阅读说明：本技术 杀线虫剂的协同组合物 (Synergistic composition of nematicides ) 是由 韦尼·波利内尼 亚历杭德罗卡尔德隆·乌雷亚 格伦达·W·波洛茨克 于 2018-03-17 设计创作，主要内容包括：本发明提供了一种杀线虫剂的协同组合物,所述杀线虫剂包括衍生自包括至少两种胜肽的CED-4蛋白序列的一杀线虫胜肽。所述杀线虫肽在1毫克/毫升的浓度下单独显示出100％的杀线虫活性,而所述以1：1的比例组合的至少两种胜肽在低至0.8毫克/毫升的浓度下显示出100％杀线虫活性并产生一协同作用。由于所述协同杀线虫组合物在100％的活性下所需要的胜肽的浓度少了20％,因此所述协同杀线虫组合物是高度经济的,此外,所述协同杀线虫组合物对环境安全的且对人及动物无毒。(The present invention provides a synergistic composition of nematicides comprising a nematicidal peptide derived from a CED-4 protein sequence comprising at least two peptides. The nematicidal peptides show 100% nematicidal activity alone at a concentration of 1 mg/ml, whereas the nematicidal peptides show 100% nematicidal activity at a concentration of 1: 1 at concentrations as low as 0.8 mg/ml, show 100% nematicidal activity and produce a synergistic effect. The synergistic nematicidal composition is highly economical since it requires 20% less concentration of peptide at 100% activity, and moreover, it is environmentally safe and non-toxic to humans and animals.)

1. A synergistic composition for controlling nematodes characterized by: the synergistic composition comprises at least two peptides derived from CED-4 protein,

wherein the plurality of peptides consists of the amino acid sequences of SEQ ID No.2 to 4;

the ratio of the plurality of peptides is 1: 1; and

the synergistic anti-nematode efficiency of the composition at a concentration of about 0.8 mg/ml is 100%.

2. The composition of claim 1, wherein: the composition comprises a peptide consisting of: an amino acid sequence of SEQ ID No.2 having a length of 15 amino acids; and all analogues, homologues, derivatives and conservative variants of said amino acid sequences.

3. The composition of claim 1, wherein: the composition comprises a peptide consisting of: an amino acid sequence of SEQ ID No.3 having a length of 16 amino acids; and all analogues, homologues, derivatives and conservative variants of said amino acid sequences.

4. The composition of claim 1, wherein: the composition comprises a peptide consisting of: an amino acid sequence of SEQ ID No.4 having a length of 12 amino acids; and all analogues, homologues, derivatives and conservative variants of said amino acid sequences.

5. The composition of claim 1, wherein: the composition further comprises at least one synergist, emulsifier and/or surfactant.

6. The composition of claim 1, wherein: the composition further comprises at least one agrochemical active compound.

7. The composition of claim 3, wherein: the agrochemical active compound is selected from, but not limited to, substances capable of treating a plurality of plants, fungicides, bactericides, insecticides, acaricides, nematicides, molluscicides, safeners, plant growth regulators, plant nutrients and biological control agents.

Technical Field

The present invention relates to nematicides in the agricultural field in compositions for killing a plurality of plant parasites, in particular a plurality of nematodes. More specifically, the present invention relates to a synergistic composition of nematicides comprising nematicidal peptides derived from CED-4 proteins.

Background

Nematodes are a major threat to agriculture, and have a major economic impact due to their multiple serious losses to yield. It is well known that nematodes are secondary to insects and cause serious damage to crops such as tomatoes and other vegetables, citrus fruits, potatoes, rice, coconut, wheat and other grains, ornamentals, and the like. Nematodes alone or in combination with other soil microorganisms have been found to attack almost every part of a plant, including: roots, stems, leaves, fruits and seeds. One predicted yield loss of nematodes worldwide is 12.3% ($1570 billion dollars), of which 4030 million dollars are reported to be from india (Singh et al, 2015).

The root knot nematodes belong to the genus Meloidogyne, which is one of three of a plurality of plant parasitic nematodes that cause the greatest damage to the economy in horticulture and crops, and are obligate parasites of the roots of various plants; and meloidogyne incognita is one of the main global pests.

Nematicides are compositions for killing the plant parasites, nematodes, and most nematicides used are chemical compositions that are highly toxic to humans and also harmful to useful soil bacteria. Various nematicides have also been shown to contaminate groundwater and cause depletion of the ozone layer. A well-known nematicide, methyl bromide, is banned in a number of countries including the united states and india. Another highly toxic nematicide widely used in crops is carbofuran; a single particle of carbofuran kills birds in a few minutes. Several nematicides such as formosan easily penetrate the soil into and contaminate groundwater.

The serious drawbacks associated with multiple chemical nematicides necessitate the development of new technologies for controlling multiple nematodes. One such method is the generation of transgenic plant lines expressing multiple transgene lines that are nematode resistant. However, this is a time consuming and expensive process, requiring a significant amount of prior research activity. In many countries, including india, the introduction of multiple transgenic lines is strongly objected to based on some ethical, ethical and unseen environmental issues.

One effective method involves the generation of multiple compositions of nematicides that are environmentally safe, non-toxic, and easy to generate and use.

US9125413B1 describes peptides derived from the CED-4 protein of C.elegans, having potent nematicidal activity. The CED-4 protein consists of SEQ ID NO.1 and belongs to the apoptosis protein family. The SEQ ID NO.1 comprises:

MLCEIECRALSTAHTRLIHDFEPRDALTYLEGKNIFTEDHSELISKMSTRLERIANFLRIYRRQASELGPLIDFFNYNNQSHLADFLEDYIDFAINEPDLLRPVVIAPQFSRQMLDRKLLLGNVPKQMTCYIREYHVDRVIKKLDEMCDLDSFFLFLHGRAGSGKSVIASQALSKSDQLIGINYDSIVWLKDSGTAPKSTFDLFTDILLMLKSEDDLLNFPSVEHVTSVVLKRMICNALIDRPNTLFVFDDVVQEETIRWAQELRLRCLVTTRDVEISNAASQTCEFIEVTSLEIDECYDFLEAYGMPMPVGEKEEDVLNKTIELSSGNPATLMMFFKSCEPKTFEKMAQLNNKLESRGLVGVECITPYSYKSLAMALQRCVEVLSDEDRSALAFAVVMPPGVDIPVKLWSCVIPVDICSNEEEQLDDEVADRLKRLSKRGALLSGKRMPVLTFKIDHIIHMFLKHVVDAQTIANGISILEQRLLEIGNNNVSVPERHIPSHFQKFRRSSASEMYPKTTEETVIRPEDFPKFMQLHQKFYDSLKNFACC。

three peptides derived from SEQ ID No.3 have been shown to have nematicidal activity at effective concentrations of about 1 milligram per milliliter (mg/ml), the peptides comprising the following sequences:

1) peptide 2, consisting of 15 amino acids DLLRPVVIAPQFSRQ (99 to 113 amino acid sequence segments, SEQ ID NO.2),

2) peptide 3 consisting of 19 amino acids RQMLDRKLLLGNVPKQMTC (112 to 130 amino acid sequence segments, SEQ ID NO.3), and

3) peptide 12, consisting of 12 amino acids FPKFMQLHQKFY (529 to 540 amino acid sequence segments, SEQ ID No. 4).

However, the synthesis of multiple peptides is an expensive technique and in order to make a composition cost effective, one needs to reduce the cost of production. Typically, the cost of synthesizing 50 milligrams (mg) of peptide in the range between 10 and 20 amino acids is between $25,000 to 30,000 manus and $240 to $ 250. Therefore, it is important to prepare a plurality of compositions that reduce production costs.

The present invention takes into account several disadvantages of the prior art and provides a method of using a plurality of antiparasitic peptides that are non-toxic and environmentally safe.

The purpose of the invention is as follows:

accordingly, it is a primary object of the present invention to provide a nematicide composition comprising a plurality of nematicidal peptides.

It is another object of the present invention to provide a synergistic nematicidal composition comprising: two peptides derived from the CED-4 protein as set forth in SEQ ID No.1, said two peptides consisting of the amino acid sequences:

a.DLLRPVVIAPQFSRQ(SEQ ID NO.2)，

RQMLDRKLLLGNVPKQMTC (SEQ ID NO.3), and

c.FPKFMQLHQKFY(SEQ ID NO.4)。

it is another object of the present invention to provide a synergistic nematicidal composition exhibiting nematicidal activity, the concentration of the plurality of peptides being less than 1 mg/ml.

It is another object of the present invention to provide a synergistic nematicidal composition with reduced production costs.

It is another object of the present invention to provide a synergistic nematicidal composition which is non-toxic to humans.

It is another object of the present invention to provide a synergistic nematicidal composition which does not have a broad spectrum of activity and is not harmful to a plurality of soil microorganisms.

It is another object of the present invention to provide a synergistic nematicidal composition which is easy to synthesize, does not require the production of multiple transgenic plants, and is very economical.

Disclosure of Invention

The present invention relates to a composition of nematicides for agricultural purposes to kill a plurality of plant parasites, in particular a plurality of nematodes. More specifically, the present invention relates to a synergistic composition of nematicides comprising a plurality of nematicidal peptides.

In its main embodiment, the present invention provides a nematicide composition comprising: a plurality of peptides derived from the CED-4 protein as set forth in SEQ ID No. 1. More particularly, the composition comprises a synergistic combination of at least two peptides consisting of the amino acid sequences:

DLLRPVVIAPIQFSRQ (SEQ ID NO.2), peptide 2, comprising 15 amino acids,

RQMLDRKLLLGNVPKQMTC (SEQ ID NO.3), peptide 3, which comprises 19 amino acids, and

fpkfmqlhqkfy (SEQ ID No.4), peptide 12, which comprises 12 amino acids.

The peptides showed 100% nematicidal activity at various concentrations as low as 1 mg/ml, respectively.

However, the combination of the at least two peptides acts in a synergistic manner, and the concentration of each of the plurality of peptides can be reduced to 0.4 mg/ml for 100% nematicidal activity. The significant reduction in the amount of peptide required for activity greatly reduces the cost of production, making the unique composition very economical.

Drawings

FIG. 1 shows the inclusion of peptide 2 and peptide 3 in a plurality of 96-well plates; peptide 2 and peptide 12; peptide 3 and peptide 12; and mixtures of peptides 2, 3 and 12 on a plurality of nematodes.

Detailed Description

The present invention will now be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be provided so that this disclosure will be thorough and will fully convey the scope of the invention to those skilled in the art.

The present invention relates to a synergistic composition having the ability to kill a plurality of plant parasites, in particular a plurality of nematodes. More particularly, the present invention relates to a synergistic composition of a nematicide comprising a plurality of nematicidal peptides derived from said CED-4 protein sequence.

In its principal embodiment, the present invention provides a synergistic composition of a nematicide comprising: at least two peptides derived from the CED-4 protein as set forth in SEQ ID No. 1. More particularly, the composition comprises a combination of the following peptides:

a. peptide 2, a 15 amino acid long peptide comprising: DLLRPVVIAPQFSRQ sequence (SEQ ID NO.2),

b. peptide 3, a 19 amino acid long peptide comprising: RQMLDRKLLLGNVPKQMTC sequence (SEQ ID NO.3), and

c. peptide 12, a 12 amino acid long peptide comprising: FPKFMQLHQKFY sequence (SEQ ID NO. 4);

at least two peptides containing a plurality of amino acids of SEQ ID No.2 to 4 in a ratio of 1: 1 as a highly effective nematicide, showed 100% nematicidal activity at total peptide concentrations as low as 0.8 mg/ml. The combination of the two peptides brings about a synergistic effect and acts at a lower concentration than the individual peptides alone. The synergistic composition reduces the total peptide concentration required by 20%, thereby making the present invention very economical. Moreover, the compositions are environmentally safer than other existing methods and are easier to produce than multiple transgenic lines with multiple nematicidal activities.

Example 1-multiple steps involved:

A) propagation of meloidogyne incognita

A plurality of tomato plants were inoculated with a plurality of larvae of meloidogyne incognita (m.incognita) and maintained in a growth chamber. After at least two months, the southern root knot nematode eggs were extracted from the plurality of roots for a number of experiments, and the following describes the steps for extracting southern root knot nematode eggs.

The plurality of root tissues may be minced by hand using a surgical blade and a sight glass, or may be minced using a food processor, and then the minced tissues are placed in a bottle and washed with a 10% diluted bleaching agent. The root solution was then poured into multiple sieves (60 mesh on top and 500 mesh on the bottom) under multiple aseptic conditions. Coarse eggs were collected from the bottom of the 500 mesh screen and 5 ml of bleach and egg mix were placed in 15 ml Falcon (Falcon) tubes, respectively. Then 5 ml of a 70% sucrose solution was placed in each falcon tube, and then 1 ml of a double distilled sterile aqueous layer was gently placed over the multiple sucrose mixtures in each falcon tube. The multiple samples were then centrifuged at 1200 revolutions per minute (rpm) for 5 minutes. A total of 3 ml of embryos suspended between the sucrose solution and 1 ml of aqueous layer were collected from each falcon duct (3 ml of the top layer of the solution) into a new 15 ml falcon duct, 10 ml of 5% bleach solution was added, and the eggs were vortexed for 10 minutes. The falcon tubes were then centrifuged at 2000rpm for 5 minutes. The supernatant was then removed and the multiple eggs were rinsed in 10 ml of sterile double distilled water and centrifuged again at 2000rpm for 5 minutes. The process was repeated two more times. After the final wash, 5 ml of supernatant was removed, and the remaining 5 ml of water was mixed with the multiple eggs and placed in a 5 ml petri dish. The eggs are then placed in an incubator at 25 to 27 ℃ and after about 10 days a plurality of larvae is hatched (stage J2) and the larvae are stored in an incubator at 25 to 27 ℃.

B) Preparing multiple test solutions and placing multiple nematodes

Multiple lyophilized peptides were dissolved in the desired amount of double distilled water to achieve multiple desired concentrations.

100 microliters of the multiple peptide solutions having multiple desired concentrations were pipetted into 30 wells of a 96-well plate, and a larva was transferred from a stock solution of extracted southern root knot nematode stage J2 into each well. For a negative control group, 30 larvae were placed in 100 microliters of sterile double distilled water in each experiment.

C) Monitoring multiple nematodes

The bioassay is intended to test the ability of the plurality of peptides and chalcones to kill the plurality of larvae (% mortality). Each test was performed in a 96-well plate with one nematode in each well (30 wells total). The plurality of nematodes is incubated in the plurality of treatment solutions for 5 days. The viability of the nematodes was examined under a dissecting microscope by examining the movement of each nematode after interference with a probe.

Example 2-efficacy of nematicidal compositions comprising a combination of multiple nematicidal peptides:

as shown in figure 1, larval nematodes in stage J2 incubated in water resulted in about 10 to 20% of multiple nematode deaths on day 5, which served as a negative control. Incubation of multiple larval nematodes in individual peptides 2, 3 or 12 at a concentration of 0.8 mg/ml resulted in approximately 60 to 66% of the multiple larval nematodes dying on day 5. However, in the case of a composition comprising a ratio of 1: 1, peptide 2 and peptide 3, peptide 2 and peptide 12, or a combination of peptide 3 and peptide 12 resulted in approximately 100% mortality of a plurality of juvenile nematodes at day 5, said total concentration of said plurality of peptides being 0.8 mg/ml. In addition, the ratio of 1: 1: 1, also caused 100% of deaths of multiple juvenile nematodes on day 5.

The results indicate that the peptides derived from the CED-4 protein act synergistically in combination with each other as a nematicidal composition.

Sequence listing

<110> private company India of Telluris Biotechnology

<120> synergistic composition of nematocides

<130> P.1505.IB

<150> 201741009365

<151> 2017-03-17

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 549

<212> PRT

<213> Caenorhabditis elegans

<400> 1

Met Leu Cys Glu Ile Glu Cys Arg Ala Leu Ser Thr Ala His Thr Arg

1 5 10 15

Leu Ile His Asp Phe Glu Pro Arg Asp Ala Leu Thr Tyr Leu Glu Gly

20 25 30

Lys Asn Ile Phe Thr Glu Asp His Ser Glu Leu Ile Ser Lys Met Ser

35 40 45

Thr Arg Leu Glu Arg Ile Ala Asn Phe Leu Arg Ile Tyr Arg Arg Gln

50 55 60

Ala Ser Glu Leu Gly Pro Leu Ile Asp Phe Phe Asn Tyr Asn Asn Gln

65 70 75 80

Ser His Leu Ala Asp Phe Leu Glu Asp Tyr Ile Asp Phe Ala Ile Asn

85 90 95

Glu Pro Asp Leu Leu Arg Pro Val Val Ile Ala Pro Gln Phe Ser Arg

100 105 110

Gln Met Leu Asp Arg Lys Leu Leu Leu Gly Asn Val Pro Lys Gln Met

115 120 125

Thr Cys Tyr Ile Arg Glu Tyr His Val Asp Arg Val Ile Lys Lys Leu

130 135 140

Asp Glu Met Cys Asp Leu Asp Ser Phe Phe Leu Phe Leu His Gly Arg

145 150 155 160

Ala Gly Ser Gly Lys Ser Val Ile Ala Ser Gln Ala Leu Ser Lys Ser

165 170 175

Asp Gln Leu Ile Gly Ile Asn Tyr Asp Ser Ile Val Trp Leu Lys Asp

180 185 190

Ser Gly Thr Ala Pro Lys Ser Thr Phe Asp Leu Phe Thr Asp Ile Leu

195 200 205

Leu Met Leu Lys Ser Glu Asp Asp Leu Leu Asn Phe Pro Ser Val Glu

210 215 220

His Val Thr Ser Val Val Leu Lys Arg Met Ile Cys Asn Ala Leu Ile

225 230 235 240

Asp Arg Pro Asn Thr Leu Phe Val Phe Asp Asp Val Val Gln Glu Glu

245 250 255

Thr Ile Arg Trp Ala Gln Glu Leu Arg Leu Arg Cys Leu Val Thr Thr

260 265 270

Arg Asp Val Glu Ile Ser Asn Ala Ala Ser Gln Thr Cys Glu Phe Ile

275 280 285

Glu Val Thr Ser Leu Glu Ile Asp Glu Cys Tyr Asp Phe Leu Glu Ala

290 295 300

Tyr Gly Met Pro Met Pro Val Gly Glu Lys Glu Glu Asp Val Leu Asn

305 310 315 320

Lys Thr Ile Glu Leu Ser Ser Gly Asn Pro Ala Thr Leu Met Met Phe

325 330 335

Phe Lys Ser Cys Glu Pro Lys Thr Phe Glu Lys Met Ala Gln Leu Asn

340 345 350

Asn Lys Leu Glu Ser Arg Gly Leu Val Gly Val Glu Cys Ile Thr Pro

355 360 365

Tyr Ser Tyr Lys Ser Leu Ala Met Ala Leu Gln Arg Cys Val Glu Val

370 375 380

Leu Ser Asp Glu Asp Arg Ser Ala Leu Ala Phe Ala Val Val Met Pro

385 390 395 400

Pro Gly Val Asp Ile Pro Val Lys Leu Trp Ser Cys Val Ile Pro Val

405 410 415

Asp Ile Cys Ser Asn Glu Glu Glu Gln Leu Asp Asp Glu Val Ala Asp

420 425 430

Arg Leu Lys Arg Leu Ser Lys Arg Gly Ala Leu Leu Ser Gly Lys Arg

435 440 445

Met Pro Val Leu Thr Phe Lys Ile Asp His Ile Ile His Met Phe Leu

450 455 460

Lys His Val Val Asp Ala Gln Thr Ile Ala Asn Gly Ile Ser Ile Leu

465 470 475 480

Glu Gln Arg Leu Leu Glu Ile Gly Asn Asn Asn Val Ser Val Pro Glu

485 490 495

Arg His Ile Pro Ser His Phe Gln Lys Phe Arg Arg Ser Ser Ala Ser

500 505 510

Glu Met Tyr Pro Lys Thr Thr Glu Glu Thr Val Ile Arg Pro Glu Asp

515 520 525

Phe Pro Lys Phe Met Gln Leu His Gln Lys Phe Tyr Asp Ser Leu Lys

530 535 540

Asn Phe Ala Cys Cys

545

<210> 2

<211> 15

<212> PRT

<213> Caenorhabditis elegans

<400> 2

Asp Leu Leu Arg Pro Val Val Ile Ala Pro Gln Phe Ser Arg Gln

1 5 10 15

<210> 3

<211> 19

<212> PRT

<213> Caenorhabditis elegans

<400> 3

Arg Gln Met Leu Asp Arg Lys Leu Leu Leu Gly Asn Val Pro Lys Gln

1 5 10 15

Met Thr Cys

<210> 4

<211> 12

<212> PRT

<213> Caenorhabditis elegans

<400> 4

Phe Pro Lys Phe Met Gln Leu His Gln Lys Phe Tyr

1 5 10