diversity of ralstonia solanacearum populations ... - Tobacco Science

DIVERSITY OF RALSTONIA SOLANACEARUM POPULATIONS AFFECTING TOBACCO CROPS IN NORTH CAROLINA M. Katawczik1, H.T. Tseng1, and A.L. Mila1*

One hundred eighty four strains of Ralstonia solanacearum isolated in 2007 and 2008 from 11 tobacco fields in North Carolina were evaluated for genotypic diversity and aggressiveness. All strains were race 1, biovar 1, and belonged to phylotype II. Genetic diversity of the strains was assessed with the use of repetitive sequence–based polymerase chain reaction. DNA primers (REP, ERIC, and BOX) were used to generate genomic fingerprints. Both REP and BOX revealed 3 patterns: Ar, Cr, and Dr and Ab, Cb, and Db, respectively. Five patterns were identified with ERIC-PCR. Pattern Ae was found in 80% of the strains collected. Pattern Be was in 4%, pattern Ce in 13%, pattern De in 2%, and pattern Ee in 1% of the strains collected. Cluster analyses showed that the

strains were 88% similar and constitute a rather homogeneous group. Aggressiveness of strains was evaluated on 3 tobacco cultivars with different levels of resistance to bacterial wilt. Overall, aggressiveness depended on the field from which the strains were collected. Within a field there was a significant difference (P , 0.0001) in aggressiveness among strains. Differences in aggressiveness were significantly associated (P , 0.0001) to genotypic patterns. In fields where patterns A and C were found, strains of pattern A were the most aggressive, whereas in fields with patterns B, C, and E, strains of pattern E were the most aggressive. Additional key words: bacterial wilt, REP-PCR, Nicotiana tabacum

INTRODUCTION

Asia, II for strains from Americas, III for those from Africa, and IV for strains from Indonesia. In North Carolina tobacco is 1 of the top 4 agricultural products in farm revenue with a production value of US$900 million (23). In 2014, approximately 2.3% of the North Carolina tobacco crop was affected by BW accounting for a loss of US$22 million (22). BW on tobacco is best managed through a combined use of rotation, soil fumigation, and resistant cultivars (5). Flue-cured tobacco cultivars available in the United States have various levels of resistance to BW that originated from a line known as TI448A (19). Cultivars with this type of resistance have been used in tobacco fields in North Carolina for several decades and successfully control the disease in most years. Previous research suggests that plant host may influence diversity in R. solanacearum strains, but information is absent on the effect of implementation of resistant cultivars on the diversity of a bacterial population (2). Robertson et al. (27) reported that strains isolated from tobacco and tomato in North Carolina, South Carolina, Georgia, and Florida were race 1, biovar 1. Race 1, biovar 3 strains isolated from pepper were reported for the first time in Florida in 2007 and found to group with strains originating in Asia (10,28). Differences in R. solanacearum populations collected from South Carolina, Georgia, North Carolina, and Florida have been found with the use of REP-PCR (28). In the same study, only a small portion of strains was evaluated for aggressiveness and only 2 strains from North Carolina (28) were included. Overall, little is known about aggressiveness of strains of R. solanacearum, especially those strains collected from tobacco crops. Knowledge of diversity of local populations of a pathogen is a key factor for successful breeding and integrated pest management programs. In the present study 184 R. solanacearum strains from flue-cured

Bacterial wilt (BW), caused by Ralstonia solanacearum, is a soil-borne disease affecting hundreds of plant species worldwide (3,20). In the United States the disease infects economically important crops such as tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), and pepper (Capsicum annuum) (10,12,21). BW occurs predominantly in the southeastern United States, causing severe losses in North Carolina and South Carolina on tobacco and tomato, whereas in Florida and Georgia it is a problem on tomato (12,14,22,27,28). Ralstonia solanacearum is a complex species with a very large diversity among strains (8). Historically, this complex species was divided into races, separated by host range; and biovars, separated based on carbohydrate utilization (8,13). Currently, 5 races and 7 biovars have been identified worldwide. Recently, molecularbased approaches have been developed to investigate the genetic diversity of strains of R. solanacearum. Repetitive sequence–based polymerase chain reaction (REP-PCR) is a technique that uses primers to amplify certain repetitive DNA sequences found throughout gramnegative bacteria, like R. solanacearum, and allows identification of genetic diversity within populations at a clonal level and in some instances between biovars (6,30). The method has also differentiated tomato from tobacco strains collected from the same state and between strains from the same host across a region (15,27). Recently, a new hierarchical classification scheme based on sequence of specific genes divides the complex into 4 phylotypes (4,24). The phylotypes are I for strains from *Corresponding author: A.L. Mila; email: [email protected] 1 Department of Plant Pathology, North Carolina State University, Campus Box 7616, Raleigh, NC 27695

Tobacco Science (2016) 53:1–11

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Table 1. Origin and genotypic diversity of Ralstonia solanacearum strains collected in North Carolina in 2007 and 2008. County

a b

Field

Edgecombe

1

Johnston

2

Johnston

3

Edgecombe Vance Johnston

4 5 6

Edgecombe

7

Johnston

8

Johnston Caswell

9 10

Duplin

11

Race Biovara

Year Collected

Tobacco Cultivarb

Number of Strains

ERIC-PCR Pattern

R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1 R1B1

2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008

K 326 K 326 K 326 K 394 SP 168 K 326 K 326 K 346 SP 168 K 326 K 326 K 346 K 346 CC 27 K 346 K 394 NC 196 SP 220 SP 227 CC 27 CC 27 K 346 K 346 K 394 NC 196 SP 220 SP 220 SP 227 K 326 CC 27 K 346 K 394 NC196 SP 220 SP 227 CC 27 CC 65 CC 700 K 346 K 394 NC 196 NC 196 PVH 1118 SP 220 SP 227

4 3 1 1 1 1 3 2 2 7 5 1 18 3 4 15 5 2 2 2 2 1 3 2 2 4 1 2 3 2 2 3 3 1 5 7 3 3 13 11 1 4 2 6 16

De Ae Ae Ae Ae Ee Ce Be Ce Ae Ae Ce Ae Ae Ae Ae Ae Ae Ae Ce Be Be Ce Be Ce Ce Be Ce Ce Ae Ae Ae Ae Ae Ae Ae Ae Ae Ae Ae Ce Ae Ae Ae Ae

Race and biovar were determined for each strain as described in the main text. Cultivars from which strains were isolated. Resistant cultivars: K 346, SP 168, SP 227, SP 220, and CC 65; moderate resistant: CC 27, NC 196, PVH 1118, CC 700; low resistant: K 326, and K 394.

tobacco grown in 5 counties and 11 fields in North Carolina were examined. One objective of this study was to compare the genetic diversity of R. solanacearum populations collected from tobacco crops in North Carolina. All strains were characterized according to race and biovar and then subjected to genomic fingerprinting with the use of REP-PCR assays. Another objective was to analyze phenotypic variation of the 2

strains by performing a comparative aggressiveness analysis with the use of tobacco cultivars differing in resistance to R. solanacearum. MATERIALS AND METHODS Collection of Strains and Identification. The R. solanacearum strains used in this study are described


in Table 1. Six naturally infested fields in Edgecombe, Johnston, and Vance counties were sampled in 2007 and 5 located in Edgecombe, Johnston, Caswell, and Duplin counties were sampled in 2008. Fields 2 and 3 in Johnston County in 2007 and the fields in Edgecombe, Johnston, Caswell, and Dublin counties in 2008 were fields where replicated on-farm trials to evaluate resistance of tobacco cultivars to BW were established during the year that strains were collected. These fields had known history of severe BW and the disease was verified on tobacco crops a year before the establishment of the on-farm trials. The other 5 fields were commercial fields where BW incidence was diagnosed the year strains were collected (Mila, unpublished). We collected 20 symptomatic plants from the commercial fields and 20 symptomatic plants (5 per replication; 4 replications) per cultivar from the farm trials. On the on-farm trials symptomatic plants were collected from susceptible (K326, K394), moderately resistant (CC27, CC700, NC196, PVH1118), and resistant (K346, SP168, SP220, SP227) cultivars. In fields with low incidence we were unable to identify and collect 20 symptomatic plants. Tobacco plants with visual typical symptoms of infection by R. solanacearum were brought to the laboratory for isolation. Stems were externally washed and surface sterilized with 70% ethanol, and a transverse section was made to uncover the xylem. Small pieces of the xylem exhibiting dark streaking (typical symptom of infection by R. solanacearum) were used to prepare microscopic specimens. When bacterial oozing was observed under the microscope, a sterile loop was used to sample this exudate, which was streaked directly onto 2,3,5-triphenyltetrazolium chloride (TZC) medium (13). Plates were incubated for 2–3 days at 29uC. Colonies with typical R. solanacearum appearance (irregularly shape colonies, fluidal with a pink center) were subcultured onto TZC medium and tested with R. solanacearum–specific immunoassay strips according to the manufacturer’s instructions (Agdia Inc., Elkhart, IN) to confirm the pathogen’s identity. Several strains with colony appearance atypical to that of R. solanacearum on TZC (small, round, dark red color colonies) were used as negative controls when tested with the immunoassay strips. All strains that had typical R. solanacearum colony appearance were stored as suspensions in 20% glycerol at 280uC for further analysis. Prior to use, strains were removed from storage and revived by growing on TZC at 29uC. Race Differentiation and Biovar Determination. The race differentiation was performed as described by Lozano and Sequeira (18). The typical duration for R. solanacearum–elicited, incompatible reaction is 10–12 hr, whereas the compatible necrotic reaction occurs at 36 hours postinoculation or later (18). Fully expanded tobacco leaves of cultivars K 326 and K 394 were syringe infiltrated with a suspension of R. solanacearum cells adjusted to 1 3 108 CFU/ml in sterile deionized water (SDW). Tomato strain K 136 was used as a positive and SDW as a negative control. Plants were maintained at 28uC with a 12-hr photoperiod and were monitored for response at 18, 36, 48, and 72 hr postinfiltration (18).

Infiltrated areas of leaves were recorded for tissue collapse, dying, and browning. The race differentiation test was performed 2 times for each strain. A variation of Hayward’s (7) biochemical test, which assays the ability of strains to oxidize a panel of sugars and sugar alcohols, was used for biovar determination of the 184 strains. The test was carried out in 96-well ELISA plates; 200 ml of a final 1% solution of Hayward’s medium amended with filter-sterilized dextrose, mannitol, sorbitol, trehalose, lactose, maltose, or d(+) cellobiose was dispensed into each well of the ELISA plate. Hayward’s medium without a carbon source served as a control. A suspension of 10 ml of 1 3 108 colonyforming units (CFU)/ml was prepared from fresh cultures of the strains grown for 24 hr prior to the test. Plates were incubated at 28uC for 28 days. The color change of the ELISA plates was visually recorded every 2 days. Acid production changed the color of the culture medium from green to yellow. The biovar test was carried out 2 times. Endoglucanase Gene PCR Amplification, Sequencing, and Analysis. A subset of 30 strains was used to amplify the endoglucanase gene (egl). PCR was performed with the MasterAmp Tfl DNA polymerase kit as recommended by the manufacturer (Epicentre Technologies, Madison, WI). PCR primers were synthesized by Integrated DNA Technology (Coralville, IA). For direct PCR from cells, about 108 cells were suspended in 100 ml of deionized water, heated to 100uC for 15 min, and then 1 ml was used as the template in a 25-ml reaction. Each 25-ml PCR reaction consisted of 1.5 mM MgCl2, 400 mM dNTPs, and primers at 50 pmol. A 750–base pair (bp) fragment of the R. solanacearum egl gene, which encodes a moderately conserved pathogenicity factor, was amplified as described by Poussier et al. (24) for each of the strains with the use of the primer pair Endo-F (59-ATGCATGCCGCTGGTCGCCGC) and Endo-R (59-GCGTTGCCCGGCACGAACACC). The thermocycler conditions using the egl primer set were 5 min of denaturation at 96uC; followed by 30 cycles of 95uC for 1 min, 69uC for 1 min, and 72uC for 1 min 30 sec; with a final extension of 10 min at 72uC. DNA sequencing was done in both directions at the North Carolina State University Genomic Science Laboratory. Phylogenetic analysis was performed on the sequences reported here by using the Biology Workbench 3.2 software (San Diego Super Computer Center, San Diego, CA). The following 5 reference sequences were retrieved from GenBank and included in the phylogenetic analysis: UW134 (DQ657373) isolated from S. tuberosum L., Kenya, K60 (AF283285) isolated from S. lycopersicum L., NC, UW551 (DQ657596) Pelargonium 3 hortorum Bailey, Kenya, JS422 (HM775373) isolated from S. melongena L., China and GMI1000 (DQ657595). Repetitive Sequence–Based Polymerase Chain Reaction (REP-PCR). Colonies of the 184 strains grown on TZC medium for 36 hr at room temperature were used for total genomic DNA extraction. Extraction was conducted by using the Qiagen DNeasy blood and tissue kit (Qiagen Inc., Chatsworth, CA) according to the manufacturer’s instructions. The DNA from each strain


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was quantified and diluted to 50 ng ml21 for further genomic applications. REP-PCR was performed employing 3 primers: REP (REP1R-1, REP2-1), ERIC (ERIC1R, ERIC2), and BOX (BOXAIR). PCR amplifications were performed with the use of a Bio-Rad iCycler (Bio-Rad Laboratories, Inc., Hercules, CA), under the conditions previously described by Louws et al. (17). To confirm reproducibility of the results, amplifications were repeated once for each strain. Amplified PCR products were separated by horizontal electrophoresis in 1.5% (wt/vol) agarose gels in 1x Tris-acetate-EDTA at 90 V for 10 hr. HyperLadder I (Bioline, Randolph, MA) was included in order to normalize the banding pattern of the PCR profiles. Gels were photographed with the use of an Alpha Imager EC (Alpha Innotech, San Leonardo, CA). Gel images were analyzed with GelCompar II (version 3.0) software (Applied Mathos, Kortrijk, Belgium). Differences in banding patterns observed were established by the presence or absence (indicated by 1 or 0, respectively) of an amplification product. Analyses were conducted with the use of band-based similarities calculated with the use of the Pearson’s product-moment correlation, a curved-based coefficient (25,26). Cluster analysis with the use of the unweighted pair-group method analysis (UPGMA) was performed (16,31). Aggressiveness Assays. Three flue-cured tobacco cultivars with different levels of resistance to R. solanacearum were used for the aggressiveness assays. Specifically, cultivars SP 168 (highly resistant), NC 71 (medium resistance), and K 326 (susceptible) were used, and they will be referred to as differential cultivars hereafter. Level of resistance of these cultivars to BW has been determined in replicated field evaluations conducted since 2000 in nurseries naturally infested with R. solanacearum in North Carolina (22). Seedlings of the cultivars were grown in 12-well tissue culture plates with 0.3 g of perlite/well. Seedlings were fertilized 10 days after seeding and then at 10-day intervals with 500 ml of 200 ppm water-soluble fertilizer (20–10–20, N–P–K). Inoculation was conducted when seedlings were 21 days old, as described in Katawczik and Mila (11). For inoculum preparation, each strain of R. solanacearum was grown for 24 hr on TZC medium and inoculum concentrations were adjusted to 1 3 108 CFU/ml in sterile deionized water. The roots of the seedlings were injured immediately before inoculation by stabbing a sterilized scalpel around each seedling in a well, effectively severing pieces of the root from each seedling. The injured seedlings were inoculated with 400 ml of the bacterial suspension applied in each plant. There were 2 replicates of 7–8 seedlings for each cultivar per strain. The experiment was repeated once for each strain. SDW and tomato strain K 136 were used as negative controls. The inoculated seedlings were kept at 29uC with a 12-hr photoperiod. Three to four tissue culture plates were placed in trays with moist paper towels and a polyvinyl-chloride wrap cover to keep moisture levels high to facilitate development of disease symptoms. Bacterial wilt incidence was evaluated starting at day 4 after inoculation and every 3–4 days up to 21 days 4

after inoculation for a total of 6 evaluations. Seedlings with BW symptoms were assessed as infected with R. solanacearum. Typical symptoms were stunting, blackening of the roots and the stem, 1-sided leaf wilting, yellowing, and necrosis between veins and leaf margins (19). The negative controls remained healthy in all repeats of the experiments. At the end of the experiments, the disease indexes (DI) for each cultivar was calculated and used to compare aggressiveness among strains. The DI is a measurement that incorporates all the disease evaluations and weighs earlier evaluations more heavily than later evaluations (1). For instance, if disease incidence has been evaluated 5 times during the assay, then the DI is DI 5 [Si 5 i 2 n Xi[100 2 (i 2 1)(100/n)]/N, where i is an ordinal evaluation number, n is the number of disease evaluations, X is the number of diseased seedlings since the last count, and N is the total number of seedlings. The data were analyzed with generalized linear models (proc GLM; SAS, version 9.1, SAS Institute, Inc., Cary, NC) to determine the effect of strain, differential cultivar, and their interaction on the DI. Factors included were experiment, repetition, strain, differential cultivar, and the interaction between strain and differential cultivar. Experiment, repetition, and differential cultivar were fixed factors and strain random factor. Subsequently the effect of strain and field on DI was examined with a nested design with strain nested within field. The same nested analysis was performed replacing the factor strain, with the factor genotypic pattern (ERIC-PCR). Field and ERIC-PCR factors were fixed. Analysis was done with the use of the GLM procedure of Statistical Analysis System (SAS, version 9.1; SAS Institute, Cary, NC). RESULTS Collection of Strains and Identification. Disease incidence was very low in 2007 and thus we were unable to collect 20 symptomatic plants per field, except for field 6 (Table 1). On the contrary, disease incidence was high in 2008. In total, 184 strains were isolated from tobacco in fields naturally infested with R. solanacearum in North Carolina in 2007 and 2008. Forty-nine strains were isolated in 2007 and 135 in 2008. In 5 fields, a large number of strains was collected, ranging from 16 to 66 and in the other 6 fields there were only a few strains collected (3–8) (Table 1). The variable number of strains per field was a result of difference in disease incidence between fields and years and failure to isolate the pathogen from symptomatic plants occasionally. All strains with typical colony appearance were confirmed to be R. solanacearum by the immunoassay strip test but not the strains with atypical appearance. Race Differentiation, Biovar, and egl Characterization. All strains induced a dark brown necrotic area surrounded by a yellow halo and were classified as race 1. Seventy-three percent of the strains induced a reaction at 36 hr and the rest at 48 hr or later. All strains were


Figure 1. Phylogenetic analysis of partial endoglucanase gene sequences of 30 Ralstonia solanacearum strains from tobacco crops in North Carolina and 5 reference strains from the species complex. Scale bar represents 1 nucleotide substitution per 100 nucleotides. Refer to the main text for details of egl sequencing and phylogenetic analysis.

classified as biovar 1 based on their biochemical profiles. A summary of these data along with the genotypic data is provided in Table 1. All strains had identical egl sequences and were assigned to phylotype II (Figure 1). The high similarity of the egl sequence indicates that the strains are probably clonal. REP-PCR Assay. Information on genetic diversity of the 184 strains was generated with REP, BOX, and ERIC-PCR assays. With the use of band-based analysis, REP and BOX-PCR contained bands ranging from

400 to 2,500 and 600 to 3,000 bp, respectively and revealed 3 patterns: Ar, Cr, Dr, and Ab, Cb, Db, respectively (Figures 2A and 2B). Interestingly, strains with a REP-PCR pattern Ar had a BOX-RPC pattern Ab; pattern Cr corresponded to Cb, and Dr to Db. The ERICPCR revealed a larger diversity than REP and BOXPCR with patterns Ae, Be, Ce, De, and Ee for a total of 5 patterns (Figure 2C). Strains with Ae corresponded to strains with pattern Ar or Ab, strains with De to strains with pattern Dr or Db, and strains with Be, Ce, and Ee to strains with pattern Cr or Cb.


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Figure 2. Agarose gel showing the 5 REP-PCR patterns of representative strains. (A) REP-PCR pattern depicting patterns Ar, Cr, and Dr. (B) BOX-PCR depicting patterns Ab, Cb, and Db. (C) ERIC-PCR depicting patterns Ae, Be, Ce, De, and Ee. Numbers above gel images are the representative strains, and the letters are the depicted patterns. Arrows represent differences between the different patterns.

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Figure 3. Cluster analysis based on repetitive sequence polymerase chain reaction (REP-PCR) of 31 strains of Ralstonia solanacearum. County and field isolated from are shown along with REP-PCR patterns. Analysis was preformed with the use of the unweighted pair-group method with the use of arithmetic averages (UPGMA) with GelCompar II (version 3.0) software. The patterns were defined by Pearson’s correlation coefficient.

Patterns Ar and Ab were found in 80%, patterns Dr and Db in 2%, and Cr and Cb in 18% of the strains. Pattern Ae was the predominant pattern and was found in 80% of the strains collected. Pattern Ce was in 13%, pattern Be in 4%, pattern De in 2%, and pattern Ee in 1% of the strains collected (Table 1). Grouping patterns by cultivar strain was isolated from did not revealed any particular trend, as pattern A was by far the predominant pattern, ranging from 66 to 100%, regardless of the cultivar (Table 1). Grouping patterns by county revealed pattern Ae as predominant in Duplin (98%), Caswell (100%), Edgecombe (91%), and Vance (100%) counties. Duplin and Edgecombe counties are in the Coastal Plain and Caswell and Vance counties in the Piedmont of North Carolina, 2 distinct regions of the state with different soil types and tobacco production practices. Fields 3, 8, and 9, where patterns Be, Ce, and Ee were found, were in Johnston County (Table 1). Strains collected in fields in Johnston County demonstrated the largest genotypic diversity, with 4 patterns: Ce (42.2%), Ae (40.4%), Be (15.4%), and Ee (2%). The race differentiation test revealed that strains with patterns Bb and Cb induced a necrotic reaction at 48 hr or later compared to those of pattern Ab, Db, and Eb, which displayed the necrotic reaction always at 36 hr.

Because of the very similar genotypic patterns of As, Bs, Cs, Ds, and Es, a subsample of 31 strains was used and a dendogram was constructed based on the results from the Pearson’s correlation with the use of a 5% tolerance level (Figure 3). There was an 88% similarity among all the strains. Curve-based analyses grouped the strains into the 3 groups that were identified by the REP and BOX-PCR as patterns A, C, and D. Pattern C was further divided into patterns B, C, and E with ERICPCR; yet the similarity among these strains was over 96% (Figure 3) Aggressiveness Assays. The factors experiment (P 5 0.6368), repetition (P , 0.5362), and strain (P , 0.4204) were not significant. On the contrary, differential cultivar, and the interaction between strain and differential cultivar, were significant (P , 0.0001), suggesting that results related to aggressiveness depend on the differential cultivar used. Overall, DI obtained with K 326 with a mean ranging between 50 and 53.6, and it was significantly higher (P , 0.0001) than the one with NC 71 that had a range of 37.5 to 41.3. The lowest mean DI was obtained with the resistant cultivar SP 168. It ranged between 31 and 34.5 and it was significantly different that the one obtained with K 326 or NC 71. The majority of the strains produced the highest DI values on K 326 regardless of the cultivar from which


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Figure 4. Frequency of strains isolated from different cultivars that produced the highest disease index (DI) in each differential cultivar (K 326, NC 71, and SP 168).

they had been isolated (Figure 4). Interestingly, as many strains produced the highest DI on K 326 as on SP 168 in case of strains isolated from K 326 (Figure 4). These strains derived from different fields and there was no association between aggressiveness on specific differential cultivar and field of origin. Field from which the strains were isolated had a significant effect on the DI (P , 0.0001) (Table 2; Figure 5). Within a field there was significant difference among strains with respect to the DI values as the term strains nested to field was also significant (P , 0.0001) (Table 2). Results depended on the differential cultivars used. For instance, when SP 168 was inoculated with the strains the highest average DI was for fields 4 and 5 and the lowest for field 9, whereas the largest range of DI values were for fields 6 and 9 (Figure 5C). On the contrary, with NC 71 or K 326 the highest average DI was for fields 7, 8, 9, 10, and 11 (Figures 5A and 5B). The highest average DI for all fields was with K 326, and the largest range of DI values were for fields 4 and 11 (Figure 5A). Differences in aggressiveness among strains based on their genotypic pattern were also observed and were field dependent (Table 3). In fields where both pattern Ae and Ce were found, strains of pattern Ae had always

the highest DI. On the other hand, in field 3 in Johnston County, where patterns Be, Ce, Ee were found, the strain of pattern Ee had the highest DI, whereas in field 8 strains of pattern Ce were more aggressive than those of pattern Be. Unfortunately, a limited number of strains from these patterns were collected and thus the significance of the funding may not hold true in other locations. DISCUSSION Diversity of tobacco strains of R. solanacearum in the southeastern United States present a special interest, as the crop has been established in this part of the United States for over a century, and until recently it could be grown only in particular fields because of the quota system (19). In this study, 184 strains of R. solanacearum collected from tobacco crops in North Carolina were evaluated to investigate genotypic diversity and aggressiveness of strains between and within tobacco fields. We found low genetic diversity among strains of R. solanacearum affecting tobacco crops in North Carolina, whereas aggressiveness differed greatly among strains, depending on the field from which the strains were collected. Aggressiveness could

Table 2. Analysis of variance for the main effects on disease index. Source SP 168 Field Strain (field) NC 71 Field Strain (field) K 326 Field Strain (field) a b c

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DFa

TIII SSb

MSc

F Value

Pr . F

10 173

33,875.0 61,280.1

3,387.5 354.2

24.84 2.60

,0.0001 ,0.0001

10 173

32,669.0 90,459.0

3,266.9 522.9

27.2 4.35

,0.0001 ,0.0001

10 173

33,131.4 90,281.9

3,313.1 521.8

25.1 3.95

,0.0001 ,0.0001

DF: Degrees of freedom. TIIISS: Type III sum square. MS: Mean sum.


Figure 5. Average disease index of bacterial wilt induced by strains of Ralstonia solanacearum when cultivars (A) K 326, (B) NC 71, and (C) SP 168 were inoculated. Strains were collected in Edgecombe County (field 1 and 4), Johnston County (fields 2, 3, 6, 8, and 9), Caswell County (field 10), Vance County (field 5), and Duplin County (field 11). Bars are 1 standard deviation of the mean. Bars with the same number of asterisks indicate average disease indices that are not significantly different from each other.

not be associated closely with cultivars from which the strains were isolated, as had been initially speculated. Field of origin had a significant effect on overall aggressiveness of strains. The most aggressive strains were from Caswell County, from a field with 1-yr rotation, where K 346 has been planted several times in the past. However, aggressiveness was also high in fields 7, 8, 9, and 11, all of which were in a 2-yr rotation schedule and where only moderately resistant cultivars had in the past deployed in those fields. In another study, R. solanacearum strains isolated from a single potato field in Florida had similarly considerable variation in aggressiveness (21). It was speculated that strains with differences in aggressiveness had been selected by 20 consecutive years of planting the particular field on a potato cultivar trial where certain resistant or tolerant cultivars were present from year to year (21). Thus, although field appears to be a significant factor for strain aggressiveness, our study cannot conclude which elements of field history, such as frequency of tobacco planted, type of cultivars planted, or rotation pattern are the ones that contribute the most. Although overall cultivars strains were isolated from were not significant with regards to aggressiveness, strains isolated from K 326 were most aggressive on K 326 or SP 168. These strains were isolated from fields 1 and/or 2, an indication that populations of R. solanacearum differ even within a field with regards to aggressiveness similar to findings of previous reports (9,21,27). A similar situation was observed in field 11 in Duplin County, where strains isolated from K 346 and CC 27 develop the highest DIs predominately on K 326 but some on NC 71 or SP 168. Our results indicate that breeding programs should take into account aggressiveness variability in the pathogen population, and thus breeding lines should be evaluated with more than 1 strain in the greenhouse or in multiple fields before they are released as commercial cultivars. REP-PCR has previously been utilized to study genetic diversity in R. solanacearum populations in the United States and other countries around the world (15,27,29). Interestingly, REP-PCR patterns and time of necrotic reaction development were correlated with each

Table 3. Analysis of variance for the main effects on disease index. Source SP 168 Field ERIC-PCR (field) NC 71 Field ERIC-PCR (field) K 326 Field ERIC-PCR (field) a b c

DFa

TIII SSb

MSc

F Value

Pr . F

10 6

27,503.4 4,196.3

2,750.3 699.3

11.75 2.99

,0.0001 0.0074

10 6

28,905.4 5,032.1

2,890.5 838.6

9.44 2.74

,0.0001 0.0130

10 6

25,638 5,299

2,563.8 883.2

8.23 2.84

,0.0001 0.0104

DF: Degrees of freedom. TIIISS: Type III sum square. MS: Mean sum.


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other. This is the first report of such a correlation as previous studies were not able to correlate genotypic with phenotypic groups (9,27) or necrotic reaction (9,17). It may prove useful to evaluate diversity of R. solanacearum strains without using REP-PCR. There was no correlation between the cultivars strains were isolated from and REP-PCR pattern; however, fields were divided into 2 groups, those with only pattern A and those with more than 1 pattern. This type of association has been reported in other studies as well (16,27). Fields such as in Caswell and Duplin Counties, where a large number of strains were collected, may safely considered as 1-pattern fields. On the other hand, in some other fields like the ones in Edgecombe, Johnston (2,207), and Vance Counties a small number of strains was isolated and thus our results may not representative of the locations. What does the segregation of different patterns mean for those fields? All fields were planted on tobacco for decades. Given the limited crop information available for those fields, we can only speculate that it might be related to total number of years tobacco was planted, or how long ago R. solanacearum was introduced in those fields and from where. For instance, fields 3, 8, and 9 are farmed by the same grower. Given the similarity in the genetic profile of these fields, one may suggest that R. solanacearum may have moved from 1 field to the other with contaminated soil on equipment or other means, as it is known that the pathogen may spread this way (19). Lastly, another question that cannot be answered is if pattern A is the initial, endogenous pattern and the other patterns were introduced later or vice versa. This would be a very interesting question to answer and would help identify the origin and longevity of R. solanacearum populations in tobacco fields in North Carolina. Our research was not very conclusive about the influence of field history on R. solanacearum population diversity; however, it demonstrated that there is significant variation with regards to REP-PCR pattern and aggressiveness. Because location may have the greatest effect, possibly because of differences in field history, further studies that investigate large population of strains from a few fields coupled with detailed crop information will provide more definite answers than the ones we could generate with our study. On the other hand, this study was the first to establish knowledge about the level of diversity of R. solanacearum in tobacco crops in North Carolina. ACKNOWLEDGMENTS We thank J. Radcliff for his technical assistance in the field experiments, F. Alayli, B. Poole, and X. Georgiou for their technical assistance, Dr. C. Arellano for assistance with statistical analysis of the data, and Dr. T. Sutton for his critical review of the manuscript before submission. We thank the North Carolina county agents for their assistance with sample collection and the North Carolina Tobacco Commission for financial support. 10

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