Management of bacterial canker of tomato caused by Clavibecter michiganensis subsp. michiganensis (Smith and Davis et al.) using different antibiotics

In an attempt to control bacterial canker of tomato, a very hard-to-control disease, both in-vitro and in-vivo studies were conducted using antibiotics alone or in combinations with other antibiotics and with copper oxychloride. Using paper disc diffusion assay and 50 ppm 400 ppm concentrations, tetracycline+streptomycin produced the largest inhibition zone (22.8 mm) followed by tetracycline alone (22.0) at highest concentration after 48 hrs incubation. The result of antibiotic combination streptomycin and gentamicin were statistically at par with those of tetracycline+streptomycin combination. However, streptomycin alone proved to be ineffective. Antibiotic concentrations of 250 ppm and higher were sufficient to inhibit the in-vitro bacterial growth. In case of screen-house studies, combination of tetracycline and copper oxychloride gave minimum % disease severity (5.40%), as compared to inoculated but untreated control in which case the % disease severity was 56.6 %. The same combination (i.e. tetracycline+copper oxychloride) also proved to be more effective than other treatments in term of enhancing yield per plant (1147.7g), plant height (102.0 cm), plant dry weight (130.8g) and other parameters. The better performance of this combination treatment could be attributed to its interactive effect. This study suggested that combination of antibiotics should be used for proper disease management and further combinations of different antibiotics in different concentrations should be evaluated.


Introduction
Tomato, Lycopersicon esculentum, belonging to family Solanaceae is a popular vegetable [1]. It is the second most consumed vegetable in the world behind potato. Tomato is an excellent source of phosphorus, iron and vitamin A, B and C. They also contain small amounts of the B complex vitamins; thiamin, niacin riboflavin [2]. Tomato fruit contain 93% water which provides conducive environment to various microorganisms including bacterial canker pathogen. Bacterial canker has been a serious problem all over the world. The causal organism of bacterial canker is Clavibacter michiganensis subsp. michiganensis (Cmm) [3].The color of the bacterial colonies when grow on nutrient agar medium could be yellow, white and pink with the most virulent forms being yellow and white [4]. On nutrient agar, the colonies are smooth but become butter like, with continued sub culturing, pale yellow becoming deeper yellow, cloudy and glistening [3]. Common name of the disease is bacterial canker. Other names used to describe bacterial canker include bird's eye spot, stem rot and stem canker. The first symptoms of bacterial canker are chlorosis (yellowing), curling and wilting of the leaflets, often on one side of the leaf. The affected leaflets become necrotic and dry but remain attached to the plant. Long brown strips may appear on stems and shoots. These strips dry and crack open to form cankers from which the disease gets its name [5]. The most distinctive symptoms of bacterial canker are fruit lesion called bird's eye lesion and the vascular discoloration at the junction between the stem and the petiole. At later stage, longitudinal cankerlike openings appear on stem, petiole and midrib resulting in stunting of plants . The bacterium is soil inhabitant and also remains in seed coat. Invasion of the cotyledons happens through stomata. The organism, after getting established in the phloem invades cortex and cause formation of lysogenous cavities [6]. Management of bacterial diseases depends mostly on "host resistance, sanitation (e.g., preventing the introduction of pathogens and removing diseased plants) and cultural practices (e.g., avoiding overhead irrigation and limiting nitrogen fertilization)". In some cases, chemical bactericides (e.g., copper compounds and antibiotics) and biological control agents are successfully integrated into the disease management program. Bacterial canker may be avoided by using certified plant material [7] or applying effective antibiotic or copper compounds in the greenhouse and the field [8].
Unfortunately no commercial varieties with resistance to C. michiganensis subsp. michiganensis have been developed to date [9]. Because of being systematic and effective, various antibiotics are used for control of many plants bacterial diseases. Antibiotics are produced by bacteria, fungi, actinomycetes and to a limited extent by other groups of microorganism [10]. Antibiotics are neither disinfectants nor antiseptic but they are selective antimicrobial agents applied to living tissue to kill or prevent the growth of susceptible microorganisms. The "selective action of antibiotics upon bacteria and other microorganisms" is known as the antibiotic spectrum. Some antibiotics are categorized as very narrow spectrum, whereas others have a wide range of activity. Some are active only against certain bacteria and not upon others, whereas some are active against fungi too. The most commonly used plant antibiotics are oxytetracycline, streptomycin, kasugamicine and gentamicin. Although bacterial canker is an important disease of tomato in KPK, no significant amount of research work has been on this disease so far. As the use of antibiotics as tomato transplant dip could be easily integrated in Cmm integrated disease management (IDM) package, therefore we were interested in investigating the role of these chemicals in Cmm management. The research was carried out to test the efficacy of different antibiotics on the inhibition of the in-vitro growth of Cmm and to investigate the effect of selected antibiotics on the control of bacterial canker under screen-house condition. Materials and methods Source of bacterial culture and pathogenicity test Already identified polymerase Chain Reaction (PCR) culture of Clavibacter michiganense subsp. michiganenses (Cmm) was obtained from the culture bank of the Department of Plant Pathology, The University of Agriculture Peshawar. For the confirmation of Cmm pathogen, pathogenicity test was carried out. Bacterial suspension, having 10 9 cfu/ml, was prepared from 2 days old bacterial culture. Thirty day old seedlings were transplanted into earthen pots having pore at bottom (to facilitate air exchange and avoid water accumulation) and were injected using hypodermic medical syringe at crown region of tomato plant. Shoot(s) and leaves showed downward turning (wilting) indicated a systemic infection. Bacterial pathogen was reisolated from wilted shoot on YDM medium and identified as Cmm.

In-Vitro
antibacterial efficacy of antibiotics A total of three antibiotics and their combinations viz streptomycin, tetracycline and gentamicin were tested for their ability to retard the growth (produce inhibition zones) of the bacterium (Cmm) on PDA under in vitro conditions [11] . Each antibiotic was tested at 8 different concentration levels (50 ppm, 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 400 ppm).Two factors (antibiotics and their concentrations) in CRD, five replicates and one paper disc (6mm) per treatment per replicate was used. For testing the antibacterial efficacy of different antibiotics and their concentrations paper disc diffusion method (PDM) was applied [12] . Bacterial lawn was first prepared on by spreading bacterial suspension (approximately 10 9 cfu/ml) on PDA (100 ul/plate) by sterile bent rod. These plates were then used for invitro tests involving bacterial growth inhibition by different antibiotics. Sterilized filter paper discs (6 mm, Advantec Tokyo , Tokyo Roshi Kaisha Ltd, Japan) were soaked in each ppm of antibiotics solutions for 15-20min, dried and then placed aseptically on pre-inoculated (for the production of bacterial lawn) PDA plates. Almost 10ul antibiotic concentration was absorbed by each disc. Disc soaked in sterile distilled water served as negative control. Plates were incubated at 28+20C. The bacterial inhibition zones (mm) around different discs were recorded after 24 and 48 hrs.

In-Vivo
antibacterial efficacy of antibiotics and copper fungicide Total 8 treatments and 3 concentrations (200 ppm, 250 ppm and 400 ppm) were used in screen house experiment. T1=Tetracycline, T2=Streptomycin, T3=Tetracycline + streptomycin, T4=Streptomycin+ copper oxychloride (COC), T5=Tetracycline+ copper oxychloride (COC), T6=Tetracycline+ Streptomycin + copper oxychloride (COC), T7= Copper oxychloride (COC) and T8= Control (sterilized distilled water).Tomato variety used was Rio Grande. Tomato nursery was raised in earthen plates filled with chemically sterilized soil (deconil @ 2g/kg). About 3 weeks old tomato seedling (one plant/pot) were transplanted into pots (12 inches, diameter) having chemically sterilized soil. Antibiotics and their concentration (ppm) that gave promising results in in-vitro tests (tetracycline and streptomycin), copper fungicide (Copper oxychloride) and their combinations were tested for their effect against Cmm under screen house conditions. Plants were sprayinoculated as well as leaf clip-inoculated by using contaminated scissors. Inoculated plants without antibiotics were included as a control. Experiment was arranged in two factors factorial experiment in CRD layouts with five replicates per treatment in screen house. Plants were uprooted after termination of experiment and data on disease severity, yield and yield parameters were taken after two months of inoculation.

Inoculation of plants
Bacterial culture grown over night on LB was scrapped off of plates and suspended in 0.85% saline (NaCl) solution or sterilized distilled water. Concentration was adjusted to 10 9 cfu/ml using spectrophotometer, OD600 = 1.0 [13] . Bacterial suspension was sprayed by using common sprayer on tomato transplant at surface run-off level.The selected antibiotics and their different concentrations (ppm) were also applied to inoculated plants by using spray method. Copper is a contact fungicide so it was sprayed before inoculation of tomato plants.
Water was used as a negative control. All other antibiotics and their combinations were applied after inoculation. Four sprays were done with the intervals of 15 days.

Data parameters and statistical analyses
Data on disease severity was recorded at the interval of 15 days and total 4 sprays were done. Disease severity was recorded on each plant according to the scale of [14] i.e. 0 = No disease (healthy plant no symptoms), 1 = Less than 50% of the plant had symptoms (wilting, chlorosis of lower leaves and brown spots on leave), 2 = More the 50% of the plant had symptoms (wilting, chlorosis of upper leaves and lower leaves, marginal necrosis and canker), 3 = Plant completely dead. Disease severity was converted to % by using the formula [15]. S=∑n/3N, where S=severity (%), ∑n=summation of individual rating, 3=highest scale and N=total no. of plants/leaves. Yield per plant (g) was recorded by taking four pickings from each tomato plant. Weight of all fruit per plant was measured. In screen house height in cm of individual plant was measured after 60 days of transplantation. Numbers of shoots were also measured from ground to upward of each plant after 60 days of transplantation. After termination of experiment plants were uprooted, washed and plant fresh weight (g), plant dry weight (g) and dry root weight (g) of individual plant was recorded by using electric balance. In case of plant and root dry weight they were dried under sunlight for 10 days. Data were analyzed by using statistix software 8.1. CRD design with 2 factorial arrangements was used. Difference between the treatments was found by using Fisher's protected Least Significant Difference test (LSD) at P<0.05.

In-Vitro
Antibacterial Efficacy of Different Antibiotics against Bacterial Canker of Tomato Different antibiotics and their concentrations were evaluated for their ability to inhibit the in-vitro growth of Cmm. Antibiotics and their concentrations significantly (P<0.05) retarded the growth of Cmm. The largest inhibition zone was produced by tetracycline+streptomycin (22.8mm) followed by tetracycline (22.0 mm) and streptomycin+gentamicin (21.20mm) at 400ppm. Smallest inhibition zone was produced by streptomycin with 11.10 mm at 400ppm. All antibiotics showed progressive increase as concentrations increased from 50 ppm to 400 ppm. Although streptomycin showed less effective results but when streptomycin was used in combination with tetracycline, it gave good results. Combination of streptomycin with gentamicin also gave significant results. However, because of being expensive gentamicin was dropped. This antibiotic irreversibly binds to ribosomal protein and interferes with the movement of the ribosome along the mRNA. As a consequence, streptomycin makes protein synthesis less accurate or blocks the synthesis of protein [21][22][23]. Tetracycline (oxytetracycline) on the other hand, reversibly binds to bacterial ribosome and interferes with the loaded tRNA (containing an amino acid) coming into the ribosome. As a result, protein synthesis remains blocked for as long as the antibiotic is bound to the ribosome [16]. Long term use of bactericides, as expected, may lead to the appearance of antibioticresistance strains. Therefore, plant bacterial management programs that are not wholly dependent on bactericides are desirable [8]. Streptomycin resistance has been found to occur in plant pathogens but no tetracyclineresistance plant pathogenic bacteria have been reported yet [24]. Two mechanisms for resistance to streptomycin have been reported in antibiotic-resistant strains. The most common mechanism is the spontaneous mutation of the chromosomal gene rpsl, which encodes the production of ribosomal protein. As a result of this mutation, streptomycin is un-able to bind to the mutated ribosomal protein. The second mechanism involves the inactivation of streptomycin by bacterial phosphotransferase, an enzyme produced by strA and strB gene. The genes strA and strB usually reside on mobile genetic elements (transposons) and have been identified in at least 17 environmental and clinical bacteria populating diverse niches. This explains why streptomycin resistance is so frequent in plant pathogen [21,22]. In order to discourage or delay the development of antibiotic-resistance in plant pathogens, antibiotics are used in combinations with each other as well as copper oxychloride or other coppercontaining chemicals. The results of our study indicated that disease severity was significantly reduced and yield and yield parameters were improved when bactericides were used in combinations. It is quite common and easy for plant pathogenic bacteria to have point mutations and develop resistance against antibiotics when they are used alone over a period of time. However, in order to develop resistance against more than one chemical (used in combination) bacteria have to develop multiple mutations at the same time which is not easy.

Conclusion
In our study some of the antibiotic concentrations proved to be very significant in terms of reducing % disease severity of bacterial canker of tomato and enhancing yield and yield parameters. However, these combinations were used only as sprays.