Abstract:
Sprayable, starch-based formulations for autoencapsulating biological control agents, such as pathogenic bacteria and viruses, incorporate a sugary material to promote adherence of the encapsulated agent to treated foliage. The autoencapsulated pathogens are characterized by high survivability and are useful in controlling insects and other pest species.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     It has been estimated that entomopathogens, mainly Bacillus thuringiensis (B.t.), will reach a $100,000,000 market by 1992, approximately 90% of which will be sold as sprayable formulated material. Effectiveness of spray formulations is dependent on attractiveness to target pest insects and retention of pathogenic activity. This invention relates to a novel spray formulation, based on a renewable resource, which satisfies these criteria. 
     2. Description of the Prior Art 
     The use of starch has many attractive properties for biocontrol agent encapsulation. First, it is inert and will not alter resting stages of most living organisms; second, particulate or liquid UV-screening agents are easily added; third, its major component is amylopectin which is readily digested by most phytophagous pests possessing α-amylase enzymes [G. M. Chippendale et al., J. Insect Physiol. 20: 751-759 (1974); K. Nishide et al., J. Fac. Agric. Tottori Univ. 11: 12-22 (1976)]; and fourth, it is abundant and inexpensive compared to most other materials currently used in encapsulation [B. S. Shasha, In Controlled Release Technologies: Methods, Theory, and Applications, Vol. 2, A. F. Kydoniens (ed.), CRC Press, Inc., Boca Raton, Fla.]. 
     Recently, Dunkle et al. [Environ. Entomol. 17: 120-126 (1988) and U.S. patent application Ser. No. 07/72,205 filed on July 10, 1987, now U.S. Pat. No. 4,859,377] prepared a granular formulation of B.t. encapsulated within a starch matrix. The advantage of this method over existing formulations is that it allows incorporation of various additives such as sunlight protectors to prevent solar inactivation and feeding stimulants to increase palatability and thereby reduce the amount of active ingredient necessary for control. Trimnell et al. [J. Controlled Release 7: 263-268 (1988)] have reported a sprayable herbicide formulation utilizing pregelatinized corn starch and flour. These sprays give a thin film of the formulation on plant leaves which autoencapsulates (encapsulates the active agent in situ) upon drying and thereby allows sustained release of active ingredient. However, within 2-3 days after application, these films peel away from the plant leaves. In general, sprayable formulations of B.t. lose activity within 2-4 days following application to plant foliage in the field [Morris, Can. Ent. 115: 1215-1227 (1983); Beegle et al., Environ. Entomol. 10: 400-401 (1981); Leong et al., Environ. Entomol. 9: 593-599 (1980)]. 
     SUMMARY OF THE INVENTION 
     We have now unexpectedly discovered that when a sugary material is incorporated into a spray formulation in combination with a pregelatinized starchy material and a biocontrol agent, the sugary material acts as a sticking agent, and the resulting formulation is retained on plant leaves for a dramatically longer period of time. 
     In accordance with this discovery, it is an object of the invention to provide a facile, universal, and industrially acceptable formulation for autoencapsulation of sensitive biocontrol agents. 
     It is also an object of the invention that the primary matrix-forming material be derived from naturally renewable resources. 
     Another object of the invention is that the resulting encapsulation be characterized by high survivability of the active agent. 
     It is a further object of the invention that the encapsulated substance be controllably released to the target pests and resistant to losses by environmental conditions. 
     Other objects and advantages of this invention will become readily apparent from the ensuing description. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Starch is a low-cost and abundant natural polymer composed of amylose and amylopectin. Amylose is essentially a linear polymer having a molecular weight in the range of 100,000-500,000, whereas amylopectin is a highly branched polymer having a molecular weight of up to several million. When starch is gelatinized in water and cooled, the amylose retrogrades to a much greater extent than the amylopectin fraction. Retrogradation is a term applied to the phenomenon whereby starch chains in dispersion associate, become insoluble, and precipitate. The rate and extent of retrogradation depend on properties of dispersion (pH, temperature, concentration) and on the amount of amylose present in the dispersion. Common corn starch (pearl) contains about 25% amylose and 75% amylopectin; whereas the waxy corn starches contain only amylopectin, and those referred to as high-amylose starch contain up to 75% amylose. 
     The starting encapsulating material for use in the invention includes any pregelatinized starch which will form a gel upon rehydration in an aqueous medium. Pregelatinized starches are commercially available and are prepared for example by cooking the starch at elevated temperatures and pressures in the presence of a lower alcohol. A preferred pregelatinized starch is a product sold commercially under the tradename &#34;MIRA-SPERSE&#34; which contains mostly amylopectin. Source materials for deriving the pregelatinized starch include pearl corn starch, potato starch, tapioca starch, flours containing these starches, as well as mixtures of these with waxy corn starch and high-amylose corn starch. 
     The sugary materials contemplated for use in the invention as sticking agents include sucrose, glucose, fructose, mannose, α-methyl glucoside, and various corn syrups. The amount of sugary material required is that amount which is effective to delay the peeling of the dried formulation from the target substrate. Ratios of starch:sugary material will typically range from about 1:2 to about 1:0.6, with ratios in the range of 1:1 to 1:0.6 being preferred. 
     The biocontrol agents contemplated for use herein include without limitation all bacteria, fungi, yeasts, viruses, microsporidians, protozoa, and other lower organisms which are pathogenic toward target pests. Of course any component of the organism or stage of its life cycle which is infective to the host upon ingestion is considered to be within the scope of the invention. For instance, in the case of B.t., the vegetative cells, spores, and proteinaceous crystals are all effective in directly or indirectly killing host insects susceptible to B.t. It is also known that naturally occurring and synthetic vectors such as plasmids, phages, and various DNA/RNA constructs have potential for functionally modifying higher organisms, and therefore are also included herein as being within the scope of the term &#34;biocontrol agent.&#34; Examples of other agronomically important pest pathogens besides B.t. are B. sphaericus, B. popillae, microsporidians such as Vairimorpha necatrix and Nosema locustae, Autographa californica nuclear polyhedrosis virus, and Heliothis spp. virus, and the fungus Beauveria bassiana. 
     The target pests contemplated for control by means of the subject encapsulated agents include all species susceptible to the above-mentioned biocontrol agents. These characteristics are typical of most phytophagous (plant-eating) insects, especially those considered to be crop or tree pests. 
     Besides the active agent itself, other additives and adjuncts may be formulated into the subject compositions. Examples of these include dispersants, feeding stimulants (phagostimulants), UV protectants, preservatives, and inert fillers. Also of interest are agronomically acceptable carriers or vehicles for the active agent or any of the other components formulated into the encapsulated compositions. 
     In accordance with one embodiment of the invention, formulation of the biocontrol agent into a sprayable liquid is performed by dry-mixing the pregelatinized starchy material with the sugary material and combining this mixture with a dispersion of the entomopathogen in water. Vigorous stirring is usually required to disperse the starch in water. Alternatively, the starch material and/or the sugary material can be predispersed in water prior to combination with the entomopathogenic agent. The pregelatinized starchy material in aqueous dispersion must have a stable but low enough viscosity to be sprayable by conventional equipment. This property is characteristic of diluted starches and flours as well as starches and flours which have been partially degraded by chemical or physical means to the extent that the amylose chains will not spontaneously reassociate to a significant degree until their concentration in dispersion is raised above a certain threshold value. Thus, gel formation is retarded until evaporation of water from the sprayed composition causes the concentration of the degraded starch molecules to exceed the threshold, and then autoencapsulation occurs. Initial concentrations of the starch in the sprayable formulation should be in the range of about 1-10% by weight. In field application, droplets of the liquid adhere to the foliage surfaces and remain bound thereto even after gelling takes place. 
     In yet another embodiment of the invention, the biocontrol agent, pregelatinized starchy material, and the sugary material can be admixed and applied to the plant foliage as a dry formulation. The hygroscopic nature of the sugar enables the mixture to absorb moisture from the ambient. Moisture provided during periods of high humidity, dew, and rain will promote in situ formation of an aqueous dispersion of the formulation and gelling of the starchy material. Upon drying, autoencapsulation occurs as previously described. 
     The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. 
     EXAMPLE 1 
     Preparation of Formulations 
     Seven formulations given in Table I, and devoid of biocontrol agent were prepared for subsequent evaluation. The single dry component of Formulations 1-3 was dispersed in 300 ml of water using a Waring blender. Formulation 4 was prepared by pasting the dry component in the glycerol before dispersion in the water (supra). The dry components of Formulations 5-7 were thoroughly mixed dry before dispersion in the water. 
     The amount of material not dissolved and therefore likely to clog spray nozzles was measured by screening (0.7-mm diameter pores) the formulations and weighing the dried residues. Viscosity of the screened formulations was measured with a &#34;Brookfield LVF&#34; viscometer at 6 rpm and 21° C., 2 hr after initial mixing. The results are reported in Table II below. &#34;MIRA-SPERSE&#34; contains mostly amylopectin, whereas &#34;MIRA-GEL&#34; contains the same level of amylose (about 25%) as found in regular corn starch. &#34;MIRA-SPERSE&#34; (formulations 2 and 6) dispersed completely, did not retrograde to form clumps, and left no residue when screened. &#34;MIRA-GEL&#34; (1) and pregelatinized flour (3) retrograded somewhat to produce clumps which resulted in a residue on the screen. Furthermore, the protein of the pregelatinized flour did not dissolve well. The addition of glycerol to &#34;MIRA-GEL&#34; (4) alleviated the residue problem of this component, and sucrose reduced the amount of residue from &#34;MIRA-GEL&#34; (5) and flour (7). The viscosity of all formulations, including those containing &#34;MIRA-SPERSE&#34; (2 and 6), was well within the range required for sprayable materials. 
     EXAMPLE 2 
     (B.t.) Viability in Formulations 
     Seven formulations were prepared as described in Example 1, then autoclaved, and cooled to room temperature. B.t. (technical powder, 80,000 IU/mg, Abbott Laboratories, North Chicago, Ill.) was suspended in sterile water, and aliquots were thoroughly mixed into each of the formulations. The formulations containing the B.t. were held for 0, 4, or 7 days at 2° C. after which samples were diluted and plated (10 μl) on the Semidefined Growth Medium for Bacillus thuringiensis of Luthy [Vierteljahrsschrift der Naturforschenden Gesellschaft in Zurich 120: 81-163 (1975)]. Following incubation for 24 hr at 28° C., colonies were counted. None of the liquid formulations tested were toxic to B.t. spores, as shown by the results in Table III, which gives average numbers of colonies from diluted samples. An increase in colony counts over a 7-day period suggests the occurrence of spore germination and growth of vegetative cells. 
     EXAMPLE 3 
     Formulation Adherence to Leaf Surfaces 
     Cotton plants were obtained approximately 3 wks after seeding, when 2-4 true leaves had expanded. Upper surfaces of the leaves were treated with the seven formulations of Example 1. Coatings were applied by brushing the formulations onto the leaves with a 2.5-cm paint brush. After the leaves had dried, plants were subjected to one of two watering regimes: (1) plants were watered only to the soil or (2) plants were watered to the soil and to the foliage. Foliage watering was accomplished every 2 days by allowing water to flow from an 8-cm diameter nozzle with 1-mm perforations until runoff occurred. Estimates of the amount of applied material adhering to each leaf were made every 1-2 days by visual examination. The same individual made all the estimates throughout the experiment. The estimates are expressed as percent of applied material in Table IV (watered to soil only) and Table V (watered to soil and foliage). 
     The results show that the &#34;MIRA-SPERSE&#34;-sucrose formulation (Formulation 6) remained on leaves longer than any of the other formulations regardless of watering regime. &#34;MIRA-SPERSE&#34; alone (Formulation 2) quickly dried and flaked off the plant leaves when watering was to the soil, but the leaves retained approximately 50% of the applied material when watering was over the leaves. &#34;MIRA-GEL&#34; and pregelatinized flour without additives (Formulations 1 and 3, respectively) both lost material quickly regardless of watering regime. When watering was to the soil only, &#34;MIRA-GEL&#34; and flour combined with sucrose (Formulations 5 and 7, respectively) remained on the leaves for a longer period of time. When watering was over the leaves, material was not lost from these formulations until the fifth day after application. This compares to a loss of material within 3 days when sucrose was not present. The largest effect of sucrose was observed with &#34;MIRA-SPERSE.&#34; When sucrose was present and watering was over the leaves, less than 5% of applied material was lost up to 13 days post-application. When watered to the soil only, material was not lost until day 20 of the experiment. 
     EXAMPLE 4 
     Effect of Sugar Type on Adherence of &#34;MIRA-SPERSE&#34; Formulations 
     Formulations of &#34;MIRA-SPERSE&#34; and the various sugars shown in Table VI in a 1:1 ratio were prepared as described in Example 1 for Formulations 5-7. The concentration of each component was 3.3%. The formulations were applied to cotton plants and evaluated for adherence by the procedure of Example 3 in which the plants were watered to the pot. The results are reported in Table VI below. 
     EXAMPLE 5 
     Effect of Sucrose Concentration on Adherence of &#34;MIRA-SPERSE&#34; Formulations 
     Formulations of &#34;MIRA-SPERSE&#34; and sucrose in various ratios were prepared as described in Example 1 for Formulations 5-7. The concentration of &#34;MIRA-SPERSE&#34; was 3%. The formulations were applied to cotton plants and evaluated for adherence by the procedure of Example 3 in which the plants were watered to the pot. The results are reported in Table VII below. 
     EXAMPLE 6 
     Effect of Corn Syrup Solids on Adherence of &#34;MIRA-SPERSE&#34; Formulations 
     Formulations of &#34;MIRA-SPERSE&#34; and corn sugars solids in various ratios were prepared as described in Example 1 for Formulations 5-7. The concentration of &#34;MIRA-SPERSE&#34; was 3%. The formulations were applied to cotton plants and evaluated for adherence by the procedure of Example 3. For each ratio, the plants were divided into two groups, one of which was watered to the soil only (pot), and the other to the soil and foliage (leaves). The results are reported in Table VIII below. 
     EXAMPLE 7-9 
     Bioassay of B.t.-Treated Leaves Against the European Corn Borer (ECB) 
     Formulations of &#34;MIRA-SPERSE&#34; and sucrose in a 1:1 ratio were prepared by dry mixing 1.5 g of each component and dispersing the mixture in 50 ml sterile water containing 3 mg B.t. technical powder (Example 7) or 20 mg &#34;Dipel 2X&#34; (Examples 8-9) (32,000 IU/B.t./mg) using a Waring blender. The formulations were applied to cotton plants as described in Example 3. As a control, additional leaves were treated with water containing similar amounts of B.t. in the absence of an encapsulating system. Plants watered to the pot were compared to plants watered to the leaves. On the day of assay, leaves were excised and trimmed to fit 9-cm diameter petri dishes. Ten ECB larvae less than 12-hr old were added, filter paper was applied to the lid to absorb excess moisture, and the dish was sealed with two wraps of &#34;Parafilm.&#34; Dishes were incubated at 28° C. in the dark for 3 days and then examined for live and dead larvae. Shapes of the individual curves were compared by analysis of variance and subsequent linear and quadratic contrasts. The results are reported in Table IX below. 
     It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention. 
     
                                           TABLE I__________________________________________________________________________Test-Sprayable Test FormulationsFormulation  &#34;MIRA-GEL&#34;.sup.a          &#34;MIRA-SPERSE&#34;.sup.b                    Flour.sup.c                            Glycerol.sup.d                                    Sucrose.sup.eNo.    (g/300 ml H.sub.2 O)          (g/300 ml H.sub.2 O)                    (g/300 ml H.sub.2 O)                            (g/300 ml H.sub.2 O)                                    (g/300 ml H.sub.2 O)__________________________________________________________________________1      102              103                        104      10                        205      10                                106              10                        107                        10              10__________________________________________________________________________ .sup.a Pregelatinized corn starch (industrial grade, C3445, A. E. Staley Co.). .sup.b Pregelatinized corn starch (industrial grade, C3444, A. E. Staley Co.). .sup.c Pregelatinized corn flour (12687, Illinois Cereal Mills, Inc.). .sup.d ACS grade, Fisher Scientific. .sup.e &#34;Domino Confectioners 10X Sugar,&#34; Amstar Sugar Corporation. 
    
     
                       TABLE II______________________________________Physical Characteristics of Sprayable FormulationsFormu-lation                    Residue   ViscosityNo.    Components         (mg)      (cp)______________________________________1      &#34;MIRA-GEL&#34;         223       502      &#34;MIRA-SPERSE&#34;       0        34003      corn-flour         235       504      &#34;MIRA-GEL + glycerol                      0        705      &#34;MIRA-GEL&#34; + sucrose                      55       3306      &#34;MIRA-SPERSE&#34; + sucrose                      0        34007      corn-flour + sucrose                     120       70______________________________________ 
    
     
                       TABLE III______________________________________Viability of  .sub.-- B. .sub.- t. in Sprayable Test FormulationsFormu-                    Days in contact withlation                    formulationNo.    Component          0       4     7______________________________________  water              24.8    56.8   55.91      &#34;MIRA-GEL&#34;         19.2    50.9  102.62      &#34;MIRA-SPERSE&#34;      49.7    92.1   83.13      corn-flour         58.5    102.5 120.04      &#34;MIRA-GEL&#34; + glycerol                     35.2    46.9   93.15      &#34;MIRA-GEL&#34; + sucrose                     38.7    44.9   77.06      &#34;MIRA-SPERSE&#34; + sucrose                     110.7   124.1 129.07      corn-flour + sucrose                     95.8    104.4 123.6______________________________________ 
    
     
                       TABLE IV______________________________________Percent of Original Material Remaining onLeaf Surface When Watered to Pot OnlyDays after    Formulationapplication    1      2      3    4    5     6     7______________________________________1        99     99     99   100  100   100   1002        99     99     99   100  100   100   1003        75     85     90   100  100   100   1004        75     25     50   90   100   100   1005        50     25     50   60   100   100   1007        40     25     25   60   100   100   1008        40     25     20   40   100   100   1009        35     25     20   30   100   100   10011       35     10     10   30    90   100   10013       35     10     10   30    90   100   10015       35     10     10   20    85   100    9518       25     10     10   20    60   100    9020       20     10     10   20    60    95    90______________________________________ 
    
     
                       TABLE V______________________________________Percent of Original Material Remainingon Leaf Surface When Watered to Pot and LeavesDays after    Formulationapplication    1      2      3    4    5     6     7______________________________________1        99     99     99   100  100   100   1002        99     99     99   100  100   100   1003        85     90     99   90   99    100   1004        50     60     50   70   98    98    1005        40     60     20   65   60    98    207        40     50     10   50   40    98    108        35     50     10   50   50    98    109        30     50      0   50   50    98    1011       30     50      0   30   30    98    1013       30     50      0   30   30    80    1015       30     50      0   25   40    70    1018       20     50      0   10   30    70    1020       20     40      0   10   30    70     5______________________________________ 
    
     
                                           TABLE VI__________________________________________________________________________Effect of Sugar Type on Adherence of &#34;MIRA-SPERSE&#34; FormulationsDays after % of Original material remaining on plantapplication M. glucoside        Glucose             Mannose                  Fructose                       Sucrose                            Mannitol                                 None__________________________________________________________________________ 5    100    100  100  100  100  95   65 7    100    100  100  100  100  60   5511    100    95   100  100  100  20   2514     95    95   100  100  100  25   2017     90    95   100  100  100  20   20__________________________________________________________________________ 
    
     
                       TABLE VII______________________________________Effect of Sucrose Concentration on Adherenceof &#34;MIRA-SPERSE&#34; Formulations     % of Original material remaining on plantDays after     &#34;MIRA-SPERSE&#34;:sucroseapplication     10:10     10:6   10:4    10:2 10:0______________________________________ 6        100       100    50      30   3010        100       100    50      30   2513        100       100    50      20   25______________________________________ 
    
     
                                           TABLE VIII__________________________________________________________________________Effect of Corn Syrup Solids on Adherence of &#34;MIRA-SPERSE&#34; Formulations% of Original material remaining on plants&#34;MIRA-SPERSE&#34;:corn syrup solidsDays after 10:10 10:7.5             10:5  10:2.5                         10:0application Pot    Leaf       Pot          Leaf             Pot                Leaf                   Pot                      Leaf                         Pot*                             Leaf__________________________________________________________________________ 5    95 100       85 100             70 100                   85 100                         . . .                             100 9    85 100       50 95 25 95 10 85 . . .                             9012    70 100       50 95 25 95 10 85 . . .                             9014    70 100       50 95 25 95 10 85 . . .                             90__________________________________________________________________________ *Not done. 
    
     
                                           TABLE IX__________________________________________________________________________Bioassay of Leaves Treated with  .sub.-- B. .sub.- t. and Exposed toDifferent watering RegimesExample  Amount    Mean % mortality (Reps).sup.1(conclusions  of     Days            Encapsulated  Not encapsulatedP &lt; 0.05).sup.2,3)   .sub.-- B. .sub.- t. (IU/ml)         PA Leaves                Pot Watered to                          Leaves                              Pot__________________________________________________________________________7      40,000 1  100(5)                100(5)    100(5)                              100(5)(A,B,C,D)     8  98(10)                95(10)     46(10)                              38(10)         15 35(5)                100(5)    15(4)                              7.5(4)8      12,800 0  94(5)                88(5)     72(5)                              66(5)(B,C,D)       7  79(10)                83(10)    20(6)                              25(10)         14 50(5)                44(5)     14(5)                              0(5)9      12,800 1  99(10)                100(10)    90(10)                              91(10)(A,B,C,D)     8  79(10)                87(10)     16(10)                               7(10)         15 24(10)                72(10)     2(10)                              10(10)__________________________________________________________________________ .sup.1 Replications consisted of individual Petri dishes containing 10 EC larvae and one leaf. .sup.2 A. Significant difference between encapsulatedleaf watered and encapsulated potwatered leaves. B. Significant difference between encapsulatedpot watered and not encapsulatedpot watered leaves. C. Significant difference between encapsulatedleaf watered and not encapsulatedleaf watered leaves. D. Lumping watering treatments, significant difference between encapsulated and not encapsulated treatments. .sup.3 No. significant differences between not encapsulatedleaf watered and not encapsulatedpot watered leaves.