First Report of a Leaf Spot on Conyza sumatrensis Caused by Phoma macrostoma in China

Conyza sumatrensis (Asteraceae), an annual or biennial plant, is native to North and South America. It is an invasive, noxious weed that is widespread in southern and southeastern China. It invades farm land and causes great losses to dry land crops, including wheat, corn, and beans. It also reduces biological diversity by crowding out native plants in the infested areas (3,4). During a search for fungal pathogens that could serve as potential biological control agents of C. sumatrensis, a leaf spot disease was observed in 2010 in Chongqing, China. An isolate (SMBC22) of a highly virulent fungus was obtained from diseased leaves. Pathogenicity tests were performed by placing 6-mm-diameter mycelial disks of 7-day-old potato dextrose agar (PDA) cultures of SMBC22 on leaves of 15 healthy greenhouse-grown plants of C. sumatrensis; the same number of control plants was treated with sterile PDA disks. Treated plants were covered with plastic bags for 24 h and maintained in a growth chamber with daily average temperatures of 24 to 26°C, continuous light (3,100 lux), and high relative humidity (>90%). Lesions similar to those observed in the field were first obvious on the SMBC22-inoculated leaves 3 days after inoculation. Symptoms became severe 7 to 9 days after inoculation. Control plants remained healthy. The fungus was reisolated from inoculated and diseased leaves and it was morphologically the same as SMBC22. The pathogenicity test was conducted three times. A survey of 10 southern and southeastern Chinese provinces revealed that the disease was widespread and it attacked leaves and stems of seedlings and mature plants of C. sumatrensis. Lesions on leaves were initially small, circular, and water soaked. The typical lesion was ovoid or fusiform, dark brown, and surrounded by a yellow halo. The spots coalesced to form large lesions and plants were often completely blighted. Fungal colonies of SMBC22 on PDA plates were initially white and turned dark gray. Colonies were circular with smooth edges with obvious rings of pycnidia on the surface. Aerial hyphae were short and dense. Pycnidia, black and immersed or semi-immersed in the medium, were visible after 12 days of incubation. Pycnidia were 72 to 140 μm in diameter. Conidia were produced in the pycnidia and were hyaline, unicellular, ellipsoidal, and 4.4 to 6.1 × 1.6 to 2.2 μm. To confirm identification of the fungus, genomic DNA was extracted from mycelia of a 7-day-old culture on PDA at 25°C (2). The internal transcribed spacer (ITS) gene of rDNA was amplified using primers ITS4/ITS5. The gene sequence was 524 bp long and registered in NCBI GenBank (No. HQ645974). BLAST analysis showed that the current sequence had 99% homology to an isolate of Phoma macrostoma (DQ 404792) from Cirsium arvense (Canada thistle) in Canada and reported to cause chlorotic symptoms on that host plant (1). To our knowledge, this is the first report of P. macrostoma causing disease on C. sumatrensis in China. P. macrostoma, thought of as a biocontrol agent of broadleaf weeds in Canada, has been patented in the United States. The current isolate of P. macrostoma is considered as a potential biocontrol agent of C. sumatrensis. References: (1) P. R. Graupner et al. J. Nat. Prod. 66:1558, 2004. (2) S. Takamatsu et al. Mycoscience 42:135, 2001. (3) W. Z. Tan et al. Page 177 in: Manual of Emergency Control Technology Invasive Pests in China. G. L. Zhang, ed. Science Press, Beijing, 2010. (4) C. Wang et al. J. Wuhan Bot. Res. 28:90, 2010.

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First Report of Leaf Spot of Rudbeckia hirta var. pulcherrima Caused by Septoria rudbeckiae in Korea

Plant Disease ◽

10.1094/pdis-02-12-0172-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 911-911 ◽

Cited By ~ 1

Author(s):

J. H. Park ◽

S. E. Cho ◽

K. S. Han ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

The United States ◽

Flowering Plant ◽

Leaf Spot Disease ◽

Morphological Identification ◽

Its Sequence ◽

Conidial Suspension ◽

First Report ◽

Rudbeckia Hirta ◽

Close Phylogenetic Relationship

Rudbeckia hirta L. var. pulcherrima Farw. (synonym R. bicolor Nutt.), known as the black-eyed Susan, is a flowering plant belonging to the family Asteraceae. The plant is native to North America and was introduced to Korea for ornamental purposes in the 1950s. In July 2011, a previously unknown leaf spot was first observed on the plants in a public garden in Namyangju, Korea. Leaf spot symptoms developed from lower leaves as small, blackish brown lesions, which enlarged to 6 mm in diameter. In the later stages of disease development, each lesion was usually surrounded with a yellow halo, detracting from the beauty of the green leaves of the plant. A number of black pycnidia were present in diseased leaf tissue. Later, the disease was observed in several locations in Korea, including Pyeongchang, Hoengseong, and Yangpyeong. Voucher specimens were deposited at the Korea University Herbarium (KUS-F25894 and KUS-F26180). An isolate was obtained from KUS-F26180 and deposited at the Korean Agricultural Culture Collection (Accession No. KACC46694). Pycnidia were amphigenous, but mostly hypogenous, scattered, dark brown-to-rusty brown, globose, embedded in host tissue or partly erumpent, 50 to 80 μm in diameter, with ostioles 15 to 25 μm in diameter. Conidia were substraight to mildly curved, guttulate, hyaline, 25 to 50 × 1.5 to 2.5 μm, and one- to three-septate. Based on the morphological characteristics, the fungus was consistent with Septoria rudbeckiae Ellis & Halst. (1,3,4). Morphological identification of the fungus was confirmed by molecular data. Genomic DNA was extracted using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA.). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1/ITS4 primers and sequenced. The resulting sequence of 528 bp was deposited in GenBank (Accession No. JQ677043). A BLAST search showed that there was no matching sequence of S. rudbeckiae; therefore, this is the first ITS sequence of the species submitted to GenBank. The ITS sequence showed >99% similarity with those of many Septoria species, indicating their close phylogenetic relationship. Pathogenicity was tested by spraying leaves of three potted young plants with a conidial suspension (2 × 105 conidia/ml), which was harvested from a 4-week-old culture on potato dextrose agar. Control leaves were sprayed with sterile water. The plants were covered with plastic bags to maintain 100% relative humidity (RH) for the first 24 h. Plants were then maintained in a greenhouse (22 to 28°C and 70 to 80% RH). After 5 days, leaf spot symptoms identical to those observed in the field started to develop on the leaves inoculated with the fungus. No symptoms were observed on control plants. S. rudbeckiae was reisolated from the lesions of inoculated plants, confirming Koch's postulates. A leaf spot disease associated with S. rudbeckiae has been reported on several species of Rudbeckia in the United States, Romania, and Bulgaria (1–4). To our knowledge, this is the first report of leaf spot on R. hirta var. pulcherrima caused by S. rudbeckiae in Korea. References: (1) J. B. Ellis and B. D. Halsted. J. Mycol. 6:33, 1890. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ February 2, 2012. (3) E. Radulescu et al. Septoriozele din Romania. Ed. Acad. Rep. Soc. Romania, Bucuresti, Romania, 1973. (4) S. G. Vanev et al. Fungi Bulgaricae 3:1, 1997.

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First Report of Leaf Spot Disease On Microstegium vimineum Caused by Bipolaris setariae in China

Plant Disease ◽

10.1094/pdis-04-21-0703-pdn ◽

2021 ◽

Author(s):

Min Tan ◽

Qiong Huang ◽

Hao Fan ◽

Yun Wu ◽

Richard C. Reardon ◽

...

Keyword(s):

United States ◽

Leaf Spot ◽

The United States ◽

Lethal Concentration ◽

Reference Sequence ◽

Microstegium Vimineum ◽

Morphological Observation ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease

Microstegium vimineum, a Poaceae annual C4 plant, occurred widely in crop fields, tea gardens, orchards, under forests and roadsides in most provinces and regions south of the Yellow River, China. It was introduced into the eastern USA causing ecological and environmental damage (Stricker, 2016). In October 2015, M. vimineum plants with leaf spots were observed on the roadside of Mingling Road (32.04521°E, 118.84323°N), Nanjing, China. In an early stage of disease development, light brown or brown, round or oval shaped lesions appeared on the upper surface of leaves. In a middle stage, the lesions gradually expanded and the edges of the diseased leaves were lightly curled. In a late stage, leaves were withered or curled and the entire plant died. Initial disease incidence was up to 85% among natural populations of the weed. Diseased leaves collected from field were surface disinfected (75% ethanol for 30s; 1% sodium hypochlorite solution for 30s; 75% ethanol for 30s; sterile deionized water for 1min) and placed on water agar (20g agar per liter) (Kleczewski et al., 2010). Plates were incubated in the dark at 28℃ for 3 days. Following incubation, leaves, spores and conidiophores were examined using light microscopy. Single spores were obtained by using the single-spore procedure, plating out a loopful of spores onto water agar, and then carving individual spores out with associated agar under a microscope. Single spores were isolated, plated onto MV-agar (30g M. vimineum leaves, 20g agar per liter), and placed under 365 nm wavelength black light. Fungal colonies were transferred onto PDA medium, after 4 days colonies measured between 83 to 86 mm in diameter, appeared flat and dark brown, with short, light gray aerial hyphae. Conidiophores were solitary or clustered, light brown to medium brown, with pale apical color and multiple septa. The upper part was usually geniculated, 5.5-9.5 μm wide. Conidia were light yellowish brown to medium yellowish brown, mostly fusiform, straight or curved, fusoid or navicular, often slightly curved, rarely straight, smooth, 5-9 (mostly 7) septa, 48-70×10-14.5 μm (average 57×12.5 μm); hilium slightly prominent, and truncated at the base. Through morphological observation, the fungus was preliminarily identified as Bipolaris sp.. Four to five seeds of M. vimineum were planted in pots (10 cm in diameter) filled with nutrient soil, placed in the greenhouse and watered regularly. Four pots were inoculated with a conidia suspension of 1×105 sp/mL, at 4-5 true stage. Inoculated seedlings were maintained under 80% humidity and 28℃ for 24h in the dark, and then transferred to a greenhouse. Three pots of uninoculated seedlings were used as controls. Two days after the inoculation, buff-colored, irregular-shaped spots appeared centered on leaf veins. Within a week, diseased leaves became crinkled and their edges were yellow to brown due to proliferation of the spots. By 15 days, large areas of brown spots appeared on the leaves, some leaves turned yellow-brown and severely curled, and 80% of the plants had died. The diseased symptoms were similar to that of the field sample. The fungus re-isolated resulted morphologically identical to the original isolate grown on PDA medium and used for inoculation, thus fulfilled Koch’s postulates. The CTAB method was used to extract DNA from isolates of diseased leaves taken directly from the field, and the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase gene (GPDH) were amplified using primer pairs of ITS1/ITS4 and GPD/GPD2 (Manamgoda et al., 2014) respectively. The ITS amplified sequence (Genbank accession MW446193) shared 100% identity with the reference sequence of Bipolaris setariae (MN215638.1) and the GPDH amplified sequence (MW464364) shared 99.83% identity with the reference sequence of B. setariae (MK144540.1). Field experiments were conducted in Laboratory Base of Nanjing Agricultural University, where M. vimineum plants were planted. Spore suspensions with concentrations of 105, 104, 103, 102, and 101 sp/mL were prepared, distilled water was used for control, and there were four replicates of each treatment. Twenty four plots were randomly arranged, the experimental unit consisted of 50 to 60 plants in an area of 0.5m×0.6m. The interval distance between plots was about 20 cm so as to prevent the mutual influence among treatments. M. vimineum plants were inoculated at 3-4 true leaf stage. Inoculation was done at sunset, and 60 mL spore suspension was sprayed onto each plot. After spraying, the waterproof-breathable black cloth was used to cover the plots, and removed 36 hours later. The outdoor temperature was 20~28℃. After 10 days, the symptoms of M. vimineum were observed and the disease index was recorded. SPSS 20 software (SPSS Inc., Chicago, IL, USA) was used for variance analysis, and Origin 9.0 (OriginLab, Hampton, MA, USA) was used to calculate the half lethal concentration (ED50) and 90% lethal concentration (ED90) of the strain MLL-1-5 on M. vimineum. Symptoms appeared on inoculated M. vimineum seedlings immediately after dark treatment. Within a week, all seedlings inoculated with the highest spore concentration were dead. Plants sprayed with water remained healthy. ED50 and ED90 of the strain MLL-1-5 was 1.9×101 and 1.4×103 sp/mL respectively, which indicated aggressiveness of the strain MLL-1-5 B. setariae. After 28 days, infected M. vimineum plants did not recover. This is the first report of leaf spot disease on M. vimineum caused by B. setariae in China. M. vimineum is a widely distributed and extremely harmful weed in China and United States. No biocontrol agents against M. vimineum are currently available. B. setariae may have potential as a biocontrol agent against M. vimineum both in China and the United States.

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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 a Leaf Spot Disease of Tobacco Caused by Pseudomonas cichorii in China

Plant Disease ◽

10.1094/pdis-10-21-2158-pdn ◽

2021 ◽

Author(s):

Dayu Lan ◽

Fangling Shu ◽

Yanhui Lu ◽

Anfa Shou ◽

Wei Lin ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Pseudomonas Cichorii ◽

Koch’S Postulates ◽

First Report ◽

Initial Symptoms ◽

Wide Range ◽

Koch's Postulates

Tobacco (Nicotiana tabacum L.), one of the chief commercial crops, is wildly cultivated worldwide. In June 2020 and 2021, an unknown bacterial leaf spot on tobacco was found in Hezhou and Hechi City, Guangxi, China. 30% of the tobacco were affected and the rate of diseased leaves reached about 10% in the field under high temperature and rainstorm. The disease mainly damaged the middle and top leaves of tobacco plants at vigorous growing stage. The initial symptoms were water-soaked spots on the frontal half of a leaf, and then expanded into circular to irregular spots with a yellow halo at the edge. The spots mostly appeared dark brown at high air humidity, while yellow brown at low humidity and exhibited a concentric pattern. In severe cases, the lesions coalesced and the whole leaf was densely covered with lesions, resulting in the loss of baking value. A bacterium was consistently isolated from diseased leaf tissues on nutrient agar (NA). Growth on NA was predominantly grayish white circular bacterial colonies with smooth margins, and the bacterium is rod-shaped, gram-negative and fluorescent on King’s B medium. Seven isolates (ND04A-ND04C and ZSXF02-ZSXF05) were selected for molecular identification and pathogenicity tests. Genomic DNA of the bacterium was extracted and the housekeeping gene of cts (encoding citrate synthase) was amplified with the primers cts-Fs/cts-Rs (forward primer cts-Fs: 5’-CCCGTCGAGCTGCCAATWCTGA-3’; reverse primer cts-Rs: 5’-ATCTCGCACGGSGTRTTGAACATC-3’) (Berge et al. 2014; Sarkar et al. 2004). 409-bp cts gene sequences were deposited in the GenBank database for seven isolates (accession no. OK105110-OK105116). Sequence of seven isolates shared 100% identity with several Pseudomonas cichorii strains within the GenBank database (accession no. KY940268 and KY940271), and the phylogenetic tree of cts genes of the seven isolates clustered with the phylogroup 11 of Pseudomonas syringae (accession no. KJ877799 and KJ878111), which was classified as P.cichorii. To satisfy Koch’s postulates, a pathogenicity test was tested by using a needle to dip a suspension of the bacterium (108 CFU/ml) and pricking three holes in the tobacco leaf. The control plants leaves were needled with sterile water. Each tobacco plant was inoculated with three leaves, and the test was repeated three times. All plants were placed in transparent plastic boxes and incubated in a greenhouse at 25 ± 3°C. The water-soaked spots appeared 24h after inoculation and quickly expanded through leaf veins. Three days after inoculation, all the inoculated leaves showed symptoms similar to those observed in the field. Control plants remained healthy. Only P. cichorii was successfully re-isolated from the lesions, confirming Koch’s postulates. Pseudomonas cichorii can infect eggplant, lettuce, tomatoand other crops, and has a wide range of hosts (Timilsina et al. 2017; Ullah et al. 2015). To our knowledge, this is the first report of P. cichorii causing leaf spot on tobacco in China.

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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 of Lettuce (Lactuca sativa L.) Caused by Phoma tropica in Italy

Plant Disease ◽

10.1094/pdis-04-12-0394-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1380-1380 ◽

Cited By ~ 4

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

G. Ortu ◽

M. L. Gullino

Keyword(s):

Relative Humidity ◽

Lactuca Sativa ◽

Leaf Spot ◽

Morphological Characteristics ◽

The United States ◽

Fluorescent Light ◽

Malt Extract ◽

Pathogenicity Test ◽

First Report ◽

Lactuca Sativa L

Lettuce (Lactuca sativa L.) is widely grown in Italy, with the production for the preparation of ready-to-eat salads becoming increasingly important. During the spring of 2011, a previously unknown leaf spot was observed on L. sativa plants, cv Rubia, grown in several plastic tunnels in Lumbardy (northern Italy), 20 to 25 days after sowing. Thirty to forty per cent of leaves of the plants growing in the part of the tunnel with the highest relative humidity were affected. Leaves of infected plants showed extensive, irregular, dark brown, necrotic lesions with a chlorotic halo. Lesions initially ranged from 0.5 to 3 mm, then eventually coalesced, reaching 2 to 3 cm, showing a well-defined, dark brown border. Affected leaves senesced and withered. The crown was not affected by the disease. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then cultured on potato dextrose agar (PDA), amended with 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed, producing a greenish grey mycelium with a white border when incubated under 12 h/day of fluorescent light at 21 to 23°C. In order to favor the production of conidia, the fungus was transferred on malt extract agar (MA) and maintained under 12 h/day of fluorescent light at 22°C. After 15 days, black pycnidia, 175 to 225 μm, developed, with hyaline, elliptical, unicellular conidia, measuring 3.21 to 6.7 × 1.08 to 3.2 (average 5.5 × 1.9) μm. On the basis of these morphological characteristics, the fungal causal agent of the disease could be related to the genus Phoma (2). The internal transcribed spacer (ITS) region of rDNA of the isolate PHT30 was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 466-bp segment showed a 99% similarity with the sequence of Phoma tropica (GenBank Accession No. JF923820.1). The nucleotide sequence has been assigned the GenBank Accession No. JQ954396. Pathogenicity tests were performed by spraying healthy 20-day-old lettuce plants, cv Rubia, with a spore suspension (1 × 105 conidia/ml) prepared from 14-day-old colonies of the strain PHT30 grown on MA cultures. Plants inoculated with water alone served as controls. Ten plants per isolate were used. Plants were covered with plastic bags for 5 days after inoculation and maintained in a growth chamber at 20°C and 80% relative humidity. The first foliar lesions, similar to those occurring on the naturally infected plants, developed on leaves 12 days after inoculation. Control plants remained healthy. The pathogen was consistently reisolated from leaf lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of P. tropica on lettuce in Italy as well as worldwide. In the United States, the presence of P. exigua was reported in 2006 (3). The economic importance of the disease at present is limited, probably also because symptoms can be confused with those caused by Botrytis cinerea. However, P. tropica could become a more significant problem because of the importance of the crop. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) G. H. Boerema. Trans. Br. Mycol. Soc. 67:289, 1976. (3) S. Y. Koike. Plant Dis. 90:1268, 2006.

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First Report of Leaf Spot Caused by Phyllosticta yuccae on Yucca filamentosa in Brazil

Plant Disease ◽

10.1094/pdis-04-13-0384-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1257-1257 ◽

Cited By ~ 1

Author(s):

A. D. A. Silva ◽

D. B. Pinho ◽

B. T. Hora Junior ◽

O. L. Pereira

Keyword(s):

Leaf Spot ◽

Its Region ◽

The United States ◽

Leaf Spot Disease ◽

Infected Leaf ◽

Single Spore ◽

First Report ◽

Leaf Spots ◽

Slime Layer ◽

Pure Cultures

Yucca filamentosa L. (Agavaceae), commonly known as Adam's needle, is known in Brazil as “agulha-de-adão.” It is an ornamental garden plant with medicinal properties (4). In 2010, 100% of Y. filamentosa seedlings and plants were observed with a severe leaf spot disease in two ornamental nurseries located in the municipality of Viçosa, Minas Gerais, Brazil. Initially, lesions were dark brown, elliptical, and scattered, and later became grayish at the center with a reddish brown margin, irregular and coalescent. Infected leaf samples were deposited in the herbarium at the Universidade Federal de Viçosa (Accession Nos. VIC32054 and VIC32055). A fungus was isolated from the leaf spots and single-spore pure cultures were obtained on potato dextrose agar (PDA). The sporulating single-spore cultures were deposited at the Coleção de Culturas de Fungos Fitopatogênicos “Prof. Maria Menezes” (CMM 1843 and CMM 1844). On the leaf, the fungus produced pycnidial conidiomata that were scattered or gregarious, usually epiphyllous, immersed, dark brown, unilocular, subglobose, and 95 to 158 × 108 to 175 μm, with a minute, subcircular ostiole. Conidiogenous cells were blastic, hyaline, conoidal, or short cylindrical. Conidia were aseptate, hyaline, smooth walled, coarsely granular, broadly ellipsoidal to subglobose or obovate, usually broadly rounded at both ends, occasionally truncate at the base or indented slightly at the apex, and 7.5 to 13.5 × 6 to 10 μm. Conidia were also surrounded by a slime layer, usually with a hyaline, flexuous, narrowly conoidal or cylindrical, mucilaginous apical appendage that was 10 to 16 μm long. Spermatia were hyaline, dumbbell shaped to cylindrical, both ends bluntly rounded, and 3 to 5 × 1 to 1.5 μm. These characteristics matched well with the description of Phyllosticta yuccae Bissett (1). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and amplified using primers ITS1 and ITS4 (2) for the ITS region (GenBank Accession Nos. JX227945 and JX227946) and EF1-F and EF2-R (3) for the TEF-1α (JX227947 and JX227948). The sequencing was performed by Macrogen, South Korea. The ITS sequence matched sequence No. JN692541, P. yuccae, with 100% identity. To confirm Koch's postulates, four leaves of Y. filamentosa (five plants) were inoculated with 6-mm-diameter plugs from a 7-day-old culture growing on PDA. The leaves were covered with plastic sack and plants were maintained at 25°C. In a similar manner, fungus-free PDA plugs were placed on five control plants. Symptoms were consistently similar to those initially observed in the nurseries and all plants developed leaf spots by 15 days after inoculation. P. yuccae was successfully reisolated from the symptomatic tissue and control plants remained symptomless. P. yuccae has been previously reported in Canada, the Dominican Republic, Guatemala, Iran, and the United States of America. To our knowledge, this is the first report of P. yuccae causing disease in Y. filamentosa in Brazil and it may become a serious problem for the nurseries, due to the severity of the disease and the lack of chemical products to control this pathogen. References: (1) J. Bissett. Can. J. Bot. 64:1720, 1986. (2) M. A. Innis et al. PCR Protocols: A guide to methods and applications. Academic Press, 1990. (3) Jacobs et al. Mycol. Res. 108:411, 2004. (4) H. Lorenzi and H. M. Souza. Plantas Ornamentais no Brasil. Instituto Plantarum, 2001.

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First Report of Leaf Spot Caused by Alternaria alternata on Marigold (Tagetes erecta) in Beijing, China

Plant Disease ◽

10.1094/pdis-09-13-1005-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1153-1153 ◽

Cited By ~ 1

Author(s):

Y. Li ◽

J. Shen ◽

B. H. Pan ◽

M. X. Guo ◽

Q. X. Wang ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Leaf Spot Disease ◽

Tagetes Erecta ◽

Pathogenicity Test ◽

First Report ◽

Leaf Spots ◽

Commercial Crop ◽

Necrotic Spots ◽

Beijing China

Marigold (Tagetes erecta) is an important commercial crop and 200 ha are planted every year in the Beijing district of China. A leaf spot disease of T. erecta was observed during 2012 and 2013 in the Beijing district. The disease was widespread, with 60 to 75% of the fields affected. Leaves of the affected plants had small, brown, necrotic spots on most of the foliage. Yield losses of flowers of up to 20 to 30% were reported. The spots gradually enlarged, becoming irregular in shape, or remained circular, and with concentric rings or zones. In the later stages of infection, the spots coalesced, and the leaves withered, dried, and fell from the plants (4). A fungus was consistently isolated on potato dextrose agar (PDA) from the infected leaves of T. erecta. After 6 days of incubation at 26°C and a 12-h photoperiod, the fungus produced colonies that were flat, with a rough upper surface (2). The conidiophores were short. Conidia varied from 18 × 6 to 47 × 15 μm and were medium to dark brown or olive-brown in color, short beaked, borne in long chains, oval and bean shaped, with 1 to 5 transverse septa and 0 to 2 longitudinal septa. The rDNA of the internal transcribed spacer regions 1 and 2 and the 5.8S gene in seven isolates were amplified using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The nucleotide sequence was the same as isolate No. 7, which was deposited in GenBank (Accession No. KF307207). A BLAST search showed 97% identity with the strain Alternaria alternata GNU-F10 (KC752593). Seven isolates were also confirmed as A. alternata by PCR identification performed by specific primers (C_for/C_rev) of A. alternata (1). Seven isolates were grown on PDA for 2 weeks and the conidia harvested. A 5-μl drop of spore suspension (1 × 105 spores/ml) was placed on each leaflet of 140 detached, surface-sterilized T. erecta leaves. Twenty leaves were inoculated with sterile distilled water as a control. The leaves were incubated in a growth chamber at 80 to 90% relative humidity, 50 to 60 klx/m2 light intensity, and a 12-h photoperiod. After 6 days, leaf spots similar to the original developed at inoculation sites for all isolates and A. alternata was consistently re-isolated. The control leaves remained symptomless. The pathogenicity test was performed three times. Leaf spot of T. erecta caused by Alternaria spp. is well known in Asian countries such as Japan (3). To our knowledge, this is the first report of A. alternata on T. erecta in the Beijing district of China. References: (1) T. Gat. Plant Dis. 96:1513, 2012. (2) E. Mirkova. J. Phytopathol. 151:323, 2003. (3) K. Tomioka. J. Gen. Plant Pathol. 66:294, 2000. (4) T. Y. Zhang. Page 284 in: Flora Fungorum Sinicorum, Volume 16: Alternaria. Science Press, Beijing, 2003.

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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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First Report of Corynespora Leaf Spot of Eucalyptus in China

Plant Disease ◽

10.1094/pdis-07-14-0697-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 419-419 ◽

Cited By ~ 1

Author(s):

C. K. Phan ◽

J. G. Wei ◽

F. Liu ◽

B. S. Chen ◽

J. T. Luo ◽

...

Keyword(s):

Leaf Spot ◽

Southern China ◽

Tap Water ◽

Pathogenic Fungi ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Commonwealth Mycological Institute ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Eucalyptus is widely planted in the tropics and subtropics, and it has become an important cash crop in Southern China because of its fast-growing nature. In the Guangxi Province of southern China, Eucalyptus is produced on approximately 2 million ha, and two dominant asexual clones, Guanglin No. 9 (E. grandis × E. urophylla) and DH3229 (E. urophylla × E. grandis), are grown. Diseases are an increasing threat to Eucalyptus production in Guangxi since vast areas are monocultured with this plant. In June 2013, a leaf spot disease was observed in eight out of 14 regions in the province on a total of approximately 0.08 million ha of Eucalyptus. Initially, the lesions appeared as water-soaked dots on leaves, which then became circular or irregular shaped with central gray-brown necrotic lesions and dark red-brown margins. The size of leaf spots ranged between 1 and 3 mm in diameter. The main vein or small veins adjacent to the spots were dark. The lesions expanded rapidly during rainy days, producing reproductive structures. In severe cases, the spots coalesced and formed large irregular necrotic areas followed by defoliation. The causal fungus was isolated from diseased leaves. Briefly, the affected leaves were washed with running tap water, sterilized with 75% ethanol (30 s) and 0.1% mercuric dichloride (3 min), and then rinsed three times with sterilized water. Small segments (0.5 to 0.6 cm2) were cut from the leading edge of the lesions and plated on PDA. The plates were incubated at 25°C for 7 to 10 days. When mycelial growth and spores were observed, a single-spore culture was placed on PDA and grown in the dark at 25°C for 10 days. A pathogenicity test was done by spraying a conidial suspension (5 × 105 conidia ml–1) of isolated fungus onto 30 3-month-old leaves of Guanglin No. 9 seedlings. The plants were covered with plain plastic sheets for 7 days to keep the humidity high. Lesions similar to those observed in the forests were observed on the inoculated leaves 7 to 10 days after incubation. The same fungus was re-isolated. Leaves of control plants (sprayed with sterilized water) were disease free. Conidiophores of the fungus were straight to slightly curved, erect, unbranched, septate, and pale to light brown. Conidia were formed in chains or singly with 4 to 15 pseudosepta, which were oblong oval to cylindrical, subhyaline to pale olivaceous brown, straight to curved, 14.5 to 92.3 μm long, and 3.5 to 7.1 μm wide. The fungus was morphologically identified as Corynespora cassiicola (1). DNA of the isolate was extracted, and the internal transcribed spacer (ITS) region (which included ITS 1, 5.8S rDNA gene of rDNA, and ITS 2) was amplified with primers ITS5 and ITS4. 529 base pair (bp) of PCR product was obtained and sequenced. The sequence was compared by BLAST search to the GenBank database and showed 99% similarity to C. cassiicola (Accession No. JX087447). Our sequence was deposited into GenBank (KF669890). The biological characters of the fungus were tested. Its minimum and maximum growth temperatures on PDA were 7 and 37°C with an optimum range of 25 to 30°C. At 25°C in 100% humidity, 90% of conidia germinated after 20 h. The optimum pH for germination was 5 to 8, and the lethal temperature of conidia was 55°C. C. cassiicola has been reported causing leaf blight on Eucalyptus in India and Brazil (2,3) and causing leaf spot on Akebia trifoliate in Guangxi (4). This is the first report of this disease on Eucalyptus in China. References: (1) M. B. Ellis and P. Holliday. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 303. Commonwealth Mycological Institute, Kew, Surrey, UK, 1971. (2) B. P. Reis, et al. New Dis. Rep. 29:7, 2014. (3) K. I. Wilson and L. R. Devi. Ind. Phytopathol. 19:393, 1966. (4) Y. F. Ye et al. Plant Dis. 97:1659, 2013.

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