Despite the urgent need for alternatives to chemicals in plant protection, biological herbicides are not widely used as biofungicides and bioinsecticides. The review is devoted to connections between fungal biology, biochemistry, their ability to survive in extreme environment and development of effective mycoherbicides. Advanced studies on the production and stabilization of mycofungicides and mycoinsecticides were analyzed too in order to obtain ideas for the improvement of efficacy and technology of mycoherbicides in the future. The analysis of research data published within last 20 years showed following trends. First, more attention is paid for production both effective and stress tolerant propagules especially based on the submerged fungal mycelium and its modifications (blastospores, chlamydospores and microsclerotia). Second, the construction of bioreactors, in particular, for solid-state fermentation is continuously being improved that allows producing highly stress tolerant fungal aerial conidia. Third, based on studies of biochemical mechanisms of viability of fungi in extreme environment, the approaches of stabilization and storage of fungal propagules were developed. However, the positive reply to the question, if biopesticides including mycoherbicides, will become a serious alternative to agrochemicals, will be possible when they demonstrate stable efficacy in the field conditions and safety for both environment and end users.
- stress tolerance
With gradual increase of restrictions for use of chemical pesticides, the role of natural regulators of pest organisms including weeds and invasive plants will grow up. The development of weed biocontrol is stimulated due to their increasing resistance to chemical herbicides and slow down development of novel herbicidal active components with new mode of action . There are a few mycoherbicides among biopesticides registered in the last years .
Despite biocontrol efficacy is generally lower than application of pesticides, biologicals have some advantages over chemicals: (1) biopesticides can be used for resistance management, especially since may have multiple modes of action, which would reduce the chance of resistance occurring in a particular crop pest; (2) many biopesticides have no or low restricted entry intervals, meaning that post-application, restricted entry into the field is very low and there are often no limitations prior to harvest and (3) there are generally exemptions of biopesticides from maximum residue limits because they are considered acceptable and relatively safe .
More than half a century had passed since the first mycoherbicide was registered. Dispute raged, and still rages today, about whether “Have bioherbicides come of age?”, “What is they really contribution to crop protection?” or “Athletes foot or Achilles heel?” [4, 5, 6, 7, 8, 9]. This is partly because the biological herbicides as distinct from chemical preparations are not “stand alone” products. There are significant differences in their origins (biological vs. chemical), modes of action (multiple vs. singular), manufacturing methods (fermentation vs. synthesis), requirements to storing and application conditions, etc. . Efficacy of mycoherbicide strategy depends on thorough understanding of host-pathogen-environment interactions. The biological herbicides are more effective when they are incorporated into integrated weed management programs . For example, it was demonstrated that the bioherbicide
Currently, it highlighted 18 of the most serious weeds in agriculture and 50 troublesome ones in cultivated crops, pastures and waterways . Mycoherbicides are mainly used to prevent and control the spread of such worst parasitic weeds as
Key features of mycoherbicides are host specificity, crop tolerance, efficacy, environmental fate, temperature and moisture spectrum, mode of action and toxicology . It is important to realize that not only the choice of the strain, but also types of propagules (conidia, mycelium, sclerotia, etc.), production and application method is influenced by mycoherbicide features. Fungal propagules are influenced by a number of environmental factors (temperature, humidity etc.) that affect their biocontrol efficacy. It was demonstrated that the propagules’ choice, formulation and application strategy potentially reduce the dew period requirement [24, 25]. Another possible approach would be a manipulation with fermentation conditions up to product infection materials with set-up parameters [7, 26]. Similarly, during fungal growth physical, chemical and nutritional conditions can be altered to manipulate endogenous reserves for production of propagules with improved stress tolerance to abiotic factors and virulence to host [7, 27, 28, 29]. Depending on production method conidia significantly differ by the content of compatible solutes and resistance to environmental influences. The maximum difference is observed when comparing conidia obtained on artificial nutrient media and in nature [28, 30].
Despite of considerable progress in technologies of production and application of mycoherbicides, biopesticides for control of phytophagous insects and plant pathogens have showed much higher commercial success. In some cases, the useful experience for development commercially viable mycoinsecticides and mycofungicides can be tested for the improvement of potential mycoherbicides. For this reason, in this review we analyzed the approaches for producing both mycoherbicides and other types of biopesticides based on fungi.
2.1. Choice of propagule types
Various kinds of fungal propagules often fulfill different purposes. In nature, the typical infectious propagules of the pathogenic Ascomycetes are the aerial conidia that facilitate distribution and spreading of these fungi. Generally, aerial conidia can be cost-effectively produced under laboratory conditions . Blastospores, submerged (microcycle) conidia, sporogenically competent mycelia and microsclerotia may be used as the infectious agents as well. They often have a higher survival capability as well as the increased genetic diversity, which probably enhances survival in unstable environments [32, 33]. The morphological and physiological features of submerged conidia can significantly differ from properties of aerial conidia produced by a solid-state culture. For example, submerged conidia and blastospores of
However, a few of successful field experiments with microsclerotia-based mycoinsecticide were described. The field efficacy of solid and liquid formulations of microslecrotia
2.2. Mass production of mycoherbicide propagules
High spore density (about 1012–1014 CFU per ha) is required for use of mycoherbicides in the field. Therefore, one of the main technological goals is to obtain cost-effective, viable and aggressive infectious material [3, 26]. The secondary use of substrates is a solution of their decontamination and utilization. For example, multi-step waste wood bio-recycling includes the cultivation of
The loss of viability of the infectious material is usually observed during its drying and storage. Moreover, in nature, the combination of temperature and humidity optimal for rapid germination of fungal spores is relatively rare. Germination of spores can be also suppressed by the action of solar irradiation. Thus, the techniques and conditions for cultivation of biocontrol fungi and the selection of the nutrient media composition should be directed both to reach high biomass yields and to improve their activity in the field [63, 64].
There are several approaches to improve fitness of biocontrol fungi: strain selection, optimization of media composition, addition protectors (compatible solutes such as trehalose, sucrose, glycine-betaine, etc.) and treatment of growing cultures with sub-lethal doses of stress factors (e.g. oxidative stress and temperature) [26, 65, 66]. However, on the practice sub-optimal water activity of the substrates are widely used and helpful [67, 68].
Propagules can be produced by solid-state and liquid fermentation or two-phase system.
2.2.1. Liquid submerged fermentation (LSF)
LSF is the most commonly used technology for microbial inoculum production. Collego and DeVine, the first commercially produced bioherbicides, had been manufactured this way. The ability to fully control the cultivation process and its relatively short duration (several days) is an undoubted advantage of LSF over solid-state fermentation. The composition of a culture medium is an important parameter in the biotechnological process because it is 30–40% of the production costs. A commercial LSF medium for
To obtain a high yield of viable and stress tolerant infectious material, the composition of the liquid nutrient medium requires optimization. Its algorithm can include three main steps: (1) selection of the basal medium with a set of vitamins and trace elements, on which the fungus grows and/or sporulate well; (2) selection of carbon and nitrogen sources and their optimal concentration and ratio determination and (3) replacement of artificial carbon and nitrogen sources by cheap natural ones . Application of factorial design and response surface methods were successfully used to optimize the growth parameters required for large scale conidia production of potential mycoherbicides based on
To obtain high titers of
The liquid nutrient medium tonicity has a significant effect on the yield and quality of propagules. Sporulation of
Non-optimal carbon sources also stimulated
At the same time, liquid substrates are uncommon one for fungal growth.
2.2.2. Solid-state fermentation (SSF)
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 . In the most cases, spore yields and viability are higher than they are produced by SSF . Hydrophobic air conidia are best suitable for oil formulations, since prolong the conidial viability and decreases UV radiation sensitivity [84, 85, 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 . 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 factors found in open fields .
A polysaccharide matrix often surrounds the spores produced by SSF and protects them during desiccation opposite the spores produced by LSF . The choice of substrate, its humidity and growing time also affect the quality of propagules . For example, dried conidia of Colletotrichum truncatum produced on vermiculite tended to retain efficacy during storage better than spores recovered from perlite culture . 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
In the case of small manufacturers, the propagules traditionally produced in the plastic bottle or perforated polypropylene carrier bags [95, 96]. This process was the first designed to meet the biological requirements of genus
It is well known the positive effect of near ultraviolet radiation on sporulation of certain phytopathogenic fungi from genera
Naturally occurring substances can be applied for bioherbicide production [106, 107]. SSF allows to obtain bioherbicides utilizing the agroindustry waste such as bagasse, soybean bran and corn steep liquor .
3. Stabilization of fungal propagules
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
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 .
There are quite simple and cheap techniques of stabilization and storage of some microorganisms. For instance, infection material of
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, 112, 113, 114, 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
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.
A liquid formulation of the biofungicide was developed on the base of the yeast
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
The mycelium of
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
In some inoculum stabilization protocols, convection drying was proposed for formulation of conidia and microsclerotia of
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
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
A method was developed for microencapsulation of
Under liophylisation, water vapors from ice under low pressure bypass the liquid state. Conidia of
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 .
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
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
Composition of alginate formulation of
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
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 (
The composition of Pesta granules can be easily modified. Shabana et al.  evaluated various compositions for
For encapsulation of conidia of potential mycopesticides (
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 . 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
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
Molecular marking of biocontrol strains is an approach for their post-application tracking and quantification. For instance, the strain
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.
The research was supported by Russian Science Foundation (project # 16-16-00085).