Pseudomonas strains and consortia thereof for use in protection against plant diseases

Abstract

The present invention relates Pseudomonas strains and consortia thereof that are useful in protecting plants against microbial plant diseases caused by pathogens such as Ralstonia, Clavibacter, Erwinia, Curtobacterium, Fusarium, Phytophthora and Helminthosporium. The Pseudomonas strains were selected on the basis of their antagonistic abilities against plant pathogens such as production of antimicrobial compounds, direct inhibition of growth of plant pathogens, competition of carbon or nitrogen sources and endophytic features such as anaerobic growth on nitrate as electron acceptor and growth on arabinose as carbon source. The invention further relates to compositions comprising the strains or consortia of the invention, preferably lyophilized compositions, and to methods wherein they are used in protecting a wide variety of plants against a wide variety of microbial plant diseases.

Claims

1. A consortium the consortium comprising at least 2, 3, 4, 5, 6, 7, or 8 strains selected from the strains 17, 55, Pr, Br, 27, 80, 20, 24, 11 and 29.

2. The consortium according to claim 1, wherein the consortium is comprised in one or more compositions, each composition comprising one or more of the strains of the consortium and an agriculturally acceptable carrier.

3. The consortium according to claim 2, wherein at least one of the compositions is a lyophilized composition.

4. The consortium according to claim 2, wherein each strain of the consortium is present at a concentration ranging from about 110.sup.2 to about 510.sup.11 CFU per gram.

5. The consortium according to claim 4, wherein each strain of the consortium is present at a concentration that is higher than about 110.sup.2 CFU per gram.

6. The consortium according to claim 1, wherein the consortium comprises at least 2, 3, 4, 5 or 6 strains selected from the strains 17, 55, Pr, Br, 11 and 29.

7. The consortium according to claim 6, wherein the consortium comprises the strains 17, 55, Pr, Br, 11 and 29.

8. The consortium according to claim 7, wherein the consortium consists of the strains 17, 55, Pr, Br, 11 and 29.

9. A method of protecting plants against a plant pathogen comprising applying to plants, plant tubers, plant seeds, plant roots or soil surrounding plants, plant tubers, plant seeds, plant roots or plant cuttings, a consortium according to claim 1, under conditions effective to protect said plants or the plants produced from said plant cuttings, tubers or seeds against the plant pathogen.

10. The method according to claim 9, wherein the plant pathogen is a selected from the Group consisting of Ralstonia, Clavibacter, Xanthomonas, Erwinia, Curtobacterium, Fusarium, Rhizoctonia, Verticillium, Pythium, Botrytis, Phytophthora and Helminthosporium.

11. The method according to claim 9, wherein the consortium is used to treat the plant by topical application, to treat soil around the plant's roots or wherein the consortium is applied to seed of the plant to be protected.

12. The consortium according to claim 6, wherein the consortium comprises at least 2 or 3 strains selected from the strains 17, 55, Pr and Br.

Description

DESCRIPTION OF THE FIGURE

(1) FIGURE.

(2) Plating of co-cultures of 10 Pseudomonas consortia with Ralstonia (mix of two strains, one strain (LMG2291 being race 3, biovar 2 and one strain RS03 being race 1) as described in the Examples. After 4 days of co-culture 4 ul per incubation was spotted to a cetrimide plate (Pseudomonas selective, the two plates on top) and a violet plate (the two plates at the bottom) selective for Ralstonia (SMSA+TTC). The plates were incubated for 2 days at 28 C. before they were photographed. The left-hand plates present from left to right, respectively, the consortia A to E, the right-hand plates present from left to right, respectively, the consortia F to E. The columns on the plates are from top to bottom, respectively, dilutions of 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4 and 10.sup.5.

Examples

(3) 1. Selection of Pseudomonas Strains for Use in Protection Against Plant Diseases

(4) We obtained some 90 microbial strains from various sources of which we assumed they would belong to the group of (fluorescent) Pseudomonads as the strains were isolated on Pseudomonas-selective cetrimide plates (Tritium microbiology, Eindhoven) to demonstrate the ability to produce fluorescent compounds at room temperature (21 C.) or at 4 C.

(5) The strains were screened using a set of fluorescent Pseudomonad-specific PCR primers as described by Kim et al. (2013, J. Agric. Chem. Environ. Vol 2, No 1. pp 8-15). Strains positively identified as Pseudomonads were further subjected to 16S ribosomal gene-sequencing in order to identify the isolates at species level. Strains identified as P. aeruginosa were eliminated as potential human pathogens.

(6) The remaining Pseudomonas strains were subjected to screening for their ability to use carbon and nitrogen sources as required for their ability to for compete on substrate level with the plant pathogen Ralstonia solaneacearum as published by Wang et al. (2015, Plant pathol. J. 14:38-34).). The strains were grown on OSG medium with the individual carbon and nitrogen sources as described for the synthetic tomato exudates as described by Wei et al (Nature communications DOI:10.1038/ncomms9413).

(7) Next, the ability to use inorganic nitrogen sources was tested. Specifically inorganic nitrogen sources often used as fertilizer, such as nitrate, nitrite, ammonia and urea were tested for competitive utilization of these sources.

(8) After this, the ability to grow well under anaerobic conditions in the presence of nitrate as electron acceptor was tested for the top 20 candidate strains that remained after the carbon and nitrogen utilization pattern preselection. 7 out of the 20 strains demonstrated good anaerobic growth using nitrate as electron acceptor. The competition in low oxygen condition is important as it is e.g. known that Ralstonia is able to utilize nitrate as electron acceptor under anaerobic or low oxygen conditions such as root zones and xyleme (Dalsing et al. (2015) mBIO 6(2):e02471-14. Doi:10.1128/mBio.02471-14). The genetic potential of the strains to produce antimicrobial compounds was tested using PCR based screening for the presence of genes necessary for the production of HCN (hcnBC genes), 2-4 DAPG (phlD gene), phenazines (phzCD genes) and pyrrolnitrin (prnD gene) and pyoluterorin (PltC). PCR primers used for detecting these genes were as described by Kim et al. (2013, supra). The presence of genes necessary for the production of lipopeptides (CLP genes) was tested using a set of primers (218_clpS/clpA_FW and 219 clpS/clpA_RV) based on a piece of DNA occurring frequently in CLP producing pseudomonads as described by Song et al. (2015, BMC Microbiology 15:29).

(9) The antimicrobial activity of the strains was further tested on agar plates detection of halo's in a field of a plant pathogen, around the inoculated Pseudomonas strains. Target plant pathogenic organisms in the screening were: Ralstonia solaneacerum, Erwinia carotovora ssp carotovora, Curtobacterium flaccumfaciens and Clavibacter michiganensis ssp michiganensis, Xanthomonas hortorum pv. Pelargonii, Fusarium oxysporum and Phytophthora infestans Botrytis cinerea, Pythium ultimum, Verticillium dahlia, Rhizoctonia solani.

(10) Based on this information a set of 12 strains was selected that demonstrated growth inhibition towards at least one of the target organisms. These 10 strains were further tested able to utilize arabinose, a property strongly correlated with endophytic lifestyle of Pseudomonads (Arch. Microbiol. (2013) 195:9-17 and Appl. Soil Ecol. 42:141-149).

(11) Table 1 present an over view of these strains and their various relevant properties. The most relevant strains were deposited on 8 Apr. 2016 (#11 and #29 were deposited on Jul. 12, 2016) at the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands), under the regulations of the Budapest Treaty. Table 2 presents the accession no's of the deposited strains.

(12) TABLE-US-00001 TABLE 1 Ralstonia Ralstonia inhibition inhibition Strain Strain RS03: LMG2291 RS03 Race 1 Race 3, 24 48 0.1 Biovar 2 0.1 Curtobacterium TSB 48 0.1TSB TSB inhibition ID Species Phe DAPG pltC prnD CLP HCN TSB ATJ TSB ATJ TSB TSB ATJ 24 P. palleroniana + + PR P. protegens + + + + + ++ ++ + + ++++ + + BR P. brassicacearum + + + + + +++ ++ + 27 P. putida + +/ +/ + + 20 P. putida + + ++ +/ 55 P. reinekei + + +/ +/ 80 P. putida + + + + + + +++ +/ +/ 17 P. moraviensis + + + +/ ++ ++ +/ 86 P. putida + + +/ + + + ++ +/ +/ 11 P. moraviensis + + + 29 Pseudomonas sp. + + ++ Anaerobic Growth X. hortorum Erwinia using pv carotocoa ssp NO.sub.3 as Growth Clavibacter pellargoni carotovora electron Phytophthora Fusarium on ID inhib-tion inhibition DSM30168 acceptor inhibition inhibitbion arabinose 24 ++ PR ++++ + + ++ BR ++++ + + + ++ ++ + 27 ++ ++ + 20 ++ + 55 + + 80 + + ++ + 17 + 86 + + + + 11 + + + + + 29 + + + ATJ: Artificial tomato exudate medium based on OSG medium containing 48 carbon sources as described in carbon and nitrogen sources as described for the synthetic tomato exudates as described by Wei et al (Nature communications DOI: 10.1038/ncomms9413)

(13) TABLE-US-00002 TABLE 2 Accession no. Strain CBS 141219 Pseudomonas moraviensis # 17 CBS 141220 Pseudomonas putida # 20 CBS 141221 Pseudomonas palleroniana # 24 CBS 141222 Pseudomonas putida # 27 CBS 141223 Pseudomonas reinekei # 55 CBS 141224 Pseudomonas putida # 80 CBS 141225 Pseudomonas putida # 86 CBS 141226 Pseudomonas brassicacearum BR01 CBS 141227 Pseudomonas protegens PR01 CBS 141646 Pseudomonas moraviensis #11 CBS 141647 Pseudomonas sp. #29* *CBS 141647 has been determined to be Pseudomonas extremaustralis strain #29
2. Consortia of Strains for Biocontrol Against Various Plant Pathogens

(14) Using the criteria of Example 1 above, a number of consortia of Pseudomonas strains were assembled to further enhance the inhibition of growth of the plant pathogenic bacteria and fungi. The consortia were tested for antagonistic activity against the plant pathogens Ralstonia solanacearum race 1 and race 3, a Curtobacterium flaccumfaciens, Clavibacter michigansis ssp michiganensis and a second Ralstonia solaneacearum race 1 (occurring in roses in the Netherlands).

(15) As a laboratory screen to demonstrate elimination of Ralstonia solanacearum we grew two Ralstonia strains for 2 days on 0.1 TSB (Tryptic soy broth) medium at 28 C., one isolated from plant material; Ralstonia solancearum RS03 (Race 1) obtained from Dummen Group, and one Ralstonia solancearum LMG2291 (Race 3). The cultures were diluted and then inoculated to a chemically defined (OSG) medium at pH 5.8 using an artificial tomato juice comprising the 48 carbon sources from Wang et al. (2015, Plant pathology journal 14, 38-34) at a concentration of 0.025 gr/L each and applying 1.5 gr/L NaNO.sub.3 as nitrogen source using a MOPS buffer at 10 gr/L to control pH between 6 and 7 upon cultivation to prevent too drastic pH increase upon growing on nitrate. OSG medium is described by Ornston and Stanier (1966, J. Biol. Chem. 241:3776-86).

(16) 8 Pseudomonads were grown individually on 0.1 TSB medium for 24 hrs at 28 C. and inoculated in different combinations. For co-culture of Pseudomonas consortia with Ralstonia (mix of two strains) both were inoculated initially at a level of 10.sup.7 cells/ml in the above described chemically defined artificial tomato juice medium, and incubated at 20-22 C. (room temperature). Then after each day 4 ul per incubation was spotted to a cetrimide plate (Pseudomonas selective) and a violet plate selective for Ralstonia (SMSA+TTC). The plates were incubated for 2 days at 28 C. before they were assessed. Table 3 presents the Pseudomonas consortia that were studied. FIG. 1 presents the data obtained after 4 days of co-cultivation. This gave the following ranking of the Pseudomonas consortia with respect to their agonistic activity against Ralstonia:

(17) 1. Consortium E

(18) 2. Consortium H

(19) 3. Consortium I

(20) 4. Consortium F

(21) 5. Consortium B

(22) 6. Consortium A

(23) 7. Consortium J

(24) 8. Consortium C

(25) 9. Consortium D

(26) 10. Consortium G

(27) The Pseudomonas moraviensis strain number 17 appears to be one of the better strains for suppression of the two Ralstonia's tested as it was present in each of the consortia in the top 3 of the ranking. After 4 days consortia E, H and I had completely eliminated 10.sup.7 Ralstonia cells/ml to less than 10 per 4 ul, as no colony appears anymore even in the 10.sup.1 dilutions.

(28) Also the possibly negative effect of the various Pseudomonas strains towards each other was tested by inoculating each the various Pseudomonas strains in separate holes in an agar layer of 0,1 TSB medium solidified with agar and then spreading one of the strains as a lawn and detecting growth inhibition in the lawn around the various holes. The following inhibitions were found: strain 80 inhibits growth of strain 27 and of strain Br; strain Br inhibits growth of strain 24; and strain 86 inhibits growth of strain Br.

(29) TABLE-US-00003 TABLE 3 Consortium Strains A Pr, Br, 17, 20, 27, 55, 86, 71 B Pr, 17, 55, 71 C Br, 20, 27, 86 D Pr, 71 E 55,17 F Br, 20 G 27, 86 H Pr, Br, 17, 27 I Pr, Br, 17, 27, 55 J Pr, Br, 17, 27, 55, 86
3. Production of Pseudomonads in Economic Amounts

(30) The organisms were produced in a fermentation process applying a high cell density glucose limited fed batch fermentation process at 28 C., pH 6.8 on a suitable chemically defined medium (see WO1998037179 A2 and OSG medium) with a duration of 24-40 hours producing a very high cell count of 5*10.sup.10-3*10.sup.12 CFU/ml that does not need centrifugation as a concentration step. Alternatively, strains of the invention can be fermented at lower cell count, after which the cells of the strain can be concentrated by microfiltration and or centrifugation. After a batch phase of 10-12 hours consuming the 20 gr/L glucose after inoculation, a concentrated glucose (>50% glucose) feed was started and was increased to maximum flow that allowed good aeration of the culture while maintaining glucose <1 g/L. Ammonium was kept sufficiently high to enable reproduction of the cells by adding ammonium salts to the batch medium and use concentrated ammonium (>12.5% NH.sub.3) as a titrant. The inoculum was a 1.5% (vol/vol) of full grown TSB medium culture prepared at 28 C for 24 hours or 48 hrs dependant on the growth rate of the strain.

(31) After the fermentation of approximately 24-48 hours, the cells were freeze dried by adding skim milk powder at 20-30% w/w % adding cryoprotectants like sucrose, glycerol, sorbitol, or any other cryoprotectant as described by Hubalek (Cryobiology 46 (2003) 205-229) and frozen to <80 C and freeze dried to >90% dry matter in 24-48 hours. The germ count of viable cells reached in this way was >10.sup.12 CFU/gr allowing economic exploitation of the strains produced despite the fact that we have only 1% survival yet, so we need an improvement here as well, and should take a position now to be further proven the coming year.

(32) When the cells in the fermentation broth with Pseudomonas putida #27 with a cell count of 3.4*10.sup.11/gr (5 gram broth as such) were mixed with 20 gram skim milk solution (20% w/w), 5 gram sucrose solution (20% w/w) and 2 gram glycerol solution (20% w/w), 6.6 gr powder at 97% dry matter was obtained at a viable cell count of 2.1*10.sup.11/gr giving a survival rate of 68%. Whereas the individual compounds skim milk, and sucrose only gave yields of appr. 1% survival and mixture of skim milk and sucrose or sorbitol gave 20-34% survival rate (see table 2).

(33) Next to this, we have done experiments to freeze the cells rapidly in liquid nitrogen (196 C.). We need to see what cryoprotectant we need to add, as so far this preferred way of operation was not successful yet with skim milk and sucrose or skim milk and sorbitol. When we added glycerol in the same mixture as above, a survival rate of 34% was found enabling also freezing the cells directly in liquid nitrogen (196 C.) allowing a fast and easy operation reducing freezing time in the freeze dryer and allowing faster drying due to the nice small frozen cell particles with a diameter of 2-4 mm giving a large sublimation surface compared to of a tray with a 1-2 cm thick ice cake.

(34) In addition, spray-drying of cells was tested. 234 gr of fermentation broth of strain Pseudomonas putida 86 with a viable cell count of 8.5*10.sup.10/ml was mixed a premix comprising of 1013 gr tap water 187.5 gr skim milk, 47 gr sucrose and 18.8 gr glycerol and was spray dried with an inlet temperature of 80 or 85 C. and outlet temperature of 40 or 45 C. on Buchi 290 spray dryer. Table 4 shows the composition before and after spray-drying. The viable cell count of the powder of 93% dry matter was 7.3*10.sup.10/gr. A survival rate of 72% was obtained.

(35) TABLE-US-00004 TABLE 4 Compositions before and after spray-drying g. as such % of total g. dry matter % of dry matter broth 234 15.6 17.6 6.7 water 1013 67.5 0.0 0.0 Skim milk 187.5 12.5 178.1 68.3 sucrose 47 3.1 47.0 18.0 glycerol 18.8 1.3 18.7 7.2 1500 261.4

(36) In addition, the effect of glycerol concentration was testes in a similar experiment as described above. Apparently the survival rate is clearly dependant on the glycerol concentration as can be seen in Table 5. When glycerol was dosed at 29.6 gr/L in the mix described in Table 4, spray drying was not feasible and a gum was obtained sticking in the spray-drying chamber. The optimum therefore might be around 10 or 15 g/L or 20 or 25 g/L, but in any case as high as possible, but as low as necessary to keep harvesting a dry product.

(37) TABLE-US-00005 TABLE 5 Effect of glycerol concentration on survival percentage. Conc. Survival Inlet Outlet glycerol percent- temp temp Polyol in mix age Strain (C.) (C.) addition (g/L) (%) Pseudomonas #86 80 45 No n.a. 1.5 Pseudomonas #86 80 45 Glycerol 0.74 3.5 Pseudomonas #86 80 45 Glycerol 2.96 14.7 Pseudomonas #86 80 45 Glycerol 7.4 23.1 Pseudomonas #86 80 45 Glycerol 14.8 47.4 Pseudomonas #86 80 45 Glycerol 29.6 n.a.* *too sticky, impossible to dry

(38) The spray drying was also tested at more elevated temperatures using the mix of Table 4 again. In Table 6 we see that a very good process can also be achieved at 100 C. inlet and 45 C. outlet with 58.8% survival rate using glycerol at 14.8 g/L. When 120 C. was used we could also still get survival and even at 160 C. inlet temperature some survival was observed demonstrating the very good protective properties of glycerol.

(39) TABLE-US-00006 TABLE 6 Effect of temperature on survival Inlet Outlet Polyol g/L % Strain temp (C.) temp (C.) added polyol survival Pseudomonas #86 80 45 No n.a. 8.3 Pseudomonas #86 80 45 Glycerol 14.8 77.1 Pseudomonas #86 80 45 Glycerol 14.8 36.4 Pseudomonas #86 90 45 Glycerol 14.8 42.4 Pseudomonas #86 100 45 Glycerol 14.8 58.8 Pseudomonas #86 120 45 Glycerol 14.8 16.9 Pseudomonas #86 120 60 Glycerol 14.8 24.1 Pseudomonas #86 160 80 Glycerol 14.8 5.2

(40) In a similar set up as described above, we tested alternative polyols and alternative organisms. As can be observed in Table 7, improved drying of other organisms than Pseudomonads was very limited in our mix with skimmed milk and sucrose. Improved drying was observed for spores of the fungus Metarhizium anisopliae and Saccharomyces cerevisiae, although the effect was rather limited/less pronounced. However, alternative polyols seemed to work even better; ethyleneglycol being the most preferred one, and 1,3 propanediol slightly less preferred. 1,2 propanediol did not improve drying of Pseudomonads.

(41) TABLE-US-00007 TABLE 7 Effect of type of polyol and type of organism on survival during spray drying Inlet Outlet Strain temp (C.) temp (C.) Polyol added g/L polyol % survival Pseudomonas protegens 80 45 No n.a. 0.3 Pseudomonas protegens 80 45 Glycerol 14.8 27.7 Pseudomonas #86 80 45 No n.a. 8.3 Pseudomonas #86 80 45 Glycerol 14.8 77.1 Sacharomyces cerevisiae S288C 120 60 No n.a. 1.5 Sacharomyces cerevisiae S288C 120 60 Glycerol 22.2 5.1 Sacharomyces cerevisiae S288C 160 80 No n.a. 0.0 Sacharomyces cerevisiae S288C 160 80 Glycerol 22.2 0.0 Pseudomonas #86 80 45 Glycerol 14.8 43.0 Pseudomonas #86 80 45 Ethyleneglycol 14.8 49.0 Pseudomonas #86 80 45 Ethyleneglycol 9.98 33.4 Pseudomonas #86 80 45 1,3-propanediol 14.8 23.2 Metarhizium anisopliae Met52 120 60 No n.a. 68.9 Metarhizium anisopliae Met52 120 60 Glycerol 14.8 84.2 Metarhizium anisopliae Met52 140 70 No n.a. 0.9 Metarhizium anisopliae Met52 140 70 Glycerol 14.8 3.2 Lindnera jandidii 120 60 No n.a. 12.2 Lindnera jandidii 120 60 Glycerol 22.2 0.1 Lindnera jandidii 120 60 Ethyleneglycol 14.8 4.2 Lindnera jandidii 120 60 1,2-propanediol 14.8 13.3 Pseudomonas #86 80 45 No n.a. 3.2 Pseudomonas #86 80 45 Glycerol 14.8 11.6 Pseudomonas #86 80 45 1,2-propanediol 14.8 3.8 Lactobacillus casei 80 45 No n.a. 103.4 Lactobacillus casei 80 45 Glycerol 14.8 94.1 Lactobacillus casei 80 45 Ethyleneglycol 14.8 104.0 Lactobacillus casei 80 45 1,2-propanediol 14.8 53.1 Lactobacillus casei 80 45 1,3-propanediol 14.8 65.8 Azospirillum brasilense 80 45 No 14.8 <0.1 Azospirillum brasilense 80 45 Glycerol 14.8 <0.1 Azospirillum brasilense 80 45 Ethyleneglycol 14.8 <0.1 Azospirillum brasilense 80 45 1,2-propanediol 14.8 <0.1 Azospirillum brasilense 80 45 1,3-propanediol 14.8 <0.1

(42) The protective mix of Table 4 was studied also for a wide range of pseudomonas species in a freeze drying experiment. Clearly the mix is very well suited to enable very high viable counts in the powders obtained.

(43) TABLE-US-00008 TABLE 8 Effect of cryoprotectant mix as described in table 4 on Germ count after freeze drying for various Pseudomonads. Strain Viable count (cfu/gr) BR01 4*10.sup.10 PR01 8*10.sup.10 #17 8*10.sup.10 #20 1*10.sup.11 #24 9*10.sup.10 #27 2*10.sup.11 #55 4*10.sup.10 #80 1*10.sup.11 #86 9*10.sup.10

(44) After 3 months of storage at room temperature appr. 70% viability retained upon storage under vacuum and upon storage cool (<6 C.) and vacuum (12 mbar) or N.sub.2 stored loss of viability could not be observed.

(45) After 235 >70% survival was seen for most Pseudomonas species when stored at (<6 C.). Storage under nitrogen did not make much difference. Pseudomonas brassicacerum Br01 was slightly lower with 45% viability after 235 days. This show that the formulation whit skim milk, sucrose and glycerol at dry matter >90% and stored vacuum (12 mbar) at <6 C. is a good and stable formulation and storage condition for Pseudomonads.

(46) 4. The Pseudomonad Consortia were Also Tested In Vivo on Flower Cuttings.

(47) 4.1 Material and Methods

(48) Cuttings of Pelargonium peltatum variety Dancing Idols Candy were rooted in water-soaked rockwool cubes (44 cm) for 4 weeks in a greenhouse compartment at 18-22 C. under 16 hr light and a relative humidity of 755%. After rooting, the rockwool cubes with rooted cuttings were transferred to a climate chamber and kept throughout the experiments at 20 C. under 18 hr light (8000 lux) and a relative humidity of >80%. Immediately after transfer to the climate chamber the rooted cuttings were inoculated with predefined mixtures of antagonistic bacteria and placed in a plastic tent. Four days later Ralstonia solanacearum race 1 was applied using 10 ml of suspended bacteria in culture medium at an OD of 0.1.

(49) For each combination of a mixture of antagonistic bacteria and R. solanacearum, 12 cubes with rooted cuttings were placed on a water-soaked cloth in a tray to avoid contaminations between treatments. To ensure all rockwool cubes stayed moist during the course of the experiments, water with nutritional salts (EC 1.0) typically used in hydroponic culture was applied twice a week.

(50) Six predefined mixtures of bacteria were tested for their antagonistic activities towards R. solanacearum: #32: Pseudomonas protegens PR01, P. brassicacearum BRO1 and P. moraviensis #33: P. protegens PR01, P. brassicacearum BRO1, P. moraviensis, P. putida #27 and P. putida #80 #34: P. moraviensis #17 #35: P. protegens PR01, P. brassicacearum BRO1, P. moraviensis #17, P. putida #27 and P. reinekei #55 #36: Control with only carrier materials as used during the drying process containing skimmed milk, glycerol and sucrose #37: P. moraviensis #17 and P. reinekei #55 #38: P. protegens PR01, P. brassicacearum BRO1, P. moraviensis #17, P. putida #27 and P. reinekei #55, P. putida #80, P. palleroniana #24, P. putida #20

(51) Per treatment, 5 ml of the predefined bacterial mixtures at a total concentration of 510.sup.8 cells/ml equally divided over the Pseudomonas species in the mixture was added to each rockwool cube with a rooted cutting. Mixture #36 served as a control.

(52) 4.2 Results

(53) Leaf symptom development induced by R. solanacearum on the inoculated plants was monitored at 3-4 days intervals during a period of three weeks post inoculation. All plants in all treatments with R. solanacearum showed leaf symptoms at the end of the experiment. None of the plants treated with the antagonistic mixtures only showed leaf symptoms.

(54) First symptoms of R. solanacearum started to develop 8 days post inoculation on plants without an antagonist pretreatment (mixture #36). On plants pretreated with antagonist mixtures #34 and #32, leaf symptoms appeared after 8 and 11 days, respectively. Up till 14 days after R. solanacearum inoculations plants pretreated with antagonist mixtures #37 and #38 remained symptomless. Thus demonstrating a marked effect on delaying symptom development of antagonist mixtures #37 and #38.

(55) Symptom development on leafs and roots, 14 and 21 days after R. solanacearum inoculation is presented in Table 9. Plants not infected with R. solanacearum did not develop any symptoms on roots or leafs.

(56) Symptom development was scored on a scale from 0 (no symptoms) to 3 (severe symptoms) on leafs and roots.

(57) TABLE-US-00009 TABLE 9 Symptom development 14 days post R. solanacearum infection on P. peltatum variety Dancing Idols Candy Symptom development score Antagonist Leafs* Leafs* Roots** Roots** mixture 14 dpi 21 dpi 14 dpi 21 dpi 32 1.5 2.5 1.2 2.6 33 2.0 2.5 2.1 2.8 34 2.0 2.5 1.9 2.6 35 1.5 2.3 1.6 2.7 36 = Mock 1.8 3.0 2.5 3.0 37 1.0 2.1 0.8 2.6 38 0 1.3 0.1 1.9 *The score represents 0 = No symptoms, 1 = Bottom leafs yellow/brown, 2 = Early systemic symptoms, leafs from the bottom of the plant up to half of the stem yellow/brown/wilting 3 = Severe systemic symptoms, at least half of the leafs are yellow, brown and/or wilting. **The score represents 0 = No Symptoms, 1 = Brown root tips, 2 = Brown root tips and a maximum of 50% of the roots is completely brown 3 = At least 50% of the roots are completely brown
5. The Pseudomonad Consortia are Also Tested In Vivo on Tomato and on Potato

(58) Consortia a) Br01, Pr01, 11, 17, 27, 29, 55 and 86; b) Br01 and 11; c) Pr01 and 11; and d) Br01, Pr01 and 11 are also tested and found to be effective in protecting tomato against Clavibacter and in protecting potato against Erwinia and/or Phytophtora.