Co-Occurrence Analysis of Citrus Root Bacterial Microbiota under Citrus Greening Disease

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB.