ITPI20070067A1 - METHOD FOR BIOFERTILIZATION AND BIOPROTECTION OF PLANT BULBS, IN PARTICULAR FOR APPLICATIONS OF PROPAGATION IN VITRO, PROTECTED CROPS, SEEDS, AND BACTERIAL FORMULA FOR IMPLEMENTING THIS METHOD. - Google Patents

Description

Description of the industrial invention entitled "METHOD FOR THE BIOFERTILIZATION AND BIOPROTECTION OF PLANTULES, IN PARTICULAR FOR APPLICATIONS OF IN VITRO PROPAGATION, PROTECTED CROPS, SEEDS, AND BACTERIAL FORMULATION TO IMPLEMENT THIS METHOD"

DESCRIPTION

Scope of the invention

The present invention relates to a process for the biofertilization and bioprotection of plant material, in particular for propagation applications:

- in vitro including the subsequent acclimatization phase (post vitro);

- in seedbeds;

- in protected crops;

- in hydroponic crops;

- with nursery techniques in general

The invention also relates to a bacterial formulation, in particular, based on Azospirillum brasilense, for such applications.

Description of the prior art

There are numerous beneficial microorganisms for plants that are known as growth promoters (PGPR, Plant Growth Promot ing Rhizobacteria) and / or biocontrol agents (BCA, Biological Control Agents).

The growth promoting effect of PGPR is mainly manifested through the release of metabolites and nitrogen fixation processes by microorganisms. Biocontrol, on the other hand, occurs through an indirect action of BCAs that interact with soil pathogens thanks to different mechanisms such as antibiosis, competition and parasitism.

Growth promotion and biocontrol can sometimes be due to the same microorganism that positively influences the development of the plant through different mechanisms, such as increasing the availability and assimilation of mineral nutrients, the release of growth stimulators and the suppression of pathogenic microorganisms in plant. This results in more resistant and lush plants. Furthermore, PGPR species are able to metabolize numerous and variable sources of carbon, to multiply quickly and above all they show a greater competence than deleterious microorganisms in colonizing the radical rhizosphere.

Much progress has been made in the use of PGPRs over the past 30 years, especially with plants such as rice and cotton, and several strategies have been developed based on the introduction of single microbial agents or consortia. Despite the potential, there have not always been significant results on the pitch. The causes are mainly due to the strong influence of environmental factors and the short survival of microorganisms.

The root is the organ of the plant where the plant-microorganism interaction is most intimate, both in terms of beneficial relationship (nodulation, phytostimulation, phytoprotection) and as a prelude to negative phenomena, such as rot. The root exudates produced by plants are a source of nourishment and support active microbial growth. In this context, the exchange of signal molecules is particularly lively, which act as modulators of the plant-microorganism interaction and contribute to making the environment around the root peculiar, whether the cultivation takes place on soil or on artificial means.

Beneficial rhizobacteria have been found in many microbial genera. Among the most common we can mention Agrobacterium, Azotobacter, Bacillus, Erwinia, Pseudomonas, Rhizobium, Bradyrhizobium, Streptomyces, Actinomyces, and Azospìrìllum.

In particular, the effects of bacteria belonging to the genus Azospìrìllum are known. These are gram-negative bacteria that live in the rhizosphere of numerous plants, positively influencing their growth, and above all the root system, through the production of substances similar to plant hormones (cytokinins, auxins and gibberellins) and processes such as the fixation of atmospheric nitrogen, thus making it assimilable by the plant. Several experimental trials were carried out with a wide range of economically important crops - such as wheat and barley with production increases of 26%. Strains of Azospìrìllum, applied on maize crops, allowed the reduction of 35-40% of the fertilizer normally adopted.

The above experiments gave limited positive results due to the fact that the bacterium is subjected to biotic and abiotic stresses frequently present in open field crops; its persistence in the soil is therefore limited in time, in particular bacterial cells can enter the VBNC state (Viable But Not Culturable) and this results in a drastic reduction of their beneficial effects.

Therefore the high operating cost and the high quantity required make it uneconomical to use in agriculture.

Micropropagation is a plant multiplication technique and is very advantageous as it allows to produce a large number of plants in very small spaces which, being genetically uniform with each other, have all those genotypic and phenotypic characteristics of improvement that have possibly been introduced such as resistance to diseases, making it possible to make a significant contribution to the improvement of the species.

However, the seedlings obtained with micropropagation have higher costs and a high mortality in post transplantation; in particular, transplant shock can cause stunting with loss of the benefits achieved. A very delicate aspect of micropropagated plants is in fact that of their adaptation to external environmental conditions (post vitro). This phase, defined as acclimatization, involves an adaptation to external factors, with stress phenomena that can seriously affect the survival of plants. The main causes of stress are generally attributable to the breaking of the roots during transplanting operations in peat, to excessive losses of water from the tissues, to heat strokes and in particular to the presence of pathogenic microorganisms. The in vitro culture conditions are normally free from microorganisms thanks to the use of sterilization procedures of the starting plant material, equipment, media and substrates used.

In the subsequent phase of post transplantation on an organic substrate (post vitro), massive use is then made of synthetic phytochemicals aimed at avoiding the presence of phytopathogens. However, it is desirable to reduce the use of chemicals in the production and defense of in vitro propagated cultures and to increase the efficiency of micropropagation.

Summary of the invention

It is therefore an aim of the present invention to provide a process for the production of micro-propagated plant material and for the subsequent adaptation to the in vivo cultivation conditions that uses microorganisms capable of simultaneously exercising stimulating and biocontrol activities, such as to favor a better condition of the plants propagated in vitro. and subsequently their better adaptation to in vivo culture conditions.

It is another object of the present invention to provide a method for obtaining more vigorous micropropagated plants and more protected from attack by pathogens in the subsequent stages of growth, obtaining, in particular, significant increases in biomass.

It is another object of the invention to provide a bacterial formulation effective on the stimulation and bioprotection of in vitro propagated plant pathogens which is easy to apply during the in vitro culture and post vitro acclimatization phases.

In particular, said bacterial formulation must be capable of:

- increase the process of rhizogenesis (PGPR) in nursery propagation techniques;

- protect the plant material (BCA) from the attack of pathogenic microorganisms thus reducing the incidence of diseases;

- improve the in vitro propagation technique making it applicable also to plant species which for reasons not yet well defined (presence of endophytes, intrinsic genetic factors, presence of viruses, viroids and phytoplasmas) are recalcitrant to vitro;

- bio-suppressing the germination of weed species of plant material in vivo on organic substrate;

- improve the vegetative vigor.

These and other purposes are achieved by the method for the biofertilization and bioprotection of plant material, in particular for in vitro propagation applications, protected crops, seedbeds, hydroponic crops, and all nursery propagation techniques in general whose characteristic is to include at least a contact phase of a portion of plant material suitable for vegetative and / or seed propagation with a bacterial formulation containing Azospirillum brasilense Sp245.

According to another aspect of the invention, a bacterial formulation for the biofertilization and bioprotection of plant material comprises:

- a culture of a bacterial strain with biofertilization, bioprotection and bio-suppression properties of pest plant species; - a liquid, in particular sterile water and / or isotonic mineral solution;

- a thickener selected from agarose and the like, cellulose and other natural fibers, pectins, gelling polysaccharides, gelogenic in general.

Said liquid, said thickener, said culture are mixed in such a way as to form said formulation in which or with which seeds and / or portions of plants can be put in contact, in particular for applications of micropropagation, protected crops, hydroponic crops, seedbeds and nursery techniques in general.

An advantageous aspect is that said bacterial formulation can include the addition of a compound to stimulate bacterial growth and their effectiveness. This compound is preferably represented by microbial growth factors such as ac. malic, tryptophan, etc.

Preferably, said bacterial strain culture with biofertilization and bioprotection properties is chosen from:

- Azospirillum brasilense, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces spp. , Azotobacter spp. , Bacillus spp. , Pseudomonas spp. Rhizobium spp. , Bradyrhizobium spp. , Paenibacillus spp. , Stenotrophomonas spp. Streptomyces spp. , Actinomyces spp. et c.

Advantageously, in vitro propagation includes the phases of removal and sterilization of the explant, in vitro implantation, proliferation, elongation, rooting, acclimatization, said wetting phase with said bacterial formulation being carried out in at least one of said phases (implantation, proliferation, elongation, rooting, acclimatization).

Brief description of the drawings

Further characteristics and advantages of the method according to the invention will become clearer with the following description of an application example given by way of non-limiting example, with reference to the attached drawings, in which:

Figure 1 schematically shows the removal of the explant from the mother plant and all the steps of the in vitro propagation technique envisaged by the method, subjected, according to the invention, to the method for biofertilization and bioprotection of plant material.

Figure 2 schematically shows the inoculation phase of the plant portion, the aforementioned inoculation phase being carried out in at least one of the phases shown in Figure 1,

- Figure 3 schematically shows the further inoculation phase to which the rooted plant of Figure 1 is subjected before being transplanted in vivo on organic substrate (in pots, and / or alveolar containers),

- Figures 4 to 7 schematically compare the results obtained in terms of number, length and weight of roots, number and length of shoots, number of nodes for portions of plants subjected to the method, according to the invention, and portions of plants not subjected to the method,

- Figure 8 shows the percentage of actively growing apexes in the inoculated and control plants;

- Figure 9A schematically shows how in an external environment, the plants of the uninoculated control show a greater number of weed plant species (in particular the Conyza canadiensis species) compared to the plants inoculated with A. brasilense Sp245,

- Figure 9B shows how the weeds developed in the pots of the inoculated plants have a reduced biomass compared to those grown in the uninoculated control pots.

EXAMPLES

Materials and methods

Bacterial strain and cultivation

Cells of A. brasilense Sp245 "wild" type (Baldani et al., 1986) were cultured at 30 ° C in a microbial growth liquid. The cells were collected by centrifugation (10 min at 6000 rpm, at 4 ° C), washed in sterile water, resuspended in sterile deionized water to obtain a suspension with 10 <7> CFU ml <-1> to be used as an inoculum. .

I croppagazi one

With reference to Figures 1 to 3, tests were carried out on plant material consisting of explants, i.e. vegetative apexes 2 and microtalee 3 taken from a mother plant 1, of clonal rootstocks Mr.S 2/5 (Prunus cerasi fera x Prunus spinosa ), GF677 (Prunus persica x Prunus amìgdalus), MM106 (Cultivar Northen Spy x MI). The first two are rootstocks of the plum and the peach tree respectively, the third of the apple tree. Said explants 2 and 3 were propagated, in vitro, on growth substrates 5 differentiated according to the clonal rootstock to be propagated and precisely:

Example 2

Mr.S2 / 5- MS proliferation medium (Murashige and Skoog, 1962) supplemented with 1 mg / 1 of Thiamine, 100 mg / 1 of Myoinositol, 30 g / 1 of Sucrose, 0.6 mg / 1 of BAP, 0.2 mg / 1 of Gibberellic Acid (GA3), 0.06 mg / 1 of Indolbutyric Acid (IBA), at pH 5.3, and 6 g / 1 of Agar. Then, explants 2 and 3 were moved to an elongation MS substrate 6 in the presence of 0.2 mg / l of BAP and 200 mg / l of charcoal before being transferred to a rooting substrate 7.

The explants of clone Mr.S 2/5, at the end of the elongation phase, were inoculated at basal level 2a with A. brasilense Sp245, dipping them in the bacterial formulation 10 present in the vessel 11, as shown in Figure 2, and transferred to a standard MS type rooting substrate 20, contained inside a test tube 15, or a culture vessel 25, to favor the process of rhizogenesis (Figures 4A-4C). In particular, the substrate 20 used in the rooting step is MS supplemented with 0.6 mg / 1 of IBA. Each treatment was replicated fifteen times and the glass tubes 15 (20 x 160 mm), each containing a single seedling, were arranged in a random configuration and incubated at 24 ± 1 ° C with a light / dark ratio of 16/8. -h, made using cold white lamps (60 pmol πΓ <2> s <_1>).

Tab. 1 Rooting phase

1 MS IBA (0.6 mg / 1) [Control]

2 MS IBA (0.6 mg / 1) I <6> cells / explant

Example 3

GF677- DKW proliferation medium (Driver & Kuniyuki, 1984) supplemented with 1 mg / 1 of Thiamine, 100 mg / 1 of Myoinositol, 30 g / 1 of Sucrose, 2.0 mg / 1 of BA, at pH 5.3, 5 g / 1 of Agar and 4 g / 1 of Carrageenan. Then, the explants were moved to a 6 'elongation DKW substrate (Fig. 1) in the presence of 0.6 mg / 1 of BA before being transferred to a rooting substrate 7.

The explants of clone GF677, at the end of the elongation phase, were inoculated at the basal level with A. brasilense Sp245, immersing them in the bacterial formulation 10 present in the vessel 11 as shown in Figure 2, and transferred to a standard rooting substrate 20 type DKW to favor the process of rhizogenesis. In particular, the substrate 20 used in the rooting step is DKW supplemented with 0.8 mg / 1 of IBA 5 mg / 1 of the polyamides Spermine and Spermidina.

Tab. 2 Rooting phase

1 DKW IBA (0.8 mg / 1) [Control]

2 DKW IBA (0.8 mg / 1) IO <6> cells / explant

Example 3

MM106- DKW proliferation medium (Driver & Kuniyuki, 1984) supplemented with 1 mg / 1 of Thiamine, 100 mg / 1 of Myoinositol, 20 g / 1 of Sucrose, 10 g / 1 Sorbitol, Fluoroglucinol 150 mg / 1, 2, 4 mg / 1 of BA, 0.2 mg / 1 of GA3, 0.06 mg / 1 of IBA at pH 5.3 and 6 g / 1 of Agar. Then, explants 2 and 3 were moved to a 6 'elongation DKW substrate in the presence of 0.2 mg / l of BA before being transferred to a rooting substrate 7 (Fig. 1).

The explants of clone MM106, at the end of the elongation phase, were inoculated at basal level 2a with A. brasilense Sp245, immersing them in the bacterial formulation 10 present in the vessel as shown in Figure 2, and transferred to a standard rooting substrate 20 type DKW to promote the process of rhizogenesis. In particular, the substrate used in the rooting phase is DKW supplemented with 0.6 mg / 1 of IBA.

Tab. 3 Rooting phase

1 DKW IBA (0.6 mg / 1) [Control]

2 DKW IBA (0.6 mg / 1) IO <6> cells / explant

Acclimatization (post vitro)

Example 4

Plants 101 of 20 days, rooted in vitro (Figure 1) were inoculated with A. brasilense Sp245 (10 <6> cells / plant), dipping them in the bacterial formulation 10 present in vessel 11 (Figure 3), and transplanted in vivo into cellular containers 105 (one plant for each cell), or in pots 104, filled with organic substrate 110 (peat TKS1 Flora Gard, 34% organic carbon, 0.2% organic nitrogen, 60% organic material, pH 5-6). During the first week, the seedlings were kept in climatic cells, with the aim of slowly reducing the relative humidity. The cells were gradually opened to allow easier acclimatization of the plants to the external environmental conditions, keeping them at a temperature of 24 ± 2 ° C with a light / dark ratio 16/8-h, provided by white and cold lamps (45 ± 5 pmol πΓ <2> s <_1>). On alternate days the pots were saturated with tap water. Uninoculated plants were considered as controls (C). Each treatment was repeated at least twenty times and the containers were arranged randomly. The number of roots, their length, the fresh weight of the roots, the number of nodes, the number, length and fresh weight of the shoots were evaluated on 50-day-old plants (see Figures). The plants were then transferred to the home (18-1012 ° C, 16 h light / 8 h dark and periodically irrigated) and the above values (number of roots, etc.) were again evaluated on 80-day old plants.

Bioprotection of plants

Example 5

The bio-control activity of A. brasilense Sp245 was evaluated in 50-day seedlings of Mr.S 2/5 infected with the pathogenic fungus Rhizoctonia spp.

The plants rooted in 20 days were transplanted into an organic substrate naturally infected with Rhizoctonia spp and inoculated with Azospirillum brasilense Sp245 (IO <6> cell / explant). The inoculation of bacteria was repeated after 3 days with the same amount of cells. Non-inoculated plants were used as a control. The plants were grown as described in the previous acclimatization phase. Thirty days after inoculation, the plants still alive were checked.

In vitro fungal inhibition test

Example 6

The test was conducted against Rhizoctonia spp, directly isolated from naturally infected peat. The bacterial suspensions were inoculated on PDA (Potato Dextrose Agar) in Petri dishes and incubated at 28 ° C. After 24 h of incubation portions of fungal mycelium were placed on the aforementioned PDA plates. An incubation at 28 ° C for 7 days was performed, recording an inhibition in the growth of ite. PDA capsules without bacterial inoculation were considered as controls.

Effect of Sp245 on the rhizospheric fungal community Example 7

The DGGE (Denaturing Gradient Gel Electrophoresis) analysis of the fungal community (Oros-Sichler et al., 2005) was performed at the end of the acclimatization experiment in 80-day-old plants on naturally fungal-infected rhizospheric soil.

RESULTS

In vitro rooting

At the end of the rooting phase (20 days after inoculation), the explants of Mr.S 2/5 propagated on the standard MS substrate and inoculated with A. brasilense Sp245, marked in the Figures with the abbreviation Sp245, showed a greater number of roots (Figure 4B) and a greater length of the same (Figure 4C) compared to the uninoculated control thesis, indicated in the Figures with the letter "C". In proportion, the percentage of rooted explants was higher than in the control thesis (Figures 4A and 5).

Acclimatization in life

New rooted (post vitro) plants of Mr.S 2/5, GF677, MM106 20 days old were inoculated with A. brasilense and transferred under live conditions on organic substrate. Surveys carried out at 30 and 60 days from inoculation on plants of 50 days and 80 days respectively showed:

1) significant differences between inoculated and uninoculated explants in terms of root length, root weight, number of nodes, stem length and stem weight (See Figures 4 to 7) 2) significant differences were found on the percentage of apices in active growth which was greater in the inoculated seedlings than in the control ones whose apical meristem remained dormant for a long time (Figure 8).

Bioprotection of plants

Azospirillum brasilense Sp245 was also tested in in vivo conditions for its ability to bioprotect Mr.S 2/5 seedlings from rhizottoniosis, during the acclimatization phase. Bacterial inoculation was significantly effective in protecting plants against Rhizoctonia spp., Naturally present in the soil. A survival rate of 100% compared to 0% in the control was observed in all inoculated treatments. Molecular analyzes carried out on fungal rhizospheric DNA extracted from medium inoculated with A. brasilense highlighted the disappearance of some dominant fungal populations which, conversely, were present in the uninoculated control medium.

Biosuppression of weeds

Acclimatized plants of Mr.S 2/5 inoculated and not inoculated with A. brasilense were transferred to outside greenhouse acclimatization. After 5 months from the transfer to the external environment, the uninoculated control plants showed a greater number of weed plant species (in particular of the Conyza canadiensis species) compared to the plants inoculated with A. brasilense Sp245 (Figure 9A). Furthermore, the weeds that developed in the pots of the inoculated plants were found to have a reduced biomass compared to those grown in the uninoculated control pots (Figure 9B).

Claims (9)

CLAIMS 1. Method for the biofertilization and bioprotection of plant material, in particular for in vitro propagation applications, protected crops, seedbeds, hydroponic crops, nursery techniques, characterized by the fact of comprising at least one contact phase of a portion of plant material suitable for vegetative and / or seed propagation with a bacterial formulation containing Azospirillum brasilense Sp245. Method for the biofertilization and bioprotection of plant material, according to claim 1, wherein said vegetative and / or seed propagation is achieved through the use of standard rooting / growth substrates comprising organic substrates, agar substrates and liquid substrates ( hydroponics) plus known bacterial formulation. Method according to claim 1, wherein said in vitro propagation comprises the steps of proliferation, elongation, rooting and acclimatization, said contact step with said bacterial formulation being carried out in at least one of said steps. 4. Method, according to claim 1, in which among said applications the micropropagation of plant species is foreseen which are recalcitrant to the in vitro growth conditions for reasons not yet well defined (presence of endophytes, intrinsic genetic factors, presence of viruses, viroids and phytoplasmas). 5. Bacterial formulation for the biofertilization and bioprotection of plant material characterized by the fact of including: - a culture of a bacterial strain with biofertilization, bioprotection and bio-suppression properties of infesting species; a liquid, in particular sterile water and / or isotonic mineral solution; a thickener selected from agarose and the like, cellulose and other natural fibers, pectins, gelling polysaccharides, gelogenic in general, said formulation comprising said culture, said liquid, and said thickener mixed, with said formulation being able to put seeds and / or portions into contact of plants, in particular for micropropagation applications, hydroponic crops, protected crops, seedbeds and nursery techniques in general. 6. Bacterial formulation according to claim 5, additionally comprising a compound for stimulating bacterial production. 7. Bacterial formulation, according to claim 6, wherein said compound able to stimulate bacterial production is selected from malic acid, tryptophan, growth factors, modulation factors of microbial growth. 8. Bacterial formulation, according to claim 5, wherein said bacterial strain culture with biofertilization and bioprotection properties is selected from: Azospirillum brasilense, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces spp. , Pseudomonas spp. , Bacillus spp. , Azotobacter spp. , Rhizobium spp. , Bradyrhizobium spp. , Actinomyces spp. , Paenibacillus spp. , spp. etc. 9. Bacterial formulation, according to claim 8, wherein said bacterial strain culture with biofertilization and bioprotection properties is Azospirillum brasilense Sp 245.