First Report of Bipolaris oryzae Causing Leaf Spot on Cultivated Wild Rice (Oryza rufipogon) in China

Wild rice (Oryza rufipogon) has been widely studied and cultivated in China in recent years due to its antioxidant activities and health-promoting effects. In December 2018, leaf spot disease on wild rice (O. rufipogon cv. Haihong-12) was observed in Zhanjiang (20.93 N, 109.79 E), China. The early symptom was small purple-brown lesions on the leaves. Then, the once-localized lesions coalesced into a larger lesion with a tan to brown necrotic center surrounded by a chlorotic halo. The diseased leaves eventually died. Disease incidence was higher than 30%. Twenty diseased leaves were collected from the fields. The margin of diseased tissues was cut into 2 × 2 mm2 pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Fifteen isolates were obtained. Two isolates (OrL-1 and OrL-2) were subjected to further morphological and molecular studies. The colonies of OrL-1 and OrL-1 on PDA were initially light gray, but it became dark gray with age. Conidiophores were single, straight to flexuous, multiseptate, and brown. Conidia were oblong, slightly curved, and light brown with four to nine septa, and measured 35.2–120.3 µm × 10.3–22.5 µm (n = 30). The morphological characteristics of OrL-1 and OrL-2 were consistent with the description on Bipolaris oryzae (Breda de Haan) Shoemaker (Manamgoda et al. 2014). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, GDF1gpp1/GDR1 gdp2 (Berbee et al. 1999), and EF-1α-F/EF-1α-R EF-1/EF-2 (O’Donnell 2000), respectively. Amplicons of OrL-1 and OrL-2 were sequenced and submitted to GenBank (accession nos. MN880261 and MN880262, MT027091 and MT027092, and MT027093 and MT027094). The sequences of the two isolates were 99.83%–100% identical to that of B. oryzae (accession nos. MF490854，MF490831，MF490810) in accordance with BLAST analysis. A phylogenetic tree was generated on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via Maximum Likelihood method, which clustered OrL-1 and OrL-2 with B. oryzae. The two isolates were determined as B. oryzae by combining morphological and molecular characteristics. Pathogenicity test was performed on OrL-1 in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Rice (cv. Haihong-12) with 3 leaves was grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on leaves after 10 days, but the controls remained healthy. The morphological characteristics and ITS sequences of the fungal isolates re-isolated from the diseased leaves were identical to those of B. oryzae. B. oryzae has been confirmed to cause leaf spot on Oryza sativa (Barnwal et al. 2013), but as an endophyte has been reported in O. rufipogon (Wang et al. 2015).. Thus, this study is the first report of B. oryzae causing leaf spot in O. rufipogon in China. This disease has become a risk for cultivated wild rice with the expansion of cultivation areas. Thus, vigilance is required.

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First Report of Leaf Spots caused by Nigrospora oryzae on Wild Rice in China

Plant Disease ◽

10.1094/pdis-11-20-2522-pdn ◽

2021 ◽

Author(s):

Yue Lian Liu ◽

Jian Rong Tang ◽

Ya Li ◽

Hong Kai Zhou

Keyword(s):

Leaf Spot ◽

Wild Rice ◽

Morphological Characteristics ◽

Oryza Rufipogon ◽

Its Sequences ◽

Likelihood Method ◽

Pathogenicity Test ◽

Sterile Water ◽

First Report ◽

Nigrospora Oryzae

In recent years, wild rice (Oryza rufipogon Griff) has been widely cultivated because of its health-promoting effects. In May 2019, leaf spot lesions on cv. Haihong-12 were observed in Zhanjiang (20.93N, 109.79E), China. Leaf symptoms were yellow-to-brown, oval or circular with a very distinctive, large yellow halo. Black spores appeared on the leaves with advanced symptoms. The lesions coalesced, causing the entire leaf to become blighted and die. Disease incidence reached approximately 10% in the fields (8 ha) surveyed. Twenty leaves with symptoms were collected and cut into pieces of 2 ×2 cm in size. They were surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water, blotted dry on sterile paper, plated on potato dextrose agar (PDA) medium, and incubated at 28°C in the dark for 4 days. Ten pure cultures were obtained by transferring hyphal tips to new PDA plates, and monosporic cultures were obtained from three isolates (Nos-1, Nos-2, and Nos-3). Those isolates exhibited very similar morphological characteristics on PDA. Colony of isolate Nos-1 was white at the early stage and became dark gray after 7 days. Conidia were produced from clusters of conidiophores, single celled, black, smooth, spherical, and 9.5 to 14.2 µm (average 10.6 µm ± 0.42) in diameter. Morphological characteristics of the isolates matched the description of Nigrospora oryzae Petch (Wang et al. 2017). The ITS region was amplified using primers ITS1 and ITS4 (White et al. 1990). Nucleotide sequences of isolates Nos-1, Nos-2, and Nos-3 deposited in GenBank under acc. nos. MW042173, MW042174, and MW042175, respectively, were 100% identical to N. oryzae (acc. nos. KX985944, KX985962; and KX986007). A phylogenetic tree generated based on the ITS sequences and using a Maximum Likelihood method with 1,000 bootstraps showed that these three isolates from wild rice were grouped with other N. oryzae isolates downloaded from GenBank (bootstrap = 100%) but away from other Nigrospora spp. Pathogenicity test was performed with these three isolates in a greenhouse at 24 to 30°C. Approximately 50 seedling of wild rice cv. Haihong-12 were grown in each pot. At the 3-leaf stage, plants in three pots were inoculated with each isolate by spraying a spore suspension (105 spores/ml) until runoff. Three pots sprayed with sterile water served as the controls. Each 3-pot treatment was separately covered with a plastic bag. The test was conducted three times. Diseased symptoms were observed on the inoculated leaves after 10 days while no disease was observed in the control plants. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased leaves were identical to those of N. oryzae. N. oryzae has been reported to cause leaf spot on O. sativa (Wang et al. 2017), but not on O. rufipogon. Thus, this is the first report of N. oryzae causing leaf spot of O. rufipogon in China. The finding provides the information important for further studies to develop management strategies for control of this disease.

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First Report of Leaf Spot caused by Curvularia lunata on Wild Rice in China

Plant Disease ◽

10.1094/pdis-01-21-0147-pdn ◽

2021 ◽

Author(s):

Hong Kai Zhou ◽

Yue Lian Liu ◽

Jian Rong Tang ◽

Fei Teng Zhong ◽

Ya Li

Keyword(s):

Leaf Spot ◽

Wild Rice ◽

Disease Incidence ◽

Its Region ◽

Oryza Rufipogon ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Curvularia Lunata ◽

Sterile Water

Wild rice (Oryza rufipogon), a species only recently cultivated in China, is an invaluable resource for rice breeding and basic research. In June 2019, a leaf spot disease on wild rice (O. rufipogon cv. ‘Haihong-12’) was observed in a 3.3 ha field in Zhanjiang (20.93 N, 109.79 E), China. The early symptoms were the presence of small, brown, and circular to oval spots that eventually turned reddish brown. The size of the spots varied from 1.0–5.0 mm × 1.0–3.0 mm. Disease incidence was higher than 20%. High temperature and high humidity climate were favorable for the disease occurrence. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, then rinsed three times with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates, namely, Cls-1, Cls-2, and Cls-3, were subjected to further morphological and molecular studies. The colonies of the three isolates on PDA were initially light gray later becoming dark green. Conidiophores were erect, dark brown, geniculate, and unbranched. Conidia were fusiform, geniculate or hook-shaped, smooth-walled, dark-brown, 3-septate, with the second curved cell about 13.4–18.2 μm × 6.5–8.6 μm in size (n = 30). These morphological features agreed with previous descriptions of Curvularia lunata (Wakker) Boed (Macri and Lenna 1974). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, gpp1/gdp2 (Berbee et al. 1999), and EF-1/EF-2 (O’Donnell 1997), respectively. Amplicons of the three isolates were sequenced and submitted to GenBank (accession nos. MW042182, MW042183, and MW042184; MW091453, MW091454, and MW091455; MW090049, MW090050, and MW090051). The sequences of the two isolates were 100% identical to those of C. lunata (accession nos. MG971304, MG979801, MG979800) according to the results of BLAST analysis. A phylogenetic tree was built on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via the maximum likelihood method. The tree clustered Cls-1, Cls-2, and Cls-3 with C. lunata. The three isolates were determined as C. lunata by combining morphological and molecular characteristics. Pathogenicity tests were performed on Cls-1 in a greenhouse at 24 °C–30 °C with 80% relative humidity. Individual rice plants (cv. ‘Haihong-12’) with three leaves were grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and another five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on the leaves after 10 days, but the controls remained healthy. C. lunata occurs on O. sativa (rice) (Liu et al. 2014; Majeed et al. 2016), but it has not been reported on O. rufipogon until now. To the best of our knowledge, this study is the first to report that C. lunata causes leaf spots on O. rufipogon in China. Thus, vigilance is required for breeding O. rufipogon.

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First Report of Leaf Spot caused by Bipolaris oryzae on Switchgrass in Tennessee

Plant Disease ◽

10.1094/pdis-01-13-0070-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1654-1654 ◽

Cited By ~ 3

Author(s):

A. L. Vu ◽

M. M. Dee ◽

J. Zale ◽

K. D. Gwinn ◽

B. H. Ownley

Keyword(s):

Leaf Spot ◽

Panicum Virgatum ◽

Morphological Characteristics ◽

Its Region ◽

Spore Production ◽

Post Inoculation ◽

Sterile Water ◽

Bipolaris Oryzae ◽

First Report ◽

Symptomatic Leaf

Knowledge of pathogens in switchgrass, a potential biofuels crop, is limited. In December 2007, dark brown to black irregularly shaped foliar spots were observed on ‘Alamo’ switchgrass (Panicum virgatum L.) on the campus of the University of Tennessee. Symptomatic leaf samples were surface-sterilized (95% ethanol, 1 min; 20% commercial bleach, 3 min; 95% ethanol, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). A sparsely sporulating, dematiaceous mitosporic fungus was observed. Fungal plugs were transferred to surface-sterilized detached ‘Alamo’ leaves on sterile filter paper in a moist chamber to increase spore production. Conidia were ovate, oblong, mostly straight to slightly curved, and light to olive-brown with 3 to 10 septa. Conidial dimensions were 12.5 to 17 × 27.5 to 95 (average 14.5 × 72) μm. Conidiophores were light brown, single, multiseptate, and geniculate. Conidial production was polytretic. Morphological characteristics and disease symptoms were similar to those described for Bipolaris oryzae (Breda de Haan) Shoemaker (2). Disease assays were done with 6-week-old ‘Alamo’ switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized in 50% bleach. Nine 9 × 9-cm square pots with approximately 20 plants per pot were inoculated with a mycelial slurry (due to low spore production) prepared from cultures grown on potato dextrose agar for 7 days. Cultures were flooded with sterile water and rubbed gently to loosen mycelium. Two additional pots were inoculated with sterile water and subjected to the same conditions to serve as controls. Plants were exposed to high humidity by enclosure in a plastic bag for 72 h. Bags were removed, and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the disease assay, plants were kept in a growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 5 to 14 days post-inoculation for eight of nine replicates. Control plants had no symptoms. Symptomatic leaf tissue was processed and plated as described above. The original fungal isolate and the pathogen recovered in the disease assay were identified using internal transcribed spacer (ITS) region sequences. The ITS region of rDNA was amplified with PCR and primer pairs ITS4 and ITS5 (4). PCR amplicons of 553 bp were sequenced, and sequences from the original isolate and the reisolated pathogen were identical (GenBank Accession No. JQ237248). The sequence had 100% nucleotide identity to B. oryzae from switchgrass in Mississippi (GU222690, GU222691, GU222692, and GU222693) and New York (JF693908). Leaf spot caused by B. oryzae on switchgrass has also been described in North Dakota (1) and was seedborne in Mississippi (3). To our knowledge, this is the first report of B. oryzae from switchgrass in Tennessee. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/, 28 June 2012. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) M. Tomaso-Peterson and C. J. Balbalian. Plant Dis. 94:643, 2010. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: a Guide to Methods and Applications. M. A. Innis et al. (eds), Acad. Press, San Diego, 1990.

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First Report of Epicoccum layuense Causing Leaf Brown Spot on Camellia oleifera in Hefei, China

Plant Disease ◽

10.1094/pdis-08-21-1726-pdn ◽

2022 ◽

Author(s):

Xiang Xie ◽

Shiqiang Zhang ◽

Qingjie Yu ◽

Xinye Li ◽

Yongsheng Liu ◽

...

Keyword(s):

Leaf Spot ◽

Brown Spot ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Original Strain ◽

Conidial Suspension ◽

Camellia Oleifera ◽

Sterile Water ◽

Beta Tubulin ◽

First Report

Camellia oleifera, a major tree species for producing edible oil, is originated in China. Its oil is also called ‘‘eastern olive oil’’ with high economic value due to richness in a variety of healthy fatty acids (Lin et al. 218). However, leaves are susceptible to leaf spot disease (Zhu et al. 2014). In May 2021, we found circular to irregular reddish-brown lesions, 4-11 mm in diameter, near the leaf veins or leaf edges on 30%-50% leaves of 1/3 oil tea trees in a garden of Hefei City, Anhui Province, China (East longitude 117.27, North latitude 31.86) (Figure S1 A). To isolate the causal agents, symptomatic leaves were cut from the junction of diseased and healthy tissues (5X5 mm) and treated with 70 % alcohol for 30 secs and 1 % NaClO for 5 min, and subsequently inoculated onto PDA medium for culture. After 3 days, hyphal tips were transferred to PDA. Eventually, five isolates were obtained. Then the isolates were cultured on PDA at 25°C for 7 days and the mycelia appeared yellow with a white edge and secreted a large amount of orange-red material to the PDA (Figure S1 B and C). Twenty days later, the mycelium appeared reddish-brown, and sub-circular (3-10 mm) raised white or yellow mycelium was commonly seen on the Petri dish, and black particles were occasionally seen. Meanwhile, the colonies on the PDA produced abundant conidia. Microscopy revealed that conidia were globular to pyriform, dark, verrucose, and multicellular with 14.2 to 25.3 μm (=19.34 μm, n = 30) diameter (Figure S1 D). The morphological characteristics of mycelial and conidia from these isolates are similar to that of Epicoccum layuense (Chen et al.2020). To further determine the species classification of the isolates, DNA was extracted from 7-day-old mycelia cultures and the PCR-amplified fragments were sequenced for internal transcribed spacer (ITS), beta-tubulin and 28S large subunit ribosomal RNA (LSU) gene regions ITS1/ITS4, Bt2a/Bt2b and LR0R/LR5, followed by sequencing and molecular phylogenetic analysis of the sequences analysis (White et al. 1990; Glass and Donaldson 1995; Vilgalys and Hester 1990). Sequence analysis revealed that ITS, beta-tubulin, and LSU divided these isolates into two groups. The isolates AAU-NCY1 and AAU-NCY2, representing the first group (AAU-NCY1 and AAU-NCY5) and the second group (AAU-NCY2, AAU-NCY3 and AAU-NCY4), respectively, were used for further studies. Based on BLASTn analysis, the ITS sequences of AAU-NCY1 (MZ477250) and AAU-NCY2 (MZ477251) showed 100 and 99.6% identity with E. layuense accessions MN396393 and KY742108, respectively. And, the beta-tubulin sequences (MZ552310; MZ552311) showed 99.03 and 99.35% identity with E. layuense accessions MN397247 and MN397248, respectively. Consistently, their LSU (MZ477254; MZ477255) showed 99.88 and 99.77% identity with E. layuense accessions MN328724 and MN396395, respectively. Phylogenetic trees were built by maximum likelihood method (1,000 replicates) using MEGA v.6.0 based on the concatenated sequences of ITS, beta-tubulin and LSU (Figure S2). Phylogenetic tree analysis confirmed that AAU-NCY1 and AAU-NCY2 are closely clustered with E. layuense stains (Figure S2). To test the pathogenicity, conidial suspension of AAU-NCY2 (106 spores/mL) was prepared and sterile water was used as the control. Twelve healthy leaves (six for each treatment) on C. oleifera tree were punched with sterile needle (0.8-1mm), the sterile water or spore suspension was added dropwise at the pinhole respectively (Figure S1 E and F). The experiment was repeated three times. By ten-day post inoculation, the leaves infected by the conidia gradually developed reddish-brown necrotic spots that were similar to those observed in the garden, while the control leaves remained asymptomatic (Figure S1 G and H). DNA sequences derived from the strain re-isolated from the infected leaves was identical to that of the original strain. E. layuense has been reported to cause leaf spot on C. sinensis (Chen et al. 2020), and similar pathogenic phenotypes were reported on Weigela florida (Tian et al. 2021) and Prunus x yedoensis Matsumura in Korea ( Han et al. 2021). To our knowledge, this is the first report of E. layuense causing leaf spot on C. oleifera in Hefei, China.

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First report of leaf spot disease caused by Stemphylium eturmiunum on garlic in Korea.

Plant Disease ◽

10.1094/pdis-03-21-0674-pdn ◽

2021 ◽

Author(s):

Walftor Dumin ◽

Mi-Jeong Park ◽

You-Kyoung Han ◽

Yeong-Seok Bae ◽

Jong-Han Park ◽

...

Keyword(s):

Leaf Spot ◽

Allium Sativum ◽

Morphological Characteristics ◽

Fungal Isolate ◽

Causal Agent ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

First Report ◽

Spot Disease ◽

Intact Leaves

Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 – 22 × 16 – 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch’s postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in Korea.

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

Plant Disease ◽

10.1094/pdis-09-21-1901-pdn ◽

2021 ◽

Author(s):

Lei Li ◽

Yishuo Huang ◽

Yanxia Shi ◽

A LI CHAI ◽

Xuewen Xie ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Citrate Synthase ◽

Morphological Characteristics ◽

Likelihood Method ◽

Bacterial Suspension ◽

Leaf Spot Disease ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

First Report

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.

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First Report of Leaf Spot on Industrial Hemp (Cannabis sativa L.) Caused by Alternaria alternata in China

Plant Disease ◽

10.1094/pdis-01-21-0130-pdn ◽

2021 ◽

Author(s):

Lili Tang ◽

Xixia Song ◽

Liguo Zhang ◽

Jing Wang ◽

Shuquan Zhang

Keyword(s):

Leaf Spot ◽

Cannabis Sativa ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Leaf Spots ◽

Industrial Hemp

Industrial hemp is an economically important plant with traditional uses for textiles, paper, building materials, food and medicine (Li 1974; Russo et al. 2008; Zlas et al. 1993). In August 2020, an estimated 80% of the industrial hemp plants with leaf spots were observed in greenhouse in Minzhu town, Harbin City, Heilongjiang Province, China (45.8554°N, 126.8167°E), resulting in yield losses of 20%. Leaf symptoms began as small spots on the upper surface of leaves and gradually developed into brown spots with light yellow halos. These irregular spots expanded gradually and eventually covered the entire leaf; the center of the spots was easily perforated. To identify the pathogen, 20 diseased leaves were collected, and small sections of (3 to 5 mm) were taken from the margins of lesions of infected leaves. The pieces were sterilized with 75% alcohol for 30 s, a 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water. Samples were then cultured on potato dextrose agar at 28℃ in darkness for 4 days. A single-spore culture was obtained by monosporic isolation. Conidiophores were simple or branched, straight or flexuous, brown, and measured 22 to 61 μm long × 4 to 5 μm wide (n = 50). Conidia were solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid. Conidia ranged from 23 to 55 μm long × 10 to 15 μm wide (n = 50) with one to eight transverse and several longitudinal septa. For molecular identification (Jayawardena et al. 2019), genomic DNA of pathogenic isolate (MZ1287) was extracted by a cetyltrimethylammonium bromide protocol. Four gene regions including the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosplate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1) and RNA polymerase II beta subunit (RPB2) were amplified with primers ITS1/ITS4, GDF1/GDR1, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR, respectively (White et al. 1990). Resulting sequences were deposited in GenBank with accession numbers of MW272539.1, MW303956.1, MW415414.1 and MW415413.1, respectively. A BLASTn analysis showed 100% homology with A. alternata (GenBank accession nos. MN615420.1, MH926018.1, MN615423.1 and KP124770.1), respectively. A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate MZ1287 clustered in the A. alternata clade with 100% bootstrap support. Thus, based on morphological (Simmons 2007) and molecular characteristics, the pathogen was identified as A. alternata. To test pathogenicity, leaves of ten healthy, 2-month-old potted industrial hemp plants were sprayed using a conidial suspension (1×106 spores/ml). Control plants were sprayed with sterile water. All plants were incubated in a greenhouse at 25℃ for a 16 h light and 8 h dark period at 90% relative humidity. The experiment was repeated three times. After two weeks, leaf spots of industrial hemp developed on the inoculated leaves while the control plants remained asymptomatic. The A. alternata pathogen was re-isolated from the diseased leaves on inoculated plants, fulfilling Koch's postulates. Based on morphology, sequencing, and pathogenicity test, the pathogen was identified as A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spot disease of industrial hemp (Cannabis sativa L.) in China and is worthy of our attention for the harm it may cause to industrial hemp production.

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Leaf Spot Disease Caused by Pestalotiopsis kenyana on Zanthoxylum schinifolium in Sichuan Province, China

Plant Disease ◽

10.1094/pdis-10-20-2247-pdn ◽

2021 ◽

Author(s):

Chang Liu ◽

Fengying Luo ◽

Tianhui Zhu ◽

Shan Han ◽

Shujiang Li

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Molecular Techniques ◽

Morphological Observation ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Sterile Water ◽

Spot Disease ◽

Severe Stage ◽

Zanthoxylum Schinifolium

Zanthoxylum schinifolium Sieb. et Zucc, a species of prickly ash, is one of the main economic plants in China and mainly grown in Southwest China. The planting area of Z. schinifolium accounts for more than 70% of the total area of prickly ash, and one of the largest plantings of Z. schinifolium is located in Jianyang City (Sichuan) with the area of 6.67 km2. Since 2018, Z. schinifolium, located in Jianyang City, have developed leaf spot disease, with approximately 50% showing disease symptoms. At the beginning of the occurrence, yellow-brown lesions formed on the leaves; in the later stages, the area of the lesions expanded. At the severe stage, multiple lesions merged into one large, dead spot, and the plants failed to blossom and bear fruit. The samples were collected from typical symptoms of Z. schinifolium leaves in Jianyang City. A total of 20 leaf samples were collected from 5 Z. schinifolium plants (4 leaves per plant), and were cut into small pieces of 2 × 2mm at the junction of infected and healthy tissues. These tissues were surface-disinfested for 30 s in 3% sodium hypochlorite and the for 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml), and incubated in a dark incubator at 25°C. Morphological observation was performed on 18 recovered isolates, 15 of which were described as Pestalotiopsis sp. The colonies were incubated on PDA at 25°C for 7 days and reached a diameter of 80-90 mm. The colonies were white with undulating edges and were similar in colors on the reverse side. After colony culture at 25°C for 10 days, gregarious black conidiomata were scattered on the mycelial mats. The conidia and appendages of the samples were measured by Leica Application Suite X 3.4.1.17822 (20 conidia per isolate), and the sizes of which were consistent with the description from Maharachchikumbura et al. Based on morphological observations, the isolates were identified as Pestalotiopsis kenyana Maharachch., K.D. Hyde & Crous. PCR was performed with primers ITS1/ITS4 for the ITS region, primers D1/D2 for the large subunit ribosomal RNA gene (LSU), primers 5f2/7cr for the RNA polymerase II second largest subunit (RPB2), primers Bt2a/Bt2b for the β-tubulin gene (TUB), and primers EF1-526F/EF2-567R for the translation elongation factor 1-alpha gene (TEF). The Sanger-sequenced PCR products were sequenced and blasted in GenBank, and the sequences showed that ITS: 99.17% (594 out of 599 bp), LSU: 100% (909 out of 909 bp), RPB2: 99.17% (832 out of 832 bp), TUB: 100% (774 out of 774 bp), TEF: 100% (485 out of 485 bp) with the type specimen of P. kenyana CBS 442.67 (ITS: GenBank accession NR147549.1, LSU: MH870724.1, PRB2: MH554958.1, TUB: KM199395.1, TEF: KM199502.1). Representative sequences were deposited in GenBank (ITS: MT509798; LSU: MT509800; RPB2: MT522448; TUB: MT522450; TEF: MT522449). To fulfill Koch's postulates, leaves on fifteen one-year-old healthy potted Z. schinifolium plants were sterilized by 75% ethanol cotton balls, and were rinsed by sterile water for three times. Then each leaf was punctured with sterile needles for two wounds (five leaves per plant). The wounds were inoculated by placing 8 mm mycelial plugs obtained from the periphery of 7-day-old single-spore cultures. An equal number of plants were wounded with the same method, and were respectively inoculated with sterile water and PDA plugs without mycelium as controls. All plants were placed in a growth chamber at 25°C under 90% relative humidity. After 7 days, all mycelial-inoculated leaves of the plants showed symptoms identical to those described above, whereas the control plants remained symptom free. P. kenyana was re-isolated from the infected leaves and confirmed to be the same as the inoculated pathogen through analyses of morphological characteristics and molecular techniques. The pathogenicity test was repeated three times with similar results. To our knowledge, this is the first report of P. kenyana as a causal agent of leaf spot disease on Z. schinifolium in China. These findings will aid the development of better preventive measures in accordance with the emergence of this new pathogen.

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First Report of Leaf Spot of Weigela florida Caused by Epicoccum layuense in China

Plant Disease ◽

10.1094/pdis-07-20-1498-pdn ◽

2021 ◽

Author(s):

Yue Tian ◽

Yingying Zhang ◽

Chaodong Qiu ◽

Zhenyu Liu

Keyword(s):

Leaf Spot ◽

Large Subunit ◽

Morphological Characteristics ◽

Likelihood Method ◽

Pathogenicity Test ◽

Distilled Water ◽

Beta Tubulin ◽

First Report ◽

Leaf Spots ◽

Weigela Florida

Weigela florida (Bunge) A. DC. is a dense, rounded, deciduous shrub commonly planted in landscapes. It is also used in Chinese medicine to treat sore throat, erysipelas, cold, and fever (Zheng et al. 2019). In May 2019, leaf spots were observed on approximately 50% of W. florida plants grown in the Wisdom Plaza Park of Anhui Agricultural University in Hefei, Anhui Province, China. Leaf spots begun as small light brown and irregular lesions, enlarged, turned reddish brown, coalesced to form large blighted areas, and eventually covered the entire leaf surface. Five pieces of tissues were removed from the lesion margins of each diseased leaf (five leaves from five different plants), chopped into several 3-4 mm2 pieces, disinfected with 1.5% NaOCl for 2 min, rinsed 3 times with sterile distilled water for 1 min, plated onto Potato Dextrose Agar (PDA) medium containing 50 μg/ml of ampicillin and kanamycin, and incubated at 25°C with a 12-hour photoperiod for 5 days. One segment of the fungal growth from the growing edge of the colony was transferred onto a fresh PDA plate for purification and incubated under the same conditions for another 5 days. The colony morphology of one representative isolate (AAU0519) was characterized by a pale orange cushion in the center surrounded by irregular pink margin, diffusing red orange pigments into the PDA medium. Isolate AAU0519 was cultured on PDA medium for 7 days at 25°C in the dark to induce sporulation. The produced conidia were globose, subglobose to pyriform, golden brown to brown, and with a diameter of 7.7 - 23.8 μm. Both cultural and morphological characteristics suggested that isolate AAU0519 was an Epicoccum species, according to the description by Chen et al. 2017. Amplification and sequencing of the internal transcribed spacer (ITS), beta-tubulin, and 28S large subunit ribosomal RNA (LSU) gene fragments from the extracted genomic DNA of AAU0519 were performed using primer sets ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and LSU1Fd/LR5 (Crous et al. 2009; Vilgalys and Hester 1990), respectively. A phylogenetic tree was constructed by the maximum-likelihood method with 1,000 bootstrapping replications based on the concatenated ITS, beta-tubulin, and LSU sequences from isolate AAU0519 and representative strains of 22 species of the genus Epicoccum (Chen et al. 2017). Isolate AAU0519 clustered with ex-holotype CGMCC 3.18362 of Epicoccum layuense Qian Chen, Crous & L. Cai (Chen et al. 2017). All obtained sequences were deposited into GenBank under accession numbers MK983497 (ITS), MN328723 (beta-tubulin), and MN328724 (LSU). A pathogenicity test was conducted on leaves of five 3-year-old W. florida cultivar “Red Prince” planted in the field (five leaves for each treatment and control per plant) by spraying 30 ml of a spore suspension (106 spores/ml) of isolate AAU0519 as treatment or sterilized distilled water as control. Before the inoculation, the leaves were disinfected with 70% ethanol. After inoculation, the leaves were wrapped with a plastic bag to keep high relative humidity. The average air temperature was about 28°C during the period of pathogenicity test. The experiment was repeated once. Ten days after inoculation, the fungal-inoculated leaves developed light brown lesions resembling those of naturally infected leaves, control leaves did not develop any symptoms. E. layuense was recovered from leaf lesions and its identity was confirmed by morphological and sequence analyses as described above. To our knowledge, E. layuense has been previously reported as a pathogen of Perilla sp. (Chen et al. 2017), oat (Avena sativa) (Chen et al. 2019), and tea (Camellia sinensis) plants (Chen et al. 2020), but this is the first report of E. layuense causing leaf spot on W. florida in China. This pathogen could pose a threat to the ornamental value of W. florida plants. Thus, it is necessary to adopt effective management strategies against leaf spot on W. florida.

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First report of leaf spot caused by Colletotrichum fructicola on Dalbergia hupeana in China

Plant Disease ◽

10.1094/pdis-08-21-1842-pdn ◽

2021 ◽

Author(s):

Yang Zhou ◽

Rou Ye ◽

Qin Ying ◽

Yang Zhang ◽

Linping Zhang

Keyword(s):

Phylogenetic Tree ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Jiangxi Province ◽

Leaf Spot Disease ◽

Sterile Water ◽

First Report ◽

Spot Disease ◽

Colletotrichum Fructicola

Dalbergia hupeana is a kind of wood and medicinal tree widely distributed in southern China. Since 2019, a leaf spot disease was observed on the leaves of D. hupeana in Gangxia village, Luoting town in Jiangxi Province, China (28°52′53″N, 115°44′58″E). The disease incidence was estimated to be above 50%. The symptoms began as small spots that gradually expanded, developing a brown central and dark brown to black margin. The spots ranged from 4 to 6 mm in diameter. Leaf pieces (5 × 5 mm) from lesion margins were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fifteen strains with similar morphological characterizations were isolated, and three representative isolates (JHT-1, JHT-2, and JHT-3) were chosen and used for further study. Colonies on PDA of three isolates were grayish-green with white edges and dark green on the reverse side. Conidia were transparent, cylindrical with rounded ends, and measured 3.6-5.3 µm × 9.5-15.2 µm (3.7 ± 0.2 × 13.6 ± 1.1 µm, n = 100). Appressoria were dark brown, globose or subcylindrical, and ranged from 6.2-9.2 µm× 5.1-6.8 µm (7.9 ± 0.4 × 5.9 ± 0.3 µm, n=100). The morphological characteristics of the three strains were consistent with the description of species in the Colletotrichum gloeosporioides complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin 2 (TUB2) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR and T1/Bt2b (Weir et al. 2012), respectively. The sequences were deposited in GenBank (Accession Nos. MZ482016 - MZ482018 for ITS; MZ463636 - MZ463638 for ACT; MZ463648- MZ463650 for CAL; MZ463639 - MZ463641 for CHS-1; MZ463642 - MZ463644 for GAPDH; MZ463645 - MZ463647 for TUB2). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (ITS, ACT, GAPDH, TUB2, CHS-1, CAL) (Kumar et al. 2016). According to the phylogenetic tree, three isolates fall within the Colletotrichum fructicola clade (boot support 99%). Based on morphological characteristics and phylogenetic analysis, three isolates were identified as C. fructicola. The pathogenicity of three isolates was conducted on two-yr-old seedlings (30 cm tall) of D. hupeana. Healthy leaves were wounded with a sterile needle and then inoculated with 10 μL spore suspension (106 conidia per mL). Controls were treated with sterile water. All plants were covered with transparent plastic bags and incubated in a greenhouse at 28°C with a 12 h photoperiod (relative humidity > 80%). Within five days, the inoculated leaves developed lesions similar to those observed in the field, whereas controls were asymptomatic. The experiments repeated three times showed similar results. The infection rate was 100%. C. fructicola was re-isolated from the lesions, whereas no fungus was isolated from control leaves. C. fructicola can cause leaf diseases in a variety of hosts, including Aesculus chinensis (Sun et al. 2020), Peucedanum praeruptorum (Ma et al. 2020), and Mandevilla × amabilis (Sun et al. 2020). C. brevisporum and C. gigasporum were also reported to infect Dalbergia odorifera (Chen et al. 2021; Wan et al. 2018). However, This is the first report of C. fructicola associated with leaf spot disease on D. hupeana in China. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

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