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ABSTRACT: BACKGROUND: Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. Previously, we reported a combination of Penicillin G and Streptomycin was effective in eliminating or suppressing the associated bacterium, 'Candidatus Liberibacter asiaticus' (Las). RESULTS: Here we report the bacterial composition and community structure in HLB-affected citrus plants during a growing season and while being treated with antibiotic combinations PS (Penicillin G and Streptomycin) and KO (Kasugamycin and Oxytetracycline) using the PhylochipTM G3 array. Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChipTM G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, of which five contained 100 or more OTUs, Proteobacteria (44.1%), Firmicutes (23.5%), Actinobacteria (12.4%), Bacteroidetes (6.6%) and Cyanobacteria (3.2%). In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599. The over-all bacterial diversity decreased with the antibiotic treatments, as did the abundance of 11 OTUs within Proteobacteria, Firmicutes, Bacteroidetes and Planctomycetes. Within the Proteobacteria, ten OTUs representing the class gamma-proteobacteria increased in abundance after four months of treatment, when the Las bacterium was at its lowest level in the HLB-affected citrus field plants. CONCLUSIONS: Our data revealed that Proteobacteria was constantly the dominant bacterial phylum recovered from citrus leaf midribs, with the alpha-proteobacterial and the gamma-proteobacterial classes vying for prevalence. In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described. Bacterial cells in close proximity may be able to modify their microenvironment, making the composition of the microbial community an important factor in the ability of Las to cause HLB progression. A low Las level was seen as an annual fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.
BMC Microbiology 05/2013; 13(1):112. · 3.04 Impact Factor
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ABSTRACT: Surface runoff water is an important non-point source of fecal pollution to downstream water; however, there is a lack of systematic studies on the microbial quality of surface runoff water from watersheds with mixed land uses. In this study water samples from 12 surface runoff holding water bodies (SRW), which collected runoff from various patterns of land use within the St. Lucie watershed along the southeastern coastline of Florida, were collected monthly for 22months. The concentration of fecal indicator bacteria (FIB) and frequency of detection of Salmonella and host specific markers (HF183, CF128, CF193, and HS-esp) were determined, and their associations with land use, rainfall, and water physico-chemical parameters were investigated. Higher FIB concentrations were observed from urban land and cattle ranch sites. Within the same primary land use pattern, different sub-patterns did not have the same level of FIB: golf communities contributed less to fecal pollution than residential areas, and plant nursery sites contained relative higher FIB concentrations than other agricultural sites. Salmonella, CF128, and CF193 markers were more frequently detected from the cattle ranch sites. In contrast the frequency of detecting human specific markers (HF183 and HS-esp) was much higher in residential sites. Rainfall positively affected the concentration of FIB and occurrence of Salmonella, possibly by providing more inputs or mobilizing the sources from sediments. Water temperature, dissolved organic carbon (DOC), and nutrient levels were positively correlated with FIB concentrations and occurrence in SRW, possibly by promoting their growth and survival. This study indicated the need for site specific mitigation strategies to improve SRW and downstream water quality.
Science of The Total Environment 02/2013; 449C:426-433. · 3.29 Impact Factor
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ABSTRACT: Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion-rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.
Phytopathology 06/2012; 102(6):567-74. · 2.80 Impact Factor
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ABSTRACT: Ten Mile Creek (TMC) is a major tributary of the Indian River Lagoon (IRL), one of the largest and most ecologically diverse
estuaries of the east coast of Florida. Recent algal blooms within the IRL have focused attention on the role of different
watersheds playing in the supply of growth-limiting nutrients. The goal of this study was to determine the nutrient-limiting
status of the TMC outflow, which is influenced by both agricultural input and urban development. Four laboratory experiments
were conducted with water samples from TMC, adding different concentrations of phosphorus (P) and nitrogen (N) under controlled
conditions. The results showed that turbidity and phytoplankton biomass (in terms of chlorophyll a concentration) in TMC water samples were responsive to N additions. Turbidity and phytoplankton biomass increased with addition
of available N, but were not affected by addition of reactive P. The results indicate that available N is the limiting nutrient
for the growth of phytoplankton in the TMC.
Hydrobiologia 04/2012; 605(1):247-258. · 1.78 Impact Factor
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ABSTRACT: Bacteroidales markers are promising indicators of fecal pollution and are now widely used in microbial source tracking (MST) studies. However, a thorough understanding of the persistence of Bacteroidales population after being released into environmental waters is lacking. We investigated the persistence of two host specific markers (HF183 and CF193) and temporal change of Bacteroidales population over 14 days in freshwater microcosms seeded with human or bovine feces. The concentrations of HF183/CF193 and Escherichia coli were determined using quantitative polymerase chain reaction (qPCR) and standard cultivation method, respectively. Shifts in the Bacteroidales population structure were fingerprinted using PCR-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing analysis targeting both 16S rDNA and rRNA-transcribed cDNA. Both HF183 and CF193 decayed significantly faster than E. coli but the decay curves fit poorly with first-order model. High diversity of Bacteroidales population was observed for both microcosms, and persistence of different species in the population varied. Sequence analysis indicated that most of the bovine Bacteroidales populations in our study are unexplored. DGGE and decay curve indicated that RNA decayed faster than DNA, further supporting the use of rRNA as indicator of metabolically active Bacteroidales population. Evaluations with more realistic scenarios are warranted prior to extending the results of this study to real field settings.
Water Research 11/2011; 46(1):247-57. · 4.86 Impact Factor
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ABSTRACT: Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide and is threatening the survival of the Floridian citrus industry. Currently, there is no established cure for this century-old and emerging disease. As a possible control strategy for citrus HLB, therapeutic compounds were screened using a propagation test system with 'Candidatus Liberibacter asiaticus'-infected periwinkle and citrus plants. The results demonstrated that the combination of penicillin and streptomycin (PS) was effective in eliminating or suppressing the 'Ca. L. asiaticus' bacterium and provided a therapeutically effective level of control for a much longer period of time than when administering either antibiotic separately. When treated with the PS, 'Ca. L. asiaticus'-infected periwinkle cuttings achieved 70% of regeneration rates versus <50% by other treatments. The 'Ca. L. asiaticus' bacterial titers in the infected periwinkle plants, as measured by quantitative real-time polymerase chain reaction, decreased significantly following root soaking or foliar spraying with PS. Application of the PS via trunk injection or root soaking also eliminated or suppressed the 'Ca. L. asiaticus' bacterium in the HLB-affected citrus plants. This may provide a useful tool for the management of citrus HLB and other Liberibacter-associated diseases.
Phytopathology 09/2011; 101(9):1097-103. · 2.80 Impact Factor
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ABSTRACT: "Candidatus Liberibacter asiaticus" is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of "Ca. Liberibacter" associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyv(I) and hyv(II)) were identified in the prophage regions of the Psy62 "Ca. Liberibacter asiaticus" genome. Sequence analyses of the hyv(I) and hyv(II) genes in 35 "Ca. Liberibacter asiaticus" DNA isolates collected globally revealed that the hyv(I) gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv(II) contains up to 2 NITRs and 4 partial repeats and shares homology with hyv(I). Frequent deletions or insertions of these repeats within the hyv(I) and hyv(II) genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of "Ca. Liberibacter asiaticus" bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single "Ca. Liberibacter asiaticus"-infected sample. This is the first evidence of different "Ca. Liberibacter asiaticus" populations coexisting in a single HLB-affected sample. The Florida "Ca. Liberibacter asiaticus" isolates contain both hyv(I) and hyv(II), while all other global "Ca. Liberibacter asiaticus" isolates contain either one or the other. Interclade assignments of the putative Hyv(I) and Hyv(II) proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple "Ca. Liberibacter asiaticus" populations in the world and a multisource introduction of the "Ca. Liberibacter asiaticus" bacterium into Florida.
Applied and environmental microbiology 07/2011; 77(18):6663-73. · 3.69 Impact Factor
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ABSTRACT: Citrus huanglongbing is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of 'Candidatus Liberibacter', of which 'Ca. L. asiaticus' is the most widely distributed. An optimized system using 'Ca. L. asiaticus'-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 microg/ml) and indole-3-butyric acid (4 microg/ml). This system allowed a plant regeneration rate of 60.6% for 'Ca. L. asiaticus'-infected cuttings in contrast to the <1% regeneration rate with water alone. Two chemical agents, penicillin G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA), were found to be effective at eliminating or suppressing the 'Ca. L. asiaticus' bacterium in this periwinkle regeneration system. When treated with penicillin G sodium at 50 microg/ml, all plants regenerated from 'Ca. L. asiaticus'-infected cuttings were 'Ca. L. asiaticus' negative as determined by both nested polymerase chain reaction (PCR) and quantitative real-time PCR. In addition, DBNPA was also able to significantly reduce the percentage of 'Ca. L. asiaticus'-positive plants and the titer of the 'Ca. L. asiaticus' bacterium at 200 microl/liter.
Phytopathology 03/2010; 100(3):239-45. · 2.80 Impact Factor
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ABSTRACT: Lead (Pb), zinc (Zn), copper (Cu), and cadmium (Cd) often seriously deteriorate water quality. Spatial and temporal fluctuations of the metal concentrations in the Ten Mile Creek (Florida) (TMC) were monitored on a weekly basis at 7 sampling sites, from June 2005 to September 2007. River sediment samples were also collected from these sites in April, June, and October 2006 and January 2007, and analyzed for water, Mehlich 1 (M1), and Mehlich 3 (M3)-extractable metals (Mehlich, 1953, 1984), to examine the role of sediments as sources or sinks of the metals. The concentrations of lead, zinc, copper, and cadmium in the water samples were <detection limit (DL)-309, < DL-102, < DL-106, and < DL-15.7 microg/L, respectively. Even though median concentrations of lead (<DL), zinc (6.45 microg/L), copper (2.53 microg/L), and cadmium (<DL) were lower than their respective U.S. Environmental Protection Agency (Washington, D.C.) limits for freshwater (2.5 microg/L for lead, 120 microg/L for zinc, 9.0 microg/L for copper, and 0.25 microg/L for cadmium), pulse concentrations of the metals significantly exceeded these limits. Correlations of lead, zinc, copper, and cadmium concentrations in the TMC to other environmental factors, including the results of principal component analysis, suggest that there are different sources of metals. These sources include surface runoff from agricultural lands or urban wastewater, geological backgrounds, and tidal flow. Water-, M1-, and M3-extractable lead, zinc, copper, and cadmium concentrations in river sediments indicate the possibility that river sediment serves as an internal source of the metals for the TMC. This hypothesis was supported by positive correlations between concentrations of copper, lead, and zinc in the river water and their respective water-extractable concentrations in the sediments.
Water Environment Research 01/2009; 81(1):40-50. · 0.88 Impact Factor
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ABSTRACT: Water quality throughout south Florida has been a major concern for many years. Nutrient enrichment in the Indian River Lagoon (IRL) is a major surface water issue and is suggested as a possible cause of symptoms of ecological degradation. In 2005-06, water samples were collected weekly from seven sites along Ten Mile Creek (TMC), which drains into the Indian River Lagoon, to investigate and analyze spatial and temporal fluctuations of nutrients nitrogen (N) and phosphorus (P). The objective of this study was to understand the relationships among chlorophyll a concentration, nutrient enrichment and hydrological parameters in the surface water body. High median concentrations of total P (TP, 0.272 mg L(-1)), PO4-P (0.122 mg L(-1)), and dissolved total P (DTP, 0.179 mg L(-1)); and total N (TN, 0.988 mg L(-1)), NO3(-)-N (0.104 mg L(-1)), NH4+-N (0.103 mg L(-1)), and total Kjeldahl N (TKN, 0.829 mg L(-1)), were measured in TMC. The concentrations of TP, PO4-P, DTP, TN, NO3(-)-N, NH4+-N, and TKN were higher in summer and fall than in winter and spring. However, chlorophyll a and pheophytin concentrations during this period in TMC varied in the range of 0.000-60.7 and 0.000-17.4 microg L(-1), with their median values of 3.54 and 3.02 microg L(-1), respectively. The greatest mean chlorophyll a (10.3 microg L(-1)) and pheophytin (5.71 microg L(-1)) concentrations occurred in spring, while the lowest chlorophyll a (1.49 microg L(-1)) and pheophytin (1.97 mug L(-1)) in fall. High concentrations of PO4-P (>0.16 mg L(-1)), DTP (>0.24 mg L(-1)), NO3(-)-N (>0.15 mg L(-1)), NH4+-N (>0.12 mg L(-1)), and TKN (>0.96 mg L(-1)), occurred in the upstream of TMC, while high concentrations of chlorophyll a (>6.8 mug L(-l)) and pheophytin (>3.9 microg L(-l)) were detected in the downstream of TMC. The highest chlorophyll a (11.8 mug L(-l)) and pheophytin (6.06 microg L(-l)) concentrations, however, were associated with static and open water conditions. Hydrological parameters (total dissolved solid, electrical conductivity, salinity, pH, and water temperature) were positively correlated with chlorophyll a and pheophytin concentrations (P < 0.01) and these factors overshadowed the relationships between N and P concentrations and chlorophyll a under field conditions. Principal component analysis and the ratios of DIN/DP and TN/TP in the water suggest that N is the limiting nutrient factor for phytoplankton growth in the TMC and elevated N relative to P is beneficial to the growth of phytoplankton, which is supported by laboratory culture experiments under controlled conditions.
Journal of Environmental Monitoring 05/2008; 10(4):508-16. · 1.99 Impact Factor
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ABSTRACT: The distribution pattern of citrus tristeza virus (CTV) T-36 isolate in leaves of infected mexican lime [Citrus aurantifolia (Christm.) Swingle] plants was visualized us-ing a whole-leaf-blot immunoassay (WLBIA) procedure in combination with a computer scanning imaging technique and CTV-specific monoclonal antibody 17G11 (CTV MAb 17G11). The distribution pattern of CTV T-36 in leaves varied with the age of the leaves and shoots of infected plants. In the young leaves, especially the about 5-day-old leaves and the completed expanded leaves, CTV T-36 was easily detected in most of the leaf veins, the main veins and the large and small primary veins. In the old leaves, CTV T-36 only was detected in the main veins, sometimes in a few of the large primary veins with weak signals, and seldom in the small primary veins. The distribution density and immunoas-say reaction signals of CTV T-36 reacted to CTV MAb 17G11 in leaves from new shoots were much higher than that in leaves from old shoots. ELISA test results using leaves with different ages from different shoots of the same mexican lime plants infected with CTV T-36 supported the visualized-test results obtained by the WLBIA in combination with computer scanning imaging technique. This is the first reported visual analysis of the distribution pattern of CTV in leaves of infected citrus plants. The results indicate that the WLBIA in combination with computer scanning imaging technique is a useful tool for studying the distribution of plant viruses in leaves of virus-infected plants. Citrus tristeza virus (CTV), a member of the genus Closterovirus with the largest genome among plant RNA viruses, occurs in most citrus producing regions, and is the most serious pathogen of citrus (Bar-Joseph et al., 1980; Ghorbel et al., 2001; Niblett et al., 2000). Control strategies for this virus focus on clean stock program, cross-protection, and breed-ing/engineering for resistance (Ghorbel et al., 2001; Lee et al., 1988; Mas and Pallas 1995; Niblett et al., 2000). Information regarding virus distribution in the plant is crucial to fully evaluate and implement these strategies. The distribution of CTV in infected citrus plants has been investigated by light and electron microscopy (Brlansky and Lee Lin et al., 2000), in situ immunofluorescence technique (Brlansky and Lee 1988; Sasaki et al., 1980), dot-im-munobinding assay (Rocha-Peña et al., 1991), uneven depending on CTV strain, citrus variety, and the sampling season (Lee et al., 1988). None of these studies assess the distribution of CTV within leaves of different ages or leaves from shoots of different ages. Polston et al. (1991) reported a whole-leaf-blot immunoassay using virus specific IgG to detect the coat proteins of viruses in leaves of infected plants. Mas and Pallas (1995) reported a nonisotopic tissue-printing hybridization technique to study the movement of plant viruses within leaves of infected plants. La-bonne et al. (1997) reported an immunoprinting technique to detect the distribution of plum pox virus in leaves of infected Prunus. Their results indicated that both whole-leaf-blot immunoas-say and whole-leaf-blot RNA-hybridization are useful tools to study the distribution pattern of viruses in leaves of infected plants. In this paper, we present the visible distribution pattern of CTV T-36 isolate in leaves of infected mexican lime plants using an improved whole-leaf-blot immunoassay in combination with computer scanning imaging technique.
HORTSCIENCE Kitajima and Costa. 01/2006; 41(41):725-728.
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Journal of Vegetable Crop Production 01/2002; 8(2):81-85.
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ABSTRACT: Summer squash (Cucurbita pepo) cultivars, three from each of the six cultivar groups, were grown under field and greenhouse conditions at Fort Pierce, Florida, U.S.A. in the presence of sweetpotato whiteflies (Bemisia tabaci) and observed for severity of leaf silvering. All cultivars were affected by leaf silvering, but some had less severe symptoms than others. The cocozelle group was the least susceptible to silvering. There appeared to be an association between geographic origin of cultivars and their susceptibility to silvering.
Euphytica 12/1992; 69(1):69-72. · 1.55 Impact Factor
