Detection of rice tungro bacilliform virus in non-vector insect speciesfollowing genomic amplification

S. R. Venkitesh and H. Koganezawa, IRRI

Rice tungro disease (RTD) caused by rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) has a semi-persistent relationship with its principal vector the green leafhopper Nephotettix virescens (Distant). This implies there is no replication of virus particles in the vector, and the active site for association of the virus is in the anterior part of the gut, namely the pharynx, cibarium, or possibly the stylets. The very low quantities of viruses acquired during feeding are only retained for a relatively short period, up to 3-5 days. To detect RTBV in single viruliferous insects, we used Ampliwax polymerase chain reaction (PCR) amplification procedure in which a small fragment (569 bp) of RTBV DNA was amplified from the total nucleic acid extract of a single viruliferous insect and detected on agarose gel.

Total nucleic acid extract of insect tissue was prepared by grinding the individual insect in a micro-centrifuge tube containing the extraction buffer, (10 mM Tris-HC1, pH 8.3, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, and 1 mg proteinase K/ml). The contents were transferred to eppindorf tubes and incubated at 65 °C for 2 hours. The samples were extracted with TE phenol/chloroform (1:1 v/v) and then with chloroform and isoamyl alcohol (24:1 v/v). Total genomic DNA was precipitated by adding 3 M ammonium acetate (1/10V), glycogen (1 μl), and 2/3 volume of cold absolute alcohol to the aqueous phase and incubated at -20 °C overnight. Pellets formed by the centrifugation at 13,000 rpm for 20 min were washed carefully with 70% alcohol. Excess alcohol was removed by vacuum drying. The pellets were suspended in 30 μl of TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8). The nucleic acid concentration was estimated by gel electrophoresis of samples on 1% agarose gel. Twenty-mer primers were designed according to the published sequence of RTBV. The plus strand primer (5'-CCTGTGAAACCCAATACTGC-3') and minus strand primer (5'-GCTGAGGTGCTACATAGGTT-3') were complementary to the region of the P 194 open reading frame of the RTBV Genome.

In PCR amplifications using wax beads (Ampliwax PCR Gem l00), the PCR reagents were mixed separately as lower and upper components. The lower reagent mix contained 1X PCR buffer (50 mM KC1, 10 mM Tris-HC1, pH 8.3), 0.2 mM of each dNTP, 2.5 mM of MgCl 2 , and 0.5 μM of each of two primers, made up to a total volume of 25 μl per reaction tube using sterile deionized water. A wax bead was added in each reaction tube. The wax was melted by incubating the reaction tubes at 80 °C for 5 minutes. Melted wax was layered over the lower reagent mix. After wax layering, the reaction tubes were either used directly for PCR reactions or stored at 4 °C until needed. The upper reagent mix contained 1X PCR buffer, 2.5 units (0.5 μl) of AmpliTaq polymerase and 100 ng (15-25 μL) of total nucleic acid extract of insect tissue, made up to a total volume of 65 μL per reaction tube. Upper reagent mix was aliquot to each reaction tube above the solid wax layer, PCR amplifications were carried out for 45 cycles with a denaturation temperature of 94 °C for 1 minute, annealing temperature of 53 °C for 2 minutes, extension temperature of 72 °C for 3 minutes, and a final extension temperature of 72 °C for 5 minutes. PCR products were examined with ethidium bromide-stained 2% agarose gel in 1X TBE buffer using size markers ranging from 100 to 1000 bp.

To obtain viruliferous insects, female adults of the four insect species reared on virus-free TN1 were exposed to RTD-infected TN1 rice plants for 3 days. Among 30 insects from each species tested, PCR detected RTBV in several individual insects of vector species such as Nephotettix virescens, N. nigropictus, and Recillia dorsalis, and in one non-vector species, Nilaparvata lugens. (Results of PCR on 10 individual insect samples from each species are shown in Figures 1 and 2.)

Amplification of RTBV DNA in the non-vector occurred presumably from RTBV particles that had entered the insect gut through ingestion of plant sap containing the virus. Absence of desired PCR products in some viruliferous insects may be because of the presence of undigested enzyme inhibitors, variability in the amount of viral DNA in individual insects, or rapid viral nucleic acid degradation. PCR is an expensive technique, particularly if the reagents are not assembled in the lab and expensive kits must be purchased. As our results show, the technique can be very sensitive, but that sensitivity does not necessarily indicate the identification of a vector. Further considering all these difficulties, we found that

PCR is not a reliable virus detection method for the routine population analyses of viruliferous vectors of rice tungro.

1. Top: PCR products resolved in 2% agarose gel, stained with ethidium bromide and visualized by UV illumination. The DNA template used was M = size marker (100- 1000 bp); lanes 1 and 2 = total genomic DNA from RTBV-infected plant (positive control); lanes 3-12 = total genomic DNA of N. virescens (top) and 10 N. nigropictus (bottom); lanes 13 and 14 = total genomic DNA from nonviruliferous insects (negative control).2. Below: PCR products resolved in 2% agarose gel, stained with ethidium bromide and visualized by UV illumination. The DNA template used was M = size marker (300- 1000 bp); top lane 1 = total genomic DNA from RTBV-infected plant (positive control); lane 2 = total genomic

DNA from nonviruliferous insect (negative control): lanes 3-14 = total genomic DNA of 12 Nilaparvata lugens. Bottom: lanes 1-2 = total genomic DNA from RTBV-infected plant (positive control); lanes 3-12 = total genomic DNA of 10 Recillia dorsalis; lanes 13 and 14 = total genomic DNA from nonviruliferous insect (negative control).

Venkitesh SR, H Koganezawa. 1995. Detection of rice tungro bacilliform virus in nonvector insect species following genomic amplification. International Rice Research Notes 20 (1) 35-36.