PLANT INOCULATION METHOD

Abstract

A method for inoculating a plant with a nitrogen-fixing bacteria such as Gluconacetobacter diazotrophicus, said method comprising administering the nitrogen-fixing bacteria to a wound of a growing plant, for example to recently cut grass. Inoculation in this manner leads to enhanced growth characteristics including increased greenness of grass. Novel compositions suitable for use in the method are also described and claimed, together with kits for producing these.

Claims

1. A method for inoculating a plant with a nitrogen-fixing bacteria, said method comprising administering the nitrogen-fixing bacteria to a wound of a growing plant.

2. The method of claim 1 wherein the nitrogen-fixing bacteria is Gluconacetobacter diazotrophicus.

3. The method of claim 1 wherein the nitrogen-fixing bacteria is combined with a strain of Terribacillus.

4. The method of claim 1 wherein the wound is the result of mowing, cutting, ratooning, pruning, consumption by livestock or harvesting.

5. The method of claim 4 wherein in a preliminary step, the plant is subjected to mowing, cutting, ratooning, pruning or harvesting, and wherein the nitrogen-fixing bacteria is applied within 1-2 hours of said preliminary step.

6. (canceled)

7. The method of claim 1 wherein the nitrogen-fixing bacteria are applied to the wound of a plant in the form of a composition.

8. The method of claim 7 wherein composition comprises from 1 to 1×10.sup.7 nitrogen-fixing bacteria per millilitre.

9-16. (canceled)

17. The method of claim 1 wherein the plant is a perennial, biennial or persistent annual plant.

18. (canceled)

19. The method of claim 1 wherein the plant is cut grass.

20. An agriculturally acceptable composition comprising Gluconacetobacter diazotrophicus, and a further component selected from a polysaccharide or a surfactant or a combination thereof.

21. The composition of claim 20 wherein the composition comprises from 1-100 bacteria per millilitre.

22. The composition of claim 20 wherein the polysaccharide is an exudate gum polysaccharide.

23. The composition of claim 22 wherein the exudate gum polysaccharide is gum Arabic.

24. The composition of claim 20 wherein the surfactant is a non-ionic detergent.

25. The composition of claim 24 wherein the surfactant is 70% composed of the fatty acid oleic acid and the remainder a combination of linoleic, palmitic and stearic acids.

26. The composition of claim 20 which comprises from 0.0005 to 10% v/v surfactant.

27. The composition of claim 20 which further comprises a nutrient for said nitrogen-fixing bacteria.

28. (canceled)

29. A kit for forming an agriculturally acceptable composition, which kit comprises (i) a nitrogen fixing bacteria and (ii) a further component comprising an agriculturally acceptable surfactant, or a polysaccharide or a combination thereof.

30. (canceled)

31. The kit of according to claim 29 wherein the further component is a concentrate which may be diluted and mixed with the nitrogen-fixing bacteria to produce an agriculturally acceptable composition.

32. The method of claim 1 which is carried out in order to increase the greenness of a plant by increasing the chlorophyll level thereof.

33. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The invention will now be particularly described by way of example with reference to the accompanying diagrams in which:

[0038] FIG. 1 is a graph showing the mean dry weights (g) of un-inoculated and inoculated cut grass;

[0039] FIG. 2 is a graph showing the above ground dry weights of inoculated cut grass treated with Gd and sucrose, Tween and/or Gum Arabic or combinations thereof;

[0040] FIGS. 3A-3B illustrate an example of preparation of vegetative tea propagation, FIG. 3A illustrates the removal of the cutting; FIG. 3B is diagrammatical representation of sub-sections of each cutting taken for DNA isolation;

[0041] FIG. 4 shows an image of a gel of PCR products obtained from samples of tea plants which had been inoculated with Gd; all bands in control plants were sequenced and confirmed as non-specific binding. Sequenced bands from inoculated plants were confirmed as Gluconacetobacter diazotrophicus;

[0042] FIG. 5 is a graph showing the effects of various treatments on the biomass of cut grass;

[0043] FIG. 6 shows the results of a test to determine the effect of Gd on the number of flower heads of grass; and

[0044] FIG. 7 is a graph showing the results of treatments with various compositions on the biomass of cut grass.

[0045] However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the invention. The following descriptions of specific embodiments of the present invention are presented for purposes of illustration and description. They are not intended to be exhaustive of or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

EXAMPLE 1

Application to Cut Grass

Methodology

[0046] Culture of G. diazotrophicus:

[0047] G. diazotrophicus strain IMI 501986 (now IMI 50998) with the pRGS561 plasmid expressing GUS, were cultured on ATGUS medium, [0.8% (w/v) agar, yeast extract (2.7 g I.sup.−1), glucose (2.7 g I.sup.−1), mannitol (1.8 g I.sup.−1), MES buffer (4.4 g I.sup.−1), K.sub.2HPO.sub.4 (4.8 g I.sup.−1), and KH.sub.2PO.sub.4 (0.65 g I.sup.−1), pH 6.5] as required. Expression of the b-glucuronidase (gusA) gene was tested by plating on ATGUS medium containing X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexylammonium salt) at 50 mg I.sup.−1; the formation of dark blue colonies indicated gusA gene expression.

Inoculation Procedures:

[0048] An aqueous suspension of the G. diazotrophicus was prepared to give an optical density at 600 nm of 1.1, c. 10.sup.9 colony forming units (CFU) per milliliter. The number of CFU was determined by serial dilution, plating on ATGUS medium (with antibiotics as appropriate) and counting bacterial colonies after 4d incubation in Petri dishes (28° C., dark). The suspension was diluted to 10.sup.−4 to produce a solution containing approximately 100 bacteria per ml ready for spraying as described below.

[0049] A standard weight of 0.5 g of grass Lolium perenne variety Cassiopeia seeds were sown in seedling trays of John Innes No. 1 compost and lightly covered with compost.

[0050] The individual trays were placed in larger trays and provided with adequate water in a growth room at 21° C./15° C. day/night 16/8 h cycle for 20 days. After which the grass was cut at a height of 2 cm above soil level using scissors (clippings were removed) and the following treatments were applied using a domestic handheld mist sprayer:

Experiment 1. Treatments

[0051] Control of water+3% sucrose [0052] Gd+water+3% sucrose

Experiment 2 Treatments

[0053] Gd+water [0054] Gd+water+3% sucrose [0055] Gd+water+0.1%Tween [0056] Gd+water+0.3% Gum Arabic [0057] Gd+water+3% sucrose+0.1% Tween [0058] Gd+water+3% sucrose+0.3% Gum Arabic [0059] Gd+water+3% sucrose+0.1% Tween+0.3% Gum Arabic

Dry Weight of Germinated Seedlings

[0060] The seedlings were removed from the agar with forceps and all remaining agar washed from the roots. Each seedling was placed in a paper bag and placed in an oven 80° C. for 48 hours and then weighed.

[0061] Results from Experiments 1 and Experiment 2 are shown in FIGS. 1 and 2 respectively.

[0062] The results in FIG. 1 show a significant increase in the mean dry weight of the grass (0.09676 g for un-inoculated and 0.1276 g for inoculated graph). These dry weights were significantly different at P<0.01. Thus, inoculation in this manner clearly leads to a significant enhancement of growth.

[0063] These results shown in FIG. 2 show a significant difference (P<0.001) between Gd/S/T/GA and the next highest dry weight (Gd/T) and Gd/S/T, demonstrating a synergistic effect of the combinaton of three components. Gd and Gd/S are not significantly different at P=0.05.

[0064] EXAMPLE 2

Colonisation of Tea (Camellia sinensis) BY Gluconacetobacter diazotrophicus (Azoticus)
Vegetative Reproduction from a Stem Cutting

[0065] The standard means of vegetative propagation of tea clones is a single-leaf cutting. From larger stems comprising of approximately four to six nodes and a shoot tip, sections of stem and leaf were selected based upon health of the tissue (i.e. free of insects and diseases). The section chosen for the cutting was between red and green wood as recommended by Yamasaki et al. Soil and Crop Management, (2008) SCM-23). Recently matured shoots containing slightly reddened bark adjacent to mature leaves with actively breaking axillary buds have been found to result in the best rooting success.

[0066] From the preferred sections, a sample was selected comprising of a 3-5 cm section of stem and one healthy leaf. Each stem section was excised using a diagonal cut (1) approximately 0.5 cm above the leaf (2) and another diagonal cut below the leaf around an internode (3) avoiding pinching or bruising of the wound site (See FIG. 3A.)

[0067] The bottom of each tea stem cutting was dipped into 1% indole-butyric acid solution and placed into individual pots; the cutting planted with the stem straight of slightly slanted so that the leaf does not touch the soil. Each pot contained sand and John Innes number 1 cutting mix in a 4:1 ratio, saturated with water. To the cut top surface of each cutting either 20 μl of water, or 20 μl of Gd at 2.5×10.sup.5 cfu/ml in water was applied, and the humidity of each sample maintained by covering each pot with a plastic sheet and sprayed lightly with water.

[0068] Following 3 months growth, and in order to confirm successful colonisation of the stem cuttings with Gd, uninoculated and inoculated stem cuttings were removed from the pots. Each cutting was sub-divided into sections which were (a) the top of shoot, including inoculation site (4) in (FIG. 3B), (b) the nodal section (5) in FIG. 3B, and (C) the lower stem section including any root tissue (6) in FIG. 3B. These sections where then snap frozen in liquid nitrogen.

[0069] DNA isolation from each section of cutting (i.e. 4, 5 and 6 in FIG. 3B) was carried out using TRIzol reagent according to the manufacturer's protocol and PCR performed. The PCR reaction carried out was a two-step reaction as described by Tian et. al., (2009); the first step using GDI-25F (5′-TAGTGGCGGACGGGTGAGTAACG-3′; SEQ ID NO:1) and GDI-923R (5′-CCTTGCGGGAAACAGCCATCTC-3′; SEQ ID NO:2) which amplified an 899bp product containing the amplicon of primers GDI139F (5′TGAGTAACGCGTAGGGATCTG-3′; SEQ ID NO:3) and GDI916R (5′-GGAAACAGCCATCTCTGACTG-3′; SEQ ID NO:4), the latter designed based upon 16S rDNA sequence information available in the GenBank database. After an initial denaturation step at 95° C. for 3 minutes, the following temperature profile was executed 32 times; denaturation for 20 seconds at 95° C., annealing for 45 seconds at 55° C., and extension for 20 seconds at 72° C., with a final extension step of 5 minutes at 72° C. One microliter of this PCR product was then taken and used as template for the second step of the PCR using GDI39F and GDI916R. Modifications to the parameters in the second round included increasing the annealing temperature to 62° C. for 15 seconds, and increasing the cycle number to 39. The PCR amplification products were analysed on a 1% agarose gel stained with ethidium bromide as well as sequenced to confirm identity of product (FIG. 4.).

[0070] Interestingly, AzGd was not detected in section 4 of the tea cutting suggesting that AzGd moved basipetally from the wound site following inoculation, being detected in sections 5 and 6 respectively.

[0071] Sequencing and subsequent BLAST results provided confirmation that the bands seen in section 1 of control plants were as a result of non-specific binding of the primer sets used, with the 4 bands observed in sections 2 and 3 of inoculated tissue being identified as Gluconacetobacter diazotrophicus Pal5 (at 100% identification, 86% query cover and an E-value of 7e-04). The results suggest that AzGd although at a low copy number in the inoculated tissue did successfully colonise Camellia sinensis following inoculation of the wound site. This is possibly the first example of colonisation of a perennial plant by Gd.

EXAMPLE 3

Investigation of Effect of Treatment on Grass Biomass

[0072] Grass was grown in a plant growth chamber (Fitotron®) (23° C./15° C. at 65% humidity) in seed trays using John Innes No. 1 compost, for 2 weeks. It was then cut to a height of 8 cm and immediately sprayed with 10 ml of treatment as set out below using a domestic sprayer.

Treatments

[0073] 1. Water [0074] 2. 3% sucrose+0.1% Tween+0.3% Gum Arabic [0075] 3. Water+Gd (2.5×10.sup.5 cfu/ml) [0076] 4. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10.sup.3 cfu/ml) [0077] 5. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10.sup.4 cfu/ml) [0078] 6. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10.sup.5 cfu/ml) [0079] 7. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10.sup.6 cfu/ml) [0080] 8. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10.sup.7 cfu/ml)

[0081] The grass was returned to the Fitotron for a further 2 weeks under similar growth conditions. 5 plants, chosen at random, were cut at the soil level to form one sample and weighed. This was repeated a further five times to give six samples in total for each treatment.

[0082] These samples were dried in the oven for 48 hours and weighed.

[0083] The results are shown in FIG. 5. These results show that, provided some sucrose is present to support the growth of Gd, the biomass of the grass increased with the addition of Gd depending upon the formulation. Furthermore, the increase was dose dependent, with an optimum growth being observed at 2.5×10.sup.6 cfu/ml. Such a dose may therefore be beneficial if the grass treated is pasture grass where maximising biomass is beneficial. However, if the grass treated is amenity or turf grass, lower biomass with enhanced greenness may be beneficial in that it may improve appearance without increasing the need for further cutting or mowing. In this case, a dosage of either less than or greater than 2.5×10.sup.6 cfu/ml may be used.

EXAMPLE 4

Field Trial

[0084] A formulation comprising water+3% sucrose+0.1% Tween+0.3% gum Arabic+Gd (2.5×10.sup.5 cfu/ml) was applied to a single 1 m.sup.2 cut grass plot (established Lolium perenne turf) relative to an 1 m.sup.2 uninoculated cut grass plot treated with water only (control).

[0085] The formulation and water were applied, within 30 minutes of the grass being freshly mown, using a household mist sprayer to run-off. The control plot was protected from the treatment plot by a plastic screen. The application was made late afternoon in still air.

[0086] The 1 m.sup.2 plots were subsampled using a 20 cm squared wire quadrant by counting the number of fully extended and fully formed flowering heads.

[0087] The results from each 20 cm square within each plot was averaged and the results are shown in FIG. 6. It is clear that the Gd treatment, applied in this way, substantially impacted on flower growth.

EXAMPLE 5

Comparison of Components of Composition

[0088] The method of Example 3 was repeated using various compositions including individual components of the composition used in that experiment. Specifically, the compositions used in this experiment were as follows:

Treatments

[0089] 1. Water [0090] 2. Water+Gd (2.5×10.sup.5 cfu/ml) [0091] 3. Water+3% sucrose+0.1% Tween+0.3% Gum Arabic+Gd (2.5×10.sup.5 cfu/ml) [0092] 4. 0.3% Gum Arabic+Gd (2.5×10.sup.5 cfu/ml) [0093] 5. 3% Sucrose+Gd (2.5×10.sup.5 cfu/ml) [0094] 6. 0.1% Tween+Gd (2.5×10.sup.5 cfu/ml)

[0095] AberGlyn grass was grown for 2 weeks in John Innes No. 1 soil in a plant growth chamber (Fitotron®) at 23/15° C., 80% humidity. The grass was cut to a height of 8 cm with scissors, the cuttings removed and the grass immediately sprayed with the 10 ml treatment using a domestic sprayer. The grass was returned to plant growth chamber for a further two weeks.

[0096] Five plants were chosen at random from the tray and pooled together to make one sample and weighed. This was repeated a further five times so a total of six samples were taken per treatment. Grass was dried for 48 hours at 80° C. and then weighed.

[0097] The results are shown in FIG. 7. This experiment shows that the component used does have an effect on the growth of the grass. In this example, the surfactant gave the greatest increase in dry weight. Gum Arabic showed a marginal improvement only over the control, possibly due to the fact that the surfactant may be required to assist in the spread of the Gd on the plant and helps the liquid enter the wounds of the grass (although on this occasion, the combination did not show the expected an improvement). Again the water+Gd treatment was similar to the control so indicates that Gd needs the addition of at least some of these components to colonise the wounds.