USE OF BACILLUS METHYLOTROPHICUS AS A STIMULANT OF PLANT GROWTH AND BIOLOGICAL CONTROL MEANS, AND ISOLATES OF SAID SPECIES

Abstract

The invention relates to the use of microorganisms as plant growth stimulants and for the biological control of bacterium, insects, fungi and phytopathogenic nematodes. More specifically, the invention relates to the use of microorganisms of the genus Bacillus, more specifically the Bacillus methylotrophicus species, as well as to cultures thereof, compositions comprising these bacteria, different culture methods and the products comprising same, as plant growth stimulants and for the biological control of bacterium, insects, fungi and phytopathogenic nematodes.

Claims

1. Use of bacteria of the species Bacillus methylotrophicus in a method for the biological control of phytopathogenic bacteria and/or phytopathogenic nematodes and/or phytopathogenic fungi no belonging to the species Magnaporthe oryzae.

2. Use of bacteria of the species Bacillus methylotrophicus according to claim 1 in a method for the biological control of phytopathogenic nematodes.

3. Use according to claim 2, characterized in that wherein the nematodes belong to the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema and Trichodorus.

4. A microorganism belonging to the species Bacillus methylotrophicus, strain XT1 (deposit number CECT8661) deposited on 23 Apr. 2014 by the Universidad de Granada in the Colección Española de Cultivos Tipo (CECT—Spanish Type Culture Collection) (Spanish Type Culture Collection) and/or microorganism with a high degree of homology with strain XT1, the 16S rRNA gene DNA sequence of which is identical by at least 99.6%, 99.7%, 99.8% or 99.9% to the 16S rRNA gene DNA sequence of strain XT1, based on the identity of all the nucleotides of said DNA sequences.

5. A microorganism belonging to Bacillus methylotrophicus strain XT2 (deposit number CECT8662) deposited on 23 Apr. 2014 in the Colección Española de Cultivos Tipo (CECT—Spanish Type Culture Collection) by the Universidad de Granada and/or microorganisms with a high degree of homology with strain XT2, the 16S rRNA gene DNA sequence of which is identical by at least 99.6%, 99.7%, 99.8% or 99.9% to the 16S rRNA gene DNA sequence of strain XT2, based on the identity of all the nucleotides of said DNA sequences.

6. A culture of microorganisms comprising microorganisms according to claim 4 or 5.

7. A culture of microorganisms consisting of microorganisms according to claim 4 or 5.

8. A composition comprising microorganisms according to claim 4 or 5.

9. A method of stimulating plant growth comprising: use of the microorganisms according to claim 4 or 5 and/or of the culture according to one or more of claims 6 to 7 and/or of the composition according to claim 8 thereby stimulating plant growth.

10. A method of biologically controlling phytopathogenic organisms comprising: use of the microorganisms according to claim 4 or 5 and/or of the culture according to one or more of claims 6 to 7 and/or of the composition according to claim 8 thereby providing biological control of phytopathogenic organisms.

11. Use according to claim 10, wherein the phytopathogenic organisms are bacteria and/or insects and/or fungi and/or nematodes.

12. Use according to claim 11, wherein the phytopathogenic organisms are insects.

13. Use according to claim 12, wherein the insect belongs to the family Aphididae or to any of the species commonly referred to as “whitefly.”

14. Use according to claim 11, wherein the phytopathogenic organisms are nematodes.

15. Use according to claim 14, wherein the nematode belongs to one of the following genera: Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema or Trichodorus.

16. Use according to claim 11, wherein the phytopathogenic organisms are fungi.

17. Use according to claim 16, wherein the fungi belong to one or more species selected from the list consisting of: Alternaria alternata, Aspergillus niger, Botrytis cynerea, Fusarium oxysporum, Phytophthora cactorum, Phytophthora cinnamomi, Rhizopus oryzae, Sclerotinia sclerotiorum, Thanatephorus cucumeris and Verticillium dahliae.

18. Use according to claim 17, wherein the fungi belong to the species Botrytis cinnerea.

19. Use according to claim 11, wherein the phytopathogenic organisms are bacteria.

20. Use according to claim 19, wherein the bacteria belong to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris.

21. A method for stimulating plant growth comprising the steps of: a. obtaining a microorganism and/or a bacteria culture and/or a composition according to claim 1; and b. putting a plant in contact with the microorganism and/or bacteria culture and/or composition obtained in step a.

22. A method for the biological control of phytopathogenic organisms comprising the steps of: a. obtaining a microorganism and/or a bacteria culture and/or a composition according to claim 1; and b. putting a plant affected by a phytopathogen in contact with the microorganism and/or bacteria culture and/or composition obtained in step a.

23. The method according to claim 22, wherein the phytopathogenic organism is a nematode.

24. The method according to claim 23, wherein the nematode belongs to one of the following genera: Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema or Trichodorus.

25. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant by foliar application.

26. The method according to claim 25, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant by means of spraying and/or dripping.

27. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant by means of the use of localized irrigation systems.

28. The method according to claim 27, wherein the localized irrigation system is a microsprinkler.

29. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant by means of the use of sprinklers.

30. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant by means of the use of drippers.

31. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant at least twice, once at t=0 and once more after at least 30 days.

32. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition have a microorganism concentration of at least 5×10.sup.8 colony forming units (UFC) per ml used at a dilution between 0.5-5% (v/v).

33. The method according to claim 32, wherein the microorganisms, the bacteria culture or composition have a microorganism concentration of 1.5% (v/v).

34. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant at least 6 times for 6 days.

35. The method according to claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant at least 2 times for 30 days.

36. The method according claim 21, wherein the microorganisms, the bacteria culture and/or composition are put in contact with the affected plant at least 2 times for 30 days, once at time t=0 days and once more at time t=30 days.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] In order to complement the description that is being made and to help improve understanding of the features characteristics of the invention according to several embodiments, the following drawings are shown herein with an illustrative and non-limiting character:

[0078] FIG. 1. Inhibition zone of XT1 and XT2 against Botrytis and inhibition zone of XT1 against Fusarium.

[0079] FIG. 2. Activity of strain XT1 against Agrobacterium tumefaciens

[0080] FIG. 3. Multiplication factor of M. javanica in tomato plants treated with strains XT1, XT2 and type strain.

[0081] FIG. 4. Average number of nematodes found per plant treated with strains XT1, XT2 and type strain.

[0082] FIG. 5. Average number of nematodes per g of root found in plants treated with strains XT1, XT2 and type strain.

[0083] FIG. 6. Nectarine tree affected by aphids before treatment with strain XT1.

[0084] FIG. 7. Pumpkin plant growth after 50 days of cultivation in pots at room temperature. Pots B1 and B2 were inoculated with strain XT1 whereas the B3 and B4 were not inoculated so they could be used as a control.

DETAILED DESCRIPTION OF THE INVENTION

[0085] The present invention relates to the use of microorganisms as plant growth stimulants and for the biological control of phytopathogenic bacteria, insects, fungi and nematodes. More specifically, the present invention relates to the use of microorganisms of the genus Bacillus, specifically of the species Bacillus methylotrophicus, to cultures thereof, to compositions comprising these bacteria, to different culture methods and to the products comprising them, as plant growth stimulants and for the biological control of phytopathogenic bacteria, insects, fungi and nematodes. Preferably, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to cultures thereof, to compositions comprising these bacteria, to different culture methods and to the products comprising them for the biological control of bacteria, insects, phytopathogenic nematodes and fungi.

[0086] Preferably, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to cultures thereof, to compositions comprising these bacteria, to different culture methods and to the products comprising them for the biological control of bacteria, insects, phytopathogenic nematodes and fungi, with the exception of the fungi belonging to the species Magnaporthe oryzae.

[0087] Preferably, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to cultures thereof, to compositions comprising these bacteria, to different culture methods and to the products comprising them for the biological control of bacteria belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestres; fungi belonging to the species Alternaria alternata, Aspergillus niger, Botrytis cynerea, Fusarium oxysporum, Phytophthora cactorum Phytophthora cinnamomi, Rhizopus oryzae, Sclerotinia sclerotiorum, Thanatephorus cucumeris and Verticillium dahliae; insects belonging to the family Aphididae, as well as insects belonging to the species commonly referred to as whitefly; and/or nematodes such as species of Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, for example, and generally all parasitic plant nematodes.

[0088] Biological control or biocontrol is defined in the present invention as a method of controlling pests, diseases and undergrowth consisting of using living organisms for the purpose of controlling populations of another organism (phytopathogenic organisms).

[0089] In a particular embodiment, the invention relates to bacteria belonging to the strain with deposit number CECT8661, deposited on 23 Apr. 2014 by the Universidad de Granada in the Colección Española de Cultivos Tipo (CECT—Spanish Type Culture Collection). Throughout the present specification, reference may be made to this strain using the term “strain XT1”.

[0090] In another particular embodiment, the invention relates to bacteria belonging to the strain with deposit number CECT8662, deposited on 23 Apr. 2014 by the Universidad de Granada in the Colección Española de Cultivos Tipo (CECT—Spanish Type Culture Collection). Throughout the present specification reference may be made to this strain using the term “strain XT2”.

[0091] One object of the present invention relates to the use of the bacteria belonging to strains XT1 and/or XT2 in a method for the biological control of phytopathogenic organisms and/or in a method for stimulating plant growth.

[0092] Strains XT1 and XT2 belong to the species Bacillus methylotrophicus. This species was described by Madhaiyan et al. in 2010. It was isolated from the rhizosphere of a rice plant (Oryza sativa). From a phylogenetic perspective, the species Bacillus methylotrophicus is very closely related to Bacillus subtilis, B. licheniformis, B. licheniformis and B. amyloliquefaciens, all of which are microorganisms with various applications in the field of agriculture. The percentage of identity with these species ranges between 98.2 and 99.2%. Strains XT1 and XT2 have 99.5% and 99.3%, respectively, identity with the type species of B. methylotrophicus. This conclusion was reached after sequencing the whole RNAr 16S gene (1500 pb).

[0093] The scientific classification of strains XT1 and XT2 of the present invention is the following: Domain: Bacterium/Phylum: Firmicutes/Class: Bacilli/Order: Bacillales/Family: Bacillaceae/Genus: Bacillus.

[0094] Both strains are sporulated Gram positive bacilli. Their size ranges between 1.5 and 3.5 μm in length by 0.5 μm in width. They generate ivory-colored colonies of with irregular edges. They are oxidase negative and catalase positive.

[0095] Strains XT1 and XT2 have peritrichous flagella giving them high mobility. They generate biofilms or films that allow being adhered to animate and inanimate substrates and act as a protection factor against predators existing in the environment. Biofilm formation makes adherence of the microorganism easier; if it is administered by drip irrigation, it will adhere to the roots. If it is administered by foliar application, it will remain in the phyllosphere. Furthermore, biofilm formation both in the roots and in the leaves and stem protects the plant from being attacked by other living beings.

[0096] Accordingly, both the presence of flagella and biofilm formation entail an advantage of these bacteria (XT1 and XT2) for colonizing the habitat.

[0097] Strains XT1 and XT2 generate ellipsoidal non-deforming spores. In the 2×SG medium, these bacteria produce more than 5×10.sup.8 spores/ml between three and five days. They are halotolerant and optimally grow in a wide range of salt concentrations [between 0 and 12% (w/v)]. They optimally grow between 20-45° C. and at a pH of 5-10. They have scarce nutritional requirements: they can grow with a wide range of organic compounds as the sole carbon source, such as citrate or sucrose. They are capable of growing with ammonium nitrate as the sole nitrogen source, without requiring the presence of yeast extract or a complex nitrogen source.

[0098] Spore formation, which allows the bacterium to remain in the habitat in adverse conditions, and the scarce nutritional requirements which allow preparing a low-cost culture medium, make strains XT1 and XT2 very appealing from the industrial viewpoint.

[0099] Strains XT1 and XT2 are facultative anaerobes. They breathe aerobically in the presence of oxygen, and in the absence thereof, for example in the roots and near the roots, they perform butanediol fermentation, producing 2,3 butanediol and acetoin. They use a number of sugars as a carbon source and energy, producing acids from said sugars. Included among the sugars these strains use are glycerol, glucose, fructose, mannitol, sorbitol, cellobiose, lactose and sucrose. It can also perform nitrogen fixation, i.e. in the absence of a nitrogen source they take up gaseous nitrogen and transform it into ammonium, which is the nitrogen source that can be used by plants. They produce dihydroxyacetone and H.sub.2S.

[0100] Strains XT1 and XT2 are capable of synthesizing chelating compounds, such as siderophore compounds, which take up Fe.sup.3+ and turn it into Fe.sup.2+. The iron ion Fe.sup.3+ has very little solubility at neutral pH and hence cannot be used by the organisms. Siderophores dissolve these ions into Fe.sup.2+ complexes, which can be assimilated by mechanisms of active transport.

[0101] Strains XT1 and XT2 are capable of producing a number of extracellular enzymes with high hydrolytic capacity, which facilitate the availability of substrates for plants. Among other effects, strains XT1 and XT2 are capable of producing amylases hydrolyzing starch, urease hydrolyzing urea generating ammonium, proteases hydrolyzing gelatin and casein, lipases hydrolyzing Tween 80 and lecithin, DNases hydrolyzing DNA, phosphatases hydrolyzing organic phosphate and inorganic phosphate and ACC deaminase.

[0102] Strains XT1 and XT2 produce in CAS medium, used for detecting siderophores, a larger clearance zone (7 and 5 mm, respectively) than the Bacillus velezensis strain of Botrybel used as a control and producing 3 mm. Both strains grow better than the control strain (a larger amount of bacterial mass is observed on the surface of the solid medium) in solid media nitrogen-free, indicating greater nitrogen fixing activity. Therefore, their activity as a fertilizer microbial is greater.

[0103] Strains XT1 and XT2 are capable of biofilm formation. This activity has not been determined in the previous commercial preparation. This capacity allows the bacteria to more readily adhere to the roots or leaves of plants to exert their plant protection or growth stimulating action.

[0104] More specifically, it has been found that strains XT1 and/or XT2 object of the present invention have greater enzyme activity than the strain of the Botrybel preparation, produce larger halos of starch hydrolysis (amylase activity, see FIG. 3), gelatin and casein (protease activity), Tween 80 and lecithin (lipase activity), and greater ACC deaminase and phosphatase activity, determined using phenolphthalein phosphate and calcium phosphate. Hydrolysis halos are observed as the occurrence of a transparent zone in the case of starch, casein and lecithin hydrolysis; for gelatin, it can be observed that liquefaction thereof occurs, i.e., it transitions from a solid to liquid state; in the case of Tween 80, there is a more opaque precipitation zone; for ACC deaminase activity growth is studied in media with aminocyclopropane carboxylic acid as the sole nitrogen source; and finally, phosphatase activity is observed along with a pink color upon adding ammonia to the plate with phenolphthalein phosphate, and calcium phosphate solubilization is analyzed seeing the transparent zone generated around the bacterial mass grown in a medium with this compound. The activities have been analyzed using as a control the Bacillus velezensis strain of the Botrybel preparation.

[0105] As for activities as biological control agents against fungi, have been determined the inhibition values of strains XT1 and XT2 and the type strain of B. methylothrophicus against Alternaria alternata, Aspergillus niger, Botrytis cynerea, Fusarium oxysporum, Phytophthora cactorum Phytophthora cinnamomi, Rhizopus oryzae, Sclerotinia sclerotiorum, Thanatephorus cucumeris and Verticillium dahliae. The growth inhibition values are very significant in the case of Botrytis cinnerea. Activity was lower against Fusarium oxysporum. Generally, the Bacillus strain of Botrybel has lower (and in some cases similar) activity with respect to strains XT1 and XT2 and the type strain (see Table 1 in Example 2 below).

[0106] Strain XT1 and B. methylothrophicus type strain also show activity against phytopathogenic bacteria such as Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris, whereas strain XT2 shows activity against P. atrosepticum and X. campestris (see Table 2 in the example 3 below).

[0107] Strains XT1, XT2 and B. methylothrophicus type strain show activity against Rhopalosiphum padi (see Table 3) and strain XT1 against whitefly (see Example 6, b1, below).

[0108] Strains XT1, XT2 and B. methylothrophicus type strain considerably reduce the multiplication factor of Meloidogyne javanica and the number of nematodes per tomato plant and the number of nematodes per g of root (see FIGS. 3, 4 and 5); likewise, treatment with strain XT1 recovered Dutch cucumber plants cultivated in greenhouse and highly infected by nematodes (see Example 4b below)

[0109] Another advantage of strains XT1 and XT2 is their high sensitivity to antimicrobial agents generally used in therapy. They are sensitive to nalidixic acid (30 μg), amoxicillin (2 μg), amoxicillin-clavulanic acid (30 μg), cefalotin (30 μg), colistin (10 μg), doxycycline 30 μg, erythromycin (15 μg), kanamycin (30 μg), nitrofurantoin (300 μg), norfloxacin (5 μg), novobiocin (30 μg), rifampicin (30 μg), trimethoprim sulfamethoxazole (1.25 μg-23.75 μg) and vancomycin (30 μg) following the diffusion in solid medium technique (Bauer and Kirby 1966). Therefore, they cannot transfer resistance genes to other microbial populations of the rhizosphere.

[0110] Strains XT1, XT2 and B. methylotrophicus type strain have an additional advantage against fungi used for biological control and as plant growth stimulants, which is how easy they are to culture and therefore how easy it is to reach an industrial level. The advantage against other bacterial strains of other genera described for the same purpose is the presence of spores on the part of strains XT1 and XT2, which entails total product stability during storage and in the environment when conditions are not suitable for handling said microorganisms.

[0111] Strains XT1 and XT2 and the B. methylotrophicus type strain produce compounds which reduce the pH, such as 2,3 butanediol and acetoin when they ferment sugars in anaerobic conditions. Furthermore, they are capable of fixing nitrogen, producing siderophores and hydrolytic enzymes. All these characteristics are mechanisms of direct plant growth stimulation (Sessitsch et al., 2002; Perrine et al., 2004; Rodriguez and Fraga, 1999; Carson et al. 2000; Essalmani and Lahlou, 2003; Choudhary and John 2009).

[0112] Strains XT1 and XT2 and type strain produce different lipopeptides, surfactants. Included among these surfactants lipopeptides are surfactin, which is similar to that produced by Bacillus subtilis. More specifically, the surfactin produced by strain XT1 does not have 12 carbon (12C) fatty acids in its lipid chain.

[0113] After extracting the lipopeptides following the method of Cooper et al. 1981, a yield of 0.12 g/I and 0.10 g/l of culture was obtained for strains XT1 and XT2, respectively. The type strain produced 0.6 g/l. Lipopeptide production has not been described in the case of the Bacillus strain of the commercial preparation Botrybel. More specifically, in addition to surfactin, strain XT1 object of the present invention produces other surfactant lipopeptides such as fengycin and lichenysin.

[0114] The cellular dry weight (CDW) of strains XT1, XT2 and of type strain is 2.7 g/I, 2.5 g/I and 2.9 g/I, respectively. In the case of strain XT1, the critical micelle concentration (CMC) is 0.0025% (0.025 mg/ml); a surface tension of 29.7 mN/m was obtained with this value. In the case of the surfactin produced by B. subtilis and marketed by Sigma®, values of 26.7 mN/m at the same CMC were obtained. In other words, strain XT1 produces very active lipopeptides surfactants showing activity similar to the surfactin available on the market.

[0115] Many lipopeptides produced by species of Bacillus show antibiotic activity, acting at the cellular membrane level in fungi and Gram negative bacteria, such as, for example, fengycins, mycobacillins, iturines, bacillomycins, surfactins, mycosubtilins, fungistatins (Volpon et al., 2000; Yilmaz et al. 2006).

[0116] More specifically, the lipopeptides produced by strain XT1 are a mixture of 13, 14 and 15 carbon atoms fatty acids which are bound to a cyclic peptide by leucine or isoleucine. The relative proportion of these fatty acids is 1, 6.5 and 5.7, respectively.

[0117] The production of enzymes (glucanases, proteases, lipases, phosphatases and urease) together with the different lipopeptides, and the release of SH.sub.2 are, according to the literature (see prior art), responsible for the action of said strains in the biological control of fungi, bacteria, insects and nematodes.

[0118] Another object of the present invention relates to a method for the biological control of phytopathogenic organisms comprising the steps of:

[0119] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0120] b. putting a plant in contact with the bacteria, bacteria cultures or compositions obtained in step a).

[0121] Another object of the present invention relates to a method for stimulating plant growth and/or for the biological control of phytopathogenic nematodes (such as the species of the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, for example, and generally all parasitic plant nematodes) comprising the steps of:

[0122] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0123] putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.

[0124] Another object of the present invention relates to a method for stimulating plant growth in plants (preferably not affected by phytopathogenic organisms) comprising the steps of:

[0125] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0126] b. putting a plant preferably not affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.

[0127] Another object of the present invention relates to a method for stimulating plant growth comprising the steps of:

[0128] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0129] b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.

[0130] One object of the present invention relates to a method for the biological control of phytopathogenic insects belonging to the family Aphididae, as well as insects belonging to the species commonly referred to as whitefly, comprising the steps of:

[0131] a. obtaining the bacteria, bacteria cultures or compositions as previously described; and

[0132] b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a).

[0133] In the methods of the present invention, the bacteria, cultures and/or composition of the present invention can be put into contact with the plant (affected) by foliar application, such as by means of spraying and/or dripping, for example, or by conventional irrigation, or by flood irrigation, etc.

[0134] The methods described in the present invention can furthermore comprise the use of a distribution system for distributing the bacteria, cultures or compositions of the present invention. For example, the methods of the present invention can comprise the use of drippers for distributing the bacteria, cultures or compositions of the present invention. For example, the methods of the present invention can comprise the use of self-compensating drippers for distributing the bacteria, cultures or compositions of the present invention. For example, the methods of the present invention can comprise the use of localized irrigation systems (such as microsprinklers, optionally with rotating or diffusing element, for example) for distributing the bacteria, cultures or compositions of the present invention. For example, the methods of the present invention can comprise the use of sprinklers for distributing the bacteria, cultures or compositions of the present invention.

[0135] The localized irrigation systems can be defined as methods of distributing fluids (water, fertilizers, or, in the case at hand, the bacteria, cultures or compositions according to the present invention) which, to maintain a suitable and constant level of the fluid distributed into the soil, applies said fluid dropwise, in a slow, localized and uniform manner in the plant root mass.

[0136] Localized irrigation systems can comprise drip, exudation and/or microsprinkling systems.

[0137] The person skilled in the art knows how localized irrigation systems work and how to use them.

[0138] A dripper according to the present invention is defined as a delivery point for the bacteria, cultures or compositions of the present invention in the vicinity of the plants to be treated. The person skilled in the art knows how a dripper works and how to use it.

[0139] Therefore, another object of the present invention relates to a method for the biological control (prevention) of phytopathogenic organisms, preferably fungi, bacteria and nematodes, comprising the steps of:

[0140] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0141] b. putting a plant in contact with bacteria, cultures or compositions obtained in step a, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention.

[0142] According to the present invention, prevention is what action is taken beforehand to minimize a risk. The purpose of prevention according to the present invention is to prevent possible damage (infection by phytopathogenic organisms) from occurring.

[0143] Therefore, another object of the present invention relates to a method for the biological control (treatment) of phytopathogenic organisms, preferably fungi, bacteria, insects and nematodes, comprising the steps of:

[0144] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0145] b. putting a plant affected by a phytopathogen in contact with the bacteria, cultures or compositions obtained in step a, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention.

[0146] In the context of the present invention, the term “treatment” is understood as the set of means the purpose of which is to cure or alleviate (palliate) diseases or symptoms.

[0147] Therefore, another object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably fungi (except the belonging to the species Magnaporthe oryzae), bacteria (preferably Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris, insects (preferably phytopathogenic insects belonging to the family Aphididae, as well as insects belonging to the species commonly referred to as whitefly), and/or nematodes (such as the species belonging to the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, and generally all parasitic plant nematodes, for example), comprising the steps of:

[0148] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0149] b. putting a plant affected by a phytopathogen in contact with the bacteria, cultures or compositions obtained in step a, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention.

[0150] Another object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably fungi belonging to the species Alternaria alternata, Aspergillus niger, Botrytis cynerea, Fusarium oxysporum, Phytophthora cactorum Phytophthora cinnamomi, Rhizopus oryzae, Sclerotinia sclerotiorum, Thanatephorus cucumeris and Verticillium dahliae, bacteria, preferably belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris, and/or nematodes, such as the species of the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, for example, and generally all parasitic plant nematodes, comprising the steps of:

[0151] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0152] b. putting a plant affected by a phytopathogen in contact with bacteria, cultures or compositions obtained in step a, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention.

[0153] The distribution systems for distributing the bacteria, cultures or compositions of the present invention can comprise localized irrigation systems, drippers, self-compensating drippers, microsprinklers, and/or sprinklers.

[0154] Furthermore, in the method of the present invention, the bacteria, cultures and/or compositions of the present invention can be put into contact with the affected plant at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more.

[0155] Furthermore, the time interval between one application of the bacteria, culture and/or composition of the present invention and the next (if they are put in contact or applied more than once) is 2 days, or 3 days, or 5 days, or 10 days, or 15 days, or 20 days, or 30 days.

[0156] Preferably the bacteria, culture and/or composition of the present invention are put in contact with the affected plant twice, once at time (t)=0 and again after 30 days.

[0157] Preferably the bacteria, culture and/or composition of the present invention are put in contact with the affected plant once every 10 days, for 60 days, or once a day for 8-12 days.

[0158] Furthermore, in the method of the present invention, the culture and/or composition of the present invention having a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml are used at a dilution between 0.5-5%. (v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example. Preferably in the method of the present invention, the culture and/or composition of the present invention have a microorganism concentration of 1.5% (v/v) of a preparation containing 5×10.sup.8 CFU/ml.

[0159] Therefore, one object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably fungi belonging to the species Alternaria alternata, Aspergillus niger, Botrytis cynerea, Fusarium oxysporum, Phytophthora cactorum Phytophthora cinnamomi, Rhizopus oryzae, Sclerotinia sclerotiorum, Thanatephorus cucumeris and Verticillium dahliae; bacteria, preferably belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris and/or nematodes, such as the species of the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, for example; insects belonging to the family Aphididae, as well as insects belonging to the species commonly referred to as whitefly, and generally all parasitic plant nematodes and insects, preferably whitefly, aphid, comprising the steps of:

[0160] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0161] b. putting a plant affected by a phytopathogen in contact with bacteria, cultures or compositions obtained in step a by means of distribution systems, and/or drippers, and/or localized irrigation systems, and/or sprinklers, for example, at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention, where the cultures or compositions have a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml, used at a dilution between 0.5-5%.(v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example, preferably once every 10 days, for 60 days, or once a day for 8-12 days.

[0162] Therefore, one object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably fungi belonging to the species Alternaria alternata, Aspergillus niger, Botrytis cynerea, Fusarium oxysporum, Phytophthora cactorum Phytophthora cinnamomi, Rhizopus oryzae, Sclerotinia sclerotiorum, Thanatephorus cucumeris and Verticillium dahliae comprising the steps of:

[0163] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0164] b. putting a plant affected by a phytopathogen in contact with bacteria, cultures or compositions obtained in step a by means of distribution systems, and/or drippers, and/or localized irrigation systems, and/or sprinklers, for example, at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention, where the cultures or compositions have a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml, used at a dilution between 0.5-5%.(v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example, preferably once every 10 days, for 60 days, or once a day for 8-12 days.

[0165] Therefore, one object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably bacteria, preferably belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris comprising the steps of:

[0166] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0167] b. putting a plant affected by a phytopathogen in contact with bacteria, cultures or compositions obtained in step a by means of distribution systems, and/or drippers, and/or localized irrigation systems, and/or sprinklers, for example, at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention, where the cultures or compositions have a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml, used at a dilution between 0.5-5%. (v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example, preferably once every 10 days, for 60 days, or once a day for 8-12 days.

[0168] Therefore, one object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably nematodes, such as the species of the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, for example, and generally all parasitic plant nematodes, comprising the steps of:

[0169] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0170] b. putting a plant affected by a phytopathogen in contact with bacteria, cultures or compositions obtained in step a by means of distribution systems, and/or drippers, and/or localized irrigation systems, and/or sprinklers, for example, at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention, where the cultures or compositions have a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml, used at a dilution between 0.5-5%.(v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example, preferably once every 10 days, for 60 days, or once a day for 8-12 days.

[0171] Therefore, one object of the present invention relates to a method for the biological control of phytopathogenic organisms, preferably insects, such as the species belonging to the family Aphididae, for example, as well as insects belonging to the species commonly referred to as whitefly, comprising the steps of:

[0172] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0173] b. putting a plant affected by a phytopathogen in contact with bacteria, cultures or compositions obtained in step a by means of distribution systems, and/or drippers, and/or localized irrigation systems, and/or sprinklers, for example, at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention, where the cultures or compositions have a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml, used at a dilution between 0.5-5%. (v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example, preferably once every 10 days, for 60 days, or once a day for 8-12 days.

[0174] Therefore, one object of the present invention relates to a method for stimulating plant growth comprising the steps of:

[0175] a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and

[0176] b. putting a plant (which may or may not be affected by a phytopathogen) in contact with bacteria, cultures or compositions obtained in step a by means of distribution systems, and/or drippers, and/or localized irrigation systems, and/or sprinklers, for example, at least once, preferably at least twice, preferably at least three times, preferably at least four times, preferably at least five times, preferably at least six times, or more, using to that end a distribution system for distributing the bacteria, cultures or compositions of the present invention, where the cultures or compositions have a microorganism concentration of at least 10.sup.8 colony forming units (CFU) per ml, used at a dilution between 0.5-5%.(v/v), such as 0.5%, 1%, 1.5%, 2%, 3% and/or 5% (v/v), for example, preferably once every 10 days, for 60 days, once a day for 8-12 days, or twice in a period of 30 days (once at time 0 days and again at time 30 days).

[0177] Throughout the description and the claims the word “comprises” and its variants do not intend to exclude other technical features, additives, components or steps. The term “comprises” also encompasses the term “consists of”. For those skilled in the art, other objects, advantages and features of the invention will be inferred in part from the description and in part from putting the invention into practice. The following examples are provided by way of illustration and do not intend to limit the present invention.

EXAMPLES

Example 1. Isolation of Strains XT1 and XT2

[0178] Strain XT1 (deposit number CECT8661), object of the invention was isolated in 1999 from a sample from the rhizosphere of a soil close to Lake Capacete, located in Fuente de Piedra, Malaga (Spain). Strain XT2 (deposit number CECT8662) was isolated in 2010 in a soil close to the mouth of the Velez River (Malaga, Spain). The medium used was MY medium (Moraine and Rogovin 1966) added with 7.5% sea salts in the case of strain XT1 and the MY medium with 3% sea salts in the case of strain XT2. Both strains were selected for their characteristics of about 5000 colonies (searching for those with higher surfactant activity by means of the method of Jain et al. 1991).

Example 2. Use of Strains XT1, XT2 and Type Strain as Antifungal Agents

[0179] Activity against fungi (Table 1) was established by seeding strains XT1, XT2, the type strain and the Botrybel strain in a small area in PDA medium (potato dextrose agar). Then was placed at the end opposite a piece of agar of about 1 cm.sup.2 with mycelium of the fungus to be tested, and after 20 days and after incubation at 25° C., the maximum and minimum radius of the mycelium of the fungus was measured to calculate the percentage of growth reduction of the fungus

TABLE-US-00002 TABLE 1 Maximum and minimum inhibition values against different fungi expressed in mm, in parenthesis percentage of reduction of the mycelium. Antifungal activity Type Fungi XT1 XT2 strain Botrybel Alternaria alternata 27 and 14 24 and 19 25 and 8 24 and 20 (49) (21) (68) (17) Aspergillus niger 30 and 22 25 and 10 ND 0 (27) (60) Botrytis cynerea 45 and 8 45 and 2 40 and 20 46 and 16 (83) (96) (50) (56) Fusarium oxysporum 30 and 28 0 32/29 0 (7) (10) Phytophthora cactorum 8 and 3 8 and 3 ND 8 and 6 (63) (63) (21) Phytophthora cinnamomi 22 and 16 22 and 15 16 and 12 20 and 14 (28) (32) (21) (30) Rhizopus oryzae 45 and 8 50 and 12 ND 40 and 7 (82) (76) (82) Sclerotinia sclerotiorum 16 and 7 16 and 12 16 and 10 ND (56) (25) (37) Thanatephorus 12 and 7 16 and 10 16 and 7 ND cucumeris (42) (37) (56) Verticillium dahliae 25 and 12 24 and 12 25 and 14 23 and 13 (52) (50) (44) (44) ND: Not determined

[0180] Among the tested fungi, the highest inhibition was achieved against Botrytis (strains XT1 and XT2) and the lowest inhibition was achieved against Fusarium (FIG. 1).

[0181] The antimicrobial activity of strains XT1 and XT2 and the type strain against Saccharomyces cerevisiae (a beneficial yeast with enormous industrial applications) was also determined and the absence thereof was observed, i.e., the inhibition zone was zero mm.

Example 3. Use of Strains XT1, XT2 and Type Strain as Antibacterial Agents

[0182] The antibacterial activity was determined by incorporating, in a Petri dish with trypticase soybean agar (TSA), an overlay with 6 ml of sterile TSA at 45° C. and 1 ml of a culture of the phytopathogenic strain to be analyzed in exponential growth phase at a concentration equivalent to 1 on the Mac Farland scale. Then once the medium solidified, it was inoculated in a well 100 μl of supernatant from the cultures. After 24 hours of incubation, the inhibition zone was measured (Table 2. FIG. 2).

TABLE-US-00003 TABLE 2 Antibacterial activity. Results expressed in mm of growth inhibition. X. P. R. A. campestris atrosepticum solanacearum tumefaciens XT1 6 3 1 4 XT2 5 2 R R Type strain 8 5 3 5 R: Resistant

Example 4. Use of Strains XT1, XT2 and Type Strain as Agents for Biological Control of Nematodes

[0183] a) Tests in tomato plants inoculated with Meloidogyne javanica (FIGS. 3, 4, and 5)

[0184] Five batches of 10 tomato plants (Solanum lycopersicum) each were used. Three batches were inoculated with the selected strains (2×10.sup.8 CFU) and then with M. javanica J2 (1500). Finally, two batches, with and without nematodes, were used as control. After 50 days the number of nematodes in the soil of each plant and in the roots was determined, and the multiplication factor (Talavera et al. 2012) was calculated. The obtained results can be seen in FIGS. 3, 4 and 5.

[0185] It is observed that all the strains reduce the nematode multiplication factor, the number of nematodes per plant and the number of nematodes per g of root, the reductions being more pronounced in the case of strains XT1 and XT2.

[0186] b) Greenhouse test with strain XT1 in a Dutch cucumber crop with recurrent problems every year due to excess moisture in the soil, difficulty in rooting and a high incidence of rot in the root, as well as infection by nematodes.

[0187] A sector of 2000 m.sup.2 was used for injection in the drip irrigation and another sector similar was used as a control. 7.5 I of culture were applied in 6 applications separated by a period of 10 days (1, 250 I of a culture with at least 10.sup.8 CFU/ml in each application). Both in the control sector and in the treatment sector the usual fertilizing and plant health treatments were maintained. The number of plants lost during this treatment in the control sector was 36, whereas that for the plants treated with strain XT1 was 6. The differences in production were also significant, a 30% higher production being obtained in the treated sector.

Example 5. Use of Strain XT1, XT2 and Type Strain as Agents for Biological Control of Insects

[0188] a) Laboratory experiments were conducted with barley aphid (Rhopalosiphum padi) and Anthocoris nemoralis for the purpose of determining the mortality percentage, generating the bacterial cultures of the three strains of B. methylotrophicus and the surfactants thereof, on these insects. Given that the first is a sucking insect, the tests are only conducted topically, whereas in the second case they are conducted both topically and by ingestion.

[0189] The activity of the bacterial cultures of strains XT1 and XT2 with 5×10.sup.8 CFU/ml, and of their surfactants at a concentration 1/1000 in distilled water, in both types of insects, were analyzed by topical use. Ten individuals were used for each treatment. In the case of Anthocoris, 5 μl were applied with a pipette on the body thereof and the aphids were impregnated with the same amount with a brush. The bacterial culture medium SG was used as a control. The obtained results expressed in mortality percentage after 48 h are the following:

TABLE-US-00004 TABLE 3 Mortality by topical use in the barley aphid Rhopalosiphum padi and Anthocoris nemoralis. Results expressed in percentage of individuals Surfactant Surfactant Control Insect XT1 XT1 XT2 XT2 (Culture medium) Anthocoris 70 100 20 ND 70 Aphids 30 60 80 60 10-30

[0190] The activity of the bacterial cultures of strains XT1, XT2 and the B. methylotrophicus type strain with 5×10.sup.8 CFU/ml, and of the three corresponding surfactants, at a concentration 1/1000 in distilled water by ingestion was likewise analyzed on Anthocoris nemoralis. Water and Tween 80 (1/1000 dilution) was used as a control. The different products were applied on a sponge moistened with water (1 ml) and by spraying (0.25 ml) on the food (Ephestia kuheniella eggs) making sure it was well covered. Four repetitions of five individuals were done for each treatment, being observed daily for 6 days. The obtained results, expressed in mortality percentage, are indicated in the following table:

TABLE-US-00005 TABLE 4 Mortality by ingestion in Anthocoris nemoralis. Results expressed in percentage of individuals. Surfac- Surfac- Type Surfactant tant tant Control Tween strain Type strain XT1 XT1 XT2 XT2 (Water) 80 5 25 15 20 15 20 10 5

[0191] b) Furthermore, a nectarine tree Prunus persica var. nectarine highly affected by green and black aphids (see FIG. 6) was treated. With a culture of XT1 with 5×10.sup.8 CFU/ml. Specifically, 50 ml of a 5% dilution of said culture was administered by foliar application every 10 days. After the second application, the population virtually disappeared although small zones still had aphids at the end of some leaves that remained rolled up and were removed by hand. After one month, the pest was controlled.

[0192] c) The disappearance of whitefly was likewise observed in a greenhouse tomato culture treated with strain XT1 (see Example 6 b1)

Example 6. Use of Strains XT1, XT2 and Type Strain as Plant Strengthening Agents

[0193] a) Pot test with pepper plants (genus Capsicum) and pumpkin plants (genus Cucurbita) with strains XT1, XT2 and type strain.

[0194] Sixteen seedlings 5 cm in height coming from each of the preceding plants that were transplanted to pots 10 cm in diameter and 15 cm in height were used and were left exposed at room temperature (temperature range of 20-38° C.) for 35 days. The pots were irrigated every each 48 h with the same amount of water (about 50 ml). Four batches of 4 pots of each type of plant were made. Five ml of a 1/100 dilution of a culture of Bacillus strain XT1, strain XT2 and type strain with 5×10.sup.8 CFU/ml were added every seven days to three batches of each type of plant, after irrigating. A batch of four pots of each type was used as a control and therefore it was not inoculated with bacterial cultures. After 35 days the aerial part was cut and dried. The obtained results are observed in the enclosed table.

TABLE-US-00006 TABLE 5 Plant strengthening effect of strains XT1, XT2, and type strain Pumpkin Pepper Increase with Increase with Weight respect to Weight respect to (g) the control (%) (g) the control (%) Type strain 6.7 ± 1.4 45.6 5.6 ± 1.5 64.7 XT1 8.15 ± 1.8  77.1 5.2 ± 1.2 52.9 XT2 5.4 ± 0.4 17.4 5.2 ± 1.3 52.9 Control 4.6 ± 0.5 0 3.4 ± 1.4 0

[0195] Note: In the cucumber crop there was a loss, due to weather conditions and pests, of 50% of the plants, in the case of the control and in those plants irrigated with the type strain, and of 25% in the plants irrigated with strain XT2; however all the plants irrigated with XT1 were maintained in optimal conditions.

[0196] b) Furthermore, the following were conducted with strain XT1:

[0197] b.1. Test in greenhouse pear tomato crops.

[0198] Treatments were performed by foliar application at three different doses of the culture broth containing at least 10.sup.8 CFU/ml (0.5, 1 and 1.5% v/v) by means of spraying and with two repetitions (at time zero and after 30 days). Six plants were planted in each treatment. An untreated control was used. During the period of the study, in the greenhouse, and therefore in the control, there were several pests: whitefly, aphid, oidium and Botrytis. The number of plants that were lost in the zones treated with the culture of strain XT1 was less than the number lost in the control zone, the most suitable dose being the 1.5% (v/v) dose. In addition, the weight of the tomatoes picked from treated plants was greater than the weight of the control plant (see Table 6).

TABLE-US-00007 TABLE 6 Tomato plants lost and weight of the tomatoes picked in two greenhouse pear tomato crop zones after treatment with XT1 Treatment 1.5% CONTROL 1.5% CONTROL Experience (1) (1) (2) (2) Plants lost 1 4 0 3 Weight of the tomatoes 3.6 3.1 3.8 3.1 (total kg)

[0199] These results showed a clear trend between the application of the product and the increase in the production of the plants with respect to the controls that can be attributed to the stimulating effect on the plant's metabolism. Furthermore, the treated plants showed a significant reduction of the whitefly and aphid pests and recovered from infection by Botrytis and oidium.

[0200] b.2. Longer-term pot test with strain XT1 with healthy and already developed tomato plant (Solanum lycopersicum), pepper plant (genus Capsicum), pumpkin plant (genus Cucurbita) and cucumber plant (Cucumis sativus) crops.

[0201] Four seedlings 10 cm in height of each of the preceding species (tomatoes (Solanum lycopersicum), peppers (genus Capsicum), pumpkin (genus Cucurbita) and cucumber (Cucumis sativus) were used. They were transplanted to pots 10 cm in diameter and 15 cm in height and were left exposed at room temperature (temperature range of 15-32° C.). The pots were irrigated every 48 h with the same amount of water (about 100 ml). Five ml of a 1/100 dilution of a culture of Bacillus XT1 were added every seven days to half the pots, after irrigating. The other half was used as a control and therefore was not inoculated. After 50 days the aerial part was cut and dried. Likewise, the number of leaves, flowers and fruits, and the height thereof, were recorded. The obtained results (mean values) are observed in the enclosed table. An increase in the aerial vegetation weight of 86%, an increase in the number of leaves of 57.6% and an increase in the number of fruits and flowers of 1 12.5 and 137.5%, respectively, are obtained (see Table 7). Furthermore, the size of the treated plant increased by 38.3%. The results in the pumpkin crop can be seen FIG. 7.

TABLE-US-00008 TABLE 7 Effect of irrigation with XT1 in pepper, cucumber, tomato and pumpkin plants. The mean is indicated. Weight of No. of aerial part Height No. No. Treatment leaves (g) (cm) fruits flowers Pepper None 12 7.7 38 0 0 Pepper XT1 14 12.6 46.5 1 1 % increase 16.7 62 22.4 100 100 Cucumber None 7 12.1 30.5 0 3.5 Cucumber XT1 14 24.3 33 0.5 8.5 % increase 100 101.2 8.2 50 142.9 Pumpkin None 8 8.9 30.5 0 3.5 Pumpkin XT1 12.5 20.4 33.5 2 5.5 % increase 56.2 128.5 9.8 200 57.1 Tomato None ND 13.2 46 0 0 Tomato XT1 ND 20.1 52 1 2.5 % increase 52.7 113 100 250 Mean overall 57.6 86 38.3 112.5 137.5 increase