Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a ‘calibrator’. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

The complete genome of Banana streak GF virus Yunnan isolate infecting Cavendish Musa AAA group in China

Banana streak virus (BSV) belongs to the members of the genus Badnavirus, family Caulimoviridae. At present, BSV contains nine species in the International Committee on Taxonomy of Viruses (ICTV) classification report (2018b release). Previous study indicated that the viral particles of Banana streak virus Acuminata Yunnan (BSV-Acum) were purified from banana (Cavendish Musa AAA group) leaves in Yunnan Province, China, and its complete genome was obtained. To further determine whether this sample infecting with Banana streak GF virus (BSGFV), the polymerase chain reaction (PCR) cloning and complete genome analysis of the Banana streak GF virus Yunnan isolate (BSGFV-YN) isolate were carried out in this study. The result showed that BSGFV-YN infecting Cavendish Musa AAA group was co-infecting this sample. Its genome contains a total of 7,325 bp in length with 42% GC content. This complete genome sequence was deposited in GenBank under accession number MN296502. Sequence analysis showed that the complete genome of BSGFV-YN was 98.14% sequence similarity to BSGFV Goldfinger, while it was 49.10–57.09% to other BSV species. Two phylogenetic trees based on the complete genome and ORFIII polyprotein indicated that BSGFV-YN and other BSV species clustered into a group, while it was the highest homology with BSGFV Goldfinger. Although BSGFV-YN and BSGFV Goldfinger were highly homologous, their cultivating bananas are different. The former cultivating banana was from Cavendish Musa AAA group, while the latter cultivating banana was from Goldfinger Musa AAAB group. Compared with BSGFV Goldfinger, the genome of BSGFV-YN has an extra multiple repetitive sequences in the intergenetic region between ORFIII and ORFI, suggesting that this region might be related to host selection. In summary, a BSGFV-YN distant from BSV-Acum was identified from the same sample, and its complete genome sequence was determined and analyzed. The study extends the polymorphism of BSVs in China and provides scientific clue for the evolutionary relationship with host selection of badnaviruses.

DETEKSI CMV DAN PYMoV PADA BENIH LADA (Piper nigrum) DENGAN TEKNIK ELISA

<p class="IsiabstrakIndonesia">Salah satu kendala penting pada budidaya tanaman lada yaitu penyakit kerdil yang disebabkan oleh <em>Piper yellow mottle virus </em>(PYMoV) dan <em>Cucumber mosaic virus </em>(CMV). Tindakan pencegahan penyebaran penyakit dapat dilakukan salah satunya dengan penggunaan benih sehat bebas dari infeksi PYMoV dan CMV. Tujuan penelitian adalah mendeteksi keberadaan PYMoV dan CMV pada benih lada siap tanam secara serologi menggunakan teknik ELISA. Benih lada yang dideteksi berumur 5 bulan, berasal dari penangkar benih di Sukabumi (Jawa Barat) dan Purbalingga (Jawa Tengah). Varietas lada yang diambil di Sukabumi adalah Natar 1, Petaling, dan Lampung Daun Kecil., masing-masing sebanyak 10 sampel, sedangkan di Purbalingga hanya ada Natar 1, diambil 30 sampel. Di setiap lokasi pembibitan juga dilakukan pengamatan terhadap gejala infeksi virus yang ditemukan. Deteksi virus dilakukan secara <em>Double Antibody Sandwich</em> (DAS)-ELISA menggunakan antiserum <em>Banana streak virus</em> (BSV) untuk PYMoV dan antiserum CMV untuk deteksi CMV. Hasil ELISA dibaca nilai absorbannya dengan elisa reader pada panjang gelombang 405 nm. Sampel dinilai positif, jika nilai absorbansinya 1,5 kali lebih besar daripada kontrol negatif. Gejala infeksi virus pada benih bervariasi, yaitu klorotik, belang, dan belang disertai perubahan bentuk daun. Hasil pengujian ELISA menunjukkan bahwa 66% benih lada dari Sukabumi dan 46% dari Purbalingga terinfeksi virus CMV. Namun, tidak ada satu pun yang menunjukkan reaksi positif terhadap antiserum PYMoV. Hal ini diduga karena konsentrasi PYMoV terlalu rendah sehingga tidak terdeteksi secara ELISA. Untuk mencegah penyebaran virus, deteksi virus pada benih lada penting dilakukan sebelum digunakan sebagai bahan tanaman. </p>