Entomopathogen Isolation | Encyclopedia MDPI

Entomopathogen Isolation: Comparison

Please note this is a comparison between Version 2 by Vivi Li and Version 1 by Lav Sharma.

Microbes that cause infections amongst insects leading to death or serious disabilities are known as entomopathogens. Entomopathogenic bacteria and fungi are quite frequently found in soils and insect cadavers. The first step in utilizing these microbes as biopesticides is to isolate them, and several culture media and insect baiting procedures have been tested in this direction. In this work, the authors review the current techniques that have been developed so far, in the last five decades, and display brief protocols which can be adopted for the isolations of these entomopathogens. Among bacteria, this study focuses on

Serratia

spp. and bacteria from the class Bacilli. Among fungi, the review focuses those from the order Hypocreales, for example, genera

Beauveria

Clonostachys

Lecanicillium

Metarhizium

, and

Purpureocillium

. The authors chose these groups of entomopathogenic bacteria and fungi based on their importance in the microbial biopesticide market.

Beauveria,Metarhizium,Hypocreales,Bacillus thuringiensis,Serratia
Beauveria
Metarhizium
Hypocreales
Bacillus thuringiensis
Serratia

1. Isolation of Entomopathogenic Bacteria

Entomopathogenic bacteria are commonly found in soils. Hence, isolating insect-pathogenic strains is quite important. Different bacterial groups, such as symbionts of entomopathogenic nematode (EPN)

Heterorhabditis

spp. and

Steinernema

spp., i.e.,

Photorhabdus

spp. and

Xenorhabdus

spp., and others, such as

Yersinia entomophaga

Pseudomonas entomophila

, and

Chromobacterium spp., exhibit entomopathogenicity [18].

spp., exhibit entomopathogenicity ^[1].

Entomopathogenic nematode symbiotic bacteria are isolated by dropping an insect’s hemolymph onto a nutrient bromothymol blue (0.0025% (

w/v

)) triphenyltetrazolium chloride (0.004% (

w/v)) agar (NBTA) and incubating the streaked plate at 25 °C, and continuously subculturing until the uniform colonies are obtained [19].

)) agar (NBTA) and incubating the streaked plate at 25 °C, and continuously subculturing until the uniform colonies are obtained ^[2].

Yersinia entomophaga

is isolated by culturing the hemolymph of diseased larvae of New Zealand grass grub,

Costelytra zealandica

White (Coleoptera: Scarabaeidae), onto Luria-Bertani (LB) agar, followed by growth on Caprylate-thallous agar (CTA) (

Appendix A

, Medium 1) and Deoxyribonuclease (DNase)-Toluidine Blue agar (

Appendix A, Medium 2), and no hemolysis on Columbia horse blood agar (Columbia agar + 5% horse blood) or Columbia sheep blood agar (Columbia agar + 5% sheep blood) [20]. Isolating

, Medium 2), and no hemolysis on Columbia horse blood agar (Columbia agar + 5% horse blood) or Columbia sheep blood agar (Columbia agar + 5% sheep blood) ^[3]. Isolating

P. entomophila

is rather tricky as the bacterium needs to elicit the systemic expression of Diptericin, an antimicrobial peptide in

Drosophila, after ingestion. However, the bacterial culture can be maintained on LB media [21]. Bacterial isolates from insects belonging to

, after ingestion. However, the bacterial culture can be maintained on LB media ^[4]. Bacterial isolates from insects belonging to

Chromobacterium exhibit violet pigment when cultured on L-agar [22]. However, EPB that are most commonly used as commercial biopesticides are further discussed in the review.

exhibit violet pigment when cultured on L-agar ^[5]. However, EPB that are most commonly used as commercial biopesticides are further discussed in the review.

1.1. Milky Disease-Causing Paenibacillus spp.

Paenibacillus popilliae

and

Paenibacillus lentimorbus are obligate pathogens of scarabs (Coleoptera) as they require the host for the growth and sporulation. In soils, they are present as endospores. These bacteria can be isolated from the hemolymph, and the methodologies may vary depending on the bacterial species. The protocols listed below have been described by Stahly et al., and more details of these protocols have been reported by Koppenhöfer et al. [23,24,25].

are obligate pathogens of scarabs (Coleoptera) as they require the host for the growth and sporulation. In soils, they are present as endospores. These bacteria can be isolated from the hemolymph, and the methodologies may vary depending on the bacterial species. The protocols listed below have been described by Stahly et al., and more details of these protocols have been reported by Koppenhöfer et al. ^[6][7][8].

(a): Disinfect the surface of the larvae of grubs (Coleoptera) with 0.5% (

(a) Disinfect the surface of the larvae of grubs (Coleoptera) with 0.5% (

v) sodium hypochlorite (NaOCl).
(b): Pinch the cadaver using a sterilized needle and collect the emerging drops in sterilized water.
(c): Culture the dilutions of the drops on St. Julian medium (J-Medium) (

) sodium hypochlorite (NaOCl).

(b) Pinch the cadaver using a sterilized needle and collect the emerging drops in sterilized water.

Appendix A, Medium 1) [26], or Mueller-Hinton broth, yeast extract, potassium phosphate, glucose, and pyruvate (MYPGP) (

, Medium 1) ^[9], or Mueller-Hinton broth, yeast extract, potassium phosphate, glucose, and pyruvate (MYPGP) (

Appendix A, Medium 2) agar [27].

, Medium 2) agar ^[10].

Note: To enhance the germination of the vegetative cells, using 0.1% (

v) tryptone solution is recommended during bacterial dilutions [26]. For spores, it is advisable to heat them for 15 min in a 1 M calcium chloride solution (pH 7.0) at 60 °C, and suspend them in the hemolymph of the cabbage looper

) tryptone solution is recommended during bacterial dilutions ^[9]. For spores, it is advisable to heat them for 15 min in a 1 M calcium chloride solution (pH 7.0) at 60 °C, and suspend them in the hemolymph of the cabbage looper

Trichoplusia ni Hübner (Lepidoptera: Noctuidae) and in tyrosine at an alkaline pH. Another way to improve the germination is to heat the spores at 75 °C for 30 min and then apply pressure using a French press [28].

Hübner (Lepidoptera: Noctuidae) and in tyrosine at an alkaline pH. Another way to improve the germination is to heat the spores at 75 °C for 30 min and then apply pressure using a French press ^[11].

1.2. Amber Disease-Causing Serratia spp.

Serratia

spp. are quite frequently isolated from soils, and some of them, being saprophytes, can also be isolated from insect cadavers. Therefore, to enhance the growth of insect pathogenic

Serratia

spp. such as

Serratia entomophila

Serratia proteamaculans

, and

Serratia marcescens, a methodology based on a selective agar medium has been described by O’Callaghan and Jackson [30].

, a methodology based on a selective agar medium has been described by O’Callaghan and Jackson ^[12].

(a): Soil inoculums or hemolymph of the diseased larvae can be isolated on Caprylate-thallous agar (CTA) (

(a) Soil inoculums or hemolymph of the diseased larvae can be isolated on Caprylate-thallous agar (CTA) (

Appendix A, Medium 3) [31].
(b): Culturing is done by pulling and separating the anterior end of the cadavers. The gut contents are then cultured on CTA plates.
(c)

, Medium 3) ^[13].

(b) Culturing is done by pulling and separating the anterior end of the cadavers. The gut contents are then cultured on CTA plates.

(c)

Serratia marcescens

produces colonies that are red in color. Cream-colured bacterial colonies formed on CTA can then be transferred into different selective media for the identification of

Serratia spp. [30].
(d): The production of a halo on a Deoxyribonuclease (DNase)-Toluidine Blue agar (

spp. ^[12].

(d) The production of a halo on a Deoxyribonuclease (DNase)-Toluidine Blue agar (

Appendix A

, Medium 4) when incubated at 30 °C for 24 h, indicates the presence of

Serratia spp. [32]. Thereafter, the production of blue or green colonies on adonitol agar (

spp. ^[14]. Thereafter, the production of blue or green colonies on adonitol agar (

Appendix A

, Medium 5) confirms

S. proteamaculans

. The formation of yellow colonies on adonitol agar hints the presence of

S. entomophila

, which can be confirmed by the growth on itaconate agar (

Appendix A, Medium 6) at 30 °C after 96 h [25]. Further molecular approaches targeting specific DNA regions can distinguish pathogenic strains from the non-pathogenic ones.

, Medium 6) at 30 °C after 96 h ^[15]. Further molecular approaches targeting specific DNA regions can distinguish pathogenic strains from the non-pathogenic ones.

2. Isolation of Entomopathogenic Fungi

Fungal entomopathogens can directly be isolated from insect cadavers in the case of visible mycosis [35]. Moreover, they can also be isolated from soils or phylloplane as they spend a considerable part of their life as saprophytes in soils or as plant endophytes. However, to our knowledge, their survival as soil saprophytes has not been proven yet [4,5,6,7,8,35,36]. In either case, the material can be cultured directly onto a medium selective for an EPF or the material can be baited with an infection-sensitive insect [37]. In case of the isolation of EPF as endophyte, proper disinfection of the material is needed. Nonetheless, different antibacterial and fungal saprophyte-inhibiting chemicals are added in the selective medium, as per the research interest. Here, different culture media used to isolate fungal entomopathogens, especially those belonging to the order Hypocreales are discussed.

Fungal entomopathogens can directly be isolated from insect cadavers in the case of visible mycosis ^[16]. Moreover, they can also be isolated from soils or phylloplane as they spend a considerable part of their life as saprophytes in soils or as plant endophytes. However, to our knowledge, their survival as soil saprophytes has not been proven yet ^{[17][18][19][20][21][16][22]}. In either case, the material can be cultured directly onto a medium selective for an EPF or the material can be baited with an infection-sensitive insect ^[23]. In case of the isolation of EPF as endophyte, proper disinfection of the material is needed. Nonetheless, different antibacterial and fungal saprophyte-inhibiting chemicals are added in the selective medium, as per the research interest. Here, different culture media used to isolate fungal entomopathogens, especially those belonging to the order Hypocreales are discussed.

2.1. Isolations from Naturally Mycosed Insect Cadavers

This method is applied to study the natural EPF infections in the fields as it relies on the collection of the dead insects from the fields. The protocol described below is similar to that employed by Sharma et al. [7].

(a): Insect cadavers are brought to the laboratory as separate entities in sterile tubes.
(b): Insects are observed under a stereomicroscope (40×) for probable mycosis.
(c): In case of a visible mycosis, the insects are surface sterilized using 70% ethanol or 1% NaOCl, for 3 min, followed by 3 distinct washes with 100 mL of sterilized water. Then, the sporulating EPF from the insect cadaver is plated directly.
(d): Cadavers are then cultured on a selective medium at 22 °C for up to 3 weeks, depending on the time taken by the fungi for germination and proliferation. In case of no germination, the cadavers can be homogenized and plated on the selective medium. Details of the different selective medium are provided later in the text.
(e): Obtained fungi are subcultured on potato dextrose agar (PDA) (

(a) Insect cadavers are brought to the laboratory as separate entities in sterile tubes.

(b) Insects are observed under a stereomicroscope (40×) for probable mycosis.

(c) In case of a visible mycosis, the insects are surface sterilized using 70% ethanol or 1% NaOCl, for 3 min, followed by 3 distinct washes with 100 mL of sterilized water. Then, the sporulating EPF from the insect cadaver is plated directly.

(d) Cadavers are then cultured on a selective medium at 22 °C for up to 3 weeks, depending on the time taken by the fungi for germination and proliferation. In case of no germination, the cadavers can be homogenized and plated on the selective medium. Details of the different selective medium are provided later in the text.

(e) Obtained fungi are subcultured on potato dextrose agar (PDA) (

Appendix A

, Medium 8) or Sabouraud dextrose agar (SDA) (

Appendix A, Medium 9) until pure culture is obtained.
(f): Fungi are identified by comparing morphological characteristics using light microscopy (400×), described in several fungal identification keys, such as Domsch et al. [38] and Humber [39].
(g): Molecular identifications can be done by extracting the DNA and performing PCR for the amplification and subsequent sequencing of the nuclear internal transcribed spacer (nrITS) region of the fungal nuclear ribosomal DNA, as described in Yurkov et al. [40].

, Medium 9) until pure culture is obtained.

(f) Fungi are identified by comparing morphological characteristics using light microscopy (400×), described in several fungal identification keys, such as Domsch et al. ^[24] and Humber ^[25].

(g) Molecular identifications can be done by extracting the DNA and performing PCR for the amplification and subsequent sequencing of the nuclear internal transcribed spacer (nrITS) region of the fungal nuclear ribosomal DNA, as described in Yurkov et al. ^[6].

2.2. Isolations from Soils

2.2.1. Soil Suspension Culture

This method is generally used to isolate a particular EPF genus of interest using different concentrations of the soil inoculums. To ensure correct isolation, the isolated EPF should also be characterized morphologically and molecularly, as described in Section 3.1. Here the authors discuss various selective media used, especially those which are useful for the isolation of the hypocrealean fungi pertaining to their dominance in fungi-based microbial pesticide market.

This method is generally used to isolate a particular EPF genus of interest using different concentrations of the soil inoculums. To ensure correct isolation, the isolated EPF should also be characterized morphologically and molecularly. Here the authors discuss various selective media used, especially those which are useful for the isolation of the hypocrealean fungi pertaining to their dominance in fungi-based microbial pesticide market.

Metarhizium spp.

Isolating EPF has always been challenged by the contamination from saprophytic fungi. In this direction, Veen and Ferron [49] suggested using dodine (

spp.

Isolating EPF has always been challenged by the contamination from saprophytic fungi. In this direction, Veen and Ferron ^[7] suggested using dodine (

-dodecylguanidine monoacetate) to inhibit the growth of saprophytes and developed Veen’s semi-selective medium to accomplish this (

Appendix A, Medium 12). Later, Chase et al. [50] and Sneh [51] also used dodine in their studies. However, Liu et al. [52] reported that the higher quantities of dodine can be inhibitory to EPF and suggested using only 10 µg/mL dodine (

, Medium 12). Later, Chase et al. ^[8] and Sneh ^[26] also used dodine in their studies. However, Liu et al. ^[27] reported that the higher quantities of dodine can be inhibitory to EPF and suggested using only 10 µg/mL dodine (

Appendix A, Medium 12). Later, Rangel et al. [53] cautioned against the use of dodine and showed the even 0.006% (

, Medium 12). Later, Rangel et al. ^[28] cautioned against the use of dodine and showed the even 0.006% (

w/v

) dodine in PDAY can completely inhibit

Metarhizium acridum

. This led to the development of CTC medium, which is made by the addition of 0.05% (

w/v

) chloramphenicol, 0.0001% (

w/v

) thiabendazole, and 0.025% (

w/v) cycloheximide in PDAY [54] (

) cycloheximide in PDAY ^[29] (

Appendix A, Medium 13). However, a recent study by Hernández-Domínguez et al. [55] suggested the use of CTC medium, along with other dodine-containing mediums, for better

, Medium 13). However, a recent study by Hernández-Domínguez et al. ^[30] suggested the use of CTC medium, along with other dodine-containing mediums, for better

Metarhizium recoveries. Posadas et al. [47] demonstrated that OM-CTAB is effective in isolating EPF while inhibiting saprophytes. Moreover, this negated the dependency on dodine, as it is not easily available in some countries.

recoveries. Posadas et al. ^[31] demonstrated that OM-CTAB is effective in isolating EPF while inhibiting saprophytes. Moreover, this negated the dependency on dodine, as it is not easily available in some countries.

Beauveria

spp.

Beauveria

spp., e.g.,

Beauveria bassiana

sensu lato (s.l.) and

Beauveria pseudobassiana, can be easily isolated using oatmeal dodine agar (ODA), as described by Chase et al. [50] (

, can be easily isolated using oatmeal dodine agar (ODA), as described by Chase et al. ^[8] (

Appendix A, Medium 14). This medium has also been used in recent studies [56,57,58,59]. Another medium, i.e., Sabouraud-2-glucose agar (S2GA), was made by Strasser et al. [60] (

, Medium 14). This medium has also been used in recent studies ^{[32][33][34][35]}. Another medium, i.e., Sabouraud-2-glucose agar (S2GA), was made by Strasser et al. ^[36] (

Appendix A

, Medium 15) for the isolation of

Beauveria brongniartii

, and was successfully used in studies concerning

B. brongniartii [61,62,63]. However, many recent studies have used S2GA, with slight modifications, to isolate

^[37][38][39]. However, many recent studies have used S2GA, with slight modifications, to isolate

B. bassiana s.l. [64,65]. A dodine-free alternative in isolating

s.l. ^[40][41]. A dodine-free alternative in isolating

B. bassiana s.l. is OM-CTAB [47]. Moreover, Ramírez-Rodríguez and Sánchez-Peña [66] suggested using PDAY with CTAB (0.015% or 0.03% (

s.l. is OM-CTAB ^[31]. Moreover, Ramírez-Rodríguez and Sánchez-Peña ^[42] suggested using PDAY with CTAB (0.015% or 0.03% (

w/v

)) and any of the antibacterial compounds, i.e., dihydrostreptomycin, oxytetracycline, or doxycycline, to isolate

Beauveria

while inhibiting fungal saprophytes.

Purpureocillium

spp.

Purpureocillium

spp., i.e.,

Purpureocillium lilacinum

and

Purpureocillium lavendulum, can easily be isolated using an agar medium containing sodium chloride, benomyl, pentachloronitrobenzene, and Tergitol [67,68] (

, can easily be isolated using an agar medium containing sodium chloride, benomyl, pentachloronitrobenzene, and Tergitol ^[43][44] (

Appendix A

, Medium 16).

Lecanicillium

spp.

Lecanicillium-selective medium (LSM) was developed by Kope et al. [69]. OM agar with 0.05% (

-selective medium (LSM) was developed by Kope et al. ^[45]. OM agar with 0.05% (

w/v

) chloramphenicol and 0.05% (

w/v) CTAB can also be used, as described recently by Xie et al. [70] (

) CTAB can also be used, as described recently by Xie et al. ^[46] (

Appendix A

, Medium 17).

Clonostachys

spp.

Clonostachys

spp., e.g.,

Clonostachys rosea

rosea

, is reported entomopathogenic and can be isolated frequently from soils. Culture medium such as DRBCA is highly effective in isolating

Clonostachys spp., at least in the case of the isolations from cadavers [7].

spp., at least in the case of the isolations from cadavers ^[20].

2.2.2. Insect Baiting

This method is arguably the most commonly used method for EPF isolation, as the bait insect specifically selects entomopathogens from other saprobes in the soils [35,71,72], although surface sterilization of the insect cadavers is needed to avoid occasional contaminations by saprophytic fungi.

This method is arguably the most commonly used method for EPF isolation, as the bait insect specifically selects entomopathogens from other saprobes in the soils ^[16][47][48], although surface sterilization of the insect cadavers is needed to avoid occasional contaminations by saprophytic fungi.

Galleria

-Bait Method or

Tenebrio

-Bait Method

The use of

Galleria mellonella

Linnaeus (Lepidoptera: Pyralidae) for isolating EPF from soil or the “

Galleria-bait method” was first described by Zimmermann [73]. Since then, it has been used for EPF isolations in many studies [74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91].

-bait method” was first described by Zimmermann ^[49]. Since then, it has been used for EPF isolations in many studies ^{[50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67]}.

Tenebrio molitor

Linnaeus (

Coleoptera: Tenebrionidae) has also been used as a bait insect in some studies [92,93,94]. Some previous studies have noticed that insect baiting is more sensitive in isolating EPF than culturing soil suspensions on selective medium [61,62,95,96]. Other studies have also used insect baiting along with soil suspension cultures [57,97,98,99,100]. Although insect baiting is a widely accepted method for EPF isolation, it should be used with caution as some lines of insect baits, such as the dark (melanic) morphs of

) has also been used as a bait insect in some studies ^[68][69][70]. Some previous studies have noticed that insect baiting is more sensitive in isolating EPF than culturing soil suspensions on selective medium ^{[37][38][71][72]}. Other studies have also used insect baiting along with soil suspension cultures ^{[33][73][74][75][76]}. Although insect baiting is a widely accepted method for EPF isolation, it should be used with caution as some lines of insect baits, such as the dark (melanic) morphs of

G. mellonella

, are more resistant to

B. bassiana

s.l., and this trait has also been observed in

T. molitor

for

M. anisopliae s.l. [101,102]. Similarly, immune-suppressed

s.l. ^[77][78]. Similarly, immune-suppressed

G. mellonella were found to be highly (~200 times) susceptible to EPF, which can lead to the isolation of a diverse set of EPF from soils, although saprophytic fungi may not induce any insect mortality [103].

were found to be highly (~200 times) susceptible to EPF, which can lead to the isolation of a diverse set of EPF from soils, although saprophytic fungi may not induce any insect mortality ^[79].

Galleria-Tenebrio

-Bait Method

As bait insects can be sensitive to infection by one particular EPF genus, some studies have used both

G. mellonella

and

T. molitor to isolate EPF, either in part or throughout their whole experiment [7,104,105,106,107]. Recently, Sharma et al. [7] suggested using the “

to isolate EPF, either in part or throughout their whole experiment ^{[20][80][81][82][83]}. Recently, Sharma et al. ^[20] suggested using the “

Galleria-Tenebrio

-bait method” to avoid any underestimation of EPF abundance and diversity, as it was found that

G. mellonella

and

T. molitor

were significantly more sensitive toward the infections by

B. bassiana

s.l. and

M. robertsii,

respectively. This method is described in

Figure 2

Figure 2.

Isolation of entomopathogenic fungi from soils using the “

Galleria-Tenebrio-bait method” The method has been described in detail by Sharma et al. [7].

-bait method” The method has been described in detail by Sharma et al. ^[20].

2.3. Isolation from Phyllosphere

Some studies have also isolated EPF from the phylloplane and other parts of the plant phyllosphere, as these fungi can also be present as plant epiphytes or endophytes ^[84]. Meyling et al. suggested a leaf imprinting methodology where the leaf is cultured onto a selective agar medium ^[40]. Petri dishes with partitions are used and the upper (adaxial), and the lower (abaxial) surface of the leaf are pressed on the separate sides of the Petri plate. Henceforth, the same leaf is put on a paper sheet and photocopied to estimate its surface area using image analysis software at a later stage. The petri plates are incubated in the dark at 23 °C to count fungal colony-forming units (CFUs) ^[40]. Surface sterilization is quite important in isolating hypocrealean fungi as endophytes. This can be done by dipping the plant part in either 70% ethanol and/or 1–5% NaOCl for 3 min. In the case of the leaves, the petiole can be first kept out of the sanitizer to avoid the chemical reaching inside the leaf, and then it can be cut to culture the sterilized part of the leaf on either of the selective mediums described above. It is always recommended to sanitize the intact plant part and then cut it into pieces for further culturing, as this avoids the sterilization of the endophytic fungi ^[85]. Different studies have isolated EPF from the phyllosphere, such as bark and branch samples ^[32][86] and leaves ^[35][87].

2.4. Molecular Identifications of the Isolated Entomopathogenic Fungi

After obtaining a single spore fungal culture on a PDA or SDA (Appendix A; Medium 8 and/or 9), the species can be resolved or identified by amplifying the regions of nuclear ribosomal DNA, such as nrITS, large (28S) subunit (nrLSU), or small (18S) subunit (nrSSU). Another, nuclear ribosomal DNA region, i.e., the intergenic spacer region between nrSSU and nrLSU or IGS, has also been used to understand Beauveria and Metarhizium speciation ^{[87][88][89][90]}. The resolution of the molecular identification can be increased by amplifying other nuclear DNA regions of interest, e.g., for Bloc for Beauveria ^[87][88][89] and the 5′ intron-containing region of translation elongation factor 1-alpha subunit (5′-tef1α) for Metarhizium ^[90][91]. Other nuclear DNA markers, such as the regions of the gene encoding for the largest subunit of RNA polymerase II (rpb1), the second largest subunit of RNA polymerase II (rpb2); β-tublin (β-tub), and the coding region of Tef1-α, can also be employed, in general, for any EPF ^[92][93].

Moreover, in the last decades, researchers have been constantly developing and validating the use of several microsatellite markers for the genotyping of Beauveria ^{[69][89][94][95][96][97]} and Metarhizium ^[98][99] isolates. For example, Oulevey et al. ^[99] described 18 small single repeats or microsatellite marker sets for Metarhizium, i.e., Ma145, Ma325, Ma307, Ma2049, Ma2054, Ma2055, Ma2056, Ma2057, Ma2060, Ma2063, Ma2069, Ma2070, Ma2077, Ma2089, Ma2283, Ma2287, Ma2292, and Ma2296. Similarly, Meyling et al. ^[69] and Goble et al. ^[97] validated the use of 17 to 18 microsatellite marker sets for Beauveria, i.e., Ba06, Ba08, and Ba12-Ba29. This methodology enables enhanced resolution among very closely related isolates which may otherwise be rendered as clones. Recently, Kepler and Rehner ^[93] developed primers for the amplification and sequencing of nuclear intergenic spacer markers for the resolution of Metarhizium isolates, i.e., BTIGS, MzFG543, MzFG546, MzIGS2, MzIGS3, MzIGS5, and MzIGS7, and Kepler et al. ^[75] successfully validated the use of MzIGS3 and MzFG543 on the Metarhizium isolated from agricultural soils.

Appendix A

Come studies have also isolated EPF from the phylloplane and other parts of the plant phyllosphere, as these fungi can also be present as plant epiphytes or endophytes [41]. Meylon culture medingum et al. suggested a leaf imprinting methodology where the leaf is cultured onto a selective agar medium [64]. Peused for the isolatri dishes with partitions are used and the upper (adaxial), and the lower (abaxial) surface of the leaf are pressed on the separate sides of the Petri plate. Henceforth, the same leaf is put on a paper sheet and photocopied to estimate its surface area using image analysis software at a later stageon of entomopathogenic bacteria.

(1) Caprylate-thallous agar (CTA).

The petri plates are incubated in the dark at 23 °C to count fungal colony-forming units (CFUs) [64]. Surface sterilization is quite is medium is mportant in isolating hypocrealean fungi as endophytes. This can be done by dipping the plant part in either 70% ethanol and/or 1–5% NaOCl for 3 min. In the case of the leaves, the petiole can be first kept out of the sanitizer to avoid the chemical reaching inside the leaf, and then it can be cut to culture the sterilized part of the leaf on either of the selectivede by mixing two solutions, i.e., A and B. Both these mediums described above. It is always recommended to sanitize the intact plant part and then cut it into pieces for further culturing, as this avoids the sterilization of the endophytic fungi [111]. Different stu should be autoclaved separately andies have isolated EPF from the phyllosphere, such as bark and branch samples [56,112] anadded leaves [59,113].

2.4. Molecular Identifications of the Isolated Entomopathogenic Fungi

Aftser obptaining a single spically.

(1a) Solution A

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Monopotassium phosphate	KH₂PO₄	0.68 g
Magnesium sulfate heptahydrate	MgSO₄.7H₂O	0.3 g
Dipotassium phosphate	K₂HPO₄	0.15 g
Thallium(I) sulphate	Tl₂SO₄	0.25 g
Yeast Extract		1 g
Calcium chloride	CaCl₂	0.1 g
Caprylic (n-octanoic) acid	CH₃(CH₂)₆.COOH	1.1 mL
Trace element solution		10 mL
Distilled water	H₂O	1 L

Norte fungal culture on a PDA or SDA (Appendix A; Med: Thallium 8 and/or 9(I), as described in the Section 3.1, sulphathe species can be resolved or identified by amplifying the regions of nuclear ribosomal DNA, such as nrITS, large (28S) sis extremely toxic so it shoubunit (nrLSU), or smalld (18S) subunit (nrSSU). Anothber, nuclear ribosomal DNA region, i.e., the intergenic spacer region between nrSSU and nrLSU or IGS,used with caution. The haspH also been usshould be adjusted to understand Beauveria and Metarhizium speciation [113,114,115,116]7.2 The resolution of the molecular identification can beeither by increased by amplifying other nuclear DNA regions of interest, e.g., fing it using K₂HPO₄ or Bloc for Beauveria [113,114,115] and the 5′ intron-containing region of translation elongation fcreasing it is using KH₂PO₄.

Tractore 1-alpha subunit (5′-tef1α) for Metarhizium [116,117]. Other nuclear DNA markers, such as the regment solutions

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Ferrous sulphate heptahydrate	FeSO₄.7H₂O	0.055 g
Trihydrogen phosphate	H₃PO₄	1.96 g
Zinc sulphate heptahydrate	ZnSO₄.7H₂O	0.0287 g
Manganese(II) sulphate monohydrate	MnSO₄.H₂O	0.0223 g
Copper(II) sulphate pentahydrate	CuSO₄.5H₂O	0.0025 g
Cobalt(II) nitrate hexahydrate	Co(NO₃)₂.6H₂O	0.003 g
Boric acid	H₃BO₃	0.0062 g
Distilled water	H₂O	1 L

Nof the gene encoding for the largest subunit of RNA polymerase II (rpb1), the: Once made the trace ele smecond largest subunit of RNA polymerase II (rpb2); β-tnt solublin (β-tub), and the coding region of Tef1-α, on can also be employed, in general, for any EPF [118,119].

Mkept for moreover, in the last decades, researchers have been cths at 4 °C.

(1b) Solution B

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Ammonium sulphate	(NH₄)₂SO₄	1.0 g
Sodium chloride	NaCl	7.0 g
Agar		15 g
Distilled water	H₂O	1 L

Nonstantly developing and validating the use of several microsatellite markers for the genotyping of Beauveria [93,115,120,121,122,123]e: Adjust the pH to 7.3–7.5 and Metarhizium [124,125] isautolatesclave. For example, Oulevey et al. [125] plate culture, adescribed 18 small single repeats or microsatellite marker sets for Metarhizium, i.e., Ma145, Ma325, Ma307, Ma2049, Ma2054, Ma2055, Ma2056, Ma2057, Ma2060, Ma2063, Ma2069, Ma2070, Ma2077, Ma2089, Ma2283, Ma2287, Ma2292, and Ma2296. Simi20 g agar. Add glucose after autoclarly, Meylving et al. [93] and G

(4) Mueller-Hinton broth, yeast extract, potassium phosphate, glucose and pyruvate (MYPGP) medium.

use of 17 to 18 microsatellite marker sets for Beauveria,t the pH to i7.e., Ba06, Ba08, and Ba12-Ba29. This methodology enables enhanced resolution among very closely related isolates which may otherwise be rendered as clones. Recently, Kepler and Rehner [119] 1 and autoclave. For plate culture, add 20 g agar. Adeveloped primers for the amplification and sequencing of nuclear intergenic spacer markers for the resolution of glucose after autoclaving.

(5) Adonitol agar.

Note: Adjust the pH to 7.4 before adding bromothymol blue solution.

Bromothymol blue solution

Reagents and Chemicals

Chemical Formula (If Applicable)

Quantity

This medium is easily available as powder and sold by the majority of the culture media suppliers.

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity	Bromothymol blue	C₂₇H₂₈Br₂O₅S
Dichloran Rose-Bengal Chloramphenicol agar	0.2 g
	32.0 g	Sodium hydroxide (0.1M)	NaOH
Distilled water	H₂O 5 mL


Distilled water	H₂O	1 L

(2) Deoxyribonuclease (DNase)-Toluidine Blue agar.

1 L

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Deoxyribonuclease test agar		37.8 g
Toluidine blue 0.1% w/v solution	NaCl	90.0 ml
L-arabinose	C₅H₁₀O₅	10.0 g
Distilled water	H₂O	900 mL

(3) St. Julian medium (J-medium).

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Yeast extract		15 g
Tryptone		5 g
Dipotassium phosphate	K₂HPO₄	3 g
Glucose (sterilized by filtration)	C₆H₁₂O₆	2.0 g	Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Dipotassium phosphate	K₂HPO₄	3.0 g
Sodium pyruvate	C₃H₃O₃Na	1.0 g
Mueller-Hinton broth		10.0 g
Glucose (sterilized by filtration)	C₆H₁₂O₆	2.0 g
Yeast Extract		10.0 g
Distilled water		1 L

Noblte et al. [123] vali: Adated the j

Distilled water



H	₂O	900 mL

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Sodium chloride	NaCl	4.17 g
Adonitol	C₅H₁₂O₅	5.0 g
Peptone		8.33 g
Bacto agar		12.5 g
Bromothymol blue solution	C₂₇H₂₈Br₂O₅S	10 mL
Distilled water	H₂O	990 mL

(6) Itaconate agar.

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Monopotassium phosphate	KH₂PO₄	3.0 g
Disodium phosphate	Na₂HPO₄	6.0 g
Sodium chloride	NaCl	0.5 g
Ammonium chloride	NH₄Cl	1.0 g
Calcium chloride solution (sterilised) (0.01M)	CaCl₂	10.0 mL
Magnesium sulfate heptahydrate (sterilised) (1M)	MgSO₄.7H₂O	1.0 mL
Itaconic acid solution (filter sterilised) (20%)	C₅H₆O₄	10 mL
Distilled water	H₂O	1 L

Note: Adjust the pH to 7.0 before autoclaving.

(7) Minimal Basal Salt (MBS) medium.

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Monopotassium phosphate	KH₂PO₄	6.8 g
Magnesium sulfate heptahydrate	MgSO₄.7H₂O	0.3 g
Manganese monohydrate sulphate	MnSO₄.1H₂O	0.02 g
Ferric sulfate	Fe₂(SO₄)₃	0.02 g
Zinc sulfate heptahydrate	ZnSO₄.7H₂O	0.02 g
Calcium chloride	CaCl₂	0.2 g
Tryptone		10 g
Yeast Extract		2 g

Note: Adjust the pH to 7.2 before autoclaving.

Common culture medium used for the isolation of entomopathogenic fungi.

(8) Potato Dextrose agar (PDA)

Reagents and Chemicals	Chemical formula (If Applicable)	Quantity
Potato dextrose agar		39.0 g
Distilled water	H₂O	1 L

(9) Sabouraud Dextrose agar (SDA)

Reagents and Chemicals	Chemical Formula (if Applicable)	Quantity
Sabouraud dextrose agar		65.0 g
Distilled water	H₂O	1 L

(10) Oatmeal Cetyl Trimethyl Ammonium Bromide (OM-CTAB) agar.

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Oatmeal (cooked in distilled water)		20.0 g
Cetyl trimethyl ammonium bromide (CTAB)	C₁₉H₄₂BrN	0.6 g
Chloramphenicol	C₁₁H₁₂Cl₂N₂O₅	0.5 g
Agar		20 g
Distilled water	H₂O	To make upto 1L

(11) Dichloran Rose-Bengal Chloramphenicol agar (DRBCA).

(12)

Metarhizium is

Medium

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Glucose	C₆H₁₂O₆	10.0 g
Peptone		10.0 g
Oxgall		15.0 g
Agar		35.0 g
Dodine (N-dodecylguanidine monoacetate)	C₁₅H₃₃N₃O₂	10 mg
Cycloheximide	C₁₅H₂₃NO₄	250 mg
Chloramphenicol	C₁₁H₁₂Cl₂N₂O₅	500 mg
Distilled water	H₂O	1 L

Note: Cyclohexamide is quite toxic and caution is needed while handling.

(13) Chloramphenicol Thiabendazole Cycloheximide (CTC) medium.

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Potato dextrose agar		39.0 g
Yeast extract		0.5 g
Chloramphenicol	C₁₁H₁₂Cl₂N₂O₅	500 mg
Thiabendazole	C₁₀H₇N₃S	1 mg
Cycloheximide	C₁₅H₂₃NO₄	250 mg
Distilled water	H₂O	1 L

(14) Oatmeal Dodine agar (ODA).

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Oatmeal infusion		20.0 g
Dodine (N-dodecylguanidine monoacetate)	C₁₅H₃₃N₃O₂	550 mg
Chlortetracycline	C₂₂H₂₃ClN₂O₈	5 mg
Crystal violet	C₂₅N₃H₃₀Cl	10 mg
Agar		20.0 g
Distilled water	H₂O	1 L

(15) Sabouraud-2-Glucose agar (S2GA).

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
Glucose	C₆H₁₂O₆	20.0 g
Peptone		10.0 g
Streptomycin sulphate	C₄₂H₈₄N₁₄O₃₆S₃	600 mg
Tetracycline	C₂₂H₂₄N₂O₈	50 mg
Cycloheximide	C₁₅H₂₃NO₄	50 mg
Dodine (N-dodecylguanidine monoacetate)	C₁₅H₃₃N₃O₂	100 mg
Agar		12.0 g
Distilled water	H₂O	1 L

(16) Purpureocillium lilacinum medium.

Reagents and Chemicals	Chemical formula (If Applicable)	Quantity
Potato dextrose agar		39.0 g
Sodium chloride	NaCl	10–30 g
Tergitol		1 g
Pentachloronitrobenzene	C₆Cl₅NO₂	500 mg
Benomyl	C₁₄H₁₈N₄O₃	500 mg
Streptomycin sulphate	C₄₂H₈₄N₁₄O₃₆S₃	100 mg
Chlortetracycline hydrochloride	C₂₂H₂₄C_l2N₂O₈	50 mg
Distilled water	H₂O	1 L

(17) Lecanicillium-specific medium.

Reagents and Chemicals	Chemical Formula (If Applicable)	Quantity
L-sorbose	C₆H₁₂O₆	2 g
L-asparagine	C₄H₈N₂O₃	2 g
Dipotassium phosphate	K₂HPO₄	1 g
Potassium chloride	KCl	1 g
Magnesium sulfate heptahydrate	MgSO₄.7H₂O	0.5 g
Ferric-sodium salt (FeNaEDTA)	C₁₀H₁₂N₂O₈FeNa	0.01 g
Agar		20 g
Streptomycin sulphate	C₄₂H₈₄N₁₄O₃₆S₃	0.3 g
Chlortetracycline hydrochloride	C₂₂H₂₄C_l2N₂O₈	0.05 g
Pentachloronitrobenzene	C₆Cl₅NO₂	0.8 g
Borax	NaB₄O₇.10H₂O	1 g
Distilled water		1 L

Nolates, i.e., BTIGS, MzFG543, MzFG546, MzIGS2, MzIGS3, MzIGS5, and MzIGS7, and Kepler et al. [99] s: Adjust the pH to 4.0 uccessfully validated the use of MzIGS3 and MzFG543 on the Metarhizium iing 10% trihydrogen phosolphated (H₃PO₄) befrom agricultural soilsore autoclaving.

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