TECHNICAL FIELD

[001] The present invention generally relates to the field of bioinoculants. More particularly, the present invention relates to methods for the preparation and application of efficient Rhizobium bioinoculants in a host-specific manner for application to leguminous plants in soils with poor nutrition thereby enhancing soil fertility, nodulation and crop growth.

BACKGROUND

[002] Nodulation in legumes provides a major conduit of available nitrogen into the biosphere. The development of nitrogen-fixing nodules results from a symbiotic interaction between nitrogen fixing bacteria and legume plants. Among the nitrogen fixing bacteria, Rhizobium is one of the most studied because it nodulates legumes, which are environmentally significant in soil nitrogen fertility management of cultivated lands. The degree of host specificity varies tremendously among the rhizobia. Some strains have a very narrow host range while others have a very broad host range. The establishment of symbiotic relationship between legume species and rhizobia is quite complex.

[003] Agroforestry has considerable potential, not as the only way to improve agricultural production, but as one important way to enhance and maintain overall productivity of the small upland farm, the agricultural unit that is becoming more prevalent in many parts of the world. Nitrogen fixing tree species (NFT) possess all the virtues of multipurpose tree species (MPT) and are given top priority in agroforestry. The relevance of microbial nitrogen fixation and especially symbiotic fixation to agricultural productivity has sustained interest in this phenomenon for a century. A large number of biotic and abiotic factors can contribute to the inability of an inoculant strain to nodulate under field conditions. Currently establishment of nitrogen fixing bacteria (NFB) in the mycorrhizosphere and manipulation of these microbial associations as a biotechnological tool to enhance plant growth are two important topics of research. Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation.

[004] An efficient Rhizobium is a strain that is able to compete in the field with other indigenous rhizobia for the colonization of the rhizosphere of its homologous legume partner, under various soil physical and chemical conditions. This efficient strain will form many large nitrogen-fixing nodules on the roots of the plant host that will supply, for most legumes, from 70% to 90% of the plant need in nitrogen. Inoculations of Rhizobium sp. causes a greater increase in growth and yield and the number of nodules per root system is significantly higher in plants inoculated with Rhizobium sp. compared to plants without Rhizobium sp. under field condition. In addition to their beneficial N2- fixing activity with legumes, rhizobia can improve plant P nutrition by mobilizing inorganic and organic P. Current methods for culturing Rhizobium in lab conditions are costlier and needs skilled personnel and not every farmer can afford such Rhizobium cultures.

[005] In the light of aforementioned discussion, there exists a need to develop easy and cost effective methods for the preparation and application of efficient host-specific Rhizobium bioinoculants that can be carried out by farmers. Furthermore, to further unravel the potential of Rhizobium sp., there exists a need for the optimization of Rhizobium bioinoculants with reference to specificity to host plants for the Rhizobium bioinoculant to function without any adaptation problems. The present invention discloses innovative methods involving preparation and application of host-specific rhizobium bioinoculant formulations which can be employed for improved nodulation and plant growth in legumes. The present invention further discloses Rhizobium bioinoculant formulations produced by such methods.

BRIEF SUMMARY

[006] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later. [007] Exemplary embodiments of the present disclosure are directed towards methods for preparation and application of host-specific efficient Rhizobium bioinoculant formulations and the process comprises of the following steps: a) Collecting a number of forest soil sample from an undisturbed forest area; b) Mixing a number of forest soil sample to form a mixture of the forest soil sample; c) Layering the mixture of forest soil sample in a suitable container with a layer of test soil in between the forest soil samples. The test soil is the soil taken from a field to be cultivated with a desired leguminous plant; d) Sowing seeds of the desired leguminous plant in the mixture of the forest soil sample layered in the suitable container; e) Growing the seeds into the desired leguminous plant under suitable conditions in the suitable container for a predetermined period; f) Selecting the best supporting root nodules from one of the desired leguminous plant, which exhibits desired growth parameters and presence of large proportion of root nodules; g) Separating the best supporting root nodules from the desired leguminous plant; h) Washing the best supporting root nodules with either water or diluted ethanol or both to get sterile Rhizobium bioinoculant formulation; and i) Applying the Rhizobium bioinoculant formulation to the test soil in the field to be cultivated with the desired leguminous plant either directly or after crushing in a predetermined quantity of distilled water.

[008] Yet another exemplary embodiment of the present disclosure is directed towards a hostspecific efficient Rhizobium bioinoculant formulations. A mixture of forest rhizospheric soil samples is layered along with a test soil for growth of a desired legume under controlled conditions. The plant that exhibits desired growth parameters and a large proportion of root nodules is selected and the best supporting root nodules are collected for producing such Rhizobium bioinoculant formulations. [009] It is an object of the present invention to disclose an innovative method which involves preparation and application of efficient rhizobium root nodules which are specific to legume tree/crop variety and is also farmer friendly in the form of a Rhizobium bioinoculant. The main advantage of the disclosed method is that instead of isolating and culturing the rhizobia in lab conditions for large scale production, the root nodules of the legume plants which contain the Rhizobium bacteria can be separated and spread in the agricultural fields directly or by crushing the root nodules, which will support better plant growth and nodulation when compared to other bioinoculants which are legume crop/tree specific. This method does not need skilled personnel but can be carried out even by layman and farmers without scientific knowledge and laborious processes. Also, the disclosed Rhizobium bioinoculant has less adaptation problems. Furthermore, selection of Rhizobium bioinoculants in relation to the specific legume plant variety results in efficient strains of Rhizobium. Another advantage is that these bioinoculants can be applied to soils with poor nutrition thereby enhancing soil fertility, nodulation and crop growth.

BRIEF DESCRIPTION OF DRAWINGS

[010] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:

[011] FIG. 1 is a diagrammatic representation of a method for preparation and application of host specific efficient Rhizobium bioinoculant formulations, in accordance with a non-limiting exemplary embodiment of the present disclosure.

[012] FIG. 2 is a graphic representation of effects of nodulation in Sesbania grandiflora by BD-2 host specific rhizobial strain in agricultural, barren and polluted soils.

DETAILED DESCRIPTION [013] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[014] The use of "including", "comprising" or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms "first", "second", and "third", and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[015] According to a non limiting exemplary embodiment of the present disclosure, methods for preparation and application of host-specific efficient Rhizobium bioinoculant formulations for enhancing soil fertility, improved nodulation and leguminous crop growth are disclosed. [016] In accordance with a non limiting exemplary embodiment of the present disclosure, hostspecific efficient Rhizobium bioinoculant formulations prepared by employing best supporting root nodules from leguminous plants is disclosed.

[017] According to a non limiting exemplary embodiment of the present disclosure, a method for the preparation and application of a host-specific efficient Rhizobium bioinoculant formulation is disclosed. The method starts with a first step of collecting a number of forest soil samples from an undisturbed forest area. Next the collected forest soil samples are mixed uniformly to form a mixture of the forest soil sample. In the next step, the mixture of forest soil sample is layered in a suitable container with a layer of test soil at the bottom and the mixture of the forest soil sample at the top. In a different embodiment, the test soil is layered between two layers of the mixture of the forest soil sample. The test soil is the soil taken from a field which is to be cultivated with a desired leguminous plant or plant barren, polluted and agricultural soils. Subsequently, a number of seeds of the desired leguminous plant are sowed in the mixture of the forest soil sample layered in the suitable container and grown under suitable conditions in the suitable container for a predetermined period. The best growth supporting root nodules are collected from one of the desired leguminous plant exhibiting good growth parameters and presence of a large proportion of root nodules. The best growth supporting root nodules are collected from the desired leguminous plant and washed with either water or diluted ethanol or both to obtain a sterile Rhizobium bioinoculant formulation. Subsequently, the Rhizobium bioinoculant formulation is applied to the test soil in the field to be cultivated with the desired leguminous plant either directly or after crushing in a predetermined quantity of distilled water.

[018] Referring to FIG. 1, it is a diagrammatic representation of a method for preparation and application of host-specific efficient Rhizobium bioinoculant formulations, in accordance with a non limiting exemplary embodiment of the present disclosure. Three different forest soil samples FSl, FS2 and FS3 were used in the method. FSl comprises of a mixture of soil samples from 15 different sites in FSl forest area; FS2 comprises of a mixture of soil samples from 15 different sites in FS2 forest area and FS3 comprises of a mixture of soil samples from 15 different sites in FS3 forest area. In separate pots, each forest soil sample (FSl, FS2 and FS3) is layered as a top layer over test soil samples and the seeds of the leguminous plants were sown in the forest soil sample layer. As can be seen in the given figure, the seedlings emerging from the forest soil samples showed enhanced growth when compared with the control. Best plant growth supporting root nodules were selected from a particular plant grown in the pot having FSl soil sample based on the plant growth and presence of large proportion of nodules. The best growth supporting root nodules were collected from the desired leguminous plant and after the desired steps of cleaning and processing, it was used as the Rhizobium bioinoculant formulation. The formulation when employed in the field was found to be the best supporter of plant growth and nodulation.

[019] In accordance with a non limiting exemplary embodiment of the present disclosure, the forest soil sample is collected from about fifteen different sites in the radius of 15 km distance. The forest soil sample is a rhizospheric soil and the rhizospheric soil is associated with legume plants. The mixture of forest rhizosphere soil was placed as a single layer of 2-3 cm on top of the test soil samples in pots and then those pots were sown with same batch seeds of the specific legume host. Forest soils have rich microbial diversity and the rhizobium present in the forest soil samples get associated with the roots of the seedlings that is being grown. When the best supporting root nodules from such a sample is used as a bioinoculant for the same host under field conditions, the host- specific Rhizobium bioinoculant formulation is very efficient and effective in supporting improved nodulation and hence improved plant growth.

[020] According to a non-limiting exemplary embodiment of the present disclosure, the Rhizobium bioinoculant formulation is applied as a liquid spray. The best supporting root nodules can be directly separated from the tree/crop, washed with water and crushed in distilled water and sprayed in the field for best growth.

[021] In accordance with a non-limiting exemplary embodiment of the present disclosure, the Rhizobium bioinoculant formulation is mass produced at the commercial level by collecting root nodules from the legume plants grown in the field followed by washing with 50% ethanol and crushing in distilled water to yield a liquid formulation. These liquid formulations that serve as biofertilizers can be stored for long period under appropriate conditions without losing their viability. The mass production may also be done in handy by farmers involving picking up 100- 150 healthy root nodules, thorough washing and crushing them in 5-10 liters of distilled water and sprayed in the large area of desirable fields. This method can be employed for any different types of legume crop/trees in barren, polluted and agricultural soils.

[022] According to a non-limiting exemplary embodiment of the present disclosure, a hostspecific efficient Rhizobium bioinoculant formulation comprising a best supporting root nodules separated from one of a desired leguminous plants followed by washing the best supporting root nodules with either water or diluted ethanol or both is disclosed. A mixture of forest rhizospheric soil samples is layered along with a test soil for growth of a desired legume under controlled conditions. The plant that exhibits desired growth parameters and a large proportion of root nodules is selected and the best supporting root nodules are collected for producing such Rhizobium bioinoculant formulations. Examples:

Effect of host-specific root nodules on Agreforestry tree species:

[023] In accordance with a non-limiting exemplary embodiment of the present disclosure, evaluation of the effectiveness of host-specific root nodules on nodulation and growth in agroforestry tree species (AFT) viz. Acacia nilotica, Albizzia lebbeck, Gliricidia maculata, Sesbania grandiflora and Pongamia pinnata from Jakaram, Eturunagaram, Mulugu, Bhadrachalam and Kothagudem forest soil samples was carried out as shown in Table 1. All best selected root nodules were tested under gnotobiotic conditions for improving nodulation in the test legume tree species, later through application in field trials for the unraveling and amelioration of crop production in barren, polluted and agricultural soils. Interestingly, it was observed that the root nodules from Bhadrachalam forest were found to be highly host specific for three tested plants (Albizzia lebbeck, Sesbania grandiflora and Pongamia pinnata) and those from Jakaram and Kothagudem forests were found to be host specific for Gliricidia maculata and Acacia nilotica, respectively and when supplied with such nodules, improved nodulation and plant growth in legume tree species was observed. This method may be employed across the globe for any type of legumes for the reclamation of vegetation in the soils that support agriculture poorly for any type of legume crop/tree.

Table 1: Screening of different indigenous rhizobial strains from host-specific agroforestry tree species from forest areas of Telangana

Name of Rhizobi Nodulation Height of the Dry weight of N the plant al strain plant (cm) the plant (gm) Conter inoculat (%) ed

No Size Shoot Root Shoot Root

weight

(mm)

(gm)

Albizzia

JK1 29 0.2 0.25 40.3 29.2 0.95 0.64 2.6 lebbeck ET1 19 0.3 0.16 33.2 25.8 0.88 0.57 2.4

MG 14 0.2 0.12 24.6 16.8 0.61 0.32 1.2

BD1 31 0.3 0.28 48.9 38.4 1.02 0.68 2.8

KT 30 0.3 0.27 45.6 33.2 1.01 0.67 2.8

Sesbania JK2 57 0.5 0.45 48.7 24.8 1.94 0.86 2.8 grandiflora ET2 45 0.6 0.39 45.3 2.8 1.63 0.74 2.5

MG 25 0.6 0.31 33.4 19.7 0.91 0.49 1

BD2 74 0.7 0.65 55.4 29.2 2.51 0.95 3.2

KT 64 0.6 0.51 51.4 27.1 2.18 0.92 3.1

JK3 36 0.7 0.56 54.4 34.3 2.17 1.37 3

Pongamia ET3 24 0.8 0.47 34.8 23.1 1.76 1.02 3.3 pinnata MG 15 0.5 0.18 21.8 17.1 0.82 0.48 1.4

BD3 30 0.6 0.51 39.7 29.4 1.95 1.13 2.5

KT 27 0.7 0.49 36.4 25.2 1.81 1.06 2.9

Gliricidia JK4 54 0.2 0.22 53.7 35.8 2.04 0.91 2.5 macu te ET4 42 0.2 0.18 49.2 30.3 1.72 0.86 2.2

MG 20 0.2 0.11 36.4 25.1 0.19 0.51 1

BD4 52 0.2 0.23 60.8 42.5 2.25 1.02 2.8

KT 32 0.3 0.12 60.2 40.6 2.12 0.98 2.7

Acacia JK5 20 0.2 0.11 47.6 28.2 1.05 0.76 2.4 nilotica ET5 12 0.2 0.06 45.3 26.8 0.92 0.66 2

MG 7 0.1 0.04 36.9 21.8 0.63 0.28 0.9

BD5 15 0.2 0.7 49.2 29.1 1.27 0.82 2.5

KT 22 0.1 0.11 50.6 29.7 1.39 0.88 2.6

Uninoculate - 07 0.1 0.03 19.4 13.4 0.28 0.42 0.8 control Effects of Nodulation in Sesbania grandiflora by BD-2 in agricultural, barren and polluted soils:

[024] Different rhizosphere soils were collected from 5 different plants in different forests and were made into a single sample and applied to Sesbania grandiflora. The rhizobial strain named BD-2 was found to be specific for Sesbania grandiflora which exhibited desired growth parameters and a large proportion of root nodules. These best supporting root nodules from that plant are collected, washed crushed and spread for the better growth of same plant in any problematic site, the results of which are shown in Table 2 and Fig. 2. Further these nodules can be crushed and stored for producing such Rhizobium bioinoculant formulations. This is an innovative farmer friendly method which is easy and cost-effective and does not involve culturing Rhizobium in lab conditions.

Table 2: Effects of Nodulation in Sesbania grandiflora by BD-2 in agricultural, barren and polluted soils

0.36 0.78

45+1 0.6+ 45.53+1. 22+1. 2.53+

PP Polluted soil +0.0 1.54+0.16 +0.0

.73 0.15 9 32 0.14

4 5

43.3 0.56 0.37 0.77

24.43 2.73+

Barren soil 3+1. +0.1 +0.0 46.1+2.11 1.80+0.08 +0.0

+1.91 0.17 76 7 5 4

0.76 0.67

25+1 0.4+ 35.33+1.0 20.96 1.53+

Agricultural soil +0.0 1.08+0.09 +0.1 .73 0.05 0 +0.69 0.31

8 0

27.6 0.76 0.44 0.58

21.53 1.07+

GM Polluted soil 6+2. +0.2 +0.0 38.2+2.97 0.97+0.05 +0.1

+1.56 0.04 40 0 6 5

0.37 0.63

26+1 0.7+ 33.96+1. 19.4+ 0.97+

Barren soil +0.0 0.85+0.05 +0.1

.15 0.11 4 1.09 0.05

5 2

77.6 0.96 0.76 1.05

58.56+1. 31.9+ 3.63+

Agricultural soil 6+2. +0.1 +0.0 2.66+0.08 +0.0

7 1.43 0.29 02 7 6 5

0.84

72+1 0.5+ 0.5+ 25.2+ 2.9+0.

SG Polluted soil 54+1.53 2.44+0.13 +0.0

.15 0.11 0.03 1.05 11

6

66.6 0.56 0.51 0.86

28.76 0.28+

Barren soil 6+1. +0.1 +0.0 54.4+2.25 2.13+0.08 +0.0

+2.07 0.04

33 4 6 6

66.3 0.73 0.58 0.99

29.5+ 3.73+

Agricultural soil 3+1. +0.0 +0.0 54.9+1.93 2.46+0.20 +0.0

1.28 0.34 45 6 3 4

55.6 0.43 0.48

26.1+ 1+0. 2.97+

AN Polluted soil 6+2. 3+0. +0.0 50.8+1.56 2.14+0.09

1.19 05 0.10 02 14 5

0.56 1.01

57+1 0.6+ 24.36 2.66+

Barren soil +0.0 53.2+0.72 1.9+0.12 +0.0

.15 0.11 +1.08 0.16

5 9

Values

[025] Although the present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive.

[026] Thus the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.