SUMMARY A runner, as an elongated branch, develops from the axillary bud (AXB) in the leaf axil and is crucial for the clonal propagation of cultivated strawberry (Fragaria × ananassa Duch.). Runner formation occurs in at least two steps: AXB initiation and AXB outgrowth. HANABA TARANU (HAN ) encodes a GATA transcription factor that affects AXB initiation in Arabidopsis and promotes branching in grass species, but the underlying mechanism is largely unknown. Here, the function of a strawberry HAN homolog FaHAN in runner formation was characterized. FaHAN transcripts can be detected in the leaf axils. Overexpression (OE) of FaHAN increased the number of runners, mainly by enhancing AXB outgrowth, in strawberry. The expression of the strawberry homolog of BRANCHED1 , a key inhibitor of AXB outgrowth in many plant species, was significantly downregulated in the AXBs of FaHAN ‐OE lines, whereas the expression of the strawberry homolog of SHOOT MERISTEMLESS, a marker gene for AXB initiation in Arabidopsis, was upregulated. Moreover, several genes of gibberellin biosynthesis and cytokinin signaling pathways were activated, whereas the auxin response pathway genes were repressed. Further assays indicated that FaHAN could be directly activated by FaNAC2, the overexpression of which in strawberry also increased the number of runners. The silencing of FaNAC2 or FaHAN inhibited AXB initiation and led to a higher proportion of dormant AXBs, confirming their roles in the control of runner formation. Taken together, our results revealed a FaNAC2–FaHAN pathway in the control of runner formation and have provided a means to enhance the vegetative propagation of cultivated strawberry.
Brassica yellows virus (BrYV; genus Polerovirus, family Solemoviridae) has an icosahedral spherical virion with a positive-sense single-stranded RNA genome and it is distinguished from turnip yellows virus (TuYV) based on differences in ORF0 and ORF5 (Xiang et al., 2011). To investigate the occurrence and distribution of viruses infecting strawberry (Fragaria ananassa) in the main production areas in China, a survey of nine greenhouses (667 m2 each) was conducted in the cities of Yantai and Beijing, China in August 2020. About 1% of strawberry plants in each greenhouse showed virus-like symptoms of chlorotic spots; 89 symptomatic leaf samples were randomly collected for virus testing. Total RNA was extracted from a pool of eight samples of four different cultivars (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) from Yantai using RNAprep Pure Plant Plus Kit (TianGen, China). A cDNA library was constructed by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) after ribosomal RNA-depletion using an Epicentre Ribo-Zero™ rRNA Removal Kit (Epicentre, USA). High-throughput sequencing was done on Illumina Hiseq 4000, generating 70,931,850 high-quality 150 bp paired-end reads. Clean reads were de novo assembled by Trinity (v2.2.0) and the resulting contigs were screened by BLASTn and BLASTx against GenBank database as described previously (Grabherr et al., 2013). A total of 1,432,164 high-quality reads unmapped to the strawberry genome were obtained and assembled into 93 contigs (ranging from 33 to 8,031 nt). Seven of these contigs (277 to 1,254 nt) shared 98.2 to 100% nt identities with BrYV-A (accession no. HQ388348) and covered 89.5% of the genome of BrYV-A. Subsequent analyses indicated the presence of Strawberry pallidosis-associated virus and Strawberry mottle virus in the analyzed sample, both have been reported in strawberry in China (Shi et al., 2018; Fan et al., 2021). To confirm BrYV infection, total RNA was isolated from the eight samples used for HTS and reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers (CP and rtp, Supplementary Table 1) designed based on the assembled contigs. PCR products with expected sizes (587 and 609 bp) were observed in one sample (cv. Mibao). BLASTn analysis indicated that the amplicons (accession no. MW548437 and MW548438) shared 98.6% and 99.3% nt identity with BrYV-A, respectively. To obtain the complete sequence of the putative BrYV isolate, the gaps were bridged and the terminal sequences were determined using 5ʹ and 3ʹ RACE kits (Clontech, China) based on the assembled contigs. The complete genome sequence of the putative BrYV isolate has a length of 5,666 nt (accession no. MZ666129) and shares more than 94.3% nt identities with other BrYV isolates. Phylogenetic analysis indicated that the isolate grouped closely with BrYV and further from TuYV (Figure S1). In addition, 11 samples (cv. Benihoppe) of the remaining 81 symptomatic strawberry samples tested positive for BrYV by RT-PCR with the two pairs of primers mentioned above. The sequences (accession no. MZ407232 and MZ407233) revealed 99.5% and 99.3% nt identities with MW548437 and MW548438. To the best of our knowledge, this is the first report of natural infection of BrYV in strawberry plants. Our findings expand the host range of BrYV, but disease association is difficult to establish due to presence of mixed infection and non-fulfillment of Koch's postulates.
Strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural plants worldwide with high economic and nutritional value. Strawberry virus 1 (StrV-1), closely related to the genus Cytorhabdovirus, was first reported from F. ananassa (cv. Čačanská raná) and F. vesca (cv. Rujana) in Czech Republic (Fránová et al., 2019). To identify viruses in four strawberry cultivars (cv. Honeoye, Mibao, 8128 and All Star), leaves showing virus-like symptoms (foliar chlorotic spots or streaks) were collected from Yantai in Shandong province, China in August 2020. 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 nucleotides (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 eight samples (used for HTS) 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: 5ʹ-CATTACTGAAGCATTCCGTG-3′/Reverse: 5ʹ-AGATATCACGCACAGTGAC-3ʹ), and StrVp2 (Forward: 5ʹ-TTGCGCGAAGCGGATGTCCG-3′/Reverse: 5ʹ-GGCTGCCAGAGCGTTGGATG-3ʹ), targeting nt positions 70-1,231 and 7,825-9,348 of StrV-1 isolate B, respectively. Fragments with expected sizes of 1,162 bp and 1,524 bp were amplified from two samples of cv. All Star. The amplicons were cloned, sequenced, and deposited into GenBank database under accession no. MW419123, MW419124, MW645247, and MW645248. The StrV-1 sequences from All Star shared 91% to 92% nt identity with the corresponding sequences of StrV-1 isolate B. Crude sap was prepared by homogenizing the StrV-1 infected strawberry leaf tissues in 0.02 mol/L sodium phosphate buffer with 0.45% (w/v) sodium diethyldithiocarbamate thihydrate, then gently rubbed onto five healthy N. 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 using RNA templates prepared from non-inoculated leaves. In addition, a survey of strawberry greenhouses was conducted in August 2020 and approximately 10% of plants in one 667 m2 greenhouse in Yantai had StrV-1 like symptoms. To the best of our knowledge, this is the first report of StrV-1 infecting strawberry in China. Our findings expand the geographic range of StrV-1 and indicate it could be a potential virus threat to strawberry production in China.
Strawberry mottle virus (SMoV) and strawberry vein banding virus (SVBV) cause diseases on strawberry plants, but the effect of coinfection of SMoV and SVBV on the growth, development, and defense system of strawberry (Fragaria × ananassa Duchesne) remains unknown. We investigated the effect of SMoV and SVBV coinfection on strawberry cultivar 'Benihope'. The results showed that stem diameter, leaf size, leaf number, relative chlorophyll content, total chlorophyll content, photosynthetic parameters, and stomatal aperture of SMoV and SVBV co-infected strawberry (VIS) plants were in a weaker level than uninfected control plants, indicating that viruses inhibited the growth and photosynthesis of strawberry plants. Furthermore, the initiation of flowering and fruiting stages of VIS plants were delayed by about three weeks compared with the controls, and the fruiting period was shortened, demonstrating that the reproduction of VIS plants was inhibited. Fruit quality was damaged in VIS plants due to a significant increase in fruit firmness and titratable acidity and decrease in total soluble solid content than control fruits. More dead cells and H 2 O 2 accumulated along the veins of VIS leaves, and the content of abscisic acid and catalase activity significantly increased, whereas anthocyanin content was lower than that of control plants. The results demonstrate that SVBV and SMoV coinfection inhibits the growth and development of 'Benihope' strawberry plants, and the plants respond to viruses by regulating stomatal aperture, the accumulation of ABA and antioxidants.To our knowledge, this study contributes information to understand how both viruses impair the strawberry growth and development for the first time.
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