First Report of a Root and Crown Disease of the Invasive Weed Lepidium draba Caused by Phoma macrostoma

The exotic rangeland perennial Lepidium draba occurs as a noxious weed in 22 states, mostly in the western United States. Because chemical control measures against this invasive perennial, a member of the Brassicaceae, have not achieved adequate results, biological control is being pursued. While inventories of arthropods that feed on L. draba have been established, little is known of soilborne pathogens for possible use as biological control agents. To address this deficiency, we have surveyed for diseases of L. draba in the United States and Eurasia to identify and test potential biocontrol agents. In intensive surveys for soilborne diseases in a single infestation that is >20 years old in a cattle pasture in south-central Montana, several chlorotic, stunted plants were noted. Roots of chlorotic plants that exhibited elongated fissures from which other soilborne fungi were isolated also had numerous prominent pycnidia embedded in the crown tissue above the lesions. Examination with a dissecting microscope revealed large ostioles made evident by the wide concave inversions in the short necks of the pycnidia. Culture of root tissue on potato dextrose agar resulted in whitish, becoming pale gray colonies, with a dull peach-to-reddish tinge at the margins, with abundant single pycnidia. Conidia in vitro were mainly unicellular, variable shape, subglobose to ellipsoidal, with several guttules averaging 6 × 2.5 μm. These morphological traits are characteristic of Phoma macrostoma, which is regarded as a weak or wound pathogen. The internal transcribed spacer region of rDNA was amplified using primers ITS1 and ITS4 and sequenced. BLAST analysis of the 575-bp fragment showed a 100% homology with the sequence of an isolate of P. macrostoma that has been investigated extensively for commercialization as a biological control agent of various agricultural weeds (1), including wild mustard (GenBank No. DQ474091). The nucleotide sequence has been assigned GenBank No. HM755951. Pathogenicity tests consisted of making four 1.4-mm-diameter holes in five NaOCl (0.1%)-sterilized root sections of L. draba and pipetting ~50 to 100 μl of a 106 CFU/ml conidial suspension into the incisions, incubating the inoculated roots at 20 to 25°C overnight and planting the root sections, one per pot, in an artificial greenhouse potting mix and placing the pots in the greenhouse at 20 to 25°C. Controls were five root sections that were treated similarly except that sterile water was injected. The experiment was repeated. After 10 days, shoots that grew from inoculated roots were chlorotic and shorter than those produced from control roots. P. macrostoma was isolated from tissue of inoculated roots that became blackened distal to the inoculation points. To examine the host range of P. macrostoma on other brassica species, crowns of 2-week-old seedlings of radish, broccoli, cauliflower, broccoli raab, turnip, kohlrabi, cabbage, Chinese cabbage, mustard greens, and canola were injected with 0.5 ml of a 106 CFU/ml conidial suspension. Plants were grown in the greenhouse at 20 to 25°C for 4 weeks after inoculation and examined for symptoms. The experiment was repeated twice. Blackened root tissue with slight chlorosis occurred only on roots of radish and crowns of broccoli, from which P. macrostoma was reisolated. To our knowledge, this the first report of a disease of L. draba caused by P. macrostoma. Reference: (1) K. L. Bailey et al. U.S. Patent Application Serial No. 60/294,475, Filed May 20, 2001.

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First Report of Pilidium concavum Causing Tan-brown Rot on Strawberry Nursery Stock in South Carolina

Plant Disease ◽

10.1094/pdis-01-14-0048-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1010-1010 ◽

Cited By ~ 1

Author(s):

D. Fernández-Ortuño ◽

P. K. Bryson ◽

G. Schnabel

Keyword(s):

United States ◽

South Carolina ◽

Brown Rot ◽

The United States ◽

Conidial Suspension ◽

Strawberry Fruit ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Nursery Stock ◽

Wide Range

Pilidium concavum (Desm.) Höhn. [synanamorph: Hainesia lythri (Desm.) Höhn.] is an opportunistic pathogen that causes leaf spots and stem necrosis in a wide range of hosts, including strawberry (Fragaria ananassa) (1,2). In October 2013, 24 strawberry plug plants (cv. Chandler) with brown to dark brown necrotic lesions on stolons were obtained from a nursery in Easley, SC. The lesions were oval shaped and varied in length from 2 to 8 mm. The tips of stolons with larger spots had died. To isolate the causal agent, 3 to 5 cm of necrotic stolon tissue was surface disinfected for 1 min with 10% bleach, rinsed with sterile distilled water, air dried, and placed on potato dextrose agar (PDA). After 7 days of incubation at 22°C, pink-orange masses of spores emerged. Single spore colonies on PDA produced a gray to brown colony with whitish aerial mycelium. Numerous discoid to hemisphaerical conidiomata (0.3 to 2.2 mm in diameter) developed with a dark base and exuded a pink, slimy mass that contained many conidia. Conidiophores (10.2 to 47.8 × 0.8 to 2.0 μm) were hyaline, unicellular, cylindrical, and filiform. Conidia (3.0 to 8.5 × 1.0 to 2.9 μm) were aseptate, fusiform, hyaline, and canoe-shaped to allantoid. On the basis of morphology, the pathogen was identified as P. concavum (3). The internal transcribed spacer region ITS1-5.8S-ITS2 was amplified by PCR and sequenced with primers ITS1 and ITS4 (4). The sequence was submitted to GenBank (Accession No. KF911079) and showed 100% homology with sequences of P. concavum. Pathogenicity was examined on strawberry fruit and leaves. Our previous efforts to achieve infection without wounding failed, which is consistent with experiences of other scientists (not cited). Thus, 24 strawberry fruit were wounded (1 cm deep) with a needle once, and submerged for 3 min in a conidial suspension (2 × 106 conidia ml−1). Controls were wounded and submerged in sterile water. After 4 days of incubation at 22°C, characteristic symptoms were observed at the wound site only on inoculated fruit. Detached leaves (about 6 cm in diameter) from 3- to 4-week-old strawberry plants cv. Chandler were surface sterilized and placed right side up in petri dishes (one leaf per dish) containing water agar. Leaves were inoculated at one site with a 50 μl conidial suspension (2 × 106 conidia ml−1) after inflicting a scraping-type injury with a needle to the surface at the point of inoculation. Control leaves received just water. After 7 days of incubation at 22°C, only the inoculated leaves showed symptoms similar to those observed on strawberry stolons. The fungus was re-isolated from symptomatic fruit and leaf lesions and identity was confirmed based on morphological features. The experiments were repeated. To our knowledge, this is the first report of P. concavum causing tan-brown rot on strawberry tissue in South Carolina. Prior to this study, the pathogen has been described from different hosts and countries including Belgium, Brazil, China, France, Iran, Poland, and the United States. Contamination of strawberry nursery stock by P. concavum could become a plant health management issue in the United States, especially if the pathogen is transferred to strawberry production areas. Further information on in-field occurrence of P. concacum is needed. References: (1) J. Debode et al. Plant Dis. 95:1029, 2011. (2) W. L. Gen et al. Plant Dis. 96:1377, 2012. (3) A. Y. Rossman et al. Mycol. Prog. 3:275, 2004. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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First Report of a Leaf Spot Caused by Cercospora bizzozeriana on Lepidium draba subsp. draba in Tunisia

Plant Disease ◽

10.1094/pd-89-0206a ◽

2005 ◽

Vol 89 (2) ◽

pp. 206-206

Author(s):

T. Souissi ◽

D. K. Berner ◽

H. J. Dubin

Keyword(s):

United States ◽

Biological Control ◽

Biological Control Agent ◽

Aqueous Suspension ◽

The United States ◽

Voucher Specimen ◽

First Report ◽

Leaf Spots ◽

White Leaf ◽

Lepidium Draba

Lepidium draba (L.) subsp. draba (synonym = Cardaria draba (L.) Desv.), commonly known as white-top or hoary-cress (1), family Brassicaceae, is a common weed and emerging problem in wheat in Tunisia. It is also a problematic invasive weed in the northwestern United States and a target of biological control efforts. During the summer of 2002, dying L. draba plants were found around Tunis, Tunisia. Plants had grayish white leaf spots on most of the leaves. In some cases, the leaf spots dropped out of the leaves producing “shot-holes”. In most cases, the leaf spots coalesced, and the leaves wilted and died. Diseased leaves were collected, air-dried, and sent to the quarantine facility of the Foreign Disease-Weed Science Research Unit (FDWSRU), USDA/ARS, Fort Detrick, MD. The air-dried leaves were observed microscopically, and numerous conidiophores and conidia were observed on both sides of the leaves within and around the lesions. The fungus isolated (DB03-009) conformed to the description of Cercospora bizzozeriana Saccardo & Berlese (2). Conidiophores were unbranched, pale olive-brown, 1 to 5 geniculate, and uniform in color and width. Conidia were hyaline, straight to slightly curved, multiseptate, and 57 to 171 × 3.8 to 6.7 µm (average 103 to 4.6 µm). Stems and leaves of 12 rosettes (10 to 15 cm in diameter) of 6-week-old L. draba plants were spray inoculated with an aqueous suspension of conidia (1 × 105/ml) harvested from 6- to 8-day-old cultures grown on carrot leaf decoction agar. Six of the plants and two noninoculated plants were placed in a dew chamber at 22°C in darkness and continuous dew. The other half of the plants and two noninoculated plants were placed on a greenhouse bench at approximately 25°C and covered with clear polyethylene bags. After 72 h, plants from the dew chamber were moved to a greenhouse bench, and the bagged plants were uncovered. All plants were watered twice daily. After 9 days, symptoms were observed on the plants that had been bagged but not on the plants from the dew chamber. Symptoms were identical to those observed in the field in Tunisia and included “shot holes”. No symptoms were observed on noninoculated plants. C. bizzozeriana was reisolated from the leaves of all symptomatic plants. Completion of Koch's postulates was repeated with an additional five plants. This isolate of C. bizzozeriana is a destructive pathogen on L. draba subsp. draba, and severe disease can be produced by inoculation of foliage with an aqueous suspension of conidia. This isolate is a good candidate for mycoherbicide development in Tunisia where the weed and pathogen are indigenous. However, some commercially grown Brassica species were found susceptible to this isolate, which will preclude its use as a classical biological control agent in the United States. To our knowledge, this is the first report of C. bizzozeriana on L. draba subsp. draba in Tunisia. A voucher specimen has been deposited at the U.S. National Fungus Collections (BPI 843753). Live cultures are being maintained at FDWSRU and the Institut National Agronomique de Tunisie, Tunis, Tunisia. References: (1) I. A. Al-Shehbaz and K. Mummenhoff. Novon 12:5, 2002. (2) C. Chupp. A Monograph of the Fungus Genus Cercospora. C. Chupp, Ithaca, New York, 1953.

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First Report of Flower Anthracnose Caused by Colletotrichum karstii in White Phalaenopsis Orchids in the United States

Plant Disease ◽

10.1094/pdis-04-12-0360-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1227-1227 ◽

Cited By ~ 9

Author(s):

I. Jadrane ◽

M. Kornievsky ◽

D. E. Desjardin ◽

Z.-H. He ◽

L. Cai ◽

...

Keyword(s):

United States ◽

Species Complex ◽

The United States ◽

Likelihood Method ◽

Conidial Suspension ◽

Necrotic Tissue ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Whole Plant ◽

Pure Cultures

In October 2010, a Colletotrichum species was isolated from white Phalaenopsis flowers growing in a greenhouse in San Francisco, CA. This Phalaenopsis is a common commercial orchid hybrid generated mostly likely from Phalaenopsis amabilis and P. aphrodite. The white petals showed anthracnose-like lesions where necrotic tissue is surrounded by a ring of green tissue. The green halo tissues around the necrotic tissue contain functional chloroplasts. One-centimeter disks were cut around the necrotic sites and surface-sterilized with 95% ethanol and 0.6% sodium hypochlorite. The disks were placed on potato dextrose agar (PDA) medium to establish cultures. Pure cultures were obtained by subculturing hyphal tips onto fresh PDA plates. The generated colonies had white aerial mycelia and orange conidial mass. The color of the reverse colony varies between colorless and pale orange. Microscopic observations identified the conidia as cylindrical, straight, and rounded at both ends. In addition, the conidia were approximately 15.0 to 18.0 μm long and 5.0 to 6.5 μm in diameter. These observed morphological features suggested that these isolates possessed the same characteristics as previously described for Colletotrichum karstii, a species considered as part of the C. boninense species complex (1). Four putative independent Colletotrichum isolates were recovered (DED9596, DED9597, DED9598, and DED9599). To confirm the Colletotrichum isolates as the causative pathogen, healthy white Phalaenopsis flowers (five total) in a whole plant were sprayed with a conidial suspension (approximately 1.2 × 106 conidia/ml) of the isolates and incubated at 20°C and 100% relative humidity with cycles of 16 h light and 8 h of darkness. Approximately 1 ml of conidial suspension solution was used for each flower. The plants were watered regularly and flowers were sprayed with sterile double-distilled water daily. As negative controls, five flowers in a whole plant were sprayed with water. Fifteen to twenty days after inoculation, lesions started to form on the petals sprayed with the putative Colletotrichum isolates. All controls remained healthy. The Colletotrichum-inoculated flowers remained alive and did not die as a result of the infection. This same experiment was repeated and the same results were obtained. DNA was extracted from the necrotic regions of the petals infected by the pure cultures of the four isolates and used to sequence the 18S rRNA ITS (internal transcribed spacer) region. All four isolates gave identical ITS sequences. Analysis of the obtained representative sequences (GenBank Accession No. JQ277352) suggested that the isolated pathogen as C. karstii. Using the published ITS data for the C. boninense species complex (1), a phylogenetic tree was generated via the maximum likelihood method. This created tree places the isolates in the same group as C. karstii. This type of C. karstii infection in Phalaenopsis orchid petals was not documented in the U.S. before, although it has been reported in China and Thailand (2). To our knowledge, this is the first report of infection and green island formation caused by C. karstii on orchid flower in the United States. References: (1) Damm et al. Studies in Mycology 73:1, 2012. (2) Yang et al. Cryptogamie Mycologie 32:229, 2011.

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First Report of Smut Caused by Microbotryum silybum on Ivory Thistle

Plant Disease ◽

10.1094/pd-89-1242a ◽

2005 ◽

Vol 89 (11) ◽

pp. 1242-1242 ◽

Cited By ~ 1

Author(s):

T. Souissi ◽

D. K. Berner ◽

E. L. Smallwood

Keyword(s):

United States ◽

Biological Control ◽

Flux Density ◽

The United States ◽

Photon Flux ◽

Photon Flux Density ◽

Internal Transcribed Spacer Region ◽

Field Inoculation ◽

Voucher Specimen ◽

First Report

Silybum eburneum Coss. & Durieu. (ivory thistle) and S. marianum (L.) Gaertn. (milk thistle) are dominant, invasive weeds in northern Tunisia (1). S. marianum is also invasive in the United States and targeted for biological control. The smut fungus Microbotryum silybum Vánky & Berner is a naturally occurring pathogen of S. marianum in Greece (2) but not in Tunisia or the United States. To assess the safety of the fungus for biological control in the United States, plants related to S. marianum were evaluated for susceptibility to M. silybum in the quarantine facility of the Foreign Disease-Weed Science Research Unit (FDWSRU), USDA/ARS, Fort Detrick, MD. Because of the close genetic relationship of S. eburneum to S. marianum, both were tested for susceptibility under greenhouse conditions at the FDWSRU. All inoculations were done by placing 5 mg of teliospores of M. silybum in the central whorl of rosettes with three to five true leaves. Individual plants in soil-filled pots were placed in a controlled chamber at 16°C with 10 h of light daily. Photon flux density in the chamber was 34 μmol·m-2·s-1 supplied by three 1.8-m long 115W fluorescent tubes and three 52W incandescent bulbs. The central whorl was misted with distilled water twice daily for 2 weeks and the temperature was then lowered to 8°C for 6 weeks. The plants were transferred to a greenhouse bench at 22 to 25°C with 14 h of light daily. Photon flux density on the bench was 620 μmol·m-2·s-1 provided by two 500W sodium vapor lamps, one 1,000W metal halide lamp, and incidental sunlight. After approximately 7 weeks, plants of each species had fully developed capitula that flowered normally, produced no flowers, or formed abnormal flowers. Abnormal capitula contained powdery masses of teliospores in the ovaries of the florets. In contrast to systemic infections that were observed in the field (2), different branches of bolted plants bore both diseased and normal capitula. In turn, diseased capitula of both species were either completely diseased (all florets filled with teliospores) or partially diseased. Four of ten S. marianum plants and six of nine S. eburneum plants were diseased. Pathogenicity tests were repeated four times with similar results. In Greece, field inoculation of S. marianum with 5 mg of teliospores produced an average of 89% diseased plants with an average of 250 g of teliospores produced per plant. A similar level of disease is possible for S. eburneum under field conditions. Teliospores from smutted ovaries of both plant species conformed to the description for M. silybum (2). Both species are annual plants that reproduce solely by seeds. Since M. silybum prevents seed production, this fungus has great potential as a biological control agent in the United States and Tunisia. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 863477). Nucleotide sequences for the internal transcribed spacer region are available in GenBank (Accession No. AY285774). To our knowledge, this is the first report of M. silybum parasitizing S. eburneum. References: (1) G. Pottier-AlaPetite. Flore de la Tunisie: Angiospermes-Dicotylédones, Gamopétales, Tunis, 1981. (2) K. Vánky and D. Berner. Mycotaxon 85:307, 2003.

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First Report of Stem Canker of Salsola tragus Caused by Diaporthe eres in Russia

Plant Disease ◽

10.1094/pdis-93-1-0110b ◽

2009 ◽

Vol 93 (1) ◽

pp. 110-110 ◽

Cited By ~ 1

Author(s):

T. Kolomiets ◽

Z. Mukhina ◽

T. Matveeva ◽

D. Bogomaz ◽

D. K. Berner ◽

...

Keyword(s):

United States ◽

Biological Control ◽

Biological Control Agent ◽

Aqueous Suspension ◽

Stem Canker ◽

The United States ◽

Invasive Weed ◽

Voucher Specimen ◽

First Report ◽

Stem Lesions

Salsola tragus L. (Russian thistle) is a problematic invasive weed in the western United States and a target of biological control efforts. In September of 2007, dying S. tragus plants were found along the Azov Sea at Chushka, Russia. Dying plants had irregular, necrotic, canker-like lesions near the base of the stems and most stems showed girdling and cracking. Stem lesions were dark brown and contained brown pycnidia within and extending along lesion-free sections of the stems and basal portions of leaves. Diseased stems were cut into 3- to 5-mm pieces and disinfested in 70% ethyl alcohol. After drying, stem pieces were placed into petri dishes on the surface of potato glucose agar. Numerous, dark, immersed erumpent pycnidia with a single ostiole were observed in all lesions after 2 to 3 days. Axenic cultures were sent to the Foreign Disease-Weed Science Research Unit, USDA, ARS, Ft. Detrick, MD for testing in quarantine. Conidiophores were simple, cylindrical, and 5 to 25 × 2 μm (mean 12 × 2 μm). Alpha conidia were biguttulate, one-celled, hyaline, nonseptate, ovoid, and 6.3 to 11.5 × 1.3 to 2.9 μm (mean 8.8 × 2.0 μm). Beta conidia were one-celled, filiform, hamate, hyaline, and 11.1 to 24.9 × 0.3 to 2.5 μm (mean 17.7 × 1.2 μm). The isolate was morphologically identified as a species of Phomopsis, the conidial state of Diaporthe (1). The teleomorph was not observed. A comparison with available sequences in GenBank using BLAST found 528 of 529 identities with the internal transcribed spacer (ITS) sequence of an authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514; BPI 748435; CBS 109767). Morphology is consistent with that of Phomopsis oblonga (Desm.) Traverso, the anamorph of D. eres (2). Healthy stems and leaves of 10 30-day-old plants of S. tragus were spray inoculated with an aqueous suspension of conidia (1.0 × 106 alpha conidia/ml plus 0.1% v/v polysorbate 20) harvested from 14-day-old cultures grown on 20% V8 juice agar. Another 10 control plants were sprayed with water and surfactant without conidia. Plants were placed in an environmental chamber at 100% humidity (rh) for 16 h with no lighting at 25°C. After approximately 24 h, plants were transferred to a greenhouse at 20 to 25°C, 30 to 50% rh, and natural light. Stem lesions developed on three inoculated plants after 14 days and another three plants after 21 days. After 70 days, all inoculated plants were diseased, four were dead, and three had more than 75% diseased tissue. No symptoms occurred on control plants. The Phomopsis state was recovered from all diseased plants. This isolate of D. eres is a potential biological control agent of S. tragus in the United States. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 878717). Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2) were deposited in GenBank (Accession No. EU805539). To our knowledge, this is the first report of stem canker on S. tragus caused by D. eres. References: (1) B. C. Sutton. Page 569 in: The Coelomycetes. CMI, Kew, Surrey, UK, 1980. (2) L. E. Wehmeyer. The Genus Diaporthe Nitschke and its Segregates. University of Michigan Press, Ann Arbor, 1933.

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First Report of Phomopsis citri Associated with Dieback of Citrus lemon in India

Plant Disease ◽

10.1094/pdis-04-14-0388-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1281-1281 ◽

Cited By ~ 2

Author(s):

S. Mahadevakumar ◽

Vandana Yadav ◽

G. S. Tejaswini ◽

S. N. Sandeep ◽

G. R. Janardhana

Keyword(s):

Fungal Pathogen ◽

The United States ◽

Apical Region ◽

Post Inoculation ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Productivity Level ◽

First Report ◽

Dieback Disease

Lemon (Citrus lemon (L.) Burm. f.) is an important fruit crop cultivated worldwide, and is grown practically in every state in India (3). During a survey conducted in 2013, a few small trees in a lemon orchard near Mysore city (Karnataka) (12°19.629′ N, 76°31.892′ E) were found affected by dieback disease. Approximately 10 to 20% of trees were affected as young shoots and branches showed progressive death from the apical region downward. Different samples were collected and diagnosed via morphological methods. The fungus was consistently isolated from the infected branches when they were surface sanitized with 1.5% NaOCl and plated on potato dextrose agar (PDA). Plates were incubated at 26 ± 2°C for 7 days at 12/12 h alternating light and dark period. Fungal colonies were whitish with pale brown stripes having an uneven margin and pycnidia were fully embedded in the culture plate. No sexual state was observed. Pycnidia were globose, dark, 158 to 320 μm in diameter, and scattered throughout the mycelial growth. Both alpha and beta conidia were present within pycnidia. Alpha conidia were single celled (5.3 to 8.7 × 2.28 to 3.96 μm) (n = 50), bigittulate, hyaline, with one end blunt and other truncated. Beta conidia (24.8 to 29.49 × 0.9 to 1.4 μm) (n = 50) were single celled, filiform, with one end rounded and the other acute and curved. Based on the morphological and cultural features, the fungal pathogen was identified as Phomopsis citri H.S. Fawc. Pathogenicity test was conducted on nine healthy 2-year-old lemon plants via foliar application of a conidial suspension (3 × 106); plants were covered with polythene bags for 6 days and maintained in the greenhouse. Sterile distilled water inoculated plants (in triplicate) served as controls and were symptomless. Development of dieback symptoms was observed after 25 days post inoculation and the fungal pathogen was re-isolated from the inoculated lemon trees. The internal transcribed spacer region (ITS) of the isolated fungal genomic DNA was amplified using universal-primer pair ITS1/ITS4 and sequenced to confirm the species-level diagnosis (4). The sequence data of the 558-bp amplicon was deposited in GenBank (Accession No. KJ477016.1) and nBLAST search showed 99% homology with Diaporthe citri (teleomorph) strain 199.39 (KC343051.1). P. citri is known for its association with melanose disease of citrus in India, the United States, and abroad. P. citri also causes stem end rot of citrus, which leads to yield loss and reduction in fruit quality (1,2). Dieback disease is of serious concern for lemon growers as it affects the overall productivity level of the tree. To the best of our knowledge, this is the first report of P. citri causing dieback of lemon in India. References: (1) I. H. Fischer et al. Sci. Agric. (Piracicaba). 66:210, 2009. (2) S. N. Mondal et al. Plant Dis. 91:387, 2007. (3) S. P. Raychaudhuri. Proc. Int. Soc. Citriculture 1:461, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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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 Alternaria tenuissima Causing Postharvest Decay on Apple Fruit from Cold Storage in the United States

Plant Disease ◽

10.1094/pdis-07-13-0802-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 690-690 ◽

Cited By ~ 1

Author(s):

L. P. Kou ◽

V. L. Gaskins ◽

Y. G. Luo ◽

W. M. Jurick

Keyword(s):

United States ◽

South Africa ◽

Cold Storage ◽

Fungal Pathogens ◽

The United States ◽

Apple Fruit ◽

Conidial Suspension ◽

Single Spore ◽

Alternaria Tenuissima ◽

First Report

Apples are grown and stored for 9 to 12 months under controlled atmosphere conditions in the United States. During storage, apples are susceptible to various fungal pathogens, including several Alternaria species (2). Alternaria tenuissima (Nees) Wiltshire causes dry core rot (DCR) on apples during storage and has recently occurred in South Africa (1). Losses range widely, but typically occur at 6 to 8% annually due to this disease (2). In February 2013, ‘Nittany’ apples with round, dark-colored, dry, spongy lesions were obtained from wooden bins in a commercial cold storage facility located in Pennsylvania. Symptomatic fruits were transported to the lab, rinsed with sterile water, and the lesions were sprayed with 70% ethanol until runoff and wiped dry. The skin was aseptically removed with a scalpel, and asymptomatic tissue was placed onto potato dextrose agar (PDA) and incubated at 25°C. Two single-spore isolates were propagated on PDA and permanent cultures were maintained as slants and stored at 4°C. The fungus produced a cottony white mycelium that turned olive-green to brown with abundant aerial hyphae and had a dark brown to black reverse on PDA. Isolates were identified as Alternaria based on conidial morphology as the spores were slightly melanized and obclavate to obpyriform catentulate with longitudinal and transverse septa attached in unbranched chains on simple short conidiophores. Conidia ranged from 10 to 70 μm long (mean 27.7 μm) and 5 to 15 μm wide (mean 5.25 μm) (n = 50) with 1 to 6 transverse and 0 to 2 longitudinal septa. Conidial beaks, when present, were short (5 μm or less) and tapered. Mycelial genomic DNA was extracted, and a portion of the histone gene (357 bp) was amplified via gene specific primers (Alt-His3-F/R) using conventional PCR (Jurick II, unpublished). The forward and reverse sequences were assembled into a consensus representing 2× coverage and MegaBLAST analysis showed that both isolates were 100% identical to Alternaria tenuissima isolates including CR27 (GenBank Accession No. AF404622.1) that caused DCR on apple fruit during storage in South Africa. Koch's postulates were conducted using 10 organic ‘Gala’ apple fruit that were surface sterilized with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were aseptically wounded with a nail to a 3 mm depth, inoculated with 50 μl of a conidial suspension (1 × 104 conidia/ml), and stored at 25°C in 80 count boxes on paper trays for 21 days. Mean lesion diameters on inoculated ‘Gala’ apple fruit were 19.1 mm (±7.4), water only controls (n = 10 fruit) were symptomless, and the experiment was repeated. Symptoms observed on artificially inoculated ‘Gala’ apple fruit were similar to the decay observed on ‘Nittany’ apples from cold storage. Based on our findings, it is possible that A. tenuissima can cause decay that originates from wounded tissue in addition to dry core rot, which has been reported (1). Since A. tenuissima produces potent mycotoxins, even low levels of the pathogen could pose a health problem for contaminated fruit destined for processing and may impact export to other countries. To the best of our knowledge, this is the first report of alternaria rot caused by A. tenuissima on apple fruit from cold storage in the United States. References: (1) J. C. Combrink et al. Decid. Fruit Grow. 34:88, 1984. (2) M. Serdani et al. Mycol. Res. 106:562, 2002. (3) E. E. Stinson et al. J. Agric. Food Chem. 28:960, 1980.

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First Report of Anthracnose of Salsola tragus Caused by Colletotrichum gloeosporioides in Greece

Plant Disease ◽

10.1094/pd-90-0971b ◽

2006 ◽

Vol 90 (7) ◽

pp. 971-971 ◽

Cited By ~ 4

Author(s):

D. K. Berner ◽

C. A. Cavin ◽

M. B. McMahon ◽

I. Loumbourdis

Keyword(s):

Biological Control ◽

Colletotrichum Gloeosporioides ◽

Type A ◽

The United States ◽

Ionic Surfactant ◽

Potential Candidate ◽

Conidial Suspension ◽

Type B ◽

First Report ◽

Stems And Leaves

In early October of 2005, dying Salsola tragus L. (Russian thistle, tumbleweed), family Chenopodiaceae, plants were found along the Aegean Sea at Kryopigi Beach, Greece (40°02′29″N, 23°29′02″E, elevation 0 m). All of the 30 to 40 plants in the area were diseased and approximately 80% were dead or dying. All plants were relatively large (approximately 1 m tall × 0.5 m diameter), and living portions of diseased plants were flowering. Dying plants had irregular, necrotic lesions extending the length of the stems. Leaves of these plants were also necrotic. Lesions on stems and leaves were dark brown and usually coalesced. Diseased stem pieces were taken to the European Biological Control Laboratory, USDA, ARS at the American Farm School in Thessaloniki, Greece. There, diseased stem pieces were surface disinfested for 15 min with 0.5% NaOCl and placed on moist filter paper in petri dishes. Numerous, waxy subepidermal acervuli with black setae were observed in all lesions after 2 to 3 days. Conidiophores were simple, short, and erect. Conidia were one-celled, hyaline, ovoid to oblong, falcate to straight, 12.9 to 18.0 × 2.8 to 5.5 μm (mode 16.1 × 4.5 μm). These characters conformed to the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. (2). Conidia were placed on modified potato carrot agar and axenic cultures from these isolations were sent to the quarantine facility of the Foreign Disease-Weed Science Research Unit, USDA, ARS, Fort Detrick, MD for testing. On the basis of DNA sequences, two variants within S. tragus have been described in California and named “Type A” and “Type B” (1). Conidia were harvested from 14-day-old cultures grown on 20% V8 juice agar, and healthy stems and leaves of 18 30-day-old plants of S. tragus Type A and 10 Type B plants were spray inoculated with an aqueous conidial suspension (1.0 × 106 conidia/ml plus 0.1% non-ionic surfactant). Three control plants of each type were sprayed with water and surfactant only. Plants were placed in an environmental chamber (18 h of dew in darkness at 25°C). After 1 day, all plants were transferred to a greenhouse (20 to 25°C, 30 to 50% relative humidity, and natural light augmented with 12-h light periods with 500-W sodium vapor lights). Lesions developed on stems of inoculated Type A plants after 5 days. After 14 days, all inoculated Type A plants were dead. Lesions on Type B plants were small and localized; all plants were diseased but no plants died. No symptoms occurred on control plants. C. gloeosporioides was reisolated 14 to 21 days after inoculation from stem pieces of all inoculated plants of both types of S. tragus. This isolate of C. gloeosporioides is a destructive pathogen on S. tragus Type A and is a potential candidate for biological control of this weed in the United States. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on S. tragus in Greece. A voucher specimen has been deposited with the U.S. National Fungus Collections, Beltsville, MD (BPI 871126). Nucleotide sequences for the internal transcribed spacers (ITS 1 and 2) were deposited in GenBank (Accession No. DQ344621) and exactly matched sequences of the teleomorph, Glomerella cingulata. References: (1) F. Ryan and D. Ayres. Can. J. Bot. 78:59, 2000. (2) B. C. Sutton. Page 15 in: Colletotrichum Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International Mycological Institute, Wallingford, UK, 1992.

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First Report of Postharvest Fruit Rot in Persimmon Caused by Phacidiopycnis washingtonensis in Italy

Plant Disease ◽

10.1094/pdis-94-6-0788b ◽

2010 ◽

Vol 94 (6) ◽

pp. 788-788 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

M. T. Amatulli ◽

M. L. Gullino

Keyword(s):

United States ◽

Its Region ◽

The United States ◽

Fruit Rot ◽

Diospyros Kaki ◽

Pathogenicity Test ◽

Conidial Suspension ◽

First Report ◽

Pathogenicity Tests ◽

Phacidiopycnis Washingtonensis

Persimmon (Diospyros kaki L.) is widely grown in Italy, the leading producer in Europe. In the fall of 2009, a previously unknown rot was observed on 3% of fruit stored at temperatures between 5 and 15°C in Torino Province (northern Italy). The decayed area was elliptical, firm, and appeared light brown to dark olive-green. It was surrounded by a soft margin. The internal decayed area appeared rotten, brown, and surrounded by bleached tissue. On the decayed tissue, black pycnidia that were partially immersed and up to 0.5 mm in diameter were observed. Light gray conidia produced in the pycnidia were unicellular, ovoid or lacriform, and measured 3.9 to 6.7 × 2.3 to 3.5 (average 5.0 × 2.9) μm. Fragments (approximately 2 mm) were taken from the margin of the internal diseased tissues, cultured on potato dextrose agar (PDA), and incubated at temperatures between 23 and 26°C under alternating light and darkness. Colonies of the fungus initially appeared ash colored and then turned to dark greenish gray. After 14 days of growth, pycnidia and conidia similar to those described on fruit were produced. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 502-bp segment showed a 100% similarity with the sequence of Phacidiopycnis washingtonensis Xiao & J.D. Rogers (GenBank Accession No. AY608648). The nucleotide sequence has been assigned the GenBank Accession No. GU949537. Pathogenicity tests were performed by inoculating three persimmon fruits after surface disinfesting in 1% sodium hypochlorite and wounding. Mycelial disks (10 mm in diameter), obtained from PDA cultures of one strain were placed on wounds. Three control fruits were inoculated with plain PDA. Fruits were incubated at 10 ± 1°C. The first symptoms developed 6 days after the artificial inoculation. After 15 days, the rot was very evident and P. washingtonensis was consistently reisolated. Noninoculated fruit remained healthy. The pathogenicity test was performed twice. Since P. washingtonensis was first identified in the United States on decayed apples (2), ‘Fuji’, ‘Gala’, ‘Golden Delicious’, ‘Granny Smith’, ‘Red Chief’, and ‘Stark Delicious’, apple fruits also were artificially inoculated with a conidial suspension (1 × 106 CFU/ml) of the pathogen obtained from PDA cultures. For each cultivar, three surface-disinfested fruit were wounded and inoculated, while three others served as mock-inoculated (sterile water) controls. Fruits were stored at temperatures ranging from 10 to 15°C. First symptoms appeared after 7 days on all the inoculated apples. After 14 days, rot was evident on all fruit inoculated with the fungus, and P. washingtonensis was consistently reisolated. Controls remained symptomless. To our knowledge, this is the first report of the presence of P. washingtonensis on persimmon in Italy, as well as worldwide. The occurrence of postharvest fruit rot on apple caused by P. washingtonensis was recently described in the United States (3). In Italy, the economic importance of the disease on persimmon fruit is currently limited, although the pathogen could represent a risk for apple. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) Y. K. Kim and C. L. Xiao. Plant Dis. 90:1376, 2006. (3) C. L. Xiao et al. Mycologia 97:473, 2005.

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