The updated PhyloChip^TM G3 were used to explore the differences in the relative abundance and phylogenetic diversity of the bacterial communities associated with HLB-affected citrus plants in the field over a growing season and those treated with antibiotic combinations of AG (Ampicillin at 1000 mg/L and Gentamicin at 100 mg/L) and PS (Penicillin at 1000 mg/L and Streptomycin at 100 mg/L). Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and their hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChip™ G3, 7,028 known OTUs in 58 phyla were detected from the field plants, and 7,407 OTUs in 53 phyla from the inoculated potted plants. Proteobacteria was the constantly dominant phylum of bacteria (38.7%~44.1%) vying for prevalence based on the season, followed by Firmicutes (23.5%~29.0%), Actinobacteria (12.4%~16.1%), Bacteroidetes (6.2%~6.6%) and Cyanobacteria (2.3%~3.2%). Circular tree comparing the Las-free and the Las-infected samples indicated that only 17 families present in the Las-free plants, such as Cyanobacteriaceae; but more than 137 families detected in the Las-infected plants, such as Staphylococcaceae and Pseudonocardiaceae. Cyanobacteria are believed to be responsible for introducing oxygen into the atmosphere and fixing nitrogen and phosphorus assimilation for plant growth. Both Staphylococcaceae and Pseudonocardiaceae were recognized as an emerging opportunistic pathogen of plant and animals. When compared to the bacterial populations in the leaves of citrus trees receiving the water control treatment, the Bacteroidete population decreased (Pr<0.05) by 59.6% and 51.8% in the plants receiving AG and PS treatments, respectively. The overall diversity of bacteria also decreased with the antibiotic treatments. Bacterial cells in close proximity may be able to modify their microenvironment; thus, 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 a both a seasonal fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.

Huanglongbing (HLB) is a highly destructive, fast spreading disease of citrus that causes substantial economic losses to the citrus industry worldwide. Nutrient levels and their cellular distribution patterns in HLB-affected grapefruit were analyzed after graft-inoculation with infected lemon scions containing ‘Candidatus Liberibacter asiaticus’, the heat-tolerant Asian type of HLB bacterium. After 12 months, infected plants showed typical HLB symptoms including leaf curling and blotchy mottle on leaves. Zinc (Zn) concentrations in young, mature, and old leaves of grapefruit significantly decreased by HLB infection. Micro-XRF imaging of Zn and other elements showed that preferential distribution of Zn was observed in the phloem tissues of leaves and stems collected from healthy grapefruit plants but was absent from HLB-affected samples. Quantitative analysis of Zn intensity in the cross-sections of leaves using standard samples revealed that Zn concentration in phloem tissues of healthy grapefruit leaves was more than 10 times higher than that in the HLB-affected leaves. No significant variation was observed in the distribution patterns of other elements such as K and Ca in stems and leaves of grapefruit plants before or after graft-inoculation withHLB infected lemon scions. These results suggest that reduced phloem transport of Zn is one of the most important constraints that contribute to HLB-induced Zn deficiency in citrus such as grapefruit. Our report provides the first in situ visualization of elements’ variation within the tissues of HLB-infected citrus at cellular level.

Objective

Design

Results

Conclusions