First Report of Diplodia seriata Causing Pear Branch Canker Dieback in California

California produces 26% of the United States pear crop on approximately 5,600 ha. A survey of seven northern California pear orchards (Pyrus communis cv. Bartlett) in summer 2010 revealed the presence of wedge-shaped cankers on 2- to 5-cm diameter branches, equating to 1- to 3-year-old wood. Many of the observed cankers occurred near pruning wounds, and there was decreased foliation on infected branches. Infected wood was surface disinfected with 95% ethanol and briefly flamed. After removing bark, small sections of diseased tissue were plated onto 4% potato dextrose agar (PDA) amended with 0.01% tetracycline and placed on the lab bench at 22°C until fungal growth emerged. Fungal colonies that were consistently isolated were transferred to fresh PDA using hyphal tip isolation. Fungal colonies were dark brown to gray with aerial mycelium and formed pycnidia after 15 days of incubation at 22°C. Conidia were brown, oval to oblong, and measured (16.5-) 20 to 24 (-26) × (7.5) 8.75 to 11 (-12.5) μm (n = 50). DNA from 14- to 21-day-old colonies was extracted and sequences of the rDNA internal transcribed spacer region and part of the β-tubulin gene were amplified using primers ITS4/ITS5 and Bt2a/Bt2b, respectively (2). The DNA sequences of fungal isolates from California showed 99 to 100% homology with the ex-type Diplodia seriata De Not. (1) CBS112555 deposited in GenBank. DNA sequences from three California isolates were submitted to GenBank with accession numbers KC937062, KC937065, KF481957, KF481598, KF481959, and KF481960. Pathogenicity tests were performed in March 2011 on 3-year-old Bartlett pear trees planted at an experimental farm in Davis, CA. A single, circular, 2-cm pruning wound at the top of the trunk was inoculated on each of three single-tree replications using 2-cm mycelial plugs from 14-day-old colonies growing on PDA. After inoculation, mycelial plugs were covered and sealed with Parafilm and aluminum foil for the duration of the trial. Three control trees were inoculated using sterile PDA plugs. Twelve months after inoculation, UCD103 and UCD105 were consistently re-isolated from the margin between necrotic and healthy tissue using the same methods described for the original isolation, and UCD102 was re-isolated in two out of three plants. The average lesion lengths of UCD102, UCD103, UCD105, and control plants were 12.5, 17.3, 23, and 1 mm, respectively. Control lesions were short and sterile, and seemed to be a physiological reaction from the plant. A second pathogenicity test was completed in 5 months beginning in June 2012. UCD105 was consistently re-isolated, and UCD102 and UCD103 were re-isolated in two out of three plants. The average lesion lengths for UCD102, UCD103, UCD105, and control plants were 2, 3, 5, and 1 mm, respectively. Compared to grapevine (Vitis vinifera), the pathogen grows more slowly in pear tissue under natural conditions. To our knowledge, this is the first report describing D. seriata as a causal agent of pear branch canker in California. Canker diseases can reduce the lifespan of perennial plants, ultimately leading to long term economic losses for growers (3). References: (1) A. J. L. Phillips et al. Fungal Diversity 25:141, 2007. (2) J. R. Urbez-Torres et al. Plant Dis. 90:1490, 2006. (3) J. R. Urbez-Torres and W. D. Gubler. Plant Dis. 93:584, 2009.

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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 Pectobacterium wasabiae Causing Soft Rot of Cabbage in Malaysia

Plant Disease ◽

10.1094/pdis-01-13-0112-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1110-1110 ◽

Cited By ~ 2

Author(s):

E. Golkhandan ◽

K. Sijam ◽

S. Meon ◽

Z. A. M. Ahmad ◽

A. Nasehi ◽

...

Keyword(s):

16S Rrna ◽

Dna Sequences ◽

Acid Production ◽

Soft Rot ◽

Economic Losses ◽

Economic Damage ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

First Report ◽

Carotovorum Subsp

Soft rot of cabbage (Brassica rapa) occurs sporadically in Malaysia, causing economic damage under the hot and wet Malaysian weather conditions that are suitable for disease development. In June 2011, 27 soft rotting bacteria were isolated from cabbage plants growing in the Cameron Highlands and Johor State in Malaysia where the economic losses exceeded 50% in severely infected fields and greenhouses. Five independent strains were initially identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and elicit hypersensitive reaction (HR) on Nicotiana tabaccum and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG-positive and positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-D-glucoside, and D-arabitol. All the strains exhibited pectolytic activity on potato slices. PCR assays were conducted to distinguish P. wasabiae from P. carotovorum subsp. brasiliensis, P. atrosepticum, and other Pectobacterium species using primers Br1f/L1r (2), Eca1f/Eca2r (1), and EXPCCF/EXPCCR, respectively. DNA from strains did not yield the expected amplicon with the Br1f/L1r and Eca1f/Eca2r, whereas a 550-bp amplicon typical of DNA from P. wasabiae was produced with primers EXPCCF/EXPCCR. ITS-RFLP using the restriction enzyme, Rsa I, produced similar patterns for the Malaysian strains and the P. wasabiae type strain (SCRI488), but differentiated it from P. carotovora subsp. carotovora, P. atrosepticum, P. carotovorum subsp. brasiliensis, and Dickeya chrysanthemi type strains. BLAST analysis of the 16S rRNA DNA sequence (GenBank Accession No. KC445633) showed 99% identity to the 16S rRNA of Pw WPP163. Phylogenetic reconstruction using concatenated DNA sequences of mdh and gapA from P. wasabiae Cc6 (KC484657) and other related taxa (4) clustered Malaysian P. wasabiae strains with P. wasabiae SCRI488, readily distinguishing it from other closely related species of Pectobacterium. Pathogenicity assays were conducted on leaves and stems of four mature cabbage plants for each strain (var. oleifera) by injecting 10 μl of a bacterial suspension (108 CFU/ml) into either stems or leaves, and incubating them in a moist chamber at 80 to 90% relative humidity at 30°C. Water-soaked lesions similar to those observed in the fields and greenhouses were observed 72 h after injection and bacteria with similar characteristics were consistently reisolated. Symptoms were not observed on water-inoculated controls. The pathogenicity test was repeated with similar results. P. wasabiae was previously reported to cause soft rot of horseradish in Japan (3). However, to our knowledge, this is the first report of P. wasabiae infecting cabbage in Malaysia. References: (1) S. H. De Boer and L. J. Ward. Phytopathology 85:854, 1995. (2) V. Duarte et al. J. Appl. Microbiol. 96:535, 2004. (3) M. Goto and K. Matsumoto. Int. J. Syst. Bacteriol. 37:130, 1987. (4) B. Ma et al. Phytopathology 97:1150, 2007.

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First Report of Botryosphaeria dothidea Causing Canker and Shoot Blight of Eucalyptus in China

Plant Disease ◽

10.1094/pdis-93-7-0764c ◽

2009 ◽

Vol 93 (7) ◽

pp. 764-764 ◽

Cited By ~ 6

Author(s):

L. Yu ◽

X. L. Chen ◽

L. L. Gao ◽

H. R. Chen ◽

Q. Huang

Keyword(s):

Aerial Mycelium ◽

The United States ◽

Eucalyptus Species ◽

Botryosphaeria Dothidea ◽

Internal Transcribed Spacer Region ◽

Middle Point ◽

First Report ◽

Eucalyptus Wood ◽

Shoot Blight ◽

And Control

Eucalyptus spp. are grown on approximately 2 million ha in China and is the most important genus in forest plantations worldwide. An outbreak of cankers and shoot blight was observed for the first time on Eucalyptus grandis in China during May of 2008. Symptoms included dieback of shoots and branches, lesions and canker formation on the stems, and clear or yellow-to-red exudates on stems and branches. Isolations were made from affected trees in Shilin County from May to July of 2008. Diseased samples were plated on potato dextrose agar (PDA) and incubated at 26°C. Fungal isolates developed copious, white, aerial mycelium that became dark gray after 5 to 6 days and formed black pycnidia after 14 days. Conidia were hyaline, aseptate, thin walled, fusiform, and measured 19 to 28 × 4 to 6 μm. Ascospores were hyaline, aseptate, and widest from the middle to upper third (17 to 28 × 6 to 13 μm). Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2) with primers ITS1 and ITS4. BLAST searches showed 99 to 100% identity with Botryosphaeria dothidea isolates from GenBank (Accession Nos. FJ358703 and EU080916). Representative sequences of B. dothidea from eucalyptus from China were deposited into GenBank (Accession Nos. FJ517657 and FJ517658). On the basis of morphological and molecular results, the fungus isolated from diseased eucalyptus wood was confirmed to be B. dothidea. Pathogenicity tests were conducted by stem inoculation of 10-month-old E. grandis seedlings. Two experiments were conducted using two inoculation techniques. In the first experiment, 2-mm-diameter actively growing mycelium plugs of B. dothidea were applied to 2-mm-long bark wounds on the middle point of the stems, and control seedlings were inoculated with sterile PDA plugs in a similar fashion as above. Inoculated and control seedlings were inoculated in a greenhouse and watered as needed. In the second experiment, segments of branches (averaging 18 mm in diameter and 30 cm long) were inoculated with 5-mm-diameter plugs of actively growing mycelium. Control segments of branches were inoculated as previously described. The branches were incubated at 26°C in moist chambers. There were five replicate seedlings per inoculation technique. After 20 days, all E. grandis seedlings showed leaf wilting, Dark, vascular stem tissue was observed. Symptoms were more abundant on the segment of branches. After 6 days, vascular necroses that developed on the inoculated plants were 5.2 ± 1.2 cm. B. dothidea was reisolated from all inoculated symptomatic tissue; no symptoms were visible in the control seedlings and no fungus was isolated from them. These results confirm previous reports of B. dothidea causing canker and dieback symptoms of Eucalyptus species in Australia (2), the United States (1), and South Africa (3). To our knowledge, this is the first report of B. dothidea causing canker disease on eucalyptus in China. References: (1) E. L. Barnard et al. Plant Dis. 71:358, 1987. (2) E. M. Davison and C. S. Tay. Plant Dis. 67:1285, 1983. (3) H. Smith et al. Plant Pathol. 43:1031, 1994.

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Presence of Colletotrichum acutatum Causing Leaf Spot on Azalea japonica in Italy

Plant Disease ◽

10.1094/pdis.2004.88.5.572a ◽

2004 ◽

Vol 88 (5) ◽

pp. 572-572

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

O. Vinnere ◽

M. L. Gullino

Keyword(s):

The United States ◽

Colletotrichum Acutatum ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Plastic Bags ◽

Commercial Farms ◽

And Control ◽

Selection Of

Rhododendron cultivation has a long history in northern Italy where a wide selection of varieties and hybrids are grown. In the fall of 2002, a previously unknown disease was observed on Azalea japonica cv. Palestrina in several commercial farms in the Province of Verbania. Leaves showed irregular necrotic areas that were 1 mm in diameter. Lesions were dark brown to black and were surrounded by a chlorotic halo. Eventually, lesions coalesced, forming large irregular spots. Heavily infected leaves fell prematurely, resulting in severe defoliation. On the infected leaves, acervuli were present from which cylindrical tapered conidia measuring 4.8 to 7.2 × 11.0 to 22.8 μm at one end were released. Fifty conidia per isolate were measured. Fungus identified as Colletotrichum acutatum was consistently recovered from infected leaves, disinfested in 1% NaOCl for 1 min, and plated on potato dextrose agar amended with 100 mg/l of streptomycin sulfate. Pathogenicity of three fungal isolates was confirmed by inoculating healthy A. japonica (cvs. Palestrina and Snow) plants grown in plastic pots (18-cm diameter, 3 liters). Plants (five per treatment) were sprayed with a conidial suspension (1 × 106 conidia per ml) of the three isolates of C. acutatum. Noninoculated plants served as a control. Inoculated and control plants were covered with plastic bags to maintain high relative humidity conditions. All plants were maintained in growth chambers at 20 ± 1°C (12 h per day of fluorescent light). Six days after the artificial inoculation, plants developed typical symptoms on the leaves. C. acutatum was consistently reisolated from infected plants. The pathogenicity test was carried out twice. Sequencing of the internal transcribed spacer region of the rDNA and a portion of the β-tubulin gene were performed, and the obtained sequences were compared with those available in GenBank. Identification of the fungus as C. acutatum, therefore was confirmed. To our knowledge, this is the first report of the presence of C. acutatum on A. japonica in Italy. Although observed presently only in a few nurseries, the disease has the potential to spread, becoming more relevant in an area where rhododendron cultivation is economically important. Anthracnose on rhododendron has already been described in several countries (1,2). References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (2) O. Vinnere et al. Mycol. Res. 106:60, 2002.

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

Plant Disease ◽

10.1094/pdis-05-14-0474-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1741-1741

Author(s):

A. C. Scruggs ◽

S. C. Butler ◽

L. M. Quesada-Ocampo

Keyword(s):

North Carolina ◽

Leaf Spot ◽

Fungal Growth ◽

The United States ◽

Hot Water ◽

Economic Losses ◽

Fluorescent Light ◽

Single Spore ◽

First Report ◽

Infected Seed

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

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First Report of Downy Mildew on Nursery-Grown Hellebore Caused by Peronospora pulveracea in California

Plant Disease ◽

10.1094/pdis-93-3-0319c ◽

2009 ◽

Vol 93 (3) ◽

pp. 319-319 ◽

Cited By ~ 1

Author(s):

C. Y. Warfield ◽

C. L. Blomquist ◽

E. E. Lovig

Keyword(s):

Downy Mildew ◽

The United States ◽

Early Spring ◽

Economic Losses ◽

Late Winter ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Potted Plants ◽

Rdna Sequences ◽

Young Leaves

Hellebore or Lenten rose (Helleborus × hybridus) is an evergreen, herbaceous perennial in the family Ranunculaceae. Hellebores are sold as decorative, potted plants and as shade-loving landscape plants favored for their attractive and prolonged blooms in late winter or early spring. In April of 2008, downy mildew-like growth was observed on the foliage of approximately 60 containerized plants of Helleborus ‘Blue Lady’, ‘Pink Lady’, ‘White Lady’, and ‘Royal Heritage’ grown outdoors in a retail nursery in coastal San Mateo County, California. Infected foliage had angular, vein-delimited, dark brown-to-black speckled lesions on adaxial leaf surfaces turning dry and necrotic with age. Young leaves were small and distorted. Affected flowers were spotted and brown. The abaxial sides of affected leaves had light brown-to-purplish downy mildew-like growth. Subhyaline conidia, globose to ellipsoid in shape, ranged from 25 to 31 × 17 to 24 μm (average 28 × 21 μm). Conidiophores ranged from 265 to 375 × 5 to 11.5 μm (average 333 × 8.9 μm), branching dichotomously four to five times in the upper half. Morphological measurements fell within the range previously described for Peronospora pulveracea and P. alpicola, which were reported on Helleborus spp. and Ranunculus aconitifolius, respectively (1,2). DNA sequence of the internal transcribed spacer region of rDNA of our isolate (Genbank Accession No. FJ384778) matched sequences of P. pulveracea (Genbank Accession No. AY198270) and P. alpicola (Genbank Accession No. AY198271) with 100% identity. These two organisms are taxonomically indistinguishable by rDNA sequences and are likely to be the same species (3). To our knowledge, this is the first report of P. pulveracea on Helleborus × hybridus in California and the United States. Lenten rose is commercially propagated by seed, which is a potential pathway for introduction of this pathogen. Mature plants are sold and shipped intra- and interstate as decorative flowering plants or nursery stock. The importance and economic impact of this disease is limited, but significant economic losses could occur during production. References: (1) E. A. Gäumann. Beitr. Kryptogamenflora Schweiz 5:113, 1923. (2) G. Hall. Mycopathologia 126:57, 1994. (3) H. Voglmayr. Mycol. Res. 107:1132, 2003.

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First Report of Crown and Stem Rot of Crested Molded Wax Agave (Echeveria agavoides) caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-08-12-0803-pdn ◽

2013 ◽

Vol 97 (2) ◽

pp. 288-288

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

P. Pensa ◽

A. Poli ◽

M. L. Gullino

Keyword(s):

Fusarium Oxysporum ◽

Apical Cell ◽

The United States ◽

Stem Rot ◽

Economic Losses ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Potato Dextrose Broth ◽

First Report ◽

Leaf Tissues

During March 2012, 95% of 24-month-old plants of crested molded wax agave (Echeveria agavoides), a succulent plant belonging to the Crassulaceae family, showed symptoms of a basal stem and leaf rot in a commercial farm near Ventimiglia (northern Italy). Affected plants showed extensive chlorosis from the crown level to the stem apex, followed by yellowing and by the appearance of a water-soaked aspect of stem and leaf tissues. As disease progressed, leaves became brown, wilted, and rotted. Wilting was at first unilateral and later affected the entire plant. Brown discoloration was observed in the vascular system of cut stems and leaves. In some cases, leaves were covered by a whitish-orange mycelium. This produced 3-septate, slightly curved macroconidia with a foot-shaped basal cell and a short apical cell, measuring 27.4 to 39.6 × 3.0 to 4.1 (average 34.2 × 3.7) μm and unicellular, ovoid to elliptical microconidia measuring 4.8 to 11.6 × 1.5 to 3.7 (avg. 7.2 × 2.7) μm. A fungus was consistently isolated from discolored vascular leaf tissues on Komada selective medium. Cultures on potato dextrose agar (PDA) and carnation leaf-piece agar (CLA) were incubated at 24 to 29°C. On PDA, a thin growth of whitish mycelium without pigments in the agar was observed. On CLA, sparse macroconidia, 18.9 to 30.7 × 3.0 to 4.2 (avg. 23.9 × 3.6) μm, microconidia, 4.7 to 7.7 × 1.7 to 3.1 (avg. 6.0 × 2.4) μm, and abundant chlamydospores that were single or paired, terminal and intercalary, rough walled, and 6.8 to 9.5 (avg. 7.7) in diameter were produced. Such characteristics are typical of Fusarium oxysporum (2). Amplification of the internal transcribed spacer (ITS) of the rDNA using primers ITS1/ITS4 (3) yielded a 486-bp band (GenBank Accession No. JX441893). Sequencing and BLASTn analysis of this band showed 100% identity and an E-value of 0.0 with the ITS sequence of F. oxysporum (JN232163). To confirm pathogenicity, five 3-month-old healthy plants of E. agavoides were inoculated by dipping unwounded roots in a conidial suspension (1.0 × 107 CFU/ml) of one isolate of F. oxysporum obtained from affected plants, grown for 10 days in potato dextrose broth. Plants were transplanted into pots filled with steam-sterilized substrate (sphagnum peat-perlite-pine bark-clay 50:20:20:10) and maintained in a glasshouse at 28 to 33°C. Five non-inoculated plants served as a control. Chlorosis and yellowing developed on the inoculated plants 15 days after the inoculation. Basal stem rot and vascular discoloration in the crown and stem developed within 30 days on inoculated plants. A whitish-orange mycelium producing macroconidia covered the affected leaves. Non-inoculated plants remained healthy. F. oxysporum was consistently reisolated from symptomatic plants. The pathogenicity test was conducted twice. A Fusarium sp. has been reported as the causal agent of a stem rot on Echeveria sp. in the U.S. (1). To our knowledge, this is the first report of F. oxysporum on E. agavoides in Italy. The disease is currently present in few nurseries, although it could spread, causing significant economic losses due to the increasing cultivation of E. agavoides in Italy. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. APS Press, St. Paul, MN, 1989. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

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First Report of Impatiens Downy Mildew Outbreaks Caused by Plasmopara obducens Throughout the Hawai'ian Islands

Plant Disease ◽

10.1094/pdis-10-13-1017-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 696-696 ◽

Cited By ~ 7

Author(s):

J. A. Crouch ◽

M. P. Ko ◽

J. M. McKemy

Keyword(s):

United States ◽

Downy Mildew ◽

The United States ◽

Molecular Data ◽

Economic Losses ◽

Voucher Specimen ◽

First Report ◽

Plastic Bags ◽

Leaf Surfaces ◽

Impatiens Walleriana

Downy mildew of impatiens (Impatiens walleriana Hook.f.) was first reported from the continental United States in 2004. In 2011 to 2012, severe and widespread outbreaks were documented across the United States mainland, resulting in considerable economic losses. On May 5, 2013, downy mildew disease symptoms were observed from I. walleriana ‘Super Elfin’ at a retail nursery in Mililani, on the Hawai'ian island of Oahu. Throughout May and June 2013, additional sightings of the disease were documented from the islands of Oahu, Kauai, Maui, and Hawai'i from nurseries, home gardens, and botanical park and landscape plantings. Symptoms of infected plants initially showed downward leaf curl, followed by a stippled chlorotic appearance on the adaxial leaf surfaces. Abaxial leaf surfaces were covered with a layer of white mycelia. Affected plants exhibited defoliation, flower drop, and stem rot as the disease progressed. Based on morphological and molecular data, the organism was identified as Plasmopara obducens (J. Schröt.) J. Schröt. Microscopic observation disclosed coenocytic mycelium and hyaline, thin-walled, tree-like (monopodial branches), straight, 94.0 to 300.0 × 3.2 to 10.8 μm sporangiophores. Ovoid, hyaline sporangia measuring 11.0 to 14.6 × 12.2 to 16.2 (average 13.2 × 14.7) μm were borne on sterigma tips of rigid branchlets (8.0 to 15.0 μm) at right angle to the main axis of the sporangiophores (1,3). Molecular identification of the pathogen was conducted by removing hyphae from the surface of three heavily infected leaves using sterile tweezers, then extracting DNA using the QIAGEN Plant DNA kit (QIAGEN, Gaithersburg, MD). The nuclear rDNA internal transcribed spacer was sequenced from each of the three samples bidirectionally from Illustra EXOStar (GE Healthcare, Piscataway, NJ) purified amplicon generated from primers ITS1-O and LR-0R (4). Resultant sequences (GenBank KF366378 to 80) shared 99 to 100% nucleotide identity with P. obducens accession DQ665666 (4). A voucher specimen (BPI892676) was deposited in the U.S. National Fungus Collections, Beltsville, MD. Pathogenicity tests were performed by spraying 6-week-old impatiens plants (I. walleriana var. Super Elfin) grown singly in 4-inch pots with a suspension of 1 × 104 P. obducens sporangia/ml until runoff using a handheld atomizer. Control plants were sprayed with distilled water. The plants were kept in high humidity by covering with black plastic bags for 48 h at 20°C, and then maintained in the greenhouse (night/day temperature of 20/24°C). The first symptoms (downward curling and chlorotic stippling of leaves) and sporulation of the pathogen on under-leaf surfaces of the inoculated plants appeared at 10 days and 21 days after inoculation, respectively. Control plants remained healthy. Morphological features and measurements matched those of the original inoculum, thus fulfilling Koch's postulates. To our knowledge, this is the first report of downy mildew on I. walleriana in Hawai'i (2). The disease appears to be widespread throughout the islands and is likely to cause considerable losses in Hawai'ian landscapes and production settings. References: (1) O. Constantinescu. Mycologia 83:473, 1991. (2) D. F. Farr and A. Y. Rossman. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ July 16, 2013. (3) P. A. Saccardo. Syllogue Fungorum 7:242, 1888. (4) M. Thines. Fungal Genet Biol 44:199, 2007.

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First Report of Stem and Foliage Blight of Chrysanthemum Caused by Phytophthora drechsleri in the United States

Plant Disease ◽

10.1094/pdis-03-21-0631-pdn ◽

2021 ◽

Author(s):

Charles Krasnow ◽

Nancy Rechcigl ◽

Jennifer Olson ◽

Linus Schmitz ◽

Steven N. Jeffers

Keyword(s):

United States ◽

South Carolina ◽

Sequence Similarity ◽

Morphological Characteristics ◽

The United States ◽

Universal Primers ◽

Broad Host Range ◽

Pathogenicity Test ◽

First Report ◽

Foliage Blight

Chrysanthemum (Chrysanthemum × morifolium) plants exhibiting stem and foliage blight were observed in a commercial nursery in eastern Oklahoma in June 2019. Disease symptoms were observed on ~10% of plants during a period of frequent rain and high temperatures (26-36°C). Dark brown lesions girdled the stems of symptomatic plants and leaves were wilted and necrotic. The crown and roots were asymptomatic and not discolored. A species of Phytophthora was consistently isolated from the stems of diseased plants on selective V8 agar (Lamour and Hausbeck 2000). The Phytophthora sp. produced ellipsoid to obpyriform sporangia that were non-papillate and persistent on V8 agar plugs submerged in distilled water for 8 h. Sporangia formed on long sporangiophores and measured 50.5 (45-60) × 29.8 (25-35) µm. Oospores and chlamydospores were not formed by individual isolates. Mycelium growth was present at 35°C. Isolates were tentatively identified as P. drechsleri using morphological characteristics and growth at 35°C (Erwin and Ribeiro 1996). DNA was extracted from mycelium of four isolates, and the internal transcribed spacer (ITS) region was amplified using universal primers ITS 4 and ITS 6. The PCR product was sequenced and a BLASTn search showed 100% sequence similarity to P. drechsleri (GenBank Accession Nos. KJ755118 and GU111625), a common species of Phytophthora that has been observed on ornamental and vegetable crops in the U.S. (Erwin and Ribeiro 1996). The gene sequences for each isolate were deposited in GenBank (accession Nos. MW315961, MW315962, MW315963, and MW315964). These four isolates were paired with known A1 and A2 isolates on super clarified V8 agar (Jeffers 2015), and all four were mating type A1. They also were sensitive to the fungicide mefenoxam at 100 ppm (Olson et al. 2013). To confirm pathogenicity, 4-week-old ‘Brandi Burgundy’ chrysanthemum plants were grown in 10-cm pots containing a peat potting medium. Plants (n = 7) were atomized with 1 ml of zoospore suspension containing 5 × 103 zoospores of each isolate. Control plants received sterile water. Plants were maintained at 100% RH for 24 h and then placed in a protected shade-structure where temperatures ranged from 19-32°C. All plants displayed symptoms of stem and foliage blight in 2-3 days. Symptoms that developed on infected plants were similar to those observed in the nursery. Several inoculated plants died, but stem blight, dieback, and foliar wilt were primarily observed. Disease severity averaged 50-60% on inoculated plants 15 days after inoculation. Control plants did not develop symptoms. The pathogen was consistently isolated from stems of symptomatic plants and verified as P. drechsleri based on morphology. The pathogenicity test was repeated with similar results. P. drechsleri has a broad host range (Erwin and Ribeiro 1996; Farr et al. 2021), including green beans (Phaseolus vulgaris), which are susceptible to seedling blight and pod rot in eastern Oklahoma. Previously, P. drechsleri has been reported on chrysanthemums in Argentina (Frezzi 1950), Pennsylvania (Molnar et al. 2020), and South Carolina (Camacho 2009). Chrysanthemums are widely grown in nurseries in the Midwest and other regions of the USA for local and national markets. This is the first report of P. drechsleri causing stem and foliage blight on chrysanthemum species in the United States. Identifying sources of primary inoculum may be necessary to limit economic loss from P. drechsleri.

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First Report of a Leaf Spot on Conyza sumatrensis Caused by Phoma macrostoma in China

Plant Disease ◽

10.1094/pdis-03-11-0228 ◽

2012 ◽

Vol 96 (1) ◽

pp. 148-148 ◽

Cited By ~ 1

Author(s):

J. Liu ◽

H. D. Luo ◽

W. Z. Tan ◽

L. Hu

Keyword(s):

Leaf Spot ◽

Fungal Pathogens ◽

Biological Diversity ◽

Biocontrol Agent ◽

Continuous Light ◽

The United States ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Dry Land ◽

First Report

Conyza sumatrensis (Asteraceae), an annual or biennial plant, is native to North and South America. It is an invasive, noxious weed that is widespread in southern and southeastern China. It invades farm land and causes great losses to dry land crops, including wheat, corn, and beans. It also reduces biological diversity by crowding out native plants in the infested areas (3,4). During a search for fungal pathogens that could serve as potential biological control agents of C. sumatrensis, a leaf spot disease was observed in 2010 in Chongqing, China. An isolate (SMBC22) of a highly virulent fungus was obtained from diseased leaves. Pathogenicity tests were performed by placing 6-mm-diameter mycelial disks of 7-day-old potato dextrose agar (PDA) cultures of SMBC22 on leaves of 15 healthy greenhouse-grown plants of C. sumatrensis; the same number of control plants was treated with sterile PDA disks. Treated plants were covered with plastic bags for 24 h and maintained in a growth chamber with daily average temperatures of 24 to 26°C, continuous light (3,100 lux), and high relative humidity (>90%). Lesions similar to those observed in the field were first obvious on the SMBC22-inoculated leaves 3 days after inoculation. Symptoms became severe 7 to 9 days after inoculation. Control plants remained healthy. The fungus was reisolated from inoculated and diseased leaves and it was morphologically the same as SMBC22. The pathogenicity test was conducted three times. A survey of 10 southern and southeastern Chinese provinces revealed that the disease was widespread and it attacked leaves and stems of seedlings and mature plants of C. sumatrensis. Lesions on leaves were initially small, circular, and water soaked. The typical lesion was ovoid or fusiform, dark brown, and surrounded by a yellow halo. The spots coalesced to form large lesions and plants were often completely blighted. Fungal colonies of SMBC22 on PDA plates were initially white and turned dark gray. Colonies were circular with smooth edges with obvious rings of pycnidia on the surface. Aerial hyphae were short and dense. Pycnidia, black and immersed or semi-immersed in the medium, were visible after 12 days of incubation. Pycnidia were 72 to 140 μm in diameter. Conidia were produced in the pycnidia and were hyaline, unicellular, ellipsoidal, and 4.4 to 6.1 × 1.6 to 2.2 μm. To confirm identification of the fungus, genomic DNA was extracted from mycelia of a 7-day-old culture on PDA at 25°C (2). The internal transcribed spacer (ITS) gene of rDNA was amplified using primers ITS4/ITS5. The gene sequence was 524 bp long and registered in NCBI GenBank (No. HQ645974). BLAST analysis showed that the current sequence had 99% homology to an isolate of Phoma macrostoma (DQ 404792) from Cirsium arvense (Canada thistle) in Canada and reported to cause chlorotic symptoms on that host plant (1). To our knowledge, this is the first report of P. macrostoma causing disease on C. sumatrensis in China. P. macrostoma, thought of as a biocontrol agent of broadleaf weeds in Canada, has been patented in the United States. The current isolate of P. macrostoma is considered as a potential biocontrol agent of C. sumatrensis. References: (1) P. R. Graupner et al. J. Nat. Prod. 66:1558, 2004. (2) S. Takamatsu et al. Mycoscience 42:135, 2001. (3) W. Z. Tan et al. Page 177 in: Manual of Emergency Control Technology Invasive Pests in China. G. L. Zhang, ed. Science Press, Beijing, 2010. (4) C. Wang et al. J. Wuhan Bot. Res. 28:90, 2010.

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