First Report of Zonate Leaf Spot of Vigna vexillata var. tsusimensis Caused by Cristulariella moricola

A zonate leaf spot disease on a wild bean variety, Vigna vexillata L. var. tsusimensis Mat., occurred in the patch fields and foothills of Chungnam and Kyonggi districts in Korea during late September, October, and early November of 1999 to 2001. The zonate lesions were particularly prevalent in October following periods of heavy dew accumulation. Initial symptoms were small, circular lesions with darkbrown marginal rings that later developed into large spots with characteristic target-shaped rings. The spots were gray to bright or blackish brown, progressed rapidly, and sometimes fused together to form lesions of up to 20 mm in diameter. Sporophores on the natural host were generally hypophyllous but sometimes amphigenous, abundant on large spots, fewer on small spots, solitary, erect, easily detachable, and up to 864 μm long. The upper portion of the sporophore is considered an individual conidium and consisted of a pyramidal head that was fusiform to ventricose and cristulate, 495 to 534 μm long, and 210 to 290 μm wide at the broadest point. Branches within the pyramidal head were short and compact, and dichotomously or trichotomously branched. The central axis within the conidium was hyaline, broad, septate, tapering toward an acute apex, and sometimes constricted at the basal septum. Conidiophores were 272 to 330 μm long and up to 24 μm wide. The fungus was identified as Cristulariella moricola (Hino) Redhead based on morphological characteristics (1,2). The fungus was isolated from Vigna leaf spots, placed on 2% water agar or potato dextrose agar (PDA), and maintained on PDA amended with 2% Vigna leaf extract. For pathogenicity tests, 4- to 5-week-old leaves of V. vexillata var. tsusimensis were surface-sterilized in 1% NaOCl. Agar disks (approximately 10 mm diameter) containing mycelia of the fungus were placed on the upper leaf surface. The inoculated plants (two leaflets per plant × 2) were then sprayed with distilled water, covered with premoistened polyethylene bags, and incubated at 15 to 25°C. Within 5 days, small leaf spots appeared that were similar to those originally observed on all inoculated leaflets. Uninoculated control leaves exposed to the same environmental conditions remained healthy. C. moricola was consistently reisolated from the infected leaves. The hyphomycete fungus C. moricola has been known to cause a bull's eye or zonate leaf spot and defoliation on woody and annual plants, including at least 73 host species and 36 families distributed in the central and eastern United States and Japan (1). In Asia, the occurrence of Cristulariella spp. on several hosts has been reported only in Taiwan and Japan (3,4). No species in the genus has ever been reported from Korea. To our knowledge, V. vexillata var. tsusimensis represents a previously unreported host for C. moricola. References: (1) M. C. Niedbalski et al. Mycologia 75:988, 1983. (2) S. A. Redhead. Mycologia 71:1248, 1979. (3). H. J. Su and S. C. Leu. Plant Dis. 67:915, 1983. (4) T. Yokoyama and K. Tubaki. Trans. Mycol. Soc. Jpn. 15:189, 1974.

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First Report of Zonate Leaf Spot of Artocarpus altilis Caused by Cristulariella moricola in Taiwan

Plant Disease ◽

10.1094/pdis.2002.86.10.1179a ◽

2002 ◽

Vol 86 (10) ◽

pp. 1179-1179 ◽

Cited By ~ 1

Author(s):

B. Y. Hu ◽

W. W. Hsiao ◽

C. H. Fu

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Eastern United States ◽

First Report ◽

Artocarpus Altilis ◽

Leaf Spots ◽

Plastic Bags ◽

Basal Septum ◽

Bull's Eye ◽

Northern Taiwan

Breadfruit (Artocarpus altilis (Parkinson) Fosberg) is an important landscape and garden tree in Taiwan. During the spring of 2002, zonate leaf spots of breadfruit were observed at a Taipei nursery in northern Taiwan. Initially, several small, brown, zonate lesions developed on leaves. As lesions enlarged, they coalesced, leading to blighting of leaves and premature defoliation. Sporophores on the host were generally hypophyllous but sometimes amphigenous, solitary, erect, easily detachable, and as much as 850 μm long. The upper portion of the sporophore is considered an individual conidium and consisted of a pyramidal head that was fusiform to ventricose and cristulate, 495 to 534 μm long and 210 to 290 μm wide at the broadest point. Branches within the pyramidal head were short and compact, and dichotomously or trichotomously branched. The conidia were hyaline, broad, septate, tapering toward an acute apex, and sometimes constricted at the basal septum. Conidiophores were 400 to 680 × 20 to 100 μm. The fungus was isolated from infected tissue and maintained on potato dextrose agar (PDA). Sclerotia were produced on PDA after 4 to 5 weeks at 20°C without light, but conidia were not observed in culture. The fungus was identified as Cristulariella moricola (Hino) Redhead based on morphological characteristics (1,2). To complete Koch's postulates, three sporophores from infected leaves or three sclerotia from cultures were placed individually on each of 10 breadfruit leaves. The plants were placed in plastic bags and incubated at 16 to 20°C. Symptoms were observed after 2 to 3 days on 100% of plants inoculated with sporophores and after 6 days on 50% of plants inoculated with sclerotia. The pathogen was reisolated from lesions on plants inoculated with sporophores and sclerotia. No symptoms were observed on the control plants. C. moricola has been known to cause a bull's eye or zonate leaf spot and defoliation on woody and annual plants, including at least 51 host species and 36 families distributed in the central and eastern United States (1). To our knowledge, this is the first report of zonate leaf spot and defoliation of breadfruit caused by C. moricola. References: (1) T. T. Chang. Bull. Taiwan For. Res. Inst. New Ser. 10 (2):235, 1995. (2) S. A. Redhead. Can. J. Bot. 53:700, 1975.

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First Report of Zonate Leaf Spot of Cinnamomum kanehirae Caused by Hinomyces moricola in Taiwan

Plant Disease ◽

10.1094/pdis-03-12-0252-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1226-1226

Author(s):

C. H. Fu ◽

F. Y. Lin

Keyword(s):

Morphological Characteristics ◽

Pathogenicity Test ◽

Annual Plants ◽

Eastern United States ◽

Transparent Plastic ◽

Leaf Spots ◽

Plastic Bags ◽

Initial Symptoms ◽

Medicinal Fungi ◽

Basal Septum

Cinnamomum kanehirae, a native tree of Taiwan, is an important tree that hosts popular medicinal fungi. In the winter of 2011, zonate leaf spots were observed at a nursery garden in Wu-Lai, Taiwan. Initial symptoms included small brown lesions on leaves that became larger leaf spots after expanding or fusing together, causing a leaf blight and eventually defoliation. Sporophores on the host were generally hypophyllous but sometimes amphigenous, solitary, erect, easily detachable. The upper portion of the sporophore was considered an individual conidium and consisted of a pyramidal head that was fusiform to ventricose, 320 to 580 μm long and 100 to 130 μm wide at the broadest point. Branches within the pyramidal head were short and compact, and dichotomously or trichotomously branched. The sporophore initials were hyaline, broad, septate, tapering toward an acute apex, and sometimes constricted at the basal septum. Sclerotia were observed in older lesions, grey or black, spherical, and 1 to 2.5 mm in diameter. The fungus was isolated from infected tissue and sporophores, maintained on potato dextrose agar (PDA) at 20°C in darkness. Sclerotia were produced on PDA after 4 to 5 weeks and were irregular or spherical, but sporophores didn't develop on agar medium. The fungus was identified as Hinomyces moricola on the basis of morphological characteristics (1). Koch's postulates were performed by inoculating four 1-year-old, asymptomatic, potted C. kanehirae plants; every plant was inoculated with sporophores from infected leaves on each of five leaves. Four noninoculated plants were kept in separate pots and served as controls. All plants were covered with transparent plastic bags individually and incubated in a growth chamber at 18 to 20°C. Symptoms were observed after 2 to 4 days on every inoculated plant but not on uninoculated plants. The leaf spots were similar to those originally observed. The pathogen was reisolated from spots of inoculated plants. The pathogenicity test was repeated once. H. moricola is known to cause severe defoliation on woody and annual plants, including at least 73 host species and 36 families distributed in the eastern United States and Japan (2). References: (1) N.-S. Tomoko et al. Mycoscience. 47:351, 2006. (2) J. C. Trolinger et al. Plant Dis. Reptr. 62:710, 1978.

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

Plant Disease ◽

10.1094/pdis.2003.87.2.203a ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203

Author(s):

S. T. Koike ◽

S. A. Tjosvold ◽

J. Z. Groenewald ◽

P. W. Crous

Keyword(s):

Leaf Spot ◽

Sequence Data ◽

Disease Incidence ◽

Leaf Spot Disease ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Initial Symptoms

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

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First Report of Leaf Spot 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 Leaf Spot Caused by Alternaria alternata on Kiwifruit in Italy

Plant Disease ◽

10.1094/pdis.1999.83.5.487d ◽

1999 ◽

Vol 83 (5) ◽

pp. 487-487 ◽

Cited By ~ 3

Author(s):

L. Corazza ◽

L. Luongo ◽

M. Parisi

Keyword(s):

New Zealand ◽

Alternaria Alternata ◽

Leaf Spot ◽

Southern Italy ◽

Morphological Characteristics ◽

Potato Dextrose Agar ◽

First Report ◽

Leaf Spots ◽

Detached Leaves ◽

Pathogenicity Tests

A leaf spot of kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferg.) leaves was recently observed on plants of the cultivar Hayward in an orchard near Salerno, in southern Italy. The affected plants showed early severe defoliation. The fungus isolated from the infected leaves was identified as Alternaria alternata (Fr.:Fr.) Keissl., based on conidial morphological characteristics. Pathogenicity tests were made by inoculating detached leaves of male pollinator cultivar Tomuri and the female cultivars Hayward and Bruno with a 7-mm disk taken from actively growing cultures of the fungus on potato dextrose agar (PDA). After 14 days, necrotic leaf spots developed and A. alternata was consistently isolated from the inoculated leaves. A. alternata has been observed as a pathogen on leaves and fruits in New Zealand. In the Mediterranean, it has been reported in Israel (2) and in the island of Crete (1). This is the first report of Alternaria leaf spot on kiwifruit in Italy. References: (1) V. A. Bourbos and M. T. Skoudridakis. Petria 7:111, 1997. (2) A. Sive and D. Resnizky. Alon Hanotea 41:409, 1987.

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Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China

Plant Disease ◽

10.1094/pdis-10-20-2287-pdn ◽

2021 ◽

Author(s):

Dongli Liu ◽

Jing Li ◽

Saisai Zhang ◽

Xiangjing Wang ◽

Wensheng Xiang ◽

...

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Its Sequence ◽

Conidial Suspension ◽

Orychophragmus Violaceus ◽

Disease Symptoms ◽

Alternaria Tenuissima ◽

Spot Disease ◽

Pathogenicity Tests

Orychophragmus violaceus (L.) O. E. Schulz, also called February orchid or Chinese violet cress, belongs to the Brassicaceae family and is widely cultivated as a green manure and garden plant in China. During the prolonged rainy period in August 2020, leaf spot disease of O. violaceus was observed in the garden of Northeast Agricultural University, Harbin, Heilongjiang province. One week after the rainy days, the disease became more serious and the disease incidence ultimately reached approximately 80%. The disease symptoms began as small brown spots on the leaves, and gradually expanded to irregular or circular spots. As the disease progressed, spots became withered with grayish-white centers and surrounded by dark brown margins. Later on, the centers collapsed into holes. For severely affected plants, the spots coalesced into large necrotic areas and resulted in premature defoliation. No conidiophores or hyphae were present, and disease symptoms were not observed on other tissues of O. violaceus. To isolate the pathogen, ten leaves with typical symptoms were collected from different individual plants. Small square leaf pieces (5×5 mm) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol solution for 1 min, rinsed in sterile distilled water, and then transferred to Petri dishes (9 cm in diameter) containing potato dextrose agar (PDA). After 3 days of incubation at 25 oC in darkness, newly grown-out mycelia were transferred onto fresh PDA and purified by single-spore isolation. Nine fungal isolates (NEAU-1 ~ NEAU-9) showing similar morphological characteristics were obtained and no other fungi were isolated. The isolation frequency from the leaves was almost 90%. On PDA plates, all colonies were grey-white with loose and cottony aerial hyphae, and then turned olive-green and eventually brown with grey-white margins. The fungus formed pale brown conidiophores with sparsely branched chains on potato carrot agar (PCA) plates after incubation at 25 oC in darkness for 7 days. Conidia were ellipsoidal or ovoid, light brown, and ranged from 18.4 to 59.1 × 9.2 to 22.3 µm in size, with zero to two longitudinal septa and one to five transverse septa and with a cylindrical light brown beak (n = 50). Based on the cultural and morphological characteristics, the fungus was tentatively identified as Alternaria tenuissima (Simmons 2007). Genomic DNA was extracted from the mycelia of five selected isolates (NEAU-1 ~ NEAU-5). The internal transcribed spacer region (ITS) was amplified and sequenced using primers ITS1/ITS4 (White et al., 1990). Blast analysis demonstrated that these five isolates had the same ITS sequence, and the ITS sequence of representative strain NEAU-5 (GenBank accession No. MW139354) showed 100% identity with the type strains of Alternaria alternata CBS916.96 and Alternaria tenuissima CBS918.96. Furthermore, the translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (GPD) of representative strain NEAU-5 were amplified and sequenced using primers EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-5R (Sung et al., 2007), and Gpd1/Gpd2 (Berbee et al., 1999), respectively. The sequences of RPB2, GPD, and TEF of strain NEAU-5 were submitted to GenBank with accession numbers of MW401634, MW165223, and MW165221, respectively. Phylogenetic trees based on ITS, RPB2, GPD, and TEF were constructed with the neighbour-joining and maximum-likelihood algorithms using MEGA software version 7.0. The results demonstrated that strain NEAU-5 formed a robust clade with A. tenuissima CBS918.96 (supported by 99% and 96% bootstrap values) in the neighbour-joining and maximum-likelihood trees. As mentioned above, strain NEAU-5 produced seldomly branched conidial chains on PCA plates. The pattern is consistent with that of A. tenuissima (Kunze) Wiltshire, but distinct from that of A. alternata which could produce abundant secondary ramification (Simmons 2007). Thus, strain NEAU-5 was identified as A. tenuissima based on its morphology and phylogeny. Pathogenicity tests were carried out by inoculating five unwounded leaves with a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) on five different healthy plants cultivated in garden, and an equal number of leaves on the same plants inoculated with sterilized ddH2O served as negative controls. Inoculated and control leaves were covered with clear plastic bags for 3 days. After 6 days, small brown and irregular or circular spots were observed on all leaves inoculated with conidial suspension, while no such symptoms were observed in the control. The tests were repeated three times. Furthermore, the pathogenicity tests were also performed using 2-month-old potted plants in a growth chamber (28 oC, 90% relative humidity, 12 h/12 h light/dark) with two repetitions. Five healthy plants were inoculated by spraying 20 ml of a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) onto unwounded leaves. Five other healthy plants were inoculated with sterilized ddH2O as controls. After 7 days, similar symptoms were observed on leaves inoculated with strain NEAU-5, whereas no symptoms were observed in the control. The pathogen was reisolated from the inoculated leaves and identified as A. tenuissima by morphological and molecular methods. In all pathogenicity tests, A. tenuissima could successfully infect unwounded leaves of O. violaceus, indicating a direct interaction between leaves and A. tenuissima. It is known that high humidity and fairly high temperatures can favor the epidemics of Alternaria leaf spot (Yang et al., 2018), and this may explain why severe leaf spot disease of O. violaceus was observed after prolonged rain. Previously, it has been reported that Alternaria brassicicola and Alternaria japonica could cause leaf blight and spot disease on O. violaceus in Hebei and Jiangsu Provinces, China, respectively (Guo et al., 2019; Sein et al., 2020). Although these pathogens could lead to similar disease symptoms on the leaves of O. violaceus, it is easy to distinguish them by the morphological characteristics of conidiophores and ITS gene sequences. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease of O. violaceus in China.

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First Report of Leaf Spot Caused by Cylindrocladium pauciramosum on Dwarf Willow Myrtle in Italy

Plant Disease ◽

10.1094/pdis-94-2-0274c ◽

2010 ◽

Vol 94 (2) ◽

pp. 274-274 ◽

Cited By ~ 2

Author(s):

G. Polizzi ◽

D. Aiello ◽

G. Parlavecchio ◽

A. Vitale ◽

F. Nigro

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Intergenic Spacer ◽

First Record ◽

Morphological Characters ◽

Leaf Spot Disease ◽

Centraalbureau Voor ◽

Potted Plants ◽

Leaf Spots ◽

Plant Pathol

Dwarf willow myrtle (Agonis flexuosa (Willd.) Sweet) cv. Nana, an evergreen ornamental shrub belonging to the Myrtaceae, is grown in Italy as an ornamental potted plant. In November 2008, a widespread new leaf spot disease was noticed on ~80% of 5,000 6-month-old potted plants. Plants were obtained from cuttings and produced by a commercial nursery in Catania Province. Symptomatic leaves showed minute, reddish brown spots that enlarged (3 to 5 mm in diameter) and then darkened, presenting a necrotic center defined by a dark purple halo. Leaf spots were surface disinfested with 0.8% NaOCl and plated on potato dextrose agar. Twenty isolates of the fungus that was consistently isolated from the spots were selected and cultured for 8 days at 25°C on carnation leaf agar (CLA). Macroconidiophores consisted of a stipe, a penicillate arrangement of fertile branches, and stipe extension terminating in an obpyriform to ellipsoidal vesicle (6 to 10 μm in diameter). Cylindrical conidia were rounded at both ends, straight, one-septate, and ranged from 44 to 60 × 4 to 5 μm. The fungus was tentatively identified as Cylindrocladium pauciramosum based on these morphological characteristics (2). All single-conidium isolates were mated with tester strains of Calonectria pauciramosa C. L. Schoch & Crous, telomorph of C. pauciramosum, on CLA and produced fertile perithecia (4). Perithecia were solitary or in groups, orange to red-brown, subglobose to ovoid, and ranged from 280 to 400 μm long × 180 to 290 μm in diameter. Further confirmation of species was obtained by amplification and sequencing of the intergenic spacer (IGS) region of rDNA, using M13 Forward (-20) and M13 Reverse primers. On the basis of the complete IGS sequence, two primer sets (218F/218R and 106F/106R) were designed and successfully used in a nested-PCR protocol for the detection of C. pauciramosum from tissues of infected plants (3). On the basis of the combination of morphological characters, mating type, and molecular data, the isolates were identified as C. pauciramosum C.L. Schoch & Crous. One representative isolate (DISTEF-Af1) was deposited at Centraalbureau voor Schimmelcultures open fungi collection (Fungal Biodiversity Centre, Utrecht, the Netherlands; CBS 124659). Pathogenicity tests were performed by adding sterile water to CLA cultures of C. pauciramosum from a single-conidium isolate (DISTEF-Af1) and spraying the resulting spore suspension (105 conidia per ml) on the leaf surface of 20 6-month-old A. flexuosa cv. Nana potted plants. The same number of plants served as noninoculated controls. Following inoculation, plants were kept in plastic bags in a growth chamber at 25 ± 1°C. All inoculated plants developed circular, brown leaf spots identical to those observed in the nursery 5 to 7 days after inoculation. Control plants remained symptomless. C. pauciramosum was always reisolated from the infected plants and identified as previously described. Leaf spotting in seedlings of A. flexuosa was previously associated with infections by C. scoparium in Australia (1). To our knowledge, this is the first record in the world of leaf spots caused by C. pauciramosum on A. flexuosa. References: (1) A. L. Bertus. Agric. Gaz. N. S. W. 87:22, 1976. (2) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul MN, 2002. (3) F. Nigro et al. J. Plant Pathol. 88:S22, 2006. (4) G. Polizzi and P. W. Crous. Eur. J. Plant Pathol. 105:407, 1999.

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

Plant Disease ◽

10.1094/pdis-09-20-1974-pdn ◽

2020 ◽

Author(s):

Lv-Yin Zhou ◽

Shuang-Feng Yang ◽

Shao-Mei Wang ◽

Jing-Wen Lv ◽

Wei Qiang Wan ◽

...

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Post Inoculation ◽

Sodium Hypochlorite Solution ◽

Transparent Plastic ◽

Bletilla Striata ◽

Spot Disease ◽

Initial Symptoms

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

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First Report of Corynespora cassiicola Causing Leaf Spot on Akebia trifoliate

Plant Disease ◽

10.1094/pdis-04-13-0454-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1659-1659 ◽

Cited By ~ 2

Author(s):

Y. F. Ye ◽

N. Jiang ◽

G. Fu ◽

W. Liu ◽

F. Y. Hu ◽

...

Keyword(s):

Leaf Spot ◽

Sequence Similarity ◽

Yangtze River Basin ◽

Morphological Characteristics ◽

Colletotrichum Acutatum ◽

Leaf Spot Disease ◽

Corynespora Cassiicola ◽

High Sequence Similarity ◽

First Report ◽

Initial Symptoms

Akebia species have been used for centuries in medicinal practices in a few Asian countries such as China and Japan. The dried stems of Akebia trifoliata are known as mutong in the Chinese pharmacopoeia (4) and mokutsu in Kampo, the traditional Chinese medicine developed in Japan (2). In China, the plant is grown in the provinces of Shandong, Hebei, Shanxi, Henan, Gansu, and some provinces in the south of the Yangtze River basin. During the summer of 2012, a leaf spot disease was detected on A. trifoliata grown in Nanning, Guangxi, China. The disease occurred and spread rapidly in July under conditions of high temperature and high humidity. The symptoms appeared on three sites that we inspected; disease incidences were higher than 80%. Initial symptoms consisted of small (less than 5 mm in diameter), circular, purple-brown leaf spots. Spots later enlarged and became elliptical, circular, or irregular with gray-white centers and dark brown rims. The centers were slightly concave. The spots could coalesce with each other, resulting in leaf desiccation and wilting. A fungal isolate was obtained from symptomatic leaf tissue that taken from a field (22°50′N, 108°22′E) in Nanning, Guangxi, China. Single-spore culture of the isolate was incubated on potato dextrose agar (PDA) for 7 days in the dark at 28°C. Conidiophores were straight to slightly curved, unbranched, and pale brown. Conidia (19.0 to 140.5 μm long and 7.0 to 11.0 μm wide) were formed singly or in chains, obclavate to cylindrical, straight or curved, pale brown, with a rounded apex and truncate base, and 1 to 13 pseudosepta. Morphological characteristics of the isolate were similar to the descriptions of Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei (1). Genomic DNA of the isolate was extracted and used for PCR amplification of rDNA-ITS (internal transcribed spacer) sequence with primers ITS1 and ITS4. The PCR products were purified and sequenced. The sequence (GenBank Accession No. KC977496) was used in BLAST searches to interrogate GenBank for sequence similarity. High sequence similarity of 100% was obtained with several C. cassiicola strains. Pathogenicity of the isolate was investigated to demonstrate Koch's postulate. Young, healthy, fully expanded green leaves of A. trifoliata were surface sterilized. Fifteen leaves were inoculated with 10-μl drops of conidia suspension (105 conidia per ml) and 10 leaves were inoculated with the same volume of sterile water to serve as controls. All the leaves were placed in a humid chamber for 5 days. Spots with similar symptoms to those observed in the field developed on all inoculated leaves. The pathogen was reisolated and identified as C. cassiicola. The controls remained symptomless. According to previous reports, A. trifoliata was infected by Alternaria tenuissima in China and by Colletotrichum acutatum in Japan (3). To our knowledge, this is the first report of C. cassiicola found on Akebia species worldwide. Furthermore, this new disease primarily affects plantations and reduces the quality and yield of the medicine. Some effective measures should be taken to control this disease. References: (1) M. B. Ellis and P. Holliday. CMI Description of Pathogenic Fungi and Bacteria, 303, 1971. (2) F. Kitaoka et al. J. Nat. Med. 63:374, 2009. (3) Y. Kobayshi et al. J. Gen. Plant Pathol. 70:295, 2004. (4) L. Li et al. HortScience 45:4, 2010.

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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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