First report of Alternaria tenuissima causing leaf black spot on pecan in China

China is one of the largest markets for pecan (Carya illinoinensis) consumption in the world, and also, pecan production in China has been increased by years since 2008 (Zhang et al. 2015). From July to September in the year 2019 and 2020, leaf black spot was observed on several pecan cultivars including Pawnee, Burkett, Kiowa and Western schley in the germplasm in Liuhe county, Nanjing, Jiangsu Province, China. Disease incidence was approximately 40% in 2019 and 50% in 2020 respectively. Small, dark brown to black spots on leaves were observed initially, and spots expanded quickly into circular or irregular when spots coalesced. In severe cases, the disease can obviously weaken the tree vigor, ultimately leading to losses in yield. Disease symptoms were not observed on the fruits. To determine the causal agent of black leaf spot, symptomatic leaves were collected and cut into pieces (approximately 3 × 3 cm2), surface sterilized with 1% sodium hypochlorite for 2 min and 75% ethanol for 30 s and rinsed twice with sterile distilled ddH2O. Dried tissues were placed on potato dextrose agar (PDA) amended with rifampin (Solarbio, Beijing, China) at a final concentration of 100 µg/mL and incubated at 25°C for 7 days in darkness. Five colonies were obtained and purified by single spore culture for morphological characterization. Colonies were initially white, turned to dark olivaceous with white margin and moderate to abundant gray aerial hyphae. Conidiophores were linear, light brown in color and appeared as individuals or in clusters. Conidia were pale brown to brown, typically obclavate or obpyriform (8 – 49 µm× 3 – 18 µm), with one to five transverse septa and zero to three longitudinal septa (n=50). Conidia of all isolates produced no beaks or a short beak. Based on conidial morphological characteristics, isolates were tentatively identified as Alternaria tenuissima (Simmons 2007). To further confirm the five isolates, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), partial region of the histone 3 (H3) gene, translation elongation factor 1-α gene (TEF) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with PCR primer sets ITS1/ITS4 (White et al. 1990), H3-1a/H3-1b (Glass and Donaldson 1995), EF1-728F/EF1-986R (Carbone and Kohn 1999), and GDF1/GDR1 (Berbee et al. 1999) respectively. The sequences were deposited in GenBank (ITS, MN822659 to MN822661 and MZ182355 to MZ182356; histone 3, MN840997 to MN840999 and MZ202355 to MZ202356; TEF, MZ246595 to MZ246599; GAPDH, MZ246590 to MZ246594). BLAST analysis of the resulting sequences showed 99% to 100 % nucleotide identical to those of A. tenuissima isolates (KP278184 [ITS]; MH824352 [H3]; MN046379 [TEF]; MK683840 [GAPDH]). Therefore, based on morphological characteristics and DNA sequences data, the five isolates were identified as A. tenuissima. To determine the pathogenicity of the five isolates, 10 µL of 105 conidia /mL suspension from each isolate was placed to three intact young leaves (Pawnee) respectively. Leaves inoculated with ddH2O in the same manner served as the controls. Inoculated leaves were placed in a growth chamber at 28°C with 95% relatively humidity (RH). Black spot symptoms appeared on all inoculated leaves by 7 days post inoculation, A. tenuissima were re-isolated from the inoculated leaves with isolates and reconfirmed by morphological characteristics, thus fulfilling the Koch’s postulates. The controls remained symptomless. Pathogenicity tests were conducted twice. To our knowledge, this is the first report of A. tenuissima causing black leaf spot on pecan in China. Although we have not found any fruit infection, we think the disease is also a potential threat to pecan fruits and production. If more reports of this pathogen are found on pecans, then it is necessary to study and develop effective control strategies.

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First Report of Alternaria alternata Causing Black Leaf Spot of Rubber Tree in China

Plant Disease ◽

10.1094/pdis-09-14-0954-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 290-290 ◽

Cited By ~ 11

Author(s):

Z. Y. Cai ◽

Y. X. Liu ◽

G. H. Li ◽

Y. F. Wang ◽

M. Zhou

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Rubber Tree ◽

Morphological Characteristics ◽

Correct Identification ◽

Post Inoculation ◽

First Report ◽

Leaf Spots ◽

Plastic Bags ◽

Black Leaf Spot

We first reported Alternaria heveae (E.G. Simmons ) to be the pathogen that caused black leaf spot of rubber tree (Hevea brasiliensis Muell. Arg) in Heikou county in July 2014 (1). Black leaf spots that resembled the symptoms caused by A. heveae were observed on the leaves of rubber trees of the whole propagule collection nursery in Jingping County (22°68′ N and 103°05′ E) of Yunnan Province. Black foliar spots (0.1 to 2 mm in diameter) surrounded by a yellow halo with lesions slightly sunken on the leaf surface were observed. To confirm whether the disease was caused by the same pathogen, 5-mm2 sections were removed from the leading edge of the lesion and were surface-sterilized in 75% ethanol, air-dried, plated on potato carrot agar (PCA), and incubated at 28°C in the dark. Colonies of the fungus on PCA had round margins and little aerial mycelia with gray-black coloration after 6 days of growth on PCA (2). Medium brown conidia were found to be in short chains of two to eight spores, ovoid, obclavate, and obpyriform, with or without a short conical or cylindrical-shaped apical beak. Conidia ranged from 22.5 to 67.5 μm long (mean 39.9 μm) × 10 to 15 μm wide (mean 12.5 μm; 100 colodia were measured), with three to six transverse septa and zero to three longitudinal or oblique septa. Morphological characteristics matched the descriptions of A. alternata [(Fries) Keissler] (4).The ITS1-5.8S-ITS2 region of one single-spore isolate, Ah02JP1, was amplified with primers ITS1 and ITS4. The PCR product was sequenced directly and deposited in GenBank (Accession No. KM111289). A BLAST search of the GenBank database revealed 100% similarity with A. alternata isolates KJ829535.1, KJ677246.1, and KF813070.1. Therefore, the pathogen was identified as A. alternata on the basis of its morphological characteristics and ITS sequence. Pathogenicity of a representative isolate, Ah02JP1 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage. Leaves were spray-inoculated (104 conidia per milliliter spore suspension) until drops were equally distributed using a manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags to maintain high relative humidity. The plastic bags were removed 2 days post-inoculation (dpi), and the plants were monitored daily for symptom development. Five days post-inoculation, spots similar to the original ones seen on the field trees developed on all inoculated leaves, while control leaves remained symptomless. A. alternata was re-isolated from spray-inoculated leaves, confirming Koch's postulates. A. alternata has been reported as the causal agent of leaf blight of rubber tree in India, which initially appeared as minute spots on leaves and enlarged with the growth of the leaves (3). However, in the present study, the symptoms (black leaf spots) remained small over time after inoculation. To our knowledge, this is the first report of A. alternata on rubber tree in China. Correct identification of pathogens is essential for disease management strategies. This report will establish a foundation for the further study of Alternaria alternata to address the disease effectively. References: (1) Z. Y. Cai et al. Plant Dis. 98:1011, 2014. (2) E. Mirkova. J. Phytopathol. 151:323, 2003. (3) C. B. Roy et al. J. Plantation Crops 34:499, 2006. (4) T. Y. Zhang. Page 32 in: Flora Fungorum Sinicorum, Vol. 16: Alternaria. Science Press, Beijing, 2003.

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

Plant Disease ◽

10.1094/pdis-01-13-0070-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1654-1654 ◽

Cited By ~ 3

Author(s):

A. L. Vu ◽

M. M. Dee ◽

J. Zale ◽

K. D. Gwinn ◽

B. H. Ownley

Keyword(s):

Leaf Spot ◽

Panicum Virgatum ◽

Morphological Characteristics ◽

Its Region ◽

Spore Production ◽

Post Inoculation ◽

Sterile Water ◽

Bipolaris Oryzae ◽

First Report ◽

Symptomatic Leaf

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

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First Report of 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 Alternaria heveae Causing Black Leaf Spot of Rubber Tree in China

Plant Disease ◽

10.1094/pdis-01-14-0065-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1011-1011 ◽

Cited By ~ 1

Author(s):

Z. Y. Cai ◽

Y. X. Liu ◽

G. X. Huang ◽

M. Zhou ◽

G. Z. Jiang ◽

...

Keyword(s):

Rubber Tree ◽

Morphological Characteristics ◽

Black Spot ◽

Its Region ◽

Post Inoculation ◽

First Report ◽

Leaf Spots ◽

Plastic Bags ◽

Manual Pressure ◽

Tree Leaf

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop of tropical areas for natural rubber production. In October 2013, foliar spots (0.1 to 0.4 mm in diameter), black surrounded by a yellow halo, and with lesions slightly sunken were observed on the rubber tree leaf in a growing area in Heikou County of Yunnan Province. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on PDA plates, and incubated at 28°C with alternating day/night cycles of light. The pathogen was observed growing out of many of the leaf pieces, and produced abundant conidia. Colonies 6.1 cm in diameter developed on potato carrot agar (PCA) after 7 days, with well-defined concentric rings of growth. Colonies on PCA were composed of fine, dark, radiating, surface and subsurface hyphae. Conidia produced in PCA culture were mostly solitary or in short chains of 2 to 5 spores, long ovoid to clavate, and light brown, 40 to 81.25 × 8 to 20 μm (200 colonies were measured), with 3 to 6 transverse septa and 0 to 2 longitudinal or oblique septa. Morphological characteristics were similar to those described for Alternaria heveae (3,4). A disease of rubber tree caused by Alternaria sp. had been reported in Mexico in 1947 (2). DNA of Ah01HK13 isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers was completed. From the BLAST analysis, the sequence of Ah01HK13 (GenBank Accession No. KF953884), had 97% similarity to A. dauci, 96% identical to A. macrospora (AY154701.1 and DQ156342.1, respectively), indicating the pathogen belonged to Alternaria genus. According to morphological characteristics, this pathogen was identified as A. heveae. Pathogenicity of representative isolate, Ah01HK13 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage and inoculated by spraying spore suspension (concentration = 104 conidia/ml) to the copper-colored leaves until drops were equally distributed on it using manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags. The plastic bags were removed after 2 days post-inoculation (dpi) and monitored daily for symptom development (1). The experiment was repeated three times. The typical 0.1 to 0.4 mm black leaf spots were observed 7 dpi. No symptoms were observed on control plants. A fungus with the same colony and conidial morphology as A. heveae were re-isolated from leaf lesions on inoculated rubber plants, but not from asymptomatic leaves of control plants, fulfilling Koch's postulates. Based on these results, the disease was identified as black spot of rubber tree caused by A. heveae. To our knowledge, this is the first report of A. heveae on rubber tree in China. References: (1) Z. Y. Cai et al. Microbiol Res. 168:340, 2013. (2) W. J. Martin. Plant Dis. Rep. 31:155, 1947. (3) E. G. Simmons. Mycotaxon 50:262, 1994. (4) T. Y. Zhang. Page 111 in: Flora Fungorum Sinicorum: Alternaria, Science Press, Beijing, 2003.

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First Report of Leaf Spot Caused by Colletotrichum fructicola on Myrica rubra in China

Plant Disease ◽

10.1094/pdis-10-21-2138-pdn ◽

2021 ◽

Author(s):

Shucheng Li ◽

Liuhua Xiao ◽

Fan Wu ◽

Yinbao Wang ◽

Mingshu Jia ◽

...

Keyword(s):

Leaf Spot ◽

Juglans Regia ◽

Morphological Characteristics ◽

Effective Control ◽

Bootstrap Support ◽

Sterile Water ◽

First Report ◽

Myrica Rubra ◽

Initial Symptoms ◽

Colletotrichum Fructicola

Myrica rubra is an important fruit tree with high nutritional and economic value, which is widely cultivated in multiple regions of China. In January 2021, an unknown disease which caused leaf spot with approximately 20% (n=100 investigated plants) of incidence was discovered on the leaves of M.rubra in Jiujiang City of Jiangxi Province (29.71° N, 115.97° E). The initial symptoms were small pale brown spots (1 to 2 mm diameter) on the leaves, which gradually expanded into round or irregular dark brown spots with the occurrence of the disease, and the lesion developed necrotic tissues in the center at later stages, eventually leading the leaves to chlorotic and wilted. Ten diseased leaves with typical symptoms were collected and the leaf tissue (5 × 5 mm) at junction of diseased and healthy portion were cut. The surfaces were disinfected with 75% ethanol for 45 s, 1% sodium hypochlorite for 1 min, and rinsed in sterile water for 3 times then transferred to potato dextrose agar (PDA) at 28 ± 1 ℃ for 3 days. Five fungal single isolates with similar morphology were purified from single spores. On PDA medium, the colonies initially appeared white with numerous aerial hyphae, and the center of the colony turned gray at later stages, less sporulation. While on modified czapek-dox medium (Peptone 3g, K2HPO4 1g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.01g, Maltose 30g, Agar 15g, Distilled water 1000 mL, pH=7.0), the mycelia of the colony were sparse and produced a large number of small bright orange particles (conidial masses). Conidia were single-celled, transparent, smooth-walled, 1-2 oil globule, cylindrical with slightly blunt rounded ends, 14.45-18.44 × 5.54-6.98 μm (av=16.27 μm × 6.19 μm, n=50) in size. These morphological characteristics of the pathogen were similar to the descriptions of Colletotrichum fructicola (Ruan et al, 2017; Yang et al, 2021). To further confirm the identity of the pathogen, genomic DNA from a representative isolate was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), and the internal transcribed spacer (ITS), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), calmodulin gene (CAL), actin (ACT) and chitin synthase 1 (CHS 1) were amplified by using the primers ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Weir et al, 2012), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999), respectively. The PCR amplified sequences were submitted to GenBank (GenBank Accession No. ITS, MW740334; GAPDH, MW759805; CAL, MW759804; ACT, MW812384; CHS-1, MW759803) and aligned with GenBank showed 100% identity with C. fructicola (GenBank Accession No. ITS, MT355821.1 (546/546 bp); GAPDH, MT374664.1 (255/255 bp); CAL, MK681354.1 (741/741 bp); ACT, MT364655.1 (262/262 bp); CHS, MT374618.1 (271/271 bp)). Phylogenetic tree using the maximum likelihood methods with Kimura 2-parameter model and combined ITS-ACT-GAPDH-CHS-CAL concatenated sequences, bootstrap nodal support for 1000 replicates in MEGA7.0, revealed that the isolate was assigned to C. fructicola strain (ICMP 18581 and CBS 125397) (Yang et al. 2021) with 98% bootstrap support. Pathogenicities of were tested on fifteen healthy M. rubra plants (five for wounded inoculation, five for nonwounded inoculation, and five for controls) in the orchard. Twenty leaves were marked from each plant, and disinfected the surface with 75% ethanol. Ten μL spore suspension (1.0 × 106 conidia/ml) of each isolate from 7-day-old culture were inoculated on the surface of 20 needle-wounded and 20 nonwounded leaves, respectively. Healthy leaves were inoculated with sterile water as controls by the same method. All inoculated leaves were sprayed with sterile water and covered with plastic film to remained humidification. After 5 days, all the wounded leaves which were inoculated with C. fructicola showed similar symptoms to those observed on the original leaves. Symptoms of nonwounded leaves were milder than the wounded inoculated leaves, while control leaves remained healthy. Finally, the C. fructicola was re-isolated from the inoculated leaves. C. fructicola has been reported on Juglans regia, Peucedanum praeruptorum, Paris polyphylla var. Chinensis in China (Wang et al, 2017; Ma et al, 2020; Zhou et al, 2020). As far as we know, this is the first report of C. fructicola causing leaf spot on M.rubra in China. This result contributes to better understand the pathogens causing diseases of M.rubra in this region of China and develop effective control strategies.

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First Report of Colletotrichum cliviicola Causing Leaf Spot on Tobacco (Nicotiana tabacum) in Hunan Province of China

Plant Disease ◽

10.1094/pdis-02-21-0409-pdn ◽

2021 ◽

Author(s):

Ya Rong Wang ◽

Zhao Hu ◽

Jie Zhong ◽

Yi Chen ◽

Jun Zi Zhu

Keyword(s):

Nicotiana Tabacum ◽

Leaf Spot ◽

Morphological Characteristics ◽

Disease Diagnosis ◽

Hunan Province ◽

Leaf Spot Disease ◽

Post Inoculation ◽

Conidial Suspension ◽

First Report ◽

Colletotrichum Species

Tobacco (Nicotiana tabacum L.) is an annual, leafy, herb of the genus Nicotiana in the family Solanaceae. It is an important commercial crop in China. In 2020, a leaf spot disease was observed on tobacco leaves in commercial fields in the Hunan Province of China. Symptoms appeared as water-soaked, yellow-green spots, then turned dark brown, and coalesced into larger necrotic lesions, often leading to leaf wilt. Approximately 20% of the plants in a 50-ha area were infected, exhibiting symptomatic spots on 60% of these leaves. Symptomatic leaf samples were collected and cut into small pieces, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40s, rinsed with sterile distilled water for three times, plated on potato dextrose agar (PDA) and incubated at 26°C in the dark. Isolates with similar morphology were developed from ten samples. Fungal isolates produced densely, white to dark green, aerial mycelium. Conidia were straight, hyaline, aseptate, cylindrical, contained oil globules, and 15 to 25 µm × 3.0 to 4.0 µm (n=50). Appressoria were dark brown, irregularly shaped, 5.5 to 10.0 μm × 4.5 to 6.5 μm (n=50). These morphological characteristics were typical of Colletotrichum cliviicola (Yang et al. 2009). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes of a representative isolate CS16-2 were amplified and sequenced using the primer pairs as described previously (Weir et al. 2012). These sequences were deposited in GenBank (GenBank Accession Nos. MW649137 for ITS, MW656181 for ACT, MW656182 for GAPDH and MW656183 for CHS-1). BLAST analysis showed that they had 99.46% to 100% identity to the corresponding sequences of C. cliviicola strains. A concatenated phylogenetic tree was generated, using the ACT, GAPDH and CHS-1 sequences of the isolate CS16-2 and other closely matching Colletotrichum species obtained from the GenBank. We found that the CS16-2 was grouped with the C. cliviicola clade with 97% bootstrap support, including the C. cliviicola strain AH1B6 (Wang et al. 2016). Pathogenicity was tested spraying 2-month-old potted tobacco plants until runoff with a conidial suspension (105 spores/ml). Leaves were mock inoculated with sterilized water. The pathogenicity tests were performed twice, with three replicate plants each. Plants were kept in humid chambers at 26°C with a 12-h photoperiod. Five days post-inoculation, the inoculated plants developed symptoms of consisting of the yellow-brown necrotic lesion resembling the symptoms that were observed in fields, while the control plants remained symptomless. C. cliviicola was re-isolated and identified by morphological and molecular methods as described above. Currently, C. cliviicola has been reported to be the causal agent of anthracnose in some plants, such as soybean (Zhou et al. 2017) and Zamioculcas zamiifolia (Barbieri et al. 2017). However, to our knowledge, this is the first report of C. cliviicola causing leaf spot on tobacco in China and even in the word. Given that the may greatly affect the yield and quality of tobacco production, growers should be prepared to manage this new disease. This work might provide further insight for disease diagnosis on tobacco as some other Colletotrichum species, such as C. fructicola (Wang et al. 2016) and C. karsti (Zhao et al. 2020), have also been responsible for anthracnose.

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First Report of Brown Leaf Spot of Juglans hybrid Caused by Ophiognomonia leptostyla in China

Plant Disease ◽

10.1094/pdis-05-21-0981-pdn ◽

2021 ◽

Author(s):

Chunlin Yang ◽

Feng Liu ◽

Qian Zeng ◽

Xiulan Xu ◽

Yicong Lv ◽

...

Keyword(s):

Basal Cell ◽

Leaf Spot ◽

Juglans Regia ◽

Apical Cell ◽

Morphological Characteristics ◽

Control Measures ◽

Effective Control ◽

First Report ◽

Brown Leaf Spot ◽

Brown Leaf

“Chuanzao 2” is a walnut variety derived from the hybridization of Juglans regia L. and J. sigillata Dode distributed in southwest China, where it is an economically important tree species in rural regions (Xiao et al. 2012). In April 2020, the variety in a walnut garden showed symptoms of brown leaf spot in Beishan Town (107°21′43.93″E, 31°28′12.34″N), Dazhou City in Sichuan, China, with 5% to 10% of leaves per plant affected (5 plants). Symptomatic leaves showed brown to dark brown spots (2 to 5 mm) with a dark brown to black halo and grayish-tan center. The spots were subcircular to irregular in shape, and gradually expanded and formed necrotic spots. A single conidium isolation was performed (Senanayake et al. 2020) and transferred to Potato Dextrose Agar (PDA). Five isolates were obtained from five different infected leaves. Colonies of five isolates were subcircular, erose or dentate, flat or effuse, white initially, gradually becoming yellowish with white margins, developed and fluffy aerial mycelia, and conidiogenensis was produced underneath mycelia after 25-days-incubation. Conidiogenous cells were subcylindrical to cylindrical, or irregular in shape, and hyaline. Macroconidia were lunate, reniform, hyaline, basal cell bluntly rounded, apical cell with acute end, 1-septate, rarely aseptate, sometimes slightly constricted at septum, basal cell equal or larger than apical cell, and measured 16.5 to 30.5 × 5 to 8.5 μm (mean = 23.2 × 6.3 μm, n = 50). Microconidia were not observed. These morphological characteristics resembled those of Ophiognomonia leptostyla (Fr.) Sogonov (Walker et al. 2012a). For molecular identification, genomic DNA (isolates SICAUCC 21-0008 and SICAUCC 21-0010) was extracted, and the internal transcribed spacers (ITS) region, guanine nucleotide-binding protein subunit beta (MS204) gene, and translation elongation factor 1-alpha (tef1-α) were amplified and sequenced by using the primers ITS5/ITS4 (White et al. 1990), E1F1/E5R1a (Walker et al. 2012a), and EF1-728F/EF1-1567R (Walker et al. 2012b), respectively. Phylogenetic analyses (maximum likelihood) based on a combined dataset showed 100% bootstrap support values in a clade with O. leptostyla. The sequences of ITS, MS204, and tef1-α genes were deposited in GenBank with accession numbers MW493111/MZ026300, MW495270/MZ031975, and MW495271/MZ031974, respectively. To fulfill Koch’s postulates, five healthy hybrid plants (2 to 3 years old) with 5 to 8 leaves per plant were spray inoculated with conidium suspensions (104 conidia/mL; isolate SICAUCC 21-0008) prepared from 40-days-old cultures onto the wounded sites via pin-prick inoculation. Similarly, five noninoculated plants sprayed with sterile water served as controls. Plants were placed in a growth chamber at 25℃ on a 12-h fluorescent light/dark regime and daily sprayed with sterile distilled water. After two weeks, observed symptoms were similar to those from natural infections. No disease symptoms were found on control plants. The fungus O. leptostyla was reisolated from the diseased leaves and characterized morphologically. O. leptostyla is a global pathogen and has been reported to cause the leaf spot in many walnut trees, viz. J. ailantifolia, J. californica, J. cinerea, and J. major, etc. To our knowledge, this is the first report of O. leptostyla causing brown leaf spot on Juglans hybrid (J. regia × J. sigillata) in China. The increasing risk of this pathogen in the walnut-growing areas of Sichuan Province of China needs a further exploration and outreach effort to develop effective control measures. Chunlin Yang, Feng Liu, and Qian Zeng contributed equally to this paper.

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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 Leaf Spot Caused by Ramularia sphaeroidea on Vicia villosa var. glabrescens in China

Plant Disease ◽

10.1094/pdis-04-21-0862-pdn ◽

2021 ◽

Author(s):

Min Shi ◽

Yan Zhong Li

Keyword(s):

Dna Sequences ◽

Leaf Spot ◽

Tween 80 ◽

The United States ◽

Diseased Plant ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Vicia Villosa ◽

First Report

Hairy vetch Vicia villosa Roth is widely grown in southwestern China for green manure and forage. In December 2019, a leaf disease occurred on 80% plants of V. villosa var. glabrescens in an eight-hectare field in Qujing(N 25°28′12″, E 103°36′22″), Yunnan Province, China. The disease leaves had irregular, brown to dark brown leaf spots with white mold. Twenty diseased leaves from five plants were randomly collected from the field. The leaf samples were sterilized with 75% ethanol for 30 s and 1% NaClO for 75 s, rinsed three times with sterile distilled water, surface water removed with sterile filter paper, and placed onto potato dextrose agar (PDA) for culture at 20oC. The obtained fungal isolates were purified by transferring 1 to 2 mm hyphal tips onto fresh PDA plates and cultured under the same temperature condition. The isolates grew slowly, at a rate of 0.7 mm/d at 20℃ for 4 weeks. A diseased plant specimen (accession MHLZU19326) and three isolates (accessions YN1931401, YN1931402, and YN1931403) were deposited in the Mycological Herbarium of Lanzhou University (MHLZU). Conidia from the PDA cultures were hyaline, spherical, smooth, aseptate, and measured 2.13 to 3.67 × 4.56 to 5.77 μm (n = 50). Conidiophores were hyaline, smooth, and straight. DNA of purified isolates was extracted and the nuclear ribosomal internal transcribed spacer (ITS), tef1-α, his3 and gapdh genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (Carbone and Kohn 1999;O’Donnell et al. 1998), CylH3F/CylH3R (Crous et al. 2004), and gpd1/gpd2 (Berbee et al. 1999), respectively. DNA sequences of isolates YN1931401, YN1931402, and YN1931403 were deposited in GenBank for the ITS (accessions MW092181, MW332205, and MW332206), tef1-α (MW448172 to MW448174), his3 (MW448175 to MW448177), and gapdh (MW448178 to MW448180). These sequences had the highest similarities with sequences of Ramularia sphaeroidea Sacc. in GenBank, 99%(514∕516, 515∕517, and 514∕517 bp) for ITS, 99% (402∕403, 403∕405, and 405∕405bp) for tef1-α, 99% (377∕378, 378∕378, and 376∕378bp) for his3, and 100% (558∕557, 557∕559 and 561∕565 bp) for gapdh . A phylogenetic tree generated with the sequences clustered the fungus closely with R. sphaeroidea. Infection experiments were carried out with 50 plants of V. villosa var. glabrescens in 10 pots. A conidial suspension of 1. 0 × 106 conidia/ml with 0.01% Tween 80 was prepared by adding sterile distilled water to the YN1931401 culture and scraping with a sterile scalpel. The leaves of 25 healthy plants were sprayed with the conidial suspension, and those of the 25 control plants were sprayed with sterile water. All plants were covered with clear polyethylene bags for 3 days to maintain high humidity and then grown in a greenhouse at diurnal cycles of 18℃ for 18h with light and 22℃ for 6 h in dark. Ten days post-inoculation, the inoculated plants exhibited brown lesions similar to the symptoms observed in the field (Fig. 1-F), whereas no symptoms appeared on the control plants. The same fungus was re-isolated and identified as described above. R. sphaeroidea has been reported on V. fabae and V. sativa in Ethiopia and Israel (Braun 1998), on various Vicia species including V. villosa in California, the United States (Koike et al. 2004) and on V. craccain China (Zhang et al. 2006), but to our knowledge, this is the first report of this fungus causing leaf spot on V. villosa in China.

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First Report of Leaf Spot in Farfugium japonicum Caused by Alternaria tenuissima in Korea

Plant Disease ◽

10.1094/pdis-03-13-0253-pdn ◽

2013 ◽

Vol 97 (10) ◽

pp. 1382-1382 ◽

Cited By ~ 1

Author(s):

J. H. Lee ◽

D. S. Kim ◽

H. J. Cho ◽

G.-H. Gang ◽

Y.-S. Kwak

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Spot Size ◽

Plant Diseases ◽

Universal Primers ◽

Leaf Spot Disease ◽

Alternaria Tenuissima ◽

First Report ◽

Spot Disease ◽

Farfugium Japonicum

Farfugium japonicum (L.) Kitam (common name: Leopard plant) is known as a medical herb and belongs to family Asteraceae (1). In June 2012, a leaf spot disease was observed on the leaf surface of F. japonicum at a forest research plot Jinju, Gyeongnam province, Korea. More than 95% of F. japonicum plants were infected and leaf spot symptom appeared in the regions under our investigation. Light brownish symptoms initially developed and the spot size gradually increased and turned dark brown with an irregular shape as the disease progressed (spot size 1 to 10 mm in diameter). At the late stage of disease, spots became hollow and completely dehydrated. The infected leaves were easily crumbled, possibly due to dryness. To isolate the causal agent, the infected leaves were surface disinfected and pieces of leaves were placed on water agar (WA). Nine isolates were isolated from 10 pieces of the infected leaves. Fungi mycelia from the WA were transferred on potato dextrose agar (PDA) and incubated at 28°C for 7 days. The colonies were purple navy to black and conidia spores developed on the media. The morphological characteristics of spores were multi-septate, dark brown, pyriform, and 6.7 to 12.8 × 22.2 to 38.4 μm. The spores had 1 to 4 transverse and 0 to 3 longitudinal septa. The morphological characteristics of the isolates showed considerably similar to well-known Alternaria tenuissima (2). The leaf spot disease caused by A. cinerariae of F. japonicum was reported from Japan (3). Spores of A. cinerariae are golden brown to brown with 3 to 9 transverse and 0 to 6 longitudinal septa and are 87.5 × 28.7 μm (avg.) (3). To verify pathogenicity of the isolate, the pure cultured fungi on the PDA medium was taken (4 mm in diameter) and placed on healthy leaves of Leopard plant. The artificially inoculated leaves were placed on wet filter paper in Petri dishes and incubated at 25°C and 80% humidity. At 7 days after inoculation, similar disease symptoms developed on 8 out of 10 infected Leopard plant leaves. The pathogen was reisolated from artificially infected leaves. To identify in molecular biology level, genomic DNA was extracted and the ITS-rDNA region was amplified using universal primers ITS1 and ITS4. The amplified PCR product was purified and sequenced (528 bp) with ITS1 and ITS4 primers for both directions and then deposited in GenBank (Accession No. KC415611.1). The BLAST search showed that it matched previously reported A. tenuissima with 100% identity. To the best of our knowledge, this is the first report of Leopard plant leaf spot disease in Korea. References: (1) E. Y. Kim et al. J. Ethnopharmacol. 146:40, 2013. (2) E. G. Simmons. Page 1 in: Alternaria Biology, Plant Diseases and Metabolites. J. Chelchowski and A. Visconti, eds. Elsevier, Amsterdam, 1992. (3) T. Sakoda et al. Res. Bull. Pl. Prot. Japan 46:73, 2010.

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