Entomopathogenic fungi

Entomopathogenic fungi are the most important group in the biological control of pest insects. Virtually all insects are susceptible to diseases caused by these fungi. When their spores come into contact with the cuticle of susceptible insects, they germinate and grow directly through it towards the inside of their host's body. Therefore, the fungus proliferates throughout the body of the insect producing toxins and consuming nutrients from the insect, and eventually destroys it. At the beginning of the infection, symptoms may or may not be observed, but the insect begins to lose mobility and appetite. After seven or ten days, it dies due to nutritional deficiency (^{Pérez, 2004}).

The diseases they cause are known as muscardinas, a term that was first applied to Beauveria bassiana (Bals.-Criv.) Vuill. The color of the conidia is very variable, hence, there are different names such as green muscardina, for Metarhizium anisopliae (Metchnikoff) Sorokin and Nomuraea rileyi (Farlow) Samson, and red muscardina for Paecilomyces fumosoroseus (Wize) Brown & Smith. The use of these organisms is one of the best alternatives used in biological control because it is economical, simple and ecologically sustainable. However, it is essential to provide adequate temperature and humidity conditions to achieve its purpose. In addition, when it is intended to be used as bioinsecticides, it is necessary to carry out an exhaustive characterization of isolates in order to select those with high virulence and good conditions for field application. This characterization includes studies referring to the mode of infection (^{Godwin and Shawgi, 2000}; ^{Arredondo et al., 2008}; ^{Caballero, 2014}). Nowadays it is important to carry out studies on the molecular and biochemical determinants related to the specificity of the fungus to the host.

It is well known that B. bassiana infects more than 200 species of insects of different orders, which include pests of economic importance such as the cogollero worm (Spodoptera frugiperda (J.E. Smith)), borer worm (Diatrea magnifactella Dyar, 1911), coffee drill (Hypothenemus hampei Ferrari, 1867), among others. While M. anisopliae, with a broader spectrum of toxicity, has been found infecting between 300 and 400 species of lepidoptera, beetles, dipterans and homoptera (Table 1).

Entomopathogenic fungi against agricultural pests

Table 1 summarizes the main research studies carried out in the most important crops in the country where entomopathogenic fungi were used. The extensive study of Beauveria bassiana strains is outstanding, especially in the fight against pests such as the coffee drill, in which ^{Gerónimo et al. (2016)} observed a pathogenic effectiveness of 100 % at 144 h in this crop; ^{Díaz and Roblero (2007)} recorded that the optimal time for the application of the fungus is in July, before the drill enters the coffee fruit.

From Mexico's interest in citrus production, ^{Pérez-González et al. (2013)} conducted studies against the Asian citrus psyllid (Diaphorina citri Kuwayama) in which they used the fungus Hirsutella citriformis Speare; ^{Mellín et al. (2016)} with Paecilomyces fumosoroseus and ^{Hernández et al. (2007)} with Lecanicillium lecanii (Zimm.) Zare & W. Gams.; in addition to studies conducted with B. bassiana and M. anisopliae by ^{Gandarilla et al. (2013)}. In all these studies, the strains analyzed were considered as promising for the development of biological control technology of D. citri, as in the case of M. anisopliae, which caused 93-100 % mortalities in nymphs, while in adults the Mortality range ranged from 40 to 95 % (^{Mellín et al., 2016}).

Phyllophaga vetula Horn, 1887, known in Mexico as blind hen and the sprout worm (Spodoptera frugiperda) are two of the main corn pests that have been studied to combat them with M. anisopliae with which the highest level of virulence was recorded and where the treatments recorded a mortality of 80 % at 30 days (^{Nájera, 2005}). ^{Buenosaires (2013)} reported an LC₅₀ of 2.0518 × 10⁸ conidia ml^-1. In a similar way, strains of the N. rileyi fungus have been explored, which parasitized 100 % of the neonatal larvae of these pests, with a TL₅₀ between 4.1 and 6.3 d, as well as strains of P. fumosoroseus that parasitized between 92.5 and 98.8 %, with a TL₅₀ between 2.5 and 4.3 d (Lezama et al., 2004); these same authors in 2006, published a more extensive study where they evaluated other strains of entomopathogenic fungi concluding that the strains of M. anisopliae, P. fumosoroseus and P. javanicus (Friederichs & Bally) Brown & Smith, were highly virulent in eggs and larvae with a mortality of 94 and 100 %, and a TL₅₀ of 1.3 to 3.3 d. The strains of B. bassiana had a very variable virulence, with a parasitism between 3-90 % in eggs and 54-100 % in larvae (^{Lezama et al., 2006b}). On the other hand, ^{Ruiz et al. (2012)} concluded in their studies that S. carpocapsae (1.500 JI plant^-1) in combination with M. anisopliae (2 × 108 spores plant^-1) are recommended for the control of larvae of P. vetula.

Entomologic fungi against livestock plagues

In studies with entomopathogenic fungi for the control of pests in the livestock sector (Table 2), it is observed that M. anisopliae and B. bassiana, mainly, have been effective for the control of pests of cattle ticks, fleas of dog, bugs bream, grasshopper and dengue transmitting mosquitoes. There are few records in which other strains of entomopathogenic fungi such as Trichoderma, Cordyceps or Isaria have been explored, to name a few.

Among the most economically important pests in this sector is the cattle tick (Rhipicephalus microplus Canestrini, 1888); according to Rivera (2013), the use of entomopathogenic fungi such as M. anisopliae and B. bassiana are found to be pathogenic for the three, eight, and 17-day-old eggs of the insect, and it is accentuated the younger the tick egg is. For ^{Bautista (2017)}, M. anisopliae and B. bassiana are an alternative for the control of adult ticks in the XIII Maya region of Chiapas and the Ríos region of the state of Tabasco, Mexico.

Entomopathogenic fungi have been tested to combat mosquitoes that transmit serious diseases. Thus, ^{Flores et al. (2016)} investigated the effects of M. anisopliae and I. fumosorosea Wize on nymphs of Meccus pallidipennis Stål, 1872, which is the main triatomine vector of Chagas disease in Mexico, in terms of insect survival and immune response. ^{Zumaquero et al. (2014)} studied an isolation of B. bassiana from San Antonio Rayón, Puebla, Mexico and its entomopathogenic effects in Meccus pallidipennis, from which they concluded that this strain was 100 % virulent. A strain of T. longibrachiatum showed high entomopathogenic activity on larvae and adult females of Aedes aegypti Linnaeus, 1762 mosquitoes, which are Dengue vectors; therefore, this fungus can be a good candidate to be developed as a bioinsecticide

^{Rivera-Ramírez et al. (2013)} docunented that B. bassiana and M. anisopliae are pathogens for dog fleas (Ctenocephalides canis Shaftesbury, 1934) under laboratory conditions; and ^{Pacheco et al. (2015)} assessed the pathogenicity of two strains of B. bassiana at concentrations of 10, 15 and 20 % mineral oil, sterile water and Tween 80, inoculated by immersion on C. canis; it turned out that the treatment formulated at 10 % recorded mycosis of 86.3 % on fleas, confirming that it was the most pathogenic.

Entomologic fungi against forest plagues

There are very few reports in Mexico regarding the use of entomopathogenic fungi to combat forest pests (Table 3). Due to the economic importance of red cedar wood, B. bassiana has been one of the most studied fungi against the combat of the meliaceous borer (Hypsipyla grandell Zeller, 1898).

^{Díaz et al. (2009)} evaluated chemical insecticides such as Novaluron, Pyrethroids, Amitraz (Ovicides) and Carbofuradan, as well as Beauveria bassiana and Metarhizium anisopliae, an organic insecticide based on Neem (Azadirachta indica A. Juss.) and a control against this forest pest. His conclusion was that the treatments with B. bassiana and M. anisopliae had the same degree of control as the chemicals, with the advantage of not being contaminants and of not representing a risk of toxicity for the personnel that applies.

For ^{Caballero (2014)}, the inoculation of B. bassiana in washed, disinfected and conserved larvae of H. grandella in an area with a controlled climate at 22 °C, was achieved as they colonized them , which means that in the laboratory stage they B. bassiana was found effective for the control of H. grandella. On the other hand, ^{Barrios et al. (2017)} selected two native strains of B. bassiana for use in the control of this pest, and the evaluation of pathogenicity of the two isolates demonstrated mortality on third instar larvae at a dose of 1 × 10⁸. According to the authors, these strains could have a high potential to be used in the nursery or in the field for the integrated management of the borer, since they recorded 92.84 % dead of after five days, on average.

Regarding the applications of Trichoderma for the biological control of the pest of debarkers in laboratory conditions, the contribution of ^{Gijón et al. (2015)} showed, by statistical analysis, that a strain of this fungus caused 100 % mortality of these insects. The following year, ^{Arriola et al. (2016)} announced their results on the same fungus to combat the same pest in the Sierra Gorda Biosphere Reserve, Qro.; there, five treatments were applied to Pinus greggii Engelm. ex Parl. trees, which consisted of three concentrations of conidia / milliliter: high 3.6 ×10⁸, average 9 × 10⁷ and low 5 × 10⁷, a control based on a commercial product with M. anisopliae (5 × 10⁹) and an absolute water control. The highest concentration was the one that recorded the most severe mortality.

Entomopatogenic nematodes

Entomopathogenic nematodes (NEP) in the Steinernema and Heterorhabditis genera are potent agents for biological control. Nematodes parasitize their hosts (in this case plague insects) by direct penetration through the cuticle to the hemocele or by penetration through natural openings (spiracles, mouth and anus). The infection can be passive or active, and the way in which the infection process continues will depend on the species of nematode that attacks the insect. In the case of Steinernema and Heterorhabditis, once the infective juvenile manages to penetrate the hemocele, it releases the associated bacteria, which reproduces in the hemolymph of the host and causes death (^{Pérez, 2004}).

In the last decade, substantial advances have been made in its research and application, since the number of target pests that are susceptible to NEPs has continued to increase (Table 4). The progress is also due to the advances in the technology of its production, which use in vivo and in vitro systems, and the new methods of application (injections, sprays, etc.), as well as advances in genomics, nematode symbiont -bacteria interactions and ecological relations.

In Mexico, Steinernema carpocapsae Weiser, 1955 has been declared efficient in the combat of larvae of the third stage of Mexican fruit fly (Anastrepha ludens Loew, 1873), which is a pest that attacks especially citrus and mango. ^{Lezama et al. (1996b)} demonstrated that this pest is susceptible to varying degrees to various tested nematodes. S. riobravis Cabanillas, Poinar & Raulston, 1994 and S. carpocapsae All strain killed 90 % of the larvae and pupae; H. bacteriophora Poinar 1975 NC strain killed 82.5 %; Steinernema feltiae Filipjev, 1934, 81.25 %; the S. carpocapsae Tecomán strain caused 76 % mortality, while H. bacteriophora Tecomán and S. glaseri Steiner, 1929, 52.5 %. These results suggest that the S. riobravis and S. carpocapsae strain species have potential as biological control agents against the fruit fly.

On the other hand, ^{Toledo et al. (2001)} assessed the parasitic capacity of S. feltiae in laboratory conditions on larvae of A. ludens of third stage, and pupae of five and 12 days of age, in three soils of different texture and three temperature regimes Their results showed that there was no difference when the nematodes were applied to the soil before or after the larvae. The pupae were not susceptible to attack. In adults who emerged and left through the treated soil, parasitism was only 10 %. Later, in 2005, the same authors studied this plague, but this time using another nematode: H. bacteriophora (^{Toledo et al., 2005}). These studies allowed them to describe the effect of temperature, soil texture and depth of the host on the ability of nematode infection in third stage larvae of Anastrepha obliqua Macquart, 1835 and, although the potential of the nematode was demonstrated to infect and kill A. obliqua larvae, it was evident that there was an important difference in the susceptibility of the six-day-old larvae, compared to those of eight days.

In a study on the control of the maize core worm with nematodes, ^{Molina et al. (1996)} identified that S. carpocapsae strain All, S. riobravis and H. megidis Poinar, Jackson & Klein, 1988 have potential as biocontrol agents against S. frugiperda. The LC₅₀ varied from 1.5 to 20.6 and 3.4 to 37.2 mL^-1 nematodes, for larvae and prepupes, respectively, and in their studies the cumulative mortality in pupae was 5-43 % with the concentration of 100 mL^-1 nematodes.

Entomopatogenic bacteria

The greatest success in the microbial control of insects in the world has been achieved by Bacillus thuringiensis Berliner, 1915 (Bt). The crystals (Cry) that it produces under stress conditions are aggregates of a large protein (130-140 kDa) that is not really active in itself (it is a protoxin) as it is insoluble. When protoxin is subjected to very basic conditions (pH > 9.5) such as those existing in the intestines of some insects, it is solubilized and transformed by means of the insect proteases into an active toxin of about 60 kDa. This is the toxin known as "δ-Bt endotoxin", which acts by binding to receptors of epithelial cells of the intestine of the insect, which leads to the formation of pores and osmotic lysis of the cells that finally they cause their death (^{Galitsky et al., 2001}).

At present, the gene responsible for the production of this endotoxin has been introduced into tobacco, corn, tomato, cotton, potato, beet and cabbage plants, among other crops, giving rise to genetically modified (GMO) crops. The wide acceptance of GM crops, especially in the United States of America, has increased the yield per hectare and the income of farmers in these countries. The first success achieved in this field was obtained in 1987 when it was possible to produce transgenic tobacco plants capable of producing by themselves a toxin of the bacteria B. thuringiensis, which had lethal effects for certain phytophagous insects (^{Rubio and Fereres, 2005}).

Some entomopathogenic bacteria have been developed for the control of commercial scale insect pests, among which the subspecies of Bacillus thuringiensis, Lysinibacillus sphaericus Neide, 1904, Paenibacillus spp. and Serratia entomophila Grimont. The subspecies B. thuringiensis kurstaki is the most commonly used for the control of insect pests of crops and forests, and the subspecies israelensis and L. sphaericus of B. thuringiensis are the main pathogens used for the control of pests of medical importance (^{Ponce et al., 2003}).These pathogens combine the advantages of chemical pesticides and biological control agents: they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systems systemic (i.e. in transgenic plants) (^{De la Rosa et al., 2005}; ^{Camacho et al., 2017}; ^{García et al., 2018}).

There is very little experience on the use of bacteria for pest control In Mexico, and these are limited to the use of Bacillus thuringiensis for tobacco worm control, the sugarcane borer worm and the coffee drill in the agricultural sector (Table 5), and for control of the mosquito vector of the dengue virus.

In the case of the coffee drill (Hypothenemus hampei Ferrari, 1867), its most susceptible stage was its first larval instar, with an average lethal time average of 6.4 ± 1.8 days (^{De la Rosa et al., 2005}). For the sugarcane borer worm (Diatraea consideta Heinrich, 1931), ^{Camacho et al. (2017)} managed to isolate eight strains of dead insects in agricultural fields, which were from B. thuringiensis and observed a high mortality with the strains of interest.

From different tobacco plants obtained from southeastern Mexico, ^{García et al (2018)} made isolates, from which they selected bacterial colonies of Bacillus thuringiensis, which caused 100 % mortality of Manduca sexta Linnaeus larvae, 1763 at 96 h of exposition. In other studies against tobacco pests the toxicity of Bacillus thuringiensis ssp Kenyae was demonstrated against eight species of Lepidoptera, one of Coleoptera and one of Diptera (^{Barboza et al., 1998}).

On the other hand, against the control of the mosquito vector of the dengue virus (Aedes aegypti), in 2003 the bioinsecticide Vectobac 12 AS was formulated based on B. thuringiensis var israelensis Barjac, 1978 (Bti). The product was applied in pipe trucks that deliver water to several communities in the metropolitan area of Monterrey, NL; in this way, the people received it with the means to interrupt the biological cycle of the virus. Bti proved effective as a larvicide against A. aegypti even in the presence of chlorine in the water. However, the results showed that the efficiency of Bti applied in pipes was reduced mainly due to water temperature, larval density, sunlight and the effect of association with filter organisms (^{Ponce et al., 2003}).

Entomopathogenic virus

Insect pathogenic viruses are an important source of microbial control agents, particularly for the control of lepidopteran pests. Baculoviruses are accepted as safe, easily mass-produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining greater market share. However, the absence of a practical in vitro mass production system, higher production costs, limited persistence after application, slow death rate and high host specificity contribute to its restricted use in control Of pests. Overcoming these limitations are key research areas for which progress could open the use of insect viruses to much larger markets. The Baculoviridae family is the most numerous and studied of entomopathogenic viruses. The use of the Anticarsia gemmatalis Hübner 1818 NPV nucleopoliedrovirus, (AgMNPV) to control A. gemmatalis in soybeans in Brazil was a successful program and was considered the most important in the world (^{Nava, 2012}).

Biopesticides based on entomopathogenic organisms

Currently, there are several companies mainly in Holland, France, Italy, Great Britain and Russia, which sell products for biological control with entomopathogenic organisms (^{Rubio and Fereres, 2005}). The fact of marketing products with living organisms has its limitations, among which stand out:

Until almost 10 years ago, the commercial production of bioinsecticides and other biological control agents in Mexico was carried out in at least 68 companies and 25 states, although these numbers have now increased; they reproduce entomopathogenic fungi (mainly Beauveria bassiana and Metarhizium anisopliae; entomopathogenic bacteria (Bacillus thuringiensis) and nematodes (Heterorhabditis bacteriophora and Steinernema carpocapsae. These microorganisms are the basic active ingredients in the formulation of bioinsecticides (Table 6); carrier, an inert material as a support, and adjuvants, as well as compounds that promote and maintain the viability of the active ingredient and protect it from UV radiation, rain, moisture and dehydration, which facilitates its handling, application and effectiveness (^{García and Mier, 2010}).

The regulations in Mexico (NOM-032-FITO-1995) (^{Sagarpa, 2015}) indicate that for the registration and commercialization of bioinsecticides, official provisions must be complied with and the opinion of different institutions that regulate crop health, the risk of the substances that are used as pesticides must be met, as well as care for the environment and human health (^{García and González, 2013}). Likewise, it is necessary to carry out biosecurity studies and risk of the environmental impact of the agents used in biological control (qualitative and quantitative evaluation), environmental monitoring of these organisms and assessment of their effect on non-target organisms.