The Potential Use of Actinomycetes as Microbial Inoculants and Biopesticides in Agriculture

Introduction

The technological development of agriculture comes with several problems and challenges. The main ones are related to the guarantee of high production rates in a global scenario with the increasing scarcity of resources and environmental degradation (1). Some estimates suggest that the need to produce agricultural sources will grow up to 70% by 2050 (2). This high demand for productivity has resulted in the search for strategies that ensure good plant performance and help optimal develop economically important crops (3). To increase plant nutrition and protection, chemical fertilizers and pesticides are commonly applied. However, when misused, these products tend to accumulate in nature and promote eutrophication of water bodies, besides the presence of compounds that may be toxic to human health at overexposure (4, 5).

Currently, producers and consumers have been gaining more ecological and food awareness. This phenomenon is consistent with the market trend of prioritizing the use of more sustainable products, aiming to reduce waste and decrease impacts on nature through the search for biodegradable, safe, and effective replacements (6). In this sense, microorganism-based products work as an efficient alternative for reducing the use of agrochemicals, combining high productivity with a responsible view of the planet and human health (7). The demand for microbial inoculants, or biofertilizers, has increased considerably in recent decades, being used both as a substitute for conventional fertilizers and as an additional and complementary resource to existing ones (8, 9). Biopesticides based on microbial biological control agents (MBCAs) show excellent results against phytopathogens of agricultural importance (10), especially due to the high specificity of targets provided by their natural molecular mechanisms, able to decrease pest population and reestablish the ecological balance of the environment (11, 12).

Among the many varieties of microorganisms that can be part of a product for agricultural application, actinomycetes stand out for their bioactive particularities (13). These are bacteria primarily found in soil with biologically important properties, capable of producing numerous metabolites of commercial interest, such as enzymes, hormones, and antibiotics (14). These compounds are generally products of secondary metabolism, which are not used in the vital stages of their development and reproduction (14).

The area surrounding plant roots, called rhizosphere, is a space richly colonized by actinomycetes. Their high synthesis of antimicrobial compounds and other metabolites gives them a competitive advantage, besides helping with the improvement of soil fertility by promoting plant growth, providing protection against phytopathogens and resisting environmental stresses (15, 16). Bacterial endophytes are other important class of microorganisms that help the host develop while using nutrients and shelter provided by the plant cells. They constitute a group of non-pathogenic bacteria capable of colonizing the interior of plant tissues, either inside the vegetable cells or between them (17). Endophytic actinomycetes produce secondary metabolites that establish beneficial relations with the plant host by protecting them from phytopathogens and stimulating plant growth, which further demonstrates their promising role in agricultural management (18). However, despite the biotechnological potential of actinomycetes, only a few products on the market take advantage of the microorganisms' abilities and a small number of bibliographic studies on the subject.

Therefore, this review aims to elucidate some of the main aspects involving the study of actinomycetes and their applications as inoculants and biodefensives in agriculture. The mechanisms behind the bioactivity of actinomycetes capable of promoting plant growth and protection, especially rhizospheric and endophytic species already characterized in the literature, are described. Furthermore, the main challenges behind the large-scale production of actinomycetes, the achievement of stable formulations and the different forms of application in the field are listed. The strategies adopted to improve this field of research are also listed, in order to contribute to the development of a more sustainable and productive agriculture.

Actinomycetes: A Rich Source of Bioactive Compounds

Actinomycetes represent one of the largest and most diverse bacterial groups described (19). They are Gram-positive bacteria belonging to the order Actinomycetales and the phylum Actinobacteria, being cosmopolitan microorganisms with habitats in both terrestrial and marine aquatic and freshwater environments (20). Its genome is enriched in G+C content. Its morphology varies from cocci and rods to complex multicellular structures similar to the mycelium of filamentous fungi, as shown in Figure 1. The mycelial structures can be aerial or adhered to the substrate (22, 23). This characteristic, together with the fact that some individuals produce spores as a form of asexual reproduction, has already led actinomycetes to be considered as transitional microorganisms between bacteria and fungi. However, actinomycetes' genetic and cellular profiles are characteristic of bacterial attributes, such as a chromosome wrapped in a prokaryotic nucleoid and a cell wall composed of peptidoglycans (20, 24).

The potential use of actinomycetes as a source of bioproducts began in the 1920's when the first report of antibiosis by a species belonging to this group was reported. Later, in the 1940's, the first molecules with antibiotic properties produced by actinomycetes were isolated, including streptomycin, which would become the first active treatment against tuberculosis (25). Currently, it is estimated that 64% of all naturally produced antibiotics come from actinomycete species (26). In fact, the number of published articles related to actinomycetes has increased considerably since the beginning of the last decade, especially the ones focused on species capable of promoting plant growth and biocontrol (Figure 2). This behavior is in line with global trends of studying microbial biodiversity for the development of biotechnological solutions.

Studies have already demonstrated actinomycetes metabolites active against bacteria (27), fungi (28), viruses (29), nematodes (30), and insects (31), as well as compounds with antitumor (32) and herbicide (33) activities. Some species can also produce enzymes of industrial interest, such as chitinases, cellulases, and amylases (34), and enzymes involved in plant growth promotion, like phosphatases and nitrogenases (35). In addition, actinomycetes also produce volatile organic compounds (VOCs), playing a significant role in their bioactivity, acting as antimicrobial agents, and even inducing changes in the gene expression of nearby microorganisms (36).

Actinomycetes are primarily found in soil, where they represent more than 30% of the entire microbial population and can vary from 10⁶ to 10⁹ bacteria per gram (24, 37, 38). In order to survive in this environment, actinomycetes developed intricate mechanisms mainly for the decomposition of organic matter by producing enzymes through which they could break soil components and obtain the nutrition required for survival (39). Cellulose and lignin, found in wood residues, and the chitin found in the exoskeletons of insects, are some of the substrates that actinomycetes enzymes are capable of hydrolyzing (34). In this process, the nutrients released become bioavailable for plants and other organisms present in the soil (39).

Actinomycetes have a strong capacity of colonizing the region near plant roots, called the rhizosphere. This area has higher nutritional values due to the roots' exudation, secretion, and deposition of important compounds (e.g., amino acids, fatty acids, organic acids, phenols, sterols, sugars, and vitamins) (37). For this reason, actinomycetes that colonize the rhizosphere are more likely to obtain nutrients, which explains the higher bacterial concentration in this region when compared to bulk soil (40). Among the various plant species that actinomycetes can colonize there are some economically important crops, such as soybean (41), tomato (42), wheat (43), and pea and chickpea (44).

Streptomyces is the most common soil actinomycetes, and some studies suggest that this genus represents more than 95% of the total actinobacteria found in the soil (37). However, other genera have already been reported as rhizoactinomycetes, like Sanguibacter, Rhodococcus, Pseudonocardia, Propionibacterium, Nocardia, Mycobacterium, Micrococcus, Microbacterium, Frankia, Corynebacterium, Clavibacter, Cellulomonas, Arthrobacter, and Actinomyces (45, 46).

Regarding important interactions among bacteria and plants, endophytic actinomycetes are another promising biotechnological source found in nature (13). They constitute a group of non-pathogenic microorganisms that colonize the interior of plant tissues during its entire or partial life cycle, from roots to aerial structures (17).

Actinobacteria is one of the most common classes of endophytic bacteria (47). The actinomycetes endophytes genera already reported are Actinomadura, Actinoplanes, Dietzia, Glycomyces, Kineosporia, Leifsonia. Microbispora, Micrococcus, Micromonospora, Nocardia, Nocardioides, Pseudonocardia, Saccharopolyspora, Streptomyces, and Streptosporangium, among others (48, 49).

Plant-Growth Promoting Actinomycetes As Microbial Inoculants

Plant growth-promoting bacteria (PGPB) are the most used microorganisms in the composition of inoculants, especially those that colonize the rhizospheric region of the plant, called plant growth-promoting rhizobacteria [PGPR; (50)]. Associations between plants and plant-growth-promoting actinomycetes (PGPA) have been extensively studied over the years. In general terms, the PGPA with the potential to constitute microbial inoculants can benefit the host plant through two main strategies: phytohormones modulation (synthesis of auxins, gibberellins, cytokines, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase) and increase in nutrients bioavailability (biological nitrogen fixation, phosphate solubilization and production of siderophores) (Figure 3).

Phytohormones Modulation

Producing phytohormones that stimulate plant growth is recurrent in many PGPA species. They are, in most cases, indole compounds belonging to the class of auxins, with indole-3-acetic acid (IAA) being the most common (51). Harikrishnan et al. (52) conducted a study in which phytohormone production by 90 isolated actinomycetes was evaluated, of which 65 tested positive. In similar work, Gopalakrishnan et al. (53) quantified the production of several biomolecules involved in plant growth promotion and biocontrol by six strains of the genus Streptomyces. The ability to synthesize IAA is common among actinomycetes, particularly endophytic and rhizospheric species of the genus Streptomyces (54–56). The PGPA that produce IAA tend to promote more significant root growth of the plants associated with it, increasing their access to soil nutrients and improving their development behavior (57).

All bacteria tested positive for IAA production, and two strains (CAI-95 and CAI-93) stood out for promoting greater root development and productivity when inoculated in rice plants. The real-time quantitative PCR test (qRT-PCR) showed an up-regulation of genes involved in producing IAA of these two strains, which explains their greater bioactivity. Additional studies have reported IAA synthesis by other species, such as S. rosealbus (58), S. scabiei (59), and S. roseoflavus (60).

Besides Streptomyces, other genera of actinomycetes also can produce IAA, as reported in a study by Shutsrirung et al. (61). IAA synthesizing species belonging to the genera Spirillospora, Streptomyces, Nocardia, Nocardiopsis, Microbispora, and Micromonospora were identified in this work. All six genera had microorganisms with positive results for phytohormone production. The actinomycetes of the genus Nocardiopsis showed expressively high levels of IAA production (from 62.23 to 222.75 μg/mL). Nimnoi et al. (62) identified species of the genera Nonomuraea, Actinomadura, and Pseudonocardia who also had significant IAA production rates (from 9.85 to 12.07 μg/mL).

Many inoculants have more than one microorganism in their composition, aiming to achieve a synergistic effect that enhances different bioactive mechanisms and increases the product's spectrum of action (63). The endophytic actinomycete S. griseoflavus is an IAA-producing species with great potential to promote plant growth. Its association with other PGPB in the development of biofertilizers has been drawing attention in recent years (64). Htwe et al. (65) reported that the combination of S. griseoflavus P4 with two distinct species of Bradyrhizobium promotes a series of beneficial increases to soybeans and mung beans, such as root elongation, more significant plant growth, improved capture of N, P, and K, in addition to a higher yield in the production of soybean seeds. Similarly, a study conducted by Soe et al. (64) demonstrated that co-inoculation of B. yuanmingense with S. griseoflavus P4 results in a symbiotic and synergistic relationship between the two bacteria, which induces greater nodule formation in the roots of soybean plants and increases biological nitrogen fixation rates. This behavior may be related to the improvement in plant performance caused by the phytohormone produced by the actinomycete, directly affecting the production of nodules and NBF by the nodulating bacteria.

Gibberellin is another class of phytohormones that can promote plant growth (66). They are tetracyclic diterpenoid carboxylic acids with C20 or C19 structures, affecting important vegetable growth processes, such as stem elongation, seed germination, flowering, and fruit setting, improving photosynthesis rate and chlorophyll content (57). Synthesis of gibberellins is less recurrent in actinomycetes, but some species have already been reported as producers of these phytohormones. The production of gibberellic acid (GA) by four actinomycetes of four different genera (Frankia, Streptomyces, Actinoplanes, and Micromonospora) was investigated by Solans et al. (67), and the results were positive for all bacteria, showing concentrations values higher than the ones for IAA production for three strains, except Micromonospora sp. GA synthesis was also reported for A. campanulatus, S. spiralis, S. olivaceoviridis, and S. rochei (68, 69).

Cytokinins are phytohormones that play an important role in plant development, modulating seed germination, plant senescence, photosynthesis, and respiration (70). Few studies relate actinomycetes with the ability to produce cytokinins. Still, it has already been demonstrated that the genus Actinoplanes, Frankia, Streptomyces, and Micromonospora can synthesize zeatine, one of the most well-known cytokinins (67).

Ethylene, a phytohormone involved in the regulation of plant development, plays a crucial role in maintaining healthy growth at low concentration levels (~0.05 μL/L) (71). Many PGPA can produce ACC deaminase, contributing to greater control of ethylene levels and plant growth (72). In addition, this enzyme can promote aerial and root elongation of plants (73).

The investigation of ACC deaminase-producing rhizosphere actinomycetes was done by Anwar et al. (74). Six strains of Streptomyces were found capable of producing the enzyme at concentrations ranging from 1.9 to 0.71 mmol/L. Species of the genera Microbacterium (75), Actinobacterium (76), Micrococcus (77), and Rhodococcus (78) have already shown the potential of ACC deaminase production.

The importance of the interaction of ACC deaminase and IAA in the inoculation of plant-growth-promoting actinomycetes was evidenced by El-Tarabily (42). In this work, strains of Streptomyces isolated from tomato rhizosphere were tested for both productions of ACC deaminase and IAA, along with evaluating their competence as root colonizers. A strain identified as S. filipinensis no. 15 produced the enzyme and the phytohormone, while the isolate S. atrovirens no. 26 produced only ACC deaminase. Both bacteria were rhizosphere-competent. When inoculated in tomato plants in greenhouse conditions, both actinomycetes induced a decrease of the endogenous levels of ACC, a precursor in ethylene biosynthesis pathway, and promoted plant growth. However, the increase in plant growth was higher in the treatment with S. filipinensis no. 15, which may be related to its ability to produce IAA together with ACC deaminase. In addition, when a strain, not rhizosphere-competent producing ACC deaminase, was inoculated under the same conditions, the treatment did not result in plant-growth promotion. These findings show that, besides the connection between ACC deaminase and IAA, it is also important to look at the ability of the strains to colonize the rhizosphere to achieve a successful inoculation.

Nutrient Availability

Nitrogen (N) is found in the atmosphere as a gas (N₂), but plants are not capable of assimilating it in this form. The biological nitrogen fixation (BNF) process turns it into NH⁴⁺, making the nutrient available for use in the biosynthesis of vital molecules in vegetable cells (79, 80). PGPB species capable of BNF are called diazotrophic bacteria, capable of forming N-fixing nodules in the roots of the plants when imposed to nitrogen-deficient environments (79–81).

Several studies have focused on bioprospecting diazotrophic actinomycetes. However, a small number of species have been reported to own the ability of BNF. The species belonging to the genus Frankia and their capacity to induce the formation of N₂-fixing nodules on more than 200 species of dicotyledonous angiosperms, called actinorhizal plants (82). These diazotrophic actinomycetes can be found in a symbiotic association with actinorhizal plants or as free-living N₂-fixing bacteria (83). Their mechanism of N₂ fixation involves the expression of nif genes and the development of vesicles named diazovesicles when exposed to a nitrogen-deprived environment, where the nitrogenase complex is protected from oxygen by laminated lipid layers (81, 84). Frankia was considered the only N-fixing representative group among actinomycetes for a long time. However, some studies in the past decades reported non-Frankia diazotrophic species belonging to the genus Streptomyces, Mycobacterium, Corynebacterium, Microbacterium, Agromyces, and Micromonospora (85).

Streptomyces thermoautotrophicus was the first diazotrophic streptomycete described (86). On the other hand, more recent studies suggest a reclassification of the bacteria since the reported genes related to nitrogenase enzymes have similarities in genomes of non-diazotrophic species (87). Dahal et al. (88) also showed species of Streptomyces capable of biological nitrogen fixation. This study demonstrated that not all strains that can grow in a nitrogen-free culture medium necessarily have the nifH genes. Of 46 Streptomyces isolated from soil and capable of growing under nitrogen-free conditions, only four strains presented gene clusters responsible for the nitrogenase complex. This shows that other gene clusters are also related to BNF (89). Wahyudi et al. (41) evaluated the biological nitrogen fixation capacity of 18 rhizospheric Streptomyces sp, isolated from the soybean (Glycine max L.) rhizosphere. 15 strains showed positive results for growth in N-free culture medium in vitro. The best results were S. panaciradicis, S. recifenses, S. polychromogenes, and S. manipurensis. They were tested in vivo on soybean, inducing a visibly, more significant growth in all treatments after 5 days of inoculation compared to controls.

Actinomycetes can also act as Rhizobia Helper Bacteria (RHB), agents that interact with natural N-fixing microbes and improve bioactivity. A strain of Micromonospora isolated from nitrogen-fixing nodules of alfalfa (Medicago sativa L.) improved plant-growth of alfalfa when associated with the rhizobium Ensifer meliloti 1021, as reported by Martínez-Hidalgo et al. (90). The results indicated that the interactions between bacteria increased roots nodulation and nitrogen nutrition by the plant, besides promoting aerial growth, a higher shoot-to-root ratio, and a raise in the level of essential nutrients, indicating its potential as an indirect plant-growth promoter.

Phosphate solubilization is a trait found among some genus of actinomycetes, such as Streptomyces, Microbacterium, Thermobifida, Angustibacter, Kocuria, Isoptericola, and Agromyces (91, 92). The exploration of this ability usually comprises bioprospecting in mines or rocky regions, where phosphate-solubilizing bacteria are most likely to occur in the soil. Phosphorus (P) is an essential element for good plant performance, being involved in functions ranging from cellular energy metabolism to the transfer of genetic characteristics through generations (50). In nature, inorganic and organic forms of phosphorus available in the soil are not assimilated by plants. To make them accessible, some plant-associated bacteria can solubilize inorganic phosphates by producing low molecular weight acids (93).

Hamdali et al. (94) screened 300 actinomycetes isolated from Moroccan phosphate mines, an area rich in insoluble rock phosphate (RP), and 18.3% of the strains were able to solubilize rock phosphate as the only phosphate source in a minimal medium. Eight selected strains from the genus Streptomyces and Micromonospora, showed mechanisms of phosphate solubilization involving siderophores production.

Aallam et al. (95) tested the ability of 10 isolates of the genus Streptomyces to use two sources of phosphate in the soil: natural rock phosphate (RP) and tricalcium phosphate (TCP). Species identified as S. bellus and S. enissocaesilis used both forms of phosphorus for their development and released the excess as soluble P in the medium at concentrations ranging from 60 to 170 μg/mL. In the same work, preliminary studies indicated that the phosphate solubilization by S. enissocaesilis is probably related to the acidification of the medium and the production of siderophores. A similar result was found by Farhat et al. (96). They identified an actinomycete (Streptomyces sp. CTM396) capable of mineral phosphate solubilization (MPS) through a mechanism that involves the expression of the genes gdh and pqq, responsible for encoding precursors in the pathway of gluconic acid, which is stimulated by the presence of humic acids. The decrease in the pH is followed by MPS, making phosphate available for plant uptake.

The effect of phosphate-solubilizing actinomycetes on plant growth and fitness was explored by Hamdali et al. (97). In this study, the phosphate-solubilizing strains Micromonospora aurantiaca and S. griseus were inoculated in seeds of the wheat plant (Triticum durum L. cv. Vitron) grown under greenhouse conditions, with sterile soil deficient in forms of soluble phosphate supplemented or not with soluble phosphate or with insoluble RP. The treatments of both actinomycetes in the soil with insoluble RP increased plant growth, with 80–78% and 50–47% weight increase of roots and shoots, respectively. This result was associated with a higher N and P content of the plant's tissue. El-Tarabily et al. (98) isolated a strain of M. endolithica from a calcareous soil deficient in available phosphorus capable of solubilizing powdered rock phosphate (PRP) along with the production of organic acids. When inoculated in plants of beans (Phaseolus vulgaris L.) grown in soil with single super-phosphate (SP) or PRP, the growth of shoots and roots significantly increased when compared to the controls, which was in accordance with the high levels of nutrients available in the soil and the tissues of the plants inoculated with the actinomycete. The inability of the strain to produce phytohormones instigated the correlation of phosphate solubilization with the stimulation of plant growth.

Siderophores are low molecular weight molecules produced by microorganisms that can chelate Fe³⁺ ions present in the environment (50, 99). Siderophore-producing microorganisms have competitive advantages in environments where there is a low occurrence of iron. Under these conditions, the ability to capture Fe³⁺ ions prioritizes the survival of these species over other components of the local microbiota, including phytopathogenic microorganisms, which can suffer losses and cause less damage (100).

Several species of actinomycetes are capable of synthesizing siderophores compounds. Lee et al. (101) evaluated the pattern of siderophore production by actinomycetes isolated from two different sites in Western Australia. The results indicated that all the isolates had a similar percentage of synthesis of general siderophores classes, being catechol the only one to show a distinguished proportion (3% of the isolates from site 1 and 17% from site 2). A strain identified as belonging to the genus Streptomyces produced an extracellularly excreted enterobactin, an important siderophore known to be common in the Enterobacteriaceae family. Further PCR analysis demonstrated that the strain had in its genome the entF gene, responsible for the final assembly of the tri-cyclic structure of enterobactin. A second Streptomyces isolated showed the ability to produce heterobactin, a siderophore molecule also found in species of the genus Rhodococcus and Nocardia.

Desferrioxamine siderophores are one of the most common classes of siderophores produced by actinomycetes, being reported in the metabolome of more than 10 species of Streptomyces (102). Desferrioxamine B, in particular, is an important siderophore produced by S. pilosus with pharmacological properties, used as a drug in the treatment of iron intoxication (103). Nocardichelins A and B are siderophores produced by species of the genus Nocardia discovered by Schneider et al. (104), capable of inhibiting human cell lines from gastric adenocarcinoma, breast, and hepatocellular carcinoma. These bioactive compounds combine the structure of mycobactin-type siderophores from mycobacteria and desferrioxamine B produced by Streptomycetes.

In order to investigate the potential influence of siderophore production on plant-growth promotion, Rungin et al. (105) inactivated the desD-like gene of the endophytic Streptomyces sp. GMKU 3100, responsible for encoding a controlling enzyme involved in the final step of a siderophore biosynthesis, inoculated both the mutant and wild strain on plants of rice and mungbean. The treatments with the wild types had the best plant development, showing a significant increase in root and shoot biomass and length. The results were even better when the wild type was exposed to ferric citrate during inoculation on both plants, indicating a key function of siderophores in improving plant growth. Similar results were found by Verma et al. (54), who isolated endophytic actinomycetes from Azadirachta indica A. Juss. and tested them for plant-growth promotion and biocontrol agents. The strain Streptomyces AzR-051 was identified as a siderophore-producing actinomycete, capable of significantly promoting plant growth and antagonizing the phytopathogen Alternaria alternata when inoculated in seeds of the tomato.

The siderophore expression may also be related to the virulence of actinomycetes' pathogenic species, as reported by Miranda-CasoLuengo et al. (106). This work demonstrated that Rhodococcus equi produces a hydroxamate siderophore, rhequichelin, expressed by the gene cluster rhbABCDE, whose transcription increases during infection in the host tissue. In addition, when the genes were deleted, the levels of the strain virulence were low. This finding highlights that, although less common, there are important roles played by siderophores other than iron uptake and host defense.

Although they possess countless abilities related to plant growth promotion, only a few actinomycete-based biofertilizers are currently commercialized. Mizorin^® is a biofertilizer based on actinomycetes used in corn, beetroot, and mustard (107). Bamil^® and Omug^® are also microbial inoculants based on actinomycetes (Micrococcus spp.), acting by increasing the nitrification process in wheat, beetroot, onion, carrot, potato, and marrow (108). The small amount of products available on the market is indirectly proportional to the potential of bioactive biodiversity of actinomycetes, which further demonstrates the importance of studies aimed at developing microbial inoculants that take advantage of this wasted potential.

Biocontrol Promoting Actinomycetes as Biopesticides

Agriculture is constantly affected by the presence of pests, which are undesirable organisms that colonize crops and cause a disbalance in plant health and productivity (109). They constitute a varied group of organisms, being insects the most common (110). Plant-parasitic nematodes and phytopathogenic microorganisms, such as fungi, bacteria, and viruses, are also considered agricultural pests of significant impacts in production, capable of causing diseases that compromise plant performance (111, 112).

Usually, pest management in agriculture is made by using pesticides, substances, or mixtures of substances, whose chemical structures interact with unwanted organisms and decrease their levels and damages (113). Therefore, it is increasingly important to look for options that allow the reduction of chemical pesticides utilization, aiming to achieve crop productivity more safely and sustainably. In this sense, microorganisms with biological control abilities are a promising and efficient way of managing agricultural pests, from insects and nematodes to phytopathogenic fungi and bacteria (114). The microbial biological control agents (MBCAs) are the main constituents of biopesticides, and the main examples of actinomycetes with potential for being MBCAs, their mechanisms of action and targets are discussed in this topic.

MBCAs can act by indirect and direct mechanisms (Figure 4). The first type is characterized by controlling unwanted species without having direct contact with them (115), while the second happens when the unwanted species are directly affected by MBCAs (116). The indirect mechanism comprises the increase in plant resistance against pathogenic infections through activation of the systemic acquired resistance (SAR) and the induced systemic resistance (ISR) (11, 117). Competition for nutrients and space is also an indirect mode of action of biopesticide's MBCAs (115), in which a site's anticipated occupation by harmless microorganisms can prevent the plant from pathogens that have the same requirement for survival (118).

Antibiosis is the most frequent type of direct biocontrol among actinomycetes, defined as a situation where the growth of a pathogen is compromised by toxic metabolites produced by an antagonistic presence (119, 120). Usually, these metabolites are antibiotics produced by bacteria as a defense mechanism, with chemical structures capable of inhibiting microbial cells' vital processes (121). Streptomyces is the actinomycete genus most known for antibiotics production, being responsible for synthesizing about 60% of all the antibiotics applied in agriculture and horticulture (122). Lytic enzymes are an important part of antibiosis biocontrol mechanisms (123), through which MBCAs can lyse vital structures of pathogen cells and inhibit their development (124). Other biocontrol direct mechanisms, like hyperparasitism (when an organism gains nutrients by colonizing a pathogen), are also potential interactions to be explored in formulations of biopesticides. However, they are more common among fungal species, with rare occurrences in bacteria (115).

Many MBCAs can either exhibit biocontrol abilities (direct or indirect) by protecting the plant against pathogen attacks and enabling better growth, or have PGPB traits that establish better plant development and make it fit to defend itself against harmful organisms (125, 126). In addition, the same microbial species can have both biocontrol mechanisms and PGPB traits, maximizing their interactions to prioritize plant protection and nutrition (126).

Biopesticides based on actinomycetes and their metabolites have already been developed and are commercialized to help control several pests in agriculture, such as fungus, insects, and nematodes (Table 1).

Fungal diseases are a major cause of agricultural crop losses, with species capable of infecting plants and soil and high ability to adapt under selection pressures (137, 138). In agriculture, this adaptable disposition turns them into effective plant pathogens with notable susceptibility for acquiring resistance to chemical pesticides (139). Furthermore, some phytopathogenic fungi are known as producers of mycotoxins highly noxious for humans, compromising food security (138).

Species belonging to the genus Streptomyces have been identified as strong biocontrol agents against both foliar and soil-borne fungal diseases caused by important agricultural pathogens, such as Phytophthora cinnamomi (140), Colletotrichum musae, Fusarium oxysporum (141), Botryts cinerea (142), Colletotrichum gloeosporioides (143), Fusarium moniliforme (144), Rhizoctonia solani, Fusarium solani, Fusarium verticillioides, Alternaria alternata (145), Sclerotinia sclerotiorum (146), Macrophomina phaseolina (147), Corynespora cassiicola (148), Pythium aphanidermatum (149), Penicillium digitatum (150), and others.

The ways through which Streptomyces control fungal diseases are diverse. Some species, like the rhizospheric S. lilacinus NRRL B-1968T, have in their genome the types I and II polyketide synthase genes (PKS-I and PKS-II), responsible for the pathway of fatty acids biosynthesis in some bacteria and encoding secondary metabolites with bioactive properties. In addition, this type of Streptomyces produces a varied amount of phenol, pyrrolizidine, hydrocarbons, esters, and acids compounds associated with fungus inhibition and cell membrane destruction (151). The non-ribosomal peptide synthetase (NRPS) gene clusters are also important players in the antibiosis of some actinomycetes against fungal phytopathogens, like S. triticiradiciss, isolated from rhizospheric soil of wheat (Triticum aestivum L.). The NRPS proteins can use non-proteinogenic amino acids as building blocks for producing peptides with multi structures and functions, including fungicide activity. Natamycin, an antibiotic used to treat fungal diseases, is also found as a bioactive compound in the metabolome of S. triticiradiciss (43).

Some other metabolites produced by Streptomyces have also been reported as active against phytopathogenic fungi, such as salvianolic acid B, able to cause damage to mycelial cells and spores of Alternaria spp., Fusarium spp., Colletotrichum spp., Cladosporium herbarum, and Botrytis cinerea (152) and 6-amino-5-nitrosopyrimidine-2,4-diol, a compound present in the crude extract of S. amritsarensis V31 with the ability to inhibit mycelium growth of R. solani (7.5–65%), A. alternata (5.5–52.7%), Aspergillu–s flavus (8–30.7%), F. oxysporum f. sp. lycopersici (25–44%), Sarocladium oryzae (11–55.5%), and S. sclerotiorum (29.7–40.5%) (153).

Streptomyces biocontrol of fungal diseases can also be accomplished by the secretion of extracellular lytic enzymes, such as chitinases, able to hydrolyze the chitin located in the fungal cell walls (154). Several chitinases with antifungal activity isolated from Streptomyces have already been described, like chitinase F (ChiF) from S. coelicolor A3(2), chitinase 30 (Chi30) from S. olivaceoviridis ATCC 11238, chitinase 35 (Chi35) from S. thermoviolaceus OPC-520, chitinase IS (ChiIS) from Streptomyces sp. MG3 and chitinase A (ChiA) from S. cyaneus SP-27 (155). Glucanases (156), cellulases (157), and proteases (158) are other enzymes produced by some Streptomyces involved in their antifungal abilities, capable of degrading vital fungal structures and limiting their growth.

Inderiati and Franco (159) identified non-streptomycete endophytic actinomycetes from the genus Actinomycetales, Microbispora, and Nonomuraea with considerable inhibition activity against A. solani, P. parasitica, R. solani, and P. irregulare, either in antagonistic assays performed in vitro and in vivo inoculation in tomato seeds. The genus Nocardia has already been associated with the biocontrol of A. brassicicola, P. dresclea, B. cinerea (160), and F. oxysporum (161). Endophytic actinomycetes belonging to the genus Saccharopolyspora and Actinopolyspora demonstrated strong antibiosis in vitro against A. brassicicola, Chaetomium globosum, F. oxysporum, P. dresclea, R. solani, and B. cinerea (160). A rare actinomycete, Saccharothrix yanglingensis, isolated from the cucumber roots, exhibited a high pattern of chitinase production, which was related to its antifungal activity against Valsa mali (162). Nocardiopsis prasina OPC-131 is another actinomycete that also produces a chitinase (ChiB) to control the growth of phytopathogenic fungi (163).

Arbuscular mycorrhizal fungi (AMF) are symbiotic fungi found in roots and soils of some plants. Their relationship with biocontrol promoting actinomycetes has been gaining attention in the last years. Reyes-Tena et al. (164) tested the synergistic effect of a mycorrhizal consortium with two strains of actinomycetes (ABV39 and ABV02) against Phytophthora capsici in pepper plants (Capsicum annuum L.). The results showed that the co-inoculation of the AMFs with the actinomycetes decreased the disease 3 points on a severity scale. Similar findings were observed by Sastrahidayat et al. (165) when inoculating soybean rhizosphere with mychorrhizal fungi and actinomycetes for the treatment of Sclerotium rolfsii. In this worl, the disease in the plants treated with both microrganisms was decreased up to 40% when compared to the controls.

In vivo performance of actinomycetes against fungal phytopathogens are often associated with positive results, demonstrating inhibiting effects that consolidate its possible application as constituents of biopesticides for plant defense (166–168). However, the in vitro behavior may not always be reflected when evaluating the antagonistic effect in vivo. Similarly, bacterial strains with low activity in vitro can show good inhibition results when applied in planta (169). This discrepancy in both types of screening may be related to the additional biotic and abiotic pressures that overcome or maximize microbial bioactivities, such as competition and other interactions between the actinomycete and the soil/foliar microbiota, climate changes (e.g., unstable temperature and humidity), the inability of proper colonization of the rhizosphere or the plant tissues and levels of nutrient bioavailability (169, 170). For this reason, the results in sterile environments and controlled parameters need to be corroborated in field conditions to assess the real performance of the studied microorganism (171).

Insects are the most biodiverse group of animals in the world, and their ability to feed on all kinds of organic matter, including plant tissues of important crops, has turn them into one of the main causes of agricultural losses and reduced yield (172, 173). Despite using traditional sustainable control strategies, such as crop rotation, healthy crop variety, and integrated pest management, insects are usually controlled by chemical insecticides (172). Although helpful, new alternatives are being seeked to prevent ecological damages and overcome insect resistance (174).

Actinomycetes are potential candidates due to their impressive synthesis of metabolites with insecticidal properties. The polyketide compounds are the main family of natural insecticides produced by actinomycetes, including avermectins, spinosyns, polynactins, tetramycins, and analogs (175).

Avermectins are found in the fermentation products of Streptomyces avermitilis and are widely commercialized for crop protection. Their molecular structure has a high ability of binding to muscular neurons of some insects, acting as agonists for γ-aminobutyric acid (GABA)-gated chloride channels, causing insect paralysis and death (176). They are active against several species from the orders Blattodea, Coleoptera, Diptera, Hymenoptera, Isoptera, Lepidoptera, and more (175). Abamectins and emamectins are insecticides derived from avermectins (176). Milbemycins are metabolites with different structures but act similarly to avermectins, produced by S. bingchenggensis (177). Recently, novel avermectins analogs have been tested to overcome insect resistance. Zhang et al. (178) evaluated the activity of more than 40 avermectin derivatives against Aphis craccivora, of which 4 had superior effect compared to avermectin (7–8 times better), with LC₅₀ (lethal dose) ranging from 5.634 to 52.234 mM. The quantitative structure-activity relationships (QSARs) analysis suggest that the avermectin analogs activity is related to their branching degree, different molecular shape and size, and the number of double bonds.

Spinosyns comprise another important class of insecticidal metabolites produced by actinomycetes, specifically Saccharopolyspora spinosa. The most common are spinosyn A and spinosyn D, molecules with more specific action mechanisms and more focused targets, like Heliothis virescens and Spodoptera eridania (179). Spinosyns act by direct contact or ingestion, modifying GABA and the nicotinic receptor (176). Salgado (180) was the fisrt to report the mechanism through which spinosyn A acts, testing it against Drosophila melanogaster, Periplaneta amerina, Heliothis virescens, and Musca domestica. The LC₅₀ was 8.0 ppm, capable of inducing involuntary muscle contractions and tremors by the excitation of insect's neurons, followed by paralysis due to neuromuscular fatigue.

Other classes of secondary metabolites with insecticidal activity can be found in actinomycetes. El-khawaga and Megahed (181) discovered the strain S. bikiniensis A11, isolated from sandy soil in Cairo, Egypt, capable of producing aminoglycosidic antibiotics active against larvae of cotton leaf worm (Spodoptera littoralis). In another study, Kim et al. (182) screened the ability of 363 actinomycetes to promote the death of Aedes albopictus and Plutella xylostella. Among them, the selected strain Streptomyces sp. AN120537 produced five different antimycins associated with its insecticidal activity. The authors suggested that the antimycins act as insect juvenile hormone antagonists (JHANs), compounds capable of promoting premature molting and larval stages by disrupting the endocrine system of insects. Liu et al. (183) isolated the strain Streptomyces sp. KN-0647 from forest soil in China identified the secondary metabolite quinomycin A, an antibiotic active against Spodoptera exigua, Dendrolimus punctatus, Plutella xylostella, Aphis glycines, and Culex pipiens. Some non-streptomycete actinomycetes have also demonstrated good results against insects, such as Micromonospora and Amycolatopsis (184).

As discussed before, actinomycetes demonstrate a high ability to produce lytic enzymes. Among them, chitinolytic species are important tools for controlling insect pests (185). Chitin is a major constituent of insects' cuticles, providing them with rigidity and shape while helping prevent moisture loss from the insect integument. Chitinase is an enzyme that can hydrolyse these chitin structures in insects, disrupting the skin that induces their death (176). Although lytic enzyme production is more often associated with fungal control, it has been demonstrated by Gadelhak et al. (185) that the chitinases produced by the chitinolytic actinomycetes Actinoplanes philippinensis, A. missouriensis, and S. clavuligerus have a role in the controlling mechanisms of Drosophila melanogaster. The application of A. missouriensi showed the lowest pulp formation percentage (31.75%), and the combinations of S. clavuligerus with A. philippinensis decreased the values to 27.35%, demonstrating a synergistic effect.

Plant-parasitic nematodes (PPNs) are soil-borne pathogens that disturb plant nutrition, compromising its physiological functions and resulting in productivity drops and economic losses (186). The endoparasitic PPNs are the most pathogenic ones for directly interfering in the absorption of water and nutrients by the plants. They comprise the most common nematodes found in crops, such as root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp., Globodera spp.), and pine wood nematode (Bursaphlenchus xylophilus) (187, 188). Management of PPNs is not easy, especially because of their hidden nature and difficulty in identifying and associating symptoms, which are not very specific. Usually, they are controlled by soil fumigation with chemical nematicides (189).

Liu et al. (190) screened 5,000 actinobacteria for nematicidal activity against the pine wood nematode (B. xylophilus), identifying the strain Streptomyces sp. AN091965 is the most active one. Further investigations found that the compound spectinabilin was responsible for the highest nematicidal activity against the nematode, with an LC₅₀ value of 0.84 μg/mL, besides suppressing the disease in 5-year-old Pinus densiflora trees under greenhouse and field conditions. Sharma et al. (135) reported the nematicidal activity of the strain Streptomyces antibioticus M7 against root-knot nematode (M. incognita), which was associated with the production of actinomycins V, X₂, and D, with LD₅₀ values ranging from 28 to 120 μg/ml in vitro. Tomato plants treated with culture supernatant/cells/solvent extract displayed a reduction in root galls and egg masses and significantly enhanced plant growth measurements. In addition, when the strain was applied to non-infected plants, it showed a significant increase in many parameters of plant development, indicating Streptomyces antibioticus M7 as a potential nematicidal biopesticide and PGPB. Fungichromin B, a metabolite produced by S. albogriseolus HA10002, was discovered as a nematicidal agent by Zeng et al. (191). In this study, the compound was isolated and investigated against second-stage juveniles (J2) of M. incognita and M. javanica, achieving LC₅₀ values of 7.64 and 7.83 μg/ml, respectively. Fervenulin, a low molecular weight compound, is a nematicidal metabolite produced by a strain of S. roseoverticillatus isolated and characterized by Ruanpanun et al. (192). In in vitro assays, the lowest minimum inhibitory concentrations (MICs) of the fervenulin against egg hatch of M. incognita was 30 μg/ml and J2 mortality was observed at 120 μg/ml.

Other metabolites produced by actinomycetes, such as volatile compounds, fatty acids, hydrogen sulfide, ammonia, alcohol, and phenolic compounds, have also been linked to nematicidal activity (193). Polyketide compounds produced by Streptomyces, such as avermectins and its derivate abamectin, are important nematicidal metabolites that constitute some of the main commercial biological nematicides (194).

The surface coat of nematodes (cuticle) and nematode egg shells targets this pest biocontrol. Their compositions are similar, with different layers of proteins, chitin, and lipids (135). Actinomycetes lytic enzymes, such as proteases, chitinases, and lipases, can degrade these structures and interfere with the motility and reproduction of nematodes, leading to their death (195, 196). Other lytic enzymes, like glucanases, cellulases, and pectinases, have also been reported to be involved in nematode biocontrol. However, their mechanisms of action are not well-elucidated yet (197).

Rashad et al. (193) identified 28 strains of Streptomyces with nematicidal activity against M. incognita, possibly associated with their ability to produce proteases, chitinases, and lipases. In another work, Yoon et al. (198) characterized the nematicidal metabolites from Streptomyces cacaoi GY525 and observed that the enzymes chitinase and glucanase presented in the bacterial culture filtrate are possibly related to the increased mortality of J2 of M. incognita and decreased eggs hatch. Furthermore, the secondary metabolite compound 3-benzyl-1,4-diaza-2,5-dioxobicyclo[4.3.0]nonane (BDDB) was also identified with nematicidal activity and the strain inoculation in tomato plants for 7 weeks decreased the number of egg masses and population of J2 in the soil.

The losses caused by phytopathogenic bacteria are usually related to crops of less economic impact (199). This factor can explain the scarcity of products based on actinomycetes to bacterial control diseases and why research in this field is occasionally related to the application in the control of post-harvest diseases of fruits and vegetables (200). However, the bactericidal activity found among actinomycetes is expressive and may be a good source for developing biocontrol strategies of phytopathogenic bacteria.

The strain Streptomyces cinereoruber can suppress infections caused by Erwinia chrysanthemi, the causal agent of the soft rot disease (201). S. misionensis, S. cacaoi subsp. Cacaoi, and S. puniceus are active against Xanthomonas campestris pv. campestris, the causal agent of the black rot disease (202). S. albidoflavus H12 and Nocardiopsis aegyptica H14 inhibit the growth of several bacterial phytopathogens, such as Pseudomonas syringae, P. corrugata, and Pectobacterium carotovorum subsp. carotovorum (203). S. ramulosus, S. axinellae, and S. drozdowiczii produce a crude extract active against Ralstonia solanacearum, the causal agent of bacterial wilt (204).

The antibacterial mechanisms of action are mainly related to the expression of antibiotic metabolites, like actinomycin (205), maklamicin (206), nomimicin (207), ansamycin (208), and much more. Encheva-Malinova et al. (209) tested 23 actinomycetes isolated from the Antarctic soils against phytopathogenic bacteria, finding that the most active strains inhibited Clavibacter michiganensis and five species of Xanthomonas. Molecular biology investigations showed that their genomes had combinations of the PKS-II, NRPS, and glycopeptides genes, possibly involved in the expression of antimicrobial metabolites.

Actinomycetes Application in Crops: Methods and Challenges

Biofertilizers are usually inoculated in plants by three different methods: seed inoculation, consisting of the seeds immersion in a microbial solution of known concentration; soil inoculation, where the active solution is mixed directly in the soil subtracted or applied to the region near rhizosphere; and roots inoculation, in which the plant roots are immersed in the biofertilizer solution and then planted in the soil for its development (210). Seed inoculation provides the microorganism with nutrients from germination, which it uses to colonize the roots as soon as they are formed. On the other hand, inoculation directly into the roots or soil allows the bacteria to colonize the already developed roots. Inoculation in the roots allows an immediate contact of the microorganism with the plant, accelerating the colonization process (210, 211). The choice of inoculation method will depend on the plant species, the ease with which the actinomycete colonizes the plant roots and what stage of plant development is desired for the plant to receive the nutritional compounds of interest. In the case of endophytic species, the foliar application may also be considered (212).

The methods for biopesticides application can vary depending on the type of the product formulation. In general, formulations are designed to minimize additional costs over conventional chemical pesticide applications (213). In this sense, biopesticides are commonly applied in crops through basic spraying techniques, covering the aerial structures of plants and the soil (214).

Biofertilizers and most biopesticides are living products. Therefore, their success is entirely interconnected with the microbial response to biotic and abiotic factors present in the environment. Soil holds a wide range of biodiversity. The interaction between the actinomycete with surrounding microbiota and other organisms and its relation with the host plant may be explored to ensure product efficiency (210). Furthermore, actinomycetes can face severe changes in pH, temperature, salinity, moisture, light incidence, inorganic, and organic constituents of soil (215, 216). The stress caused by these factors can interfere in the rhizosphere and its constituents, causing modifications in actinomycete bioactivity regarding plant growth promotion and plant defense (217). Some species can tolerate these abiotic stresses by producing osmoregulating metabolites that prevent cell membrane plasmolysis and regulate enzymatic action and homeostasis (210). Some strains of Streptomyces and Nocardiopsis produce the osmoprotectants glycine betaine, proline, and trehalose at high osmolarity situations, agents that induce osmotic adaptation and preserve bioactivity even under stress (218).

The ability of root colonization is essential for a successful inoculation of a microbial-based biofertilizer (219). Usually, actinomycetes are strong rhizospheric and root colonizers (220). It is thought to relate to their isolation source background, associated with soil and plant tissue environments. However, many bacteria demonstrate good colonization competence in vitro studies and controlled conditions but fail to replicate this behavior in field applications (221). The causes may be explained by the reduced expression of bioactive metabolites when competing with other microorganisms in the soil, which results in lower rhizosphere competence and poor colonization of root and plant tissues (222).

Rhizosphere colonization competence is also important for biopesticides that act against soil-borne pathogens, such as fungi and insects (223). For controlling leaf diseases, it may be necessary that the active ingredient of the biopesticide can colonize plant tissues (224). This can be accomplished by using endophytic actinomycetes, which produce lytic enzymes and other metabolites that improve their ability to colonize the plant (225). The chosen inoculation method is another microbial colonization impact factor (226). Usually, bacteria have little motility in the soil. For this reason, depending on the species and its activity, its application as close as possible to the rhizosphere is necessary to obtain a good result (210).

The microbial concentration in the product is directly related to proper colonization and efficacy since the number of viable cells disposed of in the crop needs to be enough to promote the nutrition and protection of the plant (227). One of the main challenges faced in developing actinomycete-based products is to achieve high culture concentrations at large-scale production, once they show a slower reproduction rate than other bacteria (228, 229). In addition, liquid cultures of actinomycetes tend to form aggregates and mycelial pellets, making homogenization difficult (230). The optimization of bioprocesses associated with the industrial production of actinobacteria spores can be achieved by submerged fermentation (SF) and solid-state fermentation (SSF). The first usually involves a more expensive operation, however allowing greater control of parameters, shorter culture time and easier industrialization. Several studies have been performed to understand and optimize cultivation variables that maximize spore production combined with the mitigation of pellet and aggregate production by this fermentation strategy (231–234). On the other hand, SSF usually involves a cheaper operation, however performing a longer growing time and labored downstream operations. Likewise, in this fermentation route, some studies have investigated the optimization of spore production by actinobacteria (235–237).

These problems are also found when it comes to on-farm production of actinobacteria, mainly regarding its cultivation through solid state fermentation (SSF) or submerged fermentation (SF), usually forming aggregates and pellets (238). These structures formed in submerged cultivation reduce the homogeneity of cellular structures dispersion, can cause clogging of spray nozzles and overcoming these obstacles can implicate in bioreactors and downstream steps that can make the operation economically unfeasible under on-farm production conditions.

Formulation may be another obstacle in developing bioproducts based on actinomycetes (239). Fresh microbial cells are not suitable for agricultural application, becoming necessary research for effective solid, liquid, or powder formulations with bacterial spores/mycelium (240). Combining the optimized production of spores, the development of formulations can be a focus of study to enable a greater number of stable commercial formulations based on actinobacteria. This development is usually linked to drying or encapsulation technologies that protect the spores from conditions of high water activity (Aw) and other abiotic factors that may compromise their viability over shelf life. In this sense, some studies have studied the stabilization strategies for actinobacteria in formulations using drying approaches combined with protectants (240–245). However, advances are still needed to achieve greater stability of formulations.

Besides the active microorganism, formulations contain other inert ingredients that help with field application and shelf-life of the product. These compounds need to interact so that the strain bioactivity is not compromised (246). Nutritional Streptomyces requirements and susceptibility to abiotic factors can become problems regarding formulation development (241, 247). For this reason, the products based on actinomycetes cells are less frequent than the ones based only on their active metabolites (36).

Conclusion and Future Perspectives

Actinomycetes, both rhizospheric and endophytic, possess a strong ability to produce metabolites of interest directly related to their interactions with the microbiome in the environment and the plant host. Production of phytohormones modulators, nutrient-uptake increasing compounds, lytic enzymes, antibiotics, and other active metabolites makes actinomycetes an undeniable promising tool for developing microbial biofertilizers and biopesticides. Streptomyces is the most recurrent actinomycetes found in the environment and the main group with agricultural applications, showing a considerable pattern of producing extracts rich in secondary metabolites active against phytopathogenic fungi, bacteria, insects, and plant-parasitic nematodes. Although many of their bioactive compounds have already been described, further studies are required to fully comprehend its diversity, chemical structures, and molecular modes of action.

Despite all this potential, only a few products based on actinomycetes are available on the market. This may be due to many factors, such as challenges in microbial large-scale production and formulations development, which highlights the importance of optimizing bioprocesses in the cultivation of actinomycetes to achieve maximum bacterial performance and improvement of formulation technologies. Furthermore, it is also imperative to better understand the interactions between actinomycetes, environmental microbiota, and host plants to target the best method for bacterial inoculation and increase its effectiveness. Thus, the entire biological treasure of actinomycetes can be used in favor of developing products that make agriculture more sustainable and productive, having better plant nutrition and protection through the application of microbial inoculants and biopesticides.

Author Contributions

GS, IK, IR, and PL designed the project, wrote the manuscript, and edited the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by grants from the São Paulo Research Foundation, FAPESP (Proc. Nos. 2015/10974-8 and 2020/11315-6); National Council for Scientific and Technological Development, CNPq; and Agrivalle Brasil Industria e Comércio de Produtos Agrícolas Ltda.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We wish to acknowledge F. P. N. Cruz, from Department of Morphology and Pathology, Federal University of São Carlos, São Carlos, Brazil, for kindly providing the photos of the Figure 1.

References