Paecilomyces and Its Importance in the Biological Control of Agricultural Pests and Diseases

Incorporating beneficial microorganisms in crop production is the most promising strategy for maintaining agricultural productivity and reducing the use of inorganic fertilizers, herbicides, and pesticides. Numerous microorganisms have been described in the literature as biological control agents for pests and diseases, although some have not yet been commercialised due to their lack of viability or efficacy in different crops. Paecilomyces is a cosmopolitan fungus that is mainly known for its nematophagous capacity, but it has also been reported as an insect parasite and biological control agent of several fungi and phytopathogenic bacteria through different mechanisms of action. In addition, species of this genus have recently been described as biostimulants of plant growth and crop yield. This review includes all the information on the genus Paecilomyces as a biological control agent for pests and diseases. Its growth rate and high spore production rate in numerous substrates ensures the production of viable, affordable, and efficient commercial formulations for agricultural use.


Introduction
The genus Paecilomyces was first described in 1907 [1] as a genus closely related to Penicillium and comprising only one species, P. variotii Bainier. The description of this genus was revised by Brown and Smith [2], and Samson [3] defined 31 species divided into two sections: Paecilomyces characterized by thermophilic, thermotolerant, and mesophilic species, with yellow-brown colonies showing teleomorphic states corresponding to the genera Byssochlamys, Talaromyces, and Thermoascus; and Isarioidea characterized by mesophilic species with purple, pink, yellow, or green colonies. The former section includes the nematophagous or entomopathogenic species, also known as Paecilomyces lilacinus or P. fumosoroseus [4,5]. The different species in Paecilomyces are related to two genera of fungi: Cordyceps and Torrubiella [3].
Studies carried out by Luangsa-ard et al. [6] and Inglis and Tigano [4] confirm the polyphyletic origin of this genus that belongs to the Sordariomycetidae and Eurotiomycetidae subclasses. The Fungorum database [7] currently includes a list of 145 Paecilomyces species. Of all the species, some retain their original name, while others have been reclassified into other genera. One clear example is Paecilomyces lilacinus (Thom) Samson, which has been assigned to the genus Purpureocillium [8]. Despite its re-assignment to a different genus, Paecilomyces lilacinus will be included in this literature review, because of its importance in disease and pest control. Given the polyphyletic nature of the genus Paecilomyces, the evolution of these taxonomic studies is of great importance for developing microbial formulations that can be used in agriculture [9].
The genus Paecilomyces has hyaline to yellowish septate hyphae, often with smooth walls and verticillated or irregularly branched conidiophores, and phialides with a wide base and an elongated neck. The conidia are unicellular; hyaline, in chains; and the youngest conidium is at the basal end [10]. The conidial thermotolerance is correlated with their size and shape. Thus, the smaller and more spherical asexual conidia or ascospores are more vulnerable to high temperature [11][12][13][14]. Paecilomyces has high growth sporulation rates and grows over a wide range of temperatures and substrates. As a result, its rapid multiplication ensures viable and affordable development of commercial formulations [15].
The genus Paecilomyces has many species, both pathogenic and saprophytic, and can be found in a wide range of habitats, including soil [16,17], decomposing plant material or food [18,19], pasteurized food products [2,20,21] Paecilomyces also plays a significant role as an endophyte in numerous plants by providing several advantages for plant development. It can be used directly or indirectly as a potential biostimulant. When used directly, Paecilomyces or its metabolites increases the plant morphological parameters and crop yield [15,[34][35][36][37]. The plant-Paecilomyces interaction improves plant health through different mechanisms and provides protection from phytopathogens [38]. This interaction showed a production of phytohormones, such as gibberellins and indole-acetic acid, that promoted growth and mitigated the effects of abiotic stress, such as salinity [39,40]. When used indirectly in combination with pathogenic agents such as nematodes or fungi, Paecilomyces has positive effects on crop growth by acting as a biological control agent [41][42][43][44].
Numerous species of the genus Paecilomyces produce a wide variety of secondary metabolites with different chemical structures and diverse biological activities, such as herbicidal [45], insecticidal [46,47], bactericidal [48], fungicidal [49], nematicidal [50][51][52] or cytotoxic [53]. There are also descriptions of metabolites with antitumour activity [54,55] or enzyme inhibitors, such as Paecilomide, which acts as an acetylcholinesterase inhibitor and can be used to control human diseases such as Alzheimer's [56]. In addition, they have a role in aromatic compound degradation [57,58], ethanol production from agro-industrial wastes [59], or removal of ammonium from synthetic media or ammonia emission reduction in poultry manure [60,61]. Nevertheless, we cannot ignore the fact that Paecilomyces has been associated with several human infectious diseases in immunosuppressed patients [62,63] and has also been described as a phytopathogen. P. variotii was described by Aminaee et al. [64], as the causal agent of pistachio dieback, although subsequent molecular and phylogenetic studies reported that it was caused by P. formosus (Sakag., May., Inoue and Tada) Houbraken and Samsoninstead [65]. According to the map shown in Figure 1, there are few species of Paecilomyces responsible for the biological control of pests and diseases.
In this review, we will analyse the significant role of Paecilomyces in plant pest and disease control. In this sense, Paecilomyces is described as a biological control agent against bacteria, phytopathogenic fungi, nematodes, and numerous pests, using its extracts, secondary metabolites, or mycelium. To our knowledge, this is the first review of the genus Paecilomyces as a biological control agent against plant pests and diseases.  VOSviewer software (version 1.6.15, Leiden University, The Netherlands) was used to map the frequency of keyword co-occurrence networks. Differences in font size imply differences in relevance. The different colors refer to the groups or clusters formed.

Biological Control Mechanisms of the Genus Paecilomyces
Although many biological control mechanisms are unknown, advances in metagenomics provide some information on the plant-pathogen-antagonist interaction [66,67]. In the genus Paecilomyces, microbial mechanisms involved in pest and disease suppression have been direct, such as parasitism, competition or antibiosis, and indirect, which involve plant protection through induced systemic resistance (ISR) mechanisms [68][69][70].
Various studies refer to the nematicidal activity of Paecilomyces. Species of this genus, namely P. lilacinus, can penetrate both the eggshells and structural components of juvenile and adult stages of different species of nematodes through spore germination and subsequent hyphal branching and appresoria formation [92,93]. Regarding the production of lytic enzymes causing a nematicidal effect, the synthesis of amylases, lipases, proteases, and chitinases associated with this species has been described [77,78,85,87,94,95]. Overexpression of genes regulating the synthesis of these enzymes increases P. lilacinus virulence and parasitic ability against Meloidogyne incognita, Panagrellus redivivus, and Caenorhabditis elegans [96,97].
While this mechanism has a direct impact on control, competition is often accompanied by other mechanisms [70]. The rapid growth of Paecilomyces species prevents the development of certain pathogens [105,106]. For instance, spraying sunflower seeds with P. variotii spores prevents penetration and infection by pathogen Macrophomina phaseolina [107]. However, this competition can sometimes have a negative impact on the rest of the beneficial microbiota [108].

Antibiosis
The production of secondary metabolites with antimicrobial effect by Paecilomyces species has been widely described. Among them, we can highlight the synthesis of alkaloids, phenolic compounds, volatile organic compounds, steroids, flavonoids, peptides, polyketides, quinones and terpenoids [109,110]. Li et al. [111] recently described a total of 148 active metabolites produced by different Paecilomyces species that can be used for drug or agrochemical development. In the following sections, we will show the importance of these metabolites in the biological control of pests and diseases.

Induced Resistance in Plants
The literature does not provide many examples on the effect of induced resistance after Paecilomyces colonizes the root system of a plant. Suárez-Estrella et al. [112] observed that inoculation of tomato plant roots with P. variotii significantly inhibited the signs caused by the bacterium Xanthomonas campestris on leaves. Similarly, López et al. [113] observed a reduction in the Aphis gossypii population in cotton plants whose seeds had been previously inoculated by being immersed in a P. lilacinus spore suspension. The combined use of P. lilacinus and salicylic acid improved the cellulose, hemicellulose, lignin, and pectin contents in cotton plants compared to inoculation treatments using Pythium debaryanum and Fusarium oxysporum, which showed that wall lignification provides a high level of protection against pathogen invasion. Likewise, concentration of soluble proteins and phenolic compounds increased in the root, which reduced the incidence of both diseases [114]. This also occurs when okra plants are inoculated with P. lilacinus [115].
Similarly, the effect of induced resistance can also be produced by Paecilomyces extracts. A commercial extract of P. variotii known as ZhiNengCong (ZNC) could also induce resistance against Xanthomonas oryzae or Pseudomonas syringae in rice plants or Arabidopsis, respectively. A dose of 500 ng/mL of ZNC could not inhibit the development of pathogens in vitro, while the use of a smaller dose of 100 ng/mL did generate immunity against said bacteria. On the other hand, reactive oxygen substances such as superoxide and hydrogen peroxide or callose also increase, compared to untreated Arabidopsis plants, in addition to activating salicylic acid synthesis, which is necessary for the defence response [36].

Biological Control of Diseases Caused by Phytopathogenic Bacteria
Few studies show the effectiveness of Paecilomyces against different species of phytopathogenic bacteria. Paecilomyces variotii isolated from municipal solid waste compost showed a reduction in 27% of diseases caused by X. campestris in melon, and a decrease in the pathogen population [112]. Nesha and Siddiqui [44] observed a reduction in soft rot and leaf blight caused by P. carotovorum pv. carotovorum and X. campestris pv. carotae after using P. lilacinus, alone or in combination with A. niger and an increase in the dry weight and chlorophyll content of a carrot crop.
Metabolites produced by this genus play a significant role in disease control due to its antagonistic effect, although there is little information on the matter compared to phytopathogenic bacteria. There are descriptions on the importance of antibacterial metabolites such as viriditoxin or betulin against non-phytopathogenic bacteria such as S. aureus, Enterococcus sp. Micrococcus sp., Aeromonas Hydrophila, Flavobacterium sp., Pseudomonas aeruginosa, and Vibrio cholera [116,117]. Sornakili et al. [83] recently reported the inhibition of Erwinia carotovora, Xanthomonas oryzae pv. oryzae, and Ralstonia solanacearum with in vitro inhibition between 13-45% using P. tenuis, an endophyte isolated from rice leaves. Various metabolites, such as octadecanoic acid, acetic acid, and 2-ethylhexyl ester, as well as enzymatic activities, xylanases, cellulases, and lipases, were involved in this control.

Biological Control of Diseases Caused by Phytopathogenic Fungi
Various Paecilomyces species have shown their antagonistic effect against phytopathogenic fungi causing root and aerial plant diseases through various mechanisms (Table 1). P. variotii and P. lilacinus species have proven to be quite effective, although most studies are in vitro. The antagonistic effect observed in most cases is explained by a competition for space and nutrients ( Figure 2). However, other mechanisms associated with secondary metabolite production have been observed, which cause plasmolysis in spore germ tubes or hyphal melanisation in Pyrenophora tritici-repentis [118], hyphal lysis in Moniliophthora roreri caused by Paecilomyces sp. [71], mycoparasitism of F. oxysporum caused by P. variotii and P. lilacinus [119], or antibiosis against R. solani [120], among others. Viriditoxin, sphingofungins E and F [121], or eicosenoic acids are reported to have an antifungal effect against various phytopathogenic fungi such as Biscogniauxia mediterranea, Phytophthora cinnamomi or Fusarium moniliforme [61]. Varioxepin A or 6-Pentyl-α-pyrone inhibits perithecia formation and mycelial growth of Fusarium graminearum [122,123] or Paecylaminol, which inhibits soft rot development in tomatoes caused by Mucor racemosus [124].

Biological Control of Diseases Caused by Phytopathogenic Bacteria
Few studies show the effectiveness of Paecilomyces against different species of phytopathogenic bacteria. Paecilomyces variotii isolated from municipal solid waste compost showed a reduction in 27% of diseases caused by X. campestris in melon, and a decrease in the pathogen population [112]. Nesha and Siddiqui [44] observed a reduction in soft rot and leaf blight caused by P. carotovorum pv. carotovorum and X. campestris pv. carotae after using P. lilacinus, alone or in combination with A. niger and an increase in the dry weight and chlorophyll content of a carrot crop.
Metabolites produced by this genus play a significant role in disease control due to its antagonistic effect, although there is little information on the matter compared to phytopathogenic bacteria. There are descriptions on the importance of antibacterial metabolites such as viriditoxin or betulin against non-phytopathogenic bacteria such as S. aureus, Enterococcus sp. Micrococcus sp., Aeromonas Hydrophila, Flavobacterium sp, Pseudomonas aeruginosa, and Vibrio cholera [116,117]. Sornakili et al. [83] recently reported the inhibition of Erwinia carotovora, Xanthomonas oryzae pv. oryzae, and Ralstonia solanacearum with in vitro inhibition between 13-45% using P. tenuis, an endophyte isolated from rice leaves. Various metabolites, such as octadecanoic acid, acetic acid, and 2-ethylhexyl ester, as well as enzymatic activities, xylanases, cellulases, and lipases, were involved in this control.

Biological Control of Diseases Caused by Phytopathogenic Fungi
Various Paecilomyces species have shown their antagonistic effect against phytopathogenic fungi causing root and aerial plant diseases through various mechanisms (Table 1). P. variotii and P. lilacinus species have proven to be quite effective, although most studies are in vitro. The antagonistic effect observed in most cases is explained by a competition for space and nutrients ( Figure 2). However, other mechanisms associated with secondary metabolite production have been observed, which cause plasmolysis in spore germ tubes or hyphal melanisation in Pyrenophora tritici-repentis [118], hyphal lysis in Moniliophthora roreri caused by Paecilomyces sp. [71], mycoparasitism of F. oxysporum caused by P. variotii and P. lilacinus [119], or antibiosis against R. solani [120], among others. Viriditoxin, sphingofungins E and F [121], or eicosenoic acids are reported to have an antifungal effect against various phytopathogenic fungi such as Biscogniauxia mediterranea, Phytophthora cinnamomi or Fusarium moniliforme [61]. Varioxepin A or 6-Pentyl-α-pyrone inhibits perithecia formation and mycelial growth of Fusarium graminearum [122,123] or Paecylaminol, which inhibits soft rot development in tomatoes caused by Mucor racemosus [124].  Some in vivo studies show a direct effect on plant growth promotion after using Paecilomyces [15], but also an indirect effect due to fungal disease control [72]. Yang et al. [125] observed inhibited S. sclerotiorum mycelial growth and sclerotia germination and a reduced disease severity after using P. lilacinus on a rapeseed crop. Results did not show differences after using spores or filtering without fungi cells, which highlighted the importance of Paecilomyces metabolites in pathogen control. In tomatoes, spraying P. variotii spores on the leaves significantly reduces damage caused by Alternaria solani [126]. On the other hand, the increase in polyphenols and antioxidant activity due to the use of P. lilacinus on okra roots improves plant development and control of various phytopathogenic fungi causing root rot [115]. Likewise, prior use of P. fumosoroseus delays the development of powdery mildew caused by Podosphaera xanthii [127], leading to mycelium and spore destruction due to the close contact of fungi and with some degree of mycoparasitism depending on the environmental conditions.

Biological Control of Diseases Caused by Nematodes
As a nematophagus fungus, Paecilomyces has been widely studied and can be found in a variety of biological formulations for agricultural use [93]. There are many examples where Paecilomyces spp. act as nematicidal agents, especially against Meloidogyne spp., but also against other genera such as Globodera [52], Rotylenchulus, Heterodera, Xiphinema or Pratylenchus [51] ( Table 2). One example is the use of P. lilacinus and P. fumosoroseus against M. incognita or M. javanica, which drastically reduces their populations [44,51,148,149], in both in vitro [43,87] and field tests [50,150]. The spores of these species must germinate on the host to penetrate and colonize its surface, in order to modify its physiology [51]. Paecilomyces acts according to the fungal and nematode species it parasitizes.
Paecilomyces spp. can act at different nematode developmental stages by infecting eggs, young or adult nematodes. Nematode eggshell is the main barrier against parasite agents and provides resistance to both chemical nematicides and biological compounds. Paecilomyces species are capable of secreting enzymes to degrade this barrier and deploying mechanisms involved in nematode parasitism [151,152]. Thus, observations have shown that Meloidogyne incognita eggs at early stages of development are more vulnerable than eggs containing fully developed juveniles, although the latter are also affected [153][154][155]. Hollan et al. [76] confirm that eggs are parasitized by P. lilacinus at all stages, including unhatched juveniles. Egg infection occurs when hyphae lie flat on the egg surface and appresoria are formed. Then, the fungus spreads and conidiophores are formed. Studies carried out by Khan et al. [92] concluded that said juveniles show various degrees of deformities and developmental abnormalities, such as reduced mobility inside the eggs. Different studies show the significant role of proteases and chitinases in the penetration of the fungus through eggshells. Thus, M. arenaria eggshells showed vitelline membrane disaggregation, and chitin and lipid layer destruction after using P. lilacinus [156].
Juvenile M. hapla eggs were highly vulnerable to serine proteases produced by P. lilacinus than eggs containing more developed juveniles. On the contrary, larvae showed no signs of damage. Jatala et al. [157] reported that P. lilacinus is capable of infecting female Meloidogyne spp. and Heterodera spp. and Globodera spp. cysts. In these cases, hyphae entered through natural openings of the body [158]. Evidence shows that various hydrolytic proteins, such as proteases (mainly serine proteases), collagenases and chitinases are involved in nematode cuticle penetration and subsequent cell degradation [77,97,[159][160][161]. Likewise, different secondary metabolites produced by Paecilomyces also play a significant role in nematode control [162].
Nematode control effectiveness using Paecilomyces depends on the crop itself, as it affects fungal activity in many cases [163]. Thus, the use of an antagonist in combination with organic substances increases parasitism by Paecilomyces in both eggs and larvae of nematodes [164]. On the other hand, it has been reported that the use of P. lilacinus on recently solarised soil does not increase control effectiveness compared to non-solarised soil. However, a certain reduction in fungal activity is observed when both techniques are applied [165]. When comparing effectiveness using chemical compounds, P. lilacinus provides adequate control during crop growth, although the combination of both techniques shows better results compared to nematode control. [119,[166][167][168][169].
As shown in Table 2, P. lilacinus is the most important nematophagous fungus, as it is capable of controlling various nematode species in different crops, though other species such as P. marquandii (Massee) S. Hughes [170][171][172] or P. variotii [173] can be equally effective. Reports by Chen et al. [171] on the use of P. marquandii against M. hapla showed an increase in lettuce weight, a decrease in gall formation by 25.7% and a reduction in egg production by 46.3%. According to Al-Assas, et al. [174], P. variotii reduces the number of galls by more than 90%, showing more effectiveness compared to chemical compounds.

Biological Control of Diseases Caused by Arthropods
The genus Paecilomyces includes multiple species described as pest control agents capable of providing natural control without the need for exogenous applications [225], many of which have been tested under controlled conditions for elaborating bioinsecticides to fight pests of great economic importance worldwide [226,227]. Among entomopathogenic fungi, Paecilomyces species are viable sources to elaborate mycoinsecticides, as affordable stable propagule substrates such as blastospores or conidia can be easily produced on a large scale [228]. According to Ruiu [229], bioformulations containing mainly P. lilacinus and P. fumosoroseus have been commercialised for pest control.
However, initial results obtained under in vitro culture conditions were not always consistent when assessing their effectiveness under field conditions. For this reason, parameters such as the application method should be assessed. In this case, most tests under controlled laboratory conditions are assessed by immersing samples in Paecilomyces spp. conidia suspensions, which provide clear results on the infectivity of the tested species [230]. Then, in planta tests are conducted under semi-natural conditions to assess effectiveness by spraying infected seedlings with conidial suspensions inside closed structures to prevent insects from going in or out [231]. Finally, mortality is assessed under field conditions by sprinkling crops showing a specific pest density with pre-commercial Paecilomyces spp. formulations [232]. In this sense, new application methods are currently being assessed, such as the one described by López et al. [113], where P. lilacinus used as an endophyte on cotton seeds provides induced resistance to plants by causing negative effects on Aphis gossypii feeding and reproduction.
Environmental conditions at the time of application are crucial and high temperatures and relative humidity are the most favourable for infection. In this sense, P. fumosoroseus caused a mortality of 60%, 80% and 85% in Myzus persicae, and of 90%, 95% and 100% in Aphis fabae, at 10 • C, 18 and 23 • C, respectively [233]. Regarding humidity, Demirci et al. [121] reported that I. farinosa showed increased pathogenicity against Planococcus citri under high relative humidity conditions at the time of application. Another aspect to bear in mind is the insects' physiological state or size. Nymphal and larval stages tend to be more vulnerable than eggs as they have defence structures in their chorion. P. fumosoroseus is capable of affecting whitefly Aleurodicus cocois at various developmental stages [234]. Similarly, the physical barriers of Leptinotarsa decemlineata pupae make them more resistant than their larvae to Isaria fumosorosea infection [235]. In terms of size, Hunter et al. [236] showed a negative correlation between insect mortality and size mainly because larger sizes are associated with thicker cuticles, as in the use of I. fumosorosea on Diaphorina citri (psyllid) and D. citri (curculionidae), in which case the latter is bigger. Insect integument sclerotisation is also important, as it has an impact on Paecilomyces spp. ease of penetration and infection [237]. In order to avoid these obstacles, the use of formulations containing Paecilomyces spp. with high conidia densities is advised, as well as a focalised and prolonged exposure, to obtain an improved control effect against insects [238].
Paecilomyces has been described to control pests by limiting insect growth as a result of reduced feeding [236,239] reproduction [240] or simply causing their death due to mycosis [241]. In addition, it has been shown that P. fumosoroseus is capable of causing more deaths than some commercial insecticides such as fipronil when used against Frankliniella occidentalis [242]. Similar to when they act as nematophagous fungi, the potential of Paecilomyces spp. as a biological control agent that parasitises insects by penetrating their cuticle and subsequently spreading through haemolymph has been described [243]. This is possible owing to the excretion of enzymes, such as protease or chitinase synthesis [17,90], or different types of toxins, such as beauvericin [244], dipicolinic acid [46] or dibutyl succinate [245], which are described as bioactive metabolites with insecticidal or insect repellent effects, which turns them into significant virulence factors. Numerous orders of arthropods that are vulnerable to the use of Paecilomyces spp. (Table 3), including hemiptera, have been described, such as aleurodids [246], aphids [238], thysanoptera [242], diptera [247], lepidoptera [73], hymenoptera [248] and coleoptera [235].  In vitro [331] Paecilomyces spp.

Conclusions
The loss of pesticide effectiveness against certain pathogens, waste limitation in harvested products, the problems that these products cause to the environment and human health, and the ineffectiveness of genetic resistance due to quick alterations in pathogen virulence require the development of new control methods. While it is currently difficult to reduce the total amount of chemical active substances without causing losses in production, their gradual decrease and the use of bioestimulants can help optimize the use of chemical products and reduce environmental pollution. This review is the first to gather information on the potential of various Paecilomyces species as biological control agents against multiple diseases and pests, using different mechanisms of action and/or specificity that can be used in combination with cultural and chemical control in agriculture.