Characterization and Control of Pineapple Mealybug Wilt Associated

Mealybug wilt of pineapple (MWP) is a devastating disease of pineapple worldwide. Pineapple mealybug wilt associated virus-2 (PMWaV-2) infection and mealybug exposure are involved in the etiology of MWP whereas PMWaV-1 is not a necessary component in MWP but correlates with reduced yields. Degenerate primers derived from conserved sequence regions of the HSP-70 homolog gene of various Ampeloviruses were used in RT-PCR assays to identify additional viruses in pineapple. Our results show that PMWaV-3 shares 64%, 73%, and 79% amino acid identity with PMWaV-1 in the RNA dependent RNA polymerase, small hydrophobic protein, and HSP-70 homolog ORFs, respectively. Shared amino acid identity with PMWaV-2 in the same ORFs is 30%, 13%, and 44%. Preliminary data have shown that PMWaV-3 is more closely related to PMWaV-1 than to PMWaV-2. The coat protein gene of PMWaV-2 was constructed as an inverted-repeat in pCAMBIA 1300 vector and used to produce transgenic pineapple plants using Agrobacterium-mediated transformation. At least five lines of putatively transgenic pineapple plants that are resistant to PMWaV-2 infection have been produced. These plants have remained resistant to PMWaV-2 infection after multiple challenges with viruliferous mealybugs and have not developed any MWP symptoms after several months. INTRODUCTION Mealybug wilt of pineapple (MWP) is a devastating disease of pineapple worldwide (Carter, 1933, 1942, 1963; Rohrbach et al., 1988). Two Ampeloviruses, Pineapple mealybug wilt associated virus-1 (PMWaV-1) and PMWaV-2, have been identified in pineapple grown in Hawaii (Gunasinghe and German, 1989; Hu et al., 1993, 1996; Melzer et al., 2001; Sether et al., 2001). Pineapple mealybug wilt associated virus2 (PMWaV-2) infection and mealybug exposure are involved in the etiology of MWP whereas PMWaV-1 is not a necessary component in MWP (Sether and Hu, 2000, 2002a,b). Both viruses correlate with reduced yields (Sether and Hu, 2001; Sether and Hu, 2002b; Sipes et al., 2002). In this study, the diversity of the ampeloviruses in pineapple was examined (Sether et al., 2005). In addition, PMWaV-2-resistant transgenic pineapple plants were produced (Perez et al., 2004). MATERIALS AND METHODS Molecular Detection and Cloning PMWaV-1and PMWaV-2-specific MAbs were used in tissue blot immunoassay (TBIA) to screen pineapple accessions maintained at the USDA-ARS NCGR as previously described (Hu et al., 1996, 1997; Sether and Hu, 2002a). Degenerate sense and Proc. V th Intl. Pineapple Symp. Ed. P.H. Joubert Acta Hort. 702, ISHS 2006 24 antisense primers, 249, 5’-GGARGTNGGNWWHGAMTTYGGNACNAC-3’ and 250, 5’-GANVHRTCRAAMGTSCCTCCNCCRAARTC-3’, respectively, were developed based on conserved regions within the HSP70 homolog genes of PMWaV-1 (Melzer et al., manuscript submitted), PMWaV-2 (Melzer et al., 2001) and Grapvine leafroll associated virus-3 (Ling et al., 1998). Total RNA from selected plants found PMWaV-1and PMWaV-2-free in TBIA were subjected to RT-PCR using primers 249 and 250 under the reaction conditions previously described (Sether et al., 2001). RT-PCR reactions were visualized in 1% agarose gels containing ethidium bromide. Amplicons were purified from the gel and cloned into pGEM-T easy vectors (Promega Inc., Madison, WI). Plasmids were purified using QIAprep Spin Mini-prep plasmid DNA extraction kits (Qiagen Inc., Valencia, CA) and sequenced with T7 and SP6 primers in an automated sequencer Model ABI373 or ABI377 at the Biotechnology/Molecular Biology Instrumentation and Training Facility, University of Hawaii, Honolulu, HI. Doublestranded RNA (dsRNA) from a plant that was RT-PCR positive for PMWaV-3 was heat denatured and first-strand cDNAs were synthesized at 37°C using Superscript II Reverse Transcriptase (Life Technologies, Grand Island, NY) with a specific antisense primer for PMWaV-3 following manufacturer’s instructions. Step by step walking procedures were performed with the first strand mixture and PCR to clone the region upstream of the hsp70 domain as previously described (Melzer et al., 2001). Sequence data was analyzed using the web-based GCG package SeqWeb 1.1 (Genetics Computer Group, University of Wisconsin, Madison WI). Basic Local Alignment Search Tool algorithm (BLASTX) was used to translate nucleotide sequences to amino acid sequences and to compare putative translation products with sequences in the National Center for Biotechnology Information website (NCBI). Amino acid identity and similarity were computed with the BLOSUM62 matrix. Sense and antisense primers 264 (5'-AGTTCACTGTAGATTTCGGA-3') and 263 (5'-ATTGATGGATGTGTATCG-3') specific for the putative PMWaV-3 were developed and used in conjunction with primers 225 (5'-ACAGGAAGGACAACACTCAC-3') 226 (5'-CGCACAAACTTCAAGCAATC-3') for PMWaV-1 (Sether et al., 2001), 224 (5'CATACGAACTAGACTCATACG-3') and 223 (5'-CCATCCACCAATTTTACTAC-3') for PMWaV-2 (Sether and Hu, 2002a) in RT-PCR assays to determine the distribution of PMWaV-1, PMWaV-2, and PMWaV-3 in USDA-ARS maintained pineapple accessions and commercially grown pineapple cultivars and hybrids in Hawaii (Sether et al., 2005). Development of Transgenic Pineapple Plants The coat protein gene of PMWaV-2 was constructed as an inverted-repeat in pCAMBIA 1300 vector and used to produce transgenic pineapple plants using Agrobacterium-mediated transformation approach. Micropropagated shoots of a commercial, proprietary pineapple hybrid were maintained in vitro. Leaf bases from in vitro cultured shoots of pineapple and the cross-sectioned stem were subjected to Agrobacteium tumefaciens AGL0 strain containing the transformation constructs. Shoots were regenerated from Agro-exposed leaf bases in selective media, SM-1 (MS + 1 mg/l thiamine hydrochloride + 30 g/l sucrose + 0.5 mg/l BAP + 16 mg/l G418 or hygromycin). Shoots regenerated from leaf bases and stem segments were transferred to liquid selection media, SM-2 (MS + 1 mg/l thiamine hydrochloride + 30 g/l sucrose + 0.5 mg/l BAP + 250 mg/l Cefotaxime + 20 mg/l G418 or 25 mg/l hygromycin B) for two, 30-day cycles on a 70 rpm platform shaker. Antibiotic resistant plants were transferred to the greenhouse for acclimatization, transplanted to soil, and grown to about 4-5 cm before being challenged with PMWaV-2 using viruliferous mealybugs. Viruliferous grey pineapple mealybugs, Dysmicoccus neobrevipes Beardsley were prepared by allowing the mealybugs to acquire PMWaV-2 from leaves of infected pineapple plants as previously described with modification (Sether et al., 1998). Briefly, the white proximal portion of infected leaves were cut into pieces (1 x 1 cm) and placed in petri plates lined with moist filter paper. Non-viruliferous mealybugs, reared on butternut or kobacha squash were transferred onto the infected leaf