Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple (Ananas comosus) worldwide. At least one Ampelovirus species, Pineapple mealybug wilt associated virus-2 (PMWaV-2), and mealybug feeding are involved in the etiology of MWP. A previously undescribed Ampelovirus sharing highest homology with PMWaV-1 and a putative deletion mutant sharing highest homology with PMWaV-2 were detected with reverse transcription-polymerase chain reaction (RT-PCR) assays using degenerate primers. Results were verified with additional sequence information and by immunosorbent electron microscopy. Sequence homology between the virus tentatively designated PMWaV-3, and PMWaV-1 and PMWaV-2, decreases toward the N-terminal across the HSP70 homolog, small hydrophobic protein, and RNA-dependent RNA polymerase open reading frames (ORF). Putative PMWaV-3 could not be detected with four different monoclonal antibodies specific for PMWaV-1 and PMWaV-2. The potential deletion mutant spanning the N-terminal of the HSP70 region was obtained from a pineapple accession from Zaire maintained at the USDA-ARS National Clonal Germplasm Repository in Hawaii. Putative PMWaV-3, like PMWaV-1 and PMWaV-2, is transmissible separately or in combination with other PMWaVs by Dysmicoccus brevipes and D. neobrevipes mealybugs. Plants infected with PMWaV-3 that were continuously exposed to mealybugs did not develop symptoms of MWP in the absence of PMWaV-2. Specific RT-PCR assays were developed for detection of putative PMWaV-3 and the deletion mutant.

Surveys for Pineapple mealybug wilt-associated virus-1 (PMWaV-1) and PMWaV-2 were conducted on pineapple samples from Hawaii and around the world. Tissue blot immunoassays (TBIAs) with two different monoclonal antibodies (MAb) specific to either PMWaV-1 or PMWaV-2 indicated that both closteroviruses are widely distributed throughout the pineapple-growing areas of the world. In the worldwide survey, PMWaV-1 was found in 80% of the mea-lybug wilt of pineapple (MWP)-symptomatic and 78% of the asymptomatic pineapple plants tested. A subset of plants was tested for PMWaV-2; 100% of the symptomatic plants and 12% of the asymptomatic plants were positive for this virus. A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to differentiate between PMWaV-1 and PMWaV-2. Oligonucleotide primers were designed using distinct regions of the HSP 70 homolog genes of the two viruses. PMWaV-specific RT-PCR assays and TBIAs were used to screen the pineapple accessions maintained at the United States Department of Agriculture-Agricultural Research Service National Clonal Germplasm Repository for PMWaV infection; 73% of the accessions were found infected with at least one PMWaV. Pineapple accessions found PMWaV-free were challenged with viruliferous mealybugs to test for immunity to PMWaV-1. No immune germ plasm was identified. Potential alternative virus hosts were screened for infection with virus-specific RT-PCR assays and TBIAs and were also challenged with viruliferous mealybugs. No alternate hosts of PMWaV-1 or PMWaV-2 were identified. PMWaV-1 infection was eliminated through axillary and apical bud propagation from infected crowns. Strategies to manage MWP are discussed.

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Tiger nut (Cyperus esculentus L.) is cultivated in Spain for the production of tiger nut milk. Over the past 5 years, important economic losses resulting from a new tuber rot have been observed near Valencia in eastern Spain. Affected tubers were covered by a white mycelium that turned black as the disease advanced, leading to a general rotting of tubers. Aboveground plant parts showed some early decay and under high-moisture environmental conditions the white mycelium was present on the soil surface. This mycelium showed pyriform swellings characteristic of Rosellinia necatrix Prill., and coremia were occasionally observed on external surfaces of tubers after incubation in a moist chamber for 1 to 2 months. Coremia produced small ellipsoid or obovoid single-celled conidia 3.7 to 5.0 × 2.0 to 2.2 μm. The teleomorph has not been observed. Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved after excess water was drained. Two fungal disks of a 2-week-old culture of R. necatrix grown on potato dextrose agar were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks, and shaken once a week to avoid clustering of inoculum. Two plastic pots (35 cm in diameter) per isolate were filled with a sterilized mixture of equal portions (vol/vol) of soil, sand, and peat moss, and inoculum was added at a concentration of 30 g of infected wheat seeds per 1,200 g of substrate (1). Healthy tubers were surface disinfested in 1.5% (vol/vol) sodium hypochlorite for 1 min, washed twice in sterile water, sown, and subsequently thinned to one per pot after emergence. Plants were grown under field conditions. Six months after inoculation, symptomatic tubers appeared similar to those originally observed in the field. The fungus was reisolated from affected tubers, confirming Koch's postulates. Pathogenicity tests were also conducted on avocado (cv. Reed) and almond (cv. Garriges) seedlings, and apple rootstock (MM-106) as very susceptible host plants for R. necatrix (1). Inoculated plants showed symptoms of wilting and death 4 weeks after inoculation. The fungus was reisolated from affected plants. This is the first report of C. esculentus as a host of R. necatrix. Reference: (1) A. Sztejnberg and Z. Madar. Plant Dis. 64: 662, 1980.

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