Encapsulated microorganisms and methods thereof

Abstract

The present disclosure relates to compositions and methods to encapsulate a microorganism, where the encapsulated fungal microorganism is metabolically active, can release a volatile chemical through a physical barrier, and has improved shelf life. Methods of treating plants, plant parts such as harvested fruits, and soil or other growing medium for a pest are also disclosed herein.

Description

ENCAPSULATED MICROORGANISMS AND METHODS THEREOF

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No.63 / 618,183, filed January 5, 2024, which is hereby incorporated by reference in its entirety. FIELD

[0002] The present disclosure is directed to encapsulation of metabolically active microorganisms and methods of their making and use. BACKGROUND

[0003] The increasing demand for sustainable and environmentally friendly agricultural practices has led to the exploration of biological control methods as alternatives to chemical pesticides. Bioactive microorganisms, including fungi, have shown promise in managing pests and diseases in agriculture due to their ability to produce antimicrobial, pesticidal, and other functional compounds, and parasitize a variety of plant pathogens and pests. These microorganisms have been utilized for their broad-spectrum antimicrobial and pesticidal activity and effectiveness against a wide range of plant pathogens and pests.

[0004] However, the use of bioactive microorganisms in biocontrol strategies presents several challenges. One of the main issues is the maintenance of their metabolic activity and viability over time, particularly when these organisms are stored or transported. This can limit their shelf life and ultimately their effectiveness in controlling pests and diseases. Moreover, the application of these microorganisms, e.g., in the field and food preservation applications can be challenging due to environmental factors that may affect their survival and activity.

[0005] There is a need to provide shelf-stable bioactive microorganisms that can be used to promote enhanced beneficial biological activity in a variety of contexts, including laboratory and research settings, medicinal development and testing settings, and plant and agricultural settings both in field and controlled environments.

[0006] This disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY

[0007] A first aspect of the present disclosure relates to an encapsulated fungal microorganism comprising a living microorganism of the genus Muscodor encapsulated in aphysical barrier, where the encapsulated fungal microorganism is metabolically active and is capable of releasing a volatile chemical through the physical barrier.

[0008] Another aspect of the present disclosure relates to a method for encapsulating a fungal microorganism. This method involves providing a whole cell broth solution comprising whole cells of a living microorganism of the genus Muscodor; contacting the whole broth solution with alginate; encapsulating the whole cells of the living microorganism in the alginate; and drying the encapsulated microorganism, where the dried encapsulated fungal microorganism is living, metabolically active, and capable of releasing a volatile chemical.

[0009] A further aspect of the present disclosure relates to a method of treating a plant or plant part for a pest. This method involves exposing or contacting a plant, plant part or a growing medium for the plant with volatile chemical from an encapsulated fungal microorganism of any of the embodiments disclosed herein, where the volatile chemical from the encapsulated fungal microorganism treats the plant or growing medium for the pest.

[0010] Disclosed herein are encapsulated microorganisms that produce bio-active compounds to provide control of plant pathogens and soil borne pests. Prior barley-based formulas of Muscodor albus were unstable and costly to produce. In order to reduce the production cost, increase stability, and retain metabolic activity, a barley-free encapsulation formulation of the active microorganisms (e.g., Muscodor albus) was developed as described herein.

[0011] The encapsulation of a bioactive microorganism creates a novel plant protection and plant growth promotion product by maintaining fungal (e.g., Muscodor albus) viability while producing antimicrobial volatiles over time. The volatiles emitted by the metabolically active encapsulated organisms control a variety of plant pathogens and soil grown pests in growing plants and in post-harvest settings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGs.1A-B are photographs of encapsulated Muscodor albus. FIG.1A is a photograph of Muscodor albus encapsulated with alginate and kaolin clay (KaMin80B). FIG.1B is a photograph of Muscodor albus encapsulated with alginate and bentonite clay (Venatural).

[0013] FIG.2 is a process flow chart including photographic examples of steps for encapsulating Muscodor albus whole cell broth (“WCB”).

[0014] FIG.3 shows photographs of Muscodor albus WCB (left photograph) in a 10L fermenter with a pH of 6.10 and the final slurry (right photograph) with all of the ingredients as shown in Table 3 and having a pH of 6.32.

[0015] FIG.4 is a scatter plot showing the low viscosity of the WCB and higher viscosity of the encapsulated formulation of FIG.3.

[0016] FIG.5 shows photographs of encapsulated Muscodor albus WCB during encapsulation (top left), after washing (bottom left), a close-up before drying (top middle) and after drying (bottom middle) and during storage at 4oC (right).

[0017] FIG.6 shows a graph of the mass (g) before drying, after drying and the moisture content (“MC”) reduction (%) during drying processing of various run numbers. The run number refers to a certain encapsulation batch that was dried with a target moisture reduction of 50%. For the first 15 batches, the batch size was 50 g. Then the batch size was increased up to 160 g to reduce the number of runs. Larger batch sizes required a longer time for the drying process. The FIG.5 samples were 4.6 kg before drying, 2.4 kg after drying resulting in a yield of 46% a MC reduction of 48.8% and a MC of 87.6%.

[0018] FIG.7 is a photograph of Muscodor albus WCB grown in either no added carbohydrate (left bottle), grown with added starch (second bottle) or grown with added sucrose (third and fourth bottles) for use in the formulations of Table 5.

[0019] FIG.8 is a photograph of the addition of various Muscodor albus WCB cultures to calcium chloride solution to create encapsulations as described in Example 3.

[0020] FIG.9 is a photograph of formulated Muscodor albus WCB grown in either no added carbohydrate (left bottle), grown with added starch (second bottle) or grown with added sucrose (third and fourth bottles) for use in the formulations of Table 5 after drying as described in Example 3.

[0021] FIG.10 is a process flow chart including photographic examples of steps for in which 2% clay is added into 2% alginate followed by mixing the alginate / clay mixture with Muscodor albus WCB in a 1:1 ratio and encapsulation.

[0022] FIG.11 is a process flow chart including photographic examples of steps for in which 1% alginate and 1% clay are added into the Muscodor albus WCB and encapsulated.

[0023] FIG.12 shows photographs of encapsulated Muscodor albus WCB in a control sample with just alginate (#7 in Table 13) before and after drying, and an encapsulated sample made with Vanatural (#4 in Table 13) before and after drying.

[0024] FIG.13 shows photographs of encapsulated Muscodor albus WCB in the control sample with just alginate (#7 in Table 13) before and after drying as well as the interior of the sliced alginate bead before and after drying.

[0025] FIG.14 shows images of encapsulated Muscodor albus WCB in a sample with Veegum® (smectite clay) before and after drying.

[0026] FIG.15 is a graph showing moisture contents of encapsulated Muscodor albus WCB made with just alginate (control) or with alginate and various clays (A-B4) corresponding to samples 1-7 of Table 13. The samples are as follows: A – KaMin 80B clay, B – attapulgite clay; B1 – bentonite clay (Fisher); B2 – Vanatural® (bentonite clay); B3 – Veegum® (smectite clay); B4 –Vangel® SX (a blend of bentonite clay (80%) and xanthan gum (20%)); and control (alginate with no clay).

[0027] FIG.16 is a graph showing the average number of germinated seeds in the soil bourn fungal pathogen Rhizoctonia solani greenhouse bioassay for plant health for the various clay formulations of Table 13.

[0028] FIG.17 is a graph showing the percent R. solani control compared to the untreated control (UTC).

[0029] FIGs.18A-B are photographs of a split plate assay of the various clay formulations of Table 13.

[0030] FIGs.19A-B are graphs showing the % of R. solani control compared to the untreated control (UTC) at the low rate (one encapsulation bead per pot) (FIG.19A) and the high rate (three encapsulation beads per pot) (FIG.19B).

[0031] FIG.20 is a photograph showing survival of the encapsulated Muscodor albus after drying using a fluid bed dryer (FBD).

[0032] FIGs.21A-B are photographs of a plate survival assay of Muscodor albus encapsulated in the various clay formulations of Table 13 when incubated at 30oC. FIG.21A shows day 0. FIG.21B shows day 7 at 30oC.

[0033] FIG.22 is a photograph of a plate survival assay of Muscodor albus encapsulated in the various clay formulations of Table 13 when incubated at 4oC or RT for 12 weeks.

[0034] FIG.23 is a photograph of a plate survival assay of Muscodor albus encapsulated in the various clay formulations of Table 13 when incubated at 4oC or RT for 12 weeks (FIG. 22A), or when incubated at 4oC or RT for 16 weeks (FIG.22B).

[0035] FIG.24 shows photographic images of encapsulated Muscodor albus WCB in alginate (control) or alginate with Vanatural® (bentonite clay) over 4-weeks when stored at different temperatures.

[0036] FIG.25 shows photographic images of encapsulated Muscodor albus WCB in alginate (control) or alginate with Vanatural® (bentonite clay) over 12-16 weeks when stored at different temperatures.

[0037] FIG.26 shows photographs of the stability of the Muscodor albus using Alginate and Vanatural® (Bentonite clay) with different MC at 30°C. Only Vanatural (Bentonite clay) w / highest mc% can survival at 30°C after 9 days.

[0038] FIG.27 shows photographs of the stability of the Muscodor albus using Alginate and Vanatural® (Bentonite clay) with different MC at 4°C and RT. Cont-2 had a short FDB process time compared to Cont-1, which survived at both 4°C and room temp. The ones that had shorter drying processing time, the higher survival rate. The grow area is much bigger for the B- II-2 and B-II-3 compared to B-II-1.

[0039] FIG.28 shows photographic images of encapsulated Muscodor albus WCB in alginate (control) or alginate with Vanatural® (bentonite clay) over when stored at 8 weeks at different temperatures.

[0040] FIG.29 shows photographic images of mixtures of 1% alginate plus 2% Veegum clay (left) and 1% alginate with 6% Veegum® clay.

[0041] FIG.30 shows photographic images of mixtures of 1% alginate plus 4% Veegum® clay.

[0042] FIG.31 shows photographic images of mixtures of 1% alginate plus various amounts of Veegum® clay.

[0043] FIG.32 shows photographs of the stability of the Muscodor albus using different concentration of Alginate & Veegum® R at different temperatures.

[0044] FIG.33 shows photographic images of mixtures of 1% alginate plus various amounts of Vangel® SX clay.

[0045] FIGs.34A-B show photographic images of mixtures of 1% alginate versus 1% alginate plus Vangel® SX clay, and a close up of an encapsulated bead before and after drying (FIG.34A) and with different moisture contents (MC) using Alginate & Vangel® SX (a blend of bentonite clay and xanthan gum) (FIG.34B).

[0046] FIGs.35A-B show photographic images of the stability of the Muscodor albus WCB using Alginate & Vangel® SX (a blend of bentonite clay and xanthan gum) at 30 °C for 1 week (FIG.35A) and at RT and 4oC (FIG.35B) for 12 weeks.

[0047] FIGs.36A-B show photographic images of encapsulated Muscodor albus using KaMin® 80B or Vanatural® clay before and after drying (FIG.36A), and clay in comparison to controls (FIG.36B)

[0048] FIG.37 shows photographic images of stability testing of the encapsulated Muscodor albus using Kamin 80B & Vanutral at 30ºC for 4 days. The upper left plate was grown at 25oC for 7 days.

[0049] FIG.38 shows photographic images of stability testing of the encapsulated Muscodor albus using Kamin 80B & Vanutral at 30ºC for 7 days. The upper left plate was grown at 25oC for 7 days.

[0050] FIG.39 shows photographic images of the survival of the encapsulated Muscodor albus using Kamin 80B & Vanutral when incubated at RT or 4oC for 8 weeks.

[0051] FIG.40 shows photographic images of survival of the encapsulated Muscodor albus formulations using Kamin 80B & Vanutral when incubated at RT for 12 weeks.

[0052] FIG.41 shows photographic images of survival of the encapsulated Muscodor albus formulations using Kamin 80B & Vanutral when incubated at 4oC for 12 weeks.

[0053] FIG.42 shows photographic images of Muscodor albus 601 WCB grown for the different times indicated.

[0054] FIG.43 is a graph of the solid % of the WCB at the various time points shown in FIG.26.

[0055] FIG.44 is a graph of the pH of the WCB at the at the various time points shown in FIG.26.

[0056] FIG.45 shows photographic images of encapsulated Muscodor albus 601 WCB after harvest at the different time points indicated.

[0057] FIG.46 is a graph showing the viscosity of formulations of alginate and Vangel® SX with Muscodor albus WCB harvested at the time points indicated.

[0058] FIG.47 is a graph showing the viscosity of formulations of alginate and Vangel® SX with Muscodor albus WCB harvested at the time points indicated.

[0059] FIG.48 shows photographic images of encapsulated Muscodor albus WCB made from cultures harvested at different time points.

[0060] FIG.49 shows a close-up photographic image of one of the encapsulated Muscodor albus WCB from FIG.48.

[0061] FIG.50 is shows photographic images of bottled encapsulated Muscodor albus WCB made from cultures harvested at different time points.

[0062] FIG.51 shows photographic images of the survival of the encapsulated Muscodor albus using WCB harvested at different time when incubated at RT for 20 weeks.

[0063] FIG.52 shows photographic images of the survival of the encapsulated Muscodor albus using WCB harvested at different time when incubated at 4oC for 20 weeks.

[0064] FIG.53 is a graph of viscosity of WCB grown for different times.

[0065] FIG.54 shows photographic images of fresh WCB versusFIG. WCB stored at 4oC for 1 week.

[0066] FIG.55 is a graph of viscosity of sodium alginate solution at different concentrations.

[0067] FIG.56 is a graph of yield with different concentrations of alginate solution.

[0068] FIG.57 is a graph showing the correlation between viscosity and yield.

[0069] FIG.58 is a graph showing the correlation between viscosity and yield.

[0070] FIG.59 is a graph with a photographic insert that shows the mortality of nematodes using the encapsulated Muscodor albus WCB that was harvested at different times.

[0071] FIG.60 is shows photographic images of the stability of encapsulated Muscodor albus WCB that was stored at 4oC for 2 weeks before encapsulation and grown at RT or 4oC for 16 weeks.

[0072] FIG.61 shows photographic images of the stability of encapsulated Muscodor albus WCB that was stored at 4oC for 2 weeks before encapsulation and grown at RT or 4oC for 24 weeks.

[0073] FIG.62 shows photographic images of encapsulated Muscodor albus using WCB w / different Nutrients.

[0074] FIG.63 shows graphs of the % solids and pH of Muscodor albus WCB grown with different nutrients.

[0075] FIG.64 is a graph of the final moisture content of encapsulated Muscodor albus WCB after fluid bed dryer (FBD).

[0076] FIG.65 shows photographic images of encapsulated Muscodor albus WCB of the various samples grown with different nutrients.

[0077] FIG.66 shows photographic images of encapsulated Muscodor albus WCB of the various samples grown with different nutrients.

[0078] FIG.67 is a graph of the plant data results showing the average number of germinated seeds using encapsulate Muscodor albus WCB grown with various nutrients.

[0079] FIG.68 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different nutrients and incubated at RT for 20 weeks.

[0080] FIG.69 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different nutrients and incubated at 4oC for 20 weeks.

[0081] FIG.70 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different nutrients and incubated at RT for 24 weeks.

[0082] FIG.71 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different nutrients and incubated at 4oC for 24 weeks.

[0083] FIG.72 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different nutrients, stored at 4oC for 2 weeks before encapsulation and incubated at RT for 20 weeks.

[0084] FIG.73 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different nutrients, stored at 4oC for 2 weeks before encapsulation and incubated at 4oC for 20 weeks.

[0085] FIG.74 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different Nutrients at the initial day.

[0086] FIG.75 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different Nutrients at two weeks.

[0087] FIG.76 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different Nutrients at twenty weeks.

[0088] FIG.77 shows photographic images of Muscodor albus WCB grown in different pH’s.

[0089] FIG.78 shows photographic images of encapsulated Muscodor albus WCB grown in different pH’s at day 0.

[0090] FIG.79 shows photographic images of encapsulated Muscodor albus WCB grown in different pH’s and stored at RT for 8 weeks.

[0091] FIG.80 shows photographic images of encapsulated Muscodor albus WCB grown in different pH’s and stored at 4oC for 8 weeks. The arrow points to a contaminant, likely a fungal mold in the pH 9.2 sample.

[0092] FIG.81 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different pH’s at 30oC for 7 days.

[0093] FIG.82 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different pH’s at RT for 16 weeks.

[0094] FIG.83 shows photographic images of stability studies of the encapsulated Muscodor albus WCB of the various samples grown with different pH’s at 4oC for 16 weeks.

[0095] FIG.84 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different pH at time 0.

[0096] FIG.85 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different pH at 8 weeks.

[0097] FIG.86 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different pH at 12 weeks.

[0098] FIG.87 is a graph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different pH at 16 weeks.

[0099] FIGs.88A-B show a graph and a photograph of the mortality of nematodes using the encapsulated Muscodor albus using WCB with different ratios of Kamin 80B and Vangel. FIG.88B top row (left to right): 89-C1, 89-K, 89-V; bottom row (left to right): 89-V-K&V1, 89- V-K&K2; and 89-V-K&V3.

[0100] FIG.89 is a graph of the mortality of nematodes using optimization of encapsulation to extend the stability of encapsulated Muscodor albus.

[0101] FIG.90 is a graph showing the average number of germinated seeds in the R. solani soil assay.

[0102] FIG.91 is a graph showing the % disease control compared to the untreated control assay.

[0103] FIG.92 is a graph of the nematicide activity using 3 pieces of different encapsulated Muscodor albus WCB at day 0 of a stability study.

[0104] FIG.93 is a graph of the nematicide activity using 1 piece of different encapsulated Muscodor albus WCB at day 0 of a stability study.

[0105] FIG.94 is a photograph showing a plant assay using R. solani inoculated soil.

[0106] FIG.95 is a graph showing the average number of germinated seeds for the plant assay.

[0107] FIG.96 is a photograph of surface treatment of encapsulated beads with anti- sticking agents.

[0108] FIG.97 is a photograph of encapsulated beads with anti-sticking agents at 0.1- 0.5%.

[0109] FIG.98 is a photograph showing the effect of different packaging on stability in mushroom growing bags (control 70.6 hours).

[0110] FIG.99 is a photograph showing the effect of different packaging on stability in mushroom growing bags (cellulose 46.6 hours).

[0111] FIG.100 is a photograph showing the different types of containers tested.

[0112] FIG.101 shows photographs of 1 versus 2 dry cycles.

[0113] FIG.102 shows photographs of stability results of the various containers at day 0 and day 4 at 30oC. C2 packed with the Ziplock bag did not survive.

[0114] FIG.103 shows photographs of stability results of the various containers at day 8 at 30oC. Only the one packed with the vented bottle survived after 8 days at 30oC.

[0115] FIG.104 shows photographs of stability results of the various containers when stored 4 weeks at 22oC (C1-1 dry cycle).

[0116] FIG.105 shows photographs of stability results of the various containers when stored 4 weeks at 22oC (C2-2 dry cycles).

[0117] FIG.106 shows photographs of stability results of the various containers when stored 8 weeks at 22oC (C1-1 dry cycle).

[0118] FIG.107 shows photographs of stability results of the various containers when stored 8 weeks at 22oC (C2-2 dry cycles).

[0119] FIG.108 shows photographs of stability results of the various containers when stored 4 or 8 weeks at 4oC.

[0120] FIG.109 shows photographs of stability results of the various containers at different temperatures.

[0121] FIG.110 is a photograph showing encapsulated Muscodor albus WCB used in greenhouse assays.

[0122] FIG.111 is a graph showing the total chlorophyll in readings taken 2 weeks post- transplant of soybean first trifoliate leaves. Soybean was inoculated with Rhizoctonia solani and treated with encapsulated Muscodor albus WCB formulations in FIG 110.

[0123] FIG.112 shows graphs of average shoot fresh weight at harvest and the % increase compared to the inoculated control of soybeans. Soybean was inoculated with Rhizoctonia solani and treated with encapsulated Muscodor albus WCB formulations in FIG 110.

[0124] FIG.113 shows graphs of average root fresh weight at harvest and the % increase compared to the inoculated control of soybeans. Soybean was inoculated with Rhizoctonia solani and treated with encapsulated Muscodor albus WCB formulations in FIG 110.

[0125] FIG.114 is a graph of average shoot fresh weight at harvest of soybeans. Soybean was inoculated with Rhizoctonia solani and treated with encapsulated Muscodor albus WCB.

[0126] FIG.115 is a graph of average root fresh weight at harvest of soybeans. Soybean was inoculated with Rhizoctonia solani and treated with encapsulated Muscodor albus WCB.

[0127] FIG.116 shows photographs of sugarbeet roots infected with Rhizoctonia solani and disease ratings. Sugarbeets were treated with encapsulated Muscodor albus WCB.

[0128] FIG.117 is a graph of sugarbeet fresh beet weight at harvest that was infected with Rhizoctonia solani and treated with encapsulated Muscodor albus WCB.

[0129] FIG.118 is a graph of sugarbeet average disease rating, based on FIG.116.

[0130] FIG.119 is a graph of sugarbeet fresh shoot weight at harvest.

[0131] FIG.120 is a graph of sugarbeet fresh shoot weight % increase compared to inoculated control.

[0132] FIG.121 is a process flow chart for the Botrytis cinerea inoculum preparation for fruit storage experiments testing the efficacy of the encapsulated Muscodor albus WCB.

[0133] FIG.122 is a process flow chart for the Penicillium digitatum inoculum preparation for fruit storage experiments testing the efficacy of the encapsulated Muscodor albus WCB.

[0134] FIG.123 is a process flow chart for the fruit preparation and inoculation for fruit storage experiments testing the efficacy of the encapsulated Muscodor albus WCB.

[0135] FIG.124 shows photographic images of reduction / prevention of infection by Botrytis cinerea of strawberries using the high rate (3 pieces) of encapsulated Muscodor albus WCB formulations of Table 50 and controls when stored at RT.

[0136] FIG.125 shows photographic images of reduction / prevention of infection by Botrytis cinerea of strawberries using the high rate (3 pieces) encapsulated Muscodor albus WCB formulations of Table 50 and controls as a result of cold storage. On day 0 the encapsulated samples are placed on PDA and the barley control was hydrated. On day 1 the fruit was sterilized and inoculated. The fruit was stored for 3 days at 4oC using industry standard storage.

[0137] FIG.126 is a graph showing the percent control of B. cinerea infection for the RT storage experiment, relating to figure 124.

[0138] FIG.127 is a graph showing the percent control of B. cinerea infection for the 4oC storage experiment, relating to figure 125.

[0139] FIG.128 shows photographic images of reduction / prevention of infection by Botrytis cinerea of strawberries using the high rate (3 pieces) encapsulated Muscodor albus WCB formulations of Table 51 and controls. On day 0 the encapsulated samples are placed on PDA and the barley control was hydrated. On day 1 the fruit was sterilized and inoculated. On day 2 the storage crispers were moved to 4oC. On day 5 the storage crispers were moved to RT.

[0140] FIG.129 is a graph showing the percent control of B. cinerea infection after storage at 4oC using the storage regime of FIG.128.

[0141] FIG.130 shows photographic images of reduction / prevention of infection by Botrytis cinerea of strawberries using the high rate (3 pieces) encapsulated Muscodor albus WCB formulations of Table 51 and controls. The fruit was left at RT for 24 hours beforerefrigeration. On day 0 the encapsulated samples are placed on PDA and the barley control was hydrated. On day 1 the fruit was sterilized and inoculated and moved to 4oC. On day 3 the storage crispers were moved to RT. On day 8 the image was recorded.

[0142] FIG.131 shows photographic images of reduction / prevention of infection by Botrytis cinerea of strawberries using the high rate (3 pieces) encapsulated Muscodor albus WCB formulations of Table 51 and controls. The fruit was left at RT for 48 hours before refrigeration. On day 0 the encapsulated samples are placed on PDA and the barley control was hydrated. On day 1 the fruit was sterilized and inoculated. On day 2 the storage crispers were moved to 4oC. On day 5 the storage crispers were moved to RT. On day 9 the image was recorded.

[0143] FIG.132 is a graph of the percent control of B. cinerea infection after storage at RT for 24 hours before refrigeration using the storage regime of FIG.130.

[0144] FIG.133 is a graph of the percent control of B. cinerea infection after storage at RT for 48 hours before refrigeration using the storage regime of FIG.131.

[0145] FIG.134 shows photographic images of reduction / prevention of infection by Botrytis cinerea of strawberries using the low rate (1 piece) encapsulated Muscodor albus WCB formulations of Table 51 and controls. On day 0 the encapsulated samples are placed on PDA and the barley control was hydrated. On day 1 the fruit was sterilized and inoculated. On day 2 the storage crispers were moved to 4oC. On day 5 the storage crispers were moved to RT. The asterisk indicates no growth of the Vanatural encapsulated Muscodor albus WCB.

[0146] FIG.135 is a graph showing the percent disease control of B. cinerea infection using the storage regime of FIG.134.

[0147] FIG.136 is a graph of the percent control of B. cinerea infection after using the storage regime of FIG.134.

[0148] FIG.137 shows photographic images of reduction / prevention of infection by Botrytis cinerea of grapes using the encapsulated Muscodor albus WCB formulations of Table 50 during RT storage. The high rate (3 pieces) is shown.

[0149] FIG.138 shows photographic images of reduction / prevention of infection by Botrytis cinerea of grapes using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage. The high rate (3 pieces) is shown.

[0150] FIG.139 shows photographic images of reduction / prevention of infection by Botrytis cinerea of grapes using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage. The high rate (3 pieces) is shown. After inoculation, fruit was stored at 4oC for 3 weeks, then stored at RT for 5 days.

[0151] FIG.140 is a close-up photograph of the grapes of FIG.139 taken after 5 days at RT. The image on the left is one of the inoculated control grapes and the image on the right is a grape incubated with the alginate encapsulation.

[0152] FIG.141 shows photographic images of reduction / prevention of infection by Botrytis cinerea of grapes of FIG.139. After storage at RT for 5 days, the encapsulated Muscodor albus WCB was removed after, and the grapes incubated at RT for 4 additional days.

[0153] FIG.142 shows photographic images of reduction / prevention of infection by Botrytis cinerea of grapes of FIG.139. After storage at RT for 5 days, the encapsulated Muscodor albus WCB was removed after, and the grapes incubated at RT for 11 additional days.

[0154] FIG.143 shows photographic images of cut open grapes of FIG.142.

[0155] FIG.144 shows photographic images of the in vitro growth inhibition effect of individual volatile organic compounds (VOC) produced by M. albus on Botrytis cinerea mycelium.

[0156] FIG.145 is a graph of the percent control of B. cinerea growth by each VOC, relating to FIG.144.

[0157] FIG.146 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations of Table 50 during RT storage for 5 days. Inoculation was at 1 x 106spores per ml. The high rate (3 pieces) treatment is shown. White mycelium indicates non-sporulating Penicillium digitatum and green mycelium indicates sporulating Penicillium digitatum.

[0158] FIG.147 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage for 5 days. Inoculation was at 1 x 104spores per ml. The high rate (3 pieces) treatment is shown.

[0159] FIG.148 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations of Table 51 after 3 weeks at 4C storage followed by RT storage for 5 days. Inoculation was at 1 x 105spores per ml. The low rate (1 piece) treatment is shown.

[0160] FIG.149 shows an in vitro split plate assay showing growth inhibition of Penicillium digitatum in the presence of 3 pieces of the formulations of Table 51.

[0161] FIG.150 is a graph of the percent control of Penicillium digitatum growth by each sample.

[0162] FIG.151 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations ofTable 51 during RT storage for 5 days. Inoculation was at 1 x 105spores per ml. A rate of 770 mg of treatment is shown.

[0163] FIG.152 is a graph showing the average mycelium radius (mm) of non- sporulating (left) and sporulating (right) P. digitatum after infection of the samples of FIG.151.

[0164] FIG.153 is a graph showing the percent disease control of non-sporulating (left) and sporulating (right) P. digitatum after infection of the samples of FIG.151.

[0165] FIG.154 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage for 7 days. Inoculation was at 1 x 105spores per ml. A rate of 770 mg of treatment is shown.

[0166] FIG.155 is a graph showing the average mycelium radius (mm) of non- sporulating (left) and sporulating (right) P. digitatum after infection of the samples of FIG.154.

[0167] FIG.156 is a graph showing the percent disease control of non-sporulating (left) and sporulating (right) P. digitatum after infection of the samples of FIG.154.

[0168] FIG.157 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations of Table 515 days after the removal of the treatment (770 mg rate). Inoculation was at 1 x 105spores per ml.

[0169] FIG.158 is a graph showing the average mycelium radius (mm) of control of sporulating P. digitatum of the samples of FIG.157.

[0170] FIG.159 is a graph showing the percent disease control of sporulating P. digitatum of the samples of FIG.157.

[0171] FIG.160 shows photographic images of the reduction of humidity in crisper boxes associated with reduction / prevention of infection by Penicillium digitatum of oranges using the encapsulated Muscodor albus WCB formulations of Table 51, 5 days after the removal of the treatment (770 mg rate). Inoculation was at 1 x 105spores per ml.

[0172] FIG.161 shows photographic images of reduction / prevention of infection by Botrytis cinerea of Granny Smith apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.5 days post infection is shown. Inoculation was at 1 x 105spores per ml. A rate of 150 mg of treatment is shown.

[0173] FIG.162 shows photographic images of reduction / prevention of infection by Botrytis cinerea of Granny Smith apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.10 days post infection is shown. Inoculation was at 1 x 105spores per ml. A rate of 150 mg of treatment is shown.

[0174] FIG.163 is a graph of the percent control of B. cinerea infection by the treatments of FIG.161.

[0175] FIG.164 shows photographic close-up images of the inoculated control apple and encapsulation treatment apple.

[0176] FIG.165 shows photographic images of reduction / prevention of infection by Botrytis cinerea of Granny Smith apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.4 days after removal of the treatments is shown. Inoculation was at 1 x 105spores per ml.

[0177] FIG.166 shows photographic images of reduction / prevention of infection by Botrytis cinerea of Granny Smith apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.10 days after removal of the treatments is shown. Inoculation was at 1 x 105spores per ml.

[0178] FIG.167 shows photographic images of cuts through the open infection wounds of the apples of FIG.16610 days after removing the treatment.

[0179] FIG.168 shows photographic images of apples cut through the open infection wounds of the apples of FIG.16610 days after removing the treatment.

[0180] FIG.169 is a graph showing the average radius (mm) of B. cinerea of the samples of FIG.166.

[0181] FIG.170 shows photographic images of reduction / prevention of infection by Botrytis cinerea of Granny Smith apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.10 days post infection is shown. Inoculation was at 1 x 105spores per ml. A rate of 50 mg of treatment was used.

[0182] FIG.171 shows photographic images of reduction / prevention of infection by Botrytis cinerea of Granny Smith apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage. The treatment was removed from the crisper at 10 days and 16 days post infection is shown. Inoculation was at 1 x 105spores per ml. A rate of 50 mg of treatment was used.

[0183] FIG.172 is a graph showing the average percent control of B. cinerea of the samples of FIG.170.

[0184] FIG.173 is a graph showing the average percent control of B. cinerea of the samples of FIG.171.

[0185] FIG.174 shows photographic images of cut open apples of FIG.171.

[0186] FIG.175 is a graph showing the internal radius of B. cinerea of the samples of FIG.174.

[0187] FIG.176 is a graph showing the average percent control of B. cinerea of the samples of FIG.174.

[0188] FIG.177 shows photographic images of reduction / prevention of infection by Penicillium expansum of Gala apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.5 days post infection is shown. Inoculation was at 1 x 105spores per ml. A rate of 770 mg of treatment is shown.

[0189] FIG.178 is a graph showing the average mycelium radius (mm) of P. expansum of the samples of FIG.177.

[0190] FIG.179 is a graph showing the percent disease control of P. expansum of the samples of FIG.177.

[0191] FIG.180 shows photographic images of reduction / prevention of infection by Penicillium expansum of Gala apples using the encapsulated Muscodor albus WCB formulations of Table 51 during RT storage.8 days post infection is shown. Inoculation was at 1 x 105spores per ml. A rate of 770 mg of treatment is shown.

[0192] FIG.181 is a graph showing the average mycelium radius (mm) of P. expansum of the samples of FIG.180.

[0193] FIG.182 is a graph showing the percent disease control of P. expansum of the samples of FIG.180.

[0194] FIG.183 shows photographic images of the apples of FIG.1805 days after the treatment was removed.

[0195] FIG.184 is a graph showing the average mycelium radius (mm) of P. expansum of the samples of FIG.183.

[0196] FIG.185 is a graph showing the average mycelium radius (mm) of sporulating P. expansum of the samples of FIG.183.

[0197] FIG.186 shows cut open apples of the samples of FIG.183.

[0198] FIG.187 is a graph showing the average interior radius (mm) of P. expansum of the samples of FIG.186.

[0199] FIG.188 is a graph showing the percent disease control of sporulating P. expansum of the samples of FIG.186.

[0200] FIG.189 is a photographic image of the crispers used in the fruit experiments.

[0201] FIGs.190A-B show a graph and photograph of encapsulated nutrients samples at 6 months that show the mortality of nematodes (nematicide in vitro bio-assay) using the encapsulated Muscodor albus WCB with different nutrients. FIG.190B top row (left to right):control-1, Sucrose-1, Sucrose-2, Cellulose-1; bottom row (left to right): Cellulose-2, Starch-1, and Starch-2.

[0202] FIGs.191A-B show a graph and photograph of encapsulated samples of Table 5 that show the mortality of nematodes (nematicide in vitro bio-assay). FIG.191B top row (left to right): Formulations 2.1, 2.2, 2.3, and 2.4 at the low rate; bottom row (left to right): Formulations 2.1, 2.2, 2.3, and 2.4 at the high rate.

[0203] FIGs.192A-B show graphs of encapsulated samples of Table 5 of the nematicide in vitro bio-assay results. FIG.192A shows the low rate and FIG.192B shows the high rate.

[0204] FIG.193 is a graph showing results of a Rhizoctonia solani in planta bioassay of sample formulations with different pH’s.

[0205] FIGs.194A-B show a graph and photograph of encapsulated samples of Table 51 that show the mortality of nematodes (nematicide in vitro bio-assay). FIG.195B top row (left to right): Samples 1-3 at the low rate; bottom row (left to right): Samples 1-3 at the high rate.

[0206] FIG.195 shows photographic images of reduction / prevention of infection by Penicillium digitatum of oranges of encapsulated Muscodor albus WCB formulations. From bottom to top: inoculated control, 2% alginate with sucrose, 2% alginate with sucrose and Kamin 80 B, 2% alginate with sucrose and Kamin 80 B and 25% glycerol. DETAILED DESCRIPTION

[0207] The present disclosure relates to compositions comprising an encapsulated microorganism, such as a fungal microorganism, in a physical barrier. In some embodiments, the encapsulated microorganism is metabolically active and is capable of releasing a volatile chemical through the physical barrier. The present disclosure also relates to methods for encapsulating a microorganism. In some embodiments, the encapsulated microorganism is metabolically active, can release a volatile chemical through a physical barrier, and has improved shelf life. Methods of treating plants, plant parts such as harvested fruits, and soil or other growing medium for a pest are also disclosed herein.

[0208] As used throughout the present disclosure, and in any of the aspects and / or embodiments described herein, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.

[0209] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0210] As used herein, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

[0211] As used herein, “derived from” means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source. In the event that the “source” is an organism, “derived from” means that it may be isolated or obtained from the organism itself or medium used to culture or grow said organism.

[0212] As used herein, “whole cell broth” or “WCB” refers to a liquid culture containing both cells and media. If microorganisms are grown on a plate the cells can be harvested in water or other liquid to form a whole cell broth.

[0213] The term “supernatant” refers to the liquid remaining when cells that are grown in broth or harvested in another liquid from an agar plate are removed by centrifugation, filtration, sedimentation, or other means well known in the art.

[0214] As used herein, “extract” refers to liquid substance removed from cells by a solvent (water, detergent, buffer, chemical such as acetone) and separated from the cells by centrifugation, filtration or other method.

[0215] As used herein, “metabolite” or “volatile” refers to a compound, substance or by product of a microorganism, or supernatant, filtrate, or extract obtained from fermentation of a microorganism or produced by the microorganism after fermentation.

[0216] The term “pest infestation” as used herein, refers to the presence of a pest in an amount that causes a harmful effect including a disease or infection in a plant or plant part, a host, a host population, or emergence of an undesired pest or weed in a growth system.

[0217] A “pesticide” as used herein, is a substance derived from a biological product or chemical substance that increases mortality or inhibits the growth rate of plant pests and includes but is not limited to nematicides, insecticides, plant fungicides, plant bactericides, and plant viricides.Encapsulated Microorganisms

[0218] One aspect of the present disclosure relates to an encapsulated microorganism comprising a living microorganism encapsulated in a physical barrier, wherein the encapsulated microorganism is metabolically active and is capable of releasing a volatile chemical through the physical barrier.

[0219] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0220] Suitable microorganisms for use in this and all other aspects of the present disclosure include bacteria, viruses, and protozoa. In some embodiments, the microorganism is a bacteria. Exemplary, bacteria from the genera Rhizobium, Azospirillum, and Nitrosomonas are known for their ability to fix atmospheric nitrogen, enhancing soil fertility and promoting plant growth. Bacillus bacteria, particularly species like Bacillus subtilis and Bacillus thuringiensis, are known for their roles in agriculture. Bacillus subtilis produces a variety of enzymes and antibiotics that can suppress harmful microorganisms, while Bacillus thuringiensis produces toxins that are lethal to certain insect pests. Beneficial protozoa also play a role in agriculture, primarily in the soil ecosystem. Protozoa, such as amoebae, ciliates, and flagellates, contribute to nutrient cycling by feeding on bacteria and other microorganisms, releasing nutrients that can be used by plants. Some protozoa also have the ability to control plant pathogens. For example, certain species of the protozoan genus Tetrahymena have been found to feed on plant pathogenic fungi, potentially reducing the incidence of plant diseases.

[0221] In some embodiments, the microorganism is a fungus. Exemplary fungal microorganisms for this and other aspects of the disclosure include, without limitation, fungi species from genera like Trichoderma, Beauveria, Metarhizium, and Muscodor, which are known for their antimicrobial, insecticidal, and plant growth-promoting properties. These and other bioactive fungi play a crucial role in various ecological and industrial processes due to their ability to produce a wide range of bioactive compounds that can suppress the growth of harmful pathogens, control pests, and enhance plant health and productivity among others. In agriculture, these fungi are used as biocontrol agents, providing an environmentally friendly alternative to chemical pesticides and fertilizers. In some embodiments, the encapsulated microorganism in the compositions of the present disclosure is of the genus Muscodor. In some embodiments, the microorganism is Muscodor albus. Muscodor is a genus of endophytic fungi that includes several species, such as Muscodor albus, Muscodor vitigenus, Muscodor crispans, Muscodor roseus, Muscodor sutura, and Muscodor yucatanensis, among others.

[0222] Accordingly, in some embodiments, the present disclosure relates to an encapsulated fungal microorganism comprising a living microorganism of the genus Muscodor encapsulated in a physical barrier, where the encapsulated fungal microorganism is metabolically active and is capable of releasing a volatile chemical through the physical barrier.

[0223] In some embodiments, the present disclosure relates to an encapsulated microorganism comprising a living microorganism other than Muscodor encapsulated in a physical barrier, where the encapsulated microorganism is metabolically active and is capable of releasing a volatile chemical through the physical barrier.

[0224] Cultures of Muscodor albus appear whitish and have an overall greasy tone (see for example, U.S. Patent No.10,869,482, which is hereby incorporated by reference in its entirety). Under a stereoscopic microscope the growing hyphae have a spear-like appearance with little or no immediate branching patterns. The organism has never been observed to produce spores in culture or on tissues of its host plant. The mycelia hyphae are intertwined and rope like in appearance and have individual hyphal diameters ranging from 1-3 μm. This characteristic is common in all Muscodor spp. (Strobel, Current Opinions in Microbiology 9:240-244 (2006); Strobel, G. A. (2012) Microbiology Today 39-2:108-111 and Strobel, G. A.2011. Phytochemistry Reviews 10:165-172, each of which is hereby incorporated by reference in its entirety).

[0225] Compounds, metabolites or volatiles may be obtained, are obtainable, or may be derived from a bioactive microorganism such as a Muscodor strain or culture. Specifically, these organisms can be cultivated to obtain certain compounds and / or compositions by isolating compounds or chemicals from the culture of these organisms. In particular, the organisms may be cultivated in nutrient medium using methods known in the art. For example and without limitation, the organisms may be cultivated by shake or non-shake cultivation, small scale or large-scale fermentation (including but not limited to continuous, batch, fed-batch, or solid-state fermentations) in a laboratory or industrial fermentation apparatus in a suitable medium and under conditions allowing cell growth or on solid substrates such as agar. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are known and available or may be available from commercial sources or prepared according to published compositions.

[0226] In some embodiments and as set forth in the Examples below, the Muscodor strain may be cultivated on agar media such as potato dextrose agar (PDA) (Ezra et al., “New Endophytic Isolates of Muscodor albus, A Volatile-Antibiotic-Producing Fungus,”Microbiology 150.12:4023-4031 (2004), which is hereby incorporated by reference in its entirety).

[0227] In some embodiments, volatile organic compounds (VOCs) are produced by the cultured microorganism and then these VOCs may be used to control plant pathogens and infestations, e.g., in treatment methods described infra. Exemplary VOCs from Muscodor albus include, without limitation: Ethanol; Propanol; 2-Butanone, 4-hydroxy-; Ethyl Acetate; Propanoic acid, ethyl ester; 1-Butanol, 3-methyl-; 1-Butanol, 2-methyl-; Propanoic acid, 2- methyl-, ethyl ester; Butanoic acid, 2-methyl-, methyl ester; Butanoic acid, 2-methyl-, ethyl ester; Propanoic acid, 2-methyl-, butyl ester; 1-Butanol, 3-methyl-, acetate; Ethyl tiglate; Phenylethyl Alcohol; Azulene, 1,2,3, 5,6,7,8, 8a-octahydro-l,4-dimethyl-7-(l- methylethenyl)-, [lS-(l.alpha.,7. alpha., 8a.beta.)]-. And at least one of: Propanoic acid, 2-methyl-, methyl ester; Acetic acid, 2-methylpropyl ester; 1-Butanol, 2-methyl-, acetate; Propanoic acid, 2-methyl-, butyl ester; Benzene, methoxy-; 3- Octanone; Propanoic acid, 2-methyl-, 3-methylbutyl ester; Acetic acid, 2-phenylethyl ester; (-) Aristolene; Cyclohexane, l-ethenyl-l-methyl-2,4-bis(l- methylethenyl)-; Azulene, l,2,3,4,5,6,7,8-octahydro-l,4-dimethyl-7-(l-methylethenyl)-,(lS- (l.alpha.,4.alpha.,7.alpha.)]-; Bicyclo[5.3.0]decane, 2-methylene-5-(l-methylvinyl)-8-methyl-; and optionally a carrier, diluent or adjuvant. In a specific embodiment, the composition comprises Ethanol; Propanol; 2- Butanone, 4-hydroxy-; Ethyl Acetate; Propanoic acid, 2-methyl- , methyl ester; Propanoic acid, ethyl ester; 1-Butanol, 3-methyl-; 1-Butanol, 2-methyl-; Propanoic acid, 2-methyl-, ethyl ester; Acetic acid, 2-methylpropyl ester; Butanoic acid, 2- methyl-, methyl ester; Butanoic acid, 2-methyl-, ethyl ester; Propanoic acid, 2-methyl-, butyl ester; 1-Butanol, 3-methyl-, acetate; 1-Butanol, 2-methyl-, acetate; Propanoic acid, 2-methyl-, butyl ester; Benzene, methoxy-; Ethyl tiglate; 3- Octanone; Propanoic acid, 2-methyl-, 3- methylbutyl ester; Phenylethyl Alcohol; Acetic acid, 2-phenylethyl ester; (-)Aristolene; Cyclohexane, 1 -ethenyl- 1 -methyl-2,4-bis( 1 -methylethenyl)- ; Azulene, 1,2,3,4,5, 6,7, 8- octahydro-l, 4-dimethyl-7-(l- methylethenyl)-,(lS-(l.alpha.,4.alpha.,7. alpha.)]-; Bicyclo [5.3.0] decane, 2-methylene-5- ( 1 -methy 1 vinyl)- 8-methyl- ; and, Azulene, 1,2,3, 5,6,7, 8, 8a- octahydro-l,4-dimethyl-7-(l- methylethenyl)-, [lS-(l.alpha.,7. alpha., 8a.beta.)]-. and optionally a carrier, diluent or adjuvant. In some embodiments the volatiles comprise ethanol; ethyl acetate; l-Propanol, 2- methyl; Propanoic acid, 2-methyl-, methyl ester; 1-Butanol, 3-methyl; 1-Butanol, 2-methyl; and Propanoic acid, 2-methyl-, and / or ethyl ester. In some embodiments, the volatile compound is one or more of 2 methyl-propanoic acid, 2 methyl-1-butyl acetate, ethanol, phenyl ethyl alcohol, and 3 methyl-1-butyl acetate. In some embodiments, the volatile compound isselected from 2 methyl-propanoic acid, 2 methyl-1-butyl acetate, ethanol, phenyl ethyl alcohol, 3 methyl-1-butyl acetate, 2 methyl-1-butanol, 3 methyl-1-butanol and combinations thereof.

[0228] Specific exemplary compositions of encapsulated microorganisms of the present disclosure are described in the Examples infra. These exemplary compositions are described with respect to their WCB content comprising a microorganism and other components that form a physical barrier to enclose or protect the microorganism and preserve its metabolic activity and / or viability. In some embodiments, the encapsulated microorganism compositions of the present disclosure comprise the exemplary compositions described in the Examples and shown in the accompanying Figures, and functional variants thereof. For example, in some embodiments, the encapsulated microorganism of the present disclosure comprises: 50% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; and 1% w / w kaolin clay. Methods of Encapsulating a Microorganism

[0229] Another aspect of the present disclosure relates to a method for encapsulating a microorganism. This method involves providing a broth solution comprising whole cells of a living microorganism, contacting the broth solution with alginate, encapsulating the whole cells of the living microorganism in the alginate, and drying the encapsulated microorganism, wherein the dried encapsulated fungal microorganism is living, metabolically active, and capable of releasing a volatile chemical.

[0230] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0231] Various encapsulation compositions that may be made according to the methods of this aspect of the present disclosure are illustrated in the Examples and Figures herein.

[0232] In some embodiments, the method of encapsulation forms a physical barrier to protect the microorganism. As used herein, the term “physical barrier” and the concept of “encapsulation” are used interchangeably. In other words, as described herein, an encapsulated microorganism refers to a microorganism encapsulated by a physical barrier. In some embodiments, an “encapsulation composition” as described herein comprises comprising a microorganism (e.g., a microorganism in a whole cell broth) and a physical barrier (e.g., alginate) encapsulating the microorganism.

[0233] In some embodiments, the microorganism encapsulated via the methods disclosed herein is a fungal microorganism that is fermented in a whole cell broth. In some embodiments, the whole cell broth comprises nutrients needed to sustain metabolic activity and / or viability ofthe microorganism. For example, in some embodiments, the whole cell broth comprises a carbohydrate selected from sucrose, cellulose, trehalose, starch, any combination thereof, or any other carbohydrate needed or used by the microorganism. In some embodiments, the carbohydrate is sucrose, starch, trehalose, or cellulose, which is present in the whole cell broth in an amount comprising 1g / L, 5g / L, 10g / L, 20 g / L, 30g / L or any number or range therein, of the WCB.

[0234] In some embodiments, the encapsulated microorganism comprises whole cell broth (WCB) comprising Muscador albus in a physical barrier. In some embodiments, the WCB comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (or any number or range therein) by total weight of the composition of the physical barrier or encapsulation composition. In some embodiments, the whole cell broth is combined with glycerol. In some embodiments, the glycerol comprises 10%- 70% by weight of the WCB or encapsulation composition, or any value or range therein. In some embodiments, the glycerol comprises 25% by weight of the WCB or encapsulation composition.

[0235] In some embodiments, the encapsulation composition comprises alginate. In some embodiments, the alginate is sodium alginate. In some embodiments, the alginate is 0.1%, 0.5%, 1%, 2%, 5%, 10%, or any value or range therein by weight of the WCB or encapsulation composition. In some embodiments the alginate is 1% by weight of the WCB or encapsulation composition.

[0236] In some embodiments, the encapsulation composition comprises clay. Presence of the clay in the encapsulation composition provides, as non-limiting examples, shelf-life extension, a rheology modifier, and aesthetic characteristics. Exemplary clays include, without limitation, bentonite, attapulgite, kaolin, montmorillonite, zeolite, smectite clay or combinations thereof. In some embodiments the clay is a combination of bentonite clay and xanthan gum. In some embodiments, the clay comprises 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 6% (or any number or range therein) by weight of the encapsulation composition. In some embodiments, the clay comprises 1% by weight of the encapsulation composition.

[0237] In some embodiments, the encapsulation composition comprises alginate (as an encapsulating agent forming a physical barrier) and clay. In some embodiments, the encapsulation composition comprises alginate and kaolin clay. In some embodiments, the encapsulation composition comprises alginate, clay, and a blend of bentonite clay and xanthan gum.

[0238] In some embodiments, the pH of the encapsulated composition is pH 2.5-9.2 (or any value or range therein). In some embodiments, the pH is 6.0 to 8.2.

[0239] The encapsulated composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, flowability, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA List 4B. In some embodiments, the surfactant is a silica solution, such as SIRPERNAT 22S. Other compatible flow aids may be incorporated. The flow aid may be part of a rinse solution or added directly the encapsulation solution. The concentration of flow aid plus surfactant may range between 0.01-35% by weight of the solution or total formulation, or any value or range therein.

[0240] In some embodiments, encapsulation occurs by contacting the alginate encapsulation composition with calcium to form calcium alginate beads that encapsulate the microorganism. In some embodiments, the calcium is calcium chloride. In some embodiments, the calcium solution is 0.1% to 5% by weight or any number or range therein.

[0241] In some embodiments, the encapsulation composition comprises water. In some embodiments, the encapsulated composition is dried to a moisture content of 40% to 99% or any number or range therein. In some embodiments, the encapsulated alginate beads are dried to a moisture content of 50% to 99% or any number or range therein. In some embodiments, the moisture content is 80% to 95%. In some embodiments, the encapsulated alginate beads are dried to a moisture content of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or any number or range therein. In other words, the encapsulated composition may comprise from 5% to 99% moisture, or any number or range therein.

[0242] In some embodiments, the yield of the encapsulated composition is 40% to 75% before drying, or any number or range therein. In some embodiments, the yield of the encapsulated alginate beads is 20% to 50% after drying, or any number or range therein. In some embodiments, yield is determined by calculating the difference in weight before and after the drying process.

[0243] In some embodiments, the encapsulated fungal microorganism is capable of remaining metabolically active in its encapsulated state for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24 weeks (or any value or range therein) or more than 24 weeks at room temperature. In some embodiments, a metabolically active or viable microorganism is metabolically active or viable in its encapsulated state or is metabolically active or viable after being in an encapsulated state and upon release from its physical barrier after any of the period of times or storage temperatures described herein. In some embodiments, the encapsulated fungal microorganism iscapable of remaining metabolically active for at least 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 11, 12, 18, or 24 months (or any value or range therein) at room temperature. In some embodiments, the encapsulated fungal microorganism is capable of remaining metabolically active for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24 weeks (or any value or range therein) or more than 24 weeks under refrigeration (4oC). In some embodiments, the encapsulated fungal microorganism is capable of remaining metabolically active for at least 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 11, 12, 18, or 24 months (or any value or range therein) under refrigeration (4oC). In some embodiments, the encapsulated fungal microorganism is capable of remaining metabolically active for at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, or 30 days (or any value or range therein) or more than 30 days at 30oC. Metabolic activity can be determined by any of the biological assays described herein or known to those of skill in the art (see e.g., Examples 1-12).

[0244] In some embodiments, the encapsulated fungal microorganism remains viable in its encapsulated state for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24 weeks (or any value or range therein) or more than 24 weeks at room temperature. In some embodiments, the encapsulated fungal microorganism remains viable for at least 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 11, 12, 18, or 24 months (or any value or range therein) at room temperature. In some embodiments, the encapsulated fungal microorganism remains viable for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24 weeks (or any value or range therein) or more than 24 weeks under refrigeration (4oC). In some embodiments, the encapsulated fungal microorganism remains viable for at least 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, 11, 12, 18, or 24 months (or any value or range therein) under refrigeration (4oC). In some embodiments, the encapsulated fungal microorganism remains viable for at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, or 30 days (or any value or range therein) or more than 30 days at 30oC.

[0245] The compositions disclosed herein (i.e., the encapsulated microorganism) may be combined with one or more additional microorganisms and / or chemicals (e.g., pesticide, nematicide, bactericide, fungicide, insecticide) in a more complex composition comprising the encapsulated microorganism and an additional useful agent. Such additional microorganism may include, but is not limited to, an agent derived from Bacillus spp., Paecilomyces spp., Pasteuria spp. Pseudomonas spp., Brevabacillus spp., Lecanicillium spp., non-Ampelomyces spp., Pseudozyma spp., Streptomyces spp, Burkholderia spp, Trichoderma spp, Gliocladium spp. or other Muscodor strains. Alternatively, the agent may be a natural oil or oil-product having nematicidal, fungicidal, bactericidal and / or insecticidal activity (e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil, pyrethrum).

[0246] In some embodiments, the pesticide may be a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine), a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil, and a quinone outside inhibitor (e.g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yet another particular embodiment, the anti-fungal agent is a quinone, e.g., quinoxyfen (5,7- dichloro-4-quinolyl 4-fluorophenyl ether). The anti-fungal agent may also be derived from a Reynoutria extract.

[0247] The fungicide can also be a multi-site non-inorganic, chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine, and cyano-acetamide oxime.

[0248] As noted above, the composition may further comprise a nematicide. This nematicide may include, but is not limited to, chemicals such as organophosphates, carbamates, and fumigants, and microbial products such as avermectin, Myrothecium spp., Biome (Bacillus firmus), Pasteuria spp., Paecilomyces spp., and organic products such as saponins and plant oils. Treatment Methods

[0249] A further aspect of the present disclosure relates to a method of treating a plant or plant part for a pest. This method comprises exposing or contacting a plant, plant part or a growing medium for the plant with a volatile chemical from an encapsulated fungal microorganism of any of the embodiments disclosed herein, wherein the volatile chemical from the encapsulated fungal microorganism treats the plant or growing medium for the pest.

[0250] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0251] In carrying out the treatment methods of the present disclosure, the compositions described herein may be applied to a plant, plant part, or growing media using methods known in the art. For example and without limitation, the compositions may be applied to and around plants or plant parts. Plants are to be understood as meaning all plants and plant populations suchas desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants which can be obtained by conventional plant breeding and optimization methods or by biotechnological and genetic engineering methods or by combinations of these methods, including the transgenic plants and including the plant cultivars protectable or not protectable by plant breeders’ rights. Plant parts are to be understood as meaning all parts and organs of plants above and below the ground, such as shoot, leaf, fruit, flower and root, non-limiting examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. The plant parts also include, but are not limited to, harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offshoots and seeds.

[0252] Plants that may be treated include but are not limited to: (A) major edible food crops, which include but are not limited to, (1) cereals, African rice, barley, durum wheat, einkorn wheat, emmer wheat, finger millet, foxtail millet, hairy crabgrass, Indian barnyard millet, Japanese barnyard millet, maize, nance, oat, pearl millet, proso millet, rice, rye, sorghum, Sorghum spp., rye, spelt wheat); (2) fruits (e.g., abiu, acerola, achacha, African mangosteen, alpine currant, ambarella, American gooseberry, American persimmon, apple, apricot, arazá, Asian palmyra palm, Asian pear, atemoya, Australian desert raisin, avocado, azarole, babaco, bael, banana, Barbados gooseberry, bergamot, betel nut, bignay, bilberry, bilimbi, binjai, biriba, bitter orange, black chokeberry, black mulberry, black sapote, blackberry, blue-berried honeysuckle, borojo, breadfruit, murmese grape, button mangosteen, cacao, calamondin, canistel, cantaloupe, cape gooseberry, cashew nut, cassabanana, cempedak, charichuelo, cherimoya, cherry, cherry of the Rio Grande, cherry plum, Chinese hawthorn, Chinese white pear, chokeberry, citron, cocona, coconut, cocoplum, coffee, coffee Arabica, coffee robusta, Costa Rica pitahaya, currants, custard apple, date, date-plum, dog rose, dragonfruit, durian, elderberry, elephant apple, Ethiopian eggplant, European nettle tree, European wild apple, feijoa, fig, gac, genipapo, giant granadilla, gooseberry, goumi, grape, grapefruit, great morinda, greengage, guava, hardy kiwi, hog plum, horned melon, horse mango, Indian fig, Indian jujube, jabuticaba, jackberry, jackfruit, Japanese persimmon, Japanese wineberry, jocote, jujube, kaffir lime, karanda, kei apple, kepel apple, key lime, kitembilla, kiwi fruit, korlan, kubal vine, kuwini mango, kwai muk, langsat, large cranberry, lemon, Liberian coffee, longan, loquat, lychee, malay apple, mamey sapote, mammee apple, mango, mangosteen, maprang, marang, medlar, melon, Mirabelle plum, miracle fruit, monkey jack, moriche palm, mountain papaya, mountain soursop, mulberry, naranjilla, natal plum, northern highbush blueberry, olive, otaheite gooseberry, oval kumquat, papaya, para guava, passion fruit, pawpaw,peach, peach-palm, pear, pepino, pineapple, pitomba Eugenia luschnathiana, pitomba talisia esculenta, plantain, plum, pomegranate, pomelo, pulasan, purple chokeberry, quince, rambutan, ramontchi, raspberry, red chokeberry, red currant, red mulberry, red-fruited strawberry guava, rhubarb, rose apple, roselle, safou, salak, salmonberry, santol, sapodilla, satsuma, seagrape, soncoya, sour cherry, soursop, Spanish lime, Spanish tamarind, star apple, starfruit, strawberry, strawberry guava, strawberry tree, sugar apple, Surinam cherry, sweet briar, sweet granadilla, sweet lime, tamarillo, tamarind, tangerine, tomatillo, tucuma palm, Vaccinium spp., velvet apple, wampee, watermelon, watery rose apple, wax apple, white currant, white mulberry, white sapote, white star apple, wolfberry (Lyceum barbarum, L.chinense), yellow mombin, yellow pitaya, yellow-fruited strawberry, guava, (3) vegetables (e.g., ackee, agate, air potato, Amaranthus spp., American groundnut, antroewa, armenian cucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ash gourd, asparagus, avocado, azuki bean, bambara groundnut, bamboo, banana, Barbados gooseberry, beet, beet root, bitter gourd, bitter vetch, bitterleaf, black mustard, black radish, black salsify, blanched celery, breadfruit, broad bean, broccoli, Brussels sprout, Buck's horn plantain, buttercup squash, butternut squash, cabbage, caigua, calabash, caraway seeds, carob, carrot, cassabanana, cassava, catjang, cauliflower, celeriac, celery, celtuce, chard, chayote, chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese water chestnut, Chinese yam, chives, chufa sedge, cole crops, common bean, common purslane, corn salad, cowpea, cress, cucumber, cushaw pumpkin, drumstick tree, eddoe, eggplant, elephant foot yam, elephant garlic, endive, enset, Ethiopian eggplant, Florence fennel, fluted gourd, gac, garden rocket, garlic, geocarpa groundnut, Good King Henry, grass pea, groundnut, guar bean, horse gram, horseradish, hyacinth bean, ice plant, Indian fig, Indian spinach, ivy gourd, Jerusalem artichoke, jacamar, jute, kale, kohlrabi, konjac, kurrat, leek, lentil, lettuce, Lima bean, lotus, luffa, maca, maize, mangel-wurzel, mashua, moso bamboo, moth bean, mung bean, napa cabbage, neem, oca, okra, Oldham's bamboo, olive, onion, parsnip, pea, pigeon pea, plantain, pointed gourd, potato, pumpkins, squashes, quinoa, radish, rapeseed, red amaranth, rhubarb, ribbed gourd, rice bean, root parsley, runner bean, rutabaga, sago palm, salsify, scallion, sea kale, shallot, snake gourd, snow pea, sorrel, soybean, spilanthes, spinach, spinach beet, sweet potato, taro, tarwi, teasle gourd, tepary bean, tinda, tomato, tuberous pea, turnip, turnip-rooted chervil, urad bean, water caltrop trapa bicornis, water caltrop trapa natans, water morning slory, watercress, welsh onion, west African okra, west Indian gherkin, white goosefoot, white yam, winged bean, winter purslane, yacón, yam, yard-long bean, zucchinietables); (4) food crops (e.g., abiu, acerola, achacha, ackee, African mangosteen, Africanrice, agate, air potato, alpine currant, Amaranthus app., Ambarrella, American gooseberry, American groundnut, American persimmon, antroewa, apple, apricot, arazá, Armenian cucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ash gourd, Asian palmyra palm, Asian pear, asparagus, atemoya, Australian desert raisin, avocado, azarole, azuki bean, babaco, bael, bambara groundnut, bamboo, banana, barbados gooseberry, barley, beet, beetroot, bergamot, betel nut, bignay, bilberry, bilimbi, binjai, biriba, bitter gourd, bitter orange, bitter vetch, bitterleaf, black chokeberry, black currant, black mulberry, black mustard, black radish, black salsify, black sapote, blackberry, blanched celery, blue-berried honeysuckle, borojó, breadfruit, broad bean, broccoli, Brussels sprout, Buck's horn plantain, buckwheat, Burmese grape, buttercup squash, butternut squash, button mangosteen, cabbage, cacao, caigua, calabash, calamondin, canistel, cantaloupe, cape gooseberry, caraway seeds, carob, carrot, cashew nut, cassava, catjang, cauliflower, celeriac, celery, celtuce, cempedak, chard, charichuelo, chayote, cherimoya, cherry, cherry of the Rio Grande, cherry plum, chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese hawthorn, Chinese water chestnut, Chinese white pear, Chinese yam, chives, chokeberry, chufa sedge, citron, cocona, coconut, cocoplum, coffee, coffee (Arabica and Robusta types), cole crops, common bean, common purslane, corn salad, Costa Rica pitahaya, cowpea, cress, cucumber, currants, cushaw pumpkin, custard apple, date, date- plum, dog rose, dragonfruit, drumstick tree, durian, durum wheat, eddoe, eggplant, einkorn wheat, elderberry, elephant apple, elephant foot yam, elephant garlic, emmer wheat, endive, enset, Ethiopian eggplant, European nettle tree, European wild apple, feijoa, fig, finger millet, Florence fennel, fluted gourd, foxtail millet, gac, garden rocket, garlic, genipapo, geocarpa groundnut, giant granadilla, good king henry, gooseberry, goumi, grape, grapefruit, grass pea, great morinda, greengage, groundnut, grumichama, guar bean, guava, hairy crabgrass, hardy kiwi, hog plum, horned melon, horse gram, horse mango, horseradish, hyacinth bean, iceplant, Indian barnyard millet, Indian fig, Indian jujube, Indian spinach, ivy gourd, jabuticaba, jackalberry, jackfruit, jambul, Japanese barnyard millet, Japanese persimmon, Japanese wineberry, Jerusalem artichoke, jocote, jujube, jute, kaffir lime, kale, karanda, kei apple, kepel apple, key lime, kitembilla, kiwifruit, kohlrabi, konjac, korlan, kubal vine, kurrat, kuwini mango, kwai muk, langsat, large cranberry, leek, lemon, lentil, lettuce, Liberian coffee, lima bean, longan, loquat, lotus, luffa, lychee, maca, maize, malay apple, mamey saptoe, mammee apple, mangel-wurzel, mango, mangosteen, maprang, marang, mashua, medlar, melon, Mirabelle plum, miracle fruit, monk fruit, monkey jack, moriche palm, moso bamboo, moth bean, mountain papaya, mountain soursop, mulberry, mung bean, mushrooms, nance, napa cabbage,naranjilla, natal plum, neem, northern highbush blueberry, oat, oca, oil palm, okra, old man's bamboo, olive, onion, orange, otaheite gooseberry, oval kumquat, papaya, para guava, parsnip, passionfruit, pawpaw, pea, peach, peach-palm, pear, pearl millet, pepino, pigeon pea, pineapple, Pitomba (Eugenia luschnathiana, Talisia esculenta), plantain, plum, pointed gourd, pomegranate, pomelo, potato, proso millet, pulasan, pumpkins and squashes, purple chokeberry, quince, quinoa, radish, rambutan, ramontchi, rapeseed, raspberry, red amaranth, red chokeberry, red currant, red mulberry, red-fruited strawberry guava, rhubarb, ribbed gourd, rice, rice bean, root parsley, rose apple, roselle, runner bean, rutabaga, rye, safou, sago palm, salak, salmonberry, salsify, santol, sapodilla, Satsuma, scallion, sea kale, seagrape, shallot, snake gourd, snow pea, soncoya, sorghum, Sorghum spp., sorrel, sour cherry, soursop, soybean, Spanish lime, Spanish tamarind, spelt wheat, spilanthes, spinach, spinach beet, star apple, starfruit, strawberry, strawberry guava, strawberry tree, sugar apple, sugar beet, sugarcane, surinam cherry, sweet briar, sweet granadilla, sweet lime, sweet potato, tamarillo, tamarind, tangerine, taro, tarwi, teasle gourd, tef, tepary bean, tinda, tomatillo, tomato, tuberous pea, tucuma palm, turnip, turnip-rooted chervil, urad bean, Vaccinium spp., velvet apple, wampee, water caltrop (Trapa bicornis, T. natans), water morning glory, watercress, watermelon, watery rose apple, wax apple, welsh onion, west African okra, west Indian gherkin, wheat, white currant, white goosefoot, white mulberry, white sapote, white star apple, white yam, winged bean, winter purslane, wolfberry (Lycium barbarum, L. chinense), yacón, yam, yangmei, yard- long bean, yellow mombin, yellow pitaya, yellow-fruited strawberry guava, zucchini; (B) other edible crops, which includes but is not limited to (1) herbs (e.g., Absinthium, alexanders, basil, bay laurel, betel nut, camomile, chervil, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, chives, cicely, common rue, common thyme, coriander, cress, culantro, curly leaf parsley, dill, epazote, fennel, flat leaf parsley, ginseng, gray santolina, herb hyssop, holy basil, hop, jasmine, kaffir lime, lavender, lemon balm, lemon basil, lemon grass, lovage, marjoram, mint, oregano, parsley, peppermint, perilla, pot marigold, rooibos, rosemary, sage, shiny-leaf buckthorn, sorrel, spearmint, summer savory, tarragon, Thai basil, valerian, watercress, wild betel, winter savory, yerba mate); (2) spices (e.g., ajowan, allspice, anise, bay laurel, black cardamom, black mustard, black pepper, caper, caraway seeds, cardamom, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, cinnamon, clove, common juniper, coriander, cumin, fennel, fenugreek, garlic, ginger, kaffir lime, liquorice, nutmeg, oregano, pandan, parsley, saffron, star anise, turmeric, vanilla, white mustard); (2) medicinal plants (e.g., absinthium, alfalfa, aloe vera, anise, artichoke, basil, bay laurel, betel leat, betel nut,bilberry, black cardamom, black mustard, black pepper, blue gum, borojo, chamomile, caper, cardamom, castor bean, chili peppers, Chinese yam, chives, cola nut, common jasmine, common lavender, common myrrh, common rue, cilantro, cumin, dill, dog rose, epazote, fennel, fenugreek, gac, garlic, ginger, gray santolina, gum Arabic, herb hyssop, holy basil, horseradish, incense tree, lavender, lemon grass, liquorice, lovage, marijuana, marjoram, monk fruit, neem, opium, oregano, peppermint, pot marigold, quinine, red acacia, red currant, rooibos, safflower, sage, shiny-leaf buckthorn, sorrel, spilanthes, star anise, tarragon, tea, turmeric, valerian, velvet bean, watercress, white mustard, white sapote, wild betel, wolfberry (Lycium barbarum, L. chinense), yerba mate); (3) Stimulants (e.g., betel leaf, betel nut, cacao, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, coffee, coffee (Arabica, Robusta), cola nut, khat, Liberian coffee, tea, tobacco, wild betel, yerba mate); (4) nuts (e.g., almond, betel nut, Brazil nut, cashew nut, chestnut, Chinese water chestnut, coconut, cola nut, common walnut, groundnut, hazelnut, Japanese stone oak, macadamia, nutmeg, paradise nut, pecan nut, pistachio nut, walnut); (5) edible seeds (e.g., black pepper, Brazil nut, chilacayote, cola nut, fluted gourd, lotus, opium, quinoa, sesame, sunflower, water caltrop (Trapa bicornis, T. natans)); (6) vegetable oils (e.g., black mustard, camelina, castor bean, coconut, cotton, linseed, maize, neem, Niger seed, oil palm, olive, opium, rapeseed, safflower, sesame, soybean, sunflower, tung tree, turnip); (7) sugar crops (e.g., Asian palmyra palm, silver date palm, sorghum, sugar beet, sugarcane); (8) pseudocereals (e.g., Amaranthus spp., buckwheat, quinoa, red amaranth); (9) aphrodisiacs (e.g., borojo, celery, durian, garden rocket, ginseng, maca, red acacia, velvet bean); (C) nonfood categories, including but not limited to (1) forage and dodder crops (e.g., agate, alfalfa, beet, broad bean, camelina, catjang, grass pea, guar bean, horse gram, Indian barnyard millet, Japanese barnyard millet, lespedeza, lupine, maize, mangel-wurzel, mulberry, Niger seed, rapeseed, rice bean, rye); (2) fiber crops (e.g., coconut, cotton, fique, hemp, henequen, jute, kapok, kenaf, linseed, manila hemp, New Zealand flax, ramie, roselle, sisal, white mulberry); (3) energy crops (e.g., blue gum, camelina, cassava, maize, rapeseed, sorghum, soybean, Sudan grass, sugar beet, sugarcane, wheat); (4) alcohol production (e.g., barley, plum, potato, sugarcane, wheat, sorghum); (5) dye crops (e.g., chay root, henna, indigo, old fustic, safflower, saffron, turmeric); (6) essential oils (e.g., allspice, bergamot, bitter orange, blue gum, camomile, citronella, clove, common jasmine, common juniper, common lavender, common myrrh, field mint, freesia, gray santolina, herb hyssop, holy basil, incense tree, jasmine, lavender, lemon, marigold, mint, orange, peppermint, pot marigold, spearmint, ylang-ylang tree); (6) green manures (e.g., alfalfa, clover, lacy Phacelia, sunn hemp, trefoil, velvet bean, vetch); (7) erosion prevention (e.g., bamboo, cocoplum); (8) soilimprovement (e.g., lupine, vetch); (9) cover crops (e.g., Alfalfa, lacy Phacelia, radish); (10) botanical pesticides (e.g., jicama, marigold, neem, pyrethrum); (11) cut flowers (e.g., carnation, chrysanthemum, daffodil, dahlia, freesia, gerbera, marigold, rose, sunflower, tulip); (12) ornamental plants (e.g., African mangosteen, aloe vera, alpine currant, aster, black chokeberry, breadfruit, calamondin, carnation, cassabanana, castor bean, cherry plum, chokeberry, chrysanthemum, cocoplum, common lavender, crocus, daffodil, dahlia, freesia, gerbera, hyacinth, Japanese stone oak, Jasmine, lacy Phacelia, lotus, lupine, marigold, New Zealand flax, opium, purple chokeberry, ramie, red chokeberry, rose, sunflower, tulip, white mulberry); (D) trees which include but are not limited to abelia, almond, apple, apricot, arborvitae nigra American, arborvitae, ash, aspen, azalea, bald cypress, beautush, beech, birch, black tupelo, blackberry, blueberry, boxwood, buckeye, butterfly bush, butternut, camellia, catalpa, cedar, cherry, chestnut, coffee tree, crab trees, crabapple, crape myrtle, cypress, dogwood, Douglas fir, ebony, elder American, elm, fir, forsythia, ginkgo, goldenraintree, hackberry, hawthorn, hazelnut, hemlock, hickory, holly, honey locust, horse chestnut, hydrangea, juniper, lilac, linden, magnolia, maple, mock orange, mountain ash, oak, olive, peach, pear, pecan, pine, pistachio, plane tree, plum, poplar, pivet, raspberry, redbud, red cedar, redwood, rhododendron, rose-of-Sharon, sassafras, sequoia, serviceberry, smoke tree, soapberry, sourwood, spruce, strawberry tree, sweet shrub, sycamore, tulip tree, ciborium, walnut, weasel, willow, winterberry, witch-hazel, zelkova; (E) turf which includes but is not limited to Kentucky bluegrass, tall fescue, Bermuda grass, zoysia grass, perennial ryegrass, fine fescues (e.g., creeping red, chewings, hard, or sheep fescue).

[0253] Treatment of plants and plant parts with the compositions described herein may be carried out directly or by allowing the compositions to act on their surroundings, habitat, or storage space by, for example, immersion, coating, dipping, spraying, evaporation, fogging, scattering, painting on, injecting, and combinations thereof, or other ways of distributing compositions that are known and practiced in the art.

[0254] Treating with compositions described herein may also be carried out by applying the composition to the soil using methods known in the art. These include but are not limited to (a) drip irrigation or chemigation; (b) soil incorporation; (c) seed treatment.

[0255] Encapsulated compositions described herein may be used as pesticides and, in particular, may be used as insecticides, nematicides, fungicides and bactericides, alone or in combination with one or more pesticidal substances set forth above and applied to plants, plant parts, substrate for growing plants or seeds.

[0256] As discussed supra, the encapsulated compositions may be combined with other enhancing compounds such as, but not limited to, amino acids, chitosan, chitin, starch, hormones, minerals, synergistic microbes to increase efficacy and promote benefits to plants.

[0257] In some embodiments, nematodes that may be controlled using the treatment methods described herein include, but are not limited to, parasitic nematodes such as root-knot, reniform, cyst, and lesion nematodes, including but not limited to Aphelenchoides spp., Belonolaimus spp., Bursaphalenchus spp., Criconema spp. Globodera spp., Meloidogyne spp., Tylenchorhynchus spp., Helicotylenchus spp., Heterodera spp., Hoplolaimus spp., Pratylenchus spp., Rotylenchulus spp., Trichodorus spp., and Xiphinema spp. In particular, the parasitic nematodes may include but are not limited to seed gall nematodes (Afrina wevelli), bentgrass nematodes (Anguina agrostis), shoot gall nematodes (Anguina spp.), seed gall nematodes (Anguina spp., A. amsinckiae, A. balsamophila; A. tritici), fescue leaf gall nematodes (A. graminis), ear-cockle (or wheat gall) nematodes (Anguina tritici), bud and leaf (or foliar) nematodes (Aphelenchoides spp., A. subtenuis), begonia leaf (or fern, or spring crimp, or strawberry foliar, or strawberry nematodes, or summer dwarf) nematodes (A. fragariae), fern nematodes (A. olesistus), rice nematodes (A. oryzae), currant nematodes (A. ribes), black currant (or chrysanthemum) nematodes (A. ritzemabosi), chrysanthemum foliar or leaf nematodes (A. ritzemabosi), rice white-tip (or spring dwarf, or strawberry bud) nematodes (A. besseyi), fungus- feeding (mushroom) nematodes (Aphelenchoides composticola), Atalodera spp. (Atalodera lonicerae, Atalodera ucri), spine nematodes (Bakernema variabile), sting nematodes (Belonolaimus spp., B. gracilis, B. longicaudatus), pine wood nematodes (Bursaphalenchus spp., B. xylophilus, B. mucronatus), sessile nematodes (Cacopaurus spp., C. epacris, C.pestis), amaranth cyst nematodes (Cactodera amaranthi), birch cyst nematodes (C. betulae), cactus cyst nematodes (C.cacti), estonian cyst nematodes (C. estonica), Thorne's cyst nematodes (C. thornei), knotweed cyst nematodes (C. weissi), ring nematodes (Criconema spp.), spine nematodes (Criconema spp., C. civellae, C. decalineatum, C.spinalineatum), ring nematodes (Criconemella axeste, C. curvata, C. macrodora, C. parva), ring nematodes (Criconemoides spp., C. citri, C. simile), spine nematodes (Crossonema fimbriatum), eucalypt cystoid nematodes (Cryphodera eucalypti), bud, stem and bulb nematodes (Ditylenchus spp., D. angustus, D. dipsaci, D. destructor, D. intermedius), Mushroom spawn nematodes (D. myceliophagus), awl nematodes (Dolichodorus spp., D. heterocephalus, D. heterocephalous), spear nematodes (Dorylaimus spp.), stunt nematodes (Geocenamus superbus), cyst nematodes (Globodera spp.), yarrow cyst nematodes (G. achilleae), milfoil cyst nematodes (G. millefolii), apple cyst nematodes (G. mali), white cyst potato nematodes (G. pallida), golden nematodes (G.rostochiensis), tobacco cyst nematodes (G. tabacum), Osborne's cyst nematodes (G. tabacum solanacearum), horsenettle cyst nematodes (G. tabacum virginiae), pin nematodes (Gracilacus spp., G. idalimus), spiral nematodes (Helicotylenchus spp., H. africanus, H. digonicus, H. dihystera, H. erythrinae, H. multicinctus, H. paragirus, H. pseudorobustus, H. solani, H. spicaudatus), sheathoid nematodes (Hemicriconemoides spp., H. biformis, H. californianus, H. chitwoodi, H. floridensis, H. wessoni), sheath nematodes (Hemicycliophora spp., H. arenaria, H. biosphaera, H. megalodiscus, H. parvana, H. poranga, H. sheri, H. similis, H. striatula), cyst nematodes (Heterodera spp.), almond cyst nematodes (H. amygdali), oat (or cereal) cyst nematodes (H. avenae), Cajanus (or pigeon pea) cyst nematodes (H. cajani), Bermuda grass (or heart-shaped, or Valentine) cyst nematodes (H. cardiolata), carrot cyst nematodes (H. carotae), cabbage cyst nematodes or brassica root eelworm (H.cruciferae), nutgrass (or sedge) cyst nematodes (H. cyperi), Japanese cyst nematodes (H. elachista), fig (or ficus, or rubber) cyst nematodes (H. fici), galeopsis cyst nematodes (H. galeopsidis), soybean cyst nematodes (H. glycines), alfalfa root (or pea cyst) nematodes (H. goettingiana), buckwheat cyst nematodes (H. graduni), barley cyst nematodes (H. hordecalis), hop cyst nematodes (H. humuli), Mediterranean cereal (or wheat) cyst nematodes (H. latipons), lespedeza cyst nematodes (H. lespedezae), Kansas cyst nematodes (H. longicolla), cereals root eelworm or oat cyst nematodes (H. major), grass cyst nematodes (H. mani), lucerne cyst nematodes (H. medicaginis), cyperus (or motha) cyst nematodes (Heterodera mothi), rice cyst nematodes (H. oryzae), Amu-Darya (or camel thorn cyst) nematodes (H. oxiana), dock cyst nematodes (H. rosii), rumex cyst nemtodes (H. rumicis), sugar beet cyst nematodes (H. schachtii), willow cyst nematodes (H. salixophila), knawel cyst nematodes (H. scleranthii), sowthistle cyst nematodes (H. sonchophila), tadzhik cyst nematodes (H. tadshikistanica), turkmen cyst nematodes (H. turcomanica), clover cyst nematodes (H. trifolii), nettle cyst nematodes (H. urticae), ustinov cyst nematodes (H. ustinovi), cowpea cyst nematodes (H. vigni), corn cyst nematodes (H. zeae), rice root nematodes (Hirschmanniella spp., H. belli, H. caudacrena, H. gracilis, H.oryzae), lance nematodes (Hoplolaimus spp.), Columbia nematodes (H. columbus), Cobb's lance nematodes (H. galeatus), crown-headed lance nematodes (H. tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis), needle nematodes (Longidorus spp., L. africanus, L. sylphus), ring nematodes (Macroposthonia (=Mesocriconema) xenoplax), cystoid nematodes (Meloidodera spp.), pine cystoid nematodes (M. floridensis), tadzhik cystoid nematodes (M. tadshikistanica), cystoid body nematodes (Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M. conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogyne spp., M. acronea, M. arenaria, M.artiellia, M. brevicauda, M. camelliae, M. carolinensis, M. chitwoodi, M. exigua, M.graminicola, M. hapla, M. hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M. javanica, M. kikuyuensis, M. konaensis, M. mali, M. microtyla, M. naasi, M. ovalis, M. platani, M. querciana, M. sasseri, M. tadshikistanica, M. thamesi), knapweed nematodes (Mesoanguina picridis), Douglas fir nematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbus aberrans, N. batatiformis, N. dorsalis), sour paste nematodes (Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes (Paralongidorus microlaimus), spiral nematodes (Pararotylenchus spp.), stubby-root nematodes (Paratrichodorus allius, P. minor, P. porosus, P. renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P. bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P. holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P. similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P. brachyurus, P. coffeae, P. convallariae, P. crenatus, P. flakkensis, P. goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P. neglectus, P.penetrans, P. pratensis, P. scribneri, P. thornei, P. vulnus, P. zeae), stem gall nematodes (Pterotylenchus cecidogenus), grass cyst nematodes (Punctodera punctate), stunt nematodes (Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholus spp.), banana-root nematodes (R. similis), rice-root nematodes (R. oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchus cocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R. parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R. christiei, R. robustus), Thorne's lance nematodes (R. uniformis), Sarisodera hydrophylla, spiral nematodes (Scutellonema spp., S. blaberum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S. conicephalum), grass root-gall nematodes (Subanguina radicicola), round cystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes (Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T. primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (or stylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T. aspericutis, T. claytoni, T.ebriensis, T. elegans, T. golden, T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T. oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulus semipenetrans), dagger nematodes (Xiphinema spp., X. americanum, X. bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X. diversicaudatum X. index, X. insigne, X. nigeriense, X. radicicola, X. setariae, X. vulgarae, X. vuittenezi). In a particular embodiment nematodes controlled are member of the Meloidogyne spp, particularly, M. hapla or M. incognita.

[0258] Phytopathogenic insects that may be controlled using treatment methods of the present disclosure include, but are not limited to, non-Culicidae larvae insects from the order (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographaspp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp, Tortrix spp., Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example, Agriotes spp., Alphitobius sp., Anomola spp., e.g., Anomala orientalis, Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Cyclocephala spp., e.g., Cyclocephala lurida, Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Otiorhynchus sulcatus, Phlyctinus spp., Popillia spp., e.g., Popilla japonica, Psylliodes spp., Rhizopertha spp., e.g., Rhizotrogus majalis, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.; (c) Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp.; (d) Isoptera, for example, Reticulitermes spp.; (e) Psocoptera, for example, Liposcelis spp.; (f) Anoplura, for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.; (g) Mallophaga, for example, Damalinea spp. and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniella spp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii; (i) Hemiptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Tniatoma spp.; Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bactericera spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseu dococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum,Triozidae spp., Trioza erytreae and Unaspis citri; (j) Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. and Vespa spp.; (k) Diptera, for example, Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Cuterebra spp., Dacus spp., Delia spp., Delia radicum, Drosophila spp., e.g., Drosophila suzukii; Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.; (l) Siphonaptera, for example, Ceratophyllus spp. and Xenopsylla cheopis; (m) from the order Thysanura, for example, Lepisma saccharina.

[0259] Phytopathogenic bacteria includes, but is not limited to, Agrobacterium spp. (e.g., Agrobacterium tumefaciens); Erwinia, Pantoea, Pectobacterium, Serratia, S. marcescens, Acidovorax, Pseudomonas, Ralstonia, Rhizobacter, Rhizomonas, Xanthomonas, Xylophilus, Agrobacterium, Rhizobium, Bacillus, Clostridium, Arthrobacter, Clavibacter, Curtobacterium, Leifsonia, Rhodococcus, Streptomyces, Xanthomonas spp. (Xanthomonas axonopodis, Xanthomonas oryzae pv. oryzae, Xanthomonas vesicatoria). In a particular embodiment, phytopathogenic bacteria includes but is not limited to Clavibacter spp., Xanthomonas spp., Pseudomonas (e.g., Pseudomonas syringae), Pectobacterium (e.g., Pectobacterium carotovorum).

[0260] Phytopathogenic fungi includes, but is not limited to, Alternaria spp. (e.g., Alternaria alternate, Alternaria solani); Aphanomyces spp. (e.g., Aphanomyces euteiches); Aspergillus spp. (e.g., Aspergillus niger, Aspergillus fumigatus); Athelia spp. (e.g., Athelia rolfsii); Aureobasidium spp. (e.g., Aureobasidium pullulans); Bipolaris spp. (e.g., Bi polaris zeicola, Bipolaris maydis); Botrytis spp. (e.g., Botrytis cinerea); Calonectria spp. (e.g., Calonectria kyotensis); Cephalosporium spp. (e.g., Cephalosporium maydis); Cercospora spp. (e.g., Cercospora medicaginis, Cercospora sojina, Colletotrichum coccodes, Colletotrichum fragariae, Colletotrichum graminicola); Coniella spp. (e.g., Coniella diplodiella); Coprinopsis spp. (e.g., Coprinopsis psychromorbida); Corynespora spp. (e.g., Corynespora cassiicola; Curvularia spp. (e.g., Curvularia pallescens); Cylindrocladium spp. (e.g., Cylindrocladium crotalariae); Diplocarpon spp. (e.g., Diplocarpon earlianum); Diplodia spp. (e.g., Diplodia gossyina); Epicoccum spp. (e.g., Epicoccum nigrum); Erysiphe spp. (Erysiphe cichoracearum); Fusarium spp. (e.g., Fusarium graminearum, Fusarium oxysporum f.sp. fragariae, Fusarium oxysporum f.sp.tuberosi, Fusarium proliferatum var. proliferatum, Fusarium solani, Fusarium verticillioides); Ganoderma spp. (e.g., Ganoderma boninense); Geotrichum spp. (e.g., Geotrichum candidum); Glomerella spp. (e.g., Glomerella tucumanensis); Guignardia spp. (e.g., Guignardia bidwellii); Kabatiella spp. (e.g., Kabatiella zeae); Leptosphaerulina spp. (e.g., Leptosphaerulina briosiana); Leptotrochila spp. (e.g., Leptotrochila medicaginis); Macrophomina spp. (e.g., Macrophomina phaseolina); Magnaporthe spp. (e.g., Magnaporthe grisea, Magnaporthe oryzae); Microsphaera spp. (e.g., Microsphaera manshurica); Monilinia spp. (e.g., Monilinia fructicola); Mucor spp.; Mycosphaerella spp. (e.g., Mycosphaerella jijiensis, Mycosphaerella fragariae); Nigrospora spp. (e.g., Nigrospora oryzae); Ophiostoma spp. (e.g., Ophiostoma ulmi); Penicillium spp. (e.g., Penicillium digitatum, Penicillium italicum, Penicillium expansum, and Penicillium allii; Peronospora spp. (e.g., Peronospora manshurica); Phakopsora (e.g., Phakopsora pachyrhizi); Phoma spp. (e.g., Phoma foveata, Phoma medicaginis); Phomopsis spp (e.g., Phomopsis longicolla); Phytophthora spp. (e.g., Phytophthora cinnamomi, Phytophthora erythroseptica, Phytophthora fragariae, Phytophthora infestans, Phytophthora medicaginis, Phytophthora megasperma, Phytophthora palmivora); Podosphaera (e.g., Podosphaera leucotricha); Pseudopeziza spp. (e.g., Pseudopeziza medicaginis); Puccinia spp. (e.g., Puccinia graminis subsp. tritici (UG99), Puccinia striiformis, Puccinia recodita, Puccinia sorghi); Pyricularia spp. (Pyricularia grisea, Pyricularia oryzae); Pythium spp. (e.g., Pythium ultimum); Rhizoctonia spp. (e.g., Rhizoctonia solani, Rhizoctonia zeae); Rosellinia spp., Sclerotinia spp. (e.g., Sclerotinia minor; Sclerotinia sclerotiorum, Sclerotinina trifoliorum); Sclerotium spp. (e.g., Sclerotium rolfsii); Septoria spp. (e.g., Septoria glycines, Septoria lycoperski); Setomelanomma spp. (e.g., Setomelanomma turcica); Sphaerotheca spp. (e.g., Sphaerotheca macularis); Spongospora spp. (e.g., Spongospora subterranean); Stemphylium spp., Synchytrium spp. (e.g., Synchytrium endobioticum), Verticillium spp. (e.g., Verticillium albo-atrum, Verticillium dahliae). In a particular embodiment, the fungus is a member of the Botrytis spp. (e.g., Botrytis cinerea), Sclerotinia spp. (Sclerotinia minor), Sclerotium spp. (e.g., Sclerotium rolfsii), Macrophomina spp. (e.g., Macrophomina phaseolina), Verticillium spp. (e.g., Verticillium dahliae), Fusarium spp. (e.g., Fusarium oxysporum f.sp. fragariae), Rhizoctonia spp. (e.g., Rhizoctonia solani), Pythium spp. (e.g., Pythium ultimum). In some embodiments, the phytopathogenic fungus is a Rhizoctonia spp, a Botrytis spp. or a Penicillium spp. In some embodiments, the phytopathogenic organism is Rhizoctonia solani, Botrytis cinerea, Penicillium digitatum, and / or Penicillium expansum.

[0261] In post-harvest applications, the encapsulated compositions of the present disclosure comprising beneficial microorganisms (e.g., a fungal microorganism or any other beneficial microorganism) can be used to treat plants and control spoilage caused by organisms and extend the shelf life of agricultural products. The encapsulated compositions of the present disclosure can be evaluated to demonstrate the potency and shelf-life of the encapsulated microorganisms. Exemplary assay methods, including methods to assess percent disease coverage and % disease control are disclosed in the Examples and Figures of the present disclosure.

[0262] In some embodiments, the encapsulated composition inhibits growth of a phytopathogenic organism. In some embodiments, the encapsulated composition increases disease control of a phytopathogenic organism. In some embodiments, the growth inhibition and / or disease control is in a plant growing media, a soil, a food container, or other storage environment. In some embodiments, phytopathogenic organism is a fungus, bacteria, nematode or an insect. In some embodiments, the encapsulated composition increases plant germination by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any number or range therein (or greater than 100%) compared to a control without an encapsulated composition. In some embodiments, the encapsulated composition increases plant fresh weight by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any number or range therein (or greater than 100%) compared to a control without an encapsulated composition. In some embodiments, plant fresh weight comprises one or more of shoot fresh weight, root fresh weight, or other plant part.

[0263] In some embodiments, the encapsulated composition inhibits growth or infection of a phytopathogenic fungus during food storage. In some embodiments, the encapsulated composition reduces phytopathogenic fungus disease coverage by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any number or range therein compared to a control without an encapsulated composition. In some embodiments, the encapsulated composition increases phytopathogenic fungus disease control by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any number or range therein (or greater than 100%) compared to a control without an encapsulated composition.

[0264] In some embodiments, the encapsulated composition inhibits growth of a phytopathogenic organism during plant growth. In some embodiments, the encapsulated composition reduces disease by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any number or range therein compared to a control without an encapsulated composition. In some embodiments, the encapsulated composition increases disease control by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any number or range therein (or greater than 100%) compared to a control without an encapsulated composition.

[0265] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof. EXAMPLES Example 1 – Muscodor albus Encapsulation Protocol

[0266] Purpose. The purpose of this protocol is to outline the steps involved in encapsulation of Muscodor albus whole cell broth to provide a stable and protective barrier for the active compound (Muscodor albus) and to extend its stability.

[0267] Muscodor albus may be cultivated by shake or non-shake cultivation, small scale or large-scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentation apparatus performed in suitable medium and under conditions allowing cell growth or on solid substrates such as agar. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available or may be available from commercial sources or prepared according to published compositions.

[0268] Table 1 shows exemplary general formula components and inclusion ranges (% w / w) for encapsulation of Muscodor albus whole cell broth. Muscodor albus whole cell broth was combined with formulation ingredients using a rotor stator homogenizer or other appropriate mixer. The bio-reactor whole cell broth (WCB) was mixed with ingredients until a uniform blend was obtained. Viscosity, pH, and other product chemistry were measured. The blend was then dripped into a calcium chloride solution for cross-linking the alginate and to create the encapsulation matrix. Alginate performs as a physical barrier and humidity controller to enhance the preservation of Muscodor albus; clay can act as a structure support and protective agent, reduce the permeability, and slow the release rate to increase the survival of the fungus. Other materials such as glycerol, silica, etc. were also applied to extend the shelf life of the encapsulated formulations and improve the process. Table 1. General Formula Outline

[0269] All procedures followed appropriate safety precautions, including the use of appropriate personal protective equipment (PPE) and adherence to proper use, storage, and disposal of all chemicals.

[0270] Muscodor albus whole cell broth (WCB). Muscodor albus WCB used for encapsulation was made by preparing Muscodor albus as described in Example 1. The “Nutrients” column in Table 2 and other Tables indicates the addition of the listed nutrient (e.g.,e.g., sucrose at 10 g / L, starch at 10g / L, and / or cellulose at 10g / L) during fermentation of Muscodor albus. Sucrose, starch and cellulose are carbon sources for Muscodor albus. Table 2. Test Numbers 1-3 FormulationsTable 3. Test Number 3 Formulation Details

[0271] Materials. The materials used for encapsulation included the following: a balance with appropriate capacity; at least 0.001g resolution; Sporeklenz: Ready-to-Use Sterilant (for sterilization of equipment) from STERIS Life Sciences; Silverson L4RT High Shear Mixer; Fluid Bed Dryer; 70% isopropanol alcohol (IPA) solution (for sanitization of equipment); 70% ethanol (for sanitization of equipment); active ingredient: Muscodor albus whole cell broth (WCB); sodium alginate powder: encapsulating agent; calcium chloride dihydrate: crosslinking agent; KaMin 80 B: water washed kaolin clay from KaMin LLC; Vangel® SX (a blend of bentonite clay and xanthan gum): a synergistic blend of natural bentonite and xanthan gum for high thickening and suspending efficiency from Vanderbilt Minerals, LLC; glycerol: a clearliquid preservative from Research Products International; SIPERNAT® 22 S: silica as a flow aid, anti-caking, anti-sticking.

[0272] Procedure. All procedures were followed using aseptic techniques. Equipment was sanitized (tube rack, balance, countertop, and lab scoops) by first applying Sporklenz, then 70% ethanol, and finish by rinsing with DI water. The equipment was allowed to dry before use. Then, a 2% (w / w) CaCl2solution was prepared by weighing out dry ingredients in a weighing plate and DI water, separately, then mixed on a magnetic stirrer. Next, 25% (w / w) glycerol was added into the WCB by weighing out the glycerol and adding it into the WCB.

[0273] 1% (w / w) alginate and 1% (w / w) clay solution were added into the WCB and glycerol mixture as follows: The dry ingredients (sodium alginate powder, KaMin 80B, and Vangel® SX) were weighed out in weighing plates respectively, and the Kamin 80B was added into the mixture of WCB and glycerol gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes, followed by addition of the Vangel SX and sodium alginate.

[0274] Encapsulation process: the above mixture containing alginate and clay was dripped with a flow rate at 4.2 ml / min controlled by a pump into a 2% (w / w) CaCl2solution at equivalent volume while agitating on a magnetic stirrer. The orifice for dripping was I.D.5mm.

[0275] Washing process: The CaCl2solution was drained after encapsulation using a funnel with a filter paper layer, and then the encapsulated beads were washed with DI water.

[0276] Coating beads with silica solution: the encapsulated beads were added into a 0.5% SIPERNAT® 22 S solution for surface treatment and then the silica solution was drained using a funnel with a filter paper layer before drying to prevent sticking during fluid bed drying.

[0277] Drying process: The beads were drained of excess solution and the beads were rinsed. Silica was added to the rinse to prevent sticking and enhance fluidization during the drying process. The beads were added to the to bed of the fluid bed dryer and drying took place under low heat with high air flow. Parameters were optimized to encourage evaporation and minimize heat exposure. The maximum flow rate of spray pump for the Glatt Midi-Glatt Fluid Bed Processor was 54 ml / min, and the max compressed air was 6 bar (100 Nm3 / h max) with an operating range from 2.2 to 2.57 bar). The set inlet temperature was 51°C, and drying time with hot air was 2 to 10 minutes depending on the load size and cooling without hot air was 1 minute. The final outlet temperature was between 20°C and 35ºC.

[0278] Encapsulated beads were collected and packaged. The beads produced were placed into a variety of assays that demonstrate the potency and shelf-life of the encapsulated Muscodor albus. The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temperature. The target moisture content was 58% to 98%.

[0279] Storage: All samples were kept at 4oC before packaging.

[0280] FIG.4 indicates the higher viscosity as a result of the post-harvest formulation w / Sucrose 10g / L as described in Table 3 versus the M. albus end of fermentation WCB, whilst FIG.5 indicates the change in moisture and size of the encapsulated material as a result of the drying process. Formulations were further optimized and evaluated in an attempt to optimize stability and bioassay performance, and are described in Table 50 and 51. Example 2 – Post-Harvest Formulation Sample Preparation

[0281] Prepare 2% CaCl2: weigh out 50 g of CaCl2powder to DI water up to 2500 g in a beaker, then mix on a magnetic stirrer, and set aside for cross linking in the encapsulation process.

[0282] Add 25% (w / w) Glycerol into WCB: glycerol was weighed out and added into the WCB. For example, 2500 g of Glycerol was added to 7300 g WCB (see Table 4).

[0283] Add 1% (w / w) alginate and 1% (w / w) Kamin 80B into the glycerol / WCB mixture: Weigh out dry ingredients (100g Sodium Alginate Powder and 100 g KaMin 80 B) in weighing plates respectively and add the Kamin 80B into the mixture of WCB and glycerol gradually while mixing with a Silverson, a high shear Mixer, to eliminate clumps and fisheyes, followed by addition of the 100 g sodium alginate powder. Table 4. Post-harvest formulation ingredients and Example

[0284] Encapsulation process: drip the above mixture containing glycerol, alginate and Kamin 80B with a flow rate at 4.2 ml / min controlled by a pump into 2% (w / w) CaCl2solution at equivalent volume while agitating on a magnetic stirrer (the pump speed may be adjusted by viscosity of the mixture). Multiple drip orifices may be used.

[0285] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0286] Coating beads with silica solution: add the encapsulated beads into a 0.5% SIPERNAT® 22 S for surface treatment and then drain the silica solution using a funnel with a filter paper layer before drying to prevent sticking during fluid bed drying.

[0287] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting inlet temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0288] Storage: keep all samples at 4oC before packaging.

[0289] The formulation described in Table 4 is shown in FIGs.128-136, 138-139, 141- 143 and 147-188. It resulted in effective control of Penicillium digitatum (FIGs.151-160). It resulted in the highest control of Penicllium expansum (FIGs.177-188). Other formulations were tested in an attempt to optimize stability and bioassay performance. Example 3 – Field Test Formulation Sample Preparation

[0290] Additional formulations were developed for field testing as shown in Tables 5-9 below. Table 5. Formulations 2.1 to 2.4 for Field Testing

[0291] Methods for Formulation 2.1. Prepare a 2% CaCl2solution by weighing out the CaCl2powder. For example, add 30 g of CaCl2powder to DI water up to 1500 g in a beaker, then mixed on a magnetic stirrer, set aside for crossing link in the encapsulation process.

[0292] Add 1% (w / w) alginate into the Formulation 2.1 mixture: Weigh out dry ingredients (15 g sodium alginate powder) in a weighing plate and add into the WCB gradually while mixing with a Silverson, a high shear mixer to eliminate clumps and fisheyes. Table 6. Field Test Formulation 2.1 Ingredients

[0293] Encapsulation process: drop the mixture containing 1 % alginate with a flow rate at 4.2 ml / min controlled by a pump into 2% (w / w) CaCl2solution at equivalent volume while agitating on a magnetic stirrer (pump speed may be adjusted by viscosity of the mixture).

[0294] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0295] Coating beads with silica solution: add the encapsulated beads into a 0.5%SIPERNAT® 22 S for surface treatment and then drain the silica solution using a funnel with a filter paper layer before drying to prevent sticking during fluid bed drying.

[0296] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0297] Storage: keep all samples at 4oC before packaging

[0298] Methods for Formulation 2.2. Prepare 2% CaCl2solution as described previously.

[0299] Add 25% (w / w) Glycerol into WCB: Weight out the glycerol in a beaker. For example, weigh out 375 g Glycerol, and add it into a 1110 g Muscodor albus WCB (w / Sucrose 10 g / L)

[0300] Add 1% (w / w) alginate into the above mixture: Weigh out dry ingredients (15 g Sodium Alginate Powder) in a weighing plate and add into the above mixture gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes. Table 7. Field Test 2.2 Formulation Ingredients and Example

[0301] Encapsulation process: drop the above mixture containing glycerol and alginate with a flow rate at 4.2 ml / min controlled by a pump into 2% (w / w) CaCl2solution at equivalent volume while agitating on a magnetic stirrer (the pump speed may be adjusted by viscosity of the mixture).

[0302] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0303] Coating beads with silica solution: add the encapsulated beads into a 0.5% SIPERNAT® 22 S for surface treatment and then drain the silica solution using a funnel with a filter paper layer before drying to prevent sticking during fluid bed drying.

[0304] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depends on the loading size and initial temp of the beads.

[0305] Storage: keep all samples at 4oC before packaging.

[0306] Methods for Formulation 2.3. Prepare 2% CaCl2as previously described.

[0307] Add 25% (w / w) Glycerol into WCB: Weight out the glycerol in a beaker. For example, weigh out 375 g Glycerol, and add it into a 1110 g Muscodor albus WCB (w / Sucrose10 g / L)

[0308] Add 1% (w / w) alginate, 0.75 % (w / w) Kamin 80B and 0.25 % (w / w) Vangel® SX into the above mixture: Weigh out dry ingredients (15 g Sodium Alginate Powder, 11.25 g KaMin 80 B, and 3.75 g Vangel® SX) in weighing plates respectively, and add Kamin 80B into the mixture gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes, then followed by addition of the Vangel SX and sodium alginate, respectively. Table 8. Field Test Formulation 2.3 Ingredients and Example

[0309] Encapsulation process: drop the above mixture of containing 1 % alginate, 0.75 % (w / w) Kamin 80B and 0.25 % (w / w) Vangel® SX with a flow rate at 4.2 ml / min controlled by a pump into 2% (w / w) CaCl2solution at equivalent volume while agitating on a magnetic stirrer (the pump speed may be adjusted by viscosity of the mixture).

[0310] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0311] Coating beads with silica solution: add the encapsulated beads into a 0.5% SIPERNAT® 22 S for surface treatment and then drain the silica solution using a funnel with a filter paper layer before drying to prevent sticking during fluid bed drying.

[0312] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0313] Storage: keep all samples at 4oC before packaging.

[0314] Methods for Formulation 2.4. Prepare 2% CaCl2as describe previously.

[0315] Add 25% (w / w) Glycerol into WCB: Weight out the glycerol in a beaker. For example, weigh out 375 g glycerol, and add it into a 1110 g Muscodor albus WCB (w / Starch 10 g / L).

[0316] Add 1% (w / w) alginate, 0.75 % (w / w) Kamin 80B and 0.25 % (w / w) Vangel® SX into the above mixture: Weigh out dry ingredients (15 g Sodium Alginate Powder, 11.25 g KaMin 80 B, and 3.75 g Vangel® SX) in weighing plates respectively, and add Kamin 80B into the 2.4.2 mixture gradually while mixing with a Silverson, a high shear Mixer toeliminate clumps and fisheyes, then followed by addition of the Vangel SX and sodium alginate, respectively. Table 9. Field Test Formulation 2.4 Ingredients and Example

[0317] Encapsulation process: drop the mixture of 2.4.3 containing 1 % alginate, 0.75 % (w / w) Kamin 80B and 0.25 % (w / w) Vangel® SX with a flow rate at 4.2 ml / min controlled by a pump into 2% (w / w) CaCl2solution at equivalent volume while agitating on a magnetic stirrer (the pump speed may be adjusted by viscosity of the 2.4.3 mixture).

[0318] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0319] Coating beads with silica solution: add the encapsulated beads into a 0.5% SIPERNAT® 22 S for surface treatment and then drain the silica solution using a funnel with a filter paper layer before drying to prevent sticking during fluid bed drying.

[0320] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0321] Storage: keep all samples at 4oC before packaging.

[0322] Table 10, FIG.9, FIGs.191A-B, and FIGs.192A-B show encapsulated Muscodor albus WCB in formulations 2.2 to 2.4. Formulation 2.1 (1% Alginate) (Tables 5, 6, and 10) at the high rate of three pieces resulted in the highest nematode mortality (nematicide in vitro assay) in FIGs.191A-B. However, at the low rate of one piece there was no difference in performance. In FIG.192A, in vitro disease control of Rhizoctonia solani was comparable (around 80%) for formulations 2.1, 2.3 and 2.4 (Table 10) when tested at both the high rate of three pieces and low rate of one piece. Other formulations were tested in an attempt to optimize stability and bioassay performance. Table 10. Yield of Encapsulated Muscodor albus WCB from Formulations 2.1 to 2.4

[0323] A stability test of triplicate of encapsulated Muscodor albus WCB formulations made with various types of clay or nutrients was performed with storage of at 4oC versus room temperature. To evaluate stability, three encapsulated Muscodor albus pieces were incubated on a PDA + Tetracycline plate for 3 days at 25oC before counting the number showing growth indicating stability. Table 11 shows that a formulation with Vangel® SX (601-230406-134- Vangel 3) had stability at 4oC through 20 weeks but was not as stable at RT. A formulation with Muscodor albus WCB grown with sucrose for 46.6 hours (601-230518-16602-10- Sucrose 46.6 hrs) had stability through 24 weeks at 4oC and through 24 weeks at RT. Table 11. Stability Test of Formulations with Various Clay or Nutrients* Indicates contamination; Y – Yes, stable; N – No, not stable Example 4 –Additional Formulations

[0324] Methods for Formulation 3.1. Prepare 2% CaCl2as previously described.

[0325] Add 1% (w / w) alginate into the WCB: Weigh dry ingredients (1 g sodium alginate powder) in a weighing plate and add into the WCB gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes.

[0326] Add 2% (w / w) PVA (Polyvinyl Alcohol) and 5% (w / w) PEG (Polyethylene Glycol) into the mixture: Add ingredients to achieve the target concentration by weight, while mixing with a Silverson, a high shear Mixer to achieve a uniform suspension.

[0327] Encapsulation process: drip the Mixture of 3.1.3 at a flow rate at 4.2 ml / min controlled by a pump into the 2% (w / w) CaCl2solution while agitating on a magnetic stirrer (pump speed may be adjusted according to the viscosity of the 3.1.3 mixture).

[0328] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0329] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0330] Storage: keep all samples at 4oC before packaging. Table 12. Experimental formula containing PVA and PEG (3.1) or PVPP (3.2)

[0331] Methods for Formulation 3.2. Prepare 2% CaCl2as previously described.

[0332] Add 1% (w / w) alginate into the WCB: Weigh dry ingredients (1 g Sodium alginate powder) in a weighing plate and add into the WCB gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes.

[0333] Add 2% PVPP (Polyvinylpolypyrrolidone) into the mixture: Add ingredients to achieve the target concentration by weight, while mixing with a Silverson, a high shear Mixer to achieve a uniform suspension.

[0334] Encapsulation process: drip the Mixture at a flow rate at 4.2 ml / min controlled by a pump into the 2% (w / w) CaCl2solution while agitating on a magnetic stirrer (pump speed may be adjusted according to the viscosity of the mixture).

[0335] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0336] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0337] Storage: keep all samples at 4oC before packaging.

[0338] Table 11 formulation Sucrose 46.6hrs had less control than a control 46.6hrs sample in the Rhizoctonia in planta bioassay (FIG.67). Other formulations were tested in an attempt to optimize stability and bioassay performance. Example 5 –Additional Formulations with Different Types of Clay

[0339] Encapsulated alginate with different types of clay including: Kamin 80 B, attapulgite clay, bentonite (MP Biomedicals, Fisher), Vanatural® (bentonite clay), Veegum® R (smectite clay), and Vangel® SX (a blend of bentonite clay and xanthan gum) were tested as shown in Table 13. An encapsulation process in which 2% clay is added into 2% alginate followed by mixing the alginate / clay mixture with Muscodor albus WCB in a 1:1 ratio and encapsulation is shown in FIG.10. Another encapsulation process in which 1% alginate and 1% clay are added into the Muscodor albus WCB and encapsulated is shown in FIG.11. Table 13. Clay Used in Various Formulations

[0340] FIG.12 shows encapsulated Muscodor albus WCB in the control sample with just alginate (#7 in Table 13) before and after drying, and the encapsulated sample made withVanatural (#4 in Table 13) before and after drying. FIG.13 shows images of encapsulated Muscodor albus WCB in the control sample with just alginate (#7 in Table 13) before and after drying as well as the interior of the sliced alginate bead before and after drying. FIG.14 shows images of encapsulated Muscodor albus WCB in a sample with clay before and after drying.

[0341] The final moisture content of each formulation is shown in FIG.15.

[0342] Plant assays were performed using the methods of Example 12 on these formulations of Muscodor albus WCB encapsulated with different clay (A, B, B1-B4, and Control) are shown in FIGs.16 and 17. FIG.16 is a graph showing the average number of germinated seeds in the soil bourn fungal pathogen Rhizoctonia solani greenhouse bioassay for plant health. Increased numbers of germinated seeds indicate improved efficacy of a specific formulation. The untreated control (no Rhizoctonia solani) shows an average germination rate of about 38 seeds from a total of 50 sowed seeds, whereas the R. solani infected control only has an average of 5 germinated seeds. The Vanatural formation shows the highest seed germination for R. solani infested samples with an average of 22 seeds. FIG.17 is a graph showing the percent R. solani control compared to the UTC, reflecting the high performance of the Vanatural formation with about 60% control of R. solani.

[0343] Split plate assays were performed according to the methods of Example 12 using the different clay formulations of Table 13. In the split plate assay, encapsulated treatments were placed onto potato dextrose agar (PDA) on one side of a split 90 mm petri dish at a rate of 50mg (low; one bead) or 150mg (high, 3 beads). After 24 hours, a 5mm mycelial plug of a 4-5 day old culture of Rhizoctonia solani AG4, grown in the dark at room temperature was placed onto the other side of the plate mycelial side down. Plates were sealed and stored in the dark at room temperature and after 48hrs, the mycelial radius was recorded, and the percentage disease control calculated. As shown in FIG.19A the Veegum® R (Smectite clay) (MC:91.12 %) performed the best at the low rate of 1 piece. In FIG.19B the KaMin80B, Veegum® R (Smectite clay) and Alginate control performed the best at the high rate of three pieces. FIGs.19A and 19B are graphs showing the % of R. solani control compared to the untreated control (UTC) at the low rate and high rate, respectively.

[0344] FIG.20 shows the survival of the encapsulated Muscodor albus formulations of Table 13 after drying using the fluid bed dryer (FBD). Encapsulated Muscodor albus was incubated on a PDA + Tetracycline plate for 3 days at 25oC. Survival was observed for all formulations except for B4 Vangel® SX.

[0345] FIGs.21A-B show survival of Muscodor albus encapsulated in the various clay formulations of Table 13 when incubated at 30oC. FIG.21A shows survival of all formulations atday 0 and FIG.21B shows only survival of Veegum (Smectite) after day 7.

[0346] FIGs.22 and 23 show survival of Muscodor albus encapsulated in the various clay formulations of Table 13 when incubated at 4oC or RT for 12 weeks or 16 weeks respectively. At the 12-week timepoint, only KaMin 80B (A), Attapulgite clay (B) and Vangel® SX (B4) showed survival at 4C, whilst only Vangel® SX (B4) showed survival at room temperature. In FIG.23, just Attapulgite clay (B) showed survival at 16 weeks 4oC, whilst only only Vangel® SX (B4) showed survival at room temperature.

[0347] Stability testing of these various formulations of encapsulated Muscodor albus WCB is shown in Table 14 below. Sample 6, the sample with Vangel® SX (a blend of bentonite clay and xanthan gum) was stable at 4oC and RT for 30 days, and 30oC for 7 days. Samples 1 and 2 were stable at 4oC for 30 days, and sample 7 (control) was stable at RT for 30 days. Table 14. Stability Testing of Encapsulated Muscodor albus WCB in Various Clay FormulationsThe plant data is the % disease control from the tested formulations compare to an untreated non-disease control.

[0348] Longer term stability studies of samples 1-7 are shown in Table 15 below. The lotnumbers for the samples are 601230210 A (1), 601230210 A (2), 601230217 B1 (3), 601230217 B2 (4), 601230217 B3 (5), 601230217 B4 (6), and 601230210 C (7). Sample 6, the sample with Vangel® SX (a blend of bentonite clay and xanthan gum) was stable at 4oC for 12 weeks, at RT for 16 weeks. Sample 5 was stable at RT for 8 weeks and at 30oC for 7 days. Samples 1 and 2 were stable at 4oC for 16 weeks, and sample 7 (control) was stable at RT for 30 days with only 1 / 3 replicates stable for 8 weeks. Sample 1 was moldy after 16 weeks and sample 2 didn’t grow mold after 16 weeks. Table 15. Long Term Stability Testing of Various Clay Formulations* Indicates contamination; Y – Yes, stable; N – No, not stable

[0349] Table 16 shows various formulations encapsulated Muscodor albus with alginate and Vanatural® (bentonite clay) compared to controls. The filter refers to the storage process of the formulations in terms of containers with (w / filter) or without (without / filter) a filter. The control samples (alginate only) w / higher MC % had the higher survival at 30oC. FIGs.16 and 17 show images of the various samples of Table 16 over time when stored at different temperatures. Table 16. Processing Conditions and Final Moisture Contents

[0350] Table 17 shows stability of the samples of Table 16 over a longer time frame. Table 17 shows that these particular lot formulations were not stable after 8 weeks at 4 °C or room temperature prompting further optimization studies. Table 17. Stability of the Muscodor albus encapsulated Using Alginate and Vanatural® (Bentonite clay)with Different MC

[0351] Table 18 shows encapsulated Muscodor albus using different concentration of Alginate & Veegum® R (a smectite clay). Various mixtures are shown in FIGs.29 and 30. FIG.30 shows that due to the high viscosity of the 1% alginate + 4% Veegum® R mixture, encapsulated particles were not spherical. Table 18. Encapsulated Muscodor albus using different concentration of Alginate & Veegum®

[0352] Table 19 shows encapsulated Muscodor albus using different concentration of Alginate & Veegum® R and their MC% and % R. solani control compared to the UTC (untreated controls). Various mixtures are shown in FIG.31. Table 19 shows that moisture content (MC) and % R. solani control in the plant bioassay are not correlated with each other. Table 19. Encapsulated Muscodor albus using different concentration of Alginate & Veegum®

[0353] Table 21 shows the yield of encapsulation of the various Veegum formulations. Table 21 highlights there was not a correlation between yield, product moisture and % R. solani control. The 1% Alginate sample had the lowest yield but highest % R. solani control. The 2% Veegum samples had the highest yuield but lowest % R. solani control. FIGs.55 and 56 show the higher viscosity and yield respectively associated with a higher concentration of sodium alginate. Table 21. Yield of encapsulation

[0354] Table 22 shows a stability study for the various encapsulated Veegum® R formulations. The control (alginate only) w / higher MC % had the higher survival rate at 30 °C. The survival rate decreased with increasing the concentration of Veegum® R clay at 30 °C. Table 22. Stability of encapsulated Muscodor albus with different concentration of Alginate & Veegum® R* Indicates contamination; Y – Yes, stable; N – No, not stable

[0355] Table 23 shows various formulations encapsulated Muscodor albus with alginate and Vangel® SX (bentonite clay and xanthan gum) compared to controls. Drying cycle denotes the difference between Control 1 and 2 and Vangel® SX 1-4. FIGs.33-34 show photographic images of various encapsulated Muscodor albus using alginate & Vangel® SX. Table 23. Encapsulated Muscodor albus using Alginate & Vangel® SX bentonite clay and xanthan gum

[0356] Table 24 shows a stability study for the various encapsulated Vangel® SX formulations. The encapsulated Vangel-4 with lowest moisture content at 83.61 survived at Room temp after 16 weeks storage duration. Table 24. Encapsulated Muscodor albus using Alginate & Vangel® SX bentonite clay and* Indicates contamination; Y – Yes, stable; N – No, not stable

[0357] Table 25 shows processing conditions for formulations with alginate (controls), alginate and Kamin 80B and alginate plus Vanatural® bentonite clay. FIGs.36A-B show photographic images of various encapsulated Muscodor albus with using alginate & Kamin 80B or Vanatural®. Table 25. Processing Conditions for Encapsulated Muscodor albus using Alginate & Kamin 80B or Vanatural®

[0358] Table 26 shows a stability study for the encapsulated alginate (controls), alginate plus Kamin 80B and alginate plus Vanatural® bentonite clay. The encapsulated Vangel-4 with lowest moisture content at 83.61 survived at Room temp after 12 weeks storage duration. Table 26. Stability of the Encapsulated Muscodor albus using Kamin 80B & Vanutral® at Different Temperatures

[0359] FIG.37 and FIG.38 show the survival of the encapsulated Muscodor albus formulations of Table 26. Encapsulated Muscodor albus was incubated on a PDA + Tetracycline plate for 4 or 7 days, respectively, at 30oC. FIG.39 shows the survival of the encapsulated Muscodor albus formulations of Table 26 when incubated at RT or 4oC for 8 weeks. And FIG. 40 and FIG.41 show survival of the encapsulated Muscodor albus formulations of Table 26 when incubated at RT or 4oC for 12 weeks. Table 26 summarizes the survival studies shown in FIGs.37-41. Control 2 in Table 26 has the longest survival to 20 weeks at 4oC and all formulations except Vanatural® have survival at 4 weeks at room temperature.

[0360] Methods for Formulation 4. Prepare 2% CaCl2as previously described, set aside for crossing link in the encapsulation process.

[0361] Alginate solution with Kamin 80 B: Weigh out dry ingredients in a weighing plate (2 g Sodium alginate powder, and 2 g Kamin 80 B) and DI water separately, then add theSodium Alginate gradually into the DI water while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes, followed by addition of 2 g KaMin 80 B.

[0362] Mix WCB with the mixture Alginate: Mix the Whole Cell broth with the mixture of 4.2.2 at the 1:1 ratio (w / w) using the Silverson, a high shear Mixer.

[0363] Encapsulation process: drip the Mixture at a flow rate 3.9 ml / min controlled by a pump into the 2% (w / w) CaCl2solution while agitating on a magnetic stirrer (pump speed may be adjusted of the viscosity of the 4.2.3 mixture).

[0364] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0365] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0367] Storage: keep all samples at 4oC before packaging. Table 27. Experimental formula using Kamin 80 BExample 6 – Testing of Muscodor albus WCB Harvested at Different Times

[0368] Muscodor albus WCB were harvested at various times and were used for formulations made with alginate and Vangel® SX. FIG.42 shows photographic images of Muscodor albus WCB grown for various times. FIGs.43 and 44 are graphs showing the % solids and pH of the WCB at the various time points. FIG.45 shows photographic images of encapsulated Muscodor albus WCB grown for various times. FIGs.46 and 47 are graphs showing the viscosity of formulations of alginate and Vangel® SX with Muscodor albus WCB harvested at the time points indicated. The control has no Vangel® SX.

[0369] The stability of encapsulated of Muscodor albus using WCB harvested at different times is shown in Table 28. Table 28. Stability of Encapsulated of Muscodor albus Using WCB Harvested at Different Times Sample 4 °C Room Temp 30 °C

[0370] FIG.51 and FIG.52 show the survival of the encapsulated Muscodor albus formulations of Table 28. C134.9hrs had survival for 1 / 3 pieces at room temperature at 20 weeks, whilst C358.5hrs, C469hrs, and V469hrs had survival for all 3 pieces at 4 °C for 20 weeks (FIGs.51 and 52).

[0371] Table 29 shows the stability of encapsulated of Muscodor albus using fresh WCB (WCB-1) and WCB grown for different times and stored at 4oC for 1 week (FIG.54) before encapsulation. The stability test goes to 16 weeks at some temperatures. FIG.59 is a graph showing the mortality of these samples. Table 29. Stability of Encapsulated Muscodor albus Using WCB Harvested at Different Times

[0372] Table 30 shows the stability of encapsulated of Muscodor albus using WCB grown for different times and stored at 4oC for 1 week (FIG.54) before encapsulation. The stability test goes to 24 weeks at some temperatures. FIG.60 is a graph showing the mortality of these samples. Table 30. Stability of Encapsulated Muscodor albus Using WCB Harvested at Different TimesExample 7 – Yield of Various Muscodor albus WCB Formulations

[0373] The yields of the various Muscodor albus WCB formulations is shown below. Table 31 shows the yield of encapsulation formulas of Example 6 using the Muscodor albus WCB grown for various times. Table 31. Yield of Encapsulation of Muscodor albus WCB Grown for Various Times

[0374] Table 32 shows the yield of various clay encapsulation formulas from Example 5. Table 32. Yield of Encapsulation of Example 5 Formulations

[0375] Table 33 shows the yield of encapsulation formulas of Example 5 using the Veegum clay. Table 33. Yield of Encapsulation of Additional Example 5 FormulationsExample 7 –Additional Formulations with Different Types of Nutrients

[0376] Formulation of encapsulated Muscodor albus with alginate and WCB containing different types of Nutrients: sucrose, starch and cellulose, and trehalose at range from 1 to 5 % to extend the shelf life of encapsulated Muscodor albus beads. FIGs.62-66 show % solids, pH, moisture content and images of the encapsulated Muscodor albus WCB with the various nutrients. FIG.67 shows a plant assay using the methods of Example 12 on these formulations of Muscodor albus WCB encapsulated with different nutrients. The average number of germinated seeds in the soil borne fungal pathogen Rhizoctonia solani greenhouse bioassay for plant health is shown. The starch is the least effective among these nutrients. At 46.6 hrs, Control > Sucrose > Cellulose > Starch; at70.7 hrs, Cellulose > Control > Starch > Sucrose; however, there was no significant difference among these nutrients. Table 34 shows the stability of these various samples over time at RT and 30oC. Table 35 shows stability at 4oC. FIGs.68-71 show photographic images of the stability assays. Table 34 shows that the cellulose 46.6hrs and 70.6hrs formulations have the greatest survival after 24 weeks at room temperature. Table 34 shows that all formulations except the control 70.6hrs had survival at 8 days of 30 °C. Table 35 shows that the 46.6hrs formulations have longer survival than the 70.6hrs formulations at 4 °C. Table 34. Stability of Encapsulated of Muscodor albus using WCB w / different Nutrients at RTTable 35. Stability of Encapsulated of Muscodor albus using WCB w / different Nutrients at 4oC

[0377] Table 36 shows stability studies on encapsulated Muscodor albus WCB that had been stored for 2 weeks before encapsulation. Sample 1 indicates Dry 1 cycle; Sample 2 indicates Dry 2 cycles. FIGs.72-73 show photographic images of the stability assays. Table 36. Stability of Encapsulated of Muscodor albus using WCB with Different Nutrients

[0378] FIGs.74-76 show results using the nematicide in vitro bioassay using the encapsulated Muscodor albus WCB grown with different nutrients. On the initial day (FIG.74) The 46.6 hr samples are more effective than the 70.6 hr samples overall, and the samples with cellulose are slightly less effective against nematodes. After 2 weeks (FIG.75), for the control and cellulose samples, the 2-dry cycles were more effective at inducing mortality. The sucrose was the least effective overall. FIG.76 shows the results after 20 weeks. FIGs.190A-B show the results after 6 months.

[0379] Methods for Formulation 5. Prepare 2% CaCl2as previously described, set aside for crossing link in the encapsulation process.

[0380] Alginate solution: Weigh out dry ingredients in a weighing plate (2 g Sodium alginate powder) and DI water separately and add the dry ingredient gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes.

[0381] Mix WCB with Alginate: Mix the Whole Cell broth containing sucrose 10 g / L with the mixture at the 1:1 ratio (w / w) using the Silverson, a high shear Mixer.

[0382] Encapsulation process: drip the Mixture at a flow rate at 3.9 ml / min controlled by a pump into the 2% (w / w) CaCl2solution while agitating on a magnetic stirrer (pump speed may be adjusted according to the viscosity of the mixture).

[0383] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0384] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0385] Storage: keep all samples at 4oC before packaging. Table 37. Experimental Formula Using WCB with SucroseExample 8 –Additional Formulations with Different pH Levels

[0386] Formulation of encapsulated Muscodor albus with alginate and WCB containing different pHs. Formulate encapsulated Muscodor albus with alginate and WCB at various pH levels from 2.5 to 9.2.

[0387] Methods for Formulation 6. Prepare 2% CaCl2as previously described, set aside for crossing link in the encapsulation process.

[0388] Alginate solution: Weigh out dry ingredients in a weighing plate (2 g Sodium alginate powder) and DI water separately and add the dry ingredient gradually while mixing with a Silverson, a high shear Mixer to eliminate clumps and fisheyes.

[0389] Mix WCB with Alginate: Mix the Whole Cell broth at pH of 2.5 with the mixture of 6.2 at the 1:1 ratio (w / w) using the Silverson, a high shear Mixer.

[0390] Encapsulation process: drip the Mixture at a flow rate at 3.9 ml / min controlled by a pump into the 2% (w / w) CaCl2solution while agitating on a magnetic stirrer (pump speed may be adjusted according to the viscosity of the mixture).

[0391] Washing process: Drain the CaCl2solution after encapsulation using a funnel with a filter paper layer, and then wash encapsulated beads with DI water.

[0392] Drying process: The encapsulated beads were loaded to a Fluid Bed Dryer with setting temp at 51oC to remove 50% of the total weight. The drying time depended on the loading size and initial temp of the beads.

[0393] Storage: keep all samples at 4oC before packaging. Table 38. Experimental Formula Using WCB at Various pH Levels

[0394] Stability testing of the different pH samples is shown in Table 39. FIGs.81-83 show photographic images of the stability studies, and FIGs.84-87 show nematode mortality assays of the various encapsulations using Muscodor albus WCB grown in different pHs. The pH 2.5 and pH 9.2 samples had very little mycelial growth compared to the others. However, pH 2.5-5.5 had around 40% mortality. From there it gradually increased in efficacy,reaching a peak of 87% at pH 8.2, then dropping back down at pH 9.2. The neutral pH’s were more effective at inducing nematode mortality, with the slightly basic pH 8.2 being significantly most effective. Table 39. Stability of the Encapsulated Muscodor albus with different pHs.* Indicates contamination. Example 9 –Additional Formulations

[0395] Additional formulations are shown below. Table 40. Encapsulated Muscodor albus with Kamin 80 B and Vangel at Different Ratios

[0396] Mortality assays of the samples in Table 40 are shown in FIGs.88A-B. The Vangel SX 2% sample and the three Kamin 80B and Vangel SX combination samples resulted in the highest nematode mortality in FIG.88A-B. Germination and Rhizoctonia assays for samples in Table 40 are shown in FIG.90 and 91. The Kamin 80B and Vangel SX combination 1 and 3 had the highest disease control in FIGs.88A-B. Table 41. Optimize the encapsulation to extend the stability of encapsulated Muscodor albusTable 42. Optimization to Extend the Stability of Encapsulated Muscodor albus

[0397] FIGs.89-91 show results for these formulations. FIG 89 shows that all formulations resulted in high nematode mortality (above 73%) with No.42% Alg+ 2% Kaomin 80 B being the highest (95%).

[0398] Formulations: a. Batch I (1200 ml): WCB w / sucrose w / 2.5 % Alginate b. Batch II (1200 ml): WCB w / sucrose w / 2 % Alginate + 25% Glycerol c. Batch III (1200 ml): WCB w / sucrose w / 2 % Alginate + 2% Clay i. Note: 2% Clay (1.5 % Kamin 80 B + 0.5 % Vangel) d. Batch IV (1200 ml): WCB w / sucrose w / 2 % Alginate + 25% Glycerol + 2% Clay i. Note: 2% Clay (1.5 % Kamin 80 B + 0.5 % Vangel). The formulations of Table 43 were evaluated for their in vitro nematicide activity as shown in FIG.92.

[0399] Testing of the concentration of Muscodor albus WCB used in the formulations are shown in Tables 43, 44 and FIGs.92-95. FIG 92 shows that samples N.4 – N.8 in Table 43 all have high in vitro nematicide mortality (>90%) when three pieces were tested. FIG.93 at a lower single rate showed that N.6 (74%) and N.8 (75%) were the highest. FIGs.94 and 95 showthat N.4 had the highest disease control of Rhizoctonia solani as illustrated by the number of germinated seeds compared to the inoculated control. Table 43. Concentration of WCB

[0400] Formulations to screen anti-sticky agent to prevent encapsulated Muscodor albus from sticking to dryer walls and improve the drying efficiency and eliminate mixing step during drying are shown in Table 44 and FIGs.96-99. The anti-sticky agents were 1) introduced into the whole cell broth or 2) used as surface treatments of encapsulated beads. Table 44. Encapsulated of Muscodor albus using Anti-sticky agents

[0401] FIGs.98 and 99 show the effect of different packaging on stability. The effect of vented culture bottles versus regular packages (bottle and bag) were tested in Tables 45-47 and FIGs.100-109. As shown in Table 46, the sample with 2 dry cycles didn’t survive after 4 days at 30ºC within the Ziplock bag (C1: 1 Dry cycle; C2: 2 Dry cycles). Only the sample within the vented bottle survived after 8 days at 30ºC. The sample packed with the regular bottle survived at higher moisture content.Table 45. Effect of vented culture bottle vs. regular packages (bottle & bag)Table 46. Stability resultsTable 47. Effect of Paper Autoclave Bag Versus Vented and Regular BottleExample 10 –Greenhouse Assay Results

[0402] Kamin and Vangel combination formulations were tested in greenhouse assays using the rates shown in Table 48. Results of the assays are shown in FIGs.110-112. Table 48. Greenhouse assay – Kamin and Vangel Combinations

[0403] Nutrient input combination formulations were tested in greenhouse assays using the rates shown in Table 49. Results of the assays are shown in FIGs.113-120. Table 49. Greenhouse assay – nutrient inputsExample 11 –Bioassay Test Results for Fruit Storage

[0404] Experiments testing the efficacy of encapsulated Muscodor albus WCB on prevention and reduction of infection of fruits such as strawberry, grape and apple by Botrytis cinerea, oranges by Penicillium digitatum, and apple by Penicillium expansum during storage were performed.

[0405] The fruit storage assay set up and inoculum preparation are shown in FIGs.121- 123. Table 50 shows treatments (Group 1) used in the strawberry storage experiments shown in FIGs.124-127. All formulations in Table 50 achieved at least 80% control of Botrytis cinerea in Strawberry at room temperature but only VEEGUM® MC (Smectite clay) achieved 50% control under cold storage. Table 50 treatments used in grape storage experiments are shown in FIG. 137. All formulations in Table 50 had high control of Botrytis cinerea in grape. Table 50 treatments used in orange storage experiments are shown in FIG.146 showing that the encapsulate formulations limited control of Penicillium digitatum sporulation. Table 50. Treatment Group 1 Tested in Fruit Storage Bioassay

[0406] The 2-R barley control refers to the following alternate formulation process of M. albus WCB.2-R barley is prepared by applying the end of M. albus fermentation whole cell broth on to sterile barley by soaking barley in the end of fermentation whole cell broth. Pearled barley is sterilized by autoclaving for a minimum of 30 minutes and added to an excess of whole cell broth from the end of fermentation. The barley is allowed to soak in the whole cell broth for 18 hours, excess liquid is removed, and the barley is allowed to dry. The purpose of including the barley is to compare this method to the described encapsulation process in terms of treatment rates and bioassay activity.

[0407] Table 51 shows treatments used in the strawberry storage experiments shown in FIGs.128-136. Table 51 treatments used in grape storage experiments are shown in FIGs.138- 143. The formulations were able to provide control of Botrytis cinerea even after their removal. Table 51 treatments used in orange storage experiments are shown in FIGs.147-160. Alginate and Kamin 80B had the greatest inhibition of Penicillium digitatum in orange. Table 51 treatments used in apple storage experiments are shown in FIGs.161-188. The formulations were able to provide effective control of Penicillium expansum and Botrytis cinerea even after their removal. Table 51. Treatment Group 2 Tested in Fruit Storage BioassayTable 52. Treatment Application RatesExample 12 –Methods for Bioassays

[0408] Methods for bioassays used in the above formulation studies are described below.

[0409] Rhizoctonia is a soil-borne fungal pathogen that causes significant crop losses worldwide. Rhizoctonia solani is known to infect a wide range of hosts, causing diseases such as damping-off, root rot, and wire stem. Several bioassays were developed to test the efficacy of various encapsulation formulations of Muscodor albus.

[0410] Rhizoctonia solani in vitro split plate bioassay. Encapsulated treatments were placed onto potato dextrose agar (PDA) on one side of a split 90 mm petri dish at a rate of 50mg or 150mg. After 24 hours, a 5mm mycelial plug of a 4-5 day old culture of Rhizoctonia solani AG4, grown in the dark at room temperature was placed onto the other side of the plate mycelial side down. Plates were sealed and stored in the dark at room temperature and after 48hrs, the mycelial radius was recorded, and the percentage disease control calculated.

[0411] Rhizoctonia solani in planta bioassay. A fresh subculture of R. solani AG4 was grown on PDA for 4-5 days in the dark at room temperature and macerated in a blender with roughly 50ml of distilled water at a rate of ¼ mycelial plate per liter of soil.100ml of infested soil was transferred to 3.5-inch pots and 2g of encapsulated treatment was thoroughly mixed with the soil. Included was an inoculated control with no treatment applied and an untreated control with just uninfested soil. Pots were bottom watered and incubated in a controlled chamber at 23oC, 70% relative humidity with 12hr light. After 24 hours, 50 organic broccoli seeds (Waltham 29) were added to each pot and a thin layer of clean soil spread over the top followed by a misting of water. After 7 days, the number of germinated seedlings were counted and the percentage disease control calculated.

[0412] Rhizoctonia solani greenhouse bioassay – plant health. R. solani was prepared for soil inoculation by culturing mycelium on sterile imbibed barley for 3 weeks. Encapsulation treatments were mixed into the top 2 inches of soil and 1 Rhizoctonia infested barley grain was placed 1 inch below the soil line. The soil was hydrated fully following the addition of the encapsulation treatment and Rhizoctonia to the soil. After 5 days, young broccoli or sugarbeet (depending on the Rhizoctonia isolate) were transplanted 1cm from where the Rhizoctonia grainwas placed. The assay ran for a minimum of 5 weeks before recording fresh aboveground biomass, root biomass and for sugarbeet a beet disease rating.

[0413] Nematicide in vitro bioassay. Encapsulated treatments were placed onto potato dextrose agar (PDA) at a rate of 50mg or 150mg and plates were placed in the center of a clean plastic crisper box (measuring 172mm x 122mm x69mm) and enclosed immediately and left at room temperature. After 24 hours, 4 petri dishes containing 2ml of distilled water were inoculated with between 400 and 450 freshly harvested root knot nematode J2s (Meloidogyne incognita) and placed into the corners of each treatment box. One plate from each treatment was analyzed under a compound microscope score living and dead nematodes for an initial count. Treatment boxes were stored for 5 days at room temperature in the dark before counting the number of living and dead nematodes to calculate percentage mortality.

[0414] Nematicide in planta bioassay. Cucumber plants were germinated in 3.5-inch pots with sand (6 plants per treatment). Once plants reached their first true leaf (1.5-2 weeks after sowing), encapsulated treatments were added to each plant and gently mixed into the top ½- inch of soil. Plants were hydrated with one acre-inch of soil (~200mL for 3.5-inch pots. After 72 hours, four holes were made around each plant and 500 freshly harvested root knot nematode J2s (Meloidogyne incognita) were inoculated per hole, for a total of 2000 per plant. The holes were covered with soil and after three weeks, a galling index rating was given to each plant.

[0415] Post-harvest bioassay. A post-harvest bioassay was developed to evaluate efficacy of encapsulated formulations in preventing or reducing post-harvest infection by pathogens in various fruits. Penicillium digitatum and Penicillium expansum are two significant post-harvest pathogens that cause substantial losses in a variety of fruits. Penicillium digitatum, often referred to as green mold, is the most common post-harvest fungal pathogen of citrus fruits. It is responsible for significant economic losses in the citrus industry worldwide. Penicillium expansum, the causal agent of blue mold, is a major post-harvest pathogen of pome and stone fruits, such as apples and peaches. It not only causes fruit decay but also produces mycotoxins that pose a risk to human health. Both pathogens are capable of rapid growth and spread under favorable conditions, making them a significant challenge in post-harvest product management.

[0416] Encapsulated treatments were placed onto potato dextrose agar (PDA) and tested at a rate of 50mg, 150mg or 770mg depending on the pathogen and host fruit. Plates were placed into the center of a clean plastic crisper box, measuring 172mm x 122mm x 69mm for strawberry and grape and 320mm x 235mm x 110mm for orange and apple and enclosed immediately. After 24 hours at room temperature, organic fruits were rinsed thoroughly under running tap water and surface sterilized by washing in 70% ethanol for 2 minutes. Fruit was then rinsed twice in sterilewater and left to air dry. A minimum of three fruits were placed into the corners of their respective crisper boxes and a 3mm wound was made per fruit with a sterile pipette tip for pathogen inoculation. Botrytis cinerea was prepared by growth on 2xV8 media for 5 days in a dark 19oC incubator and then flooding the plate with sterile water and scraping conidia. Mycelial fragments were removed via a filter and the conidia concentration was adjusted to the desired concentration using a hemocytometer. Conidia concentrations ranged from 1x10^4 conidia / ml to 1x10^6 during these tests. The post-harvest pathogens Penicillium digitatum and Penicillium expansum were also prepared in the same way except for growth on potato dextrose agar (PDA). A 20ul conidia suspension was pipetted into each wound per fruit and the crisper box immediately enclosed and stored at room temperature for the duration of the study. The length of the study depended on the pathogen and host fruit. Strawberry and grape were also tested under cold storage at 4oC immediately following pathogen inoculation. Strawberry was stored at 4oC for 3 days and grapes for 3 weeks before being moved to room temperature for 5 days. For strawberries and grapes, a visual rating was made of each individual fruit of the percentage of disease coverage. For apples, the radius of the surface lesion was measured to calculate the percentage disease control as well as the radius of the internal lesion after cutting the fruit through the center of the lesion. For oranges, the radius of the non-sporulating and sporulating mycelium on the surface of the fruit was recorded to calculate the percentage of disease coverage.

[0417] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. An encapsulated fungal microorganism comprising: a living fungus of the genus Muscodor encapsulated in a physical barrier, wherein the encapsulated fungal microorganism is metabolically active and is capable of releasing a volatile chemical through the physical barrier.

2. The encapsulated fungal microorganism of claim 1, wherein the fungus is Muscodor albus.

3. The encapsulated fungal microorganism of claim 1 or claim 2, wherein the physical barrier comprises alginate.

4. The encapsulated fungal microorganism of claim 3, wherein the alginate comprises sodium alginate.

5. The encapsulated fungal microorganism of any one of the preceding claims, wherein the physical barrier further comprises clay.

6. The encapsulated fungal microorganism of claim 5, wherein the clay comprises bentonite clay, a blend of bentonite clay and xanthan gum, attapulgite clay, kaolin clay, montmorillonite clay, and / or smectite clay or combinations thereof.

7. The encapsulated fungal microorganism of claim 5 or claim 6, wherein the clay comprises 0.1%-6% of the physical barrier.

8. The encapsulated fungal microorganism of any one of the preceding claims, wherein the volatile chemical is an antimicrobial agent.

9. The encapsulated fungal microorganism of any one of the preceding claims, wherein the volatile compound is selected from 2 methyl-propanoic acid, 2 methyl-1- butyl acetate, ethanol, phenyl ethyl alcohol, 3 methyl-1-butyl acetate, 2 methyl-1-butanol, 3 methyl-1-butanol and combinations thereof.

10. The encapsulated fungal microorganism of any one of the preceding claims, wherein the encapsulated fungal microorganism is free of barley.

11. The encapsulated fungal microorganism of any one of the preceding claims further comprising: glycerol.

12. The encapsulated fungal microorganism of any one of the preceding claims further comprising: silica.

13. The encapsulated fungal microorganism of any one of the preceding claims further comprising: a carbohydrate selected from sucrose, starch, cellulose, trehalose, and combinations thereof.

14. The encapsulated fungal microorganism of any one of the preceding claims, comprising a pH of 2.5 to 9.

2.

15. The encapsulated fungal microorganism of any one of the preceding claims, comprising a pH of 6.0 to 8.

2.

16. The encapsulated fungal microorganism of any one of the preceding claims, wherein the encapsulated fungal microorganism is capable of remaining metabolically active for at least 24 weeks at room temperature.

17. The encapsulated fungal microorganism of any one of the preceding claims, wherein the encapsulated fungal microorganism is capable of remaining metabolically active for at least 24 weeks under refrigeration.

18. The encapsulated fungal microorganism of any one of the preceding claims, wherein the encapsulated fungal microorganism is capable of preventing or reducing phytopathogenic infection in a plant or plant part.

19. The encapsulated fungal microorganism of claim 1 comprising: 50% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; and 1% w / w kaolin clay.

20. The encapsulated fungal microorganism of claim 1 comprising: 99% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus and 1% w / w sodium alginate.

21. The encapsulated fungal microorganism of claim 1 comprising: 74% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 25% w / w glycerol; and 1% w / w sodium alginate.

22. The encapsulated fungal microorganism of claim 1 comprising: 73% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 25% w / w glycerol; 1% w / w sodium alginate; 0.75% w / w kaolin clay; and 0.25% w / w a blend of bentonite clay and xanthan gum.

23. The encapsulated fungal microorganism of claim 1 comprising: 89% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; 5% w / w PVA; and 5% w / w PEG 400.

24. The encapsulated fungal microorganism of claim 1 comprising: 50% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; and 1% w / w bentonite clay.

25. The encapsulated fungal microorganism of any one of claims 19 to 24, wherein the whole cell broth (WCB) comprises a carbohydrate selected from sucrose, starch, cellulose, trehalose, and combinations thereof.

26. A method for encapsulating a fungal microorganism, said method comprising: providing a whole cell broth (WCB) solution comprising whole cells of a living microorganism of the genus Muscodor; contacting the whole cell broth (WCB) solution with alginate; encapsulating the whole cells of the living microorganism in the alginate; and drying the encapsulated microorganism, wherein the dried encapsulated fungal microorganism is living, metabolically active, and capable of releasing a volatile chemical.

27. The method of claim 26 further comprising: contacting the whole cell broth (WCB) solution with clay simultaneously with or sequentially with said contacting the whole cell broth (WCB) solution with the alginate.

28. The method of claim 25 or claim 26 further comprising: contacting the whole cell broth (WCB) solution with glycerol prior to said contacting with alginate.

29. The method of any one of claims 26-28, wherein the whole cell broth (WCB) solution further comprises a carbohydrate selected from sucrose, starch, cellulose, trehalose, and combination thereof.

30. The method of any one of claims 26-29, wherein the alginate comprises sodium alginate powder.

31. The method of any one of claims 26-30 further comprising: storing the dried, encapsulated fungal microorganism at a temperature of about 4ºC.

32. The method of any one of claims 26-31, wherein said drying is carried out to a moisture content of 50-99%.

33. The method of any one of claims 26-32, wherein said encapsulating comprises contacting the living microorganism in the alginate with calcium to form calcium alginate beads.

34. The method of any one of claims 26-33, wherein the fungus is Muscodor albus.

35. The method of any one of claims 26-34, wherein the volatile chemical is an antimicrobial agent.

36. The method of any one of claims 26-35, wherein the encapsulated fungal microorganism is free of barley.

37. The method of claim 26, wherein the encapsulated organism comprises a composition comprising: 50% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; and 1% w / w kaolin clay.

38. The method of claim 26, wherein the encapsulated organism comprises a composition comprising: 99% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; and 1% w / w sodium alginate.

39. The method of claim 26, wherein the encapsulated organism comprises a composition comprising: 74% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 25% w / w glycerol; and 1% w / w sodium alginate.

40. The method of claim 26, wherein the encapsulated organism comprises a composition comprising:73% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 25% w / w glycerol; 1% w / w sodium alginate; 0.75% w / w kaolin clay; and 0.25% w / w a blend of bentonite clay and xanthan gum.

41. The method of claim 26, wherein the encapsulated organism comprises a composition comprising: 89% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; 5% w / w PVA; and 5% w / w PEG 400.

42. The method of claim 26, wherein the encapsulated organism comprises a composition comprising: 97% w / w whole cell broth (WCB) comprising whole cells of Muscodor albus; 1% w / w sodium alginate; and 2% w / w PVPP.

43. The method of any one of claims 26-42, wherein the WCB comprises sucrose, starch, trehalose, and / or cellulose.

44. A method of treating a plant or plant part for a phytopathogenic organism, said method comprising: exposing or contacting a plant, plant part, or a growing medium for the plant with an effective amount of a volatile chemical from an encapsulated fungal microorganism of any one of claims 1-25, wherein the volatile chemical from the encapsulated fungal microorganism treats the plant, plant part, or growing medium for the phytopathogenic organism.

45. The method of claim 44, wherein the plant part is a harvested fruit or vegetable.

46. The method of claim 44 or claim 45, wherein the plant part is stored at ambient temperature or under refrigeration.

47. The method of any one of claims 44-46, wherein the phytopathogenic organism is a Rhizoctonia spp., a Botrytis spp. or a Penicillium spp.

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