Molecular Identification and Pathogenicity of Diaporthe eres and D. hongkongensis (Diaporthales, Ascomycota) Associated with Cherry Trunk Diseases in China.

This study aimed to identify fungal species associated with trunk diseases of sweet cherries (Prunus avium) in several commercial cherry orchards in Beijing, Guizhou and Shandong provinces, China. In total, eighteen fungal strains that fitted well into the species concept of Diaporthe were isolated. Based on both morphological and multi-locus phylogenetic analyses of internal transcribed spacer region (ITS), beta-tubulin (tub-2), calmodulin (Cal) and translation elongation factor 1-α (tef1-α) sequencing data, fourteen isolates were identified as Diaporthe eres, while four isolates were classified as D. hongkongensis. Here, we report D. hongkongensis causing sweet cherry branch dieback disease and, further, we confirmed the host association of D. eres with sweet cherries in China. A pathogenicity assay revealed the ability of both D. eres and D. hongkongensis to cause shoot necrosis and stem lesions on Prunus avium cv. 'Brooks' (mean lesion lengths of 1.86 cm and 1.56 cm, respectively). The optimal temperature for the growth of both Diaporthe species was tested. The optimal growth temperature for D. hongkongensis was 30 °C, and the 25-28 °C temperatures were the most favorable for the growth of D. eres strains. This research advances the understanding of fungal trunk diseases in fruit crops, particularly gummosis and branch dieback disease in Chinese cherry orchards, and will aid growers in making decisions about cultural practices and disease management.

Identification and Characterization of Colletotrichum Species Associated with Cherry Leaf Spot Disease in China.

Leaf spot is a common and serious disease of sweet cherry worldwide and has become a major concern in China. From 2018 to 2020, disease investigations were carried out in Beijing City, Sichuan, Shandong, and Liaoning Provinces in China, and 105 Colletotrichum isolates were obtained from diseased samples. Isolates were identified by morphological characterization coupled with multigene phylogenetic analyses based on six loci (internal transcribed spacer region, glyceraldehyde 3-phosphate dehydrogenase, calmodulin, actin, chitin synthase, and ß-tubulin). A total of 13 Colletotrichum species were identified, namely Colletotrichum aenigma, C. gloeosporioides, C. fructicola, C. siamense, C. temperatum, C. conoides, C. hebeiense, C. sojae, C. plurivorum, C. karsti, C. truncatum, C. incanum, and C. dematium. Among these, C. aenigma (25.7%) was the most prominent species isolated from diseased leaves, followed by C. gloeosporioides (19.0%) and C. fructicola (12.4%). Pathogenicity was tested on detached leaves of cv. 'Tieton' and 'Summit' and young seedlings of cv. 'Brooks' under greenhouse conditions. All 13 species were pathogenic to cherry leaves, and C. aenigma, C. conoides, and C. dematium showed high levels of virulence. Seedlings inoculated with the isolates developed similar symptoms to those seen in the orchards. This study provides the first reports for 11 of the 13 Colletotrichum species on sweet cherry in the world, excluding C. aenigma and C. fructicola. This is the first comprehensive study of Colletotrichum species associated with cherry leaf spot in China, and the results will provide basic knowledge to develop sustainable control measures for cherry leaf spot.

First Report of Bacterial Wilt Caused by Enterobacter mori of Strawberry in Beijing, China.

Strawberry (Fragaria × ananassa) is an economically important crop in China, and a crucial part of urban agriculture in Beijing. In November 2020, wilt symptoms were observed in strawberry seedlings in several greenhouses in the Pinggu District of Beijing city (40.14° N; 117.12° E). The average disease incidence was 20%. Water-soaked lesions appeared along the veins of diseased strawberry leaves and bacterial ooze was also present on severely affected leaves. Bisected crowns had a reddish-brown discoloration in the xylem which later turned black. Three diseased strawberry seedlings were collected for pathogen identification. Isolations were conducted from stem, crown, leaf, and roots of diseased strawberry plants. Samples were surface sterilized by immersion in 70% ethanol for 30 s and rinsed three times with sterile distilled water, before being placed on potato dextrose agar (PDA) medium and incubated at 28℃. Several bacterial colonies grew on the medium after 24 h. Colonies were then purified on Lysogeny broth (LB) agar plates using the streak plate method. Twenty-nine isolates were obtained from 36 diseased tissue samples, which were from stem(10), crown(12), leaf(2) and roots(5) separately. All isolates appeared white, round, opaque and smooth on LB plates. To identify the isolates, genomic DNA was extracted from nine purified bacterial colonies (CM1 to CM9). The fragments of atpD, gyrB, infB and rpoB gene were amplified and sequenced with primers atpD 01-F/ atpD 02-R, gyrB 01-F/ gyrB 02-R, and infB 01-F/ infB 02-R (Brady et al. 2008) and RpoB-F/ RpoB-R (Mollet et al. 1997), respectively. All atpD, gyrB, infB and rpoB sequences belonging to the isolates were identical. The sequences of atpD, gyrB, infB and rpoB gene of isolates CM1 and CM3 were deposited in GenBank under accession numbers ON055247, ON055248, ON055249, ON055250, ON055251, ON055252, OL771192 and OL771193. BLAST searches were conducted with the sequences of atpD, gyrB, infB and rpoB. The atpD, gyrB, infB and rpoB sequences of the obtained isolate showed 99.53%, 99.06%, 99.19% and 99.80% identity with the corresponding sequences of Enterobacter mori strains, respectively. Phylogenetic analysis was performed using the maximum likelihood (ML) method with the CIPRES Science Gateway platform (http://www.phylo.org/) based on the combined atpD, gyrB, infB and rpoB sequences (Brady et al. 2013; Palmer et al. 2018). In the phylogenetic tree, the isolates were clustered together with E. mori strain LMG 25706. To confirm the pathogenicity, 200 µL of bacterial suspensions (108 CFU/mL) of the two isolates were injected into the crown of six healthy Fragaria × ananassa cv. Bennihope strawberry seedlings respectively with 1 mL sterilized syringe, and the control seedlings were injected with sterile water. The seedlings were kept in a moist chamber (28°C, 16-h light and 8-h dark period) for 2 days. Then all the seedlings were transferred to the greenhouse with conditions similar to those where the diseased plants were collected. Forty days after inoculation, old leaves started to wilt and leaf midvein necrosis, along with xylem discoloration, was observed in inoculated plants. No symptoms were observed in the control group. Pathogenicity tests were conducted three times with similar results. The bacteria were re-isolated from the symptomatic diseased strawberry plants and confirmed as E. mori by morphological and sequence analyses as above, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of strawberry bacterial wilt caused by E. mori. Due to the significant crop loss from this disease, more research is needed in epidemiology and disease management.

Fusarium Species Associated with Cherry Leaf Spot in China.

Sweet cherry is an important fruit crop in China with a high economic value. From 2019 to 2020, a leaf spot disease was reported, with purplish-brown circular lesions in three cultivating regions in China. Twenty-four Fusarium isolates were obtained from diseased samples and were identified based on morphological characteristics and multi-locus phylogenetic analyses. Seven species, including F. luffae (7 isolates), F. lateritium (6 isolates), F. compactum (5 isolates), F. nygamai (2 isolates), F. citri (2 isolates), F. ipomoeae (1 isolate) and F. curvatum (1 isolate) were identified. The pathogenicity test showed that analyzed strains of all species could produce lesions on detached cherry leaves. Therefore, Fusarium was proved to be a pathogen of cherry leaf spots in China. This is the first report of F. luffae, F. compactum, F. nygamai, F. citri, F. ipomoeae and F. curvatum on sweet cherry in China.

CpG usage in RNA viruses: data and hypotheses.

CpG repression in RNA viruses has been known for decades, but a reasonable explanation has not yet been proposed to explain this phenomenon. In this study, we calculated the CpG odds ratio of all RNA viruses that have available genome sequences and analyzed the correlation with their genome polarity, base composition, synonymous codon usage, phylogenetic relationship, and host. The results indicated that the viral base composition, synonymous codon usage and host selection were the dominant factors that determined the CpG bias in RNA viruses. CpG usage variation between the different viral groups was caused by different combinations of these pressures, which also differed from each other in strength. The consistent under-representation of CpG usage in -ssRNA viruses is determined predominantly by base composition, which may be a consequence of the U/A preferred mutation bias of -ssRNA viruses, whereas the CpG usage of +ssRNA viruses is affected greatly by their hosts. As a result, most +ssRNA viruses mimic their hosts' CpG usage. Unbiased CpG usage in dsRNA viruses is most likely a result of their dsRNA genome, which allows the viruses to escape from the host-driven CpG elimination pressure. CpG was under-represented in all reverse-transcribing viruses (RT viruses), suggesting that DNA methylation is an important factor affecting the CpG usage of retroviruses. However, vertebrate-infecting RT viruses may also suffer host' CpG elimination pressure that also acts on +ssRNA viruses, which results in further under-representation of CpG in the vertebrate-infecting RT viruses.

Online profiling of triacylglycerols in plant oils by two-dimensional liquid chromatography using a single column coupled with atmospheric pressure chemical ionization mass spectrometry.

The complexity of natural triacylglycerols (TAGs) in various edible oils is high because of the hundreds of TAG compositions, which makes the profiling of TAGs quite difficult. In this investigation, a rapid and high-throughput method for online profiling of TAGs in plant oils by two-dimensional (2D) liquid chromatography using a single column coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry was reported. A novel mixed-mode 2D chromatographic column packed with silver-ion-modified octyl and sulfonic co-bonded silica was employed in this online 2D separation system. This novel 2D column combined the features of C8 column and silver-ion. In comparison with the traditional C18 column and silver-ion column, which are the two main columns used for the separation of complex TAGs in natural oil samples, this novel 2D column, could provide hydrophobic interactions as well as π-complexation interactions. It exhibited much higher selectivity for the separation of TAGs, and the separation was rapid. This online 2D separation system was successful in the separation of a large number of TAG solutes, and the TAG structures were evaluated by analyzing their APCI mass spectra information. This system was applied for the profiling of TAGs in peanut oils, corn oils, and soybean oils. 30 TAGs in peanut oils, 18 TAGs in corn oils, and 21 TAGs in soybean oils were determined and quantified. The highest relative content of TAGs was LLL, which was found in corn oil with the relative content up to 45.43 (%, w/w), and the lowest relative content of TAGs was LLS and OSS, which was found in soybean oil and corn oil respectively, with the relative content only 0.01 (%, w/w). In addition, the TAG data were analyzed by principal component analysis (PCA). Results of PCA enabled a clear identification of different plant oils. This method provided an efficient and convenient chromatographic technology for the fast characterization and quantification of complex TAGs in plant oils at high selectivity. It has great potential as a routine analytical method for analysis of edible oil quality and authenticity control.

Preparation of a novel carboxyl stationary phase by "thiol-ene" click chemistry for hydrophilic interaction chromatography.

A novel carboxyl-bonded silica stationary phase was prepared by "thiol-ene" click chemistry. The resultant Thiol-Click-COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol-Click-COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol-Click-COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol-Click-COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water-soluble vitamins was achieved.