Impact of chemicals, plant extracts and their combination on bacterial blight of cotton Muhammad Sajid Corresp., 1 , Shahabaz Talib Sahi 2 , Muhammad Atiq 1 , Muhammad Rizwan Bashir 3 , Hafiz Arslan Anwaar 2 1 2 3 2 , Muhammad Abid Corresp., 1 , Rashida Perveen Department of Plant Pathology, Faculty of Agricultural Sciences and Techology, Bahauddin Zakariya University, Multan, Pakistan Deparment of Plant Pathology, University of Agriculture Faisalabad, Pakistan Oilseeds Research Institute, Ayub Agriculture Research Institute Faisalabad, Pakistan Corresponding Authors: Muhammad Sajid, Muhammad Abid Email address: sajid1694@gmail.com, muhammad.abid@bzu.edu.pk Five chemicals, incuding Flare, Plant Protector, Mancozeb, Agrimycine, and Copper oxychloride, and five plant extracts including N. tabacum, A. indica, M. oleifera, D. alba and C. longa were evaluated against bacterial blight of cotton caused by Xanthomonas citri pv. malvacearum (a bacterium). The impact of chemicals and plant extracts on bacterial development was tested in laboratory while on disease reduction was tested in green house and field experiments. Laboratory experiments showed that maximum inhibition zone of bacterial growth was expressed by Flare (1.693cm) at all concentrations followed by Plant Protector (1.473 cm), Mancozeb (1.290 cm), Agrimycine (1.150 cm) and copper oxy-chloride (0.953) cm respectively while in case of plant extracts maximum inhibition was expressed by N. tabacum (0.650 cm) followed by A. indica (0.486), M. oleifera (0.350), D. alba (0.256 cm) and C. longa (0.168 cm). Green house experiment revealed that the best result was produced by the combination of Flare and N. tabacum by indicating lowest disease incidence (32.27%) at all the tested concentration. Same results were obtained in field experiment, where the lowest disease incidence (40.41%) was recorded when the ,Flare and N. tabacum were applied in combination although it was higher then green house. This study concludes that N. tabacum and Flare are better option against bacterial disease development and even their combination is more significant lowering the bacterial blight disease incidence on cotton. Selection of suitable formulation and method of application could be the future aspects of plant product especially N. tabacum related research. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2900v1 | CC BY 4.0 Open Access | rec: 1 Apr 2017, publ: 1 Apr 2017 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Title: Impact of chemicals, plant extracts and their combination on bacterial blight of cotton Short Title: Management of Bacterial blight of cotton Muhammad Sajid1,*, Shahbaz Talib Sahi2, Muhammad Atiq2, Muhammad Abid1, Rashida Parveen1, Muhammad Rizwan Bashir3, Hafiz Arslan Anwaar2 1Department of Plant Pathology, Bahauddin Zakariya University Multan, Pakistan 2Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan 3Oilseeds Research Institute, Ayub Agriculture Research Institute Faisalabad, Pakistan *Corresponding author:: Muhmmad Sajid, Email: sajid1694@gmail.com ABSTRACT Five chemicals, incuding Flare, Plant Protector, Mancozeb, Agrimycine, and Copper oxychloride, and five plant extracts including N. tabacum, A. indica, M. oleifera, D. alba and C. longa were evaluated against bacterial blight of cotton caused by Xanthomonas citri pv. malvacearum (a bacterium). The impact of chemicals and plant extracts on bacterial development was tested in laboratory while on disease reduction was tested in green house and field experiments. Laboratory experiments showed that maximum inhibition zone of bacterial growth was expressed by Flare (1.693cm) at all concentrations followed by Plant Protector (1.473 cm), Mancozeb (1.290 cm), Agrimycine (1.150 cm) and copper oxy-chloride (0.953) cm respectively while in case of plant extracts maximum inhibition was expressed by N. tabacum (0.650 cm) followed by A. indica (0.486), M. oleifera (0.350), D. alba (0.256 cm) and C. longa (0.168 cm). Green house experiment revealed that the best result was produced by the combination of Flare and N. tabacum by indicating lowest disease incidence (32.27%) at all the tested concentration. Same results were obtained in field experiment, where the lowest disease incidence (40.41%) was recorded when the ,Flare and N. tabacum were applied in combination although it was higher then green house. This study concludes that N. tabacum and Flare are better option against bacterial disease development and even their combination is more significant lowering the bacterial blight disease incidence on cotton. Selection of suitable formulation and method of application could be the future aspects of plant product especially N. tabacum related research. Key words: Bacterial blight, Cotton, Xanthomonas citri pv. malvacearum,Chemicals, Plant extracts, Flare, Nicotiana tabacum 1- INTRODUCTION 39 Cotton (Gossypium hirsutum) is the most important fiber crop of Pakistan which plays a 40 significant role in the economy of country. It is grown in temperate and subtropical regions of 41 the world including Pakistan (Smith, 1999). It is cultivated on an area of 33.1 million hectares in 42 the world while on 3.0 million hectares in Pakistan during 2013-14 with the production of 116.7 43 and 9.5 million bales respectively (Johnson et al., 2014). Its present area, production and yield in 44 the world depicted that Pakistan is the fourth largest producer of cotton after China, USA and 45 India (Hanif and Jafri, 2008). Cotton is unique among agricultural crops because it provides 46 food, edible oil, fiber and other byproducts for livestock food (Chaudhry and Guitchounts, 2003). 47 Bacterial blight of cotton caused by Xanthomonas axonopodis pv. malvacearum (Xam) is one of 48 the serious diseases of cotton (Saha et al., 2001) causes 26- 30% yield losses in different cotton 49 growing areas of the world (Ramapandu et al., 1979; Chidambaram and Kannan, 1989; 50 Chattannavar et al., 2006). About 37- 40% yield losses were observed in Pakistan, Faisalabad 51 district (Bhutta and Bhatti 1983; Khan et al., 1999). This bacterium enters in healthy plants 52 through stomata or wounds, initiates infection process during any stage of the growth period and 53 producing typical symptoms including small, irregular and dark water socked spots on lower 54 epidermis of leaves that later becomes dark brown (Liberato et al., 2007). 55 Use of resistant varieties is genuine method for the management of disease because it makes 56 possible to avoid other management strategies like acid-delinting of seed, use of chemicals and 57 the destruction of diseased plant residues followed by tillage operations (Thaxton and El-Zik, 58 2001; Turkkan and Dolar, 2009). Many other stratigies that are used for the management of 59 bacterial blight of cotton are being applied as an alternate source of chemicals for the 60 management of disease but absence of durable resistance in varieties, treatment with chemicals is 61 recommended quick action and readily availability (Singh et al., 2007). Numerous reports on the 62 use of active ingredeints from plants in place of chemicals are available owing to there non- 63 phytotoxic nature, more systemic and easily biodegradable behavior (Gottlieb et al., 2002). This 64 led to screen out a large number of plants for antibacterial activity against important seed borne 65 phytopathogenic Xanthomonas pathovars under in vitro and in vivo conditions, with an ultimate 66 aim of developing plant based formulations for disease management (Kiran and Raveesha, 67 2006). Although chemicals are easily available, direct and rapid in action which consiquently 68 reduce the losses caused by this disease but the continuous dependence on pesticides has proven 69 increasingly unsuitable by causing environmental pollution and degradation due to their 70 judicious use. Therefore, it is dire need to conduct in-vitro and in-vivo and research for an apt 71 management of bacterial lignt of cotton through chemicals and plant extracts. The current 72 research was conducted with an ultimate objective of integrated management of this disease, to 73 produce the optimum crop yield of high quality at minimum cost as well as to preserve the 74 environment. 75 76 77 78 2-MATERIALS AND METHODS 2.1- Isolation of X. citri pv. malvacearum 79 Cotton leaves showing typical symptoms (water soaked lesions) of bacterial blight were 80 collected from experimental area, Department of Plant Pathology, University of agriculture 81 Faisalabad. For isolation of Xcm, diseased portion of leaves were cut into small pieces (1×1 cm), 82 surface sterilized with 0.1% mercuric chloride (HgCl2) solution and then washed with distilled 83 water. Leaves were crushed with the help of pestle and mortar and distilled water was added in it 84 for preparation of bacterial suspension contained 107cfu ml-1 Then bacterial suspension was 85 transferred to nutrient agar media (NA) with inoculating loop (Nichrome, Qty/pk-12). All the 86 plates were incubated (Drucker 614B) at 28oC for 3 days. After 3 days, bacteria produced round 87 colonies of yellow color on nutrient agar medium. Bacterial colony was examined under 88 stereoscope (OLYMPUS, SZX-ILLB2-200) and was identified on the basis of morphological 89 characteristics (size, shape, texture and colony color). 90 2.2- Pathogenicity test 91 To fulfill Koch’s postulates, seeds of susceptible variety (Bt-FH 142) of cotton were sown in 92 pots (12×9 cm dia.) which were filled with sterilize soil (2kg/pot) and these pots were transferred 93 to green house by adopting CRD Design. Recommended agronomic practices were followed 94 time to time and pots were watered once in a day. Plants at the age of 5-6 weeks were inoculated 95 by using sterilized syringe (24 Gauge needle size) filled with 20 μl (local isolates) bacterial 96 suspension, contained 107-cfu ml-1, which were measured by using colony counter (SUNTEX 97 560). Suspension was injected in midrib of leaves until the leaf became water soaked. In control 98 treatment, only sterilized water was injected. After development of disease, bacterium was re- 99 isolated from diseased leaves after one week of inoculation. Morphological characteristics (size, 100 shape, texture and color of colony) of re-isolated bacterium were compared with bacterial culture 101 that was used for inoculation. Re-isolated bacteria expressed exactly same colony characteristics 102 as that of original culture. 103 2.3- Laboratory evaluation of different chemicals against X. citri pv. malvacearum 104 Five chemicals i.e Flare, Agrimycine, Copper oxychloride, plant protector and Mancozeb were 105 used in the research Table-1.These chemicals were evaluated against the growth of Xcm by using 106 inhibition zone technique (Berry et al., 1979) under complete randomized design with three 107 replications. Each chemical was tested at 0.25%, 0.30% and 0.35% concentrations. Bacterial 108 culture was multiplied by adding freshly growing aqueous bacterial suspension (1×107 cfu/ml) 109 to the Luke warm NA media in a flask (ASTM- E288). It was shaken well and poured in plates 110 (90×15 mm). These plates were wrapped with clingfilm and incubated at 30°C. When bacterial 111 colonies were formed, wells of 6 mm were made by using sterilized cork borer at the center of 112 plate. Chemical solutions of requisite concentrations were poured in petriplates with disposable 113 sterile syringe (Glass and PTFE, 1050 model, 22 gauges) and were again wrapped with clingfilm 114 and incubated at 30°C. The plates with no chemicals and only having 20 μl sterile water were 115 considered as control against treatments. Data regarding inhibition zone was recorded after three 116 days of interval. 117 Table 1 Chemical with their active ingredients used against bacterial blight of cotton Common Name Active ingredient Concentration Company Flare Streptomycin sulphate 72% w/w Kanzo Ag (Pak.) Mancozeb Ethylene Bisdithiocarbamate 88.23% w/w Dow Agro Sciences (Pak.) Copper oxychloride Copper oxychloride 850g/kg Agri Star (Pak.) Agrimicin Streptomycin sulphate 75ml/L Nufarm (Pak.) Plant protector Benzoic acid 80% w/w Top Farmers (Pak.) 118 119 120 121 2.4- Laboratory evaluation of different plant extracts against X. citri pv. malvacearum Preparation of plant extracts Five plants extracts i.e. Nicotiana tabacum, Azadirachta indica, Moringa oleifera, Datura alba 122 and Curcuma longa) were used in this experiment Table-2. Anti-bacterial efficacy of these five 123 plants extracts was evaluated against bacterial colony growth. Fresh leaves of each plant were 124 taken at flush stage, thoroughly washed with tap water and sun dried for two days. When leaves 125 giving brittle appearance, were grinded by using electric grinder (AG014, MAKUTE). Powder 126 (25 g) of each plant leaves was taken, dissolved in 100 ml of acetone solvent and was mixed 127 thoroughly by using electric stirrer (R30, UET Mixer) which then poured into plastic tubes and 128 centrifuged (Dawlance, 9170 WB) at 6000 rpm for 5 minutes. After centrifugation, supernatant 129 was taken out with the help of pipette (Nichiprt EXII, E13319791) and passed through filter 130 paper (WhatmanNo.1). The extracts were arbitrarily considered as standard stored at -4oC and 131 used further in experiments. For Turmeric, 100g piece of turmeric bulb was taken and washed 132 thoroughly in water, macerated well in mortar and pestle in 100ml of distilled water. Mixture 133 was centrifuged (Dawlance, 9170 WB) at 9000 rpm and extract was separated. Five plant 134 extracts i.e., N. tabacum, A. indica, M. oleifera, D. alba and C. longa were evaluated against 135 Xcm by using inhibition zone technique (Berry et al., 1979) at 10, 15 and 20% concentration. 136 Bacterial culture was multiplied by adding the freshly growing aqueous bacterial suspension to 137 Luke warm NA media in a flask (Erlenmeyer, GW-11). It was shaken well and poured in petri 138 plates (90 × 15 mm). These plates were wrapped with clingfilm and incubated (SANYO, 175 M) 139 at 30°C. After solidification of culture media, wells of 6 mm dia. were made by cork borer at the 140 center of the plate. Extracts of each plant with three concentrations were poured in the wells with 141 sterilized disposable syringe (Glass and PTFE, 1050 model). Overflowing was strictly avoided. 142 In control treatment, only sterile water was poured. Petri plates were carefully wrapped with 143 clingfilm and dispensed for 24 hours in refrigerator (Dawlance, 9170 WB) at (4°C). After 144 dispensing, plates were incubated (SYNYO, 175M) at 300C. Experiment was conducted in 145 Completely Randomized Design (CRD). Each treatment was replicated thrice. Data was 146 recorded by measuring radius of Inhibition zones after 24, 48 and 72 hours. 147 148 Table 2 Plants extracts used for their antibacterial potential against bacterial blight of cotton Common Name Botanical Name Sohanjna Moring oleifera Neem Azadirachta indica Datura Datura alba Tobacco Nicotiana tabacum Turmeric Curcumba longa Active ingredients Saponins (Abalaka et al., 2012) Nimbine (Terpenoids) (Lokanadhan et al.,2012) Scopolamine (Okwu and Igara, 2009) Nicotine (Bakht et al.,2012) Curcumin (Moghadamtousi et al., 2014) Plant Part used Authority Leaves Lamarck Leaves A. Jussieu Leaves C. Linnaeus Leaves C. Linnaeus Roots C. Linnaeus 149 150 151 2.5- Evaluation of Flare and N. tabacum against bacterial blight of cotton in greenhouse In labarotary, Flare and N. tabacum expressed the most effective results against Xcm. So these 152 were also evaluated in greenhouse conditions to check their efficacy against bacterial blight of 153 cotton. For this purpose seeds of susceptible variety (Bt-FH 142) with five replications, were 154 sown in pots (2 plants/pot) which were partially filled with sterilized loamy soil (2kg/pot). These 155 plants were artificially inoculated at the age of 6-7 weeks through syringe method by using20 μl 156 (local isolates) bacterial suspension, contained 107cells/ml. Flare and N. tabacum alone and in 157 combination, were sprayed against disease, with three replication under complete randomized 158 design (CRD) in greenhouse, Department of Plant Pathology, University of Agriculture, 159 Faisalabad. Control plants were sprayed with distilled water only. Data regarding disease 160 incidence was recorded after seven, fourteen and twenty one days of application 161 2.6- Evaluation of Flare and N. tabacum against bacterial blight of cotton in field 162 To evaluate the efficacy of Flare and N. tabacum in field conditions, Seeds of susceptible variety 163 (Bt-FH 142) were sown with 30 cm plant to plant (P×P) and 75cm row to row (R×R) distance 164 under randomized complete block design (RCBD) in research area, Department of Plant 165 Pathology, University of Agriculture Faisalabad. After 5-6 weeks, when typical symptoms of 166 blight appeared on the leaves, (water soaked lesions) treatments i.e. Flare at the rate of 0.75%, 167 0.80% and 0.85% and N. tabacum at the rate of 45%, 50% and 55% alone and in combination 168 were applied. Control plants were sprayed with distilled water only. Each treatment was applied 169 with three replications with one control and each replication contained thirty plants. Data 170 regarding disease incidence was recorded after seven, fourteen and twenty one days of 171 application 172 2.7- Statistical analysis 173 The statistical analysis was performed by using SAS/STAT statistical software version 6 (SAS 174 Institute, 1990). Data was analyzed statistically and means were compared by using Least 175 Significant Difference (LSD) test (Steel et al., 1997). 176 177 3- RESULTS 178 Maximum inhibition zone was expressed by Flare 1.69 cm followed by Plant Protector 1.47cm, 179 Mancozeb 1.29 cm, Agrimycine 1.15 cm and Copper oxychloride 0.95 cm respectively as 180 compared to control (Table 3). In the interaction between treatments and concentrations 181 maximum inhibition zones 1.76 cm was produced by Flare at 0.35% followed by 1.69 cm at 182 0.30% and 1.63 cm at 0.25% concentration respectively. Copper oxy chloride showed minimum 183 inhibition zones 0.90 cm, 0.95 cm and 1.01cm at 0.25%, 0.30% and 0.35% respectively, plant 3.1- Impact of chemicals on X. citri pv. malvacearum growth 184 protector expressed 1.40 cm, 1.47 cm, 1.55 cm, Mancozeb 1.24 cm, 1.29 cm, 1.34 cm. 185 Agrimycine 1.10 cm, 1.17 cm and 1.18 cm inhibition zone at 25 %, 30 % and 35 % 186 concentrations respectively as compared to control (Table.3). 187 188 Table 3. Impact of different chemicals in three different concentrtions on the growth of X. citri pv. malvacearum. Treatments Inhibition zones (cm) Mean Inhibition zones (cm) 0.25% 0.30% 0.35% Flare Plant Protector Mancozeb 1.63c 1.40f 1.24i 1.69b 1.47e 1.29h 1.76a 1.55d 1.34g 1.693a 1.473b 1.290c 189 Agrimycin 1.10k 1.17j 1.18j 1.150d Copper oxy chloride 0.90n 0.95m 1.01i 0.953e Control 0.00 o 0.00 o 0.00 o 0.000f Means in the column and rows with the dirrefent letters are significantly different at (p<0.05) 190 3.2- Impact of plant extracts on X. citri pv. malvacearum 191 Maximum inhibition zone was produced by Nicotiana tabacum 0.65 cm followed by 192 Azadirachta indica 0.48 cm, Moringa oleifera 0.35 cm, Datura alba 0.25 cm and Curcuma 193 longa 0.17 cm as compared to control (Table 4). In the interaction between treatments and 194 concentrations, maximum inhibition zone 0.70 cm was produced by Tobacco at 20 % 0.65 cm at 195 15 % and 0.60 cm at 10 % respectively. C. longa exhibited minimum inhibition zones of 0.18 196 cm, 0.17 cm and 0.15 cm, A. indica expressed 0.43 cm, 0.49 cm, 0.54 cm, M. oleifera 0.32 cm, 197 0.35 cm, 0.38 cm, D. alba 0.22 cm, 0.26 cm and 0.28 cm inhibition zone at 10 %, 15 % and 20 198 % concentration respectively as compared to control (Table 4). 199 200 Table 4 Impact of different plant extracts in three different concentrtions on the growth of X. citri pv. malvacearum Treatments Inhibition zones (cm) Mean inhibition zones (cm) 10% 15% 20% N. tabacum (Tobacco) 0.600c 0.650b 0.700a 0.650a A. indica (Neem) 0.430f 0.490e 0.540d 0.486b M. oleifera (Moringa) 0.320i 0.350h 0.380g 0.350c D. alba (Datura) 0.227 l 0.260k 0.283j 0.256d C. longa (Turmeric) 0.150 o 0.170n 0.186m 0.168e Control 0.000p 0.000p 0.000p 0.000f 201 202 203 3.3- Impact of Flare and Nicotiana tabacum against bacterial blight of cotton in greenhouse 204 Minimum disease incidence 32.27 % was observed when Flare and N. tabacum were applied in 205 combination, followed by Flare 36.91 % and N. tabacum 41.60 % as compared to control (Table 206 5). In interaction between treatments and concentration N. tabacum expressed 37.06 %, 41.83 % 207 and 45.90 % disease incidence at 40 %, 35 % and 30 % concentration and Flare showed 32.50 %, 208 37.30 % and 40.93 % disease incidence at 0.60 %, 0.55 % and 0.50 % concentration. Minimum 209 disease incidence was expressed by (Flare + N. tabacum) 27.86 %, 32.86 % and 36.10 % 210 respectively at three concentrations as compared to control (Table 5). In interaction between 211 treatments, concentrations and days, maximum reduction in disease was observed (39.40 %, 212 37.50 % and 32.60 %) after seven days, 35.40 %, 32.60 %, 28.40 % after fourteen days, and 213 33.50 %, 28.50 % and 22.60 % after twenty one days when Flare and N. tabacum was applied 214 in combination at three concentrations. Flare expressed (44.20, 41.60 and 36.50 %), (40.10, 215 37.40 and 33.50 %) and (38.50, 32.90 and 27.50 %) disease incidence after seven, fourteen and 216 twenty one days respectively at 0.30 %, 0.35 % and 0.40 % concentration. Minimum reduction in 217 disease incidence was exhibited by N. tabacum (48.40, 45.20 and 41.80 %) after seven day, 218 (45.80, 42.80 and 37.90 %) after fourteen days and (43.50, 37.50 and 31.50)% after twenty one 219 days when applied at 30 %, 35 % and 40 % as compared to control (Table 6). 220 221 Table 5 Impact of Flare and Nicotiana tabacum and their mixture in three different concertations on bacterial blight of cotton in greenhouse Disease incidence (%) Mean Disease Treatments incidence (%) C1 C2 C3 222 223 224 225 226 227 Means in the column and rows with the dirrefent letters are significantly different at (p<0.05) *Flare +N. tabacum 36.10i 32.86j 27.86 l 32.27a **Flare 40.93f 37.30g 32.50k 36.91b ***N. tabacum 45.90d 41.83e 37.06h 41.60c Control 74.00c 74.66b 75.80a 74.82d * C1=0.50% Flare + 30% N. tabacum, C2= 0.55% Flare + 35% N. tabacum, C3= 0.60% Flare + 40% N. tabacum; ** C1= 0.50%, C2= 0.55%, C3= 0.60%; *** C1= 30%, C2= 35%, C3= 40%. Means in the column and rows with the dirrefent letters are significantly different at (p<0.05) 228 229 230 231 232 233 Table.6 Impact of Flare and Nicotiana tabacum and their mixture in three different concertations on three cosective weeks on bacterial blight of cotton in green house Disease incidence (%) Treatments C1 Week 1 C2 C3 C1 Week 2 C2 C3 C1 Week 3 C2 C3 *Flare+N.tabacum 32.60 37.50 39.40 28.40 32.60 35.40 22.60 28.50 33.50 **Flare 36.50 41.60 44.20 33.50 37.40 40.10 27.50 32.90 38.50 ***N.tabacum 41.80 45.20 48.40 37.90 42.80 45.80 31.50 37.50 43.50 Control 68.40 69.30 70.40 74.60 75.40 76.30 79.00 79.30 80.70 234 235 236 237 * C1=0.50% Flare + 35% N. tabacum, C2= 0.55% Flare + 35% N. tabacum, C3= 0.60% Flare + 40% N. tabacum; ** C1= 0.50%, C2= 0.55%, C3= 0.60%; *** C1= 30%, C2= 35%, C3= 40%. 238 Minimum disease incidence (40.41 %) was observed when Flare + N. tabacum were applied in 239 combination, followed by Flare (45.74%) and N. tabacum (50.41%) as compared to control 240 (Table 7). In interaction between treatments and concentration, N. tabacum expressed (54.60%), 241 (50.26%) and (46.36%) disease incidence at 45 %, 50 % and 55 % concentration, Flare showed 242 (49.70%, 46.46% and 41.06%) disease incidence at 243 while minimum disease incidence was expressed by Flare + N. tabacum (44.23%, 41.93% and 244 35.06%) respectively at three concentrations as compared to control (Table 7). In interaction 245 between treatments, concentrations and days, maximum reduction in disease (46.50%, 46.30%, 246 39.40%) was observed after seven days, (44.30%, 41.60% and 36.50%), fourteen days, and 247 (42.10%, 37.30% and 29.30%) after twenty one days when Flare + N. tabacum was applied at 248 three concentrations. Flare expressed (52.50%, 49.60% and 45.70%), (48.90%, 46.10% and 249 41.60%) and (47.70%, 43.70% and 35.90%) disease incidence after seven, fourteen and twenty 250 one days respectively at 0.75%, 0.80% and 0.85% concentration. Minimum reduction in disease 251 incidence was exhibited by N. tabacum (56.70%, 53.90% and 51.50%) after seven day, (55.10%, 252 50.30% and 47.70%) after fourteen days and (52.00%, 46.60% and 40.30%) after twenty one 253 days when applied at 45%, 50% and 55% concentration as compared to control (Table 8). 254 255 3.4- Impact of Flare and Nicotiana tabacum against bacterial blight of cotton in field 0.75%, 0.80% and 0.85% concentration, 256 257 258 259 260 261 262 263 264 265 Table 7 Impact of Flare and Nicotiana tabacum and their mixture in three different concertations on bacteraial blight of cotton in field experiment Treatments Disease incidence (%) Mean Disease incidence (%) C1 C2 C3 *Flare +N. tabacum 44.23h 41.93i 35.06k 40.41d **Flare 49.70f 46.46g 41.06j 45.74c ***N. tabacum 54.60d 50.26e 46.36g 50.41b Control 80.96c 83.43b 84.40a 82.93a * C1= 0.75% Flare + 45% N. tabacum, C2= 0.80% Flare + 50% N. tabacum, C3= 0.85% Flare + 55% N. tabacum; ** C1= 0.75%, C2= 0.80%, C3= 0.85%; *** C1= 45%, C2=50%, C3=55%. Means in the column and rows with the dirrefent letters are significantly different at (p<0.05) Table.8 Impact of Flare and Nicotiana tabacum and their mixture in three different concertations on three cosective weeks on bacteraial blight of cotton in filed Disease incidence (%) Treatments Week 1 Week 2 Week 3 C1 C2 C3 C1 C2 C3 C1 C2 C3 *Flare + N.tabacum 39.40 46.50 46.30 36.50 41.60 29.30 44.30 37.70 42.10 ** Flare 45.70 49.60 52.50 41.60 46.10 35.90 48.90 43.70 47.70 ***N. tabacum 51.10 53.90 56.70 47.70 50.30 40.30 55.10 46.60 52.00 Control 76.70 77.60 78.60 80.70 84.40 85.50 85.70 88.30 88.90 266 267 268 269 270 * C1= 0.75% Flare + 45% N. tabacum, C2= 0.80% Flare + 50% N. tabacum, C3= 0.85% Flare + 55% N. tabacum; ** C1= 0.75%, C2= 0.80%, C3= 0.85%; *** C1= 45%, C2=50%, C3=55%. 271 cotton, is the use of resistant varieties. If resistant varieties are not available and disease appears 272 in the field suddenly and at a very rapid rate in the field, the farmers have only one option to 273 spray crop with some effective chemical. In present study, five chemicals (Flare, Plant protector, 274 Mancozeb, Agrimycine and copper oxychloride at three concentrations were evaluated against 275 Xcm. Maximum inhibition was expressed by Flare whose main ingredient is streptomycin 276 sulphate. Plant extracts were selected because, they play an important role i.e. sustainable 277 solutions in agriculture, reduce crop losses, eco-friendly, easily bio-degradable, cheaper and are 278 an important component in integrated diseases management. In present esearch above plant 4- DISCUSSION The most suitable, economical, safe, reliable and practicable management of bacterial blight of 279 extracts were used on the basis of their easily availability in local area. Similarly five plants 280 extract (N. tabacum, A. indica, M. oleifera, D. albaand C. longa) were also evaluated against 281 growth of Xcm in lab.conditions by using inhibition zone technique. Among plant extracts N. 282 tabacum expressed maximum inhibition zone, (0.650cm). Then Flare and N. tabacum were 283 evaluated in greenhouse and filed conditions against bacterial blight of cotton. Both Flare and N. 284 tabacum expressed significant results but maximum reduction in disease was expressed by 285 combination of Flare + N .tabacum both in greenhouse and field conditions. Fallouts of the 286 present study are reinforced by the work of Singh et al., (2007) who evaluated twelve fungicides 287 and two antibiotics against bacterial blight disease. Among all chemicals streptomycin sulphate 288 expressed significant results both in vivo and in vitro. Similar results were also reported by 289 Jagtap et al., (2012). A great potential of antibacterial activity is present in a number of plant 290 (Cao et al., 2001). So in present study different plant extracts were used against bacterial blight 291 disease and outcomes of the present study are supported by the work of Sajid et al., (2013) who 292 evaluated three chemicals (plant protector, agrimycine and copper oxy chloride) and three plant 293 extracts (N. tabacum, C. colocynthis and C. longa) against Xcm at different concentrations and 294 observed that N. tabacum expressed pronounced results. 295 The management of bacterial blight of cotton is achievable by plant extracts, which we selected 296 based on their well-established antibacterial activity as documented by various researchers 297 Moringa oleifera (saponins) Abalaka et al., 2012, Azadirachta indica (Nimbine) Lokanadhan et 298 al.,2012, Datura alba (scopolamine) Okwu and Igara, 2009, Nicotiana tabacum (Nicotine) Bakht 299 et al., 2012 and Curcuma longa (Curcumin) Moghadamtousi et al., 2014 as antibacterial agent 300 gave a casement for future and selection of suitable formulation and method of application of 301 plant product in vitro, green house and in vivo especially N. tabacum related research. 302 5- CONCLUSIONS 303 Flare and N. tabacum expressed maximum inhibition zone in lab and minimum disease incidence 304 under greenhouse and field conditions. 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