Viral Strain-Specific Activation of Pathogen-Associated Molecular Pattern-Triggered Immunity Enhances Symptom Severity in Broad Bean Wilt Virus 2 Infection

Broad bean wilt virus 2 (BBWV2) is an emerging virus in various economically important crops, especially pepper (Capsicum annuum L.), worldwide. Recently, the emergence of various BBWV2 strains that induce severe symptoms has increased damage to pepper crops. While the symptomatic variations among virus strains should be associated with differences in the transcriptomic reprogramming of host plants upon infection, underlying molecular mechanisms and associated genes are largely unknown. In the present study, we employed transcriptome analysis to identify responsible host factors for symptom enhancement in the BBWV2-pepper pathosystem using two distinct BBWV2 strains, PAP1 (a severe strain) and RP1 (a mild strain). Comparative analysis of the differentially expressed genes (DEGs) revealed that various genes associated with pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and ethylene signaling were significantly upregulated upon infection with the severe PAP1 strain, but not with the mild RP1 strain. Indeed, hormone analysis revealed that ethylene emission was significantly increased in pepper plants infected with PAP1. These observations imply that the activation of the PTI-associated defense responses reinforce symptom formation during BBWV2 infection in a virus strain-specific manner.

Screening for pathotype-specific resistance to broad bean wilt virus 2 and cucumber mosaic virus in pepper (Capsicum annuum L.)

Two aphid-transmitted RNA viruses, broad bean wilt virus 2 (BBWV2) and cucumber mosaic virus (CMV), are the most prevalent viruses in Korean pepper fields and cause chronic damage in pepper production. In this study, we employed a screening system for pathotype-specific resistance of pepper germplasm to BBWV2 and CMV by utilizing infectious cDNA clones of different pathotypes of the viruses (two BBWV2 strains and three CMV strains). We first examined pathogenic characteristics of the BBWV2 and CMV strains in various plant species and their phylogenetic positions in the virus population structures. We then screened 34 commercial pepper cultivars and seven accessions for resistance. While 21 pepper cultivars were resistant to CMV Fny strain, only two cultivars were resistant to CMV P1 strain. We also found only one cultivar partially resistant to BBWV2 RP1 strain. However, all tested commercial pepper cultivars were susceptible to the resistance-breaking CMV strain GTN (CMV-GTN) and BBWV2 severe strain PAP1 (BBWV2-PAP1), suggesting that breeding new cultivars resistant to these virus strains is necessary. Fortunately, we identified several pepper accessions that were resistant or partially resistant to CMV-GTN and one symptomless accession despite systemic infection with BBWV2-PAP1. These genetic resources will be useful in pepper breeding programs to deploy resistance to BBWV2 and CMV.

Metagenomic Analysis of Marigold: Mixed Infection Including Two New Viruses

Marigold plants with symptoms of mosaic, crinkle, leaf curl and necrosis were observed and small RNA and ribo-depleted total RNA deep sequencing were conducted to identify the associated viruses. Broad bean wilt virus 2, cucumber mosaic virus, turnip mosaic virus, a new potyvirus tentatively named marigold mosaic virus (MMV) and a new partitivirus named as marigold cryptic virus (MCV) were finally identified. Complete genome sequence analysis showed MMV was 9811 nt in length, encoding a large polyprotein with highest aa sequence identity (57%) with the putative potyvirus polygonatumkingianum virus 1. Phylogenetic analysis with the definite potyviruses based on the polyprotein sequence showed MMV clustered closest to plum pox virus. The complete genome of MCV comprised of dsRNA1 (1583 bp) and dsRNA2 (1459 bp), encoding the RNA-dependent RNA polymerase (RdRp), and coat protein (CP), respectively. MCV RdRp shared the highest (75.7%) aa sequence identity with the unclassified partitivirus ambrosia cryptic virus 2, and 59.0%, 57.1%, 56.1%, 54.5% and 33.7% with the corresponding region of the definite delta-partitiviruses, pepper cryptic virus 2, beet cryptic virus 3, beet cryptic virus 2, pepper cryptic virus 1 and fig cryptic virus, respectively. Phylogenetic analysis based on the RdRp aa sequence showed MCV clustered into the delta-partitivirus group. These findings enriched our knowledge of viruses infecting marigold, but the association of the observed symptom and the identified viruses and the biological characterization of the new viruses should be further investigated.

First Report of broad bean wilt virus-2 in marigold (Tagetes erecta L.) in China

Broad bean wilt virus 2 (BBWV-2), a member of the genus Fabavirus in the family Secoviridae mainly transmitted by aphids, has been recognized as a severe pathogen affecting the production of horticultural and ornamental plants worldwide (Xia et al. 2020). The virus was reported to infect many plant species mostly belonging to the family Fabaceae in China (Wang et al. 2017). In August 2018, marigold plants with the symptom of mosaic were observed in the field of Huairou, Beijing (Figure S1). Total RNA was extracted from symptomatic leaf samples from a single plant with TRIzol reagent (Invitrogen, Carlsbad, CA) with a standard procedure following the manufacturer′s instructions, and small RNAs were isolated for deep sequencing library construction with Illumina TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The high-throughput sequencing was carried out on an Illumina HiSeq2500 platform. After raw data process, 8,911,917 clean reads were gained and further de novo assembled into contigs with CLC Genomics Workbench software. BLASTN and BLASTX analysis against the GenBank database showed that 81 of the 9,495 assembled contigs shared high nucleotide (nt) sequence identity with the bipartite genome of BBWV-2 isolate Gyp (KX686589-KX686590 for RNA1 and RNA2, 89% of the genome coverage and 90% nt identity) and 34 with high nt sequence identity of cucumber mosaic virus(CMV) from Tagetes erecta (EU665000-EU665002 for RNA1-RNA3,89% of the genome coverage and 96% nt sequence identity) with sequence coverage ranging from 24-fold to 8,078-fold at different genome positions. To further confirm the presence of BBWV-2, a RNA2 specific primer pair targeting the coat protein region (F1423-1448,5-CTGACAGAGGAATACTATTTCCAAAG-3;R2692-2719,5-CCTGTAAAATTGATATCTCCGGACAAAC-3) was designed from the obtained HTS sequence and reverse transcription-polymerase chain reaction (RT-PCR) was conducted. The 1.3 kb amplicon was ligated to pMD19-T vector (TaKaRa, Dalian, China) and sequenced. Sequence analysis showed it (BBWV-2-marigold, MW322809) shared 99% nt sequence identity with the Gyp isolate infecting Gynura procumbens from South Korea (LC497425.1). Phylogenetic analysis constructed with MEGA6 with the CP nt sequence of other reported BBWV-2 isolates showed BBWV-2-marigold clustered closely with the isolates from South Korea infecting Gynura procumbens (Figure S2), in accordance with the sequence identity analysis. Further RT-PCR with primer pair targeting the RNA1 (F3025-3050 5-GACAGAGTGATATTCCTAATCGAGAT-3; R4035-4062CACTCAATGC AATAAAGGTCTGGCACCT) was conducted and specific bands with the expected size of 1.0 kb were obtained in the agarose gel (data not shown), which further confirmed the existence of BBWV-2.A total of 16 marigold leaf samples(7 from Huairou and 9 from Yanqing) with mosaic symptom were collected and tested by RT-PCR with the abovementioned primer pair, and 4 from Huairou were BBWV-2 positive. Sequence analysis showed that these 4 isolates shared 100% nt sequence identity with the former sequenced isolateBBWV-2-marigold. Furthermore, CMV specific primer pair targeting the CP (F: 5-ATGGACAAATCTGGATCTCCCAAT-3/R: 5-CTAAGTCGGG AGCATCCGTGAGAT-3) were designed to detect the existence of CMV in these samples and results showed that all these 16 samples were positive for CMV. To the best of our knowledge, this is the first report of BBWV-2 in marigold in China.These findings will assist investigations on the epidemiology of diseases caused by BBWV2 in China.

Phylodynamics and Codon Usage Pattern Analysis of Broad Bean Wilt Virus 2

Broad bean wilt virus 2 (BBWV-2), which belongs to the genus Fabavirus of the family Secoviridae, is an important pathogen that causes damage to broad bean, pepper, yam, spinach and other economically important ornamental and horticultural crops worldwide. Previously, only limited reports have shown the genetic variation of BBWV2. Meanwhile, the detailed evolutionary changes, synonymous codon usage bias and host adaptation of this virus are largely unclear. Here, we performed comprehensive analyses of the phylodynamics, reassortment, composition bias and codon usage pattern of BBWV2 using forty-two complete genome sequences of BBWV-2 isolates together with two other full-length RNA1 sequences and six full-length RNA2 sequences. Both recombination and reassortment had a significant influence on the genomic evolution of BBWV2. Through phylogenetic analysis we detected three and four lineages based on the ORF1 and ORF2 nonrecombinant sequences, respectively. The evolutionary rates of the two BBWV2 ORF coding sequences were 8.895 × 10−4 and 4.560 × 10−4 subs/site/year, respectively. We found a relatively conserved and stable genomic composition with a lower codon usage choice in the two BBWV2 protein coding sequences. ENC-plot and neutrality plot analyses showed that natural selection is the key factor shaping the codon usage pattern of BBWV2. Strong correlations between BBWV2 and broad bean and pepper were observed from similarity index (SiD), codon adaptation index (CAI) and relative codon deoptimization index (RCDI) analyses. Our study is the first to evaluate the phylodynamics, codon usage patterns and adaptive evolution of a fabavirus, and our results may be useful for the understanding of the origin of this virus.

DETEKSI SECARA SEROLOGI DAN MOLEKULER BEBERAPA JENIS VIRUS YANG BERASOSIASI DENGAN PENYAKIT MOSAIK TANAMAN NILAM (Pogostemon cablin Benth)

<p>ABSTRAK<br />Penyakit mosaik pada tanaman nilam disebabkan oleh beberapa<br />jenis virus, yaitu Potyvirus, Potexvirus, Cucumber mosaic virus (CMV),<br />dan Broad bean wilt virus 2 (BBWV2). Penelitian ini bertujuan untuk<br />mengidentifikasi secara serologi dan molekuler virus-virus yang<br />berasosiasi dengan gejala mosaik pada nilam di KP. Manoko, KP. Cicurug<br />dan lahan petani di Cijeruk. Sampel daun nilam baik yang menunjukkan<br />gejala mosaik atau pun tidak diambil dari setiap lokasi penanaman<br />masing–masing sebanyak 30 sampel. Kejadian penyakit ditentukan melalui<br />deteksi serologi dengan Direct-ELISA dan Indirect-ELISA terhadap sampel<br />menggunakan empat antiserum, yaitu CMV, Cymbidium mosaic virus<br />(CymMV), Potyvirus, dan BBWV2. Deteksi molekuler dengan RT-PCR<br />dilakukan untuk mengonfirmasi virus baru yang ditemukan. Hasil<br />penelitian menunjukkan bahwa gejala infeksi virus yang ditemukan pada<br />nilam bervariasi, yaitu mosaik lemah, mosaik kuning hijau, mosaik dengan<br />penebalan, mosaik dengan malformasi daun, dan bintik kuning. Secara<br />serologi, kejadian virus pada setiap kebun bervariasi. Di KP Manoko,<br />Potyvirus dan BBWV2 lebih dominan (100%) dibandingkan CymMV. Di<br />KP Cicurug, kejadian Potyvirus dan CMV terlihat lebih dominan (83,3 dan<br />80%) dibandingkan BBWV2 dan CymMV, sedangkan di Cijeruk, BBWV2<br />lebih dominan (90%) dari Potyvirus (50%) dan CMV (13,3%). Hasil RT-<br />PCR dengan primer degenerate BBWV, diidentifikasi BBWV2 pada<br />sampel daun nilam dari Manoko, Cicurug, dan Cijeruk, sedangkan dengan<br />primer general Potexvirus, diidentifikasi CymMV hanya dari sampel daun<br />nilam dari asal Manoko. Hasil penelitian ini merupakan laporan pertama<br />tentang BBWV2 dan CymMV pada tanaman nilam di Jawa Barat yang<br />mengindikasikan bahwa virus merupakan kendala utama pada perbenihan<br />nilam yang harus segera diatasi.<br />Kata kunci: BBWV2, CymMV, mosaik, Pogostemon cablin Benth, PCR</p><p>ABSTRACT<br />Mosaic symptoms on patchouli plant are associated with several<br />viruses, i.e. Potyvirus, Potexvirus, CMV, and BBWV2. The objective of<br />the study was to detect virus(es) associated with mosaic symptoms on<br />patchouli at the the patchouli seed nurseries, in Manoko, Cicurug, and<br />Cijeruk. Thirty leaf samples either showing typical symptomatic mosaic or<br />asymptomatic were taken from each location. Serological testing by<br />Direct-ELISA and Indirect-ELISA using four antisera namely CMV,<br />Cymbidium mosaic virus (CymMV), Potyvirus, and BBWV2 was carried<br />out to test the incidence of each virus. Molecular detection by RT-PCR was<br />performed to confirm the new virus(es). The results showed that symptoms<br />of virus infection were found vary, i.e. weak mosaic, green yellow mosaic,<br />mosaic with thickening, mosaic with leaf malformations, and yellow spot.<br />Based on the serological detection, virus(es) incidence varied at each seed<br />nurseries. In Manoko, Potyvirus, and BBWV2 were more dominant<br />(100%) compared with CymMV. In Cicurug, Potyvirus and CMV were<br />more dominat (83.3 and 80%) compared with BBWV2 and CymMV.<br />While in Cijeruk, BBWV2 was the most dominant (90%) than Potyvirus<br />(50%) and CMV(13.3%). Result of RT-PCR with degenerate primers pair<br />of BBWV was succesfully identified BBWV2 from Manoko, Cicurug, and<br />Cijeruk samples, whereas by using Potexvirus general primary, CymMV<br />was identified only from Manoko samples. BBWV2 and CymMV were<br />first reported to infect patchouli in West Java. The result indicate that<br />virus(es) are the major constraint on patchouli seed that should be<br />managed immediately.<br />Key words: BBWV2, CymMV, mosaic, Pogostemon cablin Benth, PCR</p>