Pineapple Mealybug Wilt-associated Virus Research Articles

Complete nucleotide sequence and genome organization of pineapple mealybug wilt-associated virus-1

Pineapple mealybug wilt-associated virus-1 (PMWaV-1; family Closteroviridae, genus Ampelovirus) belongs to a complex of mealybug-transmissible viruses found in pineapple worldwide. In this study, the complete genome of PMWaV-1 was sequenced and found to be 13.1 kb in length, making it the smallest in the family. The genome encoded seven open reading frames (ORFs) and was unusual for an ampelovirus due to the lack of an intergenic region between the RdRp and p6 ORFs, an ORF encoding a relatively small coat protein (CP), and the absence of an ORF encoding a coat protein duplicate (CPd). Phylogenetic analyses placed PMWaV-1, plum bark necrosis stem pitting-associated virus and some grapevine leafroll-associated viruses in a distinct clade within the genus Ampelovirus.

The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease

Pineapple mealybug wilt-associated virus 1 (PMWaV-1), 2 (PMWaV-2) and 3 (PMWaV-3) have been detected in Australian commercial pineapple crops, along with a previously undescribed ampelovirus, forwhich the name Pineapple mealybug wilt-associated virus 5 (PMWaV-5) is proposed. Partial sequences extending from open reading frame 1 b through to the heat shock protein homologue were obtained for PMWaV-1, -3 and -5. Phylogenetic analyses of selected regions of these sequences indicated that PMWaV-5 is a distinct species and most closely related to PMWaV-1. The amino acid sequence variation observed in the RNA-dependent RNA polymerase region of PMWaV-1 isolates was 95.8–98.4% and of PMWaV-3 isolates was 92.2–99.5%. p ]In surveys of mealybug wilt disease (MWD) affected crops, none of the four viruses was clearly associated with the disease at all survey sites. A statistically significant association (P <0.001) between the presence of PMWaV-2 and symptoms was observed at one survey site (site 3), but the virus was at a low incidence at the remaining three survey sites. By contrast, although PMWaV-1 and −3 were equally distributed between symptomless and MWD-affected plants at site 3, there was a statistically significant (P < 0.001) association between each of these two viruses and MWD at sites 1 and 4. At site 2, there was a statistically significant (P < 0.001) association only between PMWaV-3 and MWD. PMWaV-1 was the most commonly found of the four viruses and conversely PMWaV-5 was only occasionally found. Australian isolates of PMWaV-1, -2 and -3 were transmitted by the mealybug species Dysmicoccus brevipes.

RT-PCR DETECTION AND PROTEIN-PROTEIN INTERACTION OF VIRAL COMPONENTS OF PINEAPPLE MEALYBUG WILT-ASSOCIATED VIRUS 2 IN CUBA

SUMMARY During a search for the causal agent of symptoms of mealybug wilt in Cuban pineapple plants, Pineapple mealybug wilt-associated virus 2 (PMWaV-2) was detected. A reverse transcription polymerase chain reaction (RT-PCR) test was developed to amplify seven ORFs using primers designed on the RNA sequence of a Hawaiian isolate of PMWaV-2. Cuban and the Hawaiian PMWaV-2 isolates shared high sequence homology. Yeast two-hybrid assays showed the homodimeric nature of the 20 kDa protein (p20) and the 22 kDa protein (p22), but not of the 34 kDa coat protein (CP). This is the first report of protein-protein interactions between isolated PMWaV-2 proteins.

IDENTIFICATION OF A SECOND MEMBER OF THE FAMILY CLOSTEROVIRIDAE IN MOSAIC-DISEASED FIGS

SUMMARY A double-stranded RNA (dsRNA) ca. 19 kb in size was recovered from a fig tree of Algerian origin (F3) showing chlorotic mottling of the leaves and clearing of the second and third veins. Filamentous closteroviruslike particles up to 2100 nm in length and with distinct cross banding were observed in leaf dips. Cytopathology of thin sectioned phloem cells was similar to that elicited by members of the family Closteroviridae. RTPCR assays with primers specific to the recently described Fig leaf mottle-associated virus (FLMaV) were negative. No virus was recovered by mechanical inoculation to herbaceous hosts. Using denatured F3 dsRNA preparations as template, a 625 bp cDNA fragment was amplified by RT-PCR using degenerate primers designed on the phosphate motifs 1 and 2 of the heat shock-protein 70 homologue (HSP70) of members of the family Closteroviridae. Sequence analysis demonstrated that the sequenced 625 bp fragment was part of a closteroviral HSP70 gene, with sequence identity to FLMaV of ca. 45% and 28% at the nucleotide and amino acid level, respectively. Comparison with the HSP70 amino acid sequences of other closteroviral species showed homologies of 48%, 37% or 36% with Little cherry virus 2 (LChV-2,) Grapevine leafroll-associated virus 1 (GLRaV-1) and Pineapple mealybug wilt-associated virus 2 (PMWaV-2), respectively. In a phylogenetic tree constructed with the amino acid sequences of the HSP70 genes of several members of the family Closteroviridae, isolate F3 clustered with members of the genus Ampelovirus. In RT-PCR assays with specific primers designed to match the F3 HSP70 sequence, 4 of 57 field-grown Italian figs trees yielded amplicons of the expected size, while Multiplex-PCR proved effective for the simultaneous detection of double infections by FLMaV and isolate F3. The different sizes of dsRNA and virus particle lengths of F3 and FLMaV, the low sequence homology of the F3 HSP70 gene with that of

Diversity and Mealybug Transmissibility of Ampeloviruses in Pineapple.

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.

PINEAPPLE MEALYBUG WILT ASSOCIATED VIRUS AND MEALYBUG WILT OF PINEAPPLE

PINEAPPLE MEALYBUG WILT ASSOCIATED VIRUS AND MEALYBUG WILT OF PINEAPPLE

Interactions Between Rotylenchus reniformis and Pineapple mealybug wilt associated virus-1 in Pineapple.

The individual and combined effects of Pineapple mealybug wilt associated virus-1 (PMWaV-1) infection in pineapple, Ananas comosus, and Rotylenchus reniformis on pineapple growth were evaluated under greenhouse and field conditions. Under greenhouse conditions, no effect of PMWaV-1 infection on pineapple growth or nematode reproduction was observed. Under field conditions, the interaction of PMWaV-1 and nematodes was evaluated in plant and ratoon crops. In the plant crop, pineapple in plots treated with the nematicide 1,3-dichloropropane showed increased vegetative growth, whereas virus infection had no effect on vegetative growth. Nematodes reduced the average fruit weight (P = 0.01), whereas PMWaV-1 infection did not (P > 0.14). However, more fruit in the largest size classes (sizes 7 and 8) were produced in PMWaV-1-free plots than in PMWaV-1-infected plots (P = 0.03). The average fruit weight decreased in the presence of virus or nematodes. The smallest fruits were from PMWaV-1-infected plants infested with R. reniformis. More early-ripening fruit (30%) were produced by plants infected with PMWaV-1 than by PMWaV-free plants (P < 0.05). PMWaV-1 infection may be one of the reasons for asynchronous fruit ripening, which is a top limiting factor for pineapple production in Hawaii. In the ratoon crop, PMWaV-1 infection reduced fruit weight by 9% (P < 0.01), whereas nematode effects were similar across treatments (P > 0.10). More fruit in the three largest size categories were produced in PMWaV-1-free plots than in PMWaV-1-infected plots (P < 0.01). PMWaV-1 infection reduced the number of fruit produced in the ratoon crop (P < 0.02). An interaction (P < 0.03) between R. reniformis and PMWaV-1 infection status was detected in the ratoon crop. The fewest fruit were produced in plots with PMWaV-1-infected plants that were nematode infected.

Closterovirus Infection and Mealybug Exposure Are Necessary for the Development of Mealybug Wilt of Pineapple Disease

ABSTRACT The roles of Pineapple mealybug wilt-associated viruses (PMWaVs) and mealybug (Dysmicoccus spp.) feeding in the etiology of mealybug wilt of pineapple (MWP) were evaluated. Container-grown pineapple (Ananas comosus) plants from five commercially grown Hawaiian proprietary selections and a field study utilizing a randomized complete block design were used to test four treatments for induction of MWP: PMWaV-1-free and PMWaV-1-infected plants maintained mealybug-free, and PMWaV-1-free and PMWaV-1-infected plants that received monthly applications of nonviruliferous mealybugs. A second PMWaV, PMWaV-2, was identified in some of the test plants during the course of these studies and was shown to be an integral factor in MWP etiology. Typical MWP symptoms developed only in plants infected with PMWaV-2 and exposed to mealybugs. MWP did not develop in PMWaV-1-free or PMWaV-1-infected plants that were exposed to mealybugs, or in mealy-bug-free plants infected with PMWaV-1, PMWaV-2, or both viruses. Plants from all five Hawaiian proprietary selections developed MWP when PMWaV-2 infected plants were exposed to mealybug feeding. A PMWaV-2-specific monoclonal antibody was produced that decorated the particles in immunosorbent electron microscopy and detected the virus in tissue blot immunoassays. PMWaV-2 was acquired and transmitted by pink and gray pineapple mealybugs (Dysmicoccus spp.) to pineapple plants, and these plants subsequently developed MWP symptoms while sustaining mealybug populations.

Yield Impact and Spread of Pineapple mealybug wilt associated virus-2 and Mealybug Wilt of Pineapple in Hawaii.

The impact of mealybug feeding and Pineapple mealybug wilt associated virus-1 (PMWaV-1) and PMWaV-2 infection on pineapple fruit yield, and the spread of PMWaV-1 and mealybug wilt of pineapple (MWP) were evaluated under field conditions with a randomized complete block design. Plots of PMWaV-1-free or infected plants were maintained mealybug-free or inoculated with mealybugs (Dysmicoccus spp.) at monthly intervals. Plants infected with PMWaV-2, an integral part of MWP etiology, were nested within plots that were maintained free of mealybugs, and in the plots of PMWaV-1 infected plants exposed to mealybugs. MWP, which only developed in PMWaV-2 infected plants exposed to mealybugs, resulted in a 35% reduction in yield when compared to PMWaV-free plants. Yield reductions were dependent on time of MWP symptom development; the earlier the expression of symptoms the greater the impact on fruit yields. An interaction effect between PMWaV infection, inclusive of both PMWaV-1 and PMWaV-2 infected plants, and mealybug exposure was detected in the plant crop (P < 0.02) but not in the ratoon crop (P > 0.59). This could be explained by the presence of MWP symptom expression during the plant crop and subsequent plant recovery in the ratoon crop. Virus infection, inclusive of PMWaV-1 and PMWaV-2, suppressed yield (P < 0.01) in the ratoon crop. The commercially desirable fruit sizes were most frequently obtained from PMWaV-free plants. Spatial analysis of PMWaV-2 spread, and MWP symptom expression in mealybug inoculated plots showed patterns of aggregation within rows and within beds but not between beds over the course of the study. Initial occurrence of MWP symptom expression in mealybug-inoculated plots was underdispersed indicating random occurrence of PMWaV-2 plants. After 6 months of mealybug exposure, patterns of both PMWaV-2 incidence and MWP were overdispersed. Management strategies are discussed.

Differentiation, Distribution, and Elimination of Two Different Pineapple mealybug wilt-associated viruses Found in Pineapple.

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.

The impact of Pineapple mealybug wilt-associated virus-1 and reduced irrigation on pineapple yield

The impacts of Pineapple mealybug wilt-associated virus-1 (PMWaV-1) and reduced irrigation on pineapple fruit yield in plant and ratoon crops were evaluated in a field experiment in Hawaii. In the plant crop, PMWaV-1 infection and reduced irrigation had no significant effects on fruit weight. In the ratoon crop, plants infected with PMWaV-1 produced smaller fruit than disease-free plants and plants that received reduced irrigation also produced smaller fruit than plants that received regular irrigation. Additive effects of PMWaV-1 infection and reduced irrigation were detected; plots of infected plants that received reduced irrigation produced the fewest fruit. Frequency distributions for fruit size in the ratoon cycle showed shifts to smaller sized fruit classes when PMWaV-1 or reduced irrigation were present. Estimated yield from combined plant and ratoon crops for infected plants was 6.7% less than disease-free plants when plants received regular irrigation. Reduced irrigation correlated with a 4.2% reduction in estimated yield of disease-free plants, and both infection and irrigation reduction together reduced yield 13.4%. These results show that PMWaV-1 and reduced irrigation can have a negative and additive impact on pineapple fruit production in the first ratoon.

Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2

The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3'-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5' to 3', potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3' terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family CLOSTEROVIRIDAE: the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.

Transmission of Pineapple Mealybug Wilt-Associated Virus by Two Species of Mealybug (Dysmicoccus spp.)

ABSTRACT Closterovirus-like particles associated with mealybug wilt of pineapple were acquired and transmitted by the pink pineapple mealybug, Dysmicoccus brevipes, and the gray pineapple mealybug, D. neobrevipes. Mealybugs acquired pineapple mealybug wilt-associated virus (PMWaV) from infected pineapple plants or detached leaves. The virus was detected in plants by tissue blot immunoassay and confirmed by immunosorbent electron microscopy. Plants exposed to mealybugs reared on PMWaV-free pineapple tissue remained uninfected. The presence of ants was correlated with an increased rate of virus spread when caged with D. brevipes. All stages of D. neobrevipes acquired PMWaV, although vector efficiency decreased significantly in older adult females. The probability of a single third-instar immature transmitting the virus was 0.04. Both species of mealybug acquired and transmitted PMWaV from infected pineapple material that had been clonally propagated for decades, and both species acquired PMWaV from sources previously infected with the virus by the other mealybug species.