BR102019008109A2 - RHIZOPHAGUS CLARUS ARBUSCULAR FUNGI INOCULANT PRODUCTION PROCESS - Google Patents

Abstract

Process for producing Rhizophagus clarus arbuscular mycorrhizal fungi based inoculum The present invention is a multiplication process of rhizophagus clarus arbuscular mycorrhizal fungi under axenic conditions. This process allows time reduction and increased seedling production, as well as the mixing of an inert expanded clay or vermiculite substrate that acts as a carrier of the seedlings, optimizing the inoculation process for use in intensive agricultural systems. In addition to increasing yield and promoting plant growth, the application of the inoculant allows the total or partial replacement of phosphorus in fertilization, with proven agronomic efficiency.

Description

PROCESS OF INOCULANT PRODUCTION BASED ON ARBUSCULAR MYCORRHIZAL FUNGI RHIZOPHAGUS CLARUS

FIELD OF THE INVENTION [001] The present invention deals with the establishment of a multiplication process of arbuscular mycorrhizal fungus of the species Rhizophagus clarus associated with carrot roots transformed in vitro. This innovative process allows the reduction of time and the increase of propagation multiplication by inducing cell differentiation, as well as the introduction of an inert substrate of expanded clay or vermiculite that works as an inoculant vehicle, optimizing the inoculation process for intensive cultivation of all cultivable plants. In addition to these benefits, inoculation with arbuscular mycorrhizal fungi allows total or partial replacement of fertilizer phosphorus with proven agronomic efficiency.

[002] According to Souza, Stürmer, Carrenho and Trufem (2010), in the article “Classification and taxonomy of arbuscular mycorrhizal fungi and their diversity and occurrence in Brazil”, arbuscular mycorrhizal fungi (FMA) are mandatory biotrophic organisms, which are associated with roots of terrestrial, epiphytic, aquatic vascular plants and also with rhizoid and bryophytic stalks and other vegetables forming a mutualistic relationship called arbuscular mycorrhiza (MA).

[003] According to Andrade (2004), in the article “Role of functional groups of microorganisms on the rhizosphere microcosm dynamics” and Giri, Giang, Kimari, Prasad, Varma (2005), in the article “Microorganisms in solis: roles in genesis and functions ”, MA is the best known interaction between microorganisms and plants and has a very important role in plant productivity and nutrient cycling.

Petition 870190037847, of 22/04/2019, p. 6/20

2/6 [004] This symbiotic mutualistic association occurs in the roots of plants with the fungus colonizing the cortical cells forming arbuscules within the cells. According to Dubel and Merbach (2005), in the article “Influence of microorganisms on phosphorus biovailability in soils”, the fungus absorbs photosynthesis from the root cells and mainly transfers phosphorus from the soil to the plant through the mycelium. In turn, the plant provides the symbiotic (heterotrophic) fungus with carbonaceous compounds from photosynthesis, as well as a protected habitat, according to Harrison (1999), in the article “Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis”.

[005] According to Sylvia, Fuhrmann, Hartel and Zuberer (1999), in the article “Principles and applications of soil microbiology”. AMs generate a series of benefits for host plants, as they improve nutrition, growth and production of biomass or grains, increase tolerance to water stress and pathogens and facilitate their adaptation in soils with low nutrient availability. These benefits are the fundamental basis that allows plants to better adapt to the environment, greater competitiveness and greater productivity.

[006] The benefit of MAs in plant growth is related to a greater absorption of nutrients from the soil. FMA can help plants increase nutrient uptake directly or indirectly. Directly, mycorrhizae increase the absorption of nutrients and water from the soil through its external mycelium which explores a larger volume of soil beyond the roots. According to Barea, Azcón and Aguilar (2005), in the article “Interactions between mycorrhizal fungi and bacteria to improve plant nutrient cycling and soil structure”, the fungus extends the root absorption field beyond the zone of root influence and allows it to increase its surface catchment.

Petition 870190037847, of 22/04/2019, p. 7/20

3/6 [007] In addition, the more efficient absorption of mycorrhizal roots is also due to an acceleration of the solubilization of insoluble phosphates in the soil, as well as other elements of low mobility. The benefits of mycorrhizal fungi in capturing phosphorus (P) are of essential importance, as this is a limiting element due to its low availability and mobility in the soil, especially in tropical acid soils.

BACKGROUND OF THE INVENTION [008] Despite the large number of studies on the beneficial effect of FMA on plant development, their use at the commercial level and their application in large-scale agriculture are quite limited due to their mandatory biotrophic nature. In Brazil, the production of mycorrhizal inoculants is limited to a small scale in universities and research centers, where the multiplication of MA is normally used in pots with different types of host plants, within which grasses are the most used. According to Barreto and Siqueira (2009), in the article “Application of formonetin in the colonization and sporulation of mycorrhizal fungi in brachy plants”, they are planted in substrates or mixtures of substrates such as soil, sand, vermiculite, peat, among others.

[009] Several methods have been developed for the propagation of mycorrhizal fungi in axenic conditions, associated with Ri TDNA carrot roots transformed as host organs, making it possible to obtain axenic cultures with potential for the large-scale production of MA inoculants.

[010] The axenic culture of FMA has been greatly improved since the pioneering studies done in the early 1960s by Mosse (1962), in the article The establishment of arbuscular vesicular mycorrhiza under aseptic conditions ”.

Petition 870190037847, of 22/04/2019, p. 8/20

4/6 [011] This study was followed by several others in the 70s and 80s, such as those by Mosse and Hepper (1975), in the article Vesicular-arbuscular infections in rootorgan cultures ”; Strullu and Romand (1986, 1987), in the articles Méthode d'obtention d'endomycohizes à vésicules et arbuscules in axéniques conditions ”and Culture axénique de vésicules isolées from d'endomycohizes et ré-association in vitro to tomato races”; and Bécard and Fortin (1988), in the article Early events the vesicular-arbuscular mycorrhiza formation in Ri T-DNA transformed roots ”.

[012] This methodology has several advantages over conventional methods of cultivation in pot, allowing the production of pure, sterile inoculants, with a high amount of fungal mass (hyphae and spores) and free of contaminating phytopathogens, according to Adholeya, Tiwari and Singh ( 2005), in the article Largescale inoculums production of arbuscular mycorrhizal fungi on root organs and inoculation strategies ”.

[013] From this technique, inoculants based on arbuscular mycorrhizal fungi were developed as in the patents WO 2007/014974, an MA inoculant based on Rhizophagus irregularis and its respective in vitro and ex vitro applications; WO 2013/098829, which describes the process of biofertilizer production based on mycorrhizae however using different concentrations of gel.

SUMMARY OF THE INVENTION [014] In general terms, the main innovation of this invention is the establishment of a multiplication process of arbuscular mycorrhizal fungi in axenic conditions mixed with an inert substrate based on vermiculite or expanded clay, which allows the reduction of time, the increase in the production of propagules and serves as a vehicle for propagules, optimizing the inoculation process for intensive agricultural cultivation. This allows the total or partial replacement of fertilizer phosphorus by inoculation with arbuscular mycorrhizal fungi with proven agronomic efficiency, promoting plant growth and production.

Petition 870190037847, of 22/04/2019, p. 9/20

5/6

DETAILED DESCRIPTION OF THE INVENTION [015] The inoculum object of this invention contains especially propagules of the arbuscular mycorrhizal fungus of Rhizophagus clarus.

[016] According to Bécard and Piché (1988), in the article “Early events of vesicular-arbuscular mycorrhiza formation in Ri T-DNA transformed roots”, the adaptation of this fungus to the in vitro multiplication conditions consists of a series of steps proposed in existing methodology for the establishment of axenic cultures of MA fungi, using carrot roots transformed with plasmid Ri T-DNA.

[017] The inoculum production process includes the adaptation of the mycorrhizal fungus R. clarus in a biphasic medium (a solidified phase and a liquid phase) in glass containers with airtight lid with a capacity between 100 and 500 mL. The solid phase consists of the combination of salts and vitamins of the MRS medium with the modification of the solid substrate (vermiculite and / or expanded clay), and its concentration can vary between 0.8% - 1.5%. The liquid phase consists of a combination of salts and vitamins from the MRS medium. In this growth medium, the fungus achieves a greater development of propagules (hyphae-spores) compared to the conventional method (semi-solidified medium) in a period of 30 to 90 days, thus reducing the time required to obtain a high amount of fungal mass (Figure 1).

[018] The inoculum can also be produced using only the solidified phase of vermiculite and / or expanded clay.

[019] The application of the inoculum in agricultural systems is done through the inoculation of seeds or the use of inert substrates with vehicles of the fungal propagules and colonized roots.

[020] The seed inoculum contains a pool of propagules of Glomus clarum and particles of vermiculite and / or expanded clay from aseptic cultivation, a polysaccharide matrix of microbial and peat origin. Each 100 g to 500 g of propagule pool is mixed with an amount that

Petition 870190037847, of 22/04/2019, p. 10/20

6/6 varies between 10 to 500 g of polysaccharide matrix and 2 to 15 g of peat depending on the species of the plant and the size of the seed to be inoculated.

[021] The peat portion may include an inoculum of symbiotic nitrogen-fixing bacteria for legumes or free-living for non-legumes.

[022] The culture medium containing an inert expanded clay or vermiculite substrate that works as a propagule carrier consists of a mixture of this previously sterilized substrate with the solidified culture medium in which the fungus grows.

[023] This mixture has a proportion of substrate that can vary between 2% and 50%. Under these conditions, fungal structures develop within the expanded clay granules facilitating their subsequent recovery, inoculum preparation and application in the field (Figure 2).

[024] The inoculum has a high applicability in extensive agricultural systems of all cultivable plants. As an example of its performance in field conditions, an increase in the percentage of mycorrhizal colonization in soybean plants (Glicine max L.) from seeds inoculated with R. clarus can be observed. The inoculation of these seeds promoted an increase between 20-30% in grain production, even being more efficient than handling with fertilization. This indicates its high potential for replacing phosphate fertilizers (Figure 3).

FIGURE LEGEND

Figure 1. Production of propagules from the culture of the mycorrhizal fungus Glomus clarum

Figure 2. Development of the mycorrhizal fungus Glomus clarumnos granules of expanded clay.

Figure 3. Efficiency of colonization of the inoculum of Glomus clarum under field conditions and its effect on increasing grain production

Claims (6)

1. PROCEDURE FOR THE PRODUCTION OF INOCULANT BASED ON ARBUSCULAR MYCORRHIZAL FUNGI RHIZOPHAGUS CLARUS, characterized by obtaining mycorrhizal inoculum in two-phase medium: a solidified phase of vermiculite and / or expanded clay and a liquid phase in glass containers with airtight lid with a capacity between 100 and 500 mL (FIG 1) 2. PROCESS, according to claim 1, characterized by obtaining mycorrhizal inoculum in biphasic medium, the solid phase being characterized by containing combinations of salts and vitamins of the MRS medium with the modification of the solidifying agent including vermiculite and / or expanded clay in a concentration ranging between 0.8% -1.5% 3. PROCESS, according to claims 1 and 2, characterized by the method of large-scale application of the inoculum produced in seed inoculation, characterized by being a mixture of a pool of R. clarus propagules from aseptic cultivation, a polysaccharide matrix of microbial origin and peat (FIG 2) 4. PROCESS, according to claims 1 and 3, characterized by containing for each 100 g to 500 g of propagule pool an amount of polysaccharide matrix that varies between 10-500 g and 2-15 g of peat 5. PROCESS, according to claims 1 and 3, characterized by the method of application of the inoculum for seed inoculation and joint co-inoculation of symbiotic nitrogen-fixing bacteria in the case of legumes or free-living for non-leguminous plants 6. PROCESS, characterized by the method of large-scale inoculation of mycorrhizal fungi that uses inert substrates from growth media as vehicles of the propagules (FIG 3 and 4).