High-throughput Sequencing Analysis on Diversity of Bacterial and Fungal Endophytes on Huanglongbing-infected Citrus Plants

  Objective  Diversity of bacterial and fungal endophytes on citrus plants infected with Huanglongbing (HLB) (citrus greening disease by Candidatus Liberibacter) was determined for further study on the pathology.

  Method  The diversities of bacterial and fungal endophytes on healthy and HLB-diseased leaves of citrus plants grown in a greenhouse were compared. In addition, the diversities on the diseased leaves of different severity from a same plant were also compared. The HLB pathogen was confirmed using primer pair of OI1/OI2c. High-throughput sequencing were used to detect the 16S rRNA of bacteria and rDNA-ITS of fungi. Then the abundance and diversity analysis on the endophytes were conducted.

  Result  For the bacterial endophytes, there were 13 phyla, 22 classes, 57 orders, 101 families, and 208 genera found, while for the fungal endophytes, 2 phyla, 12 classes, 24 orders, 43 families, and 43 genera. The relative abundance of Candidatus Liberibacter increased with severity of the disease. Methylocella and 1174-901-12 were the dominant bacteria endophytes on both healthy and diseased plants; whereas, Sphingomonas only on healthy plants. The relative abundances of Alpha cluster and Hymenobacter increased with the disease infection. Strelitziana was the dominant fungal endophyte on both healthy and diseased plants; whereas, Zasmidium and Trichomerium on diseased plants only. The relative abundance of Zymoseptoria increased with the disease infection.

  Conclusion  The infection of HLB by Candidatus Liberibacter bacteria changed the abundance and diversity of bacterial and fungal endophytes on a citrus plant. There were also significant differences on the diversity of bacterial and fungal endophytes between the healthy and the diseased plants. Successful detection of the endophytes using high-throughput sequencing overcame the obstacles on the disease identification due to the unculturable pathogen and endophytes as well as the endophytes with low abundance. It also allowed a systematic study on the microbiology associated with HLB and the cure and prevention of the disease.