Mealybug Wilt Of Pineapple Research Articles

Interaction of Rotylenchulus reniformis and Meloidogyne javanica with mealybug wilt of pineapple, in microplots

Mealybug wilt of pineapple (MWP) is a disease complex whose etiology seems to involve the mealybug Dysmicoccus brevipes and pineapple mealybug wilt-associated virus-1, −2 and/or −3. MWP, the reniform and the root-knot nematodes (RN, RKN) are major problems of pineapple crop worldwide. Although these nematodes are often found in MWP-affected plantations, so far, no study has investigated possible interactions among these pests causing symptoms and affecting the development of pineapple plants. Pineapple plantlets free of diseases and pests (Vitoria cultivar) were inoculated with i) RN; ii) RKN; iii) viruliferous mealybugs; iv) RN and viruliferous mealybugs; and v) RKN and viruliferous mealybugs. Uninoculated plants served as controls. After inoculation, the plants were transplanted into microplots, where shoot symptoms and growth were assessed at 9 and 16 months after inoculation. RN, RKN and MWP induced distinct shoot symptoms, which appeared much worse in co-inoculated plants, including the collapse and death of nearly half of the experimental plants at 16 months. RKN, RN and MWP, alone or in combination, reduced the fresh weight of the root system up to 88 % and shoot weight up to 68 %. The interaction among these pests was additive. The results of this experiment emphasize the need for further studies conducted in commercial plantations to understand the interactions among RKN, RN and MWP, and their relative importance, since these are often misdiagnosed and consequently poorly managed.

Molecular diagnosis of Fusarium guttiforme and Pineapple mealybug wilt-associated virus

Background The state of Espirito Santo has in the fruit growing, one of its main economical activities. One of them, the pineapple growth, represents a good economic potential to increase income and job opportunities in the state. However, diseases such as fusariosis and mealybug wilt of pineapple are responsible for the yield losses of up to 80% [1]. The mealybug wilt of pineapple is caused by the complex Pineapple mealybug wilt-associated virus (PMWaV-1, PMWaV-2, PMWaV-3 and PMWaV-5), a single strand RNA [2], while fusariosis on pineapple is caused by the fungus Fusarium guttiforme [3]. This study aimed to develop methodologies for detection of these etiological agents in the pineapple plant.

Geographic distribution of mealybug wilt disease of pineapple and genetic diversity of viruses infecting pineapple in Cuba

Several species of ampeloviruses and badnaviruses infect pineapple plants around the world. Pineapple mealybug wilt-associated ampeloviruses have been associated with mealybug wilt of pineapple (MWP), the major viral disease threatening this crop. Conversely, infection by the badnaviruses Pineapple bacilliform comosus virus (PBCOV) and Pineapple bacilliform erectifolius virus (PBERV) is asymptomatic. To investigate the status of infection of the pineapple crop in Cuba, a diagnostic survey was developed in commercial areas during the period 2009–2012. Incidence of MWP disease was found in up to 100% of the plants in some fields of Central and Eastern regions of the island. Molecular assays revealed the presence of PMWaV-1 for the first time in the Caribbean basin and PMWaV-2, PMWaV-3, either as mixed infections or in combination with PBCOV throughout the country. Furthermore, they revealed for the first time the presence of PMWaV-2 in Bromelia pinguin L., a plant commonly used in Cuba as hedgerow. Sequence analysis of partial heat shock protein 70h and complete coat protein gene of Cuban isolates of PMWaV-1, -2 and -3 showed nucleotide identities above 97% with cognate sequences of viruses isolated from other countries. This work discloses the presence of a complex of viruses associated with the pineapple crop in Cuba, highlights the potential role of B. pinguin in the PMWaV-mealybug-pineapple pathosystem and makes available diagnostic tools for the detection of viruses affecting pineapple for a seed certified production system in Cuba.

First report of Pineapple bacilliform comosus virus (PBCoV) and endogenous Pineapple pararetrovirus‐1 (ePPRV‐1) in pineapple plants in Cuba

Mealybug wilt of pineapple (MWP) is considered one of the most destructive diseases of pineapple (Ananas comosus) crops worldwide. MWP have been related to five species of Pineapple mealybug wilt-associated virus-1 to 5 (PMWaV-1 to 5) (Closteroviridae: Ampelovirus), but an interaction with badnaviruses (Caulimoviridae: Badnavirus) has not been completely discarded (Sether et al., 6). In Cuba, where MWP has been associated with 40% of crop losses, only PMWaV-2 and PMWaV-3 have been detected (Borroto et al., 2007; Hernandez et al., 2010a, b), and there is no evidence of badnavirus presence in the country. During a survey for badnaviruses infecting pineapple cv. Red Spanish in commercial fields in 2009-2011, 78 pineapple plants showing MWP symptoms (foliar reddening, leaves with tips curved down and dieback) were collected for further testing. Two pineapple plants cv. Red Spanish derived from in vitro apical meristem tissue culture were selected as negative controls for the PCR assays. Total nucleic acid was extracted from leaf samples according to Murray & Thompson (5) and badnavirus sequences were amplified in PCR assays using the Badna-1A/Badna 4 degenerate primers as described (Gambley et al., 2). Fragments of the expected size (ca. 544 bp) were amplified from DNA extracts from forty symptom-bearing plants. However, PCR products of the same size were also obtained when DNA extracts from the two in vitro cultured plants (BCuL30 and BCuL31) were tested. Amplicons from the extracts of these two plants and from four randomly chosen MWP symptom-bearing plants (BCuL26 to 29) were selected for further characterisation. DNA fragments were gel-purified, cloned and four individual clones per infected plant sample were sequenced. Six sequences of 540 nt were recovered and showed nucleotides identities that ranged from 52.2 to 93.5% (Table 1), except for BCuLE30 and BCuLE31 that were identical to each other. BCuL26 to BCuL29 sequences were deposited in GenBank (Accession Nos. JQ390618 to JQ390621) and showed at least 80.1% nucleotide identity with Pineapple bacillliform comosus virus (PBCoV-Au) isolated in Australia (EU377664). On the other hand, the sequence of the amplicons isolated from BCuLE30 and BCuLE31 (JQ390622) showed nucleotide identities between 91.7 and 95.6% with isolates of endogenous Pineapple pararetrovirus-1 (ePPRV-1) from Australia and Hawaii (EU377674 and GQ395780). The obtained sequences confirmed the presence of PBCoV and ePPRV-1 in pineapples from Cuba. Phylogenetic analysis grouped BCuL26 to BCuL29 sequences and PBCoV within the same phylogenetic cluster, while BCuL30 grouped in an individual branch with ePPRV-1 supporting the previous results (Fig. 1). This is the first report of the PBCoV and ePPRV-1 presence in the Caribbean basin, results that highlight the need to implement certification procedures for the propagation material to reduce the risk of viral diseases affecting pineapple crop. This research was supported in part by the grant C/5032-1 conceded to L. Hernandez-Rodriguez by the International Foundation for Science, Sweden.

A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2)

An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested polymerase chain reaction (PCR) to amplify a segment of the coat protein gene of the PMWaV-2. The outer primers were designed to anneal at higher temperatures than the nested primers to prevent primer competition in consecutive amplification reactions. To reduce potential competition further, the outer primers were used at one-thousandth the concentration of the nested primers. The specificity and sensitivity of this assay are much greater than PCR using only a single primer-pair. A TaqMan(®) probe was also designed for use in quantitative PCR to detect and quantify the PCR amplification products directly in a single-tube assay. The advantages of the single-tube assays using both conventional and quantitative PCR are reduced handling time and prevention of cross contamination compared to regular nested PCR in which the reactions are carried out in two separate tubes.

TRANSMISSION CHARACTERISTICS OF PINEAPPLE MEALYBUG WILT ASSOCIATED VIRUS-2 BY THE GREY PINEAPPLE MEALYBUGS DYSMICOCCUS NEOBREVIPES IN HAWAII

Pineapple mealybug wilt associated virus-2 (PMWaV-2) is a mealybug transmitted ampelovirus associated with mealybug wilt disease of pineapple (MWP) in Hawaii. The characteristics of PMWaV-2 acquisition and persistence of grey pineapple mealybugs (GPM), Dysmicoccus neobrevipes Beardsley, were measured. Some GPM were able to acquire the virus within 36 hours. Transmission efficiency of GPM in groups of 25 was 100% following a 72-hour acquisition access period (AAP). GPM remained viruliferous for up to three days after a 3-day AAP when serially transferred to virus-free pineapple plants at 24-hour intervals over a 7-day period. Access to a kabocha squash, a non-host of the virus, reduced the transmission efficiency and persistence of the virus. These data show that PMWaV-2 is transmitted by the grey pineapple mealybug in a semi-persistent manner. A potential management strategy based on these findings is discussed.

First report of Pineapple mealybug wilt associated virus‐3 infecting pineapple in Cuba

Pineapple (Ananas comosus) is a common crop in tropical and subtropical areas of the world. Crop yields are seriously affected by mealybug wilt of pineapple (MWP), a viral disease with mealybugs (Dysmicoccus spp.) as vectors (Sether et al., 2005). Viruses associated with MWP are members of the genus Ampelovirus, family Closteroviridae. In Hawaii, Pineapple mealybug wilt-associated virus- (PMWaV-1) infection has been correlated with 5 to 15% of ratoon crop yield reduction and losses of up to 30% of production due to premature or asynchronous fruit ripeness. However, in that region, the most widespread virus species is PMWaV-2, which causes up to 100% fruit loss (Sether & Hu, 2002). Conversely, PMWaV-2 is uncommon in Australia, a country where MWP also causes major reduction of pineapple fruit yield. In Australia, MWP symptoms are strongly correlated with infections by PMWaV-3 alone or by both PMWaV-1 and -3 (Gambley et al., 2008). Due to the high nucleotide identity and the conserved genome organization between PMWaV-1 and PMWaV-3, it is considered that these two viruses have similar deleterious effects on either growth rate or pineapple fruit yield in Hawaii (Sether et al., 2005, 2009). Currently, there are 5,310 ha of pineapple orchards in Cuba, with annual fruit production that reached 28,908 tonnes in 2009. MWP disease is an economic problem for pineapple production in the island, causing up to 40% crop losses (Anonymous, 1989). PMWaV-2 was first detected in a diseased pineapple plant from Ciego de Avila in 1998, with molecular characterisation further provided by Borroto-Fernandez et al., (2007). During a survey for PMWaVs in 2009, thirty pineapple plants showing typical symptoms of MWP (foliar reddening, leaves with tips curved down and dieback) were collected in the Island of Youth, western region of the country (Fig. 1). Total RNA was extracted using the TRIzol LS Reagent kit (Invitrogen, Scotland, UK). RT-PCR assays for PMWaV-1, PMWaV-2 and PMWaV-3 detection were performed using the 225/226, 223/224 and 263/264 primer pairs, respectively; these amplify fragments corresponding to the HSP70h protein ORF of each viral species (Sether et al., 2005). Fragments of expected size for PMWaV-2 (c. 610 bp) and PMWaV-3 (c. 490 bp) were simultaneously amplified from seventeen plants with appropriate symptoms. Amplicons corresponding to PMWaV-1 were not obtained. DNA bands were purified, ligated to pGEM®-T Easy vector (Promega, Madison, USA) and two individual clones derived from each virus per infected plant were sequenced. PMWaV-3 derived amplicons shared ≥ 98% sequence identity with each other, and a representative sequence was deposited in GenBank (PMWaV-3 Cu, Accession No. GU563497). Sequence comparisons showed the closest nucleotide identity (98%) to PMWaV-3 from Hawaii (DQ399259). On the other hand, a representative sequence of PMWaV-2 Cu amplicons (FN825676) showed the closest nucleotide identity (99%) to PMWaV-2 from Taiwan (EU769115.1) and Thailand (EU016675.1). Phylogenetic analysis based on the PMWaV-3 amplicon sequence of Cuban and reference closteroviruses, grouped PMWaV-3 Cu and PMWaV-3 isolates from Hawaii and Thailand within the same phylogenetic cluster (Fig. 2). The phylogenetic tree also supported the sequence divergence of the cluster composed by PMWaV-3 and PMWaV-1 from that of PMWaV-2Hw, GLRaV-1 and GLRaV-3 (type member of the genus Ampelovirus) as previously observed (Sether et al., 2009). Gathering all these data, this is the first report of the presence of the PMWaV-3 in Cuban pineapple fields and in the Caribbean basin. Noteworthy is that pineapple plants affected by MWP were infected by both PMWaV-3 and PMWaV-2 which suggests that a complex of ampeloviruses may be widespread in Cuban pineapple fields. Results support the need to implement certification procedures for pineapple propagation materials to reduce the economic impact of MWP disease on pineapple crops in Cuba.

Spatial and Temporal Incidences of Pineapple mealybug wilt-associated viruses in Pineapple Planting Blocks.

A complex of Pineapple mealybug wilt-associated viruses (PMWaVs) that can infect pineapple (Ananas comosus) is correlated with reduced yields and mealybug wilt of pineapple. The incidences of PMWaV-1 and PMWaV-2 at planting, fruit harvest, the beginning of the ratoon crop, and ratoon fruit harvest were determined for end, side, and central regions of planting blocks in eight commercial fields. Differences in virus incidence for the three regions at ratoon harvest were highly significant (P = 0.0018). Central regions of planting blocks had lower virus incidences at the time of ratoon fruit harvest. Collection of propagation material from the central regions of planting blocks will help to minimize PMWaV incidence in fields planted with this material.

CHARACTERIZATION AND MANAGEMENT OF PINEAPPLE MEALYBUG WILT ASSOCIATED VIRUSES

CHARACTERIZATION AND MANAGEMENT OF PINEAPPLE MEALYBUG WILT ASSOCIATED VIRUSES

Genome organization and phylogenetic relationship of Pineapple mealybug wilt associated virus-3 with family Closteroviridae members

The nucleotide sequence of Pineapple mealybug wilt associated virus-3 (PMWaV-3) (Closteroviridae: Ampelovirus), spanning seven open reading frames (ORFs) and the untranslatable region of the 3' end was determined. Based on the amino acid identities with orthologous ORFs of PMWaV-1 (54%-73%) and PMWaV-2 (13%-35%), we propose PMWaV-3 is a new species in the PMWaV complex. PMWaV-3 lacks an intergenic region between ORF1b and ORF2, encodes a relatively small, 28.8 kDa, coat protein, and lacks a coat protein duplicate. Phylogenetic analyses were used to analyze seven different domains and ORFs from members of the family Closteroviridae. Two distinct clades within the recognized genus Ampelovirus were observed; one that includes PMWaV-3 and PMWaV-1 and several GLRaVs and another that includes PMWaV-2 and GLRaV-3, the type member of the genus Ampelovirus.

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.

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.

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.