Resistance of Xanthomonas euvesicatoria strains from Brazilian pepper to copper and zinc sulfates

Bacterial spot, caused by Xanthomonas spp., is one of the major bacterial diseases in pepper (Capsicum annuum L.). The infection results in reduced crop yield, particularly during periods of high rainfall and temperature, due to the low efficiency of chemical control with copper bactericides. This study evaluated the copper and zinc sulfate sensitivity of 59 pathogenic strains of Xanthomonas euvesicatoria isolated from pepper plants produced in various regions throughout Brazil. Both the respective sulfates and a mixture thereof was evaluated at 50, 100, 200 and 400 μg.mL. All the evaluated strains were found to be resistant to zinc sulfate (100 μg.mL) and 86.4% were resistant to copper sulfate (200 μg.mL). The mixture of copper (200 μg.mL) and zinc (200 μg.mL) sulfates inhibited the growth of all strains of X. euvesicatoria. To our knowledge this is the first study to report the resistance of X. euvesicatoria strains from pepper plants to copper and zinc sulfates in Brazil.


INTRODUCTION
Pepper plants (Capsicum annuum L.) are cultivated around the world and contain compounds that add flavor and aroma to foods, in addition to being a valuable source of vitamins (Fontes 2005).In the 2013 season, 472,526 tons of peppers were produced worldwide, and Brazil was responsible for the production of 42,312 tons (FAOSTAT 2016).
Bacterial spot disease, caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri, is detrimental to pepper and tomato crops in several regions of the world, particularly during seasons of high rainfall and temperature (Quezado-Durval and Lopes 2010, Osdaghi et al. 2016, Potnis et al. 2015).X. euvesicatoria (Areas et al. 2015) and X. perforans (Araújo et al. 2017) are the major etiological agents causing bacterial spot in pepper and tomato crops, respectively, in Brazil.
Bacterial spot is very difficult to control due to the high pathogen variability and low efficiency of MAYSA S. AREAS et al.
chemicals.Also, the efficiency of chemical control varies with the region, season and the intensity and frequency of spraying (Carmo et al. 2001).In Brazil, copper hydroxide and copper sulfate are currently registered for use as chemical agents to control bacterial spot in pepper crops (Agrofit 2016).
The first report of copper resistance in Xanthomonas spp.from pepper was reported in Florida, USA (Marco and Stall 1983).In Brazil, Maringoni and Kimati (1987a) reported a low in vitro sensitivity of Xanthomonas spp.from pepper and tomato plants to copper sulfate, copper oxychloride and copper hydroxide at 100 μg.mL -1 .However, these bacterial strains were sensitive to mixtures of copper and dithiocarbamate fungicides, suggesting a synergism between these chemicals.
Carmo et al. ( 2001) evaluated the effect of weekly spraying with copper oxychloride on the progress of bacterial spot infection in pepper plants in Brazil and observed varying efficiency.In most instances, the chemicals were found to be inefficient, particularly in environmental conditions favorable for disease development.Another study from Brazil by Aguiar et al. (2003), evaluated the effect of spraying copper sulfate, copper oxychloride, cuprous oxide and a mixture of cuprous oxide and mancozeb, on the control of bacterial spot infection in pepper plants.They reported that spraying with either copper oxide alone or in combination with mancozeb, reduced the epiphytic population of Xanthomonas spp., and controlled the disease to a satisfactory level.Martin and Hamilton (2004) reported Xanthomonas spp.strains that were resistant to copper sulfate in pepper crops in Australia.More recently, Araújo et al. (2012) compared their results with those of Ritchie andDittapongpitch (1991), andQuezado-Duval et al. (2003) finding that the level of copper sulfate-resistant Xanthomonas spp. in Brazilian tomato strains was lower than that detected in the USA.
Resistance to copper fungicides occurs due to continuous spraying of crops with copper chemicals, which results in the selective pressure of resistant bacteria (Martin and Hamilton 2004).Xanthomonas spp.from pepper plants may possess copper-resistant genes, which can be located in plasmids and transferred via conjugation to sensitive strains, resulting in these strains becoming resistant (Stall et al. 1986).
This study aimed to evaluate the in vitro sensitivity of 59 X. euvesicatoria strains obtained from peppers produced throughout several regions of Brazil, to copper sulfate, zinc sulfate and mixtures thereof.The results presented herein will be important for improving the management of bacterial spot infection in pepper crops in Brazil.

STRAINS OF Xanthomonas euvesicatoria
We used 59 X. euvesicatoria strains isolated from the leaves of pepper plants showing symptoms of bacterial spot from several locations in Brazil (Table I).These bacterial strains are the same as those used by Areas et al. (2015) that were identified by PCR and physiological features by these authors.

PATHOGENICITY TEST
Pepper plants (cv.Early California Wonder) were grown in 3 L plastic pots with substrate (sand, ravine clay soil and the commercial substrate Bioplant® at a ratio of 1:1:1) under greenhouse conditions (20-30 o C and 70-90% RH).The plants were inoculated with X. euvesicatoria six weeks after sowing.All bacterial strains used for the inoculation had been grown in NSA medium (Dinghra and Sinclair 1995).The colonies were suspended in sterile distilled water and the bacterial suspensions were standardized by turbidity (OD 600nm = 0.1) to 10 8 CFU.mL -1 .Four pepper plants were inoculated per bacterial strain, using the syringe infiltration method (two leaves per pepper plant).
Bacterial spot symptoms were assessed ten days after inoculation (Maringoni and Kimati 1987b).

In vitro ASSAY
The growth of all the evaluated strains was assessed for sensitivity to copper sulfate (Sigma-Aldrich Co., USA), zinc sulfate (Ecibra Co., Brazil), and mixtures thereof (50% each) at 0, 50, 100, 200 and 400 μg.mL -1 .The evaluated concentrations of sulfates were similar to those used by Ritchie and Dittapongpitch (1991), Gore andO'Garro (1999), Costa et al. (2012) and Osdaghi et al. (2016).The copper and zinc sulfates were dissolved in distilled and sterilized water and then added to the sterilized PSA medium (Dinghra and Sinclair 1995) in a melted state at 50°C, to obtain the desired concentrations.PSA medium without added sulfates was used as a control.PSA medium was selected as it has been used previously in assays developed by Ritchie and Dittapongpitch (1991), Gore and O'Garro (1999) and Mirik et al. (2007), for the same purpose.
All the studied bacterial strains were cultivated on NSA at 28°C for 48 h.The bacterial suspensions were prepared in sterile distilled water and standardized by turbidity (OD 600nm = 0.1) to 10 8 CFU.mL -1 .Aliquots (10 µL) were then seeded onto four equidistant points on the surface of the PSA medium.The plates were incubated at 28°C for 24 h, and presence (insensitive) or absence (sensitive) of bacterial growth was recorded.Five replicates were performed for each strain and at each chemical concentration.MAYSA S. AREAS et al.

RESULTS AND DISCUSSION
All the evaluated X. euvesicatoria strains were pathogenic to pepper and insensitive to copper or zinc sulfates at 50 or 100 µg.mL -1 .At 200 µg.mL - copper sulfate, 13.6% of the strains did not grow.
Furthermore, the growth of 11.9% of the strains was inhibited at 200 µg.mL - zinc sulfate.At 400 µg.mL -1 , the growth of 35.6 and 42.4% of the X. euvesicatoria strains was inhibited for copper and zinc sulfate, respectively.Treatment of the strains with a copper and zinc sulfates mixture showed increased sensitivity, with 3.4, 49.2 and 100% of the strains inhibited at 100, 200 and 400 µg.mL -1 , respectively (Figure 1).
Xanthomonas spp.from tomato and pepper plants have been shown to be resistant to copper and zinc sulfates when grown in a PSA medium containing 200 and 100 µg.mL -1 of these chemicals, respectively (Ritchie and Dittapongpitch 1991, Ward and O'Garro 1992, Gore and O'Garro 1999).The strains evaluated in this study were predominantly resistant to zinc sulfate (100%) and copper sulfate (86.4%).All strains collected from Apodi (RN), Tinguá (CE), Nova Friburgo (RJ) and Brasília (DF) were resistant to both copper and zinc sulfates, while over 80% of the strains from São Paulo (SP), Minas Gerais (MG) and Pará (PA) were resistant to copper sulfate (Table II).All the strains were resistant to zinc sulfate (Table II).The high percentage of X. euvesicatoria strains resistant to copper sulfate is due to the intensive use of copper fungicides for the control of bacterial spot over many years, which has resulted in the selection of strains resistant to copper (Ritchie and Dittapongpitch 1991, Ward and O'Garro 1992, Gore and O'Garro 1999, Aguiar et al. 2000).
Several studies have previously investigated the in vitro sensitivity and resistance, or both, of Xanthomonas spp.from tomato and pepper plants to copper and zinc sulfates.Ward and O'Garro (1992) observed that 61 and 64% of Xanthomonas spp.from tomato and pepper plants were resistant to copper (200 µg.mL -1 ) and zinc sulfates (100 µg.mL -1 ), respectively.Similar results were obtained by Ritchie and Dittapongpitch (1991), Gore and O'Garro (1999) and Costa et al. (2012), who observed that over 60% of Xanthomonas spp.from tomato and pepper plants were resistant to treatment with 200 µg.mL - copper sulfate.
X. euvesicatoria strains evaluated in our study were more sensitive to treatment with the copper and zinc sulfates mixture, than either chemical alone, due to synergism between these chemicals.Similar results were obtained by Maringoni and Kimati (1987a), using a mixture of cupric and thiocarbamate fungicides, which was found to inhibit the in vitro growth of Xanthomonas spp.obtained from pepper and tomato plants.Spraying of crops with mixtures of copper oxide and mancozeb fungicides has shown a good level of bacterial spot control in pepper plants (Aguiar et al. 2003).Thus, the use of fungicides containing both copper and thiocarbamates have been suggested for the management of bacterial spot on pepper plants in Brazil.To our knowledge this is the first study to investigate the resistance of X. euvesicatoria strains to copper and zinc sulfates obtained from pepper plants produced in various areas throughout Brazil.

Figure 1 -
Figure 1 -In vitro sensitivity of Xanthomonas euvisatoria strains from pepper to copper sulfate, zinc sulfate and a mixture of copper and zinc sulfates.
strains analyzed; b Several localities; c Percentage inhibition; d Mixture containing 50% of copper and 50% of zinc sulfates.