Biological Control 23, 285–295 (2002) doi:10.1006/bcon.2001.1015, available online at http://www.idealibrary.com on Biological Control of Phytophthora Root Rots on Alfalfa and Soybean with Streptomyces Kun Xiao,* Linda L. Kinkel,* and Deborah A. Samac*,† *Department of Plant Pathology and †USDA-ARS Plant Science Research, University of Minnesota, St. Paul, Minnesota 55108 Received May 11, 2001; accepted November 16, 2001 sojae was the second-most important yield-suppressing A collection of 53 antibiotic-producing Streptomyces disease in the north-central region of the United isolated from soils from Minnesota, Nebraska, and States. In 1994, an estimated 560,300 metric tons of Washington were evaluated for their ability to inhibit soybean were lost to P. sojae in the United States, plant pathogenic Phytophthora medicaginis and Phy- making it third among yield-suppressing diseases tophthora sojae in vitro. Eight isolates having the (Doupnik, 1993; Wrather et al., 1997). greatest pathogen-inhibitory capabilities were subse- Phytophthora root rots are difficult to control effec- quently tested for their ability to control Phytoph- tively. A variety of methods have been studied for use thora root rots on alfalfa and soybean in sterilized vermiculite and naturally infested field soil. The in control of Phytophthora root rots of alfalfa and soy- Streptomyces isolates tested significantly reduced root bean. Plant resistance is currently the major control rot severity in alfalfa and soybean caused by P. medi- method for Phytophthora root rots on both crops (Er- caginis and P. sojae, respectively (P < 0.05). On alfalfa, win and Ribeiro, 1996). Unfortunately, none of the isolates varied in their effect on plant disease severity, currently available resistant alfalfa cultivars is im- percentage dead plants, and plant biomass in the pres- mune to the disease, especially at high pathogen inoc- ence of the pathogen. The same eight isolates of Strep- ulum densities or under wet conditions (Faris and tomyces were also tested for inhibitory activities Sabo, 1981). Under conditions that are favorable to the against each other and against three strains of Brady- pathogen, even resistant cultivars can become severely rhizobium japonicum and two strains of Sinorhizo- infected by P. medicaginis (Havey and Grau, 1985; bium meliloti isolated from soybean and alfalfa, re- Erwin and Robeiro, 1996), indicating that other meth- spectively. Streptomyces isolates clustered into two ods must be integrated with resistance to control the major compatibility groups: isolates within the same disease effectively. Race-specific resistance of soybean group were noninhibitory toward one another in vitro. cultivars has been the most important method to con- The compatibility groups corresponded with group- trol P. sojae. However, race-specific resistance can pro- ings obtained based upon inhibition of B. japonicum mote the buildup of new pathogen races and result in and S. meliloti strains. © 2002 Elsevier Science (USA) the failure of resistant soybean cultivars (Tooley and Key Words: antibiosis; biological control; Glysine Bergstrom, 1984; Anderson and Buzzell, 1992; Ryley max; Medicago sativa. and Obst, 1992; Yang et al., 1996; Abney et al., 1997). Also, growers must know what races are present in INTRODUCTION their fields. This suggests that alternative or comple- mentary methods can contribute significantly to effec- Root rots of alfalfa (Medicago sativa L.) and soybean tive and consistent disease control. (Glycine max (L.) Merrill) caused by Phytophthora Cultural approaches, including enhancing soil drain- medicaginis Hansen & Maxwell and Phytophthora so- age, reducing mechanical compaction of soil, and crop jae Kauf. & Gerd., respectively, are highly destructive rotation, also contribute to successful disease manage- on these two crops throughout the world (Hansen and ment (Pulli and Tesar, 1975; Schmitthenner, 1985, Maxwell, 1991; Erwin and Ribeiro, 1996). P. medicagi- 1988; Schmitthenner and Van Doren, 1985; Erwin and nis and P. sojae can cause pre- and postemergence Ribeiro, 1996). However, Phytophthora root rot patho- damping-off diseases. They are especially damaging gens of alfalfa and soybean overwinter as oospores, when conditions are wet during the early stages of which can survive in the soil for long periods of time in plant development and can significantly reduce both the absence of their hosts. Consequently, crop rotation establishment and yield (Erwin and Ribeiro, 1996; may have only a minor effect on Phytophthora root rots Teutsch and Sulc, 1997). Between 1989 and 1991, P. (Schmitthenner and Van Doren, 1985). 285 1049-9644/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 286 XIAO, KINKEL, AND SAMAC Fungicides, including ETMT [5-ethoxy-3-(trichlorom- MATERIALS AND METHODS ethyl)-1,2,4-thiadiazole] (Papavizas et al., 1979), metal- axyl [N-(2,6-dimethyl phenyl)-N-(methylacetyl)alanine In Vitro Inhibitory Potential of Streptomyces methyl ester] (Guy et al., 1989; Rhodes and Myers, 1989), Fifty-three antibiotic-producing Streptomyces iso- and pyroxyfur [2-chloro-6-(2-fruanylmethoxy)-4-(tri- lates from Minnesota, Nebraska, and Washington were chloromethyl)pyridine] (Diatloff et al., 1983; Lazarovits, selected based on previous work (Liu, 1992; Paulsrud, 1985) have been used as seed treatments or in-furrow 1996; L. L. Kinkel, unpublished data) and tested for applications to control Phytophthora root rots. Although their ability to inhibit the growth of P. medicaginis metalaxyl, which is the major systemic fungicide used to M2019 (Jones, 1994) and P. sojae (provided by the control the Phytophthora root rots, is effective as a seed University of Minnesota Plant Disease Clinic). The treatment (Apron) and an in-furrow treatment (Ridomil), double-layer agar method (Vidaver et al., 1972) was it may not be cost effective for field crops (Schmitthenner used with modifications to evaluate inhibitory activity and Van Doren, 1985; Schmitthenner, 1989). Also, re- of each Streptomyces isolate toward the pathogens. search indicates that metalaxyl has reduced effectiveness Briefly, oatmeal agar medium (OA; 20 g/L oatmeal, 1 in some cases due to tolerance developed by Phytoph- g/L casamino acid, and 15 g/L bacto-agar) was used as thora root rot pathogens (Davidse, 1981; Hunger et al., the bottom layer for the incubation of Streptomyces 1982; Stack and Millar, 1985; Lamboy and Paxton, 1992; isolates (Liu, 1992). Streptomyces isolates were spotted Bhat et al., 1993). onto each OA plate and grown for 3 days at 28°C. In total, effective control of Phytophthora root rots Plates then were overlaid with molten corn meal agar will require integrated strategies (Schmitthenner, (CMA; about 15 ml/plate) after colonies of Streptomyces 1985, 1988; Schmitthenner and Van Doren, 1985; were killed by inversion of plates over 3 ml chloroform Rehm and Steienstra, 1993). One component of an for 1 h. When the overlaid agar became firm, a 5-mm- integrated strategy may be biological control with an- diameter disk of the agar from the center of the top tagonistic microorganisms. To date, biocontrol agents layer was removed and replaced with a 5-mm CMA against P. sojae that have shown effectiveness in im- disk from the actively growing margin of a 7- to 14- proving the establishment and yield of soybean include day-old pathogen culture. Plates then were incubated several Actinomycetes (Filonow and Lockwood, 1985), at room temperature for 7 to 14 days. The diameters of Hyphochytrium catenoides (Hsu and Lockwood, 1984), clear inhibition zones were measured 7 days after in- and Bacillus cereus UW85 (Osburn et al., 1995), which oculation for P. medicaginis isolate M2019 and 14 days also was used as a biocontrol agent against P. medi- later for P. sojae. caginis on alfalfa (Handelsman et al., 1990). In addi- tion, research has shown that Sinorhizobium meliloti Biocontrol Activity of Antibiotic-Producing and Bradyrhizobium japonicum might play a role in Streptomyces Isolates in Sterilized Vermiculite protecting alfalfa and soybean roots from P. medicagi- The 15 Streptomyces isolates having the largest av- nis and P. sojae, respectively (Tu, 1978, 1979, 1980). An erage in vitro inhibition against P. medicaginis M2019 antibiotic-producing Streptomyces isolate has also been and P. sojae (Table 1) were selected for further study. used in research to control P. medicaginis on alfalfa The test tube assay described by Handelsman et al. (Jones and Samac, 1996). Because of their inhibitory (1990) was adapted as described below for evaluation abilities, Streptomyces spp. have been actively studied and screening of these antibiotic-producing Streptomy- and utilized as biocontrol agents against various plant ces isolates against Phytophthora root rot pathogens on pathogens (Merriman et al., 1974; Liu, 1992; El-Abyad soybean and alfalfa. et al., 1993; Hiltunen et al., 1995; Jones and Samac, Surface-sterilized (10% bleach) pregerminated al- 1996; Paulsrud, 1996; Gyenis et al., 1999). falfa seeds (cv. Vernal) and surface-sterilized soybean The goals of this research were to (1) evaluate seeds (cv. McCall) that had been soaked in sterile dis- the biocontrol potential of Streptomyces antagonists tilled water for 24 h were planted in glass test tubes against Phytophthora root rots of alfalfa and soy- (15 ϫ 115 or 20 ϫ 250 mm for alfalfa and soybean, bean, (2) investigate the effect of Streptomyces antag- respectively) filled with sterilized moistened vermicu- onists on the growth of alfalfa seedlings in the