Fei Wang

Salvia miltiorrhiza Bge (Family Lamiaceae) is a traditional Chinese herbal medicine; its roots and rhizomes are used as medicines (Zhang et al. 2023). The plant has been vegetatively propagated using root tubers, and long-term asexual reproduction can lead to the accumulation of plant viruses, which may cause severe diseases. Tobacco mosaic virus and cucumber mosaic virus have been reported to infect S. miltiorrhiza (Yao et al. 2022), and their prevalence has become increasingly serious in China. From June to September 2023, 125 samples of S. miltiorrhiza symptomatic leaves were randomly collected in Henan province, China. A small portion of each leaf of above 125 collected samples was mixed into a total sample (~30 g) and sent to Berry Genomics Corporation (Beijing, China) for high-throughput sequencing (HTS) examination. Ribosomal RNA was removed by RNAprep Pure Plant Plus kit (TIANGEN Biotech, Beijing, China), and the NEBNext Ultra RNA Library Prep kit for Illumina (NEB, Ipswich, MA, USA) was used to build a cDNA library. The sequencing procedure was performed on the Illumina Nova Seq6000 sequencing system (Berry Genomics Corporation). Wuhan Biowefind Co., Ltd. (Wuhan, China) handled the subsequent processing and analysis of sequencing data. A total of 64,043,098 paired-end reads (150 nt) were obtained, and 63,559,676 clean reads were assembled by de novo (IDBA-UA, version 1.1.3) to generate 131,718 contigs (＞200 bp). There were 58,300 reads that covered 85.8% mapping to the soybean geminivirus B genome (SGVB, GenBank MH428830.1). Via BLAST analysis, 12 contigs with length from 228-882 nt were associated with CMV of a 90.4-99.7% similarity; 464 contigs with length from 202-8864 nt were associated with Betaflexiviridae of a 52.82-73.72% similarity; 2 contigs with length from 1849-6181 nt were associated with Alphaflexiviridae of a 33.4~75.3% similarity; 30 contigs with length from 205-7164 nt were associated with Fimoviridae of a 59.17~70.09% similarity. Seventeen contigs with length from 217-1046 nt were associated with SGVB of a 87.5~100% similarity. To identify the presence of the virus, the DNA of 125 samples were extracted using the EZ-10 Spin Column Plant Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The DNA samples were subjected to PCR using the SGVB-specific primer set DSSGVBF/DSSGVBR (a 457-nt fragment), designed based on the HTS results. Results revealed 80 positive samples out of 125 (detection rate, 64%); no bands were detected in ddH2O used as a negative control. A pair of back-to-back primers (DSSGVBF1/DSSGVB1) was designed based on the sequences of the 457-nt fragments to amplify the complete sequence of SGVB. Complete sequences of seven isolates (33#, 130#, 131#, 148#, 149#, 152#, and 155#) were obtained from the positive samples with the DSSGVBF1/DSSGVB1 primer set. It was obtained by sanger sequencing (Sangon Biotech, Zhengzhou, China). The genome sequences of these isolates were 2625 nt long and were deposited in GenBank (accession no. PQ141695, PQ163462-163467). BLASTn analysis revealed that the homology of these seven isolates with the soybean geminivirus B isolate Kong (MH428830.1) was 93.94-97.37%, and the consistency between the seven isolates was 94.0-99.9%. In addition, eight nucleotides were inserted between nt sites 518-525, resulting in the CP position range from 705-1184 reported in SGVB isolate Kong changed to 424-1194 in this study, and the length changing from 477 nt to 768 nt. However, the effect on CP function is unclear. As a novel virus assigned to the Mastrevirus genus, family Geminiviridae, SGVB has only been reported in soybean crops in South Korea (Choi et al. 2022). This study revealed that S. miltiorrhiza is a new host of SGVB. To the best of our knowledge, this is the first report of SGVB-infected S. miltiorrhiza and SGVB in China. In future, we will determine how this virus was likely moved from potential soybean to S. miltiorrhiza via its insect vectors and study its pathological implications on S. miltiorrhiza in China.

Epimedium [Epimedium sagittatum (Sieb. et Zucc.) Maxim., E. brevicornu Maxim., E. pubescens Maxim., E. koreanum Nakai] plants are perennial herbs and the dry leaves were used in traditional Chinese medicine with different medicinal effects (Chinese Pharmacopoeia, 2020 edition). In early August 2022 (the high temperature was 33 ℃ and the low temperature was 25 ℃ on average with above 70% relative humidity), large necrotized leaf spots were initially observed in one-year and two-year old E. sagittatum plants in Dengzhou (32°35'26.84" N, 112°11'8.37" E), Henan province, China. White mycelia, yellow and brown sclerotia were observed along the petioles and colonized to the back of leaves and also on soil-petioles interface around the diseased Epimedium plants. White mycelia spread rapidly from the disease plants as a center to neighboring plants and the infected plants eventually wilted. The disease incidence was 16.0% and 28.0% in the surveyed cultivation fields in Dengzhou (Henan) and Danjiangkou (Hubei), respectively, which resulted in above 20% of yield loss. The symptomatic tissues (petioles, n≥10) were cut into 0.5 cm sections with autoclaved scissors and surface sterilized with 75% ethanol for 30 s, followed by 3 min in 1% NaClO, rinsed three times with distilled sterile water, plated on autoclaved filter paper to remove excess moisture in a biological laminar hood, transferred onto potato dextrose agar (PDA) plates and incubated at 28℃ in incubator in the dark for 3-5 days. Twelve fungal isolates with white radial colony morphology were obtained and purified by hyphal-tip method. The isolates formed radial colonies showing abundant aerial mycelia with a growth rate of 19.25 to 22.25 mm/day (mean = 21.67 ± 0.77 mm; n = 36) and white abundant sclerotia were observed after 5-7 days post inoculation at 28℃. The hyphae of the isolates were hyaline, branched with clamp connections at septa. The isolates formed dark brown sclerotia on PDA plate post 10-14 days incubation. The diameter of mature brown sclerotia ranged from 1.31 to 3.07 mm (mean = 1.91 ± 0.39 mm; n = 40) and 13 to 45 sclerotia (mean = 28; n = 24) were observed on each Petri dish. The isolates were tentatively identified as Agroathelia rolfsii based on morphological characteristics (Yi Y., et al. 2024) and isolate YYHBJ1 was selected as representative isolate for molecular identification and pathogenicity test. The genomic DNA was extracted using CTAB method. For molecular identification, ITS region, translation elongation factor-1alpha (TEF-1α) and nuclear large-subunit ribosomal DNA (nLSU-rDNA) were amplified (White et al. 1990, Wendland and Kothe, 1997). The sequences 684-bp ITS (OR428367), 534-bp TEF1α (OR485567), and 965-bp nLSU-rDNA (OR426612) of isolate YYHBJ1 were deposited in GenBank. Sequence analysis revealed that ITS, TEF1α and nLSU-rDNA sequences of YYHBJ001 showed 100%, 99.63%, and 100% identity to ITS (MN380242 and JF819727), EF1α (MN262527) and nLSU-rDNA (MT225781 and AY635773) sequences of Agroathelia rolfsii (Sacc.) Redhead & Mullineux (Redhead and Mullineux 2023). Pathogenicity tests was confirmed by inoculating 1 year-old E. sagittatum plants grown in pots with sterile soil. PDA plug (5 mm diameter) colonized with 3 brown sclerotia and the fungal culture which was cultivated for 12 days was deposited beside the petiole on the surface of the soil. The control plants were inoculated with PDA plugs without the pathogen. Five plants were used in each treatment. The plants were cultivated in incubator at 28℃ with 80% relative humidity. Fourteen days later, white mycelia growing out from the sclerotia and spread along the petioles to the leave and the infected leaves were yellow. After 21 to 24 days, all inoculated plants displayed symptoms like those observed in field, while no symptoms were observed on the controls. The fungus was re-isolated from the inoculated plants as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. Based on morphological feature and DNA sequences, the pathogen of southern blight on Epimedium plants was identified as A. rolfsii (anamorph S. rolfsii). The pathogen A. rolfsii usually infect kinds of crops and cause southern blight, such as peanuts, chili peppers, green onion and so on (Daunde et al. 2018). To our knowledge, this is the first report of A. rolfsii causing southern blight on E. sagittatum plants in Henan in China.

The cultivated variety of Chinese yam (Dioscorea polystachya Turcz. cv. Tiegun) is an economically important plant, capable of producing tubers that are used as food and traditional Chinese medicine. The basal stem rot was found on approximately 65% of yam (tuber expansion stage) in a total of 10 ha field in Wuzhi, Wen, and Hua counties, Henan, China (Sep 2021). Dark brown fusiform lesions initially occurred at the stems basal, irregularly extending to join together and leading to loop-stem necrotic indentation. Three diseased samples from Wuzhi county were collected, cut into 5 × 5 mm pieces, surface sterilized in 75% ethanol (30 s) and 1% NaClO (1 min), washed in sterile water 3 times, and placed on PDA in the dark for 3 days at 28℃. A total of 44 isolates forming three groups of Fusarium colonies were obtained using monosporic isolation, of which 19, 8, and 17 isolates were identified as F. oxysporum, F. solani, and F. proliferatum based on colony morphology, respectively. Typical isolates SYJJ6, 9, and 10 for each group were further studied. The SYJJ6 colonies showed gray white abundant fluffy aerial mycelium with rough edges, formation of ellipsoid, unicellular microconidia without septa, 5.6 to 13.4 × 2.4 to 4.7 μm (n = 50), and sickle-shaped, slightly curved macroconidia with 2 to 4 septa, 14.0 to 23.9 × 3.4 to 5.1 μm (n = 50). Isolate SYJJ9 produced flocculent white colonies, grew in a circular pattern with a sharp edge, forming oval or oblong microconidia with zero or one septum, 11.2 to 18.8 × 3.4 to 6.2 μm (n = 50), and slightly curved macroconidia with 2 to 3 septa, 27.6 to 44.0 × 3.9 to 7.4 μm (n = 50). SYJJ10 produced whitish or pinkish white colonies with fluffy aerial mycelium and a red pigmentation, produced renal or oval microconidia with no septa, 5.1 to 11.8 × 1.8 to 4.2 μm (n = 50), and falcate, slightly curved macroconidia with 3 to 4 septa, 16.1 to 30.2 × 3.1 to 5.9 μm (n = 50). Additionally, TUB, EF-1α, and RPB2 genes were amplified with primers BT2a/BT2b, EF1/EF2, and 5f2/-7cr, respectively (Glass and Donaldson 1995; O'Donnell et al. 1998, 2010). BLASTn analysis on SYJJ6 (OR047663, OR047666, OR047669), SYJJ9 (OR047665, OR047667, OR047670), and SYJJ10 (OR047664, OR047668, OR047671) gene sequences were over 99% identical to those of F. oxysporum (100%, MK432917; 100%, MN417196; 99.61%, MN457531), F. solani (100%, MF662662; 100%, MN223440; 99.80%, CP104055), and F. proliferatum (100%, ON557521; 100%, ON458137; 99.90%, LT841266), respectively. Pathogenicity tests of three isolates were separately performed on 60-day-old yam seedlings. The basal stems were wounded using needle, and the wounds were wrapped with cotton balls soaked with conidial suspension (1 mL, 3×106 conidia/mL) or water (control). Each isolate treated three plants and repeated three times. All plants were grown at 28℃ under a 16/8-h light/dark cycle. Typical symptoms emerged on basal stems at 16, 13, and 17 days after inoculation with the conidia of isolates SYJJ6, 9, and 10, while the control basal stems appeared healthy. The re-isolated fungi were identical to the original three isolates. Fusarium species (F. oxysporum, F. commune, F. humuli, etc.)were previously reported to cause wilt or stem rot on different D. polystachya cultivars (Fang et al. 2020; Li et al. 2023; Zhao et al. 2013), or basal stem rot on Panax ginseng (Ma et al. 2020). This is the first report of Chinese yam basal stem rot caused by Fusarium species, which threatens the production of Chinese yam 'Tiegun' and should be further studied.

Rehmannia glutinosa (also known as Chinese foxglove) is a perennial dicotyledonous herb, which plays an important role in traditional Chinese medicine. Its active ingredients have a wide range of pharmacological effects on the blood system, endocrine system, immune system, cardiovascular system, and nervous system (Zhang et al. 2008). In May 2022, leaf blight was observed on 45-day-old R. glutinosa in a seedling nursery in Jiaozuo City (35°01'44.20″N, 113°05'30.63″E), Henan Province, China with an approximate disease incidence up to 54% (~1,300 plants). Irregular brown lesion initially appeared on the tips of basal leaves, then progressed to the entire leaf causing leaf drying out (Supple. Fig. 1-A, B, C). The same symptoms appeared successively in the leaves from the base to the top of the plant, which eventually caused the whole plant to die. To identify the pathogen, eight symptomatic leaves were randomly collected from eight individual plants, and cut into small pieces (5 × 5 mm) at the border of lesions. The pieces were surface disinfected in 75% ethanol for 15 s, followed by 1% NaClO for 1 min, rinsed in sterile water three times, and placed on potato dextrose agar (PDA) medium in the dark for 3 days at 25℃. Finally, 12 purified isolates (DHY1-DHY12) were obtained by using single spore method. Leaves of R. glutinosa seedlings were inoculated with conidial suspension (106 conidia/ml), three plants were inoculated per isolate. Controls were treated with sterilized water. All inoculated and control plants were incubated in a greenhouse at 25℃ under 80 ± 10% humidity and a 8-h/16-h dark/light cycle. This experiment was repeated three times. After 5 days, similar symptoms to those of diseased leaves in the seedling nursery appeared on leaves inoculated with DHY4-DHY10, while plants inoculated with DHY1-DHY3, DHY11-DHY12, and the controls remained asymptomatic (Supple. Fig.1-D, E). The same fungi were re-isolated from diseased leaves, fulfilling Koch's postulates. The causal agents DHY4 to DHY10, showed similar morphology, which were morphologically identified as Aspergillus sp. (Visagie et al. 2014). Isolate DHY5 was selected for further study. On PDA plates, the colonies were covered with white velutinous mycelia (Supple. Fig.1-F). Conidia were ochre yellow and outwards concentric circles. Vesicles were globose, and about 20.1-26.6 μm in diameter (Supple. Fig.1-G). Conidiophore stipes were smooth walled and hyaline, with conidial heads radiating. The conidia were light yellow to orange, exudate clear to orange droplets. The conidia were (2.53-3.25) μm × (2.58-3.47) μm in diameter (n=50) (Supple. Fig.1-H). For further molecular identification, the ITS and TUB gene sequences were amplified with primer pairs ITS1/ITS4 and BT2a/BT2b (Glass and Donaldson. 1995), respectively. BLASTn searches of the ITS (PP355445) and TUB (PP382788) sequences showed 100% and 98.42% similarity to those of A. westerdijkiae (OP237108 and OP700424), respectively. Phylogenetic analysis based on the concatenated sequences of ITS and TUB confirmed that the fungus was A. westerdijkiae, (Supple. Fig.2). A. westerdijkiae was mainly reported on its secondary metabolite ochratoxin A contamination of agricultural products, fruits, and various food products, such as coffee beans (Alvindia et al 2016), grapes (Díaz et al. 2009), oranges and fruit juice (Marino et al. 2009), etc. To our knowledge, this is the first report of A. westerdijkiae causing leaf blight on R. glutinosa in China.

Honeysuckle flower (Lonicera japonica Thunb.) is a traditional Chinese medicinal plant. It is perennial and widely cultivated in China, Japan and Korea. From late August to October in 2021 and 2022, leaf spots symptoms were observed on L. japonica in different planting fields in Yuzhou, Yuanyang and Fenqiu districts, Henan province, China. The disease incidence was above 85% which reduce photosynthesis. Early disease symptoms appeared as small, circular to elliptical, brown spots on the leaves and later the lesions (1 to 5 mm × 1 to 4 mm) slowly developed yellow haloes. The different brown lesions seldom merge and form larger irregular lesions. Small fragments (3 to 5 mm) of leave tissue were excised from the lesion margins and surface-sterilized in 3% NaClO for 3 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. A total number of 8 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 290 to 700 μm (560 μm on average, n = 20). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3 to 17-septate, and measuring 150 to 240 μm (180 μm on average, n = 20). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). The genomic DNA was extracted using CTAB method. The nuclear ribosomal internal transcribed spacer region (ITS), portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting 537-bp ITS, 226-bp ACT, 410-bp HIS3, and 306-bp TEF1 sequences of isolate JDJ002 were deposited in GenBank (accession nos. OR492367, OR548247, OR548248 and OR548248, respectively). Sequence analysis revealed that ITS, ACT, HIS3 and TEF1α sequences exhibited ≥99% of identity with the ITS (KP896013), ACT(KP895965), HIS3(MK991295) and TEF1 (MN180408) sequences of C. cf. flagellaris, respectively. A pathogenicity test was conducted on healthy of L. japonica leaves. The healthy leaves pricked from L. japonica plants, rinsed in autoclaved distilled water three times and dried with distilled filter paper. Then twelve healthy leave were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of two week-old colony. As negative control, leaves inoculated with PDA medium plugs. Inoculated leaves were covered with plastic bags to maintain high relative humidity and incubated at 25°C in growth chamber. After 7 days, the inoculated leaves showed symptoms identical to those observed in the field under natural conditions, whereas negative control remained symptom-free. Re-isolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was C. cf. flagellaris. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of C. cf. flagellaris except Cercospora rhamni Fack., Alternaria alternata, Corynespora cassiicola or Phomopsis sp. causing leave spots on L. japonica in China.

Plant diseases caused by pathogenic fungi pose a significant threat to agricultural production. This study reports on a strain YBS22 with broad-spectrum antifungal activity that was isolated and identified, and its active metabolites were purified and systematically studied. Based on a whole genome sequence analysis, the new strain YBS22 was identified as Streptomyces melanogenes. Furthermore, eight gene clusters were predicted in YBS22 that are responsible for the synthesis of bioactive secondary metabolites. These clusters have homologous sequences in the MIBiG database with a similarity of 100%. The antifungal effects of YBS22 and its crude extract were evaluated in vivo and vitro. Our findings revealed that treatment with the strain YBS22 and its crude extract significantly reduced the size of necrotic lesions caused by Magnaporthe oryzae on rice leaves. Further analysis led to the isolation and purification of an active compound from the crude extract of the strain YBS22, identified as N-formylantimycin acid methyl ester, an analog of antimycin, characterized by NMR and MS analyses. Consistently, the active compound can significantly inhibit the germination and development of M. oryzae spores in a manner that is both dose- and time-dependent. As a result, we propose that the strain YBS22 could serve as a novel source for the development of biological agents aimed at controlling rice blast disease.

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.

Yam (Dioscorea opposita Thunb.) is cultivated mainly as a functional food and for nutritional and medicinal purposes in China (1). It is propagated through tubers and this facilitates the spread an...

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.

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.