Description:DETAILED DESCRIPTION
[0018] The detailed description of various exemplary embodiments of the disclosure is described herein with reference to the accompanying drawings. It should be noted that the embodiments are described herein in such details
15 as to clearly communicate the disclosure. However, the number of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the scope of the present disclosure as defined by the appended claims.
20 [0019] It is also to be understood that various quantities and ratios may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.
25 [0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms “a",” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,”
30 “comprising,” “includes” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements and/or

5 components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups thereof.
[0021] It should also be noted that in some alternative implementations, the functions/acts noted may occur out of the order noted in the figures. For
10 example, two figures shown in succession may, in fact, be executed concurrently or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.
[0022] In addition, the descriptions of "first", "second", “third”, and the like in the present invention are used for the purpose of description only, and are
15 not to be construed as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include at least one of the features, either explicitly or implicitly.
[0023] Unless otherwise defined, all terms (including technical and scientific
20 terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in
25 an idealized or overly formal sense unless expressly so defined herein.
[0024] It should be noted that the description merely illustrates the principles of the present subject matter. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described herein, embody the principles of the present subject
30 matter and are included within its scope.

5 [0025] An aspect of the invention refers to a bioformulation to control Fusarium dieback disease of tea plants comprising of
i. 20% Trichoderma harzianum (v/v)
ii. Distilled water- 80%
10 iii. 0.6% mannitol solution- 0.2%
iv. 1% peptone solution- 0.1%
v. 1% carboxy methyl cellulose- 0.1%
vi. 2% glycerol- 0.2%
vii. 0.06% Sunflower oil- 0.05%
15 [0008] In preferred embodiment of the invention, Trichoderma harzianum are used in form of submerged mycelial/spore broth culture.
[0009] In preferred embodiment of the invention, method of preparation of bioformulation of the present invention comprises steps as below-
20 i. Preparing concentrate solution (20%) of submerged mycelial culture of T. harzianum.
ii. Adding 80% distilled water in the solution of step to achieve CFU count for T. harzianum of 2 × 106 CFU/ml.

[0010] In preferred embodiment of the invention, method of preparation of bioformulation of present invention comprises steps as below-

5 i. Preparing concentrate solution (20%) of submerged mycelial broth culture of T. harzianum.
ii. Adding 80% distilled water in the solution of step (i) to achieve CFU count for T. harzianum of 2 × 106 CFU/ml.
iii. Mixing 0.2% mannitol solution (0.6%), 0.1 % peptone (1%),
0.1% carboxy methyl cellulose (1%), 0.1 % glycerol (2%) and 0.05% sunflower oil (0.6%) with continuous stirring.
15 iv. Mixing the solution prepared in step (iii) with T. harzianum concentrate of step (ii). Resultant is the bioformulation of present invention.
[0011] In preferred embodiment of the invention, bioformulation prepared
20 by the aforementioned method need to be further diluted and is used at 1%, 2%, and 5% concentrations (V/V) on tea plants as foliar spray, two rounds at 15 days intervals.
[0030] In another preferred embodiment of the invention, 2% concentration of the bioformulation is able to control up to 67.5% disease reduction of Fusarium dieback over control in tea plants.

[0031] Bioformulation of the present inventions has also significant improvement in the growth of tea bushes from defoliation at 2% concentration after two rounds of spray at 15-day intervals.

30 [0032] The effectiveness of the bioformulation at such lower concentration clearly shows the potentiality of constituents/volatiles of the T. harzianum.
[0033] In an embodiment of the invention, the shelf life of the bioformulation is 6 months at room temperature.
[0034] In a preferred embodiment, T. harzianum
used in the present invention have been isolated from the rhizosphere of tea plants from Tindharia tea garden, North Bengal, India.
[0035] The ITS (OQ703058.1) and tef-1 alpha (PP496405.1) sequences of T. harzianum
10 submitted to NCBI GenBank database. The strain is available at Mycology Laboratory, TRA Nagrakata, West Bengal and Institute of Microbial Technology, Chandigarh with accession number MTCC13385.

15 [0036] EXAMPLES
Hereinafter, the present disclosure is being described in further detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.
20 Example 1
Preparation of bioformulation (Trichoderma harzianum 20% SC) of present invention-

[0037] For the preparation of the concentrate solution, 4 L of active broth culture of T. harzianum was mixed in 16 L of double distilled water to make a total volume of 20 L. Further 200 ml mannitol (0.6%), 100 ml peptone (1%), 100 ml carboxy methyl cellulose (1%), 100 ml glycerol (2%), and 50 ml sunflower oil (0.6%) were added to the solution for enhancement of shelf life of the product and spore enrichment. The product has 6 months shelf life with CFU level 11.8 ×106 which is above the CIBRC standard (2.0 ×106 for Trichoderma sp.).
Example 2
Isolation of Trichoderma harzianum

[0038] Trichoderma harzianum was isolated from tea rhizosphere soil bound with roots of tea plants from Tindharia
25 tea garden, North Bengal, India, by serial dilution method. Trichoderma harzianum (FIG 1) was isolated on potato dextrose agar plate.
The ITS (OQ703058.1) and tef-1 alpha (PP496405.1) sequences of T. harzianum were submitted
5 to NCBI GenBank database. The strain is available at Mycology Laboratory, TRA Nagrakata, West Bengal and Institute of Microbial Technology, Chandigarh with accession number MTCC13385.
10 In vivo evaluation of bioformulation of present invention-
[0039] Tea plants infected from Fusarium dieback were sprayed with 2% of bioformulation of present invention two times at an interval of 15 days under field conditions.
[0040] Result- Reduction of Fusarium dieback upto 67.5%
15 was evidently visible (FIG 2) after just two applications of bioformulation as foliar spray at the interval of 15 days.
20 [0041] Besides, application of this bioformulation improved plant growth as we 8-9% increase in crop yield was also observed. Further, it was also found that application of this bioformulation recovered tea bushes defoliated from red spider mite and red rust attack with no harmful effect on plant as well as beneficial microorganisms.
25 [0030] It is evident from the foregoing that at the bioformulation of the present invention is effective to control Fusarium dieback upto 67.5% when used at a very low concentration. Effectiveness increases with the increase in the concentrations. Bioformulation provides a safe, ecofriendly, non-toxic, cost-effective method to control fungal disease and requires no
15 specific machinery to be produced.

[0031] Moreover, it has been seen that the Fusarium species pathogens causing dieback have the tendency to attain resistance against the fungicides/ antibiotics when high concentrations of the fungicides/ antibiotics are used. The bioformulation of the present invention
20 has the least chance of encountering such resistance as it comprises of very minute quantity of the constituents such that no individual components at such concentration are able to produce fungicidal effect but when native isolate having volatile producing properties are showing potential effect to control fungus upto 67.5%.
25 [0032] Advantages of the present invention
- Reducing side effects associated with chemicals
- Non-toxic
- Increased antifungal activity
[0033] It will be understood by those within the art that, in general, terms used
30 herein, and especially in the appended claims (e.g., bodies of the appended
5 claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced
10 claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not
15 be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or
20 “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number
25 (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations).
[0034] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is
30 determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when
combined with information and knowledge available to the person having ordinary skill in the art. , Claims: I/ WE CLAIM:
1. A bioformulation to control Fusarium dieback disease of tea plants comprising of
ii. Trichoderma harzianum (v/v) – 20%
iii. Distilled water- 80%
iv. 0.6% mannitol solution- 0.2%
v. 1% peptone solution- 0.1%
vi. 1% carboxy methyl cellulose- 0.1%
vii. 2% glycerol- 0.2%
viii. (0.06%) Sunflower oil- 0.05%
2. The method of preparation of Trichoderma harzianum 20% SC bioformulation as claimed in claim1 comprises steps as below-
i. Preparing concentrate solution (20%) of submerged mycelial culture of T. harzianum.
ii. Adding 80% distilled water in the solution of step (i) to achieve CFU count for T. harzianum of 2 × 106 CFU/ml.

3. A method of preparation of Trichoderma harzianum 20% SC bioformulation to control Fusarium dieback disease of
Tea plants comprise steps as below-
i. Preparation of 20% concentrate submerged mycelial culture of T. harzianum.
ii. Adding 80% distilled water in the solution of step (i) to achieve CFU count for T. harzianum of 2 × 106 CFU/ml.
iii. Mixing 0.2% mannitol solution (0.6%), 0.1 % peptone (1%),
0.1% carboxy methyl cellulose (1%), 0.1 % glycerol (2%) and 0.05% sunflower oil (0.6%) with continuous stirring.
iv. Mixing the solution prepared in step (iii) with T. harzianum of step (ii). Resultant is the bioformulation of present invention.

4. The bioformulation to control Fusarium dieback diseases in Tea plants as claimed claim 1 wherein bioformulation is used at 1%, 2%, and 5% concentrations (V/V) on tea plants as foliar spray, two rounds at 15 days intervals.

5. The bioformulation as claimed in claim 1 wherein Trichoderma harzianum is isolated from the rhizosphere of tea plants from Tindharia tea garden, North Bengal, India.
6. The bioformulation as claimed in claim 1 wherein Trichoderma harzianum is used in form of submerged mycelial broth culture.