Use of <i>Penicillium brasilianum </i>for stimulating plant growth

Abstract

The present invention concerns the use of an inoculum of at least one strain of Penicillium brasilianum for improving plant growth conditions. It also concerns a method of increasing the availability of phosphorus and/or micronutrients for plant uptake from a plant growth substrate, said method comprising applying to the plant growth substrate an effective amount of an inoculum of Penicillium brasilianum.

Claims

1. A method comprising improving plant growth conditions by applying an effective amount of an inoculum of at least one strain of Penicillium brasilianum (i) to the plant growth substrate, (ii) in the plant to be grown, (iii) on the plant to be grown, or any combination of (i), (ii) and (iii), wherein the at least one strain of Penicillium brasilianum is selected from the group consisting of the Penicillium brasilianum strain 064 deposited under the Budapest Treaty with the Mycothèque de l'Université Catholique de Louvain (MUCL, Belgian Coordinated Collections of Microorganisms, Université catholique de Louvain, Croix du Sud 2, 1348 Louvain-la-Neuve, Belgium) on 11 Jul. 2013 under Accession number MUCL 54519, the Penicillium brasilianum strain 065 deposited under the Budapest Treaty with the Mycothèque de l'Université Catholique de Louvain (MUCL, Belgian Coordinated Collections of Microorganisms, Université catholique de Louvain, Croix du Sud 2, 1348 Louvain-la-Neuve, Belgium) on 11 Jul. 2013 under Accession number MUCL 54520, and the Penicillium brasilianum strain 0849 deposited under the Budapest Treaty with the Centralbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, Netherlands) on 27 Oct. 2016 under Accession number CBS 141988, and mixtures thereof.

2. The method according to claim 1, wherein the inoculum is a mixture of at least two strains of Penicillium brasilianum.

3. The method according to claim 1, wherein the inoculum is used in combination with at least one inoculum of Penicillium radicum, Penicillium biliae, Penicillium expansum, and Penicillium spinulosum.

4. A method comprising enhancing plant growth by applying an effective amount of an inoculum of at least one strain of Penicillium brasilianum (i) to the plant growth substrate, (ii) in the plant to be grown, (iii) on the plant to be grown, or any combination of (i), (ii) and (iii), wherein the at least one strain of Penicillium brasilianum is selected from the group consisting of the Penicillium brasilianum strain 064 deposited under the Budapest Treaty with the Mycothèque de l'Université Catholique de Louvain (MUCL, Belgian Coordinated Collections of Microorganisms, Université catholique de Louvain, Croix du Sud 2, 1348 Louvain-la-Neuve, Belgium) on 11 Jul. 2013 under Accession number MUCL 54519, the Penicillium brasilianum strain 065 deposited under the Budapest Treaty with the Mycothèque de l'Université Catholique de Louvain (MUCL, Belgian Coordinated Collections of Microorganisms, Université catholique de Louvain, Croix du Sud 2, 1348 Louvain-la-Neuve, Belgium) on 11 Jul. 2013 under Accession number MUCL 54520, and the Penicillium brasilianum strain 0849 deposited under the Budapest Treaty with the Centralbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, Netherlands) on 27 Oct. 2016 under Accession number CBS 141988, and mixtures thereof.

5. The method according to claim 4, wherein said applying to the plant growth substrate the inoculum of at least one strain of Penicillium brasilianum comprises introducing the inoculum as a seed coating.

6. The method according to claim 4, further comprising adding a source of phosphorus to the plant growth substrate.

7. The method according to claim 4, wherein the plant is a crop plant.

8. The method according to claim 7, wherein the plant is a cereal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1-3 show histograms displaying the pH level of the medium supernatant in the phosphate solubilization assay of the example with the 62 tested fungi strains.

(2) FIG. 1: pH obtained with the following strains: Penicillium brevicompactum O853, Trichoderma sp. T41, Trichoderma sp. T42, Trichoderma hamatum Th3, Trichoderma sp. T43, Trichoderma atroviride Ta1, Trichoderma atroviride Ta2, Penicillium brasilianum O64, Trichoderma sp. T44, Penicillium griseofulvum/dipodomyicola O851, Penicillium brasilianum O849, Penicillium brasilianum O65, Trichoderma koningiopsis O153, Trichoderma koningiopsis O81, Trichoderma koningiopsis O103, Penicillium expansum O854, Penicillium radicum O238, Talaromyces purpurogenus/flavus O850, Talaromyces purpurogenus/flavus O848, Penicillium cordubense/polonicum O852, and no strain.

(3) FIG. 2: pH obtained with the following strains: Trichoderma sp. T21, Trichoderma sp. T22, Trichoderma sp. T23, Trichoderma sp. T24, Trichoderma sp. T25, Trichoderma sp. T26, Trichoderma sp. T27, Trichoderma sp. T28, Trichoderma sp. T29, Trichoderma sp. T30, Trichoderma sp. T31, Trichoderma sp. T32, Trichoderma sp. T33, Trichoderma sp. T34, Trichoderma hamatum Th1, Trichoderma sp. T35, Trichoderma hamatum Th2, Trichoderma sp. T36, Trichoderma sp. T37, Trichoderma sp. T38, Trichoderma sp. T39, Trichoderma sp. T40, Penicillium brevicompactum O853, and no strain.

(4) FIG. 3: pH obtained with the following strains: Trichoderma sp. T1, Trichoderma sp. T2, Trichoderma sp. T3, Trichoderma sp. T4, Trichoderma sp. T5, Trichoderma sp. T6, Trichoderma sp. T7, Trichoderma sp. T8, Trichoderma sp. T9, Trichoderma sp. T10, Trichoderma sp. T11, Trichoderma sp. T12, Trichoderma sp. T13, Trichoderma sp. T14, Trichoderma sp. T15, Trichoderma sp. T16, Trichoderma sp. T17, Trichoderma sp. T18, Trichoderma sp. T19, Trichoderma sp. T20, Penicillium brevicompactum O853, and no strain.

(5) FIG. 4 shows box plots displaying the OD.sub.600 nm value of the medium supernatant in the phosphate solubilization assay of the example with the tested Penicillium strains. Box plots obtained with the following strains: Penicillium radicum O238, Penicillium brasilianum O64, Penicillium brasilianum O65, Talaromyces purpurogenus/flavus O848, Penicillium brasilianum O849, Talaromyces purpurogenus/flavus O850, Penicillium griseofulvum/dipodomyicola O851, Penicillium cordubense/polonicum O852 and Penicillium expansum O854.

EXAMPLES

Example 1

(6) This example shows that Penicillium brasilianum strains are capable of solubilizing phosphorus and are therefore useful to increase availability of phosphorus for plant uptake.

(7) Material and Methods

(8) Biological Material

(9) The screening was performed on 62 strains which were of the Penicillium or Trichoderma morphotype including: Penicillium radicum CBS 100489 used as positive control, Penicillium expansum used as positive control, Penicillium brasilianum strains O64, O65 and O849, one Penicillium brevicompactum strain, one strain which was identified as belonging either to the species Penicillium cordubense or to the species Penicillium polonicum (designated as Penicillium cordubense/polonicum below), one strain which was identified as belonging either to the species Penicillium griseofulum or to the species Penicillium dipodomyicola (designated as Penicillium griseofulum/dipomyicola below), two strains which were identified as belonging either to the species Talaromyces (sexual state of Penicillium) purpurogenus or to the species Talaromyces flavus (designated as Talaromyces purpurogenus/flavus below), and 52 Trichoderma strains.
Culture Media

(10) Two culture media were used in the present study: PDA AES (AEB 152052): potato infusion (5 g/l), dextrose (20 g/l), agar (17 g/l). This medium was used for pre-cultures of strains, viability assays and CFU counting. NBRIP-BPB (National Botanical Research Institute's phosphate+colored pH indicator Bromophenol Blue BPB): glucose (10 g/l), Ca.sub.3PO.sub.4 (5 g/l), MgCl.sub.2 (5 g/l), MgSO.sub.4 (2.5 g/), KCl (0.2 g/l), (NH.sub.4).sub.2SO.sub.4 (0.1 g/l), BPB (0.025 g/l), adjusted to a pH 7.0.
“NBRIP-BPB, OD.sub.600 nm Decrease” Assay

(11) This protocol is adapted from the protocol described in Mehta et al. (2001) Current Microbiology 43:51-56.

(12) The spores of the tested strains were used either from stocks stored at −80° C. in 12.5% glycerol with a minimal concentration of spores of 3×10.sup.7 spores/ml or from precultures obtained as followed.

(13) The strains were cultured in PDA medium at 25° C. and 90% humidity in Petri dishes until obtaining a good level of sporulation (conventionally 7 to 14 days for the strains screened herein). Water with 0.2% Tween was added to each Petri dish and the surface of the dish was gently scratched in order to detach spores. The spore suspension was recovered and filtered through a 40-100 μm filter in order to remove mycelium debris. The number of spores was counted in order to known the spore concentration.

(14) The spores were used to inoculate NBRIP-BPB medium in a flask at a final concentration of 1×10.sup.6 spores/ml. Simultaneously, a small amount of spores was inoculated in PDA medium in order to confirm the satisfactory growth of the strains.

(15) The flasks were cultured at 25° C. under an agitation of 180 rpm during 6 days.

(16) A sample of each flask was collected and clarified by centrifugation at 8000 rpm during 20 min. The culture supernatants were recovered and studied by spectrophotometry: the optical density at 600 nm (OD.sub.600 nm) was measured in triplicates. The OD values were analyzed by ANOVA and the results of the analysis were displayed in the form of dot plots, since the strain was identified, by the ANOVA analysis, as being the most important variability factor.

(17) “pH-pH Decrease” Assay

(18) The pH of the medium supernatant obtained above was measured.

(19) Results

(20) The inventors isolated several hundreds of new fungic strains from different sources (wheat dead plant tissue from Bresse in France or wheat plant surface from Bourgogne in France). Among these new strains, they screened 62 strains of the Penicillium or Trichoderma morphotype in order to identify new strains capable of acidifying the culture medium by secreting organic acids and thereby capable of solubilizing phosphorus from soil.

(21) This screening was performed using two methods, one adapted from the method disclosed in Mehta et al. (2001) Current Microbiology 43:51-56 and the other consisting in measuring the final pH of the culture supernatants compared to a non-inoculated control.

(22) Two strains were used as a positive control: the Penicillium radicum strain CBS 100489 disclosed in Whitelaw et al. (1999) Soil Biology Biochem 31:655-665 and the Penicillium expansum strain.

(23) The results of the assay “pH, pH decrease”, displayed on FIGS. 1-3, show that, among the 62 tested strains, only three strains show a pH decrease higher than the positive control strain Penicillium radicum and of the Penicillium expansum strain. It is the case of the Penicillium cordubense/polonicum O852 and of the Talaromyces (sexual state of Penicillium) purpurogenus/flavus strains O848 and O850. However, since P. polonicum and T. purpurogenus/flavus are pathogenic species, the inventors considered that they were not the best candidate suitable for plants or soil application, which display a higher pH decrease than the positive control strains.

(24) However, a limited number of other strains showed a decrease of pH similar to that obtained with the Penicillium radicum strain. It is the case of the Penicillium brasilianum strains O64, O65 and O849, of the Penicillium griseofulvum/dipodomyicola strain O851 (which is known as producing a dangerous mycotoxin and is thus not suitable for plants or soil application), and of the Trichoderma koningiopsis strains O81, O103 and O153 (which are strains from a Trichoderma species known to solubilize phosphorus as confirmed by Barrera et al (2014) Poster “Strains of Trichoderma sp. and their Capacity to Mobilise Phosphorus”), which are thus also usable as biofertilizers.

(25) All the other tested strains, including strains from other Penicillium species than Penicillium brasilianum, such as Penicillium brevicompactum, have a very weak pH decrease compared to the positive control, and are thus not capable to solubilize phosphorus efficiently.

(26) The results of the assay “NBRIP-BPB, OD.sub.600 nm decrease” and of the ANOVA analysis, displayed in Table 1 below and on FIGS. 4-5, show a great diversity in the capacity of the tested strains to secrete organic acids and emphasize the fact that Penicillium brasilianum species are at least as efficient (or even more efficient) in solubilizing phosphorus as the positive controls.

(27) TABLE-US-00001 TABLE 1 Results of the ANOVA analysis Y(num.) X(cat.) p-value FStat S2Btwn S2Wthn dfBtwn dfWthn n DO strain 3.11E−51 137.179453 34.119223 2.03497881 11 90 102 DO role 2.47E−15 48.1537594 17.8278926 18.3263092 2 99 102 DO series 3.73E−03 3.48200725 6.51757562 29.6366262 6 95 102 DO date 3.73E−03 3.48200725 6.51757562 29.6366262 6 95 102

(28) Indeed, the Penicillium brasilianum strains O64 and O65 and the Penicillium cordubense/polonicum strain O852 show a decrease of OD.sub.600 nm stronger than the positive control strains Penicillium radicum and Penicillium expansum.

(29) Additionally, the Penicillium brasilianum strain O849 shows a decrease of OD.sub.600 nm similar to that obtained with the Penicillium radicum strain, while the other tested strains, including strains from other Penicillium species than Penicillium brasilianum, such as Penicillium brevicompactum, have a decrease of OD.sub.600 nm weaker than the positive control, and are thus not capable to solubilize phosphorus efficiently in the present experimental conditions.

(30) Accordingly, in this example, the inventors identified one Penicillium species which is particularly interesting for use for solubilizing phosphorous in the soil.

(31) This is the case of Penicillium brasilianum for which all the tested strains showed a better or similar capacity to solubilize phosphorus than the known Penicillium radicum strain.

(32) It should be noted that not all Penicillium species have such capacities since some Penicillium species such as Penicillium brevicompactum was unable to solubilize phosphorus in the present experimental conditions.

Example 2

(33) This example shows that Penicillium brasilianum strains are capable of improving exportation of phosphorus from the soil to the foliar parts of ray-grass in culture.

(34) Materials and Methods

(35) Biological Material

(36) The assay was performed with 4 strains which were of the Penicillium and Trichoderma morphotype including: Penicillium radicum CBS 100489 used as positive control (O238), and Penicillium brasilianum strains O64, O65 and O849.

(37) The plant used for the assay is ray-grass (Lolium perenne), more particularly English ray-grass (Maisons des Gazons, mixture of 3 varieties: 30% Amadeus/40% Greenway/30% Advent).

(38) Tested Conditions

(39) 7 different conditions were tested: Non inoculated deficient dirt as control, Dirt with tricalcium phosphate at a dose equivalent to 100 ppm of P.sub.2O.sub.5, as negative control, Dirt with tricalcium phosphate at a dose equivalent to 100 ppm of P.sub.2O.sub.5, inoculated with Penicillium radicum CBS 100489 (O238) at 10.sup.5 cfu/g of dry dirt, as positive control, Dirt with tricalcium phosphate at a dose equivalent to 100 ppm of P.sub.2O.sub.5, inoculated with Penicillium brasilianum strain O64 at 10.sup.5 cfu/g of dry dirt, Dirt with tricalcium phosphate at a dose equivalent to 100 ppm of P.sub.2O.sub.5, inoculated with Penicillium brasilianum strain O65 at 10.sup.5 cfu/g of dry dirt, Dirt with tricalcium phosphate at a dose equivalent to 100 ppm of P.sub.2O.sub.5, inoculated with Penicillium brasilianum strain O849 at 10.sup.5 cfu/g of dry dirt, Dirt with monocalcium phosphate at a dose equivalent to 100 ppm of P.sub.2O.sub.5, as positive control.
Experimental Device

(40) A specific container was used for the assay. This container comprises 3 parts which enable: Controlling independently the germination conditions, Keeping humidity and aeration of the substrate at levels which are favorable for plants germination and growth, Assaying products in mixture with several types of support (sand, dirt . . . ).
The pots comprise 3 overlapped boxes: A box without any bottom (upper part) where the seedling is carried out on 300 g of sand, at 800 mg of ray-grass by pot; A box with a perforated bottom (intermediate part) enabling strands of tissues with a sufficient capillarity to keep the compartment at the desired humidity to pass through. This bow comprises 300 g of medium (dirt+product to be tested); A box with a bottom (lower part) in which water or nutritive solution is provided.

(41) Efficacy of fungus strains to improve bioavailability of phosphorus for the plant is determined by comparing the bioavailability of phosphorus of a natural phosphate (tricalcium phosphate or phytate) with or without fungus in the medium.

(42) A mineral highly soluble phosphate fertilizer, namely pure monocalcic phosphate, was added to the assay condition “Dirt+mineral P” and phytases were added to the pot (10-40 U/pot) for the assay condition “Dirt+organic P” in order to validate the system.

(43) A contact period of 2 weeks between the substrate and the phosphate products was observed. It enables solubilization of phosphorus of the product and its integration in the pool of available phosphorus. After two weeks of contact, the upper pots, which comprise 10-day seedlings, are transferred on the intermediate pots.

(44) A mineral fertilization deficient in phosphorus was brought to each pot of each condition test. It provides to ray-grass non-limitative amounts of N, K.sub.2O, MgO, CaO and oligo elements.

(45) The assays were performed in controlled conditions with a “day” temperature of 25° C. and a “night” temperature of 19° C. The photoperiod was constituted of 16 hours of lighting and 8 hours of darkness. Humidity of the substrate was kept at 100% of its retention capacity. 3 independent tests were conducted with 3 repetitions for each condition tested.

(46) Measurements

(47) One cut of ray-grass was performed 12 weeks after seedling.

(48) The phosphorus content of the dry biomass was determined after drying at 75° C. These values enable calculating the amount of phosphorus exported to the upper parts of the plant, for each pot of the assay.

(49) —Foliar Biomass (WEIDRY):

(50) The measurement of the foliar biomass by pot (dry matter) provides information on plant matter production according to the different tested doses compared to the control “Dirt” and the control “Dirt+tricalcium P”.

(51) —Phosphorus Content in the Upper Parts of the Plant (CONP):

(52) Quantification of the phosphorus content in the upper parts of ray-grass provides information on the availability of phosphorus of the experimental system according to the different tested doses compared to the control “Dirt” and the control “Dirt+tricalcium P”.

(53) —Exported Foliar Phosphorus (PEXPORT):

(54) Calculation of phosphorus exported from the experimental system to the upper parts of ray-grass enables comparing the different tested strains globally compared to the control “Dirt” and the control “Dirt+tricalcium P”.
PEXPORT=WEIDRY×CONP
Statistical Analysis

(55) All the data were interpreted by ANOVA.

(56) ANOVA validation is based on data homogeneity and the respect of the Normal law, in other words the repartition of the data according to a Gauss curve.

(57) To check these hypotheses, different tests were used:

(58) The Skewness test enables measuring the curve symmetry.

(59) The Kurtosis test enables determining the curve kurtosis.

(60) The Barlett test enables checking the equality of the standard deviations between products.

(61) The results of the treatments are indicated by a letter in the results tables. Treatments without any letter in common are considered as significatively different with a confidence level of 95%.

(62) Results

(63) Behavior of the Controls

(64) The phosphorus source (P.sub.2O.sub.5) of the assay was validated by the coherent behaviors of the positive and negative controls.

(65) Compared to the control “Dirt”, monocalcium phosphate is assimilable by the plants, whereas tricalcium phosphate is not assimilable.

(66) Thus, the provision of tricalcium phosphate does not significantly modify the growth and nutrition in phosphorus of the plants, whereas the positive control monocalcium phosphate shows a significant exportation of phosphorus to the upper parts.

(67) TABLE-US-00002 TABLE 2 Foliar biomass (WEIDRY). phosphorus content (CONP) and exported phosphorus (PEXPORT) of the negative and positive controls at 12 weeks (cut 3) after seedling. Rating Type WEIDRY CONP PEXPORT Rating Unit g % mg Sample Size. Unit 1 plot 1 plot 1 plot Description Treatment name CUT 3 CUT 3 CUT 3 DIRT 1.254 a 1.014 a 1.262 a (100.00%) (100.00%) (100.00%) DIRT + tricalcium P 1.174 a 1.066 a 1.234 a  (93.62%) (105.04%)  (97.76%) DIRT + monocalcium P 1.776 b 1.401 b 2.462 b (141.58%) (138.12%) (195.12%) Means followed by same letter or symbol do not significantly differ (P = .05. Dunnett's vs. Control) Mean comparisons performed only when AOV Treatment P(F) is significant at mean comparison OSL

(68) In view of the results of the analysis of the controls, the assay for identifying fungus strains enabling improving assimilation of soil phosphorus is coherent concerning the phosphorus source used.

(69) Selectivity

(70) No symptom of cytotoxicity was detected on the culture during the assay. No notable visual effect was observed on foliar biomass between the different treatments.

(71) Results of the Cut 3 Months after Seedling

(72) As shown in Table 3, as expected, the results of the positive control “Dirt+monocalcium phosphorus” show a statistically significant increase of phosphorus at the foliar level after 3 months of culture.

(73) None of the fungus strain has a statistically significant effect on the phosphorus exportation in the present experimental conditions compared to the positive control “Dirt+monocalcium phosphorus”. However, it can be observed from Table 3 that the Penicillium brasilianum strain O849 shows a notable improvement of 9-10% in phosphorus exportation compared to the control “Dirt+tricalcium phosphorus”, 3 months after seedling.

(74) Accordingly, in the present experimental conditions, this strain has a long term effect, mainly targeting foliar biomass increase.

(75) Furthermore, the Penicillium brasilianum strain O65 showed an improvement of about 4% in phosphorus exportation compared to the control “Dirt+tricalcium phosphorus”, after 3 months of culture, improvement which is similar to the one observed with the positive control Penicillium radicum CBS 100489.

(76) Finally, the Penicillium brasilianum strain O64 induced a 2% increase in foliar biomass, after 3 months of culture, compared to the control “ ”Dirt+tricalcium phosphorus.

(77) TABLE-US-00003 TABLE 3 Foliar biomass (WEIDRY), phosphorus content (CONP) and exported phosphorus (PEXPORT) 12 weeks after seedling. Rating Type WEIDRY WEIDRY CONP CONP PEXPORT PEXPORT Rating Unit g g % % mg mg Sample Size, Unit 1 plot 1 plot 1 plot 1 plot 1 plot 1 plot Description Treatment Name CUT 3 CUT 3 CUT 3 CUT 3 CUT 3 CUT 3 DIRT  .sup. 1.254 1.254.sup.  1.014.sup.  1.014.sup.   .sup. 1.262 1.262.sup.  (100.00%) (100.00%)  .sup.  (100.00%) DIRT + tricalcium P   .sup. 1.174 b 1.174 a 1.066 b 1.066 a   .sup. 1.234 b 1.234 a (100.00%) (100.00%)  .sup.  (100.00%) DIRT + tricalcium P + 0238 10E5   .sup. 1.227 b 1.227 a 1.051 b 1.051 a   .sup. 1.274 b 1.274 a (104.52%) (98.64%)  (103.25%) DIRT + tricalcium P + 0849 10E5   .sup. 1.308 b 1.308 a 1.038 b 1.038 a   .sup. 1.357 b 1.357 a (111.37%) (97.39%)  (109.98%) DIRT + tricalcium P + 064 10E5   .sup. 1.195 b 1.195a  1.028 b 1.028 a   .sup. 1.223 b 1.223 a (101.75%) (96.45%)   (99.16%) DIRT + tricalcium P + 065 10E5   .sup. 1.234 b 1.234 a 1.051 b 1.051 a   .sup. 1.282 b 1.282 a (105.07%) (98.64%)  (103.91%) DIRT + monocalcium P  .sup. 1.776 a 1.776.sup.  1.401 a 1.401.sup.   .sup. 2.462 a 2.462.sup.  (151.23%) (131.49%)  .sup.  (199.59%) Means followed by same letter or symbol do not significantly differ (P = .05, Student-Newman-Keuls) Mean comparisons performed only when AOV Treatment P(F) is significant at mean comparison OSL.

(78) In conclusion, the inventors demonstrated that, in a specific experimental design, the Penicillium brasilianum strains O849 and O65 had the specific advantage of improving phosphorus exportation in the upper parts of the plant, in particular of ray-grass, of respectively about 10 and about 4%, after 3 months of culture, while the Penicillium radicum strain CBS 100489, known to solubilize phosphate, only induced an about 3% improvement.