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

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

Plant Disease ◽

10.1094/pdis-11-20-2529-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 ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Sterile Water ◽

Bipolaris Oryzae ◽

First Report

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

Plant Disease ◽

10.1094/pdis-07-21-1486-pdn ◽

2021 ◽

Author(s):

Wang Hanyi ◽

Hou Xiuming ◽

Xueming Huang ◽

Meng Gao ◽

Tingting Chen ◽

...

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Post Inoculation ◽

Bletilla Striata ◽

First Report ◽

Spot Disease ◽

Severe Stage ◽

Initial Symptoms

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.

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First report of Colletotrichum fioriniae causing anthracnose on mu oil tree in China

Plant Disease ◽

10.1094/pdis-03-21-0502-pdn ◽

2021 ◽

Author(s):

Yang Zhang ◽

Guangqiang Li ◽

Dou Yang ◽

Ruoling Zhang ◽

Songze Wan

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Juglans Regia ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

First Report ◽

Spot Disease ◽

Initial Symptoms ◽

Oil Plant

Mu oil tree (Vernicia montana) is an economically important woody oil plant, which is widely distributed in southern China. In mid-May 2020, a leaf spot disease was observed on the leaves of mu oil tree in Taihe County in Jiangxi Province, China (26°55′25.55″N, 114°49′5.85″E). The disease incidence was estimated to be above 40%. Initial symptoms were circular red-brown spots which were 1-2 mm in diameter, then enlarged with red-brown center. In later stages, the spots coalesced and formed large patches, and subsequently red-brown centers of lesions gradually dried and fell out, forming a “shot hole” appearance. To identify the pathogen, diseased leaves were collected from Taihe County. Leaf tissues (5 × 5 mm) were cut from the margins of typical symptomatic lesions, surface- sterilized in 75% ethanol for 30 seconds and 3% sodium hypochlorite for 60 seconds, then rinsed with sterile distilled water three times. Leaf pieces were placed on potato dextrose agar (PDA; 1.5%, Difco-BD Diagnostics) and incubated at 25 °C in the dark. Pure cultures were obtained from individual conidia by recovering single spores. On PDA, colonies were initially white and cottony. The mycelia then became pinkish to deep-pink with time at the center on the front side and pink on the reverse side. Colonies produced pale orange conidial masses after 9 days. Conidia were fusiform with acute ends, smooth-walled, hyaline, and measured 3.6–5.5 × 8.1–14.5 µm (4.5 ± 0.5 × 10.6 ± 1.0 µm, n = 100). The morphological characteristics of the isolate matched the descriptions of Colletotrichum acutatum complex (Damm et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), beta-tubulin 2 (TUB2), and actin (ACT) were sequenced using the primers ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, T1/Bt2b, ACT-512F/ACT-783R, respectively (Weir et al. 2012). The obtained sequences were deposited into the GenBank [accession nos. MW584317 (ITS); MW656269 (GAPDH); MW656270 (TUB2); MW656268 (CHS-1); MW656267 (ACT)]. All the sequences showed 94 to 100% similarity with those of C. fioriniae. A neighbor-joining phylogenetic tree was generated by combining all the sequenced loci using MEGA7.0 (Kumar et al. 2016). The isolate TH-M4 clustered with C. fioriniae, having 99% bootstrap support. Base on the morphology and multi-gene phylogeny, isolate TH-M4 was identified as C. fioriniae (Damm et al. 2012). To confirm pathogenicity, 20 healthy leaves of 10 mu oil trees (3-year-old) grown outdoors were inoculated with a drop of spore suspension (106 conidia per mL) of the isolate TH-M4 in September 2020. Another 10 plants were inoculated with sterile water as the control. The leaves were wounded with a sterile toothpick. All the inoculated leaves were covered with black plastic bags to maintain humidity for 2 days. The pathogenicity test was repeated twice. The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic. The same fungus was re-isolated from the lesions on the inoculated plant, fulfilling Koch’s postulates. C. fioriniae has been recorded as anthracnose pathogen on Mahonia aquifolium (Garibaldi et al. 2020), Paeonia lactiflora (Park et al. 2020), Solanum melongena (Xu et al. 2020), and Juglans regia (Varjas et al. 2020). To our knowledge, this is the first report of C. fioriniae associated with leaf spot disease on mu oil tree in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this oil plant disease.

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First report of Epicoccum sorghinum causing leaf spot on Hemerocallis citrina in China

Plant Disease ◽

10.1094/pdis-10-20-2144-pdn ◽

2021 ◽

Author(s):

Ya-ming Ma ◽

Jin Lian Zhou ◽

Zhao Hu ◽

Jie Zhong ◽

Jun Zi Zhu

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Molecular Techniques ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Distilled Water ◽

First Report ◽

Small Water ◽

Ctab Method ◽

Hemerocallis Citrina

Hemerocallis citrina Baroni, also called yellow flower vegetable (huang hua cai in Chinese), is belonging to the family Xanthorrhoeaceae and is widely planted in China, the Korea Peninsula and Japan for ornamental purposes and vegetable value. In addition, they could also be used as a traditional Chinese medicinal and modern medicinal plant (Du et al. 2014). In August 2019, a leaf spot disease was observed on H. citrina plants in Zhejiang Province of China, with approximately 85% incidence in almost 700 ha. Symptoms were firstly displayed as small, water-soaked, pale chlorotic spots, with yellow halos enlarged into large fusiform spots with brown edge and gray centers. Later, infected leaves were badly damaged and became wilted. Small pieces of infected tissue were excised from the margin of necrotic lesions, surface disinfected with 70% ethanol for 8s, 0.1% HgCl2 for 1 min, rinsed with sterile distilled water for three times, and incubated on potato dextrose agar (PDA, amended with 100 mg/L streptomycin sulfate) at 26°C in the dark. Fungal colonies with similar cultural morphology were consistently obtained from repeated isolations. When cultured on PDA, colonies were villose, regular, grayish-green, and turned gray-brown, with the reverse side became reddish-brown. Chlamydospores were gray, unicellular or multicellular, nearly spherical, 11 to 27 × 10 to 23 μm. Pycnidia and conidia were produced on PDA when the fungal colonies were exposed to ultraviolet light for 12 h with a distance of 40 cm to the late source. Pycnidia were brown, mostly spheroid, and measured 90 to 138 × 120 to 210 μm. Conidia were hyaline, ellipsoidal, unicellular, aseptate, 4.3 to 5.5 × 1.8 to 2.4 μm. These morphological characteristics agreed with the descriptions of Epicoccum sorghinum (Zhou et al. 2018). The DNA of a representative strain HHC6-2 was extracted using CTAB method and the rDNA internal transcribed spacer (ITS), actin (ACT) and β-tubulin (TUB) genes were amplified and sequenced, using the primers ITS4/ITS5 (White et al. 1990), ACT512F/ACT783R (Carbone and Kohn 1999) and Bt-1/Bt-2 (Glass and Donaldson 1995), respectively. BLASTn searches of the resulting ITS, ACT and TUB sequences (accession nos. MW073403, MW080522, MW080521) revealed 98.58 to 100% identity to the E. sorghinum sequences (MT125854, MN956831 and MF987525). The pathogenicity test was carried out by inoculation of potted H. citrina plants using conidial suspensions. H. citrina seedlings were planted in pots with sterilized soil. Before inoculation, leaves were surface-disinfected with 70% ethanol and sterile distilled water. Leaves were inoculated by placing small droplets of conidial suspensions (105 conidia/ml) on one side of the midvein, and 3 to 5 drops were used per leaf. Sterile water was used as control. All the inoculated plants were placed in humid chambers at 25°C for 48h, and then maintained in a greenhouse at 25°C with a 16 h day-8 h night cycle. The pathogenicity assays were performed twice with three replications. Four days after inoculation, yellow to brown spots resembling those observed in the fields developed on the inoculated leaves. However, no symptoms were observed on the controls. E. sorghinum was re-isolated and identified based on morphological and molecular techniques as described above. To our knowledge, this is the first report of E. sorghinum causing leaf spot on H. citrina. It seems to be a threat for H. citrina planting in China and should be considered in order to reduce losses caused by this disease. This study might provide the basis for diagnosis and control of the disease.

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First Report of Leaf Spot Caused by a Phoma sp. on Schisandra chinensis in Korea

Plant Disease ◽

10.1094/pdis-05-13-0489-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 157-157 ◽

Cited By ~ 2

Author(s):

I. Y. Choi ◽

S. E. Cho ◽

J. H. Park ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Russian Far East ◽

Morphological Characteristics ◽

Plant Diseases ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Schisandra Chinensis ◽

Spot Disease

Schisandra chinensis (Turcz.) Baill. is a deciduous woody vine native to northern China and the Russian Far East. Its berries have long been used in traditional Asian medicine. In Korea, S. chinensis is one of 10 major medicinal crops and, as of 2011, the production is 6,892 metric tons from 1,749 ha of cultivation area (1). During summer to autumn of 2011 and 2012, leaf spots were observed on S. chinensis (cv. Cheongsun) with disease incidence of 100% in many locations of Jangsu County, Korea. Early symptoms appeared as small, circular, and pale brown spots. Each spot increased in size, became grayish brown and necrotic, and finally developed concentric rings with a definite margin. Some spots coalesced to cover nearly half of the leaves, often becoming torn and giving a shot hole effect. The infected leaf tissue contained blackish pycnidia from which masses of conidia were released in a humid environment. The pycnidia were brown, globose to pyriform, ostiolate, and 45 to 160 μm in diameter. Conidia were hyaline, smooth, oval to ellipsoidal, aseptate or medianly 1-septate, very occasionally 2-septate, slightly constricted at the septa, 4 to 11 × 2.5 to 5 μm, and contained small oil drops. These morphological characteristics were consistent with the generic concept of Phoma (2). Three monoconidial isolates were successfully cultured by diluting conidia mass in sterile water and streaking conidia suspension on potato dextrose agar (PDA). A representative isolate was deposited in the Korean Agricultural Culture Collection (Accession No. KACC47113) and used for pathogenicity test and molecular analysis. Inoculum for a pathogenicity test was prepared by harvesting conidia from 30-day-old cultures (12-h diurnal cycle, 25°C) and a conidial suspension in water (1.1 × 107 conidia/ml) was sprayed onto leaves of three healthy seedlings (cv. Cheongsun). Three seedlings serving as controls were sprayed until runoff with sterile distilled water. The plants were separately covered with plastic bags for 48 h in a glasshouse. After 10 days, typical leaf spot symptoms developed on the leaves inoculated with the fungus. Phoma sp. was re-isolated from those lesions, confirming Koch's postulates. No symptoms were observed on controls. The pathogenicity test was conducted twice. Fungal DNA was extracted, and the complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced directly. The resulting 520-bp sequence was deposited in GenBank (Accession No. KC928322). The sequence showed over 99% similarity with many Phoma species from various substrates, but no exact matches. Phoma leaf spot of S. chinensis was once recorded in Korea without pathogenicity test and culture deposition (3). Phoma glomerata was recorded as a causal fungus of leaf spot disease on S. chinensis in China (4). The Korean isolates differ from P. glomerata in having larger conidia and are separated from it in ITS sequence data. Therefore, we tentatively place the Korean isolates as unidentified Phoma sp. To our knowledge, this is the first confirmed report of leaf spot disease caused by a Phoma sp. in Korea. References: (1) Anonymous. Statistics of Cultivation and Production of Industrial Crops in 2011. Korean Ministry for Food, Agriculture, Forestry and Fisheries. 2012. (2) M. M. Aveskamp et al. Mycologia 101:363, 2009. (3) E. J. Lee et al. Compendium of Medicinal Plant Diseases with Color Plates. Nat. Inst. Agric. Sci., Suwon, Korea. 1991. (4) X. Wang et al. Plant Dis. 96:289, 2012.

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First report of banana (Musa acuminate L. AAA Cavendish, cv. Formosana) leaf spot disease caused by Curvularia geniculata in China

Plant Disease ◽

10.1094/pdis-09-21-2001-pdn ◽

2021 ◽

Author(s):

Yanxiang Qi ◽

Yanping Fu ◽

Jun Peng ◽

Fanyun Zeng ◽

Yanwei Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Universal Primers ◽

Leaf Spot Disease ◽

Leaf Margin ◽

Conidial Suspension ◽

Tropical Fruit ◽

First Report ◽

Spot Disease

Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards of Chengmai county (19°48ʹ41.79″ N, 109°58ʹ44.95″ E), Hainan province, China. In total, the disease incidence was about 5% of banana trees (6 000 trees). The leaf spots occurred sporadically and were mostly confined to the leaf margin, and the percentage of the leaf area covered by lesions was less than 1%. Symptoms on the leaves were initially reddish brown spots that gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a yellow halo. The conidia obtained from leaf lesions were brown, erect or curved, fusiform or elliptical, 3 to 4 septa with dimensions of 13.75 to 31.39 µm × 5.91 to 13.35 µm (avg. 22.39 × 8.83 µm). The cells of both ends were small and hyaline while the middle cells were larger and darker (Zhang et al. 2010). Morphological characteristics of the conidia matched the description of Curvularia geniculata (Tracy & Earle) Boedijn. To acquire the pathogen, tissue pieces (15 mm2) of symptomatic leaves were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) for three days at 28°C. Grayish green fungal colonies appeared, and then turned fluffy with grey and white aerial mycelium with age. Two representative isolates (CATAS-CG01 and CATAS-CG92) of single-spore cultures were selected for molecular identification. Genomic DNA was extracted from the two isolates, the internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced with universal primers ITS1/ITS4, LROR/LR5, GPD1/GPD2, EF1-983F/EF1-2218R and 5F2/7cR, respectively (Huang et al. 2017; Raza et al. 2019). The sequences were deposited in GenBank (MW186196, MW186197, OK091651, OK721009 and OK491081 for CATAS-CG01; MZ734453, MZ734465, OK091652, OK721100 and OK642748 for CATAS-CG92, respectively). For phylogenetic analysis, MEGA7.0 (Kumar et al. 2016) was used to construct a Maximum Likelihood (ML) tree with 1 000 bootstrap replicates, based on a concatenation alignment of five gene sequences of the two isolates in this study as well as sequences of other Curvularia species obtained from GenBank. The cluster analysis revealed that isolates CATAS-CG01 and CATAS-CG92 were C. geniculata. Pathogenicity assays were conducted on 7-leaf-old banana seedlings. Two leaves from potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle and placing 10 μl conidial suspension (2×106 conidia/ml) on the surface of wounded leaves and equal number of leaves were inoculated with sterile distilled water serving as control (three replicates). Inoculated plants were grown in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). Necrotic lesions on inoculated leaves appeared seven days after inoculation, whereas control leaves remained healthy. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. C. geniculata has been reported to cause leaf spot on banana in Jamaica (Meredith, 1963). To our knowledge, this is the first report of C. geniculata on banana in China.

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First Report of Leaf Spot Disease Caused by Colletotrichum gloeosporioides on Chinese Bean Tree in China

Plant Disease ◽

10.1094/pdis-03-12-0261-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 138-138 ◽

Cited By ~ 1

Author(s):

B. Z. Fu ◽

M. Yang ◽

G. Y. Li ◽

J. R. Wu ◽

J. Z. Zhang ◽

...

Keyword(s):

Leaf Spot ◽

Colletotrichum Gloeosporioides ◽

Disease Incidence ◽

Phylogenetic Analyses ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Rdna Its ◽

Its Sequence Analysis

Chinese bean tree, Catalpa fargesii f. duciouxii (Dode) Gilmour, is an ornamental arbor plant. Its roots, leaves, and flowers have long been used for medicinal purposes in China. During July 2010, severe outbreaks of leaf spot disease on this plant occurred in Kunming, Yunnan Province. The disease incidence was greater than 90%. The symptoms on leaves began as dark brown lesions surrounded by chlorotic halos, and later became larger, round or irregular spots with gray to off-white centers surrounded by dark brown margins. Leaf tissues (3 × 3 mm), cut from the margins of lesions, were surface disinfected in 0.1% HgCl2 solution for 3 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PDA, and were slightly brown on the underside of the colony. The hyphae were achromatic, branching, septate, and 4.59 (±1.38) μm in diameter on average. Perithecia were brown to black, globose in shape, and 275.9 to 379.3 × 245.3 to 344.8 μm. Asci that formed after 3 to 4 weeks in culture were eight-spored, clavate to cylindrical. The ascospores were fusiform, slightly curved, unicellular and hyaline, and 13.05 to 24.03 × 10.68 to 16.02 μm. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (2). Sequencing of the PCR products of DQ1 (GenBank Accession No. JN165746) revealed 99% similarity (100% coverage) with Colletotrichum gloeosporioides isolates (GenBank Accession No. FJ456938.1, No. EU326190.1, No. DQ682572.1, and No. AY423474.1). Phylogenetic analyses (MEGA 4.1) using the neighbor-joining (NJ) algorithm placed the isolate in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with other C. gloeosporioides isolates. The pathogen was identified as C. gloeosporioides (Penz.) Penz. & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld & H. Schrenk) based on the morphological characteristics and rDNA-ITS sequence analysis (1). To confirm pathogenicity, Koch's postulates were performed on detached leaves of C. fargesii f. duciouxii, inoculated with a solution of 1.0 × 106 conidia per ml. Symptoms similar to the original ones started to appear after 10 days, while untreated leaves remained healthy. The inoculation assay used three leaves for untreated and six leaves for treated. The experiments were repeated once. C. gloeosporioides was consistently reisolated from the diseased tissue. C. gloeosporioides is distributed worldwide causing anthracnose on a wide variety of plants (3). To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf spots on C. fargesii f. duciouxii in China. References: (1) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. CAB International. Wallingford, UK, 1992. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) J. Yan et al. Plant Dis. 95:880, 2011.

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First Report of Fusarium fujikuroi Causing Leaf spot of Dalbergia odorifera T. Chen in China

Plant Disease ◽

10.1094/pdis-02-20-0364-pdn ◽

2020 ◽

Author(s):

JiangTao Peng ◽

Yao Chen ◽

Guo ying Zhou ◽

Jun Ang Liu

Keyword(s):

Leaf Spot ◽

Histone H3 ◽

Morphological Observation ◽

Leaf Spot Disease ◽

Fusarium Fujikuroi ◽

Control Group ◽

Beta Tubulin ◽

First Report ◽

Spot Disease ◽

Dalbergia Odorifera

Dalbergia odorifera T. Chen is a national second-grade protected and one of the four famous trees in China, with high medicinal and economic value. Leaf spot disease in this plant can cause the leaves to dry up, perforate or even fall off, which affects the growth and development, and also has a great influence on its products. In May 2019, the leaf spot of Dalbergia odorifera T. Chen was found and observed in Chengmai County (N19°40′, E110°0′), Hainan Province, China, and the symptomatic leaves were brought back to the laboratory for research; According to our survey at that time, the incidence of the disease was between 10% and 15%. A sterile stainless-steel scalpel was used to cut the tissues at the junction of the leaf lesions and placed on a clean bench, soaked in alcohol (75 %) for 30 s, and rinsed thrice with sterile water. Then it was inserted obliquely onto lactic acid-containing potato dextrose agar (PDA) and incubated at 28 °C for 5 days. The growing prominent colonies were singled out and re-inoculated on PDA and SNA plates. Preliminary identification was based on morphological characteristics, followed by molecular identification of strains by evaluating genes for translation elongation factor-1α(TEF-1α), beta-tubulin, mitochondrial small subunit (mtSSU)( Duan et al. 2019; Cao et al.2019; Stenglein et al.2010), and histone H3 (Jacobs, et al. 2010) . Through morphological observation, the isolate was identified as Fusarium fujikuroi. At the initial stage of growth on PDA, the strain produced a large number of white hyphae, followed by pink and purple-brown hyphae in the center of the colony which spread to the surrounding area. The microspores were abundant, colorless, elliptic or clavate, without septum or at 1-2 septate, and the size was about 3.3 to 13.5 × 1.2 to 3.2 µm. After nine days of culturing on SNA medium, few, large conidia were observed, typically sickle-like, with 3-4 septa with a size of about 20 to 40.2 × 2.3 to 4.4 μm. The identity of the strains was determined by comparing the gene sequences of TEF-1α, mtSSU, beta-tubulin and histone H3 by NCBI BLAST. The results showed that TEF-1 α (MN958396), mtSSU (MN958394), β - tubulin (MN958395), and histone H3 (MN958397) from the target strain (jxht0302) had 100% sequence homology with F. fujikuroi (GenBank, accession numbers KF604040.1, MF984420.1, XM023575231.1, and MF356523.1 respectively). Next, the infection of D. odorifera T. Chen seedlings with and without injury was studied using a fungus block, with PDA as a control. Two days after inoculation with injury, obvious lesions were observed on the leaves, which appeared at least 5 days post- inoculation without injury, with no lesions in the control group. F. fujikuroi could be re-isolated from the leaves with lesions, but not from the control group. F. fujikuroi causes Black Rot of Macleaya cordata and maize ear rot (Yull et al.2019; Duan et al. 2019). As far as we know, this is the first report of F. fujikuroi causing leaf spot disease of D. odorifera T. Chen. Given the importance of D. odorifera T. Chen products, this disease needs more attention to tackle it.

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