Progress 08/15/05 to 08/14/08
Outputs
OUTPUTS: Universal oligonucleotides were constructed from known badnaviruses and used to identify 20 badnavirus-like sequences in the important commercially-grown pineapple varieties in Hawaii. The 20 badnavirus-like variants that were identified can be grouped into four distinct clades. PCR-based detection assays were developed to detect and distinguish the four different clades and most of the variants. Phylogenetic analyses has shown that the C clade is firmly embedded with other characterized badnaviruses, the B clade branches more closely with another Caulimoviridae genera, and the A and D clades branch with endogenous retrotransposons, and endogenous and episomal Tungroviruses. Nucleotide sequencing of two genetic variants has revealed sequences approximately 500 and 1000 base pairs 5' from the reverse transcriptase and RNase H motifs which are not badnavirus-like suggesting integration into the genome of pineapple. Virus purification from the commercially grown pineapple hybrids has yielded fewer badnavirus-like particles relative to that observed in the previously grown commercial Smooth Cayenne cvs. Surveys for badnaviruses in the low acid hybrids grown in commercially in Hawaii show clade B is present in 100% of the material tested. Less than 9% of the material tested from a hybrid from two import-sources, Costa Rica and Philippines, was infected only with clade B, the remaining material was infected with combinations of B and clades A, C, and/or D. The same plants were simultaneously tested for the presence of Pineapple mealybug wilt associated ampeloviruses 1, -2, and -3 and incidence profiles developed for each hybrid. Incidences differ greatly between hybrids but are similar within the same hybrid for plants sampled in several fields and locations in Hawaii. This new fundamental knowledge on the distribution and incidence of the four badnavirus-like clades establishes which badnaviruses and virus-like varients are currently present in Hawaii and can provide a foundation for any future importation and exportation of pineapple propagation material that may arise. The detection assays developed and the new knowledge on the incidences and distributions are necessary tools for enabling future evaluation of the impact of the viruses on yield and roles in other diseases. The detection tools are also a necessary component for screening imported pineapple material for badnavirus-like sequences. The virus detection technologies that were developed were used to educate graduate students in Plant and Environmental Protection Sciences (PEPS) at the University of Hawaii (UH). The surveys were part of a Ph.D. student's educational program. The research findings were shared with the local researchers as well as national and international researchers in oral presentations, poster, and written forms. Training and the protocols of the virus detection technologies were also shared with visiting professionals involved in pineapple production from The Philippines (Dole Standard Philippines), Malaysia (Malaysian Agrifood Corporation Berhad), and others from several pineapple producing countries or regions (Vietnam, China, Costa Rica). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The presence of four distinct badnavirus-like clades comprised of at least 20 sequence varients were identified in pineapple in Hawaii. This is the first report of badnavirus-like sequences in Hawaiian grown pineapple. Detection assays were developed for these four clades and the varients in each clade. These assays provide a means of screening imported or exported pineapple material, when the need arises as well as germplasm maintained in repositories. Knowledge of which viruses are present in pineapple grown in Hawaii is an integral part of crop production and management strategies. The incidences and distribution of the badnavirus varients and clades and the Pineapple mealybug wilt associated viruses (PMWaVs) was determined for commercially important pineapple hybrids in Hawaii. These incidences and distributions of the viruses in the hybrids of Hawaii and germplasm are critical factors in selecting or producing propagation material to avoid potential disease problems in the future. Having in-hand, rapid and reliable assays for the viruses and making those assays available locally and worldwide provides an important foundation for determining the worldwide distribution of the viruses, their disease associations, impacts, and evolutionary patterns. Knowledge about the presence and distribution of these viruses in locally grown varieties as well as stored germplasm also impacts the type of vector control strategy selected to prevent or reduce spread of the viruses. This work also provided a hand-on training opportunity for undergraduate and graduate students as well as other researchers and professional involved in pineapple production or plant virology, locally and internationally. As a result of this work and the detection technologies developed, future etiological studies can be conducted with the badnaviruses, the PMWaVs, and important pineapple hybrids and varieties, from around the world.
Publications
- Sether, D. M., and Hu, J. S. 2007. Genetic variants of badnavirus-like sequences from pineapple in Hawaii. Phytopathology 97: S106.
- Subere, C., Sether, D., Zee, F., and Hu, J. 2007. Diversity and distribution of Pineapple mealybug wilt-associated viruses (PMWaVs) and pineapple badnaviruses in Hawaii. Phytopathology 97:S112.
- Sether, D. M., and Hu, J. S. 2006. Exploiting pathogen biology: integrated pest management examples from virology. Plant Disease Management in a Sustainable Society, 14th Annual Hawaii Conservation Conference: Sustainability Mauka to Makai. July, 2006. Honolulu, Hawaii, USA.
- Subere, V. Q., Sether, D. M., and Hu, J. S. 2006. Distribution, diversity, and incidence of Pineapple mealybug wilt-associated viruses and pineapple badnaviruses in Hawaii. CTAHR Research Symposium Abstract 98.
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Progress 08/15/06 to 08/14/07
Outputs
OUTPUTS: Four badnavirus-like clades, A, B, C, and D, have been identified from five commercially grown pineapple hybrids in Hawaii. Phylogenetic analyses has shown that the C clade is firmly embedded with other characterized badnaviruses, the B clade branches more closely with another Caulimoviridae genera, and the A and D clades, which share approximately 80% homology with each other, branch with endogenous retrotransposons, and endogenous and episomal Tungroviruses. Nucleotide sequencing of two genetic variants, one from the A clade and one from the B clade, has revealed sequences approximately 500 and 1000 base pairs 5' from the reverse transcriptase and RNase H motifs which are not badnvirus-like. Such sequences may suggest integration of members of the A- or B-like clades into the genome of pineapple. Further genomic Southern analyses and sequencing are needed to confirm integration into the pineapple genome. Virus purification from the commercially grown pineapple hybrids has
yielded fewer badnvirus-like particles relative to that observed in the previously grown commercial Smooth Cayenne cvs. PCR analyses using the Universal set of oligonucleotides revealed the presence of A, B, and C clades when DNA isolated from pineapple leaves was used as a template. However, the same PCR assay repeated on a water-based resuspension of pelleted virus from the same plant did not result in amplification of the B clade, suggesting detection of the B clade may be from a genomic sequence in the pineapple. Amplicons were produced from A and C clade specific primers suggesting that A and C be present as episomal forms. Although sequencing suggests at least one A clade-like variant may be part of the pineapple genome, it may also be represented by episomal particles based on amplification from the virus pellet suspension. Additional experiments are underway to determine if episomal forms of each clade are present. Oligonucleotides (specific) to detect and distinguish the 19
genetic variants identified across the four clades have been used to characterize the "infection" profile of pineapple plants. Some of these plants were then selected as virus acquisition sources for grey pineapple mealybug. Following acquisition, the mealybugs were moved to other characterized pineapple plants with different "infection" profiles. The plants will be analyzed with specific PCR assays to determine if transmission of specific badnavirus-like variants occurred. The universal oligonucleotide sets we designed were used to screen commercial pineapple hybrids from Oahu to determine the extent of infection. Results show clade B to be present in 100% of the material tested. Less than 9% of the material tested from a hybrid from two import-sources was infected only with clade B, the remaining material was infected with combinations of B and clades A, C, and/or D. The same plants were simultaneously tested for the presence of Pineapple mealybug wilt associated ampeloviruses 1,
-2, and -3 and incidence profiles developed for each hybrid. Incidences differ greatly between hybrids.
TARGET AUDIENCES: banana growers in Hawaii, plant virologists
Impacts
Members of the Caulimovirus family have been implicated in several serious yield-reducing or synergistic relationships with other viruses that result in devastating disease losses around the world. Hawaii is home to many economically important crops such as pineapple, taro, and citrus, that can be infected by such viruses. Our recent identification of badnavirus-like virus sequences and particles in pineapple have necessitated the development and implementation of a screening assay to determine the distribution of such viruses in locally grown commodities such as pineapple and to evaluate the possible roles of these viruses on the health of the crop. Distribution, incidence, and vector transmissibility will provide information necessary for developing management strategies to control the spread of the viruses and improve yield to assist in sustaining Hawaii as a viable location for pineapple.
Publications
- Sether, D. M., and Hu, J. S. 2007. Genetic variants of badnavirus-like sequences from pineapple in Hawaii. Phytopathology 97:S106.
- Subere, C. V., Sether, D. M., Zee, F., and Hu, J. S. 2007. Diversity and distribution of Pineapple mealybug wilt-associated viruses (PMWaVs) and pineapple badnaviruses in Hawaii. Phytopathology 97: S112.
- Subere, C. V., Sether, D. M., Zee, F. T.P., and Hu, J. S. 2007. Pineapple mealybug wilt-associated viruses (PMWaVs) and pineapple badnaviruses: Diversity and distribution in commercial hybrids and various accessions in Hawaii. Abstract 51, CTAHR Student Research Symposium, University of Hawaii, p. 38.
- Subere, V. Q., Sether, D. M., and Hu, J. S. 2006. Distribution, diversity, and incidence of Pineapple mealybug wilt-associated viruses and pineapple badnaviruses in Hawaii. Abstract 98, College of Tropical Agriculture and Human Resources (CTAHR) Student Research Symposium.
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Progress 08/15/05 to 08/14/06
Outputs
Universal oligonucleotides based on the RNase H, reverse transcriptase (RT), and protease regions were designed from 16 recognized or tentative badnavirus species and rice tungro bacilliform virus. Twenty distinct badna-like clones were obtained from commercially grown pineapple in Hawaii. Based on amino acid identity, four putative badna-like clades, A, B, C, and M were identified. Amino acid sequence similarity in the reverse transcription/RNaseH region within a putative clade is above 98% and between clades ranges from 46% to 86%. ClustalX and neighbor joining analyses placed the clones on three distinct branches, with A and M sharing a branch with the rice tungro bacilliform virus, C branching near banana streak viruses (BSV), and B forming its own branch. Universal/specific PCR assays were developed to detect multiple sequences within a clade and simultaneously distinguish the four clades in pineapple. These PCR assays for each clade are being used to screen six
groups of commercial pineapple composed of four commercial pineapple hybrids, two of which are composed of planting material obained from two different countries. The B clade was detected in nearly 100% of the hundreds of plants tested from each hybrid. Further sequence analysis of the B clade suggests the sequences may be integrated badna-like sequences in the pineapple genome, similar to that observed in banana. Further genomic Southern analyses are underway to confirm this. Putative clades A, C, and M are also distributed widely in the commercial pineapple hybrids, though at less frequency than the putative B clade. PCR amplicons from the four putative clades are detected at varying incidences in the hybrids. Sequencing of the individual amplicons generated revealed diversity in the RT/RNase H regions. In the case of the two hybrids that originated from two different countries, amplicon sequences obtained from plants originating from the same sources were identical, whereas
amplicon sequences obtained from plants of the same hybrid derived from two different countries have differences of up to 5% in the RT/RNase H region within a clade. Sequencing of entire episomal badnavirus genomes infecting commercial Hawaiian pineapple is underway. PCR amplification revealed higher levels of the C clade in pineapple that has recovered from mealybug wilt of pineapple than those that had not been afflicted with mealybug wilt. The significance of this is under evaluation. Polyclonal antiserum for sugarcane bacilliform viruses and commercially available polyclonal antibodies for badnaviruses showed little to no affinity for pineapple badnavirus-like particles in immunosorbent electron microscopy conducted with preps from infected hybrids. Virus titers in the pineapple hybrids appears to be low relative to that observed in similar purification preparations conducted on the traditionally grown Smooth Cayenne pineapple cultivars, which are no longer grown. Sequencing has
also shown the presence of at least 30 distinct retro-element like sequences possessing RT/RNase regions that appear to be integrated into the genome of pineapple.
Impacts
Badnaviruses are of serious concern to Hawaiian agriculture because of yield losses associated with their presence in various crops. Furthermore, several badnaviruses have recently been shown to integrate into the genomes of their host plants. In addition, badna-like viruses have been implicated in synergistic relationships with other viruses that result in devastating diseases around the world. In Hawaii, badnaviruses have previously been identified in plants exhibiting mealybug wilt but their role in the disease is as yet, unknown. The presence of badnaviruses pose serious threats to important crops including banana, taro, and pineapple, among others, and have resulted in restrictions on the movement of germplasm in many areas around the world. Hawaii is particularly vulnerable to the spread of these invasive viruses because of the state's crop composition, and presence of mealybug vectors. Rapid and reliable detection assays have been developed to quickly identify
the previously uncharacterized badnaviruses in pineapple as well as other economically and culturally important Hawaiian crops already established or targeted for importation or exportation. Identification and determining the distribution of the badna-like viruses that are in Hawaii is an integral part of the control strategies for these viruses in the islands. In addition, it is important to determine the roles of badnaviruses in mealybug wilt of pineapple and the high value hybrids now replacing the traditional pineapple cultivars in Hawaii.
Publications
- No publications reported this period
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