Virology Closterovirus Infection and Mealybug Exposure Are Necessary for the Development of Mealybug Wilt of Pineapple Disease D. M. Sether and J. S. Hu University of Hawaii at Manoa, Plant and Environmental Protection Sciences, Honolulu 96822. Accepted for publication 2 April 2002. ABSTRACT Sether, D. M., and Hu, J. S. 2002. Closterovirus infection and mealybug MWP symptoms developed only in plants infected with PMWaV-2 and exposure are necessary for the development of mealybug wilt of exposed to mealybugs. MWP did not develop in PMWaV-1-free or pineapple disease. Phytopathology 92:928-935. PMWaV-1-infected plants that were exposed to mealybugs, or in mealy- bug-free plants infected with PMWaV-1, PMWaV-2, or both viruses. The roles of Pineapple mealybug wilt-associated viruses (PMWaVs) Plants from all five Hawaiian proprietary selections developed MWP and mealybug (Dysmicoccus spp.) feeding in the etiology of mealybug when PMWaV-2 infected plants were exposed to mealybug feeding. A wilt of pineapple (MWP) were evaluated. Container-grown pineapple PMWaV-2-specific monoclonal antibody was produced that decorated the (Ananas comosus) plants from five commercially grown Hawaiian pro- particles in immunosorbent electron microscopy and detected the virus in prietary selections and a field study utilizing a randomized complete tissue blot immunoassays. PMWaV-2 was acquired and transmitted by block design were used to test four treatments for induction of MWP: pink and gray pineapple mealybugs (Dysmicoccus spp.) to pineapple PMWaV-1-free and PMWaV-1-infected plants maintained mealybug-free, plants, and these plants subsequently developed MWP symptoms while and PMWaV-1-free and PMWaV-1-infected plants that received monthly sustaining mealybug populations. applications of nonviruliferous mealybugs. A second PMWaV, PMWaV- 2, was identified in some of the test plants during the course of these Additional keywords: badnavirus, D. brevipes, D. neobrevipes, insect studies and was shown to be an integral factor in MWP etiology. Typical transmission, pineapple closterovirus, virus vector. Mealybug wilt of pineapple (MWP), one of the most serious culture-Agricultural Research Service (USDA-ARS) National diseases of pineapple (Ananas comosus (L.) Merr.), is present in Clonal Germplasm Repository (NCGR) in Hilo, HI (22,33). The all major pineapple-growing areas of the world (4,7,8,19,27,28, presence of this virus in symptomless plants correlates with re- 37). MWP is characterized by severe tip dieback, reddening of duced growth (29) and fruit yield (32). In field samples, there is a leaves, and leaf wilting that can lead to total collapse of mature higher incidence of PMWaV-1 infection in plants with MWP plants (5,8). A recovery phase, characterized by the absence of symptoms than in symptomless plants (21). However, many symptoms in new leaves, frequently is observed in Hawaii (6,8). PMWaV-1-infected plants do not show MWP symptoms. There- Historically, MWP has been associated with mealybug feeding (5, fore, PMWaV-1 infection is not consistently associated with 9,10,23,27). Several hypotheses have been proposed to explain the MWP. These facts suggest that another factor or factors, such as etiology of MWP, including mealybug salivary toxins (6), latent mealybug feeding or another virus, may be involved in MWP transmissible factors (8,9,10) or viral infection (11–15,20,30,31). symptom development. Flexuous rod-shaped virus particles have been isolated from pine- A study with container-grown pineapple plants from five Ha- apple plants with MWP in Hawaii, Australia, and Cuba (4,12,18, waiian-grown commercial proprietary selections and a simul- 21,22,36,37). Formerly referred to as pineapple closterovirus taneous field study conducted on the island of Maui, HI, were (PCV) (19,21,22), this virus is currently referred to as Pineapple used to evaluate the involvement of PMWaV-1 and feeding by mealybug wilt-associated virus (PMWaV) (1,20,26,31,34,35). gray and pink pineapple mealybugs in MWP etiology. During the However, PMWaV is actually a complex of closteroviruses (26, course of these studies, a second PMWaV, PMWaV-2, was found 33). Based on particle morphology (15,19,22,35) and nucleic acid to be involved in MWP. PMWaV-2 has since been characterized characteristics (26), PMWaVs have been placed in the family (26) and surveys of commercially grown pineapple throughout the Closteroviridae (1,24). world and the Hawaiian Islands have shown a 99 to 100% correla- PMWaV-1, which can be detected with monoclonal antibody tion of MWP symptoms and the presence of PMWaV-2 (33). (MAb) 35-6-5 (22), is not sap transmissible but can be acquired Here, we show that PMWaV-2 is mealybug transmitted and pres- and transmitted by gray and pink pineapple mealybugs (D. ent evidence for the roles of PMWaV-2 and mealybug feeding in neobrevipes Beardsley and D. brevipes Cockerell, respectively) the etiology of MWP. (34). It is phloem limited and can be detected in roots, leaves, stems, fruit cores, and crowns of infected plants (21,22). PMWaV- MATERIALS AND METHODS 1 has been detected in pineapple plants with and without symp- toms throughout Hawaii (21) and the world (33), as well as in Container-grown plant study. Crowns used to establish plants pineapple accessions maintained at the U.S. Department of Agri- were collected from fields of mature pineapple fruits of an A. comosus clonal selection (referred to here as selection 5), and four other proprietary clonal selections (selections 1 to 4) of A. comosus Corresponding author: J. S. Hu; E-mail address: [email protected] cv. Smooth Cayenne (21,33). Agronomic selections are referred to by number only due to the proprietary nature of the commercially Publication no. P-2002-0708-01R grown selections in Hawaii. The PMWaV-1 infection status of © 2002 The American Phytopathological Society each crown was determined using PMWaV-1-specific MAb 35-6- 928 PHYTOPATHOLOGY 5 (22) in a tissue blot immunoassay (TBIA) as previously described 1 and PMWaV-2 were conducted with mixed-aged populations of (21). Crowns were tested for PMWaV-1 infection twice prior to D. brevipes and D. neobrevipes in separate studies. Mealybugs planting and subsequently at monthly intervals. The PMWaV-2 were given acquisition access periods (AAPs) of 2 to 4 days on infection status at the time of planting was unknown because a PMWaV-1-infected, PMWaV-2-infected, or PMWaV-free detach- specific assay for this virus was not yet available. Crowns were ed leaves. A minimum of 10 mealybugs then were transferred to planted in 12-liter plastic pots and grown outdoors. Irrigation and each of the 142 pineapple plants free of PMWaV-1 and PMWaV-2 fertilization (20 g of Osmocote Plus 15-9-12 per pot; Scotts-Sierra for inoculation access (IA). D. brevipes and D. neobrevipes Horticultural Products Co., Marysville, OH) were applied as mealybugs that were given AAP to PMWaV-2 were given IA to needed. Plants were divided into four treatment groups as follows: 72 and 30 plants, respectively; groups of D. brevipes and D. PMWaV-1-free and PMWaV-1-infected plants maintained free of neobrevipes given AAP to PMWaV-1 each were given IA to mealybugs, and PMWaV-1-free and PMWaV-1-infected plants 10 plants; and groups of D. brevipes and D. neobrevipes given that received monthly applications of nonviruliferous mealybugs, AAP to PMWaV-free plants were given IA to 10 plants each. reared as previously described (34). The study was conducted Mealybug populations were allowed to develop on these plants. from July 1998 to January 2000, with 10 plants from each of the Plants were grown in a greenhouse at 22 to 30°C and monitored five selections per treatment. The PMWaV-1-free treatment group for infections with PMWaV-1- and PMWaV-2-specific TBIAs consisted only of selections 1 to 4 because all crowns of selection beginning 30 days after initial mealybug exposure. To maintain 5 were PMWaV-1 infected. Each plant in mealybug-exposed treat- mealybug populations in the absence of ants, additional inocula- ments received 25 to 50 mixed-aged, nonviruliferous D. brevipes tions of 10 to 20 nonviruliferous mixed populations of D. brevipes placed near the stem base and 50 to 100 mixed-aged, nonviru- and D. neobrevipes mealybugs were made to the plants beginning liferous D. neobrevipes placed in the center leaf whorl of plants at 45 days after IA and, subsequently, at 45-day intervals for up to monthly intervals. Inoculations began 3 months after planting of 5 months. crowns and continued for 12 months. Amdro ant bait (American Virus purification and immunosorbent electron microscopy. Cyanamid Co., Parsippany, NJ) was used according to manufac- The basal white portions of leaves from entire pineapple plants turer’s instructions to prevent ant infestations. Treatment groups were ground to a fine powder in liquid nitrogen, combined (1:5) were separated with cloth barriers to eliminate cross-contamina- with purification buffer (0.5 M Tris-HCl, pH 8.4; 4% [vol/vol] tion of mealybugs between treatments. Mealybugs could move Triton X-100; 0.5% Na2SO3; and 10 mM Mg2SO4) (37), and from plant to plant within a treatment group. Plants in mealybug- stirred overnight at 40°C. Clarified extract was layered over 5-ml free treatments were sprayed at 40-day intervals with Prentox cushions of 20% sucrose in resuspension buffer (RB) (100 mM Diazinon 50W (Prentiss Inc., Floral Park, NY). Tris-HCl, pH 8.4; 0.5% Na2SO3; and 10 mM Mg2SO4) and Reverse transcription-polymerase chain reaction (RT-PCR) centrifuged at 55,000 × g for 4.25 h at 40°C. Resulting pellets assays, capable of detecting and distinguishing PMWaV-1 and were either resuspended in RB and stored at –80°C for subsequent PMWaV-2, were conducted on all plants beginning 4 months after immunosorbent electron microscopy (ISEM) studies as previously planting and thereafter at monthly intervals on plants not showing described (22) or resuspended in RB with 10% sucrose (wt/vol).