J Pest Sci (2008) 81:199–206 DOI 10.1007/s10340-008-0206-0
ORIGINAL PAPER
Genetic variation and mtCOI phylogeny for Bemisia tabaci (Hemiptera, Aleyrodidae) indicate that the ‘B’ biotype predominates in Iran H. Rajaei Shoorcheh · B. Kazemi · S. Manzari · J. K. Brown · A. Sarafrazi
Received: 29 June 2007 / Revised: 12 May 2008 / Accepted: 15 May 2008 / Published online: 4 June 2008 © Springer-Verlag 2008
Abstract Despite a large number of investigations on the molecular genetics and population structure of the whiteXy Bemisia tabaci (Gennadius) complex, no such study had been conducted in Iran. The genetic variation of B. tabaci was examined using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) for 18 Weld collections from cucumber, eggplant, and tomato in four provinces of Iran. PCR ampliWcation and restriction digestion with two enzymes detected 388 RFLP fragments, of which 16 fragments showed polymorphisms. Cluster analysis of these data placed all B. tabaci individuals within a single group, and there was no evidence for between- or within-population genetic variation. Phylogenetic (Clustal W) analysis of 42 B. tabaci mtCOI sequences (n = 21 Weld collections) from Iran, and a comparison with well-studied haplotype or biotype reference sequences available in public sequence databases, revealed that the Iranian B. tabaci
Communicated by M. Traugott. H. R. Shoorcheh (&) Biology Department, Shahid Beheshti University, G.C. Tehran, Iran e-mail:
[email protected] B. Kazemi Cellular and Molecular Biology Research Center, Shahid Beheshti University, M.C. Tehran, Iran S. Manzari · A. Sarafrazi Insect Taxonomy Research Department, Iranian Research Institute of Plant Protection, P.O. Box 1454, Tehran 19395, Iran J. K. Brown Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA
populations were most closely related to the B biotype at 0–1.2% nucleotide identity. The B biotype is a well-known member of a sister clade from the Middle East–North African region of the world, owing to its nearly worldwide distribution and invasive characteristics. This report indicates that a single major haplotype of B biotype is prevalent in Iran and that its closest relative is the B biotype. Also, given the extent of known variation in the Middle East and African continent, data indicate somewhat surprisingly that the B. tabaci collections sampled in Iran had limited genetic variation and population substructure. Knowledge that the B biotype of B. tabaci predominates in Iran is important for designing eVective pest management strategies given that biotypes of B. tabaci are known to diVer greatly with respect to insecticide resistance, host range, virus–vector interactions, and other key biological characteristics. Keywords Bemisia tabaci · Genetic variation · Iran · mtCOI · PCR-RFLP · Phylogeny
Introduction The species Bemisia tabaci was Wrst described in 1889 as a pest of tobacco in Greece, as Aleurodes tabaci (Gennadius 1889). It was described as numerous species owing to hostassociated morphological and environmental variation and in 1957, all species and variants were synonymized into the epithet B. tabaci (Basu 1995; Bedford et al. 1994; Mound 1963; Mound and Halsey 1978; Russell 1957). B. tabaci is broadly polyphagous, feeding on an estimated 600 plant species (Basu 1995; Gill 1990; Mound and Halsey 1978), however certain variants are monophagous, or nearly so (Brown 2007). In the late 1980s, the importance of B. tabaci
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as a pest and virus vector in Weld crops and in ornamental plants has increased signiWcantly on a worldwide basis (Brown 2007; Brown et al. 1995; Byrne and Bellows 1991). Heavy colonization reduces plant vigor and growth, and certain biotypes, namely the B and its’ closest relatives, can induce physiological disorders in certain hosts (Brown 2007; Costa and Brown 1991). B. tabaci also damages plants by producing ‘honeydew’ as a waste product, which serves as a substrate for sooty mould fungi. Fungal growth can reduce photosynthetic capacity, and cause defoliation and stunting (Byrne and Bellows 1991). B. tabaci is also a vector of plant viruses belonging to four genera (Jones 2003). The genus Begomovirus (family Geminiviridae) is the most problematic and widespread, often causing 20– 100% yield loss in crops (Brown and Bird 1992). The B. tabaci complex (Brown et al. 1995; Frohlich et al. 1999; Rosell et al. 1997) is recognized as an economically important pest and plant virus vector worldwide in tropical and subtropical regions. B. tabaci is considered a cryptic species in the Aleyrodidae (Hemiptera/Homoptera) owing to the absence of morphological characters on the ‘pupal case’ of the fourth instar used to identify whiteXies to the species level, for variants that clearly diVer in behavioral and/or genetic characters. (Bedford et al. 1994; Brown et al. 1995; Gill 1990; Martin 1987, Mound 1963; Rosell et al. 1997; Russell 1957). Molecular genetics analyses of B. tabaci worldwide suggest that it is either a highly divergent complex comprising divergent haplotypes (»0–24%), with certain of them characterized as ‘biological types’, (Brown 2000, 2007; Brown et al. 1995), or a group of species (De Barro et al. 2005; Perring et al. 1993). ‘Biotypes’ or ‘host races’ are recognized based on distinguishing biological characters (Brown and Bird 1992; Burban et al. 1992; Costa and Brown 1991). In the 1980s, reports of an insecticide-resistant biotype emerged as a new pest and plant virus vector, referred to as the ‘B biotype’. The B biotype is distinguished from other B. tabaci in that it is highly polyphagous, disperses long distances, is highly fecund compared to most native populations, including the A biotype, which is endemic to the southwestern US, and Wnally, it is distributed nearly worldwide owing to multiple introductions (Brown et al. 1995; Costa and Brown 1991; Costa et al. 1993). The B biotype is thought to have originated in the Old World (Brown et al. 1995; Frohlich et al. 1999], most likely in the Middle East and eastern Africa (Brown 2007). Accurate identiWcation of insect pests and virus vectors is essential for directed and eVective management of pests and diseases. Biochemical and molecular approaches have been used for identifying B. tabaci variants and assessing genetic variability at several levels. These include isozymes and esterases (Brown et al. 2000; Byrne et al. 1995; Costa and Brown 1991; Costa et al. 1993), random
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ampliWed polymorphic DNA (RAPDs) (De Barro and Driver 1997; Gawel and Bartlett 1993; Moya et al. 2001; Perring et al. 1993), and o77766 ampliWed fragment length polymorphism (AFLP) (Cervera et al. 2000). Isozymes often are insuYciently variable to resolve genetic diVerences (Murphy et al. 1996), and RAPD-PCR and AFLP can overestimate diVerences between populations (Abdullahi et al. 2004). PCR-RFLP analysis has the potential to violate assumptions about the independence of character states, and diYculties surrounding the interpretation of ‘indels’ (Nei and Kumar 2000). Even so, PCR-RFLP is informative for assessing genetic diVerentiation, particularly for intra-speciWcs or closely related species (Avise 1994), and is useful for estimating nucleotide substitution frequencies and diversity (Nei and Li 1979). The mtCOI gene of insects evolves at a low to moderate rate, is highly conserved (Carr and Wilson 1987), and has been used for diVerentiating species and higher taxonomic levels. In contrast, the B. tabaci complex mtCOI gene has proven highly informative for diVerentiating haplotypes and resolving a phylogeography (Berry et al. 2004; Brown 2000; Frohlich et al. 1999; Legg et al. 2002; Qiu et al.2007; Sseruwagi et al. 2006; Viscarret et al. 2003). MtCOI genetic diVerentiation has corroborated signiWcant diVerentiation in this coding region (Brown and Idris 2005). Based on the mtCOI sequence, the greatest genetic variation occurs in African populations, then Asia, and Wnally, the New World, inferring an origin on the African continent (Berry et al. 2004; Legg et al. 2002; Sseruwagi et al. 2006). In Iran, B. tabaci was Wrst collected in the vicinity of Kerman in 1944. It later became an important pest of cotton in the southern and central parts of the country (Habibi 1975). It was then reported from the cotton Welds in the north, Gorgan and Mazandaran, in 1980 (Javan Moghaddam 1993). This species is currently distributed almost throughout Iran feeding on diVerent host plants. B. tabaci B biotype was Wrst described in Iran in 1993 (Samii 1993). There are no published reports on the economic signiWcance of B. tabaci in Iran, but crop damage is implicit based on several reports of whiteXy-transmitted begomovirus diseases there (Massumi et al. 2007; Yousif et al. 2007; Bananej et al. 2002; Hajimorad et al. 1996). In this study, PCR-RFLP of the mtCOI was employed to assess genetic variation and reconstruct a phylogeny for B. tabaci collected from agricultural crops in Iran. Also, the mtCOI gene was ampliWed by PCR and the DNA sequence was determined for individuals from 21 of the latter Weld collections and used to carry out a second phylogenetic analysis by comparison with a suite of reference sequences for well-studied haplotypes and biotypes (Brown 2007).
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Materials and methods
and then for 95 C for 10 min before centrifugation (10 K). The supernatant was the source of the PCR template. PCR primers that amplify a 1,070 bp fragment of the COI gene (BeTab F2 5⬘- GTTGTTACTTCTCATGC TTTC-3⬘ and BeTab R2 5⬘-GACACCAGGTTATAA TTGTTT-3⬘) were designed based on the B. tabaci complete mt sequence (GenBank Accession AY521259). PCR was carried out using 20 pmol of each primer, 100 ng template DNA, 5 l of 10X PCR buVer, 1.5 mM MgCl2, 0.1 mM dNTPs, 1.2 units Taq DNA polymerase and dH2O up to 50 l. PCR parameters were as follows: initial denaturation at 96 C for 5 min, followed by 30 cycles of 94 C denaturation (30 s), 53 C annealing (60 s) and 72 C extension (40 s), and a Wnal extension for 5 min at 72 C. PCR products were fractionated by agarose electrophoresis (1%) in Tris acetate buVer (TAE), pH 8.0, stained with ethidium bromide, and DNA bands were visualized under ultraviolet light. PCR products were digested using the restriction enzymes, AluI and MvaI (SinnaGen, Iran) for RFLP analysis. The products were electrophoresed in an agarose gel (2%) with TAE buVer, pH 8.0. The RFLP binomial data, based on the presence or absence of a restriction fragment at a given position, were imported into Arlequin, v2.00 (Schneider et al. 2000) and pair-wise distances between haplotypes were calculated to evaluate genetic population structure. An analysis of molecular variance (AMOVA) was carried out to explore variation within and between B. tabaci populations.
WhiteXy collections The specimens of B. tabaci were collected from 14 agricultural stations in four provinces of Iran with 18 population codes, according to host plants (Fig. 1; Table 1). Adults were collected from plants using an aspirator and transferred to vials containing 70% alcohol. Males and females sorted. Only females were selected for the analyses. Microscopic slides were prepared using pupae present on leaves taken from the plants from which adults were collected. Key morphological characters used to identify whiteXies to species were according to Gill (1990). Representatives from each collection of B. tabaci adults were analyzed in Iran using PCR-RFLPs (Fig. 1; Table 1), and a subset were subjected to mtCOI ampliWcation and sequencing. DNA extraction, ampliWcation and PCR-RFLP analysis DNA was extracted from single specimen using the method described by Frohlich et al. (1999). DNA extraction, PCR and RFLP was replicated for each collection (n = 5 adults/ collection). Single adult females were transferred into 1.5 ml plastic tubes, washed with distilled water, and allowed to air dry. Individuals were ground with the round end of a 0.4-L microfuge tube and incubated with Lysis buVer (10 mM Tris–HCL, pH 8.0, containing 1 mM EDTA, 0.5% Nonidet P-40 and 1 mg/ml proteinase K) at 65 C for 15 min Fig. 1 Map of Iran showing the locations at which B. tabaci adults were collected
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Table 1 Sampling city, host plant, and code for the B. tabaci collections subjected to PCR-RFLP analysis, and from which populations were selected for mtCOI-DNA sequencing Location
Hormozgan
Kerman
Khorasan
Yazd
Field collection number
Host plant
Date of collections
Code
Type of Analysis
1
Tomato
24 February 2004
50
Hor-1
RFLP and DNA seq.
2
Tomato
26 February 2004
48
Hor-11
RFLP and DNA seq.
3
Eggplant
27 February 2004
37
Hor-14
RFLP and DNA seq.
21
Eggplant
27 February 2004
10
Hor-17
DNA seq.
4
Cucumber
9 March 2004
57
Ker-3
RFLP and DNA seq.
5
Cucumber
10 March 2004
43
Ker-5
RFLP and DNA seq.
6
Eggplant
11 March 2004
52
Ker-7
RFLP and DNA seq.
19
Tomato
13 March 2004
8
Ker-10
DNA seq.
20
Eggplant
13 March 2004
9
Ker-11
DNA seq.
7
Cucumber
5 May 2004
45
Kho-1c
RFLP and DNA seq.
8
Tomato
5 May 2004
39
Kho-1t
RFLP and DNA seq.
9
Verbena
7 May 2004
40
Kho-2
RFLP and DNA seq.
10
Cucumber
8 May 2004
34
Kho-3c
RFLP and DNA seq.
11
Tomato
8 May 2004
28
Kho-3t
RFLP and DNA seq.
12
Eggplant
15 July 2004
22
Yzd-1
RFLP and DNA seq.
13
Melon
15 July 2004
34
Yzd-2
RFLP and DNA seq.
14
Tomato
15 July 2004
31
Yzd-3
RFLP and DNA seq.
15
Squash
16 July 2004
27
Yzd-4
RFLP and DNA seq.
16
Tomato
17 July 2004
35
Yzd-5t
RFLP and DNA seq.
17
Eggplant
17 July 2004
31
Yzd-5e
RFLP and DNA seq.
18
Squash
17 July 2004
35
Yzd-5s
RFLP and DNA seq.
Mt COI PCR ampliWcation, sequencing, and phylogenetic analysis AmpliWcation of a »850 bp of the mtCOI gene was carried out using the primers MT10/C1-J- 2195 (5⬘-TTGATTT TTTGGTCATCCAGAAGT-3⬘) and MT12/L2-N-3014 (5⬘- TCCAATGCACTAATCTGCCATATTA-3⬘) available in the UBC Insect Mitochondrial DNA Primer Oligonucleotide Set, complied by B. J. Crespi and C. Simon (Simon et al. 1994), according to the parameters described by Frohlich et al. (1999). Electrophoresis of PCR products was carried out in a 1% agarose gel in 1X TAE buVer, pH 8.0, and bands were viewed with a UV transilluminator after ethidium bromide staining. PCR products were puriWed using a QIA-quick PCR PuriWcation Kit (Qiagen Inc, USA) and subjected to bi-directional automated (ABI 3700) sequencing using the MT10 and MT12 primers to yield two sequences having an overlap of »150–200 bases, from which a 780 bp contig was assembled for each. The mtCOI sequences were assembled using Phred and FAKtory 1.41 (Ewing et al. 1998) available at the Biotechnology Computing Facility (http://bcf.arl.Arizona.edu/ biodesk) and a consensus sequence of »800 bases obtained for each (2 per adult whiteXy). The mtCOI consensus sequence for each individual was manually edited to »780
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Total number of collected adults
bases. The sequences were aligned using Clustal W (Megalign, DNASTAR, Lasergene, Madison, Wisconsin, USA) together with B. tabaci reference mtCOI sequences available in public sequence databases. Pairwise nucleotide distance estimates were made using Clustal V (DNASTAR). The mtCOI from the B. tabaci from Australia was included as the species outgroup. Reference mtCOI sequences used here have been reported previously (Berry et al. 2004; Brown 2000; Qiu et al.2007; Sseruwagi et al. 2006; Viscarret et al. 2003).
Results DNA extraction, ampliWcation and PCR-RFLP analysis The extent and nature of intraspeciWc variation in diVerent populations of B. tabaci was surveyed using PCR-RFLP. Analysis of 18 B. tabaci collections (5 adult/collection) by PCR-RFLP with two restriction enzymes, AluI and MvaI, identiWed a total of 388 fragments, of which 16 were polymorphic. An example of RFLP variation observed when MvaI was used as the restriction enzyme (for an arbitrary sample among the 18 B. tabaci collections) is shown (Fig. 2). Haplotypic variation was found to be extremely
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Fig. 2 MvaI digestion of PCR product from representative B. tabaci, 1 Undigested PCR product, 2 YZD-5(t), 3 HOR-11, 4 KER-7, 5 KHO1(c), 6 100-bp DNA ladder, 7 HOR-14
low, and among all of the populations examined, only two individuals exhibited a distinct haplotype. Using the MvaI restriction enzyme, an individual haplotype from the Hor11 collection was observed (Fig. 2). Digestion with AluI revealed a second unique individual haplotype in populations Hor-1, Hor-11 and Hor-14. An analysis of molecular variance (AMOVA), however, revealed no within- population variation and the observed variance were attributed to between-population diVerences (Table 2). MtCOI PCR ampliWcation, sequencing, and phylogenetic analysis The DNA sequences for collections 1, 5, 10, 15, and 20 have been deposited in the NCBI GenBank database as the accession numbers: EU547768, EU547769, EU547770, EU547771 and EU547772. Distance comparisons of the mtCOI sequence for 21 Weld collections (n = 2 adults/collection) from Iran revealed that haplotypes (780 nucleotides) exhibited minimal nucleotide divergence, at 0–1.2% for 42 individuals. Phylogenetic analysis placed all of the mtCOI sequences for B. tabaci from Iran with their closest relatives, the B biotype. The B. tabaci from Iran grouped Table 2 Results of AMOVA for 18 populations (5 adults/population) of B. tabaci based on haplotype identiWcation by restriction enzyme analysis of the mtCOI PCR product for each individual Source of variation
df
Sum of squares
Variance components
Percentage of variation
Among population comparison
18
22.737
0.21053 Va
100.00
Within-population comparison
95
0.000
0.00000 Vb
0.00
Total
113
22.737
Fixation index
FST: 1.00000
0.21053
tightly with the reference sequences determined for a large number of B biotype collections from various locations worldwide (Fig. 3). This is almost in complete agreement with the results obtained by De Barro et al. (2000, 2005) and Abdullahi et al. (2003). Haplotypes in the major clade that also contains the B biotype, are most closely related to those in a large sister clade that contains the Q biotype from Spain, and closest relatives from the region (Brown 2000). Collectively, these two major haplotypes colonize eudicots in the region of the Mediterranean Sea, North Africa, and the Middle East, overlapping in distribution in certain locations. The mtCOI sequences (n = 21) for B. tabaci adults examined here exhibited extremely low divergence (0– 1.2%), indicating a high degree of homogeneity among populations in Iran.
Discussion Members of the B. tabaci complex, a biologically and genetically variable, and highly important insect pest and plant virus vector, is one of the most important constraints to agricultural production in tropical and mild climate areas. The results of this study provide the Wrst evidence regarding the haplotype composition of the B. tabaci complex in Iran. Based on two types of analyses, the B biotypelike haplotype was the only haplotype detected in vegetable crops, and so it is surmised that the B biotype is the predominant B. tabaci throughout much or all of Iran. Other close relatives of the B biotype have been described from the Middle East and eastern Africa and adjacent islands, however, is not clear whether the B biotype in Iran is endemic or has been introduced. For Iran, there is no a priori knowledge regarding the baseline composition and/or extent of genetic variability of the B. tabaci complex. However, geographic and environmental barriers in the region do not appear to be capable of hindering the dispersal of the B biotype in the region. One hypothesis is that the extant B biotype in Iran was introduced, somewhat, recently by local traders and that the observed ‘reduced’ genetic variability is due to invasive individuals capable of out-competing endemic haplotype(s). This scenario is supported by the present study has revealed that within- and between-population diVerences are not signiWcant at the levels of nucleotide divergence or genetic diVerentiation (based on DNA sequences and PCRRFLP patterns). If this were not so, greater variation would be expected for at least certain collections (haplotypes) in Iran, with a basis in either geography and/or host plant aYliation (Mills and Allendorf 1996; Wright 1969). Conditions contributing to low within-population variation are, among others, adaptation to climate and other environmental conditions, host plant composition, and restricted gene Xow.
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Fig. 3 The Clustal W tree showing the phylogenetic relationships for selected B. tabaci individuals from Iran, in relation to well-studied B. tabaci for which sequences are available in the public sequence databases. Because the nucleotide divergence (n = 21 sequences) was extremely low, at 0–1.2% nucleotide identity, the tree was reconstructed using one sequence each from the Weld collections from Iran (IRN) numbered 1, 5, 10, 15, and 20
Representative haplotypes from Iran
GC (B) Guatemala TelAviv (B) CC (B) California FC(B)Florida IS (B) Israel Turkey(B)01 AZ(B) 88 Arizona India B Taiwan B IRN1-1 IRN5-1 IRN20-1 IRN15-1 IRN10-2 SKorea (B) AZ (B) 2000 MALAYSIASonchus Morroco 1 SP92 tomato-Spain SC Sudan SP99 cuke Spain1 TC Turkey
Australia
22.6 20
15
The results indicated that the Iranian B. tabaci from crop production zones exhibited low between-population genetic variation. WhiteXies were collected from summer crops, and certain crops were heavily infested, including cucumber, eggplant, and tomato. Typically, these vegetable crops are grown in monoculture over a large continuously cropped area. Continuous colonization of these monoculture systems by B. tabaci would be expected to result in decreased gene Xow, and consequently, leads to genetic similarity. Although B. tabaci can easily be dispersed by wind (Byrne and Bellows 1991), potentially increasing gene Xow between populations, and whiteXy infested plants transported by humans between previously isolated locales, could lead to an increased genetic variability through the introduction of genetically diverse populations, we found no evidence to support either hypothesis. These results are in agreement with the report of Gocmen and Devran (2002), in which the genetic variation of B. tabaci in Antalya was examined. There, adult whiteXies collected from cucumber, eggplant, and tomato were highly similar genetically and grouped as a single cluster. In contrast, Abdullahi et al. (2004) used PCR-RFLP to characterize the ITS1 region and revealed that B. tabaci collected from diVerent host plants exhibited high variability. The genetic variability of the B. tabaci genome has not been assessed with suYcient rigor to enable identiWcation of rapidly evolving sequences. Such an undertaking could provide important clues about the low genetic variation in this clonal population in relation to non-clonal relatives and increased within- and between- population variability. And, although we suspect that the B biotype originated in or near the portion of the continent housing Iran, the precise center of it’s’ origin remains to be determined. This absence of this information is primarily due to the near impossible task
MediterraneanNorth AfricanArabian Peninsula Group
Q biotype clade & close relatives
AZ(A) 88 Arizona CAL(A)Brawlee CA CUL Mexico CAL (A) Salinas Jat PR 00 ARG1BeanSalta ABA Benin NEW-Nepal PC92-Pakistan IW India PC91-Pakistan PC95-Pakistan INDIA 34 HC China
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B biotype clade
American Tropics clade
African exemplar
Asian clades Australia
10
5
0
of sampling thoroughly throughout the region. Thus, regional sampling combined with robust population genetics approaches and additional informative molecular markers are needed to dissect the genetic structure of B. tabaci in Iran and elsewhere, in conjunction with identiWcation of speciWc selection pressures that are conducive to ‘invasiveness’ in the B. tabaci complex. Acknowledgments We thank the staV of Insect Taxonomy Research Department, Iranian Research Institute of Plant Protection; Tehran, Iran for kind help, and colleagues of Cellular and Molecular Biology Research Center of Shahid Beheshti University, M.C., Tehran, Iran for useful comments.
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