CA1308566C - Method and composition for fertilizing soils to increase the amountof available phosphorus - Google Patents

Abstract

Abstract:
The invention provides a method and composition for fertilizing soils to increase the amounts of phosphorus available for uptake by plants. The invention involves introducing an inoculum of the fungus Penicillium bilaji into (or onto) the soil in combination with a manufactured phosphorus fertilizer. The fungus has the effect of increasing the amount of phosphorus available for uptake from the manufactured fertilizer so that less of the fertilizer is required. The fungus is applied to the soil separately from the fertilizer but can be added in prox-imity to, or in combination with, plant seeds. The inven-tion can be used to increase the phosphorus uptake of plants which will increase growth rates and yields of plants, especially crop plants grown on nutrient-deficient soils.

Description

~3~3566 Method and com~ition for fertilizing soils to increase the ____ _ ______ ________ _ ___ _ _ ____ _ ___ ____ _ amount of available ~hosE~ s ___ _ __ _ ____ ___ _ This invention relates to methods and compositions for increasing the amounts of phosphorus available for uptake by plants from soils.
In order to maintain healthy growth, plants must extract a variety of elements from the soil in which they grow. These elements include phosphorus and the so-called micronutrients (e.g. copper, iron and zinc), but many soils are deficient in such elements or they contain them only in forms which cannot be readily taken up by plants. It is generally believed that essential elements cannot be readily taken up by plants unless they are present in a dissolved form in the soil.
To conteract such deficiencies, manufactured fertilizers are generally added to the soil. Such fertilizers are expen-sive and it would consequently be advantageous to reduce the quantities of these high cost fertilizers that need to be added to soils while still maintaining the crop production levels nGw attained.
In prior Canadian patent applications Serial Nos. 532,255 filed on March 17, 1987 and 542,706 filed on July 22, 1987, the inventor of the present invention described a method of solubil-izing native rock phosphates in order to make these low cost, naturally occurring phosphates suitable as a source of phos-phorus and micro-nutrients for uptake by plants. The method involves the use of a fungus known as Penicilllum bilaii as a phosphorus-solubilizing microorganism.
It has now unexpectedly been found that this micro-organism is not only useful for solubilizing native rock phosphate, but that it is also useful for increasing the ,~

2 -amount of phosphorus which can be taken up by plants from com-mercial fertilizers. As a result of this finding it is pos-sible to reduce the amount of commercial phosphate fertilizer applied to P-deficient soil and consequently to reduce the amount of money that has to be spent on the fertilization of such soils.
Thus, according to one aspect of the invention there is provided a method of increasing the fertility of phosphorus-deficient soil for plant growth, which comprises adding an effective amount of an inoculum of the fungus Penicillium bilaji to the soil as well as fertilizing the soil with an effective amount of a manufactured phosphorus fertilizer.
According to another aspect of the invention there is pro-vided a method of increasing the phosphorus uptake by plants, which comprises growing said plants in phosphorus-deficient soil to which has been added an effective amount of an inoculum of the fungus Penicillium bilaji and an effective amount of a manufactured phosphorus fertilizer.
According to yet another aspect of the invention there is provided a composition for adding to phosphorus-deficient soils in combination with a manufactured phosphorus fertili-zer, which comprises an effective amount of an inoculum of the fungus Penicillium bilaji and a soil compatible carrier therefor.
According to yet another aspect of the invention there are provided plant seeds having a coating comprising an effective amount of an inoculum of the fungus P.bilaji and a solid soil-compatible carrier therefor.
The inoculum of the fungus is preferably supported on a soil-compatible carrier capable of acting as a carbon source for the fungus.
It is theorized that the fungus increases the amount of phosphorus available for plant uptake from commercial phos-phorus fertilizers because these fertilizers are acted upon by soil components in such a way as to convert a certain propor-tion~of the phosphorus into insoluble phosphorus compounds and this proportion is then solubilized by the action of the fungus and hence does not go to waste.
Commercially available phosphate fertilizers are of many types. Some common ones are those containing mono-ammonium 130856~;

phosphate (MAP), triple super phosphate lTSP), diammonium phos-phate, ordinary superphosphate and ammonium polyphosphate.
All of these fertilizers are produced by chemical processing of insoluble natural rock phosphates in large scale fertilizer-manufacturing facilities and, as noted above, the product isexpensive. By means of the present invention, at least in its preferred forms, it is possible to reduce the amount of these fertilizers applied to the soil by up to 50% or more while still maintaining the same amount of phosphorus uptake from the soil. Bearing in mind that recent statistics have shown that expenditures for processed phosphate fertilizers in the three prairie provinces of Canada alone are roughly 75 million dollars per year, the use of the present invention has the potential for generating significant savings.
The term ~inoculum~ as used in this specification is inten-ded to mean any forms of fungus cells or spores which are capable of propagating on or in the soil when the conditions of temperature, moisture, etc., are favourable for fungal growth.
The term "soil-compatible" means any material which can be added to the soil without having a significant adverse effect on plant growth, soil structure, soil drainage or the like.
The fungus Penicillium b ~ i is a known micro-organism that has previously been deposited at the American Type Culture Collection in Rockville, Maryland, USA under the deposit number ATCC 22348 (1974 edition of the ATCC catalogue). In the 1984 catalogue, the same deposit number is used for P bilail and a further strain is identified by the deposit number 18309. It is not known whether the change of name occurred as a result of a clerical error or whether the fungus has been re-named.
In any event, the name P bila~i is used for the micro-organism throughout this specification.
The inventor has discovered a further strain of the micro-organism in soil from a location (latitude 49 48' N, longitude 113 6' W) in Southern Alberta, Canada. This strain has improved P-solubilizing activity compared with the strains previously deposited at the ATCC. A deposit of the improved strain has been made at the ATCC under the deposit number 20851 and samples are available upon request.

--` 13/t~ SIS6~

The taxonomic details of our new strain are as follows:
Czapek Yeast Extract Aqar ~CYA) ________ ______ _ __ 25 ~ daysL : 26.2 mm diam.; wide margin deep; dense; plane to radially sulcate; floccose to funiculose;
mycelium : white condidia : dull green tMethuen 25-27 D3) exudate : present, clear to amber sol. pigment: brown reverse : reddish brown (M9E-P8) Malt Extract Agar ________ _ _7 d~ysL : 20-25 mm diam.; margin low and wide; low to medium; medium; plane;
velutinous to floccose to funiculose;
mycelium : inconspicuous condidia : dull green (Methuen 25-27 D3) exudate : absent sol. pigment: brown reverse : pale 25% Glycerol Nitrate Agar (25CL_7 daysL : 13-16 mm diam.; plane to radially sulcate; floccose;
mycelium : white condidia : sparse, similar to CYA at 25C
exudate : absent sol. pigment: brown reverse : pale CYA at 5C : 0 i.e. no germination/growth ______ CYA at 37C : 10-15 mm diam; convolute, margin irregular; velutinous;
mycelium : white exudate : absent sol. pigment: brown reverse : brown

3~3566 C'PHORES : Solitary aerial ____ STIPES : length: 30-100~ m walls : smooth PENICILLI: monoverticillate : vesiculate PHIALIDES: ampulliform ; no. : 10+6 - 8~ m long collula : short CONIDIA : subspherical ; well-defined columns;
2.5-3~ m long; finely rough The fungus can be easily propagated on a suitable carbon source such as autoclaved moist ground wheat straw amended with glucose, unamended bran, etc. Propagation normally takes place for a period of about one week or more before the in~culum is ready for use. The resulting fungus propagated on a solid support may be used as such for incorporation into soil most preferably at the root level, but may be coated onto the seeds if desired. Alternatively, a liquid culture of the fungus may be prepared using a conventional nutrient solution. The liquid culture may then be used as such or dried and the dried prod~lct applied to the soil either with or without a suitable carrier and/or nutrient source.
The use of coated seeds is a particularly efficient way of introducing the inoculum into the soil. There are several ways of coating the seeds and any suitable method can be employed. For example, spores may be mixed in a carrier com-prising a mixture of soluble starch and cellulose (preferably a 50:50 mixture by weight), the resulting combination sus-pended in water, the suspension used to coat plant seeds and then the seeds may be left to dry. Alternatively, the fungus may be grown on a carbon source (e.g. moistened sterile bran) at approximately room temperature for at least about one week, the carbon source dried and sieved, and the resulting partic-les adhered to seeds by use of a sticky or adhesive material, e.g. gum arabic.

~3Q8~6~

The amount of the inoculum to be applied to the soil is not limited in any particular respect. Clearly, if insuf-ficient is used, a reduction of the required amount of fertilizer will not be obtained. On the other hand, the use of large amounts of the inoculum relative to the fertilizer will be wasteful because there is no improvement of the effect after a certain limit. Suitable proportions of fungus to fertilizer and suitable amounts of fertilizer vary according to the type of soil, the type of crop plants, the amount of insoluble phosphate already in the soil, etc. and a suitable proportion and application rate can be found without difficulty by simple trial and experimentation for each particular case. Normally, the application rate falls into the range of 102-106 colony forming units (cfu) per seed, or a few grams of inoculated carrier (containing up to about 9 x 101 cfu/g) per meter of plant row.
The fungus should not be mixed with the fertilizer prior to application to the soil because the salt ef~ect of the concentrated fertilizer kills the fungus. The ~ungus and fertilizer should be added separately to the soil for this reason. As noted above, the supported fungus can be added to the soil ~above, below or at the same level as the plant seeds) or coated seeds can be used. The fertilizer can be applied to the soil in the conventional manner and is most preferably located beneath the plant seeds. Direct contact of the seed and concentrated fertilizer within the soil should also desirably be avoided.
Other fertilizers, such as nitrogen sources, or other soil amendments may of course also be added to the soil at approximately the same time as the supported fungus or at other times, so long as the other materials are not toxic to the fungus.
Preferably, a further carbon source for fungal growth, such as soluble staech or cellulose or mixtures thereof, is applied to the soil in addition to the phosphate Fertilizer and P. bilaii. This carbon source may be additional to the one used for the initial propagation of the fungus, i.e. the ~"` 130~35~i6 one forming part of the inoculum. The additional carbon source often increases the nutrient uptake of plants grown in the soil~ presumably because of increased fungal growth rates.
It has been found that the presence of a small amount of nitrogen (introduced in the form o~ the ammonium ion) improves the P-solubilizing activity o~ P. bilaji. For this reason NH4Cl or another ammonium source is preferably applied to the soil at approximately the same time as, or in admixture with, the supported fungus or phosphate fertilizer. The amount of the ammonium source added normally falls within the range of 5-20 Kg N/ha. In the case of MAP and the like, the ammonium need not be added since it is already a component of the manufactured fertilizer.
The mechanism by which P.bilaji solubilizes insoluble phosphate is not precisely known. However, it is theorized that the fungus could conceivably operate via two separate mechanisms, one requiring the presence of the ammonium ion and a second which does not involve ammonium but involves the excretion of organic acids. The mechanism by which P.bilaii makes phorphorus available for plant uptake is thought to be through excretion of organic acids.
There is some evidence to indicate that ~ ti~ also increase the uptake of certain micronutrients (e.g. copper, iron and zinc) by plants, and so sparingly soluble sources of these element~ may be added to the soil with the P.bilaji.
In fact, manufactured phosphate fertilizers often contain such sources and so the presen~ invention offers a double benefit of increasing the uptake of these micronutrients as well as the uptake of phosphorus.
It has been found that the presence of vesicular-arbuscular mycorrhizal fungi (hereinafter referred to as VAM) in the root zone is necessary for good P-uptake, but such micro-organisms are normally present in soil, so spe~ific addition of such micro-organisms is not required unless they are absent from the soil or present only in unusually small amounts.

~3~ ;6~

The inventlon is illustrated in more detail by the following Examples in which the P.bilaji strain employed for the tests W?S the deposited ATCC 20851 strain.
Example 1 A greenhouse formulation experiment was conducted as follows. A 1:1 mixture of beach sand and soil from Purple Springs, Alberta was used. The resulting mixture (p~ 7.2 (1:1 CaC12)) was inoculated with VA mycorrhizal fungi and 1.2 Kg were added to each of 200 clay pots. The soil was watered 2 days before seeding. The soil moisture was maintained at field capacity by daily watering.
Mono-ammonium Phosphate (11-51-O) was added at either 20 mg P/kg soil (full rate - MAPF) or 10 mg P/kg soil ~half rate - MAPH~. Control pots not receiving P fertilizer were also included. The phosphorous was added to the soil in a layer 1 cm below the seed. Nitrogen as NH4Cl was added to the appropriate pots (all but those containing MAPF) so that the nitrogen in each pot would be equivalent to that in pots with MAPF. Micronutrients were added at NH4N03 (50 mg N/kg soil) 1 and 4 weeks after planting and as (NH4)2S04 (50 mg N/kg soil) 6 weeks after planting.
The P.bilaji was added either as a seed treatment or as a bran based soil applied application. Two seed treatment formulations were used. The first consisted of spores mixed in a carrier of 50:50 soluble starch and cellulose (ST-Cell).
The base material contained 5.6 x 109 colony forming units (cfu) P.bilaji/g material. Serial dilutions of this material were prepared b~ mixing the base P.bilaji containing substrate with autoclaved starch cellulose. This material was then used to coat wheat seed (cv. Neepawa). 5.0 g of material was mixed with 14 ml of ~2 and 5.0 ml of the resulting suspen-sion was added to 10 g of wheat seed. The seeds were allowed to dry overnight at room temperature. Four P.bilaji concen-trations were used. The first (SCl) used the base material and resulted in a P.bilaji concentration of 8.4 x 10 cfu/
seed. The remaining 1.46 x 104 (SC3); and 5.0 x 103 (SC4) cfu P.bilaji/seed. Control seed was coated with the starch cellulose carrier alone (no P.bilaji).

~,._ . . . ~.

:~30~356fi g The second seed treatment used P.bilaji inoculum prepared by growing the fungus on moistened bran. The bran was inocu-lated with spores of the fungus and allowed to grow for 1 week at room temperature~ This mate~ial was dried and sieved (2 mm). The sieveæ material which contained 3.5 x 109 cfu P.bilaji/g dry bran was mixed with wheat seed which had been coated with Gum Arabic sticker. This coated seed was then sieved through a 2 mm sieve to remove excess bran. Serial dilutions oE the bran were prepared by mixing bran inoculated with P.bil_i~ with uninoculated bran. Four P bilaji concen-trations were used. The first (SBl) used only inoculated bran and had a P.bil~i~ concentration of 1.74 x 106 cfu P. ~ /
seed. The remaining treatments, SB2, SB3, and SB4 had concen-trations of 2.2 x 105, 7.57 x 104, and 2.67 x 103 cfu P.bilaji/seed, respectively. Control seed was not treated.
The soil applied bran used the same P.bila ~ containing bran used in the second seed treatment. Three rates of this material were used. LBl, LB2, and LB3 were applied at 1.0, 0.1, and 0.01 g/pot, respectively. The bran was applied to the soil in a layer 1 cm below the seed.
Ten seeds were planted in each pot and were thinned to 5 seeds/pot after emergence was completed. The pots were arran-ged in a randomized block design with 5 replications. There were 27 treatments (Table la). The plants were harvested 10 weeks after planting. Total oven dry weight and P content were determined. The results are shown in Table lb.

Table la. Treatments Formulation Experiment 1. NO P.bila~i/NO P 2. NO P.bilai~/NAOG 3. NO P.bilaj'/MAPF

4. SCO/NO P 5. SC0/MAPH
6. SCl/NO P 7. SCl/MAPH
8. SC2 ~ 0 P 9. SC2/MAPH
10. SC2/N0 P 11. SC3/MAPH
12. SC4/h0 P 13. SC4/MAPH
14. SBl/NO P 15. SBl/MAPH
16. SB2/N0 P 17. SB2/MAPH
18. SB3/N0 P 19. SB3/MAPH
20. SB4 ~ 0 P 21. SB4/MAPH
22. LBl/NO P 23. LBl/MAPH
24. LB2/N0 P 25. LB2/MAPH
26. LB3/N0 P 27. LB3/MAP~

Table lb. Analysis of Inoculation Formulation Experiment Dry Matter (g/pot) Total P (mg/pot) P.bilaji inocNo P MAP No P MAP
Form Rate ~10 k~l (10 k St-C;ell 10~-2.20 3.33 4.11 5.92 1041.45 3.30 2.60 5.63 1051.72 2.50 2.66 4.63 1061.94 3.12 3.10 5.40 Bran-seed 1032.38 3.33 4.15 5.42 1042.10 2.80 3.35 5.56 10~1.73 3.09 2.67 5.05 10~1.40 3.05 2.65 5.10 Bran-row 1042.40 2.98 4.29 6.50 1052.54 3.24 4.06 6.70 1063.13 4.52 5.50 9.00 Control ~1. 77 2.93 2.73 5.04 St-Cell -1.91 2.97 2.72 4.85 MAP (20 kgP) 3.74 3.74 8.13 8.13 LSD (.05) .75 1.04 1.69 2.49 Analysis of Var.
form .01 ns .01 .01 rate ns ns . 05 ns rate*form ns ns ns ns -``` 13Q8566 Main Effect St Cell 1.80 3.06 3~18 5.40 Bran-seed 1.90 3.07 3.21 5.28 sran-row 2.69 3.58 4.61 7.40 MAP (20) 3.74 3.74 8.13 8.13 Control 1.77 2.93 2.73 5.04 LSD (.05) .56 .77 1.26 1.86 A significant difference was observed between the three inoculum forms with respect to plant growth response in the absence of added P and in the P uptake response to added MAP. The most effective treatment was the bran inoculum added in the seed row, however the starch cellu-lose inoculum was also effective in the case of pots receiving MAP.
With respect to inoculum level, the results show that the rate of inoculum addition did not significantly affect the response of the crop. In the case of the starch cell~lose inoculum, the most effective rate was 103 cfu/seed which gave a growth response to 1/2 the MAP rate equal to 23% of that resulting from the addition of the full rate of MAP (SC-cont/MAP-cont) and a P uptake response equal to 33% of that from MAP. With the bran inoculum added in the seed row, the most effective rate of addition was 106 cfu/seed which gave a plant growth response the 1/2 the MAP rate equal to 196% of that produced by the full rate of MAP, and a P uptake response equal to 128%
of that from the full rate of MAP. By comparison of the control pots with and without the addition of the 1/2 rate of MAP, it is evident that the soil was P deficient, and responded to the addition of AMP, and also that the 1/2 rate of MAP in the absence of P. ~ was not as effective as the full rate of MAP.
The results for both plots receiving the 1/2 rate of MAP and those without added P clearly show the benefits from the addition of this organism. Penicillium bilaji, in this experiment was shown to increase plant growth responses to the addition of ~AP, and to increase plant growth in the absence of added P.

~3~ 51$~6 Exam~e 2 An experiment was conducted on a Brown Chernozemic soil located on the Vauxhall substation of the Lethbridge Research Station. The soil had a pH of 7.6 (.OlM CaC12J and low levels of available P. Corn (Zea mays cv Dward 39119YP) and spring wheat (Triticum aestivum cv Fielder) were used as test crops. Each plot consisted of ~our treatment rows 6.1 m long separated from the other treatments by a guard (i.e.
untreated) row. Wheat rows were spaced 17.8 cm apart while the corn rows were separated by 35.6 cm).
Florida rock phosphate and commercial mono-ammonium phosphate (MAP) were used as P sources. The rock phosphate was granulated by heating with urea (140C) for eight hours, then cooling the mixture, and seiving out granules with sizes between 1 and 2.5 mm diameter. The final product contained 10% N and 28% P2O5. Penicillium bilaji inoculum was prepared by growing the fungus on moistened sterile bran at room temperature (20C) for one week. The colonized bran was air dried and either used directly for application as seed row inoculum or added with the seeds along with gum arabic sticker and shaken to poduce seed coat applied material. The air-dried bran contained 9 x 101 colony forming units per gram. Seed applied P b~ii was able to deliver 1.5 x 106 cfu per wheat seed and 1.1 x 106 per corn seed.
Three fertilizers (control, rock phosphate, MAP) and three fungal treatments (control, seed applied P.bilaji, bran applied P.bilai~) were used for each crop in a factorial design with five replications. Wheat plots, where applicable received 12.2 kg P.ha equivalent, as either rock phosphate or MAP, added in the seed row below the seeds. Corn plots received 20.0 kg P.ha equivalent. Treatments receiving bran applied P bilai~ received 0.9 g bran per meter of row added in the seed row. Nitrogen as ammonium nitrate was added to each crop at seeding using braodcast methods. Wheat plots received 78.4 kg N/ha equivalent while corn plate received 35~6 112 kg N/ha. Additional N as urea was added in each seed row at a rate to equal the amount of N added with the MAP for each crop. All fertilize~s, inocula and seeds were added through a mechanical seeder with attachments for adding additional materials. Plots were hand weeded. All plots received supplemental irrigation water as necessary to main-tain soil moisture tension below -450 KPa as measured with tensiometers located within the plots. Ten plants were har-vested at maturity. The results are shown in Tables 2 a and b.

Table 2a. Effect of Penicill um bilaji on wheat growth TOTAL DRY MATTER GRAIN YIELD
NO P No P.bilaii 482 b 226 b Seed P.bilaJ.ir 551 ab 249 ab Bran P.bllal~ 535 b 254 ab ROCK P No _ bilaji 511 b 229 ab Seed P.bllai~ 510 b 223 b Bran P.b ~ 546 ab 249 ab MAP No P.bilaii515 b 242 ab Seed ~ la]1533 ab 248 ab Bran P.bila ~ 617 a 277 a Main Effects Fertilizer form TOTAL DRY MATTER GRAIN YIELD
No P 523 a 243 a Rock P 522 a 234 a MAP 555 a 256 a P. bilaii TOTAL DRY MATTER GRAIN YIELD
- P.bilaji 503 a 232 a + P.bilaii seed 531 ab 240 ab + P.bilaii bran 566 b 260 b ____ ____ values in each column followed by the same letter are not sig.
diff. as determined by LSD analysis on log transformed data.

Analysis of Variance Fert ns ns P bila~i .05 .10 FxP ns ns -Table 2b. Effect of P.bilaji on Corn Growth TOTAL DRY MAT~ERGRAIN YIELD
NO P No P.bila~ 1925 bc968 bc Seed P.bilaii 2227 ~bc 1088 ab Bran P.bilaji 2295 ab 1156 ab ROCK P No P.bilaii 1893 c 843 c Seed P.bilaji 1950 bc 954 bc Bran ~ 2029 abc 1055 ab MAP No P.bila~i 2163 abc1146 ab Seed P.bila~ 2246 abc 1126 a Bran P ~ 2357 a 1196 a Main Effects Fertilizer form TOTAL DRY MATTERGRAIN YIELD
No P 2149 ab1071 a Rock P 1957 b 951 b MAP 2255 a1156 a P.~
TOTAL DRY MATTERGRAIN YIELD
- P bilaji 1994 a 986 a P. ~ seed 2141 ab1056 ab + P.bilaji bran 2227 b1136 b values in each column followed by the same letter are not sig.
diff. as determined by LSD analysis on log transformed data.

Analysis of Variance Fert .05 .01 P.bilaji , .10 .05 FxP ns ns ` 130~35~i6 P.bilaji inoculum added as a bran based material in the seed row was much more effective than the seed applied inoculum for both crops. The seed and row applied inocula were compared in the previous Example, and the seed applied form was shown there to be inferior. However, in that Example, a different form of seed applied inoculum, using starch-cellulose instead of bran, was shown to be better than the seed applied inoculum used in this experiment.
Consequently, it is concluded that it is the form, not the method, of applying the inoculum to seeds that results in poor performance.
The results for the wheat experiment show that P.bilaii, applied in the seed row, was able to increase plant yields by 20%, and grain yields by 14.5% over uninoculated wheat plants in plots receiving MAP. P bilaji was also able to increase plant and grain yields by 11% and 12% resp. over uninoculated plants in unfertilized plots. Since the increase in plant growth due to P.bilaji for plots receiving MAP was greater than that observed for plots without MAP, we may conclude that the fungus is increasing the effectiveness of MAP as well as solubilizing soil inorganic phosphate.
Similarly, in the corn experiment, P. ~ i, applied in the seed row, was able to increase plant yields by 9%, and grain yields by 4% over uninoculated wheat plants in plots receiving MAP. P.bilaji was also able to increase plant and grain yields by 19% over uninoculated plants in unfertilized plots. The percentage increases for corn plots receiving MAP
are smaller than that observed for the wheat experiment because of the greater yields of the control plots in the corn experiment. In the corn experiment, the increased yields in the MAP fertilized plots were smaller than those f, observed in the unfertilized plots. This may be due to over-fertilization of the corn plots with MAP, such that additional increases due to increased effectiveness are unlikely.

-- i3QB5~6 Exam~
__ Experiments were conducted at ten sites in Alberta, Manitoba and Saskatchewan covering srown, D. srown and slack Chernozemic soils. The soils ranged in pH from 5. 8 to 7. 3 (.OlM CaC12) and low levels of available P. Spring wheat and barl~y were used as test crops. Each plot consisted of four treat~ent rows 7. 62m long separated from the other treatments by a guard (i.e. untreated) row. Wheat and barley rows were spaced 17.8 cm apart.
Commercial mono-ammonium phosphate (MAP) was used as a P
source. Penicillium bila~l inoculum was prepared by growing the fungus on moistened sterile bran at room temperature (20C) for one week. The colonized bran was air dried and used directl~ for application as seed row inoculum at the Gibbons site. The air-dried bran contained 9 x 101 colony forming units per gram. At the other sites, the inoculum was added to the seeds along with gum arabic (10% in H2O) to provide between 6.3 x 105 and 6.1 x 106 per seed.
Five rates of fertilizer (0, 11, 22, 33, 44 kg P2O5/ha) and two fungal treatments (control, P bilaji) were used for each experiment in a factorial design with five replications.
The MAP was added in the seed row below the seeds. Treat-ments receiving bran applied P.bil~li received 2.0 g bran per meter of row added in the seed row. Nitrogen as ammonium nitrate was added to each crop at seeding using broadcast methods. Wheat and barley pots received 78.~ kg N/ha equiva-lent. All fertilizers, inocula and seeds were added through a mechanical seeder with attachments for adding additional materials.
At maturity, the central portions of the center four rows of each plot were mechanically harvested for grain yield 112.3 m row harvested total).

13(~3566 . . ~

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~ 13~85~6 The results indicate that the addition of P bilaii to wheat and barley crops significantly (p<.05) or nearly significantly (p<.10) increased grain yields in 4 out of the ten experi~ents conducted. of the remaining six exp~riments, only one site responded to the addition of MAP, so increased responses to P.bilaii should not be expected. Nonetheless, two of the responsive experiments were on sites where the crop did not respond to the addition of MAP, showing that MAP by itself is not effective in all prairie soils. This confirms previous data by other workers for Alberta soils.
Bearing this in mind, P~bilaji inoculation resulted in increased grain yields in two of the three experiments which responded to MAP and also caused increases in two other experiments which did not respond to MAP.
Overall, the addition of P.bilaji at the four responsive sites resulted in an average increase in grain yields of 5.7%
over uninoculated plots. One experiment, Oak Bluff wheat, resulted in a significant decrease in grain yield in response to P bilai~ addition, however, the reason for this is unexplained.
The increased response of crops to MAP plus P.bilaii is most likely due to increased P availability. In the two sites which did not respond to MAP, but did respond to P.bi ~ , it is likely that the fungus was able to counter-act the action of the soil in reducing MAP effectiveness, in essence, making the fertilizer work the way it is supposed to. The organism probably also solubilized soil inorganic P
forms which contribute to plant P nutrition.

13~?85~6 Example 4 Experiments were conducted at ~lstow, Saskatchewan. The soil had a pH of 5.8 t. OlM CaC12) and low levels of available P. Barley was used as test crop. Each plot consisted of four treatment rows 7.62m long separated from the other treatments by a guard (i.e. untreated) row.
Barley rows were spaced 17.8 cm apart.
Commercial mono-ammonium phosphate (MAP) was used as a P
source. Penicil1ium bilaii inoculum was prepared by growing the fungus on moistened sterile bran at room temperature (20C) for one week. The colonized bran was air dried and used directly for application as seed row inoculum at the Gibbons site. The air-dried bran contained 9 x 101 colony forming units per gram. At the other sites, the inoculum was added to the seeds along with gum arabic (10% in H2O) to provide between 6.3 x 105 and 6.1 x 106 per seed.
Five rates of fertilizer (0, 11, 22, 33, 44 kg P2O5/ha) and two fungal treatments (control, P bilaji) were used for each experiment in a factorial design with five replications.
The MAP was added in the seed row below the seeds. Treat-ments receiving bran applied P.bilaji received 2.0 g bran per meter of row added in the seed row. Nitrogen as ammonium nitrate was added to each crop at seeding using broadcast methods. Barley plots received 78.4 kg N/ha equivalent.
All fertilizers, inocula and seeds were added through a mechanical seeder with attachments for adding additional materials.
At maturity, one meter of the center two rows of each plot were hand harvested for determination of total dr~
matter production.

~30~35~6 Table 4a. Analysis of MAP trials - Elstow R_teP.bil_ii Plant_yield~ 2m 0 - 53.2 + 102.0 ~ 64.0 + 126.9 - 65.5 + 97.2 - 65.7 + 86.3 - 44.4 + 95.1 Main Effects -P.bilaji 58 6a ~P.bilali 101 5 b Analysis of Variance MAP rate ns P.bilaii 01 R*P ns The results clearly show inceeased yields of barley, even though the crop did not respond to MAP addition. The ineffectiveness of MAP in many prairie soils was discussed in the previous Example. Overall, P.b ~ inoculation resulted in an average increase of 73% over uninoculated plots. The increases in plant yield were observed over all the rates of MAP addition, however, this is not surprising since MAP, in the absence of P bilaji, was ineffective.

13Q8S~

E~ample 5 __ An experiment was conducted on black Chernozemic soils located at Plum Coolee, Manitoba and at the University of Alberta, Edmonton, Alberta. The soils had a pH of 6.0 (.OlM
CaC12) and low levels of available P. Canola (cv ~ester) was used as a test crop. Each plot consisted of four treat ment rows 7.62m long separated from the other treatments by a guard (i~e. untreated~ row. Rows were spaced 17.8 cm apart.
Florida rock phosphate and commercial mono-ammonium phosphate (MAP) were used as P sources. The rock phosphate was granulated by heating with urea (140C) for eight hours, then cooling the mixture, and seiving out granules with sizes between 1 and 2.5 mm diameter. The final product contained 10% N and 28~ P205. Penicillium ~ inoculum was prepared by growing the fungus on moistened sterile bran at room temperature (20C) for one week. The colonized bran was air dried and used directly for application as seed row inoculum. The air-dried bran contained 9 x 101 colony forming units per gram.
Four fertilizer (control, rock phosphate, 1/2 MAP, MAP) and two fungal treatments (control, P bilai~) were used in a factorial design with five replications. The full MAP and rock P treatment received 12.2 kgP/ha equivalent, while the 1/2 MAP treatment received 6.1 kgP/ha, added in the seed row below the seeds. Treatments receiving bran applied P.b~
received 2.0 g bran per meter of row added in the seed row.
Nitrogen as ammonium nitrate was added at a rate equal to 78 kgN/ha at seeding using broadcast methods. All fertiliæers, inocula and seeds were added through a mechanical seeder with attachments for adding additional materials.
Two meters of row were harvested from the center o~ the plots at maturity at Plum Coolee, and ~.0 meters of row were harvested at the University of Alberta. Total and grain weights were measured on oven dried materials from Plum Coolee. Only grain weights were measured at the University of Alberta. The results are shown in Tables 5a and 5b.

~3~ 6~

Table 5a. Analysis of Canola data, Plum Coolee TOTAL DRY MATTER GRAIN YIELD
(9~2m 3~g/2m ) NO P - P.bilaji 221 6 b 63 8 d + P.bila~ 292 0 ab88 5 abcd ROCK P - P.bilaii 232.3 b 68 . 9 cd + P.bi1aji 257.0 ab 73.7 bcd 1/2 MAP - P.bilaji 288 . 0 ab 94 . 4 abc + P.bilaji 315.0 a 102.9 a MAP - P.bila`i 292.5 ab 84.8 abcd + P.bilaii 328.8 a 96.8 ab Main Effects Fertilizer form control 260.7 ab 77.5 bc rock P 243.3 b 71.0 c 1/2 MAP 301. 5 a 98 . 6 a MAP 310.7 a 90.8 ab P bil~i~
- P.bilaji 260.6 b 78 . 8 b + P.bllajl 300.4 a 91.4 a Analysis of Variance Fert .05 .01 p bil aj i . ns5 .nsS

l3a~s~6 Table 5b. Analysis of Canola data, University of Alberta GRAIN YIELD
(9/6m) NO P - P.bilaji 508 + P.bilaji 558 ROCK P - P.bilaii 484 + P bilaii 614 1/2 MAP - P.bilaii 443 + P.bilai~ 554 MAP - P.bila i 531 + P.bila~i 550 :
Main Effects Fertilizer form control 532a rock P 556a 1/2 MAP 499a MAP 540a P . bi laj i - P.bilaii 492a + P bilaji 570 b Analysis of Variance Fert ns P.bil~i~ .01 F*P ns The results show that the addition of P.bilaji to canola crops at the Unlversity of Alberta site was able to increase the grain yields by 10~ in the absence of added P and to increase grain yields by 25~ in the case of the 1/2 rate of MAP and by 3.6% in the case of the full rate of MAP. At the Plum Coolee site, P. ~ inoculation increased crop yields of unfertilized plots by 39%, of plots receiving 1/2 rate of MAP by 9% and of plots receiving the full rate of MAP by 14%. Overall, the main effect of P.bil~i~ addition at both sites was to increase grain yields by 16% (+P.bilaji/-P.bi~
main effect).

Claims (36)

Claims:

1. A method of increasing the fertility of phosphorus-deficient soil for plant growth, which comprises adding an effective amount of an inoculum of the fungus Penicillium bilaji to the soil as well as fertilizing the soil with an effective amount of a manufactured phosphorus fertilizer.

2. A method according to Claim 1 wherein the manufactured phosphate fertilizer is added to the soil in an amount less than that normally employed for soil fertilization.

3. A method according to Claim 1 wherein the fungus is a strain of P.bilaji identified by the American Type Culture Collection No. ATCC 20851.

4. A method according to Claim 1, Claim 2 or Claim 3 wherein the manufactured phosphate fertilizer is selected from the group consisting of mono-ammonium phosphate, triple super phosphate, diammonium phosphate, ordinary superphosphate and ammonium polyphosphate.

5. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is supported on a solid soil-compatible carrier capable of acting as a carbon source for the fungus.

6. A method according to Claim 1, Claim 2 or Claim 3 wherein the inoculum is contained with a liquid solution containing a nutrient for the fungus.

7. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is supported on a solid soil-compatible carrier capable of acting as a carbon source for the fungus, said carrier being selected from the group consisting of amended wheat straw, bran, starch, cellulose and mixtures thereof.

8. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is contained in a coating on plant seeds and said seeds are planted in the soil in order to introduce the inoculum into the soil.

9. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is mixed with a carbon source for the fungus and the mixture is coated onto plant seeds, and wherein said seeds are planted in the soil in order to introduce the inoculum into the soil.

10. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is mixed with starch and cellulose and wherein the resulting mixture is applied as a coating onto plant seeds, and wherein said seeds are planted in the soil in order to introduce the inoculum into the soil.

11. A method according to Claim 1, Claim 2 or Claim 3 wherein the fungus is propagated on bran flakes and said flakes are adhered to plant seeds to form a coating, and wherein said plant seeds are planted in the soil in order to introduce an inoculum of the fungus into the soil.

12. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is supported on a solid soil-compatible carrier capable of acting as a carbon source for the fungus, and wherein the supported inoculum is introduced into the soil immediately adjacent to plants or plant seeds, or the positions where plants or plant seeds will be subsequently introduced.

13. A method according to Claim 1, Claim 2 or Claim 3 wherein a source of ammonium ion is also introduced into the soil.

14. A method according to Claim 1, Claim 2 or Claim 3 wherein said plant seeds are planted in the soil and wherein the amount of inoculum introduced into the soil falls in the range of 102-106 colony forming units per seed.

15. A method according to Claim 1, Claim 2 or Claim 3 wherein said inoculum is introduced into the soil at a level approxi-mating the root level of plants to be grown in the soil.

16. A method according to Claim 1, Claim 2 or Claim 3 wherein at least one non-phosphorus containing fertilizer is introduced into the soil.

17. A method according to Claim 1, Claim 2 or Claim 3 wherein a carbon source for the fungus is added to the soil in addition to said inoculum.

18. A method according to Claim 1, Claim 2 or Claim 3 wherein a source of micronutrients selected from the group consisting of copper, iron, zinc and mixtures thereof is added to the soil.

19. A method according to Claim 1, Claim 2 or Claim 3 wherein vesicular-arbuscular mycorrhinzal fungi are introduced into the soil.

20. A method of increasing the phosphorus uptake by plants, which comprises growing said plants in phosphorus-deficient soil to which has been added an effective amount of an inoculum of the fungus Penicillium bilaji and an effective amount of a manufactured phosphorus fertilizer.

21. A composition for adding to phosphorus-deficient soils in combination with a manufactured phosphorus fertilizer, which comprises an effective amount of an inoculum of the fungus Penicillium bilaji and a soil compatible carrier therefor.

22. A composition according to Claim 21 wherein said fungus is a strain of P.bilaji identified by the American Type Culture Collection No. ATCC 20851.

23. A composition according to Claim 21 wherein said soil-compatible carrier is capable of acting as a carbon-source for the fungus.

24. A composition according to Claim 22 wherein said soil-compatible carrier is capable of acting as a carbon-source for the fungus.

25. A composition according to Claim 21, Claim 22, Claim 23 or Claim 24 wherein said soil-compatible carrier is selected from the group consisting of amended wheat straw, bran, starch, cellulose and mixtures thereof.

26. A composition according to Claim 21 or Claim 22 wherein said carrier comprises a liquid containing a nutrient for the fungus.

27. A composition according to Claim 21, Claim 22, Claim 23 or Claim 24, in the form of a coating adhered to plant seeds.

28. A composition according to Claim 21, Claim 22, Claim 23 or Claim 24, which contains up to about 109 colony forming units of said fungus per gram of the composition.

29. A composition according to Claim 21, Claim 22, Claim 23 or Claim 24 which further comprises a source of the ammonium ion.

30. Plant seeds having a coating comprising an effective amount of an inoculum of the fungus P.bilaji and a solid soil compatible carrier therefor.

31. Plant seeds according to Claim 30 wherein the fungus is a strain of P.bilaji identified by the American Type Culture Collection No. ATCC 20851.

32. Plant seeds according to Claim 30 wherein the carrier is capable of acting as a carbon source for said fungus.

33. Plant seeds according to Claim 30, Claim 31 or Claim 32 wherein said carrier comprises a mixture of starch and cellulose.

34. Plant seeds according to Claim 30, Claim 31 or Claim 32 wherein said carrier comprises bran flakes adhered to said seeds by an adhesive material.

35. Plant seeds according to Claim 30, Claim 31 or Claim 32 wherein said seeds are those of crop plants.

36. Plant seeds having a coating comprising approximately 5.0 x 103 to 8.4 x 105 colony forming units of spores of the fungus P.bilaji and a solid soil-compatible carrier therefor.