Apple Trees In China Research Articles

First report of Diplodia mutila associated with Botryosphaeria canker and dieback of apple trees in Xinjiang, China.

Apple (Malus pumila Mill.) is an important fruit crop in Xinjiang, China. In September 2021, apple tree canker was observed in a 21-year-old commercial apple orchard cv. Fuji in Xinjiang (38°17'51.43"N, 77°9'50.81"E) , northwest of China. Of the 200 plants surveyed, 25% were symptomatic. The diseased trees showed branch dieback and cankers. The cankers observed on the wood were sunken, shriveled, and discolored. After the bark was peeled off, the diseased wood was dark brown, and the necrosis was obvious on the cross-section of the diseased branch. To identify the causal agent, five symptomatic trees were collected and analyzed in the laboratory. Apple wood samples (0.5×0.5 cm) were surface-disinfected with 1% v/v sodium hypochlorite and 75% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA), and incubated in the dark at 25 °C for 5 days. Conidia were induced on sterilized pine needles covered with 2% w/v water agar under near-UV light. The colonies of five isolates were white to gray with sparse aerial mycelium that gradually became dark olive green in the later stage. Conidia were initially hyaline but becoming brown at maturity, 1-septate, oval, rounded at both ends, and with dimensions of 24.9-32.1 × 15.1-21.5 µm (n =50) and the aspect ratio of 1.6. Based on the cultural and morphological features of Phillips (2002), the isolates were identified initially as Diplodia mutila (Fr. : Fr.) Mont. To confirm species identification, genomic DNA was extracted from the representative isolate SC-8A. The primer ITS1/ITS4, EF1-728F/EF1-986R and BT2a/BT2b were used to amplify the rDNA sequences of, respectively, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α) gene, and a portion of beta-tubulin (tub2) gene. The nucleotide sequences indicated ≥99% identity to D. mutila (CBS 112553) for three DNA regions. Consensus sequences were deposited in GenBank. as accession numbers OM618108, OM676657 and OM676658 for ITS, EF1-α and tub2, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate SC-8A on one year old branches of cv. Fuji (n=5). Wounds were created in the middle of the branches using a sterilized hole punch (5mm diameter) and were immediately inoculated with mycelial plugs of the same diameter. For the control treatment, sterile agar plugs were used (n=5) in the branches. The inoculated and control branches were wrapped with sterile parafilm. On the 10th day after inoculation, canker lesions appeared on the inoculated branches, but no lesions were observed in the negative control. D. mutila was re-isolated from 100% of the inoculated shoots and was not re-isolated from any of the negative controls, the Koch's postulates were met. Previously, D. mutila has been reported in Canada (Úrbez-Torres et al., 2016), Argentina (Lódolo et al., 2022) and Chile (Díaz et al., 2022) causing Botryosphaeria canker and dieback in apples. To our knowledge, this is the first report of D. mutila causing Botryosphaeria canker and dieback in apple trees in China.

The Effect of Temperature and Moisture on Colonization of Apple Fruit and Branches by Botryosphaeria dothidea.

Botryosphaeria dothidea causes severe disease of apple trees in China. The process of conidium germination, colonization, and infection of apple fruit and branches was examined on 'Fuji' apple and the effect of temperature, surface wetness and relative humidity (RH), and host surface washates on these processes was studied in controlled environments. Initial germ tube development and hyphal growth resulted in the colonization of the host surface without forming an infection structure. Hyphae expanded radially across the host surface and, after entering lenticels, developed into a dense mycelium mass or differentiated pseudoparenchyma. Hyphae from the bottom of the pseudoparenchyma either directly penetrated the lenticel surface intercellularly through the cell layer, or formed an undifferentiated hypha that invaded the lenticel through cracks formed during the lenticel development. Conidial germination and hyphal colonization occurred at 10 to 40°C, with an optimum of approximately 28°C. Conidial germination required an RH > 95% or surface wetness but, for hyphal colonization, an RH > 90% was sufficient. Conidia germinated and formed germ tubes within 1 h under optimum conditions. However, the pathogen required a longer period at RH > 90% or surface wetness for hyphae to colonize and form pseudoparenchyma or dense mycelia on the host surface. Hyphal colonization is a crucial stage for infection of apple tissues by B. dothidea.

The apple FERONIA receptor-like kinase MdMRLK2 negatively regulates Valsa canker resistance by suppressing defence responses and hypersensitive reaction.

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in China and other East Asian countries. The plant receptor‐like kinase FERONIA is involved in plant cell growth, development, and immunity. However, little is known about the function of FERONIA in apple defence against V. mali. In this study, we found that MdMRLK2 was highly induced by V. mali in twigs of V. mali‐susceptible Malus mellana but not in those of the resistant species Malus yunnaensis. 35S:MdMRLK2 apple plants showed compromised resistance relative to wild‐type (WT) plants. Further analyses indicated that 35S:MdMRLK2 apple plants had enhanced abscisic acid (ABA) levels and reduced salicylic acid (SA) levels relative to the WT on V. mali infection. MdMRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia‐lyase (PAL), β‐1,3‐glucanase (GLU), and chitinase (CHT) during V. mali infection. Moreover, MdMRLK2 interacted with MdHIR1, a hypersensitive‐induced response protein, and suppressed the MdHIR1‐mediated hypersensitive reaction (HR), probably by impairing MdHIR1 self‐interaction. Collectively, these findings demonstrate that overexpression of MdMRLK2 compromises Valsa canker resistance, probably by (a) altering ABA and SA levels, (b) suppressing polyphenol accumulation, (c) inhibiting PAL, GLU, and CHT activities, and (d) blocking MdHIR1‐mediated HR by disrupting MdHIR1 self‐interaction.

First Report of a New Disease Caused by Fusarium tricinctum on Apple Tree in China.

First Report of a New Disease Caused by Fusarium tricinctum on Apple Tree in China.

First report of Lasiodiplodia theobromae causing dieback in custard apple (Annona squamosa) tree in China.

Custard apple (Annona squamosa Linn) is popular for its sweet taste and rich aroma. Hainan province is the major production area of custard apple in China. In September 2020, wilting of leaves and branches, discoloration of the vascular system and dieback of trees were observed in plantings in Lingao County of Hainnan Province, China (Fig. 1a-c). The incidence of dieback in three orchards was at least 19%, and affected samples were brought to the laboratory. Fragments of approximately 5 mm in length were obtained from five diseased branches, which were collected from different plants and orchards. Fragment surface were sterilized in 75% ethanol for 1 min, and 1% mercury chloride for 1 min, then rinsed three times with sterile distilled water. These tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated at 28°C for 3 days. Fungal colonies were transferred to fresh PDA plates, and single-spore cultures were obtained. We isolated twenty-six fungal strains, of which twenty-three isolates were morphologically identified as Lasiodiplodia species (Phillips et al. 2013). The colony morphology was initially round and white, then turned to grey or black after 5-7 days at 28°C, and formed pycnidia for 20 days (Fig. 1d). The immature conidia were ellipsoid, colorless, hyaline and unicellular, becoming brown, bicellular with longitudinal striations at maturity (Fig. 1e). Mature conidial size was: 26.61±1.57×14.87±1.14 µm (n=60). For molecular identification, genomic DNA of three isolates (HSYF01, HSYF02 and HSYF03) was extracted using E.Z.N.A.® HP Plant DNA kit (Omega Bio-Tek). The internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin (TUB) regions were amplified using the primers ITS1/4, EF1-728F/986R and Bt2a/Bt2b, respectively (Carbone and Kohn 1999; Glass and Donaldson 1995; White et al. 1990). A BLAST search of ITS, EF1-α and TUB gene sequences (Accession nos. MW625913-MW625918, MW876481-MW876483) had in 99.8% (493bp out of 494 bp), 99.31% (286bp out of 288 bp) and 100% (372bp out of 372 bp) identity to CBS164.96 of the L. theobromae (Accession nos. AY640255, AY640258 and KU887532). The identity of the putative pathogen isolates was further confirmed by phylogenetic analysis (Fig. 2). Ten healthy 1-2-year-old custard apple trees were used to confirm pathogenicity. Custard apple plants were wounded approximately 15-20 cm below the tips with a sterilized scalpel. Each cut was inoculated with a 5 mm agar plugs with mycelium from the PDA cultures. The wound site was moisturized with wet cotton wool and wrapped with laboratory film (Silveira et al. 2018). Two seedlings treated with sterile agar plugs served as a controls. The pathogenicity test was repeated twice. After 14 days of incubation in a glasshouse, all inoculated seedlings had characteristic discoloration and necrotic lesions starting from the apical branches, (Figs. 1f and 1g). The stems exhibited browning and vascular streaking of the wood from the inoculation point (Figs. 1h), while the control seedlings remained symptomless. Typical colonies of L. theobromae were isolated and identified from all inoculated seedings, fulfilling Koch´s postulates. Although, postharvest fruit stem-end rot on custard apple caused by L. theobromae was previously described (Hu et al. 2003; Meng et al. 2017), this is the first report of L. theobromae causing dieback in mature custard apple trees in China.

First report of apple rubbery wood virus 1 in apple in China.

More than 30 viral and subviral pathogens infect apple (Malus domestica, an important fruit crop in China) trees and rootstocks, posing a threat to its production. With advances in diagnostic technologies, new viruses including apple rubbery wood virus 1 (ARWV-1), apple rubbery wood virus 2 (ARWV-2), apple luteovirus 1 (ALV), and citrus virus A (CiVA) have been detected (Beatriz et al. 2018; Rott et al. 2018; Hu et al. 2021). ARWV-1 (family Phenuiviridae) is a negative-sense single-stranded RNA virus with three RNA segments (large [L], medium [M], and small [S]). It causes apple rubbery wood disease (Rott et al. 2018) and is found in apple rootstocks, causing leaf yellowing and mottle symptoms in Korea (Lim et al. 2018). To determine virus prevalence in apple trees in China, 200 apple leaf and shoot samples were collected from orchards in Hebei (n = 26), Liaoning (n = 40), Shandong (n = 100), Yunnan (n = 25), Shanxi (n = 4) and Inner Mongolia (5) in 2020. Total RNA was extracted from the shoot phloem or leaf tissues (Hu et al., 2015) and subjected to reverse transcription (RT)-PCR to detect apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple necrotic mosaic virus (ApNMV), apple scar skin viroid (ASSVd), ARWV-2, ARWV-1, ALVand CiVA using primers specific to respective viruses (Supplementary Table 1). The prevalence of ACLSV, ASPV, ASGV, ApNMV, ASSVd, ARWV-2, ARWV-1, ALV and CiVA was found to be 75.5%, 85.5%, 86.0%, 43.0%, 4.0%, 48.5%, 10.5%, 0% and 0%, respectively (Supplementary Table 2). Among the 21 positive samples for ARWV-1, three, five and 13 samples were from Hebei, Liaoning and Shandong, respectively. Five ARWV-1-positive samples (cultivars Xinhongjiangjun, Xiangfu-1, Xiangfu-2 and Tianhong) showed leaf mosaic symptoms. To confirm the RT-PCR assay, the projected ARWV-1 amplicons from cvs. Xiangfu-1 and Tianhong were cloned into the pMD18-T vector (Takara, Dalian, China), and three clones of each sample were sequenced. BLASTn analyses demonstrated that the sequences (accession nos. MW507810-MW507811) shared 96.9%-98.9% identity withARWV-1 sequences (MH714536, MF062127, and MF062138) in GenBank. An lncRNA library was prepared for high-throughput sequencing (HTS) with the Illumina HiSeq platform using Xiangfu-1 RNA. A total of 71,613,294 reads were obtained. De novo assembly of the reads revealed 135 viral sequence contigs of ACLSV, ASGV, ASPV, ApNMV, ARWV-1, and ARWV-2. The sequences of contig-100_88981 (302 nt) and contig-100_25701 (834 nt) (accession nos. MW507821 and MW507820) matched those of segment S from ARWV-1, whereas the sequences of contig-100_6542 (1,660 nt) and contig-100_27 (7,364 nt) (accession nos. MW507819 and MW507818) matched those of segments M and L, respectively. To confirm the HTS results, fragments of segments L (744 bp), M (747 bp), and S (554 bp) from Xiangfu-1 and Tianhong were amplified (Supplementary Table 1) and sequenced. The sequences (accession nos. MW507812-MW507817) showed 94.8%-99.9% nucleotide identity with the corresponding segments of ARWV-1. Co-infection of ARWV-1 with ApNMV and/or ARWV-2 was confirmed in 17/21 ARWV-1-positive samples. The prevalence of ARWV-1/ApNMV, ARWV-1/ARWV-2, and ARWV-1/ApNMV/ARWV-2 infections was 61.9%, 71.4%, and 52.4%, respectively. To our knowledge, this is the first report of ARWV-1 infecting apple trees in China. Further research is needed to determine whether and how ARWV-1 affects apple yield and quality.

The Influence of Lower Temperature Induction of Valsa mali on the Infection of Apple Trees

Apple valsa canker (AVC), caused by Valsa mali (Vm), is one of the most important diseases of apple trees in China. AVC occurred severely along with cold winter or cold spring. However, the effect of lower temperature on Vm is poorly understood. This study evaluated the influence of lower temperature pre-treatment of Vm on the infection of apple twigs and leaves. The results showed that exposing of Vm at lower temperature (between -10°C and 10°C) for more than 18 h significantly increased the disease severity of apple leaves and twigs, with a higher LAR, lesion length and DI than that at 25°C. In addition, cold treatment ranging from -5°C to 10°C promoted the colony growth. Meanwhile, the relative expression of four cell wall degrading enzymes (CWDEs) related genes pre-treated at -5°C and 5°C were significantly higher than that at 25°C. The results indicated that the virulence of Vm mycelium is sensitivity for lower temperature change. After sensing lower temperature changes, Vm can adjust its infection of apple trees by regulating the expression of pathogenicity gene and growth rate. Spring is the season with very frequent temperature changes, and Vm is highly invasive in this season. Therefore, more attention should be paid in spring to protecting apple trees from infection by Vm, such as reduction of pruning wounds formation in spring, and application of protective agents to the pruning wounds in time.

Progression of Symptoms Caused byBotryosphaeria dothideaon Apple Branches

Until recently, the causal agent of Botryosphaeria canker was assumed to differ from that causing ring rot on fruit and warts on branches on apple trees in China and East Asia. However, recent research documented that Botryosphaeria dothidea caused both disease symptoms on apple. Inoculations with strains isolated from cankers and warts on branches were conducted to investigate symptom progression caused by B. dothidea and conditions inducing the two symptom types. The results confirmed that both cankers and warts are caused by B. dothidea. Warts are the results of hyperplasia and suberization of bark tissues induced by fungal infection, whereas cankers result from the rapid growth of hyphae from inside warts, lenticels, or wounds. Resistance to B. dothidea exists in living apple branches. When a living branch is infected via lenticels, the pathogen induces proliferation and suberization of cortical cells that restricts the growth and expansion of the hyphae, leading to warts. However, under certain stress conditions such as drought, the hyphae inside host tissues expand rapidly and kill cortical cells, leading to canker development. Host resistance may recover during active growth periods, which suppresses or even stops rapid expansion of the hyphae, leading to the intermediate symptom of canker warts. Abiotic factors, such as drought or high temperature in early spring, can result in rapid extension of colonized hyphae in branches and conversion of warts to cankers. Preventing this transition can be an important measure in managing Botryosphaeria canker on apple.

Optimization of water and nitrogen management for surge-root irrigated apple trees in the Loess Plateau of China

The Loess Plateau is one of the main regions for growing apple trees in China, but a shortage of water resources and low utilization of nitrogen have restricted its agricultural development. A 2-year field experiment was conducted which included three levels of soil water content (SWC), 90–75%, 75–60%, and 60–45% of field capacity, and five levels of nitrogen application (Napp), 0.7, 0.6, 0.5, 0.4 and 0.3 kg/plant. The treatments were arranged in a strip-plot design with complete randomized blocks with three replications. For both years, the water and Napp had significant (P<0.05) effects on leaf area index (LAI), yield, water use efficiency (WUE) and nitrogen partial factor productivity (NPFP) while the interaction effect of water and Napp on yield, WUE and NPFP was significant (P<0.05) in 2018, and not in 2017. For the same SWC level, WUE first increased, then decreased as Napp increased, while NPFP tended to decrease, but the trend of LAI with different Napp was closely related to SWC. At the same Napp, the LAI increased as SWC increased, while the WUE and NPFP first increased, then decreased, but the yield showed different trends as the SWC increased. The dualistic and quadric regression equations of water and Napp indicate that the yield, WUE and NPFP cannot reach the maximum at the same time. Considering the coupling effects of water and Napp on yield, WUE and NPFP in 2017 and 2018, the SWC level shall be controlled in 75–60% of field capacity and the Napp is 0.45 kg/plant, which can be as the suitable strategy of water and Napp management for the maximum comprehensive benefits of yield, WUE and NPFP for apple trees in the Loess Plateau and other regions with similar environments.

Vm-milR37 contributes to pathogenicity by regulating glutathione peroxidase gene VmGP in Valsa mali.

MicroRNAs play important roles in various biological processes by regulating their corresponding target genes. However, the function and regulatory mechanism of fungal microRNA‐like RNAs (milRNAs) are still largely unknown. In this study, a milRNA (Vm‐milR37) was isolated and identified from Valsa mali, which causes the most serious disease on the trunk of apple trees in China. Based on the results of deep sequencing and quantitative reverse transcription PCR, Vm‐milR37 was found to be expressed in the mycelium, while it was not expressed during the V. mali infection process. Overexpression of Vm‐milR37 did not affect vegetative growth, but significantly decreased pathogenicity. Based on degradome sequencing, the target of Vm‐milR37 was identified as VmGP, a glutathione peroxidase. The expression of Vm‐milR37 and VmGP showed a divergent trend in V. mali–apple interaction samples and Vm‐milR37 overexpression transformants. The expression of VmGP could be suppressed significantly by Vm‐milR37 when coexpressed in tobacco leaves. Deletion of VmGP showed significantly reduced pathogenicity compared with the wild type. VmGP deletion mutants showed more sensitivity to hydrogen peroxide. Apple leaves inoculated with Vm‐milR37 overexpression transformants and VmGP deletion mutant displayed increased accumulation of reactive oxygen species compared with the wild type. Thus, Vm‐milR37 plays a critical role in pathogenicity by regulating VmGP, which contributes to the oxidative stress response during V. mali infection. These results provide important evidence to define the roles of milRNAs and their corresponding target genes in pathogenicity.

Occurrence and molecular variability of apple-associated Luteovirus isolates from apple in China based on coat protein genes

Apple-associated Luteovirus (AaLV) was recently found in apple trees in China. How commonly AaLV occurs in apple and how much genetic diversity is present among samples from various locations and apple cultivars are not reported but are investigated here. In this study, 162 samples from 13 locations, covering 13 apple cultivars of Malus pumila and one wild apple of Malus sieversii in China were collected, their coat protein (CP) genes were amplified by reverse transcription-polymerase chain reaction and the variability of the CP genes was analysed. The occurrence ratio of AaLV was 37.04% in apple trees in China, the AaLV isolates showed little variation on their CP genes, the geographic location or host cultivars had no consistent effect on the occurrence, variation and phylogenetic characters of AaLV. The results of this study will provide information on further study of the molecular characteristics and epidemiology of AaLV.

First report of apple rubbery wood virus 2 infection of apples in China.

Apple (Malus) is one of the most widely grown fruit trees worldwide, and viral diseases can severely inhibit its growth and development. Apple rubbery wood virus 2 (ARWV-2, family Phenuiviridae) is a negative-sense single-stranded RNA virus whose genome comprises three RNA segments (large: L, medium: M, and small: S) (Rott et al. 2018). This virus is associated with apple rubbery wood disease (Rott et al. 2018) and has previously been found in pear (Pyrus spp.) in China (Wang et al. 2019). In autumn 2019, six trees (one each of cvs. Honglu, Hongzhengzhu, Jinxiuhaitang, Liquanduanfu, Huahong-1, and Huahong-2) showing mosaic disease-like symptoms in the leaves and two trees (one each of cvs. Qingming-1 and Qingming-2) showing rusty skin symptoms (i.e., a large number of irregular rust spots on the peel's surface) in the fruits were found in Xingcheng, Liaoning province, China. Shoots of the diseased plants were collected, and total RNA was extracted from the phloem of the samples as described by Hu et al. (2015). Reverse transcription (RT)-PCR was used to detect various viruses including apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple necrotic mosaic virus (ApNMV), and ARWV-2 as well as apple scar skin viroid (ASSVd) using their respective primers (Supplementary Table 1). ACLSV, ASPV and ASGV were detected in all samples. ApNMV was detected in the six trees with leaf mosaic symptoms and ASSVd was detected in the two trees with apple rusty skin symptoms. Moreover, five trees (cvs. Honglu, Hongzhengzhu, Jinxiuhaitang, Qingming-1, and Qingming-2) tested positive for ARWV-2 in the RT-PCR assay. The PCR products of ARWV-2 from Honglu and Qingming-2 were cloned into the pMD18-T vector (Takara, Dalian, China), and one clone of each of the samples was sequenced. BLASTn analyses showed that they shared 98.2%-99.2% nt identity with ARWV-2 sequences (MT901298-MT901299) deposited in the GenBank database. A small RNAs (sRNAs) library was prepared for high-throughput sequencing (HTS) with the Solexa-Illumina platform using phloem tissue collected from a Qingming-2 tree in which apples with rusty skin symptoms were observed. A total of 3,7746,671 reads were obtained from the library. De novo assembly of the reads yielded 1,378 viral sequence contigs. Of those, 20 contigs with lengths ranging from 82 to 387 nt were mapped to the reference genome of ARWV-2 (accession nos. MT733339-MT733344, MT901300-MT901313). In addition, contigs of ACLSV, ASPV, ASGV, ApNMV and ASSVd were detected. To further confirm the HTS results, partial length fragments of segments L (717 bp), M (645 bp), and S (657 bp) of the ARWV-2 genome were amplified from Qingming-2 using primers (Supplementary Table 1) and sequenced. The resulting sequences, which have been deposited in GenBank under the accession numbers MT364372-MT364374, showed 97.2%, 97.8%, and 98.0% nt identity, respectively, with the corresponding segments of ARWV-2 isolate R7 (accession nos. MF062144-MF062146). To understand the infection status of apple trees in China with regard to ARWV-2, 116 apple shoot samples were randomly collected from commercial orchards in Liaoning, Shanxi, and Shandong provinces and subjected to RT-PCR to detect ARWV-2, ACLSV, ASGV, ASPV, ASSVd and ApNMV. In total, 49 (42.2%) of the 116 samples tested positive for ARWV-2, suggesting that this virus is wide spread in apple trees in China (Supplementary Table 2). The mixed-infection rates of ARWV-2/ApNMV and ARWV-2/ASSVd were 18.1% (21/116) and 3.4% (4/116), respectively. Among the 46 ARWV-2-positive samples, seven had mosaic disease-like symptoms in the leaves and three had rusty skin symptoms in the fruits. To our knowledge, this is the first report of ARWV-2 infection in apples showing rusty skin symptoms, as well as the first report of ARWV-2 infection in domestic apples in China. Further research is needed to understand the distribution of ARWV-2 in apple orchards throughout China, to confirm the relationship of ARWV-2 with different symptoms and to evaluate how ARWV-2 affects the performance and quality of apple.

Development of simple sequence repeat markers based on whole-genome sequencing to reveal the genetic diversity of Glomerella cingulata in China

Glomerella leaf spot (GLS) has been found on apple trees in China since 2011 and severely affects apple production (Malus domestica Borkh.). The fungus that causes GLS, Glomerella cingulata (Stoneman) Spauld. & H.Schrenk, is epidemic in regions with humid climates each year. It can spread more than 200 km per year, and within the last several years has been found in most apple-producing areas in China. To investigate the origin, evolution and diversity of G. cingulata for better GLS management, we sequenced the whole genome of G. cingulata isolate 030206 and scanned simple sequence repeat (SSR) motifs in the genome. Then we constructed an SSR marker library containing 844 markers and their primers for amplifying the makers. We selected 25 prime pairs to test 96 G. cingulata isolates gathered from eight areas in Shandong Province, China, where GLS is prevalent and proved that the SSR markers were polymorphic among the tested isolates and were informative for analyzing genetic diversity. Developing SSR markers for G. cingulata using the whole-genome sequence is an efficient strategy for genetic diversity analysis, and the library of SSR marker constructed in this study should aid the identification of G. cingulata.

Genomic Analysis, Sequence Diversity, and Occurrence of Apple necrotic mosaic virus, a Novel Ilarvirus Associated with Mosaic Disease of Apple Trees in China.

China accounts for over 50% of apple production worldwide. Very recently, a novel ilarvirus, Apple necrotic mosaic virus (ApNMV), was isolated from apple trees showing mosaic symptoms in Japan. This study compared different types of mosaic symptoms observed in apple trees in China under field conditions. Complete nucleotide sequences were obtained for six isolates of ApNMV. The genomic components varied in size from 3,378 to 3,380 nt (RNA1), 2,778 to 2,786 nt (RNA2), and 1,909 to 1,955 nt (RNA3), respectively. Although nucleotide sequence similarities with subgroup 3 ilarviruses were low (49.2 to 64.3%), results of phylogenetic analysis indicated that Chinese ApNMV isolates were clustered in subgroup 3 together with Prunus necrotic ring spot virus (PNRSV) and Apple mosaic virus (ApMV). Apple mosaic disease occurred widely in apple producing areas of China with a very high percentage (92.1%, 268 out of 291) of symptomatic trees being infected with ApNMV but not with ApMV. The data suggested that ApNMV might be the main pathogen causing apple mosaic disease in China. The genomes of the six studied Chinese ApNMV isolates demonstrated substantial sequence diversity. Here, we demonstrated a strong association of ApNMV with the mosaic disease of apple trees in China.

Seasonal dynamics of Botryosphaeria dothidea infections and symptom development on apple fruits and shoots in China

Botryosphaeria dothidea is one of the most important pathogens of apple trees in China. It causes warts and cankers on shoots and fruit rot. In this study, multi-season experiments were conducted to investigate the seasonal dynamics of B. dothidea infection and symptom development on twigs and fruits of cv. Fuji through spraying and rain-splash inoculations. Fruit were most susceptible to infection from late May to mid-August. The length of the incubation period, i.e. from inoculation to the first lesion was observed, generally decreased with the age of fruit at the time of inoculation. Fruit inoculated in May–June developed visible symptoms from early August onwards, with a peak in early September. However, fruit inoculated in September developed visible symptoms within one week after inoculation. Twigs remained susceptible to infection by conidia from the initial extension stage in early May to late September. New extension shoots were most susceptible from June to mid-August and then became gradually less susceptible over time. When inoculated before the end of August, twigs developed visible lesions within the same season. In contrast, when inoculated late in the season, most lesions became visible during the subsequent season. The incubation period on twigs was shorter for inoculations conducted in June–August, with the shortest being 25 days. These results may contribute to the improvement of the management of this disease.

Occurrence of ‘Candidatus Phytoplasma ziziphi’ in apple trees in China

SummaryYoung apple trees exhibiting symptoms of little leaf, margin involute and yellows were observed in an open‐air nursery in Yangling, Shaanxi, China. Transmission electron microscopy showed typical phytoplasma bodies in the sieve tube elements of symptomatic leaf samples. In two nested polymerase chain reaction (PCR) assays, products of expected size of 1.1 and 1.2 kb were separately generated from the total DNAs of symptomatic samples. A restriction fragment length polymorphism (RFLP) analysis of the purified 1.2 kb PCR products indicated that the disease associated with apple trees was ‘Ca. Phytoplasma ziziphi’. To our knowledge, this is the first report of ‘Ca. Phytoplasma ziziphi’ infecting apple trees in China.

Detection and Molecular Variability of Apple Stem Grooving Virus in Shaanxi, China

AbstractApple stem grooving virus (ASGV) is one of the economically important latent viruses that are distributed in apple production areas worldwide. The presence of ASGV in apple trees was studied by serological assay and molecular biology methods. A total of 550 apple leaf samples from 14 different areas in Shaanxi were tested by DAS‐ELISA, and the results revealed an ASGV infection level of 55%. Those samples were also examined by RT‐PCR, and an infection level of 67% was found. Fourteen complete coat protein gene sequences of ASGV were obtained; phylogenetic analysis revealed that these 14 sequences separated into two clusters regardless of the geographic origin or host plants. To our knowledge, this is the first report of molecular variability analysis of ASGV in apple trees in China.

黄土山地苹果树树体不同方位液流速率分析

黄土山地苹果树树体不同方位液流速率分析