FIELD OF THE INVENTION

[0001] The present disclosure relates to the field of agriculture microorganism.
More particularly, the present invention relates to plant growth-promoting
rhizobacteria (biofertilizers) from Sesame indicum L for enhancing plant
growth and yield and a method for the same thereof.
BACKGROUND OF THE INVENTION
[0002] Sesame is an important oil crop cultivated in many parts of the world.
Sesame is mainly cultivated for its oil which is highly rich in protein content.
Sesame oil is used traditionally as well as in pharamaceutical sector for various
purposes such as cooking oil, in confectioners (cakes and pasteries), cosmetics,
soap industry etc. Presence of lignin antioxidants in sesame oil makes it unique
as it prevents oxidation of oil which provides long shell life to sesame oil.
Beside this, sesame also possesses many medicinal properties. Hence, sesame
is an important oil crop with medicinal potential which support need of large
scale cultivation of sesame crop.
[0003] There are several factors which limit or restrict the growth, cultivation and
productivity of sesame crop. Fluctuation in amount of rainfall has been directly
related to crop yield. However sesame plants to certain extent are drought
resistant. Mostly ordinary cultivation practices are commonly utilized for
cultivation of sesame mainly because the crop is cultivated only for 3-4 months
and in many parts is employed as a part of crop rotation. Limited use of
certified seeds and absence of modified, improved varieties also result in low
yield. Problems associated with harvesting, pests, insects, bacterial and fungal
diseases result sesame plant ultimately leading to extensive yield loss. Hence, there is need of developing cultural practices which could result in obtaining maximum yield.
[0004] In the present invention relates to overcome the obstacles of the prior art
and emphasizes on the investigations to develop an approach for the utilization
of Plant Growth Promoting Rhizobacteria as innoculum with the crops of
sesame to enhance growth and productivity.
[0005] The information disclosed in this background of the disclosure section is
only for enhancement of understanding of the general background of the
invention and should not be taken as an acknowledgement or any form of
suggestion that this information forms the prior art already known to a person
skilled in the art.
OBJECTS OF THE INVENTION
[0006] The principal object of the present invention is to overcome the
disadvantages of the prior art and provide plant growth-promoting
rhizobacteria (biofertilizers) from Sesame indicum L for enhancing plant
growth and yield and a method for the same thereof.
[0007] Another object of the present invention relates to the use of Plant Growth
Promoting Rhizobacteria as innoculum with the crops of sesame to enhance
growth and productivity.
[0008] Still another object of the present invention is to provide a method for
enhancing crop yield.
[0009] Yet another object of the present invention is to isolate bacteria from
rhizosphere of S. indicum.
[0010] Still another object of the present invention is production of indole acetic
acid (IAA) by bacteria isolated from rhizosphere of Sesame plant

[0011] These and other objects and advantages of the present subject matter will
be apparent to a person skilled in the art after consideration of the following
detailed description taken into consideration with accompanying drawings in
which preferred embodiments of the present subject matter are illustrated.

SUMMARY OF THE INVENTION
[0012] In an important embodiment, the present invention relates to provide a
plant growth-promoting rhizobacteria (biofertilizers) from Sesame indicum L
for enhancing plant growth and yield and a method for the same thereof.
[0013] In another embodiment the present invention relates to a combination
comprising plant growth promoting rhizobacteria (PGPR) PGPR, wherein the
PGPR is a consortium of PGPR; Bacillus; Pseudomonas and Azotobacter.
[0014] In a preferred embodiment the present invention relates to a method for
enhancing plant growth and yield by plant growth-promoting rhizobacteria
(PGPR) from Sesame indicum L.
[0015] In another embodiment is to provide a method for
enhancing plant growth and productivity comprising of :inoculating the soil
environment with plant growth promoting rhizobacteria (PGPR), wherein the
PGPR is a consortium of Pseudomonas aeroginosa, Bacillus subtilis and
Azotobacter; and growing a plant in said soil environment..
[0016] In another embodiment the plant growth promoting rhizobacteria(PGPR)
exhibit plant growth promoting activities like IAA production, siderophore
production, phosphate solubilizing ability and HCN production and also
possess antagonistic activity against pathogenic fungi M. phaseolina.
[0017] In yet another embodiment, plant growth promoting rhizobacteria(PGPR)
exert a growth enhancing effect on sesame plants.

[0018] The foregoing summary is illustrative only and is not intended to be in any
way limiting. In addition to the illustrative aspects, embodiments, and features
described above, further aspects, embodiments, and features will become
apparent by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
[0020] Fig. 1 illustrates the analysis of mount of IAA produced by different bacterial isolates, in accordance with an embodiment of the present invention;
[0021] Fig. 2 illustrates the effect of PGPR on growth of M. Phaseolina in Sesame rhizosphere C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T5= Pseudomonas+Azotobactor; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention;
[0022] Fig. 3 illustrates on plate 8 the effect of PGPR on germination rate of S. indicum 8(A-B) – Control, seeds with no treatment with PGPR 8C- Seeds treated with Pseudomonas spp. As PGPR 8D- Seeds treated with Azotobacter spp. As PGPR 8E- Seeds treated with consortium of Pseudomonas and Azotobacter. 8F- Seeds treated with consortium of Pseudomonas and B. subtilis, in accordance with an embodiment of the present invention;
[0023] Fig. 4 illustrates plate 9 the effect of PGPR on germination rate of S. indicum (invivo) 9A – Control, seeds with no treatment with PGPR 9B- Seeds treated with Pseudomonas spp. as PGPR 9C- Seeds treated with B. subtilis as PGPR 9D- Seeds treated with Azotobacter spp. as PGPR 9E- Seeds treated with consortium of Pseudomonas, B. Subtilis and Azotobacter, in accordance with an embodiment of the present invention;
[0024] Fig. 5 illustrates on plate 10 the difference in initial growth of control plants and plants treated with PGPR. 10(A-B) - Control plants of S.indicum 10C - Plant containing Bacillus as PGPR in rhizosphere. 10 D - Plant containing Pseudomonas as PGPR in rhizosphere. 10E- Plant containing Azotobacter as PGPR in rhizosphere. 10F- Mature plant with Pseudomonas+ Bacillus as PGPR. 10G- Mature plant with Pseudomonas+ Bacillus+ Azotobacter as PGPRs, in accordance with an embodiment of the present invention;
[0025] Fig. 6 illustrates on plate 11 the effect of PGPR on growth of Sesame plants 11 A- Control, plant with no treatment of PGPR 11 B- Plant treated with Pseudomonas as PGPR 11 C- Plant treated with Pseudomonas and Bacillus as PGPR, in accordance with an embodiment of the present invention;
[0026] Fig. 7 illustrates the effect of PGPR on no. of branches in Sesame plant C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention;
[0027] Fig. 8 illustrates the effect of PGPR on shoot length in Sesame plant C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention; and
[0028] Fig. 9 illustrates the effect of PGPR on root length of Sesame plant C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention;
[0029] The figure depicts embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] While the embodiments of the disclosure are subject to various modifications and alternative forms, specific embodiment thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the scope of the disclosure.
[0031] The terms “comprises”, “comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by “comprises… a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.
[0032] The present subject matter relates to relates to plant growth-promoting rhizobacteria (biofertilizers) from Sesame indicum L for enhancing plant growth and yield and a method for the same thereof.
[0033] Reference may be made to Figure 1 illustrating the analysis of mount of IAA produced by different bacterial isolates, in accordance with an embodiment of the present invention;
[0034] Reference may be made to Figure 2 illustrating the effect of PGPR on growth of M. Phaseolina in Sesame rhizosphere C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T5= Pseudomonas+Azotobactor; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention;
[0035] Reference may be made to Figure 3 illustrating on plate 8 the effect of PGPR on germination rate of S. indicum 8(A-B) – Control, seeds with no treatment with PGPR 8C- Seeds treated with Pseudomonas spp. As PGPR 8D- Seeds treated with Azotobacter spp. As PGPR 8E- Seeds treated with consortium of Pseudomonas and Azotobacter. 8F- Seeds treated with consortium of Pseudomonas and B. subtilis, in accordance with an embodiment of the present invention;
[0036] Reference may be made to Figure 4 illustrating on plate 9 the effect of PGPR on germination rate of S. indicum (invivo) 9A – Control, seeds with no treatment with PGPR 9B- Seeds treated with Pseudomonas spp. as PGPR 9C- Seeds treated with B. subtilis as PGPR 9D- Seeds treated with Azotobacter spp. as PGPR 9E- Seeds treated with consortium of Pseudomonas, B. Subtilis and Azotobacter, in accordance with an embodiment of the present invention;
[0037] Reference may be made to Figure 5 illustrating on plate 10 the difference in initial growth of control plants and plants treated with PGPR. 10(A-B) - Control plants of S.indicum 10C - Plant containing Bacillus as PGPR in rhizosphere. 10 D - Plant containing Pseudomonas as PGPR in rhizosphere. 10E- Plant containing Azotobacter as PGPR in rhizosphere. 10F- Mature plant with Pseudomonas+ Bacillus as PGPR. 10G- Mature plant with Pseudomonas+ Bacillus+ Azotobacter as PGPRs, in accordance with an embodiment of the present invention;
[0038] Reference may be made to Figure 6 illustrating on plate 11 the effect of PGPR on growth of Sesame plants 11 A- Control, plant with no treatment of PGPR 11 B- Plant treated with Pseudomonas as PGPR 11 C- Plant treated with Pseudomonas and Bacillus as PGPR, in accordance with an embodiment of the present invention;
[0039] Reference may be made to Figure 7 illustrating the effect of PGPR on no. of branches in Sesame plant C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention;
[0040] Reference may be made to Figure 8 illustrating the effect of PGPR on shoot length in Sesame plant C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention; and
[0041] Reference may be made to Figure 9 illustrating the effect of PGPR on root length of Sesame plant C= Control; T1= Bacillus; T2= Pseudomonas; T3=Azotobacter; T4= Pseudomonas+Bacillus; T6= Pseudomonas+Bacillus+Azotobacter, in accordance with an embodiment of the present invention;
[0042] In accordance with an embodiment of the present invention relates to the , combination and method of using plant growth-promoting rhizobacteria (biofertilizers) from Sesame indicum L for enhancing plant growth and yield.
[0043] In another embodiment the present invention relates to a combination comprising plant growth promoting rhizobacteria (PGPR) PGPR, wherein the PGPR is a consortium of PGPR; Bacillus; Pseudomonas and Azotobacter.
[0044] In a preferred embodiment the present invention relates to a method for enhancing plant growth and yield by plant growth-promoting rhizobacteria (PGPR) from Sesame indicum L. wherein the method for enhancing plant growth and productivity comprises of :inoculating the soil environment with plant growth promoting rhizobacteria (PGPR), wherein the PGPR is a consortium of Pseudomonas aeroginosa, Bacillus subtilis and Azotobacter; and growing a plant in said soil environment..
[0045] In another embodiment the plant growth promoting rhizobacteria(PGPR) exhibit plant growth promoting activities like IAA production, siderophore production, phosphate solubilizing ability and HCN production and also possess antagonistic activity against pathogenic fungi M. phaseolina. Besides, the plant growth promoting rhizobacteria(PGPR) exert a growth enhancing effect on sesame plants.
[0046] Rhizosphere is defined as the soil surrounding and under the influence of roots of the host plant. Rhizosphere is known to be inhabited by many bacterial species which are found in soil (rhizosphere region) and possess several plant growth promoting activities. Such microbial strains are referred to as PGPR?s. In the present study PGPR were isolated from rhizosphere of Sesame indicum. A total of 22 bacteria were isolated based upon their ability to grown on Tryptone soya agar, KB agar medium and Bacillus agar medium. Isolated bacteria were subjected to morphological and biochemical analysis. Results obtained from morphological an biochemical analysis were further utilized to develop dendograms to reveal genetic similarity among different bacteria isolated from rhizosphere of S. indicum and also to establish similarity between the isolates and the reference /standard bacterial cultures(Azotobacter, Bacillus, Pseudomonas).
Methodology:
[0047] Example 1: Sample preparation: Collection of sample Certified seeds of Sesame were procured from Centre Research centre, Pantnagar and plants obtained from seeds were maintained at Botanical Garden of KL DAV PG College and Chinmaya Degree College, Haridwar. The plants were watered with normal tap water as per requirement. No additional manure or fertilizers were added to the plants. Three standard bacteria strains (procured from IMTech Chandigarh, utilized in present study were as follows:
[0048] Table 1: List of reference (Standard) bacterial culture utilized in the present study

[0049] Collection of soil sample and preparation of test sample: The Sesame plants selected for the study were gently removed from soil. The plants were shaken (slowly) to remove the soil loosely attached to roots of plants completely to obtain rhizosphere. This rhizosphere was utilized as study material for further studies.
[0050] Collected soil sample from rhizosphere of Sesame plant were serially diluted from 10-2 to 10-6 diluted samples. Approximately 1 gm of roots were excised from plant and transferred to glass bottle containing 99 ml of phosphate buffer saline (PBS). The bottle containing roots and PBS were shaken to dissociate bacteria associated with the roots. Bottles were placed on a rotator shaker for 2-3 hours for the purpose. This solution was subsequently utilized for preparation of dilutions from 10-2 to 10-6 respectively.
[0051] Isolation of bacteria and maintenance of pure culture through pour plate and streak plate method Three media namely TSA, King agar medium and Bacillus agar medium were utilized in the present study for selective isolation of PGPR from rhizosphere of Sesame plant.
[0052] Example 2: Evaluation of bacterial isolates for their PGPR activities: Indole acetic acid production: For estimation of IAA activity each bacterial isolate was inoculated in nutrient broth and tubes were incubated for 12 – 14 hrs About 2 ml of the test culture was collected from each culture tube and centrifuged at 10,000 rpm for15 min. Supernatant was filtered through whatman filter paper. 1 ml of supernatant of each isolate was taken in separate test tubes and to each tube 2 ml Salper?s reagent was added dropwise but rapidly with continuous mixing. The samples were placed in dark for 30 minutes. Development of pink colour was assayed with spectrophotometer at 535 nm.
[0053] Siderophore production The isolated bacteria was evaluated for their siderophore producing ability by using blue agar medium.Standardized blue agar medium was prepared and each bacterial isolate was inoculated onto separate petriplate and incubated at 27?C for 48 hours. Siderophore production was indicated by appearance of orange halos around the colonies after incubation.
[0054] Antagonistic activities of bacterial isolate against Macrophomina phaseolina All the bacterial isolates were examined for their antagonistic ability against M. phaseolina..
[0055] Seed bacterization by PGPR strain and their effect on growth parameters A field experiment was conducted to check the effect of selected bacterial strain on plant growth. Preparation of slurry: Added 10g sugar in 100ml distilled water (sterile) and it was heated to dissolve. After dissolving sugar about 40g gum Arabic was added to hot solution. It was mixed well to make slurry, cooled and added the innoculum to slurry and mixed again.
[0056] for 3-4 times and keep seeds in sterile distilled water until inoculated.
[0057] Seed innoculation The prepared innoculum was added to seed to obtain a uniform coat of PGPR culture around the seeds. The seeds were dried in shade and then sown in pots containing sterile soil. For all the treatments the germination rate, and growth parameters in terms of shoot length, leave size were measured.
[0058] Example 3: Evaluation of plant growth promoting attributes of bacterial isolates: Indole acetic acid (IAA) production IAA is a crucial plant growth regulator which is required by plants for normal growth and development. bacteria were naturally and actively involved in indole acetic acid production.
[0059] Siderophore production Siderophore production is another important PGPR activity which is equally beneficial for the growth of plants. In the present study cultures SI01, SI04, SI05, SI06, SI08, SI11, SI12, SI13, SI15, SI16, SI18, SI19, SI20, SI22 were found positive to be production of siderophore whereas other isolates exhibited no siderophore production.
[0060] Effect of PGPR on count of M. phaseolina in sesame rhizosphere Isolate PGPR; Bacillus (T1); Pseudomonas (T2) and Azotobacter (T3) were evaluated for their effectiveness to restrict the growth of M.phaseolina. Inseparate experiments were conducted to evaluate antagonistic ability of these isolates individually as well as in combination revealed that all three bacteria possess significant amount of inhibitory effect on growth of M. phaseolina. However, antagonistic effect was more pronounced when these bacteria were utilized in combination as consortium for treatment. In this case the pathogen count was decrease to a limiting value of 0.7*1000/g of soil. The results obtained clearly indicated that the isolated bacteria inhibitor restricts the growth of pathogenic fungi and hence promote growth of plants.
[0061] Effect of PGPR on growth of Sesame: Effect of application of PGPR on growth of sesame plant In experiments conducted to analyze effect of utilization of PGPR onto germination rate of Sesame seeds, it was found that there was no significant increase in percentage of germination between control (no treatment with PGPR) seeds and seeds innoculated with isolated PGPR (Plate 8). However, seeds treated with PGPR do exhibited rapid and fast germination as compared to control seeds. Beside that enhanced growth of seedling in terms of growth of radical and plumule and eventually elongation of cotyledons was exhibited by seeds treated with PGPR as compared to control seeds (Plate 9). Results obtained in experiments conducted to evaluate the effect of PGPR isolated from rhizosphere of sesame on growth of sesame plant clearly indicated that morphological growth was improved and enhanced in the plants which were treated with PGPR as compared to that of control (Plate 10). Among different combinations utilized consortium of Bacillus, Pseudomonasand Azotobacter (T6) was found to give best results with a 7.8 branches per plant. Similarly enhanced growth of roots was also obtained in the plants treated with PGPR?s as compared to control (Plate 11).
[0062] Effect of PGPR’s of seed yield of S.indicum As seen in other cases utilization of isolated bacteria (Pseudomonas, Bacillus, and Azotobacter) as biofertilizers in sesame crops resulted in enhanced production of seeds.
[0063] Table 2: Effect of PGPR on seed yield of S. indicum
[0064]
C= Control; T1= Pseudomonas; T2= Bacillus; T3=Azotobacter; T4= Pseudomonas+Bacillus; T5= Pseudomonas+Azotobactor; T6= Pseudomonas+Bacillus+Azotobacter
[0065] Root colonization study Consortium of Pseudomonas+Bacillus (T4) and Pseudomonas+Bacillus+Azotobacter (T6) exhibited enhanced root colonization ability. About 5.21±0.23 cfu/gm were recorded in consortium T4 after 30 days which increased to 5.71±0.23 after 60 days of inoculation. Consortium T6 revealed presence of 5.01±0.18 cfu/gm after 30 days of inoculation which increased to a maximum count of 5.92±0.21 cfu/gm after 60 days on inoculation.
[0066] Table3: Root colonization in rhizosphere of S. indicum by inoculated bacterial strain

[0067] To summarize, it can be concluded from the present study that utilization of PGPR as innoculum in soil in which sesame plants were cultivated, resulted in enhanced growth of sesame plants. Increase in number of leaves, and branches, enhanced shoot and root length and rapid germination rate was obtained in the seeds treated with PGPRs as compared to control. Also, PGPRs effect on sesame growth was more pronounced when the bacteria were utilized as consortium as compared to the result obtained when PGPR’s were utilized individually. The present supported the utilization of PGPRs for obtaining better growth and yield from sesame plants. Also, all these bacteria Pseudomonas, Bacillus, and Azotobacter were found to be beneficial for sesame growth if utilized as PGPR.
[0068] Although embodiments for the present subject matter have been described in language specific to structural features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system/component of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the scope of the present subject matter.

We Claim:

1. A biofertilizer combination comprising plant growth promoting rhizobacteria (PGPR) PGPR, wherein the PGPR is a consortium of PGPRs; Bacillus; Pseudomonas and Azotobacter.
2. The biofertilizer combination as claimed in claim 1, wherein the plant growth-promoting rhizobacteria (PGPR) is isolated from the rhizosphere of Sesame indicum L.
3. A method for enhancing plant growth and productivity comprising of :inoculating the soil environment with plant growth promoting rhizobacteria (PGPR), wherein the PGPR is a consortium of Pseudomonas aeroginosa, Bacillus subtilis and Azotobacter; and growing a plant in said soil environment..
4. The combination or method for enhancing plant growth and productivity as claimed in claim 1 or 4, wherein the plant growth promoting rhizobacteria(PGPR) exhibit plant growth promoting activities like IAA production, siderophore production, phosphate solubilizing ability and HCN production.
5. The combination or method for enhancing plant growth and productivity as claimed in claim 1 or 4, wherein the consortium gives maximum antagonistic activity against pathogenic fungi M. phaseolina.
6. The combination or method for enhancing plant growth and productivity as claimed in claim 1 or 4, wherein plant growth promoting rhizobacteria(PGPR) exert a growth enhancing effect on sesame plants.