Root rot as a result of Salvia miltiorrhiza is a common root disease caused by Fusarium spp., which has become one of the main diseases affecting the production of S. miltiorrhiza. Currently, several hypovirulence-related mycoviruses have been identified in many phytopathogenic fungi, including Fusarium spp., which show potential as biological controls. In this study, we report a new mycovirus, Fusarium oxysporum partitivirus 1 (FoPV1), isolated from F. oxysporum strain FCR51, which is a causal agent of S. miltiorrhiza dry rot. The FoPV1 genome contains two double-stranded RNA segments (dsRNA1 and dsRNA2). The size of dsRNA1 is 1773 bp, and it encodes a putative RNA-dependent RNA polymerase (RdRp). The dsRNA2 is 1570 bp in length, encoding a putative capsid protein (CP). Multiple sequence alignments and phylogenetic analyses based on the amino acid sequences of the RdRp and the CP proteins indicated that FoPV1 appears to be a new member of the family Partitiviridae that is related to members of the genus Gammapartitivirus. Pathogenicity assay showed that FoPV1 confers hypervirulence to its host, F. oxysporum. This is the first report of a partitivirus infecting F. oxysporum and the first hypovirulence-related mycovirus from the causal agent of S. miltiorrhiza dry rot.

Rehmannia glutinosa (family Scrophulariaceae) is an important traditional medicinal plant, whose root is used to treat anemia, hemoptysis, and gynecological diseases in China (Matsumoto et al. 1989). This plant is native to China and cultivated in China, Korea, Japan, and northern Vietnam (Kwak et al. 2020). Viral diseases caused remarkable loss in the yield and quality of R. glutinosa (Ling et al. 2009). To date, ten viruses have been identified globally to infect R. glutinosa and seven of these viruses reported in China (Liu et al. 2018; Zhang et al. 2021). Most plants of R. glutinosa are infected with one or more of these viruses (Kwak et al. 2018; Zhang et al. 2004). In July 2020, a survey of the viral disease infecting R. glutinosa was conducted in commercial plantations of Wenxian, Wuzhi, Mengzhou, and Yuzhou counties in Henan Province, China. The disease symptoms included mosaic, chlorosis, leaf distortion, and the percentage of symptomatic plants was over 70% in the surveyed fields (n=9). Sixty leaf samples of symptomatic R. glutinosa plants were collected from nine cultivation fields in Wenxian, Wuzhi, Mengzhou, and Yuzhou counties (five to seven plants for each field). Total RNA was extracted from one pooled sample containing a portion of all above-mentioned leaf samples using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) and analyzed by high-throughput sequencing (HTS) to identify viral pathogens. A transcriptome library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), and sequenced on an Illumina NovaSeq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 27,664,949 high-quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs (n=109,180) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. BLASTn analysis showed that the R. glutinosa plants were infected with known viruses, including broad bean wilt virus, rehmannia mosaic virus, youcai mosaic virus, and cucurbit chlorotic yellows virus. In addition, one contig (6,418 nt in length) had a nucleotide sequence identity of 99.64% with the TN29 isolate of tobacco mild green mosaic virus (TMGMV, GenBank accession no. MF139550). To confirm the presence of this virus, sixty above-mentioned samples were screened by reverse transcription-polymerase chain reaction (RT-PCR) using the specific primer pairs (Supplementary Table1) TMGMG-CPF/TMGMG-CPR targeting a 545-nt fragment within the CP gene. Amplicons with expected sizes were detected from 47 of 60 samples but not from the negative control (virus-free healthy plant through the tip meristem culture). Seventeen amplicons (11#, 13#, 14#, 21#, 22#, 23#, 25#, 26#, 27#, 31#, 32#, 33#, 37#, 52#, 57#, 59#, and 60#) of TMGMV-CP were selected, and purified. The PCR products were cloned into the pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The sequences were deposited into the GenBank (accession nos. MZ395944 to MZ395960). The near-full-length genomic sequence of TMGMV-Rg14 isolate was obtained from one positive sample (sample no. 14) by RT-PCR amplification of two overlapping fragments using the following primer pairs: TMGMV-40F/TMGMV-3570R and TMGMV-3220F/TMGMV-6400R. The near-full-length genomic sequence of the TMGMV-Rg14 isolate was 6 304 nucleotides (nt) in length and deposited into GenBank (accession no. MZ395975). BLASTn analysis demonstrated that the TMGMV-Rg14 isolate shared a sequence identity ranging from 96.89% (AB078435) to 99.60% (MF139550) with the other TMGMV isolates. Furthermore, the virus-free healthy R. glutinosa plants were inoculated with sap from the positive sample (14#) to confirm the infection of TMGMV. Mosaic symptoms were induced on the systemically infected leaves of the inoculated plants 14 days post inoculation. The systemically infected leaves of inoculated plants were assayed by RT-PCR using the primer pairs TMGMV-CPF/CPR. Amplicons of expected size were detected from the inoculated plants but not from non-inoculated plants. To our knowledge, this is the first report of TMGMV infection on R. glutinosa. Further studies are necessary to select a suitable indicator plant for this TMGMV, its host range, and the symptoms it induces in single infection. Since R. glutinosa is cultivated by vegetative propagation, production of virus-free healthy plants is necessary. This study will help to generate virus-free healthy plants and prevent viral disease on R. glutinosa. Further study is needed to determine its pathological implications and economic impact on R. glutinosa in China.

Fusarium oxysporum causes vascular wilt in more than 100 plant species, resulting in massive economic losses. A deep understanding of the mechanisms of pathogenicity and symptom induction by this fungus is necessary to control crop wilt. The YjeF protein has been proven to function in cellular metabolism damage-repair in Escherichia coli and to play an important role in Edc3 (enhancer of the mRNA decapping 3) function in Candida albicans, but no studies have been reported on related functions in plant pathogenic fungi. In this work, we report how the FomYjeF gene in F. oxysporum f. sp. momordicae contributes to conidia production and virulence. The deletion of the FomYjeF gene displayed a highly improved capacity for macroconidia production, and it was shown to be involved in carbendazim’s associated stress pathway. Meanwhile, this gene caused a significant increase in virulence in bitter gourd plants with a higher disease severity index and enhanced the accumulation of glutathione peroxidase and the ability to degrade hydrogen peroxide in F. oxysporum. These findings reveal that FomYjeF affects virulence by influencing the amount of spore formation and the ROS (reactive oxygen species) pathway of F. oxysporum f. sp. momordicae. Taken together, our study shows that the FomYjeF gene affects sporulation, mycelial growth, pathogenicity, and ROS accumulation in F. oxysporum. The results of this study provide a novel insight into the function of FomYjeF participation in the pathogenicity of F. oxysporum f. sp. momordicae.

Rehmannia glutinosa is one of the most important medicinal plants in China and is affected by viral diseases. In this study, a new virus tentatively named Rehmannia Allexivirus virus (ReAV) was identified through high-throughput sequencing, reverse-transcription polymerase chain reaction (RT-PCR), and Sanger sequencing. The complete genome length was 7297 nt and it contained five open reading frames (ORFs) encoding replicase, triple gene block 1(TGB1), TGB2, TGB3, and coat protein (CP). The replicase and CP presented nucleotide homology ranges of 59.9–65.2% and 47.5–55.5% between the nine ReAV isolates and the other 12 species of the genus Allexivirus. In the nine isolates, ReAV-20 and ReAV-31 isolates showed breakpoints in the replicase and CP regions, respectively. The other isolates shared 87.2–96.5% nt with the whole genome nucleotide identity. The phylogenetic tree showed that seven ReAV isolates based on replicase, CP, and whole genome sequences were clustered in the same branch and were related to the genus Allexivirus. The ReAV detection rates for 60 R. glutinosa samples were 73.3–81.7% through RT-PCR using primers targeting the replicase or CP genes. These results demonstrate that ReAV is the dominant virus in R. glutinosa. This study provides important evidence for understanding viruses infecting R. glutinosa and for establishing efficient strategies to prevent viral spread.

Typhonium giganteum Engl. (Baifuzi ) is a perennial plant of the family Araceae. In China, its root is commonly used as an antispasmodic for stroke and cancer treatment (Chi et al. 2010; Gao et al. 2014; Khalivulla et al. 2019). Yuzhou city in Henan Province is the main producing area of T. giganteum Engl., and in July 2020, a survey of viral disease infecting T. giganteum Engl. was conducted in the city. In the surveyed fields (n =5), over 60% of plants displayed varying levels of virus-like symptoms, including mosaic, chlorotic and leaf distortion (Supplementary Figure S1) . To identify possible viral pathogens associated with the disease symptoms afflicting T. giganteum Engl., one leaf each from 25 symptomatic plants was collected and analyzed by high-throughput sequencing (HTS) as well as PCR. For HTS analysis, total RNA was extracted from one pooled sample containing a portion of all abovementioned leaves using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China). After removing ribosomal RNA with Ribo-off rRNA depletion kit (Vazyme Biotech, Nanjing, China), a sequencing library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) and sequenced on an Illumina Novaseq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 6,899,143 high-quality clean reads were obtained after trimming and used for contig assembly. BLASTn and BLASTx analyses on the contigs (n = 128,400) showed that one contig (9,245 bp in length) exhibited a sequence identity of 84.0% with the reference sequence of dasheen mosaic virus (DsMV, NCBI reference seq. NC_003537, genus Potyvirus, family Potyviridae) , suggesting infection of the plants by DsMV. No other viral sequences were detected in the sample. To confirm these results, a near full-length genomic sequence of DsMV was obtained from one sample (sample no. 39) by reverse transcription polymerase chain reaction (RT-PCR) of three overlapping fragments with the following primer pairs: DsMV-1F (5'-AAATTAAAACATCTCAACAAAACCTACA-3') /DsMV-4130R (5'-TTCATGGTCCTCGTGGAGTATA-3'), DsMV-3870F (5'-GAGGACGTGAGAATTCAAAGTCT-3')/DsMV-8250R (5'-GTCCAACCTTGCTTGATGCATGC-3'), DsMV-7690F (5'-GGAGCGACTCCTCTTCCAAAGTTGTG-3')/DsMV-10100R (5'-TGAACACCGTGCACGAAGCATCTC-3'). The PCR products were cloned into pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The near full-length genomic sequence of the isolate (DsMV-BF39) was 9,737 nt in length and deposited into GenBank under the accession no. MZ043618. BLASTn analysis of this sequence demonstrated that it shared an identity ranging from 78.6% (MG602234) to 85.6% (MG602227) with various DsMV isolates. To determine whether DsMV was closely associated with the symptoms observed in T. giganteum Engl., leaf tissues from 30 symptomatic plants and 22 asymptomatic plants were analyzed by RT-PCR using primer pairs DsMV-CPF (5'-TGTTCTGTGAACATGATGAAGTTG-3', sense) and DsMV-CPR (5'-GTAACTGTGGCCTGTTTACCAG-3', antisense) targeting a 916 bp fragment of the CP gene of DsMV. Amplicons with the expected size were detected from the 30 symptomatic plants but not from the 22 asymptomatic plants, suggesting a close association between DsMV infection and the observed symptoms. To our knowledge, this is the first report of DsMV infecting T. giganteum Engl.. Further study is needed to identify the specific symptoms induced by this virus in T. giganteum Engl. and to understand the biological characteristics, epidemiology, prevalence of this virus in China.