Achieving precise dual detection: One-tube reverse transcription-recombinase aided amplification (RT-RAA) combined with lateral flow strip (LFS) assay for RNA and DNA target genes from pepper mild mottle virus and Colletotrichum species in crude plant samples.

Ensuring the detection sensitivity of both RNA-derived and DNA-derived target genes in a single reaction has posed a significant challenge for on-site detection of plant pathogens. This challenge was addressed by developing a one-tube dual RT-RAA assay combined with LFS for the rapid on-site detection of pepper mild mottle virus (PMMoV) and four Colletotrichum species causing anthracnose in Solanaceous crops. By testing four different combinations of primer groups, two combinations were precisely adjusted within the dual RT-RAA system to balance amplification efficiency and maintain consistent levels of amplification in crude plant samples. Utilizing commercially accessible small-scale equipment and following a streamlined optimization strategy, the assay achieved a limit of detection of 0.32 copies/µL of target genes in the reaction. Importantly, it demonstrated no cross-reactivity with other plant pathogens, thereby affirming the high sensitivity and specificity of the developed dual RT-RAA-LFS detection assay. Moreover, the entire process took only 25 min from sample collection to the visible presentation of results. The assay was validated with 60 field samples and 10 seed samples, producing results consistent with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Notably, it successfully detected PMMoV in systemic leaves without visible symptoms three days post-inoculation, underscoring its effectiveness in early disease detection. This streamlined strategy offers a valuable approach for rapid, low-cost, and highly sensitive on-site simultaneous detection of RNA genome-contained PMMoV and DNA genome-contained Colletotrichum species.

First report of pepper chlorosis-associated virus infecting tobacco (Nicotiana tabacum) plants in Sichuan Province in China.

Tobacco (Nicotiana tabacum) is an economically important crop in China, and more than 30 viruses have been reported to infect tobacco (Yin et al. 2022). In July 2022, we observed interveinal necrosis on tobacco leaves in fields in Sichuan Province (N 27.9172, E 105.6662) (Fig. 1). Total RNA was isolated from multiple leaves of one plant using an RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). Total RNAs were pooled, and a TruSeq Stranded Total RNA with RiboZero Gold Kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA. An RNA-Seq library was constructed using VAHTS Universal V6 RNA-seq Library Prep (Nanjing Vazyme, China). High-throughput sequencing was performed on the Illumina DNBseq platform (BGI-ShenZhen, China), which yielded 20,102,087 reads with an average length of 150 nt (total size >6 Gb). Unaligned reads were assembled de novo using SPAdes (Bankevich et al. 2012). Contigs with length ≥200 nt were subjected to local BLASTn and BLASTx analyses against the GenBank nt and nr databases, respectively (Wang et al. 2022). A total of 23 contigs were identified through BLASTx (e-value cut-off = 10 -3), ranging from 631 to 1555 bp long, with 82% to 96% coverage to partial genomic sequences of pepper chlorosis-associated virus (PepCaV-Higashitsuno_2021; Accessions: LC719619 to LC719621) and one contig (6459 bp) with 99% similarity to tobacco mosaic virus (Accession: OP525281) isolate DSMZ PV-0109 from Germany. The complete genome sequence of PepCaV was obtained using primers based on the assembled contigs. The 5'- and 3'-terminal regions of the RNA genome were obtained by 5'- and 3'-rapid amplification of cDNA ends. These amplicons were cloned using the pEASY-Blunt Zero Cloning Kit (TRANSGEN, Nangjing, China) and sequenced by Sanger sequencing. Complete genome sequences of tripartite PepCaV from tobacco samples were 7697, 1808, and 1557 nucleotides long (Accession: OR451987 to OR451989) and showed genome organization typical of the genus Ophiovirus in the family Aspiviridae. The complete sequences of RNA1, RNA2 and RNA3 genome segments shared 92.36%, 90.43%, and 95.24%, nucleotide sequence identities, respectively, with the isolate PepCaV-Higashitsuno_2021 pepper isolate (Accession: LC719619 to LC719621) (Shimomoto et al. 2023), but PepCaV has not been reported to infect N. tabacum. In June 2023, 10 plants collected from each place of Macheng (N 27.9094, E 105.6740), Xiangyang (N 28.0936, E 105.6249) and Moni (N 27.8899, E 105.5936) showing interveinal necrosis symptoms were tested using RT-PCR using PepCaV-MP610-F (5'-TGTTCTCTGCTATGCGGTTG -3') and PepCaV-MP610-R (5'-AGCAATCTCGCACCTGAAGT-3') to product 610bp amplicon. Twenty-five tobacco plants were positive for PepCaV. Single sequence from each location was submitted to GenBank (Accession: PP728631 to PP728633). Sap extracts from the original field leaf samples collected from Sichuan Province were used to mechanically inoculate tobacco plants (10 plants) at the four-leaf stage. After 7 days, leaf samples were tested using RT-PCR assay specific to PepCaV and TMV while samples were positive only for TMV but failed to transmit PepCaV by mechanical inoculation. According to previous literature, ophioviruses may be transmitted though soilborne fungus (Jeong et al. 2014). Further research is needed to understand the transmission, epidemiology, and pathological properties of the PepCaV. To our knowledge, this is the first report documenting natural PepCaV infection of tobacco plants in China, providing a scientific basis for PepCaV infection control in tobacco plantations.

The nanovirus U2 protein suppresses RNA silencing via three conserved cysteine residues.

Nanoviruses have multipartite, circular, single-stranded DNA genomes and cause huge production losses in legumes and other crops. No viral suppressor of RNA silencing (VSR) has yet been reported from a member of the genus Nanovirus. Here, we demonstrate that the nanovirus U2 protein is a VSR. The U2 protein of milk vetch dwarf virus (MDV) suppressed the silencing of the green fluorescent protein (GFP) gene induced by single-stranded and double-stranded RNA, and the systemic spread of the GFP silencing signal. An electrophoretic mobility shift assay showed that the U2 protein was able to bind double-stranded 21-nucleotide small interfering RNA (siRNA). The cysteine residues at positions 43, 79 and 82 in the MDV U2 protein are critical to its nuclear localization, self-interaction and siRNA-binding ability, and were essential for its VSR activity. In addition, expression of the U2 protein via a potato virus X vector induced more severe necrosis symptoms in Nicotiana benthamiana leaves. The U2 proteins of other nanoviruses also acted as VSRs, and the three conserved cysteine residues were indispensable for their VSR activity.

Chloroplast clustering around the nucleus induced by OMP24 overexpression unexpectedly promoted PSTVd infection in Nicotiana benthamiana.

Chloroplast clustering around the nucleus is a well-known mechanism that occurs in response to various biotic and abiotic stresses and is believed to be a mechanism of defence against pathogens in plants. This phenomenon is accompanied by increased production of reactive oxygen species (ROS), which can help to destroy invading pathogens. However, the function of chloroplast clustering during viroid infection is unclear. Here, we report that, although the infection by potato spindle tuber viroid (PSTVd) failed to induce chloroplast clustering, chloroplast clustering caused by the overexpression of the Nicotiana benthamiana chloroplast outer membrane protein 24 (NbOMP24) promoted the infection by PSTVd, a viroid pathogen, in N. benthamiana. Interestingly, H2 O2 treatment, which caused increased ROS accumulation, showed no significant effects on PSTVd infection. Moreover, NbOMP24 protein showed no direct interaction with PSTVd. We propose that perinuclear chloroplast clustering induced by NbOMP24 provides a favourable environment for PSTVd infection. These findings highlight the complexity of chloroplast clustering-mediated plant-pathogen interactions and the need for further research to fully understand these mechanisms.

Identification and complete genome sequence of iris potyvirus A, which causes dwarfing and foliar chlorosis with mosaic or mottle disease symptoms on lily (Lilium lancifolium Thunb.) in China.

Lily plants (Lilium lancifolium Thunb.) exhibiting dwarfing and foliar chlorosis with mosaic or mottle disease symptoms were found in Anhui Province, China. We used high-throughput sequencing of small RNA to survey the virus in the lily cultivation region of Anhui Province. Here, we report the identification and complete genome sequence of the viral agent. It contains 9733 nucleotides, excluding the poly(A) tail, and encodes a polyprotein of 3063 amino acids. The complete polyprotein ORF shows 98.92% amino acid sequence identity with that of iris potyvirus A (GenBank MH898493). Phylogenetic analysis of coat protein sequences placed the viral agent close to members of the genus Potyvirus in the family Potyviridae, and it was therefore provisionally named iris potyvirus A isolate Anhui (IrPVA-Anhui). This is the first complete genome sequence of IrPVA-Anhui from lily plant, for which only a partial sequence from Iris domestica has been reported previously. Comparative analysis of this genome sequence with those of closely related potyviruses identified nine cleavage sites and the conserved motifs typical of potyviruses. Subsequent virus identification was performed using serological assays (ELISA and antibody-based lateral flow assays), molecular methods (RT-PCR), and a pathogenicity test. Virus particles with a length of about 700 nm, similar to viruses in the genus Potyvirus, were observed via transmission electron microscope (TEM). We back-inoculated healthy plants of multiple species to investigate the host range of the virus. It infected the original host, Iris domestica, and Nicotiana benthamiana but not Triticum aestivum, Pisum sativum, Chenopodium amaranticolor, or Datura stramonium. This is the first report of natural IrPVA-Anhui infection of lily plants in China, providing a scientific basis for IrPVA-Anhui control in future lily plantings.

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants.

Pepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) of C. annuum was upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing of OMP24 in either C. annuum or Nicotiana benthamiana facilitated PMMoV infection, whereas overexpression of N. benthamiana OMP24 in transgenic plants inhibited PMMoV infection. Both C. annuum OMP24 (CaOMP24) and N. benthamiana OMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression of CaOMP24 induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression of PR1 and PR2 was also upregulated significantly in plants overexpressing OMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.

Inactivating Activities and Mechanism of Imidazo[1,2-c]pyrimidin-5(6H)-one Nucleoside Derivatives Incorporating a Sulfonamide Scaffold.

Twenty-eight imidazo[1,2-c]pyrimidin-5(6H)-one nucleoside derivatives incorporating a sulfonamide scaffold with preferable inactivating activities on pepper mild mottle virus (PMMoV) were designed and synthesized. Then, compound B29 with illustrious inactivating activity against PMMoV was received on the basis of the three-dimensional quantitative structure-activity relationship (3D-QSAR) model, with the EC50 of 11.4 µg/mL, which was superior to ningnanmycin (65.8 µg/mL) and template molecule B16 (15.3 µg/mL). Furthermore, (1) transmission electron microscopy (TEM) indicated that B29 could cause severe fracture of virions; (2) microscale thermophoresis (MST) and molecular docking further demonstrated that B29 had faintish binding affinities with PMMoV CPR62A (Kd = 202.84 µM), PMMoV CPL144A (Kd = 141.57 µM), and PMMoV CPR62A,L144A (Kd = 332.06 µM) compared to PMMoV CP (Kd = 4.76 µM); and (3) western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results of pCB-GFP-PMMoV CPR62A, pCB-GFP-PMMoV CPL144A, and pCB-GFP-PMMoV CPR62A,L144A were consistent with MST and confocal. In brief, the above results indicated that the amino acids at positions 62 and 144 of PMMoV CP might be the key amino acid sites of B29 acted on.

Complete genome sequence of triticum yellow stripe virus, a new polerovirus infecting wheat (triticum aestivum) in China.

Wheat plants with yellow stripes on their leaves were collected in the city of Tai'an (Shandong province, China). High-throughput sequencing analysis of the collected plants showed that they were coinfected with wheat leaf yellowing-associated virus (WLYaV) and an unidentified polerovirus. The genome of the unidentified virus, tentatively named "triticum yellow stripe virus" (TriYSV), comprises 5,595 nucleotides and contains seven open reading frames (ORFs), with a typical polerovirus genome structure. Analysis by sequence alignment showed that TriYSV had the highest sequence similarity to wheat yellow dwarf virus (WYDV, a tentative member of the genus Polerovirus), with 87.3% nucleotide sequence identity over the whole genome. Except for P3a and the coat protein (CP), all of the proteins encoded by TriYSV showed < 90% amino acid identity to those of other poleroviruses. Phylogenetic analysis based on RNA-dependent RNA polymerase and CP amino acid sequences and complete genome nucleotide sequences showed that the poleroviruses WYDV, cereal yellow dwarf virus RPS (CYDV-RPS), CYDV-RPV, and barley yellow dwarf virus GPV are the most closely related to TriYSV. Thus, TriYSV is proposed to be a new member of the genus Polerovirus.

First detection of coccidia species in blue peafowl (Pavo cristatus) in China via both microscopic and molecular methods.

Coccidia are protozoan parasites in the class Conoidasida. To determine the prevalence and molecular characteristics of coccidia species in blue peafowl (Pavo cristatus) in Henan, China, 240 fecal specimens were collected and screened for the presence of Eimeria spp. and Isospora spp. The overall prevalence was 65.0% (156/240), and seven different coccidia species were identified: E. pavonis (51.3%, 123/240), E. arabic (40.0%, 96/240), E. riyadhae (37.1%, 89/240), E. mandali (22.9%, 55/240), E. mayurai (14.2%, 34/240), I. mayuir (10.9%, 26/240), and I. lacazei (8.5%, 21/240). E. arabic and E. riyadhae were detected for the first time in China. Additionally, we provide molecular data of the seven different coccidia species at the 18S and 28S ribosomal RNA and the COI loci. Sequence homology percentages among the five species of Eimeria at the 18S rRNA, 28S rRNA, and COI loci were 96.0%-98.6%, 90.7%-98.2%, and 85.0%-94.9%, respectively, whereas for two species of Isospora the sequence homology percentages were 98.8%, 99.1%, and 95.4% at three corresponding loci. This is the first report of the molecular data of the seven coccidia species in blue peafowl in China.

First report of bean common mosaic virus naturally infecting yam bean (Pachyrhizus erosus) in China.

Yam bean (Pachyrhizus erosus), a high-yielding leguminous root crop with good nutritional value, is widely cultivated in southern China. In 2020, P. erosus (cv. Mumashan) plants exhibiting irregular yellow leaves and malformed seed pods (Supplementary Fig S1) were observed at Ningbo city, Zhejiang Province, China. To determine the causal agent(s) of the disease, symptomatic leaves (n=4) were collected for electron microscopy negative staining. Virus particles with a length of about 700nm, similar to viruses in the genus Potyvirus, were observed via transmission electron microscope (TEM), suggesting the presence a potyvirus(es). To further confirm which potyvirus(es) infected yam bean, total RNA was extracted from leaf samples of a total of six plants, including four symptomatic plants and two asymptomatic plants using TRIzol reagent (Invitrogen Carlsbad, CA, USA) according to the manufacturer's instructions. RNA was reverse-transcribed into cDNA with M4-T as the 3'-anchoring primer by ReverTra Ace® kit (Toyobo, Japan). Sprimer/M4 Potyviridae specific primers (Chen et al., 2001) were used for PCR analysis. A ~1,700-bp-long product was amplified from four symptomatic plants using KOD FX enzyme (Toyobo, Japan). No such band was amplified from the two asymptomatic plants. The PCR product (~1.7kb) amplified from a single symptomatic plant was ligated into the pEASY®-Blunt Zero vector (TransGen Bio, Beijing, China) and sequenced (Sangon Bio, Shanghai, China). The amplicon showed 99% nucleotide sequence identities with bean common mosaic virus (BCMV) isolate NKY021 (KJ807819). Subsequently, the complete nucleotide sequences of this BCMV isolate (referred as BCMV-NB) was amplified by overlapping RT-PCR and rapid amplification of cDNA ends with primers (Supplementary Table S1) designed from the sequence of BCMV isolate NKY021. The BCMV-NB full genome (Accession No. OL871237) consists of 10,053 nucleotides excluding the poly(A) tail and contains a large open reading frame encoding a polyprotein of 3222 amino acids. BLASTn analysis showed that BCMV-NB shared a sequence identity of 96.4% with BCMV isolate HZZB011 (KJ807815). Phylogenetic tree generated by Neighbour-Joining method revealing the BCMV-NB isolate was grouped together with Chinese isolates from Glycine max (Supplementary Fig S1). To test the infectivity of BCMV-NB, virus-free yam bean (cv. Mumashan) and Nicotiana benthamiana seedlings were mechanically inoculated with sap extracted from the symptomatic leaves of a BCMV-NB-infected yam bean plant. The inoculated yam bean plants developed typical BCMV mosaic and chlorotic symptoms at 16 days post inoculation (dpi), while Nicotiana benthamiana had no obvious symptoms at 10 or 20 dpi (Supplementary Fig S1). BCMV infections were confirmed in yam bean plants (infection rate 6/6) and N. benthamiana plants (infection rate 8/8) by RT-PCR at 16 dpi and 10 dpi, respectively. Twelve further P. erosus plants (cv. Mumashan) were collected from a field in Ningbo city and tested by RT-PCR with BCMV-specific primer pair BCMV CP (+)/(-) (Supplementary Table 1). Eight out of the 12 samples tested positive for BCMV by PCR-gel electrophoresis (Supplementary Fig S1) and Sanger sequencing, suggesting a high incidence of BCMV infection in this field. BCMV infection in yam bean has been reported from Indonesia (Damayanti et al., 2008) and Peru (Fuentes et al., 2012). To the best of our knowledge, this is the first report of BCMV naturally infecting yam bean in China. Thus, special attention and appropriate management strategies are needed to minimize the damage caused by BCMV to yam bean crops in China.

Turnip mosaic virus P1 suppresses JA biosynthesis by degrading cpSRP54 that delivers AOCs onto the thylakoid membrane to facilitate viral infection.

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mottle virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.

Complete genome sequence of a putative novel ilarvirus isolated from Eleocharis dulcis.

The complete genomic sequence of a novel ilarvirus from Eleocharis dulcis, tentatively named "water chestnut virus A" (WCVA), was determined using next-generation sequencing (NGS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The three genomic RNA components of WCVA were 3578 (RNA1), 2873 (RNA2), and 2073 (RNA3) nucleotides long, with four predicted open reading frames containing conserved domains and motifs typical of ilarviruses. Phylogenetic analysis of each predicted protein consistently placed WCVA in subgroup 4 of the genus Ilarvirus, together with prune dwarf virus, viola white distortion associated virus, Fragaria chiloensis latent virus, and potato yellowing virus. The genetic distances and lack of serological reaction to antisera against other ilarviruses suggest that WCVA is a novel member of the genus.

Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China.

BACKGROUND: Captive wild animals in zoos infected with Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. can be sources of zoonotic infections and diseases. Therefore, to investigate the distribution of these pathogens in captive wild animals of zoos in Henan, China, a total of 429 fresh fecal samples were collected from six zoos in Henan, China. The infection rates of Cryptosporidium spp., G. duodenalis, E. bieneusi, and Blastocystis sp. were determined by PCR analysis of corresponding loci. Positive results for Cryptosporidium (C. parvum and C. hominis) were subtyped based on the (gp60) gene. RESULTS: The overall prevalence was 43.1% (185/429), and the prevalence of Cryptosporidium, Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. were 2.8% (12/429), 0.5% (2/429), 20.8% (89/429), and 19.1% (82/429), respectively. Five Cryptosporidium species, namely, C. hominis, C. parvum, C. muris, C. andersoni, and C. macropodum, were identified in this study. Cryptosporidium parvum was further subtyped as IIdA19G1. Two Giardia duodenalis assemblages (A and E) were also identified. A total of 20 Enterocytozoon bieneusi genotypes were detected, including 18 known (BEB6, D, HND-1, CD7, SDD1, Henan-IV, KIN-1, CHK1, Peru8, Henan-V, CHG11, CHG-1, CHS9, CHG21, Type-IV, CHC9, CM5, and CHB1) and 2 novel genotypes (CHWD1 and CHPM1). A total of nine subtypes of Blastocystis sp. (ST1, ST2, ST3, ST5, ST6, ST7, ST10, ST13, and ST14) were identified in captive wild animals in zoos in the present study. Cryptosporidium andersoni, nine Enterocytozoon bieneusi genotypes, and five Blastocystis subtypes were here first identified in new hosts. CONCLUSIONS: Our study has expanded the host ranges of these four pathogens. The data indicate that animals in zoos can commonly be infected with these four zoonotic pathogens, and animals in zoos are potential sources of zoonotic infections in humans.

Genome-Wide Identification Reveals That Nicotiana benthamiana Hypersensitive Response (HR)-Like Lesion Inducing Protein 4 (NbHRLI4) Mediates Cell Death and Salicylic Acid-Dependent Defense Responses to Turnip Mosaic Virus.

Hypersensitive response (HR)-like cell death is an important mechanism that mediates the plant response to pathogens. In our previous study, we reported that NbHIR3s regulate HR-like cell death and basal immunity. However, the host genes involved in HR have rarely been studied. Here, we used transcriptome sequencing to identify Niben101Scf02063g02012.1, an HR-like lesion inducing protein (HRLI) in Nicotiana benthamiana that was significantly reduced by turnip mosaic virus (TuMV). HRLIs are uncharacterized proteins which may regulate the HR process. We identified all six HRLIs in N. benthamiana and functionally analyzed Niben101Scf02063g02012.1, named NbHRLI4, in response to TuMV. Silencing of NbHRLI4 increased TuMV accumulation, while overexpression of NbHRLI4 conferred resistance to TuMV. Transient overexpression of NbHRLI4 caused cell death with an increase in the expression of salicylic acid (SA) pathway genes but led to less cell death level and weaker immunity in plants expressing NahG. Thus, we have characterized NbHRLI4 as an inducer of cell death and an antiviral regulator of TuMV infection in a SA-mediated manner.

Ferredoxin 1 is downregulated by the accumulation of abscisic acid in an ABI5-dependent manner to facilitate rice stripe virus infection in Nicotiana benthamiana and rice.

Ferredoxin 1 (FD1) accepts and distributes electrons in the electron transfer chain of plants. Its expression is universally downregulated by viruses and its roles in plant immunity have been brought into focus over the past decade. However, the mechanism by which viruses regulate FD1 remains to be defined. In a previous report, we found that the expression of Nicotiana benthamiana FD1 (NbFD1) was downregulated following infection with potato virus X (PVX) and that NbFD1 regulates callose deposition at plasmodesmata to play a role in defense against PVX infection. We now report that NbFD1 is downregulated by rice stripe virus (RSV) infection and that silencing of NbFD1 also facilitates RSV infection, while viral infection was inhibited in a transgenic line overexpressing NbFD1, indicating that NbFD1 also functions in defense against RSV infection. Next, a RSV-derived small interfering RNA was identified that contributes to the downregulation of FD1 transcripts. Further analysis showed that the abscisic acid (ABA) which accumulates in RSV-infected plants also represses NbFD1 transcription. It does this by stimulating expression of ABA insensitive 5 (ABI5), which binds the ABA response element motifs in the NbFD1 promoter, resulting in negative regulation. Regulation of FD1 by ABA was also confirmed in RSV-infected plants of the natural host rice. The results therefore suggest a mechanism by which virus regulates chloroplast-related genes to suppress their defense roles.

Gynura japonica: A new host of Apple stem grooving virus and Chrysanthemum virus B in China.

Gynura japonica (Thunb.) Juel [Asteraceae; syn: G. segetum (Lour.) Merr] is an important perennial medicinal herb used in China for topical treatment of trauma injuries (Lin et al. 2003). It grows naturally in the southern provinces of China and is also sometimes cultivated. During 2018-2020, wild G. japonica plants exhibiting chlorotic spots and mosaic symptoms were observed in Zhejiang province, China. To identify the possible causal agents of the disease, a single symptomatic leaf sample was collected in August 2019 and sent to Zhejiang Academy of Agricultural Sciences (Hangzhou, China) for next generation sequencing (NGS). Total RNAs extracted with TRIzol (Invitrogen, Carlsbad, USA) were subjected to high throughput sequencing on the Illumina NovaSeq 6000 platform with PE150bp and data analysis was performed by CLC Genomic Workbench 11 with default parameters (QIAGEN, Hilden, Germany). A total of 37,314,080 paired-end reads were obtained, and 11,785 contigs (961 to 10,964 bp) were generated and compared with sequences in GenBank using BLASTn or BLASTx. Of the total of 12 viral-related contigs obtained, one with a length of 6,442 nt mapped to the genomic RNA of ASGV (MN495979), seven contigs with lengths ranging from 1,034 to 2,901 nt mapped to Chrysanthemum virus B (CVB), and four mapped to broad bean wilt virus 2 (BBWV2), a virus which is known to infect G. procumbens (Kwak et al. 2017). To further confirm the presence of ASGV and CVB, primers were designed and the complete nucleotide sequences of both viruses were amplified from the original NGS sample using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) according to the manufacturer's instructions (Tiosbio, Beijing, China). BLASTn analysis revealed that the complete 6,451 nt sequence of ASGV (GenBank accession No. MW259059) shared the highest identity (81.2%) with a Chinese isolate of ASGV from citrus (MN495979). The two isolates grouped with another Chinese isolate (from pear) in phylogenetic analysis. The predicted coat protein of the virus had the highest nt identity of 93.7% (96.2% amino acid sequence identity) with that of the Chinese ASGV isolate XY from apple (KX686100). The complete genomes of two distinct molecular variants of CVB (both 8,987 nt in length) were also obtained from this sample (GenBank accession Nos. MW269552, MW269553). They shared 86.8% nt identity with each other and had 81.1% and 82.1% identity to the only known complete sequence of CVB from chrysanthemum (AB245142). Ten additional wild G. japonica plants with mosaic symptoms were collected randomly during 2019-2020 from Hangzhou (n=6) and Ningbo (n=4) in Zhejiang province and tested by RT-PCR with specific primer pairs to detect BBWV2, ASGV and CVB. RT-PCR and subsequent sequencing revealed that these three viruses were present in all the samples tested, indicating that co-infection of G. japonica by ASGV, CVB and BBWV2 is common. CVB mainly infects chrysanthemum (Singh et al. 2012), while ASGV is known as a pathogen of various fruit trees especially in the family Rosaceae, although there are recent reports that it can also infect some plants in Gramineae, Asparagaceae and Nelumbonaceae (Bhardwaj et al. 2017; Chen et al. 2019; He et al. 2019). Our results provide the first report that Gynura is a natural host of CVB and ASGV. Further surveys and biological studies are underway to evaluate the importance of Gynura as a virus reservoir for epidemics among the various hosts.

Complete genome sequence of a new achyranthes virus A isolate from Achyranthes bidentata in China.

We have determined the complete genomic sequence of a potyvirus from Achyranthes bidentata in Zhejiang, China, using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The genomic RNA is 9482 nucleotides (nt) long excluding the 3'-terminal poly(A) tail and encodes a putative large polyprotein with 3073 amino acids (aa). It has 75.4-53.5% nt sequence identity and 84.0-49.1% polyprotein sequence identity to other potyviruses and is probably a distantly related isolate of the same species as the recently reported achyranthes virus A isolate from South Korea (AcVA-SK). This is the first report of the occurrence of a potyvirus infecting A. bidentata in China.

Morphological and molecular characterization of Cystoisospora yuensis n. sp. and Cystoisospora rastegaievae (Protozoa: Eimeriidae) in amur hedgehogs, Erinaceus amurensis (Schrenk, 1859).

Twenty-four fecal samples were collected from captive amur hedgehogs (Erinaceus amurensis) in Zhengzhou, China. Based on morphological and molecular analysis, the overall prevalence of Cystoisospora was 62.5% (15/24). These samples contained two types of coccidian oocysts, including C. rastegaievae (50.0%, 12/24) and a new species named C. yuensis n. sp. (12.5%, 3/24). Sporulated oocysts (n = 30) of C. yuensis n. sp. are ovoid, (20.6 ± 1.4) µm × (20.9 ± 0.9) µm, with a shape index (length/width) of 1.0 and a smooth and bi-layered oocyst wall, 1.3 µm thick (outer layer 0.8 µm, inner 0.5 µm). A polar granule is present, but micropyle cap, micropyle, and oocyst residuum are absent. The sporocysts are ovoid-shaped, (9.3 ± 0.6) µm × (8.5 ± 1.1) µm, with a shape index (length/width) of 1.1. Stieda, substieda bodies, and refractile bodies are absent. Residuum is scattered and distributed around the entire sporocysts. At the 18S rRNA locus, C. yuensis n. sp. exhibited the highest identity to C. timoni (99.3%) from a slender-tailed meerkat. It has 98.0% identity at the 28S rRNA locus and 99.3% at the ITS locus. Based on morphological and molecular data, this isolate is a new species of Cystoisospora. Additionally, we have provided data on the prevalence of C. rastegaievae in China and sequences of the 18S rRNS, 28S rRNA, and ITS loci.

Genetic characteristics of Giardia duodenalis from sheep in Inner Mongolia, China.

Giardia duodenalis is an important zoonotic pathogen for both human and animal health. Although there have been reports on G. duodenalis infections in animals all over the world, information regarding the prevalence and genetic characteristics of G. duodenalis in sheep in Inner Mongolia, China, is limited. In this study, 209 sheep fecal specimens were collected in this autonomous region. We established that the prevalence of G. duodenalis was 64.11% (134/209), as determined using nested PCR detection and sequences analysis of the small subunit ribosomal RNA (SSU rRNA) gene. Based on the beta-giardin (bg) locus, the glutamate dehydrogenase (gdh) locus, and the triose phosphate isomerase (tpi) locus to study genetic characteristics, both assemblages A (2.99%, 4/134) and E (97.01%, 130/134) were found. Five novel nucleotide sequence of assemblage E were detected, two at the bg locus, two at the gdh locus, and one at the tpi locus. Multilocus genotyping yielded four assemblage E and two assemblage A multilocus genotypes (MLGs), including four novel assemblage E MLGs and one novel assemblage A MLG. Results of this study indicated that G. duodenalis was highly prevalent in sheep in Inner Mongolia. This study is the first to use the multilocus genotyping approach to identify G. duodenalis in sheep from this region.

TITLE: Caractéristiques génétiques de Giardia duodenalis chez les moutons en Mongolie intérieure, Chine. ABSTRACT: Giardia duodenalis est un pathogène zoonotique important pour la santé humaine et animale. Bien qu'il y ait eu des rapports sur des infections à G. duodenalis chez les animaux partout dans le monde, les informations concernant la prévalence et les caractéristiques génétiques de G. duodenalis chez les ovins en Mongolie intérieure, Chine, sont limitées. Dans cette étude, 209 spécimens fécaux de moutons ont été collectés dans cette région autonome. Nous avons établi que la prévalence de G. duodenalis était de 64,11 % (134/209), grâce à la détection par PCR imbriquée et l'analyse des séquences de la petite sous-unité du gène de l'ARN ribosomal (ARNr SSU). Sur la base du locus bêta-giardine (bg), du locus glutamate déshydrogénase (gdh) et du locus triose phosphate isomérase (tpi) pour l'étude des caractéristiques génétiques, les deux assemblages A (2,99 %, 4/134) et E (97,01 %, 130/134) ont été trouvés. Cinq nouvelles séquences nucléotidiques de l'assemblage E ont été détectées, deux au locus bg, deux au locus gdh et une au locus tpi. Le génotypage multilocus a produit quatre génotypes multilocus (GML) pour l'assemblage E et deux GML pour l'assemblage A, y compris quatre nouveaux GML pour E et un nouveau GML pour A. Les résultats de cette étude indiquent que G. duodenalis était très répandu chez les moutons en Mongolie intérieure. Cette étude est la première à utiliser l'approche de génotypage multilocus pour identifier G. duodenalis chez les moutons de cette région.