First Report of Rose rosette virus Associated with Rose Rosette Disease Infecting Knockout Roses in Florida.

Roses are one of the most popular flowering shrubs in the United States, with a total wholesale value of US$194 million. Among the major states, Florida is the fourth largest producer of roses with a total value exceeding US$20 million (4). In Florida, the roses have become especially popular in recent years with the introduction of Knock Out and other shrub roses. Virus-like symptoms including witches'-broom, excessive thorns, abnormal red discoloration of shoots and foliages, distorted leaves, and deformed buds and flowers were initially observed on Knock Out roses in a commercial nursery in Quincy, FL, in November 2013. Fifteen plants out of ~250,000 plants showed these characteristic symptoms. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from eight symptomatic and two non-symptomatic rose samples were subjected to reverse-transcription (RT) assays using SuperScript III Reverse transcriptase (Invitrogen, Life Technologies, NY) and random hexamer primers. The cDNA synthesized was then subjected to PCR assay using Platinum Taq DNA polymerase (Invitrogen, Life Technologies) and using Rose rosette virus (RRV) specific primers RRV-F and RRV-R (1), targeting the core region of the RNA1 genome of the virus. The RT-PCR assays using the specific primers produced amplicons of 375 bp, only in the symptomatic leaf samples. The obtained amplicons were PCR purified and sequenced directly (GenBank Accession Nos. KF990370 to KF990377). BLAST analysis of these sequences revealed a higher identity of 99% with the RRV (HQ871942) in the NCBI database. Pairwise comparison of the eight RRV sequences exhibited 99 to 100% identity among themselves. These results revealed the association of RRV with the symptomatic rose plants. Eight symptomatic and two non-symptomatic rose plant samples were tested for RRV using blot hybridization assay, utilizing a digoxigenin-labeled DNA probe of 511 bp, targeting the RNA1 genome of the RRV. All eight symptomatic rose plants showed a positive reaction to the RRV-specific probes, confirming the presence of RRV in the samples, while the non-symptomatic and the buffer control did not produce any reactions. Even though the virus is reported to spread by an eriophyid mite Phyllocoptes fructiphilus, thorough examination of the infected samples showed absence of the vector. The samples were also tested using RT-PCR for the presence of Rose cryptic virus (RCV) and Blackberry chlorotic ringspot virus (BCRV) using specific primers (2,3). The samples tested negative for the RCV and BCRV. This is the first report of occurrence of RRV on rose in Florida. Considering the economic importance of the rose plants and the highly destructive nature of RRV, this report underscores the need for immediate effective quarantine and management of the virus for protecting the economically important rose industry in Florida. References: (1) A. G. Laney et al. J. Gen. Virol. 92:1727, 2011. (2) S. Sabanadzovic and N. Abou Ghanem-Sabanadzovic. J. Plant Pathol. 90:287, 2008. (3) I. E. Tzanetakis et al. Plant Pathol. 55:568, 2006. (4) USDA. 2007 Census of Agriculture 3:25, Washington, DC, 2010.

First Report of Xanthomonas axonopodis Causing Bacterial Leaf Spot on Crape Myrtle.

Crape myrtle (Lagerstroemia sp.) is a popular ornamental tree in the United States and the industry produced 2,781,089 trees in 2010 with a value exceeding US $42.8 million (1,4). A new disorder of crape myrtle has been observed since 2011 in numerous nurseries in Florida, which was characterized by dark brown, angular to irregularly shaped, oily-looking spots surrounded by yellow halos. The disease primarily affects lower leaves that eventually turn yellow and can lead to rapid defoliation of susceptible cultivars. Plants examined in field surveys at the University of Florida, North Florida Research and Education Center, Quincy, FL in 2012 and 2013 also had similar symptoms on cvs. Arapaho, Carolina Beauty, Tuscarora, Whit IV Red Rocket, Whit VIII Rhapsody in Pink, and White Chocolate. The disease severity ranged from 20 to 70% and all the plants were infected. A yellow-pigmented, gram-negative, oxidase negative bacterium was consistently isolated from symptomatic leaves (two leaves from each of five plants). Pathogenicity tests were performed using five isolated bacterial strains on potted crape myrtle cv. Arapaho. Three plants were inoculated with a 108 CFU/ml suspension of bacterial strains in sterile deionized water, and covered with transparent plastic bag for 48 h. Two control plants were inoculated with sterile distilled water. The inoculated plants were then incubated in a greenhouse at 30 to 34°C for 14 days. Symptoms of dark brown, angular to irregularly shaped lesions were observed only on the inoculated plants after 7 days. The bacterium was re-isolated from the inoculated symptomatic plants as described above, thus fulfilling Koch's postulates. Fatty acid methyl ester profiling of the five isolated bacteria using GC-MIDI (Microbial IDentification Inc, Newark, DE) revealed the identity of the bacterium as Xanthomonas axonopodis with an identity index of ~0.80, but matched multiple pathovars. Total genomic DNA was extracted from the pure bacterial culture using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA). The genomic DNA was subjected to PCR assay using universal primers 27f/1492R (3) targeting the complete 16S rRNA gene and primers 16F945/23R458 (2), which target the partial 16S-23S internal transcribed spacer region. PCR amplification using primer pairs 27f/1492R and 16F945/23R458 resulted in amplicons of 1,450 and 1,500 bp, respectively. The amplicons were gel purified and sequenced directly at Florida State University. BLAST analysis of the sequences (Accession Nos. KF926678, KF926679, KF926680, KF926681, and KF926682) revealed the identity of the bacterium as X. axonopodis, ranging from 98 to 99%, with several strains in the NCBI database. Phylogenetic analysis using the neighbor-joining method showed that our strains were distantly clustered with X. axonopodis pv. dieffenbachiae when compared to other available strains in the database. To our knowledge, this is the first worldwide report of a bacterial leaf spot on crape myrtle caused by X. axonopodis. This information should aid in the development of breeding lines with resistance to bacterial leaf spot and effective disease management practices. References: (1) C. S. Furtado. Garden Bull. 24:185, 1969. (2) C. Guasp. Int. J. Syst. Evol. Microbiol. 50:1629, 2000. (3) D. J. Lane. Page 115 in: Nucleic Acid Techniques in Bacterial Systematics, 1991. (4) USDA. 2007 Census of Agriculture, Washington, DC. 3:25, 2010.

First Report of Cucumber mosaic virus Associated with Capsicum chinense var. Scotch Bonnet in Florida.

Scotch bonnet (Capsicum chinense) is a tropical hot pepper variety that is grown in South America, the Caribbean Islands, and in Florida, and is an important cash crop. In Florida, scotch bonnet is grown on ~100 acres annually. Virus-like leaf symptoms including mosaic and yellow mottling were observed on scotch bonnet plants in a field at Quincy, FL, with a disease incidence of ~5%. Two symptomatic and one non-symptomatic plant sample were collected from this field for identification of the causal agent associated with the symptoms. Viral inclusion assays (2) of the epidermal tissues of the symptomatic scotch bonnet samples using Azure A stain indicated the presence of spherical aggregates of crystalline inclusion bodies. Testing of the symptomatic samples using lateral flow immunoassays (Immunostrips, Agdia, Elkhart, IN) specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV), showed a positive reaction only to CMV. The sap from an infected leaf sample ground in 0.01 M Sorensons phosphate buffer (pH 7.0) was used to mechanically inoculate one healthy scotch bonnet plant (tested negative for CMV with Immunostrip) at the 2- to 3-leaf stage. The inoculated plant developed mild mosaic and mottling symptoms 12 to 14 days post inoculation. The presence of CMV in the mechanically inoculated plant was further verified using CMV Immunostrips. Total RNA was extracted (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from the previously collected two symptomatic and one non-symptomatic scotch bonnet samples. The samples were subjected to reverse-transcription (RT)-PCR assays using SuperScript III One-Step RT-PCR System (Invitrogen, Life Technologies, Grand Island, NY), and using multiplex RT-PCR primer sets (1). The primers were designed to differentiate the CMV subgroup I and II, targeting the partial coat protein gene and the 3'UTR. The RT-PCR assays using the multiplex primers produced an amplicon of 590 bp, with the CMV subgroup I primers. The RT-PCR product was only amplified from the symptomatic leaf samples. The obtained amplicons were gel eluted, and directly sequenced bi-directionally (GenBank Accession Nos. KF805389 and KF805390). BLAST analysis of these sequences showed 97 to 98% nucleotide identities with the CMV isolates in the NCBI database. The isolates collected in Florida exhibited highest identity (98%) with the CMV isolate from tomato (DQ302718). These results revealed the association of CMV subgroup I with symptomatic scotch bonnet leaf samples. Although CMV has been reported from scotch bonnet, this is the first report of its occurrence in Florida. References: (1) S. Chen et al. Acta Biochim Biophys Sin. 43:465, 2011. (2) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986.

First Report of Turnip mosaic virus Infecting Brassica carinata (Ethiopian Mustard) in the United States.

Brassica carinata L. Braun (Ethiopian mustard) is an annual oil seed crop currently being evaluated for its potential use as a source of biofuel. Due to its high content of erucic acid, it provides a biodegradable non-fossil fuel feedstock that has many applications ranging from biofuels to other industrial uses such as polymers, waxes, and surfactants. Moreover, high glucosinolate content adds the scope of B. carinata being used as a bio-fumigant. B. carinata is amenable to low input agriculture and has great economic potential to be used as a winter crop, especially in the southeastern United States. Virus-like leaf symptoms including mosaic, ringspot, mottling, and puckering were observed on B. carinata (cvs. 080814 EM and 080880 EM) in field trials at Quincy, FL, during spring 2013, with disease incidence of >80%. A more extensive survey of the same field location indicated that mosaic symptoms were the most common. Viral inclusion assays (1) of leaves with a range of symptoms indicated the presence of potyvirus-like inclusion bodies. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA) from six symptomatic samples and one non-symptomatic B. carinata sample were subjected to reverse transcription (RT)-PCR assays using SuperScript III One-Step RT-PCR System (Invitrogen, Life Technologies, NY), and two sets of potyvirus-specific degenerate primers MJ1-F and MJ2-R (2) and NIb2F and NIb3R (3), targeting the core region of the CP and NIb, respectively. The RT-PCR assays using the CP and NIb specific primers produced amplicons of 327 bp and 350 bp, respectively, only in the symptomatic leaf samples. The obtained amplicons were gel-eluted and sequenced directly (GenBank Accession Nos. KC899803 to KC899808 for CP and KC899809 to KC899813 for NIb). BLAST analysis of these sequences revealed that they came from Turnip mosaic virus (TuMV). Pairwise comparisons of the CP (327 bp) and NIb (350 bp) segments revealed 98 to 99% and 96 to 98% nucleotide identities, respectively, with corresponding sequences of TuMV isolates. These results revealed the association of TuMV with symptomatic B. carinata leaf samples. Although TuMV has been reported from B. carinata in Zambia (4), this is the first report of its occurrence on B. carinata in the United States. Considering the importance of B. carinata as a biofuel source, this report underscores the need for developing effective virus management strategies for the crop. References: (1) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986. (2) M. Grisoni et al. Plant Pathol. 55:523, 2006. (3) L. Zheng et al. Plant Pathol. 59:211, 2009. (4) D. S. Mingochi and A. Jensen. Acta Hortic. 218:289, 1988.

First Report of Sclerotinia Stem Rot Caused by Sclerotinia sclerotiorum on Brassica carinata in Florida.

Brassica carinata A. Braun, commonly referred to as Ethiopian rapeseed, a near relative of collards and mustard, has become the object of increasing interest as an oil crop. It has been reported that B. carinata adapts better and is more productive than B. napus (canola) in adverse conditions, such as clay and sandy soils and under low management cropping systems (1). In late February 2012, symptoms typical of sclerotinia stem rot were observed in B. carinata trials (cultivars 090867 EM and 080814 EM) at the University of Florida, North Florida Research and Education Center located in Quincy, FL. Approximately 20 to 30% of the B. carinata cultivar 090867 EM were observed to have symptoms and approximately 5% of cultivar 080814 EM displayed symptoms. Stems had white mycelia growing on the outside, plants were lodging and spherical to cylindrical, 3 to 8 mm, and black sclerotia were found outside and inside bleached stems. Sclerotia from diseased stems were surface sterilized and placed in 9-cm diameter petri plates on quarter strength potato dextrose agar (PDA) amended with 25% lactic acid. Fungal cultures consisting of white mycelia and medium-sized (mean 4 mm), black, irregular sclerotia were consistently recovered and identified as Sclerotinia sclerotiorum (Lib.) de Bary based on morphological characteristics (3). Sequence analyses were conducted on mycelium by extracting fungal DNA with the Qiagen DNeasy Plant Mini Kit (Valencia, CA). PCR amplification was performed using primers ITS1 and ITS4. The BLAST search revealed that the sequence (GenBank Accession No. JX307092) had 99 and 100% sequence identity with S. sclerotiorum GenBank accessions JN013184.1 and JN012606.1. Pathogenicity was determined by inoculating six 1-month-old B. carinata plants (cultivars 090867 EM and 080814 EM) that were grown in greenhouse pots (20 cm in diameter). Mycelia plugs (8 mm in diameter) were excised from the colony margin after 6 days of incubation at room temperature (approximately 25°C), and placed on stems, at the soil line, of B. carinata plants. Six control plants were inoculated with noncolonized PDA plugs. All plants were enclosed in plastic bags that had been sprayed with water on the inside to maintain high humidity and kept in the laboratory at room temperature (approximately 25°C). Symptoms similar to those observed in the field were evident after 3 days on inoculated plants and S. sclerotiorum was reisolated. In the controls, no symptoms developed and the fungus could not be isolated. The experiment was repeated with similar results. The majority of rapeseed production is in North Dakota, where sclerotinia stem rot caused by S. sclerotiorum is a major fungal disease affecting production (2). Currently, there is no significant B. carinata production in Florida; however, interest in biofuels could lead to an increase in planted acreage and sclerotinia stem rot could become a significant disease problem in areas of Florida were B. carinata is planted. To our knowledge, this is the first report of sclerotinia stem rot of B. carinata caused by S. sclerotiorum in Florida. References: (1) M. Cardone et al. Biomass and Bioenergy. 25:623, 2003. (2) L. E. del Río et al. Plant Dis. 91:191, 2007. (3) L. M. Kohn. Phytopathology 69:881, 1979.

First Report of Downy Mildew (Hyaloperonospora camelinae) on Camelina sativa in Florida.

Camelina sativa (L.) Crantz, Brassicaceae, whose common name is Crantz-large-seeded false flax, is an annual oilseed species. It is grown as a forage and biofuel crop in Europe and North America. False flax is an ideal low-input crop for biodiesel production, because of its low requirements for nitrogen fertilizer and pesticides. Production costs of this crop are substantially lower than those of many other oilseed crops such as rapeseed, corn, and soybean. It is an excellent rotation crop and can reduce disease and insect and weed pressure in wheat fields. During the spring of 2011, commercial and research plantings of C. sativa cultivar Calena in Liberty and Gadsden counties in north Florida developed symptoms typical of downy mildew. In spring of 2012, the same symptoms were observed in experimental plantings of false flax. A white downy mold was observed on the upper third portion of the plants, on the upper stem internodes, and on the developing seed. The affected stems exhibited a twisted growth. Conidiophores had main trunks with dichotomous branches terminating in slender curved tips. Conidia were ovoid and 28 to 45 (mean 36) µm long and 22 to 38 (mean 30) µm wide. Conidiophores were branched (three to four branches, each with six to eight curved tips) and ranged from 107 to 236 µm long and 5 to 14 µm wide. Mycelium was obtained directly from diseased plants for DNA extraction (Qiagen DNeasy Plant Mini Kit, Valencia, CA). Primers ITS1 and ITS4 were used for PCR amplification (4). The PCR product was sequenced bidirectionally with the PCR primers. A consensus nucleotide sequence (Accession JQ997103) was compared to those in the NCBI GenBank database using a BLAST search. The sequence was 99% similar to sequence from Hyaloperonospora camelinae (Gäum.) Göker, Voglmayr, Riethm, M. Weiss & Oberw. (Accession AY198249.1) with a 95% query coverage (1). Pathogenicity was established by applying white conidial masses of downy mildew from field samples to stems of 4-week-old plants grown in pots in a greenhouse maintained at 25 ± 2°C. Noninoculated plants maintained under the similar conditions served as control. Symptoms and signs of downy mildew developed after 14 days only on inoculated plants. Downy mildew constitutes a serious threat to the cultivation of C. sativa in Florida because of the humid climate favoring disease development. Diseased plants may reduce yield and disease management would increase production costs. H. camelinae was previously reported on C. sativa in Oregon, Minnesota, Montana (3), and Nebraska (2). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Florida. References: (1) M. Göker et al. Canad. J. Bot. 81:672, 2003. (2) R. M. Harveson et al. Plant Health Progress. 2011. doi: 10.1094/PHP-2011-1014-01-BR. (3) M. L. Putnam et al. Plant Health Progress. 2009. doi: 10.1094/PHP-2009-0910-01-BR. (4) 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.

Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida.

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the 'carotovora' group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.

A structured experiential curriculum in community medicine.

In order to provide experimental training in community medicine, a structured curriculum has been developed. Specific methodological skills in community medicine are identified and nine content areas are presented in seminar form during the three-year training program. Each resident is expected to participate in a community health care project and demonstrate one or more of the methodological skills identified. The experiences of two residents are reported. One involved a community health needs assessment and one, the development of a mechanism to ensure continuing consumer/provider communication.