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.

First Report of Cylindrocladiella peruviana Associated with Grapevine Black Foot Disease in China.

Black foot disease is one of the Grapevine Trunk Diseases (GTDs) that occurs in many grape growing regions and causes vine decline. Black foot disease has been reported in China in 2021 (Ye et al. 2021). In May 2022, serious root rot and nearly half brown necrosis in cross section were observed in several grapevines (Vitis vinifera cv. Cabernet Sauvignon) in Xianyang County, Shaanxi Province, China, with the incidence of 2% in 0.267 hectares. Samples (12-year-old vines) of symptomatic root were collected and taken back to the laboratory. Small fragments from the margin between healthy and diseased tissue were cut into 5 mm × 5 mm pieces. The surface was sterilized using 2% NaOCl for 2 min, followed by 75% ethanol for 30 s and rinsed three times with sterilized water. After the small pieces were dried on sterilized filter paper, they were placed onto potato dextrose agar (PDA) plates with lactic acid at 25°C for 2-3 days. The pure culture was obtained by single spore isolation. In this study, two strains were associated with black foot disease. Colony characteristics were observed in 7-day PDA plates, with cotton wool mycelium and light yellow to brown colony in reverse. Conidia were cylindrical to peanut shaped, 0-1 septa and blunt round at both ends. Size of the conidia was 7.12 to 13.89 × 2.55 to 5.16 µm (average 9.82 × 3.41 µm, n=50). For molecular phylogenetic analysis, genomic DNA of the two strains (JZB3320008 and JZB3320009) was extracted. PCR amplification was performed using four phylogenetic regions (ITS, tub2, tef1 and his3) amplified with primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (O'Donnell and Cigelnik 1997; Glass and Dnoaldson 1995), EF-688F/EF-1251R (Alves et al. 2008) and CYLH3F/CYLH3R (Crous et al. 2004), respectively. Maximum likelihood (ML) inference phylogenetic tree was constructed to confirm the identity of the two strains. ML tree reveals that the two strains clustered with Cylindrocladiella peruviana with 100% bootstrap support value. The sequences of four regions were deposited in GenBank (accession numbers OQ202205-OQ202206 for ITS, OQ225938-OQ225939 for tub2, OQ225936-OQ225937 for tef1 and OQ225934-OQ225935 for his3). Based on the morphological characteristics and molecular phylogenetic analysis, the two strains were identified as C. peruviana. To confirm the pathogenicity, the rooted cuttings (cv. Marselan) in the greenhouse were inoculated by immersing the roots in the conidial suspension (106 conidia/mL; volume, 300 mL) for 30 min, while the control was immersed in sterilized water (volume, 300 mL) for the same time. Nine plants were inoculated with C. peruviana and the same number was used as the control. These grapevines were kept in the greenhouse at 25°C. After 14 days of inoculation, the aboveground of inoculated plants showed symptoms with red leaves, while the control showed no symptoms. After 34 days of inoculation, the inoculated plants showed the reduction of the number of foliage and reduced vigor. They died with brown stem base and vascular discoloration in longitudinal section, while the control showed no symptoms. Cylindrocladiella peruviana was re-isolated by the discoloration regions and the Koch's rule was verified. To our knowledge, the pathogen has been reported in Spain and California (Agustí-Brisach et al. 2012; Koike et al. 2016). This is the first report of Cylindrocladiella peruviana causing grapevine black foot disease in China. We will better diagnose and prevent the disease in the future.

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.

First Report of Fusarium commune Associated with a Root Rot of Grapevine in China.

During last decade, species belonging to Fusarium, Rosellinia, Armillaria and Dactylonectria were confirmed as phytopathogens causing grapevine root diseases (Highet and Nair 1995; Teixeira et al. 1995; Calamit et al. 2021; Ye et al. 2021). From 2020 to 2021, grapevine decline was observed in several vineyards in Beijing region, China. Leaves turned yellow with brown necrotic patches and roots were poorly developed, which was suggesting that a root disease was affecting the vines. The disease incidence was up to 10-15% of the vineyard for sample collection. Symptomatic root samples (cv. 'Red Globe') were collected and tissue fragments were excised at the margin of the symptomatic tissue in order to isolate the potential pathogen. The surface was sterilized using 1.5% sodium hypochlorite for 3 min, followed by 70% ethanol for 30 sec, and rinsed three times with sterile distilled water (Ye et al. 2020). Tissues were dried and placed onto potato dextrose agar (PDA) plates, followed by incubation at 25°C under dark conditions for 3 d. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. Next, single spores were transferred onto new PDA plates and incubated at 25°C for 7 d. Eight isolates numbered with JZB3110172 to JZB3110179 were obtained and their culture characters were identical, and the re-isolation percentage was 100%. Colonies were white to orange, with abundant fluffy aerial mycelium. Macroconidia were fusiform with a slightly curved apical cell and a foot-shaped basal cell, and measured 16.2-43.2 µm × 2.7-4.9 µm (n=50); microconidia were cylindrical, straight to slightly curved, 5.1-13 × 2.1-3.9 µm (n=50). Morphological characters of the isolates resembled to Fusarium commune (Skovgaard et al. 2003). For phylogenetic analysis, genomic DNA of the eight isolates was extracted with a DNA extraction kit (DNeasy plant Mini Kit). PCR amplifications of two phylogenetic markers (EF-1α and RPB2) were performed using the primers EF-1/EF-2 (Geiser et al. 2004) and RPB2-5F2/RPB2-7cR (Liu et al. 1995), respectively. The sequences were deposited in GenBank ON457645 to ON457660. Comparison of base pairs on Maximum likelihood (ML) phylogenetic analysis was conducted using the RAxML-HPC2 tool on XSEDE on the CIPRES Science Gateway platform (http://www.phylo.org/). The sequences of EF-1α and RPB2 of the eight isolates showed 99 to 100% similarity to the reference isolates of F. commune. In the phylogenetic tree, the isolates from this study clustered with the representative strains of F. commune (NRRL 52764, NRRL 28387 and NRRL 52744). Based on morphological characters and the phylogenetic results, all of isolates were identified as F. commune. Koch's postulates were conducted on healthy, 3-month-old grapevine 'Marselan'. Plant roots were trimmed with sterile scissors and then soaked in a spore suspension (1.0 × 106 spores mL-1) or sterile water (as the control) for 30 min. The inoculated grapevines were transplanted into pots and kept in the greenhouse at 25°C. After 14 days, all the inoculated plants developed necrosis and turned yellow. No symptoms were observed on the control. Koch's postulates were fulfilled by re-isolating the fungus from necrotic root tissues. The isolates obtained from the artificially infected tissue were identified again as F. commune based on morphological and molecular analyses. Overall, this is the first report of F. commune associated with a grapevine root rot globally, which lays a foundation for further study and developing disease control methods.

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.

A Putative Effector LtCSEP1 from Lasiodiplodia theobromae Inhibits BAX-Triggered Cell Death and Suppresses Immunity Responses in Nicotiana benthamiana.

Lasiodiplodia theobromae is a causal agent of grapevine trunk disease, and it poses a significant threat to the grape industry worldwide. Fungal effectors play an essential role in the interaction between plants and pathogens. However, few studies have been conducted to understand the functions of individual effectors in L. theobromae. In this study, we identified and characterized a candidate secreted effector protein, LtCSEP1, in L. theobromae. Gene expression analysis suggested that transcription of LtCSEP1 in L. theobromae was induced at the early infection stages in the grapevine. Yeast secretion assay revealed that LtCSEP1 contains a functional signal peptide. Transient expression of LtCSEP1 in Nicotiana benthamiana suppresses BAX-trigged cell death and significantly inhibits the flg22-induced PTI-associated gene expression. Furthermore, the ectopic expression of LtCSEP1 in N. benthamiana enhanced disease susceptibility to L. theobromae by downregulating the defense-related genes. These results demonstrated that LtCSEP1 is a potential effector of L. theobromae, which contributes to suppressing the plant's defenses.

High Genetic Diversity and Species Complexity of Diaporthe Associated With Grapevine Dieback in China.

Grapevine trunk diseases have become one of the main threats to grape production worldwide, with Diaporthe species as an emerging group of pathogens in China. At present, relatively little is known about the taxonomy and genetic diversity of Chinese Diaporthe populations, including their relationships to other populations worldwide. Here, we conducted an extensive field survey in six provinces in China to identify and characterize Diaporthe species in grape vineyards. Ninety-four isolates were identified and analyzed using multi-locus phylogeny. The isolates belonged to eight species, including three novel taxa, Diaporthe guangxiensis (D. guangxiensis), Diaporthe hubeiensis (D. hubeiensis), Diaporthe viniferae (D. viniferae), and three new host records, Diaporthe gulyae (D. gulyae), Diaporthe pescicola (D. pescicola), and Diaporthe unshiuensis (D. unshiuensis). The most commonly isolated species was Diaporthe eres (D. eres). In addition, high genetic diversity was observed for D. eres in Chinese vineyards. Haplotype network analysis of D. eres isolates from China and Europe showed a close relationship between samples from the two geographical locations and evidence for recombination. In comparative pathogenicity testing, D. gulyae was the most aggressive taxon, whereas D. hubeiensis was the least aggressive. This study provides new insights into the Diaporthe species associated with grapevines in China, and our results can be used to develop effective disease management strategies.