Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus Allius from Greenhouse and Field Specimens

Journal of Nernatologv 41(t):60-63. 2009. © The Society of Nernatologists 2009. Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens E. Rl(;..\,' R. LARSEN, 2 K. EAsrwl:1.I.,' N. GUERRA,' L. GUERRA,3 J . M. ClossI.lN2 Abstract: The stubby root nematode, I'aratrtchodorus alms, is important to the potato industry in the Pacific Northwest of USA, because it vectors Thbarco rattle virus (TRV), the causal agent of corky ringspot disease. The current method for determining if nematodes are viruliferous for 1kV takes several weeks, requiring a glasshouse bioassay followed by a serological test. To overcome this drawback, a rapid and aftördablc molecular test was developed using revel-se transcription polymerase chain reaction (RT-PCR) to identif\ sruliferous fl adios nematodes svbluu 48 hours. Primers from the 16 kDa gene of TRV were used tO detect TR\-' in both greenhouse-reared and field collected P a/tins. TRV RNA can he detected consistently in nucleic acids equivalent to one quarter of a viruliferous adult nematode reared in the greenhouse. In order to reduce the time and expense of processing individual neniatodes from field samples. viral RNA was consistently and affordabl y detected in extracts from 5 field-collected adult P a/Ion. Key wards: Detection, Reverse Transcription PCR, Parairmehodorus alms, viruliferous stubby root nematode, tobacco rattle virus. Nematodes of the genera Paratrichodorns and Tricho- Stubby root nematodes are relatively common in the dorus are polyphagous migratory ectoparasites that feed PNW and have been found more often in potato- on roots of annual and perennial plants causing stunt- growing areas than TRV (Mojtahedi et al., 2000). ing and are referred to as the stubby root nematodes. Therefore, the presence of P allius in a field does not Nine stubby root nematode species are currently known necessarily imply that nematode control measures are to vector tobraviruses that cause economically important necessary. The development of rapid, reliable and af diseases of potatoes and ornamental bulbs (Harrison fordable diagnostic tools to identify TRV-infcted P and Robinson, 1986; Mumford et al., 2000; Taylor and alms would benefit the potato industry by allowing Brown, 1997). The most prevalent trichodorid vector of growers to make early decisions in terms of P ttllius Tobacco rattle virus ('T'RV) (genus Tobravirus) in the Pa- management practices. In this study, we developed cific Northwest (PNW) of USA is Parat-richodorus allius a molecular diagnostic test for identification of virulif- Uensen ,1963) Siddiqi 1974, and approximately 10% of erous P ullius nematodes that can he completed within field populations are viruliferous (Mojtahedi et al., 48 hours in order to meet this need. Furthermore, our 2000). In potato (Solarnim tuberosum L), TRV causes test would determine how many field stubby root nem- corky ringspot disease (CRS) (Mojtahedi et al., 2000) atodes could he used in one PCR reaction in order to which is increasing in importance in Washington State detect as few as one viruliferous nematode. (Peltei', 1997), and has been reported in several other states (Kirk et al., 2008). MATERtALS AND M,ri tons Existing biological tests that screen for viruliferous P allius are time consuming, and inaccurate. These assays Nematode and virus isolates: A viruliferous P allius require indicator plants, such as tobacco (Nicotiana to-- population was isolated from a CRS-infested field in bac-urn L.) Samsun NN', fr detecting TRV in live Pasco, WA and maintained on Ncotmiana tabacurn L. var. nematodes, often followed by an ELISA test. However, Samsun NN, a good host for both TRV and the stubby this assayrequires approximately 2 months. TRV can he root nematode (Mojtahedi and Santo, 1999). Nema- detected in potato tubers using reverse transcription- tode-infested grown on tobacco plants under green- polymerase chain reaction (RT-PCR) but has not been house conditions exhibited typical TRV symptoms, reported for the detection of TRV in P alliu,s. A real- such as light brown necrotic ring spots on the leaves. time fltiorogenic 5' nuclease PCR assay has been devel- The presence of the virus was confirmed by antigen- oped for TRV detection in two stubby root nematodes coated plate indirect enzyme-linked immunosorbent P pachyderm-us and Trichodot-us similis, which are not assay (ELISA) (Converse and Martin, 1990). Para- found in the PNW (Holeva et al., 2006). trichodorus allius was also collected from three com- mercial potato fields (Pasco and Prosser, WA; and Kla- math Falls, OR) with reported incidence of CRS dis- Received lo, publication May 10, 2008. ease; a total of 22 different soil samples (250 cm 1 each) 1Waslnrigiori Slate tTniversitv. IAREC. 24106 N. B,uin Rd., I'cosser, WA 99350, were collected from the above fields; a total of 185 USA. 2USDA-ARS, 24106 N. Bunn Rd., Pnmssei, ViA 99:150. USA. nematodes were collected from the field samples. 'WSDA, 2-I 106 N. Bonn Rd., Piosser, WA 9935>), USA. Isolation of RNA from P al/ins and reverse transcriptase E. Riga is uppoite(l hr the College of .-\gricllltulal, Iluman , and Natural Resource Sciences Agricultural Research Centre, I'PNS No. 0450, Departinemit reaction: Nematodes were collected from greenhouse- of Plant Patlmntogs and IAREC \VSU-Prosser. Project Nmm.WNP00542, Wash- grown tobacco, and field soil samples by sieving infes- ington State Univeisimv, Pullman, WA 99161-6430, LI SA. This research was funded by the Washington Stare l'omammm Commission and time Washington State ted soil followed by centrifugal flotation methods Commission on Pesticide Rcgist ratim mm. (Jenkins, 1964). Individual adult female P alms were Contact Author: E. Riga. [email protected]'dim This papem was edited by Brent Sipes. hand picked using a probe with an eyelash attached at 60 Rapid Detection of Viruliferous Stubby Root Nematode: Riga e' al. 61 its end, and total RNA was isolated based on a protocol CAAGGG,IT 3') (20 pmol), 0.2 p.1 (1 unit) of Taq obtained from Dr. R. Martin (USDA-ARS, Corvallis, Polymerase (Invitrogen, Madison, WI) and 28.3 p.1 OR) and modified as follows: RNA was extracted from sterile distilled water. After an initial denaturation for single female individuals from greenhouse raised 2 minat 94°C, the amplification consisted of 40 cycles nematodes, and from soil samples from the field, RNA Of the following: denaturation for 15 seconds at 94°C, was extracted from sets of five adult female nematodes. annealing for 2 min at 58°C, extension for 1 min at Nematodes were handpicked and placed in 1.5 ml nil- 72°C; and a final extension step for 10 min at 72°C. PCR crocentrifuge tubes containing 100 p.l sterile distilled products were resolved by electrophoresis in a 1.5% H90; 100 p.l of collagenase (Sigma-Aldrich , St Louis, agarose gel and IX TAE Buffer (1mM EDTA; 40mM MO) solution (1 Omg/ml in 50mM Tris, I mM Ca('1I 2, pH Tris; 228.4 pA/i of glacial acetic acid; pH 8.0) for 80 mm 7.4) was added to each tube and incubated at 37°C for 1 at 40 volts. Caenorhabditis elegan.s eDNA and non- hour. Then 50 mg of glass heads (Acid-washed, 425-600 viruliferous P ailius eDNA (reared on spearmint to microns, Sigma) were added followed by 200 p.1 of 2X ensure that the virus will not be present according to RNA Extraction Buffer (400 ruM Tris pH 8.5; 600 mM Mojtahedi et al., 2002) were used as negative control in LiC1; 20mM EDTA; 3% lithium dodecvl sulfate; 2% PCR reactions. In addition, 2 p.1 of TRV nucleic acid deoxycholic acid; 2% tergitol; 2% 2,13-rncrcaptoethanol extracted from a symptomatic potato tuber of the cul- (Sigma-Aldrich Milwaukee, WI). Tubes were vortexed tivar Russet Ranger collected in Pasco, WA was used for for I nun, followed by centrifugation for 30 sec at the positive control; the method used to obtain TRV 21,000 x g. The supernatant was discarded and 400 p.1 6 from potatoes was based on Dellaporta (1993). The M potassium acetate pH 6.3 were added to the pelleted expected size of the PCR-amplified TRV fragment was material; thoroughly mixed, and incubated on ice for 463 bp (Robinson, 1992). 15 min. Tubes were centrifuged at 21,000 x gfor 3 mm and the supernatant was transferred to a new tube and Ri•sti:rs ANt) DlsctisSION incubated for 15 min at -20°C, and then centrifuged again for 3 min at 21,000 x g The supernate was trans- We usedi adult females of P allius to assay for the ferred to a new tube, and 1 p.1 glycogen at a concentra- detection ofF RV particles because they are most likely tion of 20p.g/p.l (Invitrogen, Carlsbad, CA) was added to carry the virus. According to MacFarlane (2003), to each tube, followed by an equal volume of iso- tobravirus particles can be acquired by both juvenile propanol. The tubes were incubated for 13 min at - 20°C and adult nematodes and retain the virus particles for and centrifuged for 20 min at 21,000 x g. The supernate long periods. However, the virus can be lost from the was discarded without disturbing the pellet and the juveniles after molting, therefore, in our study only RNA pellet was washed twice with 300 p.1 70% ethanol. adult female nematodes were used from both the The pellet was air dined at room temperature for 10 mm greenhouse and field samples. at 20°C. Pellets were dissolved in 20 p.! dicthylpyrocar- ()ur diagnostic assay routinely detected TRV RNA in honate (DEPC) H20 (Sigma-Aldrich, St Louis, MO) nucleic acid from the equivalent of one quarter adult and placed on ice Ion immediate use or stored at -20°C.

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