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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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PhyloChip™ G3 HybScore profiles of operational taxonomic units (OTUs) identified by Prediction Analysis for Microarray (PAM). Selected OTUs from leaf samples of Huanglongbing (HLB)-affected citrus treated with different antibiotic combinations at different sampling time points. PAM identified nine Enterobacteriaceae OTUs (OTUs 5711, 5749, 5938, 4390, 4198, 4677, 5235, 4146 and 4739) with increased abundance levels in the April 2011 samples when the ‘Candidatus Liberibacter asiaticus’ (Las) bacterial titers were the lowest compared to samples collected in October of 2010 and 2011, and one Sphingomonadaceae OTU, 61276, with an increased abundance level in October 2010.
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Figure 5: PhyloChip™ G3 HybScore profiles of operational taxonomic units (OTUs) identified by Prediction Analysis for Microarray (PAM). Selected OTUs from leaf samples of Huanglongbing (HLB)-affected citrus treated with different antibiotic combinations at different sampling time points. PAM identified nine Enterobacteriaceae OTUs (OTUs 5711, 5749, 5938, 4390, 4198, 4677, 5235, 4146 and 4739) with increased abundance levels in the April 2011 samples when the ‘Candidatus Liberibacter asiaticus’ (Las) bacterial titers were the lowest compared to samples collected in October of 2010 and 2011, and one Sphingomonadaceae OTU, 61276, with an increased abundance level in October 2010.

Mentions: Principal coordinate analysis (PCoA) based on the weighted Unifrac distances between samples was performed with PhyloChip community data sets, and the results suggested that there were significant differences among the treatments and the sampling time points. The 17 OTUs selected with filter-3, which includes OTUs present in samples from one treatment but not detected in any samples of the other treatments, separated the antibiotic combinations (KO, PS) and the control group (CK). There were eight OTUs (7444, 8217, 15010, 24693, 41872, 62344, 74687 and 77432) in the KO treatment, three in the PS treatment (24114, 40218 and 49638) and six in the water control (42278, 50217, 53352, 58803, 70400 and 75179). When compared with the Antibiotic Resistance Genes Database [22], three oxytetracycline-resistant bacteria (7444, 24693 and 72432) were found in the KO treatment (Table 1). No antibiotic-resistant bacteria were found in the PS treatment. Prediction analysis for microarrays (PAM) identified Bacillus OTU48007 within Firmicutes to have increased abundance in the control samples compared to the antibiotic treatments. A total of 118 OTUs with filter-5, based on abundance metrics, partitioned the samples into distinct groups corresponding to sampling time points. Using binary metrics, 344 OTUs selected with filter-5 were found in 100% of the samples from one time point and were consistently absent in other time point samples. PAM also identified nine γ-proteobacterial OTUs (4146, 4198, 4390, 4677, 4739, 5235, 5711, 5749 and 5938) with increased abundance levels in April 2011 samples compared to samples collected in October 2010 and October 2011, and one Sphingomonadaceae, OTU61276, with an increased abundance level in October 2010 (Figure 5).


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)

PhyloChip™ G3 HybScore profiles of operational taxonomic units (OTUs) identified by Prediction Analysis for Microarray (PAM). Selected OTUs from leaf samples of Huanglongbing (HLB)-affected citrus treated with different antibiotic combinations at different sampling time points. PAM identified nine Enterobacteriaceae OTUs (OTUs 5711, 5749, 5938, 4390, 4198, 4677, 5235, 4146 and 4739) with increased abundance levels in the April 2011 samples when the ‘Candidatus Liberibacter asiaticus’ (Las) bacterial titers were the lowest compared to samples collected in October of 2010 and 2011, and one Sphingomonadaceae OTU, 61276, with an increased abundance level in October 2010.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: PhyloChip™ G3 HybScore profiles of operational taxonomic units (OTUs) identified by Prediction Analysis for Microarray (PAM). Selected OTUs from leaf samples of Huanglongbing (HLB)-affected citrus treated with different antibiotic combinations at different sampling time points. PAM identified nine Enterobacteriaceae OTUs (OTUs 5711, 5749, 5938, 4390, 4198, 4677, 5235, 4146 and 4739) with increased abundance levels in the April 2011 samples when the ‘Candidatus Liberibacter asiaticus’ (Las) bacterial titers were the lowest compared to samples collected in October of 2010 and 2011, and one Sphingomonadaceae OTU, 61276, with an increased abundance level in October 2010.
Mentions: Principal coordinate analysis (PCoA) based on the weighted Unifrac distances between samples was performed with PhyloChip community data sets, and the results suggested that there were significant differences among the treatments and the sampling time points. The 17 OTUs selected with filter-3, which includes OTUs present in samples from one treatment but not detected in any samples of the other treatments, separated the antibiotic combinations (KO, PS) and the control group (CK). There were eight OTUs (7444, 8217, 15010, 24693, 41872, 62344, 74687 and 77432) in the KO treatment, three in the PS treatment (24114, 40218 and 49638) and six in the water control (42278, 50217, 53352, 58803, 70400 and 75179). When compared with the Antibiotic Resistance Genes Database [22], three oxytetracycline-resistant bacteria (7444, 24693 and 72432) were found in the KO treatment (Table 1). No antibiotic-resistant bacteria were found in the PS treatment. Prediction analysis for microarrays (PAM) identified Bacillus OTU48007 within Firmicutes to have increased abundance in the control samples compared to the antibiotic treatments. A total of 118 OTUs with filter-5, based on abundance metrics, partitioned the samples into distinct groups corresponding to sampling time points. Using binary metrics, 344 OTUs selected with filter-5 were found in 100% of the samples from one time point and were consistently absent in other time point samples. PAM also identified nine γ-proteobacterial OTUs (4146, 4198, 4390, 4677, 4739, 5235, 5711, 5749 and 5938) with increased abundance levels in April 2011 samples compared to samples collected in October 2010 and October 2011, and one Sphingomonadaceae, OTU61276, with an increased abundance level in October 2010 (Figure 5).

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