Mealybug Wilt Research Articles

Occurrence and Molecular Characterization of Three Pineapple Mealybug Wilt-Associated Viruses in Pineapple in Taiwan

Pineapple (Ananas comosus) is one of the major fruit crops in Taiwan, accounting for 275 million U.S. dollars in 2006, following betel nut and citrus production in crop value. Tainung No. 17 is the most important cultivar, accounting for more than 70% of pineapples planted. Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple. Pineapple mealybug wilt-associated virus-1 (PMWaV-1), PMWaV-2, and PMWaV-3 were identified as three distinct species in Ampelovirus from diseased Hawaiian pineapple (1,2). In November of 2007, pineapples (cv. Tainung No. 17) planted in Pingtung County of southern Taiwan showed symptoms similar to MWP. Mealybugs (Dysmicoccus brevipes) were also found. Three primer pairs, 225/226, 223/224, and 263/264 described previously specific for the HSP70h genes of PMWaV-1 (1), -2, and -3 (2), respectively, were used to detect the presence of these three viruses by reverse transcription (RT)-PCR. Expected DNA fragments of 590, 610, and 499 nt were obtained from the total RNA isolated from the leaves of diseased pineapples with primer pairs 225/226, 223/224, and 263/264, respectively. The RT-PCR amplified fragments were cloned, sequenced, and analyzed. The 590-nt fragment (Accession No. EU769113) shared 91.6 to 99.5% nucleotide and 96.8 to 99.5% amino acid identity to those of five isolates of PMWaV-1 available in the GenBank; one each from Hawaii (Accession No. AF414119) and Thailand (Accession No. EF620774) and three from Australia (Accession Nos. EF488752, EF467923, and EF467925). The 610-nt fragment (Accession No. EU769115) showed 98.7 and 99.7% nucleotide and 98% and 100% amino acid identity to those of PMWaV-2 from Hawaii (Accession No. AF283103) and Thailand (Accession No. EU016675), respectively. The 499-nt fragment (Accession No. FJ209047) shared 86.8 to 99.0% nucleotide and 94.0 to 100.0% amino acid identity to those of five PMWaV-3 isolates available in the GenBank; one from Hawaii (Accession No. DQ399259) and four from Australia (Accession Nos. EF467918, EF467919, EF488754, and EF488755). Using primer pairs FJ08-1 (5'-ATGGCTGATTCGAGC)/FJ08-2 (5'-TTATTTGCGTCCACC), FJ08-7 (5'-AGTGAGATTGATCGT)/FJ08-8 (5'-TGCAGGTATCCGCTG), and FJ08-35 (5'-AACGACCGAACTCGC)/FJ08-36 (5'-ATACTACAGATATTG) specific to the coat protein (CP) genes of PMWaV-1, -2, and -3, respectively, expected DNA fragments of 774, 909, and 789 nt were amplified by RT-PCR. The 774-nt CP gene of PMWaV-1 (Accession No. EU769114) shared 99% nucleotide and 98.4% amino acid identity to those of Hawaiian isolate (Accession No. AF414119). The 909-nt CP gene of PMWaV-2 (Accession No. EU769116) shared 99.0 and 99.1% nucleotide identity with isolates from Hawaii (Accession No. AF283103) and Cuba (Accession No. DQ225114), respectively, and 99.3% amino acid identity with both. The 789-nt CP gene of PMWaV-3 (Accession No. FJ209048) shared 99.1% nucleotide and 98.1% amino acid identity to those of the Hawaiian isolate (Accession No. DQ399259). One to two viruses among PMWaV-1, -2, and -3 were detected in all 40 samples collected from diseased pineapples. To our knowledge, this is the first report to identify three PMWaVs in the most important and widely planted pineapple cultivar in Taiwan, Tainung No. 17, by molecular characterization of the HSP70h and CP genes.

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.

CHARACTERIZATION AND CONTROL OF PINEAPPLE MEALYBUG WILT ASSOCIATED AMPELOVIRUSES

CHARACTERIZATION AND CONTROL OF PINEAPPLE MEALYBUG WILT ASSOCIATED AMPELOVIRUSES

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

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.

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.

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.

IMPLICATIONS OF THE USE OF EXCESS COIR DUST MULCH IN PINEAPPLE CULTIVATION ON THE MEALYBUG WILT DISEASE OF PINEAPPLE

IMPLICATIONS OF THE USE OF EXCESS COIR DUST MULCH IN PINEAPPLE CULTIVATION ON THE MEALYBUG WILT DISEASE OF PINEAPPLE

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.

Analysis of Patterns of Pineapple Mealybug Wilt Disease in Sri Lanka.

Mealybug wilt disease of pineapple has a complicated etiology, which is not yet fully understood. As a result, assessments of the disease in the field, such as those that are made in the context of evaluation of disease control methods, are usually based on symptomatology. Field assessments, based on symptoms of mealybug wilt disease, were made in the Kurunegala and Gampaha districts of Sri Lanka. These data were fitted to statistical probability distributions as a method of summarizing the spatial pattern of disease incidence. Most plots showed aggregated patterns of diseased plants, but there were some exceptions. Summarizing patterns of disease incidence by means of fitting statistical probability distributions provides a basis for significance testing in cases where factors affecting disease incidence are the subject of field experimentation. Some examples of this methodology are given.

First Report of a Closterovirus-Like Particle Associated with Pineapple Plants (Ananas comosus cv. Smooth Cayenne) Affected with Pineapple Mealybug Wilt in Cuba.

Pineapple mealybug wilt (PMW) was first described in Hawaii in 1910 and has since spread wherever pineapple is cultivated. PMW is currently one of the most important field diseases of pineapple in Cuba, causing up to 40% crop loss. Leaves of plants affected with PMW turn bronze-red and lose turgidity, and the tips curve down. Root system size is decreased and fruits are unmarketable because the flesh is fibrous and sour. Leaves from wilt-affected pineapple plants were selected from the Experimental Station "Tomás Roig" at Ciego de Avila, Cuba. Asymptomatic plants derived from meristem tissue and cultivated under greenhouse conditions were also collected. Both samples were analyzed by several virus purification procedures and finally purified with a protocol modified from Gunashinge and German (1). Partially purified samples from symptomatic and asymptomatic plants were negatively stained and examined by a JEOL (JEM-2000 EX) transmission electron microscope at a magnification of ×30,000. Sixteen, long, flexuous, rod-shaped, viruslike particles (estimated length 1,200 to 1,450 nm; width about 12 nm) were observed in symptomatic leaves, using electron microscope micrographs. These particles were similar to those of the pineapple clostero-like virus found in Hawaii (1) and Australia (2). Particles were observed from symptomatic plants but not from plants propagated by primary meristem culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of viral proteins indicated that the coat protein has a molecular mass of approximately 23 kDa. This is the first report of closterovirus-like particles associated with pineapple plants (Ananas comosus cv. Smooth Cayenne) affected with PMW in Cuba.

MEALYBUG WILT OF PINEAPPLE: PINEAPPLE VIRUSES AND TWO-STEP HEAT TREATMENT OF PINEAPPLE CROWNS

MEALYBUG WILT OF PINEAPPLE: PINEAPPLE VIRUSES AND TWO-STEP HEAT TREATMENT OF PINEAPPLE CROWNS

Use of a Tissue Blotting Immunoassay to Examine the Distribution of Pineapple Closterovirus in Hawaii.

Specific monoclonal antibodies made to a pineapple closterovirus (PCV) were used in a tissue blotting immunoassay (TBIA) for the detection of PCV in pineapple. More than 2,000 samples were tested in 5 days by one person using this rapid and reliable assay. A survey was conducted using this assay to test more than 20,000 Hawaiian pineapple samples for the presence of PCV. PCV was detected in symptomless pineapple plants in the field and in the USDA pineapple germ plasm collection. Studies of the association of PCV with mealybug wilt of pineapple (MWP) suggest that PCV may be involved in MWP.

Detection of pineapple closterovirus in pineapple plants and mealybugs using monoclonal antibodies

Monoclonal antibodies (MAbs) were produced to the pineapple closterovirus (PCV) in Hawaii. These antibodies were shown to be specific for PCV by decoration of the virus particles in immunosorbent electron microscopy (ISEM) and indirect enzyme‐linked immunosorbent assay (ELISA). Several methods of ELISA were compared. An indirect DAS ELISA using a polyclonal antibody to trap virus particles followed by reaction with monoclonal antibody was shown to be the method of choice for detecting PCV in pineapple plants. Pineapple root tissue was found to be most suitable for detecting PCV in crude samples by indirect ELISA. PCV was detected in symptomatic and asymptomatic pineapple plants collected from Oahu and Maui, and pineapple collections in the USDA/ARS National Clonal Germplasm Repository, but was not detected from pineapple plants grown from seed. At least two serotypes of PCV were detected. In addition. PCV was detected from mealybugs collected from wilted pineapple plants, but not from mealybugs of the same species collected from a colony reared on squash. The role of PCV in mealybug wilt of pineapple is being investigated.

Presence of a clostero-like virus and a bacilliform virus in pineapple plants in Australia

When sap of asymptomatic or mealybug wilt-affected pineapple plants of the Smooth Cayenne group was negatively stained and examined in an electron microscope, clostero-like virus particles were occasionally seen. However, numerous clostero-like virus particles and occasionally some bacilliform particles were seen in partially purified preparations from both asymptomatic and wilted pineapple leaves. An antiserum, made by injecting partially purified preparations of clostero-like particles into a rabbit, trapped and decorated the clostero-like particles. Using this antiserum, the clostero-like particles (c. 1700 -1900x12 nm) were found in almost all plants tested of Smooth Cayenne selections C10, C13, C30 and F-180, the hybrid cv. 53-116 and a selection of the rough leaf Queen group. The particles were more readily trapped from extracts of roots of hybrid cv. 53-116 and Smooth Cayenne selection C10 than from leaves, crowns and fruits. They were not detected in seedlings of a cross between a Queen selection and the Smooth Cayenne selection C10. The clostero-like particles are similar to those reported to occur in pineapple plants in Hawaii and South Africa. This is the first report of their occurrence in Australia. Trapping and decoration tests of particles in pineapples in quarantine from Brazil, France, Malaysia and Taiwan indicated that a similar clostero-like virus occurs in all these countries. The bacilliform particles measured about 133x33 nm. They were trapped and decorated by the Queensland pineapple virus antiserum and also by an antiserum to sugarcane bacilliform badnavirus. They were detected occasionally in various smooth leaf and rough leaf pineapples in north and south Queensland and northern New South Wales. However, in one commercial planting of Smooth Cayenne selection C10 in south Queensland, bacilliform particles were trapped from 29/47 plants. This is the first report of a small bacilliform virus, probably belonging to the badnavirus group, occurring in pineapple plants. The relationship of the clostero-like and bacilliform viruses to yield loss and mealybug wilt in pineapples is unknown.

DETECTION OF PINEAPPLE CLOSTEROVIRUS, A POSSIBLE CAUSE OF MEALYBUG WILT OF PINEAPPLE

DETECTION OF PINEAPPLE CLOSTEROVIRUS, A POSSIBLE CAUSE OF MEALYBUG WILT OF PINEAPPLE

HEAT TREATMENT OF PINEAPPLE: SUBSEQUENT GROWTH AND OCCURRENCE OF MEALYBUG WILT OF PINEAPPLE

HEAT TREATMENT OF PINEAPPLE: SUBSEQUENT GROWTH AND OCCURRENCE OF MEALYBUG WILT OF PINEAPPLE