Claims:We Claim,

1. A liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant comprising an effective amount of Bacillus subtilis, Pongamia oil, and Chitosan along with other suitable carriers or excipients.

2. The liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 1 wherein the Bioformulation is in the form of foliar spray.

3. The liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 1 wherein the other suitable carriers or excipients are selected from glycerol, butylated hydroxy anisole and dextrose.

4. The liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 1 wherein the effective amount of Bacillus subtilis is ranges from 18% of Bioformulation, with 1000 ppm chitosan

5. The liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 1 wherein the effective amount of Pongamia oil is ranges from 78.95% of Bioformulation.

6. The liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 1 wherein the effective amount of chitosan is ranges from % to % of Bioformulation.

7. The liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 1 or 3 wherein the effective amount of other suitable carriers or excipients is ranges from 2% (w/v) glycerol, 0.05% (w/v)butylated hydroxy anisole and 1% (w/v) dextrose of Bioformulation.

8. A liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant comprising 18 (w/w) Bacillus subtilis, 80% (w/w) Pongamia oil and % (w/w) chitosan, 2 (w/w) glycerol, 0.05% (w/w) butylated hydroxy anisole and 1% (w/w) dextrose of Bioformulation.

9. A process for the preparation of liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant comprising:
a. preparation of active culture of Bacillus subtilis by inoculation of a single colony of Bacillus subtilis into culture media with the presence of chitosan;
b. then incubation at optimum temperature for 48 hrs until the OD reaches to 0.4 at 600 nm;
c. after culturing the concentrating of the Bacillus subtilis cells by centrifugation;
d. then the addition of other suitable carriers or excipients which are selected from glycerol, butylated hydroxy anisole and dextrose. to the concentrated Bacillus subtilis cells in broth; and
e. then to the above broth obtained at step (d), the pure Pongamia oil is added slowly to form the liquid Bioformulation for the management of Bacterial Blight disease in the Pomegranate plant.

10. The process for preparation of liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant as claimed in claim 9 wherein the amount of Bacillus subtilis ranges from 18 % (w/w)- including chitosan 1000 ppm, pongamia oil ranges from 78.95 % (w/w), chitosan ranges from % (w/w) to % (w/w) glycerol ranges from 2% (w/w), butylated hydroxy anisole ranges from 0.05% % (w/w) and dextrose ranges from 1 % (w/w) of total Bioformulation.
, Description:FORM-2

THE PATENTS ACT, 1970
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION

BIOFORMULATION OF BACILLUS SUBTILIS FOR MANAGEMENT OF BACTERIAL BLIGHT IN POMEGRANATE

1. PAVAN KUMAR
DEPARTMENT OF BIOTECHNOLOGY,
BASAVESHWAR ENGINEERING COLLEGE, BAGALKOT-587103,
KARNATAKA, INDIA.

2. MANJUNATH GIRIGOWDA
ASSISTANT PROFESSOR, COLLEGE OF HORTICULTURE,
UHS BAGALKOT, MYSORE-571130, KARNATAKA, INDIA.

AND
3. BHARATI S. METI
PROFESSOR & HoD DEPARTMENT OF BIOTECHNOLOGY,
BASAVESHWAR ENGINEERING COLLEGE, BAGALKOT-587103,
KARNATAKA, INDIA.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed

FIELD OF THE INVENTION

The present disclosure relates to the Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease. It particularly relates to the Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease in the Pomegranate plant. It specifically relates to the liquid Bioformulation of Bacillus subtilis with Pongamia oil and chitosan as a foliar spray for the management of Bacterial Blight disease in Pomegranate and when applied to pomegranate plant as a foliar spray makes the plant less susceptible against Xanthomonas axonopodis pv. punicae by inducing systemic resistance against this pathogen. The invention also relates to the process for the preparation of the Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease in the Pomegranate plant. The invention further relates to the method for the management of Bacterial Blight disease in the Pomegranate plant by using the Bioformulation of Bacillus subtilis.

BACKGROUND OF THE INVENTION
Pomegranate is an important cash crop of India, also grown in tropical and subtropical nations of the world. Pomegranate is regarded as an important functional food for its nutraceutical and health-promoting properties [Johanningsmeier, S.D., and Harris, G.K. (2011). Pomegranate as a functional food and nutraceutical source. Annu. Rev. Food. Sci. T. 2, 181-201. https://doi.org/10.1146/annurev-food-030810-153709]. India is the largest producer with a total of 246 thousand hectares of area under cultivation (Anonymous, 2019). Even though such huge land is under cultivation the annual production is not meet as per requirement. Bacterial blight diseasecaused by axonopodis pv. punicae (Xap) is one of the major hindrances in pomegranate cultivation. The bacterial blight disease can cause up to 60-8-% of yield loss under field conditions [Ramesh, C. & Ram, K. (1991). Studies on bacterial blight (Xanthomonas campestris pv. punicae) of pomegranate. Indian Phytopathology, 44, 370–372]. Management of BB relies on multiple applications of synthetic antibiotics and copper based compounds. Even, the combination of these two has proven successful for disease management [Sharma, K., Sharma, J., & Jadhav, V. (2015). In Recent Advances in Diagonosis and Management of plant Disease, 119–126. Springer Publishers]. Presently, large numbers of chemical/ synthetic fertilizers have been extensively used for disease management and high yield. Continues application of synthetic antibiotics will lead to residual toxicity and have a negative impact on the environment also leads to water eutrophication, contaminate groundwater as these antibiotics have a longer degradation period [Vessey, J. K. (2003). Plant and Soil, 255(2), 571–586. doi:10.1023/a:1026037216893]. Hence there is a need for alternate eco-friendly mean for management of the disease without any adverse effect on soil health, environment, and ensuring consumer as well. PGPR (plant growth promoting rhizobacterias) is considered to be an important approach in overcoming a problem with the excessive use of synthetic chemicals [E. Malusà, G. Sala, W. Chitarra, L. Bardi / Improvement of response to low water availability in maize plants inoculated with selected rhizospheric microbial consortia under different irrigation regimes. EQA, 12 (2013) 13-21. Doi- 10.6092/issn.2281-4485/4209; ] (Bardi and Malusà 2012; Bashan, Y., de-Bashan, L. E., Prabhu, S. R., & Hernandez, J.-P. (2013). Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013). Plant and Soil, 378(1-2), 1–33. doi:10.1007/s11104-013-1956-x]. Upon the identification of a particular beneficial microbe, it is very necessary to develop a suitable carrier material for a successful transfer of active bioagent from laboratory conditions to field conditions. Hence, developing a suitable bioformulation with an effective carrier material for any microbe is very necessary for plant protection strategies.
Bioformulation generally consists of a suitable carrier material, which helps for maintaining the viability of an active bioagent for a longer period to sustain its biological effect. The carrier material added along with the active bioagent in a bioformulation make the bioagent safe to handle, makes easy to apply, maintain cell count, make it convenient for transport and most importantly stability and storage under normal condition, without any deterioration for the longer time period. The overall aim of developing a formulation is to ensure adequate viability of a bioagent, increase the efficiency of the bioagent, and facilitate proper delivery.
Bacterial Blight caused by Xanthomonas axonopodis pv. punicae, is one of the major constrain for pomegranate production causing 60-80% of yield loss yearly [Sharma, J. et al. Pomegranate bacterial blight: symptomatology and rapid inoculation technique for Xanthomonas axonopodis pv. punicae. J. Plant. Pathol 99, 109-119, 2017]. Current management practices include frequent and excessive use of synthetic antibiotics, potentially harms the environment and health of consumers [Vassilev N., Vassileva M. 2003. Biotechnological solubilization of mineral phosphates on media containing agro-industrial wastes. Appl Microbiol Biotechnol 61:435–440].
The hazardous effect of these chemicals and their longer degradation period in the environment strongly necessitates the search for novel bioformulation for harmless means of disease management. Plant beneficial microbes are considered as an important means for overcoming the extensive use of synthetic pesticides and fertilizers. The use of active bioagents and their products are widely studied for disease management in agriculture practice. Bioagents are usually planted growth-promoting rhizobacteria (PGPR), that enhance the plant vigor, maintain the soil fertility, and develop the resistance against the pathogen through various mechanisms [Glick, B.R., 2012. Plant growth-promoting bacteria: mechanisms and applications. Scientifica (Cairo) 2012, 963-401].
Bacillus subtilis is one of the most common bioagents widely used for the management of most of the plant diseases. Bacillus species are the non-pathogenic bacterium having several advantages over other PGPRs as they produce endospores which make them survive under very high temperatures, extreme pH, and osmotic conditions [Turner, J.T, Backman, P.A. 1991. Factors relating to peanut yield increase after seed treatment with Bacillus subtilis. Plant Dis 75:347–353]. Active cultures of Bacillus can be used as foliar applications as well as for soil amendment. Greenhouse evaluation of Bacillus subtilis has been reported to reduce disease severity of Cucumber mosaic virus (CMV) on tomato by inducing ISR in plants [Zehnder, G. W., Yao, C., Wei, G., and Kloepper, J. W. 2000. Influence of methyl bromide fumigation on microbe-induced resistance in cucumber. Biocontrol Sci. Tech.10:687-693].
[Tahir, H. A. S., Gu, Q., Wu, H., Niu, Y., Huo, R., & Gao, X. (2017). Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt. Scientific Reports, 7(1). doi:10.1038/srep4048] reported species of Bacillus to stimulate the plant growth in tomato by enhanced expression of auxin synthesis gene. In another study Krause et al., 2003 [Krause, M. S., De Ceuster, T. J. J., Tiquia, S. M., Michel, F. C., Jr., Madden, L. V., and Hoitink, H. A. J. 2003. Isolation and characterization of rhizobacteria from composts that suppress the severity of bacterial leaf spot of radish. Phytopathology 93:1292-1300] reported elicitation of induced systemic resistance (ISR) against the foliar disease of radish caused by Xanthomonas campestris pv. armoraciae. Similarly, many types of research have been reported for successful management of various foliar and soil-borne pathogens using active bacillus species [Ryu, C.M., Farag, M. A., Hu, C.-H., Reddy, M. S., Wei, H.-X., Paré, P. W., and Kloepper, J. W. 2003. Bacterial volatiles promotes growth in Arabidopsis. Proc. Natl. Acad. Sci. 100:4927-4932.; and Joseph W., Kloepper, Choong-Min Ryu, and Shouan Zhang. 2003. Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp. Phytopathology. 94 (11): 1259-1266].
Jayasudha et.al., 2018 [S.M. Jayasudha, K.C. Kirankumar, R.K. Mesta and Ramesh Ippikoppa, Int. J. Curr. Microbiol. App. Sci (2018) 7(4): 317-324] reported liquid formulation of Bacillus subtilis-KK-9A, Brevibacillus borstelensis-BK-6, Brevibacillus sp-PM-2A, Lysinibacillus xylanilyticus-VK-6B, and consortium were developed using distilled water, groundnut oil, nutrient broth, Pongamia oil, and sunflower oil to enhance the shelf life and efficacy of the biocontrol agent besides easing the delivery of bio-inoculants through micro-irrigation techniques. The highest population level of Bacillus subtilis-KK-9A, Brevibacillus borstelensis-BK-6, Brevibacillus sp-PM-2A, and Lysinibacillus xylanilyticus-VK-6B were observed in Pongamia oil amended with glycerol even after 3 months of storage. Pongamia oil-based formulation was found best formulation followed by groundnut oil, sunflower oil, distilled water, nutrient broth-based formulation which retains the highest per cent survival of Bacillus subtilis (45.05), Brevibacillus borstelensis (32.93), Brevibacillus sp (48.80), Lysinibacillus xylanilyticus (60.80), and consortium 31.12 per cent even after 90 days.
Bacillus based microbial formulations: Optimization of the production process
Stojanović et. al., 2019 [Sandra Stamenković Stojanović, Ivana Karabegović, Vladimir Beškoski, Nada Nikolić and Miodrag Lazić, Hem. Ind. 73 (3) 169-182 (2019)] reported the Bacillus sp.-based microbial formulations have found wide application in many fields: from pharmacy and medicine to environmental protection and agriculture due to the ability of this species to produce various metabolites and to form endospores. Recently, these products have gained popularity as biopesticidal and phytostimulatory agents, which are a “green” alternative to overused agrochemicals. In order to obtain a high-quality and long-lasting product with desired characteristics, it is necessary to optimize the production process at each stage, which implies coordinating the microbial species, the type, and the conditions of microbial cultivation along with formulation technologies. This paper provides a concise overview of the most important findings in this area, regarding characteristics of microbial formulations and specific criteria that need to be met when such a product is formulated. It should serve as a beginning point for everyone starting new research, not just in the field of biofertilization and biological control of plant diseases, but generally in the field of biochemical engineering.
El-Hassan and Gowen, 2006 [S. A. El-Hassan and S. R. Gowen, J. Phytopathology 154, 148–155 (2006)] reported different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log10 CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log10 CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease
caused by Fusarium oxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc, and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments, the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field applications.
Schisler et al., 2004 [Schisler, D. A., Slininger, P. J., Behle, R. W., and Jackson, M. A. 2004, Phytopathology 94:1267-1271] reported that the maximizing the potential for successfully developing and deploying a biocontrol product begins with a carefully crafted microbial screening procedure, proceeds with developing mass production protocols that optimize product quantity and quality, and ends with devising a product formulation that preserves shelf-life, aids product delivery, and enhances bioactivity. Microbial selection procedures that require prospective biocontrol agents to possess both efficacy and amenability to production in liquid culture increase the likelihood of selecting agents with enhanced commercial development potential. Scale-up of biomass production procedures must optimize product quantity without compromise of product efficacy or amenability to stabilization and formulation. Formulation of Bacillus spp. for use against the plant, pathogens is an enormous topic in general terms but limited in published specifics regarding formulations used in commercially available products. Types of formulations include dry products such as wettable powders, dusts, and granules, and liquid products including cell suspensions in water, oils, and emulsions. Cells can also be microencapsulated. Considerations critical to designing successful formulations of microbial biomass are much fold and include preserving biomass viability during stabilization, drying, and rehydration; aiding biomass delivery, target coverage, and target adhesion; and enhancing biomass survival and efficacy after delivery to the target. Solutions to these formulation considerations will not necessarily be compatible. Data from several biocontrol systems including the use of B. subtilis OH 131.1 (NRRL B-30212) to reduce Fusarium head blight of wheat are used to illustrate many of these issues. Using our recently described assay for efficiently evaluating biomass production and formulation protocols, we demonstrate the effectiveness, in vitro, of UV protectant compounds lignin (PC 1307) and Blankophor BBH in reducing OH 131.1 morbidity when cells were exposed to UV light from artificial sunlight.

The United States Patent no. US 6,896,883 discloses the invention which is directed to isolated Bacillus Subtilis, Pseudomonas putida, and Sporobolomyces roseus which are useful as a biocontrol agent. These organisms are useful in a method of imparting to plants protection against plant pathogens by applying them to plants, plant Seeds, or Soil Surrounding plants under conditions effective to impart disease protection to the plants or plants produced from the plant Seeds. The biocontrol agents are also useful in a method of enhancing plant growth which involves applying them to plants, plants Seeds, or SoilSurrounding plants under conditions effective to enhance growth in the plants or plants produced from the plant Seeds.

The thesis titled “Epidemiology and management of bacterial blight of pomegranate caused by Xanthomonas axonopodis pv. punicae (Hingorani and Singh) Vauterin et al.” authored by S.T.Yenjerappa 2009 available at http://krishikosh.egranth.ac.in/handle/1/69499 discloses that by considering the magnitude and resultant losses due to bacterial blight in pomegranate, investigations were undertaken on the disease, pathogen, environment, and management aspects. The survey revealed the highest disease incidence in Chitradurga, Anantapur, and Koppal districts, and the lowest incidence and severity were recorded in Bellary district. The bacterium was rod-shaped, gram-negative and capsulated. It was positive to starch hydrolysis, gelatin, liquefaction and H2S production. Modified D-5 medium was found superior in supporting the growth of the pathogen. Cultural variability among the 20 different isolates revealed the variability in growth and colony characters. The isolates exhibited 100 percent polymorphism for OPA20, OPB03, OPF07, and OPF10 primers showing significant molecular variability. Among the different seasons, mrigbahar was found most vulnerable and hastbahar was found relatively safe in avoiding the disease. Rainfall for a longer period, maximum temperature between 29.4-35.60C, and minimum temperature between 19.5 to 27.30C, RH of 63-87 per cent were found favorable for the disease development and spread. Pathogen survived up to 20 to 22 and 18 to 20 weeks in the infected residues buried in sterilized and unsterilized soil conditions, respectively. Neem, tridax and achyranthes were the alternate hosts for the pathogen. Bordeaux mixture 1% spray was very effective in reducing the initial inoculum of the pathogen. In vitro and in vivo evaluation of bactericides indicated that bronip (0.05%) + COC (0.2%) was highly effective in managing the disease with higher yield levels. In biological control, Bacillus subtilis, Pseudomonas fluorescens and garlic extract (10%) were significantly effective in reducing the disease. The application of multi nutrient (1%) recorded the lower incidence and severity of the disease. The IDM strategy evaluated was found successful and feasible in managing the disease than farmers’ method of disease control.

Though there are few prior arts which reported for the biocontrol agent, Bacillus subtilis and its formulations but none of the prior arts in the literature reported or disclosed the liquid Bioformulation of Bacillus subtilis for management of Bacterial Blight disease in Pomegranate plant with higher efficacy rate. Also, the prior arts reported are having several drawbacks of their own for application in the fields. Thus, there is a need for a development of suitable Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease in Pomegranate with the identified candidate as bioagent (Bacillus subtilis) having antibacterial property against Xanthomonas axonopodis pv. punicae (Xap) and developed an oil-based formulation for effective management of the disease. Therefore, the present inventors developed the Bioformulation for the management of Bacterial Blight disease in Pomegranate plant with the identified candidate as bioagent (Bacillus subtilis) having antibacterial property against Xap and developed an oil-based formulation for effective management of the disease. The present bio-formulation contains the active culture of Bacillus subtilis, isolated from the native rhizospheric soil of pomegranate, along with chitosan and Pongamia oil as the main ingredients, which, when applied to pomegranate plant as a foliar spray makes the plant less susceptible against Xanthomonas axonopodis pv. punicae by inducing systemic resistance against this pathogen for better crop productivity and sustainable agriculture.

OBJECTIVES OF THE INVENTION

The primary object of the invention is to develop Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease.

The particular object of the present invention is to develop Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease in the Pomegranate plant.

The specific object of the present invention is to develop the liquid Bioformulation of Bacillus subtilis with Pongamia oil and chitosan as a foliar spray for the management of Bacterial Blight disease in Pomegranate and when applied to pomegranate plant as a foliar spray makes the plant less susceptible against Xanthomonas axonopodis pv. punicae by inducing systemic resistance against this pathogen.

The further object of this invention is to provide a natural alternative product that effectively controls Bacterial Blight disease in the Pomegranate plant.

The further object of this invention is to develop a process for preparation of the Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease in the Pomegranate plant.

The further object of this invention is to develop a method for the management of Bacterial Blight disease in the Pomegranate plant by using the Bioformulation of Bacillus subtilis.

Yet another object is to provide an effective Bioformulation of Bacillus subtilis for the management of Bacterial Blight disease in the Pomegranate plant which is safe and environmentally friendly.

Yet another object is to provide an effective Bioformulation of Bacillus subtilis for management of Bacterial Blight disease as foliar spray induces resistance against disease and enhances the fertilizer use efficacy. Similarly prophylactic foliar application resulted in reduced disease compared to chemical synthetic antibiotics, which are commonly used in pomegranate cultivation.

Yet another object is to provide an effective Bioformulation of Bacillus subtilis for management of Bacterial Blight disease which improves the reproductive parameters such as yield and productivity.

A further object is to provide an eco-friendly delivery system of active bioagent Bacillus subtilis for foliar application in bacterial blight disease management in pomegranate.

A further object is to provide a safe liquid Bioformulation of Bacillus subtilis product that does not cause any adverse effects on people/Environment.

It is a further object of the present invention to provide herbal based liquid Bioformulation of Bacillus subtilis product having a longer shelf life.

It is a further object of the present invention to develop liquid Bioformulation of Bacillus subtilis products which are inexpensive and cost effective.

It is a further object of the present invention to develop liquid Bioformulation of Bacillus subtilis product which is easy to use with little technical expertise.

STATEMENT OF THE INVENTION
A liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant comprising an effective amount of Bacillus subtilis, Pongamia oil, and Chitosan along with other ingredients, for stabilizing the enhancing storage ability of the formulation.
The Bioformulation is in the form of liquid, applied as a foliar spray.
The other suitable carriers or excipients are selected from glycerol, butylated hydroxy anisole, and dextrose.
The effective amount of Bacillus subtilis is ranges from 18 % (w/w) of Bioformulation including 1000 ppm chitosan
The effective amount of Pongamia oil is ranges from 78.97% (w/w) to % of Bioformulation.
The effective amount of chitosan ranges from % to %
The effective amount of other suitable carriers or excipients is ranges from 2 % (w/v) glycerol, 0.05% (w/w) butylated hydroxy anisole and 1% (w/w) dextrose of Bioformulation.
A liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant comprising 18% Bacillus subtilis ( including 1000ppm Chitosan), 80% (w/v) Pongamia oil and 2 % (w/w) glycerol, 0.05 % (w/w) butylated hydroxy anisole and1 % (w/w) dextrose of Bioformulation.
A process for preparation of liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant comprising:
a. preparation of active culture of Bacillus subtilis by inoculation of single colony of Bacillus subtilis into culture media with the presence of chitosan ;
b. then incubation at optimum temperature for 48 hrs until the OD reaches to 0.4 at 600 nm;
c. after culturing the concentrating of the Bacillus subtilis cells by centrifugation;
d. then addition of other suitable carriers or excipients which are selected from glycerol, butylated hydroxy anisole and dextrose. to the concentrated Bacillus subtilis cells in broth; and
e. then to the above broth obtained at step (d), the pure pongamia oil is added slowly to form the liquid Bioformulation for management of Bacterial Blight disease in Pomegranate plant.
The amount of Bacillus subtilis is 18% Bacillus subtilis ( including 1000ppm Chitosan), 80% (w/v) Pongamia oil and 2 % (w/w) glycerol, 0.05 % (w/w) butylated hydroxy anisole and1 % (w/w) dextrose of Bioformulation.

SUMMARY OF THE INVENTION

The Bioformulation contain liquid based delivery system of the active bioagent Bacillus subtilis, which is having an antibiotic property against Xanthomonas axonopodis pv. punicae a bacterial blight causing organism in pomegranate.
The bioformulation contain active bioagent Bacillus subtilis, with enhanced bio-efficacy through chitosan oligomers, glycerol as a storage material for active culture, dextrose as a sugar source for the bioagent and BHA (Butylated hydroxy anisole) for reducing toxicity and liquid carrier material Pongamia oil extract.
The primer aim of the invention is to develop an eco-friendly delivery system of active bioagent Bacillus subtilis. For foliar application in bacterial blight management of pomegranate.
The Bioformulation product developed in the present invention can be easily stored under room temperature up to 10 months from the date of production.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

Figure 1: Green house evaluation of different component of Bioformulation and their effect
on disease severity and protection over the control. T1- control, T2- Chitosan, T3- Bacillus subtilis, T4 Bacillus subtilis + Chitosan, T5- Bioformulation.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to the Bioformulation of Bacillus subtilis for management of Bacterial Blight disease. It particularly relates to the Bioformulation of Bacillus subtilis for management of Bacterial Blight disease in Pomegranate plant. It specifically relates to the liquid Bioformulation of Bacillus subtilis with Pongamia oil and chitosan as a foliar spray for the management of Bacterial Blight disease in Pomegranate and when applied to pomegranate plant as a foliar spray makes the plant less susceptible against Xanthomonas axonopodis pv. punicae by inducing systemic resistance against this pathogen. The invention also relates to the process for preparation of the Bioformulation of Bacillus subtilis for management of Bacterial Blight disease in Pomegranate plant. The invention further relates to the method for management of Bacterial Blight disease in Pomegranate plant by using Bioformulation of Bacillus subtilis.
Pomegranate is one of the important commercial fruit crops known for its rich medicinal and nutraceutical values. The crop production is limited by the bacterial blight disease caused by Xanthonomas axonopodis pv. punicae and accounting of 60-80 % of yield loss. Use of biologicals for the management of this disease is prospective option, as antibiotics and synthetics use will have residucal toxicity and consumer repercussions. In this context, a candidate bioagent Bacillus subtilis UHSBs11 (KX950664) was identified from sick plots of pomegranate, studied the antimicrobial properties against the pathogen Xanthonomas axonopodis pv. punicae. The mass production techniques were standardised and the liquid based formulation was developed impregnating plant extracts, chitosan, and other substrate molecules that are required for the stability of Bio-agent. Application of this formulation as foliar spray induces resistance against disease and enhances the fertiliser use efficacy. Similarly prophylactic foliar application resulted in reduced disease compared to chemical synthetic antibiotics, which are commonly used in pomegranate cultivation. Further, formulation improved the reproductive parameters such as yield and productivity.

The Bacillus subtilis candidate bioagent (16 rDNA partial gene sequence submitted to NCBI accession number - KX950664).
It is the first liquid based formulation for bacterial blight (BB) disease management.
The product is having the enhanced shelf life of bioagent.
The elicitations of Bacillus subtilis bio-efficacy through chitosan oligomers.
The Pongamia oil has antibiotic property against BB of Pomegranate and it also acts as insecticides and has No residual toxicity on leaf.

Table 1. Contents of the developed Bioformulation
Sl. No Content Source Purpose
1. Bacillus subtlis Soil sample Active Bioagent to induce resistance against pathogen
2. Pongamia oil Plant extract Bioagent delivery source
3. Chitosan Structural elements in the exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of fungi. Enhance plant immune activity
4. Glycerol Synthetic molecules/synthesized Naturally also Preservative
5. Butylated hydroxy anisole Synthetic molecules reduce toxicity
6. Dextrose Sugar source Sugar source for microbial culture

Table 2. Bioformulation of the Invention.

Sl. No Contents of formulation Possible range of these ingredients in the formulation Amount (in % or in weight) in formulation
1. Bacillus subtlis 180 ml – from 2000 ml broth culture
0.4 OD* at 600 nm 18% including chitosan
2. Chitosan 1000 ppm/L of broth 1 gm/L
3. Pongamia oil 789.5 ml/L 78.95
4. Glycerol 20 ml 2%
5. Butylated hydroxy anisole 500 ppm/L 0.05%
6. Dextrose 2 gm 1%

Bioformulation disclosed here is a liquid-based carrier material Pongamia oil and for delivery of Bacillus subtilis, through the foliar spray to control bacterial blight of pomegranate and enhance plant vigour.

The Bioformulation is prepared by
1. Active culture preparation - Bacillus subtilis mass production
2. Incubation at optimum temperature.
3. Concentrating the biomass
4. Addition of other supplements
5. Preparation of bioformulation
6. Packing and storage

Application of Bioformulation:

The prepared formulation must be applied to the plant as foliar application by mixing in water, 4ml of bioformulation is mixed in one litre of water.
Application of this formulation as foliar spray induces resistance against disease and enhances the fertiliser use efficacy. Similarly prophylactic foliar application resulted in reduced disease compared to chemical synthetic antibiotics, which are commonly used in pomegranate cultivation. Further, formulation improved the reproductive parameters such as yield and productivity.

WORKING EXAMPLES:

Detailed protocol for formulation development:
All the procedure mentioned below are conducted in a sterile laminar hood chamber
1. Active culture preparation: Production protocol include inoculation of single colony of bacillus subtilis using sterile nichrome loop in a 2000 ml Tryptic Soy nutrient medium ( Autoclaved at 121 ˚C for 15 minutes ).
Media composition - 34 gm Trypeptone, 6 gm Soyatone, 5 gm K2HPO4 , 5 gm Dextrose, 10 gm Nacl, and Chitosan 500 ppm to the final volume 2000 ml of water and PH 7 ± 0.5.
2. Incubation: After inoculation cells the allowed to grow under the optimum temperature of 29 ± 2˚C for 48hrs of until the OD reaches to 0.4 at 600 nm.
3. Concentrating the biomass: once the cells were developed completely or the cell density reaches to 0.4 (600 nm), the cells were concentrated by centrifuging at 4000 rpm for 10 min at 4˚C. out of 2000 ml only 1850 ml of the medium is centrifuged remaining 150 is retained all the concentrated cells were stored in the same.
4. Addition of other supplements: To the retained 150 ml of broth with active Bacillus cell mass, 50 ml of sterile glycerol is added along with 1 gm of BHA (Butylated hydroxy anisole) and 0.5 gm of dextrose to make the final volume 200 ml. all the
5. Preparation of bioformulation: To the 200 ml of active culture 800 ml of a 99 % pure pongamia oil is added slowly mixed by flipping.
6. Packing and storage: The developed formulation can be stored in an airtight container and stored in a room temperature

Experiments conducted using the present formulation

1. Greenhouse evaluation and field condition

1.1 Evaluation of Bacillus subtilis and chitosan

The experiment was conducted to evaluate the efficiency of Bacillus, Bacillus in combination with chitosan and chitosan alone and bioformulation including all the combinations at a known concentration under the greenhouse condition and compared with untreated control. The study was conducted in three replicates and average results are represented in the table. Treatment was carried out by spraying the pathogen 24 hrs prior to the treatment infection. The observance was recorded for disease severity from day 9and final observance was recorded on 15 days post pathogen inoculation. The highest disease severity was observed in water treatment control with 48 %, followed by chitosan with 16%, then Bacillus subtilis (0.35 OD), the efficiency was increased in a combination of Bacillus subtilis + Chitosan showing 8 % disease severity. The bioformulation developed shows more efficiency by recording only 6%disease severity. The highest disease protection was recorded in bioformulation developed with 86 %, followed by Bacillus subtilis+ Chitosan, treatment of Bacillus subtilis alone, and chitosan with 84, 78 and 65 % respectively.

Table 3. Effect of using different content of bioformulation and combination of bioformulation on bacterial blight of pomegranate. All the experiments were conducted in three replicates and average results were given in the table.

Treatment Disease Incidence (%) Protection (%)
T1- Water control 48.243
(43.97) 0.00
T2- Chitosan 16.547
(23.98) 65.70
T3- Bs- Bacillus subtilis 12.58
(20.69) 78.77
T4- Bs- Bacillus subtilis + Chitosan 8.293
(16.7) 84.75
T5- Bioformulation 6.661
(14.84) 86.06947
CD 3.87
Se(m) 1.717
CV 11.39

2. Field evaluation of the Bioformulation
2.1` Disease severity Analysis – The developed Bioformulations were evaluated for their efficacy under field condition and compared with the chemical synthetics for two successive seasons and disease severity was recorded for leaf blight, twig blight, and fruit blight incidence. The experiments were conducted as randomized complete block design. Disease severity on the leaf, fruit, and twigs was calculated based on the formulae demonstrated by Singh et al 2015 [Singh, N. V. et al. Genetic diversity and association mapping of bacterial blight and other horticulturally important traits with microsatellite markers in pomegranate from India. Mol. Genet. Genomics 290, 1393-1402, 2015] in each replicates. The detailed results were shown in Table 4 and 5 for two seasons.

Table 4. Effect of prepared Bioformulation on bacterial blight incidence on pomegranate leaf, fruits, and twigs under field condition, season 1.

Season 1 – disease incidence
Treatment Leaf Twig Fruit
Bio- formulation 20.79(27.10) 44(41.55) 5.45(13.46)
Streptocycline 17.27(24.54) 40(39.23) 16.29(23.78)
Water Control 75.48(60.32) 47(43.27) 90.61(72.17)
CD 4.98 3.31 1.81
CV 3.02 4.06 3.32
SEM 0.53 0.58 0.32

Table 5. Effect of prepared Bioformulation on bacterial blight incidence on pomegranate leaf, fruits, and twigs under field condition, season 2.

Season 2 – disease incidence
Treatment Leaf Fruit Twig
Dalimbe rakshaka formulation 18.92(25.77) 7.01(15.33) 41(39.83)
Streptocycline 19.7(26.34) 18.35(25.35) 42(40.399)
Water Control 71.31(57.62) 84.39(66.74) 54(47.29)
CD 2.56 2.58 1.95
CV 4.37 4.78 2.32
SEM 0.45 0.45 0.34

2.2 Effect of Bioformulation on total yield:
The developed bioformulation efficiency on total yield, yield per plant, average fruit weight, and the total number of fruit per plant were evaluated and compared with the chemical synthetics for two successive seasons. Two years of the data are shown in Table 6 and 7.

Table 6. Effect of Bioformulation on number of fruits at the time of fruit harvest, fruit yield calculated in season 1.

Season 1 – Yield parameter
Treatment Total number of fruits/plant Average weight of fruit ( gm) yield/plant
( Kg) Total yield /acre
(Tons)
Dalimbe rakshaka formulation 95.33 226.6667 21.61 8.64
Streptocycline 79.00 204.3333 16.14 6.46
Water Control 22.33 141 3.15 1.26

Table 7. Effect of Bioformulation on number of fruits at the time of fruit harvest, fruit yield calculated in season 2.

Season 2 – Yield parameter
Treatment Total number of fruits/plant Average weight of fruit ( gm) yield/plant
( Kg) Total yield /acre
(Tons)
Dalimbe rakshaka formulation 92.67 234 21.68 8.67
Streptocycline 101.67 242 24.60 9.84
Water Control 87.00 207.6667 18.07 7.23
Treatment 25.67 141 3.62 1.45

3. Effect of storage on the viability of the bioagent
For any developed formulation it is very necessary to examine the survivability status of a bioagent to examine their effect on storage. Prepared Bioformulations were studied for their viability under storage conditions for a period of 240 days. Serial dilution of the prepared Bioformulations was carried in sterile water and spread on to selective TSA medium. After 24 hrs of incubation under optimum condition, colony-forming-units/ml of the formulation were calculated. The same was repeated every two weeks with the same stored sample. The results of the total colony count of the formulation are shown in Table 8.

Table 8. Total CFU count of the developed Bioformulations.

Bioformulation ( Stored under room temperature) 30days 60days 180days 240days
Total CFU count / ml 9 ×109 8×109 8×108 7×108

Table 9. Synergistic effect demonstration of the Bioformulation of the invention for control of Bacterial blight disease of Pomegrenate.

Sl. No Ingredients Amount in % or weight or cfu Antibacterial activity
1. Bacillus subtlis * 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-
2. Pongamia oil 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-
3. Chitosan 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-
4. Glycerol 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-
5. Butylated hydroxy anisole 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-
6. Dextrose 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-
7. Bioformulation of the invention 1×109 cfu/mL or 0.4 OD at 600 nm -Nil-