WO2015001575A1

WO2015001575A1 - Plant growth promoting formulation of piriformospora indica and azotobacter chroococcum with talcum powder

Info

Publication number: WO2015001575A1
Application number: PCT/IN2014/000447
Authority: WO
Inventors: Monika ARORA; Zainul Malik ABDIN; Ajit Varma
Original assignee: Amity University
Priority date: 2013-07-05
Filing date: 2014-07-04
Publication date: 2015-01-08
Also published as: WO2015001575A9; WO2015001575A4

Abstract

The present invention relates to a plant growth promoting formulation and a method for providing plant growth promoting composition comprising mixture of microbial isolates. Particularly, present invention relates to a plant growth promoting formulation comprising Piriformospora indica and Azotobacter chroococcum with talcum powder for promoting growth of different plant varieties and for promoting the production of secondary metabolite Artemisinin in Artemisia annua.

Description

PLANT GROWTH PROMOTING FORMULATION OF PIRIFORMOSPORA INDICA AND AZOTOBACTER CHROOCOCCUM WITH TALCUM POWDER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from and the benefit of Indian Patent Application No. 2017/DEL/2013 entitled "PLANT GROWTH PROMOTING ROOT ENDOPHYTE" filed on July 5, 2013 and Indian Patent Application No. 1803/DEL/2014 entitled "PLANT GROWTH PROMOTING FORMULATION OF PIRIFORMOSPORA INDICA AND AZOTOBACTER CHROCOCCUM WITH TALCUM POWDER" filed on July 03, 2014, which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a plant growth promoting formulation and a method for providing plant growth promoting composition comprising mixture of microbial isolates. Particularly, present invention relates to a plant growth promoting formulation comprising Piriformospora indica and Azotobacter chroococcum with talcum powder for promoting growth of different plant varieties and for promoting the production of secondary metabolite Artemisinin in Artemisia annua.

[0003] Plant rhizosphere contains billions of microorganisms in one gram of soil. These are either beneficial or neutral to plant growth. A number of microorganisms are known to be present in soil ecological niche (rhizosphere) having beneficial effects on plant growth. These beneficial plant growth promoting properties are nitrogen fixation, iron chelation, phosphate solubilization, inhibition of non-beneficial microorganisms, resistance to pest, can decompose plant material in soil to increase soil organic matter.

[0004] Fungi interact with plants as pathogens or benefactors and therefore strongly influence the yields in agro-forestry and flori-horticulture. The most wide spread symbionts are the arbuscular mycorrhizal fungi (AMF) which occur on more than 80% of all land plants (Newman, New Phytol. 106 (1987), 745-751 and Tester, Can. J. Bot. 65 (1987), 419-431). As mutualists, they are able to improve the growth of crops on poor soils at the same time reducing the input of expensive and polluting chemical fertilizers and pesticides (Gianinazzi, Crit. Rev. Biotechnol. 15 (1995), 305-31 1 and Bethlenfalvay in "Mycorrhizae in sustainable agriculture"; eds. Bethlenfalvay and Linderman, American Society of Agronomy, Madison, Wisconsins, USA (1992), 1-27). Unfortunately, AMF cannot be grown in pure culture. Many studies are therefore very difficult to carry out which slows down any fundamental research and more seriously, their biotechnological application.

[0005] Piriformospora indica shows mutualistic interaction with many plants in response to increased plant growth. Many rhizospheric bacteria (PGPRs) are growth promoters, stimulating seedling growth and development, while mycorrhizal fungi provide vegetation with increased efficiency of nutrient uptake, increased productivity, drought stress and may contribute to plant diversity,

[0006] US Publication No. 20120015806 provides a high cell density novel formulation of microbial consortium of Pseudomonas fluorescens, Pseudomonas striata, Bacillus polymyxa, Bacillus subtilis, Azospirillum, Rhizobium, Azotobacter, Trichoderma herzianum and Trichoderma viride to be used as P-solubilizer, nitrogen fixer, and plant residual matter decomposer, soil rejuvenator, soil and plant health enhancer. The said microbial consortium is provided in a composition suitable for treating plants or plant seed or directly to soil. The suitable carrier used in the invention is the powder.

[0007] Publication No. WO2013090628 describes microbial compositions which include at least one biological fertilizer to enhance the growth and/or yield of a plant. For example, microbes such as but not limited to species of Achromobacter, Ampelomyces, Aureobasidium, Azospirillum, Azotobacter, Bacillus, Beauveria, Bradyrhizobium, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Delftia, Erwinia, Exophilia, Gliocladium, Herbasplrillum, Lactobacillus, Mariannaea, Microccocus, Paecilomyces, Paenibacillus, Pantoea, Pichia, Pseudomonas, at least one agriculturally acceptable carrier can be added to the seed treatment formulation such as water, solids or dry powders. The dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.

[0008] Publication No. CN 102653500 provides compound type slow release fertilizer and a preparing method thereof. The preparing method comprises the main steps of: mixing constituted raw materials including urea, cellulose, bran, a water-retaining agent, saw dust, straw ash, polypropylene, ammonium phosphate, calcium powder, potassium sulfate, attapulgite, borax, ferrous sulfate, yeast microbial fertilizer, azotobacter fertilizer and talcum powder according to certain part by weight and carrying out fermentation so as to obtain the compound type slow release fertilizer.

[0009] US Publication No. 20090308121 describes eco-friendly compositions for providing plant growth enhancing formulations comprising mixtures of microbial isolates including Azotobacter, Azospirillum, Azorhizobium, Pseudomonas, Bacillus, Rhizobium, Mycoplasma, et cetera. Wettable powders suitable for spraying can be prepared by admixing the composition with a finely divided solid, such as clays, inorganic silicates and carbonates, silicas, and incorporating wetting agents, sticking agents, and/or dispersing agents in such mixtures.

[0010] Publication No. CN 1830919 provides environment friendly release-controlled synergistic fertilizer composed of a core made of ordinary chemical fertilizer and a jacket layer made of mineral powder and functional bacteria which uses natural low-grade phosphate rock, phosphogypsum, lake sediment flour, starch, vegetable oil, polyurethane, carboxymethyl cellulose, solubilizing bacteria, fungi potassium and Azotobacter as raw materials. Talcum powder is added to the mineral fertilizer with a binder on the rolling. Rolling with a fertilizer allows a uniform powder and a binder with the surface of the fertilizer particles to form a layer coating layer.

[0011] Publication No. WO2010122501 provides a soil bio-conditioner using microorganisms reducing: the harmful effect of chemical amendments. Bacteria and fungal microorganism suitable for use in the present invention preferably are selected from one or more genera of Azotobacter, Bacilla, and Pseudomonas, in addition to specific bacteria and fungi such as Pseudomonas fluorescens, Azotobacter, Bacillus polymyxa, Trichoderma herzianum, Trichoderma viride respectively. The said microbial consortium is provided in a composition suitable to apply to the soil directly or the soil surrounding the plants preferably an inert material, powder.

[0012] The article entitled "Plant growth promotional effect of Azotobacter chroococcum, Piriformospora indica and vermicompost on rice plant" talks about the growth promotion of rice (Oryza sativa L.) due to dual inoculation of Azotobacter chroococcum and Piriformospora indica along with vermicompost (Kamil Prajapati, et al; Nepal Journal of Science and Technology; Vol. 9, Page. 85-90, 2008).

[0013] The article entitled "Effect of formulated root endophytic fungus Piriformospora indica and plant growth promoting rhizobacteria fluorescent pseudomonads R62 and R81 on Vigna mungd" talks about the effect of three beneficial organisms (root endophytic fungus Piriformospora indica (Pi) and pseudomonads strains R62 and R81) and their four different consortia' (Pi+R62, Pi+R81 , R62+R81, Pi+R62+R81) which were investigated on the plant Vigna mungo through their inorganic carrier-based (talcum powder and vermiculite) formulations. All the treatments resulted in significant increase in growth parameters under glasshouse as well as field conditions and showed a consistency in their performance on moving from glasshouse to field conditions (Kumar V, et al; World J. Microbiol Biotechnol.; 28(2):595-603, 2012).

[0014] The article entitled "Application of inorganic carrier-based formulations of fluorescent pseudomonads and Piriformospora indica on tomato plants and evaluation of their efficacy" talks about improvement of growth yields of tomato plants and control of Fusarium wilt using inorganic carrier-based formulations of two fluorescent pseudomonad strains (R62 and R81) and Pi. In field experiments the talcum-based consortium formulation of pseudomonad R81 and Pi was most effective (Sarma MV, et al; J. Appl. Microbiol.; 1 1 1(2):456-66, 2011).

[0015] The article entitled "Production of Trichoderma viride and study of its effective growth and viability under various carrier material compositions" talks about isolating Trichoderma viride and producing contamination free Trichoderma viride and also producing a compatible product to Trichoderma viride and Azotobacter. Azotocter and Trichoderma viride are compatible with each other in any of the materials like (FYM + Sand+ rice husk and Talcum powder) and if produced at large scale it would be of great benefit for the farming community (Sailash Kumar; School of Agriculture and Rural Development, Ramakrishna Mission Vivekananda University, Howrah, West Bengal, India, 2013).

[0016] The article entitled "The role of cellulose-decomposing fungi in nitrogenase activity of Azotobacter chroococcum" talks about Azotobacter chroococcum grown on cultures containing five carbon sources alone and also in co-cultures with three cellulolytic fungi {Aspergillus niger, Penicillium funiculosum and Trichoderma harzianum). The best nitrogenase activity was recorded in cultures containing faba bean straw followed by that in cultures having wheat straw, sugar ca e leaves carboxymethyl cellulose (CMC) or cellulose (M. H. Abd-Alla, et al; Folia Microbiologica; 37(3):215-218, 1992).

[0017] The article entitled "Effect of polymeric additives, adjuvants, surfactants on survival, stability and plant growth promoting ability of liquid bioinoculants" talks about the effect of liquid inoculants formulated with 2% polyvinylpyrollidone (PVP 30 K), 0.1% carboxy methylcellulose (CMC-high density) and 0.025% Polysorbate 20 which promoted long-term survival of Bacillus megaterium var. phosphaticum, Azospirillum and Azotobacter with 5.6 x 107, 1.9x108 and 3.5xl07cfu ml-1 (Leo Daniel Amalraj E, et al; Journal of Plant Physiology & Pathology; 1 :2, 2013).

[0018] Several microbial population based products are available commercially but they have their limitations in their composition and in their application to a particular crop. Most of the time, these products either contain only one plant growth property. So for getting multiple benefits, : the farmers have to apply the best choice of products. Therefore, for better cropping practices it is desirable to develop a plant growth promoting formulation with multiple properties which can be used alone. [0019] Therefore, the present invention provides carrier based consortium composition of Piriformpspora indica and Azotobacter chroococcum for promoting growth of plants of different varieties as well as promoting in its secondary metabolites.

SUMMARY OF THE INVENTION

[0020] Primary object of the present invention is to provide a plant growth promoting formulation comprising Piriformospora indica and Azotobacter chroococcum with talcum powder for enhanced plant growth and secondary metabolites production and a method of preparation thereof.

[0021] Another object of the present invention is to provide a plant growth promoting formulation which promotes growth, health, nutrition and regeneration of plants and promotes germination of plant seeds.

[0022] Yet another object of the present invention is to provide a plant growth promoting formulation with a high colony forming units (cfu) of fungal and bacterial population having longer shelf life.

[0023] Still another object of the present invention is to provide a plant growth promoting formulation which can be used for different plant varieties.

[0024] Accordingly the present invention provides a plant growth promoting formulation comprising cold shock treated Piriformospora indica and Azotobacter chroococcum with talcum powder which acts as a carrier and a method for preparation thereof. The plant growth promoting formulation of the present invention has a wide application range which includes applying the mixture to plants, plant seeds or soil directly for getting effective results in a variety of different plant species. It promotes plant growth, secondary metabolites concentration, nutrient availability and increases soil organic matter content and protects against plant pathogens. The plant growth promoting formulation of the present invention increases the concentration of Artemisinin in Artemisia annua.

[0025] In a preferred embodiment of the present invention, the P. indica is cultivated on Hill- Kaefer medium. After nine days incubation at 28°C, P. indica is pooled together and placed at 10°C for 48 hours under gentle shaking conditions in dark for cold shock treatment.

[0026] In another embodiment of the present invention, bio-formulation of Piriformospora indica ir| isolation is prepared by separating the cold shock treated P. indica from the culture filtrate then 100ml broth containing about 25 g fresh weight (0.95 g DCW/Litre) is mixed with 1 ml of carboxyl methyl cellulose (CMC) (0.1 mg/ml). 25ml of this CMC mixed fungal broth is mixed with sterilized 75g talcum powder.

[0027] In yet another embodiment of the present invention, the Azotobacter chroococcum formulation is prepared by adding 1ml glycerol (final concentration- 1% v/v) and 1ml of 0.1 mg/ml CMC is added as an additive to 98 ml culture broth containing 1 x 10⁴ - 1 x 10⁶ cfu/ml of Azotobacter chroococcum. 20ml of this suspension is mixed with 80g of sterilized talcum powder. |

[0028] Ih still another embodiment of the present invention, the plant growth promoting formulation comprising Azotobacter chroococcum-Piriformospora indica consortium is prepared by taking 10 ml of CMC mixed Azotobacter chroococcum culture (1 x 10⁴ cfu/ml) and 10ml (CMC mixed P. indica broth) and mixing it with 80g of sterilized talcum powder. This is used as bioform lation for the consortium.

[0029] In another embodiment of the present invention, the final Azotobacter chroococcum count

^! 7 0

in the bioformulation ranges between 1 x 10 - 1 x 10 cfu.

[0030] In yet another embodiment of the present invention, micro propagated Artemisia annua plants when co-cultivated with P. indica and A. chroococcum cells (109 CFU/ml) produces higher number of leaves, increased shoot length, biomass per plantlet and artemisinin content after 6 weeks of culture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The subject matter of the invention will be explained in more detail in the following text with reference to preferred exemplary embodiments which are illustrated in the attached drawings, of which

[0032] Fig. 1 illustrates the cultivation protocol of P. indica on Hill- Kaefer medium;

[0033] Fig. 2 illustrates morphological features of P. indica before and after cold shock treatment;

(a): 28°C for 10 days, (b) 10°C for 48 hours;

[0034] Fig. 3 illustrates the separation procedure of fungal biomass from the culture filtrate;

[0035] Fig. 4 illustrates co-cultivation of P. indica with Azotobacter chroococcum, showing higher, enlarged and thick walled chlamydospores;

(a): P. indica + Azotobacter chroococcum (b): P. indica alone

*

[0036] Fig. 5: illustrates the interaction of formulation with tobacco plant;

(a): control, (b): with P. indica

[0037] Fig. 6 illustrates the interaction of formulation with Coleus forskohlii;

(a): without P. indica, (b): with P. indica

[0038] Fig. 7 illustrates growth and development of A. annua when co-cultured with P. indica and A. chroococcum culture alone or dual treatment A) Control; B) Treated with P. indica; C) Treated with A. chroococcum; D) Treated with P. indica and A. Chroococcum;

[0039] Fig. 8 illustrates shoot length of A. annua L. plants treated with P. indica /A. chroococcum alone and dual treatment when grown under in vitro, Poly house and field conditions;

[0040] Fig. 9 illustrates number of leaves in A. annua L. plants treated with P. indica /A. chroococcum alone and dual treatment when grown under in vitro, Poly house and field conditions;

[0041] Fig. 10 a & b illustrates number of roots per plantlet of A. annua treated with P. indica /A. chroococcum alone and dual treatment when grown under in vitro, Poly house and field conditions;

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[0042] Fig. 1 1 a, b & c illustrates fresh and dry weight of shoot of A. annua treated with P. indica/Ai chroococcum alone and dual treatment when grown under in vitro, Poly house and field i ·

[0043] Fig. 12 illustrates total chlorophyll (a) and soluble protein content (b) in leaves of A. annua plants treated with P. indica /A. chroococcum alone and dual treatment when grown under field;

[0044] Fig. 13 illustrates artemisinin content in leaves of A. annua plants treated with P. indica /A. chroococcum alone and dual treatment when grown under in vitro, poly house and field conditions;

[0045] F g. 14 illustrates the interaction of formulation with litchee (Litchi chinensis);

(a): control, (b): P. indica+ Azotobacter chroococcum;

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[0046] Fig. 15 illustrates the interaction of formulation with mango (Mangifera indica).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0047] Accordingly, the present invention provides a plant growth promoting formulation

I comprising cold shock treated Piriformospora indica and Azotobacter chroococcum with talcum powder acting as carrier and a method for preparation thereof. The P. indica is cultivated on Hill- Kaefer medium and incubated at 28°C for nine days. After nine days of incubation period, P. indica is 'pooled together and placed at 10°C for 48 hours under gentle shaking conditions in dark for cold shock treatment for the purpose of the present invention.

[0048] The cold shock treated P. indica. is separated from the culture filtrate and 100ml broth containing about 25 g fresh weight (0.95 g DCW/Litre) is mixed with 1 ml of carboxyl methyl

I

cellulose (CMC) (0.1 mg/ml). Similarly, the Azotobacter chroococcum formulation is prepared by addinjg 1ml glycerol (final concentration- 1% v/v) and 1ml of 0.1 mg/ml. CMC as additives to 98 ml culture broth containing 1 x 10⁴ - 1 x 10⁶ cfu/ml of Azotobacter chroococcum.

[0049] The plant growth promoting formulation of the present invention comprising P. indica and Azotobacter chroococcum is prepared by taking 10 ml of CMC mixed Azotobacter chroococcum culture (1 x 10⁴ cfu/ml) and 10ml (CMC mixed P. indica broth) and mixing it with 80g of sterilized talcum powder. This is used as bioformulation for the consortium. The final Azotobacter chroococcum count in the plant growth promoting formulation is in the range of 1 x 10⁷ - 1 x 10⁹ cfu.

[0050] The plant growth promoting formulation of the present invention has a wide application range which includes applying the mixture to plants, plant seeds or soil directly for getting effective¹ results in a variety of different plant species. It promotes plant growth, nutrient availabil ty and increases soil organic matter content and protects against plant pathogens. The plant growth promoting formulation of the present invention also enhances the production of secondary metabolites in many plant varieties such as but not limited to Artemisia annua. The formulai†on has been shown to increase the production of secondary metabolite artemisinin by co-cultivation of Artemisia annua plant with the consortium of P. indica and Azotobacter i

chroocoCcum.

[0051] The invention is described in detail with reference to the examples given below. The exaitiples are provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention.

Example 1 : Cultivation of P. Indica

[0052] P. indica was cultivated on Hill- Kaefer medium (fig. 1). Each jar contained 80 ml of the liquid medium and the incubation was carried out at 28 + 2 °C. The initial pH was adjusted to 6.5. Throughout the incubation period, 80 rpm was maintained on a Gyrotary shaker.

Example; 2: Cold shock treatment of P. indica

[0053] ft^{er nme} days incubation at 28°C, jars containing P. indica were pooled together and transferred to a large 2 litre flat bottom flask. The mouth was plucked with sterile cotton. They were plaped at 10°C for 48 hours under gentle shaking conditions in dark. This shock treatment accelerated the sporulation process; the chlamydospores were enlarged with thick wall. Each spore contained 8-25 nuclei (Fig. 2). The auto fluorescence property of the wall was further enhanced. In contrast those who did not receive the cold treatment, the spores were small and thin walled. The treatment rendered a high number of sporulation. The thick wall provided a potential for long term storage at normal conditions and transport across the continents.

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Example 3: Separation of P. indica biomass and culture filtrate

[0054] The P. indica biomass was separated from the culture filtrate as shown in fig. 3. On an average 50-60 gms/litre fresh biomass was obtained. Two kinds of colonies were frequently observed depending upon the rpm used. Incubation when executed at low rpm (50-60 rpm) highly aggregated and condensed colonies were observed containing chlamydospores and hyphae. In contrast when rpm was maintained between 80-100, dispersed and explorer type colonies were recorded.

Examplej 4: Selection of Azotrophs [0055] Five Azotobacter stain were obtained from Division of Microbiology, Indian Agricultural Research! Institute, New Delhi. There is a general belief that all Azotobacter- nitrogen fixing bacteria have a synergic effect with symbiotic fungi. However, it was found that there were two i

positive (Strain W5 and WR 9) and three stains negative (Strain M4, A 41 and AS 4) (Table 1). Table 1 : Influence of A. chroococcum strains on P. indica

Strains ! Growth

Strain W5 Growth stimulation (+++)

Strain WR9 Growth stimulation (++)

Strain Mj4 Growth inhibition (+++)

Strain A|l Growth inhibition (+++)

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Strain AS4 Growth inhibition (++)

Strain WR3 No Growth

(Note: Strong: '+ +' Weak)

Example| 5:

Preparation of bio-formulation of Piriformospora indica in isolation

[0056] lPOml broth containing about 25 g fresh weight (0.95 g DCW Litre) of P. indica was mixed with 1 ml of carboxyl methyl cellulose (CMC) (0.1 mg/ml). CMC was used as an adhesive; so that the inoculum sticks to the powder. 25ml of this CMC mixed fungal broth was mixed wjth sterilized 75g talcum powder.

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Example! 6:

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Preparation of bioformulation of Azotobacter chroococcum in isolation

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[0057] For the preparation of formulations of Azotobacter chroococcum, the broth containing 1 x 104 - 1 x 10⁶ cfu/ml was used. To this, 1ml glycerol (final concentration- 1% v/v) and 1ml of 0.1 mg/ml C;MC were added as additives to 98 ml culture broth. Here, glycerol served as carbon source for keeping the cells viable while CMC acted as an adhesive. This was vortexed for uniform mixing. 20ml of this suspension was mixed with 80g of sterilized talcum powder. This could be!used as a bioformulation for Azotobacter chroococcum in isolation (20% formulation).

Example! 7: Process for preparing plant growth promoting formulation of Piriformospora indica- Azotobacter chroococcum consortium

[0058] For the preparation of formulation of Azotobacter chroococcum - Piriformospora indica consortiijm, 10 ml of CMC mixed Azotobacter chroococcum culture (1 x 10⁴ cfu/ml) and 10ml

^■ i

(CMC niixed fungal broth) were mixed with 80g of sterilized talcum powder. This could be further used as bioformulation for the consortium. Final Azotobacter chroococcum count was in the rangej of 1 x 10⁷ - 1 x 10⁹ cfu in the formulation.

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Examplej 8: Application of the plant growth promoting formulation on different plants

[0059] †he formulation promoted early flowering in plants of economic importance like Tobaccoj Brassica, and medicinal plant like Coleous, Chlorophytum, Spilanthes and several plants of! ornamental importance (Figs. 5, 6 and 7). j

[0060] Micro propagation of Artemisia annua, Co-cultivation of micro propagated plants with P. indica aijid A. chroococcum alone/or in combination, Acclimatization of tissue culture grown plantlets iand transfer in field, Analysis of physiological parameters of A. annua L. plants grown i

under in vitro, green house and field conditions, Analysis of biochemical parameters of A. annua L. plants; grown under in vitro, green house and field conditions and Root colonization are as described in reference Indian Patent Application No. 2017/DEL/2013.

Example 9: Micro propagation of Artemisia annua

[0061] Apical portion of shoots from 35 days old in vitro grown seedlings were used as explants and ino<?ulated on shoot multiplication medium comprising of MS + Sucrose (30g/l)+ Agar(8g )+NAA (0.05mg/l) + BA (1.5mg/l). After 6 weeks of inoculation, 10-15 multiple shoots Were obtained per explants. These were then separated and inoculated on rooting medium MS + Su rose (30g/l) + Agar (8g/l) + NAA (0.5mg/l).

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Example! 10: Co-cultivation of micro propagated plants with P. indica and A. chroococcum alone/or in combination

[0062] Seeds of A. annua were surface sterilized and placed on the half strength MS medium. Apical portion of shoots from four weeks old seedlings were used as explants and inoculated on shoot multiplication medium comprising NAA (0.05mg/l) and BAP (1.5mg/l). After 6 weeks of inoculation, 10-15 multiple shoots were obtained per explants. These were then separated and inoculatqd on rooting medium NAA (0.5mg/l). After 1 1 days rooting was initiated from shoots. After that micro propagated plants were co-cultivated with P. indica disc of 4mm size in diameter; and A. chroococcum cells 500 μΐ (109 CFU/ml) and were found to produce higher number hi leaves, increased shoot length, biomass per plantlet and artemisinin content after 6 weeks of culture (Figs. 8, 9, 10, 11 , 12 and 13 ; Tables 2-8).

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Example! 1 1 : Acclimatization of tissue culture grown plantlets and transfer in field i

[0063] Iri vitro grown micro propagated plantlets growing under different treatments were taken out from! the culture bottles in such a way that no damage was caused to their root system. Roots of treated and control plants were washed under running tap water to remove adhering agar and then transferred in pots containing autoclaved soil, sand, peat moss in ratio of 3:3:1. Hoagland solution iwas supplied to plants for proper growth aiid nutrition. ^Plants were covered with poly bags for maintaining humidity conditions. After 15 days small holes were made in the poly bags and subsequently bags were removed to acclimatize plants to green house conditions. Plants were allowed to grow for 8 weeks and subsequently transferred in bigger pots containing soil, sand andj peat mixture in the ratio of 3:3: 1 under field condition. Plants were maintained under field condition for 150 days (Fig. 7). Shoot length, root length, fresh weight and dry weight per plantlet, bhlorophyll content, soluble protein and artemisinin content in leaves were recorded. Example! 12: Analysis of physiological parameters of A. annua L. plants grown under in vitro, green house and field conditions

[0064] plants samples were taken for analysis of growth parameters under all three conditions. Under in vitro treatment, samples were taken after 6 weeks. Under greenhouse conditions acclimatization was carried out and after 8 weeks, growth parameters were studied. Subsequently plants were transferred to field conditions and allowed to grow for 150 days (5 months) after which physiological parameters were recorded.

A. Length of shoot

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[0065] Length of shoot and root were measured for analysis of growth. Plantlets were taken from each treajted and non- treated (control) plants. The length of roots and shoots was measured and expressed in cm.

[0066] Maximum shoot length of A. annua plants under three conditions (in-vitro, Poly house and Fielcj condition) i.e. 17.5, 48.41 & 51.54 % was the -enhancement recorded on plants treated with P.injdica and A. chroococcum culture as compared with control (Table 2, Fig. 8).

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B. Fresh land dry weight of shoot and root

[0067] Pjantlets were cut at root shoot junction and separated parts of each plantlet were weighed for fresh ;weight (mg) and then dried separately in hot air oven at 65°C for 48 hours till a constant weight Was weighed. Thereafter dry weights were determined on digital balance and expressed in g/plant. j

[0068] Significant differences in the fresh and dry weight of shoot was observed in plants inoculated with P.indica/ A. chroococcum alone or in combinations under in vitro, Poly house and field condition (Figs. 1 1 a, b & c). The maximum biomass was recorded in plants treated with dual culture as compared to the other treatments (Tables 3 and 4). Table 3: iEffect of P. indica I A. chroococcum alone and dual treatment on fresh weight and dry i

weight of stem of A. annua L. plants of in vitro, poly house and field grown plants

Treatments Stem Fresh Weight (gm) Stem Dry Weight (gm)

I

In vitro Poly House Field In vitro Poly House Field

Control j 2.78±0.1 36.2±1.9 278.5±4.2 1.64±0.2 23.64±2.3 142.68±2.7

P. indic 4.34±0.4 48±1.9 425.6±2.9 3.46±0.3 37.22±1.4 191.0±6.1

A. chroococcum 3.92±0.3 43.24±2.2 381.5±4.6 2.98±0.2 32.8±1.6 174.6±1

P. indicd and 5.12±0.2 55.42±3.1 492.18±6.8 3.74±0.2 39.86±0.9 215.96±3.9

A. chroococcum

Table 4: iEffect of P. indica I A. chroococcum alone and dual treatment on Fresh weight and Dry Weight 0f roots A. annua L. plants of in vitro, poly house and field grown plants

of in-vitijo and 82.4 leaves of Poly house A. annua plants treated with dual treatment of P. indica and A. chroococcum was recorded (Table 5, Fig. 9). Data given is the mean value of the five replications.

Table 5: IEffect of P. indica/ A. chroococcum alone and dual treatment on number of leaves per plantlet of A. annua L. plants of in vitro and Poly house grown plants Treatments Number of leaves plantlet

plants samples were taken under all three conditions. Under in vitro treatment samples were t

taken after 6 weeks. Under greenhouse conditions acclimatization was carried out and after 8 weeks growth parameters were studied. Subsequently plants were transferred to field conditions and allowed to grow for 150 days (5 months) after which artemisinin content in leaves were

I - recorded!

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A. Determination of photosynthetic pigment concentration

[0072] Chlorophyll a, b, and total chlorophyll were extracted and measured by the method of Hiscox and Israelstam, 1979 and concentration was estimated with the help of Anion's formulae

(Arnon, 1949). Chlorophyll content was expressed as mg/gm FW.

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[0073] ^"total chlorophyll content was found 6.84 mg/g in the leaves of A. annua plants co- cultivate^i with microbial consortium of P. indica and A. chroococcum. 26% enhancement was observedj in total chlorophyll content in leaves of 150 days of plants grown under field condition as compared to the untreated plants (Table 7, Fig. 12a).

Table 7: ' Effect of P. indica I A. chroococcum alone and dual treatment on total chlorophyll content ^nd total soluble protein (mg gfw-1) in leaves of A. annua L. plants of in vitro, Poly house anjd Field grown plants i

Treatments Total Chlorophyll Content Total Soluble Protein

I (mg gfw-1 ) (mg gfw-1)

Control 5.4 ± 0.12 15.1± 0.02

P. indica] 6.36 ± 0.15 19.93 ± 0.04

A. chroococcum 5.76 ± 0.11 17.47 ± 0.02

P. indica] and 6.84 ± 0.1 1 22.81 ± 1.52

A. chroococcum

B. Estimation of total soluble protein

[0074] Total soluble protein was estimated by Bradford method (1976) in leaves. The concentration was expressed in mg/g fresh weight of leaves. *^■ Leaves of A. annua plants co-cultivated with dual culture of P. indica and A. chroococcum was found tojhave 22.81 soluble protein contents. Data was collected after 150 days of plants grown under fiejld condition (Table 7, Fig. 12 b). 51.05 % enhancement of total soluble protein in leaves was recojrded in dual treatment.

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C. Extraction and quantification of artemisinin

[0075] Artemisinin was analyzed and quantified using the method described in Zhao and Zeng, 1986; Mauji et al., 2010). The quantification of artemisinin was done with the help of a standard curve prepared by High Performance Liquid Chromatography. The concentration of artemisinin was expressed as percent per gram dry weight of leaves.

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[0076] The artemisinin content in the leaves of A. annua plantlets subjected to four treatments i.e. 1) Control, 2) Treated with P. indica 3) Treated with A. chroococcum 4) Treated with P. indica and A. chroococcum was determined in the plants grown under in vitro, Poly house and field condition (Table 8, Fig. 13). 70 % enhancement was observed in artemisinin content in leaves oij in vitro, Poly house and field plants treated with microbial consortium of P. indica and A. chroococcum followed by 37 and 30% enhancement in leaves of P. indica and A. chroococcum alone treated plants.

Table 8: |E valuation of artemisinin content (%) in leaves of A. annua L. plants co cultivated with P. indic . I A. chroococcum alone and dual treatment under in vitro, poly house and field conditiorj

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Treatments Artemisinin Content (%)

In vitro % Poly House % Field %

Enhancement Enhancement Enhancement

Control 0.08 0.0 0.33 0.0 0.45 0.0

P. indica] 0.1 1 37.5 0.44 33.33 0.62 37.7

A. chroococcum 0.106 32.49 0.399 20.9 0.588 30.6

P. indica, and 0.136 70 0.57 72.72 0.77 71.11

A. chroococcum Example! 14: Root colonization

[0078] Fine roots from treated and untreated plants of A. annua were collected, rinsed well in distilled jwater and cut into 1 cm pieces. Root segments were boiled at 60-70°C with 10% KOH to soften; the root tissue. Then the tissues were neutralized with 2% HCl for 3-4 m. Roots were stained with 0.5% lactophenol blue solution (Phillips and Hayman, 1970). Slides were prepared from theise samples and bright field image was taken under Phase contrast microscope at 40 X magnification (Olympus CX41RF, model no. SN = 6K09628).

[0079] Treated plants showed inter and intracellular chlamydospores single and in chain. There was a difference between the spore size of P. indica alone treated roots and in combination with i

A. chroococcum.

Example; 15: Treatment of wheat seeds

[0080] Sjeeds were mixed with the plant growth promoting formulation as lKg/ 15 gm wheat seeds. 100 gm jaggery was suspended in 200 ml of water and was sprinkled over the seeds. They were thoiroughly mixed and left over night for better sticking of the inoculum over the seed. Precautions were taken to cover the heap of treated seed with dark tarpaulin sheet, to enhance the temperatiire and also to give heat shock treatment to seed and carbon energy source to the formulated products.

[0081] Cold and heat shock treatment before the formulation and heat treatment, while mixing with the eeds gave the much more promising results than those microbial consortium which did not receiVe temperature shock treatment (Table 9).

Table 9: Independent field trial on wheat was carried out at Farmer field ten acres, near Ambala.

Type/mojdule Yield Difference in % increase

(quintal/acre) yield w.r.t. w.r.t control

control Control (100% 12.50 00 00

chemicalj fertilizer)

(Without P. indica)

100% chemical 15.45 2.95 23.60

fertilizer !+ P. indica

75% che|nical 15.26 2.76 22.08

fertilizer \ + P. indica ^■

75% chehiical 13.96 1.46 1 1.68

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fertilizer I + Azotobacter

75% chejnical^■ 16.77 4.27 34.16

fertilizer I + P. indica +

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Azotobacter

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* + saving on chemical fertilizer

Optimum results were obtained when Azotobacter and P.^' indica were applied in combination. i

Examplej 16: Protocol for application of the plant growth promoting formulation:

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[0082] An outline of the canopy of litchee and mango adult plants were drawn. Three inches from the surface soils were removed into this plant growth promoting formulation containing Azotobacter chroococcum + P. indica were laid down. An approximately 100 gm of the plant growth promoting formulation was used for each plant. The channel was flooded with water and followed! by covering with the soil (fig. 14 and 15). Treated plants showed pro fused foliage, early infloresc nce and fruit settings.

Claims

CLAIMS:

1. A plant growth promoting formulation comprising cold shock treated Piriformospora iridica; Azotobacter chroococcum and talcum powder as a carrier.

2. The plant growth promoting formulation as claimed in claim 1, wherein the method for cbld shock treatment of P. indica comprises: incubating P. indica at 28°C for nine days tlien placing it at 10°C for 48 hours under gentle shaking conditions in the dark.

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3. The plant growth promoting formulation as claimed in claim 1, wherein the plant growth

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promoting formulation is prepared by taking 10 ml of carboxyl methyl cellulose mixed Azotobacter chroococcum culture (1 x 10⁴ cfu ml) and 10ml of carboxyl methyl cellulose mixed cold shock treated P. indica broth and mixing it with 80g of sterilized talcum powder. i

4. The plant growth promoting formulation as claimed in claim 1, wherein the bio- f (Simulation of Piriformospora indica in isolation is prepared by taking 100ml broth containing about 25 g fresh weight (0.95 g DCW/Litre) of P. indica and mixing it with 1 nil of carboxyl methyl cellulose (0.1 mg/ml).

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5. The plant growth promoting formulation as claimed in claim 1, wherein the Azotobacter chroococcum formulation is prepared by adding 1ml glycerol (1% v/v) and 1ml of 0.1 nig/ml CMC to 98 ml culture broth containing 1 x 10⁴ - 1 x 10⁶ cfu/ml of Azotobacter chroococcum.

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6. The plant growth promoting formulation as claimed in claim 1, wherein the final

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Azotobacter chroococcum count in the plant growth promoting formulation ranges between 1 x 10⁷ - 1 x 10⁹ cfu.

increases the yield of cereal crops such as wheat .