Introduction

Rice is perhaps the most widely cultivated food crop world over and it has a special benefit as it has eight of essential amino acid in delicately balanced proportion ¹.Rice production is constrained by diseases of fungal, bacterial and viral origin . Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv .Oryzae (Xoo) is one of the oldest known diseases and subsequently, its incidence has been reported from different parts of Asia, northern Australia, Africa and USA²_. Blight disease is a vascular disease resulting in a systemic infection that produces tannish-grey to white lesions along the veins ³. Symptoms are observed at the tillering stage, disease incidence increases with plant growth, peaking at the flowering stage3 ⁴ .The bacterial blight disease is known to occur in epidemic proportions in many parts of the world, incurring severe crop loss of up to 50% ⁵ . The severity and significance of damages caused by infection have necessitated the 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 and other copper and mercurial compounds were resorted to in the early fifties, environmentally safe and stable chemical control agents rendering control at very low concentrations are yet to be developed ⁶. Today, the exploitation of host resistance appears to be the only reliable method of disease management ⁷.The other novel way is to use antagonistic organisms to kill a positive agent ⁸.The present investigation is to identify antagonistic bacteria against Xanthomonas Oryzae. Pr. Oriyazae for eco-friendly control of the pathogen.

Material and Methords

Method of sterilization

Moist heat sterilization was done by autoclave for sterilization of glass wares such as beaker, test tubes, patriplates and conical flasks at 121° C at 15 PSI pressure for 15 minutes. Dry heat sterilization was done by hot air oven at different temperature for sterilization. Sprit lamp is used for sterilization, nicrome ware loop and for creating sterilizing zone.

Preparation of Different media

Different media were used for culturing microorganism by maintaining suitable condition. Potato Sucrose Agar media, Potato Dextrose Agar media and Peptone Glycerol Agar media were prepared at suitable conditions and sterilized by pouring these solution into conical flasks by raping them properly.

Plate preparation

Petri plates, Test tubes and other glasswares were rapped properly and autoclaved. After that all Autoclaved glassware’s were dried properly by hot air oven. Glassware’s then kept inside laminar flow for 15 minutes treating with UV light. Before transferring the media from conical flasks to Petri plate’s hands were washed with 70% alcohol. Under laminar floor beside sterile zone of sprite lamp media were transferred to patriplates and allow them to cool. After cooling the media the patriplates were inverted to avoid moisture.

Isolation of Bacteria from Bio product

Serial dilutions of bi product were done for detection of different bacteria by following method.

1:10 dilution

2 µl of sample were added with 18 µl of distilled water. From these above solution 10 µl were taken and 500 µl (1% agar) were added.

1:100 dilutions

2 µl of sample were added with 198 µl of distilled water. From these above solution 10 µl were taken and 500 µl (1% agar) were added.

1:1000 dilutions

5 µl of sample were added with 5ml. of distilled water. From these above solution 10 µl were taken and 50 µl (1% agar) were added.

1:10,000 dilutions

10 µl of solution were taken from 1:1000 dilutions then 90 µl of distilled water were added from this 10 µl of sample were taken and 500 µl (1% agar) were added.

Maintenances of Mother Culture 

Mother Cultures were maintained properly with suitable media like P.S.A for different isolates. Pure mother cultures were maintained inside test tube and Petri plates. These tubes or plates were wrapped properly by the help of cotton and Para film. After proper wrapping these mother cultures were kept inside incubator at 300 C for 24 hours for micro-Isolates growth properly. After complete growth of microbes these mother cultures were transferred to refrigerator for storing.

In vitro test against B.L.B

Individual isolates were taken from mother culture and allow them to grow in Petri plates by serial streaking and kept inside incubator. Bacteria Xanthomonas was also cultured in Petri plates by serial streaking method. Each individual Isolate was cultured along with Xanthomonas simultaneously by different streaking method to observe their growth rate and action. Xanthomonas was cultured one day after with each individual isolates by parallel streaking. Individual isolates were also cultured among themselves by parallel streaking to observe their different mechanism and action like antagonistic effect. Xanthomonas was placed in the middle of the plate and other isolates were placed to the surrounding of the plate to observe the growth rate.

In vivo test against B.L.B

Different bacteria were cultured in patriplates in suitable condition .Little amount of sterile water were added to patriplates carefully. Bacterial spores were brushed up carefully and transferred to glass beaker. Few amount of distilled water were added to the above solution to prepare final bacterial solution. These solutions were transferred into a test tube for proper mixing of the solution with the help of a shaker.

Inoculation of Rice plant and Disease assessment

The pot of rice plants were labeled with the isolates to be inoculated. Pair of sterile scissors was dipped into the inoculums suspension. Leaves were clipped 1-2 cm from the tip with the pair of scissors infested with bacteria.

Different clipped diseases leaves were measured by its disease length, lesion type after 14 days of inoculation. The disease reaction of the test cultivars to the 5 races.

Results

By in vitro testing, antagonistic effects of bio product bacteria’s were found in Petri plates among with Xanthomonas by parallel streaking. Different isolates produce their reaction against xanthomonas in Petri plates and able to inhibit the growth of xanthomonas in in-vitro condition. After getting antagonistic effect of bio-products isolates in vitro condition, in vivo testing was done by inoculating rice plant in net house following results were observed between test and control rice plant statistically by taking 8 disease replication of 5 different isolates and 1 control in 5 different dates .The disease progress curve of five isolates and results are given in Graph no. 1 and table no.2 .

Graph 1: Disease progress curve of five isolates.   Click here to View figure

Table 1,2: Percentage of infection recorded.

Discussion

Crop loss assessment studies have revealed that bacterial blight disease reduces grain yield to varying levels, depending on the stage of the crop, degree of cultivar susceptibility and to a great extent, the conduciveness of the environment in which it occurs . The severity and significance of damages caused by infection have necessitated the 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 and other copper and mercurial compounds were resorted to in the early fifties, environmentally safe and stable chemical control agents rendering control at very low concentrations are yet to be developed. Today, the exploitation of host resistance appears to be the only reliable method of disease management. Bacterial blight disease management can reduce the initial inoculums and subsequent development of the pathogen on host plants and this can be accomplished through chemical protection, host plant resistance, and biological control. Biological control can be done by isolating antagonistic bacteria which can completely overcome the BLB.

References

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