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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.

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.

Stunted cotton plants (Gossypium hirsutum L. cvs. PHY 375 WR and PHY 565 WR) from two separate fields near Goldsboro in Wayne County, North Carolina were collected by the NCDA&CS Agronomic Division nematode lab for nematode assay and identification in December 2011. The galls on cotton plants were very large in comparison with those commonly associated with Meloidogyne incognita Kofoid and White (Chitwood) infected cotton. In August 2012, the lab also received heavily galled roots of soybean (Glycine max (L.) Merr. cv. 7732) from Wayne and Johnston counties. Population densities of the 2nd-stage juveniles ranged from 150 to 3,800 per 500 cc soil. Female perineal patterns were similar to M. incognita, but PCR and DNA sequencing matched that of M. enterolobii Yang and Eisenback (4). DNA sequences of ribosomal DNA small subunit, internal transcribed spacer, large subunit domain 2 and 3, intergeneric spacer, RNA polymerase II large subunit, and histone gene H3, were found to be 100% homologous when comparing populations of M. enterolobii from North Carolina and China. Species identification was also confirmed using PCR by a species-specific SCAR primer set MK7-F/MK7-R (2). M. enterolobii Yang & Eisenback was described in 1983 from a population causing severe damage to pacara earpod tree (Enterolobium contortisiliquum (Vell.) Morong) in China (4). In 2004, M. mayaguensis Rammah & Hirschmann, a species described from Puerto Rico, was synonymized with M. enterolobii based on esterase phenotype and mitochondrial DNA sequence (3). M. enterolobii is considered to be a highly pathogenic species and has been reported from vegetables, ornamental plants, guava, and weeds in China, Africa, Central and South America, the Caribbean, and Florida in the United States (1,3,4). Of particular concern is its ability to develop on crop genotypes carrying root-knot-nematode resistance genes (Mi-1, Mh, Mir1, N, Tabasco, and Rk) in tobacco, tomato, soybean, potato, cowpea, sweet potato, and cotton. Consequently, this species was added to the European and Mediterranean Plant Protection Organization A2 Alert list in 2010. Two populations of M. enterolobii one from soybean and one from cotton were reared on tomato (Solanum lycopersicum L. var. lycopersicum) in a greenhouse setting. Eggs were extracted using NaOCl and inoculated, at a rate of 7,000 per 15-cm-diameter clay pot, into a sandy soil mixture (1:1 washed river sand and loamy sand). Tomato, peanut (Arachis hypogaea L.), cotton, watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), pepper (Capsicum annuum L.), and root-knot-susceptible and -resistant tobacco (Nicotiana tabacum L. cvs. K326 and NC 70, respectively) were transplanted immediately into the infested soil with four replications. Root galls on the host differentials were evaluated after 90 days. Reproduction occurred on all hosts except for peanut, which is consistent with reports for M. enterolobii and M. incognita race 4 (4). Adult females from pepper plants used in the host differential test were sequenced on partial 18S and ITS1 region and confirmed to be M. enterlobii. To our knowledge, this is the first report of a natural infection of North Carolina field crops with M. enterolobii. References: (1) J. Brito et al. J. Nematol. 36:324, 2004. (2) M. S. Tigano et al. Plant Pathol. 59:1054, 2010. (3) J. Xu et al. Eur. J. Plant Pathol. 110:309, 2004. (4) B. Yang and J. D. Eisenback. J. Nematol. 15:381, 1983.

Five viruses were previously discovered infecting soybean cyst nematodes (SCN; Heterodera glycines) from greenhouse cultures maintained in Illinois. In this study, the five viruses [ScNV, ScPV, ScRV, ScTV, and SbCNV-5] were detected within SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN from 43 greenhouse cultures and 25 field populations were analyzed using qRT-PCR. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Viruses were found at similar or lower levels within field populations of SCN compared with greenhouse cultures of North Carolina populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mixture of fewer viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Several of these SCN viruses were also detected in Heterodera trifolii (clover cyst) and Heterodera schachtii (beet cyst), but not the other cyst, root-knot, or reniform nematode species tested. Viruses were not detected within soybean host plant tissue. If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation.