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THIS ARTICLE HAS BEEN WITHDRAWN: Thrips palmi

2006; Wiley; Volume: 36; Issue: 1 Linguagem: Inglês

10.1111/j.1365-2338.2006.00917.x

1365-2338

Yeasts and Rust Fungi Studies

EPPO BulletinVolume 36, Issue 1 p. 89-94 Diagnostics†Free Access THIS ARTICLE HAS BEEN WITHDRAWN: Thrips palmi Correction(s) for this article Corrigenda Volume 41Issue 3EPPO Bulletin pages: 419-420 First Published online: November 22, 2011 First published: 30 June 2006 https://doi.org/10.1111/j.1365-2338.2006.00917.xCitations: 6AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat PM 7/3 (2) Specific scope This standard describes a diagnostic protocol for Thrips palmi. It complements and replaces EPPO Standard PM 7/3 (1) Specific approval and amendment First approved in 2000-09. Revision approved in 2005-09, incorporating material developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Introduction Thrips palmi is a polyphagous pest especially of Cucurbitaceae and Solanaceae. It originated in southern Asia and has spread from there during the 20th century. It is now present throughout Asia and is widespread in Central America and the Caribbean. Otherwise, it occurs locally in North and South America, Africa and Oceania. It is not present in Europe but there have been several outbreaks in the Netherlands and an outbreak in the UK which were subsequently eradicated. For more general information about T. palmi, see EPPO/CABI (1997). Thrips palmi is almost completely yellow coloured and its identification is hampered by its small size and great similarity with other yellow species of Thrips. Identity Name: Thrips palmi Karny, 1925. Synonyms: Thrips clarus Moulton, 1928; Thrips leucadophilus Priesner, 1936; Thrips gossypicola Ramakrishna & Margabandhu, 1939; Chloethrips aureus Ananthakrishnan & Jagadish, 1967; Thrips gracilis Ananthakrishnan & Jagadish, 1968. Taxonomic position: Insecta: Thysanoptera: Terebrantia: Thripidae: Thripini. EPPO code: THRIPL. Phytosanitary categorization: EPPO A1 list no. 175, EU Annex designation I/A1. Detection T. palmi is an outdoor pest of aubergine, Benincasa hispida, Capsicum annuum, cotton, cowpea, cucumber, Cucurbita spp., melon, pea, phaseolus bean, potato, sesame, soybean, sunflower, tobacco and watermelon. In glasshouses, economically important hosts are aubergine, Capsicum annuum, cucumber, Cyclamen, Dendranthema, Ficus and Orchidaceae. T. palmi can be carried on plants for planting and fruits of host species and on packing material. During inspection of plant material for the presence of T. palmi, attention should be paid to silvery feeding scars on leaf surfaces of host plants and especially alongside the midrib and veins. Heavily infested plants are characterized by silvered or bronzed appearance of the leaves, stunted leaves and terminals, and scarred and deformed fruits. Thrips palmi has six developmental stages, which are usually found in different parts of the plant or in the soil: • egg, in the leaf, flower and fruit tissue • larva I, on the leaves, flowers and fruits • larva II, on the leaves, flowers and fruits • pupa I, in the soil • pupa II, in the soil • adult, on leaves, flowers and fruits Individual specimens can best be collected in a fluid called AGA which is a mixture of 10 parts of 60% ethyl alcohol with 1 part glycerine and 1 part of acetic acid. To collect thrips specimens from plant parts, such as cut flowers, several methods can be used (Mantel & Vierbergen, 1996; modified): • removing from plant into tubes with a moist brush • beating from plant parts onto a white board for dark-coloured specimens and onto a black board for light-coloured specimens • dragging a light-coloured fine-mesh ground-net, resembling a butterfly net, through vegetation • collecting plant parts in a closed plastic bag containing a piece of filter paper (to absorb condensation). After 24 h, most thrips have left the plant parts and walk on the inside of the closed bag • placing plant parts (e.g. flowers) into a Berlese apparatus (funnel provided with a sieve and leading into a receptacle containing 10% alcohol). The funnel is placed under an electric lamp (60 W) and the heat and light make any thrips present in the plants escape towards the receptacle. After 8 h (for cut flowers), the content of the receptacle is checked under a stereo microscope • using coloured sticky boards (usually blue or white for T. palmi) covered with transparent plastic. Thrips specimens are removed by cutting out a fragment of the glued plastic, which is then dissolved with dichloromethane. The first two methods are suitable for collecting larvae, pre-pupae, pupae and adults. The next three can be used for collecting larvae and adults. The last is used only for sampling winged adults. For storage, specimens should be transferred to 60% ethyl alcohol and kept in the dark, preferably at temperatures well below 0°C to prevent loss of colour. Identification Morphological identification of adults Identification of thrips is performed on adults, since there are no adequate keys for the separation of species based on the characteristics of eggs, larvae or pupae. However, the presence of larvae in samples can give important additional information on development on host plants. For microscopic examination, adult thrips should be mounted on microscope slides. For routine identifications, a water-soluble mountant is rapid and relatively inexpensive. The following procedure is recommended by Mound & Kibby (1998): specimens are transferred from the collecting fluid into clean 70% ethyl alcohol; if the specimens are reasonably flexible, the wings are opened and the antennae are straightened using micropins; a thrips is placed into a drop of Hoyers Mountant (in 50 mL of water, 30 g gum Arabic, 200 g chloral hydrate, 20 mL glycerol) on a cover slip (e.g. 13 mm diameter), ventral side uppermost, and a slide is lowered onto the drop, and inverted as soon as the mountant has spread sufficiently; the slide is immediately placed in an oven at 35–40°C and left for 6 h before examination; for conservation of the material, the slide is left in the oven for about 3 weeks, then the cover slip is ringed with nail varnish. Family Thripidae T. palmi belongs to the family Thripidae, which includes about 1850 species in 260 genera. In most species, the antennae have seven or eight segments and the forewings (if present) are usually slender, with two longitudinal veins each bearing a series of setae. Genus Thrips The genus Thrips contains more than 200 species from all parts of the world. Members of the genus have only 2 pairs of ocellar setae. The antennae are 7- or 8-segmented and segments III and IV have forked sense cones. The pronotum has two pairs of major postero-angular setae. The tarsi are 2-segmented. The abdominal tergites V–VIII have paired ctenidia laterally and the sternites and pleurotergites are set with or without accessory setae (see Appendix 1). Species Thrips palmi Identification of the adults can be carried out with keys: Bhatti (1980) and Palmer (1992) for the Asian tropics; Mound & Kibby (1998) for 14 species of economic importance; Nakahara (1994) for the Americas. Detailed descriptions of T. palmi are given by Bournier (1983), Sakimura et al. (1986) and zur Strassen (1989). Sakimura et al. (1986) give seven major diagnostic characters to distinguish T. palmi from the other known species of the genus Thrips: • a clear yellow body without any greyish or brownish blotch, but with slightly thickened blackish body setae • interocellar setae standing either just outside of the ocellar triangle, or touching the outer tangent line between anterior and one of the posterior ocelli • metanotum striates in the mesial area and usually posteriorly converges mesad • abdominal tergite II with four lateral setae • abdominal sternites without accessory setae • abdominal tergite VIII with comb complete • male sternites III–VII each with a transverse glandular area. See also Appendix 1 for diagrammatic representation of these and other diagnostic features. Molecular identification Molecular biological methods can be used to identify all life stages of thrips. They can be used to confirm morphological examination of adults. In case of eggs, larvae or pupae they are the only tool for reliable identification. In this case it is recommended that at least two different molecular biological assays, preferably using different target sequences, are performed. DNA extraction DNA extraction is applied to thrips (eggs, larvae, puparia or adults) ground in lysis buffer using a micro pestle. DNA is extracted using standard DNA extraction methods, e.g. the High Pure PCR Template Preparation Kit (Roche) according to the instructions in the mammalian tissue protocol. The DNA is eluted with 50 µL of 10 mm Tris, pH 8.5. Alternatively, DNA is extracted from individual thrips using a Chelex 100 resin-based method. A single thrips is transferred to a 0.5-mL microfuge tube, 50 µL of nuclease-free water is added and the thrips is ground using a pellet pestle and electric pellet mixer. After addition of 50 µL of 1 : 1 Chelex 100 (Biorad): nuclease-free water, the homogenate is heated to 95°C for 5 min and centrifuged at 13 000 rev min−1 for 5 min. The supernatant is transferred to a new microfuge tube and stored at −20°C until ready for testing. COI-PCR The PCR primers for COI-PCR (Kox et al., 2005) are R4 (5′-CCC TCT TAA TTA TGG GTT TAT A-3′) and F5 (CAC AAA TAA TCT TAG TTT TTC TCT T) amplifying a 220-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene. These primers are specific for T. palmi. The 50 µL reaction mixture is composed as follows: 0.76 µM of each primer, 200 µM dNTPs (Promega), 1 Unit Taq DNA polymerase (Roche), 5 µL 10 × reaction buffer (with 15 mm MgCl2), 1 µL DNA. The PCR is performed in a 96-well Peltier-type thermocycler (e.g. PTC200, MJ-Research) with the following parameters: 1 min at 94°C, 40 cycles of 15 s at 94°C, 30 s at 55°C, and 45 s at 72°C, followed by a final extension for 10 min at 72°C and quickly cooled to room temperature. After amplification, 5 µL of the PCR products is subjected to electrophoresis on a 1.5% agarose gel by standard methods (Sambrook et al., 1989) along with a 100-bp DNA ladder (e.g. 100-bp ladder Fermentas) to size fragments. PCR products are viewed and photographed under UV light. COI-real-time PCR, Taqman probe The PCR primers for COI-real-time PCR (Kox et al., 2005) are R4E (5′-CCC TCT TAA TTA TGG GTT TAT ATA AAG AA-3′) and F5E (5′-TGA TCA CAC AAA TAA TCT TAG TTT TTC TCT T-3′). The Taqman MGB probe TpP is (5′- to 3′) 6-FAM-TAG CTG GGG TAT CCT CAA-MGB. The primers and Taqman MGB probe are specific for Thrips palmi. The 25-µL reaction mixture contains: 12.5 µL of 2× Taqman Universal master mix (Applied Biosystems), 0.9 µm of each primer, 0.05 µm Taqman probe TpP, 1 µL DNA. The real-time PCR is performed in a suitable instrument for detection of reporter fluorescence, for example an ABI Prism 7700 or 7900 Sequence Detection System (Applied Biosystems) using the following conditions: 10 min at 94°C; then 40 cycles of 15 s at 94°C and 60 s at 60°C. A cycle treshold (Ct) value lower than 40 means that T. palmi DNA is amplified. SCAR-based real-time PCR, Taqman probe The forward, reverse and TaqMan probe sequences (Walsh et al., 2005) are P4E8-362F (5′-CCG ACA AAA TCG GTC TCA TGA-3′), P4E8-439R (5′-GAA AAG TCT CAG GTA CAA CCC AGT TC-3′) and P4E8-385T (5′ to 3′) 6-FAM-AGA CGG ATT GAC TTA GAC GGG AAC GGT T-TAMRA, respectively. Reactions are set up in 96-well reaction plates using the TaqMan 1000 Reaction Gold Plus Buffer A Pack (Applied Biosystems). To 1 µL (10–20 ng) of DNA extract is added 2.5 µL of 10× Buffer A; 5.5 mm MgCl2; 0.2 mm each of dATP, dTTP, dCTP and dGTP; 0.025 units of Amplitaq Gold; 0.3 µm of each primer and 0.1 µm probe in a total volume of 25 µL. The reaction is carried out in a real-time PCR machine, e.g. ABI Prism 7900HT Sequence Detection System using generic system conditions (95°C for 10 min and 40 cycles of 60°C for 1 min, 95°C for 15 s) with real-time data collection. A Ct value of 40 indicates a negative result, whereas a value of less than 40 means that T. palmi DNA has been amplified. A typical Ct value for amplification of DNA from T. palmi would be between 21 and 27. COI-PCR-RFLP The PCR primers for COI-PCR-RFLP (Brunner et al., 2002) are mtD-7.2F (5′-ATT AGG AGC HCC HGA YAT AGC ATT-3′) and mtD9.2R (5′-CAG GCA AGA TTA AAA TAT AAA CTT CTG-3′). They were modified from C1-J-1751 and C1-N-2191 (Simon et al., 1994) amplifying a 433-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene. The latter primers were modified by degenerations of nucleotides to match thrips sequences better. The primers are not specific for Thrips, they amplify the COI gene of several insects. The 50-µL reaction mixture is composed as follows: 0.76 µm each primer, 200 µm dNTPs (Promega), 1 Unit Taq DNA polymerase (Roche), 5 µL 10 × reaction buffer [with 15 mm MgCl2] , 1 µL DNA. The PCR is performed in a 96-well Peltier-type thermocycler (e.g. PTC200, MJ-Research) with the following parameters: 1 min at 94°C, 40 cycles of 15 s at 94°C, 30 s at 55°C, and 45 s at 72°C, followed by a final extension for 10 min at 72°C and quickly cooled to room temperature. After amplification, 5 µL of the PCR products are subjected to electrophoresis on a 1.5% agarose gel according to standard methods (Sambrook et al., 1989) along with a 100-bp DNA ladder (e.g. 100-bp ladder Fermentas) to size fragments. PCR products are viewed and photographed under UV light. For RFLP analysis, 5 µL PCR product (without further purification) is digested with the enzymes AluI and Sau3AI in separate reactions according to the manufacturer's instructions. Digested PCR products are subjected to electrophoresis on a 2% agarose gel along with a 100-bp DNA ladder to size fragments and visualized and photographed under UV light. A specimen is identified as T. palmi when the PCR product is digested as follows: AluI: 291 and 194 bp; Sau3AI: 350 and 135 bp. ITS-PCR-RFLP The sequences of the forward and reverse PCR primers for ITS-PCR-RFLP (Toda & Komazaki, 2002) are 5′-TGTGAACTGCAGGACACATGA-3′ and 5′-GGTAATCTCACCTGAACTGAGGTC-3′, respectively, and are located in the 5.8S and 28 S regions spanning the internal trancribed spacer (ITS) 2 region of ribosomal DNA. T. palmi generates a 588-bp PCR-product. The 20-µL reaction mixture is composed as follows: 1 µm each primer, 250 µm dNTPs (Promega), 1 Unit AmpliTaq Gold DNA polymerase (Applied Biosystems), 2 µL 10 × reaction buffer (with 25 mm MgCl2), 0.5 µL DNA. The PCR is performed in a 96-well Peltier-type thermocycler (e.g. PTC200, MJ-Research) with the following parameters: 9 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C, followed by a final extension for 10 min at 72°C and quickly cooled to room temperature. After amplification, 5 µL of the PCR products are subjected to electrophoresis on a 1.5% agarose gel according to standard methods (Sambrook et al., 1989) along with a 100-bp DNA ladder (e.g. 100-bp ladder MBI Fermentas) to size fragments. PCR products are viewed and photographed under UV light. For RFLP analysis, 5 µL PCR product (without further purification) is digested with the enzyme RsaI according to the manufacturer's instructions. Digested PCR products are subjected to electrophoresis on a 2% agarose gel along with a 100-bp DNA ladder to size fragments and visualized and photographed under UV light. A specimen is identified as T. palmi when the PCR product is digested as follows: 371, 98, 61 and 58 bp. Specificity of molecular methods The specificity of both the conventional and real-time PCRs targeting the COI gene were assessed using 15 T. palmi specimens, and 61 specimens from 23 other thrips species commonly occurring in Europe including Thrips alni, Thrips tabaci, Frankliniella occidentalis, Frankliniella schultzei (Kox et al., 2005). All T. palmi specimens could be detected, while no cross-reactions with other thrips could be detected in the real-time PCR. The conventional PCR gave the same results except that Thrips major gave a positive result. The SCAR-based real-time PCR has been screened against 10 species in the genus Thrips (flavus, major, minutissimus, nigropilosus, sambuci, tabaci, trehernei/physapus, urticae, validus, vulgatissimus), a further 9 species in the family Thripidae (including Frankliniella intonsa, F. occidentalis, F. schultzei) and one species each in the families Aeolothripidae and Phlaeothripidae as outgroups. None of these thrips gave a positive reaction in the SCAR-based real-time PCR assay. The PCR-RFLP assays developed by Brunner et al. (2002) (COI) and Toda & Komazaki, (2002) (ITS) were not designed specifically to identify T. palmi. They are molecular keys to identify 9 and 8 thrips species, respectively, including T. palmi, T. tabaci and F. occidentalis. All thrips species tested produced different RFLP with the restriction enzymes used in these assays. Confusion with other Thrips species is possible. Distinction is based on morphological criteria. Very close to T. palmi are the Indian species T. alatus and T. pallidulus (Palmer, 1992). In T. alatus, antennal segment V is uniform brown, abdominal tergites III–IV with S2 much weaker than S3 in the female and male and tergite IX is missing the anterior pores (T. palmi: S2 and S3 on these tergites almost equal, Web Fig. 3.10; on tergite IX usually both medial and anterior pores present, Table 1, Web Fig. 3.8). In T. pallidulus, the metanotal sculpture is medially reticulate (T. palmi: medially with longitudinal lines only, Web Fig. 3.4). Table 1. The discriminatory characters of females of Thrips palmi forms and similar Thrips spp. (from Vierbergen, 2002) Thrips palmi‘regular’ Thrips palmi‘Serissa’ Thrips alatus Thrips alni Antennal segment IV & V completely brown – + + + Forewing darkened – + – – Metanotum sculpture converging + +/– +/– – Abd. terg. III and IV with S2 pale and shorter than S3 – – + + Abd. terg. IX with anterior pores + +/– – + Common species occurring in Europe which may be confused with T. palmi are T. alni, T. flavus and T. tabaci (zur Strassen, 1989). T. alni, only known from Alnus, differs from T. palmi in abdominal tergites III–IV with S2 much weaker than S3 in the female and male (T. palmi: S2 and S3 on these tergites almost equal, Web Fig. 3.10) and additionally the narrow oval glandular areas on the abdominal sternites in the male (T. palmi: broad transverse areas, Web Fig. 3.9). T. flavus can be discriminated in having the interocellar setae inside the ocellar triangle (T. palmi: outside the triangle, Web Fig. 3.2). Additionally the length of antennal segment VI for T. palmi is 42–48 µm, while for T. flavus it is 54–65 µm. T. tabaci has three lateral setae on tergite II (T. palmi: 4, Web Fig. 3.6) and lacks pores on the metanotum (T. palmi: two pores present, Web Fig. 3.4). Like T. tabaci, Thrips nigropilosus has three lateral setae on abdominal tergite II and has no pores on the metanotum. Additionally T. nigropilosus has dark abdominal markings (T. palmi: no dark markings) and it has abdominal tergites IV–V with 2 pairs of median setae (S1) more than 0.5 times as long as median length of these tergites (T. palmi: less than 0.3 times, Web Fig. 3.10). Another commonly encountered yellow species is Thrips urticae, which differs in the length of a major pair of long discal setae (usually more than 30 µm) on the anterior part of the pronotum (T. palmi: less than 25 µm, Web Fig. 2.3), the abdominal tergites have usually a grey area medially and abdominal tergite IX has no anterior pores (T. palmi has usually the anterior pores present, Web Fig. 3.8). In the Netherlands a form of T. palmi was found on Serissa (as bonsai), which differed in some important discriminatory characters from the regular form of T. palmi. These characters are given in Table 1. For a positive diagnosis, the procedures for detection and identification described in this protocol should have been followed. The characters of the genus Thrips, and the characters that distinguish T. palmi from similar species, listed in the section Identification and Appendix 1, should have been determined to be present. Alternatively, one of the molecular methods given above (COI-PCR, COI-real time-PCR, SCAR-based real-time PCR, COI-PCR-RFLP, ITS-PCR-RFLP) should have produced the proper signal (amplicon, Ct value, or restriction fragments). Reporting and documentation Guidelines on reporting and documentation are given in EPPO Standard PM7/– (in preparation). Further information Further information on this organism can be obtained from: Entomology Section, Department of Diagnostics, Plant Protection Service, PO Box 9102, 6700 HC Wageningen (NL) Invertebrate Identification Team, Central Science Laboratory, Sand Hutton, York YO41 1LZ (GB). Footnotes 1 The figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. Acknowledgements This protocol was originally drafted by G. Vierbergen and L.F.F. Kox, Plant Protection Service, Wageningen (NL). The protocol for the SCAR-based real-time PCR was kindly provided by D.W. Collins, Central Science Laboratory, York (GB). Appendix Appendix 1 Diagnostic features of the genus Thrips and of T. palmi Specimens can be recognized as Thrips by the following combination of characters (see Web Fig. 1 for the location of the various features): Antenna comprising seven or eight distinct segments: segments III and IV with forked (‘cow horn’) trichomes or sense cones (Web Fig. 2.1 & 3.1) Head with two pairs of ocellar setae (II and III); pair I missing (Web Fig. 2.2) Pronotum with two pairs of posteroangular setae (Web Fig. 2.3) Forewing 1st vein – row of setae with gaps (European species) (Web Fig. 2.4) Abdominal tergites V to VIII with ctenidia (combs – each comprising a series of ridges) (Web Fig. 1) Abdominal tergite VIII with ctenidia posterior and medial to the spiracles (Web Fig. 2.5) Specimens can be identified as Thrips palmi by the presence of the following characters: Body colour clear yellow body with no dark areas on the head, thorax or abdomen: antennal segments I and II are pale (Web Fig. 3.1) Antennal segment V usually yellowish at basal 1/3–1/2 (Web Fig. 3.1) Antennal segment VI length = 42–48 µm (Web Fig. 3.1) Head: ocellar setae pair III with their bases outside or tangential to the ocellar triangle (Web Fig. 3.2) Forewing: 1st vein with two or three setae in the distal half (Web Fig. 3.3) Metanotum with a pair of metanotal pores (campaniform sensillae); with striate sculpturing, usually converging posteriorly (Web Fig. 3.4) Abdominal pleurotergites ridges without microtrichia; accessory setae absent (Web Fig. 3.5) Abdominal tergite II with four lateral setae (Web Fig. 3.6) Abdominal tergites III and IV S2 almost equal to S3 (Web Fig. 3.10) Abdominal tergite VIII with complete posteromarginal comb of microtrichia (Web Fig. 3.7) Abdominal tergite IX usually with two pairs of pores (anterior and medial) (Web Fig. 3.8) Male: sternites transverse glandular areas on sternites III to VII (Web Fig. 3.9) Web Fig. 1 Location of general characters of Thrips. 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