Efficacy of Antibiotics and Biorational Pesticides against Bacterial Blight of Paddy

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Rice is the most important cereal crop in south east Asia. In Asia alone world's 96 per cent of rice is produced. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Ishiyama, 1922;Swings et al . 1990) is the major constraint for production all over the world causing 20-30 per cent yield losses (Ou, 1985;Islam andBora, 1998 andKumar et al.1999). Cultivation of high yielding varieties, intensive farming practices and application of high dose of nitrogen frequently aggravate the severity of BB. This leads to the reduced yield and grain quality. A very limited success is achieved in the control of this disease so far due to its fast spreading nature.
The severity and significance of damage caused by infection necessitated development of strategies to control and manage the disease so as to reduce crop loss and to avert an epidemic. Though the use of Bordeaux mixture, antibiotics, copper and mercurial compounds were resorted to in the early fifties, environmentally safe and stable chemical control agents rendering control at low concentrations are yet to be developed. Keeping this in view the present investigations were under taken to study management strategies against bacterial blight to reduce yield losses.

Material and Methods
An immunomodulator, Bactrinashak, antibiotics like streptocycline, tetracycline, cyclohexamide, Agrimycin 100, Copper oxychloride (COC), combination of Streptocycline + copper oxychloride and Streptocycline + Copper sulphate were tested at 250, 500, 750 and 1000 ppm under laboratory condition by paper disc or inhibition zone method against Xanthomonas oryzae pv. oryzae to know their efficacy in inhibiting the growth of bacteria. A heavy suspension of Xanthomonas oryzae pv. oryzae (7x 108 cfu/ml) was mixed with molten potato sucrose agar (PSA) contained in 500 ml Erlenmeyer flask, so as to get a thick growth of bacteria on the medium. The seeded medium was poured in sterilized petri plates and allowed to solidify.
Previously sterilized filter paper discs (Whatman No. 42) measuring five mm diameter were soaked in different antibiotic solutions for five minutes and transferred on to the surface of the seeded medium contained in petridishes. Filter paper discs dipped in sterile water served as check.The inoculated plates were placed first at 4 to 5oC in the refrigerator for 3 to 4 hrs to allow the chemicals to diffuse into the medium.Then the plates were incubated at 27 or 280 ± 10C for 48 hrs. Observations were recorded after 48 hr on the zone of inhibition produced around the filter paper discs by measuring the diameter of the inhibition using antibiotic inhibition zone scale.
Chemicals effective under in vitro were tested in farmer's field, where the experiment was laid out in randomized complete block design (RCBD) during the kharif season. The variety Sonamahsuri was grown and sprayed with different bio-rational pesticides, botanicals and bio-agent (Table 2). Two sprays were given one spray at 30 days after transplanting (DAT) as a prophylactic spray and second spray at 60 DAT. In control plot no spray was given. Disease incidence was recorded at 60 DAT and 90 DAT. The experiment included seven treatments and a check with three replications.
In each plot, 10 rice plants were selected diagonally representing the entire plot and labeled *1Corresponding author email: tgowda123@gmail.com with tags, in each plant five leaves were scored by using the 0-9 scale (Anonymous., 1996) as described below. 0= No affected area, 1= 1-5 per cent affected area, 3 = 6-12 per cent affected area, 5 = 13-25 per cent affected area, 7 = 26-50 per cent affected area, 9 = 51-100 per cent affected area, Final observation was taken on 90th day after transplanting. Per cent disease index (PDI) was worked out by using the formula given by Singh (2002).
At the time of harvesting yield returns due to chemical spray was calculated for each treatment over control by taking into consideration the cost incurred on chemicals, labour and additional yield obtained in terms of money over untreated check. Gross return (GR) = Total profit earned by selling the produce i.e., yield x selling rate of Paddy Net return (NR) = [Gross return -(cost of chemicals for two sprays + labour cost for spraying)]

Phenol Estimation
Leaf samples were collected a day after each spray. Leaf material was extracted in ethanol as per the procedure followed by Jaypal and Mahadevan (1968) and clarified with saturated solution of lead acetate. The excess lead acetate was precipitated by the addition of sufficient quantity of saturated solution of sodium hydrogen orthophosphate. The precipitate was re-separated by filtering the alcohol extract through Whatman No.1 filter paper and the 593 filtrate was made up to a known volume with 80 per cent alcohol. Phenols and ortho-dihydroxy phenols were estimated in the alcohol extract of fresh leaves.
Estimation of total phenols: Estimation of total phenols present in plant samples was carried out by Folin -Ciocalteau Reagent method and Arnow's method was used to estimate orthodihydroxy phenols.

In vitro evaluation of antibiotics
The differences among the treatments and concentrations were found to be statistically significant except between Streptocycline and Streptocycline + Copper sulphate. There was significant difference between the concentrations of treatments. Among them, streptocycline was significantly superior from the rest of the chemicals with mean inhibition zone of 22.83 mm (Table. 1) in all the tested concentrations, followed by streptomycin sulphate, streptocycline + Copper sulphate, Streptocycline + Copper oxychloride, Agrimycin 100 and Cyclohexamide (21.91 mm, 21.58 mm, 20.33 mm, 16.25 mm and 12.58 mm respectively). Copper oxychloride exhibited zero inhibition zone showing ineffectiveness. Among concentrations, 1000 ppm recorded the mean maximum inhibition zone (14.48 mm) followed by 750 and 500 ppm ( Table 1). The least inhibition was observed at 250 ppm (11.44 mm). per cent recorded the least per cent disease index Streptocycline showed the maximum inhibition at of 52.7 at 90 DAT followed by Bactrinashak @ 0.04 1000 ppm. While least was recorded at 250 ppm.
The data pertaining to grain yield are presented in Table 3. In the present study there was significant Two sprays of Streptocycline + copper oxychloride @ 0.025 % + 0.1% not only reduced the disease incidence but also gave the higher net returns Rs.12,503.00, followed by Bactrinashak @ 0.04 per cent that gave Rs.9,778.00 and Bactrinashak @ 0.03 per cent gave Rs.9,193.67.0 respectively. This was obviously due to the higher grain yield obtained in these chemical treated plots. Least net returns were obtained in Neem seed kernel extract + Cow urine + Vermiwash (Rs.-4695.00) ( Table 3).

Biochemical studies
Effectiveness of immunomodulator, biocontrol agent and botanicals on changes in total and orthodihydroxy phenols in leaf tissues of rice was studied ( Table 4). The data indicated that the amount of total phenol and ortho-dihydroxy phenol contents The maximum total phenol content of 312.3 μg / g of leaf weight was noticed in Pseudomonas fluorescens treated leaf, followed by Bactrinashak 210.4 μg / g of leaf weight after third spray. While, the least was in water sprayed control treatment (Table 4).
Similarly, the maximum total and ortho-dihydroxy phenol content of 73.95 μg / g of leaf weight was recorded in Pseudomonas fluorescens treated leaf while, the least was noticed in water spray (Table 4). The results of the present investigation are in agreement with earlier studies by Sindhan and Jaglan (1988) who reported that total phenols enhanced by application of fungicides and plants Table 4. Effect of immunomodulator, bioagents and botanical extracts on changes in total phenols and ortho-dihydroxy phenols in the leaf tissues of rice plant showed enhanced resistance to late leaf spot of groundnut. Even the higher levels of phenols were recorded in plants in which seeds were treated with non conventional chemicals like cycloheximides, cupric chloride, DL-phenyl alanine and indole-3acetic acid (Chawdhury, 1995).
Streptomycin is well known to interfere with ribosomes and protein biosynthesis (Traub, 1969). Although the mechanism of action of the antibiotics is highly variable, antibiotics are known to control plant diseases by acting on the parasites or on the host directly or after undergoing transformation with in the plant (Dutta, 1978). The harmful effect of streptomycin has been considered to be due to inhibition of translocation of accumulated starch and the absorption of manganese.
It has been widely recognized that one of the major biological properties of the aromatic compounds such as mono and dihydric phenols, phenolic glycosides, flavonoids, anthocyanins, aromatic amino acids and coumarin derivatives is their antimicrobial activity and it is often assumed that their main role in plants is to act as protective compounds against disease causing agents. Hence, in the present study effort has been made to find out the effect of botanical extracts, bioagents and chemical on the changes in phenolics.
Bactrinashak is an immuno modulator, when used as prophylactic treatment reduces the susceptibility of plant to bacterial diseases, by modifying the immune system of the plants. Bactrinashak alters the immune system of plant by changing the contents of phenols, protiens, nitrogen and certain enzymes and make the plants resistant to bacterial attack. Choloroplyll content is also increased (http://www.indofilcc.com). Mohan and Mahadevan (2001a) studied the effect of phenol on protein and amino acid content of Xanthomonas oryzae pv. oryzae and reported that phenols altered the protein content and it declined with the increasing concentration of phenol. SDS-PAGE electrophoresis of protein samples of control and phenol treated cells revealed a marked difference between them. Further, the peaks of control cells, which were of large protein molecules were broken to several smaller peaks (or small peptide molecules) in the phenol treated cells. The mode of action of phenol involves substantial changes in the protein metabolism of X. o. pv. oryzae. Fogg and Lodge (1945) proposed that the protein precipitating ability depends upon its concentration. Further, the rate of synthesis of enzymes would be reduced by the phenol, leading to decline in metabolism. The bactericidal action of phenol, according to Cooper (1912) is by precipitating proteins. Lipid profile of the cells of X. o. pv. oryzae was markedly affected by phenol treatment (Mohan and Mahadevan, 2001b) Velusamy et al. (2006) reported that certain plantassociated strains of fluorescent Pseudomonas spp. were known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It had antibacterial, antifungal, antiviral, and antihelminthic properties and played a significant role in the biological control of tobacco, wheat and sugar beet diseases, sub population of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. They also report that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%-64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl-) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight.