Simulated Leaching of Foliar Applied Copper Bactericides on the Soil Microbiome Utilizing Various Beta Diversity Resemblance Measurements.

Copper bactericides are routinely used to control Xanthomonas perforans (XP), causal agent of bacterial spot of tomato. Given the widespread tolerance to copper in XP strains in FL, USA, nanotechnology-based elemental composites have gained interest for their potential applications in agriculture in part due to their enhanced antimicrobial properties and toxicity to copper-tolerant strains. However, little is known about the potential impact of conventional copper bactericides as well as nano-based elemental composites on soil microbial communities, as determined by high-throughput sequencing of the 16S rDNA. We compared the effects of 2 and 200 µg/mL of core-shell (CS), a metallic copper composite, and a conventional copper bactericide + mancozeb (Cu+Man) on the soil microbiome. These treatments were compared to three controls, the microbial profile of the soil prior to application of copper products, a water application, and spiking the soil with a soilborne phytobacterium, Ralstonia solanacearum (RS). The RS treatment was included to determine if downstream analysis could detect the artificial inoculation. Utilizing multiple ß diversity measurements, each emphasizing various tenets of ecology, provided a greater perspective of the effects the treatments had on the microbiome. Analysis of HTS data revealed that the two treatments containing field applied rates of metallic copper, CS 200 and Cu+Man, had the largest impact on the soil microbiome at seven-days posttreatment compared to water. However, we simulated field applied rates of CS 200 entering the soil by treating soil with CS 2 and determined this concentration had a negligible effect on the soil microbiome. IMPORTANCE Nanotechnology-based elemental composites have gained popularity for their potential applications in plant disease management due to their enhanced antimicrobial properties. However, little is known about their potential impact on the environment. Foliar applications of nano metallic composites upon leaching into the soil have the potential to impact soil microbial populations that in turn influence soil health. Utilizing multiple ß diversity measurements, high-throughput sequencing analysis revealed that field applied rates of metallic copper (200 µg/mL) from an advanced copper composite (core-shell [CS]) and a conventional copper bactericide in combination with mancozeb had the largest impact on the soil microbiome compared to water and nontreated control. To simulate leaching from the leaf surface, a lower concentration (2 µg/mL) of CS was also applied to the soil and had a negligible effect on the soil microbiome. Thus, field applied rates of CS may have a minimal effect on soil microbial communities.

Particle-size dependent bactericidal activity of magnesium oxide against Xanthomonas perforans and bacterial spot of tomato.

Bacterial spot, caused by Xanthomonas spp., is a highly destructive disease of tomatoes worldwide. Copper (Cu) bactericides are often ineffective due to the presence of Cu-tolerant strains. Magnesium oxide (MgO) is an effective alternative to Cu bactericides against Xanthomonas spp. However, the effects of particle size on bactericidal activity and fruit elemental levels are unknown. In this study, nano (20 nm) and micron (0.3 and 0.6 µm) size MgO particles were compared for efficacy. Nano MgO had significantly greater in vitro bactericidal activity against Cu-tolerant X. perforans than micron MgO at 25-50 µg/ml. In field experiments nano and micron MgO applied at 200 and 1,000 µg/ml were evaluated for disease control. Nano MgO at 200 µg/ml was the only treatment that consistently reduced disease severity compared to the untreated control. Inductively Coupled Plasma Optical Emission Spectroscopy revealed that nano MgO applications did not significantly alter Mg, Cu, Ca, K, Mn, P and S accumulation compared to fruits from the untreated plots. We demonstrated that although both nano MgO and micron MgO had bactericidal activity against Cu-tolerant strains in vitro, only nano MgO was effective in bacterial spot disease management under field conditions.

Recombinase Polymerase Amplification Assay for Field Detection of Tomato Bacterial Spot Pathogens.

Bacterial spot of tomato is caused by Xanthomonas gardneri, X. euvesicatoria, X. perforans, and X. vesicatoria. Current diagnostic methods for the pathogens are not in-field assays. Recombinase polymerase amplification (RPA) is ideal for in-field detection assays, because it is an isothermal technique that is rapid and more tolerant to inhibitors compared with polymerase chain reaction. Hence, novel RPA probes and primers were designed to amplify regions of the hrcN gene of X. gardneri, X. euvesicatoria, and X. perforans. The X. gardneri RPA is specific to X. gardneri with a detection limit of 106 CFU/ml and detected X. gardneri in lesions from naturally (n = 6) or artificially (n = 18) infected plants. The X. euvesicatoria RPA detects both X. euvesicatoria and X. perforans with a detection limit of 106 CFU/ml and detected both pathogens in plants artificially infected (n = 36) or naturally infected (n = 85) with either X. euvesicatoria or X. perforans. The X. perforans RPA is specific to X. perforans with a detection limit of 107 CFU/ml. Although the X. perforans RPA assay was unable to detect X. perforans from lesions, the X. euvesicatoria RPA was successfully used in field to detect X. perforans from symptomatic field samples (n = 31). The X. perforans RPA was then used to confirm the pathogen in the laboratory. The X. euvesicatoria and X. gardneri RPA is promising for rapid, real-time in-field detection of bacterial spot and one of the first developed among plant pathogenic bacteria.

Nano-Magnesium Oxide: A Novel Bactericide Against Copper-Tolerant Xanthomonas perforans Causing Tomato Bacterial Spot.

Bacterial spot caused by Xanthomonas perforans causes significant damage on tomato in Florida. Due to the presence of copper (Cu)-tolerant X. perforans strains, Cu bactericides are not effective in disease management. Hence, there is a critical need to find alternatives for Cu. Antibacterial activity of magnesium oxide (Nano-MgO), and other metal oxide nanoparticles, were evaluated against a Cu-tolerant and -sensitive X. perforans strain. In vitro experiments demonstrated high antibacterial activity of Nano-MgO against both strains compared with the commercial Cu. The minimum inhibitory concentration of Nano-MgO is 25 µg/ml and the minimum bactericidal concentration is 100 µg/ml against a Cu-tolerant X. perforans strain after 4 h of exposure. Structural changes in the bacterial membrane following exposure to Nano-MgO treatments compared with the controls were observed using transmission electron microscopy. In two greenhouse experiments with a Cu-tolerant strain, bacterial spot severity was significantly reduced by Nano-MgO at 200 µg/ml compared with Cu-ethylene bis-dithiocarbamate (grower standard), and the untreated control (P = 0.05). In three field experiments, Nano-MgO at 200 µg/ml significantly reduced disease severity with no negative impact on yield compared with the untreated control. Inductively coupled plasma mass spectrometric analysis of the fruit confirmed that Nano-MgO application did not lead to the accumulation of Mg, Cu, Ca, K, Mn, P, and S. This study is the first to demonstrate the potential of Nano-MgO against bacterial spot of tomato.

Advanced Copper Composites Against Copper-Tolerant Xanthomonas perforans and Tomato Bacterial Spot.

Bacterial spot, caused by Xanthomonas spp., is a widespread and damaging bacterial disease of tomato (Solanum lycopersicum). For disease management, growers rely on copper bactericides, which are often ineffective due to the presence of copper-tolerant Xanthomonas strains. This study evaluated the antibacterial activity of the new copper composites core-shell copper (CS-Cu), multivalent copper (MV-Cu), and fixed quaternary ammonium copper (FQ-Cu) as potential alternatives to commercially available micron-sized copper bactericides for controlling copper-tolerant Xanthomonas perforans. In vitro, metallic copper from CS-Cu and FQ-Cu at 100 µg/ml killed the copper-tolerant X. perforans strain within 1 h of exposure. In contrast, none of the micron-sized copper rates (100 to 1,000 µg/ml) from Kocide 3000 significantly reduced copper-tolerant X. perforans populations after 48 h of exposure compared with the water control (P < 0.05). All copper-based treatments killed the copper-sensitive X. perforans strain within 1 h. Greenhouse studies demonstrated that all copper composites significantly reduced bacterial spot disease severity when compared with copper-mancozeb and water controls (P < 0.05). Although there was no significant impact on yield, copper composites significantly reduced disease severity when compared with water controls, using 80% less metallic copper in comparison with copper-mancozeb in field studies (P < 0.05). This study highlights the discovery that copper composites have the potential to manage copper-tolerant X. perforans and tomato bacterial spot.

Molecular Epidemiology of Pseudomonas syringae pv. syringae Causing Bacterial Leaf Spot of Watermelon and Squash in Florida.

From 2013 to 2014, bacterial leaf spot epidemics incited by Pseudomonas syringae pv. syringae affected an estimated 3,000 ha of watermelon and squash in Florida, and caused foliar blighting and transplant losses in severely affected fields. To investigate the diversity of the causal agent, we isolated 28 P. syringae strains from diseased plants grown in 10 Florida and Georgia counties over the course of 2 years. Strains were confirmed as P. syringae through sequence analysis of the 16S ribosomal RNA, phenotypic, and biochemical profiling; however, 20 displayed an atypical phenotype by exhibiting nonfluorescent activity on King's medium B agar and being negative for ice-nucleating activity. Multilocus sequence analysis and BOX polymerase chain reaction revealed the presence of two haplotypes among the collected strains that grouped into two distinct clades within P. syringae phylogroup 2. Pathogenicity testing showed that watermelon, cantaloupe, and squash seedlings were susceptible to a majority of these strains. Although both haplotypes were equally virulent on cantaloupe, they differed in virulence on watermelon and squash. The distribution of one haplotype in 9 of 10 Florida and Georgia counties sampled indicated that these epidemics were associated with the recent introduction of a novel clonal P. syringae lineage throughout major watermelon production areas in Florida.

Epidemiology and management of bacterial leaf spot on watermelon caused by Pseudomonas syringae.

Bacterial leaf spot of watermelon caused by Pseudomonas syringae has been an emerging disease in the southeastern United States in recent years. Disease outbreaks in Florida were widespread from 2013 to 2014 and resulted in foliar blighting at the early stages of the crop and transplant losses. We conducted a series of field trials at two locations over the course of two years to examine the chemical control options that may be effective in management of this disease, and to investigate the environmental conditions conducive for bacterial leaf spot development. Weekly applications of acibenzolar-S-methyl (ASM) foliar, ASM drip, or copper hydroxide mixed with ethylene bis-dithiocarbamate were effective in reducing the standardized area under the disease progress curve (P < 0.05). Pearson's correlation test demonstrated a negative relationship between the average weekly temperature and disease severity (-0.77, P = 0.0002). When incorporated into a multiple regression model with the square root transformed average weekly rainfall, these two variables accounted for 71% of the variability observed in the weekly disease severity (P < 0.0001). This information should be considered when choosing the planting date for watermelon seedlings as the cool conditions often encountered early in the spring season are conducive for bacterial leaf spot development.

Identification and characterization of two novel genomic RNA segments RNA5 and RNA6 in rose rosette virus infecting roses.

Rose rosette virus (RRV), a negative-strand RNA virus belonging to the genus Emaravirus, has recently been characterized to be the causal agent of rose rosette disease. Roses showing typical symptoms of RRV collected from a rose nursery in Florida were subjected to reverse transcription-PCR (RT-PCR) assay using primers corresponding to the conserved inverted 13 nucleotide long stretches found at the termini of the RRV genomic RNA segments. RT-PCR analysis yielded two novel genomic RNA segments, RNA5 and RNA6, in addition to the previously identified four RNA segments. The RNA5 is 1650 bp long and encodes for a polypeptide of 465 amino acids (54.3 K), while RNA6 is 1400 bp long and encodes for a polypeptide of 233 amino acids (27.05 K). RACE analysis showed that, both the RNA segments posses at their 5' and 3' termini, stretches of conserved inverted complementary13 nucleotides long sequence with two nucleotide mismatches as previously identified in other genomic RNA segments. Northern blot analysis as well as RT-PCR using specific primers showed the presence of the novel genomic RNA segments in infected plants, but absent in the non-infected plants. The GenBank Acc. Nos. for the sequences reported in this paper are KT007556 and KT007557.

Low Concentrations of a Silver-Based Nanocomposite to Manage Bacterial Spot of Tomato in the Greenhouse.

Bacterial spot, caused by four Xanthomonas spp., is one of the most damaging diseases of tomato worldwide. Due to limited disease management options, growers rely heavily on copper-based bactericides, which are often ineffective due to the presence of copper-resistant Xanthomonas strains. This study was undertaken to characterize the antibacterial activity of a silver-based nanocomposite, Ag-dsDNA-GO, and its potential as an alternative to copper. Ag-dsDNA-GO at rates as low as 10 µg/ml killed all bacterial cells of copper-tolerant and -sensitive Xanthomonas perforans strains in suspensions containing approximately 103 CFU/ml within 15 min of exposure in vitro, whereas equivalent rates of copper (10, 25, and 50 µg/ml) were unable to significantly reduce populations compared with the untreated control after 24 h of exposure (P = 0.05). All copper concentrations killed the copper-sensitive X. perforans strain but required exposure for ≥1 h. Ag-dsDNA-GO also exhibited antibacterial activity against copper-tolerant X. vesicatoria, X. euvesicatoria, and X. gardneri strains. In greenhouse studies, tomato plants treated with Ag-dsDNA-GO at either 75 or 100 µg/ml prior to artificial inoculation significantly reduced disease severity when compared with copper-mancozeb and negative controls (P = 0.05). This study highlights the potential of Ag-dsDNA-GO as an alternative to copper in tomato transplant production.

Angular Leaf Spot of Cucurbits is Associated With Genetically Diverse Pseudomonas syringae Strains.

Angular leaf spot of cucurbits is generally considered to be caused by Pseudomonas syringae pv. lachrymans. It has a worldwide distribution and has been observed to emerge sporadically under humid and wet conditions. Reports of multiple P. syringae pathovars associated with the disease and lack of molecular analysis has left the true diversity of populations in the United States unclear. In this study, we collected 27 P. syringae strains causing foliar lesions and blighting on watermelon, cantaloupe, and squash in Florida, Georgia, and California over several years. Strains were fluorescent on King's medium B agar and displayed the typical phenotypic and biochemical characteristics of P. syringae. P. syringae pv. lachrymans is a member of genomospecies 2. However, the genetic profiles obtained through both MLSA (gyrB, rpoD, gapA, and gltA) and BOX-PCR (BOXA1R) identified 26 of the P. syringae strains to be distributed among three clades within genomospecies 1, and phylogenetically distinct from genomospecies 2 member P. syringae pv. lachrymans. A novel MLSA haplotype of the pathogen common to all states and cucurbit hosts was identified. Considerable genetic diversity among P. syringae strains infecting cucurbits is associated with the same disease, and reflects the larger ecological diversity of P. syringae populations from genomospecies 1.

A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato.

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.

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.

Synergetic bitherapy in mice with xenografts of human prostate cancer using a methional mimic (METLICO) and an aldehyde dehydrogenase 3 inhibitor (MATE): systemic intraperitoneal (IP) and targeted intra-tumoral (IT) administration.

An intraperitoneal (IP) monotherapy in nu/nu mice with subcutaneous xenografts of a human prostate epithelial cancer cell line:DU145 was undertaken with an aldehyde dehydrogenase 3 inhibitor MATE, that is a potent apoptogen on (DU145) in culture but not on their human prostate epithelial normal counterparts [13] . Tumour growth was slowed down but treatment had to be done 5days/week. To try to potentiate the action of MATE in vivo, a bitherapy was undertaken based on the synergetic apoptotic effect that had been observed previously in culture on DU145 treated with a methional mimic METLICO and DIMATE, an inhibitor of ALDH1 and ALDH3 [19]. The bitherapy with METLICO/MATE administered IP was as effective as the monotherapy with MATE alone by IP, but at a 2-fold lower dose of MATE and at a dose of METLICO that had no growth-inhibitory effect as a monotherapy . Hence there was definite synergism with bitherapy. To try to increase the efficacy of bitherapy, it was administered by the intra-tumoral (IT) route using the recently developed 20-bars-pressurized microinjection system from CERMA [16, 17]. IT administration of the bitherapy was indeed more effective than that by IP as regards tumour volumes are concerned. Histopathological analysis of IT-treated tumours confirmed that there were many necrotized zones but intact cells were still present. Approaches for treating a wider zone of tumour tissue by IT-bitherapy are discussed.

A set of glycoproteins recognized by A2B5 antibody with major bands at 55 and 76 kDa is overexpressed in human head and neck squamous cell carcinomas.

A2B5 antibody was found to strongly label frozen sections of human head and neck squamous cell carcinomas. The low amount of glycolipids (c-series gangliosides and sulfatides) purified from the same tumors and reactive with A2B5 by immunostaining on thin-layer plates could not account for the high level of tissue labeling. Proteins were extracted from both normal tissues and squamous cell carcinomas and analyzed by Western blot with A2B5 antibody on PVDF membranes. The antibody was found to stain a set of glycoproteins with two major bands at 55 and 76 kDa present in normal tissues and overexpressed in carcinomas. Staining was abolished by prior treatment of the PVDF membranes either with Arthrobacter ureafaciens neuraminidase or with a solution of 10 mM periodate that is known to destroy carbohydrates. Our results show that the carbohydrate epitope recognized by A2B5 antibody can be displayed by both glycolipids and glycoproteins.

Outcome of patients with idiopathic pulmonary fibrosis admitted to the ICU for respiratory failure.

STUDY OBJECTIVES: To analyze the outcome of acute respiratory failure (ARF) in patients with idiopathic pulmonary fibrosis (IPF), and to evaluate the benefits of invasive and noninvasive mechanical ventilation (MV). DESIGN: Retrospective study. SETTING: University hospital. PATIENTS: Fifteen consecutive patients with IPF referred to the ICU for ARF between January 1989 and June 1998. MEASUREMENTS AND RESULTS: Fifteen patients (mean +/- SD age, 64 +/- 10 years) were included. Eight patients had clinical, functional, and radiologic features of IPF, and the remaining seven patients also had biopsy specimen-proven IPF. The mean duration between diagnosis of IPF and admission to the ICU was 26.5 +/- 28 months. At the time of ICU admission, mean arterial blood gas levels were as follows: PaO(2)/fraction of inspired oxygen, 113 +/- 95; pH, 7.32 +/- 0.10; and PaCO(2), 55 +/- 21 mm Hg. All patients received MV; 12 patients required tracheal intubation, either at the time of ICU admission (n = 10) or after failure of noninvasive ventilation (NIV; n = 2); and 3 patients only received NIV. Three of the five patients receiving NIV died of respiratory failure. Eleven patients died in the ICU, either from hypoxemia (n = 8) or from septic shock (n = 3). Four patients were discharged alive from the ICU, and two of them died shortly thereafter. CONCLUSION: The outcome of patients with IPF referred to the ICU for ARF was very poor and not improved by MV. Without a clearly identified reversible cause of ARF, these patients should not benefit from admission to the ICU.

Hyperintense benign liver lesions on spin-echo T1-weighted MR images: pathologic correlations.

BACKGROUND: To determine the incidence of hyperintensity on T1-weighted spin echo (SE) images in benign liver lesions, value of fat-suppressed magnetic resonance (MR) imaging for the detection of fat within these lesions, and the causes of hyperintensity by correlation to pathologic examinations. METHODS: Five hundred forty-nine patients with 805 benign liver lesions including 585 hemangiomas, 188 focal nodular hyperplasias (FNHs), 14 hepatic adenomas (HAs), 14 focal fatty infiltrations (FFIs), two biliary cystadenomas, and two hemorrhagic cysts were examined by T2-weighted and T1-weighted SE MR imaging. For hyperintense lesions on T1-weighted SE images, fat-suppressed images were obtained by selective presaturation of fat. RESULTS: Thirty-two lesions (four FNHs, 10 HAs, 14 FFIs, two biliary cystadenomas, and two hemorrhagic cysts) appeared hyperintense on T1-weighted SE images; 21 of these became hypointense on the fat-suppressed T1 weighted SE images (one FNH, six HAs, and 14 FFIs) and contained fat at pathological examination. The other 11 lesions remained hyperintense on fat-suppressed T1-weighted SE images and had no fat deposition. Causes of hyperintensity in these cases were sinusoidal dilatation, copper deposition, hemorrhage, and high protein content. CONCLUSION: Among benign liver lesions, hyperintensity on T1-weighted SE images is rare (3.9%). Causes of this hyperintensity are fat deposition, copper accumulation, sinusoidal dilatation, bemorrhage, and high protein content. Fat-suppressed imaging can distinguish fat deposition from other causes of hyperintensity.