Molecular detection of nematicidal crystalliferousBacillus thuringiensis strains of Iran andevaluation of their toxicity on free-living andplant-parasitic nematodes

Gholamreza Salehi Jouzani, Ali Seifinejad, Abbas Saeedizadeh, Amin Nazarian,Majid Yousefloo, Saeed Soheilivand, Maryam Mousivand, Rosa Jahangiri,Mehdi Yazdani, Reza Maali Amiri, and Sepideh Akbari

Abstract: The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) col-lection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6,cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein bandpatterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nema-todes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates(31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundantand represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 geneswere less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry geneprofiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, andcry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containingnematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolateswere selected for detailed bioassays. Isolates YD5 and KON4 at 2 � 108 CFU/mL concentrations showed 77% and 81%toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and thehighest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was re-corded for isolates SN1 and KON4 at 2 � 108 CFU/mL concentrations and resulted in 68% and 77% adults deaths of C.tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity pre-diction of nematicidal Bt isolates.

Key words: Acrobeloides enoplus, Bacillus thuringiensis, cry gene, Meloidogyne incognita, Chiloplacus tenuis.

Resume : La caracterisation de souches de Bacillus thuringiensis (Bt) efficaces contre les nematodes et des genes cry, apartir d’une collection de Bt iraniennes (70 isolats), est presentee. La caracterisation a ete realisee par une analyse en PCRa l’aide de 12 amorces specifiques aux genes cry5, cry6, cry12, cry13, cry14 et cry21 codant des proteines actives contreles nematodes, par l’analyse de la morphologie des cristaux et par les patrons de bandes proteiques, ainsi que par leur acti-vite nematicide envers un nematode des racines (Meloidogyne incognita) et deux nematodes libres (Chiloplacus tenuis etAcrobeloides enoplus). Les resultats en PCR avec les amorces correspondant a ces genes ont montre que 22 isolats(31,5 %) contiennent au moins un gene cry actif contre les nematodes. Les souches contenant le gene cry6 etaient les plusabondantes et representaient 22,8 % de tous les isolats. Les souches de Bt comportant le gene cry14 etaient aussi abondan-tes (14,2 %). Les genes cry21 et cry5 etaient les moins abondants, etant trouves chez 4,2 % et 2,8 % des souches, respecti-vement. Au total, six profils de genes cry actifs contre les nematodes ont ete detectes dans cette collection. Quatre isolatsne possedant aucun des produits PCR de tailles attendues pour les genes cry5, cry6 et cry 21 pourraient contenir de nou-veaux genes codant des proteines cristallines insecticides. Vingt-deux isolats de Bt contenant un gene cry actif contre les

Received 7 May 2008. Revision received 21 June 2008. Accepted 18 July 2008. Published on the NRC Research Press Web site atcjm.nrc.ca on 17 September 2008.

G. Salehi Jouzani,1 A. Seifinejad, A. Nazarian, S. Soheilivand, M. Mousivand, R. Jahangiri, and S. Akbari. Department ofMicrobial Biotechnology and Biosafety, Agricultural Biotechnology Research Institute of Iran, Mahdasht Road, P.O. Box 21525-1897,Karaj, Iran.A. Saeedizadeh and M. Yousefloo. Department of Plant Protection, Faculty of Horticultural Sciences and Plant Protection, UniversityCollege of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.M. Yazdani and R.M. Amiri. Department of Agronomy and Plant Breeding, Faculty of Agronomy and Animal Sciences, UniversityCollege of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

1Corresponding author (e-mail: gsalehi@abrii.ac.ir).

812

Can. J. Microbiol. 54: 812–822 (2008) doi:10.1139/W08-074 # 2008 NRC Canada

nematodes ont ete choisis pour realiser des essais preliminaires envers M. icognita. A partir de ces essais, quatre de cesisolats ont ete choisis pour des essais biologiques detailles. Les isolats YD5 et KON4 utilises a des concentrations de 2 �108 CFU/mL etaient toxiques a 77 % et 81 % respectivement envers M. incognita. Les nematodes libres C. tenuis et A.enoplus etaient plus sensibles, la mortalite la plus forte etant observee en moins de 48 h d’incubation a toutes les concen-trations testees. La mortalite maximale a ete enregistree avec les isolats SN1 et KON4 a des concentrations de 2 �108 CFU/mL qui avaient cause 68 % et 77 % de mortalite chez C. tenuis adulte et 68 % et 72 % de mortalite chez A. eno-plus, respectivement. Nous resultats demontrent que la PCR est une technique utile pour predire la toxicite d’isolats de BTnematicides.

Mots-cles : Acrobeloides enoplus, Bacillus thuringiensisi, genes cry, Meloidogynes incognita, Chiplacus tenuis.

[Traduit par la Redaction]

______________________________________________________________________________________

IntroductionPlant-parasitic nematodes are found as pests of crops

throughout the world with many having a severe economiceffect. The cost imparted on world agriculture by plant-parasitic nematodes was estimated to be around US$125 bil-lion in 2003 (Chitwood 2003). The most economically im-portant species are the sedentary endoparasites of the genusMeloidogyne, also called root-knot nematodes because oftheir characteristic galling effect on roots (Dubreuil et al.2007), and the genera Heterodera and Globodera (cyst nem-atodes) (Lilley et al. 2007).

Biological control of plant-parasitic nematodes has longbeen considered as an alternative to nematicides, especiallybecause of the environmental and health concerns associatedwith these chemicals. In the past, research into the biologi-cal control of plant-parasitic nematodes has focused mainlyon predatious (e.g., nematophagous fungi) and parasitic(e.g., Pasteuria penetrans) microorganisms (Aalten et al.1998; Khan et al. 2003; Castillo et al. 2006; Rodrıguez-Romero et al. 2008).

Previously, it was shown that some Bacillus thuringiensis(Bt) strains had significant toxicity on eggs and larvae ofplant-parasitic and free-living nematodes (Osman et al.1988; Bradfish et al. 1991; Devidas and Rehberger 1992;Mozgovaya et al. 2002; Tian et al. 2007). Bt is a Gram-positive soil bacterium that is characterized by the produc-tion of insecticidal crystal proteins, which are known to betoxic to a wide range of insect species in the orders Lepi-doptera, Diptera, Coleoptera, and Hymenoptera (Maagd etal. 2001; Seifinejad et al. 2008). Some Cry proteins are alsotoxic to other invertebrates such as nematodes, mites, andprotozoans (Feitelson et al. 1992; Schnepf et al. 1998). Todate, there are six Cry protein families (Cry5, Cry6, Cry12,Cry13, Cry14, and Cry21) known to be toxic to larvae of anumber of free-living or parasitic nematodes (Bravo et al.1998; Crickmore et al. 1998; Marroquin et al. 2000; Wei etal. 2003; Crickmore 2005; Kotze et al. 2005; Tian et al.2007). On the basis of amino acid sequence homology, thesenematode-affecting Cry proteins (except for Cry6A) wereassigned to a single cluster in the main Cry lineage, parallelto other main groups (Bravo 1997; Marroquin et al. 2000).Separate phylogenetic analysis of the three domains of Cryprotein also generated a consensus tree result. The domain Iand domain II trees showed that nematode-specific toxins(Cry5, Cry12, Cry13, Cry14, and Cry21) were arranged to-gether in a single branch. Domain III from all the nematode-specific toxin trees are also clustered together (Bravo 1997).

Nematicidal and insecticidal toxins of Bt are believed toshare similar modes of action. Cry protein exerts its effectsby forming lytic pores in the cell membrane of gut epithelialcells. After ingestion of toxin by target nematode larvae, thecrystals dissolve within the gut of the nematode, and this isfollowed by proteolytic activation (Crickmore 2005). Crytoxicity is directed against the intestinal epithelial cells ofthe midgut and leads to vacuole and pore formation, pitting,and eventual degradation of the intestine (Marroquin et al.2000). The binding of pore forming toxin to a receptor inthe epithelial cell is a major event. A detailed understandingof how the Bt toxins interact with nematodes should facili-tate the production of more effective Bt biocontrol agents.Other than Cry toxin, previous studies using Bacillus thurin-giensis var. israelensis, Bacillus thuringiensis var. kurstakiand another parasporal crystal-forming bacterium, Bacillussphaericus, showed that some strains had significant activityon the eggs and larvae of animal-parasitic nematodes(Bottjer et al. 1986; Bowen et al. 1986a, 1986b; Bowen andTinelli 1987; Meadows et al. 1989).

The search for native strains with activity against plant-parasite nematodes could have an impact on the control ofnematodes worldwide. In this study, we evaluated thenematode-active cry genes contents of the native Bt strainsand used these findings for prediction of nematicidal activityof these isolates. The selected isolates showed high toxicityon plant-parasitic (Meloidogyne incognita) and free-living(Acrobeloides enoplus and Chiloplacus tenuis) nematodes.Bt strains containing some of the previously describednematode-active cry genes as well as potentially novel Cryproteins were identified.

Materials and methods

Bacterial strainsSeventy native Bt strains stored in the GenBank collection

of the Agricultural Biotechnology Research Institute of Iranwere used. These strains were originally isolated from soilsamples collected from different fields within three climatezones of Iran, Cold or Moderate Mountainous, Caspianic,and Dry and Semidry zones as previously described (SalehiJouzani et al. 2008). Known strains, including Bacillus thur-ingiensis var. darmstadiensis, Bacillus thuringiensis var. gal-leriae, Bacillus thuringiensis var. sotto, and Bacillusthuringiensis var. roskildiensis, that served as references fornematode-specific cry genes were supplied by DSMZ Com-pany (the German Resource Centre for Biological Material,

Salehi Jouzani et al. 813

# 2008 NRC Canada

Braunschweig, Germany). Cultures of Bt strains were grownat 28 8C in nutrient broth (Difco) with vigorous shaking oron nutrient broth agar.

Oligonucleotide primers and PCR analysisFor detection of six nematode-specific active cry genes,

cry5, cry6, cry12, cry13, cry14, and cry21, six pairs of spe-cific primers from regions of related genes were synthesizedas previously described (Ejiofor and Johnson 2002; Porcarand Juarez-Perez 2003) (Table 1). Oligonucleotides weresynthesized in a DNA synthesizer (Microsyn 1450A; SystecInc.) as specified by the manufacturer. Total DNA was ex-tracted and purified following the method described byFerrandis et al. (1999). PCR was conducted for 250 ng oftotal Bt DNA with 2.5 U of Taq DNA polymerase,200 nmol/L each dNTP, 1 mmol/L each primer, and3 mmol/L MgCl2 in a final volume of 50 mL. Amplificationwas done in a Bio-Rad thermal cycler under the followingconditions: 5 min of denaturation at 94 8C followed by 25cycles of amplification with a 1 min denaturation at 94 8C,45 s of annealing at 45 8C, and 2 min of extension at 72 8C.An extra extension step of 10 min at 72 8C was added aftercompletion of the 25 cycles (Juarez-Perez et al. 1997). PCRproducts were analyzed by 1% agarose gel electrophoresis.PCR products were confirmed for genes of interest by re-striction analysis or sequencing. The distribution frequencyof a cry gene in Bt strains was defined as the percentage ofthe Bt isolates containing this gene among all of the isolatesaccording to Wang et al. (2003).

Production and characterization of parasporal inclusionsThe strains containing nematode-specific active cry genes

were cultured in the standard Bt medium (UG) containingbactopeptone (7 g/L), glucose, and salts as reported byLecadet and Dedonder (1971) at 28–30 8C for 72 h. By theend of growth, the bacterial density reached 1– 2 �109 CFU/mL. The percentage of vegetative cells was about1%. Spore–crystal mixtures were washed (10 000g for10 min at 41 8C) once in 0.5 mol/L NaCl and then twice incold sterile water. Two-millilitre samples of lysed cultureswere washed by centrifugation and resuspended once in2 mL of 0.5 mol/L NaCl and then twice in cold sterilizedwater containing 1 mmol/L protease inhibitor phenylmethyl-sulfonyl fluoride. Crystal proteins were solubilized with

10 mmol/L 2-mercaptoethanol at 37 8C for 4 h and centri-fuged at 10 000 g, suspended in sterile tap water, and finallystored at –20 8C until use. Parasporal inclusions of each iso-late were classified through phase-contrast and light micro-scopy.

Protein electrophoresisThe protein content of spore–crystal mixtures of the

strains containing nematode-specific active cry genes wasdetermined by SDS–PAGE analysis, as described byLaemmli (1970), using 10% or 12.5% acrylamide separatinggels. Samples (5–15 mg) of washed spore–crystal mixtures,prepared as described by Thomas and Ellar (1983), wereplaced in two concentrated sample buffers and heated at80 8C for 10 min, as described by Lecadet et al. (1992),and loaded onto the gel immediately before electrophoresis.Gels were stained with a solution containing 50% (v/v) etha-nol, 10% (v/v) acetic acid, and 0.1% (w/v) Coomasie bril-liant blue R250 for 40 min and then destained with asolution containing 6.75% (v/v) glacial acetic acid and9.45% (v/v) ethanol.

Preparation of M. incognita and bioassaysThe source of nematode inoculum was a culture of M. in-

cognita isolated originally from roots of eggplants (Solanummelongena) in fields of the Karaj region (Tehran Province)and maintained on tomato seedlings (Lycopersicum esculen-tum). The egg mass and adult females were separated fromthe root galls. To identify the species, perineal pattern offemales was prepared and the egg mass of identified spe-cies was first transferred to water for 24–48 h at 20–22 8Cuntil the second-stage juveniles (J2s) were hatched. Theywere then added to pots with pasteurized soil and newlyplanted tomato seedlings. Nematode eggs were extractedfrom 8-week-old nematode-infected roots, disrupted in 1%sodium hypochlorite by the procedure described by Husseyand Barker (1973), and used as inocula in the bioassays.Preliminary bioassays with high concentrations of spore–crystal suspensions (extracted spore–crystal mixtures wereused without any dilution) of the strains containingnematode-specific cry genes were performed with M. in-cognita. Four strains with high toxicity on M. incognitawere selected for detailed bioassays. Two treatments wereused in which a volume of 6 mL of the spore–crystal mix-

Table 1. Characteristics of specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes.

Primer Sequence Positions Gene(s) recognizedProductsize Accession No(s).

cry5(+) 5’-taagcaaagcgcgtaacctc-3’ 2459–2478 cry5Aa1, Ab1, Ac1, Ba1 322 L07025, L07026, I34543, U19725cry5(–) 5’-gctcccctcgatgtcaatg-3’ 2780–2762 .cry6(+) 5’-tggcgtagaggctgttcaagta-3’ 609–630 cry6Aa1, Aa2, Ba1 302 L07022, AF499736, L07024cry6(–) 5’-tgtcgagttcatcattagcagtgt-3’ 910–887 .cry12(+) 5’-ctcccccaacattccatcc-3’ 2555–2573 cry12Aa1 363 L07027cry12(–) 5’-aattacttacacgtgccatacctg-3’ 2917–2894 .cry13(+) 5’-ctttgattatttaggtttagttcaa-3’ 225–250 cry13Aa1 313 L07023cry13(–) 5’-ttgtagtacaggcttgtgattc-3’ 537–515 .cry14(+) 5’-ataatgcgcgacctactgttgt-3’ 1634–1655 cry14Aa1 456 U13955cry14(–) 5’-tgccgttatcgccgttatt-3’ 2089–2071 .cry21(+) 5’-atacagggataggatttcaag-3’ 1112–1132 cry21Aa1, Aa2, Ba1 453 I32932, I66477, AB088406cry21(–) 5’-atccccattttctataagtgtct-3 ’ 1564–1542 .

814 Can. J. Microbiol. Vol. 54, 2008

# 2008 NRC Canada

ture of each Bt strain with a concentration of 1 � 108 and2 � 108 CFU/mL was added to the 10 mL Petri dishes. Inthe control Petri dishes, sterile tap water was added. Therewere four replicates of each treatment and controls. Fivehundred eggs were placed into each Petri dish filled withthe two considered concentrations of spore–crystal mixturesand incubated at 25 8C (Franks et al. 2003). The percent-age of mortality (numbers of live J2s, dead J2s, and non-hatched eggs) was scored after 5 days in comparison witha parallel control in which eggs were dipped in sterile dis-tilled water instead of bacterial suspension. The data mor-tality were corrected with Abbott’s (1925) formula.

Preparation of free- living nematodes (C. tenuis andA. enoplus) and bioassays

Bioassays with spore–crystal suspensions of the strainscontaining nematode-specific cry genes also were performedwith field-collected free-living nematodes, including C. ten-uis and A. enoplus. For isolation of these nematodes, severalsoil samples were collected from tomato and eggplant fieldsof the Karaj region. These nematodes were extracted fromsoil samples with a centrifugal floatation technique andidentified according to De Grisse (1969). The adults wereused as inocula in the bioassays. Two treatments were usedin which a volume of 6 mL of the spore–crystal mixture ofeach Bt strain with a concentration of 1 � 108 and 2 �108 CFU/mL was added to the 10 mL Petri dishes. In thecontrol Petri dishes, sterile tap water was added. Then, 10adult nematodes from each species were transferred to10 mL Petri dishes filled with one of the two concentrationsof Bt spore–crystal extracts (Franks et al. 2003). There werefour replicates of each treatment and controls. Scoring ofpercentage of mortality (numbers of live and dead nema-todes) was started after 2 days in comparison with parallelcontrols. The data mortality were corrected with Abbott’s(1925) formula.

Statistical methodsFor all investigated parameters, ANOVA was performed

using the Minitab statistical software (version 13.20). Themeasurements of treatments were compared and grouped us-ing Duncan’s multiple range tests at the 0.01 significancelevel.

ResultsThe Bt strain collection used in this study was previously

characterized by different methodologies for detection andcharacterization of Lepidoptera- and Diptera-active strains(Salehi Jouzani et al. 2008; Seifinejad et al. 2008). In thisstudy, the distribution of nematode-effective cry genesamong these native Bt strains was evaluated, and finally,based on PCR and bioassays, the most nematode-effectivestrains were selected.

The PCR methodology described here utilized 12 primersto detect six different nematode-active cry genes, cry5, cry6,cry12, cry13, cry14, and cry21, described to codify for pro-teins active against nematodes (Table 1). Figure 1 showssome PCR products obtained with the strains. Knownstrains, including Bt var. darmstadiensis, Bt var. galleriae,Bt var. sotto, and Bt var. roskildiensis, that served as refer-

ences for nematode-specific cry genes showed the expectedPCR products with primers for the expected cry genes(Fig. 1). Each known cry gene should produce a PCR prod-uct with a unique molecular mass. Therefore, strains withPCR products of sizes other than those predicted are alsocandidates for harboring putative novel cry genes.

PCR results showed that 22 native strains contained aminimum of one nematode-active cry gene (31.5%). Fournematode-active cry genes, cry5, cry6, cry14, and cry21,were found in the isolates. Strains containing the cry6 genewere the most abundant and represent 16 of the 70 Bt iso-lates (22.8%) (Fig. 2). Bt strains harboring the cry14 genewere also abundant (14.2%). The cry21 and cry5 geneswere less abundant, found in 4.2% and 2.8% of the strains,respectively. None of the isolates contained more than twonematode-active cry genes. Six different nematode-activecry gene profiles were detected in this collection. The mostcommon profile of nematode-active cry genes containedonly the cry6 gene (11.5%) (Table 2). Finally, 68.5% of thestrains did not give any PCR product when assayed with pri-mers of the nematode-active genes (Fig. 2). It is importantto note that we previously showed that these isolates containother cry genes (Salehi Jouzani et al. 2008; Seifinejad et al.2008) (Table 2). Four native isolates containing nematode-active cry genes, S82, KH4, GON3, and GON7, did notshow the expected PCR product size for the cry5, cry6, andcry21 genes; they might contain potentially novel insecti-cidal crystal protein genes. Characterization and cloning ofthe potentially novel cry genes and the search for additionalnovel genes will be continued.

Microscopic studies of 22 nematode-active cry gene con-taining isolates showed that about 50% of the isolates con-tained more than one type of crystal. These strainscommonly showed bipyramidal, cuboidal, rhomboidal,spindly, and spherical forms of parasporal inclusions(Table 2). The diameter of the crystals was 0.7–1.5 mm.The isolates containing nematode-active cry genes were fur-ther characterized by SDS–PAGE of the crystal proteinproducts. The results revealed that these isolates synthesizeproteins with a molecular mass between 28 and 140 kDa(Fig. 3). The isolates produced two to six proteins withbands in this vicinity. All 22 isolates showed more than oneband.

Preliminary in vitro bioassays showed that high concen-trations of spore–crystal mixtures of 22 selected nativestrains had 0%–100% toxicity against M. incognita (datanot shown). The most effective strains, YD5, SN1, KON4,and GN5, were selected for more detailed bioassays. In thefollowing bioassays, two concentrations, 1 � 108 and 2 �108 CFU/mL, of spore crystal mixtures extracted from thesefour selected strains were used. After 24 h of incubation,none of the treatments affected the hatched nematodes. Theobservations were continued for 96 h. After 4 days, treatedeggs with spore–crystal mixtures either did not hatch orhatched and the larvae were dead. All preparations showednematicidal activity against M. incognita with up to 81%nematode mortality by days 3–4 (Table 3). Four strainsshowed significant nematicidal effects at probability 0.01 onM. incognita in comparison with the control. The toxicityrate of 2 � 108 CFU/mL for all isolates was about 10%more than that for 1 � 108 CFU/mL. Strains YD5 and

Salehi Jouzani et al. 815

# 2008 NRC Canada

KON4 (2 � 108 CFU/mL) showed 77% and 81% toxicity onM. incognita, respectively. It is important to note that allpreparations inhibited hatching of nematode eggs. The fourtested isolates inhibited egg hatching to varying degrees(Table 3). The highest inhibition of hatching of root-knotnematode eggs was observed for YD5 and KON4 with a46% and 45% inhibition rate, respectively.

The free-living nematodes C. tenuis and A. enoplus weremore susceptible and the highest mortality was observedwithin 48 h of incubation at all concentrations tested. Twopreparations of the four strains showed significant nemati-cidal effects, killing the adults of C. tenuis and A. enoplusto a varying degree (Table 4). Maximum mortality was re-

corded where 2 � 108 CFU/mL of strain SN1 and KON4was used and resulted in 68% and 77% adult deaths, respec-tively, of C. tenuis and 68% and 72%, respectively, for A.enoplus. Strain YD5 also produced 41% mortality of larvalC. tenuis (Table 4). It is important to note that our micro-scopic observations of inoculated nematodes showed that Bttoxins disrupted and blacked the digestive tract in threestudied nematodes after 3–4 days (data not shown).

Discussion

Recent reports on the frequency of occurrence of Bt iso-lates in natural environments indicate a high possibility of

Fig. 1. Agarose gel (1.2%) electrophoresis of PCR products amplified from native Bt strains. (A) Electrophoresis with cry5 primers: lane 1,Bt var. darmstadiensis as positive control; lanes 2–4, native Bt strains (S54, GN5, and GN3, respectively); M, molecular mass marker(1 kb); (B) electrophoresis with cry6 primers: lanes 1–5, native Bt strains (GN15, S54, GN5, GN3, and GN4, respectively); (C) electro-phoresis with cry14 primers: lane 1, QM2; lane 2, Bt var. sotto as positive control; lanes 3–6, native Bt strains (AL11, KON4, MI1, andYD5, respectively); (D) electrophoresis with cry21 primers: 1, Bt var. roskildiensis as positive control; lanes 2–4, native Bt strains (YD5,KH7, and QM2, respectively).

816 Can. J. Microbiol. Vol. 54, 2008

# 2008 NRC Canada

Tab

le2.

Cry

stal

prot

ein

and

gene

feat

ures

of22

Bt

isol

ates

cont

aini

ngne

mat

ode-

activ

ecr

yge

nes.

Stra

inC

ryst

alfo

rma

Prot

ein

size

(s)

(kD

a)b

Nem

atod

e-ac

tive

gene

(s)

Lep

idop

tera

-act

ive

gene

(s)

dete

cted

byPC

R(S

eifi

neja

det

al.

2008

)D

ipte

ra-a

ctiv

ege

ne(s

)de

tect

edby

PCR

(Sal

ehi

Jouz

ani

etal

.20

08)

S54

Rm

35,

70,

135

cry5

cry4

B,

cry1

0,cr

y11,

cry2

4K

H5

Bp,

Rm

95,

50,

35cr

y6cr

y1A

a,cr

y1A

c,cr

y1C

,cr

y1D

,cr

y1F

,cr

y1I,

cry2

Ab,

cry9

B,

cry9

D,

vip3

Aa

cry2

Ab,

cry4

B,

cry1

0,cr

y11,

cry2

4,cy

t2

KH

7B

p,Sp

135,

95,

70cr

y6,

cry2

1cr

y1A

a,cr

y1A

b,cr

y1A

c,cr

y1C

,cr

y1D

,cr

y1F

,cr

y1I,

cry2

Aa,

cry9

B,

cry9

D,

vip3

Aa

cry2

Aa,

cry4

B,

cry1

0,cr

y11,

cry2

1,cr

y24

GN

1Sp

,C

b50

,70

,80

cry6

cry2

Ab,

cry4

Cor

D,

cry1

0G

N3

Cb,

Rm

17,

28,

50,

65cr

y6cr

y2A

b,cr

y10,

cry1

1,cr

y17,

cyt2

GN

4B

p,C

b13

5,12

0,63

,60

cry6

cry1

Aa,

cry1

Ab,

cry1

Ac,

cry1

C,

cry1

D,

cry1

E,

cry1

F,

cry1

I,vi

p3A

acr

y2A

a,cr

y10,

cry1

1,cy

t2

GN

5C

b28

,30

,35

,50

,13

5cr

y5cr

y2A

b,cr

y4B

,cr

y10,

cry1

1,cr

y17,

cry1

9,cy

t1,

cyt2

GN

15C

b,R

m28

,35

,50

,70

cry6

cry9

cry2

Ab,

cry4

Cor

D,

cry1

0,cr

y11,

cry2

4,cy

t2A

GI8

Rm

,C

b,B

p28

,50

,70

,12

0cr

y6cr

y1A

a,cr

y1A

c,cr

y1C

,cr

y1D

,cr

y1F

,cr

y1I

cry2

Ab,

cry1

7,cy

t1,

cyt2

KO

N1

Bp,

Cb

120,

95,

35cr

y14,

cry2

1cr

y1A

a,cr

y1C

,cr

y1D

,cr

y1E

,cr

y1F

,cr

y1I,

cry9

B,

cry9

D,

vip3

Aa

cry1

0,cr

y21,

cry2

4

YD

5B

p,R

m70

,13

5cr

y6,

cry1

4cr

y1A

a,cr

y1A

b,cr

y1A

c,cr

y1C

,cr

y1D

,cr

y1F

,cr

y1I,

cry2

Aa,

vip3

Aa

cry4

Cor

D,

cry1

0,cr

y11,

cry2

4

KH

9E

l,B

p17

,35

,50

,90

cry6

,cr

y14

cry1

Aa,

cry1

Ab,

cry1

Ac,

cry1

C,

cry1

F,

cry1

Icr

y2A

b,cr

y10,

cry1

7A

GI2

El,

Bp

50,

70,

135

cry6

cry1

Aa,

cry1

Ab,

cry1

Ac,

cry1

C,

cry1

D,

cry1

Icr

y2A

a,cr

y17

SN1

El

130

cry6

KO

N4

Bp,

Cb

135,

95cr

y14

cry1

Aa,

cry1

Ab,

cry1

Ac,

cry1

C,

cry1

D,

cry1

E,

cry1

F,

cry1

I,cr

y2A

,cr

y9D

,vi

p3A

aK

ON

6B

p,E

l24

,35

,50

,70

,12

0cr

y6,

cry1

4G

ON

4C

b,B

p,E

l50

,70

,12

0cr

y6,

cry1

4cr

y2A

bQ

M2

Bp,

Sl70

,13

5cr

y6,

cry2

1cr

y2,

cry2

1,cr

y24

AL

10B

p,C

b,Sp

120,

70cr

y6,

cry1

4cr

y1A

a,cr

y1A

b,cr

y1A

c,cr

y1C

,cr

y1D

,cr

y1E

,cr

y1F

,cr

y1I,

cry2

Ab,

vip3

Aa

cry2

Ab

AL

11B

p,C

b13

5,64

,43

cry1

4cr

y1A

a,cr

y1A

b,cr

y1A

c,cr

y1C

,cr

y1D

,cr

y1E

,cr

y1F

,cr

y1I,

cry2

Aa,

vip3

Aa

cry2

Aa

MI1

Bp,

Sl50

,90

,12

0cr

y14

cry2

Ab

AG

I1B

p,E

l13

5,95

cry6

,cr

y14

cry1

Aa,

cry1

Ab,

cry1

Ac,

cry1

Ad,

cry1

C,

cry1

D,

cry1

E,

cry1

F,

cry1

I,cr

y2A

a,vi

p3A

acr

y2A

a,cy

t2

aR

m,

rhom

boid

al;

Bp,

bipy

ram

idal

;Sp

,sp

heri

cal;

Sl,

spin

dle;

Cb,

cubo

idal

;E

l,el

liptic

al.

bM

ajor

prot

ein

band

son

SDS–

PAG

Ege

ls.

Salehi Jouzani et al. 817

# 2008 NRC Canada

being able to isolate novel strains of cry genes (Ben-Dov etal. 1997; Hansen et al. 1998; Ejiofor and Johnson 2002;Porcar and Juarez-Perez 2003; Wang et al. 2003). In this pa-per, the characterization of nematode-active Bt strains iso-lated from different agroecological regions of Iran ispresented. These results are useful for understanding the dis-tribution of cry genes and for prediction of toxicity and maylead to the identification of novel candidate genes for bio-engineered crop protection. Many researchers have utilizeda wide variety of techniques to identify and characterizecry-gene-carrying Bt isolates. For example, Brown andWhiteley (1992) employed molecular characterization toidentify two novel crystal protein genes from Bacillus thur-ingiensis var. thompsoni. They cloned and sequenced thecrystal proteins of this strain in Escherichia coli and usedthe electrophoretic mobilities to predict the molecularmasses of amino acid sequences. This method has been use-ful to characterize fully the insecticidal crystal proteins ofisolates. Recently, many workers have turned to PCR(Juarez-Perez et al. 1997; Ejiofor and Johnson 2002; Porcarand Juarez-Perez 2003; Arrieta et al. 2004) or a combination

of this technique with other methods, such as serology (Kim2000), SDS–PAGE for the detection and characterization ofcry genes from their isolates, and insecticidal activity(Porcar and Caballero 2000). Akhurst et al. (1997) used a16S rRNA gene oligonucleotide probe in PCR experiments,which allowed them to separate Bt serovars. We have com-bined morphological, molecular, and bioassay methods toachieve this objective. Few studies have reported a detailedcharacterization of Bt strain collections in terms of nemati-cidal cry gene content (Osman et al. 1988; Marroquin et al.2000; Mozgovaya et al. 2002). In this study, we consideredall known nematode-effective cry genes, detected them inthe native Bt isolates, and used obtained the information forprediction of their toxicity on nematodes.

Our results showed that 22 native strains contained a min-imum of one nematode-active cry gene (31.5%). Four nema-tode-active cry genes, cry5, cry6, cry14, and cry21, werefound in the isolates, but cry12 and cry13 were not ob-served. The frequency of nematode-active cry genes in Ira-nian strains seems to be higher than that in Bt straincollections previously analyzed (Bravo et al. 1998; Ejioforand Johnson 2002). Bravo et al. (1998) evaluated about 496Bt strains isolated from 503 soil samples collected from fiveregions of Mexico and did not find any nematode-active crygenes in their collection. Also, Ejiofor and Johnson (2002)presented a PCR characterization of 95 Bt strains isolatedfrom soil samples collected from the south-central UnitedStates that showed a different cry gene distribution and didnot find any nematode-active cry genes in the collection.

Four strains gave PCR products of different sizes whenassayed with cry5, cry6, and cry21 primers. These strainsare candidates for harboring novel cry genes. The isolationand sequencing of novel cry genes should be encouragedonce the target insect is identified and more evidence onthe potential of novel toxins as biological control agents isavailable.

The preliminary bioassays on M. incognita showed thatsome strains harboring the same cry gene profiles have dif-ferent activity against nematodes. The results confirm thatthe presence of specific genes is not an accurate indicatorof toxicity because the genes could be inactive, under thecontrol of an inefficient promoter, or expressed in a concen-tration too low to affect toxicity (Masson et al. 1998;Ferrandis et al. 1999). The bioassay results indicated thattwo native strains (YD5 and KON4) remarkably reducedthe populations of M. incognita. Also, these strains remark-ably reduced the percentages of hatchability of eggs of thenematode. These results confirm the earlier reports ofOsman et al. (1988) and Mozgovaya et al. (2002) in whichtomatoes inoculated with M. incognita had fewer galls onroots grown in soil containing Bt than on roots grown insoil without Bt. To a lesser extent, Bt also reduced the per-centage of hatchability of the eggs of M. incognita. Theseresults are in agreement with those of Osman et al. (1988)who reported that Bt reduced the root galling and numberof egg masses produced by M. incognita. They reported thatBt also reduced the percent hatch of the eggs of M. incog-nita.

The results of bioassays on C. tenuis and A. enoplusshowed that the isolates SN1 and KON4 with more than68% mortality had the most toxicity on these nematodes.

Fig. 3. SDS–PAGE of spore crystals of some native Bt strains(KON6, SN1, GN5, YD5, and KH9) containing nematode-activecry genes; M, molecular mass marker.

Fig. 2. Distribution of nematode-active cry genes in 70 field-collected strains of Bt and identified by PCR analysis with specificprimers.

818 Can. J. Microbiol. Vol. 54, 2008

# 2008 NRC Canada

Tab

le3.

Nem

atic

idal

activ

ityof

four

nativ

eB

tst

rain

ste

sted

invi

tro

agai

nst

root

-kno

tne

mat

odes

M.

inco

gnit

a.

Dea

d(%

)N

onha

tche

deg

gs(%

)O

bser

ved

tota

lm

orta

lity

(%)

Cor

rect

edm

orta

lity

(%)

Stra

in1�

108

CFU

/mL

2�

108

CFU

/mL

1�

108

CFU

/mL

2�

108

CFU

/mL

1�

108

CFU

/mL

2�

108

CFU

/mL

1�

108

CFU

/mL

2�

108

CFU

/mL

GN

518

±3

30±

434

±435

±4

52c

65b

35c

52b

KO

N4

35±

541

±5

32±4

45±

467

a86

a55

a81

aY

D5

30±

337

±4

37±6

46±

562

b83

a48

b77

aSN

121

±3

21±

330

±642

±3

51c

63b

33c

49b

Con

trol

6±2

5±2

21±4

23±

327

d28

c0d

0d

Not

e:D

ata

are

mea

nsof

four

repl

icat

es±

SE.

With

inro

ws,

mea

nsfo

llow

edby

the

sam

ele

tter

are

not

sign

ific

antl

ydi

ffer

ent

atth

e0.

01pr

obab

ility

leve

lba

sed

onD

unca

n’s

mul

tiple

rang

ete

sts.

Tab

le4.

Tox

icity

offo

urna

tive

Bt

stra

ins

test

edin

vitr

oag

ains

tth

efr

ee-l

ivin

gne

mat

odes

C.

tenu

isan

dA

.en

oplu

s.

C.

tenu

is%

obse

rved

mor

talit

yC

.te

nuis

%co

rrec

ted

mor

talit

yA

.en

oplu

s%

obse

rved

mor

talit

yA

.en

oplu

s%

corr

ecte

dm

orta

lity

Stra

in1�

108

CFU

/mL

2�

108

CFU

/mL

1�

108

CFU

/mL

2�

108

CFU

/mL

1�

108

CFU

/mL

2�

108

CFU

/mL

1�

108

CFU

/mL

2�

108

CFU

/mL

GN

539

±3c

51±

4c20

±1c

40±

3c43

±4b

56±4

b17

±2b

43±

3bK

ON

452

±5b

74±

5b37

±3b

68±

4b60

±4a

78±5

a50

±3a

72±

4aY

D5

41±

3c52

±4c

23±2

c41

±3c

39±

7c54

±5c

23±

4c40

±3c

SN1

64±

3a81

±3a

40±

2a77

±2a

62±

6a75

±6a

52±

4a68

±5a

Con

trol

24±

2d19

±2d

0d0d

21±

3d23

±3d

0d0d

Not

e:D

ata

are

mea

nsof

four

repl

icat

es±

SE.

With

inro

ws,

mea

nsfo

llow

edby

the

sam

ele

tter

are

not

sign

ific

antl

ydi

ffer

ent

atth

e0.

01pr

obab

ility

leve

lba

sed

onD

unca

n’s

mul

tiple

rang

ete

sts.

Salehi Jouzani et al. 819

# 2008 NRC Canada

SN1 and KON4 contained only cry5 and cry14, respectively.It is important to note that other strains containing two nem-atode-active cry genes showed less toxicity than others con-taining one. This may be because of silencing or lowexpression of these genes.

The mechanisms of the nematicidal activity of Bt are notcompletely understood. It has been reported that spores of Btgerminated in the intestine of the bacteriophagous nematodePanagrellus redivivus. Subsequently, colonization of the en-tire nematode body by Bt occurred within 24 h (Borgonie etal. 1995). It has been suggested that the nematicidal factor iseffectively internalized into the cells of the nematode diges-tive tract (Devidas and Rehberger 1992; Borgonie et al.1995, 1996; Mozgovaya et al. 2002; Wei et al. 2003). Weiet al. (2003) showed that toxicity in nematodes correlateswith damage to the intestine, consistent with the mechanismof crystal toxin action in insects. Intoxication was readilyjudged by a lack of pumping, inhibition of growth (i.e.,small size), pale coloration, and lethargy. Marroquin et al.(2000) observed that nematodes fed Cry5B showed exten-sive changes in gut morphology. These changes includedthe formation of vacuole-like structures in gut cells, ashrinking of the gut away from the body wall of the animal,tight constrictions at various points along the gut, and anoverall pitted appearance. Our microscopic observationsconfirm the previous studies because deformation, discolora-tion, and darkness of the digestive tract were observed inthree studied nematodes after 3–4 days.

Another possibility is contact nematicidal activity of the tox-ins, as observed by Devidas and Rehberger (1992). These work-ers found that a partially purified preparation of b-exotoxinfrom Bt, when applied directly to the nematode body sur-face, caused 100% mortality of the free-living nematodeCaenorhabditis elegans but not of the juveniles of the root-knot nematode M. incognita. We believe that both the infec-tive process and the direct contact should be considered innematicidal studies. Further studies of the mechanisms ofthe nematicidal activity of Bt are needed to estimate its po-tential as a biological control agent and to evaluate possibleside-effects.

Management of plant-parasitic nematodes involves reduc-tion in nematode populations in the soil and roots withoutadversely affecting the plant response (yield). In this study,we examined the utility of this microbe to limit plant nema-tode populations. The concentration at which noticeable re-duction in nematode numbers became apparent was 2 �108 CFU/mL for strains YD5 and KON4. Thus, it could beconcluded that the two native isolates YD5 and KON4 havehigh toxicity on M. incognita. Furthermore, in previouswork, we showed that YD5 has high toxicity on Lepidopterapests and also contains some Diptera-active cry genes in-cluding cry4C or cry4D, cry10, cry11, and cry24 (SalehiJouzani et al. 2008; Seifinejad et al. 2008), so after green-house and field confirmations, it can be used as biocontrolagent with potential and can be an alternative to chemicalpesticides to avoid pollution and hazards to the environment.

The bioassay of these two selected isolates against otherplant-parasitic nematodes, characterization of the observedpotentially novel cry genes, and the search for additionalnovel genes will be continued. Additional areas of researchcould include genetic engineering of crop plants to incorpo-

rate the toxin genes, structural modification of the toxin it-self for improved activity, and possibly the selection of Btstrains with higher levels of nematicidal activity.

ConclusionIt appears from the present study that research on native

Bt isolates from natural environments indicates a high possi-bility of being able to isolate novel strains and cry genesthat detection of nematicidal cry genes using PCR technol-ogy is very useful for prediction of nematicidal effects ofnative Bt strains, and that the use of two selected strainsthat are toxic to both insects and root-knot nematodes couldbe developed into a valuable crop management tool to re-duce the deleterious impact of insects and plant-parasiticnematodes on plant production.

AcknowledgementsThe authors are indebted to Dr. Seyyed Mojtaba Khayam

Nekouie and Dr. Rahim Osfoori for support and criticalreading of the manuscript. This work was supported by agrant from the Agricultural Research and Education Organi-zation of Iran.

ReferencesAalten, P.M., Vitour, D., Blanvillain, D., Gowen, S.R., and Sutra,

L. 1998. Effect of rhizosphere fluorescent Pseudomonas strainson plant-parasitic nematodes Radopholus similis and Meloido-gyne spp. Lett. Appl. Microbiol. 27: 357–361. doi:10.1046/j.1472-765X.1998.00440.x.

Abbott, W.S. 1925. A method of computing the effectiveness of aninsecticide. J. Econ. Entomol. 18: 265–267.

Akhurst, R.J., Lyness, E.W., Zhang, Q.Y., Cooper, D.J., andPinnock, D.E. 1997. A 16S rRNA gene oligonucleotide probefor identification of Bacillus isolates from sheep fleece. J.Invertebr. Pathol. 69: 24–30. doi:10.1006/jipa.1996.4610.PMID:9028924.

Arrieta, G., Hernandez, A., and Espinoza, A.M. 2004. Diversity ofBacillus thuringiensis strains isolated from coffee plantations in-fested with the coffee berry borer Hypothenemus hampei. Rev.Biol. Trop. 52: 757–764. PMID:17361568.

Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R.,Manasherob, R., et al. 1997. Extended screening by PCR for sevencry-group genes from field collected strains of Bacillus thuringien-sis. Appl. Environ. Microbiol. 63: 4883–4890. PMID:9406409.

Borgonie, G., Vandriessche, R., Leyns, F., Arnaut, G., Dewaele, D.,and Coomans, A. 1995. Germination of Bacillus thuringiensisspores in bacteriophagous nematodes (Nematoda, Rhabditida).J. Invertebr. Pathol. 65: 61–67. doi:10.1006/jipa.1995.1008.PMID:7876593.

Borgonie, G., Claeys, M., Leyns, F., Arnaut, G., Dewaele, D., andCoomans, A. 1996. Effect of nematicidal Bacillus thuringiensisstrains on free-living nematodes. 2. Ultrastructural analysis ofthe intoxication process in Caenorhabditis elegans. Fundam.Appl. Nematol. 19: 407–414.

Bottjer, K.P., Bowen, L.W., and Gill, S.S. 1986. Nematoda: sus-ceptibility of the egg to Bacillus thuringiensis toxins. Exp. Para-sitol. 60: 239–244. doi:10.1016/0014-4894(85)90027-X.

Bowen, L.W., and Tinelli, R. 1987. Trichostrongylus colubriformis:larvicidal activity of toxic extracts from Bacillus sphaericus(strain 1593) spores. Exp. Parasitol. 64: 514–516. doi:10.1016/0014-4894(87)90066-X. PMID:3678452.

Bowen, L.W., Bottjer, K.P., and Gill, S.S. 1986a. Trichostrongylus

820 Can. J. Microbiol. Vol. 54, 2008

# 2008 NRC Canada

colubriformis: isolation and characterization of ovicidal activityfrom Bacillus thuringiensis israelensis. Exp. Parasitol. 62:247–253. doi:10.1016/0014-4894(86)90029-9. PMID:3743716.

Bowen, L.W., Bottjer, K.P., and Gill, S.S. 1986b. Trichostrongyluscolubriformis: egg lethality due to Bacillus thuringiensis crystaltoxin. Exp. Parasitol. 60: 314–322.

Bradfish, G.A., Hickle, L.A., Flores, R., and Schwab, G. 1991. Ne-maticidal Bacillus thuringiensis toxins: opportunities in animalhealth and plant protection. 1st Int. Conf. on Bacillus thurin-giensis. St. Catherine’s College, Oxford, U.K. p. 33.

Bravo, A. 1997. Phylogenetic relationships of Bacillus thuringien-sis delta-endotoxin family proteins and their functional domains.J. Bacteriol. 179: 2793–2801. PMID:9139891.

Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz,A., et al. 1998. Characterization of cry genes in a Mexican Ba-cillus thuringiensis strain collection. Appl. Environ. Microbiol.64: 4965–4972. PMID:9835590.

Brown, K.L., and Whiteley, H.R. 1992. Molecular characterizationof two novel crystal protein genes from Bacillus thuringiensissubsp. thompsoni. J. Bacteriol. 174: 549–557. PMID:1729243.

Castillo, P., Nico, A.I., Azcon-Aguilar, C., Del Rıo Rincon, C.,Calvet, C., and Jimenez-Dıaz, R.M. 2006. Protection of oliveplanting stocks against parasitism of root-knot nematodes byarbuscular mycorrhizal fungi. Plant Pathol. 55: 705–713. doi:10.1111/j.1365-3059.2006.01400.x.

Chitwood, D.J. 2003. Research on plant-parasitic nematode biologyconducted by the United States Department of Agriculture, Agri-cultural Research Service. Pest Manag. Sci. 59: 748–753. doi:10.1002/ps.684. PMID:12846325.

Crickmore, N. 2005. Using worms to better understand how Bacil-lus thuringiensis kills insects. Trends Microbiol. 13: 347–350.doi:10.1016/j.tim.2005.06.002. PMID:15967665.

Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Rie, J.V.,Lereclus, D., et al. 1998. Review of the nomenclature for theBacillus thuringiensis pesticidal crystal proteins. Microbiol.Mol. Biol. Rev. 62: 807–813. PMID:9729610.

De Grisse, A.T. 1969. Redescription ou modifications de quelquesthechniques utilisees dans letude des nematodes phytoparasi-taires. Meded. Fak. Landbouww. Gent. 34: 351–369.

Devidas, P., and Rehberger, L.A. 1992. The effects of exotoxin(thuringiensin) from Bacillus thuringiensis on Meloidogyne in-cognita and Caenorhabditis elegans. Plant Soil, 145: 115–120.doi:10.1007/BF00009547.

Dubreuil, G., Magliano, M., Deleury, E., Abad, P., and Rosso, M.N.2007. Transcriptome analysis of root-knot nematode functions in-duced in the early stages of parasitism. New Phytol. 176: 426–436.doi:10.1111/j.1469-8137.2007.02181.x. PMID:17692078.

Ejiofor, A.O., and Johnson, T. 2002. Physiological and moleculardetection of crystalliferous Bacillus thuringiensis strains fromhabitats in the South Central United States. J. Ind. Microbiol.Biotechnol. 28: 284–290. doi:10.1038/sj.jim.7000244. PMID:11986933.

Feitelson, J.S., Payne, J., and Kim, L. 1992. Bacillus thuringiensisinsects and beyond. Biotechnology, 10: 271–275. doi:10.1038/nbt0392-271.

Ferrandis, M.D., Juarez-Perez, V.M., Frutos, R., Bel, Y., and Ferre,J. 1999. Distribution of cryI, cryII and cryV genes within Bacil-lus thuringiensis isolates from Spain. Syst. Appl. Microbiol. 22:179–185.

Franks, T., Savocchia, S., and van Heeswijch, R. 2003. In vitrosymptoms for studying the interaction of root-knot nematodewith grapevine. Nematology, 5: 235–242. doi:10.1163/156854103767139734.

Hansen, B.M., Damgaard, P.H., Eilenberg, J., and Pedersen, J.C.

1998. Molecular and phenotypic characterization of Bacillus thur-ingiensis isolated from leaves and insects. J. Invertebr. Pathol. 71:106–114. doi:10.1006/jipa.1997.4712. PMID:9500938.

Hussey, R.S., and Barker, K.R. 1973. A comparison of methods ofcollecting inocula of Meloidogyne spp., including a new techni-que. Plant Dis. Rep. 57: 1025–1028.

Juarez-Perez, V.M., Ferrandis, M.D., and Frutos, R. 1997. PCR-based approach for detection of novel Bacillus thuringiensis crygenes. Appl. Environ. Microbiol. 63: 2997–3002. PMID:9251188.

Khan, A., Williams, K., and Nevalainen, H. 2003. Testing theematophagous biological control strain Paecilomyces lilacinus251 for paecilotoxin production. FEMS Microbiol. Lett. 227:107–111. doi:10.1016/S0378-1097(03)00654-2. PMID:14568155.

Kim, H.S. 2000. Comparative study of the frequency, flagellarserotype, crystal shape, toxicity, and cry gene contents of Bacil-lus thuringiensis from three environments. Curr. Microbiol. 41:250–256. doi:10.1007/s002840010129. PMID:10977891.

Kotze, A.C., O’Grady, J., Gough, J.M., Pearson, R., Bagnall, N.H.,Kemp, D.H., and Akhurst, R.J. 2005. Toxicity of Bacillus thur-ingiensis to parasitic and free-living life stages of nematodesparasites of livestock. Int. J. Parasitol. 35: 1013–1022. doi:10.1016/j.ijpara.2005.03.010. PMID:15964574.

Laemmli, U.K. 1970. Cleavage of structural proteins during the as-sembly of the head of basterophage T4. Nature (Lond.), 227:680–685. doi:10.1038/227680a0. PMID:5432063.

Lecadet, M.M., and Dedonder, R. 1971. Biogenesis of the crystallineinclusion of Bacillus thuringiensis during sporulation. Eur. J. Bio-chem. 23: 282–294. doi:10.1111/j.1432-1033.1971.tb01620.x.PMID:5003206.

Lecadet, M.M., Chaufaux, J., Ribier, J., and Lereclus, D. 1992.Construction of novel Bacillus thuringiensis strains with differ-ent insecticidal activities by transduction and transformation.Appl. Environ. Microbiol. 58: 840–884. PMID:16348674.

Lilley, C.J., Wayne, M.B., and Urwin, P. 2007. Recent progress inthe development of RNA interference for plant parasitic nema-todes. Mol. Plant Pathol. 8: 701–711. doi:10.1111/j.1364-3703.2007.00422.x.

Maagd, R.A., Bravo, A., and Crickmore, N. 2001. How Bacillusthuringiensis has evolved specific toxins to colonize the insectworld. Trends Genet. 17: 193–199. doi:10.1016/S0168-9525(01)02237-5. PMID:11275324.

Marroquin, L.D., Elyassnia, D., Griffitts, J.S., Feitelson, J.S., andAroian, R.V. 2000. Bacillus thuringiensis (Bt) toxin susceptibil-ity and isolation of resistance mutants in the nematode Caenor-habditis elegans. Genetics, 155: 1693–1699. PMID:10924467.

Masson, L., Erlandson, M.A., Puzstai-Carey, M., Brousseau, R.,Juarez-Perez, V., and Frutos, R. 1998. A holistic approach fordetermining the entomopathogenic potential of Bacillus thurin-giensis strains. Appl. Environ. Microbiol. 64: 4782–4788.PMID:9835562.

Meadows, J., Gill, S.S., and Bowen, L.W. 1989. Factors influen-cing lethality of Bacillus thuringiensis kurstaki toxin for eggsand larvae of Trichostrongylus colubriformis. J. Parasitol. 75:191–194. doi:10.2307/3282765. PMID:2926587.

Mozgovaya, I.N., Byzov, B.A., Ryabchenko, N.F., Romanenko,N.D., and Zvyagintsev, D.G. 2002. Nematicidal effects of theentomopathogenic bacteria Bacillus thuringiensis in soil. Pedo-biologia (Jena), 46: 558–572. doi:10.1078/0031-4056-00160.

Osman, G.Y., Salem, F.M., and Ghattas, A. 1988. Bio-efficacy oftwo bacterial insecticide strains of Bacillus thuringiensis as abiological control agent in comparison with a nematicide, Ne-macur, on certain parasitic Nematoda.. Anz. Schidlingskd. Pflan-zenschutz Umweltschutz, 61: 35–37.

Salehi Jouzani et al. 821

# 2008 NRC Canada

Porcar, M., and Caballero, P. 2000. Molecular and insecticidalcharacterization of a Bacillus thuringiensis strain isolated duringa natural epizootic. J. Appl. Microbiol. 89: 309–316. doi:10.1046/j.1365-2672.2000.01115.x. PMID:10971764.

Porcar, M., and Juarez-Perez, V.M. 2003. PCR-based identificationof Bacillus thuringiensis pesticidal crystal genes. FEMS Micro-biol. Rev. 26: 419–432. doi:10.1111/j.1574-6976.2003.tb00624.x.PMID:12586389.

Rodrıguez-Romero, A.S., Badosa, E., Montesinos, E., and Jaizme-Vega, M.C. 2008. Growth promotion and biological control ofroot-knot nematodes in micropropagated banana during the nur-sery stage by treatment with specific bacterial strains. Ann.Appl. Biol. 152: 41–48. doi:10.1111/j.1744-7348.2007.00189.x.

Salehi Jouzani, G.R., Pourjan Abad, A., Seifinejad, A., Marzban,R., Kariman, K., and Maleki, B. 2008. Distribution and diversityof dipteran-specific cry and cyt genes in native Bacillus thurin-giensis strains obtained from different ecosystems of Iran. J. Ind.Microbiol. Biotechnol. 35: 83–94. doi:10.1007/s10295-007-0269-6. PMID:17999100.

Schnepf, E., Crickmore, N., Rie, J.V., Lereclus, D., Baum, J.,Feitelson, J., et al. 1998. Bacillus thuringiensis and its pesticidalcrystal proteins. Microbiol. Mol. Biol. Rev. 62: 775–806.PMID:9729609.

Seifinejad, A., Salehi Jouzani, G.R., Hosseinzadeh, A., andAbdmishani, C. 2008. Characterization of Lepidoptera-activecry and vip genes in Iranian Bacillus thuringiensis strain collec-tion. J. Biol. Control, 44: 216–226. doi:10.1016/j.biocontrol.2007.09.010.

Thomas, W.E., and Ellar, D. 1983. Mechanism of action of Bacil-lus thuringiensis var. israelensis insecticidal delta-endotoxin.FEBS Lett. 154: 362–368. doi:10.1016/0014-5793(83)80183-5.PMID:6832375.

Tian, B., Yang, J., and Zhang, K.Q. 2007. Bacteria used in thebiological control of plant-parasitic nematodes: populations,mechanisms of action, and future prospects. FEMS Microbiol.Ecol. 61: 197–213. doi:10.1111/j.1574-6941.2007.00349.x.PMID:17651135.

Wang, J.A., Boets, J., and VanRie, G. 2003. Characterization ofcry1, cry2, and cry9 genes in Bacillus thuringiensis isolatesfrom China. J. Invertebr. Pathol. 82: 63–71. doi:10.1016/S0022-2011(02)00202-1. PMID:12581721.

Wei, J.Z., Hale, K., Carta, L., Platzer, E., Wong, C., Fang, S.C., andAroian, R.V. 2003. Bacillus thuringiensis crystal proteins thattarget nematodes. Proc. Natl. Acad. Sci. U.S.A. 100: 2760–2765.doi:10.1073/pnas.0538072100. PMID:12598644.

822 Can. J. Microbiol. Vol. 54, 2008

# 2008 NRC Canada