WO2008146153A2

WO2008146153A2 - Method for biofertilization and bioprotection of vegetal material, in particular for in vitro propagation, protected cultivations, nurseries, and bacterial formulation for carrying out said method

Info

Publication number: WO2008146153A2
Application number: PCT/IB2008/001402
Authority: WO
Inventors: Lorenzo Vettori; Anna Russo; Cristiana Felici; Stefano Morini; Annita Toffanin
Original assignee: Universita di Pisa
Current assignee: Universita di Pisa
Priority date: 2007-06-01
Filing date: 2008-05-30
Publication date: 2008-12-04
Anticipated expiration: 2009-12-01
Also published as: WO2008146153A3; ITPI20070067A1

Abstract

The method for biofertilization and bioprotection of vegetal material counts several experimental trials actuated on vegetal material including explants, vegetative apexes (2) and micropiping (3) removed from the mother plant, of clonal rootstocks Mr. S 2/5 (Prunus cerasifera x Prunus spinosa), GF677 (Prunus persica x Prunus amigdalus), MM106 (Cultivar Northen Spy x M1). The explants were propagated in in vitro conditions on different growth substrates according with the type of clonal rootstock to be propagated. Then, explants 2 and 3 were transferred to an elongation medium before being transferred to a rooting substrate 7. The explants of clone Mr. S 2/5, at the end of the elongation step were inoculated at the basal level 2a with A. brasilense Sp245, by dipping plants in the formulate 10 that was located in the container 11, as shown in Fig. 2, and transferred to a standard rooting MS substrate 20, located inside a tube 15, or in a relatively bigger glass vase 25, in order to promote the process of rhizogenesis (Fig. 4A-4C). Substrate 20 used during the rooting step is an MS medium supplemented with 0.6 mg/L of IBA. Each treatment was replicated fifteen times and the glass tubes (20 x 160 mm), each containing single seedlings, were arranged in a random configuration and incubated at 24 + 1 ° C with a light/dark 16/8 - h performed by white and cold lamps (60 µ mol m-2 s-1).

Description

TITLE

METHOD FOR BIOFERTILIZATION AND BIOPROTECTION OF VEGETAL MATERIAL, IN PARTICULAR FOR IN VITRO PROPAGATION,

PROTECTED CULTIVATIONS, NURSERIES, AND BACTERIAL FORMULATION FOR CARRYING OUT SAID METHOD

DESCRIPTION

Field of the invention

The present invention concerns a process for biofertilization and bioprotection of plant material, particularly for the any of the following applications of propagation: :

- in vitro including the following step of acclimatation (post vitro) ;

- in seedbeds; — in protected crops;

- in hydroponic crops;

- along with usual plant nursery techniques.

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

Description of the known technique

Many plant beneficial microorganisms are known to be growth promoters (PGPR, Plant Growth Promoting Rhizobacteria) and/or biocontrol agents (BCA, Biological Control Agents) .

The effect of the growth promotion by PGPRs is mainly related to the release of metabolites and nitrogen fixation processes actuated by the microorganisms. On the contrary, biocontrol occurs through an indirect action of the BCAs that interact with soil pathogens through several mechanisms such as antibiosis, competition and parasitism.

Growth promotion and biocontrol can sometimes be due to a same microorganism that positively influences the development of the plant through different mechanisms, such as the increased availability and assimilation of the mineral nutritional components, the release of growth factors and the suppression of pathogenic microorganisms. This assists obtaining more resistant and healthy plants. In addition, PGPR species are able to metabolize numerous and varying carbon sources, to multiply quickly and above all to show a greater competence in colonizing the rhizosphere in comparison to the deleterious microorganisms . In the last 30 years considerable progress in the employment of PGPRs have been made, especially with plants such as rice and cotton, and several strategies based on the introduction of single agents or microbial consortia have been developed. Despite the potential, not always the results of field applications have been significant. This is mainly due to the strong influence of environmental factors and the short survival of the microorganisms.

The root is an organ of the plant where the plant- microorganisms interaction is closer in terms of beneficial relationship (nodulation, phytopromotion, phytoprotection) as well as in terms of pathogenic interaction, such as rottenness. The root exudates produced by plants are a source of nourishment supporting an active microbial growth. In this context, the exchange of signal molecules that act as modulators of the microorganism-plant interaction is particularly intense and helps to make the environment close to the root very peculiar, either the cultivation takes place on land or on artificial supports. Beneficial rhizobacteria have been found in many microbial genera. Among the more common Agrobacterium, Azotobacter, Bacillus, Erwinia, Pseudomonas, Rhizobium, Bradyrhizobium, Streptomyces, Actinomyces and Azospirillum can be reported. In particular, the effects of bacteria belonging to the genus Azospirillum are well known. Azospirillum is a gram^negative bacteria living in the rhizosphere of numerous plants positively influencing the plant growth, especially the root, through the production of substances like plant hormones (cytokinins, auxins and gibberellins) and processes such as the biological nitrogen fixation, which makes the nitrogen available for the plant. Several experimental trials have been performed with a wide range of economically important crops, such as wheat and barley, with 26% production increases. Strains of Azospirillum, applied on corn plants, allowed a 35-40% reduction of the fertilizer normally adopted.

These experiments did not produce any significant results as a consequence of biotic and abiotic stresses which the bacteria have to cope with and which are frequently present in open field conditions; as a result the microorganism persistence in the soil is short, and in particular the bacterial cells may enter into VBNC (Viable But Not Culturable) state with a severe reduction of their beneficial effects.

Therefore, the high operative costs and the high amounts of bacterial inoculum required, make the use of beneficial microorganisms uneconomical in agriculture. Micropropagation is one of the most efficient propagation techniques; it has many advantages due to the production of high numbers of plants in a small space and the propagation of genotypes recalcitrant to standard propagation procedures. Micropropagated plants are genetically uniform and maintain the characteristics introduced by breeding.

One disadvantage of micropropagated plants is the high cost of production, often due to high mortality during post transplantation steps. In particular, transplantation stress can induce a stop of shoot growth, which in turn can be the cause of inefficiency of this technique applied to some genotypes. A critical point of micropropagated plants is represented by the adaptation of the plants to exogenous environmental conditions (post vitro) . During this step, also called plantlet acclimatation, plants have to overcome different types of stress, which sometime can compromise plantlets' survival. Stress is mainly caused by a high water loss from the tissues and by the presence of pathogenic microorganisms in the acclimatization environment. On the contrary, in vitro culture conditions are in general aseptic, as sterilization of both explants and growth media takes place at the beginning of the process. Then, to control development of pathogens during acclimatization and post-transplantation (post vitro) , a prudent use of phytochemicals is required. Nevertheless, it should be advisable to reduce such chemicals and to improve the technique efficiency.

Summary of the invention

The object of this invention is therefore to provide a process for the production of micropropagated plant material and the subsequent adaptation to in vivo conditions by using microorganisms able to stimulate plant activities and biological control processes, that promotes a better in vitro propagation and a better in vivo adaptation.

Another object of this invention is to provide a method to obtain micropropagated plants that are more vigorous and more protected from pathogenic attack in the later stages of growth, having, in particular, significant increases in biomass.

Another object of this invention is to provide a bacterial formulation effective in stimulating plant growth and in protecting from pathogens in vitro and, that could be easily applied during in vitro cultivation and post vitro acclimatation. In particular, this bacterial formulation must be able to:

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

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

— improve the in vitro propagation technique making it applicable also to plant species that for reasons still not clearly defined (endophytes presence, intrinsic genetic factors, viruses presence, viroids and phytoplasms) appear to be recalcitrant in in vitro condition;

— bio-suppress weeds species germination on organic substrate in in vivo condition; — improve the plant vigour.

These and other objects are achieved by the method for biofertilization and bioprotection of vegetal material, in particular for in vitro propagation, nurseries, hydroponic cultivations and all the plant nursery techniques whose characteristic is to have at least one step of contact of a portion of plant material for vegetative propagation and/or seed with the bacterial formulation containing Azospirillum brasilense Sρ245.

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

— cultivation of a bacterial strain with biofertilization, biocontrol and weed biosuppression abilities; - a liquid, particularly sterile water and/or isotonic mineral solution;

- an additive as a thickener and/or adesivant. Advantageously, said additive is selected from the group comprised of: agarose and agarose similars, cellulose and other natural fibres, pectins, polysaccharides, gelling substances in general.

In particular, this additive is a food thickener. This additive is preferably selected from the group comprised of: Arabic gum; Tragacanth gum; Agar; Sodic alginate. Advantageously, the mentioned or each additive has the following proportion : Arabic gum 1,2-2%; Tragacanth gum 0,2-2%; Agar 0,1 - 0,5%; Sodium alginate 0,2-2%. The liquid, the additive and the bacterial culture are mixed to form the formulation in or with which seeds and/or portions of plant can be put in contact, especially for applications in micropropagation, nurseries, hydroponic cultivations and all the plant nursery techniques.

One advantageous aspect of the invention is that a compound to stimulate bacterial growth and effectiveness can be added in the bacterial formulation. This compound is represented preferably by microbial growth factors such as malic acid, tryptophan, etc.

Advantageously, to the mentioned liquid nutrients and growth factors are added selected from the group comprised of: meat extract, yeast extract, peptone, NaCl, or a combination thereof. Preferably, the mentioned or each nutrient/growth factor has the following proportion weight: Meat extract 0.003-0,03%, Yeast extract 0.006%- 0,06, Peptone 0.02-0,2%, NaCl 0.02-0,2%.

Preferably, said bacterial strain with biofertilization and biocontrol capacities is selected from the group comprised of:

- Azospirϊllum 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. etc. Advantageously, the in vitro propagation includes the steps of withdrawal and sterilization of the explant, the in vitro step, proliferation, elongation, rooting, acclimatation, the mentioned treatment with the bacterial formulation carried out in at least one of said steps (in vitro, proliferation, elongation, rooting, acclimatation) .

Brief description of the drawings. Additional characteristics and the advantages of the method will be better understood by description of the following figures.

- Fig. 1 shows schematically the explant collection from the mother plant and the subsequent steps of in vitro culture (rooting and acclimatization) where, according to the invention, the method of biofertilization and bioprotection can be applied,

- Fig. 2 shows the step of inoculation of the shoot basal portion before rooting,

- Fig. 3 shows the inoculation step of a conventionally in vitro rooted plant before its transplanting to organic medium (into the vase and/or alveolus container) , - Figures 4 to 7 show the results obtained as root number, length and weight and shoot number and length and number of nodes of the plants submitted to the method, compared to control plants . - Fig. 8 shows the percentage of shoot apexes showing active growth in the plants submitted to the method and the control plants.

- Fig. 9A shows the significant effect of the A. brasilense Sp245 method on the weed development, recorded in exogenous conditions (particularly on Conyza canadiensis) compared to uninoculated plants .

- Fig. 9B shows that weeds developed on the vases containing inoculate plants had a biomass reduced compared to those developed on the control plants that were not inoculated. Examples

Materials and methods Example n. 1

Bacterial strain and cultivation

Cells of A. brasilense Sp245 "wild type" (Baldani et al . , 1986) were grown at 30 ° C in a liquid microbial medium. The cells were harvested by centrifugation (10 min at 6000 rpm, at 4 ° C) , washed in sterile water and resuspended in sterile deionized water to obtain a suspension with 10⁷ CFU ml^"1 to be used as a direct inoculum, which is possible with a formulation composed of meat extract 0.01%, yeast extract 0.02%, peptone 0.06%, NaCl 0.06%, arabic gum 1.5%, agar 0,3%.

Micropropagation

With reference to Figures from 1 to 3, tests were performed on plant material particularly explants, like vegetative apex (Fig. 2) or micropiping (Fig. 3) removed from the mother plant (Fig. 1) of clonal rootstocks were carried out: Mr. S 2/5 {Prunus cerasifera x Prunus spinosa)

, GF677 (Prunus persica x Prunus amigdalus) , MM106

(Cultivar Northern Spy x Ml) . The first two are plum and peach rootstocks respectively, the third is an apple rootstock. The explants GF677 and MM106 have been propagated in vitro, on different substrates according to clonal rootstocks type and precisely:

Example n. 2

Mr.S2/5- MS proliferation medium (Murashige and Skoog, 1962) supplemented with 1 mg/L of Thiamin, 100 mg/L Myoinositol, 30 g/L of Sucrose, 0.6 mg/L of BAP, 0.2 mg/L gibberellic acid (GA3) , 0.06 mg/L indolbutirric acid

(IBA), pH 5.3, and 6 grams per litre of Agar. Then, explants 2 and 3 were transferred to an elongation substrate 6 MS in the presence of 0.2 mg/L of BAP and 200 mg/L of vegetable coal before being transferred to a rooting substrate 7.

The explants of clone Mr. S 2/5, at the end of the elongation step were inoculated at the basal level 2a with A. brasilense Sp245, by dipping plants in the formulate 10 that was located in the container 11, as shown in Fig. 2, and transferred to a standard rooting MS substrate 20, located inside a tube 15, or in a bigger glass vase 25, in order to promote the process of rhizogenesis (Fig. 4A-4C) . Substrate 20 used during the rooting step is an MS medium supplemented with 0.6 mg/L of IBA. Each treatment was^' replicated fifteen times. The glass tubes 15 (20 x 160 mm) , each containing a single seedlings, were arranged in a random configuration and incubated at 24 ± 1 ° C with a light/dark 16/8 - h performed by white and cold lamps (60 μ mol m-2 s-1) . Tab.l Rooting step

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

2 MS + IBA (0.6 mg/1 ) + 10⁶ cells/explant Example n . 3

GF677- DKW proliferation medium (Driver & Kuniyuki, 1984) supplemented with 1 mg/L of Thiamin, 100 mg/L

Mioinositolo, 30 g/L of Sucrose, 2.0 mg/L of BA, pH 5.3, 5 g/L of Agar and 4 grams per litre of Carrageenan. Then, explants were transferred to an elongation DKW substrate 6

(Fig. 1) in the presence of 0.6 mg/L . of BA before being moved to a rooting substrate 7.

The explants of clone GF677, at the end of the elongation step were inoculated at basal level with A. brasilense Sp245, by dipping in the formulate 10 that was located in the container 11 as shown in Fig. 2, and transferred to a standard rooting DKW substrate 20 to facilitate the process of rhizogenesis . Substrate 20 used during the rooting step is a DKW medium supplemented with 0.8 mg/L IBA 5 mg/L of Spermine and Spermidine polyamides.

Tab. 2 Rooting step

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

2 DKW + IBA (0.8 πtg/1 ) + 10^s cells / removal

Example 3

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

The explants of clone MM106, a the end of the elongation step were inoculated at basal level 2a with A. brasilense Sp245, by dipping in the formulate 10 that was located in - li the container 11 as shown in Fig. 2, and transferred to a standard rooting DKW substrate 20 to facilitate the process of rhizogenesis . Substrate 20 used in the rooting step was supplemented with 0.6 mg/L of IBA.

Tab.3 rooting step

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

2 DKW + IBA (0.6 mg/1 ) + 10^δ cells / explant

Acclimatization step (post vitro) Example n. 4

Plants of 20 days 101, rooted in vitro (Fig. 1) were inoculated with A. brasilense Sp245 (10⁶ cells/plant) , by dipping in the formulatelO that was (located in the container 11) (Fig. 3), and transplanted in vivo in alveolate pots 105 (a plant for each alveolus) , or in potsl04 filled with organic substrate 110 (TKSl Flora Gard Peat, 34% organic carbon, 0.2% organic nitrogen, 60% organic material, pH 5-6) . During the first weeks, the seedlings were kept in climate cells, to slowly reduce the relative humidity. The cells were gradually opened to allow easier acclimatization of plants to the environmental conditions at a temperature of 24 ± 2 ° C with a light/dark 16/8-h, supplied by white and cold lamps (45 ± 5 μ mol m-2 s-1). On alternate days vessels were irrigated saturating with tap water. The plants that were not inoculated were regarded as a control (C) . Each treatment was repeated at least twenty times and containers were arranged in a randomized way. The number of roots, their length and fresh weight, the number of nodes, the number, length and weight of fresh shoots were assessed on 50 days old plants (see Figure) . Finally the plants were transferred to growth conditions (18-10 ± 2°C, 16 h luce/8h dark) and regularly irrigated before to reassess the above values (number roots, etc.) in 80 days old plants.

Plant bioprotection Example n.5

The biocontrol activity of A. brasilense Sp245 was evaluated on 50-day-old Mr . S 2/5 plants infected by the phytopatogenic fungus Rhizoctonia spp.

Plants, rooted in 20 days, were trasferred on organic substrate, naturally infected with Rhizoctonia spp. and were inoculated with A. brasilense Sp245 (10^δ cell/explants) . After three days, the bacterial inoculum was repeated at the same concentration. As a control, non- inoculated plants were used. The growth conditions were those previously described in the acclimatation step. Thirty days after inoculum the number of survived plants was recorded.

Jn vitro fungal biocontrol assay Example n.6

Biocontrol test was carried out using Rhizoctonia spp. directly isolated from soil naturally infected. Bacterial suspensions were inoculated on PDA (Potato Dextrose Agar) plates and incubated at 28°C for 24h. After incubation plugs of fugal mycelium (5 mm in diameter) were placed on the plates and were incubated at 28⁰C for an additional period of 7 days. At the end of the experiment the inhibition of fungal mycelium was recorded by measuring the diameter of fungal growth. As a control, diameter of fungal mycelium was recorded from 7-day-old PDA plates inoculated with the fungus and without bacterial inoculum.

Effect of Sp245 on rhizospheric fungal community Example n.7 DGGE analysis (Denaturing Gradient Gel Electrophoresis) of fungal community (Oros-Sichler et al., 2006) was performed on soil samples collected from rhizospheric soil naturally infected by Rhizoctonia spp . at the end of the acclimatation step of 80-day-old plants.

RESULTS

Jii vitro rooting

At the end of the rooting step (20 days after bacterial inoculation) Mr. S 2/5 explants propagated on MS standard medium and inoculated with A. brasilense Sp245 (marked in the figures as "Sp245") showed a higher number of roots (Fig. 4B) and an increase in root length (Fig. 4C) with respect to non-inoculated control treatment (marked in the figures as "C") . In comparison, the percentage of rooted explant was higher in inoculated treatment than that of control non-inoculated.

In vivo acclimatation

Twenty-day-old rooted plants of Mr. S 2/5, GF677, MM106 were inoculated with A. brasilense and transferred on organic substrate in vivo conditions (post vitro) . The analysis performed 30 and 60 days after inoculation on plants 50-day-old and 80-day-old, respectively, showed:

1) significant differences between inoculated and non-inoculated explants in terms of root length, root weight, node number, stem length and stem weight (see Figures from 4 to 7);

2) the percentage of the apical meristems in active growth was higher in the inoculated plants than in the control ones, in which they seem to remain dormant (Fig. 8) .

Plant bioprotection The biocontrol ability of A. brasilense Sp245 was also evaluated in in vivo conditions on Mr. S 2/5 plants infected by Rhizoctiona spp. during the acclimatation step. The bacterial inoculation was significantly active in protecting plants against Rhizoctonia spp. naturally present in soil. The survival of inoculated plants was 100% with respect to 0% of the control non-inoculated. The analysis of fungal community based on DNA extracted from rhizospheric soil showed that some dominant fungal populations are present in the control non-inoculated but disappears in the inoculated treatment.

Biocontrol of weeds

After the acclimatation step, plants of Mr. S 2/5 inoculated and non-inoculated with A. brasilense were transferred in field. After 5 months, the control plants showed a number of weedy species (in particular, Conyza canadiensis) higher than plants which were inoculated with the bacterium (Fig. 9A) . Moreover, the weeds grown in the pots of inoculated plants had a lower biomass as compared to the weeds grown in the pots of control plants (Fig. 9B) .

Claims

1. Method for biofertilization and bioprotection of vegetal material, particularly for application of in vitro propagation, protected crops, seedbeds, hydroponic crops, plant nursery techniques, characterized in that it comprises at least one step of contact between a portion of plant material competent for vegetative and/or seed propagation and a bacterial formulation containing Azospirillum brasilense Sp245.

2. Method for biofertilization and bioprotection of vegetal material, according to claim 1, wherein said above-mentioned vegetative propagation and/or by-seed propagation is realized through the use of standard root/growth substrates including organic substrates, agar substrates and liquid substrates (hydroponic crops) along with said bacterial formulation.

3. Method according to claim 1, wherein said in vitro propagation includes the steps of proliferation, extension, rooting and acclimatation, wherein said step of contact with said bacterial formulation is effected in at least one of said steps.

4. Method, according to claim 1, wherein between the mentioned applications a micropropagation is provided of plant species which appear to be recalcitrant to in vitro growth conditions for determined reasons (presence of endophytes, intrinsic genetic factors, presence of viruses, viroids and phytoplasmas) .

5. Bacterial formulation for biofertilization and bioprotection of vegetal material characterized in that it comprises:

- a culture of a bacterial strain with properties of biofertilization, bioprotection and bio-suppression of weedy species;

- a liquid, particularly sterile water and/or mineral isotonic solution, with added nutrients and growth factors; - a liquid, particularly sterile water and/or mineral isotonic solution;

- an additive which function as a thickener and/or an adhesive agent; said formulation including said bacterial culture, said liquid, and said additive, said formulation being adapted to be put in touch with seeds and/or portions of plants, in particular for applications of micropropagation, hydroponic crops, protected crops, seedbeds and plant nursery techniques.

6. Bacterial formulation, according to claim 5, wherein said bacterial culture with properties of biofertilization and bioprotection is selected from the group comprised of:

Azospirillum brasilense, Bacillus subtilis, Pseudomonas

Streptomyces spp., Pseudomonas spp._A Bacillus spp., Azotobacter spp. ^ Rhizobium spp. , Bradyrhizobium spp. _r Actinomyces spp., Paenibacillus spp., ecc.

7. Bacterial formulation, according to claim 5, wherein said bacterial culture with properties of biofertilization and bioprotection is Azospirillum brasilense Sp 245.

8. Bacterial formulation, according to claim 5, wherein said additive is selected from the group comprised of: agarose and analogues, cellulose and other natural fibers, pectins, gelling polysaccharides, gelling agents in general.

9. Bacterial formulation, according to claim 8, wherein said additive is a food thickening agent.

10. Bacterial formulation, according to claim 8, wherein said additive is selected from the group comprised of: arabic gum, tragacanth gum, agar, sodium alginate.

11. Bacterial formulation, according to claim 12, wherein said or each additive has the following proportional weight : Arabic gum 1,2-2%; tragacanth gum 0,2-2%; Agar 0, 1 - 0,5%; sodium alginate 0,2-2%.

12. Bacterial formulation, according to claim 5, including the addition of a compound apt to improve the bacterial growth.

13. Bacterial formulation, according to claim 12, wherein said compound that is adapted to improve the bacterial growth is selected from the group comprised of: malic acid, triptophan, growth factors, microbial growth modulators.

14. Bacterial formulation, according to claim 5, wherein said liguid is added with nutrients and growth factors is selected from the group comprised of: meat extract, yeast extract, peptone, NaCl, or a combination of them.

15. Bacterial formulation, according to claim 14, wherein said or each nutrient/growth factor has the following proportional weight: meat extract 0.003 - 0,03%; yeast extract 0.006 - 0,06%; peptone 0.02 - 0,2%; NaCl 0.02 - 0,2%.