Porous matrices for culture and formulation of agricultural biopesticides and chemicals

Abstract

The present disclosure addresses biologically active formulations for agricultural and other applications that comprise a solid growth substrate that defines an open cell matrix and an active population of one or more microorganisms adhered thereto. The formulation is configured to be applied directly to a plant growth environment and does not require additional isolation and/or processing steps that would separate the microorganisms from the solid growth substrate prior to deployment. The disclosure also addresses related methods.

Claims

1. A biologically active formulation configured to be applied directly to a seed or plant environment, comprising a solid substrate comprised of a plurality of porous particles and one or more populations of metabolically active microorganisms, wherein: each particle of the plurality of porous particles is no larger in size than 5 mm; each particle of the plurality of porous particles is comprised of a plurality of interconnected open cells and/or channels that form an open cell matrix; the porous particles contain a liquid medium, provided that the open cell matrix is impregnated with the liquid medium, the porous particles are not covered by the liquid medium, the liquid medium is at a volume of about 75% to about 85% of the water absorption capacity (WAC) of the porous particle, and a plurality of the porous particles are in direct gaseous communication with the outside environment; and the one or more populations of metabolically active microorganisms are in contact with the liquid medium and present in the open cell matrix of the individual particles.

2. The formulation of claim 1, wherein the one or more populations of metabolically active microorganisms can produce one or more biologically active compounds that affect a plant and its environment.

3. The formulation of claim 1, wherein the one or more populations of microorganisms comprise a prokaryote selected from archaebacteria, Gram negative eubacteria, Gram positive eubacteria, cyanobacteria, or any combination thereof.

4. The formulation of claim 1, wherein the one or more populations of microorganisms comprises a eukaryote selected from fungi, protozoa, or both.

5. The formulation of claim 1, wherein the one or more populations of microorganisms, or a portion of the one or more populations of microorganisms, form a biofilm within the open cell matrix.

6. The formulation of claim 1, wherein the formulation does not include a biofilm.

7. The formulation of claim 1, wherein the plurality of interconnected open cells each have a diameter ranging from about 10 μm to about 1,000 μm.

8. A method for conditioning a plant growth environment, comprising applying the biologically active formulation of claim 1 to a plant or a plant growth environment.

9. The method of claim 8, wherein the plant growth environment is soil, soil mixes, hydroponic medium, or any surface of the plant.

10. The method of claim 8, wherein the applying of the biologically active formulation is to a seed, vegetative cutting, root, rhizome, bulb, tuber, stem, flower, fruit, and/or leaf of the plant.

11. The method of claim 8, wherein the one or more populations of microorganisms comprise a microorganism capable of negatively affecting a targeted pest or weed.

12. The method of claim 8, wherein the one or more populations of microorganisms comprise a microorganism that can consume a pollutant that is toxic or inhibitory to the plant.

13. The formulation of claim 2, wherein the one or more biologically active compounds comprise an agent selected from the group consisting of a pesticide, a nutrient, a biostimulant, a chelator, an enzyme, and an antibiotic.

14. The formulation of claim 3, wherein the Gram negative eubacteria is selected from Pseudomonas, Lysobacter, Rhizobium, Serratia, Methylobacterium, Agrobacterium, Azospirillum, Azotobacter, or any combination thereof.

15. The formulation of claim 3, wherein the Gram positive eubacteria is selected from Bacillus, Paenibacillus, Streptomyces, Arthrobacter, or any combination thereof.

16. The method of claim 8, wherein the one or more populations of microorganisms compete for resources naturally utilized by other organisms present on the plant or in the plant environment.

17. The method of claim 8, wherein the one or more populations of microorganisms comprise a microorganism that is a predator microorganism of an undesired microorganism.

18. The method of claim 8, wherein the predator microorganism is a predaceous fungi.

19. The formulation of claim 1, wherein the formulation is present in a container selected from a plastic, metal, or glass container.

20. The formulation of claim 14, wherein the Gram negative eubacteria comprises Pseudomonas fluorescens Pf-5.

Description

EXAMPLES

(1) The following examples are provided to illustrate exemplary approaches for practicing aspects of the present disclosure and are not intended to limit the scope of the disclosure.

Example 1

(2) This example describes a study demonstrating the efficacy of a formulation combining different open-cell solid substrates and the active microbial culture of an additional Gram negative bacteria, Pseudomonas fluorescens, established thereon to inhibit the growth of a fungal plant pathogen.

(3) Methods and Materials

(4) Solid Substrate Preparation for Growth Experiments

(5) 100% cellulosic household sponges (Industrial Commercial Supply Co, Akron, Ohio) were used to grow bacteria and fungi (pieces of sponge in the range of 2-5 mm and irregularly shaped were generally employed). The sponges were first washed under running tap water for 20 minutes to remove chemical preservatives added by the manufacturer to prevent microbial growth following with distilled water wash and drying for several days at room temperature.

(6) Viscopearl® A model AH-2050L (Rengo Co., LTD, Japan) (also referred to herein as “Viscopearl”) cellulose beads were also tested as a potential porous substrate for bacterial growth.

(7) The sponge and Viscopearl® beads were sterilized by autoclaving prior to any inoculation with bacterial cultures. Specifically, sponge or Viscopearl was weighed in portions and placed in glass tubes covered with aluminum foil. The tubes were autoclaved for 30 minutes at 121° C. and, after cooling, the material was used in the same glass tubes or aseptically transferred into sterile 50 ml plastic tubes until further use.

(8) Solid Substrate Absorption Capacity

(9) To estimate sponge water absorption capacity, a sponge piece of known weight was impregnated with distilled water in a container placed on a scale. Water was added gradually until it started to flow out of the sponge. The ratio of water absorbed by sponge to initial weight of dry sponge was defined as water absorption capacity (WAC). For sponges used, WAC was determined to be between 14 and 18.

(10) A similar approach was used to test the water absorption capacity of the Viscopearl® A model AH-2050L cellulose beads. The WAC of the beads was estimated to be 5.

(11) Bacterial Growth in Cellulosic Substrates

(12) Pseudomonas fluorescens strain Pf-5 (also referred to herein as “Pf-5”) was obtained from USDA-NRRL and grown on King Medium B (KMB) agar plates for 2-3 days. A sterile flask (100 mL capacity) with 15 ml of liquid ⅓ KMB was inoculated from the plate and incubated static with occasional shaking at 23° C.±1° C. for 24-40 hours. The culture was diluted to 10.sup.6 CFU/ml based on absorbance at 600 nm measured using a Lambda 3A UVNIS Spectrophotometer (Perkin Elmer) and plastic cuvettes with 1 cm light path length (OD.sub.600 at 0.1 corresponds to 0.5×10.sup.8 CFU/ml); see, e.g., De La Fuente, L., et al., “phlD-Based Genetic Diversity and Detection of Genotypes of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens,” FEMS Microbiol. Ecol., 56:64-78 (2006), incorporated herein by reference in its entirety). The resulting cell suspension was used to impregnate sterile sponges or Viscopearl in 50 ml Corning plastic tubes to 75%-85% WAC. The tubes were sealed with Parafilm® and incubated static for 2-7 days in the darkness at 23° C.±1° C. After incubation, distilled water was added to sponges and containers were subjected to 2 minutes of Vortex treatment. The OD.sub.600 of resulting cell suspension was measured and CFU/ml was calculated.

(13) Long-Term Viability

(14) Bacterial cells were grown in sponge square pieces (˜50 mg dry weight, 6 pieces per 25 ml tube) as described above. The sponges were left at room temperature in Parafilm®-sealed tubes for several weeks. Single sponge pieces were then removed and placed on the surface of LB plates. Outgrowth was monitored visually. Appearance of outgrowth in 24-48 hours was considered as a positive result.

(15) Dual Plate Fungal Inhibition Assay

(16) Pseudomonas fluorescens strain Pf-5 were grown in Viscopearl, as described above. Four ⅓ PDA plates were inoculated in the center by a ˜6 mm plug of 4-6-day-old Sclerotinia sclerotiorum strain Scl 10-3 (also referred to herein as “Scl 10-3”) obtained from Dr. L. Porter (USDA-ARS, Prosser, Wash.). The plates were left at 23° C.±1° C. for 24 hours in the darkness to initiate fungal growth. Then, beads of Viscopearl with 120 hours old bacterial cultures were placed at 1 cm from the edges of plates, two per plate on the opposite sides. The plates were additionally incubated for 4-6 days until fungus front met the edge of the plate. Viscopearl beads impregnated with sterile medium were used as negative control. The fungal growth inhibition capacity of bacterial strains was determined as described previously in Ashwini N. and Srividya, S., “Potentiality of Bacillus subtilis as Biocontrol Agent for Management of Anthracnose Disease of Chilli Caused by Colletotrichum gloeosporioides OGC1,” 3 Biotech 4:127-136 (2014), incorporated herein by reference in its entirety. Between 3 and 4 plates per experiment were employed.

(17) Results

(18) Pseudomonas fluorescens strain Pf-5 was grown in shredded sponge and in Viscopearl beads. The results presented in Tables 1 and 2 show that fast and reliable growth of the bacteria was observed independent of the type of open-cell matrix upon provision of appropriate nutrients for the bacteria. After 24 hours of incubation, growth was established at ˜70-80% of growth observed at 48 hours data (data not shown). Static cultures in impregnated open-cell matrixes showed higher cell accumulation than liquid static culture (Table 1), or in Viscopearl fully covered by medium (Table 2). This reflects better gas mass transfer in structured media considering the fact that liquid cultures were carried in the same media and started with the same inoculum as those carried in sponge or air-exposed Viscopearl.

(19) TABLE-US-00001 TABLE 1 Growth of P. fluorescens strain Pf-5 in shredded sponge impregnated by liquid medium. Each 50 mL Corning tube received 500 ± 30 mg of dry sterile shredded sponge crumbs. Sponge was then impregnated by 6.5 ml of the medium inoculated with bacteria to make cell concentration at 10.sup.6 CFU/ml. All tubes (three to five for each experiment) were sealed with Parafilm ®. Two independent experiments were performed. The experiment labelled “1L” received 6.5 ml of inoculated medium but no sponge as part of experiments #1 to serve as a control. The sealed tubes were incubated for 48 hours. Experiment # OD.sub.600 AVE CFU/mL* 1 5.82 9.46 ± 0.21 2 5.86 9.47 ± 0.49 1/L 1.722  8.94 ± 0.024 *Data are the means and Standard Deviation

(20) TABLE-US-00002 TABLE 2 Growth of P. fluorescens strain Pf-5 in Viscopearl impregnated by liquid medium. Each 50 mL Corning tube received 500 ± 7 mg of sterile Viscopearl. Three independent experiments were performed (#1 through #3). There were three tubes per experiment. Viscopearl beads in each tube were impregnated with 2.0 ml of medium inoculated by 10.sup.6 CFU/ml of bacteria. Experiments labelled “1/D” received 5 ml of inoculated medium to cover all the beads with liquid as part of experiment #1. All tubes were sealed with Parafilm ® and were incubated for 48 hours. Experiment # OD600 AVE LOG CFU/ml* 1 4.28 9.33 ± 0.11  2 5.22 9.42 ± 0.092 3 4.62 9.36 ± 0.24  1/D 1.634 8.91 ± 0.029 *Data are the means and Standard Deviation

(21) Long-term survival of bacteria grown in sponge was tested as described above. Pseudomonas fluorescens strain Pf-5 was tested after 11 weeks of storage at room temperature (23° C.±3° C.). The culture produced visible colonial growth on the LB plates' surfaces after 24 hours of incubation. As can be concluded from these results, after growth in the open-cell matrix, the microbes can continue to be stored in the matrix for a significant time before deployment.

(22) Next, a dual plate inhibition assay was employed to test the effect of P. fluorescens grown in the open-cell matrix on development of the plant pathogenic fungus, S. sclerotiorum strain Sd 10-3. Results presented in Table 3 showed that P. fluorescens grown in Viscopearl inhibited pathogen growth. About the same level of inhibition was obtained with P. fluorescens grown in household sponge pieces (data not shown). These results illustrate the possibility of cultivation in open-cell matrixes and subsequent application of bacteria with pesticidal features to suppress development of plant-pathogenic microorganisms.

(23) TABLE-US-00003 TABLE 3 Dual plate inhibition of S. sclerotiorum strain Scl 10-3 by P. fluorescens strain Pf-5. Scl 10 Growth Inhibition by Experiment # P. fluorescens Pf-5, %* 1 42.46 ± 6.10  2 48.36 ± 17.18 *Data are the means and Standard Deviation

(24) Conclusion

(25) These data provide further confirmation that beneficial microorganisms can be successfully cultured on solid, porous substrates, such as cellulosic sponge and beads. In this illustrative example, an additional Gram negative bacteria, P. fluorescens, was successfully cultured on multiple forms of solid, open-celled substrates. The cultures established on the solid substrates were able to maintain viability after long-term storage. Additionally, the cultures were demonstrated to retain anti-fungal properties without requiring any further processing or isolation from the solid growth substrate. Thus, these data further demonstrate the utility and efficacy of generating an anti-fungal formulation that contains the biologic component that remains in association with its solid growth substrate.

Example 2

(26) This example describes an additional study demonstrating the efficacy of another formulation that combines an open-cell solid substrate and the active microbial culture of Gram positive bacteria, Bacillus amyloliquefaciens, established thereon to inhibit the growth of a fungal plant pathogen.

(27) Methods and Materials

(28) Solid Substrate Preparation for Growth Experiments

(29) As described above in Example 1.

(30) Solid Substrate Absorption Capacity

(31) As described above in Example 1.

(32) Bacterial Growth in Cellulosic Substrates

(33) Bacillus amyloliquefaciens strain FZB42 (also referred to herein as “FZB42”) was obtained from The Bacillus Genetic Stock Center, Columbus, Ohio, and was maintained on Luria Broth (LB) agar and grown in LB at 23° C.±1° C. (see Idris, E. E. S., et al., “Use of Bacillus subtilis as Biocontrol Agent. VI. Phytohormone Like Action of Culture Filtrates Prepared From Plant Growth-Promoting Bacillus amyloliquefaciens FZB24, FZB42, FZB45 and Bacillus subtilis FZB37,” J. Plant Dis. Prot., 111:583-597 (2004), incorporated herein by reference in its entirety) the same way as described for P. fluorescens Pf-5. OD.sub.600 for B. amyloliquefaciens strain FZB42 at 0.1 corresponds to 7×10.sup.8 CFU/ml (see Talboys, P. J., et al., “Auxin Secretion by Bacillus amyloliquefaciens FZB42 Both Stimulates Root Exudation and Limits Phosphorus Uptake in Triticum aestivum,” BMC Plant Biology 14:51 (2014), incorporated herein by reference in its entirety).

(34) Long-Term Viability

(35) As described above in Example 1.

(36) Dual Plate Fungal Inhibition Assay

(37) Bacillus amyloliquefaciens strain FZB42 were grown in Viscopearl, as described above. Four ⅓ PDA plates were inoculated in the center by a ˜6 mm plug of 4-6-day-old Sclerotinia sclerotiorum strain Scl 10-3 obtained from Dr. L. Porter (USDA-ARS, Prosser, Wash.). The plates were left at 23° C.±1° C. for 24 hours in the darkness to initiate fungal growth. Then, beads of Viscopearl with 120 hours old bacterial cultures were placed at 1 cm from the edges of plates, two per plate on the opposite sides. The plates were additionally incubated for 4-6 days until fungus front met the edge of the plate. Viscopearl beads impregnated with sterile medium were used as negative control. The fungal growth inhibition capacity of bacterial strains was determined as described previously in Ashwini N. and Srividya, S., “Potentiality of Bacillus subtilis as Biocontrol Agent for Management of Anthracnose Disease of Chilli Caused by Colletotrichum gloeosporioides OGC1,” 3 Biotech 4:127-136 (2014), incorporated herein by reference in its entirety. Between 3 and 4 plates per experiment were employed.

(38) Results

(39) Bacillus amyloliquefaciens strain FZB42 were grown in shredded sponge and in Viscopearl beads. The results presented in Tables 4 and 5 show that fast and reliable growth of the bacteria was observed independent of the type of open-cell matrix upon provision of appropriate nutrients for the bacteria. FZB42 cultures resulted in a higher CFU relative to Pseudomonas fluorescens strain Pf-5 (described above in Example 1), likely because a richer medium (LB) was used to grow the FZB42 bacteria. After 24 hours of incubation, growth was established at ˜70-80% of growth observed at 48 hours (data not shown). Static cultures in impregnated open-cell matrixes showed higher cells accumulation than liquid static culture (Table 4), or in Viscopearl fully covered by medium (Table 5). As in Example 1, this data reflects better gas mass transfer in structured media considering the fact that liquid cultures were carried in the same media and started with the same inoculum as those carried in sponge or air-exposed Viscopearl.

(40) TABLE-US-00004 TABLE 4 Growth of B. amyloliquefaciens strain FZB42 in shredded sponge impregnated by liquid medium. Each 50 mL Corning tube received 500 ± 30 mg of dry sterile shredded sponge crumbs. Sponge was then impregnated by 6.5 ml of the medium inoculated with bacteria to make cell concentration at 10.sup.6 CFU/ml. All tubes (three to five for each experiment) were sealed with Parafilm ®. Two independent experiments were performed. The experiment labelled “1L” received inoculated medium but no sponge as part of experiments #1 to serve as a control. The sealed tubes were incubated for 48 hours. Experiment # OD.sub.600 AVE CFU/mL* 1 10.5 10.87 ± 0.81 2 9.30 10.81 ± 0.73 1/L 2.32  10.21 ± 0.037 *Data are the means and Standard Deviation

(41) TABLE-US-00005 TABLE 5 Growth of B. amyloliquefaciens strain FZB42 in Viscopearl impregnated by liquid medium. Each 50 mL Corning tube received 500 ± 7 mg of sterile Viscopearl. Three independent experiments were performed (#1 through #3). There were three tubes per experiment. Viscopearl beads in each tube were impregnated with 2.0 ml of medium inoculated by 10.sup.6 CFU/ml of bacteria. Experiments labelled “1/D” received 5 ml of inoculated medium to cover all the beads with liquid as part of experiment #1. All tubes were sealed with Parafilm ® and were incubated for 48 hours. Experiment # OD.sub.600 AVE LOG CFU/ml 1 9.49 10.82 ± 0.49 2 10.91 10.88 ± 0.31 3 10.32 10.86 ± 0.83 1/D 1.93  10.13 ± 0.094 *Data are the means and Standard Deviation

(42) Long-term survival of bacteria grown in sponge was tested as described above. B. amyloliquefaciens strain FZB42 was tested after 3 weeks of storage at room temperature (23° C.±3° C.). The culture produced visible colonial growth on the LB plates surfaces after 24 hours incubation. As can be concluded from these results, after growth in the open-ccll matrix, the microbes can continue to be stored in the matrix for a significant time before deployment.

(43) Next, a dual plate inhibition assay was employed to test the effect of B. amyloliquefaciens grown in the open-cell matrix on development of the plant pathogenic fungus, S. sclerotiorum strain Scl 10-3. Results presented in Table 6 showed that B. amyloliquefaciens grown in Viscopearl inhibited pathogen growth. These results illustrate the possibility of cultivation in open-cell matrixes and subsequent application of bacteria with pesticidal features to suppress development of plant-pathogenic microorganisms.

(44) TABLE-US-00006 TABLE 6 Dual plate inhibition of S. sclerotiorum strain Scl 10-3 by B. amyloliquefaciens strain FZB42. Scl 10 Growth Inhibition by Experiment # B. amyloliquefaciens, %* 1 52.08 ± 8.57 2 50.40 ± 7.88 *Data are the means and Standard Deviation

(45) Conclusion

(46) These data provide further confirmation that beneficial microorganisms, such as the Gram positive bacteria B. amyloliquefaciens, can be successfully cultured on solid, porous substrates, such as cellulosic sponge and beads. The cultures established on the solid substrates were able to maintain viability after long-term storage. Additionally, the cultures were demonstrated to have anti-fungal properties without requiring any further processing or isolation from the solid growth substrate. Thus, these data further demonstrate the utility and efficacy of generating an anti-fungal formulation that contains the biologic component that remains in association with its solid growth substrate.

Example 3

(47) This example describes an additional study demonstrating the efficacy of a formulation that combines an open-cell solid substrate and the active fungal culture of Trichoderma sp. established thereon to inhibit the growth of a fungal plant pathogen.

(48) Methods and Materials

(49) Solid Substrate Preparation for Growth Experiments

(50) As described above in Example 1.

(51) Solid Substrate Absorption Capacity

(52) As described above in Example 1.

(53) Fungal Growth in Cellulosic Substrates

(54) Trichoderma sp. ATCC 74015 (also referred to herein as “ATCC 74015”) was maintained on Potato Dextrose Agar (PDA; ATCC Medium 336) plates. 1 g Viscopearl in 25 ml glass tubes was impregnated with 5 ml of sterile liquid Potato Dextrose Broth (PDB) inoculated with a scoop of fungal spores/mycelium from a 4-5 day plate. Tubes were sealed in Parafilm® and incubated at room temperature in dim light for 10 to 30 days before use. Culture viability was tested by placing a bead of Viscopearl on the surface of PDA. Outgrowth of mycelia in 24-48 hours was considered as positive viability.

(55) Comparing Fungus Grown from Viscopearl and from PDA Plates

(56) Ten (10) days after inoculating the Viscopearl with Trichoderma sp ATCC 74015, as described above, four beads were removed and a single bead placed in the center of each of four PDA plates. At the same time 5 mm plugs of actively growing ATCC 74015 was taken from the leading growing edge of a 3-day-old PDA plate and placed in the center of each of four PDA plates. The plates were examined every 24 hours and the diameter of the colony measured. After 96 hours, when the mycelia reached the edge of the plate, there was neither a statistical nor a visual difference between any of the plates.

(57) The same experiment was performed thirty (30) days after inoculating the Viscopearl. Again, there was no difference in colony growth.

(58) The growth of the fungus from the Viscopearl was as active as the fungus from the leading edge of 3-day-old actively growing mycelia on PDA.

(59) Dual Plate Fungal Inhibition Assay Comparing Fungus Grown on Viscopearl and on PDA Plates

(60) Dual plate inhibition assay with Trichoderma sp. ATCC 74015 and Sclerotinia sclerotiorum Scl 10-3 was performed according to known techniques. See, e.g., Matroudi S., et al., “Antagonistic Effects of Three Species of Trichoderma sp. on S. sclerotiorum, The Causal Agent of Canola Stem Rot,” Egyptian Journal of Biology, 11:37-44 (2009), incorporated herein by reference in its entirety. Briefly, Trichoderma sp. ATCC 74015 was grown in Viscopearl, as described above.

(61) For dual cultures, either a mycelial plug of actively growing Trichoderma sp. ATCC 74015 isolate (5 mm diameter) incubated on potato dextrose agar or a Viscopearl bead inoculated with ATCC 74015 was placed about 1 cm from the edge of each PDA petri dish. A mycelial plug of S. sclerotiorum strain Scl 10-3 removed from the colony margin of a 3-day-old culture grown on potato dextrose agar was placed 6 cm away from the inoculation site of ATCC 74015 in the same petri dish. Petri dishes similarly inoculated with ATCC 74015 or Scl 10-3 cultures alone were used as controls. Plates were incubated at 22° C., and were examined daily for the formation of inhibition zones between fungal cultures. Radial growth reduction was calculated in relation to growth of the control as follows:
% Inhibition of radial mycelial growth=[(C−T)/C]×100 where C is the radial growth measurement of the pathogen in control plates, and T is the radial growth of the pathogen in presence of ATCC 74015 (Matroudi, et al., 2009).

(62) Results

(63) Trichoderma sp. ATCC 74015 was grown in Viscopearl/PDB as described above. At 10 and 30 days, single beads were removed and placed on PDA surface. Visible mycelial outgrowth from the beads was clearly seen in 48 hours later. Fungal mycelium was also clearly visible in the Viscopearl mass (not shown).

(64) Dual plate inhibition assays were performed to assess competition between fungal species. Using the method described above, the percent inhibition of S. sclerotiorum strain Scl 10-3 by Trichoderma sp. ATCC 74015 using either the 10 and 30-day-old beads or a plug of actively growing mycelia was the same at 55±4%.

(65) Conclusion

(66) These data demonstrate that fungi, such as Trichoderma sp., can be successfully cultured on the disclosed solid, porous substrates, such as cellulosic sponge and beads. Furthermore, the fungal culture in association with its solid substrate served as a formulation that successfully competed with a pathogenic fungus. The formulation possessed anti-fungal properties without requiring any further processing or isolation of the fungal culture from the solid growth substrate. Thus, these data further demonstrate the utility and efficacy of generating an anti-fungal formulation that can contain a wide variety of biologic components, which remain in association with its solid growth substrate as it is applied to the plant environment.

(67) While illustrative embodiments have been described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.