First Report of Brown Rot (Monilinia fructicola) on the Dogwood, Cornelian Cherry (Cornus mas).

Cornelian cherries (Cornus mas), also called cornels, are members of the dogwood family (Cornaceae), and are not true cherries. Cornelian cherry is primarily grown as an edible landscape ornamental in the United States. Brown rot, caused by fungi in the genus Monilinia, is one of the most important diseases of stone fruit worldwide. In the United States, M. fructicola is the most commonly observed Monilina species, although M. fructigena and the European brown rot pathogen, M. laxa, may also infect stone fruit. M. fructigena is the only Monilinia species reported to infect cornelian cherry, but there is only a single report of it occurring in the United States (1,4). All three species have similar morphology and are commonly misidentified (1,3,4). In August of 2010 and 2013, in one location, brown rot was observed on fruit of the cornelian cherry cultivar Elegans. In both instances, only 'Elegans' fruit was infected while neighboring 'Sunrise' exhibited no symptoms in the field, and lesions did not appear to develop into shoot blight. In 2013, single-spore isolates from the diseased fruit were cultured on potato dextrose agar (PDA) incubated at 25°C for 5 days. Colony morphology was consistent with M. fructicola and was rapidly growing, gray, producing concentric rings, and developing smooth colony margins. Conidia were hyaline, 10 × 15 µm, and formed in branched, monilioid chains of varying lengths (1). Molecular-based species identification was performed on the 450-bp amplified ribosomal internal transcribed spacer (ITS) sequences, using primers ITS1 and ITS4. BLAST searches of the ITS sequences in GenBank showed the highest similarity (100%) with sequences of M. fructicola isolates from Italy (FJ411110), China (FJ515894), and Spain (EF207423). Pathogenicity was confirmed by inoculating surface-sterilized, mature 'Sunrise' fruit with mycelial plugs of the isolate identified with the ITS sequence. Mycelial plugs (3 mm in diameter) were removed from the periphery of a 5-day-old colony and placed upside down into five fruit that were wound-inoculated with a 3-mm cork borer, petiole hole-end inoculated, or unwounded but inoculated; control fruit for each treatment received sterile plugs of PDA as a control. All fruit was stored in a moist chamber for the duration of the experiment. Wound-inoculated fruit developed symptoms within 2 days; sporulating lesions developed within 5 days. Symptoms of infection via the petiole developed in 4 days; by day six, three of the five inoculated fruit were infected, and four of the five were infected by day eight. Unwounded, inoculated fruit showed symptoms on day six; three of the five fruit were infected by day eight. None of the control inoculations showed Monilinia infection. Pathogens were re-isolated from the inoculated fruit and confirmed to be M. fructicola on the basis of morphological characteristics. To our knowledge, this is the first fulfillment of Koch's postulates demonstrating that M. fructicola can infect cornelian cherry. A previous report by Höhnel in 1918 described infection by Lambertella corni-mas of a cornelian cherry in Austria; however, the taxonomic details presented are consistent with M. fructigena (2). References: (1) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (2) T. H. Harrison and A. F. El-Helaly. Brit. Mycol. Soc. Trans. 19:199, 1935. (3) C. R. Lane. EPPO Bulletin 32:489, 2002. (4) E. M. Sagasta. EPPO Bulletin 7:105, 1977.

A Survey of Phytophthora spp. in Midwest Nurseries, Greenhouses, and Landscapes.

A survey of nurseries, greenhouses, and landscapes was conducted from 2006 to 2008 in order to determine the prevalence and diversity of Phytophthora spp. From sites in Iowa, Michigan, Ohio, and, predominantly, Indiana, 121 Phytophthora isolates were obtained from 1,657 host samples spanning 32 host genera. Based on sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA, 11 Phytophthora spp. and two hybrid species were identified. A majority of the isolates were P. citricola (35.9%) or P. citrophthora (27.4%). Six isolates were confirmed as hybrids (four of P. cactorum × hedraiandra and two of P. nicotianae × cactorum) by cloning and sequencing the ITS region. Three P. cactorum × hedraiandra isolates were obtained from the same site, from three Rhododendron spp., which are known hosts to the parental species. The fourth isolate, however, was recovered out of a different location in a Dicentra sp., which is not a known host to either parental species, suggesting an expansion of host range of the hybrid isolate as compared with either parental species.

First Report of Goss's Bacterial Wilt and Leaf Blight on Corn Caused by Clavibacter michiganensis subsp. nebraskensis in Indiana.

In August of 2008, leaves of hybrid corn (Zea mays L.) and popcorn from Pulaski and Jasper counties in northwest Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab with symptoms characteristic of a bacterial disease. Symptomatic leaves had large, tan-to-gray necrotic lesions with dark freckling present within the lesions. Shiny bacterial exudate was present on the surface of many of the lesions. Microscopic observation revealed no fungal structures within the lesions, and bacterial streaming was observed from the cut edge of symptomatic tissue under ×100 magnification with phase contrast. A commercially available ELISA test (Agdia Inc., Elkhart, IN) determined that samples were negative for Pantoea stewartii, the causal agent of Stewart's bacterial leaf blight and wilt. A bacterial suspension was prepared from symptomatic tissue and streaked onto King's B medium and subcultured on semiselective CNS medium (1,2). Axenic, peach-colored colonies present on the CNS medium tested gram positive with a KOH test. Analysis of fatty acid methyl esters (MIDI Inc, Newark, DE) indicated that the strain was very similar (0.611) to Clavibacter michiganensis. Amplification of the 500-bp 16S rRNA region of the bacterial gene and subsequent BLAST alignments of the resulting sequence indicated a 99% match for C. michiganensis subsp. nebraskensis (GenBank Accession Nos. AM410697 and U09763; D16S2 gene bacterial library, version 2.10; MIDI Inc,). Koch's postulates were used to confirm pathogenicity of the isolated bacteria on corn inbred B73. Eighteen plants were mechanically inoculated at growth stage V1 to V2 with a bacterial suspension of approximately 1 × 108 CFU/ml prepared from cultures grown on CNS for 10 days at 28°C (2). Inoculum was rubbed onto leaves dusted with Carborundum and 0.1 ml of the bacterial suspension was injected into stems with a hypodermic needle. Nine control plants were inoculated with sterile water. Plants were kept at greenhouse conditions (24°C) with supplemental 400W high-pressure sodium light. Within 5 to 8 days, leaves and stems of all 18 inoculated plants developed water-soaked, necrotic lesions. No symptoms were observed in control plants. Bacteria were reisolated from symptomatic plants on CNS medium as described above, and gram-positive colonies were obtained. Reisolated strains were identical to C. michiganensis subsp. nebraskensis by D16S2 DNA sequence analysis, confirming the causal agent of the disease. Disease incidence in affected fields ranged from 20 to 60% and significant yield loss was reported. This confirmation is of regulatory importance because of potential export restrictions of Indiana-grown seed corn and popcorn to select countries. To our knowledge, this is the first report of Goss's bacterial wilt and leaf blight on corn in Indiana. References: (1) D. C. Gross and A. K. Vidaver. Phytopathology 69:82, 1979. (2) L. M. Shepherd. M.S. thesis. Iowa State University. Ames, 1999.

First Report of Phakopsora pachyrhizi on Kudzu (Pueraria montana var. lobata) in North Carolina and Increased Incidence of Soybean Rust on Soybean in 2006.

Asian soybean rust, caused by Phakopsora pachyrhizi H. Sydow & Sydow, was first detected in the continental United States in soybean (Glycine max (L.) Merr.) in Louisiana on 6 November 2004 (3) and in kudzu (Pueraria montana var. lobata) in Florida during February 2005 (1). Soybean rust was first confirmed in North Carolina in commercial soybean fields in Brunswick, Columbus, and Robeson counties on 25 October 2005 (2). Subsequently, the disease was detected in soybean in 18 counties, but not in kudzu, even when it was growing adjacent to infected soybean. During 2006, soybean rust was first detected in North Carolina in soybean on 14 September 2006 from a sample from Columbus County that was submitted to the North Carolina State University Plant Disease and Insect Clinic (NCSU-PDIC). Thus, the first detection of soybean rust in North Carolina occurred almost 6 weeks earlier in 2006 than in 2005. Subsequently, in 2006, soybean rust was found in soybean in 42 counties in North Carolina through survey, sentinel plot monitoring, and samples submitted to the NCSU-PDIC. In addition, what appeared to be soybean rust was observed in two samples of kudzu collected on 3 and 6 November 2006 from Moore (35.28313°N, 79.38020°W) and Johnston (35.42742°N, 78.18154°W) counties of North Carolina. The diagnosis of P. pachyrhizi in kudzu was confirmed visually and by ELISA protocol supplied with the EnviroLogix QualiPlate kit (Portland, ME). ELISA tests for each kudzu sample were run in triplicate. PCR was also conducted on infected kudzu samples with a protocol previously reported (1). The PCR master mix that was used came from a dilution scheme based on previous PCR work completed by G. Z. Abad. A total of 24 reactions were run, including four 1-kb molecular markers, four positive controls, four negative controls, and four infected kudzu leaf tissue samples. The results of all diagnostic techniques confirmed the presence of P. pachyrhizi in diseased kudzu. To our knowledge, this is the first report of P. pachyrhizi in kudzu in North Carolina. References: (1) P. F. Harmon et al. Online publication. doi:10.1094/PHP-2005-0613-01-RS. Plant Health Progress, 2005. (2) S. R. Koenning et al. Plant Dis. 90:973, 2006. (3) R. W. Schneider et al. Plant Dis. 89:774, 2005.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi in North Carolina.

Asian soybean rust, caused by Phakopsora pachyrhizi Sydow, has been known to occur in the eastern hemisphere for nearly a century. More recently, it was reported from South America in 2002 and the continental United States in Louisiana in November 2004 (1,2). Subsequently, P. pachyrhizi was confirmed in Alabama, Arkansas, Georgia, Florida, Missouri, Mississippi, South Carolina, and Tennessee in 2004. Surveys conducted in North Carolina in late November 2004 failed to detect this pathogen. Symptoms of the disease were first observed on soybean (Glycine max (L.) Merr.) in North Carolina on 25 October 2005 in farmers' fields in the counties of Brunswick, Columbus, and Robeson. Typical pustules and urediniospores were readily apparent on infected leaves when viewed with a dissecting microscope. Urediniospores were obovoid to broadly ellipsoidal, hyaline to pale yellowish brown with a minutely echinulate thin wall, and measured 18 to 37 × 15 to 24 µm. This morphology is typical of soybean rust caused by P. pachyrhizi or P. meibomiae, the latter is a less aggressive species causing soybean rust in the western hemisphere (1). DNA was extracted from leaves containing sori using the Qiagen DNeasy Plant Mini kit (Valencia, CA). P. pachyrhizi was detected using a real-time polymerase chain reaction (PCR) protocol that differentiates between P. pachyrhizi and P. meibomiae in a Cepheid thermocycler (Sunnyvale, CA) with appropriate positive and negative controls. The PCR master mix was modified to include OmniMix beads (Cepheid). Field diagnosis of P. pachyrhizi was confirmed by the USDA/APHIS on 28 October 2005. Soybean rust was identified in subsequent surveys of soybean fields and leaf samples submitted by North Carolina Cooperative Extension Agents in an additional 15 counties. These samples also were assayed using a traditional PCR protocol and by the enzyme-linked immunosorbent assay protocol included in the EnviroLogix QualiPlate kit (Portland, ME) for soybean rust. Ten soybean specimens from 10 sites were confirmed positive by these methods. Disease was not found on three kudzu samples, although one kudzu sample was adjacent to a soybean field that was positive for P. pachyrhizi. Although soybean rust was eventually detected in 18 North Carolina counties in 2005, no soybean yield loss occurred since the pathogen was detected when more than 80% of the soybean crop was mature. To our knowledge, this is the first report of P. pachyrhizi in North Carolina and the northern most find on soybean in the continental United States in 2005. References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) R. W. Schneider et al. Plant Dis. 89:774 2005.

Increased Occurrence of Target Spot of Soybean Caused by Corynespora cassiicola in the Southeastern United States.

Target spot of soybean (Glycine max (L.) Merr.) caused by Corynespora cassiicola (Berk. & Curt.), although found in most soybean-growing countries, is considered to be a disease of limited importance (1) and has never been reported to cause soybean yield loss in the southeastern United States (2,3). Soybean plants submitted to the North Carolina Plant Disease and Insect Clinic (NCPDIC) in August 2004 from Beaufort, Robeson, Wilson, and Johnston counties, NC had symptoms consistent with target spot. Symptoms consisted of roughly circular, necrotic leaf lesions from minute to 11 mm in diameter, though typically approximately 4 to 5 mm in diameter, and with a yellow margin. Large lesions occasionally exhibited a zonate pattern often associated with this disease. Microscopic examination of the lesions revealed the presence of spores (conidia) typical of C. cassiicola (1). Conidia were mostly three to five septate with a central hilum at the base and ranged in size from 7 to 22 wide × 39 to 520 µm long. Three commercial soybean fields near Blackville, SC (Barnwell County) were severely affected by this disease and it caused premature defoliation. Nineteen of twenty-seven maturity group VII and VIII genotypes in the 2004 Clemson University soybean variety trial near Blackville, SC had visible symptoms of target spot. Heavy rainfall associated with hurricanes during September 2004 probably enhanced the incidence of this disease, and yield suppression due to target spot was estimated at 20 to 40% in some fields. In 2005, 20 of 161 soybean samples submitted to the NCPDIC or collected in surveys from 16 counties were positive for target spot on the basis of microscopic examination. Target spot also was diagnosed in six counties (Baldwin, DeKalb, Elmore, Fayette, Macon, and Pickens) in Alabama and in four additional counties (Bamberg, Hampton, Orange-burg, and Calhoun) in South Carolina in 2005. Records from the NCPDIC indicate that target spot had not been diagnosed on soybean in North Carolina since 1981. The large increase in incidence of target spot in the southeast may be related to changes in weather patterns, changes in pathogen virulence, and/or the introduction of more susceptible host genotypes. References: (1) J. B. Sinclair. Target spot. Page 27 in: Compendium of Soybean Diseases. G. L. Hartman et al. eds. The American Phytopathological Society, St. Paul, MN, 1999. (2) J. A. Wrather et al. Plant Dis. 79:1076. 1995. (3) J. A. Wrather et al. On-line publication. doi:10.1094/PHP-2003-0325-01-RV. Plant Health Progress, 2003.

Appearance of Tomato yellow leaf curl virus in North Carolina.

In the summers of 2000 and 2001, tomato plants (Lycopersicon esculentum) with symptoms of stunting, curling, and marginal chlorosis of leaves, reduced leaf size, and marked reduction in fruit number, similar to those caused by Tomato yellow leaf curl virus (TYLCV), were seen in Henderson County, NC. In 2001, symptomatic plants appeared in a 40-A (18.2 ha) field in 12 foci of ≈12 plants each, at a total incidence of less than 1%. In August 2001, DNA was extracted from leaf samples from four symptomatic plants and tested by polymerase chain reaction (PCR) amplification for the presence of one or more geminiviruses. Two sets of primers were used to test for begomoviruses, AC1048 and PCRv181 (3,4), which amplify a 1,020-bp DNA product from a wide range of monopartite and bipartite (A component only) begomoviruses, and C473 and PTYC1v2406, which preferentially amplifies a 859-bp DNA product from the monopartite TYLCV (1,2). Fragments of the expected size were obtained from all four samples, and all PCR products were sequenced. The sequences of the 1,020-bp PCR product from each of the four samples were compared and found to be 100% identical. The same was found for the 859-bp products. These sequences were compared with equivalent regions of begomoviruses and were identical to sequences of TYLCV. Since the two primer sets amplify overlapping regions of the TYLCV genome, the 1,020 and 859-bp products generated by the two primer sets from one plant were combined to create a 1,464-bp sequence that represented approximately half of the TYLCV genome and encompasses the C4 ORF, the intergenic region, and most of the coat protein gene. This 1,464-bp sequence from North Carolina was 99.2 to 99.6% identical to TYLCV sequences reported from Cuba (GenBank Accession No. AJ223505), the Dominican Republic (GenBank Accession No. AF024715), and Florida, and 96.9 to 98.2% identical to TYLCV sequences reported from the Bahamas, Israel (GenBank Accession No. X15656), Jamaica (GenBank Accession No. U84146), Mexico (GenBank Accession No. AF168709), and Spain (GenBank Accession No. AF071228). Symptomatic plants appeared to be infected with an isolate of TYLCV that is most similar to TYLCV isolates reported from Florida and the northeastern Caribbean. To our knowledge, this is the first report of TYLCV in North Carolina. TYLCV may have been introduced on transplants since the infected plants showed symptoms at an early growth stage. The appearance of infected plants in clusters of limited size suggests no spread or very limited spread in the field. Reports of populations of the whitefly (Bemisia tabaci) vector in the field were not available since whiteflies are not normally a problem in this area due to the higher altitude and relatively cool temperatures characteristic of Henderson County. It is not clear at this time what threat TYLCV poses to tomato production in the county, though its appearance indicates that the geographic range of TYLCV is continuing to expand in the southeastern United States. References: (1) M. Ghanim et al. Virology 240:295, 1998. (2) M. K. Nakhla et al. Phytopathol. Mediterr. 32:163, 1993. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt et al. Phytopathology 86:1288, 1996.