Science of the Total Environment 734 (2020) 139169

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Review

Genetically modified entomopathogenic bacteria, recent developments,benefits and impacts: A review

Ugur Azizoglu a,⁎, Gholamreza Salehi Jouzani b, Nihat Yilmaz a, Ethem Baz c, Duran Ozkok a

a Department of Crop and Animal Production, Safiye Cikrikcioglu Vocational College, Kayseri University, Kayseri, Turkeyb Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iranc Laboratory and Veterinary Health Department, Safiye Cikrikcioglu Vocational College, Kayseri University, Kayseri, Turkey

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Entomopathogenic bacteria are eco-friendly alternatives to chemical insecti-cides.

• Genetic engineering has great potentialfor the development of newentomopathogens.

• GM-EPBs have many advantages overwild entomopathogens.

• Limited attention has been paid to theirpotential ecological impacts.

• The main concerns about GM-EPB aretheir potential unintended effects onbeneficial organisms.

⁎ Corresponding author at: Kayseri University, Safiye CE-mail addresses: azizoglu@erciyes.edu.tr, azizoglu@k

https://doi.org/10.1016/j.scitotenv.2020.1391690048-9697/© 2020 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 March 2020Received in revised form 10 April 2020Accepted 30 April 2020Available online 11 May 2020

Editor: Fang Wang

Keywords:Recombinant entomopathogenic bacteriaInsect pest controlNontarget organisms

Entomopathogenic bacteria (EPBs), insect pathogens that produce pest-specific toxins, areenvironmentally-friendly alternatives to chemical insecticides. However, the most important problemwith EPBs application is their limited field stability. Moreover, environmental factors such as solar radia-tion, leaf temperature, and vapor pressure can affect the pathogenicity of these pathogens and their toxins.Scientists have conducted intensive research to overcome such problems. Genetic engineering has great po-tential for the development of new engineered entomopathogens with more resistance to adverse environ-mental factors. Genetically modified entomopathogenic bacteria (GM-EPBs) have many advantages overwild EPBs, such as higher pathogenicity, lower spraying requirements and longer-term persistence. Geneticmanipulations have been mostly applied to members of the bacterial genera Bacillus, Lysinibacillus, Pseudo-monas, Serratia, Photorhabdus and Xenorhabdus. Although many researchers have found that GM-EPBs canbe used safely as plant protection bioproducts, limited attention has been paid to their potential ecologicalimpacts. The main concerns about GM-EPBs and their products are their potential unintended effects onbeneficial insects (predators, parasitoids, pollinators, etc.) and rhizospheric bacteria. This review address

ikrikcioglu Vocational College, Department of Crop and Animal Production, Kayseri, Turkey.ayseri.edu.tr, azizogluugur@hotmail.com (U. Azizoglu).

2 U. Azizoglu et al. / Science of the Total Environment 734 (2020) 139169

recent update on the significant role of GM-EPBs in biological control, examining them through differentperspectives in an attempt to generate critical discussion and aid in the understanding of their potentialecological impacts.

© 2020 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Genetically modified entomopathogenic bacteria for managing insect pests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Genetically modified Bacillus thuringiensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.1.1. General characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.1.2. Expression of cry genes in Bt strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.1.3. Expression of other genes in Bt strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.1.4. Bt as a source of pest resistance genes for plant genetic engineering programs . . . . . . . . . . . . . . . . . . . . . . . . . . 62.1.5. Bt toxin genes in other microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.2. Genetically modified Lysinibacillus (Bacillus) sphaericus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73. Other genetically modified bacteria expressing insect toxin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.1. Entomopathogenic Serratiaspp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2. Entomopathogenic Pseudomonasspp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3. Entomopathogenic Photorhabdusspp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.4. Entomopathogenic Xenorhabdusspp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Possible impacts of GM entomopathogenic bacteria and GM Bt crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.1. Effects on nontarget organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4.1.1. Risks on predators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.1.2. Risks on parasitoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.1.3. Risks on pollinators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4.2. Risks on the microbial communities in the rhizosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105. Conclusions and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Authors’ contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction

During the last two decades, eco-friendly and efficient microbial in-secticides as green alternatives for harmful chemical pesticides havebeen widely taken in the account for mass control of destructive croppests (Salehi Jouzani et al., 2017). The global biopesticides market in2021 is predicted to reach approximately 7.7 billion USD and willreach 14.1% Compound Annual Growth Rate (CAGR) (Ruiu, 2018).

Entomopathogenic bacteria (EPBs) are a group of microorgan-isms that play an important role in global pest insect control, andhave been studied in detail by many researchers worldwide(Schisler et al., 2004; Cheng et al., 2010; Wang et al., 2010; Konget al., 2016; Salehi Jouzani et al., 2017; Karabörklü et al., 2018).Most of the EPBs are within the Bacillaceae, Pseudomonadaceae, En-terobacteriaceae, Streptococcaceae, and Micrococcaceae families.The most interesting group among the EPBs is Bacillus thuringiensis(Bt). However, because of the increased insect resistance to Bt toxins,researchers are searching for alternatives to this bacterium (Bucherand Stephens, 1957; Bucher, 1960; Park and Federici, 2009). ThePseudomonadaceae and Enterobacteriaceae families have been iden-tified as promising among these alternative bacteria. Different ge-netic engineering programs have been conducted on EPBs, such asBacillus, Lysinibacillus, Pseudomonas, Serratia, Photorhabdus, andXenorhabdus, to enhance more effective insect pest control. Genesthat encode toxins have been isolated, characterized, manipulatedand expressed in different organisms (Escherichia coli, Bacillus spp.,Pseudomonas spp., Serratia spp., GM Bt crops) to form new EPBtoxin combinations and to broaden the target spectrum (Federiciet al., 2003; Jurat-Fuentes and Jackson, 2012; Azizoglu et al., 2016a;Azizoglu et al., 2017; Yilmaz et al., 2017; Peng et al., 2019).

The most important problem with EPB application is their limitedfield stability. Furthermore, adverse environmental factors can affect

the pathogenicity of these pathogens. The resistance of EPBs to adverseenvironmental conditions might be increased via genetic modification(Karabörklü et al., 2018). Genetically modified (GM) varieties of wildtype EPBs are important in terms of increasing their effectivenessagainst target insects and developing new preparations with broad in-secticidal spectra (Wang et al., 2008: Patel et al., 2015). Compared towild type EPBs, GM-EPBs have many advantages, such as higher patho-genicity, longer-term efficacy, low spraying requirements, and lower in-sect resistance (Sharma, 2009; Castagnola and Jurat-Fuentes, 2012;Karabörklü et al., 2018). However, despite their advantages, they haveled to several concerns and risks. The main concern regarding GM or-ganisms is their possible adverse effects on the environment andhuman health. The potential risks of GM-EPBs on the environment in-clude geneflows towild species, the development of resistance in targetpest insects, and their effects on nontarget beneficial speciese.g., parasitoids, predators and pollinators (Castagnola and Jurat-Fuentes, 2012; Karabörklü et al., 2018) and on microbial populationsin the rhizosphere (Amarger, 2002) (Fig. 1.).

The present review is dedicated to give an update on recent develop-ments in genetic engineering of EPBs in biological control programs ofpest insects, placing GM-EPBs in different perspectives by exploringother views to discuss, examine, and evaluate their effects on nontargetorganisms.

2. Genetically modified entomopathogenic bacteria for managinginsect pests

2.1. Genetically modified Bacillus thuringiensis

2.1.1. General characteristicsBacillus thuringiensis (Bt), the best-known fast-acting and host-

specific entomopathogenic biocontrol agent and successful

Fig. 1. The possible effects of genetically modified entomopathogenic bacteria on the environment, modified from Karabörklü et al. (2018) and Azizoglu (2019)

3U. Azizoglu et al. / Science of the Total Environment 734 (2020) 139169

bioinsecticide, is an aerobic and spore-forming gram-positive bacte-rium. Bt strains produce different types of parasporal crystal proteinsor δ-endotoxins (Cry), vegetative insecticide proteins (VIPs) and cyto-lytic proteins (Cyt). These proteins are toxic to a wide range of pestsin the orders Lepidoptera, Coleoptera, Hymenoptera, Diptera,Homoptera, Orthoptera, and Mallophaga as well as nematodes, mites,and protozoa (Schnepf et al., 1998; Salehi Jouzani et al., 2008, 2017;Karabörklü et al., 2018; Zhang et al., 2018; Peng et al., 2019; Frentzelet al., 2020). This species consists of more than 100 different subspecieswith different phylogenetic and serotyping features. Based on the lastupdate, during the last 3 decades, approximately 820 cry (cry1-cry78),147 vip (vip1, 2 and 3) and 40 cyt (cyt 1, 2 and 3) genes have been de-tected and characterized from different Bt subspecies throughout theworld (Crickmore et al., 2018). The wide diversity of Bt subspecies andtheir insecticidal genes have made this bacterium the bioinsecticideused most widely in conventional spraying and in GM crops (Jainet al., 2016; Melo et al., 2016; Salehi Jouzani et al., 2017).

Genetic modifications have provided new tools for developing newefficient entomopathogens to manage pests (Karabörklü et al., 2018).Different cry genes have been expressed/overexpressed in Bt strains toincrease the yield, titer and stability of insecticidal crystal proteins orto expand the range of their insecticidal effects against different typesof pests, such as lepidoptera and coleoptera, using recombinant

technology (Zhang et al., 2018). Currently, recombinant systems cantransfer stable genes to Bt strains, which is often accomplished by ho-mologous recombination or region-specific recombination systems(Karabörklü et al., 2018). Different cry or non-cry genes can be used tocreate recombinant Bt strains to improve their virulence and environ-mental persistence (Sankar and Reji, 2018). Moreover, cry genes havebeen expressed in other hosts (bacteria, fungi and viruses), such as Ba-cillus sp., E. coli, Pseudomonas spp., Lactococcus, Pichia, Beauveria bassiana,and Baculovirus strains, to introduce new insecticidal traits in such hosts(Agaisse and Lereclus, 1994; Theoduloz et al., 2003; Shi et al., 2004;Martins et al., 2008; Hernández-Rodríguez et al., 2013; Azizoglu et al.,2016b; Durmaz et al., 2016; Deng et al., 2019). In addition, Bt has beenwidely used as the source of toxin genes in plant genetic engineeringprograms to make GM crops resistant to different pests (Melo et al.,2016; ISAAA, 2018).

2.1.2. Expression of cry genes in Bt strainsDuring the last two decades, different studies have been dedicated to

the expression of some cry genes under the control of cry gene-specificpromoters or other promoters in Bt strains for different purposes, suchas improving insecticidal activity, expanding host ranges, studyingnovel gene activity or insecticidal mechanisms and other applications(Table 1). Based on the last update, cry1Ac is the most widely used

Table 1The list of cry genes and promoters used for genetic engineering of Bt strains and their performance.

cry gene cry gene ornon-cry genepromoter

Recombinant Btstrain

Aim/New trait/efficiency Target insectpest/cells

Reference

cry1Ab cry1A promoter 4Q7−To explore the sequential binding model of toxinsto insects midgut cell receptors

Tobacco hornworm(M. sexta)

Gomez et al., (2014)Pacheco et al., (2009)

cry1Ac

cry1Ac promoter

Cry−B

Recombinant Cry1Ac-HWTX-XI (from spider)fusion enhanced insecticidal activity by 100–200%

H. armigera and S.exigua

Sun et al., (2016)

Recombinant Cry1Ac-Av3 (neurotoxin of A.viridis) fusion enhanced insecticidal activity by 2.6fold.

H. armigera Yan et al., (2014)

Recombinant cry1Ac gene with Subtilisin-LikeProtease CDEP2 increased toxicity up to 100%

H. armigera Xia et al. (2009b)

XBU001Fusion of the cry1Ac with the Neurotoxin genehwtx-I improved LC50 (5.12 μg/mL)

P. xylostella Xia et al. (2009a)

Cry(−)BThe mutant recombinant strain showedinsecticidal activity

P. xylostella Roh et al. (2004)

4Q7 Showed toxicity against the pest P. xylostella Xia et al. (2005)

cry3A promoter BMB171The cry3A promoter more efficiently enhanced theexpression of cry1Ac gene compared to cry1Acpromoter

D. punctatus Chaoyin et al., (2007)

pexsY HD73−The recombinant Cry1Ac protein directed by thePexsY promoter was toxic against the pest

O. furnacalis Zheng et al., (2014)

amyE promoter Tt14The amylase E promoter could efficiently enhanceproduce Cry1Ac

T. ni Yang et al., (2003)

HD73–5014promoter

HD73− and HDsigK− the HD73_5014 gene promoter improved Cry1Acproduction

P. xylostella Zhang et al., (2018)

Modified cry1Ab/cry1Ac cry3A promoter 407− More toxicity P. xylostellaGarcia-Gomez et al.(2013)

cry1Ac and ChiA74Δspchitinase inclusions

pcytA-p/STAB-SDpromoter

HD73 More toxicity and enzymatic activity S. frugiperdaGonzález-Ponce et al.(2017)

cry1Ba cry8E promoter HD73− More toxicity and UV protectionO. furnacalis and P.xylostella

Zhou et al., (2014)

cry1Ccry3A promoter 407− Higher insecticidal activity

S. littoralis and O.nubilalis

Sanchis et al., (1996)

cyt1A promoter 4Q7− More toxicity S. exigua Park et al. (2000)amyE promoter cry−B More toxicity T. ni and S. exigua Chak et al. (1994)

cry2A/cry2Bcry2A/cytApromoter

Acrystaliferous strain Cry2B expression – Crickmore et al., (1994)

cry2A/cry11B cyt1A promoter 4Q7− Enhanced 4.4-fold the amount of Cry2A – Park et al., (1999)

cry2Abcry1Ac promoter SP41− and 407− 6.9-fold more toxicity

H. armigera, S. lituraand S. exigua

Somwatcharajit et al.,(2014)

cry2Aa promoter 4Q7− More toxicity H. armigera Jain et al., (2006)

cry2Aa with P20 cry2Aa HD1CryB-Mp20-Hc2Aa More toxicityE. kuehniella, A. aegyptiand C. pipiens

Elleuch et al. (2016)

cry3A

cry3A promoter HD1− Expression – De Souza et al., (1993)

cyt1Aa promoter 4Q7− 1.3-fold production cry3ACotton wood leafbeetle,C. scripta

Park et al., (1998)

cry3Aa7promoter

Bt subsp. aizawaiG033A

New toxicity against coleopteran pests (LC50

0.35 mg/mL)

P. aenescens, S. exigua,P. xylostella and H.amigera

Wang et al., (2006)

cry3A7 promoter ACE-38Four times of Cry3Aa yield compared withparental strain (LC50: 1.13 μl/mL)

P. versicolora Yu et al., (2016)

cry4B/cry4A cry4B promoter 4Q2–71 Similar toxic levels A. aegyptiRodriguez-Almazanet al., (2012)

cry5B cry5B promoter BMB171 LC50 23.7 μg/mL C. elegans Sajid et al., (2018)cry6A cry6A promoter BMB171 Pore-forming mode of action C. elegans Dementiev et al., (2016)

cry7Aa2 genecry7Aa2 genepromoter

BMB17-Cry7Aa2 More toxicity by 4 times (LC50 4.9 μg/mL) L. decemlineataDomínguez-Arrizabalagaet al., (2019)

cry8

cry1Ac/cry3Aapromoters

4Q7− Low toxicity A. gemmatalis Amadio et al., (2013)

cry3A promoter Biot 185 High toxicity against three lepidoptheran pestsH. parallela, A.corpulenta and H.oblita

Jia et al., (2014)

cry8Kb3/cry8Pa3cry8Kb3/cry8Pa3promoters

4Q7− Toxicity against Coleoptera A. grandis Navas et al., (2014)

Cry10A cyt1A promoterBt. subsp. israelensis4Q7

More insecticidal activity A. aegyptiHernández-Soto et al.,(2009)

cry9Ec1 cyt1A2 promoter BFR1 Toxicity against LepidopteraB. mori and P.xylostella.

Wasano et al., (2005)

cry11A cry1Ac promoter 4Q7− Larger and more amount of CryIVD crystals – Wu and Federici (1995)

cry11Aa/cry11Ba cyt1A promoter 4Q7− LC50% lethal concentrations of 4.8 and 2.2 μg/mLC. quinquefasciatus andA. aegypti

Sun et al., (2014)

cry11B cyt1A promoter Bt subsp. israelensis Enhanced two fold toxicityC. quinquefasciatus.and A. aegypti

Park et al., (2001)

cry19A cyt1A promoter 4Q7− 4-fold larvicidal activity, LC95 1.9 μg/mL C. quinquefasciatus,Barboza-Corona et al.,(2012)

4 U. Azizoglu et al. / Science of the Total Environment 734 (2020) 139169

Table 1 (continued)

cry gene cry gene ornon-cry genepromoter

Recombinant Btstrain

Aim/New trait/efficiency Target insectpest/cells

Reference

cry20Aa cyt1A promoter YG1 Low larvicidal activityA. aegypti and C.quinquefasciatus

Lee and Gill (1997)

cry26Aa/cry28Aa cry1Ca promoter YBT-020 Expression – Ji et al., (2009)

cry27A cyt1A promoter BFR1larvicidal activity highly specific for Anophelesstephensi

Anopheles stephensi, Saitoh et al., (2000)

cry30Ca/cry60Aa/cry60Ba cyt1A promoter 4Q7- 50% lethal concentrations: 2.9 to 7.9 μg/mL C. quinquefasciatus Sun et al., (2013)cry41Aa cyt1A promoter 4D7 Parasporin production HepG2 cells Krishnan et al., (2017)

cry64Ba/cry64Ca cry1Ca promoter HD73− High insecticidal activity against hemipteran pestsL. striatellus and S.furcifera.

Liu et al., (2018)

cry69Aa1 cry3A promoter HD73− Dipteran toxicity C. quinquefasciatus Guan et al., (2014)

5U. Azizoglu et al. / Science of the Total Environment 734 (2020) 139169

gene in genetic engineering programs for the control of different lepi-dopteran pests, such as Pectinophora gossypiella (Fabrick et al., 2020),Helicoverpa armigera (Liu et al., 2020;Wei et al., 2020), Plutella xylostella(Zhang et al., 2018), Anticarsia gemmatalis and Chrysodeixis(Pseudoplusia) includens (Bel et al., 2017), Spodoptera exigua (Sunet al., 2016), Ostrinia furnacalis (Zheng et al., 2014), Dendrolimuspunctatus (Chaoyin et al., 2007), and Trichoplusia ni (Yang et al., 2003).These studies confirmed that the overexpression or heterologous ex-pression of cry1Ac significantly increases the toxicity of Bt strainsagainst such pests (Table 1).

Yan et al., (2014) indicated that the coexpression of Cry1Ac-Av3 (aneurotoxin of Anemonia viridis) fusion enhances insecticidal activityagainst H. armigera by 2.6-fold compared with original Cry1Ac. Anotherstudy confirmed that the coexpression of Cry1Ac-HWTX-XI (from a spi-der) fusion enhances insecticidal activity against H. armigera andS. exigua compared with Cry1Ac alone (Sun et al., 2016). HWTX-XI(Huwentoxin-XI) is a peptide toxin obtained from venom ofOrnithoctonus huwena. Such 55-residue peptide is a very potentKuntiz-type trypsin inhibitor (approximately 30-fold stronger than bo-vine pancreatic trypsin inhibitor) and a weak potassium channelblocker with neurotoxic activity (Jiang et al., 2014; Sun et al., 2016).

Another widely used gene is cry3A, which has been transferred indifferent Bt strains for the control of the cottonwood leaf beetlesChrysomela scripta (Park et al., 1998), Pyrrhalta aenescens (Wang et al.,2006) and Plagiodera versicolora (Yu et al., 2016). Other cry genes, in-cluding cry1Ab (Pacheco et al., 2009; Gomez et al., 2014), cry1Ba(Zhou et al., 2014), cry1C (Sanchis et al., 1996), cry2A (Crickmoreet al., 1994; Park et al., 1999), cry2B (Park et al., 1999), cry11B (Parket al., 1999), cry4B/cry4A (Rodriguez-Almazan et al., 2012: for dipterancontrol), cry5B (Sajid et al., 2018: for nematode control), cry6A(Dementiev et al., 2016: for nematode control), cry7Aa (Domínguez-Arrizabalaga et al., 2019: for Colorado potato beetle control), cry8(Amadio et al., 2013; Jia et al., 2014: for lepidopteran pest control,Navas et al., 2014: for coleopteran control), cry9Ec1 (Wasano et al.,2005), cry10A (Hernández-Soto et al., 2009), cry11A (Wu and Federici,1995; Sun et al., 2014), cry11B (Park et al., 2001; Sun et al., 2014),cry19A (Barboza-Corona et al., 2012: against Diptera), cry20Aa (Leeand Gill, 1997: against Diptera), cry26Aa/cry28Aa (Ji et al., 2009),cry27A (Saitoh et al., 2000: against Diptera), cry30Ca/cry60Aa/cry60Ba(Sun et al., 2013: against Diptera), cry41Aa (Krishnan et al., 2017:against cancer cells), cry64Ba/cry64Ca (Liu et al., 2018: againstHemiptera) and cry69Aa1 (Guan et al., 2014: against Diptera) havebeen transferred to different Bt strains and improved their insecticidalactivities (Table 1).

Based on the report of Peng et al., (2019), the cry1Ac promoter is themost commonly used promoter for the regulation of the expression ofdifferent cry genes. This promoter has been used for the expression ofcry1Ac (Yan et al., 2014; Sun et al., 2016), cry2Ab27 (Somwatcharajitet al., 2014), cry11A (Wu and Federici, 1995), cry8 (Amadio et al.,2013), cry64Ba and cry64Ca (Liu et al., 2018). Other cry gene promoterscommonly used for the construction of recombinant Bt strains include

the cry3A (de Souza et al., 1993; Chaoyin et al., 2007; Jia et al., 2014),cry1Ca (Ji et al., 2009), cry8E (Zhou et al., 2014), cry2Aa (Jain et al.,2006), cry4B (Rodriguez-Almazan et al., 2012), cry5B (Sajid et al.,2018) and cry6A (Dementiev et al., 2016) promoters (Table 1).

During the last two decades, some recombinant Bt strains have beentested in field trials and registered as commercial Bt products. They in-clude the recombinant Bt strains Raven®, producing Cry1Ac(2) Cry3Aand Cry3BbR; CRYMAX®, producing Cry1Ac(3), Cry2A, Cry1CaR; andLepinox®, producing Cry1Aa, Cry1Ac(2), Cry2A and Cry1Fa/1AcR(Ecogen company). Moreover, some other recombinant Bt products,such as Foil, Cutlass, Condor, MVP®, M-Trak®, M-Peril®, M-One® andMYX1896®, have been developed by the Ecogen and Mycogen compa-nies (Karabörklü et al., 2018).

2.1.3. Expression of other genes in Bt strainsIn addition to insecticidal activity, Bthas shownother characteristics,

such as antagonistic effects against phytopathogenic fungi and bacteria,plant growth-promoting activities and bioremediation (Salehi Jouzaniet al., 2017). Therefore, some Bt genetic engineering programs havebeen dedicated to the development of Bt strains with new traits(Table 2). Genes encoding different types of chitinases have been de-tected from different strains of Bt subsp. kenyea, pakistani, colmeneri,canadiensis, entomocidus, kurstaki, israelensis and konkukian(Karabörklü et al., 2018). These enzymes have high potential to beused for biologically controlling plant pathogens, digesting shrimpwaste and increasing the insecticidal activity of Cry proteins. Differentrecent studies have confirmed that overexpression of Bt homologchitinases could increase Bt insecticidal activities against differentpests, such as Tuta absoluta (Atia et al., 2019), Galleria mellonella andDrosophila melanogaster (Ozgen et al., 2013), Ephestia kuehniella (Drisset al., 2011), Spodoptera frugiperda (Hu et al., 2009; González-Ponceet al., 2019), and H. armigera (Hu et al., 2009). Furthermore, chitinasesof Bt show inhibitory activity against plant pathogenic fungi (Moralesde la Vega et al., 2006). Different chitinase (chi) genes have been iso-lated and characterized from a diverse range of microorganisms andhave been transffered to Bt in order to improve its insecticidal and fun-gicidal properties. For instance, the chiB and chiC genes have been trans-ferred to Bt strains, which resulted in significant increase of biocidalactivities of such recombinant Bt strains (Ozgen et al., 2013;Karabörklü et al., 2018). Moreover, Tang et al. (2017) recently trans-ferred the chitinase gene (Chi9602ΔSP) into the Bt strain BMB171.Their results confirmed high production of this enzyme in thetransformants and strong antagonistic effects on Fusarium oxysporumand Physalospora piricola.

Doruk et al. (2013) transferred the kinase gene (ppk) to Bt subsp.israelensis (Bti) and showed that the expression of this enzyme couldimprove insecticidal activity against Culex quinquefasciatus by 7.7times (LC50 5.8 ± 0.6 ng/ml). Elleuch et al. (2014) coexpressed the fu-sion of a cytolytic protein (Cyt1A98) and the accessory protein (P20)genes in the Bt strain BNS3 and confirmed that such expression couldenhance insecticidal activity against lepidopteran pests. They confirmed

Table 2The list of non-cry genes transferred to Bt strains and their performance.

Non-cry gene Recombinant Bt strain(s) Aim/New trait/efficiency Target insect/pathogen Reference

Chitinase gene 4A7 and 4Q1 Chitinase activity/more insecticidal activity T. absoluta Atia et al., (2019)Chitinase B (chiB) and C (chiC) genes Bt subsp. kurstaki and

israelensis 5724Chitinase activity/more insecticidal activity G. mellonella and adult

of D. melanogasterOzgen et al.,(2013)

The fusion Chitinase Chi255 and the carboxy-terminalhalf ofcry1Ac(chi255Δsp–CTcry1Ac)

BNS3/pF andacrystalliferous strainBNS3Cry−

Chitinase activity/more insecticidal activity by1.5–2.5 folds (LC50 144.06 μg/g)

E. kuehniella Driss et al.,(2011)

The endochitinase gene chiA74 under the control ofpromoter (pcytA)

HD-73 More chitinase activity by 362-fold – Barboza-Coronaet al. (2009)

Chitinas gene (chiA74Δsp) of Bt HD1 More chitinase activity by 7–12 fold and 20% to40% increase in the yield of Cry1A

S. frugiperda González-Ponceet al., (2019)

Chitinase gene Cry−B More insecticidal activity S. exigua and H.armigera

Hu et al., (2009)

Kinase gene (ppk) Bt subsp. israelensis (Bti) More insecticidal activity by 7.7 times (LC50 5.8± 0.6 ng /mL)

C. quinquefasciatus Doruk et al.,(2013)

The fusion of cytolytic protein (Cyt1A98) and theaccessory protein(P20) genes

BNS3 More insecticidal activity E. kuehniella and S.littoralis

Elleuch et al.,(2014)

hknA encoding histidine protein kinase Bt subsp. morrisoni strainEG1351

CrylIlA Overproduction – Malvar et al.,(1994)

Chitinase gene (CHIA) from S.marcescens BN 10 Coleoptera-specific Btstrain 3023

Chitinase activity was 6.3 fold higher – Okay (2005)

thurincin H A thurincin H-sensitivestrain of Bt

More antibacterial activity against V.parahaemolyticus

V. parahaemolyticus Oros-Flores et al.,(2018)

cyt1Aa promoter combinations and plasmid copynumber on synthesis ofCyt1Aa

4Q7 Yielded Cyt1A crystals tenfold larger – Park et al. (2016)

Tyrosinase (melanin) gene (mel) 171 (Cry-) The survival rate of Bt at conditions with U·Vradiation was increased by two times

– Ruan et al.,(2002)

Vip2A(c) and Vip1A(c) genes HD201 and T48 High expression of both toxins – Shi et al., (2004)Chitinase gene (Chi9602ΔSP) BMB171 Enhanced antifungal effects F. oxysporum and P.

piricolaTang et al.(2017)

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that P20 serves as molecular chaperone protecting Cyt1A98 from pro-teolytic activity. Morever, they indicated that BNS3pHTp20 showedhigher activity than negative control (BNS3pHTBlue) againstMediterra-nean flour moth (Ephestia kuehniella), but exibited a similar insecticidalactivity against cotton leafworm (Spodoptera littoralis).

Malvar et al. (1994) indicated that the expression of the hknA geneencoding histidine protein kinases in Bt subsp. morrisoni strainEG1351 resulted in the overproduction of the Cry3A protein. Theyshowed that hknA encodes a novel histidine protein kinase involved inBt sporulation. Morever, they inferred that the Cry3A-overproducing isprobably due to a defect in the phosphorylation of spo0A and verifiedthat Cry3A production is not dependent on sporulation. Recently,Oros-Flores et al. (2018) transferred a thurincin H gene into a thurincinH-sensitive strain of Bt and confirmed that this genetic manipulationcould improve Bt antibacterial activity against Vibrio parahaemolyticus,the causing agent of gastrointestinal illness in humans. Expression ofthe tyrosinase (melanin) gene (mel) in the Bt strain 171 (Cry-) im-proved the survival rate of Bt under UV radiation by two times (Ruanet al., 2002). Moreover, another study showed that the expression ofvip2A(c) and vip1A(c) genes in the HD201 and T48 strains resulted inthe high expression of both toxins (Shi et al., 2004).

2.1.4. Bt as a source of pest resistance genes for plant genetic engineeringprograms

Since 1996, the global acreage of pest- and herbicide-resistant GMcrops has drastically increased due to their socioeconomic and environ-mental advantages. In 2018, the acreage reached 191.7million hectares,and 825 GM varieties were confirmed for intended environmental re-lease. The acreage of Bt crops was approximately 103 million hectaresin 2018, which included 80 million hectares of GM crops containingstacked Bt/herbicide tolerance genes and 23 million hectares of thosecrops containing only Bt genes for resistance to lepidopteran and/or co-leopteran pests (ISAAA, 2018).

Since 1996, 304 Bt-GM varieties and lines of 10 plant species, includ-ing 208 maize (Zea mays), 30 potato (Solanum tuberosum), 49 cotton(Gossypium hirsutum), 6 soybean (Glycine max), 3 rice (Oryza sativa),3 sugarcane (Saccharum sp.), 2 poplar (Populus sp.), 1 tomato

(Lycopersicon esculentum), 1 eggplant (Solanummelongena) and 1 cow-pea (Vigna unguiculata) variety, have been authorized for commercialrelease in 27 different countries around the world (ISAAA's GM Ap-proval Database, 2020). Among them, 243 crop varieties are resistantto lepidopteran pests and contain anti-lepidopteran cry and vip genes,including cry1Ab, cry1Ac, cry1C, cry1F, cry1Fa2, cry2Ab2, cry2Ae, cry9Cand vip3A.

The cry1Ab, cry1Fa2, vip3Aa20 and cry1Ac have been widely used inGM-Bt crops to enhance their resistance against lepidopteran pests.Based on the recent update, 95, 81, 46 and 30 GM varieties containingthese genes, have been authorized for commercial production, respec-tively. Recently, gene pyramiding strategy, in which Bt crops withmore than one cry or vip gene are produced, have been widely per-formed to postpone potential pest resistance to recombinant Bt toxinswhich are expressed in the GM plants. About 160 Bt maize and potatovarieties containing anti-coleopteran genes have been authorized forcommercial production through theworld. Some of these crops containboth anti-coleopteran and anti-lepidopteran pest genes. The cry3Aa,cry3B, cry3Bb1, cry34Ab1 and cry35Ab1 have been transferred to suchcrops to enhance anti- coleopteran resistance properties. The cry3A(91 varieties) and cry34Ab1-cry35Ab1 (59 varieties) are themostwidelyused genes in GM Bt-crops with coleopteran pest-resistance, respec-tively. Moreover, recently, GM cotton containing the mcry51Aa2 genefor hemipteran insect resistance has been authorized for release(ISAAA's GM Approval Database, 2020).

2.1.5. Bt toxin genes in other microorganismsTo introduce insecticidal activity to other bacteria, fungi and viruses,

Bt cry genes have also been expressed in other microbial hosts, such asBeauveria bassiana (Deng et al., 2019), E. coli (Shi et al., 2004; Azizogluet al., 2016b), Pseudomonas spp., (Hernández-Rodríguez et al., 2013),Lactococcus lactis (Durmaz et al., 2016), Bacillus velezensis, Bacillussubtilis (Agaisse and Lereclus, 1994), Bacillus licheniformis (Theodulozet al., 2003) and baculoviruses (Martins et al., 2008). Azizoglu et al.(2016b) cloned a cry1Ab gene of a previously characterized lepidop-teran, dipteran and coleopteran active Bt SY49–1 strain, expressed itin E. coli BL21 (DE3), and individually tested it on two lepidopteran

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pests. The recombinant Cry1Ab with concentration of 1000 μg toxin pergram feed resulted in 40 and 64% mortality of Plodia interpunctella andE. kuehniella larvae, respectively. Deng et al. (2019) constructed a trans-genic B. bassiana fungus expressing the Cyt2Ba toxin (Bb-Cyt2Ba, andevaluated its toxicity against larval and adult Aedes mosquitoes (Aedesaegypti and Aedes albopictus). The bioassay tests indicated that recombi-nant Bb-Cyt2Ba significantly improved virulence of the transgenic fun-gus against larval and adult Aedes mosquitoes, and the median lethaltime (LT50) and insect fecundity was decreased by 30–42%. Durmazet al. (2016) transferred the full-length and truncated forms of cry5B,a nematode active gene, to L. lactisusing the high-copy-number plasmidpMSP3535H3 carrying a nisin-inducible promoter. The intracellular ly-sates of recombinant L. lactis cultures showed nematicidal activityagainst Caenorhabditis elegansworms (Durmaz et al., 2016).

Gurkan (2002) evaluated the oncolytic potential of membrane-acting immunotoxins (ITs) based on synthetic cyt2Aa1 (with opti-mized codon usage) as a novel antitumor agent using an in vitromodel system for human cancer. The recombinant production ofthese ITs was assessed using the Pichia pastoris expression system.The toxin intracellular expression was highly toxic to the host cells.In vitro cytotoxicity and hemolysis assays indicated that the recom-binant Cyt2Aa1-based IT-A was lytic to the target p185HER-2-overexpressing human cancer cell lines (SK-BR-3 and SK-OV-3). Toobtain sprayable insecticides containing Vip3 or Cry1I toxins,Hernández-Rodríguez et al. (2013) expressed these proteins in Pseu-domonas fluorescens as a protectant agent in insecticide formula-tions. The LC50 of recombinant Vip3Aa against S. frugiperda was89 ng/cm2in the diet, while the LC50 value of Cry1Ia purified fromP. fluorescens against Lobesia botrana was 10.7 μg/ml. To expand theusage of endophytes in biocontrol pests for agriculture and forestry,the Bt insecticidal gene cry218was transferred into the poplar bacte-rial endophyte Burkholderia pyrrocinia JK-SH007 (Li et al., 2017). Thetoxicity (LC50) of the expressed insecticidal protein was 0.77 g/L at72 h against second instar silkworms. Moreover, Bt cry genes havebeen transferred to different Bacillus species, such as B. velezensis(Roh et al., 2009), B. subtilis (Agaisse and Lereclus, 1994; Theodulozet al., 2003) and B. licheniformis (Theoduloz et al., 2003). To obtaintransgenic B. velezensis with antifungal and insecticidal activity, theBt cry1Ac gene under the control of its endogenous promoter wastransferred into a B. velezensis isolate with strong antifungal activityagainst different phytopathogens. The recombinant B. velezensisstrain produced a protein approximately 130 kDa in size as well asparasporal inclusion bodies and exhibited high insecticidal activityagainst a lepidopteran pest, P. xylostella (Roh et al., 2009).Theoduloz et al. (2003) expressed cry1Ab in B. subtilis (Bs-007) andB. licheniformis (Bl-012). The recombinant strains of both species ex-hibited a similar LC50 value as the Bt strain LM-466 against larvae ofT. absoluta. Agaisse and Lereclus (1994) expressed cry3A in theB. subtilis spo0A strain. Their results confirmed the correct produc-tion of the toxin and its activity against coleopteran pests. Martinset al. (2008) isolated and characterized the cry1Ia gene from the Btstrain S1451 and transferred it into the genome of a baculovirus.The recombinant Cry1Ia protein exhibited high toxicity against thecotton pests S. frugiperda and Anthonomus grandis.

2.2. Genetically modified Lysinibacillus (Bacillus) sphaericus

Lysinibacillus (Bacillus) sphaericus is a gram-positive species that hasbeen known to produce antimalarial and larvicidal compounds such asCry48/Cry49 and the S-layer protein, which kill mosquito larvae. Thisbacterium has been used for vector control programs against malaria,filariasis, yellow fever, dengue fever, and West Nile virus (Jones et al.,2007; Lozano et al., 2011; Berry, 2012; Lozano and Dussán, 2013;Naureen et al., 2017).Moreover, it has been confirmed that some strainsof this bacterium are able to promote plant growth and produce a wide

range of antifungal metabolites that can control phytopathogens(Naureen et al., 2017).

The anti-dipteran cry and cyt genes have been transferred to L.sphaericus to improve its insecticidal activity against disease vectorsand mosquitoes. For instance, the coexpression of the mosquitocidalCry and Cyt proteins in L. sphaericus cells could improve the efficacy ofL. sphaericus against Culex species by 10-fold (Federici et al., 2000).Abdel-Salam et al. (2018) improved the nematicidal activity of L.sphaericus against Meloidogyne incognita by using protoplast fusiontechnology for this bacterium and Bacillus amyloliquefaciens. Thenewly constructed strain produced a high amount of chitinase, signifi-cantly reduced nematode counts and provided better results for shootlength and fresh and dry weights than the control.

3. Other genetically modified bacteria expressing insect toxin genes

3.1. Entomopathogenic Serratiaspp.

Members of the Serratia species are some of the most important in-sect pathogens (Grimont and Grimont, 2006; Petersen and Tisa, 2012).Serratia marcescens is a gram-negative, facultative anaerobicentomopathogen that produces a high level of chitinolytic enzymes.They harbor chitinase genes, such as chiA, chiB, and chiC in their genome(Nawani and Kapadnis, 2001; Suzuki et al., 2002; Danışmazoğlu et al.,2015). In recent years, the expression of chitinase through cloning andGM-EPB strains with high chitinase activity has been popular topics ofstudy. It has been confirmed that recombinant E. colimodified by trans-ferring the chi A, B and C genes of S. marcescens show high chitinolyticactivity (45–80%) against the cotton bollworm H. armigera(Danışmazoğlu et al., 2015). When the chiA gene of S. marcescens wastransferred to the anti-coleopteran Bt 3023 strain and expressed withCry3Aa toxin, theGM-Bt 3023 strain produced a higher level of chitinasethan the source bacteria (S. marcescens) with the synergic interactionsof the ChiA-Cry3Aa proteins. They are promising for use against notonly pest insect larvae but also adult pest insects (Okay et al., 2008). Ser-ratia entomophila and Serratia proteamaculans cause amber disease ingrass grub (Costelytra zealandica). The symptoms of this disease arethat larvae stop feeding; void their intestines, turn amber in color, andeventually die. The 155 kb pADAP (Amber Disease-Associated Plasmid)of S. entomophila carries the sepA, sepB, and sepC genes. It has been de-termined that recombinant E. coli containing pADAP (47 kb) producesstrong anti-feeding activity that leads to the rapid death of larvae(Hurst et al., 2004).

3.2. Entomopathogenic Pseudomonasspp.

Pseudomonas spp. are bacteria that exist almost everywhere, includ-ing soil, water, and plant surfaces (Bucher and Stephens, 1957; Bucher,1960; Vodovar et al., 2006; Park and Federici, 2009; Liu et al., 2010).P. fluorescens is known to play an important role in plant growth byshowing antagonistic activity against plant pathogens such as Fusariumspp., Pythium spp., Rhizoctonia spp., and Sclerotium spp. (Peighami-Ashnaei et al., 2009; Trabelsi and Mhamdi, 2013). It is possible to im-prove the biological control potential of Pseudomonas spp. through ge-netic modification (Cook, 1993; Kong et al., 2016). The insecticidalactivity of GM-Pseudomonas can be stronger than that of its wild form(Kong et al., 2016).

A research group in South Africa integrated the transposable ele-ment “Omegon-Km”, designed specifically for chromosomal insertioninto their genome, to express Bt Cry1Ac7 toxins in P. fluorescens isolatedfrom sugar cane. It was also found that GM-P. fluorescens showed lowmortality against Eldana saccharina, which is harmful to sugar cane.However, it was found that the Bt cry1Ac7 and S. marcescens chiAgenes, when expressed together under the tac promoter in the GM-P. fluorescens 14:ptac-tox strain, show increased toxic effects againstE. saccharina compared to those with the expression of the genes

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individually (Downing et al., 2000). The GM-Pseudomonas-cry9Aa con-structed by transferring the insecticidal cry9Aa gene from Bt subsp.galleriaewas confirmed to be effective againstG.mellonella andpine cat-erpillar, Thaumetopoea pityocampa (Alberghini et al., 2005; Alberghiniet al., 2008).

The toxin complex (Tc) has oral activity against certain insects(ffrench-Constant et al., 2007; Liu et al., 2010). These toxins have ahigh molecular weight, are multiunits, and require three components(A, B, C) for complete toxicity. The Tc genes are found in some Pseudo-monas spp., such as insect-associated Pseudomonas entomophila, unas-sociated Pseudomonas syringae pv. tomato, and P. fluorescens strains(Liu et al., 2010). P. entomophila was the first species identified as apathogen in D. melanogaster (Vodovar et al., 2005). The insecticidal ac-tivity of P. entomophila TccC and TcdB toxins is made possible by thecoding of three TccC and one TcdB Tc toxin in its genome (Vodovaret al., 2006). The Pseudomonas sp. TKU015 strain isolated from the soilhas been defined as a new species, Pseudomonas taiwanensis, and the re-combinant TccC toxin of the tccC gene of this strain expressed in E. coliwas found to cause 60% mortality in 72 h in Drosophila larvae (Liuet al., 2010).

3.3. Entomopathogenic Photorhabdusspp.

Entomopathogenic nematodes (EPNs) are very important in biolog-ical pest control since they can survive for a long time and activelysearch for soil hosts (Şahin and Gözel, 2019). The generaHeterorhabditisand Steinernema are more successful in pest control than other EPNs(Hazir et al., 2003; Kaya et al., 2006; Lacey and Georgis, 2012; Yukseland Canhilal, 2019). Photorhabdus and Xenorhabdus have a symbiotic re-lationship with Heterorhabditis and Steinernema. This symbiotic rela-tionship creates strong pathogenicity against pests (Karabörklü et al.,2018). Photorhabdus luminescens is an entomopathogenic bacterium.The toxin complex (Tc) of P. luminescens is a heterotrimeric proteincomplex. The Tc consists of three components termed A, B, and C (orTcA, TcB, and TcC) (Waterfield et al., 2005a; Sheets and Aktories,2017) and is functional in mammalian cells as well as insects (Hareset al., 2008; Lang et al., 2010: Ng’ang’a et al., 2019). The direct use ofPhotorhabdus as an insecticide is severely limited. However, the recom-binant Tc toxins expressed in E. coli and transgenic plants have beenfound to show oral insecticidal activity against pest insects such asC. zealandica, Pieris brassicae, P. xylostella, Phaedon cochleariae G.mellonella, and M. sexta (Hurst et al., 2000; Morgan et al., 2001;Waterfield et al., 2001; Lee et al., 2004; Zhao et al., 2008).

Waterfield et al. (2005a) stated that tcdA, tcdB, and tccC in combi-nation show oral toxicity. TcdA1 and tcdB1 of the P. luminescens strainTT01 were transferred to the chromosomal DNA of Enterobacter clo-acae (the gut bacteria of a Formosan subterranean termite) via theTn5 transposon vector and were determined to produce approxi-mately 280 and 160-kDa recombinant proteins, respectively. GM-E. cloacae was found to be used successfully in the biological controlof Coptotermes formosanus and Solenopsis invicta (Zhao et al., 2008).In addition, Photorhabdus insect-related (Pir) proteins showed insec-ticidal activity. It was confirmed that these proteins (Pir A and PirB) encoded by two loci (plu4093-plu4092 and plu4437-plu4436) inthe genome of P. luminescens strain TT01 exhibited insecticidal activ-ity against moth and mosquito larvae (Waterfield et al., 2005b).When the mixture of recombinant Pir A (45 kDa) and Pir B(14 kDa) toxin expressed in E. coli EC100 was injected into theG. mellonella hemocoel, these larvae were observed to die within72 h. However, these toxins do not show insecticidal activity whenadministered alone. These toxins were further shown to have nooral activity when mixed with the artificial diet of M. sexta(Waterfield et al., 2005b). Pir toxins may be distantly related to BtCry toxins. Therefore, the action of these interesting and new bacte-rial toxins shows that they are worth studying in more detail.

3.4. Entomopathogenic Xenorhabdusspp.

Xenorhabdus nematophilus is a gram-negative entomopathogenicbacterium that has insecticidal activity on host larvae (Zhang et al.,2019). These bacteria have a symbiotic relationship with Steinernemacarpocapsae, an entomopathogenic nematode (Akhurst and Dunphy,1993). X. nematophilus, which is found in the intestinal sac of the infec-tive juveniles of EPNs, is carried to the hemocoel and intestine of thehost insect, causing the death of the insect in a short time(Khandelwal et al., 2004). The 537-bp DNA fragment ofX. nematophilus that encodes the Pilin subunit toxin protein (17 kDa)was cloned into E. coli BL21 (DE3) to produce a recombinant protein.This toxinwas found to showoral toxicity against the fourth andfifth in-star larvae of H. armigera and to cause major damage to the midgut ep-ithelial membrane (Khandelwal et al., 2004). Furthermore, the xptgenes of the X. nematophilus PMFI296 strain produced the toxin proteinresponsible for insecticidal activity. Sergeant et al. (2003) tested the in-secticidal activity of the recombinant toxin proteins produced by trans-ferring the xptA1, xptA2, xptB1, and xptC1 genes to E. coli againstP. brassicae, P. rapae, and Heliothis virescens pests alone and in combina-tion. It was reported that the three toxin combinations (XptA1, XptB1,and XptC1 combination in P. brassicae and P. rapae; XptA2, XptB1, andXptC1 combination in H. virescens) should be administered together,since the toxin combinations of XptA1-XptA2 and XptB1-XptC1 havelow insecticidal activity. Lee et al., (2004) stated that E. coli Rosetta(DE3) producing the TccC1 toxin proteins (110-kDa) of X. nematophilusdemonstrated 80%mortality within two days when the cell-free extractwas injected into G. mellonella larvae. The wild strains of Photorhabdusand Xenorhabdus can be used successfully for pest insect control, but ad-ditional new pathogenic toxins with broader host spectra can be pro-duced via genetic modification.

4. Possible impacts of GM entomopathogenic bacteria and GM Btcrops

4.1. Effects on nontarget organisms

4.1.1. Risks on predatorsThe main benefit of GM microbial insecticides that contain insect

toxins is that they reduce the use of chemical insecticides for pest con-trol. Chemical insecticides, which are applied generally by spraying, af-fect most beneficial insects (predators, parasitoids, pollinators, etc.) aswell as the target species (Downes, 2004). Most GM products are spe-cific to an insect order and may not have a direct effect on natural ene-mies. These beneficial arthropods may be indirectly affected as theyfeed on insects that take up toxins. However, Wang et al., (2008)showed that the GM-Bt UV173A strain (Cry3Aa7) had high insecticidalactivity against P. xylostella and Leptinotarsa decemlineata (Colorado po-tato beetle), and determined that it did not affect nontarget insects be-longing to different insect orders (Diptera, Hymenoptera, Coleoptera,Lepidoptera, Hemiptera, Homoptera, Thysanoptera, Orthoptera, andNeuroptera) in the field. Similarly, in a study by Rodrigo-Simón et al.(2006), it was reported that the predator Chrysoperla carnea, whenfeeding on H. armigera larvae that were exposed to the GM-BtEG11070 strain (Cry1Ac toxin), the GM Bt EG7077 strain (Cry1Abtoxin), and the GM Bt EG7699 strain (Cry2Ab toxin), was not adverselyaffected; moreover, no harmful effects were observed at the toxin bind-ing sites in the midgut epithelium of this predator when it was directlyexposed to the Cry1A toxin.

Abbas (2018) indicated that predators on GM-Bt cotton played animportant role for insect pest control on cotton, maize and peanutcrops adjacent to GM-Bt plants. The development, growth, proliferation,and longevity of the hemipteran predator Geocoris punctipes reared onH. zea and S. exigua pests fed GM-Bt cotton (Cry1Ac) were shown tobe unaffected (Torres and Ruberson, 2006). The survival, developmenttime, adult weight, and fecundity of the predator Coleomegilla maculata

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fed on T. ni (cabbage looper) larvae that were fed GM-Bt corn (Cry1Acand Cry2Ab)were reported to be unaffected (Li et al., 2011). The hemip-teran predators Geocoris punctipes, Podisus maculiventris, Nabisroseipennis, and Orius insidiosus were reported to be unaffected whenthey were fed nontarget herbivores that were interacting with GM-Btcotton (Cry1Ac) (Torres and Ruberson, 2008). Furthermore, Tian et al.(2014) confirmed that GM-Bt crops producing Cry1Ac, Cry2Ab andCry1F toxins have no effects on G. punctipes and O. insidiosus predators.However, the results might be different for other predators. Zhang et al.(2006) reported a decrease in the body weight and survival of Propyleajaponicawhen its prey, second instar S. litura, was reared on GM-Bt cot-ton. Romeis et al. (2019) reported that GM-Btmaize (Cry1Ab, Bt11) pol-len on Amblyseius cucumeris (predatory mite) caused significantadverse effects.

Finally, it could be concluded that although GM-EPBs and their re-combinant toxins have been reported to have no harmful effects onpredators in many studies, the mechanism of action of these toxinscan be changed by the toxin type, concentration, mode of administra-tion, and predator type. Moreover, there might be a risk of prey prefer-ence change, since prey toxin consumption leads to low-quality prey forpredators.

4.1.2. Risks on parasitoidsReducing the use of chemical pesticides has become a policy in

modern plant protection strategies and integrating pest manage-ment (IPM) programs (TAGEM, 2019). Not only the target organismbut also beneficial insects such as parasitoids are directly or indi-rectly affected by the insecticides that are used against agriculturalpests. Parasitoids are indispensable agents of biological control be-cause they are able to search for and find hosts (Kilinçer et al.,2010). So, evaluation of the possible risks of GM EPBs on parasitoidsis of importance. Recently, some studies have been focused on thissubject. The effect of the GM-Pseudomonas-cry9Aa strain carryingcry9Aa from the Bt ssp. galleriae (Btg) on Exorista larvarum, a larvalparasitoid of the pine processionary (Thaumetopoea pityocampa),was investigated. Marchetti et al., (2009) reported that the host lar-vae (G. mellonella) treated with GM-Pseudomonas-cry9Aa had no ad-verse effect on the egg laying, parasitism rate, and pupal weight ofE. larvarum. However, interestingly, there was a significant increasein the adult emergence of E. larvarum in host larvae treated withwild Pseudomonas compared to that of control larvae and larvaetreated with GM-Pseudomonas. Nontoxic, wild-type, epiphytic Pseu-domonas may have a stimulating effect on the parasitoid; therefore,this effect should be investigated in more detail (Marchetti et al.,2009). In a doctoral thesis by Steinbrecher (2004), GM-Bt corn(Cry1Ab) was reported to have no negative effect on Aphidiusrhopalosiphi, an aphid parasitoid, in terms of foraging efficiency andegg laying, and no evidence has been found that these herbivore par-asitoids are able to distinguish between GM-Bt corn and non-Bt corn.Similarly, it has been shown that GM-Bt canola (Cry1Ac) has no det-rimental effect on the development of Diaeretiella rapae (aphid par-asitoid), and GM-Bt eggplant has no negative effect on the adultemergence, development, or longevity of Aphidius ervi or Encarsiaformosa parasitoids. However, GM-Bt corn has been reported tocause low parasitism efficiency, as it is a low quality host site forTrichogramma brassicae reared in host eggs (H. armigera) affectedby GM-Bt corn. Moreover, Vojtech et al. (2005) reported that the sur-vival, development duration, and pupal cocoon weights of Cotesiamarginiventris parasitizing S. littoralis larvae were negatively af-fected when the larvae were fed GM-Bt corn. In another study, Xuet al. (2019) reported that GM-Bt maize had no effect on the attrac-tiveness of Trichogramma ostriniae compared to that of non-Bt iso-genic plants. Many studies have emphasized that GM-Bt crops donot directly affect herbivore parasitoids. Nevertheless, it should benoted that GM microbial agents and products might indirectly affectthese beneficial insects.

4.1.3. Risks on pollinatorsPollination is not only a process of biological interaction between

species but also a struggle for survival in the ecosystem. Approximately,75–95% of flowering plants on earth require pollination and pollinators(Crane andWalker, 1984; Buchmann and Nabhan, 1996; Ollerton et al.,2011). Pollinators provide continuity to the terrestrial ecosystems thatsupport natural life as well as growing the foods we consume(Costanza et al., 1997). They play a vital role in the conservation of bio-diversity by pollinating wild plants in ecosystems and transformingthem into food sources for wild animals and birds. Primary pollinatorsinclude honey bee species (Apis), bumblebee species (Bombus);, alkalibees (Nomia), alfalfa leafcutting bees (Megachilidae), orchard Masonbees (Osmia), carpenter bees (Xylocopa), solitary bee species, pollenwasps (Masarinae), ants (Formicidae), bee flies (Bombyliidae),hoverflies (Eupeodes), mosquitoes (Diptera), butterflies (lepidop-terans), flower beetles (Episyrphus), bird bats (Rhinolophus), insects,other bees, and some other animals (Bohart, 1972; McGregor, 1976;Torchio, 1981; Ranta and Tiainen, 1982; Torchio, 1987; Crane, 1990;Plowright and Plowright, 1997; Fairey et al., 1992; Konrad andBabendreier, 2006; Baskar et al., 2017). Honeybees are the best pollina-tors of plants and have been a standard test organism for evaluating thenontarget effects of chemical insecticides used for agricultural purposesfor many years (Duan et al., 2008). Insecticides are themost commonlyused chemicals for pest insect control in crop production. The applica-tion of chemicals affects not only pests but also beneficial insects suchas pollinators, predators, parasitoids, etc. (Johnson, 2010; Naranjo,2014). In pesticide applications, most pollinator poisonings occurwhen the pollinators visit flowers in treated areas. Such exposures canbe more dangerous than directly spraying into a hive or nest(Delaplane and Mayer, 2000). Fast-acting toxins rapidly kill bees inthe field, but bees exposed to slow-acting toxins survive long enoughto return to the nest. Furthermore, there is evidence that even low-dose insecticides disrupt the behavior of field bees in finding nests(Vandame et al., 1995). GM plant protection products are the most im-portant agent for reducing pesticide use because chemicals kill pollina-tors as well as pests (Johnson, 2010; Naranjo, 2014). It has beenreported that Bt toxins used in commercial GMproducts are not harmfulto bees, so their use can protect pollinator populations by reducing theuse of chemical pesticides. Recently, the most studied GM toxins havebeen Cry proteins, chitinase, β-1,3 glucanase, cowpea trypsin inhibitor(CpTI), serine protease inhibitors, cysteine protease inhibitors, biotin-binding protein (avidin), and snowdrop (Galanthus nivalis) lectin. Ameta-analysis of 25 studies evaluating the potential effects of GM Bttoxins on honeybees reported that Bt-Cry proteins had no adverse ef-fects on the survival of honeybee larvae and adults (Babendreier et al.,2005; Duan et al., 2008). In field studies, several researchers reportedthat Bt maize (Cry1Ab), Bt sweetcorn (Cry1Ab), and Bt oilseed rape(Cry1Ac) did not affect larval development, offspring quantity, foragingactivity, adult weight, adult survival, frequency and duration of visits toflowers, or frequency of movements among flowers in honey bees(Schur et al., 2000; Tesoriero et al., 2004; Rose et al., 2007). Additionally,it was reported that GM-Bt maize pollen (Cry1Ab, Cry1F) and GM-Btcotton pollen (Cry1Ac) had no effect on larval and pupal survival,pupalweight, hemolymphprotein concentration of young adults, bacte-rial flora of adults, or enzyme activity (superoxide dismutase) in bees(Hanley et al., 2003; Liu et al., 2005; Babendreier et al., 2007). Similarly,Yi et al., (2018) reported that GM-Bt cabbage (Cry1Ba3) pollen underlaboratory conditions did not affect pollen consumption, midgut en-zyme activity, weight and survival in A. mellifera. Similar studies wereconducted on bumblebee species (Picard-Nizou et al., 1997; Morandinand Winston, 2003; Arpaia et al., 2004; Babendreier et al., 2007; Yuet al., 2011) and Mason bees (Konrad and Babendreier, 2006), and noadverse effects were reported. Many excellent studies indicated thatGM-Bt crops had no detrimental effects on the honey bees (Steijvenet al., 2016; Wang et al., 2017; Niu et al., 2017; Ricroch et al., 2018; Yiet al., 2018). However, the tissue, nectar or pollen of GM-Bt plants can

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accumulate huge amounts of Bt toxins and can harm bee populations(colony collapse disorder (CCD)) (Poppy, 1998; Duan et al., 2008;Lemaux, 2009). In addition, in a study on the possible causes of CCD, itwas reported that therewasnot a sufficient geographical correlation be-tween GM plant-grown regions and regions with CCD (Lemaux, 2009).

In conclusion, current GM products are less toxic to wild pollina-tors than many conventional pesticides. In some cases, these prod-ucts may contribute to efficient pollination by reducing the use ofpesticides and contributing to the formation of healthy bee popula-tions. Although these products seem unlikely to have direct effectson bees, unintended changes in phenotypes resulting from geneticmodifications in GM-Bt plants, such as low nectar quality and vol-ume, may indirectly affect pollinators (O'Brien and Arathi, 2018;Gebretsadik and Ashenafi, 2018). In addition, the genes transferredto these plants do not cause a specific phenotypic change, but theexpressed gene products may alter the biochemical pathways inthe plant (Malone et al., 2001). GM-Bt proteins can be specificallyexpressed in pollen, which is the only source of protein for pollinatorinsects. The result may be chronic toxin exposure from the pollen.Further studies should investigate the long-term effects of thesetoxins on these beneficial organisms.

4.2. Risks on the microbial communities in the rhizosphere

In recent years,with the help of biotechnology and genetic engineer-ing, many microorganisms, such as EPBs which are in symbiotic rela-tionships with plants have been genetically modified and used for thecontrol of pest insects and phytopathogens (Singh et al., 2011;Carmona-Hernandez et al., 2019). However, the risk that such GM mi-croorganismsmay significantly change the existing rhizosphere popula-tion should not be ignored. In this respect, researchers have warnedabout the changes inmicrobial populations and the rate of microbial ac-tivity in the rhizosphere due to the increase in the use of manymicroor-ganisms, especially GM bacteria, that are released into the soil andrhizosphere (Wang et al., 2008).

Wang et al., (2008) observed the GM-Bt strain UV173A in the soil fortwo years and found that the effect of the bacterial concentration at thattime (Bacillus spp., and Pseudomonas spp.) on the population in the soilwas not statistically significant. Moreover, they reported that popula-tion dynamics in some nontarget invertebrates was not significantly af-fected by the GM-Bt strain, and the overall impacts of such GM bacteriashould be evaluated.

GM-P. fluorescens “pc78” and “pc78–48” strains used as biologicalcontrol agents in the rhizosphere of tomato plants did not affect thepopulation or diversity of bacteria living in the phyllosphere (Konget al., 2016). Glandorf et al. (2001) examined the effects of rifampicin-resistant GM-P. putida WCS358r, a genetically enhanced derivative ofa wild-type biocontrol species, on the fungal rhizosphere microflora offield-grown wheat and compared them to the effects of bacteria thatproduce phenazine. The strains did not affect the metabolic activity ofthe soil microbial population, the soil nitrification potential, cellulosedegradation, or plant yield. In the evaluation of such GM strains, the ef-fects of these species not only on the subsoil but also on vertebrate andinvertebrate organisms have emerged. After the release of GM-EPBs,there is a risk for the possibility of gene transfer among indigenous bac-teria, meaning that the environmental effects of such bacteria on thenatural ecological balance should be investigated and demonstrated.Among these effects, the most important is the transitions of genes be-tween different microorganisms. The concept of gene transitions iscalled gene transfer or transmission from one population to other pop-ulations (Keese, 2008; Ramzan et al., 2014). This transition occurs intwo different ways, horizontal gene transfer (HGT) and vertical genetransfer (VGT), which co-exist in the natural environment (Li et al.,2019). In vertical gene transfer, examining the microorganisms or par-ent populations for the desired traits can involve testing for the pres-ence of certain genes (Vogan and Higgs, 2011). These examinations

can provide information about the dangers that could occur due to ge-netic modifications. This information can be used for comparisons, andthemost important gene transitions to focus on are the landscape (hor-izontal = lateral) gene transfers. Horizontal gene transitions betweendifferent species may cause unpredictable changes and/or risks to theenvironment and to microorganisms. Particularly in different microor-ganism types, such as viruses and bacteria, there may be a possiblebackflow of transgenic bacteria. In different field population studies,there is evidence that gene transfer may take place among soil bacteria(Dröge et al., 1999; Amarger, 2002). Plasmids are extra-chromosomalDNA that replicate independently of the bacterial genome and are acommon feature of bacteria (Doghaither andGull, 2019).Mobile geneticelements (MGEs) such as plasmids are continually changing so the var-iability carried by plasmids increases the rapidity at which modifiedstrains arise (Maheshwari et al., 2017). However, E. cloacae carryingthe toxin complex “Tc” gene was environmentally-friendly, and thegreen fluorescent protein (GFP plasmid) in GM bacteria was not trans-ferred to other soil bacteria (Husseneder and Grace, 2005; Zhao et al.,2008).

The effect of GM crops onnontarget soilmicroorganisms depends onthe type of recombinant proteins and level of exposure. The decomposi-tion of GM crops residues releases modified biomolecules into the rhi-zosphere. Additionally, gene flow may occur when GM-Bt crops genesare transferred from GM crops to nontarget organisms. The impact ofgene flow depends on many factors such as the environmental condi-tion, applied microflora, microfauna, soil ecology, and soil microorgan-ism populations (Mandal et al., 2020). Xie et al. (2016) examined theGM-Bt cotton in the field for three years and found that GM-Bt cottondid not show significant changes in fungal community in the rhizo-sphere. Similarly, many researchers reported that GM-Bt cotton didnot significantly affect bacterial communities and soil dynamics in therhizosphere (Li et al., 2018; Qi et al., 2018; Zhaolei et al., 2018; Mandalet al., 2020).

However, evaluating such studies in general may be the wrong ap-proach. In the evaluation of such studies, many factors such as culture,environment, method,microbial communities, soil ecology, and soil dy-namics should be considered and future in situ studies should representonly one area.

5. Conclusions and recommendations

Due to the high importance of EPBs in pest management, re-searchers throughout the world carry out detailed studies on suchmicroorganisms to develop new engineered strains with more hostrange, higher toxicity and less problems in the filed conditions. Thisstrategy has led to achievement of novel engineered with higher ac-tivities against different pests. Compared with wild EPBs, GM-EPBshave many advantages, such as lower spraying requirements, long-term persistence, lower insect resistance, and higher efficiency.However; social concerns about the release of such GM-EPBs intothe environment have led to various controversies. The main con-cern about GM-EPBs is that people have doubts regarding their po-tential adverse effects on the environment, which include effect onnon-target organisms, gene flow to wild species and the develop-ment of resistance in target pests. So, following recommendationsshould be taken into consideration:

• Further analysis may be required to determine whether microbial di-versity changes following the release of GM-EPBs. In particular, thepossible effects of gene flow to indigenous bacteria and microorgan-isms should be evaluated;

• The possible changes that GM-EPBs may cause in rhizosphere micro-organism populations should be identified;

• The adverse effects that may occur in microflora due to the increaseduse of recombinant microorganisms should be investigated not onlyin vertebrates but also in invertebrates;

11U. Azizoglu et al. / Science of the Total Environment 734 (2020) 139169

• Biosafety values and human and environmental health issues shouldbe taken into consideration; in situ studies should be especiallyemphasized.

As a result, it is necessary to focus on how to make GM-EPBs morecompatible with the environment. There might be a need for more ex-tensive training for ecologists, agricultural scientists, and molecular bi-ologists to address the recommendations given above. We stronglyrecommend further multidisciplinary training and collaborative re-search efforts on the environmental risks and benefits of GM-EPBs.

Authors’ contributions

UA designed the study. UA: Entomopathogenic Photorhabdus spp,Entomopathogenic Xenorhabdus spp, Risks on predators, and Risks onparasitoids. GSJ: Genetically modified Bacillus thuringiensis and Genet-ically modified Lysinibacillus (Bacillus) sphaericus. NY: Risks on themi-crobial communities in the rhizosphere. EB: Entomopathogenic Serratiaspp., and Entomopathogenic Pseudomonas spp. DO: Risks on pollina-tors. All authors read and approved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgements

The authors would like to thank the Erciyes University Proofreading& Editing Office for their support provided in the English revision of themanuscript.

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