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Characterization of the microbial community structure in Candidatus Liberibacter asiaticus-infected citrus plants treated with antibiotics in the field.

Zhang M, Powell CA, Guo Y, Benyon L, Duan Y - BMC Microbiol. (2013)

Bottom Line: Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores.In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599.In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described.

View Article: PubMed Central - HTML - PubMed

Affiliation: Indian River Research and Education Center, IFAS-UF, Fort Pierce, FL 34945, USA.

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 Phylochip™ 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 PhyloChip™ 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 γ-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 α-proteobacterial and the γ-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.

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PhylochipTM HybScores of ‘Candidatus Liberibacter asiaticus’ (Las) from Huanglongbing (HLB)-affected citrus. The citrus plants were treated with antibiotic combinations and sampled at different times (October 2010, April 2011 and October 2011) over a year. (i) Severe HLB-like symptoms with Ct values <26, and Las bacterial titers of more than 770,000 cells per gram plant tissue; (ii) no symptoms with Ct values ≥36.0, and Las bacterial titers of less than 1,060 cells per gram plant tissue. A HybScore change of 1000 indicated a doubling in the fluorescence intensity of the OTU. PS: 5 g/tree penicillin G potassium and 0.5 g/tree streptomycin; KO: 2 g/tree oxytetracycline and 1.0 g/tree kasugamycin; and CK: water as control.
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Figure 2: PhylochipTM HybScores of ‘Candidatus Liberibacter asiaticus’ (Las) from Huanglongbing (HLB)-affected citrus. The citrus plants were treated with antibiotic combinations and sampled at different times (October 2010, April 2011 and October 2011) over a year. (i) Severe HLB-like symptoms with Ct values <26, and Las bacterial titers of more than 770,000 cells per gram plant tissue; (ii) no symptoms with Ct values ≥36.0, and Las bacterial titers of less than 1,060 cells per gram plant tissue. A HybScore change of 1000 indicated a doubling in the fluorescence intensity of the OTU. PS: 5 g/tree penicillin G potassium and 0.5 g/tree streptomycin; KO: 2 g/tree oxytetracycline and 1.0 g/tree kasugamycin; and CK: water as control.

Mentions: The dynamic variations of Las bacterial titers from August 2010 to October 2011 at the USHRL farm, Fort Pierce, FL are presented in Figure 1. The results showed that the Las bacterial population fluctuated throughout the year in HLB-affected citrus plants with or without antibiotic treatments. The highest Las bacterial titers (lowest Ct values) were observed in December 2010, and the lowest Las bacterial titers (highest Ct values) were recorded in April 2011. This variation generally coincided with HLB-symptoms in the field. There were no significant differences among the antibiotic treatments and the water control before the initial applications in August 2010. Two months after the initial applications, significant differences (Pr<0.05) existed between the antibiotic treatments and the controls. By April 2011, the titers had decreased by more than 13-fold in the water control, 259-fold in the KO treated citrus and 97-fold in the PS treated citrus. The HybScore of OTU63806, which represented Candidatus Liberibacter from PhyloChip™ G3, coincided with the Las bacterial titers detected by qPCR (r=0.812). HybScores averaged 12,186±1,320 in the untreated trees (water control, CK) compared to 11,226±1,458 and 11,037±678 in the HLB-affected trees treated with KO and PS, respectively. HybScores were the lowest in April 2011 when the HLB-bacterial population was also at its lowest level (Figure 2).


Characterization of the microbial community structure in Candidatus Liberibacter asiaticus-infected citrus plants treated with antibiotics in the field.

Zhang M, Powell CA, Guo Y, Benyon L, Duan Y - BMC Microbiol. (2013)

PhylochipTM HybScores of ‘Candidatus Liberibacter asiaticus’ (Las) from Huanglongbing (HLB)-affected citrus. The citrus plants were treated with antibiotic combinations and sampled at different times (October 2010, April 2011 and October 2011) over a year. (i) Severe HLB-like symptoms with Ct values <26, and Las bacterial titers of more than 770,000 cells per gram plant tissue; (ii) no symptoms with Ct values ≥36.0, and Las bacterial titers of less than 1,060 cells per gram plant tissue. A HybScore change of 1000 indicated a doubling in the fluorescence intensity of the OTU. PS: 5 g/tree penicillin G potassium and 0.5 g/tree streptomycin; KO: 2 g/tree oxytetracycline and 1.0 g/tree kasugamycin; and CK: water as control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672075&req=5

Figure 2: PhylochipTM HybScores of ‘Candidatus Liberibacter asiaticus’ (Las) from Huanglongbing (HLB)-affected citrus. The citrus plants were treated with antibiotic combinations and sampled at different times (October 2010, April 2011 and October 2011) over a year. (i) Severe HLB-like symptoms with Ct values <26, and Las bacterial titers of more than 770,000 cells per gram plant tissue; (ii) no symptoms with Ct values ≥36.0, and Las bacterial titers of less than 1,060 cells per gram plant tissue. A HybScore change of 1000 indicated a doubling in the fluorescence intensity of the OTU. PS: 5 g/tree penicillin G potassium and 0.5 g/tree streptomycin; KO: 2 g/tree oxytetracycline and 1.0 g/tree kasugamycin; and CK: water as control.
Mentions: The dynamic variations of Las bacterial titers from August 2010 to October 2011 at the USHRL farm, Fort Pierce, FL are presented in Figure 1. The results showed that the Las bacterial population fluctuated throughout the year in HLB-affected citrus plants with or without antibiotic treatments. The highest Las bacterial titers (lowest Ct values) were observed in December 2010, and the lowest Las bacterial titers (highest Ct values) were recorded in April 2011. This variation generally coincided with HLB-symptoms in the field. There were no significant differences among the antibiotic treatments and the water control before the initial applications in August 2010. Two months after the initial applications, significant differences (Pr<0.05) existed between the antibiotic treatments and the controls. By April 2011, the titers had decreased by more than 13-fold in the water control, 259-fold in the KO treated citrus and 97-fold in the PS treated citrus. The HybScore of OTU63806, which represented Candidatus Liberibacter from PhyloChip™ G3, coincided with the Las bacterial titers detected by qPCR (r=0.812). HybScores averaged 12,186±1,320 in the untreated trees (water control, CK) compared to 11,226±1,458 and 11,037±678 in the HLB-affected trees treated with KO and PS, respectively. HybScores were the lowest in April 2011 when the HLB-bacterial population was also at its lowest level (Figure 2).

Bottom Line: Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores.In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599.In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described.

View Article: PubMed Central - HTML - PubMed

Affiliation: Indian River Research and Education Center, IFAS-UF, Fort Pierce, FL 34945, USA.

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 Phylochip™ 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 PhyloChip™ 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 γ-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 α-proteobacterial and the γ-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.

Show MeSH
Related in: MedlinePlus