the majority being conidia. The population of antagonists in CFU per milliliter (based on the amount of water [in milliliters] contained in a cube) was then calculated. Values were compared with those for the initial population of antagonists applied to cubes in CFU per milliliter. Cube colonization by R. solani. Colonization of rooting cubes by R. solani was determined in the antagonist-treated cubes and the infested, nontreated controls by processing the adjoining half of the sampled cube as follows. (i) The outer few millimeters of a half cube was aseptically removed, leaving a cube approximately 15 by 2 by 27 mm. (ii) This was then aseptically dissected into four sections from the outside to the middle (each section measuring about 15 mm wide by 5 mm thick by 27 mm tall). (iii) These sections were then cut into 9 uniform sections measuring 5 by 5 by 9 mm (a total of 36

3 2854 CARTWRGHT AND BENSON sections per half cube were assayed for the presence of R. solani. (iv) Sections were placed on acidified one-half strength PDA and observed for the presence of R. solani 24 h later. Colonization by R. solani of sequentially numbered sections allowed determination of R. solani progress from the outside of the cube (where a rice grain colonized with R. solani was placed) towards the middle of the cube (where the cutting was placed). Data presented are based on colonization of the entire cube or only the top 9 mm of the processed half cube. Dissection of a cube for colonization assay is illustrated in Fig. 1. Experimental design and statistical analysis. Each treatment consisted of four replications, with five cubes per replication (2 plants total). Treatments were arranged in a randomized, complete block design. One cube per replication was randomly sampled (four cubes per treatment at each sampling time) on days 3, 7, 14, and 21 after application of antagonists. nfection and mortality of cuttings were evaluated daily and at each sample period. All population values were loglo transformed before analysis. The percent of initial population remaining was calculated by the formula /O x 1, where is the population at a given sample time and O is the initial population. All experiments were performed at least twice. Data analysis was performed by PC SAS (SAS nstitute, Cary, N.C.). The standard error of the mean for population and colonization was determined for each treatment by sample period. Analysis of variance was determined by sampling period and overall test period with PROC GLM. To generate Pearson correlation coefficients between the population of antagonists and colonization of cubes by R. solani, means for population and colonization were calculated for each replication over all sample periods and subtracted from individual observations. Correlation coefficients were determined by PROC CORR. For clarity, data from each antagonist treatment were pooled from separate experiments and analyzed for comparison with data on the colonization of the cubes by R. solani. Data from different experiments were pooled on the basis of homogeneity of variances. RESULTS The population of strain 5.5B of P. cepacia declined by 69, 9, 95, and 97% from the initial population of log 9.1 CFU per ml to log 8.6, 8.1, 7.7, and 7.5 CFU per ml after 3, 7, 14, and 21 days, respectively (Fig. 2). n the bacterium-treated cubes, colonization of the entire cube by R. solani was, 6, 21, and 27% after 3, 7, 14, and 21 days, respectively (Fig. 3A). This was less (P =.5) than colonization of cubes in the infested controls (nutrient- and water-based controls) that ranged from 1 to 16, 69 to 77, 94 to 1, and 97 to 1% after 3, 7, 14, and 21 days, respectively (Fig. 3A). n the top 9 mm of the treated cubes, R. solani spread more rapidly, colonizing an average of, 18, 58, and 8% of the cubes after 3, 7, 14, and 21 days, respectively (Fig. 3B). n both infested controls, colonization increased significantly, with 3 to 49, 94 to 1, 1, and 1% of the top 9 mm of cubes colonized during the same period (Fig. 3B). Percent infection of cuttings remaining at each sample period in treated cubes was,, 2, and 38% after 3, 7, 14, and 21 days, respectively. During the same period, infection of cuttings in infested controls was greater (P =.5), with averages of, 84, 96 to 1, and 94 to 1% after 3, 7, 14, and 21 days, respectively. Persistence of P. lilacinus in cubes was much greater than that of P. cepacia. Population fluctuation between sampled cubes was also much less variable. From the initial population of about log 7.8 CFU per ml, the population levels were log SB indigenous bacteria Days after applying R cepacia, strain 5.5B FG. 2. Survival of strain 5.5B of P. cepacia and increase of population of indigenous bacteria in polyfoam rooting cubes used in propagation of poinsettia cuttings. Datum points are means from two experiments for strain 5.5B (n = 8). Datum points are means from three experiments for indigenous bacteria (n = 12). Bars represent standard errors of the mean. 7.3, 7.8, 7.4, and 6.98 after 3, 7, 14, and 21 days, reductions of 11,, 9, and 21%, respectively (Fig. 4). n treated cubes, the entire cube was colonized by R. solani at rates of, 3, 12, and 17% after 3, 7, 14, and 21 days. This was less (P =.5) than in the infested controls, where colonization was 13, 73 to 76, 84 to 94, and 91 to 94%, respectively (Fig. 5A). Colonization of the top 9 mm of treated cubes by R. solani was, 8, 33, and 44%, which was significantly lower than in infested controls where colonization levels were 37 to 38, 1, 1, and 1% after 3, 7, 14, and 21 days, respectively (Fig. SB). Percent infection of cuttings in cubes treated with P. lilacinus was, 4, 17, and 23%. n both infested controls, percent infection of cuttings was, 83 to 91, 1, and 1%. Populations of indigenous bacteria were first detected 3 days after application of antagonists, with an average population of log 6.1 (Fig. 2). Numbers gradually increased throughout the test period, reaching a population of log 7.6 after 21 days (Fig. 2). The correlation coefficient between population decline and colonization of the rooting cubes by R. solani was high for strain 5.5B (r = -.821) but not for isolate 6.2F where the coefficient was near zero (r = -.114). DSCUSSON APPL. ENVRON. MCROBOL. P. cepacia (5.5B) did not survive well in polyfoam rooting cubes in these experiments. Despite application in a dilute nutrient solution, the bacterial population in cubes declined by as much as 7% after 3 days and typically more than 9% after 7 days. The population of indigenous bacteria increased to about log 7 CFU per ml after 21 days, approximately the same as the population of P. cepacia. This suggests a limited (i.e., carrying capacity) ability of the cube environment, with only minimal nutrients from the initial preparation, to sustain populations. n other systems, apparent carrying capacities exist for strains of biocontrol bacteria (18). Parke (23) observed that applying P. cepacia for biocontrol of Pythium damping-off of pea in excess of log 8.4 CFU per seed (carrying capacity) resulted in population decline. Knudsen and Spurr (17), monitoring populations of P. cepacia on peanut leaves,

4 VOL. 6, 1994 EFFECT OF POPULATON DYNAMCS loa Days after applying P cepacia, strain 5.5B B Days after applying P lilacinus, isolate 6.2F FG. 4. Survival of isolate 6.2F of P. lilacinus in polyfoam rooting cubes used in propagation of poinsettia cuttings. Datum points are means from three experiments (n = 12). Bars represent standard errors of the mean o Days after applying R cepacia, strain 5.51B FG. 3. Percent colonization by R. solani in rooting cubes left untreated or treated with strain 5.5B (a) of P. cepacia. (A) Colonization of the entire cube by R. solani. (B) Colonization of the top 9 mm of the cube by R. solani. O, nfested, water-based control; *, infested, nutrient-based control. Datum points are means from two experiments (n = 8). Bars represent standard errors of the mean. demonstrated that population decline ranged from 1 to 99% over a 2-week period. Populations of introduced biocontrol bacteria are variable depending on location and availability of nutrients. Populations of P. cepacia and Pseudomonas fluorescens applied to corn and wheat roots, respectively, varied by several log units between different roots and along the axes of individual roots (13, 34). ntroduced bacteria are typically found in greater numbers in close proximity to the inoculum source and decline as distance from the initial source increases (35). n this study, the population of strain S.SB was often very variable between replications at the same sampling time. Some of this variation may be attributable to dry spots due to varying mist coverage near the edge of greenhouse benches and/or the lack of nutrients available for bacterial growth. The population of isolate 6.2F of P. lilacinus in rooting cubes was much more stable in these experiments than that of strain S.SB. The average reduction in population of P. lilacinus was only 21% after 3 weeks. Other isolates of P. lilacinus are able to colonize substrate and survive for extended periods of time in the soil (7). Biocontrol fungi applied to soil and soilless mixes were persistent and may increase substantially, primarily in the presence of a nutrient source that serves as a food base (3, 4, 22). solate 6.2F appears to maintain a relatively stable population in the rooting cubes despite low nutrient levels. The long-term survival of P. lilacinus in rooting cubes is advantageous, particularly for prolonged efficacy of control and the critical period during the first 2 to 3 weeks after cuttings are placed, when they are more susceptible to infection (Sa). With strain 5.5B, colonization of the entire cube by R. solani was significantly less than colonization of cubes in infested controls. However, there was a negative correlation between colonization of rooting cubes by R. solani and the population of the bacterium. As population declined, colonization typically increased, but never to a level of more than about 3%. Colonization of cubes of the infested controls was as much as 1% after 21 days. Both colonization and infection of cuttings seemed to increase during the latter part of the trial period. The initial inundation of the cubes with the bacterium, therefore, seems to be an important process in prevention of colonization of cubes and infection of cuttings by R. solani. Benson (5) demonstrated that the effectiveness of fungicides for stem rot control was correlated with a lack of colonization of rooting cubes. The increased colonization after a prolonged period suggests that additional applications of the bacterium to repopulate the cubes, or specific nutrient amendments to increase numbers from residual populations, may be necessary for satisfactory control over extended periods. n contrast to colonization of the whole cube, colonization of the top of the cube by R. solani was much more rapid in all treatments. There appeared to be a relation between increased colonization of the top of the cube by R. solani and infection of the cuttings, especially after a 2-week period. The infection court of poinsettia cuttings is at the junction of the top of the cube and the cutting stem. Most isolates of R. solani infect plants at or near the soil surface. solate RS-3 appears to colonize the surface of the cubes more effectively than the inside of the cube matrix. On this basis, a successful biocontrol agent should be capable of remaining persistent near the surface of the cube. However,

5 2856 CARTWRGHT AND BENSON F u H 1 8 io w 6 * 6 A (~~~~~~~~~~~~~~ Days after applying P lilacinus, isolate 6.2F Days after applying P lilacinus, isolate 6.2F FG. 5. Percent colonization by R. solani in rooting cubes left untreated or treated with isolate 6.2F (a) of P. lilacinus. (A) Colonization of the entire cube by R. solani. (B) Colonization of the top 9 mm of the cube by R. solani. *, nfested, water-based control;, infested, nutrient-based control. Datum points are means from three experiments (n = 12). Bars represent standard errors of the mean. 21 the more extreme alternating conditions (air circulation, drying, and light) at the cube surface make survival more difficult in this area, especially for bacteria. Reapplication or manipulation of amendments may be necessary to extend survival of P. cepacia near the surface and improve long-term control. The lack of colonization of the cube by R. solani, specifically in the top of the cube, relates well to the efficacy of control exhibited by P. lilacinus in these tests. Other fungi, including Trichoderma spp. and Gliocladium virens, can reduce colonization and propagules of R. solani when applied to the soil (4). The persistence of P. lilacinus during the entire 3-week period relates well to the lack of colonization by R. solani and subsequent low level of infection of cuttings. Understanding the relation between antagonist and target pathogen is important for improving effectiveness and reliability of a potential biocontrol agent. These data demonstrate that population of strain 5.5B of P. cepacia declines rapidly in rooting cubes over an extended period, with subsequent increased colonization of cubes by R. solani, while isolate 6.2F of P. lilacinus remains more stable. From a commercial standpoint, these results help provide a necessary basis for examining the reduction of these introduced antagonists to background levels after application. These data are also important for further evaluations of application scheduling, formulations, and predictability of these antagonists as biocontrol agents of Rhizoctonia stem rot of poinsettia. ACKNOWLEDGMENTS APPL. ENVRON. MCROBOL. We thank Billy. Daughtry for his technical assistance. We also thank Smithers-Oasis U.S.A. for providing root cubes and Fairview Greenhouses and Garden Center, Raleigh, N.C., for providing poinsettia stock plants. This research was supported by the North Carolina Agricultural Research Service, North Carolina State University, Raleigh, and in part by a grant from Ciba-Geigy Agricultural Division, Greensboro, N.C. REFERENCES 1. Bailey, D. A Cultural practices and PM for poinsettias. N. C. Flower Growers' Bull. 35: Baker, K. 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