Methods for production, stabilization and use of fungal blastospores

Abstract

 

Description

1 / 45 “METHODS FOR PRODUCTION, STABILIZATION AND USE OF FUNGAL BLASTOSPORES” [1] This application claims priority for U.S. Provisional Application No. 62 / 050473, filed on September 15, 2014. BACKGROUND OF THE INVENTION [2] Field of Invention [3] The present invention relates generally to methods and compositions related to the control of arthropod pests involving the use of entomopathogenic fungi. Specifically described are methodologies and compositions for the production and drying of desiccation-tolerant blastospores (vegetative cells) of Beauveria bassiana and Cordyceps fumosorosea to control soft-bodied insect pests. [4] Background [5] Plant diseases and damage caused by arthropod pests result in significant economic losses to agriculture and agro-industry worldwide. Many and varied approaches have been sought in order to control pests that attack plants, especially plants with commercial value. Despite this, the destruction of these pests from plants and plant-derived products still represents a major challenge. [6] Traditionally, arthropod pest control has been carried out through the application of chemical insecticides. The use of chemicals is subject to a number of disadvantages. Insect pests can and have developed resistance to chemicals over time, culminating in the selection of resistant populations. In fact, resistance to chemical pesticides constitutes a major obstacle to the viability of chemical pest control in the agricultural and horticultural industries. Furthermore, chemical pesticides are not always selective against their targets, as they often negatively affect beneficial species, such as pollinating insects. Due to this and other adverse effects, such as deleterious effects on human health and the environment, other alternative control methods, such as biological control, have been investigated and encouraged. One such approach consists of the use of certain entomopathogenic fungi as microbial biological control agents.The resurgence of interest in biological control, such as microbial insecticides, over the past 20 years has stemmed directly from public opinion pressure in response to... Petition 870260076623, dated 07 / 31 / 2026, page 11 / 58 2 / 45 concerns about the toxicity of chemical pesticides. Biological control presents an alternative means of controlling arthropod pests, being potentially more effective and specific than the methods currently practiced, as well as contributing to a reduction in dependence on chemical products. [7] Insect pathogenic fungi are useful as biological control agents due to their ability to infect a wide range of insect pests and their potential for mass production. Production and formulation are key components for their success as commercial products. There are different methods for mass production, including solid substrate fermentation (SSF) for aerial conidia and liquid culture fermentation (LCF) for yeast-like blastospores, as well as microcyclic conidia and microsclerotia. To date, most entomopathogenic ascomycete fungi (Ascomycota: Hypocreales) deployed in flood biological control strategies include Beauveria bassiana sensu lato (Bals). Vuill., B. brongniartii (Sacc.) Petch, Cordyceps fumosorosea Wise (formerly Paecilomyces futuroroseus), Lecanicillium longisporum and L. muscarium (Petch) R. Zare and W. Gams (formerly Verticillium lecanii) and Metarhizium anisopliae sensu lato (Metsch.Sorokin, and its aerial conidia comprise the main active ingredient of these mycopesticides, which are mostly produced using solid substrate cultivation techniques. Unfortunately, the fermentation time to reach the sporulation phase on solid substrates generally requires weeks, and the process is also labor-intensive, with a high risk of contamination, resulting in high production costs. Liquid fermentation technology, on the other hand, can overcome these obstacles of solid-media production and provide more economical scale with the ability to produce different fungal propagules under more controlled nutritional and environmental conditions during the fermentation process.Due to the short fermentation time (comprising only a few days), the ease of product recovery, the automation of the process, and the availability of inexpensive media components, submerged liquid fermentation is considered the most cost-effective method for the production of fungal biological control agents. [8] Thus, a method for producing infective propagules of the fungus in liquid culture is desirable. However, there are technical obstacles to overcome. Firstly, anamorphic entomopathogenic fungi of the order Hypocreales, such as B. Petition 870260076623, dated 07 / 31 / 2026, page 12 / 58 3 / 45 bassiana and C. fumosorosea produce yeast-like cells of the blastospore type, not conidia (asexual spores), in liquid culture. Although called blastospores, these cells are actually vegetative yeast-like cells that are not desiccation-tolerant and have a relatively limited shelf life (ChongRodriguez et al., 2011; Lohse et al., 2014). Thus, the development of methodologies for blastospore production using submerged liquid culture techniques that exhibit desiccation tolerance and long stability during storage could be used for pest control. Therefore, we describe such methodologies here. [9] Fungi are well known for their ability to metabolize a diverse range of compounds, expressing catabolic enzymes and permeases. After carbon and oxygen, nitrogen is the most abundant element in fungal cells and is one of the most expensive nutrients in the liquid fermentation medium. Identifying low-cost nitrogen sources is fundamental in developing an economically viable liquid medium for the production of a suitable biopesticide. Low-cost nitrogen sources, such as cottonseed and soybean meal, are generally unrefined and contain mainly proteins and oligopeptides. These processed byproducts of the agricultural and food industry are less expensive than highly refined nitrogen sources, such as acid- or enzymatically hydrolyzed casein, soybean hydrolysate, or meat proteins, which contain high amounts of free amino acids.Previous studies have demonstrated the feasibility of producing C. fumosorosea blastospores in short fermentation times (< 3 days), with good tolerance to desiccation, using more refined nitrogen sources, such as acid hydrolyzed casein.

[10] Since entomopathogenic fungi have a wide genetic variability and respond differently to growth in liquid media, specific parameters of the appropriate strain must be considered when evaluating and optimizing production parameters in liquid culture. A study with C. fumosorosea defined the nutritional and environmental conditions that support the rapid production of high concentrations of desiccation-tolerant blastospores with a reasonable shelf life. However, the commercial use of B. bassiana blastospores is nonexistent due to the requirement for long fermentation times and the low desiccation tolerance and ephemeral shelf life of its blastospore-based preparations. The production of desiccation-tolerant blastospores Petition 870260076623, dated 07 / 31 / 2026, page 13 / 58 4 / 45 desiccation, which exhibit stability during storage and proven bioefficacy simultaneously, has not been achieved previously and is therefore desired. SUMMARY OF THE INVENTION

[11] In one of the preferred embodiments of the invention, a method for producing a composition containing blastospores is provided, wherein the composition comprises desiccation-tolerant blastospores of a Beauveria species or a Cordyceps species produced by inoculating a liquid culture medium, comprising a carbon source and a nitrogen source, with propagules of the Beauveria fungus of a species or a Cordyceps species, incubating said propagules under culture conditions permitting a dissolved oxygen level above zero and an osmotic pressure greater than 0.5 MPa, incubating the propagules for a time sufficient to produce blastospores, collecting / extracting the blastospores; and drying the blastospores, thereby producing desiccation-tolerant blastospores. In one of the preferred embodiments of the invention, the Beauveria species is B. bassiana.In other preferred embodiments of the invention, the Cordyceps species is C. fumosorosea. In one particular embodiment, the carbon source is present in the liquid culture medium at an initial concentration of at least six percent. The carbon source may be glucose (dextrose). In another particular embodiment, the nitrogen source is present in the liquid culture medium at an initial concentration of at least one and a half percent. The nitrogen source may be cottonseed meal or hydrolyzed casein. In one specific embodiment, the carbon source is glucose and the nitrogen source is cottonseed meal.

[12] In a preferred embodiment of the invention, an insecticidal composition is provided herein, comprising an agronomically acceptable carrier (inert carrier) and desiccation-tolerant blastospores of a Beauveria species or a Cordyceps species, wherein the inert carrier and the blastospores are contained in a vacuum-sealed package, wherein the blastospores are produced by Petition 870260076623, dated 07 / 31 / 2026, p. 14 / 58 5 / 45 Inoculation of a liquid culture medium, comprising a carbon source and a nitrogen source, with propagules of the fungus of a Beauveria species or a Cordyceps species, incubating the propagules under culture conditions that allow a dissolved oxygen level above zero and under an osmotic pressure greater than 0.5 MPa, incubating the propagules for a time sufficient to produce blastospores, collecting / recovering the blastospores, and drying the blastospores, thus producing desiccation-tolerant blastospores. In some embodiments, the Beauveria species is B. bassiana. In other embodiments, the Cordyceps species is C. fumosorosea. In some cases, an insecticidal composition also contains an oxygen-scavenging (absorber / extractor) compound, a moisture-scavenging (absorber / extractor) compound, or a combination of both.In some embodiments, more than 60% of blastospores are viable when rehydrated after storage for more than six months. In one particular embodiment, the insecticidal composition can be stored at temperatures of 28 °C or lower.

[13] In yet another embodiment of the present invention, a method for controlling insects is provided, the method comprising applying to the habitat of said insects an 'effective amount' of microbial insecticide based on desiccation-tolerant blastospores of a species of Beauveria or a species of Cordyceps. In some cases, desiccation-tolerant blastospores are produced by the method described above. In some embodiments, the Beauveria species is Beauveria bassiana. In other embodiments, the Cordyceps species is C. fumosorosea. In a particular embodiment, during blastospore growth, the carbon source is glucose and the nitrogen source is cottonseed meal or hydrolyzed casein. The insect habitat may be an agricultural crop. Incorporation by Reference

[14] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS Petition 870260076623, dated 07 / 31 / 2026, page 15 / 58 6 / 45

[15] The new features of the invention are presented in particular in the claims. The features and advantages of the present invention are referred to in the detailed description that follows, and the accompanying drawings, of which:

[16] Figure 1 provides a graphical representation of blastospore production by B. bassiana isolates GHA and ESALQ 1432, as well as their tolerance to desiccation, when produced with different nitrogen sources in submerged liquid culture.

[17] Figure 2 provides a graphical representation of blastospore yield with greater tolerance to desiccation when grown in the presence of increasing levels of nitrogen.

[18] Figure 3 presents a graph showing the relationship between water activity (aw) and water content (% moisture) in dehydrated blastospores under continuous airflow of two genera of fungi.

[19] Figure 4 provides graphs that demonstrate the long-term survival rates for various strains of B. bassiana stored at 4oC.

[20] Figure 5 provides graphs that demonstrate the long-term survival rates for various strains of C. fumosorosea stored at 4oC.

[21] Figure 6 presents a graph showing B. bassiana and the survival rates of blastospores stored at 28 °C in the presence of oxygen-absorbing sachets and moisture.

[22] Figure 7 provides a graph showing the survival rates of B. bassiana blastospores in the presence of oxygen absorbers and moisture. The cells were dehydrated by spray-drying, with or without the addition of ascorbic acid (ASA).

[23] Figure 8 provides a graph showing the blastospore production rate for B. bassiana ESALQ 1432 cultures at increasing glucose (dextrose) concentrations, also related to the respective osmotic pressure points.

[24] Figure 9 provides a morphological analysis of blastospores grown in the presence of increased osmotic pressure.

[25] Figure 10 provides a graph showing that dissolved oxygen levels are affected by liquid culture volume. Petition 870260076623, dated 07 / 31 / 2026, page 16 / 58 7 / 45

[26] Figure 11 provides two panels showing blastospore yield and their tolerance to desiccation when grown in different volumes of liquid medium and different nitrogen sources.

[27] Figure 12 provides two panels that demonstrate the effects of blastospores grown in medium with high osmotic pressure on their entomopathogenic effectiveness (virulence). DETAILED DESCRIPTION OF THE INVENTION

[28] Preferred embodiments of the present invention are shown and described herein. It will be obvious to those skilled in the art that such embodiments are provided by way of examples only. Numerous variations, alterations and substitutions will occur to those skilled in the art without departing from the invention. Various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the included claims define the scope of the invention and that the methods and structures within the scope of these claims and their equivalents are therefore encompassed. Numerous variations, alterations and substitutions will occur to those skilled in the art without departing from the invention.

[29] Scientific techniques and terms used herein have the meaning normally understood by a person skilled in the subject matter to which the invention pertains, unless otherwise defined. The terminology used in the description of the invention herein is to describe only particular embodiments and is not intended to be restrictive of the invention. As used in the description of the invention and the appended claims, the singular forms a, an, and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[30] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, as used in the specification and claims, shall be understood as being modified in all cases by the term approximately. Therefore, unless otherwise indicated, the numerical properties indicated in the following specification and claims are approximations that may vary depending on the properties desired, in an attempt to obtain them in the embodiments of the present invention. Notwithstanding the numerical ranges and parameters that are Petition 870260076623, dated 07 / 31 / 2026, page 17 / 58 8 / 45 establish the broad scope of the invention; these are approximations, the numerical values ​​set forth in the specific examples are presented as to the greatest possible precision. Any numerical values, however, inherently contain certain errors necessarily resulting from errors found in their respective measurements. The term "about" is defined as plus or minus ten percent of a given value. For example, about 1.0 g means 0.9 g to 1.1 g.

[31] The terms isolated, purified, or biologically pure, as used herein, refer to material that is substantially or essentially free of components that normally accompany the referenced material in its native state.

[32] The term shelf-stable and grammatical variations thereof, as described herein, generally refers to the air-flow or spray-drying method (less than 5% moisture) of blastospores, demonstrating viability for one year or more when stored at 4 °C, where viability is measured as at least about 60% germination after rehydration and incubating the dried blastospores in an appropriate liquid medium for 6-7 hours.

[33] The term desiccation tolerant and its grammatical variations, as described here, generally refers to dry blastospores that exhibit 50% or more germination when dehydrated and cultured for rehydration in a suitable medium, such as dextrose and potato broth, within six to seven hours of incubation.

[34] Entomopathogenic fungi are considered the first line of biorational tools for regulating populations of numerous insect pests, including whiteflies, with the potential to be integrated with the application of synthetic chemical insecticides in order to mitigate the development of insect resistance to insecticides. A large volume of literature has been dedicated to the use of aerial conidia of B. bassiana and C. fumosorosea implanted as contact bioinsecticides for whitefly control. Several studies have demonstrated that the yeast phase (blastospore) of C. fumosorosea was more effective than its aerial conidia in controlling whiteflies, subterranean termites, leafhoppers, aphids, and beetles. In our recent study, on whitefly control using aerial conidia, Brazilian isolates of B. bassiana and C. fumosorosea were identified.virulent and effective fumosorosea, which infected and killed various life stages of the whitefly Bemisia tabaci biotype B (Hemiptera: Aleyrodidae). Petition 870260076623, dated 07 / 31 / 2026, p. 18 / 58 9 / 45

[35] Here is described a liquid fermentation culture method for the production of desiccation-tolerant and shelf-stable blastospores. In a preferred embodiment, the culture conditions include high aeration rates, osmotic pressure of at least 0.5 Megapascal (MPa), at least 1.5% of a suitable nitrogen source, and at least 6% of a suitable carbon source. Liquid culture techniques can be used in the growth of entomopathogenic fungal species of the genus Beauveria to promote the aforementioned qualities of shelf stability, desiccation tolerance, and improved bioefficacy against insect pests. Beauveria species that can be used with the liquid culture fermentation method, as described, include Beauveria alba, Beauveria amorpha, Beauveria arenaria, Beauveria asiatica, Beauveria australis, Beauveria bassiana, Beauveria brongniartii, Beauveria brumptii, Beauveria caledonica, Beauveria chiromensis,Beauveria coccorum, Beauveria cretacea, Beauveria cylindrospora, Beauveria delacroixii, Beauveria densa, Beauveria dependens, Beauveria doryphorae, Beauveria effusa, Beauveria epigaea, Beauveria felina, Beauveria geodes, Beauveria globulifera, Beauveria heimii, Beauveria kipukae, Beauveria laxa, Beauveria malawiensis, Beauveria melolonthae, Beauveria nubicola, Beauveria oryzae, Beauveria paradoxa, Beauveria paranensis, Beauveria parasitica, Beauveria petelotii, Beauveria pseudobassiana, Beauveria rileyi, Beauveria rubra, Beauveria shiotae, Beauveria sobolifera, Beauveria spicata, Beauveria stephanoderis, Beauveria sulfurescens, Beauveria sungii, Beauveria tenella, Beauveria tundrensis, Beauveria velata, Beauveria varroae, Beauveria vermiconia, Beauveria vexans, Beauveria viannai, Beauveria virella. Other entomopathogenic filamentous fungi that exhibit dimorphic growth, including Metarhizium spp., Hirsutella spp., Lecanicillium spp., Cordyceps spp., and Nomuraea spp.,They will likely produce blastospores effectively and abundantly using the present conditions, which consist of a combination of high aeration, high osmotic pressure, and an adequate source and concentration of nitrogen.

[36] The blastospores of the entomopathogenic fungi of the present invention can be used to infect and kill a wide variety of economically important arthropods, including subterranean pests, soil insects and inhabitants of the phyllosphere (aerial part of plants). Without being limited to these, the arthropods that can be controlled by the compositions of the present invention include root weevils, root larvae, wireworms, fruit flies, ground grubs, termites, Petition 870260076623, dated 07 / 31 / 2026, page 19 / 58 10 / 45 ticks and mites, fleas, grasshoppers, ants, and a variety of other insects of economic importance to agriculture, horticulture, medicine, and veterinary medicine. Some non-restrictive examples of target insects include corn rootworm (Diabrotica spp.), black grapevine weevil (Otiorhynchus sulcatus), citrus root weevil (Diaprepes abbreviatus), sweet potato weevil (Cylas formicarius), sugar beet rootworm (Tetanops myopaeformis), cabbage rootworm (Delia radicum), onion rootworm (Delia antigua), turnip rootworm (Delia floralis), corn rootworm (Delia platura), carrot rust fly (Psila rosae), Japanese beetle (Popillia japonica), European white grub (Rhizotrogus majalis), subterranean termite (Reticulitermes and Coptotermes spp.).Emerald weevil (Agrilus planipennis), gypsy moth (Lymantria dispar), pecan weevil (Curculio caryae), tobacco budworm (Spodoptera litura), cotton bollworm (Helicoverpa armigera), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), European maize borer (Ostrinia nubilalis), Asian maize borer (Ostrinia furnacalis Guenee), sorghum stem borer (Chilo partellus, Coniesta ignefusalis, Busseola fusca, Chilo spp.), yellow rice stem borer (Scirpophaga incertulas), rice leafroller (Cnaphalocrocis medinalisi), brown rice leafhopper (Nilaparvata lugens), rice thrips (Stenchaetothrips biformis), leafhopper (Hishimonus) phycitis), grasshoppers (Melanoplus spp.Cactus weevil (Metamasius spinolaei), silvery whitefly (Bemisia argentifolii), beetroot caterpillar (Spodoptera exigua), cucumber beetle (Diabrotica undecimpunctata), alfalfa looper (Autographa californica), cotton aphid (Aphis gossypii), termites (Odontotermes obesus, Odontotermes spp., Trinervitermes biformis), green leafhopper (Empoasca kerri), thrips (Frankliniella schultzei, Scirtothrips dorsalis, Podothrips bicolor), cabbage moth (Plutella xylostella), aphid (Aphis gossypii), potato aphid (Macrosiphum euphorbiae), thrips (Anaphothrips dorsalis, Thrips palmi), scale insect (Maconellicoccus hirsutus), grasshopper (Melanoplus spp.), whitefly (Bemisia tabaci), shoot borer (Leucinodes orbonalis), stem borers, beet caterpillar (Spodoptera exigua, Spodoptera spp.), cabbage caterpillar (Trichoplusia ni, Trichoplusia spp.), spiny caterpillar (Earias insulana), spotted caterpillar (Earias vitella), leafroller (Sylepta derogata), mites (Tetranychus telarius, Tetranychus urticae), okra leafhopper (Amrasca biguttula), mosquitoes (Anopheles gambiae, Culex quinquefasciatus), housefly (Musca domestica), cockroaches (Periplanata americana) and ticks. Petition 870260076623, dated 07 / 31 / 2026, page 20 / 58 11 / 45 (Ixodes dammini), Triatoma infestans, Rhodnius prolixus, soft-bodied ticks (Argas persicargas persicus), cattle tick (Rhipicephalus microplus), black-legged tick or deer tick (Ixodes scapularis), sheep scab mite (Psoroptes ovis), tropical bird mite (Ornithonyssus bursa), fleas - Siphonaptera, cat flea (Ctenocephalides felis) and cattle louse (Haematopinus eurysternus).

[37] Growing conditions

[38] The fungi useful in the practice of the present invention, specifically B. bassiana and C. fumosorosea, exhibit polymorphic forms ranging from conidia to pseudohyphae, hyphae, and blastospores (unicellular structures similar to yeast cells), depending on the isolate characteristics, age, form, and culture conditions. As with most fungi, the culture conditions in which they are grown affect multiple aspects of the organism's biology, including morphological form and spectrum of bioproducts.

[39] Therefore, a person skilled in the art will recognize that various culture conditions can be modified in the practice of the invention disclosed herein. Examples of culture conditions that can be modified during the application and practice of the inventions described herein include: 1) temperature; 2) primary carbon source; 3) oxygen concentration; 4) primary nitrogen source; 5) pH; 6) mineral salts and other ion concentrations; 7) age and growth phase of the culture; 8) organization / design of an industrial fermenter; and 9) predominant morphological form. A person skilled in the art will recognize that other culture parameters affecting the desired yield and production of the bioproduct can be modified.

[40] In one aspect of the invention, cultures of fungal strains described herein can be grown at any temperature that facilitates the production of one or more bioproducts. For example, a crop may be grown at a temperature of 15°C to 30°C, or a whole or partial degree within that range, including but not limited to 15.0°C, 15.5°C, 16.0°C, 16.5°C, 17.0°C, 17.5°C, 18.0°C, 18.5°C, 19.0°C, 19.5°C, 20.0°C, 20.5°C, 21.0°C, 21.5°C, 22.0°C, 22.5°C, 23.0°C, 23.5°C, 24.0°C, 24.5°C, 25.0°C, 25.5°C, 26.0°C, 26.5°C, 27.0°C, 27.5oC, 28.0oC, 28.5oC, 29.0oC, 29.5oC, and 30.0oC,

[41] In some embodiments, the fungal strains described herein can be grown under conditions in which the culture pH facilitates the production of one or more Petition 870260076623, dated 07 / 31 / 2026, page 21 / 58 12 / 45 morphological forms of interest. For example, a culture can be grown in media where the pH is between 3.0 and 8.5, 4.5 and 6.5, or any value within that range, including but not limited to pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3,9 , 4,0 , 4,1 , 4,2 , 4,3 , 4,4 , 4,5 , 4,6 , 4,7 , 4,8 , 4,9 , 5,0 , 5,1 , 5,2 , 5,3 , 5,4 , 5,5 , 5,6 , 5,7 ,5,8 5,9 , 6,0, 6,1, 6,2, 6,3, 6,4, 6,5, 6,6, 6,7, 6,8, 6,9, 7,0, 7,1, 7,2, 7,3, 7,4, 7,5, 7,6, 7,7,7,8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5. A person skilled in the art will recognize that a stable pH does not need to be maintained throughout the entire growth process for the production of the strain of interest in the bioproduct. Thus, in some embodiments, the pH of a microbial culture of the present invention will vary. In other embodiments, pH buffering solutions may be added to maintain a relatively stable pH whereby the pH of the culture medium during the life of the culture does not vary from a chosen starting point by more than ± 0.5.

[42] In some embodiments, the microbial strains of the present invention can be cultivated in the presence of particular carbon sources. For example, a culture can be cultivated in the presence of simple carbon sources such as (D- or L-) arabitol, sucrose, fructose, glucose, mannose, galactose, arabinose, xylose, mannitol, glucitol, galactitol, xylitol, ribitol, threitol, glycerol, gluconic acid, glucosamine, or meso-erythritol. Alternatively, a culture can be cultivated in the presence of complex carbon sources such as cellulose, starch, beet molasses, locust bean pods, corn bran hydrolysates, corn syrup (corn syrup), residue from alcoholic fermentation for fuel, grape skin pulp, vegetable oils, peat hydrolysate, potato starch hydrolysate, and sulfite liquor residue. Carbon sources, which are also sources of other nutritional requirements such as nitrogen, can be used.For example, means for use in the present invention may include amino acids such as aspartate, threonine, lysine, methionine, isoleucine, asparagine, glutamic acid, glutamine, proline, alanine, valine, leucine, tryptophan, tyrosine, phenylalanine and their metabolic intermediates. These lists are not limiting and it is well within the capabilities of a person skilled in the art to utilize other carbon sources in the practice of the present invention. Any carbon source may be used alone or in combination with other carbon sources.

[43] Other nutritional parameters may also vary, including nitrogen sources. Non-limiting examples of nitrogen sources include organic nitrogen sources (e.g., cottonseed meal, casein hydrolyzed by Petition 870260076623, dated 07 / 31 / 2026, page 22 / 58 13 / 45 Acidification, autolyzed yeast, glutamic acid, peptone, soybean meal, yeast extract, food sauce, malt extract, corn liquor (corn syrup) and soybean meal) and inorganic nitrogen (e.g., urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be included in the growth medium used in the practice of the present invention. In some embodiments, a nitrogen source is present in an amount of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15% or more. Preferably, the concentration is between 1.5% and 9%.

[44] Phosphate sources such as monobasic potassium phosphate, dibasic potassium phosphate and their corresponding sodium-containing salts may be included in the growth medium as needed. Metal and mineral salts such as zinc, iron, magnesium, manganese, calcium and copper salts may be included as needed. Other nutritional supplements such as vitamins (e.g., biotin, thiamine) may also be included. An expert in the field will recognize that varying the nutritional composition of the culture can be used to maximize the production of a bioproduct of interest and decrease the production of undesirable byproducts. Any of these nutrients may be used alone or in combination with any other nutrient.

[45] In some embodiments, osmolytes are used to control the osmotic pressure of the culture liquid. Osmolytes such as sugars (glucose / dextrose, galactose, fructose, trehalose, etc.), polyols (mannitol, glycerol, erythritol, etc.), proteins, amino acids, salts, polymers (polyvinylpyrrolidone, polyethylene glycol, etc.), and any compound that can be used to alter the osmotic pressure in a liquid culture medium for submerged fermentation can be used. Generally, according to the present invention, improvements in blastospore production are achieved by increasing the osmotic pressure > 0.5 MPa.

[46] In a particular embodiment of the present invention, liquid culture techniques for the production of desiccation-tolerant spores use concentrations of at least 1.5% - 9% of a nitrogen source and about 4% - 25% of a carbon source, with at least 0.5 MPa of osmotic pressure. Petition 870260076623, dated 07 / 31 / 2026, page 23 / 58 14 / 45

[47] Nutrients can be added to liquid culture in any feeding regime, including but not limited to high cell density culture, batch culture, continuous fed batch culture, discontinuous fed culture, exponentially fed batch culture, continuous culture, or a mixture of these approaches for different nutrients.

[48] ​​In some cases, the time a crop is grown can be modified to increase or initiate the production of a bioproduct of interest. For example, a crop can be grown for 10-3 hours, or more, or any time point within the range of, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 1, 110, 120, 130, 140, 150, 160, 170, 180, 190, 2, 210, 220, 230, 240, 250, 260, 270, 280, 290, 3 hours, or more, before the start of the harvesting or recovery process of a bioproduct.

[49] In liquid cultures with agitation, cultures of the present invention can be cultivated in order to increase dissolved oxygen. Factors affecting dissolved oxygen levels include culture volume, vessel volume, rotation speed and, in a fermenter, aeration rate, agitator speed, mixing impeller design, fermentation tank design, pressure at the top of the tank, and aeration gas mixing. In some embodiments, a culture can be agitated in an incubator with an orbital shaker table at any feasible speed of 50-4 rpm. Aeration rates for fermenters can be at any feasible rate from zero to 7 standard liters per minute (slpm), including but not limited to 50, 1, 150, 2, 250, 3, 350, 4, 450, 5, 550, 6, 650, 7 or more slpm. Agitator speed can be 50-1 rpm, depending on the size of the fermenter. The pressure at the top of the fermenter can vary from 0-30 psi to increase dissolved oxygen levels.Aeration gas mixtures can vary in oxygen content from normal air, which is 21% oxygen, by injecting pure oxygen. Dissolved oxygen levels in preferred embodiments will be maintained above zero.

[50] Furthermore, optimizing fungal cell production may depend on growing a culture from a specific point in its life cycle. For example, a culture may be grown to the early latency phase (lag phase), intermediate latency phase, late latency phase, exponential growth phase (log phase), intermediate exponential growth phase, late exponential growth phase, early stationary phase, intermediate stationary phase, or death (decline) phase. In Petition 870260076623, dated 07 / 31 / 2026, page 24 / 58 15 / 45 In some cases, cultures can be maintained at a specific growth stage (for example, through batch fermentation with continuous feeding) in order to maintain a particular growth phase for the culture.

[51] In other cases, culture conditions can be altered so that one morphological form of the fungal strains predominates over other morphological forms. For example, culture conditions can be controlled so that yeast-like forms (blastospores) predominate, conidia predominate, or hyphae / pseudo-hyphae predominate.

[52] In some preferred embodiments, blastospores produced using the present invention can be dried before use as a biological control agent. For blastospores subjected to air drying, blastospores (2-5 days of fermentation) are recovered from the culture broth by filtration, centrifugation, or any other means known in the art to remove or separate the liquid medium from these propagules. Blastospores can be mixed with suitable filtration aids, such as, but not limited to, clay, diatomaceous earth, talc, silicon dioxide, or calcium silicate. This mixture can be used to remove the fermented liquid medium and form a filter cake (biomass + aid) obtained by vacuum filtration. Filter cakes can be broken up using any appropriate granulation equipment.The filtered and granulated cake composed of blastospores is then dehydrated via a continuous air stream drying system, vacuum drying, fluidized bed drying, or to a final moisture content of less than 5%, or with a water activity (aw) of less than 0.3. Alternatively, spray-drying or fluidized bed drying of blastospores is performed by spray-drying these blastospores containing culture broth or previously separated blastospore suspensions, with or without inert carriers or stabilizers. Inert carriers or stabilizers may include compounds that improve the flowability, resuspension, desiccation tolerance, and / or storage stability of the blastospore-containing formulation and may include, but are not limited to, proteins, carbohydrates, skim milk, maltodextrin, disaccharides, simple sugars, molasses, PVP, clays, diatomaceous earth, talc, silicon dioxide, or calcium silicate.Spray drying is conducted at inlet / outlet temperatures that minimize exposure of blastospores to high temperatures, resulting in a fluid, dry blastospore formulation with less than 6% moisture. Protocols of... Petition 870260076623, dated 07 / 31 / 2026, p. 25 / 58 16 / 45 spray drying, such as using 90°C inlet / 50°C outlet temperatures, are preferred. Other spray drying inlet / outlet temperature protocols are suitable, provided the blastospore formulation is only briefly exposed to high temperatures. Fluidized bed drying could employ the inert carriers described above and be operated at convenient temperatures favoring spore survival.

[53] Biological control compositions

[54] In some embodiments, blastospores produced by methods of the present invention are combined with one or more components to produce a biological control composition, typically for use as a microbial insecticide. In some cases, the biological control agent composition comprises at least one agronomically acceptable inert carrier (i.e., adjuvant). Some non-limiting examples of such inert carriers include filler stimulants, anti-caking agents, wetting or spreading agents (surfactants), emulsifiers, nutritional additives, and antioxidants. Such additives may be used alone or in any combination. A person skilled in the art is able to select appropriate adjuvants for particular applications. Filler stimulants may be a source of carbohydrates, such as a disaccharide, including, for example, sucrose and trehalose, or monosaccharides, such as fructose or glucose.Potential anti-caking agents include talc, silicon dioxide, calcium silicate, kaolinite (or kaolin), and other types of clay. Wetting agents (also known as surfactants) include, but are not limited to, surfactants or skimmed milk powder. Emulsifiers may include soy-based emulsifiers, such as lecithin, or vegetable-based emulsifiers, such as mono- and diglycerides. Antioxidants may include, but are not limited to, sodium glutamate, ascorbic acid, and citric acid.

[55] In various embodiments, the composition of this biocontrol is a stable composition, capable of supporting the reproductive viability of the fungal cell component, or capable of retaining insecticidal efficacy for a period between 2 weeks and 2 years. In some cases, this period is about 2 weeks, about 3 weeks, about 4 weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven Petition 870260076623, dated 07 / 31 / 2026, p. 26 / 58 17 / 45 months, approximately eight months, approximately nine months, approximately ten months, approximately eleven months, approximately twelve months, approximately thirteen months, approximately fourteen months, approximately fifteen months, approximately sixteen months, approximately seventeen months, approximately eighteen months, approximately nineteen months, approximately twenty months, approximately twenty months -one, approximately twenty-two months, approximately twenty-three months, approximately twenty-four months, or more. Such storage periods can be achieved at any storage temperature in the range of 0-35°C.

[56] To facilitate long-term storage, in some embodiments, components such as absorbent sachets or oxygen scavengers, and moisture absorbers (desiccants) may be used in conjunction with the packaging of the composition. Typically, the packaging is waterproof and gas-tight. The particular packaging material is not important for the present invention and any commercially available material may be used for packaging, such as aluminum foil or sealable mylar-type films. A desiccant is a hygroscopic substance usually in a porous bag or sachet, which is placed inside a hermetically sealed package.Desirable desiccants are those that are chemically stable or chemically inert and include silica gel, activated carbon, calcium sulfate (Drierite®), calcium chloride, molecular sieves (typically zeolites), INCOZOL 2, Loxanol®, BYK®-2616, Trixene AS, Tyzor® Ibay, AMP 95, AMPD, Sylosiv®A3, Silquest®A-171, NEWOTECH®547, USI®-SL25, Modarez®, Zeolum®, TAFT™, and the like. Oxygen absorbers may include any substance that absorbs oxygen molecules effectively and typically through an oxidation reaction. Examples of oxygen absorbers that can be used in the present invention include enzyme-mediated oxidation (Bioka®S1, Bioka®S-75), iron-based oxidation (Ageless®FX-1, FreshPax®), sodium ascorbate, sodium carbonate, and the like.

[57] The biocontrol compositions of the present invention can be used in combination with other components, which may be collectively referred to as agrochemicals. Examples of categories of agrochemicals that can be combined with the propagules of the fungus of the present invention include, but are not limited to, germicides, herbicides, insecticides and acaricides, plant growth regulators, fungicides, biostimulants and the like. Such additional components may be used alone or in any combination. Petition 870260076623, dated 07 / 31 / 2026, page 27 / 58 18 / 45

[58] In some cases, a biocontrol composition of the present invention also comprises blastospores of an entomopathogenic fungus, and one or more germicides may include: captan, thiuram, ziram, zineb, maneb, mancozeb, propineb, polycarbamate, chlorothalonil, quintozene, captafol, iprodione, procymidone, fluorine, mepronil, flutolanil, pencycuron, oxycarboxin, fosetyl-aluminum, propamocarb, triadimephon, triadimenol, propiconazole, diclobutrazole, bitertanol, hexaconazole, myclobutanil, flusilazole, etaconazole, fluotrimazole, flutriafen, penconazole, diniconazole, cyproconazole, fenarimol, triflumizole, prochloraz, imazalil, perfurazoate, tridemorph, phenpropimorph, trifolin, buthiobate, pirifenox, anilazine, polyoxin, metalaxyl, oxadixil, furalaxyl, isoprothiolane, probenazole, pyrrolnitrin, blasticidin S, kasugamycin, validamycin, dihydrostreptomycin sulfate, benomyl, carbendazim, thiophanate-methyl, himexazole, basic copper chloride, basic copper sulfate, fentin acetate,triphenyltin hydroxide, dietofencarb, quinomethionate, to kill mites, lecithin, sodium bicarbonate, ditianone, dinocape, phenaminosulf, diclomezine, guazatine, dodine, IBP, edifenfos, mepaniprim, ferimzone, trichlamide, methasulfocarb, fluazinam, ethoquinolac, dimethomorph, phylloquinone, tecloftalam, phthalide, phenazine oxide, thiabendazole, tricyclazole, vinclozolin, cymoxanil, cyclobutyl, guazatine, propamocarb hydrochloride, oxolinic acid, ciflufenamide, iminoctadine, cresoximmethyl, triazine, phenhexamide, cyazofamide, cyprodinil, prothioconazole, fenbuconazole, trifloxystrobin, azoxystrobin, hexaconazole, Imibenconazole, tebuconazole, difenoconazole, carpropamide, and the like. A person skilled in the art will recognize that this list is not exhaustive and any agronomically or horticulturally acceptable germicide may be used.

[59] In some cases, a biocontrol composition of the present invention comprises blastospores of an entomopathogenic fungus and one or more herbicides, including, but not limited to: 2,4-D, MCPA, clomeprop, dicamba, chlorotoluron, diuron, linuron, isourou, linuron, neburon, simazine, atrazine, symmethrin, promethrin, hexazinone, propazine, desmetryne, terbumeton, propanil, bromoxynil, ioxynil, pyridate, chloridazone, bentazone, clomethoxyphene, bifenox, acifluorfen sodium, flumioxazone, thiadiazine, oxadiazone, sulfentrazone, pentoxazone, pyraclonil, pyrazolinate, pyrazoxyphene, benzofenap, mesotrione, isoxaflutole, isoxaflutole, amitrol, aclonifena, diflufenican, benzobicyclon, diclofop-methyl, fluazifop-butyl, sodium aloxidim, clethodim, sethoxydim, tralcoxidime, tepraloxidime, bensulfuron-methyl, metosulfam pyrazine Petition 870260076623, dated 07 / 31 / 2026, page 28 / 58 19 / 45 imazapyr, imazaquin, sodium pyrithiobac, bispiribac-sodium, piriminobac-methyl, flucarbazone, propoxycarbazone, glyphosate, ammonium glyphosate salt, glufosinate, trifluralin, pendimethalin, benfluralin, prodiamine, profame, dithiopyr, alachlor, metolachlor, petoxamide, acetochlor, propachlor, dimethenamide, diphenamide, napropamide, mefenacet, fentrazamide, molinate, dimepiperate, cycloate, esprocarb, thiobencarb, thiocarbazil, bensulide, dalapan, asulam, DNOC, ethyl sulfuron, rimsulfuron, imazosulfuron, prosulfuron, fulmetsulam, diclosulam, dinoseb, Floxam, triazolam, quinclorac, cinmethylin, dazomet, dymon, etobenzanide, oxaziclomefona, piributicarb, and the like. An expert in the art will recognize that this list is not exhaustive and any agronomically or horticulturally acceptable herbicide may be used.

[60] In some cases, a biocontrol composition of the present invention comprises blastospores of an entomopathogenic fungus and one or more plant growth regulators, which include gibberellins (e.g., gibberellin A3, gibberellin A4, and gibberellin A7), IAA (indoleacetic acid), NAA (naphthaleneacetic acid), and the like. A person skilled in the art will recognize that this list is not exhaustive and any agronomically or horticulturally acceptable plant growth regulator may be used.

[61] In some cases, a biocontrol composition of the present invention comprises blastospores of an entomopathogenic fungus and one or more chemical insecticides. A person skilled in the art is able to select one or more chemical insecticides as appropriate for the control of one or more species of pest insects. Such chemical insecticides may include the following: acetylcholinesterase inhibitors (carbamates, organophosphates), gamma-aminobutyric acid (GABA) chloride channel blockers (organochlorines, cyclodienes, phenylpyrazoles), sodium channel modulators (pyrethroids, pyrethrins), competitive modulators of the nicotinic acetylcholine receptor (neonicotinoids, nicotine, sulfoxaflor, butenolides), allosteric modulators of the nicotinic acetylcholine receptor (spinosyns), allosteric modulators of glutamate-coupled chloride channels, juvenile hormone agonists (hormonal analogs, fenoxycarb, pyriproxyfen), non-specific (multi-site) inhibitors (alkyl halides,chloropicrin, sulfuryl fluoride, borates, tartar emetic, methyl isothiocyanate generators), chordotonal organ modulators (pimetrozine, flonicamide), mite growth inhibitors (clofentezine, diflovidazine, hexythiazox, ethoxazole), mitochondrial ATP synthesis inhibitors (diafenthiuron, organotin acaricides, propargite, tetradifon), chloride channel activators (avermectins, milbemycin), Petition 870260076623, dated 07 / 31 / 2026, page 29 / 58 20 / 45 Proton gradient inhibitors and oxidative phosphorylation uncouplers (chlorfenapyr, DNOC, sulfluramide), nicotinic acetylcholine channel blockers (nereistoxin analogs), chitin biosynthesis inhibitors (benzoylureas, ibuprofen), ecdysis inhibitors (cryomazine), ecdysone receptor agonists (diacylhydrazines), octopamine receptor agonists (amitraz), electron transport inhibitors (hydramethylnon, acequinocil, fluacrypyrim, METI acaricides, rotenone, phosphine, acetonitrile cyanide derivatives, carboxylics), voltage-dependent sodium channel blockers (indoxacarb, metaflumizone), acetyl-CoA carboxylase inhibitors (tetranic and tetramic acid derivatives), receptor modulators of ryanodine (diamides), and compounds with uncertain / unknown modes of action (azadirachtin, benzoximate, bifenazate, bromopropylate, chinomethionate, cryolite, dicofol, pyridalil, pyrifluquinazon, sulfur, lime sulfur).An expert in the field will recognize that this is a non-exhaustive list of possible insecticides, and that any agronomically or horticulturally acceptable pesticide can be used.

[62] In other embodiments, a biocontrol composition of the present invention comprises blastospores of an entomopathogenic fungus and one or more fungicides, for example: mancozeb, tricyclazole, carbendazim, hexaconazole, metalaxyl, benomyl, difenoconazole, propiconazole, kitazin, tebuconazole, copper oxychloride, copper hydroxide, tridemorph, propineb, Safin, sporran, blastin, bio-virulence and the like. Typically, a fungicide used will have little or no activity against blastospore-based fungi in the composition. A person skilled in the art will recognize that this list is not exhaustive and any agronomically or horticulturally acceptable plant fungicide may be used.

[63] Biocontrol composition formulations for use as an insect control agent can be prepared from blastospores that have been harvested from the fermented culture medium, as described above. As a practical matter, it is anticipated that such formulations can be prepared directly from the culture, thus avoiding the need for any purification steps. While liquid cultures can be used directly, in the preferred embodiment, water is removed from the fungal cultures by partial or complete drying, as described above, and the dried culture is fragmented or ground into small particles suitable for application by means of conventional granular applicators, using conventional techniques within the scope of the art. Drying can be carried out by Petition 870260076623, dated 07 / 31 / 2026, page 30 / 58 21 / 45 air drying method (such as mixing collected blastospores with a desiccating agent like diatomaceous earth) or spray drying.

[64] To facilitate application and subsequent fungal growth, the harvested blastospores may alternatively be formulated into an appropriate adjuvant, acceptable in agronomy, nutritional or inert, for application as wettable powders, dry powders, granules, baits, solutions, emulsifiable concentrates, emulsions, suspension concentrates and aerosols. For example, for liquid applications, biological control compositions may be formulated as a suspension or emulsion. In such embodiments, preferred vehicles include, but are not limited to, oils, water, buffers, or vegetable oils.As an alternative to the preferred embodiment particularly suitable for solid granular applications, biological control compositions may be formulated with inert solid carriers or diluents such as diatomaceous earth, talc, clay, vermiculite, CaCO3, corn cob granules, alginate gels, starch matrices or synthetic polymers, or may be incorporated into conventional controlled-release microparticles or microcapsules. Those skilled in the art will recognize that fungi may also be formulated in combination with conventional additives such as binding or adhesive agents, emulsifying agents, surfactants, foams, humectants, or wetting agents, antioxidants, UV protectors, nutritive additives, fertilizers, insecticides, or even with fungicides that exhibit low toxicity to the fungi of interest.For application to the bark or canopy of trees and plants, biocontrol compositions can also be formulated with a hygroscopic or hydrophilic adjuvant.

[65] The absolute quantity of blastospores and their concentration in the final composition can be selected to provide an effective reduction in the target insect population compared to an untreated control. The actual quantity is not critical and is a function of practical considerations such as the properties of the vehicle or adjuvant, the density of the target insect population, and the method and location of application, and can be readily determined by routine tests. Since the blastospores of the present invention serve to produce and release a high concentration of vegetative and infective fungal cells to control target insects by infection and death, for formulation and application purposes, an effective quantity is defined as any quantity of blastospores sufficient to subsequently produce cells. Petition 870260076623, dated 07 / 31 / 2026, page 31 / 58 22 / 45 sufficient in the target habitat to infect and subsequently kill the target insect, compared to a control without treatment. By way of example and without limitation, suitable formulations are expected to typically contain about 1 x 10⁶ or more blastospores per gram of biomass recovered from the liquid culture (based on the dry weight of the biomass), preferably at least 1.5 x 10⁷ blastospores per gram of biomass. For application to typical row crops, but not limited to them, suitable application rates are expected to be at least 1 x 10⁷ blastospores per hectare. In other embodiments, the application rate may be at least 1x10⁶, 5x10⁶, 1x10⁷, 5x10⁷, 1x10⁸, 5x10⁸, 1x10⁹, 5x10⁹, 1x10¹⁰, 5x10¹⁰, 1x10¹¹, 5x10¹¹, 1x10¹², 5x10¹², 1x10¹³, 5x10¹³, 1x10¹⁴, 5x10¹⁴, 1x10¹⁵, 5x10¹⁵, 1x10¹⁶, 5x10¹⁶, or more blastospores per hectare.

[66] In use, the biocontrol compositions of the present invention can be applied in situ or in the vicinity of the environment in which the target insects live, or on the surface of the plants to be protected, for example, on tree bark, or as a seed coating, using conventional techniques. In a preferred embodiment, the blastospores are applied to the soil, or to soilless nutrient substrates used in vegetable cultivation in greenhouses or shade houses, in a granular form, and to the aerial part (phyllosphere) of the plants in a sprayable suspension. Depending on the target pest, blastospores can be applied in agricultural fields, orchards, greenhouses, gardens or lawns, or on or near ornamental plants, trees or commercial or residential structures. The biological control compositions can be applied to the soil, to substrate mixtures for potted plants, to the surface of plants, or a combination thereof.

[67] Having described the invention in general, below are examples that illustrate the creation and effectiveness of the invention. Neither the examples nor the preceding general description should be interpreted as limiting the scope of this invention. Examples

[68] Preparation of fungi and inoculum

[69] Five isolates of B. bassiana and five isolates of C. fumosorosea were tested in this study. Most of the fungal isolates were from Brazil, with details presented in Table 1. The ARSEF 3581 isolate of C. fumosorosea and GHA Petition 870260076623, dated 07 / 31 / 2026, page 32 / 58 23 / 45 (ARSEF 6444) of B. bassiana, currently designated as the active ingredient in the commercial bioinsecticide known as Mycotrol® (LAVERLAM, Butte, MT, USA), served as standards for North American strains in submerged liquid culture studies. Brazilian fungal isolates were previously identified using a molecular technique based on domain gene sequencing. Stock cultures of these fungi were prepared by growing them in potato-dextrose-agar medium ([PDA] DIFCO®, Detroit, MI, USA), poured into Petri dishes, for 2-3 weeks at 22 ± 2 °C with a 12:12 h photoperiod (L:D) until the sporulation phase (conidiogenesis); Next, pieces of the fungus-medium were cut into 1 mm² pieces and stored in 10% glycerol in cryotubes at -80 °C.To produce conidial inoculum, frozen stock cultures were used to inoculate new PDA plates, which were incubated for 2-3 weeks to obtain sporulated cultures. Table 1. Isolates of B. bassiana and C. fumosorosea Fungus Isolate code Host / Source B. bassiana ESALQ-PL63 Atta sp. ESALQ447 Solenopsis invicta ESALQ1432 Diaphorina citri CG1229 Rupela albinella GHA (ARSEF6444) Diabrotica undecimpunctata C. fumosorosea ESALQ1296 Bemisia tabaci ESAL11364 Myzus persicae ESALQ1409 B. tabaci CG1228 R. albinella ARSEF3581 B. tabaci

[70] Media and conditions of cultivation

[71] The liquid medium used for the production of pre-cultures and, consequently, blastospores contained the following basal salts per liter of medium: KH2PO4, 2.0 g; CaCh^HiO, 0.4 g; MgSO4UH2O, 0.3 g; CoCU6H2O, 37 mg; FeSO4UH2O, 50 mg; Petition 870260076623, dated 07 / 31 / 2026, page 33 / 58 24 / 45 MnSO4'H2O, 16 mg; ZnSO4'7H2O, 14 mg; thiamine, riboflavin, pantothenate, niacin, pyridoxamine, thioctic acid, 5 pg each; folic acid, biotin, vitamin B12, 50 pg each. All chemicals used were sourced from the manufacturer Sigma® (St. Louis, MO, USA), unless otherwise indicated. A pre-culture medium containing basal salts was supplemented with glucose (Fisher Scientific®, Pittsburgh, PA, USA) at 80 g / L (40% carbon) and acid-hydrolyzed casein (derived from bovine milk, Hy-case™ MSF, Kerry Bioscience, New York, NY, USA) at 25 g / L (8.5% N and 53% C), providing a medium with a carbon-to-nitrogen (C:N) ratio of 23:1. The pre-culture medium had an initial pH of 5.8, and the pH was not adjusted during culture growth. Glucose solutions (20% w / v) were autoclaved separately and added before inoculation of the media. Sterilization of liquid cultures and glucose solutions was performed at 123 °C for 20 min.All media were prepared with distilled deionized water (ddH2O). The conidial inoculum of pre-cultures was prepared by scraping plates containing PDA+sporulated fungus with 10 mL of sterile aqueous solution of 0.04% polyoxyethylene sorbitan monooleate (Tween 80, Sigma®). Pre-cultures were inoculated with conidial suspensions to provide a final concentration of 5 x 10⁵ conidia per mL for C. fumosorosea and 1 x 10⁶ conidia per mL for B. bassiana in the liquid culture medium. One hundred mL of pre-cultures of all fungi were grown in 250 mL baffled Erlenmeyer flasks (Bellco Glass, Vineland, NJ, USA) for 3 days at 28 °C and 350 rpm, using an incubator with an orbital shaker table (INNOVA 4, New Brunswick Scientific, Edison, NJ, USA). Blastospore and conidia concentrations were measured microscopically using a Neubauer hemocytometer (4x magnification) with a light microscope with optical DIC (BH2, America Olympus, Center Valley, PA, USA).

[72] Experimental design for liquid fermentation studies

[73] To test optimal nutritional components for further experimentation, several nitrogen sources were tested for their ability to induce blastospore production associated with their subsequent desiccation tolerance. The nitrogen sources tested were, in the same proportion, 3% acid hydrolyzed casein, cottonseed meal, soybean meal, autolyzed yeast, corn infusion liquor (corn syrup), L-glutamic acid, and 1% ammonium nitrate [NH4NO3]. All culture media contained 12% glucose (120 g / L). Fifty mL of liquid cultures were Petition 870260076623, dated 07 / 31 / 2026, page 34 / 58 25 / 45 cultures were grown in 250 mL baffled Erlenmeyer flasks and incubated at 28 °C and 350 rpm for 3 days. Blastospores were separated from the culture broth, mixed with diatomaceous earth (7.5% w / v or w / v) and then air-dried to a final moisture content of <4% (w / w). The desiccation tolerance of blastospores was evaluated by measuring the germination of dehydrated blastospores once rehydrated in potato and dextrose broth and incubated for 7 hours at 28 °C and 3 rpm.

[74] An experimental assay in a factorial arrangement was used to investigate the impact of the nitrogen source on different isolates of B. bassiana and C. fumosorosea grown in submerged liquid cultures. Cordyceps and Beauveria cultures were grown in a volume of 1 mL in 250 mL Erlenmeyer flasks and incubated at 28 °C and 350 rpm in an incubator with an orbital shaker. The blastospore production medium contained the previously described basal salt medium supplemented with glucose (1 g / L) and either acid hydrolyzed casein or cottonseed meal (9.4% nitrogen N and 40% C; Pharmamedia®, Traders Protein, Memphis, TN, USA) at a concentration of 25 g / L. The medium had an initial pH of 5.5 and a C:N ratio of 21:1. Blastospore-based inoculum was obtained from 3-day-old precultures (exponential phase) to provide a final inoculum concentration of 5 x 10⁶ blastospores / mL.The flasks were manually shaken at least once a day during the fermentation process to minimize mycelial growth and sporulation on the flask walls. In all experiments, the pH was not controlled during culture growth.

[75] During broth sampling and dilution, blastospore suspensions were constantly agitated to ensure homogeneity. Dry weight was used as a measure of dry biomass accumulation. In duplicate 1 mL samples of broth collected from flasks, fungal biomass was separated from the medium by vacuum filtration (model 1225, Millipore®) through pre-weighed glass fiber filter paper discs (2.4 cm diameter) (G6, Fisher Scientific®, Pittsburgh, PA, USA), and then this filtered material was dried at 80 °C for 24 h until constant weight before measurement. At the end of the fermentation process, residual or remaining glucose in the medium (g / L) was measured using an electronic glucose meter (GlucCell®, CESCO, Atlanta, GA, USA) as a means of determining glucose consumption by the fungi. In addition, the final pH from Petition 870260076623, dated 07 / 31 / 2026, page 35 / 58 26 / 45 all culture broths were recorded. All shaken flask experiments were duplicated and all experiments were repeated at least three times on different dates.

[76] The effects of other culture conditions on blastospore production, as well as their tolerance to desiccation, storage, and bioefficacy (virulence), were also sought to be determined. Among the parameters tested, osmotic pressure and increased aeration stood out. To test the effects of variable osmotic pressure, cultures were grown in a basal salt medium supplemented with 2.5% cottonseed meal at 28 °C, 350 rpm, and 50 mL of medium in 250 mL Erlenmeyer baffled flasks. Glucose concentrations from 20 g / L to 200 g / L were tested, as well as media containing glucose (10%) and supplemented with 2%, 4%, or 6% polyethylene glycol (PEG 2) and non-toxic salts, NaCl and KCl, both at 0.25 mol / L (14.32 g / L and 18.64 g / L, respectively).

[77] To test the effects of variable aeration rates, the liquid culture production of blastospores by several B. bassiana isolates, grown in a basal salt medium supplemented with 2.5% cottonseed meal (A) or acid hydrolyzed casein (B) and 10% glucose was analyzed. Fifty-1 mL of cultures were grown in 250 mL baffled flasks in an incubator with shaking at 175 rpm or 350 rpm at 28 °C. Liquid culture volumes were altered to increase (50 mL) or decrease (1 mL) the aeration rate. Three-day-old blastospore suspensions were mixed with diatomaceous earth, this mixture being filtered to remove the fermented broth from the propagules, and subsequently air-dried to <4% final moisture. Blastospore viability was measured by evaluating germination after rehydration and incubation of dehydrated blastospores in potato-dextrose broth for 7 hours at 28 °C and 3 rpm in a shaker incubator.

[78] Extraction, drying and storage studies

[79] After B. bassiana and C. fumosorosea had grown for 3 days, the entire fermented fungal culture was mixed with 7.5% (w / v) diatomaceous earth [DT (HYFLO®, Celite Corp, Lompoc, CA, USA)]. The blastospore-DT mixtures were composed using a Buchner funnel with 12.5 cm diameter filter paper discs (Whatman #1, Maidstone, England) for vacuum filtration. The resulting biomass+DT cake from each flask (replicate) was disintegrated in a grinder, placed Petition 870260076623, dated 07 / 31 / 2026, page 36 / 58 27 / 45 on a 10 cm Petri dish and subjected to airflow drying with controlled relative humidity (RH ~ 50-60%) for 16-20 hours at ~ 22 °C until a final moisture content of less than 4% was achieved. Dried blastospore preparations were ground in a mini food processor (Mini Prep Plus, Cuisinart, East Windsor, NJ, USA), vacuum-packed (Multivac Inc., Kansas City, MO, USA) in polyethylene and nylon bags (15.3 x 21.8 cm), and stored at 4 °C. The water content (on a wet basis [WB]) and water activity (aw) of these formulations were measured before storage with a moisture analyzer (Mark II, Denver Instruments, Arvada, CO, USA) and a water activity analyzer (AquaLab 4TEV, decagon Devices, Inc., Pullman, WA, USA), respectively, and considered as potential covariates.

[80] At least 4 packets of each fungal isolate were obtained from different fermentation batches and monitored over time to assess the survival of blastospores dehydrated by continuous air drying. The viability of all air-dried blastospore preparations was determined immediately after drying and during storage using a previously described germination assay. Briefly, the germination assay was performed by adding ~25 mg of dried blastospore preparation to 25 mL of potato dextrose broth [BD (DIFCO®)] in 125 mL baffled Erlenmeyer flasks. After 6 h of incubation for C. fumosorosea and 7 h of incubation for B. bassiana at 28 °C and 3 rpm in a rotary shaker incubator, the percentage of viability was determined microscopically by examining 2 blastospores per replicate flask, based on germ tube formation.Germination was not evaluated in cell clusters, but rather where blastospores were discrete, and these were considered germinated when the germ tube length was at least half the cell diameter. Stability studies were conducted on samples stored under refrigeration conditions (4 °C), and blastospore viability was monitored monthly over a 13-month period, using the protocol described above for the aforementioned germination test.

[81] To determine the relationship between aw content and moisture, sorption isotherms were established for B. bassiana and C. fumosorosea formulated with 7.5% TD (w / v). Different saturated salt solutions were prepared to create different equilibrium relative humidities (ERH) using the following salts: sodium hydroxide (NaOH), Petition 870260076623, dated 07 / 31 / 2026, page 37 / 58 28 / 45 lithium chloride (LiCl), magnesium chloride (MgCl2.6H2O), potassium carbonate (K2CO3), sodium chloride (NaCl), potassium chloride (KCl), and potassium sulfate (K2SO4), which corresponded to aw values ​​of 0.082, 0.113, 0.328, 0.432, 0.753, 0.843, and 0.973, respectively. All salts were purchased from Sigma®. To achieve a very low ERH, Drierite® (anhydrous calcium sulfate, 8 mesh, WA Hammond Drierite Company, Xenia, OH, USA) was used as a standard desiccant and provided an aw of 0.0221. Saturated saline solutions were added to the bottom of vacuum desiccators (206 mm high x 149 mm internal diameter) (Bel-Art Products®, Wayne, NJ, USA), and the samples were incubated at 25 °C for seven days before reading. Insects

[83] The B. tabaci biotype B colony was originally obtained from a pre-established population in the city of Apopka, FL, USA, from 2013 and was maintained on cabbage cv. 'Bravo' (Brassica oleracea L.; Harris Seeds, Rochester, NY, USA) and snap bean cv. Lagoa Azul (Phaseolus vulgaris L. [Kelly Seed Co., Peoria, IL, USA]) with plants confined in 0.6 m3 PVC cages covered with fine mesh fabric (off-white dracon chiffon material, BioQuip Products Inc., Rancho Dominguez, CA, USA), maintained in a glass greenhouse (temperature range: 24-30 °C). All plants were grown in a commercial seedling substrate with 77.5 L of pasteurized Redi Earth® substrate (Sun Gro Horticulture Canada Ltd. Vancouver, Canada) supplemented with 60 g of MicroMax granular fertilizer and 4 g of Osmocote 1414-14 NPK. Untreated seeds were used and the plants were grown free of chemical pesticides.

[84] The virulence of fungal spores against whiteflies

[85] To compare the virulence between blastospores and aerial conidia of B. bassiana and I. fumosorosea, insect bioassays were performed on newly emerged second-instar nymphs of B. tabaci biotype B, as described in previous studies. Laboratory bioassays were performed with the fungal isolates B. bassiana (ESALQ 1432) and C. fumosorosea (CG1228). Blastospores were produced in a liquid culture medium, as previously described, containing 10% glucose and 2.5% cottonseed meal, and subsequently harvested after 3 days of incubation at 28 °C and 350 rpm. Preparations of these blastospores (size range 3-11 µm) were formulated with 7.5% TD and subjected to air drying until they reached Petition 870260076623, dated 07 / 31 / 2026, page 38 / 58 29 / 45 final moisture content <4%, while aerial conidia (with a size range of 1.8-5.0 μm) were cultured on plates containing PDA medium for 10-14 days at 22 °C Ce 12:12 h (L:D) photoperiod, before use in bioassays. Original suspensions of both fungal propagules were prepared with a 0.01% Tween 80 solution and then filtered once through a double layer of sterile gauze. Desired concentrations were adjusted through serial dilutions using Tween 80 solution (0.01%) at 1 x 10⁵, 5 x 10⁵, 2.5 x 10⁶, 1.25 x 10⁷, and 6.25 x 10⁷ propagules / mL, which in turn corresponded to deposition rates (doses) of 1.26 x 10², 6.82 x 10², 3.7 x 10³, 2.0 x 10⁴, and 1.09 x 10⁵ propagules / cm², respectively. Untreated controls consisted of nymphs sprayed with a 0.01% aqueous solution of Tween 80. The viability of blastospores used in all bioassays was always greater than 75% after 6 h (for C.For *fumosorosea*, viability was reduced after 7 h (for *B. bassiana*) of incubation in BD broth, while aerial conidia had >90% viability after 17 h of incubation in PDA at 25 °C.

[86] Individual bean leaves were placed on 20 mL of water-agar (2% w / v) in polystyrene Petri dishes (FALCON®, 1 x 15 mm), which were hereinafter designated as “ventilated plates”. Each bean leaf, infested with 50-70 second-instar nymphs, was sprayed with a micro-spray tower at 10 psi for 3 sec. Five replicates were prepared per fungal concentration, and all concentrations were tested in the same assay. The entire experiment was repeated at least twice on different dates, using different fungal batches and insect generations. After spraying the treatments, the ‘ventilated’ plates were placed inverted so that the abaxial side of the leaf was facing down and then incubated in a growth chamber at 27 ± 1 °C, 70% (48-78%) RH and 14: 10 h (L: E) photoperiod for six days before assessing mortality.Only nymphs showing signs of infection or symptoms of fungal diseases (i.e., mycoses) were recorded as dead individuals six days after the application of the fungi, thus making it possible to estimate the median lethal concentration (LC50) of each treatment, expressed as propagules / cm2. To compare the mortality rate between blastospores and aerial conidia, the same protocol previously mentioned was used, but in this bioassay the fungi were applied at a fixed concentration of 1.25 x 107 propagules / mL (i.e., 2.7 x 104 propagules / cm2), and mortality was recorded at... Petition 870260076623, dated 07 / 31 / 2026, page 39 / 58 30 / 45 intervals of 24 h after spraying, over six days, allowing estimation of the median lethal time (LT50) of each fungal propagule.

[87] We also decided to compare the mortality rate and lethal dose of blastospores produced in media with low and high osmotic pressure to determine any possible effect of this variable. Blastospores of B. bassiana ESALQ (1432) were produced in liquid medium composed of 2.5% cottonseed meal and 4%, 10%, or 14% glucose. Blastospores were isolated as described above after 3 days of growth. Blastospores were separated from the culture broth with diatomaceous earth and dried in an air stream. Exposure of nymphs (B. tabaci) to these blastospore preparations followed the experimental protocol described above.

[88] Statistical analysis

[89] The experiments were carried out with a completely randomized experimental design and repeated two to three times over time to ensure the reproducibility of the results. Generalized linear mixed models (GLMM) were used to fit the data on blastospore count with Poisson distribution and biomass accumulation with normal distribution, using the statistical program SAS macro PROC GLIMMIX. Tested isolate, nitrogen source, and fermentation day (time) were implemented as fixed factors, while shaking flasks (i.e., repeated measurements over time) and experimental replicates were declared as random effects in these mixed models. Blastospore viability ratio data from desiccation tolerance assays were fitted to a generalized linear model (GLM) with a binomial distribution for errors, where experimental replicate was included as a random term, while tested isolates and nitrogen sources comprised the fixed factors. Statistics for fixed effects and their interaction terms were also determined by Wald's type III F-test. Post-C multiple comparisons of treatment means were performed using Tukey's test at P < 0.05 for fixed effects and their interaction terms, once significance was detected.Blastospore survival data (% germination) over storage time at 4 °C were fitted to a non-linear logistic model with 4 parameters to estimate the effect of nitrogen sources on the half-life (ti / 2) of air-dried blastospores. The model had the following notation: S. Petition 870260076623, dated 07 / 31 / 2026, pages 40 / 58 31 / 45 — So + (α / (1 + (t / to) β)), where S is blastospore survival (% germination), t is storage time (in months), and α, β (slope or gradient coefficient), So, and to are the best-fit constants obtained by the interactive analysis performed in SAS using the PROC NLIN macro. To test the hypothesis that the nitrogen source can affect the storage stability of air-dried blastospores over time, the sum-of-squares reduction test was used to compare non-linear regression curves. The relationship between water content (moisture) and water activity (aw) was determined based on fitting the experimental data to the GAB (Guggenheim-Anderson-de Boer) model in order to construct a graph of sorption isotherm curves. It was also verified whether water content and water activity (aw) could have any effect on the percentage of blastospore viability, based on Spearman's non-parametric correlation (PROC CORR).Abbott's formula was applied to correct the percentage mortality generated by fungal treatments based on the natural mortality found in the control (untreated) in order to estimate the dose-response relationships between different concentrations of fungal propagules and whitefly nymph mortality by fitting these data to a logistic model (Proc PROBIT). The logistic model was chosen because it provided the best fit due to its lower deviance value. Median lethal concentration (LC50), with its corresponding confidence limits (95% LC) and slope (angular coefficient) were consecutively calculated for each type of fungal propagule tested. Virulence between propagule types was compared using the pairwise ratio test with a significance level α = 5% applied to LC50.Differences in speed of action (median lethal time, LT50) between blastospores and aerial conidia were analyzed using the prior logistic model to plot cumulative survival functions by treatment with pairwise comparisons over the Student's t-test at P < 0.05. All analyses were performed on the v.9.2 Statistical Analysis System (SAS Institute Inc., Cary, NC).

[90] Experimental results

[91] Blastospore yield and biomass accumulation Petition 870260076623, dated 07 / 31 / 2026, page 41 / 58 32 / 45

[92] Fungal isolates had significantly distinct filamentous growth patterns when grown in liquid medium supplemented with different nitrogen sources (Figure 1 - results shown for two B. bassiana isolates). In general, the results demonstrate that B. bassiana isolates grew in yeast form with higher blastospore yield when the nitrogen source was cottonseed meal, soybean meal, autolyzed yeast or acid hydrolyzed casein (CHA). Furthermore, most nitrogen sources resulted in an adequate level of desiccation tolerance. Similar results were obtained for C. fumosorosea (data not shown). Two of these nitrogen sources (CHA and cottonseed meal) were chosen as the nitrogen sources for further experiments.

[93] Liquid cultures supplemented with cottonseed meal or cottonseed meal at 25 g / L were grown as described above and their growth parameters analyzed. Based on daily observations, there was a significant increase in blastospore yield, as well as in dry biomass increment from day 2 to day 3 of growth for all B. bassiana and C. fumosorosea isolates, regardless of the nitrogen source (Tables 2, 3 - lowercase letters refer to comparisons between fungal isolates and nitrogen sources within each fermentation day (in columns), while uppercase letters refer to comparisons between fermentation days (in rows) for each fungal isolate. Table 2. Productivity of liquid cultures with different isolates of B. bassiana B. bassiana isolates Nitrogen source Yield Biomass (mg / mL) (x 10⁸ blastospores / mL) Day 2 Day 3 Day 2 Day 3 CG1229 CHA 11.7±0.6 B' b, 15.3±0.2 e, 3.4±1.3 AB c, 5.5±1.4 b, A Cottonseed meal 25.1±0.5 A a, 26.0±0.7 8.8±1.8 be, AA ab, 9.11±1.2 ab, A ESALQ1432 CHA 12.9±1.7 B b, 20.5±0.7 d, 4.4±0.3 AB bc, 7.9±0.8 ab, A Cottonseed meal 28.5±1.9 a, 32.8±0.3 a, 8.3±0.5 ab, 12.4±1.0 a, Petition 870260076623, dated 07 / 31 / 2026, pages 42 / 58 33 / 45 Cottonseed meal BABA ESALQ447 CHA 12.9±0.9 b, 16.4±0.5 e, 2.4±0.2 c, 6.8±0.8 ab, BABA Meal 26.1±1.6 a, 24.2±0.9 c, 5.1±0.4 7.8±0.5 ab, Cottonseed meal AA abc, BA ESALQ- CFTA CHA 14.3±0.9 b, 15.5±0.3 e, 0.34±0.06 d, 0.95±0.1 c, PL63 AABA Meal 25.9±0.9 a, 24.0±0.6 c, 0.30±0.02 d, 1.2±0.1 c, Cottonseed meal AABA 10.5±0.4 b, 16.2±0.5 e, 2.7±0.5 c, 4.9±0.5 b, GHA CHA ,,, ,, , , , , , , BABA Bran 25.9±0.7 a, 28.0±1.8 b, 10.0±0.6 a, 11.6±0.6 a, cotton AAAA Table 3. Productivity of liquid cultures with different isolates of C. fumosorosea C. fumosorosea Tension Nitrogen Source Biomass Yield (mg / mL) (x 10⁸ blastospores / mL) Day 2 Day 3 Day 2 Day 3 CG1228 CHA 22.1±1.5 a, 33.3±2.8 a, 14.4±1.7 ab, 26.0±0.9 BAB ab, A Cottonseed Meal 31.8±1.5 a, 40.9±1.1 a, 15.4±1.5 a, 27.9±2.2 a, BABA ESALQ1296 CHA 21.7±0.5 B a, 34.6±2.0 a, 9.6±1.1 abc, AB 16.6±0.7 bc, A Cottonseed Meal 30.6±1.5 a, 40.6±2.2 a, 10.0±0.9 13.0±1.1 c, cotton BA abc, AA ESALQ1364 CHA 20.5±1.2 B a, 32.2±2.5 a, 9.6±0.5 abc, AB 15.3±1.7 A c, 29.1±0.7 a, 36.9±0.5 a, 6.7±0.6 b, AA 6.9±0.9 d, cotton BA ESALQ1409 CHA 20.4±1.4 B a, 33.2±2.6 a, 8.7±0.6 abc, AB 13.8±1.9 A c, Petition 870260076623, dated 07 / 31 / 2026, pages 43 / 58 34 / 45 Cottonseed meal 31.6±0.8 a, 38.7±1.8 a, 9.4±0.3 abc, 15.3±1.5 c BABA ARSEF3581 CHA 23.7±1.0 a, 33.9±2.1 , BA 11.2±2.4 14.3±1.4 c abc, AA Cottonseed meal 31.2±1.5 a, 37.6±1.4 , BA 8.9±1.9 bc, 10.4±1.5 A cd, A

[94] B. bassiana isolates exhibited different growth rates, measured as blastospore concentration over time, as the isolate-fermentation day interaction term was significant (F4,105 = 5.97, P = 0.2), while blastospore production increased at the same rate in C. fumosorosea liquid cultures (F4,62 = 2.28, P = 0.0708). Blastospore counts generally reached higher concentrations on day 3 of growth for both B. bassiana (F 1,105 = 71.89, P = 0.1) and C. fumosorosea (F 1,62 = 53.29, P < 0.1), although some isolates did not produce significantly higher blastospore counts on day 3 compared to day 2. The interaction between fungal isolate and nitrogen source exerted a strong impact on blastospore yields for C. fumosorosea (F4,62 = 5.53, P = 0.7), but there was only a marginal effect for B. bassiana (F4,105 = 2.42, P = 0.0531).The effect of the nitrogen source alone was more pronounced on blastospore production for B. bassiana (F1,105 = 16.97, P < 0.1), whose isolates reached higher concentrations when cultured with cottonseed meal, except for the ESALQ-PL63 isolate, which was not responsive. An opposite result was observed for the ESALQ1364 and ARSERF3581 isolates of C. fumosorosea, which yielded higher blastospore counts when cultured with casein acid hydrolysate instead of cottonseed meal (F1,62 = 13.61, P = 0.5), although the other isolates did not respond differently when cultured with these nitrogen sources. Comparisons between Cordyceps isolates revealed that CG1228 reached the highest blastospore concentration (2.6-2.8 x 10⁹ blastospores / mL) after three days of fermentation, regardless of the nitrogen source tested (F4,62 = 37.04, P < 0.1). Under the same fermentation conditions, the best B. isolates...Bassiana blastospore-producing isolates were CG1229, GHA, and ESALQ1432, grown in liquid medium with cottonseed meal instead of casein acid hydrolysate, achieving yields of 0.9–1.2 x 10⁹ blastospores / mL in 3 days of growth (F₄,10⁵ = 21.2, P < 0.1). Interestingly, all C. fumosorosea isolates were able to... Petition 870260076623, dated 07 / 31 / 2026, pages 44 / 58 35 / 45 produced more than 1 x 10⁹ blastospores / mL on day 3, whereas only 2 / 5 of the B. bassiana isolates reached this high blastospore concentration. Furthermore, C. fumosorosea isolates demonstrated faster yeast-like growth compared to B. bassiana isolates. Among all fungal isolates tested, ESALQ-PL63 showed more abundant hyphal growth at the expense of yeast-like growth (low blastospore production), resulting in a more viscous fermented broth.

[95] Regarding the accumulation or gain of dry biomass, the data were analyzed separately for each nitrogen source, since media containing cottonseed meal presented, before fungal inoculation, a higher amount of solids (15.54 ± 0.08 mg / mL), compared to casein acid hydrolysate (0.07 ± 0.01 mg / mL), making any direct comparison between these nitrogen sources difficult. Regardless of the nitrogen source, the total dry biomass did not vary among the C. fumosorosea isolates (F4,62 = 1.81, P = 0.1383), although most of these isolates increased their biomass over time (F 1,62 = 20.43, P < 0.1) (Tables 2, 3). When B. bassiana isolates were cultivated in a medium supplemented with casein acid hydrolysate, the biomass of the fungal cultures increased considerably over time in contrast to cottonseed meal (F4,105 = 7.77, P < 0.1). Both B. bassiana and C.*C. fumosorosea* accumulated more dry biomass on day 3 than on day 2 of fermentation (F 1,105 = 43.77, P < 0.1; F 1,62 = 583.36, P < 0.1, respectively). Regardless of the nitrogen source, ESALQ1432 of *B. bassiana* showed the highest dry biomass production of all isolates tested (F4,105 = 15.04, P < 0.1). Finally, the final pH values ​​of the liquid cultures of *B. bassiana* and *C. fumosorosea* on the last day of fermentation were similar and showed an increase in acidity, with variations of 3.8-4.8 and 3.6-4.9, respectively. Blastospore production, as well as their desiccation tolerance, increased with increasing concentrations of the nitrogen source added to the liquid medium, possibly reaching plateau stability at higher N levels (Figure 2).

[96] Desiccation tolerance

[97] Drying blastospores aged 3 days at moisture content <4% (aw <0.3) revealed a significant effect for the interaction of nitrogen sources and fungal isolates on the viability of C. fumosorosea blastospores (F4,29 = 4.90, P = 0.38) Petition 870260076623, dated 07 / 31 / 2026, pp. 45 / 58 36 / 45 and B. bassiana (F4,56 = 5.26, P = 0.11). Desiccation tolerance was evaluated by measuring the percentage of germinated blastospores after drying in a continuous air stream and subsequently under rehydration in potato-dextrose broth (BD), and incubated for 7 hours and 6 hours at 28 °C and 3 rpm for B. bassiana and C. fumosorosea, respectively. When examining viability within the B. bassiana group, blastospores from isolates ESALQ1432 and ESALQ447 were more tolerant to desiccation, exhibiting higher blastospore viability (71-79%) when produced with cottonseed meal than with casein acid hydrolysate (F 1,56 = 14.06, P = 0.4) (Table 4 - non-corresponding letters denote statistical differences, with lowercase letters referring to comparisons between fungal isolates within each nitrogen source (columns), while uppercase letters refer to comparisons between nitrogen sources (rows) within each isolate). Table 4 - Desiccation tolerance of blastospores of B. bassiana and C. fumosorose a. Fungal isolate Desiccation tolerance (% blastospore survival) * Acid hydrolyzed casein Cottonseed meal Beauveria bassiana CG1229 80.8±2.1 a, A 78.2±2.3 c, A ESALQ1432 73.0±2.8 b, B 79.2±2.4 bc, A ESALQ447 73.9±2.7 b, B 84.0±2.0 ab, A ESALQ-PL63 85.3±2.0 a, A B ESALQ1364 74.1±2.9 ab, A 65.3±3.3 b, B ESALQ1409 62.3±3.4 b, B 70.6±3.1 ab, A Petition 870260076623, dated 07 / 31 / 2026, pp. 46 / 58 37 / 45 ARSEF3581 82.4±2.4 a, A 79.1±2.6 a, A

[98] On the other hand, C. fumosorosea isolates showed better tolerance to desiccation when grown in media containing acid hydrolyzed casein instead of cottonseed meal (F 1,29 = 8.37, P = 0.72), except for isolates ESALQ1409 and ARSEF3581. Blastospore survival after air drying varied significantly between B. bassiana (F4,56 = 16.5, P < 0.1) and C. fumosorosea (F4,29 = 12.96, P < 0.1) isolates. Analysis within B. bassiana indicated that both nitrogen sources supported higher viability rates (70-86%) after air drying, while C. fumosorosea isolates had an initial survival of 62-82% of germinated blastospores.

[99] After air drying, moisture sorption isotherms were determined at 25 °C to describe the relationship between the aw level of the fungal biomass (blastospores + mycelium) formulated with 7.5% TD and moisture (%, wet weight basis). The experimental data were significantly explained by the GAB model (B. bassiana: R2= 0.99, F3,28 = 3666.55, P < 0.1; C. fumosorosea: R2= 1,, F3,21 = 2738.01, P < 0.1), assuming a sigmoidal curve shape (Figure 3). Water activity (aw) of dried blastospore formulations of B. bassiana ranged from 0.251-0.364, corresponding to 1.38-2.70% moisture, while dried formulations of C. fumosorosea had aw values ​​ranging from 0.270-0.323, corresponding to water contents of 1.76-2.63%. Neither water activity nor moisture showed a significant correlation with the survival rates of dehydrated blastospores for both fungal species tested (Spearman correlation: -0.02 < r < 0.30, 0.06 < P < 0.86).Blastospores of B. bassiana cultivated under the conditions described here survive spray drying (when mixed with skimmed milk powder (SMP) or SMP + 2.5% ascorbic acid), and also survive equally well slow drying in an air stream with controlled relative humidity (when mixed with diatomaceous earth). These results are presented in Table 5 and indicate that there was no significant difference in survival after drying as a function of the formulation process or drying method tested. Desiccation tolerance was represented by the percentage of blastospores that germinated after rehydration in BD broth after 7 hours of incubation at 28 °C and 3 rpm. Petition 870260076623, dated 07 / 31 / 2026, pages 47 / 58 38 / 45 Table 5. Air drying and spray-drying of B. bassiana blastospores Drying Method Formulation Moisture (%) Water Activity (aw) Desiccation Tolerance (%) Germination) 20% LDP Drying 4.48 5.79) (2.85- 0.195 0.2827) (0.077- 83.1 ± 1.2 Spray Drying 17.5% LDP + 2.5% Ascorbic Acid 4.47 5.75) (3.84- 0.253 0.324) (0.184- 82.6 ± 0.7 Air Drying Diatomaceous Earth 0.84 1.31) (0.56- 0.246 0.2876) (0.2142- 80.9 ± 1.0 Storage

[101] The long-term stability of air-dried blastospores under refrigerated storage conditions varied with the fungal isolate and the nitrogen source (Figures 4; 5). The four-parameter logistic model adequately fit (R2= 0.74-0.99, P < 0.01) the experimental data regarding blastospore survival throughout the evaluated storage period for all B. bassiana and C. fumosorosea isolates produced with any nitrogen source. According to the least sum of squares test used to compare the non-linear regressions of the blastospore survival time curves, it was found that the type of nitrogen did not influence the stability of air-dried B. bassiana blastospores during 13 months of refrigerated storage (4 °C), however, isolates CG1229 and GHA survived much longer when produced with cottonseed meal and casein acid hydrolysate, respectively (Figure 4).Specifically for C. fumosorosea, blastospores of isolates ARSEF3581, ESALQ1296, and ESALQ1364 exhibited greater viability over a longer period of time when cultured in liquid medium containing cottonseed meal, while the long-term viability of all other isolates was not influenced by the nitrogen source (Figure 5). Generally, the survival curves of C. fumosorosea blastospores demonstrate a faster pattern of decrease or drop in viability compared to the curves of B. bassiana. Petition 870260076623, dated 07 / 31 / 2026, pages 48 / 58 39 / 45 regardless of the nitrogen source tested in the production medium of these propagules. Half-life estimates of air-dried blastospores of B. bassiana stored at 4 °C showed a minimum half-life of 14.1 months for GHA when grown on cottonseed meal, while most isolates retained a half-life greater than 14 months (Table 5). In contrast, air-dried blastospores of C. fumosorosea showed a shorter half-life (9.2 months) with the isolate ESALQ1296 grown on casein acid hydrolysate. The longest half-life obtained for C. fumosorosea (13.1 months) was attributed to the isolate ARSEF3581, grown on cottonseed meal. In most cases, cottonseed meal provided higher half-life estimates for C. fumosorosea isolates.

[102] Various packaging and storage methods were also tested for the insecticidal compositions of the present invention, specifically to test storage conditions that would favor long-term “shelf life” under elevated ambient temperature conditions (herein referred to as a constant 28 °C). For the results presented in Table 6, the half-life was calculated based on the equation ln(2) / b, where b is the rate of decay of the viability of an exponential decaying function. All vacuum-sealed packages were subjected to a sealing pressure of 999 mbar, providing concentrations of <0.021% atmospheric oxygen within the aluminized (mylar) packages. Silica gel (SG) was used as a moisture absorbent or simply a desiccant. ZM-1 and ZPT-50 were used as oxygen absorbers, separately. RP-3A is an oxygen absorber combined with a desiccant. Table 6. Effects of high storage temperature with various forms of packaging. Drying Method Formulation Packaging Storage Temperature (°C) Half-life t½ (weeks) LDP Drying Vacuum Sealed 28 Spray Drying 4 Air Drying TD Vacuum Sealed 28 4 Petition 870260076623, dated 07 / 31 / 2026, pages 49 / 58 40 / 45 LDP Drying Control 28 Atomization RP-3A 28 5 SG 28 LDP + ASA Control 28 RP-3A 28 2 ZM-1 28 ZPT-50 28 SG 28 LDP Drying Control 28 Atomization ZM-1 + SG 28 ZPT-50 + SG 28 ZM-1 28 ZPT-50 28 SG 28 Air Drying TD Control 28 RP-3A 28 6 ZM-1 28 ZPT-50 28 SG 28 1

[103] To investigate in more detail the differences found between the storage methods, the different oxygen and moisture absorbers were analyzed. Petition 870260076623, dated 07 / 31 / 2026, pages 50 / 58 41 / 45

[104] The effect of oxygen and moisture absorbers on the storage stability of air-dried and 28 °C stored B. bassiana blastospores was determined with each of the above packaging combinations. The results are presented in Figure 6 (RP-3A = O2 absorber + desiccant; ZM-1 = type A O2 absorber; ZPT-50 = type B O2 absorber; silica gel = desiccant). Survival was expressed as a percentage of viable blastospores relative to freshly dehydrated blastospore samples. Germination was measured based on germ tube protrusion and elongation after 7 hours of incubation in potato-dextrose broth (BD) at 28 °C and 3 rpm agitation. The results of a similar analysis for B. bassiana blastospores dried by spraying with LD (with and without ascorbic acid [ASA]) show that the addition of ASA to the formulation increases the survival of spray-dried formulations (Figure 7).

[105] Osmotic pressure

[106] Our results show that increased osmotic pressure resulted in higher blastospore yield, a fact not simply related to increased glucose availability for yeast cells (Figure 8, Table 7 - Means (± SE) followed by different letters indicate statistical differences (Tukey test, P < 0.05). For the osmolytes listed in Table 7, the concentrations are equivalent to 200, 14.32, and 18.64 g / L of glucose, NaCl, and KCl, respectively. Media containing NaCl and KCl were supplemented with an initial concentration of 1 g of glucose / L (0.56 M) as a carbon source. Lowercase letters refer to comparisons within each fungal isolate for each fermentation day (columns), while uppercase letters refer to comparisons between fermentation days within each fungal isolate (rows).Blastospores of this invention produced under high osmotic pressure (> 0.5 MPa) exhibited a spherical morphology that is smaller and uneven compared to the oblong blastospores produced in normal growth medium (Figure 9). This spherical blastospore shape is associated with greater virulence (infectivity) of target pests by the fungus and represents a unique blastospore shape that had not been previously reported (see below). Table 7. The increase in blastospore production induced by increased osmotic pressure. Petition 870260076623, dated 07 / 31 / 2026, pp. 51 / 58 42 / 45 B. bassiana isolate Osmolyte (mol / L) Osmotic pressure (MPa) Blastospore concentration (x 108 mL-1) Desiccation tolerance (% germination) Day 2 Day 3 ESALQ1432 Glucose (1.1) 2.7 17.3 ± aB 1.2 29.0 aA ± 1.1 66.2 ± 2.1 NaCl (0.25) 2.5 12.1 ± bB 0.8 33.1 aA ± 1.4 76.1 ± 2.8 KCl (0.25) 2.5 11.1 ± bB 1.2 29.6 aA ± 1.8 70.7 ± 2.0 GHA Glucose (1.1) 2.7 15.6 ± aB 1.2 29.4 aA ± 2.0 58.6 ± 2.2 NaCl (0.25) 2.5 12.7 ± aB 0.5 24.1 aA ± 1.7 57.4 ± 1.1 KCl (0.25) 2.5 15.1 ± aB 0.9 26.1 abA ± 1.9 54.2 ± 1.4

[107] High aeration

[108] Although previous studies indicated that higher aeration rates were obtained with baffled-type flasks and using high agitation speeds to provide more oxygen to liquid cultures and consequently increase blastospore yields of C. fumosorosea, the opposite trend has been shown based on the fact that there is no increase in blastospore production of B. bassiana above 2 rpm (Pham et al., 29 - Mycobiology).

[109] Initial results suggest that culture volume is the key factor. When we compared 50 mL of culture with 100 mL of culture (liquid medium containing basal salts, 2.5% cottonseed meal, 10% glucose in 250 mL baffled Erlenmeyer flasks, 28 °C and 350 rpm), we observed that dissolved oxygen levels were higher in the smaller volume of liquid medium (Figure 10). We also tested varying agitation speeds and found that higher speeds significantly increased blastospore production in both culture volumes, 50 Petition 870260076623, dated 07 / 31 / 2026, pages 52 / 58 43 / 45 and 1 mL (Table 8 - For each fungal isolate and each evaluation day, the mean values ​​followed by different letters are significantly different (Tukey test, P < 0.05). High blastospore yields of B. bassiana using stirred tank bioreactor fermentation further support the requirement for high levels of dissolved oxygen (data not shown). Table 8. Effects of high aeration rates on blastospore yield. Beauveria Bassiana Volume of medium Production of blastospores (rpm) (mL) (blastospores L-1 x 1011) isolated Day 2 Day 3 ESALQ1432 350 100 7.6±1.0 b 9.9±1.1 a 50 11.0±0.6 a 14.0±1.5 b 175 100 3.3±0.1 c 3.3±0.3 c 50 4.9±0.2 c 5.7±0.5 c

[110] The results demonstrate that cultures of B. bassiana, as well as C. fumosorosea (data not shown), grown under highly aerated conditions (dissolved oxygen level above zero for most of the fermentation period) with appropriate concentrations of a nitrogen source (> 15 g / L), carbon source (> 60 g / L), and osmotic pressure greater than 0.5 MPa, achieve rapid production of high blastospore concentrations (Figure 11). These blastospore production conditions provide a viable production and stabilization process for Beauveria blastospores using low-cost media components. Previous attempts at producing Beauveria blastospores using submerged liquid fermentation processes required longer fermentation times (6-8 days) to maximize yields, while the cells produced had lower survival after desiccation and low stability in storage.

[111] Efficacy against whitefly

[112] To compare the virulence between blastospores and aerial conidia, we conducted bioassays with second-stage nymphs of B. tabaci biotype B. Virulence tests revealed that B. bassiana blastospores require a spore concentration four times lower to kill 50% of the nymphs compared to aerial conidia. Petition 870260076623, dated 07 / 31 / 2026, pages 53 / 58 44 / 45 (Table 9). A total of ten (10) replicates, each containing more than fifty (50) whitefly nymphs, were tested for each fungal concentration. The median lethal concentration deposited on the nymphs (LC50) is expressed in propagules / cm2 and estimated by the logistic model. Cumulative mortality was monitored until day 6 after application. Mean mortality for nymphs in the control group was 3.7 ± 1.3%. Relative potency (RP) is a measure of the relative efficacy of blastospores in relation to aerial conidia within each fungal species: (RP = LC50 of blastospores / LC50 of conidia). Comparisons were made within each fungal species and, if the 95% confidence interval for the ratio of LC50 values ​​does not contain 1, it is concluded that the LC50 values ​​are significantly different. The values ​​of χ2 and P represent the probability of the slope (angular coefficient) being ^ 0, rather than referring to the fit of the logistic model. Table 9. Blastospores of B. bassiana (ESALQ1423) are more effective than aerial conidia for whitefly control. Propagule type n CL50 (propagules / cm2) (95% CI) PR50 (95% LC) Lower Upper χ2 (p-value) Lower Upper Blastospores 3113 485 353 643 4.6 3.25 6.50 109.35 (< 0.1) Conidia 3059 2230 1362 3393 - - - 72.22 (< 0.1)

[113] For the TL50 bioassays, ten insects per treatment (2.0 x 104 spores / cm2) were tested, while nymphs from the control group (untreated) were sprayed with a 0.01% Tween 80 solution. Corroborating previous observations for B. bassiana, the median lethal time (TL50, time required to kill 50% of the nymphs) for aerial conidia was significantly longer (t = 9.88, P < 0.1) than that calculated for blastospores, resulting in a significant increase in the mortality rate (> 37% faster) of nymphs exposed to blastospores (Table 10). The mean mortality in the control group was 11.5 ± 1.6% after 6 days. Blastospores of C. fumosorosea require 70% fewer propagules to induce mortality in 50% of nymphs, and also induce faster nymph mortality (t = 2.52, P = 0.0215) when compared to conidia (data not shown). Most corpses Petition 870260076623, dated 07 / 31 / 2026, pages 54 / 58 45 / 45 nymphs infected by both types of propagules supported fungal growth which subsequently sporulated. Table 10. Blastospores of B. bassiana (ESALQ1423) kill whiteflies faster than aerial conidia. Fungi Propagule Type Median 95% CI (days) survival time (TL50, days)b Lower Upper B. bassiana Blastospores 512 2.45 ± 0.05 b 2.34 2.55 Conidia 561 3.35 ± 0.07 a 3.21 3.49

[114] To examine whether blastospores cultivated under high osmotic pressure showed good insecticidal performance against whitefly nymphs, blastospores of B. bassiana (ESALQ1432) were produced in a liquid medium with a fixed value (2.5%) of cottonseed meal and glucose at 4%, 10%, and 14% after 3 days of fermentation. Blastospores were separated from the culture broth with diatomaceous earth and dried in an air stream, or encapsulated in a skimmed milk powder (SMP) matrix and spray-dried. The results shown in Figure 12 indicate that the smaller, rounded cells produced under high osmotic pressure (with 10% or 14% glucose) showed greater biocontrol efficacy, causing insect mortality more rapidly. Petition 870260076623, dated 07 / 31 / 2026, pages 55 / 58

Claims

1 / 3 CLAIMS 1. A method for producing a composition wherein said composition contains desiccation-tolerant blastospores of Beauveria bassiana or Cordyceps fumosorosea characterized by comprising the steps of: a) inoculating a liquid culture medium comprising a carbon source and a nitrogen source with propagules of the fungus Beauveria bassiana or Cordyceps fumosorosea with an initial inoculum density in the range of 1x10³ to 5x10⁷ blastospores / mL, wherein said carbon and nitrogen sources are present in said liquid culture medium with a carbon-nitrogen (C:N) ratio in the range of 10:1 to 50:1, wherein the carbon source is defined as glucose, in concentrations greater than 10%, and the nitrogen source is defined as cottonseed meal, soybean meal, autolyzed yeast or hydrolyzed casein, in the range of 1.5% to 9.5%;b) incubation of said propagules under culture conditions in bioreactors that allow a dissolved oxygen level of 5% to 100% saturation, an aeration rate of 0.2 to 10 L / min, an agitation speed of 50 to 1000 rpm, and an osmotic pressure in the range of 0.5 to 3.0 MPa; c) incubation of said propagules for 2 to 5 days to produce blastospores at a temperature of 15 °C to 30 °C; d) collection / recovery of said blastospores by filtration method, with or without vacuum aid, or by centrifugation; e) drying by rapid atomization (spray-drying) or by slow drying (fluidized bed) of said blastospores to a water activity range of 0.01 to 0.30, thus producing desiccation-tolerant blastospores; (e) storage of said desiccation-tolerant blastospores for at least six months during which said desiccation-tolerant blastospores have at least 60% viability when rehydrated; 2. A method for producing a composition according to claim 1 wherein said liquid culture medium has a pH in the range of 3.0 to 8.

5.

3. A method for producing a composition according to claim 1, characterized in that the incubation time of said propagules is 2 to 5 days. Petition 870260076623, dated 07 / 31 / 2026, pp. 56 / 58 2 / 3 4. A method for producing a composition according to claim 1, characterized in that the temperature during the incubation period is in the range of 15 °C to 30 °C.

5. A method for producing a composition according to claim 1 characterized in that after drying said blastospores the water activity is in the range of 0.01 to 0.

30.

6. An insecticidal composition comprising a carrier (adjuvant / excipient) and desiccation-tolerant blastospores of Beauveria bassiana or Cordyceps fumorosea, wherein said adjuvant / excipient and said blastospores are contained in a vacuum-sealed package and wherein said blastospores are produced by a method characterized by comprising the steps of: a) inoculating a liquid culture medium, according to claim 1, comprising a carbon source and a nitrogen source with fungal propagules of Beauveria bassiana or Cordyceps fumorosea; b) incubating said propagules under culture conditions providing dissolved oxygen levels above zero and osmotic pressure in the range of 0.5 to 3.0 MPa; c) incubating said propagules for 2 to 5 days to produce blastospores at a temperature of 15 °C to 30 °C; d) collection / recovery of these blastospores by filtration method, with or without vacuum aid, or by centrifugation;e) spray-drying or fluidized bed drying of said blastospores to a water activity range of 0.01 to 0.30, thereby producing desiccation-tolerant blastospores; and f) storage of said desiccation-tolerant blastospores for at least six months, during which said desiccation-tolerant blastospores have at least 60% viability when rehydrated after storage exceeding six months at a temperature range of 0 °C to 35 °C.

7. An insecticidal composition according to claim 6 characterized by comprising an oxygen extractor / absorber compound, a moisture extractor / absorber compound, or a combination of both, wherein the oxygen extractor / absorber compound may be chosen from a group consisting of enzyme-mediated oxidation, iron-based oxidation, sodium ascorbate, sodium carbonate, ZM-1, ZPT-50 and RP-3A; and the moisture extractor / absorber compound may be chosen from a group consisting of silica gel, activated carbon, calcium sulfate, calcium chloride, molecular sieves, zeolites, RP-3A and other inert desiccants.

8. An insecticidal composition according to claim 6 or 7, characterized in that the composition is stored at a temperature in the range of 0 °C to 35 °C.

9. A non-therapeutic method for insect control characterized by comprising the application to the site of said insects of an effective quantity of insecticide based on desiccation-tolerant blastospores of Beauveria bassiana or Cordyceps fumosorosea.

10. A non-therapeutic method according to claim 9 characterized in that said desiccation-tolerant blastospores are produced by the method according to claim 1.

11. A non-therapeutic method according to claim 9 or 10 characterized by the said location being an agricultural crop. Petition 870260076623, dated 07 / 31 / 2026, p. 58 / 58

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