US20210076683A1

US20210076683A1 - Use of bacillus methylotrophicus as a stimulant of plant growth and biological control means, and isolates of said species

Info

Publication number: US20210076683A1
Application number: US17/092,154
Authority: US
Inventors: María Victoria BÉJAR LUQUE; Inmaculada Llamas Company; Cristina RUÍZ GARCÍA; Emilia Quesada Arroquia
Original assignee: Universidad de Granada
Current assignee: Universidad de Granada
Priority date: 2014-07-31
Filing date: 2020-11-06
Publication date: 2021-03-18
Also published as: US10856551B2; WO2016016508A1; EP3178325A1; ES2561908B2; CL2017000236A1; ES2639375A1; ES2561908A1; CA2991678A1; CN106686983B; US20170215429A1; ES2639375B1; BR112017001910A2; CN106686983A; EP3178325A4

Abstract

The invention relates to the use of microorganisms as plant growth stimulants and for the biological control of bacteria, 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 bacteria, insects, fungi and phytopathogenic nematodes.

Description

This application is a continuation application of U.S. application Ser. No. 15/500,440, which is a National Stage Application of PCT/ES2015/070600, filed Jul. 31, 2015, which claims the benefit of Serial No. P201431158, filed Jul. 31, 2014 in Spain and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 480386_401C1_SEQUENCE_LISTING.txt. The text file is 20.51 KB, was created on Nov. 5, 2020, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD OF THE PRESENT INVENTION

The main application of the present invention is in agriculture. The bacteria of the present invention, as well as the products produced by said bacteria, are useful as plant growth stimulants and for the control of plant pathogens such as bacteria, insects, fungi and nematodes.

PRIOR ART

1. Phytopathogenic Organisms

Phytopathogenic organisms are an important cause of diseases in plants; they determine changes in their shape, function or integrity and can lead to plant death. Nematodes, bacteria, insects, fungi and virus are included among such phytopathogenic organisms.
One of the most important groups are phytopathogenic fungi including, inter alia, species of the genera Botrytis, Pythium, Alternaria, Fusarium, Phytophthora, Rhizoctonia, Colleotrichium, Eutypa, Rhizopus, Penicillium, Sclerotinia and Verticillium. They cause local damage, such as leaf spots, tissue hypertrophy or smut, or widespread damage when they affect the root or vascular system, causing the plant to wilt and die. There are more than 8000 species that attack plants, some of which are very specific, and other of which have a wide range of hosts. The economic impact these organisms have is very important. By way of example, when Botrytis cinnerea affects vineyards, thousands of tons of wastage are produced in the wine-making industry.
Plant diseases caused by bacteria have a lower incidence than plant diseases caused by fungi or viruses (Vidhyasekaran 2002). Phytopathogenic bacteria include: Erwinia amylovora, responsible for the disease referred to as fire blight, particularly affecting pear trees, apple trees, medlar trees, quince trees and ornamental rosaceae; Erwinia carotovora (Pectobacterium carotovorum), soft rot producer; Ralstonia solanacearum, which causes rot and wilting in cultivated nightshades, but also in plants of more than fifty families; the different Pseudomonas syringae pathovars, which cause spots, burns and ulcerations;
Agrobacterium tumefaciens, a bacterium causing tumors in the collar, roots, and less often in stem and having a broad spectrum of guests, including more than 700 species; and Xanthomonas campestris, responsible for spots and burns in plants, inter alia.
Nematodes are also extremely important in agriculture. They are microscopic worms measuring between 0.2-1 mm with a stinger in the upper part they use to feed off the plant. The larvae enter through any part of the plant in contact with the moist soil, but primarily through the tip of the absorbent root hairs, because their stinger is not very strong. Once they are housed in the tissues, they neither move nor change from position. Symptoms present through the occurrence of typical nodules or thickening in the roots. This damage causes vessels to become obstructed and prevents absorption by the roots, which translates into less plant development and symptoms of wilting, chlorosis and stunting. Phytopathogenic nematodes include species of the genera Meloidogyne, Heterodera, Radopholus and Pratylenchus.
Insect pests are also an important problem in plants. In some cases they are bacterial and/or viral infection vectors. In other cases, they can weaken the plant and even cut off its growth and development, and controlling or eliminating insect pests is extremely complex. This is the case of borer beetles, which cut through the bark and get under it to build their tunnels and where the females lay eggs, or olive thrips, which suck the sap from the plant by eating buds, leaves, flowers, fruits and young shoots while at the same time injecting toxins into the plant, stopping the growth and deforming the affected organs. Another example is the whitefly, which absorb the sap until the leaves start to show yellow mottled spots and finally dry up. Furthermore, the secretions produced by this insect favor fungal proliferation. Aphids, leaf cutter ants, scale insects, sawflies, geranium bronzes, flower beetle, etc. are other examples of the main insects causing damage to plants.

2. Phytopathogen Control

To control the aforementioned the plant pathogens and increase crop yields, chemicals are generally used, these chemicals having a high associated toxicity in some cases, which negatively affects the environment. Both the water and the dirt are being contaminated; living beings that are fundamental for agriculture, such as pollinating insects, are being destroyed, and animal and human health are being affected. Directive 2009/128/EC of the European Parliament and of the Council, of 21 Oct. 2009, establishes the framework for action to achieve the sustainable use of said compounds and stresses priority areas for starting up ecologically sustainable innovative actions increasing agricultural productivity and resource efficiency.
An alternative to the indiscriminate use of chemicals in agriculture is the use of beneficial microorganisms, including both fungi and bacteria. It is a non-contaminating and environmentally-friendly method that considerably reduces the risks of pathogens becoming resistant.

2.1 Plant Growth-Promoting Rhizobacteria (PGPR)

Rhizobacteria are microorganisms in the soil living near the roots and they are also widely used in biocontrol. Since these bacteria are most often found in nature in the rhizosphere, they do not usually damage other beneficial organisms, and furthermore in many cases they benefit the ecosystem by stimulating plant growth and making agricultural production more sustainable. In turn, their effects on human health are minimal or nil. These microorganisms, also called plant growth-promoting rhizobacteria, or PGPR, colonize plant roots, compete with and control plant pathogens, and act as fertilizers.
PGPRs are characterized by their capacity to stimulate plant growth through direct or indirect mechanisms. Direct stimulation includes nitrogen fixation (Sessitsch et al., 2002); the production of hormones, such as auxins, gibberellins and cytokinins increasing the root elongation, division and size (Perrine et al., 2004; Garcia de Salamone et al., 2001); phosphate solubilization (Rodriguez and Fraga, 1999); and siderophore secretion (Carson et al., 2000), inter alia. The indirect plant growth stimulation includes various mechanisms relating to biocontrol of the phytopathogenic organisms (bacteria, fungi and nematodes, inter alia), which include: competition for ecological niches or substrates; production of hydrolytic enzymes (proteases, lipases, chitinases, collagenases and glucanases); the production of antibiotics (Hassan et al., 1997; Essalmani and Lahlou, 2003); root colonization, turning them into “biological casings” which delay nematode invasion (Rodriguez-Kabana 1997; Loeppler 1997); modification of root exudates, making them less appealing for nematodes (Oostendorp and Sikora, 1990); the production of siderophores and the production by some PGPRs of volatile compounds, such as acetoin and 2,3 butanediol, which cause an increase in plant resistance to infections (also called induced systemic resistance or ISR) (Choudhary and Johri 2009) and the production of H₂S which prevents the development of nematodes (Mena 2004 and 2005).
Through all the described mechanisms, at given concentrations in the soil (at least 10⁶microorganisms/ml are required) PGPRs act not only as plant growth stimulants (plant strengtheners or plant fertilizers), as biological control agents, preventing the development of phytopathogenic bacteria, fungi and nematodes.
Among the most widely used PGPR bacteria are the species of the genera Arthrobacter, Azospirillum, Bacillus, Pseudomonas, Rhizobium and Serratia, inter alia (Kloepper et al. 2004) Others such as Tsukamurelia paurometabola are also used, for example, in the bionematicide HeberNem®, effective in the control of Meloidogyne spp., Radopholus similis and Pratylenchus spp. Their mode of action is related to the release of hydrogen sulfide and chitinases (Mena, 2004 and 2005).

2.1.1. Bacteria of the Genus Bacillus

One of the most widely used bacteria in agriculture in the control of pathogens or as a PGPR are the bacteria of the genus Bacillus.
The bacteria from this genus have many of the aforementioned characteristics, and on the other hand spore formation provides for the long-term viability of this genus in commercial preparations, unlike other rhizobacteria such as Pseudomonas, Rhizobium or Serratia.
There are various commercial products of the genus Bacillus on the market. These include, inter alia:


Product	Company	Composition	Control

Serenade ®	AgraQuest	B. subtilis	Fungi and bacteria
		QST713	in fruits
Ecoguard ®	Novozyme	B. licheniformis	Sclerotinia
		SB3088	homoeocarpa
Kodiak ®	Gustafson	B. subtilis	Fungi in cotton,
		GB03	soybean and
			legumes
Yield	Gustafson	B. pumilus GB34	Fungi in soybean
Shield ®
Bio Yield ®	Gustafson	B. amyloliquefaciens	Fungi
		GB99 + B. subtilis
		GB122
Subtilex ®	Beker	B. subtilis	Fungi in legumes,
	Underwood	MB1600	cotton and soybean
Hi Stick	Beker	B. subtilis	Fungi in soybean
L +	Underwood	MB1600 + Rhizobium
Subtilex ®

Other patents/patent applications describing the use of microorganisms of the genus Bacillus for biological control are:

- Strain of B. velezensis as a biofungicide (US 2010/0179060 A1)
- Strain of B. pumilus as a nematicide (WO 2013/067275).
- Strain of B. pumilus as an insecticide (WO2010146204).
- Strain of B. thuringiensis as a nematicide (WO 2010/078708 A1, U.S. Pat. No. 6,077,506)
- Strain of B. thuringiensis, its genes and toxins as a nematicide (EP 0853671).
- Three strains of Bacillus subtilis, Pseudomonas putida and Sporobolomyces roseus for the control of phytopathogens (WO 2004/024865 A2).
- Several strains of Bacillus spp. and Brevibacillus parabrevis with a fungicidal and bactericidal effect (WO 2010/142055).

Other bacteria used as PGPRs and in biological control are described in the following patents/patent applications:

- Strain of Serratia for the control of pathogens (CA2822178 A1).
- Three strains of Bacillus thuringiensis, Bacillus mojavensis and Azospirillum brasilense as plant growth stimulants and bionematicides (WO2011121408 A1).
- A preparation containing different strains of Pseudomonas as a plant growth stimulant and as a biological control agent (U.S. Pat. No. 6,048,713 A).
- A sulfur-oxidizing and plant growth promoting culture of Delftia acidovorans (US20080242543 A1).
- Strains of Rhizobium to increase plant growth (WO2003089640 A2).
Shan et al. (Crop Protection 44 (2013), 29-37) describe use of Bacillus methylotrophicus strain BC79 in biocontrol of the disease in rice, also known as rice blast, caused by the fungus Magnaporthe oryzae.
Madhaiyan et al. (International Journal of Systematic and Evolutionary Microbiology (2010), 60, 2490-2495) describe Bacillus methylotrophicus sp., isolated from the rice rhizosphere.
Khusro et al. (Indian Journal of Research (2013) Vol. 2, Issue 11, pages 243-244) describe the improvement of a new Bacillus methylotrophicus strain for the increased production of antimicrobial metabolites.

Despite numerous attempts to develop plant growth stimulation strategies and/or strategies for the biological control of bacteria, fungi and/or phytopathogenic nematodes, there is pressing need today to develop alternative strategies or better strategies than those currently in place, which are capable of stimulating plant growth and/or biologically controlling the presence of phytopathogens, such as bacteria, fungi and/or nematodes, for example.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of microorganisms as plant growth stimulants and/or for the biological control of phytopathogenic bacteria, insects, fungi and/or nematodes. More specifically, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to different culture methods with respect to said microorganisms and to the products comprising them, as plant growth stimulants and/or for the biological control of phytopathogens such as bacteria, insects, fungi and/or nematodes. Preferably, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to different culture methods and to the products comprising them for the biological control of phytopathogens such as bacteria, insects, nematodes and phytopathogenic fungi.
In a particular embodiment, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to different culture methods with respect to said microorganisms and to the products comprising them, as plant growth stimulants and for the biological control of phytopathogens such as phytopathogenic bacteria, insects, fungi and nematodes. Preferably, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to different culture methods and to the products comprising them for the biological control of phytopathogenic bacteria, phytopathogenic nematodes, phytopathogenic insects and phytopathogenic fungi, with the exception of fungi belonging to the species Magnaporthe oryzae.
Preferably, the present invention relates to the use of microorganisms of the species Bacillus methylotrophicus, to different culture methods and to the products comprising them for the biological control of bacteria, such as those belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris, for example; fungi, such as oidium, Botrytis or those 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, for example; insects, such as the whitefly and aphid, for example; and/or nematodes such as the species belonging to the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema, Trichodorus, for example, and generally all parasitic plant nematodes.
Another object of the present invention relates to microorganisms belonging to Bacillus methylotrophicus strain XT1 (deposit number CECT8661) 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 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; and/or microorganisms 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.
Another object of the present invention is a bacteria culture comprising or consisting of microorganisms belonging to Bacillus methylotrophicus strain XT1 and/or microorganisms 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; and/or microorganisms belonging to Bacillus methylotrophicus strain XT2 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.
A further object of the present invention relates to a composition comprising microorganisms belonging to Bacillus methylotrophicus strain XT1 and/or microorganisms 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; and/or microorganisms belonging to Bacillus methylotrophicus strain XT2 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.
Another object of the present invention relates to the use of the bacteria, cultures and/or compositions described above in a method for stimulating plant growth and/or in a method for the biological control of phytopathogenic organisms.
More specifically, one object of the present invention is use of the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains, as described above), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof as plant growth stimulants and/or for the biological control of plant pathogens, such as bacteria, insects, fungi and/or nematodes.
More specifically, one object of the present invention is use of the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains, as described above), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof as plant growth stimulants and/or for the biological control of plant pathogens, such as fungi (with the exception of fungi belonging to the species Magnaporthe oryzae) and/or nematodes.
Preferably, the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof are used as plant growth stimulants and/or for the biological control of plant pathogens, selected from the list comprising or consisting of:
Bacteria belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris, 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 those belonging to the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema and/or Trichodorus, for example.
Preferably, the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains, as described above), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof are used as plant growth stimulants and/or for the biological control of nematodes, preferably nematodes belonging to one of the following genera: Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema and/or Trichodorus.
Preferably, the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains, as described above), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof are used as plant growth stimulants and/or for the biological control of Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris.
Preferably, the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains, as described above), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof are used as plant growth stimulants and/or for the biological control of 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, even more preferably for the biological control of Botrytis cinnerea.
Preferably, the bacteria belonging to strains XT1 or XT2 (or those microorganisms having a high degree of homology with these strains, as described above), the cultures or compositions thereof comprising them, as well as the products comprising one of them or the products that can be obtained from them or from the culture thereof are used as plant growth stimulants and/or for the biological control of insects belonging to the family Aphididae, as well as insects belonging to the species commonly referred to as “whitefly”, more specifically, the species Trialeurodes vaporariorum, predominant in greenhouses.
Another object of the present invention relates to a method for stimulating plant growth comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant in contact with the bacteria, bacteria cultures or compositions obtained in step a).
Another object of the present invention relates to a method for stimulating plant growth comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a).
Another object of the present invention relates to a method for the biological control of phytopathogenic organisms comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a).
Another object of the present invention relates to a method for the biological control of phytopathogenic 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, preferably belonging to the species Botrytis cinnerea, comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.
Another object of the present invention relates to a method for the biological control of phytopathogenic bacteria belonging to the species Agrobacterium tumefaciens, Pectobacterium atrosepticum, Ralstonia solanacearum and Xanthomonas campestris comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.
One object of the present invention relates to a method for the biological control of phytopathogenic nematodes (such as for example Meloidogyne, Heterodera, Globodera, Pratylenchus, Paratylenchus, Ratylenchus, Xiphinema and/or Trichodorus species) comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a).
Another 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:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a).
The methods described in the present invention can furthermore comprise the use of a 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 with rotating or diffusing elements 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.
In a preferred embodiment, the aforementioned methods use bacteria, bacteria cultures or compositions comprising Bacillus methylotrophicus, strains XT1 and/or XT2, deposited in the Colección Española de Cultivos Tipo (CECT—Spanish Type Culture Collection) with deposit number CECT8661 and CECT8662, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

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:

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

FIG. 2. Activity of strain XT1 against Agrobacterium tumefaciens

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

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

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

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

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

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.
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.
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.
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).
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”.
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”.
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.
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).
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.
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.
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.
Accordingly, both the presence of flagella and biofilm formation entail an advantage of these bacteria (XT1 and XT2) for colonizing the habitat.
Strains XT1 and XT2 generate ellipsoidal non-deforming spores. In the 2×SG medium, these bacteria produce more than 5×10⁸spores/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.
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.
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 H₂S.
Strains XT1 and XT2 are capable of synthesizing chelating compounds, such as siderophore compounds, which take up Fe³⁺ and turn it into Fe²⁺. The iron ion Fe³⁺ has very little solubility at neutral pH and hence cannot be used by the organisms. Siderophores dissolve these ions into Fe²⁺ complexes, which can be assimilated by mechanisms of active transport.
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.
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.
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.
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.
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).
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).
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).
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)
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 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.
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.
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 Johri 2009).
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.
After extracting the lipopeptides following the method of Cooper et al. 1981, a yield of 0.12 g/l 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.
The cellular dry weight (CDW) of strains XT1, XT2 and of type strain is 2.7 g/l, 2.5 g/l and 2.9 g/l, 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.
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).
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.
The production of enzymes (glucanases, proteases, lipases, phosphatases and urease) together with the different lipopeptides, and the release of SH₂are, according to the literature (see prior art), responsible for the action of said strains in the biological control of fungi, bacteria, insects and nematodes.
Another object of the present invention relates to a method for the biological control of phytopathogenic organisms comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
b. putting a plant in contact with the bacteria, bacteria cultures or compositions obtained in step a).
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
b. putting a plant preferably not affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.
Another object of the present invention relates to a method for stimulating plant growth comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a.
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:
a. obtaining the bacteria, bacteria cultures or compositions as previously described; and
b. putting a plant affected by a phytopathogen in contact with the bacteria, bacteria cultures or compositions obtained in step a).
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.
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.
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.
Localized irrigation systems can comprise drip, exudation and/or microsprinkling systems.
The person skilled in the art knows how localized irrigation systems work and how to use them.
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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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.
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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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.
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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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.
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.
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.
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.
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.
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.
Furthermore, in the method of the present invention, the culture and/or composition of the present invention having a microorganism concentration of at least 10⁸colony 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 5×10⁸CFU/ml.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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 10⁸colony 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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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 10⁸colony 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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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 10⁸colony 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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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 10⁸colony 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.
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:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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 10⁸colony 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.
Therefore, one object of the present invention relates to a method for stimulating plant growth comprising the steps of:
a. obtaining the bacteria, bacteria cultures or compositions according to the present invention; and
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 10⁸colony 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).
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

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

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 cm²with 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 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

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).
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

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 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

a) Tests in tomato plants inoculated with Meloidogyne javanica (FIGS. 3, 4, and 5)
Five batches of 10 tomato plants (Solanum lycopersicum) each were used. Three batches were inoculated with the selected strains (2×10⁸CFU) 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.
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.
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.
A sector of 2000 m²was used for injection in the drip irrigation and another sector similar was used as a control. 7.5 l of culture were applied in 6 applications separated by a period of 10 days (1, 250 I of a culture with at least 10⁸CFU/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

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.
The activity of the bacterial cultures of strains XT1 and XT2 with 5×10⁸CFU/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 μI 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 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

The activity of the bacterial cultures of strains XT1, XT2 and the B. methylotrophicus type strain with 5×10⁸CFU/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 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

b) Furthermore, a nectarine tree Prunus persica var. nectarina highly affected by green and black aphids (see FIG. 6) was treated. With a culture of XT1 with 5×10⁸CFU/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.
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

a) Pot test with pepper plants (genus Capsicum) and pumpkin plants (genus Cucurbita) with strains XT1, XT2 and type strain.
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 5×10⁸CFU/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 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

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.
b) Furthermore, the following were conducted with strain XT1:
b. 1. Test in greenhouse pear tomato crops.
Treatments were performed by foliar application at three different doses of the culture broth containing at least 10⁸CFU/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 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)

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.
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.
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 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

Claims

1. A method of providing phytopathogen protection to a plant, the method comprising applying to at least a portion of the plant a composition comprising lipopeptides of a microorganism, wherein the microorganism comprises Bacillus methylotrophicus strain XT1 (deposit number CECT8661) or a related strain having a 16S rRNA gene sequence with at least 99.7% identity to the 16S rRNA gene sequence of the B. methylotrophicus strain XT1.

2. The method of claim 1, wherein the portion of the plant is selected from a root, a leaf, and a stem.

3. The method of claim 1, wherein the microorganism is the B. methylotrophicus strain XT1.

4. The method of claim 1, wherein the applying comprises foliar application.

5. The method of claim 1, wherein the applying comprises irrigation.

6. The method of claim 1, wherein the lipopeptides comprise surfactin, fengycin and lichenysin.

7. The method of claim 1, wherein the composition further comprises enzymes of the microorganism.

8. The method of claim 7, wherein the enzymes are selected from glucanases, proteases, lipases, phosphatases and ureases.

9. The method of claim 1, wherein the composition comprises a culture of the microorganism.

10. The method of claim 9, wherein the culture of the microorganism is at a concentration of at least 10⁸colony forming units (CFU) per ml and is at a dilution between 0.5-5% (v/v).

11. The method of claim 10, wherein the culture of the microorganism is at a concentration of at least 5×10⁸CFU per ml and is at a dilution of about 1.5% (v/v).

12. The method of claim 1, wherein the composition comprises an isolated extract of the lipopeptides.

13. The method of claim 1, wherein the applying comprises an application at a first time point, and an application at a second time point that is about thirty days after the first time point.

14. The method of claim 1, wherein the applying comprises application once every 10 days, for 60 days.

15. The method of claim 1, wherein the applying comprises application once a day for 8-12 days.

16. A composition comprising an isolated lipopeptide extract derived from a culture of a microorganism, wherein the microorganism comprises Bacillus methylotrophicus strain XT1 (deposit number CECT8661) or a related strain having a 16S rRNA gene sequence with at least 99.7% identity to the 16S rRNA gene sequence of the B. methylotrophicus strain XT1.

17. The composition of claim 16, wherein the composition comprises the one or more lipopeptides at a concentration of at least about 0.12 g/l.

18. The composition of claim 16, wherein the isolated lipopeptide extract comprises a mixture of 13, 14 and 15 carbon atoms fatty acids, which are bound to a cyclic peptide by leucine or isoleucine.

19. The composition of claim 17, wherein the relative proportion of the 13, 14, and 15 carbon fatty acids is a ratio of 1:6.5:5.7, respectively.

20. The composition of claim 16, wherein the isolated lipopeptide extract comprises one or more of surfactin, fengycin and lichenysin.

21. The composition of claim 16, wherein the isolated lipopeptide extract comprises surfactin, fengycin and lichenysin.