Irregular Brown Spots Research Articles

First report of Fusarium equiseti causing leaf spots of Bitter gourd (Momordica charantia) in Pakistan.

Bitter gourd (Momordica charantia L.) is an important vegetable crop of the Cucurbitaceae family widely cultivated in Pakistan and around the world. In October 2020, a nutrition management trial of Bitter gourd cv. Seminis-200) was conducted on an area of 10,860 sq. ft. (99×110 feet) at the Agricultural Research farm of Bahauddin Zakariya University, Multan (30.2601° N, 71.5158° E), Pakistan. Symptoms of large, brown necrotic leaf spots were observed on the leaves of bitter gourd vines. The disease started from the yellowing of leaves within the reticulate venation and turned brown. Irregular brown leaf spots coalesced to form large necrotic areas followed by foliar chlorosis then wilting that occurred very late. There were no crown rot symptoms although there was slight discoloration of roots and when cut longitudinally, browning of tissues was observed. The disease was assessed visually with 37% incidence which resulted in poor quality and yield in terms of reduced size and yellowing of fruit. Infected vines along with the roots were collected for the isolation of pathogen. A total of 34 leaves and 22 root samples were collected from the field for isolation. The leaf, collar and root portions were cut into 0.5 to 1 cm in length and surface disinfected with 1% sodium hypochlorite (NaOCl) for 2-3 minutes followed by washing twice with autoclaved distilled water and after drying, placed on potato dextrose agar (PDA) medium, and incubated at 25±2 °C for one week. The fungal colonies of fluffy white growth with light orange pigment were isolated. For morphological characterization, a total of 4 pure cultures were isolated from leaves, collar region and root by single spore technique on carnation leaf agar (CLA) medium after 15 days of incubation at 25±2℃. Curved and thick-walled macroconidia with elongated or pointed apical characteristic foot-shaped basal cells were produced in sporodochia. Macroconidia with 5-7 septa measured 22.50-41.80 μm × 2.90-4.20 μm (n = 60). Thick, brown with roughened walls and subglobose ellipsoidal chlamydospores were observed in clumps or chains with the dimension of 5.8 to 10.8 μm (n = 20). On morphological characteristics, the fungus was identified as Fusarium equiseti (Corda) Sacc. according to Leslie and Summerell (2006). Two single spore isolates were used for molecular identification by amplifying ribosomal DNA of the internal transcribed spacer (ITS) region with ITS1/ITS4 primers (White et al. 1990) and for β-tubulin gene region, primers T1/Bt-2b (O'Donnell and Cigelnik, 1997) were used. The obtained sequences were deposited in GenBank with accession numbers MW880179 and MW880198 from the ITS region and BLAST search in GenBank showed 100 and 98.11% alignment with previously published sequences of F. equiseti with accessions OM992323.1and MT558569.1 respectively. Accession number OM867571from the β-tubulin region showed 100% sequence similarity with F. equiseti with accession MN653163.1. For pathogenicity, macroconidia from 2-week-old cultures on CLA medium were harvested to prepare spore suspension (1 × 106 conidia/ml). Koch's postulates were confirmed on nine bitter gourd plants (cv. Seminis-200) by applying spore suspension of fungal inoculum at 3-4 leaf stage separately on leaves by automizer, on collar region after making incision spore suspension was applied and in the root zone, 20ml spore suspension was added whereas distilled water was used as a control with three replications. Plants were kept under controlled conditions in the greenhouse with 65% to 75% humidity and the temperature was maintained at 32±2 °C for one week. After 7-8 days, inoculated plants began to exhibit symptoms of brown, necrotic leaf spots on the leaves of bitter gourd vines followed by yellowing of leaves that eventually turned brown. Roots showed slight discoloration and browning of vascular bundles and finally, the plants wilted after four weeks. while control plants remained symptomless. The symptoms resembled those noticed in the field. The fungus was re-isolated from leaves, collar region and roots, followed by morphological identification, and finally confirmed as F. equiseti. To the best of our knowledge, this is the first report of a leaf spot caused by F. equiseti in a bitter gourd from Pakistan. If the disease is not managed properly, it may cause a drastic effect on yield under favorable environmental conditions. The pathogen may also damage other cucurbitaceous crops cultivated in the area.

First Report of Neopestalotiopsis piceana Causing Gray Blight in Camellia sinensis L. in China.

Tea plants (Camellia sinensis L.) are an important cash crop and are cultivated worldwide for their commercial value (Palanisamy et al. 2014). Tea gray blight is an important tea plant disease as it can cause a decline in tea quality and reduce yields by 20-30% (Sanjay et al. 2008). In August 2018, a disease survey was conducted on 400 ha of organic tea plantations in the Pu'er area of Yunnan Province (22.48° N, 100.58° E). The survey found that widespread disease was causing damage to 40% of the tea plantations and that the most seriously affected tea variety was Yunkang No. 10, which had an average disease incidence of 30-35%. The affected leaves grew small yellow-green spots on their tips or margins in the early stage that expanded into round or irregular brown spots with distinct concentric whorls and black conidial disks arranged in whorls when the humidity was high (Fig. 1A-B), which is consistent with tea gray blight disease (Zheng et al. 2021). Twenty-four diseased leaf samples were collected from four different tea plantations and transported to the Pu-Erh Tea Research Laboratory. Leaves with disease spots were cut into 4 mm ×4 mm square pieces, surface-sterilized with 75% alcohol for 1 min, disinfected with 1% sodium hypochlorite for 3 min, and washed thrice with sterile water. The tissue pieces were placed on potato dextrose agar (PDA) plates containing 100 µg ml-1 of chloramphenicol (Wang et al. 2021). After 3 d of culturing in the dark at 28 C, twenty pure cultures with similar morphology were obtained, and two representative isolates were selected and transferred into new PDA media. After 7 d, circular fungal colonies with dense aerial mycelium produced black, wet spore masses that grew on the PDA media (Fig. 1C-D). The conidia were spindle-shaped with four septa, measuring 25.0 (21.0-26.0) × 6.0 (4.5-7.0) µm (n=15). The conidia had three median cells, two of which were dark brown in color with unclear separations, with a single basal hyaline appendage 3.8 (3.5-4.5) µm (n=30) in length and 2-3 apical hyaline appendages 31 (27-35) µm in length (n=30) (Fig. 1E), similar to the conidial characteristics of Neopestalotiopsis piceana (Maharachchikumbura et al. 2014). Two isolates were selected for DNA extraction. The internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha (tef1-α) gene, and partial β-tubulin (tub2) gene were amplified using the ITS1F-ITS4 primer set (White et al 1990), the EF-1α-F and EF-1α-R primer sets (Li et al. 2018), and the tub1 and tub2 primers, respectively (Chauhan et al. 2007). The ITS (OP535632 to OP535632), tef1-α (OP589285,OP589287), and tub2 (OP589286,OP589288) sequences were submitted to NCBI GenBank. Basic Local Alignment Search Tool analysis demonstrated that these sequences were 100% similar to those of N. piceana isolates available in GenBank. The sequences were compared using the Mafft software package, and sequences with the same ID were concatenated using scripts. A maximum likelihood phylogenetic tree was constructed using the MEGA (ver. 5.1) software package based on the concatenated sequences (ITS, tef1-α, and tub2). Phylogenetic analysis revealed that C-5 and B-3 showed 95% bootstrap support with N. piceana isolates in references (Fig. 2). According to the morphology and molecular characterization, C-5 and B-3 were identified as N. piceana. Pathogenicity tests on these two isolates were conducted using 36 healthy tea plants. The leaves were scratched slightly with sterile toothpick tips, after which pathogen cakes (6 mm diameter) were placed on the wounds with the mycelial side facing down and covered with sterile absorbent cotton to maintain a moist environment. Control leaves were wounded and covered with sterile PDA plugs (three replicates per treatment, three plants per replicate). Seven days later, the inoculated leaves exhibited similar symptoms observed under natural conditions, whereas the control leaves exhibited no symptoms. The same isolates as the introduced strains were isolated from the diseased tea leaves, completing Koch's postulates. To our knowledge, this is the first report of N. piceana causing gray blight on tea leaves in China. These results provide valuable information for the prevention and management of gray blight on tea leaves.

First Report of Leaf Blight Caused by Fusarium pernambucanum and Fusarium sulawesiense on Plum in Sichuan, China.

Plum (Prunus salicina Lindl.) is widely cultivated for its rich nutrients and flavor in China. In August 2020, leaf blight symptoms were observed on plum in Meishan, Sichuan, China (N29°24', E104°30'). Irregular brown spots initially appeared on the edge or tip of the leaf, then extended to larger taupe lesions that were surrounded by a chlorotic halo. In the late stage, grey-brown blighted tissue covered the entire leaf causing leaves to wither, curl and abscise. The leaves with blight were collected from three different towns in Meishan where the disease incidence was found on 15-30% of plum plants. The margin of diseased leaves was cut into small pieces (3×3 mm), surface disinfected with 75% ethanol solution for 10 s, 2% NaOCl for 1 min, and rinsed in sterile distilled water three times. Tissue pieces were plated on potato dextrose agar (PDA) and incubated at 25°C. Forty-nine morphologically similar colonies were observed on PDA plates after 3-5 days and three of these (TEY9-1, TEY12-1, TEY15A-1) were selected for intensive study. The colonies produced abundant whitish to yellowish aerial mycelium after 7 days incubation at 25°C in the dark. Macroconidia on carnation leaf agar (CLA) were falcate, hyaline, straight to slightly curved, smooth to slightly rough with 3 to 6 septa, the apical cell was blunt or hooked, and the basal cell was barely notched, 31.6 ± 2.4 μm × 4.7 ± 0.4 μm, 28.9 ± 3.0 μm × 4.5 ± 0.5 μm, 32.5 ± 3.4 μm × 4.5 ± 0.5 for TEY9-1, TEY12-1, TEY15A-1, respectively. Microconidia were hyaline, fusoid or ovoid, nonseptate or one-septate, 14.4 ± 3.9 μm × 4.3 ± 0.6 μm, 13.0 ± 3.0 μm × 4.0 ± 0.4 μm, 11.0 ± 2.4 μm × 3.7 ± 0.5 for TEY9-1, TEY12-1, TEY15A-1, respectively. Genomic DNA was extracted from 7-day-old aerial mycelia of these isolates. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM) and partial RNA polymerase second largest subunit (RPB2) were amplified using primers ITS4/ITS1, EF1/EF2, CL1/CL2A, and 5f2/7cr, respectively (White et al. 1990; O'Donnell et al. 2000, 2010). Sequences were deposited in GenBank (ITS: OK315638-OK315640; TEF1: OK338756-OK338758; CAM: OK338759-OK338761; RPB2: OK338762-OK338764). A maximum Likelihood (ML) phylogenetic tree was constructed with RAxML version 8.2.10 based on the concatenated sequences (ITS, TEF1, CAM, RPB2). According to morphology and phylogenetic analysis, TEY9-1 and TEY15A-1 were identified as Fusarium pernambucanum, and TEY12-1 was identified as Fusarium sulawesiense. Pathogenicity tests were conducted on young healthy leaves of 12 two-year-old plum plants in a 28°C greenhouse in Nanning, Guangxi, China. The epidermis of tested leaves was slightly scratched with sterile toothpick-tips forming a 3-mm-diameter cross-shaped wound, followed by inoculation with a 10 μl conidial suspension (106 spores /ml in 0.1% sterile Tween 20). Control leaves were wounded in the same way and treated with 0.1% sterile Tween 20. Plants were covered with polythene bags to maintain high humidity for 5 days. Inoculated leaves showed light brown to dark brown lesions, whereas control leaves were symptomless. Both species were re-isolated from symptomatic leaves, completing Koch's postulates. To our knowledge, this is the first report of F. pernambucanum and F. sulawesiense causing leaf blight on plum trees in China. These results will provide valuable information for prevention and management of leaf blight on plum trees.

First Report of Neopestalotiopsis cubana Causing leaf blight on Ixora chinensis in Malaysia.

Ixora chinensis (family Rubiaceae), locally known as 'Bunga Jejarum', is widely grown as an ornamental shrub and as sources for phytochemicals with medicinal properties in Malaysia. In May 2021, irregular brown spots were found on the leaves of some 'Bunga Jejarum' in Universiti Malaysia Sabah (6°02'01.0"N 116°07'20.2"E) located in Sabah province. As the disease progressed, the spots enlarged and coalesced into large necrotic areas giving rise to drying of infected leaves. The disease severity was about 70% with 20% incidence. Five symptomatic leaves (5 x 5 mm) from five plants were excised and sterilized based on Khoo et al. (2022) before plated on five potato dextrose agar (PDA) and cultured at 25°C. After 5 days, white to pale honey and dense mycelia with lobate edge were observed on all PDA plates. Globose, black conidiomata semi-immersed on PDA were observed after a week. Two to four hyaline filamentous appendages 7.7 to 17.6 μm long attached to fusoid conidia (11.8 to 20.9 x 5.7 to 7.6 μm, n = 20), which consisted of a hyaline apical cell, basal cell, and three versicolored median cells. The upper two median cells were dark brown, while the lowest median cell was pale brown. The isolate of the causal pathogen was characterized molecularly. Genomic DNA of isolate UMS01 was extracted based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS), tubulin (TUB) and translation elongation factor 1-α (TEF) region was performed based on Khoo et al. (2022) using primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) and EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999), respectively. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The isolate's sequences were deposited in GenBank as OM320626 (ITS), OM339539 (TUB) and OM339540 (TEF). They were 99% to 100% identical to ITS(KM199347) (545 out of 545 bp), TUB (KM199438) (768 out of 769 bp) and TEF (KM199521) (480 out of 481 bp) of the type sequences (CBS 600.96). Phylogenetic analysis using the maximum likelihood method based on the combined ITS, TEF and TUB sequences placed the isolate UMS01 in the same clade as the isolate CBS 600.96 of Neopestalotiopsis cubana. Thus, the pathogen was identified as N. cubanabased on the morphological description from Pornsuriya et al. (2020), molecular data in Genbank database and multigene sequence analysis. To further confirm its pathogenicity, the first and second leaves of three 'Bunga Jejarum' plants were inoculated by pipetting 1 ml aliquots of a 1 × 106 conidia/ml spore suspension. Three additional 'Bunga Jejarum' plants were mock inoculated by pipetting 1 ml of sterile distilled water on similar age leaves. The plants were covered with plastic bags after inoculation for 48 h before placing them in a glasshouse under room temperature. The leaves were sprayed with water to keep the leaf surfaces moist along the experiment. The incubation and disease observation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). After 7 days post-inoculation, all infected leaves exhibited the symptoms observed in the field, whereas the controls showed no symptoms. The same fungus was isolated from the diseased leaves and, thus confirmed Koch's postulates. The experiment was repeated two more times. The reisolated fungi were visually and genetically identical to the original isolate obtained from the field samples. To our knowledge, this is the first report of N. cubana causing leaf blight on 'Bunga Jejarum' in Malaysia, as well as the world. Our finding has broadened the distribution and host range of N. cubana, indicating that it poses potential damage to the medicinal plant Bunga Jejarum in Malaysia.

The First Report of Juglans regia Leaf Spot Caused by Fusarium proliferatum in China.

Juglans regia is planted widely in China because of its significant economic value as a woody oil species. In August 2019, leaf spots were discovered in a walnut orchard in Tang County, Baoding City, Hebei Province. In the early stages of the disease, irregular brown spots appeared on the edges of leaves, causing them to shrivel; in a later stage, the disease spread inward along the edges to the whole leaves, which then withered. The diseased leaves were collected from the walnut orchard. Select 3-4 mm2 of diseased and healthy junction for each diseased leaf, and the surfaces were sterilized with 75% ethanol for 30 s and 0.1% HgCl2. The leaves were incubated on potato dextrose agar (PDA) at 28 ℃ in darkness, each diseased leaf was repeated 10 times, and purified after 6 days. The diameters of the suspected Fusarium isolates reached 8.3 cm, and the aerial mycelia were white to cream and wool-like, later turning violet and dark purple with age. On PDA, the macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). For molecular analysis, DNA was extracted from the mycelium and used the specific primers to amplify the translation elongation factor EF-1α, and RNA polymerase II largest-subunit (RPB2) further. The amplicons were sequenced and deposited in GenBank under accession numbers MZ891582, and MZ891583. BLAST analysis revealed that all these sequences showed more than 99% sequence identification with F. proliferatum. We used Fusarium MLST for the identification of this strain and the results showed that EF-1α and RPB2 genes have more than 99% similarity with the database F. proliferatum data, with 100% sequence duplication(Deposited of EF-1α in GenBank under accession numbers: MH582346, and MH582347; Deposited of RPB2 in GenBank under accession numbers: MH582193, MH582151, MH582196, and MH582172). We created a multigenic tree using Fusarium MLST. Based on the results of the morphology tests and the molecular sequences, the isolates from the diseased leaves were identified as F. proliferatum. To confirm the pathogenicity, healthy walnut leaves of tree planted in pot were inoculated using 50 µL of conidia suspension (1.0×107 CFU) from a pure clone, and those inoculated using only distilled water were taken as controls. All treatments were repeated three times and placed under 28°C, RH 100% for 5 days. The inoculated plants showed the same symptoms as those observed in the original diseased plants, while the control plants were asymptomatic. The same fungus was isolated again from the inoculated leaves of tree planted in pot. The re-isolated pathogen from diseased plants was identical to the purified strain used for inoculation, confirmed Koch's postulates. According to the above results, F. proliferatum was identified as the pathogen behind the Juglans regia leaf spots, and it is clearly different from other pathogens, such as Colletotrichum gloeosporioides. It is reported that F. proliferatum can lead to necrotic leaf lesions and the bulb rot of stored onions (Beck et al., 2020), as well as light brown discoloration and dryness on palm leaves and branches (Alananbeh et al., 2021). At the same time, F. proliferatum has been found to have the highest colonization ability on jujube leaves and is becoming a serious pathogen on jujube (Saleh et al., 2016). To our knowledge, this is the first report of walnut leaf spots caused by F. proliferatum in China.

First Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in China.

HomePlant DiseaseVol. 106, No. 6First Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in ChinaYue Pan, Pengfei Wang, Wandong Chen, Guichai Yang, Yingjie Gu, Junwei Wang, and Juan WangYue Panhttps://orcid.org/0000-0002-2092-2366Eco-development Academy, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Pengfei WangCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Wandong ChenCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Guichai YangCollege of Garden and Horticulture, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Yingjie GuCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Junwei WangSchool of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, and Juan Wang†Corresponding author: J. Wang; E-mail Address: schima@163.comEco-development Academy, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Yue Pan1 Pengfei Wang2 Wandong Chen2 Guichai Yang3 Yingjie Gu2 Junwei Wang4 Juan Wang1 † 1Eco-development Academy, Southwest Forestry University, Kunming 650224, China 2College of Forestry, Southwest Forestry University, Kunming 650224, China 3College of Garden and Horticulture, Southwest Forestry University, Kunming 650224, China 4School of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, China Published Online:11 May 2022https://doi.org/10.1094/PDIS-09-21-2038-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleMalania oleifera Chun & S. K. Lee is an endangered plant species in China with rich nervonic acid in seeds, and, thus, has important ecological and economic value (Fu 1992; Tang et al. 2013). Brown leaf spots were observed on 3-year-old M. oleifera plants in summer 2019 in Guangnan County, Yunnan Province, China (24°05′26″N, 105°05′07″E). A total of 100 trees within 0.3 ha of mountainous area were investigated, and eight (8%) showed disease symptoms. Initially, small, brown irregular spots appeared on the tip and margin of the leaf, and then gradually expanded to the whole leaf. Ten symptomatic leaves were randomly collected from different plants for fungal isolation. They were washed with running tap water, sterilized with 75% ethanol for 3 min and 1% NaOCl for 2 min, and rinsed in sterile distilled water three times. The leaves were cut into 5 × 5 mm2 segments and cultured on potato dextrose agar (PDA) at 25°C in the dark after air drying. In total, 12 morphologically similar fungal isolates were obtained. Colonies of the fungal culture initially appeared white, turned gray from the center between 3 to 6 days, and then became dark gray with cottony aerial mycelium 9 days later. To induce sporulation, the cultures were placed on 2% water agar (WA) with autoclaved pine needles and were incubated at 25°C under near-ultraviolet light as described by Chen et al. (2011). Globose conidiomata developed on pine needle surfaces after 10 days. Conidia were hyaline, unicellular, aseptate, fusiform with apex acute and base truncate, and 18.62 to 26.14 × 4.80 to 7.63 μm (average 21.77 × 5.91 μm, n = 50). The morphological characteristics resembled those of Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves (Phillips et al. 2013). The internal transcribed spacer (ITS) region and the translation elongation factor 1-α gene (EF1-α) regions of a representative isolate (CGMCC3.20320) were amplified using primers ITS1F/ITS4 and EF1-728F/EF1-986R (Carbone and Kohn 1999; White et al. 1990), respectively. The obtained sequences (GenBank accession nos. MW642163 and MW651965) showed 99.80% (identity = 505/506) and 99.27% (identity = 272/274) similarity with the corresponding genes of ex-type CMW 27094 of Fusicoccum fabicercianum S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou, now B. fabicerciana (GenBank accession nos. HQ332197 and HQ332213). The pathogenicity of the selected isolate (CGMCC3.20320) was evaluated by leaf inoculation of 1-year-old M. oleifera seedlings. Mycelial plugs (5 mm) of B. fabicerciana from a 7-day-old culture were applied on leaves wounded with sterile scalpels. Controls were inoculated with sterile PDA plugs. Each treatment was repeated six times. Inoculated and control plants were kept in a greenhouse at 25°C and 80% relative humidity, with a 12-h photoperiod. After 10 days, irregular brown spots appeared on all inoculated leaves, whereas there were no spots from control plants. Botryosphaeria fabicerciana was reisolated only from symptomatic leaves, fulfilling Koch’s postulates. Previous studies indicated that this fungus can cause stem lesions on Eucalyptus and trunk cankers on Carya cathayensis in China (Chen et al. 2011; Zhuang et al. 2021). To our knowledge, this represents the first report of B. fabicerciana causing leaf spot on M. oleifera in China.The author(s) declare no conflict of interest.

First Report of Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China.

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander fields in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King's B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer's recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml-1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

First Report of Coniella castaneicola Causing Leaf Blight on Blueberry (Vaccinium virgatum) in China.

Blueberry (Vaccinium virgatum), an economically important small fruit crop, is characterized by its highly nutritive compounds and high content and wide diversity of bioactive compounds (Miller et al. 2019). In September 2020, an unknown leaf blight disease was observed on Rabbiteye blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E). Disease surveys were conducted at that time, the results showed that disease incidence was 90% from a sampled population of 100 plants in the field, and this disease had not been found at other cultivation fields in Nanchang. Leaf blight disease on blueberry caused the leaves to shrivel and curl, or even fall off, which hindered floral bud development and subsequent yield potential. Symptoms of the disease initially appeared as irregular brown spots (1 to 7 mm in diameter) on the leaves, subsequently coalescing to form large irregular taupe lesions (4 to 15 mm in diameter) which became curly. As the disease progressed, irregular grey-brown and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath and finally caused dieback and even shoot blight. To identify the causal agent, 15 small pieces (5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface-sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, and then incubated on potato dextrose agar (PDA) at 28°C for 5-7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were white with sparse creeping hyphae. Pycnidia were spherical, light brown, and produced numerous conidia. Conidia were 10.60 to 20.12 × 1.98 to 3.11 µm (average 15.27 × 2.52 µm, n = 100), fusiform, sickle-shaped, light brown, without septa. Based on morphological characteristics, the fungal isolates were suspected to be Coniella castaneicola (Cui 2015). To further confirm the identity of this putative pathogen, two representative isolates LGZ2 and LGZ3 were selected for molecular identification. The internal transcribed spacer region (ITS) and large subunit (LSU) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004) and LROR/LR7 (Castlebury and Rossman 2002). The sequences of ITS region (GenBank accession nos. MW672530 and MW856809) showed 100% identity with accessions numbers KF564280 (576/576 bp), MW208111 (544/544 bp), MW208112 (544/544 bp) of C. castaneicola. LSU gene sequences (GenBank accession nos. MW856810 to 11) was 99.85% (1324/1326 bp, 1329/1331 bp) identical to the sequences of C. castaneicola (KY473971, KR232683 to 84). Pathogenicity was tested on three blueberry varieties ('Rabbiteye', 'Double Peak' and 'Pink Lemonade'), and four healthy young leaves of a potted blueberry of each variety with and without injury were inoculated with 20 μl suspension of prepared spores (106 conidia/mL) derived from 7-day-old cultures of LGZ2, respectively. In addition, four leaves of each variety with and without injury were sprayed with sterile distilled water as a control, respectively. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH greater than 80%). After 4 days, all the inoculated leaves started showing disease symptoms (large irregular grey-brown lesions) as those observed in the field and there was no difference in severity recorded between the blueberry varieties, whereas the control leaves showed no symptoms. The fungus was reisolated from the inoculated leaves and confirmed as C. castaneicola by morphological and molecular identification, fulfilling Koch's postulates. To our knowledge, this is the first report of C. castaneicola causing leaf blight on blueberries in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing effective control strategies, reducing economic losses in blueberry production, and promoting the development of the blueberry industry.

First Report of Tomato Brown Rugose Fruit Virus Infecting the Tomato Crop in Saudi Arabia.

Tomato (Solanum lycopersicum L.) is the most economically important member of family Solanaceae and cultivated worldwide and one of the most important crops in Saudi Arabia. The aim of this study is screening of the most common viruses in Riyadh region and identified the presence of tomato brown rugose fruit virus (ToBRFV) in Saudi Arabia. In January 2021, unusual fruit and leaf symptoms were observed in several greenhouses cultivating tomatoes commercially in Riyadh Region, Saudi Arabia. Fruit symptoms showed irregular brown spots, deformation, and yellowing spots which render the fruits non-marketable, while the leaf symptoms included mottling, mosaic with dark green wrinkled and narrowing. These plants presented the symptoms similar to those described in other studies (Salem et al., 2015, Luria et al., 2017). A total 45 Symptomatic leaf samples were collected and tested serologically against suspected important tomato viruses including: tomato chlorosis virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato chlorotic spot virus, tomato aspermy virus, tomato bushy stunt virus, tomato black ring virus, tomato ringspot virus, tomato mosaic virus, pepino mosaic virus and ToBRFV using Enzyme linked immunosorbent assay (ELISA) test (LOEWE®, Biochemica, Germany), according to the manufacturers' instructions. The obtained results showed that 84.4% (38/45) of symptomatic tomato samples were infected with at least one of the detected viruses. The obtained results showed that 55.5% (25/45) of symptomatic tomato samples were found positive to ToBRFV, three out of 25 samples (12%) were singly infected, however 22 out of 45 (48.8%) had mixed infection between ToBRFV and with at least one of tested viruses. A sample with a single infection of ToBRFV was mechanically inoculated into different host range including: Chenopodium amaranticolor, C. quinoa, C. album, C. glaucum, Nicotiana glutinosa, N. benthamiana, N. tabacum, N. occidentalis, Gomphrena globosa, Datura stramonium, Solanum lycopersicum, S. nigrum, petunia hybrida and symptoms were observed weekly and the systemic presence of the ToBRFV was confirmed by RT-PCR and partial nucleotide sequence. A Total RNA was extracted from DAS-ELISA positive samples using Thermo Scientific GeneJET Plant RNA Purification Mini Kit. Reverse transcription-Polymerase chain reaction (RT-PCR) was carried out using specific primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 bp of Open Reading Frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp). (Luria et al. 2017). RT-PCR products were analyzed using 1.5 % agarose gel electrophoresis. RT-PCR products were sequenced in both directions by Macrogen Inc. Seoul, South Korea. Partial nucleotide sequences obtained from selected samples were submitted to GenBank and assigned the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 98.99 % to 99.50 % among them and 98.87-99.87 % identity with ToBRFV isolates from Palestine (MK881101 and MN013187), Turkey (MK888980, MT118666, MN065184, and MT107885), United Kingdom (MN182533), Egypt (MN882030 and MN882031), Jordan (KT383474), USA (MT002973), Mexico (MK273183 and MK273190), Canada (MN549395) and Netherlands (MN882017, MN882018, MN882042, MN882023, MN882024, and MN882045). To our knowledge, this is the first report of occurrence of ToBRFV infecting tomato in Saudi Arabia which suggests its likely introduction by commercial seeds from countries reported this virus and spread in greenhouses through mechanical means. The author(s) declare no conflict of interest. Keywords: Tomato brown rugose fruit virus, tomato, ELISA, RT-PCR, Saudi Arabia

Fiber-optic laser energy for pulmonary artery denervation: results of an experimental study

Abstract Background/Introduction Pulmonary artery denervation (PADN) is an interventional procedure aiming to modulate and correct pulmonary artery (PA) pressure. New technologies and energy sources are being tested currently. However, the effects of laser energy on the PA wall are unknown. Purpose To assess the acute effects and impact of laser energy for PADN procedure in normotensive sheep by applying different power settings and session times. Methods A total of 10 normotensive Katumsky sheep were included in the experiment. Percutaneous vascular access was performed under general anesthesia. A fiberoptic open-irrigated non-steerable catheter was introduced via a femoral vein through a steerable sheath. Laser applications were applied in the PA trunk and the proximal areas of the right and left PA under fluoroscopic guidance with a 5mm distance between points in the anterior, posterior and lateral walls. Applications were delivered with power 10–30W, 10–35s in duration; irrigation flow 40ml/min. After the procedure, experimental animals were euthanized and underwent an autopsy. PA samples were obtained regardless of the absence of visible laser-related lesions for histological analysis (hematoxylin staining) and immunohistochemical labelling (S100). Results A total of 108 ablation sessions were performed, 33 in the right PA, 30 in the left PA and 42 in the PA trunk. During macroscopic examination, laser-related lesions described as irregular brown hemorrhage spots and rough defects observed in the PA endothelium were not homogenous in all experimental animals. Thermal injuries either in the left or right lung lobes were identified in 5 (50%) experimental animals when using 30W during 10–20sec. In 5 (50%) sheep no collateral lung injuries were identified when using 10–20W from 20–35s. A total of 64 PA fragments underwent microscopic examination, acute thermal tissue lesions were observed in all experimental animals despite the absence of laser-related lesions in the PA endothelium; dissection, edema, disruption trough tunica layers, hemorrhage and necrosis at different depth walls. The most frequent nerve damage was obtained with 20W ablation: 5/8 PAs vs, 1/6 with 10–15W and 1/14 with 25–30W (P=0.01). At the same time, there was no difference in intima necrosis between the groups (1/6, 2/8 and 4/14 for 10–15, 20 and 25–30W groups, respectively). Conclusion(s) Percutaneous PA laser ablation is feasible, reduction of perivascular nerve expression is seen most frequently, when 20W/20–35 s ablation is performed. PA lesions may differ in depth and characteristics, and perivascular nerve damage might be seen in cases with preserved intima. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant of the Ministry of Science and Higher Education of the Russian Federation

First Report of Pestalotiopsis microspora Causing Leaf Blight of Banana in Bangladesh.

Banana (Musa sp.) is one of the most important fruit crops in the world. It is also a very popular fruit crop and cultivated widely in Bangladesh. Banana diseases are one of the limiting factors for its production. Recently, a new banana leaf blight was detected in various districts of Gazipur, Bangladesh, with an incidence of 5 to 10% in June 2020 and 15 to 20% in January 2021. The initial symptoms of the disease were narrow dark brown lesions (2 to 5 x 5 to 12 mm) that later became irregular brown spots (10 to 15 x 5 to 7 mm). The lesions gradually spread from the middle of the leaf to the margin and dark brown spots appeared on the diseased leaves. There was a clear golden yellowish boundary around the spot. Blighted tissue often covered one-thirds to one-half of the infected leaves. To isolate the pathogen, 100 diseased leaves were collected from five different locations in Gazipur, Bangladesh. Small pieces (5x5 mm) were cut from the margins of lesions and surface sterilized with 75% ethanol for 30 s followed by 1% NaClO for 1 min. The samples were then rinsed three times with sterile distilled water, dried on sterilized filter paper, and placed on 1% water agar at 25°C for 7 days. Hyphal tips were then excised, placed on PDA and incubated at 25°C for 7 days. From the 20 isolates obtained, the colony characteristics and conidial morphology of three isolates, BLS_BU1, BLS_BU2, and BLS_BU3, were examined. The colony of all of three isolates was white, cottony, and circular in growth. The acervuli were black and covered all the culture plate. Conidia of all the isolates were 5-celled, fusiform, and wider in the middle than the apical and basal cells. The three internal cells were olivaceous, concolourous, and slightly constricted at the septae; the terminal cells were hyaline (Shi et al. 2015). The conidium measured 20.3 ± 1.2 to 22.7 ± 1.8 by 7.2 ± 0.6 to 8.6 ± 0.4 μm. The mean lengths of the two to four hyaline apical appendages were 20.5 to 30.25 μm and the hyaline basal appendage was 6.1 to 7.6 μm (Han et al. 2019). DNA was extracted from the fungal colony using a DNeasy Plant Mini Kit (Qiagen). PCR was performed with primers ITS1/ITS4 ITS region and Sanger sequenced by Macrogen (Seoul, Korea) (White et al. 1990). Isolates BLS_BU1 (MH707065), BLS_BU2 (MH707065), and BLS_BU3 (MH712283) had a sequence similarity of over 99% with GenBank reference Pestalotiopsis microspora (MH707065) of National Center for Biotechnology Information (NCBI) database. The reconstructed phylogenetic tree also confirmed their phylogenetic position. To assess pathogenicity, the leaves of four 2-month-old healthy plants were needle wounded and inoculated with a spore suspension (106 spores/ ml) harvested from 10-day-old cultures of isolate BLS_BU1. As a control treatment, five leaves of two healthy plants were inoculated with sterile distilled water. Treated plants were maintained in the field with average temperature ranging from 28°C to 30°C and relative humidity from 75% to 80%. After 2 weeks, all inoculated leaves produced characteristic symptoms similar to those observed in the field. However, the control plants did not develop any symptoms. Pestalotiopsis microspora from the inoculated leaves was successfully reisolated, thus confirmed that P. microspora was the cause of leaf blight of banana. To our knowledge, banana appears to be a new host of P. microspora. this This is the first report of leaf blight of banana caused by P. microspora in Bangladesh.

First report of bacterial leaf spot disease on sunflower (Helianthus annuus) caused by Pseudomonas viridiflava in South Korea.

In September 2019, bacterial leaf spot symptoms were observed on sunflowers in an experimental field in Eumseong, South Korea. The leaves of infected plants initially showed irregular brown spots surrounded by haloes; as the disease progressed, the spots became enlarged and darkened (eXtra Fig. 1a). At the flowering stage, leaves became dry and showed signs of blight including defoliation; dark brown spots were also observed on sunflower stems and petioles but not on floral discs (eXtra Fig. 1b). Disease incidence ranged from 5% to 30% in three surveyed plots of the field. Symptomatic leaf tissue was surface-sterilized, macerated with sterile distilled water, and cultured on nutrient agar plates at 28°C for 48 h. After incubation, nine bacterial isolates, representing individually collected samples from each field, were selected for further study. All nine isolates were Gram-negative and fluorescent pigments produced under UV on King's medium B. With the LOPAT test, the isolates were levan negative, oxidase negative, positive for pectinolytic activity, arginine dihydrolase negative, and positive for tobacco hypersensitivity. Based on 16s rRNA sequences, all isolates shared 100% identity with Pseudomonas viridiflava strain KNOX209.1 (GenBank accession no. AY604847). The 16s rRNA sequences of nine isolates were deposited in GeneBank (accession nos. MT393747, MW446479 to MW446486). Based on the phylogenetic analysis of the 16s rRNA, all isolates were grouped with P. viridiflava strains isolated from globe artichoke, Chinese cabbage, and rape (Myung et al. 2010, Sanver et al. 2019, and Liu et al. 2019). The isolates CPB 19362, CPB 19366 and CPB 19372, which represent each plot were selected for further phylogenetic analysis and pathogenicity assays. The identity of these isolates was confirmed by sequences of housekeeping genes of the gyrase B subunit (gyrB) and RNA polymerase σ70 factor (rpoD) (Yamamoto et al. 2000) (GenBank accession nos. MT409400, MW446487 and MW446494 for gyrB and MT409401, MW446495 and MW446502 for rpoD). Based on the phylogenetic analyses of gyrB and rpoD, the three isolates belong to the same clade as the P. viridiflava pathotypes and were distinguished from P. syringae complex (eXtra Fig. 2). These results indicated that the bacteria isolated from the spots on the sunflower plants were P. viridiflava strains. To confirm the pathogenicity, bacterial suspensions (approximately 108 CFU/mL) of three representative isolates sprayed onto 4-week-old sunflower (cv. Common) seedlings separately until runoff occurred. Sterile distilled water was used as a control and inoculated in the same manner. After inoculation, plants were covered with transparent plastic bags at room temperature for 24 h. Plastic bags were then removed and plants were grown on a plant growth shelf at 25°C in 50% relative humidity. The leaves of plants inoculated with the bacterial suspensions developed small brown spots after 24 h. After 3 days, brown spots surrounded by chlorotic or necrotic areas were observed on infected leaves (eXtra Fig. 1c). These spots gradually increased in size and formed brown lesions with haloes similar to those of infected field-grown plants (eXtra Fig. 1d), but not on the controls treated with sterile water. The pathogenicity test was repeated three times. Isolates recovered from infected leaves showed the same morphological, biochemical, and molecular characteristics as the original isolates from field samples. To our knowledge, this is the first report of bacterial leaf spot on sunflower caused by P. viridiflava in South Korea.

First report of leaf spot caused by Colletotrichum siamense on Michelia alba in China.

Michelia alba (common name: whitechampaca), native to Indonesia, is a preciously ornamental and medicinal plant in the west and southeast of China and widely distributed in Nanning, Guangxi, China (Hou et al. 2018). In May 2020, a foliar disease of M. alba was observed in Nanning (22°51' N; 108°17' E), Guangxi, China, present on ca. 20-30% of the leaves. The disease began to develop from the margins of leaves in most cases. The symptoms recorded were light yellow spots, which gradually developed into ellipsoidal to irregular brown spots, surrounded by a wide yellow halo. The spots gradually enlarged in size and became grey-brown, with the dimension of 3.5 × 2.8 to 11.0 × 3.5 cm, even more than half of leaf area. In the later stage of infection, these spots coalesced resulting in necrosis and early shedding of the leaves. Sometimes black acervuli were observed on some lesions. For isolation of the fungus, ten symptomatic leaves were randomly sampled from five trees and washed with sterile water. Small pieces of infected tissue (about 4 mm2) were surface disinfected in 75% alcohol for 30 s and in 0.1% aqueous solution of mercury chloride for 1 min. Finally these tissue pieces were rinsed three times with sterile water, plated on potato dextrose agar (PDA) and then incubated for 7 days at 28℃ with a photoperiod of 12 h. Fifteen strains with similar morphological characterizations were isolated, and five representative isolates (BL-1 to BL-5) were purified. These cultures gave rise to grey-white colonies with bright orange conidial masses with contained one-celled, hyaline, guttulate conidia, measuring 12.68-20.70 × 4.27-7.84 µm (average 15.36 × 5.35 µm, n=100). Appressoria formed from conidia were brown, ellipsoidal or inverted trapezoid and measured 6.36-12.13 × 5.07-7.39 µm (average 8.29 × 6.36 µm, n=30). These morphological characteristics were similar to those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm identification, genomic DNA from mycelium of these five isolates was extracted, and the sequence of internal transcribed spacer (ITS), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), calmodulin (CAL) and β-tubulin (TUB2) were amplified (Zhang et al. 2020), and the GenBank accession numbers for the sequences were MW186173 to MW186177 (ITS), MW161290 to 161294 (CHS-1), MW161295 to MW161299 (GAPDH), MW161285 to 161289 (ACT), MW084710 to 084714 (CAL) and MW161300 to MW161304 (TUB2). The phylogenetic tree of six combined genes of the five isolates clustered with Colletotrichum siamense strains (CBS 125378, ICMP 17795 and ICMP 18121). Therefore, the isolates were identified as C. siamense. Five isolates (BL-1 to BL-5) were tested for pathogenicity. Wounded and unwounded detached healthy leaves were inoculated using mycelial discs (5 mm in diameter) and conidial suspensions (with the concentration of 1 × 105 conidia/ml) at the same time, incubated in a growth chamber at 25-30℃ (85-90% relative humidity, with a photoperiod of 12 h). Three leaves (wounded left half blade and unwounded right half blade) were inoculated with different methods for each isolate, and the tests were repeated three times. Four days after inoculation, leaf spots were observed on all wounded leaves, while 5-10% of the unwounded leaves showed lesions. Control leaves inoculated with PDA discs and sterile water remained symptomless. Colletotrichum. siamense was re-isolated from the lesions, confirming Koch's postulates. At least 60 plant species have been reported to be infected by C. siamense worldwide (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. albain China.

Morphological and structural study of crystals in black-to-brown glazes of Yaozhou ware (Song dynasty) using imaging and spectroscopic techniques

The Yaozhou kiln complex (Shaanxi, China) is a famous black-to-brown ceramic production center due to its significant volume of production, long manufacturing history (from Tang Dynasty to Yuan Dynasty) and variety of decorations. In this work, four representative types of black-to-brown ware from the Yaozhou kilns were selected to study the morphology, structure and distribution of the precipitated crystals in the glazes using a series of imaging and analytical techniques (optical microscopy, XRF, SEM-EDS, TEM-EDS, μRS and UV–vis-IR). The results show that the rare metastable ε-Fe2O3 phase, already observed in typical sauce glaze, was also detected in other types of brown glaze decorations, such as light brown stripes on a darker background or irregular brown spots on a black background. Cross-section analyses also showed a three well-separated layered crystalline distribution in the glazes of lighter brown colors as it has been noted in glazes from Qilizhen kilns. Raman analyses revealed that the crystals contained in third layer are of varying nature: ε-Fe2O3 in the sauce glaze and magnetite in the light brown stripes. The peak shifts and line broadenings observed in the Raman spectra of ε-Fe2O3 crystals were also investigated. They are the result of Al, Ti and Mg substitutions, which were identified using TEM-EDS. Such ionic substitutions would stabilize the metastable ε-Fe2O3 crystals by increasing the cationic disorder. In addition, Mg-, Ca- and Fe-rich spinel crystals were observed for the first time in a black glaze of Yaozhou wares.

First Report of Alternaria tenuissimain Causing Leaf Spot of Celery (Apium graveolens) in China.

Celery (Apium graveolens) is one of the most widely grown vegetables in the world. A survey in Anding District of Gansu Province in 2019 showed that the incidence of celery leaf spot was 25%-45%. The disease mainly occurs in late June and July. The leaf spot is conducive to the onset at high temperature and humidity environment. The initial symptoms were many small light brown, irregular-shaped on the leaves. The lesions gradually enlarged in the later stage of the disease, and multiple lesions coalesced to form large irregular brown spots, eventually the whole leaves died. A 3~4mm leaf tissue was cut from the junction of the diseased leaf and the healthy area, the leaf tisse was surface-sterilized in 1.5% NaClO for 1 min and washed with sterile water. Then, it was incubated on potato dextrose agar (PDA) and obtained the pure culture (Q1). After 5 days of cultivation at 25°C, the fungal colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. The conidia were obclavate or ellipsoid, pale brown, with 3~4 longitudinal septa and 2~7 transverse septa, and measured 20.0 to 50.0 × 3.5 to 14.0μm (n=50). Conidiophores were septate, arising singly, and measured 3.5 to 40.0 × 2.5 to 4.5 μm (n=50). Based on morphological characteristics, the fungus was preliminarily identified as A.tenuissima (Simmons 2007). To further confirm the identification, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), endopolygalacturonase gene (endoPG), anonymous gene region (OPA10-2) and glyceraldehyde 3-phos-phatedehydrogenase (GAPDH) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), RPB2-5F2/RPB2-5R (Sung et al. 2007), Alt-for/Alt-rev (Hong et al. 2005), EPG-specific/EPG-3b (Peever et al. 2004), OPA10-2R/OPA10-2L (Peever et al. 2004) and Gpd1/Gpd2 (Berbee et al. 1999) (GenBank accession no.MN046364, MW016001, MW016002, MW016003, MW016004, MW016005, MW016006). DNA sequences of TEF, RPB2, endoPG, OPA10-2 and GAPDH were 100% identical to those of A. tenuissima (MN256108, MK605866, KP789503, JQ859829 and MK683802), but ITS and Alt a 1 were 100% similarity with A. tenuissima (MN615420, JQ282277) and A. alternate (MT626589, KP123847). The ITS and Alt a 1 sequence did not distinguish A. tenuissima from the A. alternate complex. Maximum likelihood phylogenetic analyses were performed for the combined data set with TEF, RPB2, and endoPG using MEGA6 under the Tamura-Nei model (Kumar et al. 2016). The isolate Q1 clustered with type strain A. tenuissima CBS 918.96. The 20 celery plants of 4-7 leaf age were used test the pathogenicity of Q1, the ten plants were sprayed with 20ml of spore suspension (1×105 spores/ml), the control was sprayed with 20mL sterile water, which were placed in a growth chamber (25℃, a 14h light and 10h dark period, RH > 80%). Eight days after inoculation, 40% of the leaves formed lesions, which were consistent with the field observation，the control group was asymptomatic. The pathogen was reisolated from infected leaves to fulfill Koch's postulates. To our knowledge, this is the first report of A. tenuissima causing leaf spot on celery in China.

Identification of Fusarium ipomoeae as the causative agent of leaf spot disease in Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is traditionally used for hemostasis and detumescence in China. In April 2019, a leaf spot disease on B. striata was observed in plant nurseries in Guilin, Guangxi Province, China, with an estimated incidence of ~30%. Initial symptoms include the appearance of circular or irregular brown spots on leaf surfaces, which progressively expand into large, dark brown, necrotic areas. As lesions coalesce, large areas of the leaf die, ultimately resulting in abscission. To isolate the pathogen, representative samples exhibiting symptoms were collected, leaf tissues (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface-disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA) medium, and incubated at 28°C (12-h light-dark cycle) for 3 days. Hyphal tips from recently germinated spores were transferred to PDA to obtain pure cultures. Nine fungal isolates with similar morphological characteristics were obtained. Colonies on PDA were villose, had a dense growth of aerial mycelia and appeared pinkish white from above and greyish orange at the center and pinkish-white at the margin on the underside. Macroconidia were smooth, and hyaline, with a dorsiventral curvature, hooked to tapering apical cells, and 3- to 5-septate. Three-septate macroconidia were 21.2 to 32.1 × 2.4 to 3.9 μm (mean ± SD: 26.9 ± 2.5 × 3.2 ± 0.4 μm, n = 30); 4-septate macroconidia were 29.5 to 38.9 × 3.0 to 4.3 μm (mean ± SD: 33.5 ± 2.6 × 3.6 ± 0.3 μm, n = 40); and 5-septate macroconidia were 39.3 to 55.6 × 4.0 to 5.4 μm (mean ± SD: 48.0 ± 3.9 × 4.5 ± 0.3 μm, n = 50). These morphological characteristics were consistent with F. ipomoeae, a member of the Fusarium incarnatum-equiseti species complex (FIESC) (Wang et al. 2019). To confirm the fungal isolate's identification, the genomic DNA of the single-spore isolate BJ-22.3 was extracted using the CTAB method (Guo et al. 2000). The internal transcribed space (ITS) region of rDNA, translation elongation factor-1 alpha (TEF-1α), and partial RNA polymerase second largest subunit (RPB2) were amplified using primer pairs [ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu, Whelen et al. 1999, Reeb, Lutzoni et al. 2004), respectively]. The ITS (MT939248), TEF-1α (MT946880), and RPB2 (MT946881) sequences of the BJ-22.3 isolate were deposited in GenBank. BLASTN analysis of these sequences showed over 99% nucleotide sequence identity with members of the FIESC: the ITS sequence showed 99.6% identity (544/546 bp) to F. lacertarum strain NRRL 20423 (GQ505682); the TEF-1α sequence showed 99.4% similarity (673/677 bp) to F. ipomoeae strain NRRL 43637 (GQ505664); and the RPB2 sequence showed 99.6% identity (1883/1901 bp) to F. equiseti strain GZUA.1657 (MG839492). Phylogenetic analysis using concatenated sequences of ITS, TEF-1α, and RPB2 showed that BJ-22.3 clustered monophyletically with strains of F. ipomoeae. Therefore, based on morphological and molecular characteristics, the isolate BJ-22.3 was identified as F. ipomoeae. To verify the F. ipomoeae isolate's pathogenicity, nine 1.5-year-old B. striata plants were inoculated with three 5 × 5 mm mycelial discs of strain BJ-22.3 from 4-day-old PDA cultures. Additionally, three control plants were inoculated with sterile PDA discs. The experiments were replicated three times. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 26°C for 14 days. Four days post-inoculation, leaf spot symptoms appeared on the inoculated leaves, while no symptoms were observed in control plants. Finally, F. ipomoeae was consistently re-isolated from leaf lesions from the infected plants. To our knowledge, this is the first report of F. ipomoeae causing leaf spot disease on B. striata in China. The spread of this disease might pose a serious threat to the production of B. striata. Growers should implement disease management to minimize the risks posed by this pathogen.

Leaf spots of calibrachoa caused by Nigrospora oryzae

Abstract Calibrachoa hybrida (calibrachoa, million bells) is a flowering ornamental with increasing importance due to the existence of many successful cultivars for growing indoors in containers and planting in the garden and landscape. The outstanding characteristic is a profuse flowering and intense colour. In October 2019, a fungal isolate was obtained from basal calibrachoa leaves with irregular brown leaf spots, in plants cultivated in Buenos Aires, Argentina. The aim of the present study was to identify the cause of the disease in this ornamental genus, to expand knowledge about prevalent pathologies. The isolate was identified using morphological and molecular markers, and the pathogenicity tests were fulfilled. This paper reports that Nigrospora oryzae is pathogenic to calibrachoa, which seems to be the first record of this leaf spot disease in the world.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 μm (avg. 13.8 μm) × 4.1 to 6.7 μm (avg. 5.1 μm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.

First Report of Purple Spot on Dandelion in China Caused by Alternaria tenuissima.

Dandelion (Taraxacum mongolicum) is a perennial herb of the family Asteraceae, with a high edible and medicinal value and widely grown at medium and low altitudes in China. In July 2019, purple spot of dandelion was found in a field near Harbin City, Heilongjiang province, China. Thediseaseincidenceregionallyreached 95% in fields with yield losses between 10 and 20%, seriously reducing the economic and food value of dandelion. Multiple, irregular brown spots were first observed on the leaves of this plant- that later developed into circular or near-circular purple spots with raised centers, or purple lesions along the veins. When the leaf spots coalesced, the value of the commodity was lost. To isolate the pathogen, 5 × 5 mm pieces of leaf tissue from the margins of lesions were surface disinfected in 75% alcohol, rinsed in distilled water, and incubated on potato dextrose agar (PDA) plates at 28℃ until sporulation. Using single-spore isolation, a pure culture (YY-1) was obtained with abundant grayish white hyphae that later turned olive green. The underside of the colonies were brown. Conidia were typically obclavate, had a short beak with 1 to 6, but usually 3, transverse septa, and up to 3 longitudinal septa. The transverse septum was thicker and the wall of the conidium appeared brick-like. Conidia were pale brown, catenulate, and measured from 25 to 42 μm long by 6 to 10 μm wide. YY-1 was identified as Alternaria sp. based on morphological characteristics (Simmons 2007). Molecular identification was performed to detect the fungal species, and included the Internal Transcribed Spacer (ITS), translation elongation factor 1-alpha (EF1), actin gene (ACT), plasma membrane ATPase gene (ATP), and the calmodulin gene (CAL), which were respectively amplified with primers ITS4/ITS5 (Guo et al. 2012), EF1-728F/EF1-986R (Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999), and ATPDF1/ATPDR1 and CALDF1/CALDR1 (Lawrence 2013) (GenBank Accession Nos. MN746334, MT627208, MT627209, MT558864, MT558865). The species of Alternaria could not be confirmed by sequencing the above genes, as described previously (Zheng et al. 2015). Hence, a partial coding sequence of the histone 3 gene (GenBank Accession No. MN744235) was amplified using primers H3-1a/H3-1b (Zheng et al. 2015) and it shared 98.09% sequence identity with A. tenuissima (KP267543). The ITS sequence (MN746334) was 99.81% similar to the reference sequences of A. tenuissima (KT223327) in GenBank. A Maximum-likelihood tree was then reconstructed based on the ITS, CAL, and ATP sequences by MEGA7, which showed that YY-1 was most closely related to A. tenuissima. Therefore, YY-1 was identified as A. tenuissima based on its morphological and molecular characteristics. To perform Koch's postulates, 20 healthy leaves from greenhouse-grown dandelion were inoculated with 5-μL drops of a conidial suspension (1 x 105 conidia/ml) of isolate YY-1. Sterile water was used as a control. The inoculated plants were placed in a growth chamber at 28℃ and 80 to 90% relative humidity. After 10 days, similar symptoms were observed on plants inoculated with YY-1, while control plants did not produce symptoms. The pathogen was reisolated from the inoculated leaves and identified by morphological and molecular methods as A. tenuissima. To our knowledge, this is the first report of A. tenuissima causing purple spot on T. mongolicum in China.

First Report of Botryosphaeria dothidea Causing Leaf Wilt on Hedera helix in China

Hedera helix L. (common ivy) grows worldwide and is an evergreen root-climbing liana commonly used in parks, green belts (Metcalfe 2005), indoors as an ornamental, and as a traditional medicinal plant in China. In July 2019, a leaf wilt was observed on H. helix in Xiaofuhe wetland of Zibo, Shandong Province, China (36.79°N, 117.98°E). More than 100 m² of planting area was investigated, and the disease incidence reached about 30%. The place where the disease was found was a wetland park, mainly in a watery and humid environment. Initial symptoms on leaves appeared as small water-soaked lesions, yellow or brown in color and round or oval in shape. As the disease progressed, the area of the lesions gradually increased, forming round or irregular brown necrotic spots surrounded by a yellow halo, which eventually resulted in a wilt and death. To determine the causal pathogen, small pieces of the diseased tissue were collected and surface sterilized by 75% ethanol for 30 s followed by 5% NaClO for 1 min and then rinsed three times with sterile water. The treated samples were plated on potato dextrose agar (PDA) and incubated at 25°C for 2 days, and nine fungal isolates were obtained from 10 diseased leaves. The colonies were initially white, gradually turning gray-green to dark gray after 7 days, and the margin of the colony was regular, with abundant gray aerial mycelium. To verify identity, the partial coding genes of the internal transcribed spacer region, beta-tubulin, and translation elongation factor 1 alpha genes from the original isolate were amplified with primers ITS1/ITS4 (White et al. 1990), BT-2a/BT-2b (Glass et al. 1995), and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The sequenced genes (GenBank accession nos. MN633360, MN642587, and MN642588) exhibited 99.46, 100.00, and 99.00% homology with type specimen of Botryosphaeria dothidea CBS115476 (GenBank accession nos. KF293866, EU673106, and AY573218), respectively. The isolates were confirmed as B. dothidea on the basis of morphological and molecular results (Zhai et al. 2014). To assess pathogenicity, five healthy leaves from each of three 6-month-old healthy H. helix plants were wound inoculated with a sterilized needle. Mycelial plugs (about 5 mm in diameter) of B. dothidea from a 7-day-old culture on PDA were placed on the wounds, and five other sterile PDA plugs served as negative controls on the same leaves. All treated leaves and the whole plants were covered by sterilized plastic film and sprayed with water as needed. Ten days later, all inoculated plants developed similar symptoms to those previously observed outdoors, and the reisolated pathogen was identified as B. dothidea based on morphological and molecular results, whereas no symptoms appeared and no pathogen was isolated on negative controls. The pathogenicity test was repeated three times and showed similar results, confirming Koch’s postulates. To our knowledge, this is the first report on the presence of B. dothidea affecting H. helix plants in China. The discovery of this new disease is beneficial to the application and protection of H. helix, a common landscape and medicinal plant.