Uses, methods and biological compositions of the genus Paecilomyces in the control, prevention and eradication of plant parasites in Solanaceae cultures

Abstract

The invention relates to the method for producing the fungus Paecilomyces spp., and to the uses, methods and nematicide compositions for the prevention and/or control and/or eradication of nematodes that form cysts developing in solanaceae cultures. Said invention includes a system for the application of the fungus combined with the rotation of cultures for effectively and efficiently reducing the viability of the cysts of nematodes in solanaceae cultures.

Claims

1. A method of controlling or eradicating nematodes in a culture parasited by a cyst-forming nematode, comprising applying a composition comprising: Paecilomyces carneus fungus; either ampicillin or chloramphenicol; oats, carrot juice, and water to the culture.

2. The method of claim 1, in which the culture is a Solanaceae culture.

3. The method of claim 1, in which the Paecilomyces carneus fungus is applied to the culture in combination with at least one carrier.

4. The method of claim 1, in which the Paecilomyces carneus fungus is applied to a culture simultaneously with a culture rotation.

5. The method of claim 1, in which the Paecilomyces carneus fungus is applied prior to the culture, during the culture and/or after every culture cycle.

6. A bio-nematicide composition for the control, or eradication of nematodes comprising: a living cell suspension of a Paecilomyces carneus fungus; either ampicillin or chloramphenicol; oats, carrot juice, and water.

7. The method of claim 1, in which nematode is a Globodera spp.

8. The method of claim 1, in which a living cell suspension of a Paecilomyces carneus fungus is applied combined with at least one carrier.

9. The composition of claim 6, in which the composition comprises chloramphenicol.

10. The composition of claim 6, wherein the composition comprises 500-1000 mg of ampicillin or chloramphenicol, 50-150 ml/l of carrot juice, 20-50 g/l of oats, and 0.5-5 g/l of yeast.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. A comparative study of the effect of Paecilomyces carneus and Paecilomyces lilacinus on Globodera rostochiensis.

(2) FIG. 2. A microphotography of strain IE-431 of Paecilomyces carneus.

(3) FIG. 3. A microphotography of Globodera rostochiensis.

(4) FIG. 4. Globodera rostochiensis cysts.

(5) FIG. 5. J2 of Globodera rostochiensis infected with Paecilomyces carneus strain IE-431, inside the egg.

DETAILED DESCRIPTION OF THE INVENTION

(6) This novel invention disclosed herein below comprises uses, methods and biological compositions of Paecilomyces carneus for the control and/or prevention and/or eradication of plant parasites in Solanaceae cultures.

(7) Paecilomyces carneus strain IE-431 is a fungus produced according to the methods of this invention. Said strain is presently maintained and registered in the fungal collection of the Instituto de Ecologia (INECOL), and is registered with the World Federation Culture Collection (WFCC) under No. IEWDCM782. The isolation and/or maintain of the Paecilomyces carneus strain IE-431 are carried out in one or more solid culture means, constituted by at least a nitrogen source and/or at least a carbon source and/or one or more mineral salts and/or at least a suitable vehicle and/or at least an antibiotic agent and/or at least a growing helper agent. Said culture is incubated within a temperature within the range of from 12 C. to 35 C. for a time enough to obtain a pertinent concentration.

(8) The massive reproduction of the fungus consists in the inoculation of living cells of Paecilomyces carneus in liquid media comprising one or more amino acids and/or one or more carbohydrates and/or one or more mineral salts and/or at least one suitable vehicle and/or at least an antibiotic agent and/or at least a growing helper agent. Said culture is incubated at a temperature within a range of from 12 C. to 35 C. for a time enough to obtain a pertinent concentration (FIG. 3).

(9) Said fungus Paecilomyces carneus strain IE-431obtained from a novel process of isolation and/or maintain and/or massive reproduction disclosed in this novel investigation, exhibits a series of genetic characteristics that are conserved without representative structural changes, which make it different from any other species of Paecilomyces and never observed before with any other species belonging to genus Paecilomyces spp., characterized in that presents a colony diameter of from 15 to 21 mm in 10 days on agar-oats (AA) media, of from 15 to 19 mm on Agar-dextrose-potato (PDA), while on Agar-carrot-potato (PZA) it grows from 15 to 17 mm. The mycelium thereof is of white to hyaline color (1A1) in the color chart of Farver and Faver of Wanscher and Kornerup (1961); it does not stain the culture media tested both on the front and reverse of the agar plate. The texture thereof is filamentous accented on PZA; it grows very close to the surface of the media (sparse) and becomes dusty when sporulates. Said mycelium deforms the PDA with the appearance of notorious grooves at the reverse of the box. It is shaped as erect, macronematous, simple conidiophores, with no branches with catenulated equinulated conidia of subglobe shape, and some of them have ends slightly pointed at the ends, dimensioned to 2.4-4 m2-3.2 m.

(10) In the solid culture medium, 86% of the conidia germinate at 16 hours, the germ tube varies between 6 and 54 micrometers in length. At 24 hrs 100% of the spores germinate and the mycelium is greater than 30 and less than 110 micrometers in length.

(11) Under the light of the above discussed technical knowledge developed for this invention, we are filing the DNA nucleotide sequence coding Paecilomyces carneus strain IE-431, as disclosed at the end hereof.

(12) In this novel investigation, it was surprisingly found that, when living cells of Paecilomyces carneus IE-431 were placed in contact with cysts of the nematode Globodera rostochiensis, substantial damages were produced on the cysts, such that the propagation of this parasite is avoided. During our investigations, we found that the interaction between Paecilomyces carneus and Globodera rostochiensis occurs through the fungus germination which, when contacts the cysts (FIG. 4) said cysts are colonized by the mycelium at the interior thereof (FIG. 5) thus surprisingly affecting the whole amount of eggs and larvae J1 and J2 contained within said cyst. The cyst infection starts through an enzymatic process when Paecilomyces carneus permeates the cyst wall and then the mycelium grows on the surface of the eggs and the interior of juvenile J1 and J2; therefore, the development of said mycelium becomes invasive to the point of completely degrade the eggs and/or juveniles J1 and J2.

(13) During the investigation a study was carried out on the nematicide effect of Paecilomyces carneus strain IE-431 against the effect of Paecilomyces lilacinus IE-430 on the Globodera rostochiensis cysts, and It was surprisingly found that Paecilomyces carneus IE-431 acts in a more rapid and noxious manner against the cysts of said nematode; i.e., there is a significant difference between to pathogenicity of one species and the other against said nematode. This study was carried to laboratory level, evaluating the degree of infection of said cysts with a concentration of living cells of either fungus of 110.sup.6 living cells/mL, and a third control group with the only addition of distilled sterile water. Said pathogenicity was evaluated for a period of 14 days, according to the macroscopic and microscopic aspects of cysts, and a significant unexpected difference was obtained relating the effect of fungus Paecilomyces carneus strain IE-431, compared to Paecilomyces lilacinus. Results are shown in FIG. 5.

(14) The next test stage of the use of fungus Paecilomyces carneus strain IE-431 as a nematicide was applied singly (TP) and jointly with lima bean (HP) and peas (ChP) crops rotation, with an absolute witness (TA) where neither control nor crop were applied. Said lima bean and peas crops with no treatment with said fungus, are considered as witnesses (H) and (Ch). When the population density is above 200 cysts/100 g of soil (viability average 10-40 and up to 100 eggs/g soil) the first application is effected before seeding; a second application is made during the seeding and a third application is carried out after the harvest of lima bean and peas in order to provide for a decreasing in the golden nematode population down to practically zero (Table 1 and Table 2).

(15) TABLE-US-00001 TABLE 1 Effect of Paecilomyces carneus on the control of the potato golden nematode, compared to the lima bean crop in rotation and non-farmed plots of land. Treatments parameters Monitoring H HP TP TA Cysts 1 227 272 171 180 100 g 2 155 144 200 189 3 140 102 154 183 4 133 101 153 211 Viability 1 40 64 13 15 1 g 2 14 14 20 13 3 12 7 11 14 4 15 5 9 15 J2-Gr 1 4 14 3 4 100 g 2 7 9 4 3 3 21 6 12 16 4 17 2 3 20 Free-living 1 62 131 119 124 100 g 2 148 137 78 70 3 72 82 61 52 4 42 72 57 43

(16) TABLE-US-00002 TABLE 2 Effect of Paecilomyces carneus on the control of the potato golden nematode, compared to the peas crop in rotation and non-farmed plots of land. Treatments parameters Monitoring cH chP TP TAC Cysts 1 151 173 250 237 100 g 2 139 134 139 109 3 146 95 126 130 4 128 118 124 99 Viability 1 24 19 17 26 1 g 2 17 19 15 19 3 15 13 14 15 4 8 7 9 10 J2-Gr 1 5 3 11 10 100 g 2 1 3 11 8 3 9.8 13 8 18 4 6 8 4 16 Free-living 1 73 105 84 70 100 g 2 73 101 80 69 3 61 55 69 72 4 62 56 115 50

(17) As can be seen from the above, Paecilomyces carneus strain IE-341 has a nematicide effect on the viability of the potato golden nematode cysts but no action on free life nematodes, which are to be kept on farming soils. Under this investigation, we found a synergic behavior for reducing the population density of golden nematode when the application of Paecilomyces carneus is combined with the rotation of cultures that are not favorable to the bursting of J2 Globodera rostochiensis.

(18) It is to be said that the use of rotation with lima bean and peas without the application of Paecilomyces carneus as a bionematicide is also effective, but requires of a period of time greater than 4 years.

(19) Following an exhaustive investigation, a method is provided for isolation, maintaining, massive reproduction of Paecilomyces carneus strain IE-431, as well as the use thereof in the control and/or prevention and/or eradication of cyst-forming nematodes or having characteristics similar to Globodera rostochiensis on cultures of the Solanaceae family, such as potato, chili, tomato, eggplant, among other plants where this type of parasite grows, such as Avena sativa and Vicia villosa (Winter Veza), and tobacco.

(20) Further to the products of this invention, nematicide compositions are provided comprising living cells of Paecilomyces carneus strain IE-431 and at least a living cells carrier and optionally a device for the application of said nematicide. The composition preferred for application to farmings is in a suspension shape; however, these compositions can be developed and applied in the shape of pellets, powder, capsules, emulsion, microemulsion, solution or any other form of application existing in the state of the art.

EXAMPLES

(21) Herein below, intended to be descriptive rather than restrictive, are provided compositions employed for the propagation of fungus Paecilomyces carneus strain IE-431 and the nematicide compositions obtained.

Example 1: Composition A for Mass Propagation of Paecilomyces Carneus Strain IE-431

(22) TABLE-US-00003 INGREDIENT AMOUNT Carrot juice 80-100 mL Ampicilina 500 mg Water q.s. 1000 mL

Example 2: Composition B for Mass Propagation of Paecilomyces Carneus Strain EI-431

(23) TABLE-US-00004 INGREDIENT AMOUNT Oats 20-50 gL yeast 0.1-5.0 g/l Carrot juice 50-150 mL7L Chloramphenicol 1000 mg Water q.s. 1000 mL

Example 3: Composition C for Isolation and Maintain of Paecilomyces Carneus Strain IE-431

(24) TABLE-US-00005 INGREDIENT AMOUNT Carrot 50-160 g/L Oats 20-50 g/L Chloramphenicol 1000 mg Water c.b.p. 1000 mL

Example 4: Nematicide Composition A-1

(25) TABLE-US-00006 INGREDIENT AMOUNT Living cells of P. carneus 0.5 10.sup.7/mL Composition A 1000 mL

Example 5: Nematicide Composition B1

(26) TABLE-US-00007 INGREDIENT AMOUNT Living cells of P. carneus 3 10.sup.7/mL Composition B 1000 mL

Example 6: Nematicide Composition C-1

(27) TABLE-US-00008 INGREDIENT AMOUNT Living cells of P. carneus 0.3 10.sup.7/mL Composition C 1000 mL

(28) In an integral manner, this invention provides the following advantages:

(29) 1.A method of agricultural control, based on clean and supporting technologies.

(30) 2.Said method produces an improvement on the soil quality since allows for the recovery of soils used in Solanaceae cultures.

(31) 3.The time to eradicate nematodes is one cycle, whereby it may stop the permanent interior quarantine number 17 against the potato golden nematode Globodera rostochiensis.

(32) 4.An immediate improvement is produced on the quality of the obtained product, enlarging the shelf life thereof.

(33) 5.The nematicide compositions obtained are innocuous for both humans and animals.

(34) 6.The population of the golden nematode is reduced from the very first application.

(35) 7.The cysts are directly attacked whereby better agricultural products are obtained from the very first application.

(36) 8.It can be combined with other control methods, such as rotation.