First Report of a Pestalotiopsis sp. Causing Leaf Spot of Blueberry in China

In August 2006, leaf spots were observed on half-high blueberry (Vaccinium corymbosum) in a plant nursery in Dalian, China. The symptomatic potted 1-year-old blueberry plants were located in parts of a plant nursery with poor ventilation. The primary symptom was a leaf spot, 0.4 to 0.8 cm in diameter, with brown margins that enlarged and coalesced. Mycelium grew from symptomatic and green leaf tissue removed from the margin of a necrotic leaf spot. Plant tissues were surface disinfested with 0.1% mercuric chloride for 3 min and 70% ethyl alcohol for 30 s before plating onto potato dextrose agar. The resulting colonies were white with a regular margin and a rough surface. The cultures were covered with black and globular acervuli with a diameter of 100 to 200 μm. The base of each conidiophore was swollen and globose with phialides growing from the apical end. Mature conidia were straight to fusiform, measuring 19.0 to 27.5 × 6.3 to 9.2 μm, and five-celled with the three middle cells brown and darker than the end cells. The apical cell was triangular and hyaline with three simple setulae that were 17.2 to 29.7 μm long. The base cell terminated in a point 4.0 to 8.6 μm long. Koch's postulates were fulfilled for the fungus by spray inoculating two healthy young plants with 2 × 105 conidia per ml of sterile distilled water. As a control, two similar plants were sprayed with sterile 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 at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. More than 20 days after inoculation, symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Cultures isolated from the lesions were similar to those isolated previously from plants in the nursery. The morphological descriptions and measurements were similar to Pestalotiopsis clavispora (1). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and partial β-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 and T1/Bt2b primers (2) respectively, and sequenced (GenBank Accession Nos. EF119336 and EF152585). The ITS sequences were most similar to the ITS regions of P. clavispora TA-8 (98%; GenBank Accession No. AY924264), P. clavispora TA-6 (98%; GenBank Accession No. AY924263), and P. clavispora PSHI 2002 Endo 389 (96%; GenBank Accession No. AY682929). The partial β-tubulin gene sequence was identical to Pestalotiopsis sp. isolate PSHI 2004 Endo 86 (100%; GenBank Accession No. DQ657901). The morphology and sequence data support the identity of the causal fungus as P. clavispora. To our knowledge, this is the first report on the presence of a Pestalotiopsis sp. causing a disease of blueberry in China. References: (1) E. F. Guba. Monograph of Monochaetia and Pestalotia. Harvard University Press, Cambridge, MA, 1961. (2) W. Tao et al. Mol. Cell Biol. 27:689, 2007.

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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.

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First Report of Fusarium Wilt in Blueberry (Vaccinium corymbosum) Caused by Fusarium oxysporum in China

Plant Disease ◽

10.1094/pdis-02-14-0167-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1158-1158 ◽

Cited By ~ 1

Author(s):

Y. H. Liu ◽

T. Lin ◽

C. S. Ye ◽

C. Q. Zhang

Keyword(s):

Fusarium Oxysporum ◽

Infected Plant ◽

Morphological Characteristics ◽

Vaccinium Corymbosum ◽

Morphological Characterization ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Conidial Suspension ◽

First Report

Blueberry (Vaccinium corymbosum) production is developing quickly in China with about 20,000 ha presently cultivated. In 2010 in Lin'an, Zhejiang Province, plants developed an apparently new disease of blueberry (cv. Duke) with symptoms consisting of wilting of foliage, stunting of plants, and reduced fruit yields. Internal vascular and cortical tissues of plant crowns showed a brown to orange discoloration. Approximately 3% of the plants in the commercial plantings were affected and eventually died after 50 to 60 days. Infected plant samples (stems and roots) collected from different fields were surface sterilized with 1.5% sodium hypochlorite for 2 min, rinsed in water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Single conidium cultures were consistently isolated and cultured on acidified PDA (APDA) for morphological characterization (1,2). Colonies were light with purple mycelia, and beige or orange reverse colony colors developed after 7 days incubation at 25°C. Colonies producing abundant microconidia and macroconidia. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.9 to 9.6 × 1.1 to 3.4 μm). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (28.6 to 37.5 × 3.3 to 4.2 μm). Morphology and development of macroconidia and microconida were consistent with a description of Fusarium oxysporum Schltdl (1,2). The ribosomal internal transcribed spacers ITS1 and ITS2 of eight isolates were amplified using primers ITS1/ITS4 on DNA extracted from mycelium and nucleotide sequences showed 100% similarity to that of F. oxysporum. To confirm pathogenicity, 20 blueberry plants (cv. Duke) were inoculated by dipping the roots into a conidial suspension (107 conidia per ml) for 30 min. The inoculated plants were transplanted into pots containing sterilized peat and maintained at 25°C and 100% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. The pathogenicity test was conducted twice. Within 40 days, all inoculated plants developed wilt symptoms similar to that observed in the field. No symptoms were observed on plants dipped into distilled water. The fungus was successfully re-isolated from crowns and roots cultured on APDA, exhibiting morphological characteristics identical to F. oxysporum (1,2), confirming Koch's postulates. To our knowledge, this is the first report of blueberry wilt caused by Fusarium. References: (1) P. M. Kirk et al. The Dictionary of the Fungi, 10th edition, page 159. CABI Bioscience, Wallingford, UK, 2008. (2) W. C. Snyder and H. N. Hansen. Am. J. Bot. 27:64, 1940.

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First Report of Alternaria tenuissima Causing Disease on Blueberry in China

Plant Disease ◽

10.1094/pdis-91-4-0464a ◽

2007 ◽

Vol 91 (4) ◽

pp. 464-464 ◽

Cited By ~ 15

Author(s):

Y. S. Luan ◽

L. Feng ◽

X. Y. Xia ◽

L. J. An

Keyword(s):

Morphological Traits ◽

Vaccinium Corymbosum ◽

Histone Gene ◽

Internal Transcribed Spacers ◽

Sterile Water ◽

Single Spore ◽

Alternaria Tenuissima ◽

First Report ◽

Leaf Spots ◽

Plastic Bags

During September 2006, disease symptoms were observed on mature highbush blueberry (Vaccinium corymbosum L.) cvs. Bluecrop and Covoille in a blueberry commercial field in Dalian, China. The maximum and minimum rainfalls in June to September are 3,111.9 and 1,745.6 ml, respectively. The highest temperature during the summer is 35.3°C and relative humidity may achieve 90%. Circular to irregular, light brown-to-gray leaf spots with brownish red borders, initially 3 to 7 mm in diameter, enlarged and coalesced. Reddish, circular spots appeared on stems, developing small, insignificant cankers. A fungus was recovered on potato dextrose agar (PDA, pH nature) from the margin of necrotic leaf spots. Morphological traits of the strain that developed from a single-spore culture were as follows: colonies were regular and flat, with a rough upper surface that peripherally was olive-green with a black center and dull white spots; short conidiophores arising singly and measuring 81.6 to 163.2 × 4.1 to 8.2 μm; conidia was abundant, ovoid, and obclavate muriformly septate, which horizontal and vertical septations varied from 1 to 6 and 0 to 2, respectively, and its size varied from 26 to 48.8 × 9.7 to 16.3 μm with an average beak length of 9.6 μm, and sporulation pattern is budding. Conidia derived from conidiophores. Koch's postulates were fulfilled for the isolates by spray inoculating two healthy mature plants with 2 × 105 conidia per ml homogenized in sterile water. As a control, two plants were sprayed with sterile 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 for 30 days. Symptoms on inoculated plants were similar to those previously observed. Symptoms were not observed on control plants. Cultures isolated from inoculated plants had the same morphological traits as those that were isolated previously from the field plants. The morphological descriptions and measurements were similar to Alternaria tenuissima (2). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and partial cds histone gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 and H3-1a/H3-1b primers, respectively, and sequenced (GenBank Accession No. EF031053) (1,3). The ITS sequence was identical to the ITS regions of A. tenuissima strain EGS34-015 (100%; GenBank Accession No. AY751455), the partial cds histone gene sequence was similar to A. tenuissima isolate MA6 (99%; GenBank Accession No. AF404634). The morphology, secondary conidiation, and sequences of ITS and partial cds histone gene identify the causal fungus as A. tenuissima. To our knowledge, this is the first report on the presence of A. tenuissima affecting blueberry plants in China. References: (1) J. C. Kang et al. Mycol. Res. 106:1151, 2002. (2) E. G. Simmons. Mycotaxon 70:325, 1999. (3) T. J. White et al. Pages 315–322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Pyricularia grisea Causing Gray Leaf Spot on Lily in Korea

Plant Disease ◽

10.1094/pdis-94-2-0280a ◽

2010 ◽

Vol 94 (2) ◽

pp. 280-280

Author(s):

Y. P. Zhang ◽

J. H. Wang ◽

L. F. Wu ◽

X. W. Wu ◽

G. F. Cui ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Gray Leaf Spot ◽

Tween 20 ◽

Pyricularia Grisea ◽

First Report ◽

Plastic Bags ◽

Initial Symptoms ◽

Plant Nursery

Lily is an economically important ornamental crop in Korea. In August 2008, severe leaf spot symptoms were observed on an oriental Lily ‘Action’ in a plant nursery in Daegu, Korea. Disease incidence was 20 to 30%. Initial symptoms were olive green-to-brown lesions on the leaf that developed into tan, elliptical, necrotic lesions. On severely infected leaves, lesions coalesced and killed the entire leaf blade. Infected leaves were surface disinfested with 70% ethanol for 30 s and 2% chlorox for 15 min before plating 1 cm2 sections onto potato dextrose agar. Hyphae appeared 5 days after inoculation and pure culture. Conidia were hyaline, transversely septate with one to three septa; most had two. Conidia were obpyriform and measured 29 to 46 μm long and 7 to 17 μm wide. Mycelia morphology and conidia production were consistent with that described previously for Pyricularia grisea (1). Koch's postulates were fulfilled by spraying five, healthy, vegetative-stage plants with 2 × 105 conidia per ml of sterile distilled water plus 0.05% Tween 20. As a control, five similar plants were sprayed with sterile water plus 0.05% Tween 20 only. Plants were placed inside plastic bags to maintain high relative humidity and incubated in a growth chamber at 25°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. Initial symptoms were observed 7 days after inoculation. Ten days after inoculation, disease symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Fungi isolated from these lesions had the same morphological characteristics as the ones isolated previously from plants in the nursery. To our knowledge, this is the first report of gray leaf spot on lily caused by P. grisea in Korea. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971.

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First Report of Pestalotiopsis microspora Causing Leaf Spot on Moyeam in China

Plant Disease ◽

10.1094/pdis-04-21-0859-pdn ◽

2021 ◽

Author(s):

Si-Qi Yuan ◽

icai Wang ◽

Ling Lei ◽

Ju-Yun Hong ◽

Tuyong Yi ◽

...

Keyword(s):

Nucleotide Sequence ◽

Leaf Spot ◽

Disease Incidence ◽

Universal Primers ◽

Molecular Characteristics ◽

Conidial Suspension ◽

Tubulin Gene ◽

First Report ◽

Wide Range ◽

Β Tubulin

Ampelopsis grossedentata, commonly known as moyeam, has been widely used as health care herbal tea since it contains natural plant protein cream, 17 amino acids, 14 micronutrients and lots of functional flavonoid and provides a wide range of pharmaceutical functions such as antioxidant, anti-inflammatory, antitumor (Carneiro et al. 2021; Zhang et al. 2020). Moyeam is primarily produced in Zhangjiajie, stretching over the area from between 109’40 to 110’20E to between 28’52 to 29’48N, at 1300 to1890 meter above the sea level, with subtropical humid monsoon climate. Its economic value surpasses $1.25 billion in China (Liang et al. 2020). In July 2020, leaf spots were observed on some moyeam plants in Zhangjiajie. Initial spots were pinhead-sized with a yellow halo margin. The spots developed into light brown necrotic spots 6 to 8 mm in diameter, often with a dark brown margin. After 4 days of development, the spots enlarged and coalesced into irregular shape, frequently falling out and giving the leaves a tattered appearance. The infected plants eventually died with disease incidence ranging from 18 to 23%. This disease resulted in production losses of up to $1.7 million in 2020. One fungal isolate was isolated from the symptomatic leaves based on our previously published methods (Yi et al. 2019). Colonies on potato dextrose agar (PDA) were thick and villous with white at the front of the plate and yellowish at the back. After 1 week, the fungus produced conidia, which were spindle-shaped, straight or slightly curved, with 5 cells, 4-euseptates and 2-3 apical accessory filaments. Morphologically, the fungus was similar to Pestalotiopsis spp. Aerial hyphae with vigorous growth were collected for molecular identification. ITS nucleotide sequence of the rDNA and β-tubulin gene were amplified and sequenced with universal primers ITS1/ITS4 and self-designed primers based on β-tubulin gene conserved motif. BLAST searches against GenBank indicated that the ITS nucleotide sequence shared 99% similarity with that of P. microspora (MG808374.1) and the β-tubulin gene sequence shared 99% similarity with that of P. microspora (AF115396.1). Based on morphological and molecular characteristics, the fungus was identified as P. microspora. ITS and the β-tubulin nucleotide sequences were deposited in GenBank (accession no. MW350011 and MW816914). Pathogenicity tests were carried out with the following procedure. Three healthy moyeam seedlings were sprayed with a conidial suspension of 1 x106 conidia/ml while the other three seedlings were sprayed with distilled water as the controls. Plants were maintained in a greenhouse at 28±1°C. After one day of inoculation, symptoms identical to those in the field developed in the plants inoculated with the fungus. In contrast, no symptoms developed on the control plants. P. microspora has been reported to cause diseases in many crops in China. However, this is the first report of P. microspora causing leaf spot in moyeam in China. Identifying the pathogen causing the disease is important to the development of effective disease management strategies for control of this disease.

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First Report of Leptosphaeria biglobosa (Blackleg) on Brassica oleracea (Cabbage) in Mexico

Plant Disease ◽

10.1094/pdis-94-6-0791c ◽

2010 ◽

Vol 94 (6) ◽

pp. 791-791 ◽

Cited By ~ 10

Author(s):

A. Dilmaghani ◽

M. H. Balesdent ◽

T. Rouxel ◽

O. Moreno-Rico

Keyword(s):

Brassica Oleracea ◽

Sequence Data ◽

Leptosphaeria Maculans ◽

The United States ◽

Internal Transcribed Spacers ◽

First Report ◽

5.8S Rdna ◽

Single Location ◽

Leptosphaeria Biglobosa ◽

Β Tubulin

Broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis), and cabbage (B. oleracea var. capitata) have been grown in central Mexico since 1970, with 21,000 ha cropped in 2001. In contrast, areas grown with oilseed rape (B. napus) are very limited in Mexico (<8,000 ha). Blackleg, a destructive disease of B. napus in most parts of the world, was first observed in Mexico in Zacatecas and Aguascalientes in 1988 on B. oleracea, causing as much as 70% yield loss. A species complex of two closely related Dothideomycete species, Leptosphaeria maculans and L. biglobosa, is associated with this disease of crucifers (1), but leaf symptoms on susceptible plants are different, with L. maculans typically causing >15-mm pale gray lesions with numerous pycnidia, whereas L. biglobosa causes dark and smaller lesions only containing a few pycnidia. Having a similar epidemiology, both species can be present on the same plants at the same time, and symptom confusion can occur as a function of the physiological condition of the plant or expression of plant resistance responses. A total of 209 isolates from symptomatic B. oleracea leaves were collected from three fields in central states of Mexico (58 to 71 isolates per location). All leaves showed similar symptoms, including a 10- to 15-mm tissue collapse with an occasional dark margin. Cotyledons of seven B. napus differentials were inoculated with conidia of all the isolates as described by Dilmaghani et al. (1). Two hundred isolates caused tissue collapse typical of L. maculans. However, nine obtained from white cabbage in a single location in Aguascalientes caused <5-mm dark lesions. When inoculated onto cotyledons of three B. oleracea genotypes commonly grown in Mexico (cvs. Domador, Monaco, and Iron Man), the nine isolates caused a range of symptoms characterized by tissue collapse (maximum 10 to 15 mm), showing the presence of patches of black necrotic spots within the collapse. The occasional presence of a few pycnidia allowed us to reisolate the fungus for molecular identification. ITS1-5.8S-ITS2, (internal transcribed spacers and 5.8S rDNA), actin, and β-tubulin sequences were obtained as described previously (4). Multiple gene genealogies based on these sequence data showed two subclades of L. biglobosa: L. biglobosa ‘occiaustralensis’ (one isolate; ITS [AM410082], actin [AM410084], and β-tubulin [AM410083]) and L. biglobosa ‘canadensis’ (eight isolates; ITS [AJ550868], actin [AY748956], and β-tubulin [AY749004]) (3,4), which were previously described on B. napus in the United States, Canada, and Chile. To our knowledge, this is the first report of L. biglobosa in Mexico. Previously, this species has only been reported once on B. oleracea without discrimination into subclades (2). In the Aguascalientes sampling, 24% of the isolates were L. biglobosa, similar to Canadian locations where this species is still common as compared with L. maculans (1). The large proportion of sampled L. biglobosa ‘canadensis’, highlights the prevalence of this subclade throughout the American continent (1). References: (1) A. Dilmaghani et al. Plant Pathol. 58:1044, 2009. (2) E. Koch et al. Mol. Plant-Microbe Interact. 4:341, 1991. (3) E. Mendes-Pereira et al. Mycol Res. 107:1287, 2003. (4) L. Vincenot et al. Phytopathology 98:321, 2008.

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First Report of Alternaria Leaf Spot Caused by Alternaria sp. on Highbush Blueberry (Vaccinium corymbosum) in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0344-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1434-1434

Author(s):

J.-H. Kwon ◽

D.-W. Kang ◽

M.-G. Cheon ◽

J. Kim

Keyword(s):

South Korea ◽

Leaf Spot ◽

Disease Incidence ◽

Vaccinium Corymbosum ◽

Its Rdna ◽

Universal Primers ◽

Conidial Suspension ◽

First Report ◽

Representative Isolate ◽

Alternaria Sp

In South Korea, the culture, production, and consumption of blueberry (Vaccinium corymbosum) have increased rapidly over the past 10 years. In June and July 2012, blueberry plants with leaf spots (~10% of disease incidence) were sampled from a blueberry orchard in Jinju, South Korea. Leaf symptoms included small (1 to 5 mm in diameter) brown spots that were circular to irregular in shape. The spots expanded and fused into irregularly shaped, large lesions with distinct dark, brownish-red borders. The leaves with severe infection dropped early. A fungus was recovered consistently from sections of surface-disinfested (1% NaOCl) symptomatic leaf tissue after transfer onto water agar and sub-culture on PDA at 25°C. Fungal colonies were dark olive and produced loose, aerial hyphae on the culture surfaces. Conidia, which had 3 to 6 transverse septa, 1 to 2 longitudinal septa, and sometimes also a few oblique septa, were pale brown to golden brown, ellipsoid to ovoid, obclavate to obpyriform, and 16 to 42 × 7 to 16 μm (n = 50). Conidiophores were pale to mid-brown, solitary or fasciculate, and 28 to 116 × 3 to 5 μm (n = 50). The species was placed in the Alternaria alternata group (1). To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA region of a representative isolate, AAVC-01, was amplified using ITS1 and ITS4 primers (2). The DNA products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and the resulting pOR13 plasmid was sequenced using universal primers. The resulting 570-bp sequence was deposited in GenBank (Accession No. KJ636460). Comparison of ITS rDNA sequences with other Alternaria spp. using ClustalX showed ≥99% similarity with the sequences of A. alternata causing blight on Jatropha curcas (JQ660842) from Mexico and Cajannus cajan (JQ074093) from India, citrus black rot (AF404664) from South Africa, and other Alternaria species, including A. tenuissima (WAC13639) (3), A. lini (Y17071), and A. longipes (AF267137). Two base substitutions, C to T at positions 345 and 426, were found in the 570-bp amplicon. Phylogenetic analysis revealed that the present Alternaria sp. infecting blueberry grouped separately from A. tenuissima and A. alternata reported from other hosts. A representative isolate of the pathogen was used to inoculate V. corymbosum Northland leaves for pathogenicity testing. A conidial suspension (2 × 104 conidia/ml) from a single spore culture and 0.025% Tween was spot inoculated onto 30 leaves, ranging from recently emerged to oldest, of 2-year-old V. corymbosum Northland plants. Ten leaves were treated with sterilized distilled water and 0.025% Tween as a control. The plants were kept in a moist chamber with >90% relative humidity at 25°C for 48 h and then moved to a greenhouse. After 15 days, leaf spot symptoms similar to those observed in the field developed on the inoculated leaves, whereas the control plants remained asymptomatic. The causal fungus was re-isolated from the lesions of the inoculated plants to fulfill Koch's postulates. To our knowledge, this is the first report of Alternaria sp. on V. corymbosum in South Korea. References: (1) E. G. Simmons. Page 1797 in: Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) M. P. You et al. Plant Dis. 98:423, 2014.

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First Report of Lasiodiplodia theobromae Causing Inflorescence Blight and Fruit Rot of Longan (Dimocarpus longan L.) in Puerto Rico

Plant Disease ◽

10.1094/pdis-05-13-0473-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 279-279 ◽

Cited By ~ 4

Author(s):

L. M. Serrato-Diaz ◽

L. I. Rivera-Vargas ◽

R. Goenaga ◽

R. D. French-Monar

Keyword(s):

Puerto Rico ◽

Fruit Rot ◽

Fluorescent Light ◽

Its2 Region ◽

Tropical Fruit ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Wide Range ◽

Dimocarpus Longan ◽

Β Tubulin

Dimocarpus longan L., commonly known as longan, is a tropical fruit tree of the Sapindaceae family. From 2008 to 2010, a disease survey for longan was conducted in March and October in Puerto Rico. Fruit rot and inflorescence blight (rotting of the rachis, rachilla, and flowers) were observed in fields of longan at the USDA-ARS Research Farm in Isabela, and two commercial orchards in Puerto Rico. Tissue sections (1 mm2) of diseased inflorescences and surface of the fruit were disinfested with 70% ethanol, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Three isolates of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Lt) were isolated from symptomatic tissue and identified morpho-molecularly using a taxonomic key for the Botryosphaeriaceae and DNA sequence analysis (1). In APDA, colonies of Lt had initial greenish-gray aerial mycelia that turned dark brown with age. Pycnidia were dark brown to black. Immature conidia were sub-ovoid to ellipsoid, apex rounded, truncate at the base, thick-walled, hyaline, and one-celled, becoming dark brown, two-celled, and with irregular longitudinal striations when mature. Conidia (n = 50) for all the isolates averaged 26.9 μm long by 13 μm wide. For molecular identification, the ITS1-5.8S-ITS2 region and fragments of the β-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and BLASTn searches done in GenBank. Accession numbers of gene sequences of Lt submitted to GenBank were KC964546, KC964547, and KC964548 for ITS region; KC964549, KC964550, and KC964551 for β-tubulin; and KC964552, KC964553, and KC964554 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of Lt reported in GenBank (accessions AY640255, EU673110, and AY640258). Pathogenicity tests were conducted on six random healthy non-detached inflorescences of longan and six healthy detached fruits per isolate. Unwounded inflorescences and fruit were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA. Inflorescences were enclosed in plastic bags for 5 days under field conditions while fruits were kept in a humid chamber using plastic boxes for 5 days under laboratory conditions of 25°C and 12 h of fluorescent light. Untreated controls were inoculated with APDA disks only. The experiment was repeated once. Five days after inoculation, isolates of Lt caused inflorescence blight, fruit rot, and aril (flesh) rot. Inflorescences turned brown and flower mummification was observed on the inflorescences. The exocarp (peel) and endocarp (aril) turned dark brown and mycelial growth and pycnidia of Lt were observed on fruits. Untreated controls did not show any symptoms and no fungi were re-isolated from tissue. In diseased inflorescences and fruits, Lt was re-isolated from diseased tissue and identified using morphological and molecular parameters, thus fulfilling Koch's postulates. Lt has been reported to cause dieback, stem end rot, and fruit rot on a wide range of plants host (2,4). In longan, Lt has been reported causing fruit rot in Thailand (3). To our knowledge, this is the first time that Lt has been reported causing inflorescence blight in longan and the first report of Lt causing fruit rot in Puerto Rico. References: (1) A. J. L. Phillips. Key to the various lineages in “Botryosphaeria” Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 26 November 2013. (2) B. Slippers et al. Mycologia 97:99, 2005. (3) P. Suwanakood et al. Asian J. Biol. Ed. 3:47, 2007. (4) A. F. Wright and P. F. Harmon. Plant Dis. 93:962, 2009.

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First Report of Stem Blight Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Greenhouse-Grown Blueberries in the United States

Plant Disease ◽

10.1094/pdis-02-11-0117 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1187-1187

Author(s):

J. J. Sadowsky ◽

T. D. Miles ◽

A. M. C. Schilder

Keyword(s):

United States ◽

North Carolina ◽

Root Rot ◽

The United States ◽

Vaccinium Corymbosum ◽

Conidial Suspension ◽

Centraalbureau Voor ◽

First Report ◽

Stem Lesions ◽

Β Tubulin

Necrotic stems and leaves were observed on 2- to 4-month-old, rooted microshoot plants (Vaccinium corymbosum L. ‘Liberty’ and ‘Bluecrop’, V. angustifolium Aiton ‘Putte’, and V. corymbosum × V. angustifolium ‘Polaris’) in a Michigan greenhouse in 2008 and 2009. As the disease progressed, leaves fell off and 80 to 100% of the plants died in some cases. Root rot symptoms were also observed. A fungus was isolated from stem lesions. On potato dextrose agar (PDA), cultures first appeared light tan to orange, then rusty brown and zonate with irregular margins. Chains of orange-brown chlamydospores were abundant in the medium. Macroconidiophores were penicillately branched and had a stipe extension of 220 to 275 × 2.5 μm with a narrowly clavate vesicle, 3 to 4 μm wide at the tip. Conidia were hyaline and cylindrical with rounded ends, (1-)3-septate, 48 to 73 × 5 to 7 (average 60 × 5.5) μm and were held together in parallel clusters. Perithecia were globose to subglobose, yellow, 290 to 320 μm high, and 255 to 295 μm in diameter. Ascospores were hyaline, 2- to 3-septate, guttulate, fusoid with rounded ends, slightly curved, and 30 to 88 × 5 to 7.5 (average 57 × 5.3) μm. On the basis of morphology, the fungus was identified as Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii Peerally) (1,2). The internal transcribed spacer region (ITS1 and ITS2) of the ribosomal DNA and the β-tubulin gene were sequenced (GenBank Accession Nos. HQ909028 and JF826867, respectively) and compared with existing sequences using BLASTn. The ITS sequence shared 99% maximum identity with that of Ca. colhounii CBS 293.79 (GQ280565) from Java, Indonesia, and the β-tubulin sequence shared 97% maximum identity with that of Ca. colhounii CBS 114036 (DQ190560) isolated from leaf spots on Rhododendron sp. in North Carolina. The isolate was submitted to the Centraalbureau voor Schimmelcultures in the Netherlands (CBS 129628). To confirm pathogenicity, 5 ml of a conidial suspension (1 × 105/ml) were applied as a foliar spray or soil drench to four healthy ‘Bluecrop’ plants each in 10-cm plastic pots. Two water-sprayed and two water-drenched plants served as controls. Plants were misted intermittently for 2 days after inoculation. After 7 days at 25 ± 3°C, drench-inoculated plants developed necrotic, sporulating stem lesions at the soil line, while spray-inoculated plants showed reddish brown leaf and stem lesions. At 28 days, three drench-inoculated and one spray-inoculated plant had died, while others showed stem necrosis and wilting. No symptoms were observed on control plants. Fungal colonies reisolated from surface-disinfested symptomatic stem, leaf, and root segments appeared identical to the original isolate. Cy. colhounii was reported to cause a leaf spot on blueberry plants in nurseries in China (3), while Ca. crotalariae (Loos) D.K. Bell & Sobers (= Ca. ilicicola Boedijn & Reitsma) causes stem and root rot of blueberries in North Carolina (4). To our knowledge, this is the first report of Ca. colhounii causing a disease of blueberry in Michigan or the United States. Because of its destructive potential, this pathogen may pose a significant threat in blueberry nurseries. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) L. Lombard et al. Stud. Mycol. 66:31, 2010. (3) Y. S. Luan et al. Plant Dis. 90:1553, 2006. (4) R. D. Milholland. Phytopathology 64:831, 1974.

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First Report of Pestalotiopsis mangiferae and P. vismiae Causing Twig Dieback of Myrica rubra in China

Plant Disease ◽

10.1094/pdis-12-11-1054-pdn ◽

2012 ◽

Vol 96 (4) ◽

pp. 588-588 ◽

Cited By ~ 6

Author(s):

F. Y. Chen ◽

L. M. Lu ◽

H. Z. Ni ◽

Y. Wang ◽

Y. G. Wang ◽

...

Keyword(s):

Basal Cell ◽

Southern China ◽

Apical Cell ◽

Morphological Characteristics ◽

Fluorescent Light ◽

Water Control ◽

First Report ◽

Myrica Rubra ◽

Potted Plants ◽

Detached Leaves

Chinese bayberry (Myrica rubra Siebold & Zucc.), an evergreen fruit tree, is widely grown in southern China. In 1999, severe twig dieback was observed on M. rubra in Taizhou and it spread to several major M. Rubra-producing areas of Zhejiang covering more than 6,000 ha by 2011. Symptoms were usually observed from June to November and first appeared as chlorosis of leaves and leaf drop, followed by the formation of dark brown lesions covered with white mycelia surrounding leaf scars. The lesions can extend to the whole twig and tree causing discoloration of the xylem. In most cases, infected trees die within 1 to 4 years. Two distinct fungi totaling 46 isolates were isolated from the surface-disinfested diseased twigs and cultured on potato dextrose agar (PDA) at 28°C. An isolate of each fungus, designated as C1 and B1, was characterized further following 10 days of growth on PDA at 28°C. C1 formed zonate, white colonies and black, acervular conidiomata with the conidia aggregated on acervuli as a creamy mass. Isolate B1 formed nonzonate, white colonies and black, acervular conidiomata with the conidia aggregated on acervuli as droplets. Conidia for each isolate were fusiform with five cells; one hyaline apical cell, one hyaline basal cell, and three, dark brown median cells. Conidia ranged from 17.8 to 25.2 × 6.7 to 9.2 μm for C1 and 21.2 to 27.8 × 4.3 to 7.5 μm for B1. There were two to three hyaline, filamentous appendages (9.8 to 23.5 μm long for C1 and 10.5 to 25.5 μm long for B1) attached to each apical cell, and one hyaline appendage (3.5 to 7.2 μm long for C1 and 3.0 to 6.8 μm long for B1) attached to each basal cell. The cultural and morphological characteristics of C1 (16 isolates) matched the description for Pestalotiopsis mangiferae while B1 (27 isolates) matched the description for P. vismiae (2). The PCR-amplified and sequenced internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) for isolate C1 (GenBank Accession No. JQ281542) and B1 (GenBank Accession No. JQ281543) were 99 and 100% homologous to that of the P. mangiferae isolate MM 102 (GenBank Accession No. GU722595) and P. vismiae isolate xsd08116 (GenBank Accession No. FJ481027), respectively. For pathogenicity tests, nine healthy detached leaves and 12 potted plants of M. rubra were wound inoculated with sterile water (control) or conidial suspensions (105 conidia per ml; 20 μl on each site) of C1 and B1, respectively, and maintained with relative humidity of more than 90% under fluorescent light at 28°C. Tests were performed twice. Necrotic lesions, resembling those that occurred in the field, were observed on all inoculated detached leaves and 33.3% of C1 and 25% of B1 inoculated potted plants 10 and 30 days following inoculation, respectively, while the controls remained healthy. Two fungi were reisolated from the lesions with identical morphology to the initial C1 and B1 inoculums. Therefore, P. mangiferae and P. vismiae were determined to be the causal agent for twig dieback of M. rubra in China. Pestalotiopsis spp. were previously reported as pathogens of loquat (4), mango (3), and blueberry (1) causing economic loss. To our knowledge, this is the first report of twig dieback disease of M. rubra caused by P. mangiferae and P. vismiae. References: (1) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (2) Q. X. Ge et al. Flora Fungorum Sinicorum. Vol. 38, Pestalotiopsis. Science Press, Beijing, 2009. (3). Y. Ko et al. Plant Dis. 91:1684, 2007. (4). A. E. Perelló and S. Larran. Plant Dis. 83:695, 1999.

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