Abstract
Bacterial spot, caused by Xanthomonas spp., is one of the most important diseases of tomato in Illinois. Field surveys were conducted during 2017–19 to assess occurrence of bacterial spot in commercial tomato fields. Severity of foliage and fruit infection was recorded, and symptomatic samples were collected from three-to-five cultivars in three different farms in each of northern, central, and southern regions of Illinois. Severity of symptomatic foliage ranged from 0% to 91% (average 36.7%) and incidence of symptomatic fruit ranges from 0% to 30% (average 10.8%). During the surveys, 266 Xanthomonas isolates were collected and identified as Xanthomonas gardneri and X. perforans using Xanthomonas-specific hrp primers. Eighty-six percent of the isolates from the northern region were identified as X. gardneri, whereas 73% of the isolates from southern region were identified as X. perforans. Isolates from the central region were identified as X. perforans and X. gardneri 53% and 47% of the time, respectively. Multilocus sequence analysis using six housekeeping genes (fusA, gap-1, gltA, gyrB, lepA, and lacF) revealed the endemic population of X. gardneri and X. perforans. In addition to Xanthomonas, nine non-Xanthomonas bacterial genera were isolated from the samples, with most of the isolates classified as Microbacterium, Pantoea, and Pseudomonas.
Bacterial spot of tomato (Solanum lycopersicum L.) was first identified in South Africa (Doidge, 1921). Originally, bacterial spot was thought to be caused by only one species, Xanthomonas campestris pv. vesicatoria (Stall et al., 1994; Vauterin et al., 1995); however, subsequent studies divided it into two species: X. axonopodis pv. vesicatoria (group A) and X. vesicatoria (group B) (Vauterin et al., 1995). Currently, the following species are considered to be the casual agents of the bacterial spot disease complex of tomato: X. euvesicatoria (group A), X. vesicatoria (group B), X. perforans (group C), and X. gardneri (group D) (Jones et al., 2000, 2004).
Bacterial spot of tomato is characterized by necrotic lesions on leaves, stems, flowers, and fruits (Egel et al., 2018; Jones, 1991). During the initial stages of disease development, symptoms develop as circular water-soaked lesions that dry out to give a greasy appearance, and eventually turn dark-brown to black (Jones, 1991). Some species-specific symptoms include shot-hole type lesions caused by X. perforans and lesions with a more water-soaked appearance caused by X. gardneri (Stall et al., 2009). X. euvesicatoria and X. vesicatoria have been generally associated with lesion development on fruits, though recent publications have also shown X. gardneri causing large, deep fruit lesions (Ma et al., 2011). Generally, the disease is favored by warmer and more humid conditions for progression and spread (Araujo et al., 2010); however, X. gardneri appears more often in cooler temperatures (Jones et al., 1988) and causes more severe disease at 20 °C than the other three species (Araujo et al., 2010). Xanthomonas bacteria are disseminated within a field by wind-driven rain and by mechanical means such as grafting, clipping, tying, harvesting, and spraying pesticides (Lindeman and Upper, 1985; McInnes et al., 1988).
Researchers have used diagnostic methods for identification of bacterial species, which are based on the hrp gene clusters that are highly conserved among several phytopathogenic bacteria (Fenselau et al., 1992; Hwang et al., 1992). Leite et al. (1994) was the first to use hrp gene clusters as a diagnostic tool for identification of species and pathovars of incitants of bacterial spot. Fragments of different hrp genes were amplified using a polymerase chain reaction (PCR) assay, followed by restriction digestion using endonucleases to allow detection of 28 different X. campestris pathovars. Obradovic et al. (2004) developed specific PCR primers that amplify a 420 base pair (bp) fragment of hrpB7 from four bacterial spot pathogens causing disease in tomato. This diagnostic method is still used in routine diagnostic tests.
Genotypic characterization of prokaryotes can be performed using multilocus sequence analysis (MLSA), which differentiates between bacterial strains using a small number of allelic mismatches found in housekeeping genes (Maiden et al., 1998). Six housekeeping genes (fusA, gap-1, gtlA, gyrB, lacF, and lepA) were used to create a MLSA database of Xanthomonas strains (Almeida et al., 2010).
The objectives of this study were to 1) assess the incidence and severity of bacterial spot disease of tomato in Illinois; 2) identify the Xanthomonas spp. causing the bacterial spot disease; 3) determine the genetic diversity among Xanthomonas isolates and examine the phylogenetic relationships using housekeeping genes; and 4) identify the non-Xanthomonas bacteria associated with bacterial spot caused by Xanthomonas spp.
Materials and Methods
Field surveys.
In 2017, 2018, and 2019, field surveys were conducted to assess the severity of bacterial spot disease on tomato cultivars grown in Illinois. Each year, commercial tomato farms located in the northern, central, and southern regions of Illinois (Fig. 1) were visited three or four times throughout the growing seasons (Table 1). A total of 13 different tomato cultivars were evaluated, including Biltmore, Brandywine, Carolina Gold, Chef’s Choice, Dixie Red, Heirloom, Jolene, Phoenix, Primo Red, Pony Express Plum, Red Deuce, Red Morning, and Rocky Top.
Stars indicate Illinois counties where commercial tomato fields were surveyed for the occurrence of bacterial spot disease. Kane and McHenry, Douglas and Moultrie, and Fayette counties are reported as northern, central, and southern regions, respectively.
Citation: HortScience horts 56, 1; 10.21273/HORTSCI15215-20
Stars indicate Illinois counties where commercial tomato fields were surveyed for the occurrence of bacterial spot disease. Kane and McHenry, Douglas and Moultrie, and Fayette counties are reported as northern, central, and southern regions, respectively.
Citation: HortScience horts 56, 1; 10.21273/HORTSCI15215-20
Stars indicate Illinois counties where commercial tomato fields were surveyed for the occurrence of bacterial spot disease. Kane and McHenry, Douglas and Moultrie, and Fayette counties are reported as northern, central, and southern regions, respectively.
Citation: HortScience horts 56, 1; 10.21273/HORTSCI15215-20
Plant samples collected from symptomatic foliage and fruits from different regions of Illinois during 2017–19.
At each tomato field, 10 randomly selected plants from each cultivar were evaluated for severity of bacterial spot on leaves and stems (foliage) and fruits. The scale 0–11 developed by Horsfall and Barratt (1945) was used for the evaluation of severity of the disease on foliage. Symptomatic samples of foliage and fruits were collected for isolation of bacteria associated with the affected tissues (Table 1). Samples were stored at 4 °C until bacterial isolation was completed within 72 h post collection.
Isolation and maintenance of bacterial isolates.
Bacteria were isolated from collected samples using the procedure reported by Schaad et al. (2001). Plant tissues were washed with tap water to remove soil and other particles, then 5- × 5-mm tissue sections containing lesions were cut and sections were surface-disinfested using 99% ethanol for 60 s. The sections were then washed three times (1 min each time) with sterile distilled water (SDW). Each section was inserted into a 15-mL glass test tube containing 10 mL of SDW and shaken by hand for 20 s to prepare a bacterial suspension. Then, 100 μL of the suspension was transferred onto nutrient agar (NA) and yeast dextrose calcium agar (YDC) media in petri plates. The plates were incubated at 28 °C for 72 h, at which time well-developed colonies on both culture media were subcultured onto NA. Isolates were stored in 4 mm sucrose and 20% glycerol (v:v) at −80 °C.
Identification of bacterial isolates.
Isolated bacterial colonies were grown on YDC and then grouped based on their colony morphology and color. Consistent with Schaad et al. (2001), the colonies with yellow mucoid characteristics on YDC, which were suspected to be Xanthomonas spp., were subjected to polymerase chain reaction (PCR) using RST 65/69 (hrpB7) primers. Colonies with other colors (i.e., orange, white, pink) were subjected to PCR using different primers for amplifying 16S rRNA.
Species of the isolates with yellow mucoid colonies were identified based on amplicon sequencing from the PCR assay. Isolates were streaked onto NA and incubated at 28 °C for 72 h The Xanthomonas-specific primers RST 65 (5′-GTCGTCGTTACGGCAAGGTGGTCG-3′) and RST 69 (5′-TCGCCCAGCGTCATCAGGCCATC-3′), which amplify a 420 bp fragment of hrpB7 (Obradovic et al., 2004), were used for PCR amplification. For each isolate, a 25-μL mixture containing Taq DNA polymerase (Omega Bio-Tek Inc., Norcross, GA), RST 65/69 primers, deoxyribonucleotide triphosphates (dNTPs), and a small number of bacterial cells (Leite et al., 1994) were used for the reaction. PCR amplification was performed using a ProFlex thermal cycler (Thermo Fisher Scientific, Waltham, MA), with initial denaturation at 95 °C for 5 min; 35 cycles of denaturing at 95 °C for 30 s, annealing at 63 °C for 30 s, and extension at 72 °C for 45 s; followed by a final extension at 72 °C for 5 min (Obradovic et al., 2004). Then, 5 μL of each PCR reaction was analyzed using gel electrophoresis with 1% agarose gel containing EZ-vision DNA dye (VWR Life Sciences, Radnor, PA), run at 100 V for 30 min, and visualized at 470 nm by the Azure Biosystems c400 imager (Dublin, CA).
The remaining PCR product was purified according to the manufacturer’s recommendation using EXOSAP-IT (Thermo Fisher Scientific) and sent for Sanger sequencing at the DNA Services Laboratory, Roy J. Carver Biotechnology Center, University of Illinois. Sequences were analyzed using the BLASTN database from the National Center for Biotechnology Information (NCBI).
Pathogenicity tests.
Pathogenicity of Xanthomonas isolates was conducted on four tomato cultivars (Brandywine, Dixie Red, Primo Red, and Red Deuce) in a greenhouse, using two isolates from each of the northern, central, and southern regions. Tomato seeds were sown in plastic pots (20 cm in diameter) filled with a growth medium mix of soil:peat:perlite (1:1:1; v:v). The pots were placed in a greenhouse at 26 °C during day (14 h) and 24 °C at night (10 h). The bacterial isolates were grown on NA for 48 h, then colonies were washed with SDW, and a suspension with 5 × 108 CFU/mL (OD600 = 0.3) was prepared (Kyeon et al., 2016). Eight- to ten-week-old plants were inoculated by infiltrating 0.3 mL of the bacterial suspension into each leaflet using a needleless syringe. Three plants of each cultivar and four leaves of each plant were inoculated with each isolate. Control plants were infiltrated with SDW. The development of the symptoms was monitored and recorded at 3, 5, and 7 d post inoculation. Tissues from inoculated and control plants were processed for isolation of bacteria, as previously described.
Phylogenetic analysis.
Isolates identified as Xanthomonas were used for the development of a phylogenetic tree using MLSA. The isolates were evaluated using the six housekeeping genes fusA, gap-1, gltA, gyrB, lacF, and lepA (Almeida et al., 2010). Gene fragments obtained from the Sanger sequencing center were compared with the following whole genome sequences found in NCBI: X. gardneri ATCC 19865 (NZ_AEQX00000000); X. gardneri ICMP7383 (NZ_CP018731.1); X. perforans 91-118 (NZ_CP019725, representative Florida group 1, Xp-FL1); X. perforans Xp3-15 (JZVG01, representative Florida group 2, Xp-FL2); X. perforans Xp4-20 (JZUZ01, representative Florida group 3, Xp-FL3); and X. euvesicatoria 85-10 (NC_007508.1). Stenotrophomonas maltophilia K279a (NC_010943.1) was used as the outgroup.
All the gene sequence fragments, either obtained via Sanger sequencing or extracted from whole genome sequences, were aligned using CLUSTALW within MEGA 10.0.5 (Kumar et al., 2018) and trimmed to the same length using the same starting position. For each isolate, the trimmed sequence for the six genes were concatenated for a total of 3092 nucleotides. All concatenated sequences were aligned, and the Akaike Information Criterion (AIC) within jModeltest 1.1 (Posada and Buckley, 2004) was used to select the nucleotide substitution model that best fit the aligned sequences. The Tamura 3-parameter model with gamma distribution and with invariant sites (T92 + G + I) was used for constructing the phylogenetic trees. The maximum likelihood tree was determined using the concatenated sequences with 1000 bootstrap samples. Genetic groups were defined as isolates having less than 10 nucleotide differences.
Identification of non-Xanthomonas isolates.
Isolates that tested negative for hrpB7 amplification were further analyzed for genus identification and their pathogenicity on tomato. Identification of non-Xanthomonas isolates was achieved by using 16S rRNA sequencing. PCR amplification of 16S rRNA was performed using the primer set 27F (5′-AGAGTTTGATCMGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) (Lane, 1991). PCR amplification, purification, and sequencing was performed as described above for Xanthomonas isolates, except for the PCR amplification conditions, which consisted of 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s. Sequences were analyzed using the BLASTN database from the NCBI to assign each isolate to a particular genus. Isolates that did not have a match in BLASTN database were labeled as “unknown.”
Two isolates of each of non-Xanthomonas bacteria, collected in 2018 and 2019, were tested for their pathogenicity on tomatoes in the greenhouse using the same method described above for Xanthomonas isolates. However, only two tomato cultivars, Red Deuce and Brandywine, were used with non-Xanthomonas bacteria.
Results
Incidence and severity of bacterial spot.
Bacterial spot was observed in 26 of 27 fields visited during 2017–19. In 2017 and 2019, average disease severity on foliage was highest in southern Illinois, while in 2018 it was the highest in northern Illinois (Table 2). The highest range of disease severity (0% to 91%) was observed in northern Illinois in 2019, and the lowest range was observed in central Illinois in 2017 (2% to 4.5%). Overall, disease severity of foliage was highest in the 2019 season (Table 2). Incidence of symptomatic fruit was generally low, with the overall highest incidence occurring in 2019 and the lowest in 2017 (Table 3).
Severity of bacterial spot symptoms on foliage in commercial tomato fields located in different regions of Illinois.
Incidence of tomato fruits with bacterial spot symptoms in commercial fields in Illinois.
Identification of Xanthomonas spp.
A total of 266 isolates that produced the 420 bp hrpB7 amplicon in PCR tests were identified as Xanthomonas spp. Of the 266 isolates, 221 were obtained from foliar tissues and 45 from fruits (Table 4). Of 91 isolates collected from the northern region, 13 and 78 isolates were X. perforans and X. gardneri, respectively. In contrast, of the 139 isolates collected from the southern region, 101 and 38 isolates were identified as X. perforans and X. gardneri, respectively. Among the 36 isolates collected from the central region, 19 and 17 isolates were X. perforans and X. gardneri, respectively (Table 4).
Number of Xanthomonas isolates collected from different regions of Illinois.
Pathogenicity test.
All tested Xanthomonas isolates produced symptoms of bacterial spot as observed in the fields. Bacteria were re-isolated from the infiltrated leaves and produced the hrpB7 gene fragment in PCR assays. Plants infiltrated with SDW did not develop any lesions, and no bacteria were isolated from tissues infiltrated with SDW.
Multilocus sequence analysis of isolated Xanthomonas spp.
X. perforans Xp-IL1 was the most prevalent (90 isolates) among the genetic groups of X. perforans (Fig. 1) and was like the reference strain X. perforans Xp-FL1 (Fig. 2). X. perforans Xp-IL1 was collected from seven of the nine farms. X. perforans Xp-IL2 (36 isolates) was found only in a single farm in the southern region and was like the reference strain X. perforans Xp-FL2. X. gardneri Xg-IL was the most prevalent (119 isolates) isolate and was like the reference isolate X. gardneri ATCC 19865 (Fig. 2). X. gardneri Xg-IL isolates were found in all nine farms across all three regions. Tissue samples infected with both X. perforans and X. gardneri were collected from six of nine farms. Of the original 266 isolates, 7 X. perforans and 14 X. gardneri isolates were not fully sequenced and were removed from the analysis.
Clustering of isolates of Xanthomonas perforans and X. gardneri from commercial tomato fields in Illinois during 2017–19. Multilocus sequence analysis of six housekeeping genes (fusA, gap-1, gltA, gyrB, lepA, and lacF) were used to group the isolates. Isolates of X. perforans Xp-IL1, X. perforans Xp-IL2, and X. gardneri Xg-IL were from Illinois; X. perforans Xp-FL1, X. perforans Xp-FL2, X. perforans Xp-FL3, X. euvesicatoria 85-10, X. gardneri ICMP7383, and X. gardneri ATCC 19865 are reference isolates in the NCBI; and S. maltophilia = Stenotrophomonas maltophilia. Numbers inside the parentheses indicate the numbers of the isolates classified within that particular genetic group, with Illinois genetic groups defined as having fewer than 10 different nucleotides over the total 3092 nucleotides. The scale bar represents the number of substitutions per site, and values on the branches indicate Bayesian posterior probabilities expressed as the percentage of trees based on 1000 bootstrap replicates.
Citation: HortScience horts 56, 1; 10.21273/HORTSCI15215-20
Clustering of isolates of Xanthomonas perforans and X. gardneri from commercial tomato fields in Illinois during 2017–19. Multilocus sequence analysis of six housekeeping genes (fusA, gap-1, gltA, gyrB, lepA, and lacF) were used to group the isolates. Isolates of X. perforans Xp-IL1, X. perforans Xp-IL2, and X. gardneri Xg-IL were from Illinois; X. perforans Xp-FL1, X. perforans Xp-FL2, X. perforans Xp-FL3, X. euvesicatoria 85-10, X. gardneri ICMP7383, and X. gardneri ATCC 19865 are reference isolates in the NCBI; and S. maltophilia = Stenotrophomonas maltophilia. Numbers inside the parentheses indicate the numbers of the isolates classified within that particular genetic group, with Illinois genetic groups defined as having fewer than 10 different nucleotides over the total 3092 nucleotides. The scale bar represents the number of substitutions per site, and values on the branches indicate Bayesian posterior probabilities expressed as the percentage of trees based on 1000 bootstrap replicates.
Citation: HortScience horts 56, 1; 10.21273/HORTSCI15215-20
Clustering of isolates of Xanthomonas perforans and X. gardneri from commercial tomato fields in Illinois during 2017–19. Multilocus sequence analysis of six housekeeping genes (fusA, gap-1, gltA, gyrB, lepA, and lacF) were used to group the isolates. Isolates of X. perforans Xp-IL1, X. perforans Xp-IL2, and X. gardneri Xg-IL were from Illinois; X. perforans Xp-FL1, X. perforans Xp-FL2, X. perforans Xp-FL3, X. euvesicatoria 85-10, X. gardneri ICMP7383, and X. gardneri ATCC 19865 are reference isolates in the NCBI; and S. maltophilia = Stenotrophomonas maltophilia. Numbers inside the parentheses indicate the numbers of the isolates classified within that particular genetic group, with Illinois genetic groups defined as having fewer than 10 different nucleotides over the total 3092 nucleotides. The scale bar represents the number of substitutions per site, and values on the branches indicate Bayesian posterior probabilities expressed as the percentage of trees based on 1000 bootstrap replicates.
Citation: HortScience horts 56, 1; 10.21273/HORTSCI15215-20
Identification of non-Xanthomonas bacteria.
A total of 412 bacterial isolates were collected from symptomatic tomato tissues that did not produce yellow colonies characteristic of Xanthomonas on YDC. Furthermore, none of these colonies produced the 420 bp amplicon in the PCR assay with RST 65/69 primers, nor did they cause symptoms in infiltrated tomato leaves. Most of these non-Xanthomonas isolates belonged to nine bacterial genera, including Agrobacterium, Bacillus, Curtobacterium, Microbacterium, Paenibacillus, Pantoea, Psuedomonas, Rhizobium, and Stenotrophomonas (Table 5). Most identified isolates were Microbacterium, Pantoea, and Pseudomonas spp.
Non-Xanthomonas bacteria isolated from symptomatic tomato foliage and fruits.
Discussion
This was the first survey of Illinois fields to assess the severity and identify the causal agents of bacterial spot disease on tomato plants and fruits. X. perforans and X. gardneri were identified as the incitants of the disease in Illinois. X. perforans was more prevalent in the southern region, while X. gardneri was the main species found in the northern region. In central Illinois, the distribution of X. perforans and X. gardneri was roughly equal at 55% and 45%, respectively. A previous study conducted in Ohio showed that X. perforans and X. gardneri were both present in tomato production fields (Ma et al., 2011), while other studies conducted in cooler climate regions have reported X. gardneri as the dominant species (Araujo et al., 2010; Cuppels et al., 2006; Kim et al., 2010). The findings in our study agree with these reports—that the prevalence of X. gardneri in northern Illinois is likely related to cooler weather conditions in this region. In addition, the results from our study showed that the populations of X. perforans in Illinois are genetically like those found in Florida, which has a warmer climate (Potnis et al., 2011). Moreover, a report from Brazil (Araujo et al., 2017), a place with warmer conditions, shows widespread distribution of X. performance and a limited presence of X. gardneri, which is like the findings in our study.
Multilocus sequence analysis revealed endemic populations of X. gardneri and X. perforans throughout Illinois. The widespread occurrence of genetically similar isolates likely originated from recurring infections caused by infected plant materials in previous years that subsequently spread between nearby fields, or via contaminated materials shared between production sites. The presence of multiple genetic groups of X. perforans has been reported in various tomato-growing regions such as North Carolina and Florida (Adhikari et al., 2019; Timilsina et al., 2015, 2019). About 71% of X. perforans isolates in our study (X. perforans Xp-IL1) grouped with X. perforans Xp-FL1 (a reference isolate) suggested endemic dispersal of this genetic group. About 29% of X. perforans isolates (X. perforans Xp-IL2) were grouped with X. perforans Xp-FL2 strains (Timilsina et al., 2015); however, these were isolated from a single farm, which suggests initial infection from different source that, likely over a period of some years, led to the endemic presence of Xp-IL2 isolates at this particular location. Previous studies found that a single genetic strain of X. gardneri has been distributed around the world (Kebede et al., 2014; Timilsina et al., 2015). Consistent with these findings, the X. gardneri Xg-IL isolates from Illinois tomato fields were identical to isolates from this genetic group.
Identification of pathogens has been a driving factor in the development of resistant cultivars for disease management. Because all tomato cultivars grown at the nine Illinois commercial farms included in this study were found to be susceptible to bacterial spot disease, and with evidence of endemic spread of the pathogens across the state, future research should focus on developing more effective management strategies, including breeding for resistance, inducing resistance by using chemicals or biopesticides, cultural practices, and chemical use.
In this study, Xanthomonas and non-Xanthomonas bacteria were isolated from symptomatic tomato foliage and fruits. All Xanthomonas isolates tested were pathogenic on tomato leaves, while none of the non-Xanthomonas isolates caused any symptoms. We concluded that the non-Xanthomonas bacteria isolated from tomato foliage and fruits are likely nonpathogenic. However, our findings do not preclude the possibility that some of these bacteria may act synergistically with Xanthomonas to cause greater disease symptoms, or that some may be antagonistic to the Xanthomonas bacteria. Future research would be needed to evaluate whether co-occurrence of these bacteria during tomato infection by Xanthomonas affects the development of the disease symptoms.
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