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Predicting Changes in the Concentrations

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Predicting Changes in the Concentrations DIVERSITY OF ROOT BACTERIA 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 Tobacco Science (2001/2002) 45:15-20 15 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 Micrococcus+ 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.
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