Bacterial-based volatiles composition as jassid attractant

Abstract

The present invention is related to a jassid attractant. Specifically, the present invention relates to a jassid attractant composition consisting of synthetic analogs of bacterial-based volatile compounds consisting of alkanes (Eicosane, Tetrapentacontane, Heneicosane, Tetracosane), and a pyrazine (2-Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine-1,4 (3H-5 6H)-dione), or a combination thereof. The present invention relates to a method for preparing a jassid attractant composition comprising synthetic analogs of bacterial-based volatile compounds. Furthermore, the jassid attractant composition along with yellow sticky traps (YST-existing mechanical control method for jassid) enhanced the jassid attraction to a maximum of 197% compared with the control treatment (YST alone). Furthermore, the jassid attractant is cost effective, does not have any harmful effects on soil nutrient availability and soil biology, and is environmentally friendly. It can reduce chemical pesticide usage and insect resurgence in cotton cultivation, thus having wider commercial application prospects in agriculture.

Description

BACTERIAL-BASED VOLATILES COMPOSITION AS JASSID ATTRACTANTFIELD OF THE INVENTION

[0001] The present invention relates to jassid attractant. Specifically, the present invention relates to a jassid attractant composition comprising of synthetic analogs of bacterial-based volatile compounds. Also, the present invention relates to a method for the preparation of a jassid attractant composition comprising of synthetic analogs of bacterialbased volatile compounds. Further, the jassid attractant is cost-effective, does not have any harmful effects on soil nutrient status and soil biology, environmentally friendly, can reduce chemical pesticide usage, insect resurgence in cotton cultivation, and thus have wider commercial application prospects in agriculture.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Cotton is an important natural fiber crop that provides fiber to the textile industries and supplies oil and feed to the society. Sucking pests are one of the major biotic factors which affect cotton production in India. Although cotton is affected by several sucking pests, jassids are devastating pests that cause significant yield loss.

[0004] Jassid Amrasca biguttula (Hemiptera: Cicadellidae) is a cosmopolitan, broadspectrum phloem-feeding insect. It causes severe damage to cotton plants through sap sucking and subsequent decline in plant photosynthetic activity (Saeed et al. 2016). Both nymphs and adults cause economic damage to cotton plants through the injection of toxic compounds into the leaf tissues, which results in burning of leaves, drying, and shedding (Murugesan and Kavitha, 2010). Since jassids affect the photosynthetic activity of leaves, they affect the developing bolls, resulting in a reduction in yield. Besides cotton, the cotton jassids also feed on okra, potato, brinjal, and some wild plants. The adult jassids are about 3 mm long and greenish yellow. Normally, the female lays about 15 eggs on the lower surface of the leaves. The jassids complete seven generations in a year.

[0005] To manage jassids, farmers largely depend on conventional groups of insecticides, including methyl demeton, dimethoate, acetamiprid and seed treatment chemicals such as phorate, Imidacloprid, Thiomethoxam. Among mechanical control methods, yellow stickytraps (YST) are commonly used to monitor and trap field populations of jassids. Besides conventional insecticides, several biopesticides and naturally occurring substances have been used to manage jassids in cotton. However, effective control of jassids was never achieved.

[0006] The continuous and indiscriminate use of insecticides to manage jassids has resulted in jassid resistance to insecticides in the recent past. Apart from resistance development in jassids, environmental pollution in terms of accumulation of pesticide residues in soils, adverse effects on natural enemies (predators and parasitoids) and resurgence of minor pests have also been reported in cotton cultivation in India (Kranthi et al. 2002).

[0007] Though introduction and adoption of Bt cotton have been found to successful in the management of bollworm complex in cotton, it has invited several other insect pests, especially jassids, because of the reduction in insecticidal sprays at early stages (Jeyakumar et al. 2008). Further, the sucking pests, which are insusceptible to CrylAc showed resurgence in many parts of the world due to the reduction of insecticide sprays (Kranthi et al. 2002). Presently, more than 90% area in India under Bt cotton are susceptible to sucking pests (Hofs et al. 2004; Sharma and Pampapathy, 2006). Presently, the sucking pests have attained the status of major pests from minor pests after the Bt cotton cultivation.

[0008] In this present situation, farmers are looking for alternative eco-friendly, cheap and effective jassid management options in cotton. Further, India is a major player in organic cotton production in the world. The worldwide demand for organically grown fiber also forces the cotton farmers to use eco-friendly and non-chemical approaches to manage jassids in cotton.

[0009] Microbial volatile organic compounds (mVOCs) represent a new frontier in bioprospecting, where they produce several complex and dynamic volatile compounds (Effinert et al. 2012), which are defined as compounds that have high enough vapour pressures under normal conditions to significantly vaporize and enter the atmosphere (Chaparro et al. 2014). Although several thousand volatiles emitted by plants have been identified so far, only several hundred compounds have been identified from microorganisms (Penuelas et al. 2014). Chemical ecologists consider mVOCs as potential semiochemicals that function as attractants and repellents to insects. Some mVOCs attract or repel insects, inhibit the growth of microorganisms competing with associated insects, stimulate oviposition, mimic plant hormones, or even induce plant resistance (Ryu et al. 2003, 2004). The mVOC emission, which was first reported by Ryu et al. (2003), is now recognized as an important aspect of plant-microorganism interactions (Bitas et al. 2013; Farag et al. 2013;Kanchiswamy et al. 2015a, b). Recently, a few studies have shown the wealth of mVOCs for the modulation of crop growth, development, defense, and inter- and intraspecific communication (Kanchiswamy et al. 2015a). Insects exhibit strong aggregation behaviors to specific microbial communities, although few researchers have reported mVOCs directly as pheromonal communications as a signal for food sources, oviposition sites, or mating opportunities (Symonds and Wertheim, 2005).

[0010] Although substantial advancement has been made in our understanding of mVOCs and their multifunctional roles in crop growth promotion, including biocontrol of insects under laboratory conditions, we remain far from implementing that knowledge under field conditions. To date, no microbial-based volatiles have been used for jassid attraction or its management at the field scale. Although yellow sticky traps (YST) were popularly used as mechanical control options for jassid management in cotton, there is a need for further enhancement in the attraction of jassids to YST may be more useful to the farming community in tackling the jassid problem.

[0011] Therefore, there is an unmet need in the art to enhance the attraction of jassids using a microbial-based volatile attractant toward the yellow sticky traps, thus decreasing chemical pesticide usage in cotton, associated pest resurgence, and environmental pollution.OBJECTIVE OF THE INVENTION

[0012] An objective of the present invention is to provide a jassid attractant composition comprising of synthetic analogs of bacterial-based volatile compounds.

[0013] Another objective of the present invention is to provide a method for the preparation of a jassid attractant composition comprising of synthetic analogs of bacterialbased volatile compounds.

[0014] Another objective of the present invention is to provide a jassid attractant that is cost-effective, does not have any harmful effects on soil nutrient status and soil biology, environmentally friendly, can reduce chemical pesticide usage, insect resurgence in cotton cultivation, and thus have wider commercial application prospects in agriculture.

[0015] Another objective of the present invention is to provide jassid attractant that also attracts other major sucking pests of cotton, including whiteflies, aphids, and thrips.SUMMARY OF THE INVENTION

[0016] The present invention relates to a jassid attractant composition comprising of synthetic analogs of bacterial -based volatile compounds. Also, the present invention relates to a method for the preparation of a jassid attractant composition comprising of synthetic analogs of bacterial -based volatile compounds. Further, the jassid attractant is cost-effective, does not have any harmful effects on soil nutrient status and soil biology, environmentally friendly, can reduce chemical pesticide usage, insect resurgence in cotton cultivation, and thus have wider commercial application prospects in agriculture.

[0017] In an aspect, the present invention relates to a jassid attractant composition comprising an active ingredient as a mixture of synthetic attractant compounds, in which the attractant compounds are identified from the bacterial-based volatiles consisting of alkanes and a pyrazine.

[0018] In another aspect of the present invention, the said attractant compounds were selected from the alkane group comprising of Eicosane, Tetrapentacontane, Heneicosane, Tetracosane or a combination thereof.

[0019] In another aspect of the present invention, the said attractant compound is selected from a pyrazine group comprises 2-Methyltetrahydro- 2H-Pyrazino ( 1 ,2-a) Pyrazine- 1,4 (3H- 6H) -dione.

[0020] In another aspect of the present invention, wherein the attractant compounds are synthetic analogs of bacterial -based volatile compounds.

[0021] In another aspect of the present invention, the alkanes and pyrazine were identified from the following bacterial sources: Bacillus subtilis (NAIMCC-B-02312), Bacillus mojavensis (NAIMCC-B-02307), Delftia lacustris (NAIMCC-B-02315), Pseudomonas putida (NAIMCC-B-02309), Enterobacter hormaechei (NAIMCC-B-02308), Bacillus thaonhiensis (NAIMCC-B-02313), which were isolated and identified from the cotton rhizosphere of the ICAR-Central Institute for Cotton Research, Nagpur, and were proved to act as plant growth promoters and biocontrol agents against cotton pests.

[0022] In an aspect, the present invention utilized these bacterial sources only for the identification of the major volatile compounds involved in the attraction of jassids under field conditions. These bacteria or their cells / extracts were not directly used in our field / pot study.

[0023] The representative samples of the standardized cultures of bacterial sources have been deposited at the National Agriculturally Important Microbial Culture Centre (NAIMCC) located at the ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM, which was recognized as the International Depository Authority (IDA) by the WorldIntellectual Property Organization (WIPO), Geneva), Kushmaur Maunath Bhanjan 275 103 Uttar Pradesh, India and have been identified and assigned the accession numbers as follows:

[0024] In another aspect of the present invention, wherein the said attractant composition is a liquid, preferably a volatile liquid.

[0025] In another aspect of the present invention, the attractant composition is a slow- and / or controlled-release formulation.

[0026] In another aspect of the present invention, the target plant is cotton, and the target insect pest is jassids (Amrasca biguttula).

[0027] In another aspect of the present invention, the attractant composition provides an additional advantage in monitoring, controlling, or regulating other sucking pests, including whiteflies (Bemisia tabaci}. aphids (Aphis gossypii), and thrips (Thrips tabaci).

[0028] In another aspect, the present invention relates to a method for the preparation of a jassid attractant composition comprising alkanes and a pyrazine.

[0029] In another aspect, the jassid attractant was prepared using alkane, especially Eicosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using alkane, especially Tetrapentacontane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using alkane, especially Tetracosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using alkane, especially Heneicosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using a pyrazine, especially 2- Methyltetrahydro- 2H-Pyrazino ( 1,2-a) Pyrazine- 1,4 (3H-6H)-dione, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm.

[0030] In another aspect, the stock solutions of the synthetic analogs of the above mentioned bacterial-based volatile compounds were prepared separately using absoluteethanol, 99.8% (Sigma Aldrich), and later, from the stock solutions, the synthetic analogs of the volatile compounds were blended into an attractant formulation having concentrations of at least 0.005 g / L or 0.0005% or 5 ppm using distilled water and a solvent mixture

[0031] In another aspect of the present invention is that the solvent mixture comprises methanol (99.8%; Sigma Aldrich) and acetone (99.5%; Sigma Aldrich) in a ratio of 1: 1.

[0032] In another aspect of the present invention is that the synthetic analogs of selected volatile compounds were constituted in a liquid-based support material.

[0033] In another aspect, the attractant formulation was prepared and tested at concentrations of 0.002-0.01 g / L or 0.0002-0.001% or 2 ppm to 10 ppm on jassid attraction under field conditions.

[0034] In another aspect of the present invention, the attractant formulation was found to be more effective at 0.005 g / L or 0.0005% or 5 ppm concentration in jassid attraction.

[0035] In another aspect, the jassid attractant may comprise a mixture of synthetic analogs of bacterial-based volatile compounds comprising alkanes and a pyrazine in a suitable amount or concentration to attract the jassid to the holding device, i.e., yellow sticky trap (YST; 20 x 15 cm size).

[0036] In another aspect, the final jassid attractant composition comprising of Eicosane (100 pL), Tetrapentacontane (100 pL). Tetracosane (100 pL), Heneicosane (100 pL), and 2- Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione (100 pL) were dispensed in a dispenser, especially in a tube, such as a tube having, for example, a volume of 2 ml containing other components (carrier) of the attractant (composition), such as solvent mixture (distilled water + methanol + acetone in a ratio of 2: 1: 1, i.e., 600 pL: 300 pL: 300 pL), but which themselves have no significant biological activity, at least not on the host plant, i.e., cotton.

[0037] In another aspect of the present invention, the method comprises providing a jassid attractant in a holding device, wherein the attractant attracts jassid to the holding device, i.e., sticky trap, preferably a yellow sticky trap ((YST; 20 x 15 cm size), which includes means for immobilizing or killing jassids.

[0038] In another aspect of the present invention, the attraction efficiency of jassids is assessed by counting the number of jassids on the holding device, i.e., YST with attractant and on control.

[0039] In another aspect of the present invention, the attractant composition enhanced jassid attraction by 197% (attractive index of 1.50) to the target area, i.e., YST with attractant, compared with the control (only YST).

[0040] In another aspect of the present invention, a method for attracting field populations of jassids comprises monitoring, controlling, or regulating jassid infestation in cotton

[0041] In another aspect of the present invention, the jassid attractant does not have any harmful effects on cotton plants, soil nutrient availability, or soil biology.

[0042] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF FIGURES

[0043] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0044] Figure 1 illustrates the methodology flowchart of the present invention.

[0045] Figure 2 illustrates the confirmation of the attractant effect of the present invention against jassid in field E-l.

[0046] Figure 3 illustrates the confirmation of the attractant effect of the present invention against jassid in field E-19.

[0047] Figure 4 illustrates the confirmation of the attractant effect of the present invention against jassid in field E-36.

[0048] Figure 5 illustrates the confirmation of the attractant effect of the present invention against jassid in field E-37.

[0049] Figure 6 illustrates the confirmation of the attractant effect of the present invention against jassid in field E-38.

[0050] Figure 7 illustrates the confirmation of the attractant effect of the present invention against jassid in field E-42.

[0051] Figure 8 illustrates the confirmation of the attractant effect of the present invention against jassid (A sum of all field trials i.e., E-l, E-19, E-36, E-37, E-38 and E-42).

[0052] Figure 9 illustrates the confirmation of the attractant effect of the present invention against whiteflies (A sum of all field trials i.e., E-l, E-19, E-36, E-37, E-38 and E- 42).

[0053] Figure 10 illustrates the confirmation of the attractant effect of the present invention against aphids (A sum of all field trials i.e., E-l, E-19, E-36, E-37, E-38 and E-42).

[0054] Figure 11 illustrates the confirmation of the attractant effect of the present invention against thrips (A sum of all field trials i.e., E-l, E-19, E-36, E-37, E-38 and E-42).

[0055] Figure 12 illustrates the confirmation of additive attractant effect of the present invention when used along with yellow sticky traps (YST) compared with blue (BST) or white (WST) sticky traps on jassid attraction.

[0056] Figure 13 illustrates the field installation of attractants along with yellow sticky traps.DETAILED DESCRIPTION OF THE INVENTION

[0057] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

[0058] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0059] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0060] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0061] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be constructed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0062] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0063] All processes described herein can be performed in suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0064] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0065] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0066] In a general embodiment, the present invention relates to a jassid attractant composition comprising of synthetic analogs of bacterial-based volatile compounds. Also, the present invention relates to a method for the preparation of a jassid attractant composition comprising of synthetic analogs of bacterial-based volatile compounds. Further, the jassid attractant is cost-effective, does not have any harmful effects on soil nutrient status and soil biology, environmentally friendly, can reduce chemical pesticide usage and insect resurgence in cotton cultivation, thus having wider commercial application prospects in agriculture.

[0067] In an embodiment, the present invention relates to a jassid attractant composition comprising of an active ingredient as a mixture of attractant compounds, wherein the attractant compounds are identified from the bacterial-based volatiles consisting of alkanes and a pyrazine.

[0068] In another embodiment of the present invention, the said attractant compounds were selected from the alkane group comprising of Eicosane, Tetrapentacontane, Heneicosane, Tetracosane or a combination thereof.

[0069] In another embodiment of the present invention, the said attractant compound is selected from a pyrazine group comprises 2-Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione.

[0070] In another embodiment of the present invention, the synthetic analogs of five bacterial-based volatile compounds as shown below were selected for the preparation of jassid attractant composition.

[0071] In another embodiment of the present invention, wherein the attractant compounds are synthetic analogs of bacterial -based volatile compounds.

[0072] In another embodiment of the present invention, alkanes and pyrazine were identified from the following bacterial sources: Bacillus subtilis (NAIMCC-B-02312), Bacillus mojavensis (NAIMCC-B-02307), Delftia lacustris (NAIMCC-B-02315), Pseudomonas putida (NAIMCC-B-02309), Enterobacter hormaechei (NAIMCC-B-02308), Bacillus thaonhiensis (NAIMCC-B-02313), which were isolated and identified from the cotton rhizosphere of the ICAR-Central Institute for Cotton Research, Nagpur, and were proved to act as plant growth promoters and biocontrol agents against cotton pests.

[0073] In another embodiment of the present invention, these bacterial sources were only used for preliminary identification of the major volatile compounds involved in the attraction of jassids under field conditions. These bacteria or their cells / extracts were not directly used in our field / pot study.

[0074] The representative samples of the standardized cultures of bacterial sources have been deposited at National Agriculturally Important Microbial Culture Centre (NAIMCC)located at ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM- recognized as the International Depository Authority (IDA) by the World Intellectual Property Organization (WIPO), Geneva), Kushmaur Maunath Bhanjan 275 103 Uttar Pradesh, India and have been identified and assigned the accession numbers as follows:

[0075] In another embodiment of the present invention, wherein the said attractant composition is a liquid, preferably a volatile liquid.

[0076] In another embodiment of the present invention, the attractant composition is a slow- and / or controlled-release formulation.

[0077] In another embodiment of this invention, the target plant is cotton, and the target insect pest is jassids (Amrasca biguttula).

[0078] In another embodiment of the present invention, the attractant composition provides an additional advantage for monitoring, controlling, or regulating other sucking pests, including whiteflies (Bemisia tabaci), aphids (Aphis gossypii), and thrips (Thrips tabaci).

[0079] In another embodiment of the present invention is related to a method for the preparation of a jassid attractant composition comprising alkanes and a pyrazine.

[0080] In another aspect, the jassid attractant was prepared using alkane, especially Eicosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using alkane, especially Tetrapentacontane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using alkane, especially Tetracosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using alkane, especially Heneicosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the jassid attractant was prepared using a pyrazine, especially 2-Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm.

[0081] In another embodiment of the present invention, the stock solutions of the synthetic analogs of the above mentioned bacterial-based volatile compounds were prepared separately using absolute ethanol (99.8%; Sigma Aldrich), and later, from the stock solutions, the synthetic analogs of the volatile compounds were blended into an attractant formulation having concentrations of at least 0.005 g / L or 0.0005% or 5 ppm using distilled water and solvent mixture.

[0082] In another embodiment of the present invention, is that the solvent mixture comprises methanol (99.8%; Sigma Aldrich) and acetone (99.5%; Sigma Aldrich) in a ratio of 1: 1.

[0083] In another embodiment of the present invention, the synthetic analogs of selected volatile compounds were constituted in a liquid-based support material.

[0084] In another embodiment of the present invention, the attractant formulation was prepared and tested at concentrations of 0.002-0.01 g / L or 0.0002-0.001% or 2 ppm to 10 ppm on jassid attraction under field conditions.

[0085] In another embodiment of the present invention, the attractant formulation was found to be more effective at 0.005 g / L or 0.0005% or 5 ppm concentration in jassid attraction.

[0086] In another embodiment of the present invention, the jassid attractant may comprise a mixture of synthetic analogs of bacterial-based volatile compounds comprising alkanes and a pyrazine in a suitable amount or concentration to attract the jassid to the holding device, i.e., YST.

[0087] In another aspect, the final jassid attractant composition comprising of Eicosane (100 pL), Tetrapentacontane (100 pL). Tetracosane (100 pL), Heneicosane (100 pL), and 2- Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione (100 pL) were dispensed in a dispenser, especially in a tube, such as a tube having, for example, a volume of 2 ml containing other components (carrier) of the attractant (composition), such as solvent mixture (distilled water + methanol + acetone in a ratio of 2: 1: 1, i.e., 600 pL: 300 pL: 300 pL), but which themselves have no significant biological activity, at least not on the host plant, i.e. cotton.

[0088] In another embodiment of the present invention, the method comprises providing a jassid attractant in a holding device (Figure 13), wherein the attractant attracts jassids to the holding device, i.e., a sticky trap, preferably a yellow sticky trap (YST), which includesmeans for immobilizing or killing jassids.

[0089] In another embodiment of the present invention, the attraction efficiency of the jassids was assessed by counting the number of jassids on the holding device, i.e., YST with attractant and on control.

[0090] In another embodiment of the present invention, the attractant composition enhanced jassid attraction by 197% (attractive index of 1.50) to the target area, i.e., YST with attractant, compared with the control (only YST).

[0091] In another embodiment of the present invention, a method for attracting field populations of jassids comprises monitoring, controlling, or regulating jassid infestation in cotton.

[0092] In another aspect of the present invention, the jassid attractant does not have any harmful effects on cotton plants, soil nutrient availability, or soil biology.

[0093] In another embodiment of the present invention, a detailed methodology flowchart of the present invention is presented in Figure 1 and explained in detail using examples of the present application. The invention refers to the systematic development of a jassid attractant from laboratory to field testing and evaluation.

[0094] In another embodiment of the present invention, the attractant composition shows significant additive effects on jassid attraction, when used along with yellow sticky traps (YST) compared with blue (BST) or white (WST) sticky traps as shown in Figure 12.

[0095] In yet another embodiment of the present invention, the attractant apart from attracting jassids (Figure 8), also attracts other major sucking pests, including whiteflies (Figure 9), aphids (Figure 10), and thrips (Figure 11). In particular, it attracts whiteflies (Bemisia tabaci), aphids (Aphis gossypii), and thrips (Thrips tabaci) in addition to the targeted sucking pest, i.e., jassids in cotton.

[0096] In another embodiment of the present invention, the attractant significantly increases cotton growth, seedling vigour and further enhances the elicitation of plant defense enzyme activities (peroxidase, polyphenol oxidase, catalase, L-phenylalanine ammonia lyase, and phenols) in shoots and roots of cotton plants. Also, the attractant is safe and does not have any adverse effects on soil chemical properties (soil reaction and macro, secondary and micronutrient availability). Further, the attractant is safe and does not have any adverse effects on soil biological properties (soil respiration, microbial biomass carbon, glomalin content, soil enzyme activities). Thus, the attractant is safe, eco-friendly and does not cause any environmental pollution, and can reduce chemical pesticide usage and insect resurgence in cotton cultivation, thus has wider commercial application prospects in agriculture. Also,the attractant developed will help the farming community to save costs on chemical pesticides used for jassid management in cotton. Thus, the invention disclosed here describes the utility of synthetic analogs of bacterial-based volatile attractant for management of jassids infestation in cotton.

[0097] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. 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.EXAMPLES

[0098] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although processes and materials similar or equivalent to those described herein can be used in the practice of the disclosed processes and compositions, the exemplary processes, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular processes, and experimental conditions described, as such processes and conditions may vary.

[0099] Example 1: Bacterial-based volatile formulation preparationA. Identification of bacterial-based volatiles compounds

[0100] The bacterial-based volatile compounds disclosed in this invention were identified for their jassid attraction through replicated field testing of native bacterial crude extracts [Bacillus subtilis (NAIMCC- B-02312), Bacillus mojavensis (NAIMCC- B-02307), Delftia lacustris (NAIMCC- B-02315), Pseudomonas putida (NAIMCC- B-02309), Enterobacter hormaechei (NAIMCC- B-02308), Bacillus thaonhiensis (NAIMCC- B-02313)] using yellow sticky traps. However, these bacterial sources were only used for the preliminary identification of the major volatile compounds involved in the attraction of jassids under field conditions. These bacteria or their cells / extracts were not used directly for our further field / pot study. The major bacterial-based volatiles involved in jassid attraction were identified through Gas chromatography-mass spectrometry (GC-MS).B. Optimizing the combinations and concentrations of synthetic analogs of bacterialbased volatiles for jassid attraction

[0101] The attractant was prepared using alkane, especially hexatriacontane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Tetrapentacontane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Tetracosane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Dotriacontane, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Eicosane at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Heneicosane at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Pentadecane at a concentration of at least 0.005 ml / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using alkane, especially Tetradecane at a concentration of at least 0.005 ml / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using phthalate, especially Dibutyl phthalate at a concentration of at least 0.005 ml / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using carboxylic acid, especially Hexanedioic acid at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using a phenol derivative, especially Butylated Hydroxy Toluene, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm. In another aspect, the attractant was prepared using a pyrazine, especially 2- Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione, at a concentration of at least 0.005 g / L or 0.0005% or 5 ppm.

[0102] The stock solutions of the synthetic analogs of the above mentioned bacterial-based volatile compounds were prepared separately using absolute ethanol, 99.8% (Sigma Aldrich), and later, from the stock solutions, the synthetic analogs of the volatile compounds were blended into an attractant formulation having concentrations of at least 0.005 g / L or 0.0005% or 5 ppm using distilled water and solvent mixture [(methanol, 99.8% (Sigma Aldrich) and acetone, 99.5% (Sigma Aldrich) in a ratio of 1: 1)] in a ratio of 2: 1: 1 (distilled water + methanol + acetone), and tested for their synergistic field efficacy in attracting jassids at different combinations (Table 1) using the holding device, i.e., yellow sticky traps (YST) through a multi -choice assay.

[0103] Based on the field evaluation of 250 volatile combinations, (Eicosane (EC) + Heneicosane (HC) + Tetracosane (TC) + Tetrapentacontane (TPC) + 2-Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione (PY)) was found to be optimal for attracting the maximum number of jassids to the YST (Table 1). Further, to standardize the concentration of attractant for maximum jassid attraction, the identified volatile combination(EC + HC + TC + TPC + PY) was tested at different concentrations (0.002 g / L or 0.0002% or 2 ppm to 0.01 g / L or 0.001% or 10 ppm) (Table 2) using yellow sticky traps (YST).

[0104] Based on the replicated large-scale field evaluation, the synthetic volatiles combination EC + HC + TC + TPC + PY at 5 ppm concentration was found to be optimal for attracting the maximum number of j assids to the YST (T able 2) .Table 1. Synergistic studies on volatile (attractant) combinations on Jassid attraction towards YST for shortlisting of volatile combinations for further studies (All the values are mean of 3 independent replicated field trials)*BHT, Butylated Hydroxy Toluene; DBP, Dibutyl Phthalate; DCT, Dotriacontane; EC, Eicosane; HC, Heneicosane; HDC, Hexadecanoic acid; HTC, Hexatriacontane; PD, Pentadecane; PY, 2-Methyltetrahydro-2H- Pyrazino[l,2-a] Pyrazine-1,4 (3H, 6H)-Dione; TC, Tetracosane; TD, Tetradecane; TPC, Tetrapentaco ntane **S1. No. 167 (EC X HC X TC X PY X TPC) with higher jassid attraction efficiency (277% over the positive control) was identified for further large-scale field evaluations.Table 2: Effect of the attractant on jassids at different concentrations. (All the values are mean of 3 independent replicated field trials)♦Positive Control (Distilled water + solvent mixture + YST); **Negative Control (Only Yellow Sticky Trap)♦♦♦Based on the field evaluation, the 5-ppm concentration was found to be optimum for attracting the maximum number of jassid to the YSTC. Synthetic analogs of Bacterial-based volatile formulation

[0105] Based on the preliminary field trials with 250 volatile combinations (Table 1) to test for efficacy and possible synergistic effects, the combination designated as CICR-BVJ, comprising of five synthetic analogs of bacterial-based volatiles (EC x HC x TC x TPC x PY), was found to be most effective in attracting jassids. Based on the results, following five six synthetic analogs of bacterial -based volatile compounds i.e. Tetrapentacontane 96% (Linear formula: CE^CEfc^CHs; CAS: 5856-66-6; Sigma-Aldrich), Tetracosane 99% (Linear Formula: CE^CEb^CEE; CAS: 646-31-1; Sigma-Aldrich), Heneicosane 98% (Linear formula: CH3(CH2)i9CH3; CAS: 629-94-7; Sigma-Aldrich), Eicosane 99% (Linear formula:CH3(CH2)i8CH3;CAS: 112-95-8; Sigma-Aldrich), 2-Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione (Empirical Formula: CsHnN^C^Sigma- Aldrich) were procured from Sigma Aldrich.

[0106] The stock solutions of the synthetic analogs of the above mentioned bacterial-based volatile compounds were prepared separately using absolute ethanol (99.8%; Sigma Aldrich),and later, from the stock solutions, the synthetic analogs of the volatile compounds were blended into an attractant formulation having concentrations of at least 0.005 g / L or 0.0005% or 5 ppm using distilled water and solvent mixture comprising distilled water + methanol + acetone in a ratio of 2: 1 : 1.

[0107] The final jassid attractant composition comprising of Eicosane (100 pL), Tetrapentacontane (100 pL), Tetracosane (100 pL), Heneicosane (100 pL), and 2- Methyltetrahydro- 2H-Pyrazino (1,2-a) Pyrazine- 1,4 (3H-6H)-dione (100 pL) were dispensed in a dispenser, especially in a tube, such as a tube having, for example, a volume of 2 ml containing other components (carrier) of the attractant (composition), such as solvent mixture (distilled water + methanol + acetone in a ratio of 2: 1: 1, i.e., 600 pL: 300 pL: 300 pL), and evaluated under field conditions for jassid attraction using YST as holding device.

[0108] Example 2: Field tests

[0109] Independent field trapping trials (6 fields: E-l, E-19, E-36, E-37, E-38, E-42; 1 acre each) were conducted in cotton (Bt cotton - Gossypium hirsutum) fields at ICAR - Central Institute for cotton Research, Panjari farm, Nagpur (21° 04' 71" N and 79° 04' 40" E), Maharashtra, India. A 1.7 ml of the attractant (CICR-BVJ) @ 5 ppm in plastic vials (2ml capacity) was tied and hung from the YST attached to bamboo sticks 60-80 cm above the ground. A control treatment included YST + watersolvent mixture. Within a replicate (n=5), traps were set at least 10 m apart. The YST’s were replaced with new traps once every 10 days, whereas the attractant formulation was replaced once in 40 days. Before the field installation, 2 pin holes were made on the top of the vials containing the attractant to dissipate the contents into the surroundings. The trap catches were counted on both sides of the YSTs, starting from the sucking pest window i.e., 40 days after sowing and throughout the cotton growth period at an interval of 10 days. The jassid attraction efficiency was expressed in percent increase over the control, and the jassid attractive index (Al) was calculated using a formula 2*treatment group / (treatment group + Control group). The values of Al, >1 indicate that the attractant is effective as an attractant of targeted pest.

[0110] As can be seen in the Figure 2 to Figure 7, it was confirmed that the jassid (Amrasca biguttula) attractant (CICR-BVJ) of this invention had a significant attractant effect against the field populations of jassid compared with the control treatment. A 246% (1.55), 152% (1.43), 222% (1.53), 178% (1.47), 189% (1.49), 168% (1.46) attraction efficiencies (attractive index) were recorded in the fields E-l, E-19, E-36, E-37, E-38, E-42, respectively. The overall attraction efficiency (attractive index) was found to be 197% (1.50) (Figure 8).

[0111] Similar to jassid trap catch, the attraction of other major sucking pests of cotton (whiteflies, aphids and thrips) were counted on the same YSTs, where jassid counts were made. The other sucking pest’s attraction efficiency were expressed in percentage increase over the control trap, and the pest attractive index was calculated formula 2*treatment group / (treatment group + Control group). As can be seen in the Figure 9 to Figure 11, it was confirmed that the attractant (CICR-BVJ) of this invention had a significant attractant effect against the field populations other sucking pests such as whiteflies (98% increase & 1.32 Al) (Figure 9), aphids (117% increase & 1.37 Al) (Figure 10), and thrips (106% increase & 1.35 Al) (Figure 11) compared with control treatment (YST + solvent mixture)).

[0112] The jassid attractant (CICR-BVJ) were also tested separately under field conditions for their additive effect along with yellow (YST), blue (BST), and white (WST) sticky traps for jassids attraction. As can be seen in the Figure 12, it was confirmed that the attractant (CICR-BVJ) of this invention had a significant additive attractant effect against the field populations jassids, when used along with yellow sticky traps (YST) compared with blue (BST) or white (WST) sticky traps. CICR-BVJ + YST had an attraction efficiency and attractive index (Al) of 243% & 1.55, respectively as compared to BST (170% & 1.46) and WST (159% & 1.44).

[0113] Example 3: Pot study

[0114] Independent pot studies were conducted to determine the effects of the attractant (CICR-BVJ) on cotton growth, plant defense enzyme activity, soil chemical and soil biological properties, and soil volatile content following seed treatment. Standard analytical assays and procedures were used to analyze changes in the treatment and control groups. All experimental data were statistically analyzed using one-way analysis of variance with WASP version 2.0 (Web Agri Stat Package, Indian Council of Agricultural Research, India).

[0115] Effects of Jassid attractant on plant growth, defense enzyme activity, soil biology, and soil nutrient availabilityAs can be seen in Table 3, it was confirmed that the attractant (CICR-BVJ) of this invention had significant growth promoting effect on cotton in terms of germination (3% increase), shoot length (32% increase), root length (67% increase), root girth (33% increase), root shoot ratio (24% increase), root surface area (122% increase), and seedling vigour index (44% increase) as compared to control treatment.Table 3: Effect of attractant of the present invention on cotton growth attributes (30-day-old seedlings, values are mean of 10 replicates)As can be seen in Table 4, it was confirmed that the attractant (CICR-BVJ) of this invention had a significant positive effect on the elicitation of natural plant defense and antioxidant enzyme activities including peroxidase (20.5% & 14.9% increase), polyphenol oxidase (75% & 47.2% increase), catalase (16.6% & 11.7% increase), L-phenylalanine ammonia lyase (25.6% & 36.4% increase), and total phenols (28.2% & 102.5% increase) in cotton shoots and roots, respectively, as compared to control treatment. Table 4: Effect of attractant of the present invention on elicitation of plant defense enzyme activities in cotton (30 days-old cotton seedlings; all the values are mean of 10 replications)As can be seen in Table 5, it was confirmed that the attractant (CICR-BVJ) of this invention had a non-significant effect on soil biology, indicating non-harmful effects of attractant on soil biological functions.Table 5: Effect of attractant of the present invention on the soil biological properties (30-day- old seedlings, values are mean of 10 replicates)As can be seen in Table 6, it was confirmed that the attractant (CICR-BVJ) of this invention had a positive effect on the soil chemical and nutrient availability, indicating non-harmful effects of attractant on soil chemical properties and nutrient dynamics. Table 6: Effect of attractant of the present invention on the soil chemical properties and nutrient availability (30 dayold seedlings, values are mean of 10 replicates)As can be seen in Table 7, it was confirmed that the attractant (CICR-BVJ) of this invention had a non-significant effect on the soil volatiles, indicating a non-harmful effect of the attractant on soil volatiles and associated soil biological functions.Table 7: Effect of attractant of the present invention on the soil volatiles (30-day-old seedlings, values are mean of 3 replicates)

[0116] A skilled artisan will appreciate that the quantity and type of each ingredient can be used in different combinations or singly. All such variations and combinations would be falling within the scope of present disclosure.

[0117] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.ADVANTAGES OF THE PRESENT INVENTION1. The present invention provides an attractant composition that comprises synthetic analogs of bacterial-based volatiles for enhanced attraction of jassids to yellow sticky traps, thereby helps in management of jassid infestation in cotton compared with the use of yellow sticky traps alone.2. The present invention provides an attractant composition that significantly increases cotton growth, seedling vigour and further enhances the elicitation of plant defense enzyme activities in cotton.3. The present invention provides an attractant composition that is cost-effective, does not have any harmful effects on soil nutrient status and soil biology, environmentally friendly, and can reduce chemical pesticide usage and insect resurgence in cotton cultivation, thus has wider commercial application prospects in agriculture.4. The present invention provides an attractant composition that is eco-friendly and simple to use, helping the farming community to save costs on chemical pesticides used for jassid management in cotton.REFERENCES1. Bitas V, Kim H-S, Bennett JW, Kang S. 2013. Sniffing on microbes: diverse roles of microbial volatile organic compounds in plant health. Mol. Plant Microbe Interact. 26:835-843.2. Chaparro JM et al. 2014. Rhizosphere microbiome assemblage is affected by plant development. ISME J. 8:790-803.3. Effinert U, Kalderas J, Wamke R, Piechulla B. 2012.Volatile mediated interactions between bacteria and fungi in the soil. J. Chem. Ecol. 38:665-703.4. Farag MA, Zhang H, Ryu CM. 2013. Dynamic chemical communication between plants and bacteria through airborne signals: induced resistance by bacterial volatiles.J. Chem. Ecol. 39: 1007-1018.5. Hofs JL, Schoeman A, Vaissayre M. 2004. Effect of Bt cotton on arthropod biodiversity in South African cotton fields. Comm. Agri. Appl. Biol. Sci. 69: 191-194.6. Jeyakumar P, Tanwar RK, Chand M, Singh A, Monga D, Bambawale OM. 2008. Performance of Bt cotton against sucking pests. J. Biopesticides. 1:223-225.7. Kanchiswamy CN, Malnoy M, Maffei ME. 2015a. Bioprospecting bacterial and fungal volatiles for sustainable agriculture. Trends Plant Sci. 20:206-211.Kanchiswamy CN, Malnoy M, Maffei ME. 2015b. Chemical diversity of microbial volatiles and their potential for plant growth and productivity. Front.Plant Sci. 6: 151. Kranthi KR, Jadhav DR, Kranthi S, Wanjari RR, Ali S, Russell D. 2002. Insecticide resistance in five major insect pests of cotton in India. Crop Prot. 21:449-460. Murugesan, Kavitha A. 2010. Host plant resistance in cotton accessions to the leafhopper, Amrasca devastans (Distant). J. Biopesticides 3: 526-533. Penuelas J, Asensio D, Tholl D,Wenke K, Rosenkranz M, Piechulla B, et al. 2014. Biogenic volatile emissions from the soil. Plant Cell Environ. 37: 1866-1891. Ryu CM, Farag MA, Hu CH, Reddy MS, Kloepper JW, Pare PW. 2004. Bacterial volatiles induce systemic resistance in Arabidopsis . Plant Physiol. 134: 1017-1026. Ryu C-M, Farag MA, Hu C-H, Reddy MS, Wei HX, Pare PW. 2003. Bacterial volatiles promote growth in Arabidopsis . PNASUSA. 100:4927-4932 Saeed R, Razaq M, Rafiq M, Naveed M. 2016. Evaluating insecticide spray regimes to manage Cotton Leafhopper, Amrasca devastans (Distant): Their impact on natural enemies, yield and fiber characteristics of transgenic Bt cotton. Pakitan J. Zool. 48:703-711. Sharma HC, Pampapathy G. 2006. Influence of transgenic cotton on the relative abundance and damage by target and non-target insect pests under different protection regimes in India. Crop Pro. 25:800-813. Symonds MRE, Wertheim B. 2005. The mode of evolution of aggregation pheromones in Drosophila species. J. Evol. Biol. 18: 1253-1263.

Claims

We Claim:

1. A jassid attractant composition comprising of a mixture of attractant compounds in a concentration of 2 to 10 ppm as active ingredient, wherein the attractant compounds are bacterial -based synthetic volatile compounds consisting of an alkane and a pyrazine.

2. The jassid attractant composition as claimed in claim 1, wherein the said alkanes are selected from the group consisting of Eicosane, Tetrapentacontane, Heneicosane, Tetracosane and a combination thereof.

3. The jassid attractant composition as claimed in claim 1, wherein the said pyrazine is 2- Methyltetrahydro- 2H-Pyrazino ( 1,2-a) Pyrazine- 1,4 (3H-6H)-dione.

4. The jassid attractant composition as claimed in claim 1, wherein the attractant compounds are synthetic analogs of bacterial based synthetic volatile compounds.

5. The jassid attractant composition as claimed in claim 1, wherein the jassid attractant composition is a slow or controlled release formulation.

6. The jassid attractant composition as claimed in claim 1, wherein the jassid are Amrasca biguttula.

7. The jassid attractant composition as claimed in claim 1, wherein the jassid attractant composition is formulated as a liquid formulation, wherein the jassid attractant composition as claimed in claim 1 is constituted in a solvent containing distilled water and an organic solvent mixture.

8. The jassid attractant composition as claimed in claim 1, wherein the synthetic volatile compounds are present in the concentration of 5 ppm.

9. The jassid attractant composition as claimed in claim 1, wherein the organic solvent mixture comprises methanol and acetone in the ratio of 1 : 1.

10. A method for attracting the jassids comprises of (a) hanging the jassid attractant composition as claimed in anyone of claims 1- 9 in a vial and a yellow sticky traps (YST) side-by-side in a field; and (b) effecting the attraction of field population of jassids and sticking of jassids in the YST.

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