WO2017037500A1

WO2017037500A1 - Bacterial inoculant to boost phenological growth of coffee plants

Info

Publication number: WO2017037500A1
Application number: PCT/IB2015/056547
Authority: WO
Inventors: Teresita JIMÉNEZ-SALGADO; Flor GUTIERREZ-CONTRERAS; Ana Line GARCÍA-TORRES; Amparo MAURICIO-GUTIERREZ; Refugio Armando TAPÍA-HERNÁNDEZ
Original assignee: Benemérita Universidad Autónoma De Puebla
Priority date: 2015-08-29
Filing date: 2015-08-29
Publication date: 2017-03-09
Also published as: MX2015014809A

Abstract

A process for developing a biofertilizer bacterial inoculant called Biocaferti-BUAP to boost growth in the cultivation of coffee plants at their different stages, wherein. the process is obtained based on three isolated strains of the genus Azospirillum, which are as follows: CaBUAP1, CaBUAP2 y CaBUAP3; wherein: the phenological phases of germination, sprouting and "chapola" during the coffee's juvenile period (Coffea arábica L.) are boosted, bringing about a high coffee yield in less time.

Description

BACTERIAL INOCULANT TO BOOST PHENOLOGICAL GROWTH OF COFFEE PLANTS

BACKGROUND OF THE INVENTION

5

Technical Field of the invention.

The present invention relates to the development of a biofertilizer bacterial inoculant called Biocaferti-BUAP based on three strains of the genus Azospirillum 10 spp (CaBUAPI , CaBUAP2, and CaBUAP3) to boost growth in the cultivation of coffee plants at their different stages.

BACKGROUND OF THE INVENTION

15 In Mexico, coffee bean is considered one of the most important crops in an economic, socio-cultural and environmental sense. The most important coffee- producing states are: Chiapas, Veracruz, Puebla and Oaxaca [SAGARPA, 2014].

Coffee production in Mexico is one of the highlights of the country for its 20 economic and social importance, employing more than 500,000 producers and directly or indirectly links about 3 million people; with a market value of 20 billion pesos a year and exports amounting to USD$ 897 million a year. Agricultural exploitation of coffee in the country has a cultivated area of 690,000 hectares in 12 states and 391 municipalities. 84% of the cultivated area in the states of 25 Chiapas, Veracruz, Oaxaca and Puebla are high-yield; followed by Guerrero, San Luis Potosi, Nayarit and Hidalgo with an average production; and the states of Jalisco, Queretaro, Colima and Tabasco with a low yield [SAGARPA, 2014].

The global coffee market is constituted by 82 countries, with the supply 30 provided by 48 exporting nations and the demand by 34 importing countries. The main coffee-producing countries are: Brazil, Vietnam, Indonesia, Colombia, Ethiopia, India, Honduras, Peru and Mexico (International Coffee Organization, 2014). Therefore, one of the Mexican economy strengths is agriculture, with coffee being a potential crop as it has promoted economic development in important regions for many years and it involves exports of USD$ 897 million/year, it is the leading producer of organic coffee in the world [SAGARPA, 2014].

Weather conditions to be considered for the growth of high quality coffee are: relative humidity between 70%- 80%, temperatures ranging between 16°C and 25°C, rainfall between 1000 mm and 2300 mm; and from 15,000 to 25,000 hours of effective brightness and a good nutritious soil quality [Haarer, 1984; Menaz et al., 1983]. Coffee has different growth stages as juveniles, such as germination ranging between 50 to 60 days, followed by a sprouting phase ranging between 65 to 70 days; "chapola" phase between 85-90, transplanting of seedlings of about 1 year, transplanting of saplings for about 2 years and the adult stage (production stage) [Regalado, 1996].

Coffee is a perennial plant and it requires great care during the early stages of growth to allow it to begin the production of cherries; therefore, the nutritional conditions of the soil are fundamental. Coffee demands macro- and micronutrients at different stages of growth to achieve high yields of coffee, where nitrogen is essential for increased production and branching of fruit- bearing shoots and for the formation of green glossy leaves. This feature is hardly available in coffee soils; therefore, the use of nitrogen chemical fertilizers is required. Nitrogen is one of the nutrients that are lost in the soil due to volatilization, leaching, erosion, and similar processes [Caballero et al., 1998; Boddey et al., 1992]. Therefore, the use of chemical fertilizers for a high yield is necessary, bringing about ecological consequences. In nitrogen fertilizers such as urea, ammonium and nitrates that are used in agriculture, only 50% to 60% of the nitrogen applied as mineral fertilizer is found in the plant, 20% is lost due to the high solubility and negatively-charged nitrates, these are leached into the aquifers, which in turn are carried towards superficial bodies of water and 10% to 15% is lost due to de-nitrification, thereby releasing N2, NO and N2O into the atmosphere [Caballero et al., 1998]. The end result becomes an economic cost and has an environmental impact on the environment due to the potential increase of N2O [Bohool et al., 1992].

Given the need to increase crop yields without high economic and environmental costs, the use of microorganisms in the rhizosphere such as biofertilizers (biostimulants or inoculants) are an alternative for farmers to reduce the use of chemical fertilizers, without detriment in the yield [Caballero, 2001 ]. Another alternative is the application of biofertilizers in agricultural fields that grow coffee {Coffea arabica L). Biofertilizers are products containing microorganisms that, when inoculated, can live associated or symbiotically with plants and help the same in their nutrition and protection [Grageda-Cabrera et al., 2012]. One of the most common genera is Azospirillum and has been reported in a variety of plants [Mehnaz et al., 2007; Perez, 2007; Garcia et al., 2006].

The genus Azospirillum is considered a promoter bacteria for Plant Growth-Promoting Bacteria (PGPB), having a positive effect on the growth of crops due to the biological nitrogen fixation mechanisms (BNF) - a process responsible for reducing molecular nitrogen (N2) to ammonium -; hormone production and plant protection [de-Bashan et al., 2007]. Worldwide, it has been shown that Azospirillum improves the growth and yield of crops, thereby reducing costs [Perrig et al., 2007].

The present invention addresses the issue of developing a biofertilizer bacterial inoculant called Biocaferti-BUAP based on three strains of the genus Azospirillum spp (CaBUAPI , CaBUAP2, and CaBUAP3) to boost growth in the cultivation of coffee plants at their different stages.

Ukrainian patent application UA86178 (GYOERGY ET AL), published on April 10^th, 2009, specifies: preparations and a method for soil cultivation and plant seeds comprising living organisms or microorganisms capable of multiplying in different soil types in the plant surroundings, wherein it comprises the cultivation of Azotobacter vinelandii spp. M657 (NCAIM/P/B 001292) in an amount of 5 10 10, preferably 10-10 cells/hectare, and unnecessarily, one or more of the following microorganisms: Azospirillum brasilense ssp. SW51 (NCAIM/P/B 001 293); Pseudomonas fluorescens var. SW1 1 (NCAIM/P/B 001 296); Bacillus var polymyxa. SW17 (NCAIM/P/B 001 295); Bacillus megaterium var. M326 (NCAIM/ P/B 001 291 ), Micrococcus roseus ssp (NCAIM/P/B 001 294); Var Bradyrhizobium japonicum. PH25 (NCAIM/P/P 001302); Var albus Streptomyces. 0003 LP (NCAIM/P/B 001301 ). The aforementioned microorganisms breed at a low temperature, preferably below 20°C, and also in soils with a low pN value, preferably less than 5.0. The invention also relates to a method for improving and maintaining the soil structure, wherein microbial polysaccharides preferably obtained from the microorganisms applied through biosynthesis of succinoglucone are applied to the soil.

Mexican patent application No. PA 04001383 A (OTT) published on June 6^th, 2005, corresponding to document WO 03/016241 , discloses: preparations suitable for treatment of the soil and plant seeds, which contain living microorganisms or microorganisms that may proliferate in different types of soil around a plant, wherein they contain in an amount of 5 x 10⁶ - 10¹¹ , preferably 10⁷ - 10¹⁰ cells/g, the cultivation of at least one of the following microorganisms: Azospirillum brasilense ssp. SW51 (NCAIM/P/B/ 001293), Azotobacter vinelandii ssp. M657 (NCAIM/P/B 001292), Pseudomas fluorescens var. SW1 1 (NCAIM/P/B 001296), Bacillus polymyxa var. SW17 (NCAIM/P/B 001295, Bacillus megaterium var. M326 (NCAIM/P/B 001291 ), Micrococcus roseus ssp. A21 (NCAIM/P/B 001294), Bradyrhizobium japonicum var. PH25 (NCAIM/P/B 001302) and Streptomyces albus var. 0003 LP (NCAIM/P/B 001301 ) as microorganisms that proliferate at low temperatures as well, preferably below 20^SC and in soils with low pH, preferably lower than pH 5.0, and deposited with the National Deposit of Agricultural and Industrial Microorganisms, with agriculturally-acceptable wet or dry carriers and non-toxic to microorganisms.

Patent application US 2013/0239633 A1 US (HALOS ET AL), published on September 19^th, 2013, discloses: methods of producing microbial preparations for crop production and microbial preparations produced by such methods. These formulations have microbial inoculants such as Azospirillum spp, which is grown in a complex medium with freeze-dried powdered milk supplemented with minerals and vitamins, starch and inert substances such as talc, chalk and diatomaceous earth. The products made through these methods have a shelf life of more than three years. They are used to inoculate seeds, plantations and planting materials in different agricultural and forest species to improve the root, root hairs and the formation of sprouts, thereby allowing the plants to efficiently use nutrients from the soil and providing the plants with nitrogen from the air to withstand drought and protect plants from root pathogens.

International application WO 2008/146153 A2 (VETTORI ET AL) published on December 4^th, 2008, discloses: a method for biofertilization and bioprotection of plant material, particularly for application of the propagation in vitro, protected crops, nurseries, hydroponic crops, plant nursery techniques, wherein it comprises at least one contact stage between a portion of proper plant material for vegetation and/or seed propagation and a bacterial formulation containing Azospirillum brasilense SP245.

Chinese patent application CN103004350 (A) (TIANQUAN ET AL), published on March 3^rd, 2013, discloses: a balanced nutrition fertilization technology in plants, in particular a method of balanced fertilization for an economical Coffea arabica plant in the cultivation process. The method of balanced fertilization for growing Coffea arabica comprises the following steps: A, sowing in the field and basic fertilization of fertilizers; B, detection of soil sample; C, preparation and application of additional fertilizer; and D, leaf surface top- dressing. Compared with the traditional fertilization method, the method of balanced fertilization performs fertilization, respectively, in different periods according to the features of the fertilizers required by Coffea arabica, and improves the effectiveness of fertilizers; and microelements are added, so that a favorable nutrition for the growth of the coffee plant is provided, and a high and stable yield is guaranteed provided, such yield can be increased by 15%-18% based on the initial yield. Balanced fertilization method is combined with the soil surface to apply several elements and the surface of the leaf of pulverized microelements, to meet the Coffea arabica needs of different elements, greatly improves the efficiency of fertilizers, it obviously improves the performance of Coffea arabica, reduces fertilizer losses, and saves the cost of production.

There exist developments, however the state of the art has no process for the development of a biofertilizer bacterial inoculant process called Biocaferti- BUAP based on three strains of the genus Azospirillum spp (CaBUAPI , CaBUAP2, CaBUAP3) to boost growth in the cultivation of coffee plants at their different stages, as claimed in the present invention.

SUMMARY OF THE INVENTION One example of an objective of the present invention is to achieve the development of a biofertilizer bacterial inoculant called Biocaferti-BUAP based on three strains of the genus Azospirillum spp (CaBUAPI , CaBUAP2, CaBUAP3) to boost growth in the cultivation of coffee plants at their different stages. Yet another example of an objective of the present invention contemplates the formulation of a biofertilizer called Biocaferti-BUAP based on three strains of the genus Azospirillum spp (CaBUAPI , CaBUAP2, CaBUAP3) that boosts the phenological phases such as germination, sprouting and "chapola" phase during the coffee's juvenile period {Coffea arabica L); thereby resulting in a high yield in less time.

Furthermore, another example of an objective of the present invention contemplates achieving through the formulation of a biofertilizer called Biocaferti- BUAP based on three strains of the genus Azospirillum spp (CaBUAPI , CaBUAP2, CaBUAP3), the production stage in a reduced time in order to respond to national and international demands of this crop.

The above objectives are achieved by a process for developing a biofertilizer bacterial inoculant called Biocaferti-BUAP to boost growth in the cultivation of coffee plants at their different stages, wherein, the process is obtained based on three isolated strains of the genus Azospirillum, which are as follows: CaBUAPI , CaBUAP2 y CaBUAP3; wherein: the phenological phases of germination, sprouting and "chapola" during the coffee's juvenile period {Coffea arabica L.) are boosted, bringing about a high coffee yield in less time. Other features and advantages will become apparent from the following detailed description, taken together with the attached drawings, which illustrate by way of example the characteristics of various embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be completely understood by the detailed description given below and in the attached drawings, which are given only by way of illustration and example and therefore do not limit the aspects of the present invention. In the drawings, identical reference numbers identify similar elements or actions. The sizes and relative positions of the elements in the drawings are not necessarily drawn to scale. For example, the forms of the various elements and angles are not drawn to scale, and some of these elements are enlarged and located arbitrarily to improve the understanding of the drawing. In addition, the particular forms of the elements as drawn do not intend to convey any information concerning the real shape of the particular elements and only have been selected to facilitate its recognition in the drawings, wherein:

Figure 1 graphically shows the percentages of the different growth stages of coffee inoculated with Azospirillum spp, in accordance with an embodiment of the present invention; and

Figure 2 shows a table containing information related to the evaluation of the response to inoculation with different strains of Azospirillum spp, on coffee plants {coffea arabica), in accordance with one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the present invention are described in more detail below, with reference to the attached drawings (figures, diagrams, tables and graphs), in which the variations and the aspects of the present invention are shown. Several examples of aspects of the present invention may, however, be realized of many different forms and should not be construed as limitations to the variations in the present invention; on the other hand, the variations are provided so that this description is complete in the illustrative embodiments, and the scope thereof is fully conveyed to those skilled in the art.

Unless otherwise defined, all the technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which aspects of the present invention belong. The methods, processes, and examples provided in this document are for illustrative purposes only and are not intended to be limiting. To the extent that the methods and developments are capable of reproducing the magnitudes reported in experiments, they can be still be considered for modeling various natural processes.

The prior art describes Azospirillum spp as a bacterium associated with crops, and has been found in coffee plants but not evaluated as biofertilizer. Co- inoculations reported include Azotobacter PACHAZ08, Azospirillum 1 1 B and Glomus intraradices Schenck and Smith, which promote growth in terms of biomass and nutritional content in the coffee, and do not boost the phenological stages of the crop.

Therefore, the present invention contemplates the formulation of a biofertilizer called Biocaferti-BUAP based on three strains of the genus Azospirillum spp (CaBUAPI , CaBUAP2, CaBUAP3) that boosts the phenological phases such as germination, sprouting and "chapola" phase during the coffee's juvenile period (Coffea arabica L.) and allows it to reach the production stage in less time, thereby responding to national and international demands of this crop. The biofertilizer for growing coffee is prepared with strains of Azospirillum spp., which are isolated from the same crop of rhizospheric zones and endophytic areas, which are in the collection of the Soil Microbiology Laboratory "Dr. Jesus Caballero Mellado" CICM-ICUAP and identified as CaBUAPI , CaBUAP2 and CaBUAP3.

1. Methodology.

Preparation of Biofertilizer (Biocaferti-BUAP). Each one of the strains were grown in a Nfb liquid medium and incubated at 30°C/220 rpm/36 hrs., after the time elapsed the population was adjusted to 1 x10⁸ CFU/ mL to tyndallize and mix these strains on a sterile support of peat (under 10 lb/2 hrs. every other day, three times); this carrier was stored in polyethylene bags for a period of 4 days at 32°C with a moisture content of 60% to mature.

2. Example.

Assessment of biological activity. Caturra and garnica coffee seeds were washed and disinfected with 1 %

Ca(CIO)2 and rinsed until elimination of chlorine to be inoculated with the biofertilizer (Biocaferti-BUAP) with a population of 2x10⁷ cfu/g and were added an adhesive (40% sugar solution); afterwards they were pelleted with the biofertilizer and sown 1 cm deep in soil from the coffee zone of Sierra Norte, in the municipality of Huitzian de Serdan, Puebla; the soil was sterilized and distributed in nurseries, relative humidity 60%, where 100 seeds of coffee were sown, each treatment was performed in triplicate. Germinators were placed in a greenhouse with a temperature of 25 to 28°C, and a relative humidity of 60%. Evaluations were performed at different juvenile phenological stages of coffee (nursery and plantraiser). 180 days after inoculation of the coffee beans, 8 plants were taken randomly to measure the following: a) Root weight, b) Root length, c) Weight of the above-ground part, d) Length of the above-ground part. Results.

The strains of Azospirillum spp, CaBUAP3, CaBUAPI and CaBUAP2 were individually tested or in combination in coffee seeds of both varieties. The inoculation response with different Azospirillum spp treatments were evaluated in the early growth stages of caturra coffee, and it was found that the CaBUAP2- CaBUAP3 mixture had 91 .7% germination at 25 days after seeds were inoculated, 82.5% in the sprouting stage 35 days and 70% in "chapola" at 55 days, compared with the control which recorded 37%, 29% and 32% respectively for each of the stages. In the garnica variety, the best response was with CaBUAPI treatment, with 94% germination at 31 days; 60% sprouting at 45 days and 74.6% "chapola" at 60 days compared to the control. The largest populations of Azospirillum recovered after 180 days were obtained from the rhizosphere and rhizoplane. Besides the increase in the germination percentage, time was reduced from 22% to 48% in each of the stages by using different strains of Azospirilum spp.

After 180 days of inoculation, evaluation of the plants was conducted, and it was found that CaUAP160 strain showed an statistically significant difference (SSD) in weight and root length, as well as in the weight of the above-ground part, whereas the strain CaUAP290 had a SSD in pairs of leaves and % of nitrogen, relative to the other treatments and the control.

Conclusions. Inoculation of bacteria of the genus Azospirillum spp on coffee seeds helps their growth, thereby reducing the time of each of their phenological stages (germination, sprouting and "chapola").

An effective strain (CaUAP160) was found to be applied as biofertilizer for Garnica variety.

An effective strain (CaUAP290-38) was found to be applied as biofertilizer for cultivation of caturra coffee. The largest population of Azospirillum spp was found in the rhizosphere after 180 days. Figure 1 graphically shows the results of the percentages from the different growth stages of inoculated coffee ("chapola" 55 days, sprouting 35 days and germination 21 days) with different strains of Azospirillum spp: CaBUAPI , CaBUAP2 and CaBUAP3.

Figure 2 shows a table of results from different strains of Azospirillum spp CaBUAPI , CaBUAP2 and CaBUAP3 with treatments for root weight (g); root length (cm); weight of the above-ground part (g); length of the above-ground part (g), pairs of leaves; % N of plant and % P of plant.

The description of the invention is given by way of example only and is not intended in limiting in any way the scope of the invention. The previous specification is provided to allow any skilled artisan realize the various achievements described herein. Various modifications to these embodiments will be readily apparent to the skilled artisans, and the generic principles defined herein can be applied to other embodiments. Therefore, it is not intended that the claims are limited to the embodiments shown here, but they must be granted a full scope consistent with the wording of the claims, where reference to an element in particular is not intended to mean "one and only one", unless specifically stated, but rather "one or more". All the structural and functional equivalents of the elements of the various embodiments described throughout this specification, which are known or that will be subsequently known by artisans in the art are expressly incorporated herein by reference, and are intended to be covered by the claims. Therefore, it must be understood that numerous and varied modifications can be made without departing from the spirit of the present invention.

Claims

1.- A process for preparing a biofertilizer bacterial inoculant called Biocaferti-BUAP to boost growth in the cultivation of coffee plants at their different stages, wherein:

the process is obtained based on three isolated strains from the genus Azospirillum spp, which are: CaBUAPI , CaBUAP2 and CaBUAP3; wherein:

the phenological phases of germination, sprouting and "chapola" during the coffee's juvenile period {Coffea arabica L.) are boosted, bringing about a high coffee yield in less time.

2.- The process for preparing a biofertilizer bacterial inoculant called

Biocaferti-BUAP of claim 1 , wherein each one of the strains were grown in a Nfb liquid medium and incubated at 30°C/220 rpm/36 hrs., after the time elapsed the population was adjusted to 1 X10⁸ CFU/ ml_ to tyndallize and mix these strains on a sterile support of peat, which were submitted to 10 lb/2 hrs. every other day and stored in polyethylene bags for a period of 4 days at 32°C with a moisture content of 60% to mature.