Strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural plants worldwide with high economic and nutritional value. Strawberry associated virus 1 (SaV1) is a putative Cytorhabdovirus isolated from strawberry in Fujian province, China (Ding et al., 2019). Strawberry virus 1 (StrV-1) is another putative Cytorhabdovirus characterized from F. ananassa and F. vesca in Czech Republic (Fránová et al., 2019). The complete genomes of isolates of SaV1 and StrV-1 share 79 to 98% nucleotide (nt) identities. In August 2020, foliar chlorotic spots or streaks were observed in four strawberry cultivars (cv. Honeoye, Mibao, 8128 and All Star) in Yantai, Shandong province, China. To identify the associated viruses, symptomatic leaves from two plants of each cultivar (8 samples) were pooled for high-throughput sequencing (HTS). Total RNA was extracted from the composite sample and used for constructing a cDNA library after ribosomal RNA (rRNA)-depletion. Sequencing was carried out on Illumina Hiseq 4000 (Novogene, China). Raw reads were filtered, trimmed and de novo assembled as described previously (Grabherr et al., 2013; Zhou et al. 2020). The resulting contigs were screened by BLASTn and BLASTx against GenBank database. Subsequent analyses indicated the presence of strawberry vein banding virus, strawberry pallidosis associated virus and strawberry mottle virus in the analyzed sample, which had been reported previously in strawberry (Martin and Tzanetakis, 2013; Shi et al., 2018; Bhagwat et al., 2016). Besides, five contigs ranging from 266 to 6,057 nt were obtained. They shared 87 to 91% nt sequence identity with StrV-1 isolate B (GenBank accession no. MK211271). To confirm StrV-1 infection in the strawberry plants, total RNA was isolated from all eight samples using RNAprep Pure Plant Plus Kit (Tiangen, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers StrVp1 (Forward: 5'-CATTACTGAAGCATTCCGTG-3'/Reverse: 5'-AGATATCACGCACAGTGAC-3'), and StrVp2 (Forward: 5'-TTGCGCGAAGCGGATGTCCG-3'/Reverse: 5'-GGCTGCCAGAGCGTTGGATG-3'), targeting nt positions 70-1,231 and 7,825-9,348 of StrV-1 isolate B, respectively. Fragments with the expected sizes were amplified from two samples of cv. All Star. The amplicons were cloned, sequenced, and deposited in GenBank under accession no. MW419123-124 and MW645247-248. Both protein encoding sequences shared 91 to 92% and 80 to 84% nt identities with the corresponding sequences of StrV-1 isolate B and SaV1, respectively, indicating that the isolates from this study are genetic variants of StrV-1 and distantly related to SaV1. Crude sap was prepared by homogenizing leaf tissues of StrV-1 infected strawberry in 0.02 mol/L sodium phosphate buffer with 0.45% (w/v) sodium diethyldithiocarbamate thihydrate, then gently rubbed onto five healthy Nicotiana benthamiana plants. Neither the inoculated leaves nor the systemically infected leaves showed obvious symptoms seven days post inoculation. However, StrV-1 was detected by RT-PCR in all five N. benthamiana plants as described above. In addition, a survey of strawberry greenhouses was conducted in August 2020 and approximately 10% of plants in a 667 m2 greenhouse in Yantai had StrV-1-like symptoms. To the best of our knowledge, this is the first report of the occurrence of StrV-1 infecting strawberry in Shandong province, China. Our findings expand the geographic range and genetic diversity of StrV-1 and indicate it could be a potential virus threat to strawberry production in China.

Apple mosaic disease is widespread in the major apple-producing areas in China and is frequently associated with the presence of the newly identified Apple necrotic mosaic virus (ApNMV), belonging to subgroup 3 of Ilarvirus genus in the family of Bromoviridae. Mosaic symptoms were also observed in a hawthorn tree. Deep sequencing revealed the hawthorn tree with mosaic symptom was infected by ApNMV, which was confirmed by RT-PCR. The complete nucleotide sequences of RNA1 (3,378 nt), RNA2 (2,778 nt), and RNA3 (1,917 nt) of ApNMV from the hawthorn were obtained, sharing 93.8 to 96.8%, 89.7 to 96.1%, and 89.8 to 94.6% nucleotide identities with those from apples and crabapples, respectively. Two hypervariable regions were found, which showed 59.2 to 85.7% and 64.0 to 89.3% sequence identities at position 142 to 198 aa and at position 780 to 864 aa in the POL protein, respectively, between the hawthorn isolate and other isolates (apple, crabapple). A grafting test demonstrated that ApNMV was easily transmissible from hawthorns to apple trees, with severe chlorosis, yellowing, mosaic, curling, and necrosis. In addition, a total of 11,685 hawthorn trees were surveyed for the incidence of mosaic disease from five provinces in China, and only six were found showing typical mosaic symptoms. A total of 145 individual trees (six symptomatic, 68 asymptomatic, and 71 other symptoms) were tested for the presence or absence of ApNMV by RT-PCR. Among them, six symptomatic, four asymptomatic, and 10 other symptomatic trees tested positive for ApNMV. Taken together, these results demonstrated that the hawthorn tree was identified as a new natural host for ApNMV with a relatively low frequency (13.8%, 20 out of 145) in the main producing areas, and it was likely to be the causal pathogen of hawthorn mosaic disease.

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

Apple (Malus domestica) is an important fruit crop in Ethiopia. Among the viruses reported to infect apple trees, apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem groo...

Apple mosaic disease (AMD) is a widespread viral disease affecting apple-growing regions around the world. Recent studies have identified a novel ilarvirus, apple necrotic mosaic virus (ApNMV), as the major causal agent of AMD in China. However, the molecular mechanisms underlying AMD pathogenesis and the global gene expression changes during mosaic symptom development remain largely unknown. In this study, we performed transcriptome analysis to investigate apple gene responses to AMD. A total of 815 differentially expressed genes (DEGs) were identified in mosaic leaves compared to healthy controls, while 1050 DEGs were found between symptomless leaves (infected with ApNMV) and mosaic leaves. Functional enrichment analysis revealed that these DEGs were predominantly involved in carbohydrate metabolism, oxidation-reduction processes, secondary metabolite biosynthesis, and plant hormone signal transduction. Further biological assays demonstrated that the manifestation of mosaic symptoms in apple leaves was associated with reactive oxygen species (ROS) accumulation and downregulation of ROS-scavenging genes. Collectively, our findings provide new insights into the molecular basis of ApNMV-induced mosaic symptom development in apple and offer potential targets for the management of AMD.

SMoV and SVBV are two major viruses that pose significant threats to the global strawberry industry. Both are latent viruses, making early detection difficult due to their uneven distribution and low concentration in host tissues. Traditional RT-PCR techniques are insufficient for precise and quantitative detection. In this study, TaqMan RT-qPCR detection systems for SMoV and SVBV were established for application in practical production settings, enabling accurate, rapid, and efficient detection of strawberry viruses. When viral accumulation in plants is low, the highly sensitive TaqMan RT-qPCR technique allows for accurate quantification, facilitating the early identification of infected plants and preventing large-scale outbreaks in cultivation areas. The development of a duplex TaqMan RT-qPCR assay enables simultaneous quantification of SMoV and SVBV in a single reaction, improving detection efficiency and providing technical support for risk assessment and effective control of strawberry viral diseases.