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1 Journal home page: INTERNATIONAL JOURNAL OF INNOVATIVE AND APPLIED RESEARCH RESEARCH ARTICLE Management of bacterial leaf blight of Basmati rice caused by Xanthomonas oryzae pv. oryzae with some available antibiotics and plant products Rahul Singh 1, *Ramesh Singh Yadav 1 and Shaily Javeria 2 1. Centre of Excellence for SPS, SVPUA&T, Meerut. 2. Division of Plant Pathology, IARI, New Delhi-12. Abstract: Rice (Oriza sativa L.) belongs to family Poaceae (Gramineae) and sub family Oryzoidea. Rice provides 21% of energy and 15% of protein requirements to human populations globally. Rice is the major crop of Uttar Pradesh and grown in almost all the districts and contributes 13.5 % of total rice production in India. Among the rice, Basmati varieties of rice are attacked by number of pathogens (fungi, bacteria, virus and nematodes etc.). Bacterial leaf blight is serious problems in basmati rice production. The disease is caused by the pathogens Xanthomonas oryzae pv. oryzae. Bacterial Leaf Blight of rice posses two phase of disease symptoms i.e. (Kresek phase) wilting and (leaf blight) pale yellowing of leaves. In rice bacterial leaf blight occurs at all stages. In present investigation antibiotics and secondary metabolites of higher plants were evaluate for their effectiveness in management of bacterial leaf blight. Among the used treatments, maximum plant height (67.23cm and 88.23cm) was found with T1 (oxytetracycline + copper oxychloride) after 20 and 40 days spray in compression to control (57.34 and 78.12cm) respectively. Maximum panicle length, panicle weight and percent increase yield was also recorded with T1 (oxytetracycline + copper oxychloride) as 27.63cm, 2.84g and 16.67% respectively. Disease intensity in different treatment was observed in range from 55.10% to 68.25%. Minimum disease intensity (55.10%) was observed with T1 whereas 67.50% disease intensity with T5 (spray of lemon grass oil). Maximum disease intensity (68.25%) was observed in control. These antibiotics and plant products also tested in vitro to test their efficacy on growth of Xanthomonas oryzae pv. oryzae at 1%, 2% and 4% concentration. When bacterial leaf blight pathogen i.e. Xanthomonas oryzae pv. oryzae. was treated with T1 (oxytetracycline + copper oxychloride) it showed maximum inhibition zone i.e. 2.83cm, 3.33cm and 3.53cm at 1%, 2% and 4% concentration respectively. Introduction Rice is an important cereal crop and growing all over the world. Rice occupies around 20% of total land area covered under cereal crop. More than half rice produced and consumed by Asian country in which million hectares of rice planted annually and approximately 95 % of rice consuming people belongs to Asian country. China and India both are major rice producing country which covers 49.1 % of total rice. Rice grown in India is primarily divided into Basmati rice and Non-Basmati rice. India is the major producer and exporter of basmati rice to the world. Basmati rice is attacked by many fungal, bacterial, viral and nematode pathogens. Among the bacterial diseases, bacterial leaf blight is serious problems in basmati rice production. Bacterial Leaf Blight (BLB) caused by Xanthomonas oryzae pv. oryzae has been ranked in the top-10 list of bacterial plant pathogens (Mansfield et. al., 2012). The development of BLB of rice posses three phase of disease symptoms i.e. leaf blight, wilting (Kresek phase) and pale yellowing of leaves. Irrigated and rainfed lowland ecosystems favours this disease. BLB occurs at all the growth stages of rice and is manifested by either leaf blight or Kresek symptoms. Yield loss of the crop due to bacterial disease depends on the virulence of pathogen, host-pathogen relationship and different environmental factors. In India, the damage has been estimated to vary from per cent (Rajarajeswari and Muralidharan, 2006). Bacterial leaf blight is devastating and can cause yield losses from 20 to 74% in Southeast Asia and India (Ahmed and Singh, 1975; Srivastava, 1967; Ou, 1985). The earlier studies have identified some chemicals and antibiotics with relative efficacy against the disease. However, effective control of the disease has not been recorded (Reissingn et.al., 1986). As there is no single most effective control measure available against this disease, some cultural practices may minimize disease incidence (Chaudhary et. al., 2009). The present investigation was to bring together comprehensive update to the research on the bacterial disease of rice and the management strategies in Indian context. Henceforth, the present work was undertaken to evaluate the effectiveness of some antibiotics and plant extract single and in combination, for the control of BLB. 1

2 Material and methods In-vivo experiment was conducted at Crop Research Centre (CRC) and In-vitro experiment was conducted at Centre of Excellence for Sanitary and Phytosanitary (SPS) of the Department of Plant Pathology, SVP University of Agriculture & Technology, Meerut (U.P.). Collection of disease The rice leaves naturally infected with bacterial leaf blight were collected by Crop Research Centre (CRC), SVP University of Agriculture & Technology, Meerut (U.P.). Isolation and identification of Xanthomonas oryzae pv. Oryzae The bacteria was isolated from infected leaves of rice and identified on the basis of morphological and cultural characters of Xanthomonas oryzae pv. oryzae. The identification of pathogen was based on the findings of Suresh et al., (2013) and Sacchad et. al., (2000). The overnight culture of isolated pathogen was used for Gram s staining. The similar morphological characters of Xanthomonas oryzae pv. oryzae were recorded which were reported by Samanta et. al., (2014). In vitro evaluation of antibiotics and plant products Commonly available antibiotics and plant product viz., Streptomycin, oxytetracycline, copper oxychloride and neem oil, lemon grass oil (extracted by essential oil distiller) were evaluated against the Xanthomonas oryzae pv. oryzae. A stock solution of each antibiotic and plant product separately was made using (w/v, 1μg/ml) and different concentrations viz., 1%, 2%, and 4% were made by using the stock solution to examine the effectiveness of antibiotics and plant product against the pathogen by disc diffusion method given by Beuer and Kirby (1985) was used to evaluate the effectiveness of the antibiotics and plant product against the Xanthomonas oryzae pv. oryzae. About 6mm paper discs were impregnated with 50 μl of each antibiotic and plant product concentrations were placed on the surface of the Nutrient agar medium inoculated with Xanthomonas oryzae by spread plate method separately. All the petri plates were incubated at 37 ºC. After 72 hrs the diameter of inhibition zone were measured including the petri plate where disc soaked in water serves as control. In vivo evaluation of antibiotics and plant product Commonly available antibiotics and plant product were evaluated against the bacterial leaf blight disease. Plant extract and antibiotics were used in following doses:- T1- 75g/ ha +500g Copper oxychloride, 15g/ha +500g Copper oxychloride, T g/ ha, T4- Neem 0.03% T5-2 ml lemon grass oil T6- Neem 0.03% + 2 ml lemon grass oil T7- + Neem 0.03% T8- 75g/ ha + 2 ml lemon grass oil T9-Neem 0.03% + 75g/ ha T10- water The antibiotics and plant products were mixed with distilled water to make a solution of 2 Liter for each treatment having three replications and sprayed over affected leaf surface of rice. Data recorded To test the effects of different treatments (antibiotics and plant products) on plant height, panicle length and panicle weight of rice was recorded at 20 days after spray and at 40 days after spray. Percent disease intensity and percent control The Percent disease intensity of rice plants were scored by using the scale of 0-9 as per the standard evaluation of IRRI. The scale used for BLB assessment was as follows: Scale Lesion area 0 0% 1 1-5% % 2

3 % % % The percent disease index (PDI) was calculated for each treatment Sum of all disease rating Percent Disease intensity (%) = X 100 Total number of rating X Maximum disease grade Harvesting and grain yield Each plot was harvested separately leaving borders rows from all sides to record yield and other observations. Threshing was done separately of each plot and grain yield per plot was obtained. The final grain yield was adjusted to 14% moisture. On the basis of grain yield from the net area of each plot, yield per hectare was calculated. One thousand grain weight was obtained by weighing 1000 filled grain obtained from each plot separately. The per cent increase in yield was calculated by using following formula: T - C Y= 100 C x Where, Y= Per cent loss in yield T=Yield of treated plot C= Yield in the check plot Statistical analysis Data was recorded with the help of analysis of variance table wherever required.the F value was tested and critical difference (CD) was calculated at 5 per cent level of significance for comparing treatment means (Gomez, 1996 and Chandel, 2002). Result and discussion Pathogen Xanthomonas oryzae pv. oryzae was isolated on nutrient agar medium from infected leaves of rice plants and identified on the basis of morphological and cultural characters. The colonies of the pathogen were circular, slightly raised, flattened and convex with yellow color to bright yellow color on Nutrient Agar medium. It is a slow growing bacteria having circular form, convex margin with entire elevation. Bacterium was purified and proved its Pathogenicity. The pathogen was Gram s negative, rod shaped bacterium with a polar flagellum, cells appear red, singly or in pairs, or in chains on the basis of Gram staining. In vitro evaluation of antibiotics and plant products The effectiveness of different antibiotics and plant Product against Xanthomonas oryzae were tested in laboratory at 1%, 2% and 4% concentration. Each treatment was replicated three times. All used antibiotics were found effective and significantly suppress the pathogen growth. In comparison to antibiotics, essential oil of neem and lemon were observed less effective and suppress minimum growth of pathogen. It was observed that at 1% concentration maximum (2.83 cm) inhibition zone was recorded in oxytetracycline + copper oxychloride followed by streptomycin + copper oxychloride (2.23 cm). At 2% concentration, oxytetracycline + copper oxychloride treated Petri plates were observed maximum (3.33 cm) inhibition zone followed by streptomycin + copper oxychloride (2.43 cm). At 4 %, maximum (3.53 cm) inhibition zone was recorded in oxytetracycline +copper oxychloride followed by streptomycin + copper oxychloride (2.97cm) which were significantly differ from essential oil treated Petri plate that were observed by neem oil (1.83 cm) and (1.70 cm) inhibition zone in lemon grass oil. While no inhibition zone was recorded in control (table. 1). Similar reports of inhibition zone were reported by many researchers. Thimmegowda et. al., (2012) reported that, the application of streptocycline at 1000ppm was effective against bacterial blight under in vitro condition, with maximum inhibition zone (25.0 mm), followed by streptocycline + Copper sulphate 1000 ppm (24.33mm).Result concluded that, the streptocycline + copper oxychloride was found best with least percent disease index. Bibiana et. al., (2012) reported the efficacy of lemon grass oil having good antibacterial effects against the tested gram positive and gram negative bacteria. The antimicrobial activity of the oil is exhibited by its major constituent geraniol. 3

4 In vivo evaluation of antibiotics and plant product Effect of treatment of plant morphology The result of different treatments was clearly indicate that the plots sprayed with Oxytetracycline +copper oxychloride, streptomycin + copper oxychloride and oxytetracycline + streptomycin were found better as compare to essential oil of neem and lemon. All antibiotics significantly increased the growth of plant. Plant height under different treatment ranged from cm to cm. Among the treatment, Oxytetracycline + copper oxychloride was recorded with maximum (67.23 cm) plant height followed by streptomycin + copper oxychloride (65.43cm), oxytetracycline + streptomycin (64.11 cm) and oxytetracycline + neem oil (63.17 cm) at 20 days after spray At the 40 days after spray Maximum plant height (88.23 cm) was recorded in plots which were sprayed with oxytetracycline + copper oxychloride followed by streptomycin + copper oxychloride (87.18 cm). The minimum plant height (57.34 cm and cm) was recorded in control at 20 and 40 days after spray. The significant effect of these antibiotics was also observed on number of tillers, panicle length and plant weight. Maximum panicle length (27.63 cm) was recorded in plots which were sprayed with oxytetracycline + copper oxychloride followed by streptomycin + copper oxychloride (26.77 cm). In respect to increase in panicle weight maximum results (2.84 gm) was observed in plots which were sprayed with oxytetracycline + copper oxychloride followed by streptomycin + copper oxychloride (2.74 gm) and oxytetracycline + streptomycin (2.59 gm). Observations were made to check the increment in average and total yield of treatments as compare to control. The maximum average and total yield (5.40kg and 16.2kg) were obtained in plots which were sprayed with oxytetracycline + copper oxychloride followed by streptomycin + copper oxychloride (5.30kg and 15.9kg) and oxytetracycline + streptomycin (5.17kg and15.5kg) respective. Patel et. al. (2009) found streptomycin sulphate + copper oxychloride was superior over rest of the treatments and increase grain and straw yield. Thimmegowda et. al., (2012) reported the effectiveness of streptocycline and streptocycline + copper oxychloride against bacterial blight under in vitro and in vivo condition. They were concluded that the streptocycline + copper oxychloride was found best with least percent disease index and highest grain yield returns. Prophylactic and curative spraying of streptocycline (500 ppm), streptomycin + oxytetracycline (1:9, 250 and 500 ppm), bactrinol-100 (500 ppm) and cow dung extract (20 g/litre) on rice for observing the effectiveness against bacterial leaf blight and grain and straw yield recorded by Mary et. al., (2001). Effect of different treatments on percent disease intensity Uses of antibiotics are important component of crop protection in framework of management of bacterial leaf blight of rice. In present investigation antibiotics and essential oil were used to observe the effect on target disease at different field doses. In used antibiotics, Oxytetracycline + copper oxychloride, streptomycine + copper oxychloride and oxytetracycline + streptomycin were significantly reduced the plant disease intensity. The minimum (55.10%) disease intensity was recorded in plots which were sprayed with oxytetracycline + copper oxychloride followed by streptomycin + copper oxychloride (55.90 %) and oxytetracycline + streptomycin (59.90). Whereas, the maximum disease intensity was recorded in control with water spray i.e %. According to data of disease intensity, it was concluded that highest (19.26%) disease control was observed by oxytetracycline + copper oxychloride which was followed by streptomycine + copper oxychloride (18.09%), oxytetracycline + streptomycin (12.23%) and oxytetracycline + neem oil (8.42 %). Lemon grass oil was observed less effective with reduces minimum (1.09%) disease intensity (Table. 2). Chaudhary et al., (2012) studied the effectiveness of Bordeaux mixture, copper oxychloride 50%, oxytetracycline and streptomycin (0.50 kg/ha) and their combinations. They recorded significantly low incidence of bacterial leaf blight when Bordeaux mixture alone (40.89 and %), in combination with oxytetracycline (38.67 and %) and streptomycin (36.89 and %) were applied as compared to control (56.00 and 63.45%) respectively. Biswas (2009) evaluated the efficacy of the antibiotics with the copper containing compound against bacterial blight of rice and concluded that all the treatments significantly reduced the disease severity. Seed treatment with streptocycline (100 ppm) + FS of streptocycline in combination with copper oxychloride ( ppm) was the most effective against the disease. Suresh et al., (2014) used different antibiotics with the combination of copper oxy- chloride against BLB disease of rice under field condition. Seed treatment with Streptocycline (100 ppm) along with foliar spray in combination with copper oxy- chloride (100 ppm ppm) gave best control of bacterial blight. Table-1 Effect of different treatments on the growth of X. oryzae pv. oryzae (in vitro). Treatment Inhibition zone (cm) after 24 hours of inoculation At 1% conc. At 2% conc. At 4% conc. T1-Oxytetracycline + Copper oxychloride T2-Streptomycin+Copper oxychloride

5 T3- Streptomycin + Oxytetracycline T4-Neem oil T5-Lemon grass oil T6-Neem oil+ Lemon grass oil T7-Streptomycin+Neem oil T8-Oxytetracyclin+Lemon grass oil T9-Neem oil + Oxytetracycline T10 (Control) C.D (P=5%) Table-2 effect of antibiotics and plant products on plant morphological characters and disease intensity of BLB disease of rice Treatment Plant height (cm) Panicle 20 days after spray 40 days after spray length (cm) Panicle weight (gm) Yield (Kg) Total yield (Kg) Disease intensity (%) T T T T T T T T T Control C.P kg 2.83 Conclusion In the present investigation it can be concluded that antibiotics oxytetracycline and streptomycin with copper oxychloride integrated with plant products may proved more economical effective than only antibiotics for the control of bacterial leaf of rice. However, there is a need to investigate and explore the plant products for integration with antibiotics for the management of bacterial leaf blight. References Ahmad KM and Singh RA. (1975): Disease development and yield loss in rice varieties by bacterial leaf blight. Indian Phytopath. 28: Bauer AW, Kirby WMM, Sherris JC and Turck M. (1985): Antimicrobial susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45: Biswas SK, Rai M and Srivastava SSL. (2009): Evaluation of antibiotics and their suitable use against bacterial blight of paddy [Xanthomonas oryzae pv. oryzae (Ishiyama) Dye]. Indian Phytopathology. 62(1): Bibiana MA, Selvamani P and Latha S. (2012): In-Vitro antimicrobial evaluation of extracts, oil and fractionated geraniol of cymbopogan citratus-an aromatic grass international journal of environment science Volume 3, No 1. Chaudhary SU, Hussain M, Iqbal J and Ali MA. (2009): Effect of nitrogen doses on incidence of bacterial leaf blight in rice. J. Agric. Res. 47(3):

6 Chaudhary SU, Iqbal J and Hussain M. (2012): Effectiveness of different fungicides and antibiotics against bacterial leaf blight in rice. J. Agric. Res. 50(1). Goto M. (1964): Kresek and pale yellow leaf, systemic symptoms of bacterial leaf blight of rice. Plant Dis. Reptr. 48: Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G and Foster GD. (2012): Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13 (6): Mary CA, Nair SK and Saraswathy P. ( 2001): Efficacy of prophylactic and curative spraying on the control of bacterial leaf blight of rice.thrissur, India: Kerala Agricultural University. Journal of Tropical Agriculture. 39 (1): Patel SJ, Chauhan BL, Mehta AN and Gohil NM. (2009): Management of bacterial leaf blight of rice with chemicals,botanicals and antagonists. J. Pl. Dis. Sci. Vol 4(2): Rajarajeswari NVL and Muralidharan K. (2006): Assessments of farm yield and district production loss from bacterial leaf blight epidemics in rice. Crop Protection 25 (3): Reissing WH, Heinrichs EA, Litsinger LA, Moody K, Fiedler L, Mew TW and Barrian AT. (1986): Illustrated Guide to Integrated Pest Management in Rice Tropical Asia, IRRI, Manila, Philippines., p Samanta TT, Samanta P and Das A. (2014): Isolation and characterization of Xanthomanas oryzae isolation from different regions of midnapore district of west bangals and their ecofriendly management by some medicinal plant. International Journal of Phytomedicine. 6: Schaad BW, Jones JB and Chun B. (2000): Laboratory guide for identification of plant pathogenic bacteria, APS Press, The American Phytopathological Society, St. Paul, Minnesota Suresh SR, Yenjerappa ST and Pavithra S. (2014): Field Evaluation of Bacterial Blight of Paddy by Chemicals and Biological Agents. Trends in Biosciences. 7(23): Suresh SR, Yenjerappa ST, Naik MK, Mallesh SB and Kalibavi CM. (2013): Studies On Cultural And Physiological Characters Of Xanthomonas Oryzae Pv. Oryzae causing Bacterial blight of Rice Karnataka. J. Agric. Sci.,26 (2) : Thimmegowda PR, Arun S, Ambika DS, Prasad PS and Chandrashekar M. (2012): Efficacy of antibiotics and biorational pesticides against bacterial blight of paddy. Madras Agricultural Journal. 99 (7/9):

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