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PLANT HEALTH EFFECT OF PURPUREOCILLIUM LILACINUM

20190313647 · 2019-10-17

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a method for promoting or improving plant health and/or plant growth of agricultural plants wherein the plants, the plant propagules, the seed of the plants and/or the locus where the plants are growing or are intended to grow are treated with an effective amount of a composition comprising the fungus Purpureocillium lilacinum or spores thereof. Further aspects of the invention relate to uses of a composition comprising the fungus Purpureocillium lilacinum or spores thereof for promoting or improving plant health and/or plant growth.

    Claims

    1. A method for promoting or improving plant health and/or plant growth of agricultural plants wherein the plants, the plant propagules, the seed of the plants and/or the locus where the plants are growing or are intended to grow are treated with an effective amount of a composition comprising the fungus Purpureocillium lilacinum or spores thereof essentially in the absence of pathogenic nematode pressure or independent of pathogenic nematode pressure.

    2. The method of claim 1, wherein promoting or improving plant health comprises and/or manifests in improved stress tolerance, less dead basal leaves, greener leaf color, pigment content, photosynthetic activity and enhanced plant vigor.

    3. The method of claim 1, wherein promoting or improving plant growth comprises or manifests in tillering increase, increase in plant height, bigger leaf blade, bigger leaf surface, stronger tillers, earlier flowering, early grain maturity, less plant verse (lodging), increased shoot growth, increased plant stand and early and better germination, earlier emergence, improved crop yield, improved total vegetative weight or whole plant biomass, improved protein content, improved oil content, improved starch content, improved root growth, improved root size, improved root weight, increased root weight, increased plant biomass and/or improved root effectiveness, improved shoot weight and improved fruit weight.

    4. The method of claim 2, wherein improved stress tolerance comprises improved tolerance to drought, heat, salt, UV, water, cold and/or xenobiotic conditions.

    5. The method of claim 1, wherein the P. lilacinum strain is strain 251.

    6. The method of claim 1, wherein plant growth refers to root growth, root size, root weight, fruit weight, shoot weight, leaf surface, plant biomass and/or crop yield.

    7-8. (canceled)

    9. The method of claim 1, wherein the spores are conidia.

    10. The method of claim 1, wherein seed is treated.

    11. The method of claim 1, wherein the treatment is carried out in-furrow, by drip application, soil incorporation, drench application, sprinkler irrigation or micro injection.

    12. The method of claim 11, wherein said treatment is carried out in the soil prior to germination of a seed and/or in the soil in contact with a seed or root of said plant or where a plant is intended to grow.

    13. The method of claim 1, wherein the treatment is carried out at least twice.

    14. The method of claim 1, further comprising applying, simultaneously or sequentially, at least one further plant protection agent.

    15. The method of claim 13, wherein said at least one further plant protection agent is selected from the group consisting of fluopyram, B. firmus strain CNCM I-1582, B. subtilis, abamectin, aldicarb, fenamiphos, fluensulfone, fluazaindolizine, oxamyl and a fumigant.

    16. The method of claim 1, wherein the agricultural plant is selected from soybean, corn, wheat, triticale, barley, oat, rye, rape, millet, rice, sunflower, cotton, sugar beet, pome fruit, stone fruit, citrus, banana, strawberry, blueberry, almond, grape/grapevine, mango, papaya, peanut, potato, tomato, pepper, cucurbit, cucumber, melon, watermelon, garlic, onion, broccoli, carrot, cabbage, bean, dry bean, canola, pea, lentil, alfalfa, trefoil, clover, flax, elephant grass, grass, lettuce, sugarcane, tea, tobacco and coffee; each in its natural or genetically modified form.

    17. The method of claim 1, wherein the agricultural plant is tomato, cucumber or corn.

    18. The method of claim 1, wherein said composition further comprises at least 75% polyether-modified trisiloxane.

    19. The method of claim 18, wherein said polyether-modified trisiloxane is BREAK-THRU 5240.

    20. The method of claim 1, wherein said composition further comprises up to 8% fumed silica.

    21. The method of claim 20, wherein said fumed silica is AEROSIL.

    22. (canceled)

    23. The method of claim 15 wherein said plant protection agent is B. subtilis QST713.

    Description

    [0160] The figures show:

    [0161] FIG. 1A: Nematicidal activity of Purpureocillium lilacinum strain 251 and fluopyram against Meloidogyne incongita. Mean of 3 replicates+standard error (SE).

    [0162] FIG. 1B: Plant health activity of Purpureocillium lilacinum strain 251 and fluopyram in presence of Meloidogyne incongita. Mean of 3 replicates+standard error (SE). Significant differences (p<0.05) are indicated by asterisks.

    [0163] FIG. 2A: Total root surface area of tomato treated with a liquid formulation of P. lilacinum strain 251 (BIOACT liquid); 4 reps per treatment. Run unpaired T Test, confidence level of 95%, examining threshold of significance, p<0.05.

    [0164] FIG. 2B: Whole plant biomass of tomato treated with a liquid formulation of P. lilacinum strain 251 (BIOACT liquid); Statistically significant with a p-value of 0.0249, t value of 2.973 and df=6 degrees of freedom).

    [0165] FIG. 2C: Nematode efficacy showing average number of egg masses. Statistically significant with a P<0.0001, t value of 13.36, df=6.

    [0166] FIG. 3A: Whole plant biomass of tomato treated with different formulations of P. lilacinum strain 2514 reps per treatment; One-Way analysis of variance (ANOVA).

    [0167] FIGS. 3B, 3C, 3D: Average root weight, total root surface area (cm.sup.2) (p-value of 0.0270) and M. javanica egg masses (p-value of 0.0007) for the treatments shown in 3A.

    [0168] FIG. 4: Leaf surface area of tomatoes treated with formulations of P. lilacinum strain 251 as compared to other fungal strains.

    [0169] FIG. 5A: Fresh whole plant biomass of corn plants treated with a liquid formulation of P. lilacinum strain 251; *p=0.05 or less; **p=0.01 or less.

    [0170] FIGS. 5B and 5C: Root length (% B) and total number of tip, forks and crossings (TFC) of the trials shown in FIG. 5A.

    [0171] FIG. 6: Trial tomato treatment with a liquid formulation of P. lilacinum strain 251 in 80% sand and 20% Sunshine #3. A: Gall rating; B: Egg count; C: dry shoot weight; D: dry root weight; E: total vegetative weight.

    [0172] FIG. 7: Trial tomato treatment with a liquid formulation of P. lilacinum strain 251 in 80% Profile and 20% Sunshine #3. A: Gall rating; B: total fruit weight; C: dry shoot weight; D: total vegetative weight.

    [0173] FIG. 8: Trial cucumber treatment with a liquid formulation of P. lilacinum strain 251 in 80% sand and 20% Sunshine #3. A: Gall rating; B: Egg count; C: dry root weight.

    [0174] FIG. 9: Total fruit weight from trial tomato treatment with a liquid formulation of P. lilacinum strain 251 in 80% Profile and 20% Sunshine #3.

    [0175] FIG. 10: Efficacy (bars) and yield (dots) of trials with PL251 in cucumber (10A) and tomato (10B).

    [0176] FIG. 11: Total root length (cm, FIG. 11A) and root surface area (cm.sup.2, FIG. 11B) of tomato plantlets treated with spores of three P. lilacinum strains in comparison with untreated control plantlets.

    [0177] The examples further illustrate the invention in a non-limiting fashion.

    EXAMPLE 1: DRENCH APPLICATION OF PURPUREOCILLIUM LILACINUM STRAIN 251 (PL 251) IN TOMATO

    [0178] To prepare a suitable dosage form the formulated product PL251 (liquid formulation of P. lilacinum comprising BREAK-THRU 5240 and AEROSIL) is diluted with water to the desired concentration.

    [0179] A quantity of 5,000 cm.sup.3 of sandy loam soil, pH 6.8 per pot is mixed with 150,000 infective units (mixed population of eggs and juveniles) of the Southern Root Knot Nematode (Meloidogyne incognita). The desired concentration of Purpureocillium lilacinum strain 251 is drenched in 400 mL of solution (application A) to obtain 80% field capacity. Pots are incubated at 25 C. until transplanting of tomato seedlings (Lycopersicon lycopersicum) at 7 days after drench application A. At the day of transplanting a second drench application (application B) is carried out with 400 mL of solution.

    [0180] After transplanting of tomato seedlings several post-plant application patterns are tested using 400 mL of solution per application (Table A).

    [0181] In addition, 10 mg of the nematicide fluopyram (VELUM PRIME, suspension concentrate SC 400) is applied at transplanting and used as a chemical reference. The non-treated control (UTC) is drenched with 400 mL of water at each application. The experiment is kept for 7 weeks after transplanting at 25 C.

    TABLE-US-00001 TABLE A Purpureocillium lilacinum strain 251 drench test in tomato Application A B C D Application timing 7 d prior to at transplanting 4 weeks after 6 weeks after transplanting transplanting transplanting Treatment UTC water water water water 1 5.5 10.sup.5 viable 5.5 10.sup.5 viable 5.5 10.sup.5 viable water spores per cm.sup.3 spores per cm.sup.3 spores per cm.sup.3 substrate substrate substrate 2 5.5 10.sup.5 viable 5.5 10.sup.5 viable water 5.5 10.sup.5 viable spores per cm.sup.3 spores per cm.sup.3 spores per cm.sup.3 substrate substrate substrate 3 5.5 10.sup.5 viable 5.5 10.sup.5 viable 5.5 10.sup.5 viable 5.5 10.sup.5 viable spores per cm.sup.3 spores per cm.sup.3 spores per cm.sup.3 spores per cm.sup.3 substrate substrate substrate substrate 4 water 10 mg fluopyram/ water water plant

    [0182] After the specified period the nematicidal activity is determined on the basis of the percentage of gall reduction. 100% means that no galls were found; 0% means that the number of galls found on the roots of treated plants was equal to that in untreated control plants. Moreover the shoot biomass is also determined to assess the overall plant health status.

    [0183] The experimental set up is fully randomized and comprised three replicates per treatment. One-way analysis of variance (ANOVA) is carried out for shoot biomass using a threshold for significance of p<0.05 and a Bonferroni posttest to compare all treatments against the UTC.

    [0184] The bionematcide PL251 shows no to only weak nematicidal activity at a rate of 5.510.sup.5 spores per cm.sup.3 of substrate and at the used nematode pressure of 3,000 Meloidogyne incognita per 100 cm.sup.3 of soil. This finding is independent of the application patterns tested in this experiment (FIG. 1A). The chemical nematicide fluopyram shows excellent biocontrol activity at 10 mg per plant.

    [0185] The lacking performance of PL251 is likely because of the high nematode pressure used in the present study. Kiewnick et al. 2011.sup.1 reported approx. 50% biocontrol efficacy at 400 infective units of Meloidogyne incognita per 100 cm.sup.3 of soil. At a higher nematode density of 1,600 infective units, only 22% biocontrol were reported. However, the nematode pressure used in the present study (3,000 infective units per 100 cm.sup.3 soil) was considerably higher compared to that of Kiewnick et al. 2011. .sup.1 Kiewnick, S.; Neumann, S.; Sikora, R. A.; Frey, J. E. 2011: Effect of Meloidogyne incognita Inoculum Density and Application Rate of Paecilomyces lilacinus Strain 251 on Biocontrol Efficacy and Colonization of Egg Masses Analyzed by Real-Time Quantitative PCR. Phytopathology, Vol. 101, No. 1, 2011

    [0186] Despite its weak nematicidal performance PL251 improves tomato shoot fresh weight by 12-26% depending on the post-plant application pattern.

    [0187] Statistical analysis reveals a significant improvement of shoot fresh weight after nematicide treatment and a highly significant shoot fresh weight increase following application of PL251 at ABC (ANOVA, p=0.0222, df=14; Bonferroni multiple comparison test, t=4.191 for UTC vs. PL251 at ABC). The results clearly show that PL251 displays an additional plant health effect independent of its nematicidal potential leading to improved shoot weight in tomato.

    EXAMPLE 2: GROWTH CHAMBER TOMATO IN-PLANTA JAR ASSAY TO EVALUATE THE EFFICACY OF BIOACT AGAINST ROOT-KNOT-NEMATODES AS WELL AS ITS PLANT GROWTH PROMOTION (PGP) EFFECT

    Treatments

    [0188] 1. UTC-75 mLs of water [0189] 2. BIOACT applied at planting 1 L (1.08 mg) per 100 mL of soil, 5.510.sup.5 viable spores per cm.sup.3 substrate [0190] Soil Combination added 225 mL to the plastic jar

    Procedure:

    [0191] Eight 300 mL (10 oz) polypropylene flip top jars for this assay were prepared to examine fungal colonization of a liquid formulation of P. lilacinum strain 251, PGP effects, and to determine nematode reduction. Each jar was filled with soil combination (type of soil to mimic field soil conditions). Planted tomato seeds, ACE 55 Tomato Variety, Mountain Valley Seed Co. This assay is to examine Purpureocillium lilacinum fungal growth in-planta, determining a soil type that would provide a carbon source for the fungus to grow, colonize the soil and protect the roots from root-knot nematode infection.

    [0192] The treatment jars received 75 mL of drench solution. Each UTC jar was watered with 75 mL of tap water at time of planting. Each treatment was placed in sterile greenhouse flats to eliminate cross-contamination. All treatments were placed in a plant growth chamber. Experiment duration was 7 week trial, the settings for the growth chamber were set for photoperiod of 12 hrs of light and 12 hrs of dark, light intensity 320 Mol, temperature of 25 C. for light period, 20 C. for dark period.

    Tomato in-Planta Jar Assay Takedown:

    [0193] Each root system was washed from the 225 mLs of soil in a plastic 3 quart pitcher. As roots were cleaned, they were placed on paper towels to dry excess water running off.

    Analysis of Tomato Roots:

    [0194] Tomato roots were analyzed using the program WinRhizo, Regent Instruments, Inc. (Arsenault et al, 1995). This program provides for a complete plant root measurement and analysis. WinRhizo allows looking at the length, area, volume, topology, and architecture of the plant roots. Each UTC and Treatment were scanned to examine the total root surface area (cm.sup.2).

    Staining RKN Egg Masses:

    [0195] Once roots were scanned using WinRhizo, the roots were stained with Erioglaucine 1 mg/L solution for 15 mins. Each root system was submerged in the solution. The Erioglaucine Blue Solution stains the egg masses in bright blue (making it easier to visualize and count the fully developed females). The stain sticks to the gelatinous matrix that surrounds the RKN egg masses from the posterior end of the female nematode.

    Results:

    Total Root Surface Area:

    [0196] Comparing untreated with BIOACT DC drench, on average an increase in root surface area is visible in the treatment. The treatment average measurement was 19.916 cm.sup.2, untreated average was 18.669 cm.sup.2. (see FIG. 2A)

    Whole Plant Biomass:

    [0197] Total fresh shoot (whole plant biomass) of Untreated compared to BIOACT Treatment was evaluated. BIOACT treatment had significantly higher fresh whole plant biomass than UTC. Larger tomato roots treated with BIOACT drench showed an increase in lateral root growth as compared to UTC (see FIG. 2B).

    Nematode Efficacy Showing Average Number of Egg Masses:

    [0198] Nematicidal activity of Purpureocillium lilacinum strain 251 against Meloidogyne javanica. Overall, there was a decrease in the number of egg masses developed on BIOACT treatment compared to UTC. Effects of BIOACT treatment were significantly different from the UTC. One application of BIOACT treatment showed a significant reduction of M. javanica egg masses. The average number of egg masses in the UTC was 161 compared to that in the BIOACT treatment of 65.25 (see FIG. 2C). Evaluation of the number of egg masses definitely illustrates a smaller number of J2s penetrating and fully developing into a female adult, decreasing the number of egg mass production.

    EXAMPLE 3: GROWTH CHAMBER TOMATO IN-PLANTA JAR ASSAY TO EVALUATE THE EFFICACY OF TWO DIFFERENT FORMULATIONS OF BIOACT (LIQUID AND WG) AGAINST ROOT-KNOT NEMATODES AND TO COMPARE THEIR PGP PERFORMANCE

    Treatments

    [0199] 3. UTC-75 mLs of water [0200] 4. BIOACT applied at planting 5.510.sup.5 viable spores per cm.sup.3 substrate [0201] 5. BIOACT WG applied at planting 5.0 mg per 100 mL of soil (corresponding to 1.7510.sup.6 viable spores per 100 mL of soil [0202] 6. Blank Formulation applied at planting 1.08 mg per 100 mL of soil

    [0203] *Soil Combination added 225 mL to the plastic jar

    Procedure:

    [0204] Twenty 300 mL (10 oz) polypropylene flip top jars were prepared to examine BIOACT fungal colonization, PGP effects, and to determine nematode reduction. Each jar was filled with soil combination (type of soil to mimic field soil conditions). Planted tomato seeds were from the variety ACE 55, Mountain Valley Seed Co.

    [0205] The treatment jars received 75 mLs of drench solution. Each UTC jar was watered with 75 mLs of tap water at time of planting. The blank formulation comprised the formulants of the liquid formulation, i.e., BREAK-THRU 5240 and AEROSIL, each jar received the same amount as the one treated with the liquid formulation of BIOACT, 2.7 mg in 75 mLs of water. Pots treated with BIOACT WG (wettable granule received 125 mg in 75 mLs of water per treatment. Each treatment was placed in sterile greenhouse flats to eliminate cross-contamination. All treatments were placed in a plant growth chamber. The experiment duration was 7 weeks, the settings for the growth chamber were set for photoperiod of 12 hrs of light and 12 hrs of dark, light intensity 320 Mol, temperature of 25 C. for light period, 20 C. for dark period.

    [0206] Two weeks after planting tomato seeds, inoculated each jar with 300 active infective stage juveniles of Meloidogyne javanica (freshly hatched J2s from our root knot nematode tomato cultures).

    Tomato in-Planta Jar Assay Takedown:

    [0207] Each root system was washed from the 225 mLs of soil in a plastic 3 quart pitcher. As roots were cleaned, they were placed on paper towels to dry excess water running off. Each tomato plant was weighed to determine the total plant biomass. Afterwards, the shoots were cut off and discarded. Each root system was weighed per treatment to determine the weight of treatment.

    Staining RKN Egg Masses:

    [0208] Roots were stained with Erioglaucine 1 mg/L solution for 15 mins. Each root system was submerged in the solution.

    Statistical Analysis:

    [0209] Experiment set up is randomized and comprises 4 reps per treatment, One-Way analysis of variance (ANOVA) was performed.

    Results:

    [0210] There is a significant increase in whole plant biomass in BIOACT liquid treatment compared to UTC and blank. BIOACT WG also performed better than Blank and untreated (see FIG. 3A).

    [0211] The weight of root mass was examined for each treatment. There is a significant increase in root weight of BIOACT liquid compared to UTC and Blank. It is evident that PGP present with BIOACT compared to Blank, and UTC (see FIG. 3B).

    [0212] The total root surface area (cm.sup.2) of each treatment was examined using the WhinRhizo root analysis program. BIOACT treatment results in significantly more average root weight as compared to UTC and Blank (see FIG. 3C).

    [0213] The number of RKN egg masses were counted for each treatment (4 reps per treatment). There is significance in the reduction of the number of egg masses in BIOACT liquid treatment compared to UTC and Blank treatments.

    EXAMPLE 4: TOMATO DRENCH ASSAY TO EVALUATE PGP PROPERTIES OF P. LILACINUM STRAIN 251 AS COMPARED TO OTHER FUNGAL STRAINS

    Assay:

    [0214] 30 day evaluation to allow tomatoes to grow and examine the foliar canopy, determination of increase in leaf surface area.

    Treatment List:

    [0215]

    TABLE-US-00002 # TREATMENT RATES 1 Untreated Control Sterile DI Water 2 Blank Formulation 1.08 mg/100 mL of soil (formulants of liquid formulation) 3 Purpureocillium lilacinum strain 1.7 10.sup.8 spores per mL 251 4 BIOACT liquid 1x 1.08 mg/100 mL of soil 5.5 10.sup.5 viable spores per cm.sup.3 substrate 5 BIOACT liquid 10x 10.8 mg/100 mL of soil 5.5 10.sup.6 viable spores per cm.sup.3 6 Penicillium bilaii 1.11 10.sup.9 spores per mL 7 Trichoderma virens 1.27 10.sup.8 spores per mL

    Protocol:

    [0216] 67 plug trays were cut from a 1020 (200) cell plug tray (Hummert, Int.) Each cell tray was filled with potting soil and every other cell was seeded with one tomato seed (Washington Cherry Tomato Variety). Each seeded cell in the tray was treated with 2 mL of material using a serological pipette. Plug trays were watered from the bottom by flooding a clam shell tray. Flats were then placed under high intensity lights (300 Einsteins, set to 16 hour light/8 hour dark schedule) and watered once a day. Samples were compared based on volume. Every 2-3 days, clam shells were randomized. Plants were rated 30 days after planting. Three replicates per treatment were prepared.

    Drench Solution Preparation:

    [0217] Fungal strains (Penicillium, Trichoderma and P. lilacinum) were streaked onto Potato Dextrose Agar plates to enable fungal spores to grow. [0218] 1. Set up day: 10 mL of sterile DI water was poured on fungal plate. [0219] 2. Fungal spores were scrapped with an L-shaped rod to break away the spores from the agar, prepare spore suspension. [0220] 3. The spore suspension was collected in a falcon tube, and then passed through a sterile piece of cheesecloth to ensure only spores (no agar) were present. [0221] 4. Spore suspension was quantified using a hemocytometer and then diluted to the desired concentrations. [0222] 5. 2 mL of spore suspension was added to each seeded cell in a tray.

    Measurements:

    [0223] Leaf surface area was examined using Image J software and documented by taking top view images of each treatment rep block using a Nikon Camera and tripod. In each picture a ruler placed next to each tomato tray treatment which is used as reference to calibrate the software's scale.

    Results

    Leaf Surface Area (See FIG. 4):

    [0224] The following strains and rates had significant increases in leaf surface area compared to the Untreated Water Control trays: Trichoderma virens, Purpuroecillium lilacinum, and both rates of BIOACT liquid (Standard and 10). Overall, BIOACT liquid 1 and 10 as well as the unformulated P. lilacinum strain 251 had a larger increase in leaf surface foliar canopy compared to UTC, and P. bilaii.

    [0225] BIOACT liquid 1 and BIOACT liquid 10 have a larger leaf surface area compared to all other treatments. Examining BIOACT liquid treatments, the surfactant in the formulation could be aiding in the movement of Purpuroecillium lilacinum strain 251 spores in the soil mix.

    EXAMPLE 5: CORN DRENCH ASSAY WITH A LIQUID FORMULATION OF SPORES OF P. LILACINUM STRAIN 251 (BIOACT LIQUID)

    [0226] Different doses of a liquid formulation of Purpureocillium lilacinum strain 251 (BIOACT liquid) were tested in a small pot drench assay on corn to evaluate plant growth promotion.

    Start Date:

    [0227] Aug. 19, 2015: Set up experiment, Sep. 2, 2015: Take down experiment (14 days experiment).

    Method:

    [0228] 333 cubic inch small pots were filled with autoclaved 3:1 Soil:Sand mix. There were 4 pots per treatment replicate (12 pots total per treatment). 2 sterilized GP7169 GT corn seeds were planted 1-inch deep in each pot, 2 inches apart. 50 milliliters of drench solution were applied per pot at the rate specified for each treatment (see Calculations for Drench section).

    Environment:

    [0229] Light Racks [0230] Light Temperature Range=20 C. low to 30 C. high [0231] Relative Humidity: low 21% to high 62%

    Treatments:

    [0232] 1. UTC [0233] 2. VOTIVO 5% (Assay Positive Control) [0234] 3. BIOACT 2.1310.sup.8 spores/pot [0235] 4. BIOACT 2.1310.sup.9 spores/pot

    Calculations for Drench:

    [0236] All BIOACT drench solutions were made at 600 mL per treatment.

    For BIOACT DC

    [0237] 1. Standard rate: 1.08 mg/100 mL soil4.25=4.59 mg/pot 2.1310.sup.8 spores/pot [0238] 2. 10 rate: 10.8 mg/100 mL soil4.25=45.9 mg/pot 2.1310.sup.9 spores/pot [0239] 3. For VOTIVO 5%, 35 mL of VOTIVO were added into 665 mL of DI water, equivalent to 9.1510.sup.7 CFU/pot.

    Observations Notes and Measurements:

    [0240] 1. Germination (poor, normal), Phytotoxicity (if any) [0241] 2. Whole Plant Biomass (fresh) [0242] 3. WinRhizo Root Analysis

    Results:

    [0243] Germination: normal. Phytotoxicity: none observed

    Fresh Whole Plant Biomass (FWPB) (FIG. 5A):

    [0244] Up to 24 plants were measured. Plants that were not measured include non-germinated seedlings and incomplete plants (incomplete plants are ones without roots or shoots). A dose response was observed with the BIOACT treatments with the highest rate (BIOACT 10) having the best PGE (plant growth enhancement) (according to t-test analysis, if p<0.1, then there is a significant difference). The difference between the highest rate and standard rate was 15% although the standard rate of BIOACT was also significantly better UTC.

    WinRHIZO Analysis of 6 Roots Per Treatment (Sub-Sample of Up to 24 Possible Roots).

    [0245] Results are shown in FIGS. 5B and 5C. Overall, BIOACT liquid 1 and BIOACT liquid 10 treatments had higher values of RL and total number of TFC.

    [0246] BIOACT dose rates were tested in the small pots drench assay for plant growth enhancement. BIOACT doses were tested at standard rate (4.59 mg or 2.1310.sup.8 spores/pot) and 10 times the standard rate (45.9 mg or 2.1310.sup.9 spores/pot) alongside UTC (negative control), and VOTIVO 5% (positive control). Fresh whole plant biomass (FWPB) was taken 14 days after planting. A dose response with BIOACT was observed in the assay. BIOACT standard rate and 10 rate showed significantly higher FWPB than UTC. 6 roots of each treatment were analyzed using the WinRHIZO. A dose rate was observed and similarly to the FWPB results, BIOACT standard and 10 rate had the best root length (RL), and total number of tips (TFC), forks, and crossings. VOTIVO 5% roots generally did not show better root architecture than UTC. In conclusion, plant growth enhancement with BIOACT was shown in a 14-day corn drench bioassay.

    EXAMPLE 6: NEMATODE AND GREENHOUSE TRIALS TO IDENTIFY DIFFERENCES IN PGP (PLANT GROWTH PROMOTION) AND NEMATODE GALLING IN PLANTS TREATED WITH A LIQUID FORMULATION OF P. LILACINUS STRAIN 251 (BIOACT) IN COMPARISON WITH THE UTC IN TWO DIFFERENT SUBSTRATES

    General Materials and Methods:

    Tomatoes:

    [0247] Variety: Ace 55

    [0248] Tomatoes were planted into 200 cell plug trays. The seeds were distributed in a checkerboard pattern to allow sufficient spacing for the plants to expand prior to transplanting. The tomatoes were transplanted into 5.5 pots after two weeks.

    Cucumbers:

    [0249] Variety: Sultan

    [0250] The cucumbers were direct seeded into 32 oz. cups.

    Potting Mixes:

    [0251] Sand: Coarse Play Sand

    [0252] Potting soil

    [0253] Kaolin clay

    [0254] The plants were grown in two different potting mixes either comprising sand and soil or a clay-based additive and soil.

    Experimental Design:

    Treatments:

    [0255] 1. UTUI-Untreated uninfested

    [0256] 2. UTC-Untreated Control, infested with RKN (root knot nematodes)

    [0257] 3. BIOACT 1One application of BIOACT, infested with RKN nematodes

    [0258] 4. BIOACT 2Two applications of BIOACT, infested with RKN nematodes

    Replicate Number: 10

    BIOACT Drench Applications:

    [0259] Prepared BIOACT drench solutions for each 4 trials, each plant received 30 mLs of drench solution. The spore load per application was 5.510.sup.5 viable spores per cm.sup.3 substrate.

    Pesticide Applications:

    [0260] Cucumbers received fungicide treatment in addition

    Tomato Trial in Sandy Soil

    [0261] Plant Date: Jul. 11, 2016

    [0262] Transplant Date: Jul. 25, 2016

    [0263] Infestation Date: Aug. 9, 2016

    [0264] 1st Treatment Date: Jul. 29, 2016

    [0265] 2nd Treatment Date: Aug. 16, 2016

    [0266] Take Down: Oct. 3, 2016

    Results:

    [0267] There was a significant difference in dry shoot weight, dry root weight and total vegetative weight between the UTC and the BIOACT treatments (FIGS. 6C, 6D and 6E). Also gall rating and egg count were significantly different (FIGS. 6A and 6B).

    Tomato Trial in Soil Mixed with Clay Soil

    [0268] Plant Date: Jul. 18, 2016

    [0269] Transplant Date: Aug. 1, 2016

    [0270] Infestation Date: Aug. 17, 2016

    [0271] 1st Treatment Date: Aug. 9, 2016

    [0272] 2nd Treatment Date: Aug. 23, 2016

    [0273] Take Down: Sep. 30, 2016

    Results:

    [0274] Significant reduction in gall development visible in BIOACT treatments compared to control (UTC) (FIG. 7A), there was good nematode infection (galling) seen in the UTC. Differences in total fruit weight (FIG. 7B) were also observed between UTUI and treated plants. There was a significant difference in dry shoot weight between the UTC and the BIOACT 2 (FIG. 7C) and total vegetative weight (FIG. 7D).

    Cucumber Trial in Sandy Soil

    [0275] Plant Date: Jul. 18, 2016

    [0276] Infestation Date: Aug. 9, 2016

    [0277] 1st Treatment Date: Jul. 29, 2016

    [0278] 2nd Treatment Date: Aug. 16, 2016

    [0279] Take Down: Oct. 3, 2016

    Results:

    [0280] Significant differences were observed between BIOACT treatments and UTC (with nematodes) in gall rating and egg count (FIGS. 8A and B). The reduction in the number of eggs compared to UTC was visible. Differences were also observed in dry root weight (FIG. 8C).

    Cucumber Trial in Soil Mixed with Clay

    [0281] Plant Date: Jul. 25, 2016

    [0282] Infestation Date: Aug. 17, 2016

    [0283] 1st Treatment Date: Aug. 9, 2016

    [0284] 2nd Treatment Date: Aug. 23, 2016

    [0285] Take Down: Oct. 3, 2016

    Results:

    [0286] BIOACT liquid 1 performed better than the other three treatments. There was significant increase in fruit yield with one application of BIOACT DC compared to UTUI and UTC. Significant differences in the total fruit weight were observed/FIG. 9A).

    EXAMPLE 7: TRIALS TO EVALUATE NEMATICIDAL EFFICACY AND YIELD INCREASE BY P. LILACINUM STRAIN 251 IN CUCUMBER AND TOMATO IN GREENHOUSE AREA

    General Materials and Methods:

    [0287] This yield study was carried out in high commercial greenhouses representative for most important cucumber and tomato areas in Spain and Italy, respectively. In total 6 trials were conducted in following provinces and trial sites:

    Yield Program for Cucumber:

    [0288] 1. Spain: [0289] Granada/Carchuna [0290] 2. Italy: [0291] Lazio/Sabaudia [0292] Sicily/Vittoria

    Yield Program for Tomatoes:

    [0293] 1. Spain: [0294] Granada/Salobrea [0295] Cadiz/Zahora [0296] 2. Italy: [0297] Puglia/Palagiano

    [0298] The yield program indicated splitting of trials in each crop throughout short crop cycle (spring) as well as long-term crop cycle for each country and experimental site. The selection for variety followed agronomic practice and market requests.

    Experimental Design:

    [0299] The trial sites were selected with a history of root knot nematode population; preferably medium nematode population. To assess initial nematode population and their distribution before trial start soil samples from 4 areas in the experimental field were selected. In each area, 10 vertical core subsamples, discarding the top 10 cm of soil, were sampled within the 10 to 30 cm feeder-root zone of plant, respectively.

    [0300] The experimental set up was fully randomized and comprised 6 treatments with 5 replicates for each trial. Application of the formulated product PL251 (liquid formulation of P. liliacinum comprising BREAK-THRU 5240 and AEROSIL) was applied sequentially with 0.75 L/ha (with 5.410.sup.10 viable spores/mL throughout cropping period.

    [0301] Following agronomic practice one dripper per plant at two lines or one line irrigation system was set up to guarantee best chemigation and daily drip irrigation. Throughout season water amount of daily drip irrigation were adjusted according environmental conditions (air temperature C.) and developmental stage of plants (BBCH).

    [0302] To keep sufficient moisture for fungal growth soil were kept moist after first application of PL 251 liquid at 14 days (d) prior to transplanting. For every further application via drip system (see Table 10A and 10B) the following chemigation cycle was used:

    [0303] of the total water volume (water only)

    [0304] of the total water volume (water+PL 251)

    [0305] 1/3 of the total water volume (water only)

    [0306] Fertilizer management followed local recommendation and farmer practice. To guarantee best pollination for yield thus, bumble bee colonies were used in each trial and greenhouse.

    TABLE-US-00003 TABLE A Treatments for cucumber trial setup: A C D E F Application 14 d B 2 d 4 weeks 8 weeks 20 d Application prior to at after after after after Treatment timing transplanting transplanting transplanting transplanting transplanting transplanting 1 UTC 2 PL 251 0.75 L/ha 0.75 L/ha 0.75 L/ha 0.75 L/ha 3 VELUM 250 G A/ha SC 4 PL 251 0.75 L/ha 0.75 L/ha 0.75 L/ha 0.75 L/ha 4 VELUM 250 G A/ha SC 5 PL 251 0.75 L/ha 0.75 L/ha 5 VELUM 250 G A/ha SC

    TABLE-US-00004 TABLE B Treatments for tomato trial setup: A C D E F G Application 14 d B 2 d 12 days 22 days 4 weeks 8 weeks Application prior to at after after after after after Treatment timing transplanting transplanting transplanting transplanting transplanting transplanting transplanting 1 UTC 2 PL 251 0.75 L/ha 0.75 L/ha 0.75 L/ha 0.75 L/ha 3 VELUM 250 G SC A/ha 4 PL 251 0.75 L/ha 0.75 L/ha 0.75 L/ha 0.75 L/ha 4 VELUM 250 G SC A/ha 5 PL 251 0.75 L/ha 0.75 L/ha 5 VELUM 250 G SC A/ha

    Efficacy Assessment:

    [0307] To investigate root galls development and efficacy of product, 15 plants were randomly selected at harvest in each plot, respectively. Hereunto, roots were digged with whole root system and washed carefully to bare root knot infestation, respectively. Based on root galls nematicidal activity was determined on the basis of the percentage (%) of gall reduction and/or damage of attack on each plant. Following assessment 100% indicate that no galls were found; 0% means that number of galls found on the roots of treated plants was equal to that in untreated control plants. Additionally crop safety was evaluated by estimating percentage of phytotoxicity on the whole plot.

    Yield Assessment:

    [0308] For yield assessment each harvest and/or pick of fruits were recorded throughout cropping period. Pickings were executed as many times as necessary following each variety dependent characteristic. The harvest foresaw first pickings 2 weeks after last application of PL 251, respectively. At each picking date fresh weight of fruits in kilogram per plot were recorded. Further fruits were assessed by size and weight following variety characteristics.

    Results

    Efficacy:

    [0309] PL251 solo treatment indicated reduction in gall development compared to UTC. For the cucumber trial program (FIG. 10A) efficacy for the biologic solo program showed highest efficacy in cucumber with approximately 30% compared to 24% in tomato (FIG. 10B), respectively, VELUM SC (Fluopyram) as chemical solo program however indicated similar efficacy levels in cucumber and tomato. The sequential application of VELUM SC and PL251 throughout cropping period clearly indicated increased efficacy values in both crops.

    Yield:

    [0310] For yield increase significant differences in fruit weight and quantity between UTC and BIOACT DC treated plants were observed. Statistical analysis reveals a significant improvement of fruit weight (kg) in cucumber and tomato following application of PL251, respectively. The results in both crops clearly indicate that PL 251 displays an additional plant health effect independent of its nematicidal potential leading to improved shoot weight in fruiting vegetables.

    EXAMPLE 8: COMPARISON OF PGP EFFECT OF DIFFERENT P. LILACINUM STRAINS

    [0311] To compare the PGP effect, in particular an effect on root growth, of different P. lilacinum strains spore suspensions (each containing 110.sup.7 spores per ml) of three different strains were tested on tomato seedlings. Suspensions contain: [0312] 1. Sterile water [0313] 2. Spores of Purpureocillium lilacinum strain 251 extracted from product BioAct DC [0314] 3. Spores of Purpureocillium lilacinum strain extracted from product Lila-Sin WG [0315] 4. Spores of Purpureocillium lilacinum strain extracted from product Hocusia

    [0316] Tomato seeds were placed on 1% agar plates and the plates were incubated in a vertical position in a Conviron growth chamber set for a period of 12 hrs light and 12 hrs dark, light intensity 320 Mol, temperature 25 C. during the light period, 20 C. during the dark period for 7 days. On day 7, root lengths were measured and treatments were effected afterwards (4 plantlets per treatment) by dipping the roots of the seedlings into the respective solution/suspension 1 to 4 for 15 s. The treated plantlets were placed on 1% agar plates and the roots of each plantlet were placed on the surface of the agar plates. The plates were placed in the fume hood for 5 min to allow for the treatment to dry on the roots. The plates were incubated again in a vertical position in the Conviron growth chamber for another 7 days.

    [0317] The tomato roots were analysed using the program WinRhizo which provides for a complete plant root measurement and analysis, such as length, area, volume, topology, and architecture of plant roots. Each tomato root was scanned to determine a total root surface area (in cm.sup.2) and root length (cm). The results are displayed in FIGS. 11A and 11B.

    [0318] The total root length of each Purpureocillium lilacinum strain was evaluated. As to be seen in FIG. 11A, an increase in root length (P value 0.0007) was observed with a spore suspension based on spores of P. lilacinum strain 251 as compared to a suspension based on P. lilacinum strains isolated from the products Lila-Sin and Hocusia.

    [0319] The total root surface area of each Purpureocillium lilacinum strain was evaluated. As shown in FIG. 11B, a significant increase in lateral root growth was only observed after treatment with P. lilacinum strain 251 (P value 0.0410).