First Report of Leaf Spot Caused by Nigrospora oryzae on Blueberry in Shanghai, China

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

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First Report of Leaf Spot of Lotus (Nelumbo nucifera) Caused by Nigrospora oryzae in China

Plant Disease ◽

10.1094/pdis-10-17-1676-pdn ◽

2018 ◽

Vol 102 (5) ◽

pp. 1038-1038 ◽

Cited By ~ 1

Author(s):

Q. H. Zhang ◽

L. L. Huang ◽

Y. J. Liu ◽

Y. Ai ◽

D. H. Peng

Keyword(s):

Leaf Spot ◽

Nelumbo Nucifera ◽

First Report ◽

Nigrospora Oryzae

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First Report of Leaf Spot Caused by Nigrospora oryzae on Dendrobium candidum in China

Plant Disease ◽

10.1094/pdis-09-13-1006-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 996-996 ◽

Cited By ~ 6

Author(s):

J. B. Wu ◽

C. L. Zhang ◽

P. P. Mao ◽

Y. S. Qian ◽

H. Z. Wang

Keyword(s):

Leaf Spot ◽

Perennial Herb ◽

Its Sequence ◽

Factors Affecting ◽

First Report ◽

Kanamycin Sulfate ◽

Plastic Bags ◽

Symptomatic Leaf ◽

Dendrobium Candidum ◽

Nigrospora Oryzae

Dendrobium (Dendrobium candidum Wall. ex Lindl.) is a perennial herb in the Orchidaceae family. It has been used as traditional medicinal plant in China, Malaysia, Laos, and Thailand (2). Fungal disease is one of the most important factors affecting the development of Dendrobium production. During summer 2012, chocolate brown spots were observed on leaves of 2-year-old Dendrobium seedlings in a greenhouse in Hangzhou, Zhejiang Province, China, situated at 30.26°N and 120.19°E. Approximately 80% of the plants in each greenhouse were symptomatic. Diseased leaves exhibited irregular, chocolate brown, and necrotic lesions with a chlorotic halo, reaching 0.8 to 3.2 cm in diameter. Affected leaves began to senesce and withered in autumn, and all leaves of diseased plants fell off in the following spring. Symptomatic leaf tissues were cut into small pieces (4 to 5 mm long), surface-sterilized (immersed in 75% ethanol for 30 s, and then 1% sodium hypochlorite for 60 s), rinsed three times in sterilized distilled water, and then cultured on potato dextrose agar (PDA) amended with 30 mg/liter of kanamycin sulfate (dissolved in ddH2O). Petri plates were incubated in darkness at 25 ± 0.5°C, and a grey mycelium with a white border developed after 4 days. Fast-growing white mycelia were isolated from symptomatic leaf samples, and the mycelia became gray-brown with the onset of sporulation after 5 days. Conidia were unicellular, black, elliptical, and 11.4 to 14.3 μm (average 13.1 μm) in diameter. Based on these morphological and pathogenic characteristics, the isolates were tentatively identified as Nigrospora oryzae (1). Genomic DNA was extracted from a representative isolate F12-F, and a ~600-bp fragment was amplified and sequenced using the primers ITS1 and ITS4 (4). BLAST analysis showed that F12-F ITS sequence (Accession No. KF516962) had 99% similarity with the ITS sequence of an N. oryzae isolate (JQ863242.1). Healthy Dendrobium seedlings (4 months old) were used in pathogenicity tests under greenhouse conditions. Leaves were inoculated with mycelial plugs (5 mm in diameter) from a 5-day-old culture of strain F12-F, and sterile PDA plugs served as controls. Seedlings were covered with plastic bags for 5 days and maintained at 25 ± 0.5°C and 80 ± 5% relative humidity. Eight seedlings were used in each experiment, which was repeated three times. After 5 days, typical chocolate brown spots and black lesions were observed on inoculated leaves, whereas no symptoms developed on controls, which fulfilled Koch's postulates. This shows that N. oryzae can cause leaf spot of D. candidum. N. oryzae is a known pathogen for several hosts but has not been previously reported on any species of Dendrobium in China (3). To our knowledge, on the basis of literature, this is the first report of leaf spot of D. candidum caused by N. oryzae in China. References: (1) H. J. Hudson. Trans. Br. Mycol. Soc. 46:355, 1963. (2) Q. Jin et al. PLoS One. 8(4):e62352, 2013. (3) P. Sharma et al. J. Phytopathol. 161:439, 2013. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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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 Leaf Spot of Kentucky Bluegrass (Poa pratensis) Caused by Nigrospora oryzae in Ontario

Plant Disease ◽

10.1094/pdis-02-12-0127-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 909-909 ◽

Cited By ~ 8

Author(s):

L. Zheng ◽

F. Shi ◽

D. Kelly ◽

T. Hsiang

Keyword(s):

Leaf Spot ◽

Poa Pratensis ◽

Kentucky Bluegrass ◽

Back Side ◽

Cool Season ◽

First Report ◽

Surface Sterilization ◽

Agar Plug ◽

Pathogenicity Tests ◽

Nigrospora Oryzae

Kentucky bluegrass (Poa pratensis L.) is an important cool-season perennial grass in Ontario. It is native to Europe and can form an attractive and durable turf. In late September 2011, distinct leaf spots were observed on a Kentucky bluegrass lawn in Guelph, ON. Symptoms ranged from small lesions that were chocolate brown and oval or circular up to withered leaves. On potato dextrose agar (PDA) amended with streptomycin and tetracycline, a fungus was consistently recovered from symptomatic leaf samples after surface sterilization for 1 min in 1% sodium hypochlorite. On PDA, cultures were gray with an irregularly distributed, wool-like, fastgrowing aerial mycelium, showing a dark back side as the colony changed to darker brown after 7 days at 25°C. On diseased leaves, conidia were observed after moist incubation, borne on a hyaline vesicle at the tip of each conidiophore. Conidia were single celled, black, smooth, spherical, and 11.2 to 15.5 μm (average 13.8 μm) in diameter. The pathogen was identified as Nigrospora oryzae based on previous descriptions (1,2). Genomic DNA was extracted from a representative isolate, 11201, and the internal transcribed spacer (ITS) region of the ribosomal DNA was amplified by the primers ITS1 and ITS4 (4). The ITS sequence showed 99.8% similarity in the overlapping 508-bp portion with N. oryzae (GenBank No. GQ328855). Pathogenicity tests were performed in the laboratory with the isolate on 3-week-old, sand-based, Magenta box-grown plants of three cool-season turfgrass species, P. pratensis, Agrostis stolonifera, and Lolium perenne, by inoculating with fungal plugs. A 5-mm-diameter plug from 5-day-old PDA cultures was directly placed onto leaves in each of four replicate boxes per species, and then removed after 48 h of incubation. Plants treated with sterile agar plugs served as controls. Magenta boxes containing treated turf were covered loosely with their plastic lids and incubated at 23°C. Three days after inoculation and 1 day after inoculum removal, typical chocolate brown spots were observed on inoculated leaves from all three turfgrass species, but no symptoms were seen on agar plug-treated control plants. Koch's postulates were fulfilled by reisolation of N. oryzae from diseased leaves. The pathogenicity tests were carried out twice with the same results. This is an indication that N. oryzae causing leaf spot of Kentucky bluegrass in Ontario was not hostspecific, and could potentially affect other cool-season turfgrass species. Review of the literature revealed that N. oryzae is known as a pathogen on maize, rice, sorghum, cotton, weeds, and several other hosts, but has not been reported on any species of turfgrass (3). To our knowledge, this is the first report of N. oryzae infecting Kentucky bluegrass in Ontario or worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes, CAB, Kew, Surrey, England, 1971. (2) H. J. Hudson. Trans. Brit. Mycol. Soc. 46:355, 1963. (3) R. W. Smiley et al. Compendium of Turfgrass Diseases. 3rd ed. APS Press, St Paul, MN, 2005. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Leaf Spot Disease in Aloe vera Caused by Nigrospora oryzae in Bangladesh

Plant Disease ◽

10.1094/pdis-11-17-1857-pdn ◽

2018 ◽

Vol 102 (7) ◽

pp. 1461 ◽

Cited By ~ 1

Author(s):

M. Begum ◽

A. Hamza ◽

T. Tanny ◽

K. C. Das ◽

M. T. Mahmud ◽

...

Keyword(s):

Leaf Spot ◽

Aloe Vera ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Nigrospora Oryzae

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First Report of Nigrospora Leaf Spot Caused by Nigrospora oryzae on Watermelon in China

Plant Disease ◽

10.1094/pdis-02-18-0284-pdn ◽

2019 ◽

Vol 103 (5) ◽

pp. 1019 ◽

Cited By ~ 3

Author(s):

X. Chen ◽

N. Wang ◽

M.-F. Yang ◽

H.-X. Li

Keyword(s):

Leaf Spot ◽

First Report ◽

Nigrospora Oryzae

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First Report of Nigrospora oryzae Causing Leaf Spot of Cotton in China

Plant Disease ◽

10.1094/pdis-04-12-0349-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1379-1379 ◽

Cited By ~ 9

Author(s):

L. X. Zhang ◽

S. S. Li ◽

G. J. Tan ◽

J. T. Shen ◽

T. He

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Morphological Data ◽

Its Sequence ◽

Pathogenicity Test ◽

First Report ◽

Wide Range ◽

Cotton Plants ◽

Symptomatic Leaf ◽

Nigrospora Oryzae

Cotton (Gossypium hirsutum L.) is widely cultivated for the important economic value of the fiber. In the summer of 2011, a leaf spot of cotton plants cv. Wanza40 was observed in 11 fields (total of about 4 ha) in Qianshan County in southwest Anhui Province, China. Approximately 30% of the plants in each field were symptomatic. Affected plants exhibited brown to reddish, irregular foliar lesions, each with a brown border near the vein of the leaves. A sign of fungal infection was a dark leaf mold observed on lesions on the abaxial surface of leaves. Sections of symptomatic leaf tissues were surface-sterilized (in 75% ethanol for 30 s, then 1% NaOCl for 1 min), rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). A fungus consistently recovered from symptomatic leaf samples produced colonies that were initially white and then became grayish brown with the onset of sporulation. Black, spherical to subspherical, single-celled conidia (10 to 12 × 14 to 16 μm) were borne on a hyaline vesicle at the tip of each conidiophore. Morphological characteristics of the fungus were similar to that of Nigrospora oryzae (2). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) from a representative strain of the fungus, AHC-1, was amplified using the primers ITS1/ITS4 (4) and sequenced (GenBank Accession No. JQ864579). The ITS sequence had 99% identity with >553 bp of the ITS sequence of an N. oryzae isolate (GenBank Accession No. EU918714.1). On the basis of morphological data and ITS rDNA sequence, the isolate was determined to be N. oryzae. A pathogenicity test was performed on detached, young leaves of 4-month-old healthy cotton plants of cv. Wanza40. Six leaves were inoculated by placing a colonized agar piece (5 mm in diameter) from 7-day-old cultures of the fungus on pushpin-wounded leaves. Another six leaves treated with sterile PDA plugs served as a negative control treatment. Leaves were incubated in petri dishes and maintained at 25°C in a growth chamber programmed for 12 hours of fluorescent white light/day. After 5 days, brown to black lesions were observed on all inoculated leaves, whereas no symptoms developed on control leaves. N. oryzae was consistently reisolated from symptomatic leaves but not from the control leaves. N. oryzae is a weak pathogen on a wide range of plants, and has been described as the causal agent of lint rot on cotton (1,3), but to our knowledge this is the first report of N. oryzae causing a leaf spot of cotton in China. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA, Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , April 8, 2012. (2) H. J. Hudson. Trans. Br. Mycol. Soc. 46:355, 1963. (3) A. J. Palmatter et al. Plant Dis. 87:873, 2003. (4) T. J. White et al. In: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Nigrospora oryzae Causing Leaf Spot on Ginger in China

Plant Disease ◽

10.1094/pdis-02-21-0260-pdn ◽

2021 ◽

Author(s):

Zeng-Liang LIU ◽

Shuangyun Zhou ◽

Liangliang Qi ◽

Xiaoguo Wang ◽

Juan Song ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Control Measures ◽

Likelihood Method ◽

Effective Control ◽

Economic Losses ◽

Leaf Spot Disease ◽

First Report ◽

Nigrospora Oryzae

Ginger (Zingiber officinale Rosc.) is an herbal crop widely grown in China for its medicinal and savory qualities of rhizomes. In August 2018, leaf spot symptoms were observed on ginger plants grown in a field in Nanning, Guangxi Province (E108°3'54", N23°14'48"). Disease incidence was above 50%, and in a Nanning field, rhizome yield loss was almost 30%. Early symptoms appeared as circular, necrotic areas that later developed into circular or irregular spots. The centers of the lesions were white and often surrounded by chlorotic halos (Figure S1A). In severe infections, the spots frequently coalesced, causing the entire leaf to become withered and curved. Small pieces (3 to 4 mm2) from the margin of infected lesions were surface sterilized in 75% ethanol for 40 s followed by 1% NaOCl for 90 s, placed on potato dextrose agar (PDA) and incubated at 28°C in the dark for 4 days. Hyphal tips from the leading edge of colonies were transferred to fresh PDA plates to obtain pure cultures. Fungal colonies were initially white, then turned black/grayish brown when maintained in the dark at 28°C after 5 days (Figure S1B). Conidia were single-celled, brown, or black, smooth, spherical, or subspherical with diameters varying from 9.5 to 15 μm (mean = 13.5 ± 0.72 µm, n = 50) (Figure S1C). Based on these morphological characteristics, the isolates were provisionally identified as Nigrospora oryzae (Ellis 1971; Hudson 1963). Genomic DNA was extracted from a representative isolate Sjb-2. The internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The obtained ITS sequence (GenBank accession no. MW555242), TUB2 sequence (MZ048644), and TEF1-α sequence (MZ048645) showed >99% similarity with several GenBank sequences of N. oryzae (KF516962 for ITS; MK550707 for TUB2; and KY019425 for TEF1-α, respectively). Based on the combined sequences of ITS, TUB2 and TEF1-α sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that the isolates were N. oryzae (Figure S2). Pathogenicity of the isolate was confirmed by fulfilling Koch’s postulates. Agar blocks (3 mm diameter) containing a fungal mycelium were placed on detached healthy leaves of ginger. The leaves were then wrapped with sterile polyethylene and incubated in a greenhouse at 25°C with 60% RH. Within 7 days, symptoms appeared on inoculated leaves similar to spots observed in the field, whereas controls remained symptomless. The same pathogen was reisolated from the spots. Pathogenicity tests were performed twice with three replications, indicating that N. oryzae is responsible for leaf spot disease on ginger. The disease in ginger caused by N. oryzae had been reported in Southern Africa (Grech et al. 1989). To our knowledge, this is the first report of N. oryzae causing leaf spot of ginger in China. In the field, this pathogen can substantially affect ginger's health and rhizome yield if no effective control measures are implemented. Therefore, management of the disease should be further investigated to avoid major economic losses.

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