Biocontrol Efficacies of Bacillus Species Against Cylindrocarpon Destructans Causing Ginseng Root Rot
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Plant Pathol. J. 27(4) : 333-341 (2011) http://dx.doi.org/10.5423/PPJ.2011.27.4.333 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 © The Korean Society of Plant Pathology Open Access Biocontrol Efficacies of Bacillus Species Against Cylindrocarpon destructans Causing Ginseng Root Rot Yelim Jang, Sang Gyu Kim and Young Ho Kim* Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea (Received on July 19, 2011; Revised on October 4, 2011; Accepted on October 4, 2011) Two antifungal bacteria were selected from forest soils Cylindrocarpon destructans is most responsible for ginseng during the screening of microorganisms antagonistic to root rot and is one of the main causes of long-term replant Cylindrocarpon destructans, a cause of ginseng root rot. problems (Chung, 1975; Jeon et al., 2008; Kim et al., 2006; The antifungal bacteria were identified as Bacillus Lee, 2004; Ohh et al., 1983; Park, 2001; Yu, 1987; Yu and subtilis B. amyloliquefaciens (I4) and (yD16) based on Ohh, 1993). One of conventional control methods for physiological and cultural characteristics, the Biolog fungal diseases is the use of chemical fungicides (Agrios, program, and 16S rRNA gene sequencing analyses. 2005). However, chemical control of soil-borne diseases is Antagonistic activity of both bacterial isolates to C. destructans increased with increasing temperature. expensive and rarely achieves full disease control; other More rapid starch hydrolytic activity of the bacteria disadvantages include potential environmental hazards, was seen on starch agar at higher temperatures than at toxicity to crops, and development of fungicide-resistant lower temperatures, and in the higher density inoculum strains (Hass and Defago, 2005; Handelsman and Stabb, treatment than in the lower density inoculum treatment. 1996). Biocontrol of plant pathogens is an alternative The bacterial isolates failed to colonize ginseng root the means of reducing the incidence and severity of diseases root tissues inoculated with the bacteria alone at an with no or few negative impacts on the environment 6 inoculum density of 1 × 10 cfu/ml, but succeeded in compared to chemical controls with fungicides. colonizing the root tissues co-inoculated with the bacteria Over a hundred microbial products are currently re- C. destructans. and Scanning electron microscopy showed gistered or marketed as biocontrol agents worldwide that the pathogen was damaged by the low-density (Whipps and McQuiken, 2009), and presently 14 microbial inoculum treatment with the bacterial isolates as much as by the high-density inoculum treatment. Both bac- fungicides are commercially registered in Korea (Kim et terial isolates were more effective in reducing root rot al., 2010). Four bacterial strains were reported to be effec- when they were treated at a concentration of 1 × 106 cfu/ tive for control of ginseng diseases such as Phytophthora ml than at 1 × 108 cfu/ml. Also, only the former treatment blight caused by Phytophthora cactorum and root rot induced prominent wound periderm formation, related caused by C. destructans (Bae et al., 2004; Sang et al., to structural defense against pathogen infection. The 2006). Two bacterial isolates strongly antagonistic to C. results suggest that the bacterial antagonists may have destructans were isolated during the survey of biocontrol high potential as biocontrol agents against ginseng root agents for ginseng root rot. They were identified and rot at relatively low-inoculum concentrations. exaimned their biocontrol activity for use in practical applications to control ginseng root rot. Keywords : Bacillus species, biological control, Cylindro- carpon destructans, ginseng root rot Materials and Methods Ginseng (Korean ginseng) (Panax ginseng Meyer, Araliaceae) Pathogen preparation for disease development. Cylindro- is recognized in the Orient a very important medicinal carpon destructans KACC 41077 was obtained from The plant. During ginseng cultivation, plants may be attacked Korean Agricultural Culture Collection (KACC), Rural by a variety of biotic agents, especially soil-borne patho- Development Administration (RDA), Suwon, Korea, and gens, such as fungi, bacteria, and nematodes, among which used in this experiment after confirming its pathogenicity to ginseng roots, of which the inoculation methods and *Corresponding author. resulting disease severity were almost identical to those for Phone) +82-2-880-4675, FAX) +82-2-873-2317 the inoculation control in pot experiments in this study. The E-mail) [email protected] fungus was cultured on potato-dextrose agar (PDA) at 334 Yelim Jang et al. 21 ºC for 14 days. This fungal culture was used directly as a KACC 41077 grown on PDA at 21 ºC for 2 weeks were pathogen inoculum or cultured again on rice hull medium placed in the center of Czapek-Dox agar plates and spotted (rice hull 1: sand 4: distilled water 1.5) for use as a at 3 cm intervals with 10 μl bacterial suspension, grown in pathogen inoculum for pot experiments. BHI broth at 28 ºC for 2 days with shaking at 200 rpm. These plates were incubated at 15, 18, 21, 25, and 28 ºC. In vitro screening of bacteria with antifungal activity SDW was used as the control. After incubation for 14 days, against C. destructans. For biological control of C. the inhibitory activity was measured for each bacterial destructans, 174 bacterial isolates were collected from isolate as mycelial growth of C. destructans. Three forest soils in various mountain areas, mushrooms, and replications were used for each treatment. crop fields in Korea and screened for antifungal activity against C. destructans. Mycelial discs (1-cm in diameter) of Starch hydrolytic activity of antifungal bacteria. Starch the fungus grown for 14 days on PDA as described above hydrolytic activity of the bacterial isolates was tested as were placed in the center of PDA plates. The bacterial enzyme activity related to ginseng root rotting in previous isolates were grown in brain heart infusion (BHI) studies (Jeon et al., 2003, 2010). For this, aliquots (10 μl) of (CONDA, Madrid, Spain) broth at 28 ºC for 2 days with each bacterial suspension at inoculum densities of 1 × 104 shaking at 200 rpm, and 10 μl of the bacterial suspensions cfu/ml, 1 × 106 cfu/ml, and 1 × 108 cfu/ml were spotted on were spotted on PDA plates 3 cm apart from the mycelial modified starch agar plates (3 g beef extract, 5 g peptone, 2 discs. Sterile distilled water (SDW) was used as the g soluble starch, and 15 g agar per 1 L), and incubated at untreated control. After incubation for 14 days, the temperatures of 18, 21, 25, and 28ºC. At 6-h intervals after inhibitory activity was measured for each bacterial isolate inoculation, the agar media were stained with Gram’s as the mycelial growth of C. destructans. Three replications iodine to examine starch hydrolysis by the formation of were used for each treatment. clear halos around bacterial colonies due to enzyme activity. A definite clear zone formed around a bacterial Identification of antifungal bacteria. Among the bacteria, colony was considered to be a positive response for starch two isolates with the highest antifungal activity against C. hydrolytic activity (+) and no formation of the clear zone destructans were selected for further biocontrol study. negative (−). These isolates were identified based on physiological and culture characteristics, and analysis with the Biolog pro- Effect of bacterial treatment on inhibition of root rot gram. To confirm bacterial identities, 16s rRNA gene caused by C. destructans on ginseng root discs and in sequences of the isolates were analyzed using the 27 mF pots. Two bacterial isolates were grown in BHI broth at (5'-AGAGTTTGATCM-TGGCTCAG-3') and 1492 mR 28 ºC for 2 days with shaking at 200 rpm. C. destructans (5'-GGYTACCTTGTTACG-ACTT-3') primers (Brosius et KACC 41077 was grown on PDA at 21 ºC for 2 weeks. al., 1978; Weisburg et al., 1991) after PCR amplification of Four-year-old ginseng roots were surface-sterilized with the 16s rRNA gene. The resulting sequences were analyzed 70% EtOH for 5 min, 1% NaOCl for 5 min, and rinsed using Blast search program in GenBank and compared with twice with SDW. These ginseng roots were cut to discs other sequences listed in GenBank. about 0.5-cm thick and 2.5-cm wide and placed in Petri dishes with sufficient moisture provided by inclusion of Antifungal activities of bacteria with different nutrients water-soaked cotton swabs. Twenty microliters of bacterial and temperatures. C. destructans KACC 41077 was suspensions at 106 cfu/ml and 108 cfu/ml were spotted on grown on PDA at 21 ºC for 2 weeks and mycelial discs of the center of the root discs and dried for 5 min in air in a 1-cm diameter were placed in the center of modified PDA clean bench. Mycelial plugs of C. destructans culture were plates with various PD broth concentrations (0, 25, 50, 75, inoculated on the central region of root discs and incubated and 100%). The bacterial suspensions (10 μl) from the at 21 ºC. Roots were examined daily for development of bacterial cultures grown in BHI broth at 28 ºC for 2 days root rot symptoms. Three days after inoculation, the degree with shaking at 200 rpm were spotted on PDA plates 3 cm of rotting was scored using the following disease severity apart from the mycelial discs. Then, the plates were rating system: 0, no discoloration; 1, yellowish discolora- incubated at 21 ºC. SDW was used as the control. After tion; 2, mild brown rot; 3, brown rot; 4, severe rot (Jeon et incubation for 14 days, inhibitory activity was measured for al., 2003). each bacterial isolate as % inhibition of mycelial growth of For the pathogen inoculum for pot experiment, rice hull C. destructans relative to the mycelia growth in the control.