Method for producing plant seed containing endophytic micro-organisms

Abstract

The invention discloses a method for producing plant seed containing endophytic microorganisms characterised in by the following steps: contacting a flowering plant with a preparation of endophytic microorganisms, whereby the endophytic microorganisms enter the plant via the flowers and are conveyed to seed produced by the plant; and obtaining the plant seed containing endophytic microorganisms from the plant.

Claims

1. A method for producing a bacterial colonized plant seed comprising inoculant endophytic microorganisms inside the seed, the method comprising: contacting at least one flower of a flowering plant in the course of a flowering phase of the flowering plant with a preparation comprising a population of inoculant endophytic microorganisms, wherein the preparation comprises 10.sup.6 to 10.sup.10 cfu per mL and thereby causing the inoculant endophytic microorganisms to enter the flowering plant via the at least one flower; growing the flowering plant until it forms seeds and thereby forming at least one bacterial colonized seed in which the inoculant endophytic microorganisms are established inside of the seed; and obtaining from the contacted flowering plant the bacterial colonized plant seed comprising inoculant endophytic microorganisms inside the seed.

2. The method of claim 1, wherein the inoculant endophytic microorganism is an endophytic bacterium selected from Burkholderia, Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, Herbaspirillum, Azospirillum, Acetobacter, Arthrobacter, Bacillus, Paenibacillus, Streptomyces, Pantoea, Enterobacter, and Pseudomonas.

3. The method of claim 1, wherein the inoculant endophytic microorganism is Burkholderia phytofirmans.

4. The method of claim 1, wherein contacting the at least one flower with the preparation is performed via spraying the preparation on the flower at the time of flowering.

5. The method of claim 1, wherein the preparation comprises 10.sup.8 to 10.sup.9 cfu/mL of the inoculant endophytic microorganisms.

6. The method of claim 1, wherein the bacterial colonized plant seed containing the inoculant endophytic microorganisms is stored for at least 1 month, for at least 3 months, for at least 6 months, for at least 12 months, for at least 2 years, or for at least 3 years.

7. The method of claim 1, wherein the inoculant endophytic microorganism is a recombinantly produced bacterium.

8. The method of claim 1, wherein the flowering plant is of genus Zea.

9. The method of claim 1, wherein a plant grown from the bacterial colonized plant seed comprising the inoculant endophytic microorganisms has at least one improved plant effect compared to a plant grown from an untreated seed, the plant effect selected from tolerance to drought, tolerance to metals, tolerance to disease, herbivory, growth, yield, nutrient acquisition, production of phytohormones, production of antibiotics, production of siderophores, production of pesticides, and biological nitrogen fixation.

10. The method of claim 1, wherein the preparation comprising a population of the inoculant endophytic microorganisms is administered to the flower with a spatula, a syringe, an inoculation loop or a pollen-feeding insect.

11. A bacterial colonized plant seed produced by the method of claim 1.

12. The bacterial colonized plant seed of claim 11, wherein the inoculant endophytic microorganism population is more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% of a population of endophytic microorganisms in the bacterial colonized plant seed.

13. The bacterial colonized plant seed of claim 11, wherein the inoculant endophytic microorganisms are selected from Burkholderia phytofirmans, Burkholderia phytofirmans PsJN, Pantoea sp. FD17 Paenibacillus sp. S10, Actinobacter sp. S9, Bradyrhizobium sp. NC92 and Bradyrhizobium japonicum TAL379.

14. The bacterial colonized plant seed of claim 11, wherein the flowering plant is maize and the inoculant endophytic microorganisms are Burkholderia phytofirmans, in a population density of 10.sup.2 to 10.sup.5 cfu per gram fresh weight of bacterial colonized plant seed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows cob sheath, grain and cob interior colonization of Burkholderia phytofirmans strain PsJN in maize cvs Peso and Morignon (x-axis shows CFU/g dry weight);

(3) FIG. 2A, FIG. 2B, and FIG. 2C show light microscopy images of a mature seed colonized by Burkholderia phytofirmans strain PsJN::gusA; the blue colour is due to gusA-marked bacterial cells; strain PsJN is present inside the embryo (FIG. 2A and FIG. 2B) and in radicals (FIG. 2C); PsJN starts moving from embryo to germinated parts (FIG. 2C);

(4) FIG. 3 shows Burkholderia phytofirmans strain PsJN recovery from the grain interior at different time intervals after harvesting (DAH; Days after harvesting);

(5) FIG. 4A, FIG. 4B, and FIG. 4C show the effect of Burkholderia phytofirmans strain PsJN colonized/non-colonized seeds on germination and seedling growth of maize.

(6) FIG. 5A, FIG. 5B, and FIG. 5C show the effect of Burkholderia phytofirmans strain PsJN colonized/non-colonized seeds on shoot growth of maize (30, 45, 60 days after sowing).

(7) FIG. 6 shows representative results of GUS-staining in pepper treated with PsJN::gusA110 15 days p.i. GUS activity was found in all fruit parts including seeds.

(8) FIG. 7 shows FISH analyses of pepper seeds colonized by B. phytofirmans PsJN using a general probe targeting eubacteria and a 23S rDNA probe specific for B. phytofirmans. Bacteria other than B. phytofirmans (eubmix-FITC) are indicated with a small arrow and B. phytofirmans PsJN is indicated with a longer arrow.

DETAILED DESCRIPTION

EXAMPLES

Example 1: Introducing Burkholderia phytofirmans Strain PsJN into Maize Seeds

(9) The concept of internal seed colonization with plant growth promoting microorganisms according to the present invention was tested with the endophytic bacterium Burkholderia phytofirmans stain PsJN and two plant varieties of maize. Strain PsJN was applied by spraying female flowers with a suspension of 10.sup.8-10.sup.9 cfu mL.sup.−1. At maturity, PsJN cells were detected within maize seeds at viable population densities that ranged from 10.sup.2-10.sup.5 CFU g.sup.−1 fresh weight. Strain PsJN was not recovered from plants of the seed inoculation trial. After 12 months of storage 10.sup.2 viable cells per g seeds were still recovered. Experiments were performed to determine the effects of internally colonized maize seeds on offspring plant biomass and vigor as compared to non-treated controls and external application of the same bacterial strain.

(10) Experimental Description

(11) The present invention provides seeds having beneficial microorganisms, especially bacteria, inside, enabling improved plant biomass equally over control as employing the same microorganisms (in the present case: bacteria) exogenously to seeds. A variant of the bacterium Burkholderia phytofirmans strain PsJN chromosomally tagged with the beta-glucuronidase gene (gusA, reporter gene for detection and monitoring of the strain by color formation) was used as a test strain in to maize cultivars (Peso and Morignon). For this, series of experiments were performed and the experimental setup was divided into two categories (1st and 2nd year experiments). A) Evaluation of strain PsJN colonization potential in different tissues of maize plants (particularly grains). B) Follow-up evaluation of strain PsJN colonized seed and strain PsJN inoculation (exogenously) to improve plant productivity over control.
Growth of PsJN Strain as Bacterial Inoculum

(12) The bacterial strain was grown by loop-inoculating one single colony in LB broth amended with spectinomycin (100 μg mL-1) in 100 mL flasks. The bacterial culture was incubated at 28±2° C. for 2 days at 180 rpm in a shaking incubator. The bacterial inoculum was applied in two different ways i.e. seed soaking and spraying inoculum at flowering stage. Maize seeds were surface sterilized by dipping for 5 and 3 min in 70% ethanol and NaOCl following 3 washings with sterilized water. There were three treatments, 1) seed inoculation 2) specific spraying of flowers and 3) seed inoculation combined with flower inoculation. Plants grown from seeds treated with sterile culture broth only served as control. For inoculation, seeds of two maize cultivars were dipped for 3-4 hours in bacterial inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1). Likewise, bacterial inoculum was specifically sprayed to the female flower when the crop reached flowering stage. Seeds were sown in plastic trays filled with soil and 12 days old seedlings were transferred into 50 kg soil container (2 plants in each container) under wirehouse conditions.

(13) Endophytic Colonization by PsJN Strain (Particularly Grain Colonization)

(14) The rhizosphere and endophytic colonization of root, stem and leaves by the gusA-labeled variant of B. phytofirmans strains PsJN was determined by plate counting using LB plates amended with 5-Bromo-4-chloro-3-indolyl D-D-glucuronide (X-glcA, 50 μg mL-1), IPTG (50 μg mL-1) and the antibiotic spectinomycine (100 μg mL-1). Root, stem and leaf samples were washed, surface sterilized (as described above) and used for PsJN recovery (colonization). For this, samples were crushed in 0.9% saline buffer, subjected to oscillation in a pulsifier for 30 sec and dilution series were spread on agar plates. Beta-glucuronidase positive cells appear blue on media containing X-glcA. The blue colonies were counted after 3 days of incubation at 30° C. and the original cell number per g plant tissue was calculated. Similarly, PsJN colonization was also observed from different cob parts i.e. sheath, grains and cob interior. The identity of the blue colonies was further confirmed by RFLP analysis of the 16S-23S rRNA intergenic spacer region.

(15) Follow-up experiments were performed in the 2nd year to evaluate the

(16) 1. Viability, activation and colonization ability of strain PsJN colonizing maize seeds. 2. Effect of strain PsJN colonized seed on germination and seedling vigor compared to untreated control (plastic tray assay). 3. Effect of strain PsJN colonized seed on plant biomass compared to untreated control (pot trials).

(17) Prior to the plant experiments, PsJN colonized seeds of both cultivars were tested to see whether PsJN cells are present and still alive inside. For this purpose, 20 seeds were imbibed in saline buffer for 2-3 days and subsequently crushed in 0.9% saline buffer, shaken for 45 second with a pulsifier and spread in dilutions on LB plates amended with X-glcA, IPTG and spectinomycin.

(18) Bacterial inoculum was prepared as described above and three experiments were performed with four treatments i.e. control, seed inoculation with strain PsJN (exogenously), PsJN colonized seeds (produced in 1st year by spraying), PsJN colonized seed+inoculation.

(19) For testing the germination performance, seeds (45) were surface sterilized and inoculated as described earlier, and were sown in plastic tray (diameter 30 cm) with three replicates. Data regarding time to start germination, mean germination time, time to 50% and final germination, germination index and energy, coefficient of uniform germination, and skewness were recorded of PsJN colonized over control.

(20) Two pot experiments were performed to evaluate the performance of PsJN colonized seeds concerning plant biomass production as compared to control. Surface sterilized seeds were directly sown in pots with soil (first pot trial) or alternatively sown in plastic trays, and after 10 days seedlings were transferred to 5 kg pots (2nd pot trial). All plants were harvested after 60 days and data of plant height, number of leaves per plant and root-shoot biomass were recorded. The data were subjected to analyses of variance using SPSS software package version 19 (SPSS Ink, Chicago, Ill.).

(21) Results

(22) Experiment a (1st Year): Seed Colonization by Strain PsJN

(23) The ability of strain PsJN to colonize maize cobs (cob sheath, cob interior and grains) was analyzed in plants treated by specific flower inoculation (by spraying) only or by seed inoculation (FIG. 1). Only inoculation of flowers resulted in internal colonization of seeds. Internal seed colonization by strain PsJN was observed in both cultivars and both flower inoculation treatments. PsJN cells were detected in maize seeds at viable population densities that ranged from 10.sup.2-10.sup.5 CFU g.sup.−1 fresh weight.

(24) Experiment B1 (2nd Year): Viability, Activation and Colonization Ability of Strain PsJN Colonizing Maize Seeds.

(25) PsJN colonized seeds, recovered from the first year experiment were tested to see whether PsJN cells survive inside of dormant seed and have the ability to colonize the plants emerging from the seeds what is very important as seeds may be stored for several months till planting. 10.sup.2 viable cells were detected in two months old dormant seeds (FIG. 1). Imbibing in saline buffer for 2-3 days activated the 6 months old seeds and together with the seeds beginning to germinate PsJN started to proliferate resulting in a recovery of 10.sup.4 viable cells (FIG. 4). Sprouts the emerged of 420 day old seeds were colonized by 10.sup.5 PsJN cells and the bacteria was found all over inside the sprouts (FIGS. 1 and 2).

(26) Experiment B2 (2nd Year): Effect of PsJN Colonized Seeds on Germination and Seedling Vigor as Compared to Untreated Control

(27) The data summarized in table 1 and 4 revealed that PsJN colonized seeds showed significant improved germination ability. PsJN colonized seeds of both cultivars started to germinate 36-48 hours early than the control. PsJN colonized seed showed almost 100% final germination rate and required less mean germination time as compared to the control seeds. Consequently, the colonized seeds have better germination index as compared to control.

(28) Moreover PsJN colonized seeds of both cultivars showed significantly higher maize seedling biomass as compared to untreated control seeds (Tables 2 and 5; FIGS. 3 and 4) but non-significantly higher seedling biomass as compared to seeds exogenously inoculated with PsJN.

(29) Experiment B3 (2nd Year): Effect of PsJN Colonized Seed on Plant Biomass Compared to Untreated Control (Pot Trials)

(30) The data of the pot trials (Table 3 and 6) revealed that PsJN colonized maize seeds had a positive effect on plant biomass production comparable to seeds externally coated with PsJN cells with cv Morignon being more responsive than cv Peso in both treatments (Tables 3 and 6; FIG. 5). The PsJN colonized seeds showed 38% increase in plant biomass production and a significant increase in root biomass as compared to the control. Moreover, the number of leaves per plant was higher in plants of PsJN colonized seed as compared to the control.

CONCLUSIONS

(31) Burkholderia phytofirmans PsJN can be introduced into maize seeds by spraying cells onto flowers.

(32) Seed inoculation only does not allow colonization of maize seeds of the next generation.

(33) Strain PsJN can survive inside maize seeds for at least 12 months

(34) Seed-colonizing bacterial cells are rapidly activated, proliferate and colonize emerging sprouts

(35) Seed-colonizing PsJN shows substantial plant growth promotion

(36) The present example therefore shows that the method according to the present invention enables an effective and reliable way to generate seeds with endophytes in a controlled and reproducible manner.

Example 2: Introducing B. phytofirmans PsJN and Enterobacter sp. FD17 into Wheat and Barley Seeds

(37) Experimental Description

(38) Seeds of wheat (Triticum spp. cvs Collada and Monsun) and barley (Hordeum vulgare L. cvs Victoriana and Totum) were surface sterilized by dipping for 5 and 3 min in 70% ethanol and NaOCl following 3 washings with sterilized water. Seeds were sown in plastic trays and 12 days old seedlings were transferred into 20 kg soil containers and grown under green-house conditions. The soil has been collected from an agricultural field in Tulln, Lower Austria, and sieved to remove plant material. Bacterial strains (gusA-labelled variants of B. phytofirmans PsJN and Enterobacter sp. FD17) were grown by loop inoculation in LB broth amended with spectinomycin (100 μg mL-1) in 100 mL Erlenmeyer flask. Bacterial cultures were incubated at 28±20 C for 2 days at 180 rpm in a shaking incubator. Bacterial inoculum was applied by spraying exclusively flowers. Control plants were treated with sterilized broth.

(39) Endophytic Colonization of Wheat and Barley Seeds

(40) Plants were harvested at ripening stage and seeds were collected. Seed colonization by the inoculant stains was determined by GUS-staining. Therefore, seeds were cut in two pieces and incubated in GUS-staining solution (1 mM EDTA, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 100 mM sodium phosphate, pH 7.0, 1% Triton-X-100, 0.1 mg/mL X-Gluc predissolved in 5 μL/mg N,N-dimethylformamide, 0.1% IPTG) directly after harvesting at 37° C. for 20 hours. Afterwards, samples were rinsed with 70% ethanol. The ethanol was then discarded and samples were fixed in paraformaldehyde solution (4% paraformaldehyde dissolved in PBS at 60° C. with constant stirring until clarifying of the solution) overnight at 4° C. Finally, the fixed samples were rinsed 3 times in PBS and stored in the last rinse at 4° C. until further processing. In parallel, seeds were manually crushed under sterile conditions and used for bacterial community DNA isolation employing standard procedures. The presence of the inoculant strains was confirmed by sequence analysis of the 16S-23S rRNA intergenic spacer region (IGS) of single clones and subsequent comparison with those from the inoculants strains.

(41) Results

(42) Experiment a (1st Year):

(43) Both seeds of wheat and barley were found to be internally colonized by the inoculants strains. Sequence analysis of the IGS-region confirmed the presence of Enterobacter sp. FD17 and B. phytofirmans PsJN.

(44) Conclusions

(45) Burkholderia phytofirmans PsJN and Enterobacter sp. FD17 can be introduced into barley and wheat seeds by spraying cells onto flowers.

Example 3: Introducing B. phytofirmans PsJN into Tomato and Pepper Seeds

(46) Experimental Description

(47) The colonization behavior of Burkholderia phytofirmans PsJN during transmission from flowers to seeds was studied with tomato (Solanum lycopersicum cv. Micro Tom and Matina) and pepper (Capsicum annuum cv. Feher). Presence of PsJN was investigated at 3 different time points. Detection of bacteria in the seed interior of harvested samples was conducted by GUS-staining and microscopy on the one hand and strain-specific quantitative PCR on the other hand. For detection by visual observation of staining and microscopy, the gusA-labelled variant of the strain PsJN, Burkholderia phytofirmans PsJN::gusA110, was used in parallel to the wild-strain that was detected via qPCR.

(48) The ability of PsJN to survive in the seed and proliferate with the emerging seedling was studied in a subsequent germination experiment. Hereby, the harvested seeds from the previously treated plants were sown and nursed for a certain period. Afterwards the seedlings were examined regarding their presence of PsJN by GUS-staining and quantitative PCR of PsJN-specific genes.

(49) The bacterial strains were grown by loop-inoculating one single colony in LB broth containing 0.1% of the antibiotic spectinomycin in case of B. phytofirmans PsJN::gusA110 and without antibiotics in case of the wild-type strain and incubated at 28° C. on a shaker (160 rpm) overnight. The overnight culture was transferred to 500 mL Erlenmeyer flasks containing 250 mL liquid LB medium. They were incubated on a shaker (120 rpm) at 28° C. for 2 days to allow for growth of bacteria. Subsequently, aliquots of 40 mL of the incubated medium containing the bacterial culture were filled in 50 mL plastic tubes and centrifuged at 4500 rpm and 4° C. for 10 minutes (Megafuge 40R, Heraeus, Hanau, Germany). Afterwards, the supernatant was discarded and the bacterial pellet re-suspended by vortexing in 20 mL PBS (0.2 g/L KCl, 1.44 g/L Na2HPO4 and 0.24 g/L KH2PO4, in dH2O, pH 7.4, autoclaved). The control suspension was treated accordingly. The aliquots of each bacterial suspension were then pooled in 500 mL Schott bottles. The concentration of the suspensions was measured by help of spectrophotometry (NanoDrop 1000 3.7.1., Wilmington, Del., USA) and adjusted to 108 CFU/mL.

(50) Specific inoculation of tomato and pepper flowers was conducted when the plants reached growth stage 61-63 on the BBCH scale (for tomato: first inflorescence: first flower open—third inflorescence: first flower open; for pepper: first flower open—third flower open) (FELLER et al., 1995b).

(51) The bacterial inoculants and buffer only for the control were filled in a 50 mL glass pump spray bottle previously sterilized with 70% ethanol. The plants to be inoculated were spatially separated from the others to avoid contamination by drift. One single flower or 2 to 3 immediately adjacent flowers were sprayed with 675 μL of the inoculum. A filter paper was used to shield the surrounding plant parts such as leaves and stem from drift and take up surplus inoculum to avoid dripping on the soil. The treated inflorescences/flowers were marked with a twist tie to allow for later identification.

(52) Six replicates of the inoculated plants were analyzed at 3 different developmental stages. Pepper samples were taken 3 days and 15 days after spraying as well as at full ripeness. The plant material (buds, flowers, fertilized flowers, developing fruits, immature fruits, ripe fruits and seeds) was cut with a sterile scalpel and subsequently incubated in GUS-staining solution (1 mM EDTA, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 100 mM sodium phosphate, pH 7.0, 1% Triton-X-100, 0.1 mg/mL X-Gluc predissolved in 5 μL/mg N,N-dimethylformamide, 0.1% IPTG) directly after harvesting at 37° C. for 20 hours. Afterwards, destaining was done by rinsing the samples with 70% ethanol. The ethanol was then discarded and the samples fixed in paraformaldehyde solution (4% paraformaldehyde dissolved in PBS at 60° C. with constant stirring until clarifying of the solution) overnight at 4° C. Finally, the fixed samples were rinsed 3 times in PBS and stored in the last rinse at 4° C. until further processing.

(53) Material of plants inoculated with PsJN wild-type and control samples were immediately after harvest frozen in liquid nitrogen and transferred for storage at −80° C. Afterwards, DNA was isolated using standard procedures and used as described above for Example 2.

(54) Results

(55) Experiment a (1st Year):

(56) Upon flower spraying B. phytofirmans PsJN colonized seeds and pericarp of fruits of tomato and pepper (FIG. 6). The colonization process was monitored by GUS-staining and microscopy. The cell number of strain B. phytofirmans PsJN during transmission from flowers into seeds was tested by TaqMan-quantitative PCR using primers and probe targeting a gene encoding glutamine synthetase. The amount of B. phytofirmans PsJN cells applied on one flower was roughly 108 and the cell number calculated per mg plant material during the process of colonization dropped from about 3000 cells per flowers to a few dozen cells in seeds. Results were confirmed by fluorescence in situ hybridization (FIG. 7).

CONCLUSIONS

(57) Burkholderia phytofirmans PsJN can be introduced into tomato and pepper by spraying cells onto flowers.

(58) TABLE-US-00001 TABLE 1 Comparative performance of PsJN colonized seed and PsJN inoculation (exogenously) on germination of maize cv Peso (Data are average of three replicate) Time to Coefficient of Time to 50% Mean Final Germination uniform Start Germination emergence Germination % Energy emergence Germination Treatment Germination (T50) Time (MET) (FGP) (GE) (CUE) index (GI) Skewness Control.sup.‡ 4a† 5.20 b 6.74 a  83.33 bc 72.92 ab 0.80 NS 6.45 bc 0.77 bc PsJN Inoculation.sup.‡ 3.33 ab 4.80 c 6.55 a 100 a 85.42 a 0.67 8.82 a 0.73 c Control.sup.§ 4 a 5.60 a 6.83 a  77.08 c 64.58 b 0.85 5.45 c 0.82 a PsJN Inoculation.sup.§ 3.33 ab 5.30 ab 6.73 a  89.58 b 68.75 ab 0.74 6.85 b 0.78 ab PsJN colonized seed‡ 2.33 bc 4.33 d 5.49 b 100 a 69 ab 0.77 8.75 a 0.79 ab †Values sharing similar letter(s) do not differ significantly at P < 0.05, according to Duncan's Multiple Range Test. ‡Seeds prepared by spraying PsJN inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1) .sup.‡Parent seed used for first year experiment .sup.§Offspring seed produced from first year experiment

(59) TABLE-US-00002 TABLE 2 Comparative difference of PsJN inoculated and PsJN colonized seed on biomass of maize cv Peso in plastic tray experiment (data are average of three replicate). Fresh Plant biomass (g) Dry Plant biomass (g) Plant No. of Total Total height leaves Treatment Stem Leaves Root biomass Stem Leaves Root biomass (cm) per plant Control 79.37 c†  95.70 b 37.20 b 212.27 c 3.63 c  9.65 b 1.39 b 14.67 c 93.37 b 6.58 c PsJN Inoculation 93.77 b 111.03 a 38.4 ab 244.43 b 4.22 b 10.65 ab 1.73 a 16.90 b 95.87 a 7.04 b PsJN colonized seed‡ 99.70 b 113.33 a 39.63 a 251.43 ab 4.39 b 11.17 a 1.79 a 17.35 b 97.33 a 7.20 b †Values sharing similar letter(s) do not differ significantly at P < 0.05, according to Duncan's Multiple Range Test. ‡Seeds prepared by spraying PsJN inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1)

(60) TABLE-US-00003 TABLE 3 Comparative performance of PsJN colonized seed and PsJN inoculation (exogenously) on plant biomass of maize cv Peso under potted conditions (data are average of three replicate). Pot trial I (Direct sowing) No. of Pot trial II (Nursery sowing) Plant height leaves Shoot Root Shoot Root Treatment (cm) per plant biomass biomass biomass biomass Control  96.42 c† 6.98 c 5.32 c 0.82 c 1.29 c 0.28 c  PsJN Inoculation 108.01 ab 9.04 ab 8.80 ab 1.42 a 2.37 b 0.423 ab PsJN colonized seed‡ 104.62 b 8.42 b 7.17 b 1.12 b 2.16 b 0.358 b †Values sharing similar letter(s) do not differ significantly at P < 0.05, according to Duncan's Multiple Range Test. ‡Seeds prepared by spraying PsJN inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1)

(61) TABLE-US-00004 TABLE 4 Comparative performance of PsJN colonized seed and PsJN inoculation (exogenously) on germination of maize cv Morignon (data are average of three replicate). Time to Coefficient of Time to 50% Mean Final Germination uniform Start Germination emergence Germination % Energy emergence Germination Treatment Germination (T50) Time (MET) (FGP) (GE) (CUE) index (GI) Skewness Control.sup.‡ 4.33 a† 4.98 a 6.72 a  85.42 bc 79.17 ab 0.81 NS 6.66 b 0.74 NS PsJN Inoculation.sup.‡ 3.67 a-c 4.96 a 6.65 a  95.83 ab 89.58 a 0.78 8.25 a 0.75 Control.sup.§ 4 ab 5.02 a 6.65 a  79.17 c 75 b 0.74 6.65 b 0.76 PsJN Inoculation.sup.§ 3.33 bc 5.07 a 6.59 a  91.67 ab 75 b 0.65 7.88 ab 0.77 PsJN colonized seed.sup.‡ 3 c 4.10 b 5.69 b 100 a 83.33 ab 0.69 9.06 a 0.72 †Values sharing similar letter(s) do not differ significantly at P < 0.05, according to Duncan's Multiple Range Test. ‡Seeds prepared by spraying PsJN inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1) .sup.‡Parent seed used for first year experiment .sup.§Offspring seed produced from first year experiment

(62) TABLE-US-00005 TABLE 5 Comparative performance of PsJN colonized seed and PsJN inoculation (exogenously) on seedling biomass of maize cv Morignon in plastic tray experiment (data are average of three replicate). Fresh Plant biomass (g) Dry Plant biomass (g) Plant No. of Total Total height leaves Treatment Stem Leaves Root biomass Stem Leaves Root biomass (cm) per plant Control 81.07 c†  97.70 b 38.43 b 215.93 c 3.83 c  9.67 c 1.76 b 15.26 c 94.76NS 6.53 c PsJN Inoculation 92.67 b 104.80 a 42.40 a 239.23 b 4.64 b 10.57 b 2.34 a 17.67 b 95.00 6.87 b PsJN colonized 92.90 b 105.07 a 41.93 a 240.13 b 4.66 b 11.25 ab 2.35 a 18.24 ab 95.02 6.84 b seed‡ †Values sharing similar letter(s) do not differ significantly at P < 0.05, according to Duncan's Multiple Range Test. ‡Seeds prepared by spraying PsJN inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1)

(63) TABLE-US-00006 TABLE 6 Comparative performance of PsJN colonized seed vs PsJN inoculation (exogenously) on plant biomass of maize cv Morignon under potted conditions (data are average of three replicate). Pot trial I (Direct sowing) No. of Pot trial II (Nursery sowing) Plant height leaves Shoot Root Shoot Root Treatment (cm) per plant biomass biomass biomass biomass Control 101.42 c† 7.98 c 6.36 c 1.12 c 3.29 c 0.41 c PsJN Inoculation 110.67 b 9.47 b 8.17 b 1.42 b 4.37 b 0.623 ab PsJN colonized seed‡ 113.01 ab 9.83 b 8.80 b 1.56 ab 4.26 b 0.558 b †Values sharing similar letter(s) do not differ significantly at P < 0.05, according to Duncan's Multiple Range Test. ‡Seeds prepared by spraying PsJN inoculum (10.sup.8-10.sup.9 cfu mL.sup.−1)