Prevalence of Xanthomonas euvesicatoria (formally X. perforans) associated with bacterial spot severity in Capsicum annuum crops in South Central Chihuahua, Mexico

Samples collection

Thirty commercial pepper cultivation lots from the South Central region of Chihuahua State were selected, collecting five samples per lot from plants with bacterial spot symptoms. Samples were then bagged in zip-lock bags, labeled, and transported at 4 °C to our facilities for laboratory processing.

Causative agent isolation

Leaves with signs of disease were surface sterilized by dipping in 0.5% sodium hypochlorite for 2 min, followed by three rinses in sterile distilled water. Small leaf sections were cut from the spot margin and individually macerated in 500 µL of sterile distilled water, using a sterile mortar and pestle. Resulting suspensions were streaked onto nutrient agar (NA; BD Difco Laboratories, Sparks Maryland, MD, USA) (Shaad, Jones & Chun, 2001) and plates incubated at 27 °C for 48 h, after which they were examined for bacterial colonies development. Pure cultures of bacterial strains were obtained by colony sub-culturing. Collected bacteria cultures were suspended in sterile 0.85% saline solution and stored at 4 °C, until use.

Phenotypic characterization

Bacterial isolates were subjected to standard biochemical and physiological testing (Shaad, Jones & Chun, 2001). Strains were cultured in semi-selective SX medium (10 g starch, 1 g beef extract, 5 g ammonium chloride, and 2 g K₂HPO₄), 1 mL of methyl violet 2B (1% solution in 20% ethanol), 2 mL of methyl green (1% aqueous solution), 15 g agar, and 5 mL of cycloheximide (5.0 g to 10 mL methanol and adjusted to 100 mL with sterile water), and were negative for anaerobic growth and fluorescence development on King’s B medium (20 g proteose peptone, 1.5 g K₂PO_4, 1.5 g MgSO₄.7H₂O, 15 mL glycerol, and 15 g agar).

Amylolytic activity of bacterial cultures was tested on nutrient agar plates (BD Difco Laboratories, Sparks Maryland, MD, USA), containing 0.2% soluble starch (Jones et al., 2004). In addition, we performed Gram staining (Hycel^®, Zapopan, Jalisco, México), oxidase and catalase activities, aerobic/anaerobic growth, and colony characteristics on yeast extract dextrose calcium carbonate agar medium (YDC), containing 10 g/L yeast extract (BD Difco Laboratories, Sparks Maryland, MD, USA), 20 g/L dextrose, 20 g/L calcium carbonate (Fagalab, Sinaloa, México), and 15 g/L agar (BD Difco Laboratories, Sparks Maryland, MD, USA). In YDC medium, Xanthomonas species develop yellow, convex, and mucoid colonies. X. campestris ATCC 1395 strain was used as a positive control in all tests.

DNA extraction

DNA extraction was achieved by the phenol-chloroform modified method (Bardakci & Skibinski, 1994). In brief, 1.5 mL of each bacterial isolate grown in nutrient broth medium for 24 h, was centrifuged twice at 12,000 rpm for 5 min at 4 °C, after which supernatant was discarded. Next, 100 μL of 10% sodium dodecyl sulfate, 5 M NaCl, and 10% cetyltrimethylammonium bromide were added, and mixture was homogenized by vortexing at 3,400 rpm (VX-200; Labnet International, Inc., Edison, NJ, USA), followed by incubation for 10 min at 65 °C in a water bath. After this, 750 μL of phenol/chloroform/isoamyl alcohol (25:24:1) mixture was added, mixed by vortexing at 3,400 rpm and centrifuged at 12,000 rpm for 5 min at 4 °C. In order to precipitate the DNA, supernatant was collected, and 500 μL isopropanol were added. The mixture was then carefully stirred by vortexing at low speed 2,000 rpm and placed in the freezer at 20 °C for 24 h, after which solution was centrifuged at 12,000 rpm for 10 min at 4 °C and supernatant was discarded. The precipitate was then washed twice with 300 μL of 70% ethanol, centrifuged for 10 min, dried at 25 °C for 30 min, and suspended in 50 μL of Tris-EDTA (TE) buffer (10 mM Tris-HCl and 1 mM disodium ethylenediaminetetraacetic acid (EDTA) solution at pH 8.

PCR protocol

Samples concentration was obtained with the Eppendorf BioSpectrometer basic (Eppendorf do Brasil Ltda., Mexico City) and adjusted to 5 ng/μL. To determine the causative agent of spot disease, a multiplex PCR was performed, using the specific primers pairs BsXeF/BsXeR, BsXvF/BsXvR, BsXgF/BsXgR, and BsXpF/BsXpR for X. euvesicatoria, X. vesicatoria, X. gardneri, and X. perforans, respectively (Koenraadt et al., 2009) (Table 1) and the following amplification program: an initial denaturation cycle at 94 °C for 5 min, then 35 cycles of denaturation at 94 °C for 30 s, alignment at 64 °C for 1 min, and extension at 72 °C for 1 min, and a final extension cycle at 72 °C for 7 min. Results were analyzed by electrophoresis in a 1.5% agarose gel, using 1X ethidium bromide as an intercalating agent, for 120 min and observing in a GenLogic 200 photodocumenter (Kodak, New York, NY, USA).

Pathogenicity test

Bacterial isolates were evaluated for their pathogenicity on pepper seedlings (Capsicum annuum var M Southern Star Seeds S. de R.L. de C.V., Mexico City). Seedlings were grown in 10-cm diameter pots with sterile expanded perlite particles, under a 16 h light at 28 °C and 8 h dark at 18 °C. Pathogenicity tests were conducted on 30-d-old pepper seedlings. Before and after testing, seedlings were conditioned two days in darkness, with relative humidity above 90% in a closed 27-gallon plastic chamber (Plastic Trends, Jalisco, Mexico). Bacterial suspensions were prepared from a 24-h-old culture grown on nutrient broth medium at a concentration of 1 × 10⁸ colony forming units (CFU) per milliliter, corresponding to an optical density (OD) of 0.01 at 600 nm by UV-visible spectrophotometry (Evolution 60 S; Thermo Fisher Scientific Inc., Waltham, MA, USA) (Mbega et al., 2012). Next, 10 µL of each bacterial suspension were infiltrated into leaves using Plastipak SFP 1 mL syringes (Becton-Dickinson, Brooklyn, NY, USA), and sterile distilled water as a control. Three leaves were infiltrated with two dots in five seedlings for each bacterial isolate. Disease severity was determined after 5 and 10 d, calculating the leaf damage percentage, using the Canopeo app (https://canopeoapp.com). Canopeo is an application that uses a red, green, and blue (RGB) system. It produces pixels analysis according to R/G, B/G ratios, and the green index excess. Results are interpreted in binary images where white pixels represent the pixels that satisfied the selection criteria (green canopy), whereas black pixels represent the pixels that did not comply with the selection criteria (not green canopy) (Patrignani & Ochsner, 2015). Severity was evaluated considering green percentage present in leaves. A 100% value was associated with healthy leaves, whereas lower percentages were considered as Xanthomonas damaged leaves.

Copper, gentamicin, and oxytetracycline susceptibility testing

Susceptibility of 13 isolates to antibacterial products was evaluated by the broth dilution method (Wiegand, Hilpert & Hancock, 2008).

Tested isolates were selected based on the highest disease severity evidenced in the pathogenicity test, as well as five strains with medium, and other four showing low severity. The evaluated products were Glucob Plus^® (8% copper gluconate + 1% citric acid (CuG)), Coboxy^® (45% copper oxychloride + 37.7% oxytetracycline hydrochloride (Cu + Ox)), Final Bacter (2% gentamicin + 6% oxytetracycline hydrochloride (Gen + Ox)), gentamicin sulphate (GenS), and copper sulphate pentahydrate (CuS). For minimum inhibitory concentration (MIC) analysis, antimicrobial dilutions were prepared at 4, 8, 16, 32, 64, 128, 256, 512, and 640 µg/mL in tryptone soy broth (TSB). Microbial resistance was defined by Xanthomonas growth on copper compounds at 200 µg/mL (Zhang et al., 2013; Roach et al., 2020), on Ox at 25 µg/mL (Farfán, Benítez & Carvajal, 2014), and on gentamicin at 25 µg/mL (Rojas, Peña & Peña-Vera, 2019). In addition, pH values from all the antimicrobial tested and their dilutions were determined.

Bacterial suspensions were adjusted to 0.5 McFarland standard (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) by turbidimetry, which is equivalent to 1 × 10⁸ CFU/mL in TSB. Bacterial suspension was mixed and diluted to 1:100. Next, 50 μL of bacterial suspension plus 50 μL of antimicrobial solution were placed in a microplate, using 100 µL of TSB as a negative control, and 50 µL TSB + 50 µL of each antimicrobial as blank and TSB + bacterial suspension as positive control. Final inoculum contained 5 × 10⁵ CFU/mL. Microplates were then sealed with Parafilm and incubated at 28 °C for 48 h under constant shaking at 225 rpm. After incubation, ODs were read in a Varioskan Flash microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 600 nm for MICs determination. Bacterial growth was confirmed by seeding 10 µL of each treatment plus 990 µL of TSB in NA and incubating at 28 °C for 48 h. Tests were performed in triplicate.

Data analysis

Results from pathogenicity tests were statistically analyzed by the Minitab Statistical Software v.16. Bacterial spot severity results were evaluated by cluster analysis, using the Spearman average linked method. Euclidean distance measure was performed with the InfoStat software (InfoStat version 2009; Grupo InfoStat, Córdoba, Argentina). Phytopathogens were grouped (clusters) into the following categories: (1) isolates that cause high severity, (2) isolates causing medium severity, and (3) isolates causing low severity. From these categories, some representative microorganisms were selected for antimicrobial susceptibility testing.

Antibacterial agents susceptibility data were analyzed by comparing the ODs of the interactions between Xanthomonas and antimicrobials at different concentrations, testing concentrations of resistance MIC values for gentamicin, cupper and oxytetracycline, reported by Rojas, Peña & Peña-Vera (2019), Roach et al. (2020) and Farfán, Benítez & Carvajal (2014), respectively.

Isolation and identification of the causative agent

In the present study, we isolated 43 Xanthomonas spp. strains from chili pepper leaves with characteristic infection of bacterial spots. Colonial morphology on YDC medium showed yellow, mucoid, smooth, circular, and convex colonies with entire margins (Fig. S1). Xanthomonads were gramnegative, and positive for catalase and KOH test. According to biochemical tests, these bacterial isolates belong to the Xanthomonas genus. In addition, 37 isolates (87%) showed strong amylolytic activity, associated with X. euvesicatoria (formally X. perforans) species, whereas 6 isolates (14%) did not possess amylolytic activity and were associated with X. euvesicatoria species.

Molecular characterization of the causative agent

The amplification of 197 bp fragment of 37 isolates with species-specific primers Bs-XpF/Bs-XpR, confirmed them as X. euvesicatoria (formally X. perforans). In addition, the amplification of 197 bp fragment of six isolates with species-specific primers Bs-XeF/Bs-XeR, confirmed them as X. euvesicatoria. No amplification was observed for negative controls and other species-specific primer pairs did not amplify DNA, which confirmed that the isolates do not belong to X. vesicatoria or X. gardneri (Fig. S2).

Pathogenicity tests

All inoculated pepper leaves with Xanthomonas isolates showed signs of bacterial spot. About 5 to 10 d after inoculation, infection was characterized by watery spots, leaves with chlorosis and perforation of leaf lamina, and necrotic spots with chlorosis and defoliation. Negative control plants remained healthy (Fig. 1).

Cluster analysis of the severity of bacteria spot Xanthomonads were classified into three conglomerates, according to their virulence on both evaluations (5 d and 10 d); 9.3% of isolates [all X. euvesicatoria (formally X. perforans)] caused the highest damage to pepper leaves, from 5 d to the end of the evaluation period, with a final severity of 92.6%. In addition, 25.6% of isolates (ten X. perforans and one X. euvesicatoria) caused medium damage with 76.8% final severity, and 65.1% (23 isolates were identified as X. perforans and 5 as X. euvesicatoria) induced low damage with 29.8% severity (Table 2). Medium-severity and low-severity groups showed 54.0% and 13.9% increases in damaged pepper leaves, respectively (Fig. 1).

Antimicrobial susceptibility testing

Evaluated Xanthomonas strains were resistant to CuS and CuG at all concentrations tested. However, all strains were susceptible to Cu + Ox at ≥ 32 µg/mL. In addition, strains did not grow in Gen + Ox or Gen at 32 µg/mL. Furthermore, microbial growth from Xp47 and Xp60 (the most virulent strains) was inhibited by Cu + Ox at 4 µg/mL. In general, 47% of strains did not grow when exposed to Gen at > 4 µg/mL. Since Xanthomonas strains were resistant to CuS and CuG, the MIC could not be determined. Calculated MIC of Cu + Ox was from 4 µg/mL to 32 µg/mL, MIC of GenS was from 4 µg/mL to 32 µg/mL, and MIC of Gen + Ox was 32 µg/mL (Table 3). After analyzing the pH values of the antimicrobial agents tested and their dilutions, pH ranged from 6.48 to 7.38 (Table S1).

Copper resistance genes detection

Most Xhantomonas strains showed resistance to copper, since copLAB genes were detected. The copL, copA, and copB genes had a molecular weight of 356, 870, and 531 bp, respectively (Fig. 2). However, copLAB genes were not detected in the Xp3 strain.