CHARACTERIZATION OF THE VIROME IN PINEAPPLE (Ananas sp.) AND FLOWERING GINGER (Alpinia purpurata) IN HAWAII

Abstract

Pineapple (Ananas comosus) and flowering ginger (Alpinia purpurata) are two of Hawaii’s most economic and culturally valuable crops. Plant viruses that affect food and horticultural crops are important threats to Hawaii’s economy. They can have a significant negative impact on their host by reducing both decorative value and quality of propagated material and by causing great economic damage. Because of the relevance of new viruses with a potential threat to Hawaii’s agriculture, characterization of their host range, genetic diversity, and tri-trophic virus-host-vector relationships are essential to evaluating strategies for disease management. Pineapple is a pantropic crop whose production has been severely affected by a viral disease, mealybug wilt of pineapple (MWP). MWP is associated with members of the genus Ampelovirus known as the pineapple mealybug wilt-associated virus (PMWaV) complex. PMWaVs are spread by several mealybug species of Dysmicoccus sp. Pineapple mealybug wilt-associated virus 2 (PMWaV-2) is associated with MWP in Hawaii, Cuba, and Brazil, but not in Australia or China. This may suggest that there might be uncharacterized virus or viruses infecting pineapple and associated with the disease. Here, we report the use of RNA-sequencing technologies to characterize five new virus species in pineapple belonging to two families. Four (+) ssRNA viruses, with bipartite genome and each RNA segment encoding one large open reading frame (ORF), were clustered as members within the subgenus Cholivirus, genus Sadwavirus (family Secoviridae). The four viruses were characterized infecting commercial and germplasm pineapple accessions, and a public domain of the Transcriptome Shotgun Assembly (TSA) in the GenBank database. They belong to the pineapple secovirus (PSV) complex including PSV-A, PSV-B, PSV-C, and PSV-D. Additionally, another member in the PMWaV complex, PMWaV-6, was characterized from MWP-symptomatic field samples, and was also found infecting germplasm accessions. Being closely related to PMWaV-2, PMWaV-6 was clustered as a member in the genus Ampelovirus, subgroup I (family Closteroviridae). RNA-seq was also used to study the occurrence of viral populations associated with pineapple germplasm accessions maintained in the USDA-ARS NPGR at PBARC in Hilo, Hawaii. We discovered 69 viral sequences representing 10 members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the PMWaV complex, as were PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. The high recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for specific identification and detection of viruses infecting pineapple. The RT-PCR assays were designed based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events and diversity found in these viruses in the Ananas germplasm, the reported and validated RT-PCR assays bring an important advance in the surveillance of viral infections of pineapple. Finally, the pineapple mealybug, Dysmicoccus spp., and the pineapple red mite, Dolichotetranychus floridanus, were identified as potential vectors of the ampelovirus PMWaV-6, and the two secoviruses PSV-A and PSV-B, respectively in our preliminary studies. A. purpurata is a perennial plant in the family Zingiberaceae with origins in the South Pacific Islands. As an ornamental and cut flower crop, flowering ginger generates an impact on the Hawaiian floriculture and plant nurseries which in 2020 reported sales estimated at around $81 million (356,000 for cut ginger stems). During the last decade, virus-like symptoms observed in A. purpurata in Hawaii were reported to be caused by banana bract mosaic virus (BBrMV, genus Potyvirus) and Canna yellow mottle virus (CaYMV, genus badnavirus). Symptoms caused by a single infection with BBrMV include chlorotic yellow streaks on leaves and stems and red-brown spindle-shaped streaks on the bracts. Although single and mixed infectious have been observed for CaYMV, the virus has not been clearly associated with specific viral-like symptoms. In 2018, a second badnavirus, banana streak GF virus (BSGFV) was identified infecting flowering ginger on Oahu, Hawaii. In the last five years, flowering ginger production in Hawaii has been declining. Stakeholders have reported a decline in crop vigor and yield affecting the production of flowering ginger and associated to outbreaks of a severe dieback syndrome. To study the association of viral infection with the slow decline syndrome observed in flowering ginger in Hawaii, we used RNA-seq and bioinformatic analyses, and virus indexing on samples collected from four islands. Viral sequences corresponding to six viruses were recovered from samples with virus-like symptoms. Three of these viruses, CaYMV (genus Badnavirus), and two novel viruses, Alpinia vein clearing virus (ApVCV, genus Ampelovirus) and Alpinia vein streaking virus (ApVSV; genus betanucleorhabdovirus), were associated with the slow decline syndrome. The other three viruses included the badnavirus, BSGFV and two potyviruses, BBrMV and the newly reported bean common mosaic virus (BCMV), were found in low incidence, and were thus not associated with the slow decline symptoms. Virus detection from potential vectors in tandem with transmission assays identified the mealybug Planococcus citri as vector of CaYMV and ApVCV, while the aphid Pentalonia caladii was identified as a vector of the novel ApVSV. Both P. citri and P. caladii are common pests of flowering ginger in Hawaii. Transmission of ApVSV was achieved using aphid colonies naturally feeding on ApVSV-infected flowering ginger plants and detached leaves experiments although transmission studies using aphids reared on an alternate host were not successful. This may suggest that host switch of the vector P. caladii may have a negative influence on the transmission of ApVSV. Results reported in this dissertation provide an insight into the association of viral infections with the slow decline syndrome observed in flowering ginger in Hawaii. The discovery and characterization of new viruses contribute to our knowledge of the diversity and characteristics of the virome of many crops. Our multiple-approach methodology using RNA-seq and PCR-based diagnostics were useful to characterize the virome present in two economically important crops in Hawaii. Virus characterization and development of robust detection methods, most often PCR-based, were undertaken simultaneously and offered a fundamental screening tool to index propagative material and field collected samples of flowering ginger and pineapple. The diagnostic PCR-based methods implemented in this study will be a foundational indexing resource for the obtention of virus-free plants. Such “clean plants” are the cornerstone of plant production for all propagated crops, including pineapple and flowering ginger.