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Assessment of copper resistance in Pseudomonas syringae pv. phaseolicola, the pathogen of halo blight on snap bean
Crop Protection 98 (2017) 8e15
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Assessment of copper resistance in Pseudomonas syringae pv. phaseolicola, the pathogen of halo blight on snap bean Shouan Zhang a, *, Yuqing Fu a, Zelalem Mersha a, 1, Ken Pernezny b a b
Tropical Research and Education Center, Department of Plant Pathology, University of Florida, IFAS, Homestead, FL 33031, USA Everglades Research and Education Center, University of Florida, IFAS, Belle Glade, FL 33430, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2016 Received in revised form 3 March 2017 Accepted 11 March 2017
Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Psp), is one of major bacterial diseases on beans worldwide. The disease infects leaves and pods of beans and is most destructive in areas such as south Florida where temperatures are moderate, rainfalls are frequent, and abundant inoculum is present. Application of fixed copper is the primary method currently used by growers to reduce halo blight damage. However, the development of copper resistance in the pathogen populations has become a primary concern in managing this disease. A total of 35 strains of Psp were isolated from snap bean samples collected from commercial fields in 2009 and 2010 in Homestead, Florida. Results from this study indicate that 80% of the Psp strains (28 of 35) from commercial snap bean fields were resistant to copper, based on bacterial populations after 24-h exposure in liquid NB media. Growth of the Psp strains on CYE agar was similar to that in liquid NB, both amended with copper, suggesting that assays on CYE agar amended with copper can be used for rapid assessment of copper resistance in Psp populations. Data from greenhouse experiments indicated that addition of mancozeb improved the efficacy of copper hydroxide for management of halo blight on snap bean caused by copper-resistant Psp strains. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas syringae pv. phaseolicola Halo blight Snap bean Copper resistance Disease management
1. Introduction Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Psp) (Burkholder) Young et al. is one of the major bacterial diseases of beans worldwide (Schwartz et al., 2005). The bacterial pathogen Psp colonizes the seed of dry bean and becomes infective after germination (Burkholder, 1930), and can survive as both a parasite within plant tissue and as an epiphyte on plant surfaces. Psp is spread in the field by rain splash, irrigation water, plant-to-plant contact, and field workers (Schwartz et al., 2005). Halo blight is most severe in early spring in south Florida when temperatures are lower than 25 C with high moisture, and can cause considerable losses to the growers when the epidemics begin early in the growing season. Halo blight has been reported to cause up to 43% reductions in total yield and further losses occurred due to the poor quality of infected pods (Arnold et al., 2011).
* Corresponding author. E-mail addresses: szhang0007@ufl.edu (S. Zhang), [email protected] (Z. Mersha), kenp@ufl.edu (K. Pernezny). 1 Present address: Lincoln University Cooperative Extension, Jefferson City, MO 65101, USA. http://dx.doi.org/10.1016/j.cropro.2017.03.009 0261-2194/© 2017 Elsevier Ltd. All rights reserved.
Halo blight is primarily managed by selecting resistant varieties, planting pathogen-free seed, and crop rotation (Schwartz et al., 2005). The use of fixed copper-based bactericides can reduce epiphytic populations of bacterial pathogens on bean leaves, and also suppress disease development when they are applied as a preventative practice (Schwartz et al., 2005). However, management of bacterial diseases by using copper compounds is often not satisfactory (Montesinos and Vilardell, 2001). Many studies indicate that lack of efficacy appears to be associated with the frequent occurrence of copper-resistant strains of bacterial pathogens (Adaskaveg and Hine, 1985; Jones et al., 1991; Pohronezny et al., 1994; Pernezny et al., 2008; Ritchie and Dittapongpitch, 1991). To improve efficacy of copper-based bactericides, adding mancozeb to copper products usually increased mortality of the bacterial pathogens. The improvement was first reported that a tank mix of copper and mancozeb was more effective in bacterial disease management on vegetable crops than copper alone (Conover and Gerhold, 1981). Snap bean (Phaseolus vulgaris L.) is an economically important vegetable crop in Florida with a total of 33,338 acres harvested in 2012 (USDANASSCensus of Agriculture, 2012). Miami-Dade County ranks second in snap bean production in the United States with a
S. Zhang et al. / Crop Protection 98 (2017) 8e15
Abbreviations CFU CYE GNA NA NB Psp
colony-forming unit casitone-yeast extract glycerol-nutrient agar nutrient agar nutrient broth Pseudomonas syringae pv. phaseolicola
total of 11,126 acres. During the 2008e2010 growing seasons, bean growers in Homestead, Florida reported that some commercial copper bactericides were not as effective in management of foliar bacterial diseases in snap bean as they were previously (Mary Lamberts, personal communications). Psp was consistently isolated from snap bean samples submitted to the Plant Diagnostic Clinic at the Tropical Research and Education Center of University of Florida. In a preliminary experiment in which the bacterial strains were exposed for 2 h to recommended rates of the copper bactericide Kocide® 3000, a marginal difference was detected in the populations of a Psp strain Xcp2 between the treatment and the water control (Pernezny, unpublished data). Different types of culture agar media and laboratory protocols have been developed to assess sensitivity of bacterial strains to copper (Pernezny et al., 2008). By using casitone-yeast extract (CYE) agar amended with CuSO4, strains of P. cichorii (Swingle) Stapp, the pathogen of bacterial blight in celery (Apium graveolens L.) from seedbeds, were classified as sensitive, moderately resistant, and highly resistant to copper, based on their growth on this agar medium (Pohronezny et al., 1994). In a study of copper resistance of P. syringae van Hall strains from citrus (Citrus spp.) and almond (Prunus dulcis (Mill.) Webb) orchards (Andersen et al., 1991), a similar technique was employed for identifying a range of responses to copper depending on the dosage of copper used. Stall et al. (1986) reported that nutrient agar (NA) amended with 200 mg/mL of CuSO4$5H2O was useful for screening copper resistance among pepper (Capsicum spp.) strains of Xanthomonas campestris pv. vesicatoria (Doidge) Dye (now renamed X. euvesicatoria Jones et al.). In Florida, approximately 97% of X. euvesicatoria strains from commercial pepper fields during 1989e1991 were proven to be copper resistant (Pohronezny et al., 1992). Due to the effectiveness of NA in screening Xanthomonas strains for their sensitivity to copper, the same procedure was utilized to evaluate tomato (Solanum lycopersicum L.) strains of X. perforans Jones et al. (1991) and lettuce strains of X. campestris pv. vitians (Brown) Dye (Pernezny et al., 1995) for their copper sensitivity. The objectives of this study were to (i) determine whether resistance to copper has developed in the strains of Psp from snap bean plants collected during 2009e2010 in commercial production fields in Homestead, Florida, (ii) compare the usefulness of different culture media (NA, glucose nutrient agar (GNA) and CYE agar) in rapid assessment of copper resistance in Psp strains, and (iii) investigate if improved efficacy can be achieved by incorporating mancozeb with a copper bactericide for effective management of halo blight caused by resistant strains of Psp.
2. Materials and methods 2.1. Isolation of bacterial strains, identification, and pathogenicity test Fifteen and thirty-five samples of snap bean (cv. Inspiration)
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with typical symptoms of halo blight on leaves were collected in 2009 and 2010, respectively, from a total of ten commercial snap bean fields near Homestead, FL. The samples were sealed in plastic bags, maintained in a cooler with ice, and brought to the laboratory for processing. The snap bean leaves were washed with tap water to remove soil adhering to the foliage before the leaves were cut into small pieces (~0.5 cm2) containing diseased and healthy tissues. The small pieces of leaves were sterilized in 70% ethanol for 1 min, then in 0.6% sodium hypochlorite for 10 min, and finally rinsed in sterile water. The sterilized leaf tissues were placed on nutrient agar (Thermo Fisher Scientific Inc., Lenexa, KS) plates after they were air dried in a sterile laminar flow hood, and incubated in the dark at 28 C for 2e3 days. Individual bacterial colonies were streaked on NA plates to ensure isolation of pure cultures. White to cream-colored single colonies were selected and saved in 15% glycerol at 80 C for further experiments. Pathogenicity tests were carried out using bean pods and entire snap bean plants. Bacterial cell suspensions were prepared by streaking bacterial strains from ultra-cold storage onto NA plates and incubating at 28 C for 24 h to check for purity, then transferring single colonies to nutrient broth (NB) at 150 rpm overnight. Bacterial cells were washed twice by centrifuging at 3000 rpm each for 10 min in sterile water. The bacterial suspensions were adjusted with sterile water with a spectrophotometer (ƛ ¼ 600 nm, A ¼ 0.22) to 1 108 CFU/mL, then further diluted to 1 107 CFU/mL for inoculation. In the pod inoculation assays, pods of snap bean surface sterilized with 70% ethanol were wounded with a sterilized needle and 50 ml of bacterial suspensions (1 107 CFU/mL) were pipetted on each wound site. Inoculated pods were placed in a tray sealed with a plastic film containing moist tissue paper at the bottom. Wounded pods receiving 50 ml of sterile water served as a non-treated control. The bacterial strains causing water-soaked lesions after 24 h around the wound sites on snap bean pods were saved. For entire plant inoculation, five 3-week old plants of the susceptible snap bean cv. ‘Inspiration’ were used for each isolate. Suspensions (1 107 CFU/mL) of bacterial strains were sprayed onto the leaves of snap bean plants until run-off using a handheld sprayer. Inoculated plants were covered with plastic bags overnight to maintain high humidity, and placed on greenhouse benches for 5e7 days prior to disease observation. The temperatures of the greenhouse was set 25 C with a 14-h photoperiod. Plants sprayed with water served as a non-treated control. Bacterial strains inciting typical symptoms, i.e. small, water-soaked spots and chlorosis on the leaves that rapidly became necrotic lesions, were saved for further identification by 16S rRNA gene sequencing. Each bacterial strain was tested twice. Further confirmation of the identity of the bacterial strains from the pod and entire plant assays was conducted by 16S rRNA gene sequencing using the universal primers 8F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1492R (50 GGT TAC CTT GTT ACG ACT T-30 ) (Eden et al., 1991). 2.2. Sensitivity of Psp exposed to copper hydroxide in liquid culture media Forty-five Psp strains obtained above were evaluated in liquid NB for their sensitivity to copper hydroxide (Kocide® 3000, DuPont Co., Wilmington, DE) over a period of 24 h. The rate of copper hydroxide at 0.55 mg/mL used in this experiment was based on label rates of Kocide® 3000 applied on commercial bean crops. The suspension of Kocide® 3000 was freshly prepared with sterile water at 1.2 mg/mL (equivalent to 0.55 mg/mL copper hydroxide), which was added and suspended in 50 mL of NB in a 125-mL flask. About 0.5 mL of the suspension at 1 106 CFU/mL of each bacterial isolate was added, and the initial bacterial concentration was approximately 1 104 CFU/mL. For each bacterial isolate, 0.5 mL of the
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bacterial suspension added into NB without addition of copper hydroxide served as a non-treated control. Each isolate was tested in three flasks as three replications, and the flasks were incubated at 28 C on a rotary shaker at 150 rpm. The experiment was a completely randomized design. Bacterial samples were collected at 0, 9, and 24 h after incubation and the concentrations of Psp were determined by serial dilutions and pipetting 20 mL of appropriate dilutions on NA plates (three drops for each isolate). Bacterial colonies were counted on plates with less than 50 colonies per drop, and data were expressed as log10 CFU/mL. This experiment was repeated once. The number one was added to all bacterial population data before counts were converted to log10 equivalents (Pernezny et al., 2008). Bacterial populations were recorded as zero when no colonies were detected on plates from samples drawn directly from the undiluted solutions. Log equivalents of bacterial populations from each isolate were subjected to analysis of variance. Means of treatments were separated by Fisher's protected LSD test at P ¼ 0.05 using the Statistical Analysis System (SAS) package version 9.4 (2014, SAS Institute, Inc., Cary, NC). 2.3. Assessment of Psp strains for copper resistance on media agar plates NA amended with 0.5% (w/v) glucose (GNA) (pH 7.0) and CYE agar (1.7 g casitone, 0.35 g yeast extract, 5 g glucose, and 17 g Difco agar per Liter of distilled water) (pH 6.0 and 7.0) were first used for the comparison of bacterial growth with and without copper amendment. Copper sulfate (CuSO4$5H2O) (Fisher Scientific, Fair Lawn, NJ) at appropriate concentrations was prepared by incorporating it from a filter-sterilized solution of CuSO4 into autoclaved media cooled to approximately 50 C. Also, the commercial copper hydroxide bactericide Kocide® 3000 was also added at 1.2 mg/mL into NA and CYE and tested for the growth of bacterial strains in the same manner. A total of 35 strains of Psp from snap bean were tested in this study for sensitivity to copper. Treatments were GNA (pH 7.0), GNA (pH 7.0) þ 200 mg/mL CuSO4$5H2O, CYE (pH 6.0), CYE (pH 6.0) þ 100 mg/mL CuSO4$5H2O, CYE (pH 6.0) þ 161 mg/mL CuSO4$5H2O, and CYE (pH 6.0) þ 200 mg/mL CuSO4$5H2O, CYE (pH 7.0), CYE (pH 7.0) þ 100 mg/mL CuSO4$5H2O, CYE (pH 7.0) þ 161 mg/ mL CuSO4$5H2O, and CYE (pH 7.0) þ 200 mg/mL CuSO4$5H2O. The concentration of 161 mg/mL of CuSO4$5H2O was equivalent to the originally published concentration of 0.64 mM CuSO4 (Andersen et al., 1991). Bacterial cells were taken using a sterile inoculation loop from 3-day-old cultures on NA and streaked on triple plates of each culture medium (Pohronezny et al., 1994). The experiment was a completely randomized design. Plates were incubated at 28 C for 72 h when a visual estimate was made of the growth on copper-containing culture media and expressed as a percentage of growth on control plates without copper amendments (Pernezny et al., 2008), in this case where 0 ¼ 0, 1 ¼ 1e10%, 2 ¼ 11e25%, 3 ¼ 26e50%, 4 ¼ 51e75%, 5 ¼ 76e100%. Psp strains with ratings of 0 were considered sensitive to copper, those with ratings of 1, 2, 3 and 4 were moderately resistant (MR), and those with ratings of 5 were highly resistant (HR). All bacterial strains were evaluated in the same manner for a second time. Results from the agar plate test were compared to the growth of the Psp strains in liquid NB which was used as the criterion to identify the types of agar media that were best suited for studies on copper resistance. The numbers of resistant including highly resistant and moderately resistant, and susceptible Psp strains from the agar plate test, along with the numbers from the liquid NB assays were subjected to analysis of variance (ANOVA). Means of strain numbers from each agar and liquid medium were separated by Fisher's protected LSD test at P ¼ 0.05 using the Statistical Analysis System (SAS) package version
9.4 (2014, SAS Institute, Inc., Cary, NC). 2.4. Efficacy of copper hydroxide and addition of mancozeb for management of halo blight Four experiments were conducted in the greenhouse to assess the efficacy of copper hydroxide (Kocide® 3000, DuPont Co., Wilmington, DE) for the management of halo blight caused by a resistant isolate of Psp with and without addition of mancozeb (Manzate Pro-Stick, United Phosphorus, Inc., King of Prussia, PA). Susceptible snap bean seeds cv. ‘Inspiration’ (experiments 1 & 2) and ‘Pony Express’ (experiments 3 & 4) were planted in 10-cmdiameter plastic pots containing soilless Pro-mix growing medium (Miracle-Gro Lawn Products, Inc., Marysville, OH) and watered daily. A controlled-release fertilizer, Osmocote PLUS (15-9-12; Scotts-Sierra Horticultural Products Company, Marysville, OH), was mixed into the soilless potting Pro-mix medium at a rate of 1.3% (wt/wt) prior to seed planting. Experiments were conducted in an air-conditioned greenhouse with a maximum daytime temperature of 25 Ce28 C and a typical light intensity at bench height of 780 mE/m2/s. Snap bean plants were exposed to natural light with photoperiods of 14 h light and 10 h dark with no supplemental artificial lighting. Copper-resistant Psp strain 28 was grown on NA for 3 days at 28 C. The inoculum of Psp strain 28 was prepared as previously described in pathogenicity tests under 2.1. Bacterial suspensions (1 107 CFU/mL) were applied to plants 17 days after planting through misting both adaxial and abaxial surfaces of the leaves until run-off (approximately 15 of suspension per plant) using a handheld spray bottle. In order to enhance wetting of leaf surfaces and full coverage, the surfactant Silwet-77 (Setre Chemical Co., Memphis, TN) was added to the inoculum suspensions at a final rate of 0.1%. A total of five treatments were included in this experiment: copper hydroxide (Kocide® 3000, 1.2 mg/mL), mancozeb (Manzate Pro-Stick, 0.24 mg/mL), copper hydroxide (Kocide® 3000, 1.2 mg/ mL) þ mancozeb (Manzate Pro-Stick, 0.24 mg/mL), copper hydroxide þ mancozeb [ManKocide®, 1.2 mg/mL], and non-treated water control (CK). Treatments with chemicals were applied 2 weeks after planting (V2 growth stage) by spraying solutions onto the plants until runoff using a handheld spray bottle. Three days after the treatment, snap bean plants were inoculated with the Psp strain 28 as described previously. One week following the inoculation, plants were treated with the chemical compounds for the second time. Disease severity was rated 2e3 weeks after inoculation as the percentage of leaf area covered with bacterial lesions. The experiment was arranged in a randomized complete block design with eight replications for each treatment, each plant in a pot served as a replication within each treatment. The experiment was repeated once. The values of disease severity were transformed by square root, and were analyzed separately for each repeated experiment. Data of disease were pooled for Exp. 1 & 2, Exp. 3& 4 after statistical analysis demonstrated that experiment by treatment interactions were not significant (P 0.05). Means of treatments were separated by Fisher's protected LSD test at P ¼ 0.05 using the Statistical Analysis System (SAS) package version 9.4 (2014, SAS Institute, Inc., Cary, NC). 2.5. In vitro effect of copper hydroxide and mancozeb on populations of copper-resistant Psp strain 28 Suspensions of the treatments used in the efficacy trial were freshly prepared with sterile water, which were added into 50 mL NB in a 125-mL flask. About 0.5 mL of the suspension at 1 106 CFU/ mL of the Psp strain 28 was added and the initial bacterial
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concentration was approximately 1 104 CFU/mL. Three flasks for each treatment including the control served as three replications, and the flasks were incubated at 28 C on a rotary shaker at 150 rpm. The experiment was a randomized complete block design. Bacterial samples were collected at 0 and 24 h after incubation and the concentrations of Psp were determined by serial dilutions and plating of 20 mL of appropriate dilutions on NA plates (three drops for each isolate). Bacterial colonies were counted on plates after 3 days, and data were expressed as log10 CFU/mL. Data analyses were conducted by using SAS as described previously in 2.2. 3. Results 3.1. Isolation and identification of Psp strains from snap bean A total of 45 bacterial strains of white to cream color on NA were obtained from fifty snap bean samples collected in 2009 and 2010 from commercial fields near Homestead, Florida. Thirty-five strains incited water-soaked lesions on inoculated bean pods and typical
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symptoms of halo blight on leaves of plants in the greenhouse pathogenicity tests. The identity of these strains was further verified as Pseudomonas syringae pv. phaseolicola, the pathogen of halo blight of beans, by 16S rRNA gene sequencing (data not shown). 3.2. Population dynamics of bacterial strains exposed to copper hydroxide After 9-h exposure, populations of eight highly resistant strains, i.e. strains 19, 21, 26, 28, 34, 35, 36, 42, and 45, reached log values of 4.5e5.6, comprising the group with highest populations among 35 strains (Fig. 1A). Those classified as moderately resistant strains, i.e. strains 2, 3, 4, 5, 9, 11, 22, 25, 32, 33, 37, 40, and 44, had log values between 1.0 and 2.0 with an exception of isolate 44, which was not detectable after 9-h exposure (Fig. 1B). However, for those classified as sensitive strains, only three strains, i.e. 6, 8, and 10, had no detected growth after 9-h exposure. Populations of the other four strains (1, 7, 20, and 38) reached log values of 1.8e3.3, even greater than those of moderately resistant strains in this assay.
Fig. 1. Populations of P. syringae pv. phaseolicola strains in NB after (A) 9-h and (B) 24-h exposure to copper hydroxide. Samples of strains 13, 14, 18, 30, 31, and 46 were not taken for assessing cell populations after 9-h exposure.
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Based on the bacterial population after 24-h exposure to copper hydroxide at the label rate, 20% of Psp strains (7 of 35 strains) were classified as copper sensitive with no bacterial cells detected, 43% (15 of 35 strains) were highly resistant to copper with log values between 7.2 and 8.8, and 37% (13 of 35 strains) were moderately resistant with log values lower than 3.7 (Fig. 1B). The log values of these Pseudomonas strains were in the range of 8.5e9.2 with no addition of copper hydroxide depending on each individual isolate (data not shown). For example, the population of a copper susceptible strain 7 down to a log value of 1.3 after 9-h exposure, and no bacterial cells were detected after 24 h (Fig. 2A). However, the population of a copper resistant strain 28 reached an equivalent level to the untreated control (no addition of copper) after 24-h exposure (Fig. 2B). Strain 37, defined as a moderately resistant strain, reached a log value of 1.8 and 2.4 after 9-h and 24-h exposure, respectively (Fig. 2C).
sensitive (Fig. 1B). Compared to the results in liquid NB, CYE agar plates with pH 7.0 amended with CuSO4 at 161 mg/mL, 200 mg/mL, and copper hydroxide (Kocide® 3000, 1.2 mg/mL) had comparable results in evaluation of Psp strains for resistance to copper (Table 1, Fig. 3). Similar results were also obtained on CYE agar of pH 6.0 with addition of CuSO4 at 161 mg/mL and 200 mg/mL. Other tested agar media, i. e. NA þ copper hydroxide (Kocide® 3000), GNA þ CuSO4 (200 mg/mL), detected significantly lower numbers of Psp strains
3.3. Assessment of Psp strains for copper resistance on agar culture media The sensitivity of Psp strains to copper was dependent on the agar media used in qualitatively visual assays (Tables 1 and 2). In this case, bacterial strains were classified as highly resistant, moderately resistant, and sensitive to copper, based on the growth on media plates. On CYE plates, 17e28% of Psp strains (6e10 of 35 strains) were classified as copper sensitive, and 72e83% (25e29 strains) strains were identified as resistant to copper (Table 1). At pH 7.0, the growth of bacterial strains resistant to copper was similar on agar YCE plates amended with CuSO4 at varying concentrations, i. e. 28, 29, and 27 Psp strains, were copper resistant when the concentration of copper in the media was 100, 161, and 200 mg/mL, respectively, which was not significantly different from the liquid NB. Psp strains of 28, 30, 31, 35, 36, 42, 45, and 46 were consistently rated as highly resistant in the tests on YCE plates. However, among the resistant strains, the number of highly resistant strains differed with copper concentration in the agar media. When concentrations of CuSO4 were 161 and 200 mg/mL, 18 and 12 strains were highly resistant, and 11 and 15 were moderately resistant, respectively. At 100 mg/mL of CuSO4, none of the Psp strains were classified as moderately resistant, and all 28 resistant strains were categorized as highly resistant to copper. Therefore, 100 mg/mL was not included in assays on CYE agar with pH 6.0, which was conducted afterwards. On CYE agar media at pH 6.0, amended with copper of 161 mg/ mL, eight strains were evaluated as highly resistant, 19 were moderately resistant, and eight were sensitive to copper. When the copper concentration was increased to 200 mg/mL, no Psp strains were classified as highly resistant, but 25 strains were assessed as moderately resistant and 10 were sensitive to copper. When copper hydroxide (Kocide® 3000) was added at a label rate (equivalent to 1.2 mg/mL) to NA media, 27 strains were detected to be highly resistant, six were moderately resistant, and two were sensitive to copper. However, on CYE agar media, 10 strains were highly resistant to copper, 16 were moderately resistant, and nine were sensitive. The Psp strains of 14, 18, 28, 30, 31, 35, 36, 42, 45, and 46 were rated as highly resistant in all the tests on NA and CYE agar plates with copper hydroxide. 3.4. Comparison of culture agar media for resistance assessment of Psp strains to copper Based on the bacterial population after 24-h exposure to copper hydroxide at the label rate, 15 of the 35 Psp strains were classified as highly resistant to copper, 13 were moderately resistant, and 7 were
Fig. 2. Population dynamics of representative strains of P. syringae pv. phaseolicola classified as (A) sensitive (strain 7), (B) highly resistant (strain 28), and (C) moderately resistant (strain 37) to copper after 9- and 24-h exposure to copper hydroxide in NB.
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Table 1 Assessment of 35 strains of P. syringae pv. phaseolicola on agar culture media for their response to copper.a Response to copper
Resistantb HR MR Sensitive
No. of strains resistant or sensitive to copper CYE agar pH 7.0
pH 6.0
CuSO4$5H2O (mg/mL)
CuSO4$5H2O (mg/mL)
100
161
200
161
200
28 bcc 28 a 0d 7b
29 b 18 b 11 bc 6 bc
27 bc 12 bc 15 b 8 ab
27 bc 8 cd 19 ab 8 ab
25 c 0d 25 a 10 a
CYE (7.0) agar þ Kocide® 3000 (1.2 mg/mL)
NA þ Kocide® 3000 (1.2 mg/mL)
GNA þ CuSO4$5H2O (200 mg/mL)
NB þ copper hydroxide (24-h exposure)
26 bc 10 bc 16 b 9 ab
33 a 27 a 6 cd 2 cd
35 a 33 a 2d 0d
28 bc 15 bc 13 bc 7b
a Evaluation of resistance based on visual estimates of growth of Psp strains on copper-containing agar media compared with growth on agar media without copper amendments (control). A visual estimate was made of the growth on copper-containing culture media and expressed as a percentage of growth on control plates without copper amendments, where 0 ¼ 0, 1 ¼ 1e10%, 2 ¼ 11e25%, 3 ¼ 26e50%, 4 ¼ 51e75%, 5 ¼ 76e100%. b Resistance category is a sum of HR and MR isolates. HR ¼ highly resistant (rating ¼ 5), MR ¼ moderately resistant (rating ¼ 1, 2, 3, 4), Sensitive ¼ 0. c In each category, means of strain numbers across all agar and liquid NB media within a cell followed by the same letter are not significantly different (P ¼ 0.05) according to the Fisher's protected LSD test.
Table 2 Efficacy of copper hydroxide and addition of mancozeb for management of halo blight in snap bean caused by copper-resistant strain 28 of P. syringae pv. phaseolicola.a Treatmentb
Product (rate)
Disease Severity (%)c
Copper hydroxide Mancozeb Copper hydroxide þ Mancozeb Copper hydroxide þ Mancozeb Non-treated Control (CK)
Kocide® 3000 (1.2 mg/mL) Manzate Pro-Stick (0.24 mg/mL) Kocide® 3000 (1.2 mg/mL) þ Manzate Pro-Stick (0.24 mg/mL) ManKocide® (1.2 mg/mL) e
3.8 9.1 0.8 2.1 7.1
Exp. 1 þ 2 bd a c b a
Exp. 3 þ 4 6.3 9.1 3.5 4.4 8.7
ab a c c a
a
Snap bean seeds cv. ‘Inspiration’ (experiments 1 & 2) and ‘Pony Express’ (experiments 3 & 4) were planted in plastic pots containing soilless Pro-mix growing medium. Treatments with chemicals were applied at 2 weeks after planting by spraying solutions onto the plants until runoff. Three days after the treatment, the plants were inoculated with the Psp strain 28 (1 107 CFU/mL) by spraying the inoculum onto both surfaces of the leaves until runoff. One week following the inoculation, plants were treated with chemical compounds for the second time. c Disease severity was rated 2e3 weeks after inoculation as the percentage of leaf area covered with bacterial lesions. d The values of disease severity were transformed by square root prior to analysis. The table contains de-transformed values. Means within a column followed by the same letter are not significantly different (P ¼ 0.05) according to Fisher's protected LSD test. b
Fig. 3. Percentage of Psp strains that were resistant, moderately resistant, and sensitive to copper on NA, GNA and CYE agar media.
that were sensitive to copper, compared to liquid NB. 3.5. Efficacy of copper hydroxide and the addition of mancozeb for management of halo blight in snap bean Mancozeb alone did not reduce severity of halo blight in any of
these experiments. However, addition of mancozeb to copper hydroxide significantly (P < 0.05) improved the efficacy of copper hydroxide. The combination of copper hydroxide (Kocide® 3000) þ mancozeb (Manzata Pro-Stick) achieved equivalent or better levels in halo blight management to a commercial product ManKocide®, which is a pre-formulated combination product of
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copper hydroxide and mancozeb. 3.6. In vitro effect of copper hydroxide and mancozeb on populations of copper resistant Psp strain 28 There was no effect of copper hydroxide on populations of the Psp strain 28 compared to the control (Table 3). Mancozeb alone significantly (P < 0.05) inhibited the growth of the strain 28 after 24 h. However, no bacterial cells were detected in the treatment of a mixture of copper hydroxide þ mancozeb or Mankocide®. 4. Discussion Results from this study revealed that 80% of the Psp strains (28 of 35) from commercial snap bean fields in Homestead, Florida were resistant to copper, based on bacterial populations after 24-h exposure in liquid NB media (Fig. 1B). Growth of the Pseudomonas strains on CYE agar at pH 6.0 or 7.0 was similar to that in liquid NB, both amended with an appropriate rate of copper such as 161 mg/ mL CuSo4, suggesting that assays on CYE agar amended with copper can be used for rapid assessment of copper resistance in Psp populations (Fig. 3). The results from greenhouse experiments indicated that addition of mancozeb improved the efficacy of copper hydroxide for management of halo blight on snap bean caused by copper-resistant Psp. Results from this study indicate that CYE agar þ CuSO4 is more appropriate for screening Psp strains for copper resistance than other media such as NA or GNA, based on populations of these strains in liquid nutrient broth after 24-h exposure (Fig. 1B). Screening of Psp on CYE agar broadly classified Psp strains as sensitive, moderately resistant, or highly resistant to copper; whereas, 100% (35 strains) and 94.3% (33 strains) Psp strains were classified as copper resistant on GNA containing CuSO4$5H2O and NA amended with copper hydroxide (Table 1). The classifications in this study corresponded to those from the in vitro population studies based on liquid assays in the current study, which were similar to previous reports from other researchers. CYE media amended with copper was reported to be useful for copper resistance studies among P. syringae strains from fruit trees (Andersen et al., 1991) and ornamentals (Scheck and Pscheidt, 1998). Pohronezny et al. (1994) concluded that CYE agar with copper was extremely useful for screening P. cichorii strains recovered from celery seedbeds. Results from the present study could be very important and useful in disease management in that copper resistance is a major concern for growers because it impacts the efficacy of copper bactericides for management of halo blight. Rapid assessment of Psp strains will aid growers by commercial services or research institutes in making wise decisions on effective disease management. In this study, CYE agar was shown to be the most appropriate one among those evaluated for rapid assessment of Psp for their resistance to copper (Fig. 3). Compared to NB with copper hydroxide (Kocide® 3000), Psp strains exhibited similar responses to copper on CYE agar with exceptions of i) CuSO4$5H2O at 200 mg/mL at pH 6.0, which failed to detect highly resistant strains of Psp, and ii) CuSO4$5H2O at 100 mg/mL at pH 7.0, where no moderately resistant strains were detected. NA, a commonly used medium for bacterial culture, detected a significantly lower numbers of sensitive Psp strains and higher numbers of highly resistant strains. GNA, amended with 200 mg/mL CuSO4$5H2O in this study, failed to detect sensitive Psp strains. Therefore, it is recommended that the intermediate rate of 161 mg/mL be an appropriate rate of CuSO4$5H2O for Psp in screening for copper resistance on CYE agar at pH 6.0 or 7.0. It has been clearly documented that the development of copper resistance in populations of Xanthomonas causing bacterial spot on
Table 3 Populations of copper-resistant P. syringae pv. phaseolicola strain 28 after 24-h exposure to copper hydroxide (Kocide® 3000), mancozeb, and mixture of copper hydroxide þ mancozeb.a Treatment
Log10 CFU/mLb
Non-treated control Copper hydroxide (1.2 mg/mL) Mancozeb (0.24 mg/mL) ManKocide® (1.2 mg/mL) Copper hydroxide (1.2 mg/mL) þ Mancozeb (0.24 mg/mL)
8.84 ac 8.75 ab 5.12 c 0d 0d
a About 0.5 mL of the suspension at 1 106 CFU/mL of strain 28 was added to 50 mL NB containing test chemicals in a 125-mL flask. Three flasks for each treatment, including the non-treated control, served as three replications, and the flasks were incubated at 28 C on a rotary shaker at 150 rpm. b Bacterial samples were collected at 0 and 24 h after incubation, and the concentrations of Psp were determined by serial dilutions and plating appropriate dilutions on NA plates. Bacterial colonies were counted on plates after 3 days, and data were expressed as log10 CFU/mL. The digit one was added to all bacterial population data before counts were converted to log10 equivalents. Bacterial populations were recorded as zero when no colonies were detected on plates from samples drawn directly from the undiluted solutions. c Means within a column followed by the same letter are not significantly different (P ¼ 0.05) according to Fisher's protected LSD test.
tomatoes and peppers is related to frequent use of copper (Pernezny et al., 2008). Copper has been used in Florida for over fifty years, and nearly 100% of the X. euvesicatoria and X. perforans strains were resistant to copper (Pohronezny et al., 1992). All strains of X. campestris pv. vitians causing foliar bacterial disease of lettuce were sensitive to copper because copper was not applied frequently (Pernezny et al., 1995). Snap bean is an economically important vegetable crop in south Florida. Diseases caused by bacterial pathogens on snap bean occur periodically. Usually, only a few sprays of copper bactericides during a growing season in south Florida are made on snap bean for management of bacterial diseases. Surprisingly, in this study, 80% of Psp strains from south Florida were resistant to copper. At this time, the reasons for the discrepancy between copper application frequency and copper resistance development in Psp strains are not known. In recent years, however, more frequent applications of copper bactericides have been made on snap bean in south Florida due to increased prevalence of bacterial diseases (Ken Pernezny, personal communications). During 2009e2010, snap bean growers in Homestead, FL reported that copper bactericides were not as effective in management of bacterial diseases in snap bean as they were previously. In November 2016, halo blight outbreaks occurred in some fields in Homestead, FL due to cool temperatures and several rainy days in late October. As a result, these snap bean plantings were destroyed in order to reduce the bacterial inoculum because no other choices were available for growers for managing the disease. Results from this study indicated that the reduced efficacy of copper bactericides may be, at least in part, due to the development of copper resistance in bacterial populations. Growers should use copper bactericides judiciously to mitigate the widespread appearance of resistance in pathogen populations. In four repeated experiments during this study, copper hydroxide had lower disease severity in three experiments compared to the non-treated control (Table 2). This reduction is not thought to be adequate for successful management of the disease. Addition of mancozeb to copper hydroxide usually resulted in increased mortality of the bacterial pathogen. Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide commonly used as a protectant against a broad spectrum of fungal pathogens on many crops including snap beans (Wells and Fishel, 2011). In an in vitro experiment, no bacterial cells were detected in the copper hydroxide þ mancozeb treatment of the copper-resistant Psp strain 28 (Table 3). This may be due to increases in concentration of Cu2þ
S. Zhang et al. / Crop Protection 98 (2017) 8e15
ions in the mixed solution (Marco and Stall, 1983; Menkissoglu and Lindow, 1991), resulting in increased mortality of bacterial cells (Scheck and Pscheidt, 1998). Another possibility is that mancozeb may chelate copper ions, so copper ions become more available at the sites in bacterial cells that are detrimentally affected by copper (Medhekar and Boparai, 1981). Conover and Gerhold (1981) first reported that a tank mix of copper and mancozeb was more effective in management of bacterial diseases on vegetable crops than copper alone. In the greenhouse experiments of this study, neither copper hydroxide nor mancozeb alone adequately reduced halo blight disease caused by the copper-resistant Psp strain 28. However, in vitro toxicity of mancozeb to Psp was detected in this study after 24-h exposure in liquid NB (Table 3). This finding is in agreement with Colin and McCarter (1983) who reported that mancozeb was toxic to strains of P. syringae. It is likely that the zinc cation, part of the coordinated moiety of mancozeb, is involved in mortality of Pseudomonas cells. Acknowledgments We thank Xiaodan Mo and Jiebin Guo for their technical assistance, and Xiaohui Fan for his help with the statistical analysis. This study was funded in part by DuPont Co., Wilmington, DE. References Adaskaveg, J.E., Hine, R.B., 1985. Copper tolerance and zinc sensitivity of Mexican strains of Xanthomonas campestris pv. vesicatoria, causal agent of bacterial spot of pepper. Plant Dis. 69, 993e996. Andersen, G.L., Menkissoglou, O., Lindow, S.E., 1991. Occurrence and properties of copper- tolerant strains of Pseudomonas syringae isolated from fruit trees in California. Phytopathology 81, 648e656. Arnold, D.L., Lovell, H.C., Jackson, R.W., Mansfield, J.W., 2011. Pseudomonas syringae pv. phaseolicola: from ‘has bean’ to supermodel. Mol. Plant Pathol. 12, 617e627. Burkholder, W.H., 1930. The bacterial diseases of the bean: a comparative study. Memoirs, Cornell Univ. Agric. Exp. Stn. 127, 1e88. Colin, K.C., McCarter, S.M., 1983. Effectiveness of selected chemicals in inhibiting Pseudomonas syringae pv. tomato in vitro and in controlling bacterial speck. Plant Dis. 67, 639e644. Conover, R.A., Gerhold, N.R., 1981. Mixtures of copper and maneb or mancozeb for
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control of bacterial spot of tomato and their compatibility for control of fungus diseases. Proc. Fla. State Hortic. Soc. 94, 154e156. Eden, P.A., Schmidt, T.M., Blakemore, R.P., Pace, N.R., 1991. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA- specific DNA. Int. J. Syst. Bacteriol. 41 (2), 324e325. Jones, J.B., Woltz, S.S., Jones, J.P., Portier, K.L., 1991. Population dynamics of Xanthomonas campestris pv. vesicatoria in tomato leaves treated with copper bactericides. Phytopathology 81, 714e719. Marco, G.M., Stall, R.E., 1983. Control of bacterial spot of pepper initiated by strains of Xanthomonas campestris pv. vesicatoria that differ in sensitivity to copper. Plant Dis. 67, 779e781. Medhekar, S., Boparai, K.S., 1981. Fungicidal bis (1-amidino-Oethylisourea) copper (II) carbamates. J. Agric. Food Chem. 29, 421e422. Menkissoglu, O., Lindow, S.E., 1991. Relationship of free ionic copper and toxicity to bacteria in solutions of organic compounds. Phytopathology 81, 1258e1263. Montesinos, E., Vilardell, P., 2001. Effect of bactericides, phosphonates and nutrient amendments on blast of dormant flower buds of pear: a field evaluation for disease control. Eur. J. Plant Pathol. 107, 787e794. Pernezny, K., Nagata, R., Havranek, N., Sanchez, J., 2008. Comparison of two culture media for determination of the copper resistance of Xanthomonas strains and their usefulness for prediction of control with copper bactericides. Crop Prot. 27, 256e262. Pernezny, K., Raid, R.N., Stall, R.E., Hodge, N.C., Collins, J., 1995. An outbreak of bacterial spot of lettuce in Florida caused by Xanthomonas campestris pv. vitians. Plant Dis. 79, 359e360. Pohronezny, K., Sommerfeld, M.L., Raid, R.N., 1994. Streptomycin resistance and copper tolerance among strains of Pseudomonas cichorii in celery seedbeds. Plant Dis. 78, 150e153. Pohronezny, K., Stall, R.E., Canteros, B.I., Kegley, M., Datnoff, L.E., Subramanya, R., 1992. Sudden shift in the prevalent race of Xanthomonas campestris pv. vesicatoria in pepper fields in southern Florida. Plant Dis. 76, 118e120. Ritchie, D.F., Dittapongpitch, U., 1991. Copper-and-streptomycin resistant strains and host- differentiated races of Xanthomonas campestris pv. vesicatoria in North Carolina. Plant Dis. 75, 733e736. Scheck, H.J., Pscheidt, J.W., 1998. Effect of copper bactericides on copper resistant and sensitive strains of Pseudomonas syringae pv. syringae. Plant Dis. 82, 397e406. Schwartz, H.F., Steadman, J.R., Hall, R., Forster, R.L., 2005. Compendium of Bean Diseases, second ed. The American Phytopathological Society Press, St. Paul, pp. 49e50. Stall, R.E., Loschke, D.C., Jones, J.B., 1986. Linkage of copper resistance and avirulence loci on a self-transmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology 76, 240e243. USDA- NASS-census of Agriculture, 2012. Available at: https://www.agcensus.usda. gov/Publications/2012/index.php (Accessed 15 October 2016). Wells, B., Fishel, F.M., 2011. Agricultural Pesticide Use in Florida: a Summary, 20072009. http://edis.ifas.ufl.edu/pi235 (Accessed 15 October 2016).
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Assessment of copper resistance in Pseudomonas syringae pv. phaseolicola, the pathogen of halo blight on snap bean Shouan Zhang a, *, Yuqing Fu a, Zelalem Mersha a, 1, Ken Pernezny b a b
Tropical Research and Education Center, Department of Plant Pathology, University of Florida, IFAS, Homestead, FL 33031, USA Everglades Research and Education Center, University of Florida, IFAS, Belle Glade, FL 33430, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2016 Received in revised form 3 March 2017 Accepted 11 March 2017
Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Psp), is one of major bacterial diseases on beans worldwide. The disease infects leaves and pods of beans and is most destructive in areas such as south Florida where temperatures are moderate, rainfalls are frequent, and abundant inoculum is present. Application of fixed copper is the primary method currently used by growers to reduce halo blight damage. However, the development of copper resistance in the pathogen populations has become a primary concern in managing this disease. A total of 35 strains of Psp were isolated from snap bean samples collected from commercial fields in 2009 and 2010 in Homestead, Florida. Results from this study indicate that 80% of the Psp strains (28 of 35) from commercial snap bean fields were resistant to copper, based on bacterial populations after 24-h exposure in liquid NB media. Growth of the Psp strains on CYE agar was similar to that in liquid NB, both amended with copper, suggesting that assays on CYE agar amended with copper can be used for rapid assessment of copper resistance in Psp populations. Data from greenhouse experiments indicated that addition of mancozeb improved the efficacy of copper hydroxide for management of halo blight on snap bean caused by copper-resistant Psp strains. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas syringae pv. phaseolicola Halo blight Snap bean Copper resistance Disease management
1. Introduction Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Psp) (Burkholder) Young et al. is one of the major bacterial diseases of beans worldwide (Schwartz et al., 2005). The bacterial pathogen Psp colonizes the seed of dry bean and becomes infective after germination (Burkholder, 1930), and can survive as both a parasite within plant tissue and as an epiphyte on plant surfaces. Psp is spread in the field by rain splash, irrigation water, plant-to-plant contact, and field workers (Schwartz et al., 2005). Halo blight is most severe in early spring in south Florida when temperatures are lower than 25 C with high moisture, and can cause considerable losses to the growers when the epidemics begin early in the growing season. Halo blight has been reported to cause up to 43% reductions in total yield and further losses occurred due to the poor quality of infected pods (Arnold et al., 2011).
* Corresponding author. E-mail addresses: szhang0007@ufl.edu (S. Zhang), [email protected] (Z. Mersha), kenp@ufl.edu (K. Pernezny). 1 Present address: Lincoln University Cooperative Extension, Jefferson City, MO 65101, USA. http://dx.doi.org/10.1016/j.cropro.2017.03.009 0261-2194/© 2017 Elsevier Ltd. All rights reserved.
Halo blight is primarily managed by selecting resistant varieties, planting pathogen-free seed, and crop rotation (Schwartz et al., 2005). The use of fixed copper-based bactericides can reduce epiphytic populations of bacterial pathogens on bean leaves, and also suppress disease development when they are applied as a preventative practice (Schwartz et al., 2005). However, management of bacterial diseases by using copper compounds is often not satisfactory (Montesinos and Vilardell, 2001). Many studies indicate that lack of efficacy appears to be associated with the frequent occurrence of copper-resistant strains of bacterial pathogens (Adaskaveg and Hine, 1985; Jones et al., 1991; Pohronezny et al., 1994; Pernezny et al., 2008; Ritchie and Dittapongpitch, 1991). To improve efficacy of copper-based bactericides, adding mancozeb to copper products usually increased mortality of the bacterial pathogens. The improvement was first reported that a tank mix of copper and mancozeb was more effective in bacterial disease management on vegetable crops than copper alone (Conover and Gerhold, 1981). Snap bean (Phaseolus vulgaris L.) is an economically important vegetable crop in Florida with a total of 33,338 acres harvested in 2012 (USDANASSCensus of Agriculture, 2012). Miami-Dade County ranks second in snap bean production in the United States with a
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Abbreviations CFU CYE GNA NA NB Psp
colony-forming unit casitone-yeast extract glycerol-nutrient agar nutrient agar nutrient broth Pseudomonas syringae pv. phaseolicola
total of 11,126 acres. During the 2008e2010 growing seasons, bean growers in Homestead, Florida reported that some commercial copper bactericides were not as effective in management of foliar bacterial diseases in snap bean as they were previously (Mary Lamberts, personal communications). Psp was consistently isolated from snap bean samples submitted to the Plant Diagnostic Clinic at the Tropical Research and Education Center of University of Florida. In a preliminary experiment in which the bacterial strains were exposed for 2 h to recommended rates of the copper bactericide Kocide® 3000, a marginal difference was detected in the populations of a Psp strain Xcp2 between the treatment and the water control (Pernezny, unpublished data). Different types of culture agar media and laboratory protocols have been developed to assess sensitivity of bacterial strains to copper (Pernezny et al., 2008). By using casitone-yeast extract (CYE) agar amended with CuSO4, strains of P. cichorii (Swingle) Stapp, the pathogen of bacterial blight in celery (Apium graveolens L.) from seedbeds, were classified as sensitive, moderately resistant, and highly resistant to copper, based on their growth on this agar medium (Pohronezny et al., 1994). In a study of copper resistance of P. syringae van Hall strains from citrus (Citrus spp.) and almond (Prunus dulcis (Mill.) Webb) orchards (Andersen et al., 1991), a similar technique was employed for identifying a range of responses to copper depending on the dosage of copper used. Stall et al. (1986) reported that nutrient agar (NA) amended with 200 mg/mL of CuSO4$5H2O was useful for screening copper resistance among pepper (Capsicum spp.) strains of Xanthomonas campestris pv. vesicatoria (Doidge) Dye (now renamed X. euvesicatoria Jones et al.). In Florida, approximately 97% of X. euvesicatoria strains from commercial pepper fields during 1989e1991 were proven to be copper resistant (Pohronezny et al., 1992). Due to the effectiveness of NA in screening Xanthomonas strains for their sensitivity to copper, the same procedure was utilized to evaluate tomato (Solanum lycopersicum L.) strains of X. perforans Jones et al. (1991) and lettuce strains of X. campestris pv. vitians (Brown) Dye (Pernezny et al., 1995) for their copper sensitivity. The objectives of this study were to (i) determine whether resistance to copper has developed in the strains of Psp from snap bean plants collected during 2009e2010 in commercial production fields in Homestead, Florida, (ii) compare the usefulness of different culture media (NA, glucose nutrient agar (GNA) and CYE agar) in rapid assessment of copper resistance in Psp strains, and (iii) investigate if improved efficacy can be achieved by incorporating mancozeb with a copper bactericide for effective management of halo blight caused by resistant strains of Psp.
2. Materials and methods 2.1. Isolation of bacterial strains, identification, and pathogenicity test Fifteen and thirty-five samples of snap bean (cv. Inspiration)
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with typical symptoms of halo blight on leaves were collected in 2009 and 2010, respectively, from a total of ten commercial snap bean fields near Homestead, FL. The samples were sealed in plastic bags, maintained in a cooler with ice, and brought to the laboratory for processing. The snap bean leaves were washed with tap water to remove soil adhering to the foliage before the leaves were cut into small pieces (~0.5 cm2) containing diseased and healthy tissues. The small pieces of leaves were sterilized in 70% ethanol for 1 min, then in 0.6% sodium hypochlorite for 10 min, and finally rinsed in sterile water. The sterilized leaf tissues were placed on nutrient agar (Thermo Fisher Scientific Inc., Lenexa, KS) plates after they were air dried in a sterile laminar flow hood, and incubated in the dark at 28 C for 2e3 days. Individual bacterial colonies were streaked on NA plates to ensure isolation of pure cultures. White to cream-colored single colonies were selected and saved in 15% glycerol at 80 C for further experiments. Pathogenicity tests were carried out using bean pods and entire snap bean plants. Bacterial cell suspensions were prepared by streaking bacterial strains from ultra-cold storage onto NA plates and incubating at 28 C for 24 h to check for purity, then transferring single colonies to nutrient broth (NB) at 150 rpm overnight. Bacterial cells were washed twice by centrifuging at 3000 rpm each for 10 min in sterile water. The bacterial suspensions were adjusted with sterile water with a spectrophotometer (ƛ ¼ 600 nm, A ¼ 0.22) to 1 108 CFU/mL, then further diluted to 1 107 CFU/mL for inoculation. In the pod inoculation assays, pods of snap bean surface sterilized with 70% ethanol were wounded with a sterilized needle and 50 ml of bacterial suspensions (1 107 CFU/mL) were pipetted on each wound site. Inoculated pods were placed in a tray sealed with a plastic film containing moist tissue paper at the bottom. Wounded pods receiving 50 ml of sterile water served as a non-treated control. The bacterial strains causing water-soaked lesions after 24 h around the wound sites on snap bean pods were saved. For entire plant inoculation, five 3-week old plants of the susceptible snap bean cv. ‘Inspiration’ were used for each isolate. Suspensions (1 107 CFU/mL) of bacterial strains were sprayed onto the leaves of snap bean plants until run-off using a handheld sprayer. Inoculated plants were covered with plastic bags overnight to maintain high humidity, and placed on greenhouse benches for 5e7 days prior to disease observation. The temperatures of the greenhouse was set 25 C with a 14-h photoperiod. Plants sprayed with water served as a non-treated control. Bacterial strains inciting typical symptoms, i.e. small, water-soaked spots and chlorosis on the leaves that rapidly became necrotic lesions, were saved for further identification by 16S rRNA gene sequencing. Each bacterial strain was tested twice. Further confirmation of the identity of the bacterial strains from the pod and entire plant assays was conducted by 16S rRNA gene sequencing using the universal primers 8F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1492R (50 GGT TAC CTT GTT ACG ACT T-30 ) (Eden et al., 1991). 2.2. Sensitivity of Psp exposed to copper hydroxide in liquid culture media Forty-five Psp strains obtained above were evaluated in liquid NB for their sensitivity to copper hydroxide (Kocide® 3000, DuPont Co., Wilmington, DE) over a period of 24 h. The rate of copper hydroxide at 0.55 mg/mL used in this experiment was based on label rates of Kocide® 3000 applied on commercial bean crops. The suspension of Kocide® 3000 was freshly prepared with sterile water at 1.2 mg/mL (equivalent to 0.55 mg/mL copper hydroxide), which was added and suspended in 50 mL of NB in a 125-mL flask. About 0.5 mL of the suspension at 1 106 CFU/mL of each bacterial isolate was added, and the initial bacterial concentration was approximately 1 104 CFU/mL. For each bacterial isolate, 0.5 mL of the
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bacterial suspension added into NB without addition of copper hydroxide served as a non-treated control. Each isolate was tested in three flasks as three replications, and the flasks were incubated at 28 C on a rotary shaker at 150 rpm. The experiment was a completely randomized design. Bacterial samples were collected at 0, 9, and 24 h after incubation and the concentrations of Psp were determined by serial dilutions and pipetting 20 mL of appropriate dilutions on NA plates (three drops for each isolate). Bacterial colonies were counted on plates with less than 50 colonies per drop, and data were expressed as log10 CFU/mL. This experiment was repeated once. The number one was added to all bacterial population data before counts were converted to log10 equivalents (Pernezny et al., 2008). Bacterial populations were recorded as zero when no colonies were detected on plates from samples drawn directly from the undiluted solutions. Log equivalents of bacterial populations from each isolate were subjected to analysis of variance. Means of treatments were separated by Fisher's protected LSD test at P ¼ 0.05 using the Statistical Analysis System (SAS) package version 9.4 (2014, SAS Institute, Inc., Cary, NC). 2.3. Assessment of Psp strains for copper resistance on media agar plates NA amended with 0.5% (w/v) glucose (GNA) (pH 7.0) and CYE agar (1.7 g casitone, 0.35 g yeast extract, 5 g glucose, and 17 g Difco agar per Liter of distilled water) (pH 6.0 and 7.0) were first used for the comparison of bacterial growth with and without copper amendment. Copper sulfate (CuSO4$5H2O) (Fisher Scientific, Fair Lawn, NJ) at appropriate concentrations was prepared by incorporating it from a filter-sterilized solution of CuSO4 into autoclaved media cooled to approximately 50 C. Also, the commercial copper hydroxide bactericide Kocide® 3000 was also added at 1.2 mg/mL into NA and CYE and tested for the growth of bacterial strains in the same manner. A total of 35 strains of Psp from snap bean were tested in this study for sensitivity to copper. Treatments were GNA (pH 7.0), GNA (pH 7.0) þ 200 mg/mL CuSO4$5H2O, CYE (pH 6.0), CYE (pH 6.0) þ 100 mg/mL CuSO4$5H2O, CYE (pH 6.0) þ 161 mg/mL CuSO4$5H2O, and CYE (pH 6.0) þ 200 mg/mL CuSO4$5H2O, CYE (pH 7.0), CYE (pH 7.0) þ 100 mg/mL CuSO4$5H2O, CYE (pH 7.0) þ 161 mg/ mL CuSO4$5H2O, and CYE (pH 7.0) þ 200 mg/mL CuSO4$5H2O. The concentration of 161 mg/mL of CuSO4$5H2O was equivalent to the originally published concentration of 0.64 mM CuSO4 (Andersen et al., 1991). Bacterial cells were taken using a sterile inoculation loop from 3-day-old cultures on NA and streaked on triple plates of each culture medium (Pohronezny et al., 1994). The experiment was a completely randomized design. Plates were incubated at 28 C for 72 h when a visual estimate was made of the growth on copper-containing culture media and expressed as a percentage of growth on control plates without copper amendments (Pernezny et al., 2008), in this case where 0 ¼ 0, 1 ¼ 1e10%, 2 ¼ 11e25%, 3 ¼ 26e50%, 4 ¼ 51e75%, 5 ¼ 76e100%. Psp strains with ratings of 0 were considered sensitive to copper, those with ratings of 1, 2, 3 and 4 were moderately resistant (MR), and those with ratings of 5 were highly resistant (HR). All bacterial strains were evaluated in the same manner for a second time. Results from the agar plate test were compared to the growth of the Psp strains in liquid NB which was used as the criterion to identify the types of agar media that were best suited for studies on copper resistance. The numbers of resistant including highly resistant and moderately resistant, and susceptible Psp strains from the agar plate test, along with the numbers from the liquid NB assays were subjected to analysis of variance (ANOVA). Means of strain numbers from each agar and liquid medium were separated by Fisher's protected LSD test at P ¼ 0.05 using the Statistical Analysis System (SAS) package version
9.4 (2014, SAS Institute, Inc., Cary, NC). 2.4. Efficacy of copper hydroxide and addition of mancozeb for management of halo blight Four experiments were conducted in the greenhouse to assess the efficacy of copper hydroxide (Kocide® 3000, DuPont Co., Wilmington, DE) for the management of halo blight caused by a resistant isolate of Psp with and without addition of mancozeb (Manzate Pro-Stick, United Phosphorus, Inc., King of Prussia, PA). Susceptible snap bean seeds cv. ‘Inspiration’ (experiments 1 & 2) and ‘Pony Express’ (experiments 3 & 4) were planted in 10-cmdiameter plastic pots containing soilless Pro-mix growing medium (Miracle-Gro Lawn Products, Inc., Marysville, OH) and watered daily. A controlled-release fertilizer, Osmocote PLUS (15-9-12; Scotts-Sierra Horticultural Products Company, Marysville, OH), was mixed into the soilless potting Pro-mix medium at a rate of 1.3% (wt/wt) prior to seed planting. Experiments were conducted in an air-conditioned greenhouse with a maximum daytime temperature of 25 Ce28 C and a typical light intensity at bench height of 780 mE/m2/s. Snap bean plants were exposed to natural light with photoperiods of 14 h light and 10 h dark with no supplemental artificial lighting. Copper-resistant Psp strain 28 was grown on NA for 3 days at 28 C. The inoculum of Psp strain 28 was prepared as previously described in pathogenicity tests under 2.1. Bacterial suspensions (1 107 CFU/mL) were applied to plants 17 days after planting through misting both adaxial and abaxial surfaces of the leaves until run-off (approximately 15 of suspension per plant) using a handheld spray bottle. In order to enhance wetting of leaf surfaces and full coverage, the surfactant Silwet-77 (Setre Chemical Co., Memphis, TN) was added to the inoculum suspensions at a final rate of 0.1%. A total of five treatments were included in this experiment: copper hydroxide (Kocide® 3000, 1.2 mg/mL), mancozeb (Manzate Pro-Stick, 0.24 mg/mL), copper hydroxide (Kocide® 3000, 1.2 mg/ mL) þ mancozeb (Manzate Pro-Stick, 0.24 mg/mL), copper hydroxide þ mancozeb [ManKocide®, 1.2 mg/mL], and non-treated water control (CK). Treatments with chemicals were applied 2 weeks after planting (V2 growth stage) by spraying solutions onto the plants until runoff using a handheld spray bottle. Three days after the treatment, snap bean plants were inoculated with the Psp strain 28 as described previously. One week following the inoculation, plants were treated with the chemical compounds for the second time. Disease severity was rated 2e3 weeks after inoculation as the percentage of leaf area covered with bacterial lesions. The experiment was arranged in a randomized complete block design with eight replications for each treatment, each plant in a pot served as a replication within each treatment. The experiment was repeated once. The values of disease severity were transformed by square root, and were analyzed separately for each repeated experiment. Data of disease were pooled for Exp. 1 & 2, Exp. 3& 4 after statistical analysis demonstrated that experiment by treatment interactions were not significant (P 0.05). Means of treatments were separated by Fisher's protected LSD test at P ¼ 0.05 using the Statistical Analysis System (SAS) package version 9.4 (2014, SAS Institute, Inc., Cary, NC). 2.5. In vitro effect of copper hydroxide and mancozeb on populations of copper-resistant Psp strain 28 Suspensions of the treatments used in the efficacy trial were freshly prepared with sterile water, which were added into 50 mL NB in a 125-mL flask. About 0.5 mL of the suspension at 1 106 CFU/ mL of the Psp strain 28 was added and the initial bacterial
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concentration was approximately 1 104 CFU/mL. Three flasks for each treatment including the control served as three replications, and the flasks were incubated at 28 C on a rotary shaker at 150 rpm. The experiment was a randomized complete block design. Bacterial samples were collected at 0 and 24 h after incubation and the concentrations of Psp were determined by serial dilutions and plating of 20 mL of appropriate dilutions on NA plates (three drops for each isolate). Bacterial colonies were counted on plates after 3 days, and data were expressed as log10 CFU/mL. Data analyses were conducted by using SAS as described previously in 2.2. 3. Results 3.1. Isolation and identification of Psp strains from snap bean A total of 45 bacterial strains of white to cream color on NA were obtained from fifty snap bean samples collected in 2009 and 2010 from commercial fields near Homestead, Florida. Thirty-five strains incited water-soaked lesions on inoculated bean pods and typical
11
symptoms of halo blight on leaves of plants in the greenhouse pathogenicity tests. The identity of these strains was further verified as Pseudomonas syringae pv. phaseolicola, the pathogen of halo blight of beans, by 16S rRNA gene sequencing (data not shown). 3.2. Population dynamics of bacterial strains exposed to copper hydroxide After 9-h exposure, populations of eight highly resistant strains, i.e. strains 19, 21, 26, 28, 34, 35, 36, 42, and 45, reached log values of 4.5e5.6, comprising the group with highest populations among 35 strains (Fig. 1A). Those classified as moderately resistant strains, i.e. strains 2, 3, 4, 5, 9, 11, 22, 25, 32, 33, 37, 40, and 44, had log values between 1.0 and 2.0 with an exception of isolate 44, which was not detectable after 9-h exposure (Fig. 1B). However, for those classified as sensitive strains, only three strains, i.e. 6, 8, and 10, had no detected growth after 9-h exposure. Populations of the other four strains (1, 7, 20, and 38) reached log values of 1.8e3.3, even greater than those of moderately resistant strains in this assay.
Fig. 1. Populations of P. syringae pv. phaseolicola strains in NB after (A) 9-h and (B) 24-h exposure to copper hydroxide. Samples of strains 13, 14, 18, 30, 31, and 46 were not taken for assessing cell populations after 9-h exposure.
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S. Zhang et al. / Crop Protection 98 (2017) 8e15
Based on the bacterial population after 24-h exposure to copper hydroxide at the label rate, 20% of Psp strains (7 of 35 strains) were classified as copper sensitive with no bacterial cells detected, 43% (15 of 35 strains) were highly resistant to copper with log values between 7.2 and 8.8, and 37% (13 of 35 strains) were moderately resistant with log values lower than 3.7 (Fig. 1B). The log values of these Pseudomonas strains were in the range of 8.5e9.2 with no addition of copper hydroxide depending on each individual isolate (data not shown). For example, the population of a copper susceptible strain 7 down to a log value of 1.3 after 9-h exposure, and no bacterial cells were detected after 24 h (Fig. 2A). However, the population of a copper resistant strain 28 reached an equivalent level to the untreated control (no addition of copper) after 24-h exposure (Fig. 2B). Strain 37, defined as a moderately resistant strain, reached a log value of 1.8 and 2.4 after 9-h and 24-h exposure, respectively (Fig. 2C).
sensitive (Fig. 1B). Compared to the results in liquid NB, CYE agar plates with pH 7.0 amended with CuSO4 at 161 mg/mL, 200 mg/mL, and copper hydroxide (Kocide® 3000, 1.2 mg/mL) had comparable results in evaluation of Psp strains for resistance to copper (Table 1, Fig. 3). Similar results were also obtained on CYE agar of pH 6.0 with addition of CuSO4 at 161 mg/mL and 200 mg/mL. Other tested agar media, i. e. NA þ copper hydroxide (Kocide® 3000), GNA þ CuSO4 (200 mg/mL), detected significantly lower numbers of Psp strains
3.3. Assessment of Psp strains for copper resistance on agar culture media The sensitivity of Psp strains to copper was dependent on the agar media used in qualitatively visual assays (Tables 1 and 2). In this case, bacterial strains were classified as highly resistant, moderately resistant, and sensitive to copper, based on the growth on media plates. On CYE plates, 17e28% of Psp strains (6e10 of 35 strains) were classified as copper sensitive, and 72e83% (25e29 strains) strains were identified as resistant to copper (Table 1). At pH 7.0, the growth of bacterial strains resistant to copper was similar on agar YCE plates amended with CuSO4 at varying concentrations, i. e. 28, 29, and 27 Psp strains, were copper resistant when the concentration of copper in the media was 100, 161, and 200 mg/mL, respectively, which was not significantly different from the liquid NB. Psp strains of 28, 30, 31, 35, 36, 42, 45, and 46 were consistently rated as highly resistant in the tests on YCE plates. However, among the resistant strains, the number of highly resistant strains differed with copper concentration in the agar media. When concentrations of CuSO4 were 161 and 200 mg/mL, 18 and 12 strains were highly resistant, and 11 and 15 were moderately resistant, respectively. At 100 mg/mL of CuSO4, none of the Psp strains were classified as moderately resistant, and all 28 resistant strains were categorized as highly resistant to copper. Therefore, 100 mg/mL was not included in assays on CYE agar with pH 6.0, which was conducted afterwards. On CYE agar media at pH 6.0, amended with copper of 161 mg/ mL, eight strains were evaluated as highly resistant, 19 were moderately resistant, and eight were sensitive to copper. When the copper concentration was increased to 200 mg/mL, no Psp strains were classified as highly resistant, but 25 strains were assessed as moderately resistant and 10 were sensitive to copper. When copper hydroxide (Kocide® 3000) was added at a label rate (equivalent to 1.2 mg/mL) to NA media, 27 strains were detected to be highly resistant, six were moderately resistant, and two were sensitive to copper. However, on CYE agar media, 10 strains were highly resistant to copper, 16 were moderately resistant, and nine were sensitive. The Psp strains of 14, 18, 28, 30, 31, 35, 36, 42, 45, and 46 were rated as highly resistant in all the tests on NA and CYE agar plates with copper hydroxide. 3.4. Comparison of culture agar media for resistance assessment of Psp strains to copper Based on the bacterial population after 24-h exposure to copper hydroxide at the label rate, 15 of the 35 Psp strains were classified as highly resistant to copper, 13 were moderately resistant, and 7 were
Fig. 2. Population dynamics of representative strains of P. syringae pv. phaseolicola classified as (A) sensitive (strain 7), (B) highly resistant (strain 28), and (C) moderately resistant (strain 37) to copper after 9- and 24-h exposure to copper hydroxide in NB.
S. Zhang et al. / Crop Protection 98 (2017) 8e15
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Table 1 Assessment of 35 strains of P. syringae pv. phaseolicola on agar culture media for their response to copper.a Response to copper
Resistantb HR MR Sensitive
No. of strains resistant or sensitive to copper CYE agar pH 7.0
pH 6.0
CuSO4$5H2O (mg/mL)
CuSO4$5H2O (mg/mL)
100
161
200
161
200
28 bcc 28 a 0d 7b
29 b 18 b 11 bc 6 bc
27 bc 12 bc 15 b 8 ab
27 bc 8 cd 19 ab 8 ab
25 c 0d 25 a 10 a
CYE (7.0) agar þ Kocide® 3000 (1.2 mg/mL)
NA þ Kocide® 3000 (1.2 mg/mL)
GNA þ CuSO4$5H2O (200 mg/mL)
NB þ copper hydroxide (24-h exposure)
26 bc 10 bc 16 b 9 ab
33 a 27 a 6 cd 2 cd
35 a 33 a 2d 0d
28 bc 15 bc 13 bc 7b
a Evaluation of resistance based on visual estimates of growth of Psp strains on copper-containing agar media compared with growth on agar media without copper amendments (control). A visual estimate was made of the growth on copper-containing culture media and expressed as a percentage of growth on control plates without copper amendments, where 0 ¼ 0, 1 ¼ 1e10%, 2 ¼ 11e25%, 3 ¼ 26e50%, 4 ¼ 51e75%, 5 ¼ 76e100%. b Resistance category is a sum of HR and MR isolates. HR ¼ highly resistant (rating ¼ 5), MR ¼ moderately resistant (rating ¼ 1, 2, 3, 4), Sensitive ¼ 0. c In each category, means of strain numbers across all agar and liquid NB media within a cell followed by the same letter are not significantly different (P ¼ 0.05) according to the Fisher's protected LSD test.
Table 2 Efficacy of copper hydroxide and addition of mancozeb for management of halo blight in snap bean caused by copper-resistant strain 28 of P. syringae pv. phaseolicola.a Treatmentb
Product (rate)
Disease Severity (%)c
Copper hydroxide Mancozeb Copper hydroxide þ Mancozeb Copper hydroxide þ Mancozeb Non-treated Control (CK)
Kocide® 3000 (1.2 mg/mL) Manzate Pro-Stick (0.24 mg/mL) Kocide® 3000 (1.2 mg/mL) þ Manzate Pro-Stick (0.24 mg/mL) ManKocide® (1.2 mg/mL) e
3.8 9.1 0.8 2.1 7.1
Exp. 1 þ 2 bd a c b a
Exp. 3 þ 4 6.3 9.1 3.5 4.4 8.7
ab a c c a
a
Snap bean seeds cv. ‘Inspiration’ (experiments 1 & 2) and ‘Pony Express’ (experiments 3 & 4) were planted in plastic pots containing soilless Pro-mix growing medium. Treatments with chemicals were applied at 2 weeks after planting by spraying solutions onto the plants until runoff. Three days after the treatment, the plants were inoculated with the Psp strain 28 (1 107 CFU/mL) by spraying the inoculum onto both surfaces of the leaves until runoff. One week following the inoculation, plants were treated with chemical compounds for the second time. c Disease severity was rated 2e3 weeks after inoculation as the percentage of leaf area covered with bacterial lesions. d The values of disease severity were transformed by square root prior to analysis. The table contains de-transformed values. Means within a column followed by the same letter are not significantly different (P ¼ 0.05) according to Fisher's protected LSD test. b
Fig. 3. Percentage of Psp strains that were resistant, moderately resistant, and sensitive to copper on NA, GNA and CYE agar media.
that were sensitive to copper, compared to liquid NB. 3.5. Efficacy of copper hydroxide and the addition of mancozeb for management of halo blight in snap bean Mancozeb alone did not reduce severity of halo blight in any of
these experiments. However, addition of mancozeb to copper hydroxide significantly (P < 0.05) improved the efficacy of copper hydroxide. The combination of copper hydroxide (Kocide® 3000) þ mancozeb (Manzata Pro-Stick) achieved equivalent or better levels in halo blight management to a commercial product ManKocide®, which is a pre-formulated combination product of
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copper hydroxide and mancozeb. 3.6. In vitro effect of copper hydroxide and mancozeb on populations of copper resistant Psp strain 28 There was no effect of copper hydroxide on populations of the Psp strain 28 compared to the control (Table 3). Mancozeb alone significantly (P < 0.05) inhibited the growth of the strain 28 after 24 h. However, no bacterial cells were detected in the treatment of a mixture of copper hydroxide þ mancozeb or Mankocide®. 4. Discussion Results from this study revealed that 80% of the Psp strains (28 of 35) from commercial snap bean fields in Homestead, Florida were resistant to copper, based on bacterial populations after 24-h exposure in liquid NB media (Fig. 1B). Growth of the Pseudomonas strains on CYE agar at pH 6.0 or 7.0 was similar to that in liquid NB, both amended with an appropriate rate of copper such as 161 mg/ mL CuSo4, suggesting that assays on CYE agar amended with copper can be used for rapid assessment of copper resistance in Psp populations (Fig. 3). The results from greenhouse experiments indicated that addition of mancozeb improved the efficacy of copper hydroxide for management of halo blight on snap bean caused by copper-resistant Psp. Results from this study indicate that CYE agar þ CuSO4 is more appropriate for screening Psp strains for copper resistance than other media such as NA or GNA, based on populations of these strains in liquid nutrient broth after 24-h exposure (Fig. 1B). Screening of Psp on CYE agar broadly classified Psp strains as sensitive, moderately resistant, or highly resistant to copper; whereas, 100% (35 strains) and 94.3% (33 strains) Psp strains were classified as copper resistant on GNA containing CuSO4$5H2O and NA amended with copper hydroxide (Table 1). The classifications in this study corresponded to those from the in vitro population studies based on liquid assays in the current study, which were similar to previous reports from other researchers. CYE media amended with copper was reported to be useful for copper resistance studies among P. syringae strains from fruit trees (Andersen et al., 1991) and ornamentals (Scheck and Pscheidt, 1998). Pohronezny et al. (1994) concluded that CYE agar with copper was extremely useful for screening P. cichorii strains recovered from celery seedbeds. Results from the present study could be very important and useful in disease management in that copper resistance is a major concern for growers because it impacts the efficacy of copper bactericides for management of halo blight. Rapid assessment of Psp strains will aid growers by commercial services or research institutes in making wise decisions on effective disease management. In this study, CYE agar was shown to be the most appropriate one among those evaluated for rapid assessment of Psp for their resistance to copper (Fig. 3). Compared to NB with copper hydroxide (Kocide® 3000), Psp strains exhibited similar responses to copper on CYE agar with exceptions of i) CuSO4$5H2O at 200 mg/mL at pH 6.0, which failed to detect highly resistant strains of Psp, and ii) CuSO4$5H2O at 100 mg/mL at pH 7.0, where no moderately resistant strains were detected. NA, a commonly used medium for bacterial culture, detected a significantly lower numbers of sensitive Psp strains and higher numbers of highly resistant strains. GNA, amended with 200 mg/mL CuSO4$5H2O in this study, failed to detect sensitive Psp strains. Therefore, it is recommended that the intermediate rate of 161 mg/mL be an appropriate rate of CuSO4$5H2O for Psp in screening for copper resistance on CYE agar at pH 6.0 or 7.0. It has been clearly documented that the development of copper resistance in populations of Xanthomonas causing bacterial spot on
Table 3 Populations of copper-resistant P. syringae pv. phaseolicola strain 28 after 24-h exposure to copper hydroxide (Kocide® 3000), mancozeb, and mixture of copper hydroxide þ mancozeb.a Treatment
Log10 CFU/mLb
Non-treated control Copper hydroxide (1.2 mg/mL) Mancozeb (0.24 mg/mL) ManKocide® (1.2 mg/mL) Copper hydroxide (1.2 mg/mL) þ Mancozeb (0.24 mg/mL)
8.84 ac 8.75 ab 5.12 c 0d 0d
a About 0.5 mL of the suspension at 1 106 CFU/mL of strain 28 was added to 50 mL NB containing test chemicals in a 125-mL flask. Three flasks for each treatment, including the non-treated control, served as three replications, and the flasks were incubated at 28 C on a rotary shaker at 150 rpm. b Bacterial samples were collected at 0 and 24 h after incubation, and the concentrations of Psp were determined by serial dilutions and plating appropriate dilutions on NA plates. Bacterial colonies were counted on plates after 3 days, and data were expressed as log10 CFU/mL. The digit one was added to all bacterial population data before counts were converted to log10 equivalents. Bacterial populations were recorded as zero when no colonies were detected on plates from samples drawn directly from the undiluted solutions. c Means within a column followed by the same letter are not significantly different (P ¼ 0.05) according to Fisher's protected LSD test.
tomatoes and peppers is related to frequent use of copper (Pernezny et al., 2008). Copper has been used in Florida for over fifty years, and nearly 100% of the X. euvesicatoria and X. perforans strains were resistant to copper (Pohronezny et al., 1992). All strains of X. campestris pv. vitians causing foliar bacterial disease of lettuce were sensitive to copper because copper was not applied frequently (Pernezny et al., 1995). Snap bean is an economically important vegetable crop in south Florida. Diseases caused by bacterial pathogens on snap bean occur periodically. Usually, only a few sprays of copper bactericides during a growing season in south Florida are made on snap bean for management of bacterial diseases. Surprisingly, in this study, 80% of Psp strains from south Florida were resistant to copper. At this time, the reasons for the discrepancy between copper application frequency and copper resistance development in Psp strains are not known. In recent years, however, more frequent applications of copper bactericides have been made on snap bean in south Florida due to increased prevalence of bacterial diseases (Ken Pernezny, personal communications). During 2009e2010, snap bean growers in Homestead, FL reported that copper bactericides were not as effective in management of bacterial diseases in snap bean as they were previously. In November 2016, halo blight outbreaks occurred in some fields in Homestead, FL due to cool temperatures and several rainy days in late October. As a result, these snap bean plantings were destroyed in order to reduce the bacterial inoculum because no other choices were available for growers for managing the disease. Results from this study indicated that the reduced efficacy of copper bactericides may be, at least in part, due to the development of copper resistance in bacterial populations. Growers should use copper bactericides judiciously to mitigate the widespread appearance of resistance in pathogen populations. In four repeated experiments during this study, copper hydroxide had lower disease severity in three experiments compared to the non-treated control (Table 2). This reduction is not thought to be adequate for successful management of the disease. Addition of mancozeb to copper hydroxide usually resulted in increased mortality of the bacterial pathogen. Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide commonly used as a protectant against a broad spectrum of fungal pathogens on many crops including snap beans (Wells and Fishel, 2011). In an in vitro experiment, no bacterial cells were detected in the copper hydroxide þ mancozeb treatment of the copper-resistant Psp strain 28 (Table 3). This may be due to increases in concentration of Cu2þ
S. Zhang et al. / Crop Protection 98 (2017) 8e15
ions in the mixed solution (Marco and Stall, 1983; Menkissoglu and Lindow, 1991), resulting in increased mortality of bacterial cells (Scheck and Pscheidt, 1998). Another possibility is that mancozeb may chelate copper ions, so copper ions become more available at the sites in bacterial cells that are detrimentally affected by copper (Medhekar and Boparai, 1981). Conover and Gerhold (1981) first reported that a tank mix of copper and mancozeb was more effective in management of bacterial diseases on vegetable crops than copper alone. In the greenhouse experiments of this study, neither copper hydroxide nor mancozeb alone adequately reduced halo blight disease caused by the copper-resistant Psp strain 28. However, in vitro toxicity of mancozeb to Psp was detected in this study after 24-h exposure in liquid NB (Table 3). This finding is in agreement with Colin and McCarter (1983) who reported that mancozeb was toxic to strains of P. syringae. It is likely that the zinc cation, part of the coordinated moiety of mancozeb, is involved in mortality of Pseudomonas cells. Acknowledgments We thank Xiaodan Mo and Jiebin Guo for their technical assistance, and Xiaohui Fan for his help with the statistical analysis. This study was funded in part by DuPont Co., Wilmington, DE. References Adaskaveg, J.E., Hine, R.B., 1985. Copper tolerance and zinc sensitivity of Mexican strains of Xanthomonas campestris pv. vesicatoria, causal agent of bacterial spot of pepper. Plant Dis. 69, 993e996. Andersen, G.L., Menkissoglou, O., Lindow, S.E., 1991. Occurrence and properties of copper- tolerant strains of Pseudomonas syringae isolated from fruit trees in California. Phytopathology 81, 648e656. Arnold, D.L., Lovell, H.C., Jackson, R.W., Mansfield, J.W., 2011. Pseudomonas syringae pv. phaseolicola: from ‘has bean’ to supermodel. Mol. Plant Pathol. 12, 617e627. Burkholder, W.H., 1930. The bacterial diseases of the bean: a comparative study. Memoirs, Cornell Univ. Agric. Exp. Stn. 127, 1e88. Colin, K.C., McCarter, S.M., 1983. Effectiveness of selected chemicals in inhibiting Pseudomonas syringae pv. tomato in vitro and in controlling bacterial speck. Plant Dis. 67, 639e644. Conover, R.A., Gerhold, N.R., 1981. Mixtures of copper and maneb or mancozeb for
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control of bacterial spot of tomato and their compatibility for control of fungus diseases. Proc. Fla. State Hortic. Soc. 94, 154e156. Eden, P.A., Schmidt, T.M., Blakemore, R.P., Pace, N.R., 1991. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA- specific DNA. Int. J. Syst. Bacteriol. 41 (2), 324e325. Jones, J.B., Woltz, S.S., Jones, J.P., Portier, K.L., 1991. Population dynamics of Xanthomonas campestris pv. vesicatoria in tomato leaves treated with copper bactericides. Phytopathology 81, 714e719. Marco, G.M., Stall, R.E., 1983. Control of bacterial spot of pepper initiated by strains of Xanthomonas campestris pv. vesicatoria that differ in sensitivity to copper. Plant Dis. 67, 779e781. Medhekar, S., Boparai, K.S., 1981. Fungicidal bis (1-amidino-Oethylisourea) copper (II) carbamates. J. Agric. Food Chem. 29, 421e422. Menkissoglu, O., Lindow, S.E., 1991. Relationship of free ionic copper and toxicity to bacteria in solutions of organic compounds. Phytopathology 81, 1258e1263. Montesinos, E., Vilardell, P., 2001. Effect of bactericides, phosphonates and nutrient amendments on blast of dormant flower buds of pear: a field evaluation for disease control. Eur. J. Plant Pathol. 107, 787e794. Pernezny, K., Nagata, R., Havranek, N., Sanchez, J., 2008. Comparison of two culture media for determination of the copper resistance of Xanthomonas strains and their usefulness for prediction of control with copper bactericides. Crop Prot. 27, 256e262. Pernezny, K., Raid, R.N., Stall, R.E., Hodge, N.C., Collins, J., 1995. An outbreak of bacterial spot of lettuce in Florida caused by Xanthomonas campestris pv. vitians. Plant Dis. 79, 359e360. Pohronezny, K., Sommerfeld, M.L., Raid, R.N., 1994. Streptomycin resistance and copper tolerance among strains of Pseudomonas cichorii in celery seedbeds. Plant Dis. 78, 150e153. Pohronezny, K., Stall, R.E., Canteros, B.I., Kegley, M., Datnoff, L.E., Subramanya, R., 1992. Sudden shift in the prevalent race of Xanthomonas campestris pv. vesicatoria in pepper fields in southern Florida. Plant Dis. 76, 118e120. Ritchie, D.F., Dittapongpitch, U., 1991. Copper-and-streptomycin resistant strains and host- differentiated races of Xanthomonas campestris pv. vesicatoria in North Carolina. Plant Dis. 75, 733e736. Scheck, H.J., Pscheidt, J.W., 1998. Effect of copper bactericides on copper resistant and sensitive strains of Pseudomonas syringae pv. syringae. Plant Dis. 82, 397e406. Schwartz, H.F., Steadman, J.R., Hall, R., Forster, R.L., 2005. Compendium of Bean Diseases, second ed. The American Phytopathological Society Press, St. Paul, pp. 49e50. Stall, R.E., Loschke, D.C., Jones, J.B., 1986. Linkage of copper resistance and avirulence loci on a self-transmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology 76, 240e243. USDA- NASS-census of Agriculture, 2012. Available at: https://www.agcensus.usda. gov/Publications/2012/index.php (Accessed 15 October 2016). Wells, B., Fishel, F.M., 2011. Agricultural Pesticide Use in Florida: a Summary, 20072009. http://edis.ifas.ufl.edu/pi235 (Accessed 15 October 2016).