Strawberry In China Research Articles

First Report of Leaf Spot Caused by Botrytis cinerea on Strawberry in China.

In the winter of 2022, circular or irregular leaf spots were observed on strawberry (Fragaria × ananassa) planted in commercial fields (cultivar 'xuetu', 'mengzhifu') in Yinzhou, Ningbo, Zhejiang, China (N29°48'48″, E121°39'47″), with disease incidence ranging from 10 to 15% in a field approximately 0.67 ha in size. The estimated crop loss associated with this disease was ~10%. Symptoms included circular or irregular lesions with brown halos and wheel marks, which eventually developed into leaf blight and petiole decay, but spore masses were seldom found on the leaf surface. In severe cases, leaves withered and abscissed. To isolate the causal agent, ten diseased leaves from ten different plants were collected, surface-sterilized with 75% ethanol for 50 s, rinsed twice with sterile distilled water, cut into small pieces (0.5 cm × 0.5 cm), and plated on potato dextrose agar (PDA), then incubated at 25°C in darkness for 5 days. Isolates , which displayed one kind of colony morphology were consistently obtained from each of the ten samples, and 58 single-conidium isolates with the same colony morphology were obtained. The isolation frequency was 58 of 60 samples. The colonies that grew on PDA produced white mycelia, which sporulated after 1 week, producing typical Botrytis-like gray spores. Three isolates (NBCM-1, NBCM-2, NBCM-3) were selected for identification and pathogenicity assays. Conidia were round to ellipsoid, 9.2 to 14.3 μm long (n=50), and 6.4 to 9.2 μm wide (n=50). Sclerotia were not observed on PDA. Based on these characteristics, the pathogen was tentatively identified as Botrytis cinerea (Zhang 2001). PCR was conducted for each of the three isolates to amplify the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, which are typically used for molecular identification of Botrytis species (Staats et al. 2005; Liu et al. 2016). The resulting amplicons were sequenced, and the sequences were processed using BLAST in the National Center for Biotechnology Information. Sequences of the three isolates were deposited in GenBank (accession nos. OR052082 to OR052086, OR493405 to OR493414). BLASTn analyses showed that isolates were 99 to 100% identical to B.cinerea reported causing leaf spot on strawberry in California; accession numbers MK919496 (G3PDH, 883/883 bp), MK919494 (HSP60, 992/992 bp), and MK919495 (RPB2, 1081/1081 bp). The resulting concatenated data set of G3PDH-HSP60-RPB2-NEP1-NEP2 was used to conduct a multilocus phylogenetic analysis (MLSA) using the maximum likelihood method. The MLSA tree indicated that the three isolates belonged to Botrytis cinerea. To test for pathogenicity, three 1-month-old strawberry (cultivar 'xuetu') plants were inoculated with each isolate (NBCM-1, NBCM-2, NBCM-3). A noninoculated control (sterile water only) was also included. The strawberry plants were inoculated by spraying with conidia suspension (1.0 × 105/ml) until run-off. Inoculations with sterile water served as controls. All plants were kept at 28/25°C (day/night), under a 12:12-h light/dark photoperiod. All plants were covered with transparent plastic bags to maintain humidity for the first 48 h, after which the bags were removed. After 4 to 7 days, leaf spot symptoms similar to those observed in the field were observed in all inoculated plants, while the controls remained healthy. The experiment was repeated three times. The pathogen was reisolated from the inoculated leaves and again identified as B. cinerea, with the same methodology used for the initial identification. Leaf spot caused by B. cinerea on strawberry was recently reportedin California (Mansouripour and Holmes 2020) and Florida (Marin and Peres 2022). To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in China. The pathogen is also the causal agent of Botrytis fruit rot on strawberry. Given the high variability of this pathogen (Marin and Peres 2022), further studies on its occurrence, spread, management, and control are required. The identification of this pathogen provides a basis for further research on its management and control.

Systemic Colonization of Xanthomonas fragariae Strain YL19 Causing Dry Cavity Rot of Strawberry Crown Tissue in China.

Xanthomonas fragariae usually causes angular leaf spot (ALS) of strawberry, a serious bacterial disease in many strawberry-producing regions worldwide. Recently, a new strain of X. fragariae (YL19) was isolated from strawberry in China and has been shown to cause dry cavity rot in strawberry crown. In this study, we constructed a green fluorescent protein (GFP)-labeled Xf YL19 (YL19-GFP) to visualize the infection process and pathogen colonization in strawberries. Foliar inoculation of YL19-GFP resulted in the pathogen migrating from the leaves to the crown, whereas dip inoculation of wounded crowns or roots resulted in the migration of bacteria from the crowns or roots to the leaves. These two invasion types both resulted in the systematic spread of YL19-GFP, but inoculation of a wounded crown was more harmful to the strawberry plant than foliar inoculation. Results increased our understanding of the systemic invasion of X. fragariae, and the resultant crown cavity caused by Xf YL19.

First report of Neopestalotiopsis clavispora causing leaf blight on Aegicerascorniculatum in China.

Aegicerascorniculatum is an important mangrove plant that mainly grows in tropical and subtropical regions. Ithasimportantecological,economicandsocialbenefits (Bandaranayake 1998). In April 2021, a leaf disease on A.corniculatum was observed in Zhanjiang (21.21° N, 110.41° E), Guangdong province, China. Diseaseincidenceandseverity were 15%and 20% (n = 100 investigated plants), respectively. The disease symptoms on leaves primarily appeared as small brown spots, then enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. At the late stage of symptom development, black acervuli appeared on the necrotic lesions. Ten symptomatic leaves from 10 plants were collected. Small pieces of tissue (4 × 4 mm) were cut from lesion borders and were surfaced disinfected in 75% ethanol for 30 s, followed by 1 min in 1% NaClO, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. After 7 days, a total of 10 fungal isolates with 100% isolation frequency were obtained and three representative strains (THS-1, THS-2, and THS-3) were used for morphological and molecular characterization.Colonies were white with cottony aerial mycelium and irregular margins. Black viscous acervuli were scattered on the colony surface 10 days after incubation. Conidia were spindle shaped, five cells, four septa, 18.77-28.70 × 4.53-6.80μm (mean 23.13 × 5.14μm) in size (n = 50). Basal and apical cells were colorless while the three medium cells were dark brown and lightly versicolor. All conidia had one basal appendage of 3.51-7.27μm (mean 5.49 μm; n = 50) and two to three apical appendages of 15.80-33.64μm (mean 25.87 μm; n = 50). These morphological characteristics are consistent with those of Neopestalotiopsis clavispora(Maharachchikumbura et al. 2012). The ITS (OM698813-15), tub2 (OM810165-67) andtef1α(OM810186-88) sequences were 99.38%, 99.09%, and 99.17% identical to the type N.clavispora strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis, respectively. A phylogenetic tree was generated using the concatenated sequences of ITS, tub2, and tef1α. The result showed that these three isolates were clustered with N.clavispora strains including the type MFLUCC12-0281. To perform pathogenicity tests, 20 healthy potted seedlings of A.corniculatum (2-year-old) were selected. Ten surface-sterilized leavesof 10 seedlings were wounded and inoculated by spraying conidial suspension ( 105conidia/ml). The same number wounded leaves of the other 10 plants treated with sterile water served as controls. All plants were wrapped in polyethylene bags for 24 h and incubated at 28°C in a growth chamber (at 90% relative humidity). After 10 days, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic. N. clavispora was reisolated from the lesions in terms of morphology and molecular characterization, whereas no fungus was isolated from the control leaves. The pathogenicity test was repeated three times under the same conditions. Thus, Koch's postulates were fulfilled. This pathogen has been reported on a wide host range worldwide, such as leaf spot on strawberry in China (Zhao et al. 2016) and twig blight on blueberry in Spain (Borrero et al. 2018). This is the first report of N. clavispora causing leaf blight on A.corniculatum in China.This study provides valuable information for the identification and control of the disease.

First worldwide report of Exserohilum rostratum causing leaf spot on strawberry (Fragaria × ananassa Duch.).

During April 2022, leaf spot was observed on strawberry (Fragaria × ananassa Duch.) with a disease incidence of approximately 45% among 100 plants. Strawberry was cultivated in a nursery at Huzhou University (30.87゜N, 120.13゜E), Zhejiang Province, China. In the strawberry greenhouse, the average temperature was 15-18 degrees, 40%-60% humidity. Early symptoms appeared as dark brown or black spotted necrotic lesions, which expanded from 2 to 6 mm in diameter. Dark brown spots with yellow halos occupied half of the leaf area and eventually developed leaf blight with large yellow halos. To isolate the causal agent, 0.5 cm x 0.5 cm fragments were cut from three symptomatic leaves, and were surface sterilized with 75% ethanol for 30 s and then rinsed three times with sterilized water. The airdried leaf fragments were placed on PDA with 50 μg/ml ampicillin and incubated in the dark at 25℃ for two days. Isolates were obtained by transferring hyphal plugs of 1 mm in diameter onto PDA. The colony morphology was circular and dark brown on the upperside and black on the underside, with cottony mycelium and an large amount of gray aerial mycelium. Conidia were large, light olive-brown to dark olive-brown and light olive-black and septate. The typical conidia were oval or rod-shaped, rarely curved, and dark septa defined the basal and apical cells. In the two typical forms of conidia, the average size of oval conidia was approximately 18.77 × 54.92 μm (11.99 to 26.97 × 35.13 to 74.59 μm, n = 20), and the average size of the rod-shaped conidia was approximately 14.80 × 103.24 μm (11.24 to 24.64 × 73.11 to 131.51 μm, n = 20). The morphological characteristics matched well with previous descriptions of Exserohilum rostratum (Sharma et al. 2014; Liu et al. 2021). The identity of C1-L and C1-S from symptomatic tissues was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted from the mycelium using the CTAB (cetyltrimethylammonium bromide) method (Griffith & Shaw 1998). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, and partial beta-tubulin 2 (TUB2), using the primers ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT512F/ACT783R (Carbone and Kohn 1999), T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson, 1995). The obtained sequences of C1-L and C1-S were the same. Moreover, the sequences have been deposited in GenBank under accession numbers ON982516 (ITS), ON996915 (GAPDH), ON996916 (ACT), and ON996917 (TUB2). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, and ACT had 100% identity with the sequences of E. rostratum (GenBank Accession No. LT837834, LT883550, and LT837672, respectively), the TUB2 had 99.61% similarity with BLAST sequences of E. rostratum (LT899391). These morphological characteristics and molecular analyses allowed the identification of the pathogen as E. rostratum. Koch's postulates were performed with five healthy detached strawberry leaves with three inoculations per leaf of the 'Akihime' strawberry variety. Surface-sterilized leaves were wounded with an aseptic needle, and inoculated with 2 mm diameter mycelial plugs from 5-day-old cultures of E. rostratum. Control leaves were also wounded with the aseptic needle, and inoculated with a sterile PDA agar plug. The leaves were incubated at 25℃ in Petri plates with petioles wrapped in moist sterile cotton. The diseased symptoms included black spots on the epidermis of the wounded leaves within 5, 10, and 20 days after inoculation. Mock-inoculated controls remained asymptomatic, and three biological repetitions were conducted. The fungus reisolated from the diseased leaves was confirmed as E. rostratum by sequencing. Abundant reports have shown that E. rostratum can infect many economically important crops such as maize, rice, and pineapple (Sun et al. 2021; Kabore et al. 2022; Luo et al. 2012). To the best of our knowledge, this is the first report of E. rostratum on strawberry in China and worldwide.

Lysobacter changpingensis sp. nov., a novel species of the genus Lysobacter isolated from a rhizosphere soil of strawberry in China

In the present work, we characterized in detail strain CM-3-T8T, which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8T were Gram-negative, non-spore-forming, aerobic, short rod. Growth occurred at 25–37 °C, pH 5.0–10.0, and in the presence of 0–8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CM-3-T8T formed a stable clade with Lysobacter soli DCY21T and Lysobacter panacisoli CJ29T, with the 16S rRNA gene sequence similarities of 98.91% and 98.50%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG-8 T and the two reference type strains listed above were 76.3%, 79.6%, and 34.3%, 27%, respectively. The DNA G + C content was 68.4% (mol/mol). The major cellular fatty acids were comprised of C15:0 iso (36.15%), C17:0 iso (8.40%), and C11:0 iso 3OH (8.28%). The major quinone system was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylethanolamine (PME), diphosphatidylglycerol (DPG), and aminophospholipid (APL). On the basis of phenotypic, genotypic, and phylogenetic evidence, strain CM-3-T8T (= ACCC 61714 T = JCM 34576 T) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension ofP. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused byPantoea ananatishas been reportedin Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

First Report of Neopestalotiopsis clavispora Causing Calyx and Receptacle Blight on Strawberry in China.

Strawberry (Fragaria × ananassa) is widely cultivated in Liaoning Province, the most important strawberry production area in China. In November 2020, extensive brown spotting on the calyces and receptacles and dark brown necrotic lesions on the leaves of 'YanLi' strawberry plants were observed in the greenhouse, College of Horticulture, in Shenyang Agriculture University of Liaoning Province, China. The incidence ranged from 15% to 20% in the greenhouse. As the disease progressed, the spots extended to cover the entire receptacles and entire leaves. Fresh samples (5 mm2) were cut from the margin of healthy and infected tissues. The isolation and purification of methods were as described previously (Obregón et al. 2018). Fungal isolations are suborbicular with irregular edges. The colonies showed white aerial mycelium at the early stage and formed concentric circles after 5 days culture at 28°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 7~10 days culture. Acervulus was 162.4 µm in average diameter, n= 30. Conidia were spindle shape, five cells, four septa, 7.0 µm (average width) and 24.0 µm (average length), n=30. The apical and basal cells are lighter in color, most of them are hyaline, the middle three cells are darker in color, mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 30.8 µm in average length, and the basal cell has a small appendage, 9.0 µm in average length, n=30 (Fig. 1c-g). For fungal identification to species level, genomic DNA was extracted by the 2% CTAB method. Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. Purified PCR products were sequenced in single direction. BLASTn searches of GenBank, using the partial ITS (MW861730), β-tubulin (MW856302) and TEF-1α (MW861729) sequences of representative isolate NP102 as the queries, revealed 97%, 100% and 100% sequence identity, respectively, to Neopestalotiopsis clavispora strain WJ31 accessions MG729690, MG740736 and MG740759. The phylogenetic tree was constructed by MEGA X (Fig. 2) (Kumar et al. 2018). Pathogenicity assay was carried out by spraying a spore suspension (1 × 106 spores/ml) of 8-day-old culture of fungus on six healthy 'YanLi' strawberry plants. An additional six control plants were only sprayed with sterile distilled water, and the inoculated plants were maintained in a light incubator at 25 ± 1°C, 90% relative humidity, with a photoperiod of 12 h. Calyx and receptacle blight and leaf spot symptoms resembled those observed in the greenhouse on all inoculated tissues after 10 days (Fig. 1h and i), whereas calyces, receptacles and leaves on control plants remained asymptomatic. The experiment was repeated twice. Neopestalotiopsis clavispora was successfully reisolated from the infected tissues, fulfilling Koch's postulates. Researchers in Spain (Chamorro et al. 2016) reported N. clavispora for the first time on strawberry. After two years, N. clavispora was found in Argentina (Obregón et al. 2018). The reports characterized it mainly as a root rot pathogen. In January 2021, Neopestalotiopsis spp. were reported that it is a pathogen of strawberry leaves and fruits in Florida strawberry fields (Juliana et al. 2021). Our studies indicated that isolate NP102 was more aggressive. In the warm and humid greenhouse, the disease struck suddenly at the early stages of strawberry fruiting. Unlike previous reports, symptoms were a calyx and receptacle blight as well as a leaf spot. This pathogen can cause serious economic losses to strawberry growers. To the best of our knowledge, this is the first report of calyx and receptacle blight by N. clavispora on strawberry in China.

Strawberry, a New Natural Host of Brassica Yellows Virus in China.

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 5' and 3' 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.

The occurrence of strawberry virus 1 infecting strawberry in Shandong province, China.

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.

First Report of Strawberry Crown Rot Caused by Xanthomonas fragariae in China.

Strawberry (Fragaria × ananassa Duch.) is a kind of fruit with great economic importance and widely cultivated in the world. From 2019 to 2020, a serious crown rot disease was sporadically observed in several strawberry cultivars including 'Zhang Ji', 'Hong Yan' and 'Yue Xiu' in Shanghai, China. Initially, water-soaked rot appeared in inner tissue of strawberry crown, then progressed into browning and hollowing symptoms accompanied with yellow discolorations of young leaves. To isolate and identify the causal agent, small pieces of tissue taken from ten diseased crowns were sterilized by 70% alcohol. The cut-up pieces were macerated and serially diluted. The dilutions were placed on nutrient agar (NA) medium. After incubation at 25°C for 4-5 days, the yellow bacterial colonies were tiny and were streaked on NA plate for purification. The colonies were yellow, mucoid, smooth-margined, and five independent representative colonies were used for further confirmation. To confirm the species identity of the bacterial, genomic DNA was extracted from the five representative isolates, and 16S rRNA gene was amplified and sequenced using universal primers 27F/1492R. The 16S rRNA sequence was deposited in GenBank (MW725235) and showed 99% nucleotide similarity with Xanthomonas fragariae strain LMG 708 (NR_026318). The isolate's identity was further confirmed by X. fragariae-specific primers XF9/XF12 (Roberts et al. 1996). All five isolates could be detected by XF9/XF12 primer. To confirm Koch's postulates, five healthy strawberry plants were placed in 1000 ml glass beakers by submerging the cutting wound in 50 ml the bacterial suspension of 108 CFU/ml. Five additional strawberry plants treated with sterilized water served as a control. The beakers containing inoculated plants were sealed with plastic film at 25°C. Water-soaked rot appeared on internal tissue of crown similar to those observed in the field within 10-12 days after inoculation, while the control samples remained healthy. The bacteria were re-isolated from rot of inoculated crowns, and confirmed by X. fragariae-specific primers XF9/XF12. X. fragariae has been reported to cause angular leaf spot on strawberry in China (Wang et al. 2017; Wu et al., 2020). It's also found that X. fragariae could systematically infect crown tissue (Milholland et al. 1996; Mahuku and Goodwin, 1997). To our knowledge, this is the first report of X. fragariae causing strawberry crown rot in China. This report increased our understanding of X. fragariae, and showed that the spread of this disease might seriously threaten the development of strawberry industry in the future.

First Report of Xanthomonas fragariae strain YL19 causing crown infection pockets in strawberry in Liaoning Province, China.

Strawberry (Fragaria × ananassa Duch.) is an important fruit crop in China. Typical crown infection pockets symptoms were observed on the infected strawberry in Liaoning province, China (121°60'E, 38°90'N) in the autumn of 2017. The disease incidence was estimated to be around 5 to 10 %, but could reach 30 to 40 % in some heavily affected plastic tunnels. The infected plants early displayed water-soaked symptoms on the abaxial leaf surface and subsequently developed reddish-brown shaped stripes and coalesced lesions on the adaxial leaf surface around the main veins (Fig. 1-A, 1-B). Several variable-size (0.3-0.8 mm in diameter) pockets were observed inside the crown tissues after dissection (Fig. 1-C). The diseased plants rarely reached fruiting and were easily broken between the crown tissue and the stem, and would eventually die. To identify the causal agent of this disease, the several surface-disinfested infected main veins and crown tissues were individually ground in sterile water and plated on sucrose peptone agar(SPA) medium (Hayward 1960) with 10-fold serial dilutions and incubated at 25℃. A number of yellow colonies grew on the medium at the 10-4 dilution 7 days after plating (Fig. 1-D) in all specimens. The colonies were aerobic, yellow, viscous, smooth, and gram-negative, which is a typical characteristic of Xanthomonas. To confirm identity of the causal bacteria, 18 colonies selected randomly were subjected to polymerase chain reactions (PCR) for the amplification of the cpn60 (Sahin et al. 2010), gyrB, rpoD, and fyuA (C Manceau et al. 2011), respectively. The results showed that the 18 colonies are identical. The cpn60, gyrB, rpoD, and fyuA sequences of this isolate were deposited in GenBank with accession numbers MT513132.1, MW233896, MW233897, and MW233895, respectively. BLAST searches with sequences of this isolate cpn60, gyrB, rpoD, and fyuA revealed 97.7%, 96.4%, 97.8%, and 97.3% similarity with the corresponding sequences of X. fragariae strain NBC2815 (LT853880.1), respectively. The resulting concatenated data set of cpn60-gyrB-rpoD-fyuA was used to build a Multilocus Sequence Analysis (MLSA) by maximum likelihood criteria (Fig. 2). The cpn60-gyrB-rpoD-fyuA sequences of the isolate from Liaoning clustered in the clade containing the type strain of X. fragariae NBC2815, indicating that it belongs to X. fragariae. Thus, the bacterial strain from Liaoning was designated as X. fragariae strain YL19. To fulfill Koch's postulates, the base of leaf petioles of disease-free strawberry plants were syringe-infiltrated inoculated with bacterial suspension (2×108 CFU) prepared from colonies of X. fragariae YL19 washed from SPA plates. The inoculated and control (treated with sterile water) were placed in a chamber (25/20℃day/night,≥90% relative humidity(RH), 12/12 h photoperiod) for three months. After one month, water-soaked symptoms were observed in the crown tissues of all X. fragariae YL19-inoculated plants. Two months after inoculation, a significant crown pocket similarly to initial symptoms observed in the field was developed on all inoculated plants. No symptoms were observed in the control plants. The bacteria were re-isolated from the symptomatic leaves, petioles and crowns, and confirmed as X. fragariae YL19 by the above mentioned morphological and molecular analyses. Pathogenicity tests were conducted three times and the same results were obtained. It was reported that X. fragariae usually causes angular leaf spot, a serious bacterial disease in many strawberry production regions worldwide. The typical symptoms of angular leaf spot caused by X. fragariae include reddish-brown, irregular spots on the upper leaf surface, water-soaked lesions developed along leaf veins. Although angular leaf spot caused by X. fragariae has been reported in Tianjin and Taiwan province, China (Wang et al. 2017; Wu et al. 2020), there is no report about the symptoms that infection pockets on crowns caused by X. fragariae strain YL19 as described above. This result indicated that YL19 is different from the other two X. fragariae strains reported in China or the disease caused by YL19 could be a severe case of angular leaf spot and vascular decline or collapse in strawberry (Bradbury, 1977). . To the best of our knowledge, these results showed a previously unreported new strain YL19 of X. fragariae is the causal agent of crown infecton pocket in strawberry in China, it may lead to serious losses to the local strawberry industry. This report will assist in developing management measures for this disease promptly.

Dactylonectria species associated with black root rot of strawberry in China

Dactylonectria species associated with black root rot of strawberry in China

First report on Neofusicoccum kwambonambiense causing strawberry root rot on strawberry in China

First report on Neofusicoccum kwambonambiense causing strawberry root rot on strawberry in China

Characterization of Colletotrichum spp. Sensitivity to Carbendazim for Isolates Causing Strawberry Anthracnose in China.

Strawberry anthracnose caused by Colletotrichum species is an important disease that may cause significant economic losses. Based on multilocus sequence analyses and morphological characteristics, 64 isolates from strawberry anthracnose samples collected from nine Chinese provinces and municipalities were identified as three species: Colletotrichum fructicola (29 isolates), Colletotrichum siamense (23 isolates), and Colletotrichum nymphaeae (12 isolates). Isolates of C. siamense showed strong aggressiveness to fruit and leaves. Isolates of C. fructicola showed strong aggressiveness to crowns. Isolates of C. nymphaeae were weakly or not pathogenic to fruit, leaves, or crowns. Sensitivity to carbendazim was determined for a total of 75 isolates, including 11 previously preserved. Two isolates of C. siamense were highly resistant (HR) and 21 were moderately resistant (MR). Nine isolates of C. fructicola were sensitive (S), 24 were HR, and four were MR. All 15 isolates of C. nymphaeae were insensitive, and their mycelial growth was not completely inhibited on potato dextrose agar amended with 500 μg/ml carbendazim. Beta-tubulin (TUB2) of representative isolates was amplified and sequenced, revealing a glutamic acid substituted by alanine at codon 198 in HR isolates of C. siamense and C. fructicola. MR isolates of C. siamense and C. fructicola had a point mutation at codon 200, causing a replacement of phenylalanine acid by tyrosine. No point mutation was detected at codons 50, 167, 198, 200, or 240 in TUB2 of C. nymphaeae insensitive isolates. Overall, this study revealed that C. fructicola was the dominant species causing anthracnose on strawberry and could improve the understanding of the management of fungicide resistance in Colletotrichum species on strawberry in China.

First Report of Phytopythium helicoides Causing Root and Crown Rot on Strawberry in China

Strawberry (Fragaria × ananassa Duch.) is an important cash crop in China. According to the 2016 China Agricultural Yearbook, Jiangsu Province is an important player in China’s strawberry market, with a cultivation area of 18,100 ha and production of 460,000 metric tons per year. We investigated a commercial field in the Jiangsu Province where the strawberry cultivar HongYan had been cultivated for more than 5 years. Between September and December 2017, symptoms of root and crown rot appeared in strawberry plants: plants developed disease symptoms characterized by yellowing of lower leaves, wilt, blight, and often death. The roots had a dark brown rot, and crowns were also rotted. The average incidence of the disease was up to 25%, and it even led to destruction and closure of a certain strawberry greenhouse. Root tissues (5 mm²) from 25 infected plants were excised from the central rotted root, surfaced sterilized by soaking in 75% ethyl alcohol for 30 s, rinsed three times in sterilized distilled water, and plated onto potato dextrose agar (PDA). All 25 isolates from each infected plant had the same morphological characteristics. They were able to grow at temperatures between 15 and 40°C, and there were no distinctive colony growth patterns on PDA. The sporangia were obovate or subglobose, papillate, and 21 to 34 × 20 to 28 µm in diameter. Oogonia were smooth and 22 to 30 µm in diameter. To confirm the identity of the pathogen, the rDNA internal transcribed spacer region (ITS, NCBI accession no. MT179561), cytochrome c oxidase I gene (cox I, MT188654), and cytochrome c oxidase II gene (cox II, MT188655) of three isolates were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), FM35/FM59, and FM66/FM58 (Martin 2000), respectively. BLAST analysis showed in all cases 98 to 100% identity with segments of Phytopythium helicoides (GenBank accession nos. MG597190.1 for ITS, KT258906.1 for cox I, and KT258907.1 for cox II). Based on the morphological features and the phylogenetic analyses of DNA sequences, the isolates were identified as P. helicoides. Pathogenicity assays were conducted on 3-week-old HongYan strawberry seedlings in September 2018. The roots of each seedling were dipped for 10 min into zoospore suspension of each isolate (1 × 10⁶ zoospores/ml), and the control seedlings were inoculated using the same procedure with sterilized distilled water. Five replicate seedlings were inoculated and planted individually into 1.8-liter free-draining pots containing sterilized soil. All the seedlings were incubated at 22°C under a 16-h light/dark regime and watered with 50 ml every 5 days. All inoculated plants showed root necrosis and crown rot 20 days after inoculation. The symptoms of inoculated seedlings were similar to the naturally infected plants. The same pathogen was reisolated from symptomatic roots and crowns. No symptoms were observed in the control seedlings. Although P. helicoides has been reported causing root and crown rot on strawberry in Japan and America (Ishiguro et al. 2014; Marin et al. 2019), rhizome rot on Asian lotus, and stem rot on Shatangju mandarin in China (Chen et al. 2016; Yin et al. 2016), it has not been reported in China from strawberry. Thus, strawberry growers should consider this pathogen when diagnosing and managing plant diseases in this crop.

Fungicide resistance of Botrytis cinerea from strawberry to procymidone and zoxamide in Hubei, China

Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of strawberry in China. For resistance monitoring, 198 B. cinerea isolates were collected from strawberry greenhouses at 10 locations in Hubei Province. The isolates were screened for resistance to fungicides procymidone and zoxamide. In mycelium growth assays for procymidone, the mean values of effective concentration at which mycelium growth is inhibited by 50% (EC50) for sensitive (ProS) and resistant (ProR) isolates were 0.25 μg/mL and 2.21 μg/mL, respectively. The frequency of ProR isolates was 14%, and the highest frequency (48%) was observed in Yichang. Positive cross-resistance was detected for ProR isolates to other dicarboximide fungicides, but not to phenylpyrroles. Comparative analysis of fitness parameters revealed increased osmotic sensitivity of ProR isolates compared to ProS ones. Sequence analysis of the dicarboximide target gene BcOs1 revealed that ProR isolates carried either a single point mutation at codon 365 (I365S) or a pair of point mutations (Q369P and N373S). The mean EC50 values for zoxamide sensitive (ZoxS) and resistant (ZoxR) isolates were 0.31 μg/mL and 7.76 μg/mL, respectively. Only six (3%) isolates from three locations were found to be resistant to zoxamide. All ZoxR isolates were found resistant to carbendazim. Fitness parameters did not show significant difference between ZoxR and ZoxS isolates. Sequence analysis of the beta-tubulin gene in resistant isolates revealed four previously reported point mutations (E198A, E198K, F200Y and T351I). The mutation T351I was detected only in the isolates possessing E198K point mutation. Mutation F200Y was detected in a highly resistant isolate. Results of this study will be helpful for the management of fungicide resistance in B. cinerea.

First Report of Hop Stunt Viroid Infecting Strawberry in China

First Report of Hop Stunt Viroid Infecting Strawberry in China

Gray mold of strawberry (Fragaria ananassa) caused by a rare pink-colored isolate of Botrytis cinerea in China

Botrytis cinerea is a major pathogen causing gray mold disease on strawberry (Fragaria ananassa). Here, one pink-colored B. cinerea strain GS5–15 was isolated from diseased strawberry fruit, and its identity was confirmed by both morphological and molecular methods. Besides showing typical culture morphology of B. cinerea, strain GS5–15 produced pink mycelium on potato dextrose agar. A 354-bp DNA fragment was detected in strain GS5–15 by using PCR with B. cinerea-specific primers Bc-f/Bc-r, indicating that it belonged to B. cinerea. BLASTn analysis showed that the sequences of ITS, G3PDH, HSP60, and RPB2 were 99–100% identical to the corresponding sequences of other B. cinerea strains, and the phylogenetic analysis based on concatenated G3PDH, HSP60 and RPB2 sequences also showed that strain GS5–15 was clustered with reference strains of B. cinerea. Re-inoculation of strain GS5–15 on strawberry fruit displayed typical gray mold symptom, and a fungus showing colony morphology similar to the original strain was re-isolated from the diseased fruits. Therefore, B. cinerea strain GS5–15 was determined to be the causal agent of gray mold on strawberry in China. To our knowledge, this is the first record of pink-colored B. cinerea infecting strawberry in China.

Occurrence of Outbreak of Leaf Spot Caused by Corynespora cassiicola in Strawberry in China.

Occurrence of Outbreak of Leaf Spot Caused by Corynespora cassiicola in Strawberry in China.

Detection and fitness comparison of target-based highly fludioxonil-resistant isolates of Botrytis cinerea from strawberry and cucumber in China

Botrytis cinerea has a high risk of developing resistance to fungicides. Fludioxonil, belonging to phenylpyrroles, has been used for more than three decades, however, only few cases of field resistance against phenylpyrroles have been reported. In this study, the highly fludioxonil-resistant (HR) isolates of Botrytis cinerea were firstly detected in the commercial greenhouses of strawberry in China in 2015, and biochemical characterization differences in high fludioxonil-resistance from strawberry and cucumber were compared. All of the five HR isolates from greenhouses of strawberry and cucumber could grow on PDA amended with 100 μg/mL fludioxonil, and exhibited a positive correlation between the resistance of dicarboximide fungicides and fludioxonil. Sporulation and sclerotium production of the strawberry-originated HR isolates were increased in comparison with the cucumber-originated HR isolates. No matter how the HR isolates were from strawberry and cucumber, all the HR isolates showed enhanced sensitivity to the osmotic agents, but with significant difference. Based on sequence alignment of the BcOS1 which codes protein bound by fludioxonil, two genotypes of the strawberry-originated HR isolates were observed, i.e., (F127S + I365N + S426P) and (G538R + A1259T), which were totally different from those of the cucumber-originated HR isolates. Molecular docking of fludioxonil to the binding site of BcOS1 protein from the five HR isolates illustrated that all the HR isolates had less affinity than the sensitive isolates. Our data indicated that genotypes of the HR isolates match the corresponding fludioxonil-selection pressure on the field populations of B. cinerea in the commercial greenhouses of the two host plants.