Int.J.Curr.Microbiol.App.Sci (2020) 9(6): 3570-3575 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 6 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.906.420 Bioefficacy of Chemicals against Bacterial Leaf Blight Disease of Rice A. Khandual1,2, M. K. Mishra1, H. Swain2, S. Mohanty1,2, P. C. Rath3 and A. K. Mukherjee2* 1 Department of Plant Pathology, OUAT, Bhubaneswar, India 2 Molecular Plant Pathology Laboratory, Crop Protection Division, ICAR-NRRI, Cuttack, India 3 Crop Protection Division, ICAR-NRRI, Cuttack, India *Corresponding author ABSTRACT Keywords Bacterial leaf blight, Rice, Chemicals Article Info Accepted: 26 May 2020 Available Online: 10 June 2020 Bacterial leaf blight of rice, caused by Xanthomonas oryzae pv. oryzae is not only a problem in India but also worldwide. Management using chemicals is undoubtedly quick in action, restricting the severity and spread of the disease. Both in vitro and in vivo studies were conducted to investigate the inhibitory potential of different chemicals against the bacterial pathogen. Out of eight chemicals comprising both antibiotics and fungicides, Streptomycin sulphate 90% + Tetracycline hydrochloride 10% exhibited highest inhibitory effect in lowest concentration of 100 ppm followed by 2-bromo-2-nitropropane 1,3-diol (15.5%). Streptomycin sulphate 90% + Tetracycline hydrochloride 10% @ 100 ppm proved best in controlling BLB in pot condition with 42.2% disease reduction and 121 % yield increase over control. Introduction Rice, the golden cereal, is an important part of daily human food and provides carbohydrates, proteins, minerals and vitamins (Pradhan et al., 2019). With the global population expanding at a rapid rate, it warrants a solution to food scarcity and hunger. To provide food for the billions, the productivity of rice needs to be increased. But yield losses tend to rise with swelling pest population which is the consequence of changing climate and agroecological systems. A number of diseases pose threat to the rice industry and bacterial leaf blight (BLB) is a major one which can hamper more than 70% yield (Reddy et al., 1979). BLB is caused by Xanthomonas oryzae pv. oryzae (Xoo), which enters the plant system via hydathodes (Nino 3570 Int.J.Curr.Microbiol.App.Sci (2020) 9(6): 3570-3575 et al., 2006). Xoo attacks the crop mainly at two stages viz., nursery and tillering. Kresek is a symptom associated with wilting of seedlings. Leaf blight occurs mainly from tillering to flowering stage. The intensity and type of blight symptoms vary with varieties, environmental conditions and crop growth stages. Stock solutions of 10,000 ppm were prepared for each chemical using sterile distilled water. For each chemical, seven concentrations consisting of 5000, 2500, 1000, 750, 500, 250 and 100 ppm were prepared employing serial dilution. A homogenous bacterial suspension was prepared from three days- old culture and sterile distilled water. A number of management strategies such as host resistance, biological control and chemical control are employed to overcome the disease. But sometimes, sudden appearance of this disease in the field puzzles the farmer. In this situation, the use of chemicals becomes indispensable for a quick and effective solution. An investigation was conducted in ICAR-NRRI, Cuttack using some antibiotics and fungicides, both in vitro and in vivo to find out the best chemical for management of BLB. A lawn of inoculum was spread onto solidified MWA plates and allowed to dry for 30 minutes. Sterile disc (HiMedia Laboratories) of 6 mm diameter impregnated with 30 µl of chemical was placed at the centre of the plate with inoculum lawn and incubated. The experiment was conducted with three replications per treatment. After 72 hours, the zone of inhibition (excluding the diameter of the disc) was recorded and percent inhibition was calculated as follows: % 𝑖𝑛𝑕𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = Materials and Methods Collection, isolation, purification pathogenicity of the pathogen and Fifty-two disease samples were collected from few states of Eastern India. The pathogen was isolated by ooze method (Kotasthane, 2003) and purified on modified Wakimoto’s agar (MWA). Pathogenicity tests were performed on Taichung Native1 plants (BLB susceptible cultivar) by leaf clipping (Kauffman et al., 1973). The isolates were subjected to virulence profiling using differentials and a representative isolate from the most virulent pathotype was used for this study. Antimicrobial conditions assay under in vitro 𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑡𝑕𝑒 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛𝑕𝑖𝑏𝑖𝑡𝑖𝑜𝑛 × 100 𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑡𝑕𝑒 𝑝𝑒𝑡𝑟𝑖𝑝𝑙𝑎𝑡𝑒 Antimicrobial conditions assay under in vivo From the in vitro experiment, two lower concentrations of all the test chemicals showing inhibitory effect were chosen. An experiment with a total of 17 treatments including control and three replications each was designed. TN1 cultivar was used for the trial. Standard agronomic practices were followed. Disease was clip-inoculated to the plants at 45 days after sowing. After 10 days, a foliar spray of the chemicals was given while the control was sprayed with sterile water only. The disease score (IRRI,1996) was recorded after 21 days of inoculation. Percent disease index was computed as follows: The entire procedure of agar disc diffusion (Baeur et al., 1966) was done aseptically. 3571 PDI % = 𝑆𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 𝑏𝑙𝑖𝑔𝑕𝑡 𝑠𝑐𝑜𝑟𝑒𝑠 × 100 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑒𝑎𝑣𝑒𝑠 𝑠𝑐𝑜𝑟𝑒𝑑 × 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑟𝑎𝑡𝑖𝑛𝑔 Int.J.Curr.Microbiol.App.Sci (2020) 9(6): 3570-3575 Results and Discussion Antimicrobial conditions assay under in vitro As presented in Table 1, the sensitivity of the Xoo isolate had a varied response to the different chemicals used as well as their concentrations. Among all, a maximum of 5.5 cm zone of inhibition was recorded for chloramphenicol at 5000 ppm. But chloramphenicol was isolate specific in inhibitory effect (Khan et al., 2012). Copper oxychloride and copper hydroxide were totally ineffective at 500, 250 and 100 ppm concentrations. The former chemical was equally effective as Captan at 5000 and 1000 ppm strength. Interestingly, inhibitions by chloramphenicol were at par with 2-bromo-2-nitropropane 1,3diol at 500 ppm and with streptomycin sulphate at 100 ppm respectively. A ranking with a descending trend in the efficacy of chloramphenicol, 2-bromo-2nitropropane 1,3-diol, streptocycline and streptomycin sulphate was observed from 5000 to 1000 ppm. However, at the two lower concentrations (250 and 100 ppm), streptocycline was the most effective of all, followed by chloramphenicol, 2-bromo-2-nitropropane 1,3-diol and streptomycin sulphate. Streptocycline had promising effect to check Xoo (Mahto et al., 1988). The chemical, 2-bromo-2-nitropropane 1,3diol produced excellent control over the pathogen under in vitro conditions (Praveen et al., 2019). Surprisingly, all the chemicals and their concentrations failed to exhibit any uniform trend in inhibition of the pathogen. It partially corroborated with the previous studies, where increase in inhibition with rise in concentration followed a regular trend (Khan et al., 2012 and Ashrafuzzaman, 1987). This indicated the presence of a specific type of interaction of the microbe with varying chemicals and concentrations. Antimicrobial conditions assay under in vivo At both the concentrations, chloramphenicol, 2-bromo-2-nitropropane 1,3-diol, streptocycline and streptomycin sulphate were able to restrict the disease by 19-25% over control (Table 2). A maximum yield (26.36 g/pot) was attained using streptocycline spray @ 250 ppm, followed by chloramphenicol (24.80 g/pot) and 2-bromo-2-nitropropane 1,3-diol (24.10 g/pot) at the same concentration. Satisfactory disease control and yield was experienced when 2-bromo-2-nitropropane 1,3-diol was used against the disease (Pramesh et al., 2017). Eight treatments belonging to Mancozeb, Captan, copper hydroxide and copper oxychloride were ineffective in restricting the disease. However, they produced equal yield among themselves, but superior to control. Treatment with copper oxychloride produced fair yield performance in rice against BLB (Shahbaz et al., 2016 and Chaudhary et al., 2012). 3572 Int.J.Curr.Microbiol.App.Sci (2020) 9(6): 3570-3575 Table.1 Evaluation of in vitro efficacy of chemicals against Xanthomonas oryzae pv. Oryzae Treatm ent Chemical name Trade name T1 Chloramphe nicol 500mg Chloramphe nicol T2 2-bromo-2nitropropan e 1,3-diol Streptomyci n sulphate 90% + Tetracycline hydrochlori de 10% Mancozeb 75% WP Captan 50% WP Copper hydroxide 77% WP Copper oxychloride 50% WP Streptomyci n sulphate Control Bactinash200 T3 T4 T5 T6 T7 T8 T9 SE(m)± CD (0.05) 5000 Mean % zone inhibit of ion inhibit ion (cm) 5.5 65.5 * (2.5) 5.2 61.9 (2.4) 2500 Mean % zone inhibit of ion inhibit ion (cm) 5.1 60.3 (2.4) 1000 Mean % zone inhibit of ion inhibit ion (cm) 4.3 51.2 (2.2) Concentration (ppm) 750 500 Mean % Mean % zone inhibit zone inhibit of ion of ion inhibit inhibit ion ion (cm) (cm) 4.1 48.4 3.2 38.1 (2.1) (1.9) 250 Mean % zone inhibit of ion inhibit ion (cm) 2.5 30.2 (1.7) 100 Mean % zone inhibit of ion inhibit ion (cm) 1.1 13.5 (1.3) 4.7 (2.3) 56.0 3.8 (2.1) 45.3 3.7 (2.1) 44.4 3.2 (1.9) 38.1 2.2 (1.7) 26.6 1.3 (1.3) 15.5 Streptocycli ne 4.4 (2.2) 52.4 3.7 (2.1) 44.4 3.4 (2.0) 40.5 3.0 (1.9) 36.1 3.0 (1.9) 35.7 2.7 (1.8) 31.8 1.6 (1.5) 19.4 Mancozeb 3.0 (1.9) 1.8 (1.5) 1.6 (1.4) 36.1 2.8 (1.8) 1.3 (1.3) 1.0 (1.2) 33.7 2.4 (1.7) 1.2 (1.3) 0.7 (1.1) 29.0 2.0 (1.6) 0.9 (1.2) 0.6 (1.0) 23.8 1.6 (1.5) 0.8 (1.1) 0.0 (0.7) 19.4 1.3 (1.3) 0.8 (1.1) 0.0 (0.7) 15.5 1.0 (1.2) 0.6 (1.0) 0.0 (0.7) 12.3 Blitox50 1.8 (1.5) 21.0 1.5 (1.4) 17.9 1.2 (1.4) 14.7 1.1 (1.3) 13.1 0.0 (0.7) 0.0 0.0 (0.7) 0.0 0.0 (0.7) 0.0 Streptomyci n sulphate Control 3.5 (2.0) 0.0 (0.7) 0.01 0.03 41.7 3.3 (2.0) 0.0 (0.7) 0.01 0.03 39.7 3.2 (1.9) 0.0 (0.7) 0.01 0.03 38.5 3.2 (1.9) 0.0 (0.7) 0.01 0.03 38.1 3.0 (1.9) 0.0 (0.7) 0.01 0.02 35.7 1.8 (1.5) 0.0 (0.7) 0.01 0.03 21.8 1.2 (1.3) 0.0 (0.7) 0.01 0.03 13.9 Captan Kocide 21.0 18.7 15.5 11.9 * Data in parenthesis represent √ (x + 0.5) transformed values 3573 14.3 8.7 11.1 6.8 9.5 0.0 9.1 0.0 7.1 0.0 Int.J.Curr.Microbiol.App.Sci (2020) 9(6): 3570-3575 Table.2 Evaluation of in vivo efficacy of chemicals against Xanthomonas oryzae pv. Oryzae Treatment Chemical name Trade name Mean PDI (%) T1 Chloramphenicol 500mg 77.8(61.9) T2 Chloramphenicol 500mg T3 2-bromo-2-nitropropane 1,3diol 2-bromo-2-nitropropane 1,3diol Streptomycin sulphate 90% + Tetracycline hydrochloride 10% Streptomycin sulphate 90% + Tetracycline hydrochloride 10% Mancozeb 75% WP Chloramphenicol @ 250ppm Chloramphenicol @ 100ppm Bactinash @ 250ppm Bactinash @ 100ppm Streptocycline @ 250ppm Streptocycline @ 100ppm T4 T5 T6 Mancozeb @ 250ppm Mancozeb 75% WP Mancozeb @ T8 100ppm Captan 50% WP Captan @ 250ppm T9 Captan 50% WP Captan @ 100ppm T10 Copper hydroxide 77% WP Kocide @ 1000ppm T11 Copper hydroxide 77% WP Kocide @ 750ppm T12 Copper oxychloride 50% WP Blitox50 @ T13 1000ppm Copper oxychloride 50% WP Blitox50 @ 750ppm T14 Streptomycin sulphate Streptomycin T15 Sulphate @ 250ppm Streptomycin sulphate Streptomycin T16 Sulphate @ 100ppm Control T17 SE(m)± C.D. 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Int.J.Curr.Microbiol.App.Sci. 9(06): 3570-3575. doi: https://doi.org/10.20546/ijcmas.2020.906.420 3575