First report of a new disease caused by Fusarium tricinctum on apple tree in China

Wilting of branches and leaves was observed on 4-5 year old apple trees of the varieties Delicious and Fuji in orchards located in Wushan, Gansu Province, China in April 2018. Subsequently, the stem vascular tissue and woody xylem became discolored and necrotic. The stem dieback expanded rapidly to the entire vasculature of the branches. Finally, the epidermis of the stem bases split and was covered with light pink mold. For the pathogen isolation, 25 symptomatic stems were collected from 25 symptomatic trees in 3 individual orchards. Fragments (approximately 0.5 cm in length × 0.5 cm in width) of symptomatic stems were surface sterilized and individually transferred to Petri dishes containing potato dextrose agar (PDA), and incubated for 4 days at 25°C. Five types of isolates with distinct morphological characteristics (PJ1 to PJ5) were obtained from the 25 symptomatic stems after the single spore inoculation and sub-culture. The isolation frequency of PJ1, PJ2, PJ3, PJ4 and PJ5 types was 11%, 8%, 100%, 4% and 13%, respectively, in the 25 symptomatic stems. A spore suspension of PJ1, PJ2, PJ3, PJ4 and PJ5 types was prepared by adding 5 ml of sterile distilled water in the 14-day old culture colonies and filtered through 0.22 mm Millipore membranes, and the final concentration was adjusted to 108 per ml for inoculation. Detached healthy apple stems (15 cm in length) were surface-disinfested and used to evaluate the pathogenicity of PJ1 (7 isolates), PJ2 (5 isolates), PJ3 (32 isolates), PJ4 (2 isolates) and PJ5 (9 isolates) by dipping the stems into sterilised tubs containing the spore suspension (108 per ml) of each isolate. Apple stems dipped in sterile distilled water served as the control. Each control and treatment were repeated 3 times. At day 15 and 35, the stems infected with the spore suspension of PJ3 isolates developed symptoms that were similar to those observed in the apple orchards. However, the other four types (PJ1, PJ2, PJ4 and PJ5) exhibited either no symptoms or different symptoms from those observed in the apple orchards. There were no symptoms on the control stems. After the colony of the pathogen (PJ3 type) was re-isolated from the infected stem bases 35 days inoculation. The PJ3 type isolates with same morphological characteristics as the original PJ3 type isolates were used for further examination and identification. After 4 days of incubation on PDA, the colonies of PJ3 type isolates developed velvety aerial mycelia with white or light pink color when they were viewed from the front/top side of the PDA and orange-red color when they were viewed from the reverse/bottom side. After 14 days of incubation, the color in the centre of the colonies changed to yellow green in the front view and carmine red in the reverse view of the plates. Three types of spores (microconidia, macroconidia and chlamydospores) were observed after incubation of PJ3 type isolates for 14 days. The size (width and length) of 30 conidia in each of PJ3 type isolates was measured and averaged. The microconidia were abundant on aerial mycelia and limoniform, oval or pyriform with 0-1 septa. Their size ranged from 1.94 μm to 8.05 μm in length and 1.48 μm to 3.62 μm in width. The macroconidia were falciform and curved in shape, mostly with 3-5 septa and a size ranging from 13.52 μm to 22.43 μm in length and 2.31 μm to 3.87 μm in width. The chlamydospores were spherical, intercalary and formed in chains on PDA plates. These morphological characteristics indicate that the PJ3 type isolates could be Fusarium tricinctum (Chen et al. 2019; Aktaruzzaman et al. 2018). To confirm the morphological identification, the sequences of internal transcribed spacer (ITS), transcriptional enhancer factor-1 (TEF-lα) and ribosomal RNA large subunit gene (LSU) of the representative isolate PJ3-3 selected from the PJ3 type isolates with same morphological characteristics were sequenced and used for molecular identification (Laurence et al. 2011; Abd-Elsalam et al. 2003; Miller et al. 1996). The sequences of ITS, TEF-lα and LSU of the PJ3-3 isolate were deposited in NCBI database with the accession numbers of MZ799356, MZ820045 and MZ820044, respectively. In BLAST analyses, the obtained sequences of the PJ3-3 isolate showed 99.47%, 100% and 99.01% identity to the corresponding region of F. tricinctum ITS (JX179207.1: 3-566 Fusarium tricinctum isolate Fyx 1), TEF-lα (MK032320.1 F. tricinctum isolate ZD3) and LSU (KC311496.1 Fusarium tricinctum isolate L12), respectively. The phylogenetic analysis clustered the PJ3-3 isolate sequences within the same clade with ITS, TEF-lα and LSU sequences of F. tricinctum isolates. Thus, the PJ3-3 isolate was identified as F. tricinctum based on the pathogenicity tests, morphological characteristics and molecular analyses. Previously, the symptoms of xylem browning and dieback were observed in the twigs of wild apple trees that were collected from wild apple forests, and the species F. avenaceum, F. solani, F. tricinctum, F. proliferatum, and F. sporotrichioides were isolated from diseased wild apple trees (Chen et al. 2019). Only F. avenaceum, F. solani, F. proliferatum, and F. sporotrichioides were reported as the pathogens causing the disease symptoms of xylem browning and dieback in wild apple trees in Xinjiang, China (Chen et al. 2019). In our present study, we found that F. tricinctum can cause stem vascular and woody xylem browning, wilting, and dieback in the apple tree varieties Delicious and Fuji. These are new symptoms discovered in our present research and different from the previous paper (Chen et al. 2019). Therefore, to our knowledge, this study is the first report of F. tricinctum causing a new disease on apple trees in China following Koch’s postulates. Our findings are important for the management of apple disease and protect apple trees in the future.

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First Report of Wilt of Rubber Tree Caused by Chalaropsis thielavioides in China

Plant Disease ◽

10.1094/pdis-09-20-2066-pdn ◽

2020 ◽

Author(s):

Xue Li ◽

Jie Li ◽

Hua Yong Bai ◽

Kecheng Xu ◽

Ruiqi Zhang ◽

...

Keyword(s):

Rubber Tree ◽

Morphological Characteristics ◽

Spore Suspension ◽

Malt Extract ◽

Pathogenicity Test ◽

Distilled Water ◽

Ceratocystis Fimbriata ◽

First Report ◽

Mould Fungus ◽

Its Sequence Analysis

Rubber tree (Hevea brasiliensis (Willd. ex Adr. Juss) Müll. Arg.) is used for the extraction of natural rubber and is an economically and socially important estate crop commodity in many Asian countries such as Indonesia, Malaysia, Thailand, India, Sri Lanka, China and several countries in Africa (Pu et al, 2007). Xishuangbanna City and Wenshan City are the main rubber cultivation areas in Yunnan Province, China. In November 2012, rubber tree showing typical wilt symptoms (Fig. 1 A) and vascular stains (Fig. 1 B) were found in Mengla County, Xishuangbanna City. This disease was destructive in these trees and plant wilt death rate reached 5%. The diseased wood pieces (0.5cm long) from trunk of rubber was surface disinfected with 75% ethanol for 30s and 0.1% mercuric chloride (HgCl2) for 2min, rinsed three times with sterile distilled water, plated onto malt extract agar medium (MEA), and incubated at 28℃. After 7 days, fungal-like filaments were growing from the diseased trunk. Six cultures from 6 rubber trunk were obtained and incubated on MEA at 28℃, after 7 days to observe the cultural features. The mycelium of each culture was white initially on MEA, and then became dark green. Cylindrical endoconidia apices rounded, non-septate, smooth, single or borne in chains (8.9 to 23.6 × 3.81 to 6.3μm) (Fig. 1 C). Chlamydospores (Fig. 1 D) were abundant, thick walled, smooth, forming singly or in chains (11.1 to 19.2 × 9.4 to 12.0μm). The mould fungus was identifed as Chalaropsis based on morphology (Paulin-Mahady et al. 2002). PCR amplification was carried out for 3 isolates, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (Thorpe et al. 2005). The nucleotide sequences were deposited in the GenBank data base and used in a Blast search of GenBank. Blast analysis of sequenced isolates XJm8-2-6, XJm8-2 and XJm10-2-6 (accessions KJ511486, KJ511487, KJ511489 respectively) had 99% identity to Ch. thielavioides strains hy (KF356186) and C1630 (AF275491). Thus the pathogen was identified as Ch. thielavioides based on morphological characteristics and rDNA-ITS sequence analysis. Pathogenicity test of the isolate (XJm8-2) was conducted on five 1-year-old rubber seedlings. The soil of 5 rubber seedlings was inoculated by drenching with 40 ml spore suspension (106 spores / ml). Five control seedlings were inoculated with 40 ml of sterile distilled water. All the seedlings were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculated 6 weeks, all the seedlings with spore suspension produced wilt symptoms, as disease progressed, inoculated leaves withered (Fig. 1 E) and vascular stains (Fig. 1 F) by 4 months. While control seedlings inoculated with sterile distilled water remained healthy. The pathogen re-isolated from all inoculated symptomatic trunk was identical to the isolates by morphology and ITS analysis. But no pathogen was isolated from the control seedlings. The pathogenicity assay showed that Ch. thielavioides was pathogenic to rubber trees. Blight caused on rubber tree by Ceratocystis fimbriata previously in Brazil (Valdetaro et al. 2015), and wilt by Ch. thielavioides was not reported. The asexual states of most species in Ceratocystis are “chalara” or “thielaviopsis” (de Beer et al. 2014). To our knowledge, this is the first report of this fungus causing wilt of rubber in China. The spread of this disease may pose a threat to rubber production in China.

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First Report of Colletotrichum clavatum Causing Quince Anthracnose in Serbia

Plant Disease ◽

10.1094/pdis-01-14-0052-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1272-1272

Author(s):

S. Živković ◽

V. Gavrilović ◽

T. Popović ◽

N. Dolovac ◽

N. Trkulja

Keyword(s):

Management Practices ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Fruit Trees ◽

Morphological Identification ◽

Distilled Water ◽

First Report ◽

Mediterranean Countries ◽

Wide Range ◽

Plant Pathol

Quince (Cydonia oblonga Mill.) tree is traditionally grown in Serbia. The fruits are used for compote, marmalade, and brandy production. In December 2012, quince fruits cv. Leskovacka with symptoms of postharvest anthracnose were collected in a storage facility in the area of Sabac, western Serbia. The symptoms were observed on fruits approximately 2 months after harvest. The incidence of the disease was about 3%, but the symptoms were severe. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. Small pieces (3 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphology characteristics of three isolates were observed after growth on PDA for 7 days at 25°C under a 12-h photoperiod. Fungal colonies developed white to gray dense aerial mycelium with orange conidial masses in the center of the colony. Conidia were hyaline, aseptate, clavate with rounded distal apices, 15.2 (12.8 to 16.8) × 4.5 (4.0 to 5.2) μm (mean L/W ratio = 3.3, n = 100). Morphological characteristics are consistent with the description of Colletotrichum clavatum (2). Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1/IT4 primers and β-tubuline 2 gene using T1/T2 primers. The nucleotide sequences were deposited in GenBank (ITS Accession Nos. KF908866, KF908867, and KF908868; β-tubuline 2 gene KF908869, KF908870, and KF908871). BLAST analyses of ITS and β-tubuline 2 gene sequences showed that isolates from quince were 100% identical to other C. clavatum in GenBank (ITS JN121126, JN121130, JN121132, and JN121180; β-tubuline 2 gene JN121213 to 17, JN121219, JN121228, JN121261 to 62, and JN121266 to 69), thus confirming the morphological identification. To fulfill Koch's postulates, asymptomatic fruits of quince cv. Leskovacka (five fruits per isolate) were surface sterilized with 70% ethanol, wounded with a sterile needle, and inoculated with 50 μl of a spore suspension (1 × 106 conidia/ml). Five control fruits were inoculated with 50 μl of sterile distilled water. The experiment was repeated twice. After 10 days of incubation in plastic containers, under high humidity (>90% RH) at 25°C, typical anthracnose symptoms developed on inoculated fruits, while control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as original isolates. C. clavatum previously indicated as group B (3), or genetic group A4 within the C. acutatum sensu lato complex (4), is responsible for olive anthracnose in some Mediterranean countries (1,2), and has been reported as causal agent of anthracnose on a wide range of other hosts including woody and herbaceous plants, ornamentals, and fruit trees worldwide (4). To our knowledge, this is the first report of C. clavatum in Serbia, and the first report of quince anthracnose caused by this pathogen in Europe. Anthracnose caused by C. clavatum can endanger the production and storage of quince in the future, and may require investigation of new disease management practices to control this fungus. References: (1) S. O. Cacciola et al. J. Plant Pathol. 94:29, 2012. (2) R. Faedda et al. Phytopathol. Mediterr. 50:283, 2011. (3) R. Lardner et al. Mycol. Res. 103:275, 1999. (4) S. Sreenivasaprasad and P. Talhinhas. Mol. Plant Pathol. 6:361, 2005.

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First Report of Botryosphaeria dothidea Causing White Rot of Apple Fruit in Serbia

Plant Disease ◽

10.1094/pdis-05-13-0493-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1659-1659 ◽

Cited By ~ 4

Author(s):

M. Vasić ◽

N. Duduk ◽

I. Vico ◽

M. S. Ivanović

Keyword(s):

Morphological Characteristics ◽

Its Region ◽

Apple Fruit ◽

White Rot ◽

Morphological Identification ◽

Botryosphaeria Dothidea ◽

Apple Trees ◽

First Report ◽

Surface Sterilization ◽

Control Fruit

Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not has a worldwide distribution infecting species from over 80 genera of plants (1). Apart from being an important pathogen of apple trees in many countries, B. dothidea can cause pre- and postharvest decay on apple fruit (2). It has been known to cause canker and dieback of forest trees in Serbia (3), but has not been recorded either on apple trees or apple fruit. In December 2010, apple fruit cv. Idared (Malus × domestica Borkh.) with symptoms of white rot were collected from one storage in the area of Svilajnac in Serbia. The incidence of the disease was low but the symptoms were severe. Affected fruit were brown, soft, and almost completely decayed, while the internal decayed tissue appeared watery and brown. A fungus was isolated from symptomatic tissue of one fruit after surface sterilization with 70% ethanol (without rinsing) and aseptic removal of the skin. Small fragments of decayed tissue were placed on potato dextrose agar (PDA) and incubated in a chamber at 22°C under alternating light and dark conditions (12/12 h). Fungal colonies were initially whitish, but started turning dark gray to black after 5 to 6 days. Pycnidia were produced after 20 to 25 days of incubation at 22°C and contained one-celled, elliptical, hyaline conidia. Conidia were 17.19 to 23.74 μm (mean 18.93) × 3.72 to 4.93 μm (mean 4.45) (n = 50). These morphological characteristics are in accordance with those described for the fungus B. dothidea (4). Genomic DNA was isolated from the fungus and internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The nucleotide sequence has been assigned to GenBank Accession No. KC994640. BLAST analysis of the 528-bp segment showed a 100% similarity with several sequences of B. dothidea deposited in NCBI GenBank, which confirmed morphological identification. Pathogenicity was tested by wound inoculation of five surface-sterilized, mature apple fruit cv. Idared with mycelium plugs (5 mm in diameter) of the isolate grown on PDA. Five control fruit were inoculated with sterile PDA plugs. After 5 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22°C, typical symptoms of white rot developed on inoculated fruit, while wounded, uninoculated, control fruit remained symptomless. The isolate recovered from symptomatic fruit showed the same morphological features as original isolate. To the best of our knowledge, this is the first report of B. dothidea on apple fruit in Serbia. Apple is widely grown in Serbia and it is important to further investigate the presence of this pathogen in apple storage, as well as in orchards since B. dothidea may cause rapid disease outbreaks that result in severe losses. References: (1) G. H. Hapting Agriculture Handbook 386, USDA, Forest Service, 1971. (2) A. L. Jones and H. S. Aldwinckle Compendium of Apple and Pear Diseases. APS Press, St. Paul, MN, 1990. (3) D. Karadžic et al. Glasnik Šumarskog Fakulteta 83:87, 2000. (4) B. Slippers et al. Mycologia 96:83, 2004.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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First report of Coniella castaneicola causing leaf blight on blueberry (Vaccinium virgatum) in China

Plant Disease ◽

10.1094/pdis-05-21-1122-pdn ◽

2021 ◽

Author(s):

Jiahao Lai ◽

Guihong Xiong ◽

Bing Liu ◽

Weigang Kuang ◽

Shuilin Song

Keyword(s):

Molecular Identification ◽

Morphological Characteristics ◽

Leaf Blight ◽

Yield Potential ◽

Effective Control ◽

Economic Losses ◽

Distilled Water ◽

First Report ◽

Fungal Isolates ◽

Blight Disease

Blueberry (Vaccinium virgatum), an economically important small fruit crop, is characterized by its highly nutritive compounds and high content and wide diversity of bioactive compounds (Miller et al. 2019). In September 2020, an unknown leaf blight disease was observed on Rabbiteye blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E). Disease surveys were conducted at that time, the results showed that disease incidence was 90% from a sampled population of 100 plants in the field, and this disease had not been found at other cultivation fields in Nanchang. Leaf blight disease on blueberry caused the leaves to shrivel and curl, or even fall off, which hindered floral bud development and subsequent yield potential. Symptoms of the disease initially appeared as irregular brown spots (1 to 7 mm in diameter) on the leaves, subsequently coalescing to form large irregular taupe lesions (4 to 15 mm in diameter) which became curly. As the disease progressed, irregular grey-brown and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath and finally caused dieback and even shoot blight. To identify the causal agent, 15 small pieces (5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface-sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, and then incubated on potato dextrose agar (PDA) at 28°C for 5-7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were white with sparse creeping hyphae. Pycnidia were spherical, light brown, and produced numerous conidia. Conidia were 10.60 to 20.12 × 1.98 to 3.11 µm (average 15.27 × 2.52 µm, n = 100), fusiform, sickle-shaped, light brown, without septa. Based on morphological characteristics, the fungal isolates were suspected to be Coniella castaneicola (Cui 2015). To further confirm the identity of this putative pathogen, two representative isolates LGZ2 and LGZ3 were selected for molecular identification. The internal transcribed spacer region (ITS) and large subunit (LSU) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004) and LROR/LR7 (Castlebury and Rossman 2002). The sequences of ITS region (GenBank accession nos. MW672530 and MW856809) showed 100% identity with accessions numbers KF564280 (576/576 bp), MW208111 (544/544 bp), MW208112 (544/544 bp) of C. castaneicola. LSU gene sequences (GenBank accession nos. MW856810 to 11) was 99.85% (1324/1326 bp, 1329/1331 bp) identical to the sequences of C. castaneicola (KY473971, KR232683 to 84). Pathogenicity was tested on three blueberry varieties (‘Rabbiteye’, ‘Double Peak’ and ‘Pink Lemonade’), and four healthy young leaves of a potted blueberry of each variety with and without injury were inoculated with 20 μl suspension of prepared spores (106 conidia/mL) derived from 7-day-old cultures of LGZ2, respectively. In addition, four leaves of each variety with and without injury were sprayed with sterile distilled water as a control, respectively. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH greater than 80%). After 4 days, all the inoculated leaves started showing disease symptoms (large irregular grey-brown lesions) as those observed in the field and there was no difference in severity recorded between the blueberry varieties, whereas the control leaves showed no symptoms. The fungus was reisolated from the inoculated leaves and confirmed as C. castaneicola by morphological and molecular identification, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. castaneicola causing leaf blight on blueberries in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing effective control strategies, reducing economic losses in blueberry production, and promoting the development of the blueberry industry.

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First Report of Leaf Spot of Sweet Basil Caused by Cercospora guatemalensis in Korea

Plant Disease ◽

10.1094/pdis-05-12-0436-pdn ◽

2012 ◽

Vol 96 (10) ◽

pp. 1580-1580

Author(s):

J. H. Park ◽

K. S. Han ◽

J. Y. Kim ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Its Region ◽

Culture Collection ◽

Distilled Water ◽

First Report ◽

Sweet Basil ◽

Organic Farm ◽

Leaf Spots ◽

Close Phylogenetic Relationship

Sweet basil, Ocimum basilicum L., is a fragrant herb belonging to the family Lamiaceae. Originated in India 5,000 years ago, sweet basil plays a significant role in diverse cuisines across the world, especially in Asian and Italian cooking. In October 2008, hundreds of plants showing symptoms of leaf spot with nearly 100% incidence were found in polyethylene tunnels at an organic farm in Icheon, Korea. Leaf spots were circular to subcircular, water-soaked, dark brown with grayish center, and reached 10 mm or more in diameter. Diseased leaves defoliated prematurely. The damage purportedly due to this disease has reappeared every year with confirmation of the causal agent made again in 2011. A cercosporoid fungus was consistently associated with disease symptoms. Stromata were brown, consisting of brown cells, and 10 to 40 μm in width. Conidiophores were fasciculate (n = 2 to 10), olivaceous brown, paler upwards, straight to mildly curved, not geniculate in shorter ones or one to two times geniculate in longer ones, 40 to 200 μm long, occasionally reaching up to 350 μm long, 3.5 to 6 μm wide, and two- to six-septate. Conidia were hyaline, acicular to cylindric, straight in shorter ones, flexuous to curved in longer ones, truncate to obconically truncate at the base, three- to 16-septate, and 50 to 300 × 3.5 to 4.5 μm. Morphological characteristics of the fungus were consistent with the previous reports of Cercospora guatemalensis A.S. Mull. & Chupp (1,3). Voucher specimens were housed at Korea University herbarium (KUS). An isolate from KUS-F23757 was deposited in the Korean Agricultural Culture Collection (Accession No. KACC43980). Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequence of 548 bp was deposited in GenBank (Accession No. JQ995781). This showed >99% similarity with sequences of many Cercospora species, indicating their close phylogenetic relationship. Isolate of KACC43980 was used in the pathogenicity tests. Hyphal suspensions were prepared by grinding 3-week-old colonies grown on PDA with distilled water using a mortar and pestle. Five plants were inoculated with hyphal suspensions and five plants were sprayed with sterile distilled water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 24 h and then transferred to a 25 ± 2°C greenhouse with a 12-h photoperiod. Typical symptoms of necrotic spots appeared on the inoculated leaves 6 days after inoculation, and were identical to the ones observed in the field. C. guatemalensis was reisolated from symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in Malawi, India, China, and Japan (2,3), but not in Korea. To our knowledge, this is the first report of C. guatemalensis on sweet basil in Korea. Since farming of sweet basil has recently started on a commercial scale in Korea, the disease poses a serious threat to safe production of this herb, especially in organic farming. References: (1) C. Chupp. A Monograph of the Fungus Genus Cercospora. Ithaca, NY, 1953. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology & Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , May 5, 2012. (3) J. Nishikawa et al. J. Gen. Plant Pathol. 68:46, 2002.

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First Report of Phytophthora drechsleri Associated with Stem and Foliar Blight of Gynura bicolor in Taiwan

Plant Disease ◽

10.1094/pdis-12-10-0931 ◽

2011 ◽

Vol 95 (7) ◽

pp. 874-874 ◽

Cited By ~ 1

Author(s):

Y. M. Shen ◽

C. H. Chao ◽

H. L. Liu

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Hyphal Growth ◽

Pathogenicity Test ◽

Distilled Water ◽

First Report ◽

Foliar Blight ◽

Dual Cultures ◽

Phytophthora Drechsleri ◽

Gynura Bicolor

Gynura bicolor (Roxb. ex Willd.) DC., known as Okinawa spinach or hong-feng-cai, is a commonly consumed vegetable in Asian countries. In May 2010, plants with blight and wilt symptoms were observed in commercial vegetable farms in Changhua, Taiwan. Light brown-to-black blight lesions developed from the top of the stems to the petioles and extended to the base of the leaves. Severely infected plants declined and eventually died. Disease incidence was approximately 20%. Samples of symptomatic tissues were surface sterilized in 0.6% NaOCl and plated on water agar. A Phytophthora sp. was consistently isolated and further plated on 10% unclarified V8 juice agar, with daily radial growths of 7.6, 8.6, 5.7, and 2.4 mm at 25, 30, 35, and 37°C, respectively. Four replicates were measured for each temperature. No hyphal growth was observed at 39°C. Intercalary hyphal swellings and proliferating sporangia were produced in culture plates flooded with sterile distilled water. Sporangia were nonpapillate, obpyriform to ellipsoid, base tapered or rounded, and 43.3 (27.5 to 59.3) × 27.6 (18.5 to 36.3) μm. Clamydospores and oospores were not observed. Oospores were present in dual cultures with an isolate of P. nicotianae (p731) (1) A2 mating type, indicating that the isolate was heterothallic. A portion of the internal transcribed spacer sequence was deposited in GenBank (Accession No. HQ717146). The sequence was 99% identical to that of P. drechsleri SCRP232 (ATCC46724) (3), a type isolate of the species. The pathogen was identified as P. drechsleri Tucker based on temperature growth, morphological characteristics, and ITS sequence homology (3). To evaluate pathogenicity, the isolated P. drechsleri was inoculated on greenhouse-potted G. bicolor plants. Inoculum was obtained by grinding two dishes of the pathogen cultured on potato dextrose agar (PDA) with sterile distilled water in a blender. After filtering through a gauze layer, the filtrate was aliquoted to 240 ml. The inoculum (approximately 180 sporangia/ml) was sprayed on 24 plants of G. bicolor. An equal number of plants treated with sterile PDA processed in the same way served as controls. After 1 week, incubation at an average temperature of 29°C, blight and wilt symptoms similar to those observed in the fields appeared on 12 inoculated plants. The pathogen was reisolated from the lesions of diseased stems and leaves, fulfilling Koch's postulates. The controls remained symptomless. The pathogenicity test was repeated once with similar results. G. bicolor in Taiwan has been recorded to be infected by P. cryptogea (1,2), a species that resembles P. drechsleri. The recorded isolates of P. cryptogea did not have a maximal growth temperature at or above 35°C (1,2), a distinctive characteristic to discriminate between the two species (3). To our knowledge, this is the first report of P. drechsleri being associated with stem and foliar blight of G. bicolor. References: (1) P. J. Ann. Plant Pathol. Bull. 5:146, 1996. (2) H. H. Ho et al. The Genus Phytophthora in Taiwan. Institute of Botany, Academia Sinica, Taipei, 1995. (3) R. Mostowfizadeh-Ghalamfarsa et al. Fungal Biol. 114:325, 2010.

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First Report of Phytophthora hydropathica Causing Wilting and Shoot Dieback on Viburnum in Italy

Plant Disease ◽

10.1094/pdis-03-14-0308-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1582-1582 ◽

Cited By ~ 5

Author(s):

S. Vitale ◽

L. Luongo ◽

M. Galli ◽

A. Belisario

Keyword(s):

Natural Infection ◽

Morphological Characteristics ◽

Leaf Tissue ◽

The United States ◽

Selective Medium ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Width Ratio ◽

Morphological Identification ◽

First Report

The genus Viburnum comprises over 150 species of shrubs and small trees such as Laurustinus (Viburnum tinus L.), which is one of the most widely used ornamental plants in private and public gardens. Furthermore, it commonly forms stands of natural woodland in the Mediterranean area. In autumn 2012, a survey was conducted to determine the presence of Phytophthora ramorum on Viburnum in commercial nurseries in the Latium region where wilting, dieback, and death of twigs were observed on 30% of the Laurustinus plants. A Phytophthora species was consistently recovered from soil rich in feeder roots from potted Laurustinus plants showing symptoms. Soil samples were baited with rhododendron leaves. Small pieces of leaf tissue cut from the margin of lesions were plated on P5ARPH selective medium (4). Pure cultures, obtained by single-hypha transfers on potato dextrose agar (PDA), were petaloid. Sporangia formation was induced on pepper seeds (3). Sporangia were almost spherical, ovoid or obpyriform, non-papillate and non-caducous, measuring 36.6 to 71.4 × 33.4 to 48.3 μm (average 53.3 × 37.4 μm) with a length/width ratio of 1.4. Chlamydospores were terminal and 25.2 to 37.9 μm in diameter. Isolates were considered heterothallic because they did not produce gametangia in culture or on the host. All isolates examined had 30 to 35°C as optimum temperatures. Based on these morphological characteristics, the isolates were identified as Phytophthora hydropathica (2). Morphological identification was confirmed by internal transcribed spacer (ITS), and mitochondrial partial cytochrome oxidase subunit 2 (CoxII) with BLAST analysis in the NCBI database revealing 99% identity with ITS and 100% identity with CoxII. The sequences of the three isolates AB234, AB235, and AB236 were deposited in European Nucleotide Archive (ENA) with the accession nos. HG934148, HG934149, and HG934150 for ITS and HG934151, HG934152, and HG934153 for CoxII, respectively. Pathogenicity tests were conducted in the greenhouse on a total of six 1-year-old shoots cut from V. tinus plants with two inoculation points each. Mycelial plugs cut from the margins of actively growing 8-day-old cultures on PDA were inserted through the epidermis into the phloem. Controls were treated as described above except that sterile PDA plugs replaced the inoculum. Shoots were incubated in test tubes with sterile water in the dark at 24 ± 2°C. After 2 weeks, lesions were evident at the inoculation points and symptoms were similar to those caused by natural infection. P. hydropathica was consistently re-isolated from the margin of lesions, while controls remained symptomless. In the United States in 2008, P. hydropathica was described as spreading from irrigation water to Rhododendron catawbiense and Kalmia latifolia (2). This pathogen can also attack several other horticultural crops (1), but to our knowledge, this is the first report of P. hydropathica causing wilting and shoot dieback on V. tinus. References: (1) C. X. Hong et al. Plant Dis. 92:1201, 2008. (2) C. X. Hong et al. Plant Pathol. 59:913, 2010. (3) E. Ilieva et al. Eur. J. Plant Path. 101:623, 1995. (4) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986.

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First report of Curvularia plantarum causing leaf spot on sweet potato (Ipomoea batatas) in China

Plant Disease ◽

10.1094/pdis-08-21-1665-pdn ◽

2021 ◽

Author(s):

Jiahao Lai ◽

Tongke Liu ◽

Bing Liu ◽

Weigang Kuang ◽

Shuilin Song

Keyword(s):

Sweet Potato ◽

Molecular Identification ◽

Leaf Spot ◽

Ipomoea Batatas ◽

Morphological Characteristics ◽

Distilled Water ◽

Gene Sequences ◽

First Report ◽

Potted Plants ◽

Fungal Isolates

Sweet potato [Ipomoea batatas (L.) Lam], is an extremely versatile vegetable that possesses high nutritional values. It is also a valuable medicinal plant having anti-cancer, antidiabetic, and anti-inflammatory activities. In July 2020, leaf spot was observed on leaves of sweet potato in Nanchang, China (28°45'51"N, 115°50'52"E), which affected the growth and development of the crop and caused tuberous roots yield losses of 25%. The disease incidence (total number of diseased plants / total number of surveyed plants × 100%) was 57% from a sampled population of 100 plants in the field. Symptomatic plants initially exhibited small, light brown, irregular-shaped spots on the leaves, subsequently coalescing to form large irregular brown lesions and some lesions finally fell off. Fifteen small pieces (each 5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water and incubated on potato dextrose agar (PDA) plates at 28°C in darkness. A total of seven fungal isolates with similar morphological characteristics were obtained as pure cultures by single-spore isolation. After 5 days of cultivation at 28°C, dark brown or blackish green colonies were observed, which developed brown, thick-walled, simple, or branched, and septate conidiophores. Conidia were 18.28 to 24.91 × 7.46 to 11.69 µm (average 21.27 × 9.48 µm, n = 100) in size, straight or slightly curved, middle cell unequally enlarged, brown to dark brown, apical, and basal cells slightly paler than the middle cells, with three septa. Based on morphological characteristics, the fungal isolates were suspected to be Curvularia plantarum (Raza et al. 2019). To further confirm the identification, three isolates (LGZ1, LGZ4 and LGZ5) were selected for molecular identification. The internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and translation elongation factor 1-alpha (EF1-α) genes were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004), gpd1/gpd2 (Berbee et al. 1999), EF-983F/EF-2218R (Rehner and Buckley 2005), respectively. The sequences of ITS region of the three isolates (accession nos. MW581905, MZ209268, and MZ227555) shared 100% identity with those of C. plantarum (accession nos. MT410571-72, MN044754-55). Their GAPDH gene sequences were identical (accession nos. MZ224017-19) and shared 100% identity with C. plantarum (accession nos. MN264120, MT432926, and MN053037-38). Similarly, EF1-α gene sequences were identical (accession nos. MZ224020-22) and had 100% identity with C. plantarum (accession nos. MT628901, MN263982-83). A maximum likelihood phylogenetic tree was built based on concatenated data from the sequences of ITS, GAPDH, and EF-1α by using MEGA 5. The three isolates LGZ1, LGZ4, and LGZ5 clustered with C. plantarum. The fungus was identified as C. plantarum by combining morphological and molecular characteristics. Pathogenicity tests were conducted by inoculating a conidial suspension (106 conidia/ml) on three healthy potted I. batatas plants (five leaves wounded with sterile needle of each potted plant were inoculated). In addition, fifteen wounded leaves of three potted plants were sprayed with sterile distilled water as a control. All plants were maintained in a climate box (12 h light/dark) at 25°C with 80% relative humidity. All the inoculated leaves started showing light brown flecks after 7 days, whereas the control leaves showed no symptoms. The pathogenicity test was conducted three times. The fungus was reisolated from all infected leaves of potted plants and confirmed as C. plantarum by morphological and molecular identification, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. plantarum causing leaf spot on sweet potato in China. The discovery of this new disease and the identification of the pathogen will contribute to the disease management, provide useful information for reducing economic losses caused by C. plantarum, and lay a foundation for the further research of resistance breeding.

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First Report of Cladobotryum protrusum causing Cobweb Disease on the Edible Mushroom Coprinus comatus

Plant Disease ◽

10.1094/pdis-07-14-0757-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 287-287 ◽

Cited By ~ 6

Author(s):

G. Z. Wang ◽

M. P. Guo ◽

Y. B. Bian

Keyword(s):

Fruiting Body ◽

Morphological Characteristics ◽

Edible Mushroom ◽

Its2 Region ◽

Pathogenicity Test ◽

Distilled Water ◽

Fruiting Bodies ◽

Coprinus Comatus ◽

First Report ◽

Medicinal Value

Coprinus comatus is one of the most commercially important mushrooms in China. Its fruiting body possesses rich nutritional and medicinal value. In November 2013, unusual symptoms were observed on C. comatus on a mushroom farm in Wuhan, Hubei, China. At first, fruiting bodies were covered by white and cobweb-like mycelia. Later, the cap and stipe turned brown or dark before rotting and cracking. The pathogen was isolated from infected tissue of C. comatus. Colonies of the pathogen on potato dextrose agar (PDA) medium first appeared yellowish, followed by an obvious ochraceous or pinkish color. Aerial mycelia grew along the plate wall, cottony, 1 to 4 mm high. Conidiophores were borne on the tops of hyphae, had two to four branches, and were cylindrical, long clavate, or fusiform. Conidia were borne on the tops of the branches of conidiophores, had one to two separates, and were long and clavate. The spores ranged from 15.3 to 22.1 μm long and were 5.1 to 8.3 μm wide, which was consistent with the characteristics of Cladobotryum protrusum (1). The species was identified by ribosomal internal transcribed spacer sequencing. The ribosomal ITS1-5.8S-ITS2 region was amplified from the isolated strain using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to C. protrusum (GenBank Accession Nos. FN859408.1 and FN859413.1). The pathogen was grown on PDA at 25°C for 3 days, and the inoculation suspension was prepared by flooding the agar surface with sterilized double-distilled water for spore suspension (1 × 105 conidia/ml). In one treatment, the suspension was sprayed on casing soil (106 conidia/m2) and mixed thoroughly with it, then cased with treated soil for 2 to 3 cm thickness on the surface of compost in cultivation pots (35 × 25× 12 cm), with sterile distilled water as a control (2). Eight biological replicates were included in this treatment. In the second treatment, mycelia plugs (0.3 × 0.3 cm) without spore production were added to 20 fruiting bodies. Mushrooms treated with blank agar plugs (0.3 × 0.3 cm) were used as a control. The plugs were covered with sterilized cotton balls to avoid loss of moisture. Tested cultivation pots were maintained at 18°C and 85 to 95% relative humidity. In the samples where casing soil was sprayed with conidia suspension, white mildew developed on the pileus, and a young fruiting body grew out from the casing soil. Eventually, the surface of the mushroom was overwhelmed by the mycelia of the pathogen and the pileus turned brown or black. For the other group inoculated with mycelia plugs, only the stipe and pileus inoculated with mycelia turned brown or dark; it rotted and cracked 2 to 3 days later. The symptoms were similar to those observed on the C. comatus cultivation farm. Pathogens re-isolated from pathogenic fruiting bodies were confirmed to be C. protrusum based on morphological characteristics and ITS sequence. To our knowledge, this is the first report of the occurrence of C. protrusum on the edible mushroom C. comatus (3). Based on the pathogenicity test results, C. protrusum has the ability to severely infect the fruiting body of C. comatus. References: (1) K. Põldmaa. Stud. Mycol. 68:1, 2011. (2) F. J. Gea et al. Plant Dis. 96:1067, 2012. (3) W. H. Dong et al. Plant Dis. 97:1507, 2013.

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