**3. Stabilization of fungal propagules**

potential (Ψ = −2.1 MPa) by adding glycerol (7.3% w/v) and calcium chloride (20 mM) to the medium. Biomass from liquid cultures responded to water-stress by accumulating increased concentrations of polyols (glycerol) and trehalose [79]. Increased liquid nutrient medium tonicity (osmolality 804–1454 mOsm) of the made by 50–150 g/l of PEG 200 polyethylene glycol increased the yield of submerged *Metarhizium anisopliae* var. *acridum* conidia up to 25%. Spores from high osmolality medium had increased pathogenicity and tolerance to drying. Interestingly, relative drying stability did not appear to be the result of differences in polyol or trehalose concentra-

Non-optimal carbon sources also stimulated *M. anisopliae* formatting resistant to long-range ultraviolet (<290 nm) and accumulating about two times more mannitol and trehalose conidia [80]. The effect of alkane-growth induction of the entomopathogenic fungus *Beauveria bassiana* on the virulence was demonstrated. That alkane supplementation of culture media does not affect the fatty acid composition but change the unsaturated/saturated ratio. However, the

Solid-state fermentation is the most suitable for cultivation of fungi because their habitats are chiefly solid substrates. In fact, SSF imitates the yields aerial conidia as the final product of conidiation processes. For example, 98% of marine fungi were isolated from submerged solid substrates [82]. In the most cases, spore yields and viability are higher than they are produced by SSF [83]. Hydrophobic air conidia are best suitable for oil formulations, since prolong the conidial viability and decreases UV radiation sensitivity [84–86]. Indeed, numerous studies have shown that conidia produced in an SSF culture are tolerant toward environmental factors (dehydration, drop of temperature and solar irradiation) than spores obtained by SmF [87]. Conidia and blastospores are the main infective units used in biological control with entomopathogenic fungi. There is no absolute advantage between both infective units. However, most formulations of mycoinsecticides are based on aerial conidia obtained in solid-state culture, since these propagules are more resistant to abiotic

A polysaccharide matrix often surrounds the spores produced by SSF and protects them during desiccation opposite the spores produced by LSF [89]. The choice of substrate, its humidity and growing time also affect the quality of propagules [90]. For example, dried conidia of Colletotrichum truncatum produced on vermiculite tended to retain efficacy during storage better than spores recovered from perlite culture [91]. Sometimes the fermentation can be terminated after the fungus has penetrated the nutritive substrate but before conidiation has begun [92, 93]. Dried grain kernels colonized by *Beauveria* or *Metarhizium* remain competent for regrowth and sporulation upon rehydration. The colonized grains are also viable for lengthy periods in the soil, germinating when suitable conditions arise. For example, after such granules are applied into soil or mixed into horticultural soil the conidia were produced within the habitat of the target insect [92, 93]. At the same time, SSF is not widely used earlier in bioherbicide production due to higher costs, more chances of contamination and the com-

unsaturated/saturated ratio diminished markedly from 4.32 to 2.47 [81]. At the same time, liquid substrates are uncommon one for fungal growth.

tions [35].

*2.2.2. Solid-state fermentation (SSF)*

68 Biological Approaches for Controlling Weeds

factors found in open fields [88].

plexity of spores' recovery from the substrate [94].

Biological material produced by fermentation and separation from a substrate as a rule cannot be stored for a long time. Even at a low temperature of the storage fungal spores, the mycelium can germinate slowly under appropriate wetness that is unpromising without a plant substrate. Many locally produced biopesticides should be used within several weeks after fermentation was finished as DeVine™, a mycoherbicide based on spores of *Phytophthora palmivora* [64, 109].

At the high-productivity biotech companies, the microorganisms should be stabilized to prevent germination of propagules for a long time (months, years). This can be achieved basically by concentration, drying or encapsulation of biomaterial on polymer layer and storage under appropriate conditions. In the ideal situation, the modern biopesticides can be stored not less than 2 years at the temperature 4°С, 3 months at 30°C and several days at 40–50°С [64].

(5%) to the concentrated spore suspension (10<sup>9</sup> CFU/mL) prevented a preparation contamination and cell sedimentation. At the temperature 4°C, CFU number was decreased 100 times after 6 months of the storage. For stabilization of a bioinsecticide based on the mycelium of *Lagenidium giganteum* a concentrated emulsion was developed containing 40% of refined corn oil and 0.5% AEROSIL (Fumed Silica, R974). The latter prevented mycelium sedimentation and aggregation. This formulation can be stored under room conditions for 12 weeks without

Production and Stabilization of Mycoherbicides http://dx.doi.org/10.5772/intechopen.76936 71

Some components of emulsion concentrates (for instance, plant or paraffinic oils) affect efficacy of biopesticides including mycoherbicides. They prevent fast water evaporation from spray droplets and improve thermotolerance of fungal cells as it was shown for *Metarhizium anisopliae* s.l. (IP 46) and *Metarhizium robertsii* (ARSEF2575) [120]. Application of *Microsphaeropsis amaranthi* against the weed, *Microsphaeropsis amaranthi* in Sunspray 6E oil (10% v/v) resulted

The mycelium of *Trichoderma asperellum* GSS 03-35 produced by submerged liquid fermentation was stabilized by concentration to 6–10% of dry matter into paste containing corn starch (5%) as stiffener. The paste had pH 3 and contained copper sulfate (20 mg/L). During the course of storage, the fungus formed chlamydospores and conidia. After 6 months of storage at the temperature 20°C the fungus remained effective against head blight of wheat [122].

The drying is the most popular technique of inoculum stabilization. Besides simple drying by warm heat on trays (convection drying), spray drying, fluid bed drying and lyophilisation (freeze-drying) are used. The selection of the drying technique depends on availability, costs

The biomaterial mixed with preservatives and fillers is dried on trays in thin layer. This technique is used for production of the biofungicide *Trichodermin* (Biotechmash, the Ukraine) as a wettable powder. This simple technique can be applied for drying of low-scale amounts of the biomass and for preliminary experiments. Corn starch, rice flour, talc, diatomaceous earth and kaolin were evaluated as preservatives and fillers during drying of blastospores of *Beauveria bassiana*. Kaolin (at the concentration of 5% w/v of spore concentrate) allowed to maintain satisfactory viability of spores (≥50%) for 7 weeks storage at 4°C [123]. Conidia of *Stagonospora* 

air flow kept high viability (>70%) and pathogenicity about 5 months under the temperature 3°C. After 17 months of the storage just 5% of total conidia were viable, when the conidia were stored under the temperature 20°C conidia their viability decrease to 20% for a week [124].

In some inoculum stabilization protocols, convection drying was proposed for formulation of conidia and microsclerotia of *Beauveria*, *Metarhizium*, *Colletotrichum*, *Mycoleptodiscus* and *Trichoderma*. Some useful additives can be used to improve of viability of the infection units:

conidia) by

*convolvuli* LA39 produced on V8 agar and dried with kaolin as a filler (1 g per 10<sup>9</sup>

loss of efficacy against mosquitos [119].

*3.1.2. Pastes*

**3.2. Drying**

and sensitivity of the biomaterial.

*3.2.1. Convection drying*

in improved disease impact under low moisture conditions [121].

There are quite simple and cheap techniques of stabilization and storage of some microorganisms. For instance, infection material of *Fusarium oxysporum* antagonistic strains is produced, dried and stored in the peat. The fungal spores did not lose viability for several years [110]. There are no universal recipes. An optimal stabilization technique should be developed for any fungal biocontrol agent.

It is well known that fungal growth and development are depend on temperature, free water availability, pH and oxygen concentration. For stabilization of the fungal propagules, these factors are manipulated by lowering pH, water activity, temperature and oxygen concentration [67, 68].

In many fungi, spores or spore matrix contains the inhibitors that prevent their germination in fruiting bodies, conidiomata, pustules even at the favorable wetness and temperature. These compounds isolated from some rust and anthracnose fungi were demonstrated to be fungistatic [111–115]. Probably, they can be used as natural preservatives and for stabilization of spores of biocontrol fungi.

Spores of many different fungi aggregated in conidiomata can survive over a season and longer under stress and varied environmental conditions including drying, UV-irradiation and low winter temperature. As a rule, such spores are pigmented or/and surrounded by thin shell (as teliospores of rust and smut fungi) or incorporated into spore matrix (as in coelomycetous fungi). Chemical analysis of the matrix in *Ascochyta* and *Phoma* spp. showed that it consists of pigments, glucose, polysaccharides, tyrosine and proteins [116, 117].

Protective compounds, such as pigments and compatible solutes, in fungal cells as well thickness of cell wall and plasma membrane lipid composition play important role in their resistance to artificial drying. Pigments, especially phenolic ones, utilize reactive oxygen species (ROS) which production is induced in drying process [28, 46]. Taking in account this consideration protective compounds are added to the biomaterial (at the concentration about 5–20%) before drying to prevent deleterious effects of ROS and to regulate osmotic pressure. Dried biomass should be stored at the darkness and lower oxygen concentrations. The rehydration is the important step too. It should be gradual and be made in wet atmosphere, warm water (30–37°C) in order to prevent the injury of fungal plasma membranes [46, 118].

#### **3.1. Biomass concentration and preservation**

The preparation of the concentrated suspensions or emulsions, pastes with addition of preservatives (germination inhibitors, antibiotics, etc.) is the simple techniques of stabilization and storage of fungal propagules, especially, if the it sensitive to drying.

#### *3.1.1. Concentrates*

A liquid formulation of the biofungicide was developed on the base of the yeast *Rhodotorula minuta* for biocontrol of mango anthracnose. The addition of glycerol (20%) and xanthan (5%) to the concentrated spore suspension (10<sup>9</sup> CFU/mL) prevented a preparation contamination and cell sedimentation. At the temperature 4°C, CFU number was decreased 100 times after 6 months of the storage. For stabilization of a bioinsecticide based on the mycelium of *Lagenidium giganteum* a concentrated emulsion was developed containing 40% of refined corn oil and 0.5% AEROSIL (Fumed Silica, R974). The latter prevented mycelium sedimentation and aggregation. This formulation can be stored under room conditions for 12 weeks without loss of efficacy against mosquitos [119].

Some components of emulsion concentrates (for instance, plant or paraffinic oils) affect efficacy of biopesticides including mycoherbicides. They prevent fast water evaporation from spray droplets and improve thermotolerance of fungal cells as it was shown for *Metarhizium anisopliae* s.l. (IP 46) and *Metarhizium robertsii* (ARSEF2575) [120]. Application of *Microsphaeropsis amaranthi* against the weed, *Microsphaeropsis amaranthi* in Sunspray 6E oil (10% v/v) resulted in improved disease impact under low moisture conditions [121].

#### *3.1.2. Pastes*

by concentration, drying or encapsulation of biomaterial on polymer layer and storage under appropriate conditions. In the ideal situation, the modern biopesticides can be stored not less than 2 years at the temperature 4°С, 3 months at 30°C and several days at 40–50°С [64].

There are quite simple and cheap techniques of stabilization and storage of some microorganisms. For instance, infection material of *Fusarium oxysporum* antagonistic strains is produced, dried and stored in the peat. The fungal spores did not lose viability for several years [110]. There are no universal recipes. An optimal stabilization technique should be developed for

It is well known that fungal growth and development are depend on temperature, free water availability, pH and oxygen concentration. For stabilization of the fungal propagules, these factors are manipulated by lowering pH, water activity, temperature and oxygen concentra-

In many fungi, spores or spore matrix contains the inhibitors that prevent their germination in fruiting bodies, conidiomata, pustules even at the favorable wetness and temperature. These compounds isolated from some rust and anthracnose fungi were demonstrated to be fungistatic [111–115]. Probably, they can be used as natural preservatives and for stabilization of

Spores of many different fungi aggregated in conidiomata can survive over a season and longer under stress and varied environmental conditions including drying, UV-irradiation and low winter temperature. As a rule, such spores are pigmented or/and surrounded by thin shell (as teliospores of rust and smut fungi) or incorporated into spore matrix (as in coelomycetous fungi). Chemical analysis of the matrix in *Ascochyta* and *Phoma* spp. showed that it

Protective compounds, such as pigments and compatible solutes, in fungal cells as well thickness of cell wall and plasma membrane lipid composition play important role in their resistance to artificial drying. Pigments, especially phenolic ones, utilize reactive oxygen species (ROS) which production is induced in drying process [28, 46]. Taking in account this consideration protective compounds are added to the biomaterial (at the concentration about 5–20%) before drying to prevent deleterious effects of ROS and to regulate osmotic pressure. Dried biomass should be stored at the darkness and lower oxygen concentrations. The rehydration is the important step too. It should be gradual and be made in wet atmosphere, warm water

The preparation of the concentrated suspensions or emulsions, pastes with addition of preservatives (germination inhibitors, antibiotics, etc.) is the simple techniques of stabilization and

A liquid formulation of the biofungicide was developed on the base of the yeast *Rhodotorula minuta* for biocontrol of mango anthracnose. The addition of glycerol (20%) and xanthan

consists of pigments, glucose, polysaccharides, tyrosine and proteins [116, 117].

(30–37°C) in order to prevent the injury of fungal plasma membranes [46, 118].

storage of fungal propagules, especially, if the it sensitive to drying.

**3.1. Biomass concentration and preservation**

*3.1.1. Concentrates*

any fungal biocontrol agent.

70 Biological Approaches for Controlling Weeds

spores of biocontrol fungi.

tion [67, 68].

The mycelium of *Trichoderma asperellum* GSS 03-35 produced by submerged liquid fermentation was stabilized by concentration to 6–10% of dry matter into paste containing corn starch (5%) as stiffener. The paste had pH 3 and contained copper sulfate (20 mg/L). During the course of storage, the fungus formed chlamydospores and conidia. After 6 months of storage at the temperature 20°C the fungus remained effective against head blight of wheat [122].

#### **3.2. Drying**

The drying is the most popular technique of inoculum stabilization. Besides simple drying by warm heat on trays (convection drying), spray drying, fluid bed drying and lyophilisation (freeze-drying) are used. The selection of the drying technique depends on availability, costs and sensitivity of the biomaterial.

#### *3.2.1. Convection drying*

The biomaterial mixed with preservatives and fillers is dried on trays in thin layer. This technique is used for production of the biofungicide *Trichodermin* (Biotechmash, the Ukraine) as a wettable powder. This simple technique can be applied for drying of low-scale amounts of the biomass and for preliminary experiments. Corn starch, rice flour, talc, diatomaceous earth and kaolin were evaluated as preservatives and fillers during drying of blastospores of *Beauveria bassiana*. Kaolin (at the concentration of 5% w/v of spore concentrate) allowed to maintain satisfactory viability of spores (≥50%) for 7 weeks storage at 4°C [123]. Conidia of *Stagonospora convolvuli* LA39 produced on V8 agar and dried with kaolin as a filler (1 g per 10<sup>9</sup> conidia) by air flow kept high viability (>70%) and pathogenicity about 5 months under the temperature 3°C. After 17 months of the storage just 5% of total conidia were viable, when the conidia were stored under the temperature 20°C conidia their viability decrease to 20% for a week [124].

In some inoculum stabilization protocols, convection drying was proposed for formulation of conidia and microsclerotia of *Beauveria*, *Metarhizium*, *Colletotrichum*, *Mycoleptodiscus* and *Trichoderma*. Some useful additives can be used to improve of viability of the infection units: skimmed milk or/and glycerol (ca. 1–2%, nutrient sources, humectants), clay (ca. 5%, kaolin or peat to protect conidia against UV) and plant oil (4–10%, adhesive) [126, 127].

**3.3. Encapsulation**

*3.3.1. Alginate granules*

*3.3.2. Microencapsulation*

*sorokiniana* [10–141].

kaolin can be effectively replaced by corn flour [136].

Concentrated biomaterial can be incorporated into different polymer matrices that protect fungal cells from effects of some factors such as UV-irradiation and microbial contamination. Products that are resulted from encapsulation process include gel, granules, capsules and

Production and Stabilization of Mycoherbicides http://dx.doi.org/10.5772/intechopen.76936 73

The process is based on the polymerization of sodium alginate in the solution calcium chloride. For instance, the suspension of the biomaterial (1 part) is mixed with sodium alginate (1.3% solution, 4 parts) and kaolin (5% of total weight); the mixture is dropped into 0.25 M solution of calcium chloride; the resulted granules are filtered and dried. The technique was used for the first time to formulate conidia of *Alternaria cassiae* [135]. Intensity of the fungal sporulation on the granules depended on inoculum production method, fillers and adjuvants;

Various compositions of alginate granules were evaluated for many potential and commercial biopesticides. Chitin (2% of granules weight) together with wheat bran (2%) significantly increased spore production of *Beauveria bassiana* on the granules [137]. Starch addition accelerated the rupture of granules and colonization of the peat substrate by *Trichoderma* sp. [138]. For field experiments of biofungicide based on the non-toxigenic *Aspergillus flavus* different adjuvants (1% of granules weight) and fungicides (0.5–1.25 mg per 50 g of the mixture of sodium alginate and 2.5 g of corn flour) were evaluated. Triptone and peptone addition significantly stimulated spore production of the fungus on the granules. The fungicides did not inhibited the antagonist development and preserve the granules against contamination [136]. Composition of alginate formulation of *Trichoderma* sp. conidia optimized by factorial design experiments included glycerol (2% w/v), sodium polyphosphate 2% (w/v) and citrus pectin that allows to maintain the satisfactory titer of conidia for 14 weeks at 28°C. Formulation quality was monitored by Fourier-transform infrared spectroscopy and some chemical interactions between polymers were found [138]. For production of complex mycoinsecticides ("attract and kill") based on *Saccharomyces cerevisiae* (used as an attractant for wireworms) and *Metarhizium brunneum* (as an insect killer), the technical scale technology was developed that included jet cutting of droplets and bed drying of granules at 40–50°C till aw 0.1–0.2 [139].

Fungal biomaterial (e.g. conidia and mycelia) suspended in sodium alginate solution or in the mixture of agar-agar (1%) and gelatin (1:1, v/v) is emulsified in corn oil with n-hexadecan (6:4) and lecithin as emulsifier. Gelatin-agar globules were gelated in the emulsion while alginate microcapsules were polymerized when dropped into calcium chloride solution. The size of microcapsules varied from 10 to 400 μm depending on ratio of the mentioned components. The microcapsules were separated from the liquids by vacuum filtration and used by spraying. The microencapsulation technique was successfully used in model experiments for development of artificial conidia based on conidia of *Fusarium avenaceum* and mycelium of *Bipolaris* 

microcapsules. There are various industrial equipment for their production [134].

The drying technique "Stareze" is based on the addition of a membrane stabilizer (sucrose) during the fermentation. High concentration of sucrose (400 g/L) was added to 96-h submerged culture of *Metarhizium anisopliae*. The fermentation was stopped after 168 h and a filler (silica, HiSil™-233, 35 g/L) was added. The filtered product was dried on the trays at ambient temperature. The blastospores of *M. anisopliae* stayed alive for 6 months at 2–4°C [128].

## *3.2.2. Spray drying*

The spore suspension with some adjuvants and additives is sprayed in heated air followed by fast drying (5–30 s). In the case of fluid bed drying, the suspension follows to the bed from dried material babbling by air that forms pseudo-boiling layer. Particles of the drying material stick to gradually form granules (www.niroinc.com).

Submerged conidia of *M. anisopliae* mixed with defatted milk (20%) and sucrose (2.5%) survived better spray drying than freeze-drying process. However, inlet and outlet temperature caused significant effect on their viability [129]. Granules of the commercial biofungicide Contans® are produced by drying conidia of *Coniothyrium minitans* in glucose solution in a spray drier; the product contains about 95% of glucose and 5% of conidia (ca. 1 × 1013 conidia/ kg) and remains effective for 2 years when stored at 4°C [96]. In some cases, this technique of drying is not appropriate. Conidia of the epiphytic fungus, *Epicoccum nigrum*, produced by solid-state fermentation lost viability after spray drying at inlet temperature 150°C. However, fluid bed drying was favorable: dried at 30–40°С conidia remained viable even without any preservative and can be stored for 90 days and more [130].

A method was developed for microencapsulation of *Trichoderma* conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10<sup>10</sup> CFU in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31°C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of *T. harzianum* was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations [131].

## *3.2.3. Freeze-drying*

Under liophylisation, water vapors from ice under low pressure bypass the liquid state. Conidia of *Septoria passiflorae* survived well after freeze-drying when 10% of skimmed milk was added to the conidial suspension. The fungus stayed viable for >1 year when stored in a vacuum package at 1°C [132]. Blastospores of *Paecilomyces fumosoroseus* together with protectors (10% lactose + 1% bovine albumin, or composition of starch, vegetable oil, sucrose and milk) remain viable after freeze-drying at the level 75% for 50 weeks at −20°C, while at 4°C their viability decreased to the level of 10% [133].

#### **3.3. Encapsulation**

skimmed milk or/and glycerol (ca. 1–2%, nutrient sources, humectants), clay (ca. 5%, kaolin or

The drying technique "Stareze" is based on the addition of a membrane stabilizer (sucrose) during the fermentation. High concentration of sucrose (400 g/L) was added to 96-h submerged culture of *Metarhizium anisopliae*. The fermentation was stopped after 168 h and a filler (silica, HiSil™-233, 35 g/L) was added. The filtered product was dried on the trays at ambient temperature. The blastospores of *M. anisopliae* stayed alive for 6 months at 2–4°C [128].

The spore suspension with some adjuvants and additives is sprayed in heated air followed by fast drying (5–30 s). In the case of fluid bed drying, the suspension follows to the bed from dried material babbling by air that forms pseudo-boiling layer. Particles of the drying mate-

Submerged conidia of *M. anisopliae* mixed with defatted milk (20%) and sucrose (2.5%) survived better spray drying than freeze-drying process. However, inlet and outlet temperature caused significant effect on their viability [129]. Granules of the commercial biofungicide Contans® are produced by drying conidia of *Coniothyrium minitans* in glucose solution in a spray drier; the product contains about 95% of glucose and 5% of conidia (ca. 1 × 1013 conidia/ kg) and remains effective for 2 years when stored at 4°C [96]. In some cases, this technique of drying is not appropriate. Conidia of the epiphytic fungus, *Epicoccum nigrum*, produced by solid-state fermentation lost viability after spray drying at inlet temperature 150°C. However, fluid bed drying was favorable: dried at 30–40°С conidia remained viable even without any

A method was developed for microencapsulation of *Trichoderma* conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10<sup>10</sup> CFU in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31°C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of *T. harzianum* was a flow-able powder with over 99% conidia, which could be used in a variety

Under liophylisation, water vapors from ice under low pressure bypass the liquid state. Conidia of *Septoria passiflorae* survived well after freeze-drying when 10% of skimmed milk was added to the conidial suspension. The fungus stayed viable for >1 year when stored in a vacuum package at 1°C [132]. Blastospores of *Paecilomyces fumosoroseus* together with protectors (10% lactose + 1% bovine albumin, or composition of starch, vegetable oil, sucrose and milk) remain viable after freeze-drying at the level 75% for 50 weeks at −20°C, while at 4°C

of formulation developments from seed coatings to sprayable formulations [131].

peat to protect conidia against UV) and plant oil (4–10%, adhesive) [126, 127].

rial stick to gradually form granules (www.niroinc.com).

preservative and can be stored for 90 days and more [130].

their viability decreased to the level of 10% [133].

*3.2.2. Spray drying*

72 Biological Approaches for Controlling Weeds

*3.2.3. Freeze-drying*

Concentrated biomaterial can be incorporated into different polymer matrices that protect fungal cells from effects of some factors such as UV-irradiation and microbial contamination. Products that are resulted from encapsulation process include gel, granules, capsules and microcapsules. There are various industrial equipment for their production [134].

#### *3.3.1. Alginate granules*

The process is based on the polymerization of sodium alginate in the solution calcium chloride. For instance, the suspension of the biomaterial (1 part) is mixed with sodium alginate (1.3% solution, 4 parts) and kaolin (5% of total weight); the mixture is dropped into 0.25 M solution of calcium chloride; the resulted granules are filtered and dried. The technique was used for the first time to formulate conidia of *Alternaria cassiae* [135]. Intensity of the fungal sporulation on the granules depended on inoculum production method, fillers and adjuvants; kaolin can be effectively replaced by corn flour [136].

Various compositions of alginate granules were evaluated for many potential and commercial biopesticides. Chitin (2% of granules weight) together with wheat bran (2%) significantly increased spore production of *Beauveria bassiana* on the granules [137]. Starch addition accelerated the rupture of granules and colonization of the peat substrate by *Trichoderma* sp. [138]. For field experiments of biofungicide based on the non-toxigenic *Aspergillus flavus* different adjuvants (1% of granules weight) and fungicides (0.5–1.25 mg per 50 g of the mixture of sodium alginate and 2.5 g of corn flour) were evaluated. Triptone and peptone addition significantly stimulated spore production of the fungus on the granules. The fungicides did not inhibited the antagonist development and preserve the granules against contamination [136].

Composition of alginate formulation of *Trichoderma* sp. conidia optimized by factorial design experiments included glycerol (2% w/v), sodium polyphosphate 2% (w/v) and citrus pectin that allows to maintain the satisfactory titer of conidia for 14 weeks at 28°C. Formulation quality was monitored by Fourier-transform infrared spectroscopy and some chemical interactions between polymers were found [138]. For production of complex mycoinsecticides ("attract and kill") based on *Saccharomyces cerevisiae* (used as an attractant for wireworms) and *Metarhizium brunneum* (as an insect killer), the technical scale technology was developed that included jet cutting of droplets and bed drying of granules at 40–50°C till aw 0.1–0.2 [139].

#### *3.3.2. Microencapsulation*

Fungal biomaterial (e.g. conidia and mycelia) suspended in sodium alginate solution or in the mixture of agar-agar (1%) and gelatin (1:1, v/v) is emulsified in corn oil with n-hexadecan (6:4) and lecithin as emulsifier. Gelatin-agar globules were gelated in the emulsion while alginate microcapsules were polymerized when dropped into calcium chloride solution. The size of microcapsules varied from 10 to 400 μm depending on ratio of the mentioned components. The microcapsules were separated from the liquids by vacuum filtration and used by spraying. The microencapsulation technique was successfully used in model experiments for development of artificial conidia based on conidia of *Fusarium avenaceum* and mycelium of *Bipolaris sorokiniana* [10–141].

#### *3.3.3. "Pesta" granules*

The production of Pesta granules is based on the technology of pasta production. Inoculum suspension (52 mL), wheat semolina flour (80 g) and kaolin (20 g) are mixed to produce dough. The dough is passed through a pasta maker after that it is dried, crashed and sieved. The technique was tried for encapsulation of conidia of potential mycoherbicides (*Alternaria cassiae*, *A. crassa*, *Colletotrichum truncatum* and *Fusarium lateritium*) as well for stabilization of entomopathogenic nematodes [142, 143]. Melanized fungal structures as pigmented conidia, chlamydospores, microsclerotia and sclerotia granules are generally compatible to Pesta process while non-pigmented conidia of *F. oxysporum*, *C. truncatum*, *Trematophoma lignicola* were not viable in the final product [49, 57, 143–145].

crashed or milled. This technique was successfully used for potential bioherbicides based on *Fusarium oxysporum* (microconidia and mycelium) and *Pseudomonas* spp. that remain viable for a long time [152–154]. However, submerged conidia of *Metarhizium anisopliae* (the producer of the bioinsecticide Green Muscle™) survived better when the above-mentioned process Satreze

Production and Stabilization of Mycoherbicides http://dx.doi.org/10.5772/intechopen.76936 75

The safety and evaluation of postponed risks of mycopesticides are still under question. An agroecosystem is inundated by a fungus at very high concentrations and there is a risk of the crop injury. Some plant pathogens can survive in the soil or plant debris. They are able of producing biologically active compounds (mycotoxins, antibiotics, phytotoxins, etc.). The number of safety research on the safety of mycoherbicides is limited to *Sclerotinia sclerotiorum*, *S. minor*, *Colletotrichum coccodes*, *Fusarium oxysporum* f. sp. *strigae*, *Phoma macrostoma* and *Stagonospora convolvuli* [7, 23, 55, 155–159]. The experience of field observations is limited to

Molecular marking of biocontrol strains is an approach for their post-application tracking and quantification. For instance, the strain *Fusarium oxysporum* f. sp. *strigae* F2, which is potential mycoherbicide against *Striga* spp., was compared with several strains *F. oxysporum* using fluorescent AFLP. Based on this comparison a specific PCR primer was developed for making F2

In conclusion, the approaches for stabilization and storage of biopesticides based on fungal propagules were discussed in this review. In order to produce both virulent and stress tolerant propagules for mycoherbicides based on the submerged fungal mycelium as well as on conidia, chlamydospores and microsclerotia a liquid medium should be optimized. The construction of bioreactors, in particular, for solid-state fermentation is continuously being improved that allows of producing highly stress tolerant fungal aerial conidia. Various recipes for liquid (e.g. suspension and emulsion concentrates) and solid (like alginate and stabilize granules) formulation of mycoherbicides were developed to be stored for a long time and effectively used. However, the efficacy of mycoherbicides is still unstable and their safety is

The research was supported by Russian Science Foundation (project # 16-16-00085).

was used [128].

several years.

only in the soil [20, 21, 160].

**Acknowledgements**

**Author details**

not proved clearly to be widely commercialized.

Alexander Berestetskiy\* and Sofia Sokornova

\*Address all correspondence to: aberestetskiy@vizr.spb.ru

All-Russian Institute of Plant Protection, Saint-Petersburg, Russia

Microslecrotia of *C. truncatum* survived in Pesta granules and remained to produce virulent conidia (for biocontrol of weed *Sesbania exaltata*) for 52 weeks at 25°С low water activity (aw 0.18–0.29), and for 10 years at 4°С [57, 146] while the fungal conidia can be stored no more than 32 weeks [147]. Interestingly, that during the process of encapsulation of *Alternaria alternata* conidia with Pesta process, the number of colony forming units increased due to destroying their aggregations. The virulence of the fungus was stable at a low relative air humidity (12%) for more than 2 years [145].

The composition of Pesta granules can be easily modified. Shabana et al. [148] evaluated various compositions for *Fusarium oxysporum* f. sp. *orthoceras* using 3% (w/w) sucrose, corn flour, glycerol, starch WaterLock B209, cellulose and yeast extract. The last component improved viability of chlamydospores as well as of microconidia in the granules. However, the prepared samples showed appropriate viability (60–80% for 12 months) under 25°C and relative humidity 11–12%; under higher temperature (25°C) and humidity (51–53%) viability of the fungus dramatically decreased by the 4–8th month of storage [148]. The biocontrol efficacy of *Aspergillus alliaceus* against parasitic weeds (*Orobanche* spp.) incorporated in Pesta granules was improved by addition of potato broth or sorghum meal [149].

For encapsulation of conidia of potential mycopesticides (*C. truncatum*, *Alternaria* sp., *Paecilomyces fumoroseus*, *Aspergillius flavus*, *A. parasiticus*) produced by solid and liquid state fermentation the twin-screw extrusion was successfully tested. Ingredients were mixed in the mixer of an extruder and resulted Pesta granules were dried by fluid bed drying at 50°С. The inoculum produced by solid-state fermentation was shown to be less sensitive to whole the stabilization process than the biomaterial from the liquid culture [150].

#### *3.3.4. Stabilize granules*

The main components of these granules are a membrane stabilizer (for instance, sucrose at the concentration 10–65% from granules weight), a water absorbance agent (starch), a filler (diathomaceous earth, silica Hi-Sil® at the concentration 5–20%). Additionally, the granules can include vegetable oil (ca. 20%), UV-protectant, preservatives and other inert fillers [151]. For example, sucrose (4 parts), starch (1 part), unrefined vegetable oil (1 part), silica gel (1.5 parts) and biological suspension (4 parts) are mixed and extruded; the resulted pasta is conventionally dried and crashed or milled. This technique was successfully used for potential bioherbicides based on *Fusarium oxysporum* (microconidia and mycelium) and *Pseudomonas* spp. that remain viable for a long time [152–154]. However, submerged conidia of *Metarhizium anisopliae* (the producer of the bioinsecticide Green Muscle™) survived better when the above-mentioned process Satreze was used [128].

The safety and evaluation of postponed risks of mycopesticides are still under question. An agroecosystem is inundated by a fungus at very high concentrations and there is a risk of the crop injury. Some plant pathogens can survive in the soil or plant debris. They are able of producing biologically active compounds (mycotoxins, antibiotics, phytotoxins, etc.). The number of safety research on the safety of mycoherbicides is limited to *Sclerotinia sclerotiorum*, *S. minor*, *Colletotrichum coccodes*, *Fusarium oxysporum* f. sp. *strigae*, *Phoma macrostoma* and *Stagonospora convolvuli* [7, 23, 55, 155–159]. The experience of field observations is limited to several years.

Molecular marking of biocontrol strains is an approach for their post-application tracking and quantification. For instance, the strain *Fusarium oxysporum* f. sp. *strigae* F2, which is potential mycoherbicide against *Striga* spp., was compared with several strains *F. oxysporum* using fluorescent AFLP. Based on this comparison a specific PCR primer was developed for making F2 only in the soil [20, 21, 160].

In conclusion, the approaches for stabilization and storage of biopesticides based on fungal propagules were discussed in this review. In order to produce both virulent and stress tolerant propagules for mycoherbicides based on the submerged fungal mycelium as well as on conidia, chlamydospores and microsclerotia a liquid medium should be optimized. The construction of bioreactors, in particular, for solid-state fermentation is continuously being improved that allows of producing highly stress tolerant fungal aerial conidia. Various recipes for liquid (e.g. suspension and emulsion concentrates) and solid (like alginate and stabilize granules) formulation of mycoherbicides were developed to be stored for a long time and effectively used. However, the efficacy of mycoherbicides is still unstable and their safety is not proved clearly to be widely commercialized.
