First Report of Leaf Spot Caused by Colletotrichum spaethianum on Peucedanum praeruptorum in China

Peucedanum praeruptorum Dunn, a traditional Chinese medicinal herb, is an important crop in Ningguo, China. Since 2010, leaf spot symptoms were observed yearly starting in June. Blighted leaf areas on individual plants ranged from 10 to 25% in many fields, and up to 200 ha were affected each year. Symptoms consisted of small, brown, necrotic spots uniformly distributed on the 1- to 2-week-old leaves. Small tissue pieces from the edges of lesions were disinfected in 2% NaClO for 3 min, rinsed twice in distilled water, plated on potato dextrose agar (PDA), and incubated at 25°C in darkness for 4 days. Single spore isolations were obtained for six strains. When inoculated on SNA media, the six strains produced typical septate mycelium, with the young hyphae hyaline and aged ones white greyish. Setae of the strains on SNA were brown, tip acute, 2- to 3-septate, and 32.5 to 85.6 μm long. Conidiogenous cells were hyaline, cylindrical, 2- to 3-septate, 6.2 to 16.5 μm in length, and 2.8 to 4.3 μm in width. The mature conidia were slightly curved, with round apex and truncate base, 1 to 5 oil globules, and were 13.3 to 23.8 μm in length and 3.0 to 3.9 μm in width, respectively. Appressoria were solitary or in loose groups, dark brown, irregular shapes, and were 6.8 to 9.2 μm in length and 4.3 to 7.1 μm in width. PCR amplification was carried out by utilizing the universal rDNA-ITS primer pair ITS4/ITS5 (1) and the actin gene primer pair ACT-512F and ACT-783R (2). The PCR products of ITS (GenBank Accession No. KC913201) and actin gene (KC913202) from six isolates were identical, respectively, and shared 100% identity to the ITS sequence of strain CBS 167.49 of Colletotrichum spaethianum (GU227807.1) and 99% similarity to the actin gene of strain CBS 167.49 of C. spaethianum (GU227905.1), which was isolated from Hosta sieboldiana in Germany (3). Based on the above, the isolates were identified as C. spaethianum. To confirm pathogenicity, conidial suspensions (105 conidia ml–1) of each of the six isolates were sprayed on four leaves per plant on five 6-month-old P. praeruptorum plants. Control plants were sprayed with water. Plants were maintained at 28°C in a greenhouse with constant humidity (RH 90%) and a 12-h photoperiod of fluorescent light. Symptoms similar to the original ones started to appear after 10 days, while the control plants remained healthy. The tests were repeated three times and the fungus was recovered and identified as C. spaethianum by both morphology and molecular characterization. To our knowledge, this is the first report of C. spaethianum causing leaf spot on P. praeruptorum in China. Since the C. spaethianum infections pose a serious threat to P. praeruptorum production, this disease needs to be considered for developing effective control strategies. References: (1) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (2) U. Damm et al. Fung. Divers. 39:45, 2009. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Download Full-text

First Report of Blueberry Leaf Spot Caused by Cylindrocladium colhounii in China

Plant Disease ◽

10.1094/pd-90-1553a ◽

2006 ◽

Vol 90 (12) ◽

pp. 1553-1553 ◽

Cited By ~ 2

Author(s):

Y. S. Luan ◽

L. Feng ◽

L. J. An

Keyword(s):

Leaf Spot ◽

Morphological Traits ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Adaxial Side ◽

Single Spore ◽

Tubulin Gene ◽

First Report ◽

Plastic Bags ◽

Β Tubulin

During late July and early August of 2005, leaf spot symptoms were observed in a blueberry nursery at a plantation in Dalian, which to our knowledge, lies within the largest blueberry-production area in China. Symptoms were observed primarily on lowbush species, for example Blomidon, as well as half-highbush cultivars. A slow-growing, white mycelium from the margin of necrotic leaf spots was recovered on potato dextrose agar (PDA). The following morphological traits were observed: erect conidiophores that branch twice and were terminated in a stiped, clavate phialide; hyaline, cylindrical, four-celled conidia; and globose, reddish brown, aggregated chlamydospores. Conidiophores (including stipes and terminal phialides) were 305 to 420 × 5 to 9 μm; primary branches were 9 to 45 × 5 to 6.3 μm; secondary branches were 9 to 17.3 × 3.1 to 4.5 μm; phialides were 7.8 to 17.5 × 2.5 to 6 μm; stipes (from the highest branch area to vesicle) were 150 to 270 μm long; and vesicles were 13 to 30 × 2 to 4.5 μm. Conidia were 50 to 72 × 4 to 5.5 μm. Chlamydospores were 15 to 20 μm in diameter. Koch's postulates were fulfilled by spray inoculating two healthy cultivars with conidiophores homogenized in axenic water. As a control, two healthy plants were sprayed with axenic water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and 20°C in darkness for 10 h. After 2 days, the plastic bags were removed and plants were maintained under the same conditions. After 4 days, small-to-medium brown spots with purplish margins were observed on the adaxial side of leaves from inoculated plants, but not from control plants. Fungi isolated from these lesions had the same morphological traits as the ones isolated previously from field plants. The morphological descriptions and measurements were similar to Cylindorocladium colhounii (2). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and the β-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 primers and T1/Bt2b primers, respectively, and sequenced (1). The ITS and β-tubulin gene sequences were similar to C. colhounii STE-U 1237 (99%; GenBank Accession No. AF231953) and C. colhounii STE-U 705 (99%; GenBank Accession No. AF231954), respectively. The morphology, secondary conidiation, and sequences of ITS and β-tubulin gene identify the causal fungus as C. colhounii. To our knowledge, this is the first report of C. colhounii on blueberry in China or in the world. References: (1) P. W. Crous et al. Can. J. Bot. 77:1813, 1999. (2) T. Watanabe. Page 222 in: Dictorial Atlas of Soil and Seed Fungi. CRC Press, Inc., Boca Raton, Fl, 1994.

Download Full-text

First Report of Passion Fruit Anthracnose Caused by Colletotrichum constrictum

Plant Disease ◽

10.1094/pdis-04-21-0754-pdn ◽

2021 ◽

Author(s):

Na Wang ◽

Fumei Chi ◽

Zhirui Ji ◽

Zongshan Zhou ◽

Junxiang Zhang

Keyword(s):

Sequence Data ◽

Control Strategies ◽

Pcr Amplification ◽

Morphological Characters ◽

Fungal Species ◽

Passion Fruit ◽

Effective Control ◽

First Report ◽

Sterile Needle ◽

Morphologic Data

Passion fruit (Passiflora edulis) is widely cultivated in tropic and subtropic regions. Because of its unique and intense flavour and high acidity, passion fruit juice concentrate is used in making delectable sauces, desserts, candy, ice cream, sherbet, or blending with other fruit juices. Anthracnose of passion fruit is favored by frequent rainfall and average temperatures above 27°C. In August 2018, anthracnose on passion fruit was observed in commercial plantings in Lincang, Yunnan, China (23.88 N, 100.08 E). Symptoms included lesions of oval to irregular shapes with brown to dark brown borders. Infection covered most of the fruit surface with pink-to-dark sporulation as reported by Tarnowski and Ploetz (2010). A conidial mass from an individual sorus observed on an infected fruit was isolated and cultured on potato dextrose agar (PDA) supplemented with 50 μg ml-1 of streptomycin. From a single microscopic field, two monospore isolates were dissected using a sterile needle, subcultured, and referred to as BXG-1 and BXG-2. Morphological characters including conidia colour, size, and shape were similar between the two isolates. Conidia were aseptate and cylindrical with apex and rounded base. Conidial length ranged from 12.3 to 16.1 µm (avg. 13.5) and width ranged from 5.5 to 6.2 µm (avg. 5.7). Morphologic data were consistent with Colletotrichum constrictum (Damm et al., 2012). To further confirm the fungal species, the ribosomal internal transcribed spacer (ITS), partial sequences of actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin 2 (TUB2) were amplified and sequenced. Primers and PCR amplification were described by Damm et al. (2012). The sequences were compared to type sequences in GenBank. The results showed the ITS (GenBank accession MW828148 and MW828149), ACT (MW855882 and MW855883), CHS-1 (MW855884 and MW855885), GAPDH (MW855886 and MW855887), and TUB2 (MW855888 and MW855889) sequences of the isolates BXG-1 and BXG-2 were 98% identical with sequence data from strain CBS:128504 of C. constrictum. A maximum likelihood tree was constructed using MEGA-X version 10.1.6 (Kumar et al., 2018) based on a combined dataset of the ITS, ACT, CHS-1, GAPDH, and TUB2 sequences of BXG-1 and BXG-2, and those of 18 Colletotrichum spp. previously deposited in GenBank (Damm et al., 2012). The phylogenetic analysis showed that BXG-1 and BXG-2 belong to the C. constrictum clade. Based on morphology and DNA sequencing, BXG-1 and BXG-2 were identified as C. constrictum. To verify pathogenicity, passion fruit were sprayed with a suspension of 1 × 105 conidia ml–1. Control fruit were sprayed with sterilized water. After inoculation, fruit were incubated in an Artificial Climate Box at 27°C and 80% RH. Necrotic symptoms appeared 8 days after inoculation and were similar to those observed on fruit form the field. The pathogen was reisolated from lesions thus fulfilling Koch’s postulates. C. constrictum has been reported to cause anthracnose of citrus from Australia (Wang et al., 2021) and mango from Italy (Ismail et al., 2015). To our knowledge, this is the first report of C. constrictum causing anthracnose on passion fruit worldwide, and these data will provide useful information for developing effective control strategies.

Download Full-text

First Report of a Leaf Spot on Hazel Leaves Caused by Phyllosticta coryli in Liaoning Province of China

Plant Disease ◽

10.1094/pdis-02-13-0213-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1254-1254 ◽

Cited By ~ 10

Author(s):

J. Sun ◽

D.-M. Wang ◽

X.-Y. Huang ◽

Z.-H. Liu

Keyword(s):

Leaf Spot ◽

Control Strategies ◽

Morphological Characteristics ◽

Leaf Tissue ◽

Its Region ◽

Liaoning Province ◽

Single Spore ◽

First Report ◽

Leaf Spots ◽

Microscopic Morphology

Hazel (Corylus heterophylla Fischl) is an important nut tree grown in China, especially in Liaoning Province, and is rich in nutritional and medicinal values. In August 2011, leaf spotting was observed on hybrid hazel (Dawei) leaves in Paotai Town, Wafangdian County of Liaoning Province. By August 2012, the disease had spread to Zhangdang Town, Fushun County. Symptoms initially appeared on both sides of leaves as pinpoint brown spots, which enlarged and developed into regular, dark brown lesions, 3 to 9 mm in diameter. The lesions were lighter in color in the center compared to the margin. To identify the pathogen, leaf pieces (3 to 5 mm) taken from the margins, including both symptomatic and healthy portions of leaf tissue, were surface-disinfected first in 75% ethanol for 5 s, next in 0.1% aqueous mercuric chloride for 50 s, and then rinsed with sterilized water three times. Leaf pieces were incubated on potato dextrose agar (PDA) at 25°C for 14 days in darkness. Single spore isolates were obtained from individual conidia. For studies of microscopic morphology, isolates were grown on synthetic nutrient agar (SNA) in slide cultures. Colonies grew up to 45 to 48 mm in diameter on PDA after 14 days. Pycnidia appeared on the colonies after 12 days. Conidiophores were short. Pycnidia were dark brown, subglobose, and 150 to 205 μm in diameter. Conidia were unicellular, colorless, ovoid to oval, and from 2.4 to 4.5 × 1.6 to 2.4 μm. On the basis of these morphological characteristics, the isolates were tentatively identified as Phyllosticta coryli Westend (2). The rDNA internal transcribed spacer (ITS) region was amplified using primers ITS1 and ITS4 and sequenced (GenBank Accession No. KC196068). The 490-bp amplicons had 100% identity to an undescribed Phyllosticta species isolated from Cornus macrophylla in Gansu, Tianshui, China (AB470897). On the basis of morphological characteristics and nucleotide homology, the isolate was tentatively identified as P. coryli. Koch's postulates were fulfilled in the growth chamber on hazelnut leaves inoculated with P. coryli conidial suspensions (107 conidia ml–1). Eight inoculated 1-year-old seedlings (Dawei) were incubated under moist conditions for 8 to 10 days at 25°C. All leaf spots that developed on inoculated leaves were similar in appearance to those observed on diseased hazel leaves in the field. P. coryli was recovered from lesions and its identity was confirmed by morphological characteristics. P. coryli was first reported as a pathogen of hazel leaves in Bull of Belgium (2). In China, P. coryli was first reported on Corylus heterophylla Fisch. in Jilin Province (1). To our knowledge, this is the first report of P. coryli causing leaf spot on hybrid hazel in Liaoning Province of China. The outbreak and spread of this disease may decrease the yield of hazelnut in northern regions of China. More studies are needed on control strategies, including the possible resistance of hazel cultivars to P. coryli. References: (1) Y. Li et al. J. Shenyang Agric. Univ. 25:153, 1994. (2) P. A. Saccardo. Sylloge Fungorum Vol. III, page 31, 1884.

Download Full-text

First Report of Leaf Spot Caused by Colletotrichum siamense on Magnolia grandiflora in China

Plant Disease ◽

10.1094/pdis-08-21-1698-pdn ◽

2021 ◽

Author(s):

Yang Zhou ◽

Dou Yang ◽

Lingping Zhang ◽

Yong-Chun Zeng ◽

Yang Zhang

Keyword(s):

Phylogenetic Tree ◽

Leaf Spot ◽

Control Strategies ◽

Epidemiologic Studies ◽

Leaf Spot Disease ◽

Single Spore ◽

Individual Tree ◽

First Report ◽

Magnolia Grandiflora ◽

Colletotrichum Siamense

Magnolia grandiflora (Southern magnolia) is a popular evergreen tree, planted especially as an ornamental for landscaping. In September 2019, leaf spots were observed on M. grandiflora at the campus of Jiangxi Agricultural University (28°45′56″N, 115°50′21″E). Approximately 64% (23 out of 36) M. grandiflora trees (most 24-year-old) occurred leaf spot disease at the campus. On average, 40% of the leaves per individual tree were affected. Foliar symptoms began as small dark brown lesions formed along the leaf margins. As the disease developed, the lesions’ center was sunken with a dark brown border. Symptomatic leaves were collected and cut into 5 × 5 mm pieces. Leaf pieces from the margin of the necrotic tissue were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed in sterile water three times. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Of more than 35 isolates, most shared a similar morphology, with an isolation rate of 85%. Three isolates (JNG-1, JNG-2, and JNG-3) were chosen for single-spore purification and used for morphological characterization and identification. Colonies on PDA of the three isolates were white, cottony, and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 4.4-5.6 × 13.2-17.8 µm (4.7 ± 0.3 × 14.6 ± 1.0 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.5-9.2 × 4.6-6.5 µm (7.3 ± 0.4 × 5.4 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum siamense as previously described (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-345R and GDF/GDR (Weir et al. 2012), respectively and sequenced. All sequences were deposited into GenBank (ITS, MZ325948-MZ325950; ACT, MZ461477 - MZ461479; GAPDH, MZ461483 - MZ461485; TUB2, MZ461486 - MZ461488; CHS-1, MZ441182 - MZ441184; CAL, MZ461480 - MZ461482). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (Kumar et al. 2016). According to the phylogenetic tree, all three isolates fall within the C. siamense clade (boot support 96%). The pathogenicity of three isolates were tested on M. grandiflora plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of spore suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). All the inoculated leaves were covered with black plastic bags to keep a high-humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in field, whereas control leaves were asymptomatic for 10 days. The infection rate was 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. It was confirmed that C. gloeosporioides is the causal agent of leaf spot on Magnolia virginiana in America (Xiao et al. 2004). However, this is the first report of C. siamense causing leaf spot on M. grandiflora in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Download Full-text

First Report of Twig Canker on Peach Caused by Phomopsis amygdali in China

Plant Disease ◽

10.1094/pdis-04-11-0321 ◽

2012 ◽

Vol 96 (2) ◽

pp. 288-288 ◽

Cited By ~ 4

Author(s):

F. M. Dai ◽

R. Zeng ◽

J. P. Lu

Keyword(s):

Control Strategies ◽

Morphological Characteristics ◽

Universal Primers ◽

Effective Control ◽

Fluorescent Light ◽

First Report ◽

Ctab Method ◽

Pcr Products ◽

Dna Fragment ◽

Potato Sucrose Agar

During May and June of 2009, canker and twig dieback were observed with 30 to 40% incidence in trees in one peach orchard in Nanhui of Shanghai (cv. YuLu juicy peach) and one orchard (cv. JingXiu yellow peach) in Jiaxin of Zhejiang Province, China. Cankers were generally centrally positioned on the nodes at the base of shoots with sunken, reddish brown/tan-to-silver symptoms. Blight was also observed on a few shoots (1). Five samples were collected from each orchard and isolations were conducted on potato sucrose agar (PSA). Ten isolates were obtained and all had white mycelia on PSA. Black pycnidia, formed in culture, produced two types of conidia: hyaline, fusiform alpha conidia and hyaline, string-like beta conidia. Alpha conidia varied from 5.0 to 6.3 × 1.5 to 2.5 μm and beta conidia ranged from 20 to 25 × 1.2 to 1.5 μm. Morphological characteristics suggested the identity of the fungal isolates to be Phomopsis amygdali. To confirm pathogenicity, an inoculum suspension was made from one isolate (106 conidia/ml) and was sprayed until runoff onto five twigs with buds. Inoculated twigs were maintained at 26°C and 100% relative humidity in a growth chamber with a 12-h period of fluorescent light daily. Twigs inoculated with sterilized water were included as noninoculated controls. After 4 days, dark brown lesions appeared around buds on inoculated twigs. No symptoms were observed on the control twigs. Constriction cankers were reproduced and P. amygdali was reisolated from the lesions. To confirm the identity of the pathogen, total genomic DNA was extracted with the cetyltrimethylammoniumbromide (CTAB) method from the mycelia of two isolates from YuLu juicy peach and Jinxiu yellow peach (2). PCR was performed with universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) to amplify a DNA fragment of approximately 550 bp. The PCR products were purified and sequenced in both directions (Sangon Biotech (Shanghai) Co., Ltd., China). The sequences (GenBank Accession Nos. HQ632013 and HQ632014) shared 98.9% identity with each other (MegAlign software; DNASTAR, Madison, WI). A comparison of these two sequences with those in GenBank showed that the sequences had the highest nucleotide similarity (99%) with P. amygdali isolate FAU1052 from peach in the southeastern United States (Accession No. AF102998). To our knowledge, this is the first report of P. amygdali causing twig canker on peach in China and will provide useful information for developing effective control strategies. References: (1) D. F. Farr et al. Mycologia 91:1008, 1999. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

Download Full-text

First Report of Leaf Spot Caused by Alternaria alternata on Chinese Dwarf Banana in China

Plant Disease ◽

10.1094/pdis-08-13-0831-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 691-691 ◽

Cited By ~ 3

Author(s):

B. Z. Fu ◽

Z. H. Zhang ◽

L. H. Wang ◽

G. Y. Li ◽

J. Z. Zhang ◽

...

Keyword(s):

Alternaria Alternata ◽

Leaf Spot ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Its Region ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Rdna Its ◽

Its Sequence Analysis

The Chinese dwarf banana (Ensete lasiocarpum) is one of the ornamental bananas that belongs to Musaceae family. The plant is native to the southwestern China, where it grows semi-wild in the mountains between 1,500 and 2,500 m above sea level. During July 2011, a leaf spot disease on this plant was observed in the campus and parks in Kunming, Yunnan Province. The incidence level was about 22%, mainly on the old leaves. The leaf symptoms were irregular spots with gray to off-white centers surrounded by dark brown margins, and usually also surrounded by chlorotic halos. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected (95% ethanol for 3 min, 0.1% HgCl2 for 2 min, rinsed three times with sterile water), plated on potato sucrose agar (PSA), and incubated at 26°C under natural lights. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PSA that were black on the underside. The colonies were further identified as Alternaria sp. based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 5.26 to 30.26 μm long and 3.95 to 15.79 μm wide, averaging 10.21 (±3.17) × 20.02 (±5.75) μm (n = 50), with a beak length of 0 to 7.89 μm, and had 3 to 8 transverse and 0 to 3 longitudinal septa. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (1). The ITS region of isolate DY1 (GenBank Accession No. KF516556) was 572 bp in length. BLAST search revealed 99% identity with two Alternaria alternata isolates (JF440581.1 and GQ121322.2). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the isolate in a well-supported cluster with other A. alternata isolates. The pathogen was identified as A. alternate (Fr.:Fr.) Keissler based on the morphological characteristics and rDNA-ITS sequence analysis. To confirm pathogenicity, Koch's postulates were performed on detached leaves of E. lasiocarpum inoculated with mycelial plugs with ddH2O and agar plugs as a control. Leaf spots identical to those observed in the field developed in 9 days on the inoculated leaves but not on the control. The inoculation assay used three leaves, totaling 72 spots for control and 36 spots for inoculation. The experiments were repeated once. A. alternata was consistently re-isolated from the inoculated leaves. The symptom developed easier with wounds. To our knowledge, this is the first report of E. lasiocarpum leaf spot disease caused by A. alternata in China and the world. References: (1) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (2) T. Y. Zhang. Flora Fungorum Sinicorum, Vol. 16: Alternaria. Science Press, Beijing, China, 2003.

Download Full-text

First Report of Cladosporium Leaf Spot of Spinach Caused by Cladosporium variabile in North Carolina

Plant Disease ◽

10.1094/pdis-05-14-0474-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1741-1741

Author(s):

A. C. Scruggs ◽

S. C. Butler ◽

L. M. Quesada-Ocampo

Keyword(s):

North Carolina ◽

Leaf Spot ◽

Fungal Growth ◽

The United States ◽

Hot Water ◽

Economic Losses ◽

Fluorescent Light ◽

Single Spore ◽

First Report ◽

Infected Seed

Cladosporium leaf spot of spinach, caused by Cladosporium variabile, can result in significant economic losses in the United States (2). In March 2014, symptoms consistent with Cladosporium leaf spot (4) appeared on the spinach cultivar Tyee in a greenhouse located in Rowan County, NC. Of 1,080 spinach plants, 90 to 100% were infected. Symptoms consisted of small (1 to 3 mm in diameter), circular, tan lesions each outlined with a dark margin on the adaxial surface of the leaf. On severely infected foliage, lesions coalesced to produce relatively large necrotic regions. Profuse fungal sporulation was observed on the lesion surface with a dissecting microscope at 40× magnification. Using a dissecting microscope, conidia were collected with a sterile needle and transferred to petri plates containing potato dextrose agar. Plates were then incubated at 23 ± 2°C under continuous fluorescent light, and fungal growth was apparent after 24 h. Isolations from leaves of six infected plants produced slow-growing, dark green to brown fungal colonies that reached only 31 mm in diameter after 14 days, which is characteristic of C. variabile (4). Colonies contained dense masses of dematiaceous, septate, unbranched conidiophores with conidial chains, each containing up to five conidia. Conidia were ovate to elongate, with some being septate. The length of individual conidia ranged from 10 to 19 μm. Conidial septa were distinctly dark when observed at 100× magnification, which is a defining feature of C. variabile vs. the conidia of C. macrocarpum (4). The surface of the conidia appeared verrucose at 100× magnification, and conidia were each distinctly darkened toward the base. A single isolate obtained through single-spore transfer was used for DNA extraction, and the histone 3 (H3) gene sequence was amplified using the primers CYLH3F and CYLH3R (1). Sequence analysis of the amplified product using BLAST analysis indicated that the H3 sequences had 100% identity to that of a C. variabile isolate (GenBank Accession No. EF679710.1), and 99% identity to a C. macrocarpum isolate (EF679687.1). The H3 sequence from a representative isolate was deposited in GenBank (KJ769146). To our knowledge, this is the first report of Cladosporium leaf spot on spinach in North Carolina based on morphological evaluation and H3 sequencing results. C. variabile is a seedborne pathogen, so it is possible inoculum was introduced into the greenhouses in North Carolina on infected seed (3). Seeds can be treated with hot water or chlorine to reduce the risk of disease outbreaks caused by infected seed (2). Furthermore, Cladosporium leaf spot may be controlled with the use of fungicides (3). References: (1) P. Crous et al. Stud. Mycol. 50:415, 2004. (2) L. J. du Toit and P. Hernandez-Perez. Plant Dis. 89:1305, 2005. (3) L. J. du Toit et al. Fung. Nemat. Tests 59:V115, 2004. (4) Schubert et al. Stud. Mycol. 58:105, 2007.

Download Full-text

First Report of Alternaria Leaf Spot of Banana Caused by Alternaria alternata in the United States

Plant Disease ◽

10.1094/pdis-01-13-0007-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1116-1116 ◽

Cited By ~ 3

Author(s):

V. Parkunan ◽

S. Li ◽

E. G. Fonsah ◽

P. Ji

Keyword(s):

United States ◽

Alternaria Alternata ◽

Leaf Spot ◽

Morphological Characteristics ◽

The United States ◽

Its Sequencing ◽

Single Spore ◽

First Report ◽

Alternaria Leaf Spot ◽

Leaf Spots

Research efforts were initiated in 2003 to identify and introduce banana (Musa spp.) cultivars suitable for production in Georgia (1). Selected cultivars have been evaluated since 2009 in Tifton Banana Garden, Tifton, GA, comprising of cold hardy, short cycle, and ornamental types. In spring and summer of 2012, 7 out of 13 cultivars (African Red, Blue Torres Island, Cacambou, Chinese Cavendish, Novaria, Raja Puri, and Veinte Cohol) showed tiny, oval (0.5 to 1.0 mm long and 0.3 to 0.9 mm wide), light to dark brown spots on the adaxial surface of the leaves. Spots were more concentrated along the midrib than the rest of the leaf and occurred on all except the newly emerged leaves. Leaf spots did not expand much in size, but the numbers approximately doubled during the season. Disease incidences on the seven cultivars ranged from 10 to 63% (10% on Blue Torres Island and 63% on Novaria), with an average of 35% when a total of 52 plants were evaluated. Six cultivars including Belle, Ice Cream, Dwarf Namwah, Kandarian, Praying Hands, and Saba did not show any spots. Tissue from infected leaves of the seven cultivars were surface sterilized with 0.5% NaOCl, plated onto potato dextrose agar (PDA) media and incubated at 25°C in the dark for 5 days. The plates were then incubated at room temperature (23 ± 2°C) under a 12-hour photoperiod for 3 days. Grayish black colonies developed from all the samples, which were further identified as Alternaria spp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 23 to 73 μm long and 15 to 35 μm wide, with a beak length of 5 to 10 μm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures of four isolates from four different cultivars were obtained and genomic DNA was extracted and the internal transcribed spacer (ITS1-5.8S-ITS2) regions of rDNA (562 bp) were amplified and sequenced with primers ITS1 and ITS4. MegaBLAST analysis of the four sequences showed that they were 100% identical to two Alternaria alternata isolates (GQ916545 and GQ169766). ITS sequence of a representative isolate VCT1FT1 from cv. Veinte Cohol was submitted to GenBank (JX985742). Pathogenicity assay was conducted using 1-month-old banana plants (cv. Veinte Cohol) grown in pots under greenhouse conditions (25 to 27°C). Three plants were spray inoculated with the isolate VCT1FT1 (100 ml suspension per plant containing 105 spores per ml) and incubated under 100% humidity for 2 days and then kept in the greenhouse. Three plants sprayed with water were used as a control. Leaf spots identical to those observed in the field were developed in a week on the inoculated plants but not on the non-inoculated control. The fungus was reisolated from the inoculated plants and the identity was confirmed by morphological characteristics and ITS sequencing. To our knowledge, this is the first report of Alternaria leaf spot caused by A. alternata on banana in the United States. Occurrence of the disease on some banana cultivars in Georgia provides useful information to potential producers, and the cultivars that were observed to be resistant to the disease may be more suitable for production. References: (1) E. G. Fonsah et al. J. Food Distrib. Res. 37:2, 2006. (2) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

Download Full-text

First Report of Ramularia coleosporii causing Leaf Spot on Perilla frutescens in Korea

Plant Disease ◽

10.1094/pdis-01-21-0223-pdn ◽

2021 ◽

Author(s):

Md Aktaruzzaman ◽

Tania Afroz ◽

Hyo-Won Choi ◽

Byung Sup Kim

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Rust Fungus ◽

Morphological Characteristics ◽

Perilla Frutescens ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Perilla (Perilla frutescens var. japonica), a member of the family Labiatae, is an annual herbaceous plant native to Asia. Its fresh leaves are directly consumed and its seeds are used for cooking oil. In July 2018, leaf spots symptoms were observed in an experimental field at Gangneung-Wonju National University, Gangneung, Gangwon province, Korea. Approximately 30% of the perilla plants growing in an area of about 0.1 ha were affected. Small, circular to oval, necrotic spots with yellow borders were scattered across upper leaves. Masses of white spores were observed on the leaf underside. Ten small pieces of tissue were removed from the lesion margins of the lesions, surface disinfected with NaOCl (1% v/v) for 30 s, and then rinsed three times with distilled water for 60 s. The tissue pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Five single spore isolates were obtained and cultured on PDA. The fungus was slow-growing and produced 30-50 mm diameter, whitish colonies on PDA when incubated at 25ºC for 15 days. Conidia (n= 50) ranged from 5.5 to 21.3 × 3.5 to 5.8 μm, were catenate, in simple or branched chains, ellipsoid-ovoid, fusiform, and old conidia sometimes had 1 to 3 conspicuous hila. Conidiophores (n= 10) were 21.3 to 125.8 × 1.3 to 3.6 μm in size, unbranched, straight or flexuous, and hyaline. The morphological characteristics of five isolates were similar. Morphological characteristics were consistent with those described for Ramularia coleosporii (Braun, 1998). Two representative isolates (PLS 001 & PLS003) were deposited in the Korean Agricultural Culture Collection (KACC48670 & KACC 48671). For molecular identification, a multi-locus sequence analysis was conducted. The internal transcribed spacer (ITS) regions of the rDNA, partial actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using primer sets ITS1/4, ACT-512F/ACT-783R and gpd1/gpd2, respectively (Videira et al. 2016). Sequences obtained from each of the three loci for isolate PLS001 and PLS003 were deposited in GenBank with accession numbers MH974744, MW470869 (ITS); MW470867, MW470870 (ACT); and MW470868, MW470871 (GAPDH), respectively. Sequences for all three genes exhibited 100% identity with R. coleosporii, GenBank accession nos. GU214692 (ITS), KX287643 (ACT), and 288200 (GAPDH) for both isolates. A multi-locus phylogenetic tree, constructed by the neighbor-joining method with closely related reference sequences downloaded from the GenBank database and these two isolates demonstrated alignment with R. coleosporii. To confirm pathogenicity, 150 mL of a conidial suspension (2 × 105 spores per mL) was sprayed on five, 45 days old perilla plants. An additional five plants, to serve as controls, were sprayed with sterile water. All plants were placed in a humidity chamber (>90% relative humidity) at 25°C for 48 h after inoculation and then placed in a greenhouse at 22/28°C (night/day). After 15 days leaf spot symptoms, similar to the original symptoms, developed on the leaves of the inoculated plants, whereas the control plants remained symptomless. The pathogenicity test was repeated twice with similar results. A fungus was re-isolated from the leaf lesions on the inoculated plants which exhibited the same morphological characteristics as the original isolates, fulfilling Koch’s postulates. R. coleosporii has been reported as a hyperparasite on the rust fungus Coleosporium plumeriae in India & Thailand and also as a pathogen infecting leaves of Campanula rapunculoides in Armenia, Clematis gouriana in Taiwan, Ipomoea batatas in Puerto Rico, and Perilla frutescens var. acuta in China (Baiswar et al. 2015; Farr and Rossman 2021). To the best of our knowledge, this is the first report of R. coleosporii causing leaf spot on P. frutescens var. japonica in Korea. This disease poses a threat to production and management strategies to minimize leaf spot should be developed.

Download Full-text

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

Plant Disease ◽

10.1094/pdis-03-12-0261-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 138-138 ◽

Cited By ~ 1

Author(s):

B. Z. Fu ◽

M. Yang ◽

G. Y. Li ◽

J. R. Wu ◽

J. Z. Zhang ◽

...

Keyword(s):

Leaf Spot ◽

Colletotrichum Gloeosporioides ◽

Disease Incidence ◽

Phylogenetic Analyses ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease ◽

Rdna Its ◽

Its Sequence Analysis

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

Download Full-text