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 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 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.