DIVERSITY OF ROOT FROM TOBACCO CROPPING SYSTEMS

J. H. Kim, H. D. Skipper1, D. T. Gooden, and K. Xiong

Little is known of the effects of various cropping systems on genera recovered from non-rhizosphere soils. Four to seven the rhizobacteria associated with tobacco. Our objective was genera of rhizobacteria accounted for the predominant to develop a database on the rhizobacteria present in organisms in both cropping systems; the total number of

continuous and rotational fields of tobacco by sampling in genera ranged from 17 to 22. Under monoculture tobacco, Downloaded from http://meridian.allenpress.com/tobacco-science/article-pdf/doi/10.3381/0082-4623-45.1.15/2324838/0082-4623-45_1_15.pdf by guest on 28 September 2021 field plots over a 4-year period. Plots were established in a gram-negative rhizobacteria were dominant in July, whereas, Norfolk soil near Florence, SC. For continuous culture plots, gram-positive root bacteria were the major components in tobacco was planted in monoculture for four years. In June. The ecological shift in rhizobacteria in just 30 days rotational plots, tobacco, soybean, corn, and tobacco were may be a result of environmental factors, especially root planted during the 4-year test. Rhizobacteria were isolated exudates. This initial database on rhizobacteria for tobacco from the roots of tobacco and rotation plants and identified will be useful in future ecological studies. by fatty acids composition using gas chromatography ADDITIONAL KEY WORDS: bacterial ecology, crop (GC/FAME). Arthrobacter and Bacillus were the primary rotations, root exudates

INTRODUCTION rhizobacteria is limited. Recently, Maurhofer et al. (11) found the bacterial production of salicylic acid induced The zone of soil immediately around the root, the systemic resistance in tobacco plants against Tobacco rhizosphere, frequently supports a greater number of Necrosis Virus. Two genes that encode for the biosynthesis microorganisms than soil just a few millimeters away from of salicylic acid in Pseudomonas aeruginosa, pchA and the root (14). Numerous methods have been developed for pchB, were introduced into a root-colonizing Pseudomonas studying the rhizosphere (3,9,14). For example, after fluorescens strain P3, a strain that lacks the salicylic acid approximately 20 years of intense research on the biosynthetic capability. The recombinant strain P3 produced rhizosphere, Rovira (14) found over 2,000 publications on salicylic acid in vitro and induced resistance in tobacco. In this topic and concluded that managing the rhizosphere addition, when Phyllobacterium was cultivated with callus microflora may be a viable approach to increasing plant tissue of tobacco, the bacterium produced indoleacetic acid, growth. He also concluded that frustrations would continue which induced auxin-like effects, resulting in root unless more thought and effort were put into understanding elongation (10). In contrast, some species of Pseudomonas the microbial ecology of the rhizosphere (14). are pathogenic on tobacco (17). The rhizosphere microflora is composed of many Bolton et al. (3) support the need for research in groups of organisms that are capable of affecting plant rhizosphere ecology to increase the productivity of crop health, with both beneficial (8,13) and deleterious plants. They suggest that the interactions must be (4,6,15,16,18) effects. For example, four genera isolated investigated at smaller scales to understand the many from the rhizoplane (root surface) of canola (Brassica concurrent processes that occur in the rhizosphere. napus), Agrobacterium, Phyllobacterium, Pseudomonas, The objectives of this study were to establish a database and Variovorax, induced an increase of root dry weight up for the major bacteria associated with tobacco roots and to to 52% (2). In contrast, the cell-free culture filtrate of monitor ecological shifts of these rhizobacteria during a Pseudomonas fluorescens, a plant-growth inhibiting specific crop rotation over the 4-year period from 1997 to bacterium, showed a strong inhibitory effect on wheat root 2000. elongation and the inhibitory substance(s) was synthesized when the bacterium was grown in wheat root exudates (1). MATERIALS AND METHODS In a soybean-corn rotation, Acidovorax avenae, a weak pathogen, was the dominant rhizobacterium from Soybean was the last crop grown before initiation of continuous soybean roots and a significant yield reduction this tobacco study at the Pee Dee Research and Education was associated with the presence of this organism (6). Center near Florence, SC. The soil type was a Norfolk Tobacco is still an important crop in the United States loamy sand (fine-loamy, kaolinitic, thermic Typic and may become a major crop for nutraceutical production. Kandiudult). Tobacco and other crops were grown per Like many plant-rhizobacteria relationship studies, the standard recommended practices for the state (7). Soil- information on interactions between tobacco roots and their applied pesticides used for tobacco were sulfentrazone and clomazone for weeds; chlorpyrifos, for insects; metalaxyl for diseases; and 1,3-dichloropene for nematodes. All Department of Crop and Soil Environmental Science, Clemson University, Clemson, SC 29634-0359. materials were applied at recommended rates. Technical Contribution No. 4907 of the Clemson University Experiment Roots from tobacco and other crop plants and Station. This material is based upon work supported by the associated non-rhizosphere soil samples were obtained in CSREES/USDA, under project number SC-1700137 and SC-1000146. 1 June and July each year. Tobacco was planted for four years Corresponding author: H. D. Skipper; E-mail: [email protected] in the continuous tobacco plots, whereas, in the rotational

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plots, tobacco, soybean, corn, and tobacco were planted in 200 rpm on a rotary shaker. Soil without roots was used for 1997, 1998, 1999, and 2000, respectively. Two root samples the non-rhizosphere control. The resulting suspensions were were collected and pooled during the summer of each year subjected to serial dilution and were plated using from each of two replicates that contained 4-rows of crop. A standardized techniques and medium (5, Figure 1). A 0.1 mixture of root and soil within a 15-cm radius and depth strength tryptic soy broth agar (TSBA) supplemented with around each plant was collected for each tobacco/crop plant. cycloheximide (100 mg/L) to inhibit fungi was used to For non-rhizosphere control soil, soil samples were determine total bacterial populations. From the 0.1 strength collected from soil without vegetation. The samples were TSBA plates, we randomly selected 40 bacterial isolates to kept on "blue ice" until being processed within 48 hours. represent each plot or non-rhizosphere control. These

Tobacco/crop roots were separated from soil, placed in isolates were identified using gas chromatographic analysis Downloaded from http://meridian.allenpress.com/tobacco-science/article-pdf/doi/10.3381/0082-4623-45.1.15/2324838/0082-4623-45_1_15.pdf by guest on 28 September 2021 a sterile dilution buffer (Na4P2O7, 1.0 g; 6N HCl, 0.69 ml; of fatty acid methyl esters (GC/FAME analysis, Figure 1) in glycerol, 10 ml; H2O, 1,000 ml), and shaken for 30 min at the Multiuser Laboratory at Clemson University (12). The data presented represent an average for 2 replicates or 80 Table 1. Percentage of genera present in non- isolates per sampling for each treatment. rhizosphere Norfolk soil samples collected during summer in 1998, 1999, and 2000. Blank RESULTS AND DISCUSSION indicates that the genus was not detected or the percentage was less than 5% The major genera recovered from non-rhizosphere Genus 6/98 7/98 6/99 7/99 6/00 7/00 Norfolk soil samples were Arthrobacter and Bacillus, and Arthrobacter+ 30 8 25 14 11 11 together they accounted for 46% to 93% of the 80 isolates at Bacillus+ 50 85 21 57 46 46 each sampling. In addition, more than 70% of the bacterial Brevibacillus+ 6 8 isolates identified were gram-positive. This abundance of Burkholderia 8 gram-positive bacteria in non-rhizosphere samples could be Cellulomonas+ 11 the result of high temperature and decreased soil moisture Kocuria+ 5 6 content (Table 1). Under such stress conditions, the major + 9 10 5 genus in the Norfolk soil, Bacillus, could form spores that Paenibacillus+ 5 15 5 5 allow long-term survival during unfavorable conditions. Other genera 3(1)a 5(2)a 18(8)a 14(5)a 13(8)a 8(6)a In July 1997, a total of 21 genera (Table 2) and 32 No match 4 2 12 4 7 8 species (Table 3) of root bacteria were identified from aThe numbers in the parentheses indicate the total number of tobacco roots in the continuous tobacco fields. Based on genera with percentages below 5%. + GC-FAME identification, 19 tobacco isolates (24%) did not The plus sign indicates gram-positive and no sign indicates match any of the genera or species in the MIDI library (12). gram-negative bacteria. Of the 61 total tobacco isolates identified, Acidovorax Table 2. Percentage of rhizobacterial genera isolated from continuous (13%), Arthrobacter (10%), Phyllobacterium tobacco fields during summer in 1997, 1998, 1999, and 2000. (6%), Pseudomonas (6%), and Xanthobacter Blank indicates that the genus was not detected or the (5%) were the major genera in the first year percentage was less than 5% (Table 2). Acidovorax avenae (13%), Phyllobacterium rubiacearum (6%), and Genus 7/97 6/98 7/98 6/99 6/00 7/00 Xanthobacter agilis (5%) were the major Acidovorax 13 9 species in July 1997 (Table 3). From tobacco Arthrobacter+ 10 28 5 23 10 roots in plots assigned to the rotational plots, a Aureobacterium+ 10 total of 22 genera and 30 species of Bacillus+ 5 8 15 rhizobacteria were identified and 26 (33%) of Brevibacillus+ 5 the tobacco isolates did not match with any of Burkholderia 13 5 the species in the MIDI library. Of the 54 total Chryseobacterium 5 tobacco isolates identified, Acidovorax (11%), Clavibacter+ 5 5 Arthrobacter (9%), Phyllobacterium (6%), and Microbacterium+ 11 Pseudomonas (5%) were the major genera, and Micrococcus+ 13 6 9 Acidovorax avenae (11%), Arthrobacter ilicis Paenibacillus+ 9 (6%), and Phyllobacterium rubiacearum (6%) Phyllobacterium 6 5 11 were the major species (Tables 4 and 5). Since Pseudomonas 6 5 24 tobacco was planted in both the continuous and Ralstonia 5 rotational plots in 1997, no large variations Stenotrophomonas 6 were expected or observed in the total number Variovorax 6 of genera and species identified, or in the major Xanthobacter 5 genera and species present. Other genera 36(16)a 21(13)a 23(14)a 25(13)a 19(10)a 30(15)a In June 1998, 18 genera and 31 species of No match 24 18 34 26 17 24 aThe numbers in the parentheses indicate the total number of genera with rhizobacteria were identified from the percentages below 5%. continuous tobacco fields and 15 tobacco +The plus sign indicates gram-positive and no sign indicates gram-negative isolates (19%) did not match any of the species bacteria. in the MIDI library. Of the 65 total tobacco

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isolates identified, Arthrobacter (28%), Micrococcus (13%), (9%), Micrococcus luteus (9%), Arthrobacter ilicis (6%), Aureobacterium (10%), Clavibacter (5%), and and Clavibacter michiganense (5 %) were the major species Phyllobacterium (5%) were the major genera (Table 2). in June 1998 (Table 3). In July 1998, a total of 20 genera Arthrobacter globiformis (11%), Aureobacterium barkeri and 25 species were identified from the continuous tobacco fields. Based on FAME Table 3. Percentage of rhizobacterial species isolated from continuous tobacco identification, 27 tobacco isolates fields during summer in 1997, 1998, 1999, and 2000. Blank indicates that (33%) did not match with any of the the species was not detected or the percentage was less than 5% species in the MIDI library. Of the Species 7/97 6/98 7/98 6/99 6/00 7/00 53 total tobacco isolates identified,

Acidovorax avenae 13 9 Phyllobacterium (11%), Acidovorax Downloaded from http://meridian.allenpress.com/tobacco-science/article-pdf/doi/10.3381/0082-4623-45.1.15/2324838/0082-4623-45_1_15.pdf by guest on 28 September 2021 Arthrobacter globiformis+ 11 (9%), Micrococcus (6%), Arthrobacter ilicis+ 6 Arthrobacter (5%), Bacillus (5%), Aureobacterium barkeri+ 9 and Ralstonia (5%) were the major Bacillus megaterium+ 9 genera (Table 2). Acidovorax avenae Burkholderia gladioli 10 5 (9%), Phyllobacterium rubiacearum Chryseobacterium indologenes 5 (9%), and Ralstonia pickettii (5%) Clavibacter michiganense+ 5 5 were the major species in July 1998 Microbacterium liquefaciens+ 6 (Table 3). Soybean root samples Micrococcus luteus+ 9 9 from the rotational fields were not + 8 collected in June and July of 1998 Phyllobacterium rubiacearum 6 9 due to severe damage by deer. Pseudomonas putida 5 24 In June 1999, 17 genera and 29 Ralstonia pickettii 5 species of root bacteria were Stenotrophomonas maltophilia 6 identified from the continuous Variovorax paradoxus 6 tobacco roots and 23 tobacco isolates Xanthobacter agilis 5 (28%) did not match with any of the Other species 52(29)a 41(26)a 44(22)a 55(27)a 48(25)a 29(16)a species in the MIDI library. Of the No match 24 19 33 28 17 25 57 total tobacco isolates identified, aNumbers in parentheses indicate the total number of species with percentages below 5%. Arthrobacter (23%), Micrococcus +The plus sign indicates gram-positive and no sign indicates gram-negative bacteria. (9%), Paenibacillus (9%), and Bacillus (8%) were the major genera (Table 2). Micrococcus luteus (9%) and Paenibacillus Table 4. Percentage of rhizobacterial genera isolated from rotational tobacco fields during summer in 1997, polymyxa (8%) were the major species in June 1999 1999, and 2000. Blank indicates that the genus was (Table 3). From corn roots in the rotational plots in May not detected or the percentage was less than 5% 1999, a total of 18 genera and 29 species were identified and seven corn root isolates (8%) did not match with 7/97 5/99 6/99 6/00 7/00 any of the species in the MIDI library. Of the 73 total Genus tobacco corn corn tobacco tobacco corn isolates identified, Ralstonia (20%), Bacillus Acidovorax 11 (19%), Burkholderia (19%), and Sphingobacterium Acinetobacter 15 + (6%) were the major genera. With respect to species, Arthrobacter 9 8 Ralstonia pickettii (18%), Burkholderia cepacia (11%), Bacillus+ 19 23 10 15 + Bacillus megaterium (9%), and Bacillus cereus (5%) Brevibacillus 8 were the major species (Tables 4 and 5). In June 1999, Burkholderia 19 10 11 20 genera and 35 species were identified from the corn Chryseobacterium 6 roots and only two isolates were not identified. Of the Clavibacter+ 5 + 78 corn isolates identified, Bacillus (23%), Ralstonia Corynebacterium 8 (14%), Burkholderia (10%), Brevibacillus (8%), Enterobacter 5 Chryseobacterium (6%), Flavobacterium (6%), Flavobacterium 6 + Enterobacter (5%), and Pseudomonas (5%) were the Microbacterium 5 major genera. Ralstonia pickettii (13%), Bacillus Micrococcus+ 10 + megaterium (11%), Brevibacillus brevis (8%), Bacillus Paenibacillus 6 lentimorbus (6%), Burkholderia cepacia (6%), and Phyllobacterium 6 Flavobacterium johnsoniae (6%) were the major Pseudomonas 5 5 10 species. Bacillus was the only genus found on both corn Ralstonia 20 14 and tobacco roots in June 1999; whereas, corn had a Sphingobacterium 6 a a a a a slightly more diverse population of rhizobacteria than Other genera 36(18) 28(14) 21(12) 14(8) 35(18) tobacco. No match 33 8 2 22 26 a In June 2000, a total of 17 genera and 30 species The numbers in the parentheses indicate the total number of genera with percentages below 5%. were identified from the continuous tobacco fields. +The plus sign indicates gram-positive and no sign indicates gram- Based on FAME identification, 14 tobacco isolates (17 negative bacteria. %) did not match with any of the species in the MIDI

Tobacco Science (2001/2002) 45:15-20 17 library. Of the 66 total tobacco isolates identified, Bacillus Burkholderia gladioli (10%), Bacillus megaterium (9%), (15%), Burkholderia (13%), Microbacterium (11%), Microbacterium liquefaciens (6%), Clavibacter Arthrobacter (10%), Brevibacillus (5%), Clavibacter (5%), michiganense (5%), and Pseudomonas putida (5%) were the and Pseudomonas (5%) were the major genera (Table 2). major species in June 2000 (Table 3). From the rotational tobacco fields that were now back in tobacco, Table 5. Percentage of rhizobacterial species isolated from rotational a total of 15 genera (Table 4) and 26 species tobacco fields during summer in 1997, 1999, and 2000. Blank (Table 5) were identified and 18 tobacco indicates that the genus was not detected or the percentage isolates did not match with any of the species was less than 5% in the MIDI library. Of the 62 total tobacco

7/97 5/99 6/99 6/00 7/00 isolates identified, Acinetobacter (15%), Downloaded from http://meridian.allenpress.com/tobacco-science/article-pdf/doi/10.3381/0082-4623-45.1.15/2324838/0082-4623-45_1_15.pdf by guest on 28 September 2021 Species tobacco corn corn tobacco tobacco Burkholderia (11%), Bacillus (10%), Acidovorax avenae 11 Micrococcus (10%), Arthrobacter (8%), Acinetobacter baumannii 14 Clavibacter (5%), and Microbacterium (5%) Arthrobacter ilicis+ 6 were the major genera. Acinetobacter Bacillus cereus+ 5 baumannii (14%), Burkholderia gladioli Bacillus lentimorbus+ 6 (11%), Micrococcus luteus (10%), Bacillus Bacillus megaterium+ 9 11 5 9 megaterium (5%), and Clavibacter Brevibacillus brevis+ 8 michiganense (5%) were the major species Burkholderia cepacia 11 6 (Tables 4 and 5). Except for Brevibacillus and Burkholderia gladioli 11 Pseudomonas in the continuous fields and Clavibacter michiganense+ 5 Acinetobacter and Micrococcus in the Corynebacterium aquaticum+ 8 rotational fields, the genera from both fields Enterobacter cancerogen were the same. Flavobacterium johnsoniae 6 In July 2000, 20 genera and 21 species of Micrococcus luteus+ 10 root bacteria were identified from the Paenibacillus macerans+ 5 continuous tobacco fields. Based on GC- Phyllobacterium rubiacearum 6 FAME identification, 20 tobacco isolates Pseudomonas huttiensis (25%) did not match with any of the species Pseudomonas putida 8 in the MIDI library. Of the 60 total tobacco Ralstonia pickettii 18 13 isolates identified, Pseudomonas (24%), Other species 44(27)a 49(25)a 40(29)a 33(21)a 45(27)a Stenotrophomonas (6%), Variovorax (6%), No match 33 8 10 22 25 Burkholderia (5%), and Chryseobacterium aThe numbers in the parentheses indicate the total number of species with (5%) were the major genera (Table 2). With percentages below 5%. respect to species, Pseudomonas putida + The plus sign indicates gram-positive and no sign indicates gram-negative (24%), Stenotrophomonas maltophilia (6%), bacteria. Variovorax paradoxus (6%), Burkholderia gladioli (5%), and Chryseobacterium

Figure 1. Flow chart for the enumeration, isolation and identification of rhizobacteria from plant rhizosphere

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indologenes (5%) were the major species (Table 3). From pseudomonad and its inhibitory metabolite(s). J. Appl. the rotational tobacco fields, a total of 22 genera and 31 Bacteriol. 74:20-28. species were identified and 21 tobacco isolates (25%) did 2. Bertrand, H., R. Nalin, R. Bally, and J.C. Cleyet- not match with any of the species in the MIDI library. Of Marcel. 2001. Isolation and identification of the most the 59 total tobacco isolates identified, Bacillus (15%), efficient plant growth-promoting bacteria associated with Pseudomonas (10%), Corynebacterium (8%), and canola (Brassica napus). Boil. Fertil. Soils 33:152-156. Paenibacillus (6%) were the major genera; and Bacillus 3. Bolton, H., Jr., J.K. Fredrickson, and L.F. Elliott. megaterium (9%), Corynebacterium aquaticum (8%), 1993. Microbial ecology of the rhizosphere. Pages 27-63, in: Pseudomonas putida (8%), and Paenibacillus macerans Soil Microbial Ecology F.B. Metting, Jr., ed. Marcel

(5%) were the major species (Tables 4 and 5). Pseudomonas Dekker, Inc., NY. Downloaded from http://meridian.allenpress.com/tobacco-science/article-pdf/doi/10.3381/0082-4623-45.1.15/2324838/0082-4623-45_1_15.pdf by guest on 28 September 2021 was the only common genus in both fields. 4. Elliott, L.F., and J.M. Lynch. 1985. Plant growth- Arthrobacter was isolated from tobacco rhizospheres in inhibitory pseudomonads colonizing winter wheat (Triticum all-sampling dates; however, since the percentage was less aestivum L.) roots. Plant Soil. 84:57-65. than 5% in both continuous and rotational fields in July 5. Elliott, M.L., E.A. Guertal, E.A. Des Jardin, and 2000, it is not listed in the tables (Tables 2 and 4). H.D. Skipper. 2003. Effect of nitrogen rate and root-zone When the gram-negative and gram-positive bacteria mix on rhizosphere bacterial populations and root mass in from the continuous plots were compared, gram-negative creeping bentgrass putting greens. Biol. Fertil. Soils 37:348- bacteria were predominant in July and gram-positive 354. bacteria were predominant in June (Table 2). Continuous 6. Frederick, J.R., H.D. Skipper, J.H. Kim, and S.J. tobacco might create the soil/root environment favorable for Robinson. 2001. Rhizobacteria from soybean and corn in gram-negative bacteria and gram-positive bacteria in July rotation. Presented at ASA Annu. Meeting. Madison, WI. and June, respectively. A shift in bacterial genera was 7. Gooden, D.T. 2000. Tobacco Production. Pages 7-67 observed when the isolates in June were compared with in: South Carolina Tobacco Growers Guide. Clemson Univ. those of July. This fluctuation may be a result of Ext. Circ. 569. environmental factors, such as different nutrient levels due 8. Hodges, C.F., D.A. Campbell, and N. Christians. to changes in the chemical components of tobacco root 1993. Evaluation of Streptomyces for biocontrol of exudates, temperature, and moisture content. Bipolaris sorokiniana and Sclerotinia homoeocarpa on the Even though Acidovorax avenae, a weak pathogen on phylloplane of Poa pratensis. J. Phytopathol. 139:103-109. some plants (6), was a dominant rhizobacterium isolated in 9. Kloepper, J.W., and C.J. Beauchamp. 1992. A review the first year of this study, it did not persist in either of issues related to measuring colonization of plant roots by cropping systems over time. The pathogenicity of this bacteria. Can. J. Microbiol. 38:1219-1232. organism on tobacco merits evaluation. Tobacco yield in 10. Lambert, B., H. Joos, S. Dierickx, R. Vantomme, J. 2000 was 3621 kg/ha in rotational plots and 2576 kg/ha in Swings, K. Kersters, and M. van Montagu. 1990. continuous tobacco plots. This was a significant reduction at Identification and plant interaction of a Phyllobacterium sp., the 5% level and was attributed to a strong infestation of a predominant rhizobacterium of young sugar beet plants. root-knot nematode in the continuous tobacco system that Appl. Environ. Microbiol. 56:1093-1102. was not controlled with an in-row application of 1,3- 11. Maurhofer, M., C. Reimmann, P. Schidli-Sacherer, dichloropene at 98.2 L ha-1 (10.5 gal A-1). S. Heeb, D. Haas, and G. Defago. 1998. Salicylic acid Since rhizobacteria may play an important role in biosynthetic genes expressed in Pseudomonas fluorescens sustainable agriculture via effects on plant growth and strain P3 improve the induction of systemic resistance in biological control of pests, a critical research need in crop tobacco against tobacco necrosis virus. Phytopathology management is to understand the interactions of crop 88:678-684. rotation and rhizobacteria on tobacco production. The 12. MIDI. 2000. Microbial identification system, database on rhizobacteria for tobacco developed in this Operation manual, Version 5. Microbioal ID, Inc., Newark, study is limited to one location and it needs to be expanded DE. to more locations in the future and with more intense 13. Nelson, E.B., and C.M. Craft. 1991. Introduction sampling times. Even with its limitations, the database is an and establishment of strains of Enterobacter cloacae in golf essential first step to further ecological studies with tobacco. course turf for the biological control of dollar spot. Plant Dis. 75:510-514. ACKNOWLEDGMENT 14. Rovira, A.D. 1991. Rhizosphere research - 85 years of progress and frustration. Pages 3-13 in: The Rhizosphere This project was partially funded by the Clemson and Plant Growth. D.S. Keister and P.B. Cregan, eds. University Agroecology Program. We appreciate Dr. M.B. Kluwer Academic Publishers, Boston, MA. Riley for professional assistance with the GC/MIDI 15. Schippers, B., A.W. Bakker, and P.A. Bakker. Microbial Identification System. 1987. Interaction of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Annu. LITERATURE CITED Rev. Phytopathol. 25:339-358. 16. Suslow, T.V., and M.N. Schroth. 1982. Role of 1. Astrom, B., A. Gustafsson, and B. Gerhardson. 1993. deleterious rhizobacteria as minor pathogens in reducing Characteristics of a plant deleterious rhizosphere crop growth. Phytopathology 72:111-115.

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17. Tso, T.C. 1990. Pest and pest control. Page 136 in: Production, Physiology, and Biochemistry of Tobacco Plant. Ideals, Inc., Beltsville, Maryland. 18. Wang. G., and H.D. Skipper. 2004. Identification of denitrifing rhizobacteria from bentgrass and bermudagrass golf greens. J. Appl. Microbiol. 97:827-837. Downloaded from http://meridian.allenpress.com/tobacco-science/article-pdf/doi/10.3381/0082-4623-45.1.15/2324838/0082-4623-45_1_15.pdf by guest on 28 September 2021

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