Sterile Potato Dextrose Agar Discs Research Articles

First Report of Diaporthe tectonendophytica Causing Leaf Spot on Dalbergia odorifera in Guangxi, China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is a national level II protected plant in China, with extremely high economic value and medical properties (Zhao et al. 2020). In June 2023, an unknown leaf spot was found in a garden land of Pingxiang city, Guangxi, China, and approximately 80% of the plants covered an area of 500 m2 displayed similar symptoms. The spots were grey to white, 4~6 mm in diameter (n=30) with black pycnida on the spots surface (Fig S1, A-D). Multiple disease spots were observed on a single leaf. The pycnida on the lesion were picked and mashed, to make a conidia suspension using sterile water. The conidial solution was then spread onto a potato dextrose agar (PDA) plate containing streptomycin, with 10 mg of streptomycin per 100 mL, and incubated for 3 days at 28°C with a 12 hour photoperiod. Three isolates (GXPX01, GXPX02 and GXPX03) were obtained by re-culturing the colonies on fresh PDA plates. The colony on PDA were white with aerial mycelia (Fig S1, E-F). Black conidiomata developed at 28°C with a 12 hour photoperiod in 20 days (Fig S1, G-H). Alpha conidia were 4.2~6.4 μm × 1.8~2.6 μm (average =5.1 × 2.3 μm, n = 30), mostly bi-guttulate, hyaline, ellipsoid, apex bluntly rounded, base obtuse to subtruncate, smooth (Fig S1, I). Beta conidia were 15.1~33.5 μm × 1~1.8 μm (average = 24.5 × 1.5 μm, n = 30), filiform, hyaline, curved or hamate, aseptate, base subtruncate (Fig S1, J). Morphological characteristics of the three isolates matched those of Diaporthe spp.(Gomes et al. 2013). The rDNA internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1), the calmodulin (CAL), the histone H3 (HIS) and the β-tubulin (TUB2) genes of the three isolates were amplified using the primer pairs ITS4/ITS5, EF1-728F/EF1-986R, CAL-228F/CAL2Rd, CYLH3F/H3-1B, and T1 /CYLTUB1R, respectively (Crous et al. 2004, Sun et al. 2021). The sequences were all deposited in GenBank (accession numbers OR437511 to OR437513 for ITS, OR454965 to OR454967 for TEF1, OR454968 to OR454970 for CAL, OR454971 to OR454973 for TUB2, OR454974 to OR454976 for H3). Sequences had 98.36% to 100% homology with the corresponding sequences of known Diaporthe tectonendophytica strains MFLUCC 13-0471 in the NCBI database. Phylogenetic analysis was based on combined ITS, TEF1, TUB2 and CAL sequences data using MEGA 11 software to construct phylogenetic tree with Maximum Likelihood (Doilom et al. 2017). In the phylogenetic tree, the combined sequences attributed the three isolates to the D. tectonendophytica (Fig S2). The pathogenicity was tested on leaves of 1.5-year-old D. odorifera seedlings. Three leaves were wounded with a sterile needle and individually inoculated with a 5 mm mycelial disk of PDA culture from each isolate. Sterile PDA disks inoculated leaves as a control. The test was repeated three times. The inoculated plants were placed in a greenhouse at 25℃ and 90% humidity, with a photoperiod of 12 hours. Five days after inoculation, necrotic lesions appeared on inoculated leaves and symptoms from all three isolates were the same as those form natural infections ( Fig S1, K-N), whereas all the control remained symptomless (Fig S1, P). The pathogen was reisolated from the inoculated leaves and again identified as D. tectonendophytica, with the same methodology used for the initial identification. D. tectonendophytica was reported to cause plant diseases, such as stem gray blight of red-fleshed dragon fruit (Hylocereus polyrhizus) (Rahim et al. 2021), leaf spots disease on Elaeagnus conferta and Pometia pinnata (Sun et al. 2021). To our knowledge, this is the first report of D. ctonendophytica causing leaf spot disease on D. odorifera.

First Report of Colletotrichum fructicola Causing Leaf Spot on Smilax glabra Roxb in China.

Smilax glabra Roxb is a medicinal plant distributed in 17 countries and used in the production of food and tea (Wu et al. 2022). In May 2021, a leaf spot disease was observed on ~60% of S. glabra plants in a field (∼0.4 ha) in Qinzhou City, Guangxi Province. Initially, small, circular, brown spots appeared on the leaf surfaces, which then gradually expanded into large, sunken, dark brown necrotic areas. As disease progressed, lesions merged into large spots, eventually leading to defoliation. To determine the causal agent, six symptomatic plants were collected from the field. Small pieces (∼5 mm2) were cut from the infected leaves (n = 12), sterilized for two min in 1% NaOCl, and rinsed three times in sterile water. Then, the leaf tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/liter) and incubated for 3 days at 28°C (12-h photoperiod). Pure cultures were obtained by transferring hyphal tips from recently germinated spores or colony edges onto PDA. Among the 17 isolates, 15 exhibited similar morphologies. Two single-spore isolates (TFL45.1 and TFL46.2) were subjected to further morphological and molecular characterization. Colonies on PDA were grayish green with a white outer ring and cottony surface, and pale blackish green on the reverse side. Conidia were hyaline, aseptate, straight, and cylindrical, with rounded ends, and 11.4 to 16.5 μm × 4.1 to 6.1 μm (average 13.9 × 4.8 μm, n = 100). Appressoria were brown to dark brown, with a smooth edge and different shapes such as ovoid, elliptical or irregular, and 6.8 to 8.9 μm × 5.9 to 7.8 μm (average 7.7 × 6.6 μm, n = 25). For molecular identification, eight target gene sequences, internal transcribed spacer (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPHD), calmodulin (CAL), partial actin (ACT), chitin synthase (CHS-1), glutamine synthetase (GS), manganese superoxide dismutase (SOD2), and β-tubulin (TUB) were selected for PCR amplification (Weir et al. 2012). The resulting sequences were deposited in GenBank (OR399160-61 and OR432537-50). BLASTn analysis of the obtained sequences showed 99-100% identity with those of the ex-type strain C. fructicola ICMP:18581 (JX010165, JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405) (Weir et al. 2012). In addition, a phylogenetic analysis confirmed the isolates as C. fructicola. Therefore, based on morphological and molecular characteristics (Park et al. 2018; Weir et al. 2012), the isolates were identified as C. fructicola. To verify pathogenicity, three healthy leaves on each of six two-year-old S. glabra plants were inoculated with ∼5 mm2 mycelial discs or aliquots of 10 μl suspension (106 conidia/ml) of the strain TFL46.2, and six control plants were inoculated with sterile PDA discs or sterile water. All plants were enclosed in plastic bags and incubated in a greenhouse at 25°C (12-h photoperiod). Six days post-inoculation, leaf spot symptoms appeared on the inoculated leaves. No symptoms were detected in the controls. Experiments were replicated three times with similar results. To fulfill Koch's postulates, C. fructicola was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing of the eight genes, whereas no fungus was isolated from the control plants. To our knowledge, this is the first report of C. fructicola causing leaf spot disease on S. glabra. Further studies will be needed to develop strategies against this disease based on the identification of this pathogen.

First report of stem-end rot caused by Botryosphaeria fabicerciana on mango fruits in China.

Mango (Mangifera indica L.), belongs to the family Anacardiacea, and is one of the most popular tropical fruits in the world. Stem-end rot is a major postharvest disease of mango fruit, causing severe losses during storage in China (Chen et al., 2015). In July 2021, the mango fruits harvested from Baise Municipal National Agricultural Science and Technology Park (23.683568 N, 106.986325 E) of Guangxi province in China developed stem-end rot during storage. The disease incidence reached ca. 8.3%. The initial symptoms appeared as light brown lesions surrounding the peduncle, which quickly expanded becoming large dark-brown lesions. Small pieces of epidermis (5 mm × 5 mm) from 8 typical diseased friuts were cut from the edges of lesions surface-sterilized with 2% sodium hypochlorite and rinsed with sterile distilled water. The tissue was plated on potato dextrose agar (PDA) and incubated at 28 ℃ in the dark for 3 days. Fifteen, similarcolonies were isolated from the symptomatic tissue. The representative isolates DF-1, DF-2 and DF-3 were selected for morphological characterization, molecular identification, and pathogenicity testing. The colonies were circular with fluffy aerial mycelium, initially white turning to smoke-gray from the center in upper side and greenish black in reverse side, covering the 90 mm diameter Petri dish after 4 days of incubationon PDAat 28 ℃in dark. Pycnidia were produced on the surface of the colony after 30 days. Conidia were fusiform, aseptate, hyaline, thin-walled with granular contents, apex sub-obtuse, base subtruncate to bluntly rounded, 14.0-20.3 (16.8±1.6) μm × 3.1-7.2 (5.1±0.9) μm (n=50). The sexual stage was absent. Based on morphology, isolates were preliminarilyidentified asBotryosphaeriaspeices.To accurately identify the pathogen, genomic DNA was extracted from the mycelium of the three isolates DF-1, DF-2 and DF-3. The internal transcribed spacer of rDNA region (ITS), elongation factor 1-alpha (EF-1α) and beta-tubulin gene (TUB) genes were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b, respectively (Slippers et al., 2004). The nucleotide sequences were all deposited in GenBank (ITS: OP729176-OP729178 EF-1α: OP758194-OP758196 and TUB: OP758197-OP758199). Based on the BLASTn analysis, the ITS, EF1-α and TUB sequences of three isolates were 100%, 99% and 99% similar to the Botryosphaeria fabicerciana MFLUCC 10-0098 sequences (ITS: JX646789, EF-1α: JX646854 and TUB: JX646839). Multi-locus phylogenetic analyses (ITS, EF-1α and TUB) showed that the isolate DF-1, DF-2 and DF-3 were clustered within Botryosphaeria fabicerciana clade based on the maximum likelihood , Bayesian inference, and maximum parsimony methods. The pathogenicity test was performed by placing discs mycelium around the peduncle of mature mango fruits by pin-prick method. Each treatment carried out with 12 fruits. The inoculated fruits were placed in plastic boxes at 28 ℃ with three replicates. Three days after inoculation, typical symptoms of stem-end rot were observed. The control fruits were inoculated with sterile PDA discs, and remained symptomless. The same fungus was re-isolated from the symptomatic tissue to complete Koch's postulate. Botryosphaeria fabicerciana (basionym: Fusicoccum fabicercianum) was first reported as pathogen causing senescent twig of Eucalyptus spp. in China (Chen et al., 2011; Phillips et al., 2013). To our knowledge, this is the first report of Botryosphaeria fabicerciana causing stem-end rot of Mangifera indica in China.

First Report of Chestnut Blight Caused by Cryphonectria parasitica on Chestnut (Castanea seguinii) in Gansu Province, China.

In China, chestnut blight usually causes insignificant damage to fruit production of Chinese chestnut (Castanea mollissima Blume) and no serious disease epidemics occur, due to the high resistance to Cryphonectria parasitica (Huang et al. 1998). According to recent surveys, chestnut blight was mainly found in sixteen provinces including Shandong, Hebei, Anhui, Hunan, Jiangxi, Beijing, and Fujian, with severe cases occurring occasionally (Guo et al. 2005). The disease incidence has been aggravated with increasing monoculture of newly improved chestnut cultivars in chestnut-producing areas (Yan et al. 2007), though it was not detected in Gansu Province. In September 2021, some chestnut trees (Castanea seguinii) showing symptoms of crown dieback and diffuse sunken cankers on the trunk with swelled margins and subsequent cracking of the outer bark, were collected in mountains of Hui County in Longnan City, Gansu Province (E 104° 15' 5.76″ ，N 35° 11' 30.84″). Symptomatic branches were washed using tap water and dried on sterilized tissue paper. The Junction between diseased and healthy tissue was cut from the bark and sterilized with NaClO (2.5 %) for 2 minutes, then plated on potato dextrose agar (PDA) and incubated at 25 ℃ for 3 to 4 days. After fungal colonies formed, mycelia were transferred and subcultured onto new PDA media and then purified using single spore culture. After 7 days, colonies turned yellow white. Uninucleate conidia were formed in orange pycnidia and the orange pigments could turn purple if in 2% KOH. Conidia were straight or slightly curved, hyaline, with 2.5-3.5 × 1.2-1.5 μm in size. The characteristics of the culture and morphology were similar with those of C. parasitica (Tziros et al. 2016). Perithecia were not found on culture medium. In accordance with previous findings, the sexual stage of C. parasitica appears on diseased trees in late October. For molecular identification, genomic DNA was extracted from mycelium using a Fungal Genomic DNA Extraction Kit (Tsingke Biotech Co. Ltd, Xi'an, China), the ITS region was amplified with primers ITS1/ITS4 (Sorrentino et al. 2019), and the TEF1-α region was amplified with primers TEF-1H/TEF-2T (O'Donnel et al. 1998). Cloning and sequencing of PCR products were carried out by Tsingke Biotech Co. Ltd, Xi'an, China. The resulting sequences were deposited in GenBank (ITS sequence accession number: OM033734, TEF sequence accession number: OM12254). BLAST results revealed that the sequences of ITS and TEF shared identity over 99% with those of C. parasitica strains (GenBank accession number: AY308953, KP524763, KP824756 and KF220299). Based on morphological and molecular characteristic, the fungal isolates were identified as C. parasitica. To verify pathogenicity, thirty 3-year-old chestnut seeding (70 cm high, 1 cm diameter) of Castanea seguinii were used for inoculation. Chestnut branches were wounded (five wounds per sapling) using a hole punch and inoculated with a mycelial plug (5 mm in diameter) from the edge of 7-day-old, actively growing colonies. Pathogen-free PDA plugs were used as controls. To prevent desiccation, inoculated wounds were sealed with parafilm, and saplings were incubated in a greenhouse at 25℃. Each treatment consisted of 5 seedling and the pathogenicity tests were repeated three times. After inoculation for 5 weeks, symptoms of bark cankers were observed on branches similar to those of diseased chestnut trees in the field. Control saplings with sterile PDA discs did not display symptoms. C. parasitica was reisolated from inoculated branches. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Gansu Province, one of the few areas in the China thought to be free of the disease. The specimens were found in the westernmost part of the natural distribution of chestnuts in China. There are more than 2.6 million chestnut trees, which constitute one of the most important economic forests in Hui County Gansu Province (Yang et al. 2005). The occurrence of chestnut blight could be a restricting factor for chestnut forests.

First Report of Stem and Leaf Blight Disease on Mesona chinensis Caused by Rhizoctonia solani in China.

Mesona chinensis is an important medicinal and edible plant resource distributed in eight provinces in southern China. In December 2021, an unknown stem and leaf blight disease was found in M. chinensis cultivation areas in Longzhou County, Guangxi, China. Sixty days after transplanting, the incidence of this disease was 10%. Leaf spots mostly appeared from the leaf edge, were irregular, brown to dark brown, causing more than half of the leaf or the whole leaf to die. The infected stem first showed dark brown spots, then constricted slightly, became necrotic and rotted with the expansion of the spots, resulting in the death of the whole plant. Loose cobweb-like mycelia, which resembled Rhizoctonia, could be seen on the diseased tissues in conditions of high humidity. To identify the pathogen, diseased stems and leaves with typical symptoms from Longzhou County were collected and surface-sterilized with 75% ethanol for 30 s. Small fragments (5×5 mm) at the junction of diseased and healthy tissues were disinfected with 1% NaClO for 1min, washed with sterile water three times, transferred to potato dextrose agar (PDA), and incubated at 28°C for 3 days. Mycelial tips were removed, and six isolates (No. R1-R6) were obtained. The colonies were initially gray white and later light brown. Many nearly round to irregular sclerotia appeared after 7 days of culture. The sclerotia turned from light brown to deep brown and were 1 to 5 mm in diameter. The mycelium branched at a 90° angle, with septa near the branches and a constriction of the mycelium at the base of the branch. These morphological characteristics were consistent with Rhizoctonia. For molecular identification, genomic DNA of the six isolates was obtained using an extraction kit (Biocolor, Shanghai, China), and primers ITS4/ITS5 were used to amplify the internal transcribed spacers (ITS) and 5.8S rRNA (White et al. 1990). A 750 bp DNA fragment was obtained and the sequences were deposited in GenBank (OM095383-OM095388). All isolates had ≥ 99% identity with anastomosis group AG1-1B (HG934429 and HQ185364) of R. solani. A phylogenetic tree showed that the isolates and those from anastomosis group AG1-1B clustered into one branch. To satisfy Koch's postulates, the isolates from diseased leaf (No. R1, R2, and R3) and diseased stem (No. R4, R5, and R6) were inoculated on leaves and stems of 45-day-old M. chinensis plants. Five leaves and stems were inoculated with mycelial plugs of each isolate without wounding and another five leaves and stems were inoculated with mycelial plugs of each isolate after pinprick wounding. Control wounded leaves and stems were inoculated with sterile PDA discs. To maintain high humidity, the plants were incubated at 28°C and covered with transparent plastic covers. Diseased spots first appeared 24 h after inoculation. Three days post-inoculation, all inoculated leaves and stems showed symptoms like those observed in the field, whereas controls were asymptomatic. The pathogen was re-isolated from the diseased inoculated tissues using the method described above, and isolated fungi had the morphological characteristics of R. solani. Thus, the pathogen causing stem and leaf blight disease of M. chinensis was determined to be R. solani. The host range of R. solani is wide, and anastomosis group AG1-1B has been reported to infect plants such as rice, bean, fig, cabbage, and lettuce (Sneh et al. 1991). To our knowledge, this is the first report of R. solani causing a stem and leaf blight on M. chinensis, and provides a basis for diagnosis and control of the disease.

First report of Calonectria canadiana causing peach fruit rot in China.

Peach (Prunus persica (L.) Batsch) is one of the most popular fruits grown in Northern China. In July 2021, a fruit rot outbreak on the peach cultivar "Yonglian Sweet" occurred after unusual rains in Baoding, Hebei Province, China. Sixty peach trees from three orchards were assessed, and a 30% disease incidence was estimated. The disease initiated as a small concave spot on the fruit surface expanding circularly rotting the fruit (3-5 cm deep) with the appearance of grayish-white mycelia (Figure S1A). The infected fruit did not disintegrate but turned light brown. To identify the pathogen, 20 infected fruits were collected, and fruit tissues from lesion margins were inoculated on the potato dextrose agar (PDA) medium. A total of 15 fungal pure cultures with highly similar morphological characteristics were obtained by the hyphal-tipping method. The fungal culture formed smooth-edged colonies of extensive, dense, wooly aerial mycelium, with color changing from sienna to luteous, and to grayish-white along the radius of colonies (Figure S1B) Chlamydospores were extensive and developed micro-sclerotia after 20 d of growth. The conidiophore produced three branches in a "broom" shape, with the primary branch ranging 7.5-25.0 μm in length, the secondary branch 5.5-15.5 μm, and the tertiary branch 10-12.5 μm (N = 30). The top of the tertiary branch tapered and produced conidia. Conidia were colorless and culm-like, 40.0-57.5 μm long and 3.8-6.25 μm wide (N = 30). Hyphae occasionally produced spherical chlamydospores with a diameter of around 7.5 μm (N = 30). Conidia germinated after 12 h in moist conditions, and germ tubes originated from multiple points on the conidia. Based on these morphological features, the isolated fungus was identified as Calonectria spp. (Lombard et al. 2010). Six loci, including ITS, act, cmdA, his3, tef1, and tub2, were amplified and sequenced for molecular identification of an isolate F099 using primers listed in Table S1. The obtained ITS (528 bp, GenBank accession no. OL635556), act (263 bp, OL694221), cmdA (470 bp, OL694222), his3 (432 bp, OL694223), tef1 (487 bp, OL694224), and tub2 (535 bp, OL694225) sequences showed 100% similarity to the ex-type strain of Calonectria canadiana, CMW 23673 (accession nos. MT359667, MT334976, MT335206, MT335446, MT412737, and MT412958, respectively; Figure S1D) (Kang et al. 2001, Lechat et al. 2010, Liu et al. 2020). The isolate F099 of C. canadiana was further subjected to pathogenicity tests. Koch's postulates were performed by placing three mycelial disks (ten-day old, 5 mm) with conidia on the sterile needle-acupunctured surface of healthy fruits of the peach cultivar "Yonglian Sweet" (N= 10). Mock inoculations with sterile PDA disks were served as a control. All the inoculated fruits were kept in a moist chamber (25℃, 16-h light and 8-h dark period). The inoculation assay was repeated twice. Rotting symptoms developed on all the inoculated fruits about 5 days post-inoculation (dpi) and grayish-white mycelia appeared around ten days post inoculation while mock inoculated fruits did not show any rotting. The pathogen of interest was re-isolated from the inoculated fruits and validated as C. canadiana by ITS and tef1 sequences. All above evidence collectively indicates that the fungal pathogen causing the peach fruit rot is C. canadiana. The new host plant and new geographic distribution reported here will inform future management of this fungal species.

First Report of Stem Rot Caused by Fusarium kyushuense on Dendrobium officinale in China.

Dendrobium officinale Kimura et Migo is a Chinese medicinal plant within the Orchidaceae. In September 2021, D. officinale seedlings in a greenhouse farm in the region of Wenzhou City (N28.11°, E120.98°), Zhejiang Province, China, were affected by stem rot. Symptoms initially occurred at the stem base with dark gray to black lesions, vascular discoloration, wilting and death. Approximately 30% of the surveyed plants were affected. To determine the causal agent, 10 symptomatic plant samples were collected. The necrotic tissue pieces (3×3 mm) from symptomatic stems were disinfected with 10% sodium hypochlorite for 1 min, rinsed three times with sterile water, and dried on sterile paper. Samples were then placed on potato dextrose agar (PDA), and incubated at 25℃ in the dark. After 3 days, reddish-white to yellow aerial mycelium with deep red pigments developed on PDA. A total of 15 single-spore isolates from all ten infected tissues were identified as Fusarium kyushuense based on morphological features (Aoki and O'Donnell 1998). DDO-S1 was chosen as a representative isolate for further study. Microconidia were ellipsoidal to clavate, 0 to 1 septate, and 5.2 to 17.6 × 2.3 to 4.7 μm (n = 100; length × width). Hyaline and straight or slightly curved macroconidia were observed with 3 to 5 septate, 32.4 to 51.7 × 3.9 to 5.3 μm (n = 100; length × width). In order to identify the isolate, a portion of RNA polymerase second largest subunit (RPB2) (Liu et al. 1999) and translation elongation factor-1α (TEF-1α) (Geiser et al. 2004) were amplified by PCR. The gene sequences from isolate DDO-S1 were deposited in the NCBI GenBank nucleotide database with accession numbers OL548912 and OL548913. BLASTn analysis showed highest similarity with F. kyushuense strain MRC 2534 99.9% (RPB2) and 99.2% (TEF-1α) nucleotide sequence identity, which indicated that the pathogen was F. kyushuense. A maximum-likelihood phylogenetic analysis based on multi-locus sequence analysis using MEGA X showed similar results (Kumar et al. 2018). Pathogenicity of this isolate was confirmed by following Koch's postulates. To verify pathogenicity, 30 healthy D. officinale seedlings (1-year-old) were used for inoculation tests. Fifteen surface sterilized stems were stab inoculated with 5 × 5 mm colonized agar discs of strain F. kyushuense DDO-S1 pierced at three points using a 1-mm sterile needle, and another 15 samples that were mock inoculated with sterile PDA disks as negative controls (Xiao et al. 2021). Each experimental seedling was inoculated with three agar disks from a colony of DDO-S1. Seedlings were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber at 25°C and a daily 12 h photoperiod for 8 days. All inoculated stems showed symptoms identical to those observed in the field. No disease was observed on the controls. The pathogen was reisolated from all 15 diseased stems, and species identification was confirmed by the morphological and molecular methods described above. Fusarium kyushuense has been reported to cause diseases on a broad range of plants in China, such as tobacco (Wang et al. 2013), maize (Wang et al. 2014) and rice (Zhao et al. 2007). To the best of our knowledge, this is the first report of F. kyushuense on D. officinale in China. D. officinale is native to Wenzhou, and spread of the disease may seriously affect the local economy and food security. This report will help diagnose stem rot of D. officinale and provide a foundation for development of management tools.

First Report of Leaf Spot Caused by Alternaria alternata on Yucca gloriosa in China.

Yucca gloriosa L. is introduced to China as a garden plant because of its attractive tubular flowers (Ding et al. 2020). In 2020 and 2021, a foliar disease occurred on approximately 10% of the Y. gloriosa plants in the campus of Henan Institute of Science and Technology, Xinxiang (35°18'N, 113°54'E), Henan Province, China. At the early stages, symptoms appeared as small brown spots on the tip of the leaves. As the disease developed, the spots gradually expanded and turned into necrotic tissue with a clear brown border. The length of lesions ranged from 1 to 3 cm. Infected tissue samples were cut into small pieces, surface sterilized with 75% ethanol for 30 s followed by 0.5% NaClO for 2 min, rinsed thrice with sterile water and plated on potato dextrose agar (PDA). After incubation at 25℃ for 3 days, five fungal isolates were collected and purified using single spore culturing. Morphological observations were made on the 7-day-old cultures. Colonies on PDA were white at first and then turned to dark olive or black along with profuse sporulation. Conidia were borne on branched conidiophores, light brown to dark brown, ellipsoidal to obpyriform, and 20.5 to 43.6 ×7.5 to 15.4 μm in size, with 2-6 transverse septa and 0-3 longitudinal septa (n = 50). The morphological characteristics of the five isolates were consistent with the description for Alternaria alternata (Simmons 2007). One representative isolate (ZQ20) was selected for molecular identification. The internal transcribed spacers (ITS)-rDNA, translation elongation factor-1 alpha (TEF-1α), Alternaria major allergen (Alt a1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EFl-728F/ EFI-986R (Carbone and Kohn, 1999), Alt-for/Alt-rev (Hong et al. 2005), and gpd1/gpd2 (Berbee et al. 1999), respectively. Their sequences were submitted to GenBank (ITS, MW832377; TEF-1α, MW848791; Alt a1, MW848792; GAPDH, MW848793). BLAST searches showed ≥99% nucleotide identity to the sequences of A. alternata (ITS, 100% to KF465761; TEF-1α, 100% to MT133312; Alt a1, 100% to KY923227; and GAPDH, 99% to MK683863). Thus, the fungus was identified as A. alternata based on its morphological and molecular characteristics. To confirm its pathogenicity, 25 healthy leaves of five 2-year-old Y. gloriosa plants were used. Leaves were wounded with one sterile needle and inoculated with 5-mm-diameter fungal agar disks obtained from 5-day-old cultures. Sterile PDA disks of the same size were used as the controls. Treated plants were covered with a plastic bag at 12 to 25℃ for 48 h to ensure a high level of moisture. After 15 days, the inoculated plants developed the symptoms similar to those observed in naturally infected plants, whereas the control plants were symptomless. The fungus was reisolated from the symptomatic leaves with the same morphological and molecular characteristics as the original isolates, fulfilling the Koch's postulates. Leaf spot caused by A. alternata in the Yucca plants has been reported in India (Pandey 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on Y. gloriosa in China. Identification of the cause of the disease is important to developing effective disease management strategies.

First Report of Fusarium commune Causing Leaf Spot Disease on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae family, known as Baiji in Chinese) is a perennial herb and has been traditionally used for hemostasis and detumescence in China. In April of 2020, a leaf spot disease on B. striata was observed in plant nurseries (∼0.2 h) in Guilin, Guangxi Province, China. Approximately 20% of the plants were symptomatic, of which 150 plants were randomly selected for investigation. Initial symptoms include the appearance of small, circular or irregular light brown spots, randomly scattered on the edges and surfaces of the leaves, which progressively expand into large, suborbicular or irregular-shaped dark brown, necrotic areas. At the severe stage, the lesions coalesced into large necrotic areas and ultimately resulted in leaf abscission. To isolate the pathogen, three representative plants exhibiting symptoms were collected from the nurseries. Leaf tissues (5 × 5 mm) were cut from the margin of necrotic lesions (n = 18), surface-disinfected in 1% sodium hypochlorite (NaOCl) solution for 2 min, then rinsed three times in sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium, and incubated at 28°C (12-h photoperiod) 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. Three single-spore isolates, BJ23.1, BJ55.1, and BJ91.3, were subjected to further morphological and molecular characterisation. Colonies on PDA plates were villose, had a dense growth of aerial mycelia and appeared white (1A1) to yellowish white (3A2). Macroconidia were smooth, hyaline, straight to slightly curved, usually contained three or five septa, and measuring 23.3 to 42.1 × 3.0 to 6.2 μm (mean ± SD: 31.2 ± 5.1 × 4.2 ± 0.6 μm, n = 50). Microconidia were generally cylindrical, straight to slightly curved, aseptate, and measuring 7.2 to 18.8 × 2.5 to 4.3 μm (mean ± SD: 12.1 ± 2.8 × 3.3 ± 0.5 μm, n = 62). Morphological characteristics are similar to those of F. commune (Skovgaard et al. 2003). For molecular identification, the genomic DNA of the isolates BJ23.1, BJ55.1, and BJ91.3 were extracted using the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region of rDNA, partial translation elongation factor-1 alpha (TEF-1α), RNA polymerase second largest subunit (RPB2), and the mitochondrial small subunit rDNA (mtSSU) genes were amplified using primer pairs [ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu et al. 1999, Reeb et al. 2004), MS1/MS2 (Li et al. 1994), respectively]. The obtained sequences were deposited in NCBI GenBank under the following accession numbers: ITS (MZ424697 to MZ424699), TEF-1α (MZ513467 to MZ513469), RPB2 (MZ513473 to MZ513475), and mtSSU (MZ513470 to MZ513472). BLAST® analysis of the deposited sequences showed 99 to 100% identity with those of F. commune present in GenBank (Accession numbers: DQ016205, MH582348, MH582181, AF077383). In addition, a phylogenetic analysis using concatenated sequences of ITS, TEF-1α, mtSSU genes showed that BJ23.1, BJ55.1, and BJ91.3 located on the same clade with strains of F. commune. Therefore, based on morphological and molecular characteristics, the isolates were identified as F. commune (Skovgaard et al. 2003, Stewart et al. 2006). Pathogenicity was tested using 1.5-year-old B. striata plants. Healthy leaves on plants were inoculated with 5 × 5 mm mycelial discs of strains BJ23.1, BJ55.1, and BJ91.3 from 3-day-old PDA cultures, each isolate was inoculated onto three plants; three other plants inoculated with sterile PDA discs served as controls. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 28°C for 14 days (12-h photoperiod). Three days post-inoculation, leaf spot symptoms appeared on the inoculated leaves. No symptoms were detected on control plants. Experiments were replicated three times with similar results. To fulfill Koch's postulates, F. commune was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing, whereas no fungus was isolated from the control plants. F. commune has been reported to cause diseases on some plants, including sugarcane (Wang et al. 2018), maize (Xi et al. 2019) and Wax Gourd (Zeng et al. 2020). To our knowledge, this is the first report of F. commune causing leaf spot disease on B. striata in China. Identification of this pathogen provides the information for further studies to develop management strategies to control the disease.

First Report of Phytopythium helicoides Causing Stalk Rot on Corn in China.

Corn (Zea mays L.) is one of the most important grain crops in the world, especially in China. Besides, corn stalks are often used in production of bio-fuels (Xue et al., 2017). Recently, the production and quality of corn have been severely influenced by corn stalk rot in China caused by Fusarium spp. (Yu et al., 2017). At the end of June of 2019, a field survey of corn was carried out in Tai'an City, western Shandong Province, China. During the survey, the average day time temperature ranged between 22-28°C with intermittent rainfall, the relative humidity was 50-70%. In this survey, the symptomatic corn plants showed signs of necrosis and rotting on stalks and root collars. Five fields were surveyed and symptomatic corn plants were observed in three fields. The incidence rate of disease was about 5%, and the disease was more of a problem in low-lying areas. A total of twenty-eight symptomatic corn plants (7-12 per field), hybrid Denghai-618, at the 3-4 leaf stage were collected and tested for the presence of pathogens. The diseased tissues were excised, surface-sterilized with 75% ethanol for 30 seconds, rinsed for 3 to 5 times with sterile distilled water, and plated on potato dextrose agar (PDA). All plates were incubated at 28°C for 48 hours, emerging colonies were sub-cultured onto PDA plates. Forty-two isolates were obtained, and twenty-seven isolates were identified as Fusarium spp. The remaining fifteen isolates had similar morphology, with colonies that were white and cottony in texture after incubation at 28°C for three days on PDA. The suitable temperature range for growth of hyphae was between 15°C to 40°C, and sporangia were ellipsoidal, papillate, and 23 - 34×21 - 31 µm in diameter. Oogonia (smooth, 22 - 30 μm in diameter) were present in the cultures after 28 days at 28°C. The isolates were identified using both morphological characteristics and DNA sequencing. Identity of the oomycete was confirmed using the BLAST algorithm available through the GenBank with the DNA sequences of rDNA internal transcribed spacer region (ITS), cytochrome c oxidase Ⅰ (coxⅠ) gene and cytochrome c oxidase Ⅱ (coxⅡ) gene, which were amplified using the primers ITS1/ITS4 (White et al. 1990), FM35/FM59 and FM66/FM58 (Martin 2000), respectively. The fifteen isolates selected for sequence analysis had identical gene sequences, and hence, only sequences for isolate RMSD1 were submitted to GenBank (ITS - MW440691, coxI - MW450815 and cox II - MW450816). The ITS, coxI and coxII sequences of the isolate RMSD1 showed 97% identity (751/774 bp), 99% identity (1087/1098 bp) and 99% identity (548/554 bp) with Phytopythium helicoides Accession nos: HQ643382, FR774199, and AB108014, respectively. The pathogenicity of RMSD1 was tested on the corn hybrid Denghai-618. Three-leaf-stage corn plants (N = 15) were inoculated with mycelial agar disks (3 to 4 mm in diameter) colonized with RMSD1 placed on their root-collars. Sterile PDA disks (3 to 4 mm in diameter) served as the negative control (N = 9). Inoculated plants were placed in the growth chamber at 28°C, 60% relative humidity, 16 h / 8 h light regime cycle. Ten days post-inoculation, the inoculated plants showed necrosis, with symptoms of stem rot similar to those observed in the field. The inoculation experiments were repeated twice with the same results, fulfilling Koch's postulates. The root-collars and stems of negative control remained asymptomatic, and P. helicoides was not isolated. Previously, P. helicoides has been reported as a pathogen of strawberry (Zhan et al. 2020) and kiwi fruits (Wang et al. 2015) from China, but not from corn. To our knowledge, it is the first report of P. helicoides causing corn stalk rot in China. In the future, P. helicoides can be considered as a potential candidate causing stem and collar-rot of corn in China, but not the only one. There are other microbes that can produce similar symptoms on corn, and control methods for pathogenic oomycetes differ from those for fungi.

First Report of Leaf Spot on White Chrysanthemum (Chrysanthemum morifolium) Caused by Epicoccum sorghinum in Hubei province, China.

White Chrysanthemum (Chrysanthemum morifolium), a perennial herb of the Compositae family, is used for traditional medicine. The planting area of white chrysanthemum in Macheng city, Hubei Province is about 3333 ha and the annual output can reach more than 5000 tons. In 2019, leaf spot disease appeared on almost all middle and lower leaves of white chrysanthemum in most fields of Fengshumiao county, Macheng city (N31°29'57″, E115°05'49″). This county has 33 acres white chrysanthemum planting area, and most of the plants in the county were infected with the leaf spot disease. The average incidence of leaf spot disease was 65%, and incidence in some areas was 100%. In our observations, leaf spot disease can occur throughout the whole growth period of white chrysanthemum, and it will become more serious under the high temperature and humidity condition. Usually, the diseased leaves account for 30 to 80% of the total leaves on the plant. Leaf spot initially manifests as necrotic lesions on the edge and tip of the leaf, and then the lesions coalesce and gradually expand to form irregular light-brown to brown-black spots, eventually leading to necrosis and curling of the entire leaf. This disease seriously affects the growth and development of plants, resulting in the decline of yield and quality of white chrysanthemum. Ten symptomatic leaf samples were collected, the surfaces were disinfected with 0.1% mercuric chloride (HgCl2) for 3 min, and washed with sterile distilled water three times. Ten tissue samples at the junction of diseased and healthy areas (0.5 × 0.5 cm2) were cut and placed on potato dextrose agar (PDA) medium containing 100 µg/ml cefotaxime sodium and incubated in a dark chamber at 28°C. After 2 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Finally, eight isolates were obtained and these isolates were similar in morphology. The color of purified isolates was initially white to pale yellow. After six days of incubation, colonies had a diameter of 8 cm and the cultures were pale gray and starting to secrete scarlet pigment. After 15 days incubation, the colonies were grayish brown, while the backside was reddish-brown. Gray to tan chlamydospores were observed, nearly spherical, with a wart-like surface. Unicellular chlamydospores were 7.91 to 32.23 × 12.03 to 38.42 µm (n=30) and multicellular chlamydospores were 6.32 to 25.10 × 21.75 to 100.05 µm (n=30). The morphological characteristics were similar to Epicoccum sorghinum (Kang et al. 2019). The isolate FDY-5 was chosen for molecular identification. The sequence of rDNA-ITS, TUB, and LSU of the FDY-5 were amplified (GenBank MT800929, MT799852, and MT800935, respectively) (White et al. 1990; Carbone and Kohn 1999; Lumbsch et al. 2000). BLAST results showed that the rDNA-ITS sequences, the TUB gene sequences, and LSU gene sequences of strain FDY-5 shared 99% identity with the sequences of E. sorghinum (syn. Phoma sorghina) in GenBank (MN555348.1, MF987525.1, MK516207.1, respectively). Moreover, a phylogenetic tree of the LSU gene sequence of FDY-5 was constructed based on the Neighbor-Joining (NJ) method in MEGA6 software (Tamura et al. 2013) and revealed that strain FDY-5 was closest to E. sorghinum. Based on morphological and molecular characteristics, the fungus was identified as E. sorghinum. Pathogenicity tests were conducted on two-month-old white chrysanthemum plants. The upper three leaves of three plants were randomly selected for stab treatment and were inoculated with 5 × 5 mm mycelial discs produced from a fifteen-day-old colony on PDA. The inoculated and control (treated with sterile PDA disks) plants were incubated in a moist chamber (25 ± 2 °C, RH 85%). The first lesions appeared 1 day after inoculation on leaves, and the necrotic lesion area expanded outward and showed typical symptoms 3 days later. To fulfill Koch's postulates, the pathogen was reisolated from nine inoculated leaves by repeating the above isolating operation, and confirmed as E. sorghinum by morphology. To the best of our knowledge, this is the first report of E. sorghinum causing leaf spot on white chrysanthemum in China. E. sorghinum has a wide host range worldwide and often causes crop yield reduction. This report will facilitate the diagnosis of white chrysanthemum leaf spot of white chrysanthemum allowing control measures to be adopted to manage this disease in a timely manner. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Kang, Y., et al. 2019. Plant Dis. 103 (7):1787. Lumbsch, H., et al. 2000. Plant Biol. 2:525. Tamura, K., et al. 2013. Mol. Biol. Evol. 30:2725-2729. White, T. J., et al. 1990. Page 315 in:PCR protocols:a guide to methods and applications. Academic Press, San Diego, CA. Funding Funding was supported by Major Increase and Decrease Projects at the Central Level of China (2060302) and the National Key Research and Development Program (2017FYC1700704).

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.

First Report of Epicoccum sorghinum Causing Leaf Blight on Kadsura coccinea in China

HomePlant DiseaseVol. 104, No. 12First Report of Epicoccum sorghinum Causing Leaf Blight on Kadsura coccinea in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Epicoccum sorghinum Causing Leaf Blight on Kadsura coccinea in ChinaHuiyun Fan, Weiming Chen, Yicheng Huang, Qingqian Zeng, and Yong HuangHuiyun Fan†Corresponding author: H. Y. Fan; E-mail Address: fhy1262006@126.comhttps://orcid.org/0000-0002-3457-8658Guangdong Institute of Traditional Chinese Medicine, Guangzhou 510640, ChinaSearch for more papers by this author, Weiming ChenGuangdong Institute of Traditional Chinese Medicine, Guangzhou 510640, ChinaSearch for more papers by this author, Yicheng HuangGuangdong Institute of Traditional Chinese Medicine, Guangzhou 510640, ChinaSearch for more papers by this author, Qingqian ZengGuangdong Institute of Traditional Chinese Medicine, Guangzhou 510640, ChinaSearch for more papers by this author, and Yong HuangGuangdong Institute of Traditional Chinese Medicine, Guangzhou 510640, ChinaSearch for more papers by this author AffiliationsAuthors and Affiliations Huiyun Fan † Weiming Chen Yicheng Huang Qingqian Zeng Yong Huang Guangdong Institute of Traditional Chinese Medicine, Guangzhou 510640, China Published Online:13 Oct 2020https://doi.org/10.1094/PDIS-04-20-0884-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleKadsura coccinea (Lem.) A. C. Smith, a perennial medicinal woody vine in the family Schisandraceae, is mainly cultivated in Jiangxi, Guangdong, Sichuan, Guizhou, and Yunnan Provinces in China. The roots and canes of K. coccinea have been used for the treatment of chronic gastritis, bruises, rheumatism, and dysmenorrhea by local residents in China (Xu et al. 2018), and the fruit also can be eaten as a dessert. In October 2019, severe leaf blight symptoms were observed on the second year growth of six of 13 K. coccinea vines in the private garden of Guangdong Institute of Traditional Chinese Medicine (N23°10′3.09″, E113°23′47.31″) in Guangzhou, China. Zonate lesions of alternating light brown and dark brown started from the leaf tips. After surface sterilization, samples of leaf tissue taken from the margin of the lesion and healthy leaf tissue were plated on potato dextrose agar (PDA) and incubated at 28°C in the dark for 2 days. Mycelia growing from the edges of the tissue samples were transferred to new PDA plates and cultured at 28°C in the dark for 6 days. A total of 11 isolates were obtained from six diseased leaves. The colonies of the isolates were regular round and approximately 6.0 cm in diameter after 6 days of culture, and the reverse side was light brown. Aerial hyphae were villose and white. After 12 days of incubation, the colonies became pale brown and secreted a scarlet diffusible pigment that colored the underside of the colony reddish brown. Many small pycnidia (89.1 to 215.0 × 111.2 to 244.4 μm) were visible in the colony. Unicellular chlamydospores were brown to black and were 2.4 to 7.8 × 4.9 to 12.4 μm, and multicellular chlamydospores were 4.8 to 22.5 × 13.7 to 54.6 μm. Based on the above morphological characteristics, these isolates were identified as Epicoccum sp. (Zhou et al. 2018). Genomic DNA was extracted using a Fungal DNA Kit (Omega Bio-tek, Guangzhou, China). Polymerase chain reaction amplifications of the gene fragments of ribosomal internal transcribed spacer (ITS), β-tubulin (TUB), 28S nrDNA (LSU), and actin (ACT) of strain GDTCMF19 were amplified with primers ITS1/ITS4, T1/Bt2b, LR7/LROR, and ACT-512F/ACT-783R, respectively (Aveskamp et al. 2010; Carbone and Kohn 1999; Glass et al. 1995; O’Donnell et al. 1997). Sequences from isolate GDTCMF19 were deposited in GenBank (accession nos. MN915133 for ITS, MN917744 for TUB, MN915134 for LSU, and MN917745 for ACT). The identification of the isolate as Epicoccum sorghinum was validated by constructing a phylogenetic tree and that showed high congruence to E. sorghinum H10, E. sorghinum SCCZ16, and E. sorghinum SCCZ09 (shown in the supplementary materials). Pathogenicity tests were performed by inoculating unwounded leaves of six healthy second-year transplants with 6 × 6-mm mycelial discs of E. sorghinum grown on PDA. All control plants were inoculated with sterile PDA discs. All plants were maintained in a chamber at 25 to 28°C and relative air humidity of 50 to 65%. After 2 weeks, typical symptoms were observed. Small brown spots that gradually expanded were observed at the leaf margins 7 days after inoculation. After 2 weeks, zonate lesions similar to those observed in the garden were observed on 1/3 of the inoculated leaf. No symptoms appeared on control plants. The experiments were repeated three times. To fulfill Koch’s postulates, the fungus was reisolated from the inoculated leaves on PDA and confirmed as E. sorghinum. To our knowledge, this is the first report of E. sorghinum causing leaf blight on K. coccinea in China. Because K. coccinea is used medicinally by local residents, the leaf blight is of concern, and this report will assist with monitoring distribution of the disease to assist with developing management recommendations.The author(s) declare no conflict of interest.

First Report of Stagonosporopsis vannaccii Causing Leaf Spot on Crassocephalum crepidioides in China.

Crassocephalum crepidioides (Benth.) S. Moore, native to tropical Africa, is an important invasive weed in many countries, seriously threatening the safety of agricultural ecosystem. During December 2018, 100% of C. crepidioides plants exhibited leaf spots in the Kudzu (Pueraria lobata) garden in Tianlin County, Baise City, Guangxi, China (24°40'20.42″N, 106°11'33.51″E), but Kudzu was not affected by this disease. The leaf spots appeared as small brown spots surrounded by a yellow-green halo initially, enlarged to subrotund or irregular in shape, slightly sunken, then developed as a dark brown to dark spot with grey-white necrotic center (Supplementary Fig. 1 a,b), and exuded an orange droplet under high humidity conditions (Supplementary Fig. 1 c). Symptomatic leaf tissues were cut into small pieces (5 x 5 mm) from the junction of necrotic and healthy tissues, and small pieces were disinfected in 75% ethanol solution for 30 s and 0.1% mercury dichloride for 30 sec, then rinsed with sterile water 3 times. These tissues were plated onto potato dextrose agar (PDA) medium and incubated in a thermostatic incubator at 28°C under natural sunlight conditions. Four isolates with similar morphological features were obtained after purification. Colonies of these isolates exhibited creme-orange margins and aerial mycelium was sparse. The colonies formed concentric circles on the surface that were fusco-black, violet-slate and vinaceus-grey (from centre to edge), fusco-black on the reverse after 7 days (Supplementary Fig. 2 a,b), and then the pycnidia and conidia produced for about 30 days (Supplementary Fig. 2 c). Pycnidia of representative isolate YTH-12 were black, subglobose, and unilocular, 95.60-168.27 μm (average 128.32 µm) (n = 40) in diameter. The ostiole was single and central, slightly papillate to papillate and occasionally rostrate (Supplementary Fig. 2 d). Conidia were hyaline, oval to elliptical, aseptate, 2.30 to 5.83 × 1.42 to 3.50 µm (average, 4.36 × 2.03 µm) (n = 50) (Supplementary Fig. 2 e). These morphological characters are consistent with those described for Stagonosporopsis vannaccii (Crous et al. 2019). To further identify the isolate YTH-12, the rDNA internal transcribed spacer (ITS) region, 28s large subunit ribosomal RNA (LSU), RNA polymerase II second largest subunit (RPB2) gene and β-tubulin (TUB2) gene were amplified by polymerase chain reaction using the primer pairs ITS1/ITS4 (White et al. 1990), LR0R (Rehner and Samuels 1994)/LR7 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/fRPB2-7cR (Liu et al. 1999), respectively. The PCR products were purified and sequenced by Sangon Biotech Co. Ltd. (Shanghai, China). The sequences were deposited in GenBank (accession nos. MN892355, MN893911, MN905510 and MN905511). The ITS (522 bp), LSU (1313 bp), TUB2 (380 bp) and RPB2 (1193 bp) nucleotide sequences showed 100% identity to S. vannaccii strain LFNO148 (accession nos. MK519453, MK519452, MK519454 and MN534891). Phylogenetic analysis based on the multi-locus sequences of ITS, LSU, RPB2 and TUB2 was performed in MEGA version 6.0 (Chen et al. 2015). The relative stability of the branches was evaluated by bootstrapping with 1000 replications. The isolate YTH-12 was placed in the same clade as S. vannaccii with 100% bootstrap support. Based on morphology and molecular analyses, this pathogen was identified as S. vannaccii. To satisfy Koch's postulates, the isolate YTH-12 was inoculated on leaves of C. crepidioides plants. Twenty punctured leaves and twenty unwounded leaves were inoculated with a 5-mm-diameter mycelial disc, respectively. Leaves inoculated with sterile PDA discs were used as blank controls. Plants were maintained in a growth chamber (25°C-28°C and relative humidity 80%-90%). Brown spots were observed on inoculated leaves (both punctured and unwounded) about 30 hours after inoculation and typical symptoms appeared about 55 hours after inoculation (Supplementary Fig. 1 d), and the diseased leaves produced black pycnidia and orange droplet 10 days after inoculation (Supplementary Fig. 1 e). All inoculated leaves developed symptoms similar to those on the naturally infected plants in the garden and the disease incidence reached 100%, whereas the control leaves remained symptomless (Supplementary Fig. 1 f). The same fungus was re-isolated from inoculated leaves. To our knowledge, this is the first report of S. vannaccii causing leaf spot on C. crepidioides in China. So far, Stagonosporopsis vannaccii has been reported as a plant pathogenic fungus only in Brazil, causing anthracnose symptoms on pods of soybean (Crous et al. 2019). Crassocephalum crepidioides is a widely distributed weed. If S. vannaccii has strong host specificity, it is possible to be used as a biocontrol fungus to control the weed. Conversely, if the fungus has a wider host range, C. crepidioides may act as a good bridge to spread the pathogen. This study helps to deepen the understanding of S. vannaccii and its associated plant diseases.

First Report of Leaf Spot Caused by Alternaria alternata on Sonchus asper in China.

Sonchus asper (Spiny sowthistle), belonging to the Asteraceae, is a problematic weed in grain crops, orchards as well as turf (Cho et al., 2019). In April 2016, about 90% of the S. asper plants infesting a pear orchard had symptomatic with black spots densely distributed on the leaves and stems in Da Yang Town, Luyang District, Hefei, Anhui (N31°55'43″, E117°11'40″). The foliar lesions were mostly circular (3.0 mm to 1.5 cm in diameter) or irregular in shape; lesions on the stem were elliptical to irregular in shape running along the vascular bundle and about 3 to 6mm long × 2 to 3mm wide. Severely affected plants had their leaves become completely blighted and eventually the plants died. Fifteen tissue pieces from five symptomatic leaves and stems were surface sterilized with 0.5% NaClO for two min, rinsed three times with sterile water, and then plated on 1/4-strength potato dextrose agar (PDA) medium. Fourteen fungal isolated were obtained from the tissues, the isolation frequency from the plant tissue pieces was almost 93%. Fungal colonies were circular, initially white, and eventually turned to dark olive or black along with profuse sporulation. The conidia were tawny to brownish green, obclavate to obpyriform, with a cylindrical or coniform short beak, ranging from 16.8 to 39.8 μm long × 6.3 to 14.7 μm wide with two to five transverse and zero to two longitudinal septa (n = 50) and were borne on branched conidiophores. These morphological characteristics were similar to those described for Alternaria alternata (Simmons, 2007). For one representative isolate, the internal transcribed spacer (ITS) region of rDNA, partial coding sequence of the actin (ACT) gene, partial Alt a 1 major allergen (ALT) gene and partial Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced with primers ITS1/ITS4 (White et al., 1990), ACT512F/ACT783R (Carbone and Kohn, 1999), Alt-4for/Alt-4rev and gpd1/gpd2 (Lawrence et al., 2013) respectively. Sequences were submitted to GenBank (ITS: MH886523; ACT: MH892480; ALT: MN655781; GAPDH: MN655782). Based on a BLAST analysis, ITS, ACT gene and GAPDH gene had 100%-99.8% identity with the existing sequences of the ex-type CBS 916.96 of A. alternata (Fries) Keissler (ITS: AF347031; ACT: JQ671702; GAPDH: AY278808, respectively) and ALT gene showed 100% identity with the A. alternata isolate SCWC10 (MG199093). Thus the pathogen was identified as A. alternata based on its morphological and molecular characteristics. Pathogenicity tests were conducted on six to seven-leaf stage potted S. asper plants (one plant per pot). Five mm diameter fungal disks, which were excised from the edge of a three-day old A. alternata colony, were placed on healthy leaves of five plants. Same size sterile PDA disks were used as controls. In addition, a conidial suspension (107 conidia/ml) was also inoculated on healthy leaves of five plants, sterile distilled water was used as control. Both inoculation experiments were conducted in the greenhouse at 25 ± 2°C, 90 % relative humidity, 12 h light/dark photoperiod and repeated at least three times. Lesions were observed on all the leaves within 12 h after fungal disk inoculation. All pathogen inoculated plants developed lesions, similar to those observed on the symptomatic leaves in the field 12 days after conidial inoculation. No symptoms were observed on both control treatments. A. alternata was re-isolated from the inoculated leaves and confirmed by both morphological and molecular techniques, confirming Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf spot on S. asper in China. The pathogen could cause severe disease in S. asper, and has the potential to be further studied as a fungal weed biocontrol.

First Report of Leaf Brown Spot Caused by Epicoccum sorghinum on Digitaria sanguinalis in China

Digitaria sanguinalis (L.) Scop. is a cosmopolitan malignant weed that infests autumn harvest crops and vegetable fields. In September 2017, leaf spots were observed on more than 70% of leaves from D. sanguinalis in a paddy bund located in Chaohu city, Anhui. The spots were primarily circular or elliptical, straw colored, and had brown well-defined margins. Small pieces of symptomatic leaf tissue were surface sterilized with 0.5% NaClO for 2 min, rinsed three times with sterile water, plated on quarter-strength potato dextrose agar (PDA) medium, and cultured in the dark at 28°C. The fungal colonies were transferred to PDA medium after 2 days of cultivation. The colonies were villose and regular and initially white to pale yellow. The colonies then turned gray and had chlamydospore formation at the colony surface. The chlamydospores were gray and nearly spherical, unicellular or multicellular, ranging from 11 to 27 × 10 to 23 μm. The mycelium could secrete pigment and caused the medium to eventually turn crimson. Scratched fungal colonies grown on PDA medium that were irradiated under an 8-W ultraviolet lamp for 12 h at a distance of 30 cm could be induced to produce numerous pycnidia. The pycnidia were brown, spherical, had a cylindrical or coniform ostiole, and ranged from 110 to 183 × 80 to 143 μm. The conidia were hyaline, ellipsoidal, unicellular, aseptate, and ranged from 4.6 to 5.4 × 1.9 to 2.3 μm. These morphological characteristics were consistent with the characteristics described for Epicoccum sorghinum (Sacc.) Aveskamp (Zhou et al. 2018). The internal transcribed spacer (ITS) regions of rDNA, actin (ACT), and β-tubulin (TUB) genes were amplified with the primers ITS1/ITS4, ACT512F/ACT783R, and TUB2FD/TUB4RD (Aveskamp et al. 2009; Carbone and Kohn 1999; White et al. 1990), respectively. The sequence results were submitted to GenBank (ITS, MK039445; ACT, MK044832; TUB, MK044833). BLAST analysis showed that the sequences of the three genes showed 99 to 100% identity with the existing sequences of E. sorghinum in GenBank (MF948994.1, MF987526.1, and MF987525.1). Based on these morphological and molecular analyses, this pathogen was identified as E. sorghinum. Pathogenicity tests were performed on a five-leaf-stage D. sanguinalis plant. For inoculation with mycelium discs, 5-mm-diameter mycelium discs that were cut from the edge of a 5-day-old E. sorghinum colony were inoculated on 10 healthy leaves from D. sanguinalis. Each leaf was inoculated with one disc. Sterile PDA disks of the same size were inoculated as a control. In addition, mycelium suspensions were sprayed on potted D. sanguinalis plants (25 to 30 plants per pot). Control plants were mock inoculated with sterile water, and five pots were used for each treatment. The tests were performed under laboratory conditions of 25 ± 2°C, 90% relative humidity, and 12-h light/dark photoperiod. Obvious lesions were observed 24 h after mycelium disc inoculation, whereas control leaves remained asymptomatic for the next 3 days. Severe symptoms developed on whole plants that were sprayed with the mycelium suspension 5 days later, whereas control plants remained healthy. The same E. sorghinum strain was consistently reisolated from inoculated symptomatic leaves, confirming Koch’s postulates. To our knowledge, this is the first report of E. sorghinum causing leaf brown spots on D. sanguinalis in China. This pathogen could be considered as a potential biocontrol fungus to control D. sanguinalis pending further study.

First Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in Italy

HomePlant DiseaseVol. 103, No. 4First Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in Italy PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in ItalyI. Camele and S. M. MangI. Camele†Corresponding author: I. Camele; E-mail: E-mail Address: ippolito.camele@unibas.ithttp://orcid.org/0000-0001-8099-8234School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, ItalySearch for more papers by this author and S. M. MangSchool of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, ItalySearch for more papers by this authorAffiliationsAuthors and Affiliations I. Camele † S. M. Mang School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, Italy Published Online:13 Feb 2019https://doi.org/10.1094/PDIS-09-18-1686-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In spring 2018, in a vineyard (7 ha) located in Matera province (southern Italy), during a survey to verify the sanitary status of grapevines in Basilicata Region, Vitis vinifera plants (25-year-old, cv. Sangiovese) were found with typical “esca disease” symptoms on the leaves (tiger stripes). In addition, trunk cankers and internal wood necrosis were observed. More than 30% of the living plants were symptomatic and 10% more were dead. To isolate the likely pathogen, small pieces from trunk discs of living, symptomatic plants were surface sterilized with 70% alcohol, plated in Petri dishes containing potato dextrose agar (PDA), and incubated at 24°C for 14 days in darkness. From all samples, only a single fungal species was isolated, and this was identified as Seimatosporium vitis X.P. Xiao, Camporesi E. & K.D. Hyde through morphological comparison (Senanayake et al. 2015) and a molecular analysis based on three genes including the rDNA-ITS region (ITS), β-tubulin-2 (TUB2), and translation elongation factor 1-α (TEF1-alpha). The sequences of four pure-culture isolates were deposited in EMBL GenBank under accession numbers LS991528 to LS991531 (ITS), LS997596 to LS997599 (TUB-2), and LS999502 to LS999505 (TEF1-alpha). All sequences showed 99 to 100% identity with authentic S. vitis sequences already present in the GenBank database under the following accession numbers: ITS (KY632661, KX555652, and KU721882), TUB-2 (KY706248, KY706254, and KY706257), and TEF1-alpha (KY06328, KY06331, and KY06332). A phylogenetic tree, based on combined ITS, TUB-2, and TEF1-alpha data, was obtained using MEGA version 6.0 with the maximum parsimony analyses revealing that all isolates fell in the S. vitis clade, confirming the species identification. Koch’s postulates were fulfilled using four S. vitis isolates inoculated on woody stems of 2-year-old potted cultivar Sangiovese. Shoots were surface sterilized with 70% alcohol and then wounded with a sterile scalpel. Discs (0.5-mm diameter) from 14-day-old agar cultures were applied to the wound and sealed with Parafilm, and then the plants were kept in a growth chamber at 24°C and 90% humidity and 16-h light. Sterile PDA discs were used as controls. Each isolate was inoculated onto 10 plants, and five others were used as controls (45 plants in total). The pathogenicity test was carried out twice. About 45 days after inoculation, each inoculated shoot presented a canker lesion about 3 to 4 cm long around the inoculation point and interveinal chlorosis on leaves. Control plants were always symptomless. Only S. vitis was consistently reisolated from inoculated tissues. Pathogenicity assays confirmed that S. vitis from V. vinifera plants is the causal agent of the observed symptoms in grapevine. S. vitis was reported to be associated with grapevine trunk diseases (GTDs) in Hungary (Váczy 2017) and in California (Lawrence et al. 2018). Senanayake et al. (2015) had earlier reported S. vitis from Italy but not in association with GTDs and without pathogenicity tests. This is the first report of S. vitis associated with the GTDs in Italy.

First Report of Curvularia asianensis Causing Leaf Blotch of Epipremnum pinnatum in Guangxi Autonomous Region of China

Epipremnum pinnatum Engl. is a flowering plant in the family Araceae. In southern China, it is often cultivated for medicinal and ornamental purposes. A distinct leaf blotch was observed on E. pinnatum in October 2017. Symptoms included gray or tan irregular blotches, with reddish to yellow margins. Portions of the symptomatic leaves were surface sterilized in 70% ethanol for 30 s and then in 2% NaClO for 1 min, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). Seven strains (GUCC 8601 to GUCC 8607) from four diseased E. pinnatum plants were obtained by single spore selection. Fungal colonies on PDA reached 70 to 75 mm in diameter after 7 days, were velvety to slightly powdery, and were gray olivaceous to olivaceous black both top and reverse. Conidiophores were single or in groups, septate, straight or flexuous, and geniculate. Conidiogenous cells were smooth walled to finely verruculose, terminal or intercalary, proliferating sympodially, pale brown to brown, subcylindrical to swollen, and 6 to 14 × 3 to 5 μm (n = 30). Conidia were curved, pale brown to brown, 3 to 4 distoseptate, 23 × 9 μm (n = 50) with a protuberant hilum; morphology was consistent with Curvularia asianensis Manamgoda, L. Cai & K. D. Hyde, isolated from a dried Panicum sp. leaf in northern Thailand (Manamgoda et al. 2012). The internal transcribed spacer (ITS) region and partial sequences of two protein-coding genes, namely, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-α (TEF1), were amplified using primers ITS1 and ITS4 (White et al. 1990), gpd1 and gpd2 (Berbee et al. 1999), and EF983F and 2218R (Schoch et al. 2009). The ITS, GAPDH, and TEF1 sequences were compared with entries in GenBank database, through nucleotide BLAST search, and our isolates showed higher than 99% identity to the ex-type strain (MFLUCC 10-0711) of C. asianensis (ITS, JX256424; GAPDH, JX276436; and TEF1, JX266593) for these three gene regions. The three gene regions were combined into a multigene phylogenetic tree. On the basis of morphological characteristics and nucleotide homology, our isolates were confirmed as C. asianensis. For pathogenicity tests, mycelial disks (5 mm in diameter) cut from 1-week-old PDA cultures of two strains (GUCC 8601 and GUCC 8602) were placed on four healthy leaves (two inoculated points on every leaf) of an E. pinnatum plant under natural conditions on 20 November 2017 and removed after 48 h. Leaves inoculated with sterile PDA disks served as controls. Within 24 h after discs were removed, tan lesions began to appear on the leaves. Seven days after inoculation, all inoculated leaves developed blotch symptoms consistent with what was observed in nature. C. asianensis strains were reisolated from infected tissues and confirmed by the methods mentioned above. Controls remained healthy. To our knowledge, this is the first report of C. asianensis causing leaf blotch disease on E. pinnatum in China.

First Report of Sclerotium hydrophilum Causing Stem Rot of Rice in Northeast China

In August 2016, rice plants with brown to black lesions with irregular shape and water-soaked margins in leaf sheaths were observed in Baoqing county in Heilongjiang province, Northeast China. To isolate the causal pathogen, pieces of symptomatic leaf sheaths were treated with 1% NaClO for 1 min, rinsed with sterile distilled water for 2 min, and then transferred to potato dextrose agar (PDA) medium for incubation at 28°C. The cultivated mycelium was transferred to new PDA medium. Colonies of pure cultures on new PDA medium were initially white and turned brown about 3 weeks later. The hyphal width was measured with a range of 4.5 to 6.2 μm. Large numbers of small and globose sclerotia were observed on surface of the colonies at 3 days after subculturing. The sclerotia were white at first and then turned black over time. The diameters of sclerotia ranged from 0.31 to 0.54 mm with an average of 0.41 mm (n = 50). DNA of a representative isolate named Hbq001 was extracted, and the 18S rDNA region was amplified by PCR with universal primer pair NS1/NS6 (White et al. 1990). The 18S rDNA sequence of isolate Hbq001 (GenBank accession no. KY995575) shared 99.7% nucleotide identities with those of Sclerotium hydrophilum (GenBank accession no. KC354147). Therefore, based on these cultural characteristics and rDNA sequence, isolate Hbq001 was identified as S. hydrophilum (Demirci et al. 2009). To complete Koch’s postulates, 4 mm diameter PDA disks containing mycelium and sclerotia of S. hydrophilum isolate Hbq001 were placed on the stem of 4-week-old healthy rice seedlings and wrapped with Parafilm. Sterile PDA disks were used as controls. After 4 days at 28°C, symptoms similar to those in the field were observed on the inoculated seedlings but not on control plants. Finally, the fungus S. hydrophilum, which showed the same cultural characteristics as described above, was reisolated from the inoculated seedlings. In China, S. hydrophilum was first identified in Yunnan province and was found to be distributed in southern China (Hu et al. 2008; Ruan and Chen 2002; Wang et al. 2013). Northeast China is a major rice-producing region. Differing from the rice-producing areas of southern China, this region has specific climatic conditions with cold temperature and abounds in high-quality japonica rice. To our knowledge, this is the first report of S. hydrophilum in the cold northern region of China.

FIRST REPORT OF LEAF SPOT OF CAMPANULA GLOMERATA CAUSED BY ALTERNARIA sp. IN ITALY

During Summer 2014, extensive necrosis were observed on Campanula glomerata plants grown in a private garden near the city of Biella (northern Italy). Pale-brown, irregular lesions were present on the leaves that wilted and rotted. When also the stems were affected, the plants died. A fungus was consistently isolated on potato dextrose agar (PDA) which, on potato carrot agar (PCA) produced a greenish mycelium, with brown, septate, ovoid, elliptical or obclavate conidia, measuring 8-33×6-11 μm (average: 15×8 μm). These morphometric traits allowed the identification of the pathogen as Alternaria sp. (Simmons, 2007). DNA was extracted using the NucleoSpin Plant kit (Macherey Nagel, Germany) and PCR amplification carried out using ITS1/ITS4 primers. The 484 bp amplified product was sequenced (GenBank accession No. KP225273) and a BLASTn search (Altschul et al., 1997) confirmed that the sequence corresponded to that of Alternaria sp. For pathogenicity tests, leaves of three healthy plants of C. glomerata were inoculated with disks excised from fungal colonies grown on PDA. Plants inoculated only with sterile PDA disks served as controls. All plants were covered with plastic bags and maintained at a temperature of 19 to 25°C. Seven days post inoculation, lesions developed only on inoculated leaves from which the same fungus used for inoculation was consistently reisolated. To the best of our knowledge this the first report of Alternaria sp. on C. glomerata in Italy and in the world.