First Report of Leaf Spot Disease on Cinnamomum camphora (Camphor tree) Caused by Epicoccum poaceicola in China

As an important industrial, pharmaceutical and evergreen shade tree (Singh and Jawaid 2012), the camphor tree (Cinnamomum camphora) has been coppiced in Jiangxi Province, China. From 2017 to 2020, we noticed many camphor trees with leaf spots, with an incidence estimated at 50 to 75%, which could severely inhibit leaf growth and reduce their biomass. A dark-green circle with a watery spot appeared on the infected leaves at the initial stage, and necrosis with forming shot-spots surrounded by yellow halos occurred (Figure 1 A). Five leaves with typical symptoms were sampled and washed with tap water for ca. 15 min. Isolation and morphological analysis were performed following the method of Bao et al. (2019). Among 61 fungal isolates, 49 showed the same culture characters. Colonies on PDA were villose and regular, the reverse was scarlet at the edge of the colony, which was ca. 8.75 cm after 7 days of inoculation (Figure 1 I). Chlamydospores were aseptate, dark brown, smooth, in chains or solitary, ellipsoidal to ovoid, 4.8–9.6 × 4.8–11.1 μm (Figure 1 J). The pycnidia were produced on PDA and varied from 47.4 to 85.8 µm (mean 60.2 µm) × 38.6 to 66.8 μm (mean 49.7 μm) (n = 17) (Figure 1 K). Conidia were hyaline, unicellular, elliptical to ovoid, 4.3-6.4 µm (mean 5.1 µm) × 2.3-3.3 µm (mean 2.8 µm) (n = 52) (Figure 1 L). Pathogenicity tests of isolate XW-9 was carried out in the field. Ten leaves were wounded with a sterilized insect needle and inoculated with mycelium plugs (7-mm diameter). Non-colonized PDA plugs served as the negative controlIn addition, conidial suspensions (105 conidia/mL) of isolate XW-9 were sprayed on surface-sterilized leaves with a further ten leaves being sprayed with sterile water as the control. Symptoms described in this study appeared in 100% of the mycelium-inoculated leaves and more than 80% of the conidium-inoculated leaves after 7 days post-inoculation (Figure 1 B-E). No symptoms were seen in the controls (Figure 1 C). Three days after inoculation, brown spots resembling those observed in the field developed on the inoculated leaves, and some lesions turned into shot holes on the infected leaves (Figure 1 G & H). However, no symptoms were observed on the controls (Figure 1 F). The fungus was re-isolated from the margins of the leaf spots and labelled P-XW-9A. The gene regions for ITS, LSU, tub2, RPB2 and ACT of isolates XW-9 and P-XW-9A were amplified and sequenced. The sequences of rDNA-ITS, LSU, tub2, RPB2 and ACT of XW-9 were GenBank MW142397, MW130844, MW165322, MW446945 and MW165324, respectively and those of P-XW-9A were GenBank MW142398, MW130845, MW165323, MW446946 and MW165325, respectively (Lumbsch, et al. 2000; Aveskamp, et al. 2009; Hou et al. 2020). Phylogenetic analysis using concatenated sequences of ITS, LSU, RPB2, and tub2 showed that isolates XW-9 and P-XW-9A formed a single clade with the reference strain of E. poaceicola CBS 987.95 (Figure 2). Thus, XW-9 was identified as E. poaceicola based on its morphological and molecular characteristics. Significantly, the recovered isolate P-XW-9A also aligned with E. poaceicola fulfilling the criteria for Koch's Postulates. E. poaceicola was only reported as a fungal pathogen of Phyllostachys viridis in China (Liu et al. 2020). To our knowledge, this is the first report of leaf spot disease on camphor trees caused by E. poaceicola in China and our findings will be useful for its management.

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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 a Leaf Spot Disease of Bells-of-Ireland (Moluccella laevis) Caused by Cercospora apii in California

Plant Disease ◽

10.1094/pdis.2003.87.2.203a ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203

Author(s):

S. T. Koike ◽

S. A. Tjosvold ◽

J. Z. Groenewald ◽

P. W. Crous

Keyword(s):

Leaf Spot ◽

Sequence Data ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Initial Symptoms

Bells-of-Ireland (Moluccella laevis) (Lamiaceae) is an annual plant that is field planted in coastal California (Santa Cruz County) for commercial cutflower production. In 2001, a new leaf spot disease was found in these commercially grown cutflowers. The disease was most serious in the winter-grown crops in 2001 and 2002, with a few plantings having as much as 100% disease incidence. All other plantings that were surveyed during this time had at least 50% disease. Initial symptoms consisted of gray-green leaf spots. Spots were generally oval in shape, often delimited by the major leaf veins, and later turned tan. Lesions were apparent on both adaxial and abaxial sides of the leaves. A cercosporoid fungus having fasciculate conidiophores, which formed primarily on the abaxial leaf surface, was consistently associated with the spots. Based on morphology and its host, this fungus was initially considered to be Cercospora molucellae Bremer & Petr., which was previously reported on leaves of M. laevis in Turkey (1). However, sequence data obtained from the internal transcribed spacer region (ITS1, ITS2) and the 5.8S gene (STE-U 5110, 5111; GenBank Accession Nos. AY156918 and AY156919) indicated there were no base pair differences between the bells-of-Ireland isolates from California, our own reference isolates of C. apii, as well as GenBank sequences deposited as C. apii. Based on these data, the fungus was subsequently identified as C. apii sensu lato. Pathogenicity was confirmed by spraying a conidial suspension (1.0 × 105 conidia/ml) on leaves of potted bells-of-Ireland plants, incubating the plants in a dew chamber for 24 h, and maintaining them in a greenhouse (23 to 25°C). After 2 weeks, all inoculated plants developed leaf spots that were identical to those observed in the field. C. apii was again associated with all leaf spots. Control plants, which were treated with water, did not develop any symptoms. The test was repeated and the results were similar. To our knowledge this is the first report of C. apii as a pathogen of bells-of-Ireland in California. Reference: (1) C. Chupp. A Monograph of the Fungus Genus Cercospora. Cornell University Press, Ithaca, New York, 1954.

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

Plant Disease ◽

10.1094/pdis-09-20-1896-pdn ◽

2020 ◽

Author(s):

Dahui Liu ◽

Qiaohuan Chen ◽

Yuhuan Miao ◽

Jinxin Li ◽

Qi Yang

Keyword(s):

Leaf Spot ◽

Hubei Province ◽

Chrysanthemum Morifolium ◽

Yield Reduction ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Gene Sequences ◽

First Report ◽

Spot Disease ◽

Rdna Its

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

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First Report of Leaf Spot Disease Caused by Epicoccum nigrum on Lablab purpureus in India

Plant Disease ◽

10.1094/pdis-07-13-0798-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 284-284 ◽

Cited By ~ 12

Author(s):

S. Mahadevakumar ◽

K. M. Jayaramaiah ◽

G. R. Janardhana

Keyword(s):

Leaf Spot ◽

Leaf Surface ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

Lablab Purpureus ◽

First Report ◽

Spot Disease ◽

Epicoccum Nigrum

Lablab purpureus (L.) Sweet (Indian bean) is an important pulse crop grown in arid and semi-arid regions of India. It is one of the most widely cultivated legume species and has multiple uses. During a September 2010 survey, we recorded a new leaf spot disease on L. purpureus in and around Mysore district (Karnataka state) with 40 to 80% disease incidence in 130 ha of field crop studied, which accounted for 20 to 35% estimated yield loss. The symptoms appeared as small necrotic spots on the upper leaf surface. The leaf spots were persistent under mild infection throughout the season with production of conidia in clusters on abaxial leaf surface. A Dueteromyceteous fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl2 solution then washed thrice, dried, inoculated on potato dextrose agar (PDA) medium, and incubated at 28 ± 2°C at 12 h alternate light and dark period for 7 days. The fungal colony with aerial mycelia interspersed with dark cushion-shaped sporodochia consists of short, compact conidiophores bearing large isodiametric, solitary, muricate, brown, globular to pear shaped conidia (29.43 to 23.92 μm). Fungal isolate was identified as Epicoccum sp. based on micro-morphological and cultural features (1). Further authenticity of the fungus was confirmed by PCR amplification of the internal transcribed spacer (ITS) region using ITS1/ITS4 universal primer. The amplified PCR product was purified, sequenced directly, and BLASTn search revealed 100% homology to Epicoccum nigrum Link. (DQ093668.1 and JX914480.1). A representative sequence of E. nigrum was deposited in GenBank (Accession No. KC568289.1). The isolated fungus was further tested for its pathogenicity on 30-day-old healthy L. purpureus plants under greenhouse conditions. A conidial suspension (106 conidia/ml) was applied as foliar spray (three replicates of 15 plants each) along with suitable controls. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). The appearance of leaf spot symptoms were observed after 25 days post inoculation. Further, the pathogen was re-isolated and confirmed by micro-morphological characteristics. E. nigrum has been reported to cause post-harvest decay of cantaloupe in Oklahoma (2). It has also been reported as an endophyte (3). Occurrence as a pathogen on lablab bean has not been previously reported. To our knowledge, this is the first report of the occurrence of E. nigrum on L. purpureus in India causing leaf spot disease. References: (1) H. L. Barnet and B. B. Hunter. Page 150 in: Illustrated Genera of Imperfect Fungi, 1972. (2) B. D. Bruten et al. Plant Dis. 77:1060, 1993. (3) L. C. Fávaro et al. PLoS One 7(6):e36826, 2012.

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First Report of Leaf Spot Disease on Walnut Caused by Colletotrichum fioriniae in China

Plant Disease ◽

10.1094/pdis-09-14-0938-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 289-289 ◽

Cited By ~ 9

Author(s):

Y. Z. Zhu ◽

W. J. Liao ◽

D. X. Zou ◽

Y. J. Wu ◽

Y. Zhou

Keyword(s):

Dna Sequences ◽

Leaf Spot ◽

Juglans Regia ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Koch’S Postulates ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Koch's Postulates

In May 2014, a severe leaf spot disease was observed on walnut tree (Juglans regia L.) in Hechi, Guangxi, China. Leaf spots were circular to semicircular in shape, water-soaked, later becoming grayish white in the center with a dark brown margin and bordered by a tan halo. Necrotic lesions were approximately 3 to 4 mm in diameter. Diseased leaves were collected from 10 trees in each of five commercial orchards. The diseased leaves were cut into 5 × 5 mm slices, dipped in 75% ethanol for 30 s, washed three times in sterilized water, sterilized with 0.1% (w/v) HgCl2 for 3 min, and then rinsed five times with sterile distilled water. These slices were placed on potato dextrose agar (PDA), followed by incubating at 28°C for about 3 to 4 days. Fungal isolates were obtained from these diseased tissues, transferred onto PDA plates, and incubated at 28°C. These isolates produced gray aerial mycelium and then became pinkish gray with age. Moreover, the reverse of the colony was pink. The growth rate was 8.21 to 8.41 mm per day (average = 8.29 ± 0.11, n = 3) at 28°C. The colonies produced pale orange conidial masses and were fusiform with acute ends, hyaline, sometimes guttulate, 4.02 to 5.25 × 13.71 to 15.72 μm (average = 4.56 ± 0.31 × 14.87 ± 1.14 μm, n = 25). The morphological characteristics and measurements of this fungal isolate matched the previous descriptions of Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan (2). Meanwhile, these characterizations were further confirmed by analysis of the partial sequence of five genes: the internal transcribed spacer (ITS) of the ribosomal DNA, beta-tubulin (β-tub) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, chitin synthase 3(CHS-1) gene, and actin (ACT) gene, with universal primers ITS4/ITS5, T1/βt2b, GDF1/GDR1, CHS1-79F/CHS1-354R, and ACT-512F/ACT-783R, respectively (1). BLAST of these DNA sequences using the nucleotide database of GenBank showed a high identify (ITS, 99%; β-tub, 99%; GAPDH, 99%; CHS-1, 99%; and ACT, 100%) with the previously deposited sequences of C. fioriniae (ITS, KF278459.1, NR111747.1; β-tub, AB744079.1, AB690809.1; GAPDH, KF944355.1, KF944354.1; CHS-1, JQ948987.1, JQ949005.1; and ACT, JQ949625.1, JQ949626.1). Koch's postulates were fulfilled by inoculating six healthy 1-year-old walnut trees in July 2014 with maximum and minimum temperatures of 33 and 26°C. The 6-mm mycelial plug, which was cut from the margin of a 5-day-old colony of the fungus on PDA, was placed onto each pin-wounded leaf, ensuring good contact between the mycelium and the wound. Non-colonized PDA plugs were placed onto pin-wounds as negative controls. Following inoculation, both inoculated and control plants were covered with plastic bags. Leaf spots, similar to those on naturally infected plants, were observed on the leaves inoculated with C. fioriniae within 5 days. No symptoms were observed on the negative control leaves. Finally, C. fioriniae was re-isolated from symptomatic leaves; in contrast, no fungus was isolated from the control, which confirmed Koch's postulates. To our knowledge, this is the first report of leaf disease on walnut caused by C. fioriniae. References: (1) L. Cai et al. Fungal Divers. 39:183, 2009. (2) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009.

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First Report of Embellisia hyacinthi Causing a Leaf Spot and Bulb Skin Spot Disease on Scilla peruviana in California

Plant Disease ◽

10.1094/pdis-11-10-0845 ◽

2011 ◽

Vol 95 (3) ◽

pp. 356-356

Author(s):

S. Rooney-Latham ◽

C. L. Blomquist ◽

D. G. Fogle ◽

E. G. Simmons

Keyword(s):

Leaf Spot ◽

Apical Cell ◽

Leaf Spot Disease ◽

Adaxial Side ◽

Internal Transcribed Spacer Region ◽

California Department ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Food And Agriculture

The genus Scilla (Hyacinthaceae) includes more than 50 species of perennial, flowering bulbs grown in landscapes worldwide. In December 2000 and May 2009, an unknown leaf spot disease on Scilla peruviana was submitted to the California Department of Food and Agriculture Plant Pest Diagnostic Lab. Samples were collected during routine phytosanitary inspections of production fields in Santa Cruz County in 2000 and Monterey County in 2009. The disease was detected before plants flowered in one field at each location each year and appeared to have a scattered distribution. Foliar spots were large, elliptical to oblong with grayish black centers and brown margins. Yellow halos surrounded many of the spots. Examination of the bulb material revealed small necrotic patches on the outer bulb scales. A rapidly growing fungus was isolated on one-half-strength acidified potato dextrose agar (APDA) from the sporulating leaf spots and necrotic patches on the bulbs. The colonies were greenish gray and became dark olivaceous with age. Dictyospores, which formed on simple to branched, geniculate conidiophores, were oblong, fusiform or obclavate and usually had a triangular apical cell. They were initially hyaline, turning olivaceous brown with age. Conidia measured 14 to 39 × 8 to 13 μm (average 24.6 × 9.9 μm) typically with two to four (but up to seven) thick, transverse septa and one to two longitudinal septa. Morphologically, the fungus matched the description of Embellisia hyacinthi de Hoog & Miller (1,3). To confirm pathogenicity, four leaves of four S. peruviana plants were inoculated by taking colonized mycelial plugs from 2-week-old cultures and placing them in a plastic screw-cap lid filled with sterile water. The water plus mycelial plug suspension in the lid was then clipped to the adaxial side of a pushpin-wounded leaf (4). Plants were placed in a dark dew chamber at 20°C for 48 h and then moved to a growth chamber at 20°C with a 12-h photoperiod. After 48 h, the clips, caps, and plugs were removed. An equal number of control plants were wounded and mock inoculated with noncolonized APDA agar plugs and the experiment was repeated. Leaf lesions were visible 3 days after clip removal and expanded to an average of 26 × 10 mm, 14 days after inoculation. Sporulation was observed in the lesions after 5 to 7 days and the fungus was isolated from all inoculated leaves. No symptoms developed on the control leaves. DNA sequencing of the internal transcribed spacer region of the isolate (GenBank Accession No. HQ425562) using primers ITS1 and ITS4 matched the identity of E. hyacinthi (2,4). E. hyacinthi has been reported as a foliar and bulb pathogen on Hyacinthus, Freesia, and Scilla in Japan and Europe including Great Britain. Bulbs infected with E. hyacinthi are generally less sound and less valuable than noninfected bulbs (1). To our knowledge, this is the first report of the disease on S. peruviana in California. References: (1) G. S. de Hoog and P. J. Muller. Neth. J. Plant Pathol. 79:85, 1973. (2) B. Pryor and D. M. Bigelow. Mycologia 95:1141, 2003. (3) E. Simmons. Mycotaxon 17:216, 1983. (4) L. E. Yakabe et al. Plant Dis. 93:883, 2009.

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

Plant Disease ◽

10.1094/pdis-09-20-1974-pdn ◽

2020 ◽

Author(s):

Lv-Yin Zhou ◽

Shuang-Feng Yang ◽

Shao-Mei Wang ◽

Jing-Wen Lv ◽

Wei Qiang Wan ◽

...

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Post Inoculation ◽

Sodium Hypochlorite Solution ◽

Transparent Plastic ◽

Bletilla Striata ◽

Spot Disease ◽

Initial Symptoms

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

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The First Report of Leaf Spots in Aloe vera Caused by Nigrospora oryzae in China

Plant Disease ◽

10.1094/pdis-03-13-0314-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1256-1256 ◽

Cited By ~ 12

Author(s):

L. F. Zhai ◽

J. Liu ◽

M. X. Zhang ◽

N. Hong ◽

G. P. Wang ◽

...

Keyword(s):

Leaf Spot ◽

Aloe Vera ◽

Kentucky Bluegrass ◽

The United States ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Pathogenicity Tests ◽

Nigrospora Oryzae

Aloe vera L. var Chinese (Haw) Berg is a popular ornamental plant cultivated worldwide, whose extracts are used in cosmetics and medicine. Aloe plants are commonly affected by leaf spot disease caused by Alternaria alternata in Pakistan, India, and the United States (1). An outbreak of Alternaria leaf spot recently threatened aloe gel production and the value of ornamental commerce in Louisiana (1). During the summer of 2011, leaf spot symptoms were observed on A. vera plants growing in several greenhouses and ornamental gardens in Wuhan, Hubei Province, China. In two of the greenhouses, disease incidence reached 50 to 60%. The initial symptoms included chlorotic and brown spots that expanded to 2 to 4 mm in diameter and became darker with age. Lesions also developed on the tips of 30 to 50% of the leaves per plant. In severe infections, the lesions coalesced causing the entire leaf to become blighted and die. In September of 2012 and February of 2013, 10 symptomatic A. vera leaves were collected randomly from two greenhouses and gardens in Wuhan. A fungus was consistently recovered from approximately 80% of the tissue samples using conventional sterile protocols, and cultured on potato dextrose agar (PDA). The colonies were initially white, becoming grey to black, wool-like, and growing aerial mycelium covering the entire petri dish (9 cm in diameter) plate within 5 days when maintained in the dark at 25°C. The conidia were brown or black, spherical to subspherical, single celled (9 to 13 μm long × 11 to 15 μm wide), borne on hyaline vesicles at the tip of conidiophores. The conidiophores were short and rarely branched. These colonies were identified as Nigrospora oryzae based on the described morphological characteristics of N. oryzae (2). Genomic DNA was extracted from a representative isolate, LH-1, and the internal transcribed spacer region was amplified using primer pair ITS1/ITS4 (3). A 553-bp amplicon was obtained and sequenced. The resulting nucleotide sequence (GenBank Accession No. KC519728) had a high similarity of 99% to that of strain AHC-1 of N. oryzae (JQ864579). Pathogenicity tests for strain LH-1 were conducted in triplicate by placing agar pieces (5 mm in diameter) containing 5-day-old cultures on A. vera leaves. Four discs were placed on each punctured surface of each leaf. Noncolonized PDA agar pieces were inoculated as controls. Leaves were placed in moist chambers at 25°C with a 12-h photoperiod. After 3 days, the inoculated leaves showed symptoms similar to those observed in the greenhouses. N. oryzae was reisolated from these spots on the inoculated leaves. No visible symptoms developed on the control leaves. The pathogenicity tests were performed twice with the same results. Based on the results, N. oryzae was determined as a pathogen responsible for the leaf spots disease on A. vera. N. oryzae has been described as a leaf pathogen on fig (Ficus religiosa), cotton (Gossypium hirsutum) and Kentucky bluegrass (Poa pratensis) (4), and to our knowledge, this is the first report of N. oryae causing leaf spot disease on A. vera worldwide. References: (1) W. L. da Silva and R. Singh. Plant Dis. 86:1379, 2012. (2) M. B. Ellis. Dematiaceous Hyphomycetes, CAB, Kew, Surrey, England, 1971. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (4) L. X. Zhang et al. Plant Dis. 96:1379, 2012.

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First report of Alternaria alternata causing leaf spot on Oat (Avena sativa) in India

Plant Disease ◽

10.1094/pdis-02-21-0322-pdn ◽

2021 ◽

Author(s):

Boda Praveen ◽

Prasanna Kumar M.K. ◽

Devanna Pramesh ◽

PALANNA K B ◽

Buela P.P. ◽

...

Keyword(s):

Avena Sativa ◽

Leaf Spot ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

Cultural Characteristics ◽

Leaf Quality ◽

First Report ◽

Spot Disease ◽

Pathogenicity Assay

Oat (Avena sativa L.) is an important cereal crop grown worldwide primarily for food and animal feed. In November 2019, a leaf spot disease was observed on the oat plants at Mandya (12.5218° N, 76.8951° E), Karnataka, India. The disease incidence on plants was ranged between 43 to 57 percent. Initially, the symptoms appeared on leaves as small dark-brown spots surrounded by a yellow halo later turned to irregular necrotic spots with a yellow halo. A total of thirty leaves showing typical symptoms were collected from ten plants (three leaves per plant), cut into an area of 4-5 mm pieces at the junction of infected and healthy tissues. Cut tissues were soaked in 75% ethanol for 45 seconds, followed by 1% sodium hypochlorite solution for 1 min, rinsed five times in sterile distilled water, air dried, and placed on PDA and incubated at 25 ± 1 ℃. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for five isolates. The colonies initially produced light-greyish aerial mycelia, then turned to dark-greyish upon maturity. Conidia were obclavate to pyriform and measured 17.34 to 46.97 µm long, 5.38 to 14.31 µm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2.73 to 10.17µm) (n = 50.) Based on the morpho-cultural characteristics, the isolates were identified as Alternaria spp., and PCR assay using species-specific primers (AAF2/AAR3; Konstantinova, et al. 2002) confirmed the taxonomic identity of all five isolates as A. alternata. To further confirm the identity, the internal transcribed spacer (ITS), small subunit (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a1), endopolygalacturonase (endoPG), an anonymous gene region OPA10-2, KOG1058 and translation elongation factor 1-alpha (tef1) of two isolates MAAS-1 and MAAS-2 were PCR amplified using the primers described previously (Woudenberg et al. 2015; Praveen et al. 2020) and the resultant PCR products were sequenced and deposited in NCBI GenBank (ITS: MW487388, MW741962, SSU: MW506220, MW752854, gapdh: MW506221, MW752855, rpb2: MW506222, MW752856, Alt a1: MW506223, MW752857, endoPG: MW506224, MW752858, OPA10-2: MW506225, MW752859, KOG1058: MW506226, MW752860, and tef1: MW506227, MW752861) which showed (99.62%, 99.81%), (100%, 100%), (100%, 99.66%), (100%, 100%), (99.58%, 99.15%), (99.55%, 99.32%), (99.53%, 99.68%), (99.23%, 99.56%) and (99.17%, 99.58%) identity with ITS (AF347031), SSU (KC584507), gapdh (AY278808), rpb2 (KC584375), Alt a1 (AY563301), endoPG (JQ811978), OPA10-2 (KP124632), KOG1058 (KP125233) and tef1 (KC584634) genes/genomic regions of type strain CBS916.96 of A. alternata respectively, confirming the identity of MAAS-1 as A. alternata. For pathogenicity assay, the conidial suspension (2 × 106 conidia/ml) of MAAS-1 isolate was artificially sprayed until runoff on ten healthy oat plants (cv. Kent, 35 days old) and ten plants sprayed with sterile water served as control. All plants were covered with polyethylene covers and kept under the greenhouse at 28 ± 1 ℃. The pathogenicity assay was repeated three times. After six days post-inoculation, small dark-brown spots with light-yellow halo appeared on leaves of MAAS-1inoculated plants. In comparison, no symptoms were observed on control plants. The fungal pathogen was re-isolated from the artificially infected plants and confirmed as A. alternata based on morpho-cultural characteristics and PCR assays. The leaf spot disease of Oat caused by A. alternata has already been reported from China (Chen et al. 2020); to our knowledge, it is the first report of A. alternata causing leaf spot on Oat in India. The leaf spot disease is an emerging threat to oat cultivation, and it reduces the grain yield and leaf quality; therefore, its management is essential for increasing productivity.

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