Key messages

  • Current and novel fungi-based biopesticide applications for crop pest control are critically described.

  • The overview identified no reports on formulation approaches retaining efficacy and improving shelf life.

  • Current research and approaches to product formulations are critically synthesized.

  • Innovative formulations, emerging pest control technologies, and challenges are critically presented.

Introduction

Abiotic and biotic factors can limit the agro-industry yields needed to succeed commercially and, most importantly, to meet the growing worldwide needs for food, fiber, and renewable resources. Abiotic factors refer to environmental conditions such as temperature, water availability, and soil nutrient content which are strongly influenced by geographic location and season (Basilico et al. 2007). Biotic factors refer almost exclusively to pests such as weeds, insects, and pathogenic microorganisms, which negatively affect plants and farm animals. Under field conditions, crops are affected by a combination of abiotic and biotic factors. Abiotic factors such as drought and high temperature are not only detrimental to agricultural productivity, but also significantly influence the occurrence and spread of phytopathogens and weeds (Haile 2000; Pandey et al. 2017). Projections for the year 2050 indicate that crop yield will need to be doubled to cover worldwide demands (Ray et al. 2013; Technavio 2015, 2017). Land clearing is no longer an option to increase crop production due to detrimental CO2 emission effects. Crop protection improvement, particularly reduction in crop losses due to pest and diseases, is a more viable option (Oerke and Dehne 2004; Technavio 2015; Tilman et al. 2011; Villaverde et al. 2016).

Currently, chemical pesticides (CPs) represent about 95% of the world pesticides market and prevent an estimated 50% crop loss (BBC Research 2012; Marrone 2014; Mishra et al. 2015; Technavio 2015). These overall crop loss rates have not significantly diminished over the past four decades (Jallow et al. 2017). Instead, a rising trend has been observed (Gasic and Tanovic 2013). These findings and reports of potential undesirable effects of CPs on natural resources, and on animal and human health (Benuszak et al. 2017; Bro-Rasmussen 1996; Heard et al. 2017; Schade and Heinzow 1998; Voldner and Li 1995), are driving a search for alternatives to CPs. Among the biological control strategies under development, most important are biopesticides (BPs), i.e., formulations based on biological control agents (BCAs) such as bacteria, fungi, viruses, and their combinations (Saito and Brownbridge 2018; Syed Ab Rahman et al. 2018). The frequent use of fungi in BPs reflects their role in nature as balancers of the populations of aphids, ticks, and other insects, and many other pests affecting plants and animals (Becher et al. 2018; Jaber and Ownley 2018). Complex pest control mechanisms make unlikely the development of pest resistance to fungi-based BPs (Fernandes et al. 2012; Jackson et al. 2010; Khan et al. 2012). These mechanisms include hyperparasitism, substrate competition, production of antibiotics and lytic enzymes, and secretion of substances reinforcing the host immune system or promoting growth (Junaid et al. 2013). For example, when entomopathogenic fungi contact and adhere to the insect cuticle, their spores germinate, and then assisted by the secretion of lytic enzymes, the germ tube penetrates the cuticle (24–48 h). Fungi grow inside the insect, finally taking over its hosts’ body completely (5–7 days). Once infected, the insect body supports the development of conidia which eventually spread new spores reaching new hosts (Fernandes et al. 2012; Pinnamaneni and Potineni 2010; Schrank and Vainstein 2010; Shah and Pell 2012). Biocontrol of pathogenic fungi by Trichoderma sp. relies on many of the above mechanisms (Harman 2006). In addition, early plant exposure to Trichoderma sp. enhances its immunological response against pathogens (Howell 2003), while long-term applications act even as a plant growth stimulant (Singh and Nautiyal 2012), enhancing root growth and development, conferring tolerance to abiotic stresses, and improving uptake and use efficiency of nutrients, and thus increasing crop productivity (Mehetre and Mukherjee 2015). High pathogen specificity (Glare et al. 2016), infrequent pest resistance development (Leng et al. 2011a), suitability for use in integrated pest management (IPM) schemes (Mishra et al. 2015), rapid biodegradation of residues (Glare et al. 2016), and minor or no health risk to workers (Koul 2011) are key BP advantages. Biocontrol efficacy comparisons of BPs have yielded similar or better results than CPs, making them an attractive option in conventional and organic farming (Crozier et al. 2015; Jayaraj et al. 2006; Lopes et al. 2011; Marcic et al. 2012; Meena et al. 2014; Thakore 2006).

Although a comprehensive overview of microbial formulations including those containing fungi was recently presented by Keswani et al. (2016), a summarized assessment of the factors affecting the shelf life of fungi-based BPs is currently unavailable. Formulation methodologies used to preserve functionality during storage and distribution have not been fully overviewed (Sarwar 2015). The main problem of BPs with short shelf life is not only the reduction in their efficiency against the target pest, but also the impact on their market competitiveness. CPs can be stored for a longer time without significant changes in their activity against pests. Thus, this overview focuses on storage conditions and formulation approaches aiming at enhancing the efficacy and shelf life of fungi-based BPs. Current approaches to product formulations and their advantages and limitations are included. Finally, emerging technologies for pest control and current BP challenges and opportunities are also critically presented.

Fungi-based biopesticides

Commercial BPs are most frequently based on Beauveria, Metarhizium, and Trichoderma strains (Agosin and Aguilera 1998; Muñiz-Paredes et al. 2017; Nascimento et al. 2017; Ríos-Moreno et al. 2018; Rossoni et al. 2014; Woo et al. 2014). For example, Trichoderma strains are reported as biocontrol for the damping-off disease caused by Rhizoctonia solani (Kakvan et al. 2013) and Fusarium (Javanshir Javid et al. 2016). Even though Khan et al. (2012) showed that most formulated and unformulated B. bassiana BPs achieve target pest control, only about half of the publications cited included field efficacy tests. This is significant because studies conducted by Jandricic et al. (2014) on commercial and non-commercial strains, primarily fungi from the genera Beauveria, Metarhizium, and Isaria, identified disparities in the efficacy claimed, particularly in the case of commercial products.

BP formulations, adjuvants, and application methods

BP formulations are classified into dry and liquid formulations with examples listed in Table 1. Dry formulations for direct application include powders, powders for seed dressing (DS), granules (G), microgranules (MG), water-dispersible granules (WDG), and wettable powders (WP). Liquid formulations include emulsifiable concentrates (EC), suspension concentrates (SC), oil dispersions (OD), suspo-emulsions (SE), and capsule suspensions (CS).

Table 1 Reported efficacy and shelf life of different fungi-based formulations
Full size table

Formulation designs and application methodologies directly affect the efficacy, consistency, shelf life, and virulence of commercial fungi-based BPs (Jackson et al. 2010; Leggett et al. 2011). For instance, formulations applied as sprays are generally expected to exert an immediate effect on plant pests and infections; thus, adjuvants such as surfactants are added to enhance adherence to the treated plant or insect surface (Rumbos and Athanassiou 2017). Since granular formulations usually applied to soils are expected to generate antagonistic reactions over an extended time, stabilizers and nutrient sources are included too. Finally, all fungi-based BP adjuvants, including oils, surfactants, stabilizers, solvents, and hygroscopic control agents improving desiccation resistance, spreading capability, and plant intake, must not interfere with BP effectiveness and in some cases they have a synergistic effect improving the biocontrol performance (Keswani et al. 2016).

BP shelf life and application effectiveness

Shelf life is a key concern in the design and formulation of fungi-based BPs (Jackson et al. 2010). Faria et al. (2012) suggested that preservation methodologies should aim at improving shelf life under worst-case scenario conditions such as tropical weather and long transportation times at 30 to 50 °C. Given their stability during storage and transportation, WP followed by WDG formulations are therefore preferred. Many authors have recommended challenging fungi-based BPs under field rather than laboratory conditions to provide more accurate information on formulation efficacy (Junaid et al. 2013; Wilson and Jackson 2013). Unfortunately, few studies include field formulation and efficacy tests after storage (John and Jeeva 2014; Mascarin et al. 2016; Navaneetha et al. 2015). The approaches used to extend the shelf life of fungi-based BPs include optimal nutrient content designs, oxygen and moisture content control by vacuum and active packaging, use of humidity and oxygen scavengers, and application of optimized drying technologies (Mascarin and Jaronski 2016). Since fungi strains differ in preservation needs, screening for optimal conditions is always required. Moreover, conidia produced by liquid fermentations are more prone to suffer desiccation viability losses than those obtained by solid cultures (Mascarin and Jaronski 2016).

Table 1, summarizing the effectiveness evidences for the most used BPs, shows that several Trichoderma species appear in marketable products. Among them, T. harzianum, followed by T. atroviridae, T. asperellum, T. gamsii, and T. polysporum, are species most frequently reported as the active biocontrol agent. Five products from three different companies include T. harzianum T22, evidencing the frequent use of this strain in BPs. Plant decay caused by fungi pests such as Pythium, Rhizoctonia, Fusarium, Sclerotinia, Cylindrocladium, Armillaria, Phytophthora, Alternaria, Botrytis, Verticillium, Rosellina, and Thielaviopsis, are controlled by the Trichoderma strains listed above. Its protection targets include fruits, vegetables, legumes, cereals, ornamental plants, grass, herbs, spices, trees, and landscape plants. Beauveria bassiana is an entomopathogenic fungus against pest insects with high demand among food producers controlling and killing whiteflies, aphids, weevils, thrips, mites, moths, stinkbugs, tingids, mealybugs, and specifically against Bemisia tabaci, Hypothenemus hampei, Tetranychus urticae, and Gonipterus scutellatus. Although this entomopathogenic fungus has been recommended for use in vegetables, fruits, legumes, cucurbits, herbs, spices, and ornamental flowers production, it could be applied to control the same pests in other crops. Other entomopathogenic fungi such as Metarhizium anisopliae and Isaria fumosorosea are also frequently listed as active ingredients in BPs protecting vegetables, fruits, cereals, spices, ornamentals, and flowers against damage caused by whiteflies, aphids, thrips, termites, ants, beetles, caterpillars, leaf miners, mealybugs, psyllids, root grub, locusts, root weevils, and rootworms. The principal fungi strain to control plant pathogenic nematodes is Paecilomyces lilacinus. Highly sensitive nematodes include the root-knot, remiform, cyst, golden cyst, and citrus groups, and particularly Meloidogyne, Radopholus similis, Heterodera, Globodera, reniformis, Nacobbus, Belonolaimus, and Pratylenchus. Several entomopathogenic (Verticillium lecanii, Lecanicillium muscarium) and mycopathogenic (Ampelomyces quisqualis, non-toxigenic Aspergillus flavus, Coniothyrium minitans, Ulocladium oudemansii) fungi are marking a new trend in the global trade. Although Table 1 lists information on the efficacy of the strains used as active ingredient in the most frequently used BPs, several brands with important global sales and including BASF (TrichoPlus™, Green Muscle™, and Green Guard™), Agri Life (Biokuprum™, Downycare™, Powderycare™, and Racer™), Dudutech (Trichotech™, Beauvitech™, Lecatech™, and Mytech™), LAM International (Mycostar™), and Verdera (Rotstop™) do not disclose key information on the strains used in their products.

Perspectives, trends, and challenges for improving shelf life of fungi-based BPs

Genetic engineering approaches

Although the control of important pests is possible by BPs, important application limits remain to be overcome, particularly low virulence and variable efficacy, even when the product is correctly applied (Zhao et al. 2016). Genetic engineering technologies have focused on virulence improvements by the expression of insecticidal proteins/peptides. Fungi species, particularly Metarhizium sp. and Beauveria sp. strains, have been genetically modified (Nai et al. 2017; Qin et al. 2010). Modifications such as the overexpression of lytic enzyme genes have yielded more virulent strains (Qin et al. 2010). Increasing the utilization of host nutrients by entomopathogens is another promising way to improve the virulence of fungi-based BPs. For example, Peng et al. (2015) achieved by genetic modification of Metarhizium acridum the overexpression of the gene ATM1, which codes for an endogenous hydrolase of trehalose, the main carbon source for insect hemolymphs. This modification, achieving a 60.7% pest reduction by using host nutrients, accelerated fungi growth and improved proliferation into the insect hemolymph. The chit42 gene (from Metarhizium anisopliae) is important for chitinase production reflecting its role in binding insoluble chitin. Li et al. (2013) integrated this gene in Trichoderma koningii, achieving a higher chitinase activity and increasing from 10 to 30% the mortality rate of corn borer larvae. Lecanicillium attenuatum has been modified by the integration of the Cdep1 gene coding for a cuticle-degrading protease from Beauveria bassiana (Xie et al. 2016). The modified strain showed improved effectiveness against cyst nematodes (Heterodera glycines).

Genetic engineering approaches focusing on enhancing fungi resistance against UV radiation, high temperature, and other environmental factors affecting the shelf life of BPs are considered a high priority (Leng et al. 2011b; Shang et al. 2012). Product stability improvements have achieved increases in tolerance of fungi-based BPs to environmental stress factors, particularly UV radiation (Zhao et al. 2016). Liao et al. (2014) reported that overexpressing the hsp25 gene in Metarhizium robertsii enhanced its growth under heat stress and also its soil persistence. Fang and Leger (2012) evaluated the transformation of Metarhizium robertsii and Beauveria bassiana to express a highly efficient archaeal Halobacterium salinarum CPD photolyase, which increased their photorepair characteristics. The modification resulted in an improved resistance to sunlight while retaining virulence and, therefore, more efficient pest control over a longer time when tested against Anopheles gambiae. Although genetically modified organisms (GMOs) have been shown to be significantly more resistant to the environmental conditions than the wild type, having higher effectiveness and shorter killing times at lower doses, commercial BPs utilizing them are not yet available in the market (Leger and Wang 2010; Nai et al. 2017). The release into the environment of genetically modified BPs that are more virulent against insects and eukaryotic microorganisms represents a potential dissemination risk to nontarget hosts (Arora and Douglas 2017). This has led to rigorous GMO regulations and a slowing down of their registration for commercial applications. Additional information on the authorization procedures for GMOs and methodologies for risk assessment and safety mechanisms can be found in Directive 2001/18/EC (Mellado Ruiz 2001).

Encapsulation polymers and attractants

An alternative to genetic manipulations is immobilization of fungal propagules (as spores, conidia, mycelia, etc.) in polymer carriers offering protection for prolonged periods, virulence retention, and facilitating their manipulation and application (Sarioglu et al. 2017). Although natural and synthetic polymers are frequently used for this purpose, biodegradable polymers are more suitable for ecology-friendly bioformulations (Hosseini Ravandi et al. 2013). Microencapsulation biopolymers used include pectin (Stănciuc et al. 2018), skim milk powder (Würth et al. 2018), dextrin (Miravet et al. 2016), sucrose (Le et al. 2017), molasses (Jin and Custis 2011), and starch (Zanjani et al. 2018). The use of carriers in fungi-based BP formulations is a relatively new and attractive alternative to improve BPs. However, operational conditions must be carefully controlled when high spray-drying temperatures are used for microencapsulation as such conditions can damage and diminish the viability of most BPs (Acuña-Jiménez et al. 2015; Jin and Custis 2011). However, when the carrier and the time–temperature conditions used are properly selected, encapsulation can improve the shelf life and field efficacy of BPs. Unfortunately, the understanding required to select the best carrier for BP bioformulations is still poor (John et al. 2011), and in some cases this technique can present a slow hydration and release of the active material when it is required to be fast (Lumsden and Walter 1996).

Aziz Qureshi et al. (2015) reported the microencapsulation of Beauveria bassiana spores with sodium humate as a carrier. The encapsulation was performed by spray drying without affecting fungi germination, retaining viability at 30 °C for 6 months, and achieving in 5 days a 93% Helicoverpa armigera mortality. Trichoderma harzianum spores microencapsulated in maltodextrin and gum Arabic showed an 86% survival rate after 8 weeks at 4 °C (Muñoz-Celaya et al. 2012). Spasova et al. (2011) incorporated spores of Trichoderma viride into chitosan biohybrid nanofibrous mats to retain fungi viability and effectively inhibit phytopathogenic fungi such as Fusarium and Alternaria. A chitosan/poly(acrylic acid-co-maleic acid) complex was also evaluated as a carrier for Trichoderma viride spores (Gicheva et al. 2012). Accinelli et al. (2012) entrapped non-aflatoxigenic Aspergillus spores into Mater-Bi® (bioplastic polymer) extending its shelf life for more than 6 months when stored at 5 to 25 °C. De Corato et al. (2018) have developed a simple, innovative, and inexpensive system using Na alginate for the microencapsulation of lyophilized yeast cells into gel beads of two strains of Pichia guilliermondii and Candida oleophila used as biocontrol agents of Penicillium digitatum, the main causal agent of postharvest green mold disease on citrus fruit. The approach preserved their vitality, morphological stability, and biological activity at 0–4 °C for up to 14 months.

In addition to biodegradability and toxicity tests, screenings of encapsulation polymers should include hydrophilicity tests to assess the compatibility of encapsulated BPs with irrigation systems, aerial sprayers, backpack sprayers, and other systems used to apply agrochemicals. They should have low cost and high efficiency, provide thermal desiccation and UV protection, and enhance the BP adhesion to plants and pests. Synthetic colloidal layered silicate could protect BP formulations and are non-toxic, nonflammable, stable to UV, resistant to microbial attack, and unaffected by high temperature (Jagtap et al. 2016; Wang et al. 2012). Low-cost agro-industry residues containing fiber, polysaccharide, oligosaccharide, gum, mucilage, and other structure-forming compounds could be used to formulate water-soluble formulations (Paz-Samaniego et al. 2016; Podgórna et al. 2017).

Finally, attractants used in the microencapsulation of spores can enhance the pest proximity to the virulent propagule. Przyklenk et al. (2014) co-encapsulated Metarhizium brunneum, Saccharomyces cerevisiae, and starch in Ca alginate beads. CO2 emitted by the synergistic action of fungi lytic enzymes and yeast metabolism worked to attract soil-dwelling insect pests (Johnson and Nielsen 2012). Beauveria bassiana was similarly evaluated by Vemmer et al. (2016). Encapsulation of Beauveria bassiana mixed with pheromones resulted in a successful formulation attracting banana weevil Cosmopolites sordidus and achieving a 96.9% in vitro mortality rate (Lopes et al. 2014). In a similar study, Kabaluk et al. (2015) reported a 100% mortality of adult Agriotes obscurus L. beetles when applying an encapsulated formula containing a mix of Metarhizium anisopliae and pheromones. Even though attractant/killing fungi-based BP formulations are interesting, issues such as the degree of attraction, species-specific compound limitations, and field conditions efficacy require more attention in depth.

Conclusions

Recent multi-billion acquisitions, and ongoing collaborations by large corporations with agrobiotechnology companies and research centers, suggest that BPs have the potential to be a sector with high future annual growth. Microbial BPs, particularly fungi-based BPs, stand out as an attractive crop protection alternative with features such as high specificity, low probability of pest resistance development, low or no known health risks, low and rapidly biodegradable residues, and suitability for incorporation into IPM schemes. However, factors hindering the full adoption of fungi-based BPs include the lack of suitable mass production procedures, heterogeneity in field test results, commercial products with short shelf life, legislations constraining the registry of new products, and end-user resistance or unawareness of the availability of these novel crop protection technologies. Regulatory framework changes in progress are expected to facilitate the registry of commercial BPs. Training is expected to reduce the end-user resistance and lack of knowledge, and considerable ongoing research on innovative formulation and preservation methodologies could improve the efficacy of commercial fungi-based BPs. Studies here overviewed focusing on improving the propagule stability, infectivity, and shelf life of BPs indicate that: (1) solid formulations, particularly in granular form, work better for extended infectivity effect on soilborne pathogens; (2) wettable powders and water-dispersible granules in topical and spray applications work better for insect and nematode biocontrol; and (3) liquid formulations, particularly oil suspensions, emulsions and/or oil dispersion work better for insect pest biocontrol improving stability and infectivity due to improved insect cuticle adherence.

In terms of shelf life, this overview shows that most wettable-dispersible granule formulations should be stored at 0–5 °C to reach 6–12 months of viability. Granular products remain effective for 4–12 months when stored at 8–23 °C. Trichoderma in granular formulations are reported to retain viability for 12 months when stored at 4–21 °C. Wettable powders, frequently used in commercial formulations, allow viability retention as in the case of the 4–15 months reported for Trichoderma BPs when stored at 0–23 °C, and that of Beauveria strains for 6–12 months at 4 to 23 °C. Although Metarhizium in wettable powder formulations can remain viable for 12 months at − 4 °C, storage at 24 °C reduces viability by more than 90%. Emulsion for seeds and oil dispersion formulations have shown a viability from 12 to 18 months at 23 to 25 °C, reflecting the enhanced desiccation protection provided by oils. Shelf life can be significantly improved by the incorporation of additives into the culture media and by using storage containers with O2/H2O scavengers. Despite the extensive fungi-based BP literature overviewed in this work, additional studies reporting field formulation tests and efficacy after storage are necessary. In addition to improvements in the shelf life and efficacy of formulations, future studies should emphasize efficacy after storage because it will provide valuable information about the field performance of formulation and application methodologies. Despite research on genetically modified fungi with improved resistance and virulence, commercial preference for the simpler registration of non-GMO products is likely to remain as the social and legal pressure to avoid GMO-based products is likely to continue and increase. Conversely, recent interest in attracting and killing co-formulation schemes will certainly expand. Finally, BPs, including fungi-based BPs, will compete successfully with CPs if the preference of the final consumer of agricultural products for green technologies continues to grow as expected.

Author contribution statement

RCQ conceived the original manuscript concept. RCQ, JJCM, and JAT wrote the manuscript. RCQ, JJCM, and MJRA analyzed the data. JAT interpreted the published data. JAT and RPS critically revised the manuscript. All authors read and approved the manuscript.