A previously undescribed polerovirus (solemoviridae) infecting theobroma cacao germplasm

Authors: ADEGBOLA, RAPHAEL - University Of Arizona; KEITH, CORY - University Of Arizona; Gutierrez, Osman; Goenaga, Ricardo; BROWN, JUDITH - University Of Arizona

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2022
Publication Date: 8/23/2022
Citation: Adegbola, R.O., Keith, C.V., Gutierrez, O.A., Goenaga, R.J., Brown, J.K. 2022. A previously undescribed polerovirus (solemoviridae) infecting theobroma cacao germplasm. Plant Disease. 107(3). https://doi.org/10.1094/PDIS-06-22-1449-PDN.
DOI: https://doi.org/10.1094/PDIS-06-22-1449-PDN

Interpretive Summary: Cacao production worldwide is severely affected by diseases and pests. The exchange of germplasm across production areas is needed to introduce new sources of disease resistance. However, the detection of pathogens during the germplasm quarantine process is of paramount importance to avoid the introduction of diseases not previously present in production areas. In 2020, leaves with virus-like symptoms that consist of leaf discoloration and distortion with downward rolling at leaf edges, and light-yellow speckling or mottling were observed on cacao grafted trees of the CCN 51, CATIE-R1, CC-137, and CATIE-R4 genotypes that were undergoing quarantine process at the Subtropical Horticulture Research Station of the USDA-ARS in Miami, Florida. Due to the previously detection on this site of Cacao mild mosaic virus (CaMMV), ARS scientists in Miami, FL, collected leaf samples of symptomatic plants and shipped them to the University of Arizona for further analysis. DNA and RNA was extracted from the samples and tests for the presence of cacao Badnaviruses were conducted and resulted negative. Subsequently, RNA sequencing was conducted, and results compared with plant virus databases. Sequencing results indicated that a Polerovirus was responsible for the observed symptoms, and it was named Cacao Leafroll Virus since it was different from other Poleroviruses that affect other crops. This is the first report of a Polerovirus in cacao and underscores the importance of the development of molecular tools for the detection of viruses before releasing the germplasm from quarantine. Further sampling of leaf material at cacao germplasm collection and quarantine stations are needed to determine the presence of Cacao leafroll virus worldwide.

Technical Abstract: Cacao (Theobroma cacao L., Malvaceae) is an economically important crop cultivated in tropical climates primarily for the beans from which chocolate is produced (Zarrillo et al., 2018). Virus-like symptoms were observed in leaves of young cacao plants (10-12 leaf stage) consisting of bluish discoloration, leaf distortion with downward rolling at leaf edges, and light yellow speckling or mottling (Fig S1) imported from the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) in Turrialba, Costa Rica cacao collection and maintained in the USDA-ARS cacao quarantine greenhouse, Miami, FL. Total RNA was isolated from leaf samples collected from four symptomatic plants using a modified silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA) depleted RNA samples were used for cDNA library preparation and sequenced on the Illumina© NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered 150-bp paired-end reads numbering between 2,601,293-3,104,474 were de novo assembled using SPAdes v.3.14.1 (Nurk et al., 2013) resulting in 200,799-276,851 contigs. Contigs were queried against the NCBI viral reference sequence database using discontiguous megablast. From the four samples, 1,344 contigs ranging in length from 150-nt to 1463-nt per sample, with k-mer coverages of 6.3x to 26,721.7x had hits to potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae) with a pairwise nucleotide (nt) identity (PNI) of 69.1%-72.8%. The contigs were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds resulted in hits to thirteen other polerovirus species ranging from 66.2% (e-value: 9e-141) to 100.0% (e-value: 1e-05) PNI, with hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), respectively. Total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA) followed by RT-PCR using generic polerovirus-specific primers PoconF/PoconcpR (Xiang et al., 2008), yielding amplicons of ~1,400-bp. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI), and were bi-directionally Sanger-sequenced (Eton Bio, Research Triangle Park, NC). The cacao polerovirus isolates (xxx to xxx) shared the greatest PNIs of 71% and 73% with potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), while the CP amino acid (aa) shared the greatest aa sequence similarity of 53% with cereal yellow dwarf virus RPV (NP_840023) whereas the MP aa shared the greatest aa sequence similarity of 38% with wheat yellow leaf dwarf virus-GPV (YP_003029842). These results suggest the association of a previously undescribed polerovirus with symptomatic cacao trees, herein, Cacao leafroll virus (Solemoviridae; Polerovirus). To our knowledge, this is the first report of a cacao-infecting polerovirus.