First Report of Crown Gall Caused by Agrobacterium tumefaciens on Euphorbia esula/virgata in Europe

Hypertrophy and hyperplasia resembling crown galls were found on roots of Euphorbia esula/virgata at a single site in east-central Hungary in 2005. E. esula/virgata, known as leafy spurge in North America, is an invasive species causing substantial economic losses to the value of grazing lands in the Northern Great Plains of the United States and is the target of biological control. E. esula/virgata is widely distributed throughout Eurasia and is found on ditch banks, along roadsides, and in other noncultivated areas in its native range. Large galls on roots resembling crown gall were first noted in 1992 on plants collected for phylogenetic studies from three locations in east-central Hungary. One of these sites was relocated during a 2005 survey and galls were collected from infected plants. Galls were diced and incubated overnight in tubes containing 3 ml of sterile water at room temperature (20 to 25°C). The supernatant was streaked onto plates of potato dextrose agar (PDA), medium 1A, medium 2E, and Roy/Sasser medium. After 7 days, colonies were picked and streaked and subsequently purified on PDA. Of 104 isolates used to inoculate three sunflower plants each (by puncturing roots just below the soil line with a sterile dissecting needle holding a drop of fluid matrix containing bacterial cells), 35 caused galls. Thirty-three isolates were randomly selected from the 104 and used to inoculate three tomato plants each at the soil line. Seventeen caused galls, including two isolates that did not cause galls on sunflower. Finally, none of 20 randomly selected isolates caused galls on kalanchoe plants (Kalanchoe blossfeldiana). Three isolates, which formed the largest galls on sunflower, were used to inoculate five plants of E. esula/virgata growing in a 1:1:1 (peat/sand/Bozeman silt loam) potting mix. The tests were repeated. Galls were visible on inoculated plants within 6 weeks. Diagnostic biochemical tests done prior to and after reisolation indicated that the causal agent was Agrobacterium tumefaciens, which differed from A. rhizogenes in the production of alkali from litmus milk, a positive reaction for the ferric ammonium citrate and 3-ketolactose tests, and negative reactions for tests to detect the production of acid from erythritol and alkali from malonic, l-tartaric, and mucic acid. The three isolates of A. tumefaciens from E. esula/virgata had identical sequences and clustered most closely (99.8 to 99.9% similarity) with five isolates of A. tumefaciens from Tibet and Japan on the basis of cluster analysis using 16S rRNA sequences. Crown gall of E. esula/virgata has also been found in Montana and western North Dakota, and isolates were identified as A. tumefaciens biovars 1 and 2 (1) (the latter is now known as A. rhizogenes). To our knowledge, this the first report of crown gall on E. esula/virgata in Europe. Reference: (1) A. J. Caesar. Plant Dis. 78:796, 1994.

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First Report of Agrobacterium vitis as the Causal Agent of Grapevine Crown Gall in Serbia

Plant Disease ◽

10.1094/pdis-07-11-0600 ◽

2012 ◽

Vol 96 (2) ◽

pp. 286-286 ◽

Cited By ~ 2

Author(s):

N. Kuzmanović ◽

A. Ćalić ◽

M. Ivanović ◽

K. Gašić ◽

J. Pulawska ◽

...

Keyword(s):

Crown Gall ◽

Causal Agent ◽

Ammonium Citrate ◽

Ferric Ammonium Citrate ◽

23S Rrna ◽

Rrna Gene ◽

Agrobacterium Vitis ◽

First Report ◽

Crown Gall Disease ◽

Grapevine Crown Gall

In November 2010, a serious outbreak of crown gall disease was observed on 3-year-old grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon grafted onto Kober 5BB rootstock in two commercial vineyards located in the South Banat District in Serbia. Large, aerial tumors were visible above the grafting point on grapevine trunks, and in most cases, the tumors completely girdled the trunk. From the gall tissues, white, circular, and glistening bacterial colonies were isolated on yeast mannitol agar medium. Eight, nonfluorescent, gram-negative, and oxidase-positive strains were isolated from seven tumor samples and selected for further identification. PCR assays with A/C′ (1) and VCF3/VCR3 (4) primers corresponding to the virD2 and virC genes yielded 224- and 414-bp fragments, respectively, confirming that the strains harbored the plasmid responsible for pathogenicity. The strains were differentiated to the species/biovar level with a multiplex PCR assay targeting 23S rRNA gene sequences (3) and were identified as Agrobacterium vitis. The 16S rDNA gene sequence from one isolate (GenBank Accession No. JN185718) showed 99% identity to the sequences of A. vitis previously deposited in NCBI GenBank database. The physiological and biochemical test results corresponded to the results of genetic analysis (2). The strains grew at 35°C and in nutrient broth supplemented with 2% NaCl. They were negative in 3-ketolactose, acid clearing on PDA supplemented with CaCO3, and ferric ammonium citrate tests; nonmotile at pH 7.0; pectolytic at pH 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Pathogenicity was confirmed by inoculation of three plants per bacterial strain on grapevine cv. Cabernet Franc and on a local cultivar of tomato (Lycopersicon esculentum L.). The plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as a negative control. Inoculated plants were maintained in a greenhouse at 24 ± 3°C. Typical tumors developed at the inoculation sites on tomatoes 3 weeks after inoculation and on grapevine 6 weeks after inoculation. No symptoms were observed on the control plants. Bacteria were reisolated from tumorigenic tissues and identified as pathogenic A. vitis by PCR. Crown gall disease was sporadically observed in vineyards in Serbia in previous years, but did not cause significant damage. Therefore, the causal agent was not studied in detail. To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Serbia. References: (1) J. H. Haas et al. Appl. Environ. Microbiol. 61:2879, 1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) K. Suzaki et al. J. Gen. Plant Pathol. 70:342, 2004.

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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 Okra yellow mosaic Mexico virus in Okra in the United States

Plant Disease ◽

10.1094/pdis-94-7-0924b ◽

2010 ◽

Vol 94 (7) ◽

pp. 924-924 ◽

Cited By ~ 7

Author(s):

C. Hernandez-Zepeda ◽

T. Isakeit ◽

A. Scott ◽

J. K. Brown

Keyword(s):

United States ◽

The United States ◽

Full Length ◽

Bipartite Begomoviruses ◽

Economic Losses ◽

Growth Stages ◽

First Report ◽

Total Dna ◽

Hidalgo County ◽

Whitefly Vector

During the okra growing season from August to November of 2009, symptoms reminiscent of geminivirus infection were observed on 75% of ‘Green Emerald’ Abelmoschus esculentus (L.) Moench, plants in a 0.2-km2 field in Hidalgo County, TX. Visible symptoms consisted of irregular yellow patches on leaves, distinctive yellow borders on leaf edges, and chlorosis of subsequently developing leaves. The whitefly vector of begomoviruses, Bemisia tabaci (Genn.), infested okra plants in the early growth stages during late July 2009. Total DNA was isolated from the leaves of three symptomatic okra plant samples (1) and used as the PCR template to amplify a 575-bp fragment of the coat protein gene (CP) using the universal begomovirus primers AV494 and AC1048 (2). PCR products of the expected size were cloned into the pGEM-T Easy (Promega, Madison, WI) and sequenced using the universal M13F and M13 R primers. ClustalV alignment indicated 99 to 100% shared nucleotide (nt) identity, and BLAST analysis revealed that the closest relative was Okra yellow mosaic Mexico virus - Tetekalitla (OkYMMV) (GenBank Accession No. EF591631) at 98%. To amplify the full-length DNA-A and a possible cognate DNA-B component, one plant that was positive by CP-PCR and DNA sequencing was selected for further analysis. Total DNA from this plant was used as template for a second detection method that consisted of rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA is a non-sequence-specific approach that permits amplification of circular DNA. The RCA products were linearized to release unit length ~2.6 kb DNA-A and DNA-B components using BamHI, and EcoRI, respectively. These products were cloned into pGEM3zf+ (Promega) and sequenced using M13F and M13 R primers and then by primer walking (>300 base overlap). Full-length DNA-A and DNA-B components were obtained, respectively, at 2,613 bp (GenBank Accession No. HM035059) and 2,594 bp (GenBank Accession No HM035060). Alignment of the DNA-A component using ClustalV (MegAlign, DNASTAR, Madison, WI) with begomoviral sequences available in GenBank indicated that it was 99% identical to OkYMMV DNA-A (GenBank Accession No. DQ022611). The closest relative to the DNA-B component (ClustalV) was Sida golden mosaic virus (SiGMV) (GenBank Accession No. AJ250731) at 73%. The nt identity of the 172-nt ‘common region’ present in the DNA-A and DNA-B components was 99%, and the iterons (predicted Rep binding motif) were identical for the two components, indicating that they are a cognate pair. The genome organization was typical of other New World bipartite begomoviruses. The economic losses due to infection by this virus could not be determined because an early freeze killed the plants. Hidalgo County is adjacent to Tamaulipas, Mexico, where ~50 km2 of okra are grown and the whitefly vector is also present. The identification of OkYMMV based on two independent detection methods, and the presence of begomovirus-like symptoms together with the whitefly vector, provide robust evidence for the association of OkYMMV-TX with diseased okra plants. To our knowledge, this is the first report of OkYMMV-TX infecting okra crops in Texas and in the continental United States. References: (1) J. J. Doyle and J. L. Doyle. Focus 12:13, 1990. (2) S. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

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First Report of Crown Gall, Caused by Agrobacterium tumefaciens on Soapberry (Sapindus delavayi) in China

Plant Disease ◽

10.1094/pdis-10-12-0988-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 685-685

Author(s):

Y. J. Wang ◽

Y. Y. He ◽

Z. Xie ◽

L. Q. Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

16S Rdna ◽

Crown Gall ◽

Maximum Parsimony Analysis ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Identification ◽

Poor Growth ◽

First Report

Soapberry (Sapindus delavayi (Franch.) Radlk.,) plants are widely grown as shade trees in the subtropical to tropical regions of China. In July 2011, large, aerial galls were observed on the above-ground trunks of 5-year-old soapberry plants in two commercial nursery gardens located in Zhejiang Province. Disease incidence was estimated to be 75%. The galls varied in weight from 2 to 24 g and in texture from soft and spongy to hard, and in some cases, the galls completely girdled the trunk. The trees with galls exhibited poor growth compared with healthy trees. Isolations from the grinded and macerated galls yielded nearly pure white, circular, and glistening bacterial colonies on Roy Sauer medium (2). Six random colonies from different galls were selected for bacterial identification, and showed the same morphological, physiological, and biochemical characters and 16S rDNA sequences. All six isolates (isolate SD01 to SD06) were gram negative, rod-shaped bacteria. Carbon source utilization testing with the Biolog GN Bacterial Identification System (version 3.50) confirmed the bacteria as Agrobacterium tumefaciens with a similarity of 0.90. The most-parsimonious tree from the maximum parsimony analysis (PHYLIP package, version 3.68, 500 replicates) of bacterial 16S rDNA gene sequences showed that A. tumefaciens SD01 (GenBank Accession No. JX997939) clustered phylogenetically most closely (99.5% similarity) with A. tumefaciens C58 (AE007870.2). Pathogenicity was confirmed by injecting 3- to 5-week old tomato and sunflower plants and 2-year-old soapberry with approximately 5 μl of the bacterial suspension (108 CFU/ml) in sterile, distilled water. Sterile distilled water was used as a negative control. Ten plants of each treatment were inoculated. Inoculated plants were then transferred to a greenhouse at 25°C. Typical tumors developed at the inoculation sites on tomatoes and sunflower plants 3 weeks after inoculation and on soapberry 6 weeks after inoculation. No symptoms were observed on the control plants. The bacteria that were readily reisolated from the inoculated plants exhibited the same morphological, physiological characters and 16S rDNA sequence as the original culture and were confirmed as A. tumefaciens, fulfilling Koch's postulates. A. tumefaciens is endemic to China and has a very wide host range (1). However, crown gall of soapberry has never been found in China and other countries. To our knowledge, this is the first report of A. tumefaciens on soapberry plants in China. References: (1) M. A. Escobar and A. M. Dandekar. Trends Plant Sci. 8:380, 2003. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Anthracnose of Sunflower Sprouts Caused by Colletotrichum acutatum in New Mexico

Plant Disease ◽

10.1094/pdis-08-12-0805-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 838-838

Author(s):

J. M. French ◽

J. J. Randall ◽

R. A. Stamler ◽

A. C. Segura ◽

N. P. Goldberg

Keyword(s):

New Mexico ◽

Sequence Analysis ◽

The United States ◽

Disease Diagnosis ◽

Colletotrichum Acutatum ◽

Economic Losses ◽

Distilled Water ◽

Koch’S Postulates ◽

First Report ◽

Koch's Postulates

In December 2011, edible sunflower sprouts (Helianthus annus) of two different commercially grown cultivars (Sungrown and Tiensvold) exhibiting stem and cotyledon lesions were submitted to the New Mexico State University Plant Clinic for disease diagnosis. The sample originated from an organic farm in Santa Fe County where the grower utilizes a small indoor growing facility. Stem lesions were elongate, reddish brown, and often constricted, resulting in stem girdling. Lesions on the cotyledons were dark brown with tan centers and round to irregular in shape. In some cases, the entire cotyledon was blighted. Fungal hyphae were observed on some lesions using a dissecting microscope. Colletotrichum acutatum was isolated from stem and cotyledon lesions when symptomatic tissue was plated on water agar. Conidia were fusiform ranging from 6.4 to 18.4 μm long and 2.1 to 5.1 μm wide and averaged 11.9 μm × 3.4 μm. Spores were measured from cream-colored colonies produced on acidified potato dextrose agar. PCR amplification and sequence analysis of 5.8S ribosomal DNA and internal transcribed spacers I and II was performed using primers ITS4 and ITS6 (2). An amplification product of approximately 600 base pairs in size was directly sequenced (GenBank Accession No. JX444690). A BLAST search of the NCBI total nucleotide collection revealed a 99% identity to multiple C. acutatum (syn: C. simmondsii) isolates. Four isolates were identified as C. acutatum based on morphological characteristics and DNA analysis. Koch's postulates were performed using four isolates of the pathogen and the two commercial sunflower cultivars (Sungrown and Tiensvold) originally submitted for disease analysis. Sunflower seeds were imbibed in distilled water for 24 h then sewn into peat plugs. Prior to seed germination, 5 ml of a C. acutatum spore solution (1 × 106/ml) from each isolate was applied to five peat plugs using an atomizer. Control plants were inoculated with distilled water and otherwise treated identically. Both sunflower cultivars were inoculated with each isolate of the pathogen and the test was replicated twice. The sewn peat plugs were incubated for 5 days at 21°C and 50% relative humidity. Symptoms similar to the original samples were present on 100% of the sprouts after 5 days. PCR and sequence analysis performed on cultures obtained from lesions showed a 100% match to the original New Mexico isolates fulfilling Koch's postulates. In an indoor organic facility, such as the one in NM, this disease has the potential to be very difficult to manage and the potential to infect a high percentage of the crop resulting in significant economic losses. To our knowledge, this is the second report of C. acutatum on sunflower sprouts in the United States (1) and the first report in New Mexico. References: (1) S. T. Koike et al. Plant Dis. 93:1351, 2009. (2) T. J. White et al. Page 315 in: 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 Colletotrichum higginsianum Causing Anthracnose of Arugula (Eruca sativa) in Florida

Plant Disease ◽

10.1094/pdis-09-13-0926-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1269-1269 ◽

Cited By ~ 2

Author(s):

J. S. Patel ◽

M. I. Costa de Novaes ◽

S. Zhang

Keyword(s):

Sequence Data ◽

Morphological Characteristics ◽

Its Region ◽

The United States ◽

Economic Losses ◽

Infected Leaf ◽

Eruca Sativa ◽

First Report ◽

Small Water ◽

Colletotrichum Higginsianum

Arugula (Eruca sativa) is grown in Florida and is an important component in packaged salad products. During spring 2013, leaf lesions on arugula caused significant economic losses in Miami-Dade County, Florida. Symptoms initially appeared as small water-soaked lesions that later became circular, sunken, and white in the center with a dark brown to black halo, up to 4 mm in diameter. Acervuli were found under a dissecting microscope on infected leaf lesions with black spines or setae. Occasionally, small, circular, often longitudinal dark brown spots appeared on leaf branches. Leaf tissues (5 × 5 mm) from lesion margins were surface sterilized in 0.9% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). PDA plates were incubated at 21°C under 24-h fluorescent lights for 4 to 6 days. The fungus initially produced gray mycelium followed by orange conidial mass. Hyphae of the fungus were septate and hyaline. After 5 to 7 days, the fungus produced acervuli with dark brown to black setae (75 to 130 μm long) (n = 20). Conidia were found in the colonies, which were single celled, oblong, hyaline, and 12 to 25 × 4 to 6 μm (n = 20). The cultural and morphological characteristics of the conidia were similar to those for Colletotrichum higginsianum Sacc (1). To further confirm the species of the isolates, the sequence of the ITS region of rDNA, chitin synthase 1 (CHS1), and actin (ACT) was amplified from isolates 05131 and 05132 using primer pairs ITS 1 and ITS 4 (4), CHS-79F and CHS-354R, and ACT-512F and ACT-783R (3), respectively. The sequenced data of each locus were deposited in GenBank with accessions KF550281.1, KF550282.1, KJ159904, KJ159905, KJ159906, and KJ159907. The resulting sequence of ITS showed 100% identity with sequences of C. higginsianum in JQ005760.1, and sequence of ACT gene showed 100% identity with C. higginsianum in JQ005823.1. The sequence of ACT gene and ITS region had ≤99% identity with other closely related Colletotrichum spp. CHS1 gene had 100% identity with JQ005781.1 belonging to C. higginsianum, and one accession JQ005783.1 belonging to C. fuscum. However, ACT gene and ITS region does not share 100% identity with C. fuscum and therefore, sequence data from three loci proves that isolated pathogen is C. higginsianum. All the above mentioned accessions that shared 100% identity with sequences of isolates used in our study have been previously used to represent the species in the C. destructivum clade in a systematics study (2). To confirm its pathogenicity, a suspension of isolate 05132 at 5 × 105 conidia/ml was sprayed on leaves of five arugula plants until runoff. The other five arugula plants sprayed with water served as non-inoculated controls. Both inoculated and non-inoculated plants were separately covered with a plastic bag to maintain high humidity for 24 h at 27 ± 5°C under natural day/night conditions in the greenhouse. Symptoms first appeared 3 to 4 days after inoculation as small water-soaked lesions, which became sunken with dark brown to black margins. Small circular and longitudinal dark brown spots were also seen on leaf branches as seen initially on naturally infected arugula. No symptoms developed on non-inoculated control plants. C. higginsianum was re-isolated from the lesions with the same morphological characteristics as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of C. higginsianum causing anthracnose of arugula in Florida. This pathogen may potentially affect the salad industry in the United States. References: (1) A. J. Caesar et al. Plant Dis. 94:1166, 2010. (2) P. F. Cannon et al. Stud. Mycol. 73:181, 2012. (3) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Five Sooty Blotch and Flyspeck Fungi on Prunus americana in the United States

Plant Disease ◽

10.1094/pdis-91-12-1685b ◽

2007 ◽

Vol 91 (12) ◽

pp. 1685-1685 ◽

Cited By ~ 5

Author(s):

J. Latinović ◽

J. C. Batzer ◽

K. B. Duttweiler ◽

M. L. Gleason ◽

G. Sun

Keyword(s):

United States ◽

The United States ◽

Apple Fruit ◽

Economic Losses ◽

Research Station ◽

First Report ◽

Sooty Blotch ◽

Sooty Blotch And Flyspeck ◽

Pcr Products ◽

Pure Cultures

The sooty blotch and flyspeck (SBFS) complex includes more than 30 fungi that blemish the cuticle of apple fruit, causing economic losses in humid regions worldwide (1). In August 2005, we sampled SBFS-infested wild plum (Prunus americana) fruit growing in hedgerows in Iowa. Colonies were categorized according to mycelial type (1), and isolates were made from representative colonies onto acidified water agar (AWA). Plum skins with SBFS signs were excised, pressed, and photographed. DNA was obtained from purified isolates and also from mycelium and fruiting bodies scraped directly from plum fruit skins. Extracted DNA was amplified using primer pair ITS1-F/Myc1-R (ACTCGTCGAAGGAGCTACG) and PCR products were sequenced using primer pair ITS-1F/ITS4. Six sequences were obtained from pure cultures and seven from colonies on plum fruit skin. BLAST analysis of the 470-bp sequences showed 100% homology to five known species in the SBFS complex: Zygophiala cryptogama, Zygophiala wisconsinensis, Pseudocercosporella sp. RH1, and Stomiopeltis spp. RS1 and RS2 (GenBank Accession Nos. AY598854, AY598853, AY5988645, AY598882, and AY598883, respectively). Observations of colony and fruiting structure morphology from cultures on potato dextrose agar (PDA) and colonies on plums confirmed species identity. A modified version of Koch's postulates was conducted to verify that these fungi caused the signs observed on plum and could also infest apple fruit. In June 2006, 1-month-old cultures on PDA were pulverized in a blender with sterile distilled water, passed through four layers of sterile cheesecloth, and transferred to sterile jars. Each isolate was inoculated onto 20 fruit on plum trees (P. americana) on the Iowa State University (ISU) campus and 20 fruit on cv. Golden Delicious apple trees at the ISU Research Station, Gilbert, IA. Each fruit was disinfested with 70% ethanol, air dried, swabbed with inoculum, and covered with a Fuji bag. At harvest, fungal colonies on fruit were reisolated onto AWA. DNA was extracted from pure cultures; when isolations on agar were unsuccessful, DNA was extracted directly from colonies on fruit. PCR was conducted using ITS1-F/Myc1-R, and PCR products were sequenced using ITS1-F/ITS4. All five species were reisolated and sequenced from apple. Pseudocercosporella sp. RH1 and Stomiopeltis sp. RS1 were sequenced from inoculated plums. Although flyspeck, presumably caused by Schizothyrium pomi, was reported on Japanese plum (P. salicina) in Japan (2) and black cherry (P. serotina) in the United States (3), to our knowledge this is the first report of SBFS fungi on plum in the United States and the first confirmation that fungi from plum can produce SBFS signs on apple fruit. Wild plum may therefore act as a reservoir host, providing inoculum for SBFS infestations on apple. References: (1) J. Batzer et al. Mycologia 97:1268, 2005. (2) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (3) T. B. Sutton. Plant Dis. 72:801, 1988.

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First Report of Potato mop top virus Causing Potato Tuber Necrosis in Colorado and New Mexico

Plant Disease ◽

10.1094/pdis-08-14-0819-pdn ◽

2015 ◽

Vol 99 (1) ◽

pp. 164-164 ◽

Cited By ~ 6

Author(s):

I. Mallik ◽

N. C. Gudmestad

Keyword(s):

New Mexico ◽

North Dakota ◽

Consensus Sequence ◽

The United States ◽

Economic Losses ◽

Acid Extraction ◽

Rt Pcr ◽

First Report ◽

Tuber Necrosis ◽

Potato Mop Top Virus

Potato mop top virus (PMTV) is considered the type member of the genus Pomovirus. PMTV is an important pathogen of potato vectored by the plasmodiophorid Spongospora subterranea f. sp. subterranea (Sss), which causes powdery scab of potato (1). Sss and PMTV are usually associated with cool and humid environments. PMTV-infected potato tubers generally exhibit internal hollow necrotic spots or concentric rings, and the virus is known to cause significant economic losses in Northern Europe, North and South America, and Asia (4). PMTV in the United States was first reported in Maine (2). Potato (Solanum tuberosum L.) tubers cv. FL2048 and cv. Atlantic were sent to our laboratory from fields in Saguache County in Colorado and in San Juan County in New Mexico, respectively, during the spring of 2013. The tubers from both locations had multiple, internal, concentric, necrotic arcs and circles. Internal tissue with necrotic lesion from six symptomatic tubers from each location were crushed in liquid nitrogen followed by ribonucleic acid extraction using a Total RNA Isolation kit (Promega Corp., Madison, WI). These extracts were tested by reverse transcription (RT)-PCR using three different sets of previously published primers for molecular detection of PMTV. The primer set H360/C819 targeting the coat protein (CP) on RNA 3 of PMTV yielded an amplicon (H360-CO and H360-NM) of 460 bp (4). The second set of primers, pmtF4/pmtR4 (5), amplified a 417-bp product (PMTF-CO and PMTF-NM) in RNA 2, and the third set, PMTV-P9/PMTV-M9 (3), designed to amplify the region encoding an 8-KD cysteine-rich protein in RNA 3 of PMTV, yielded a 507-bp amplicon (PMTV9-CO and PMTV9-NM). The amplicons generated from RT-PCR using all three sets were cloned (PGEMT-easy) and sequenced. Since the sequences from symptomatic tuber extracts from each location were identical to their respective primer sets, a consensus sequence from each primer set was submitted to National Center for Biotechnology Information (NCBI) GenBank. Sequences obtained from the H360/C819 primer set (GenBank Accession Nos. KM207013 and KM207014 for H360-CO and H360-NM, respectively) were 100% identical to the corresponding CP regions of PMTV isolates from North Dakota (HM776172). Sequences from the pmtF4/pmtR4 primer set (KM207015 and KM207016 for PMTF-CO and PMTF-NM, respectively) were 100% identical to the corresponding protein in RNA2 of PMTV isolates from North Dakota (GenBank HM776171), and sequences from the PMTV-P9/PMTV-M9 primer set (KM207017 and KM207018 for PMTV9-CO and PMTV9-NM respectively) were 99% identical to the corresponding protein in RNA3 of PMTV isolates (AY187010). The 100-99% homology of the sequences from this study to the corresponding PMTV sequences published in NCBI confirmed the occurrence of symptoms in the tubers from both Colorado and New Mexico due to PMTV. None of the symptomatic tubers tested positive for Tobacco rattle virus, Tomato spotted wilt virus, Alfalfa mosaic virus, Potato leafroll virus, or the necrotic strains of Potato virus Y by RT-PCR. To our knowledge, this is the first report of PMTV in potato in states of Colorado and New Mexico. References: (1) R. A. C. Jones and B. D. Harrsion. Ann. Appl. Biol. 63:1, 1969. (2) D. H. Lambert et al. Plant Dis. 87:872, 2003. (3) T. Nakayama et al. Am. J. Pot. Res. 87:218, 2010. (4) J. Santala et al. Ann. Appl. Biol. Online publication. DOI: 10.1111/j.1744-7348.2010.00423.x (5) H. Xu et al. Plant Dis. 88:363, 2004.

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First report of Agrobacterium tumefaciens causing crown gall disease of roselle (Hibiscus sabdariffa L.) in Taiwan

Plant Disease ◽

10.1094/pdis-05-21-1007-pdn ◽

2021 ◽

Author(s):

Huan-Yu Chen ◽

Chun-Chi Lin ◽

Chih-Wei Wang ◽

NAI-CHUN LIN

Keyword(s):

Agrobacterium Tumefaciens ◽

Sequence Analysis ◽

Crown Gall ◽

Causal Agent ◽

Reca Gene ◽

Bacterial Suspension ◽

Hibiscus Sabdariffa ◽

Agrobacterium Vitis ◽

First Report ◽

Crown Gall Disease

Roselle (Hibiscus sabdariffa L.) plants, whose calyces are used for production of beverages or jams, are mainly cultivated in Taitung County of eastern Taiwan. Since 2016, large crown galls were observed on the roselle plants in the commercial plantations at Taimali and Jinfong Townships of Taitung County. A follow-up survey in July and August of 2017 revealed spreading of this disease to the neighboring areas including Beinan and Dawu Townships. Disease incidence was estimated to be 0.6-10%. Galls of varying sizes (2-15 cm in diameter) were usually found on the roots and crowns of the roselle plants, starting with small swellings at the infection sites. Galls were light-colored, and smooth and tender in texture at the early stage, but later turned dark-colored, and appeared rough and woody. In some cases, adventitious roots extruding from the larger crown galls could be seen. Isolation of the causal agent was performed by quadrantally streaking bacterial suspension made from surface-sterilized, macerated galls on trypticase soy agar (TSA). After incubating at 28°C for 5 days, single colonies were transferred onto new TSA plates for further cultivation at 28°C. Finally, circular, convex, viscous and milky white colonies with smooth surface similar to colony morphology of Agrobacterium tumefaciens C58 were obtained for further identification. First, all six candidate isolates (TZ-1, TL1-2, TL2-1, TD1-1, TD1-24 and TD2-1) were identified as Agrobacterium spp. using the partial sequences of the 16S rRNA gene (accession numbers MW205820 to MW205825 in the GenBank database). The selected isolates also showed some biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. Moreover, they were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with calcium carbonate. Under a transmission electron microscope, the bacterium was rod-shaped and possessed peritrichous flagella. By means of multiplex PCR using primers designed for differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2, a 184 bp product was detected in all six isolates, indicating that they all belong to Agrobacterium biovar 1. Furthermore, the recA allele of each isolate was PCR amplified using primers F2898/F2899, and recA sequence analysis assigned all six isolates to A. tumefaciens genomospecies G7 (GenBank accession numbers MZ570905-MZ570910). Pathogenicity assay was carried out by inoculating the stems of 2-week-old roselle seedlings through wounds made with a sterile needle with bacteria on it. The inoculated seedlings were kept in plastic bags to maintain high humidity. Symptoms similar to those observed in the field developed at the inoculation sites after 7 days, and Koch’s postulates were fulfilled when the bacteria re-isolated from the galls were also identified as A. tumefaciens genomospecies G7 using recA gene sequence analysis. To our knowledge, this is the first report of crown gall disease caused by A. tumefaciens on Hibiscus sabdariffa in Taiwan. This disease may potentially damage the roselle industry if no action is taken to stop its spreading. Identification of the causal agent of roselle crown gall disease could help us further investigate its ecology and develop integrated pest management strategies for prevention of this disease in the future.

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First Report of Peronospora farinosa f. sp. spinaciae Race 5 Causing Downy Mildew on Spinach in Florida

Plant Disease ◽

10.1094/pdis.2004.88.1.84c ◽

2004 ◽

Vol 88 (1) ◽

pp. 84-84 ◽

Cited By ~ 3

Author(s):

B. M. Irish ◽

J. C. Correll ◽

R. N. Raid ◽

T. E. Morelock

Keyword(s):

Downy Mildew ◽

Disease Incidence ◽

Field Isolate ◽

The United States ◽

Economic Losses ◽

Palm Beach County ◽

First Report ◽

Leaf Spots ◽

Plant Densities ◽

Production Area

Downy mildew, caused by Peronospora farinosa f. sp. spinaciae, is an economically important disease in most areas where spinach is grown. This disease has become increasingly important in production fields for prepackaged salad mixes where plant densities typically are very high. In Florida, spinach production for these markets has reached approximately 200 ha. Currently, seven physiological races of the downy mildew pathogen have been described (1). Downy mildew was observed in several commercial spinach fields in the Everglades agricultural area of Palm Beach County, Florida in January 2003 on cvs. Unipak 151 and Merlo Nero. Symptoms appeared as chlorotic and necrotic leaf spots. Disease incidence reached approximately 25% in some field locations. Economic losses were significant, since entire plantings in several fields were not harvested as a result of diminished quality. The race of a field isolate recovered from the cv. Unipak 151 was determined following greenhouse inoculation procedures and using differentials outlined by Irish et al (1). Greenhouse inoculation tests were conducted twice. Disease reactions on a U.S. and international set of differentials indicated that the isolate was race 5. To our knowledge, this is the first report of race 5 occurring outside of the California/Arizona spinach production area in the United States. There are commercial spinach lines with resistance to race 5, as well as the other described races (1). References: (1) B. M. Irish et al. Plant Dis. 87:567, 2003.

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