Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2–8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining “hard-to-get” transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC resistant and are promising in genetic increases in citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)-labeled Xcc. The results demonstrated that eYFP does not affect the growth and virulence of Xcc. Xcc-eYFP allows measurement of bacterial titers but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms (‘Ichang’ papeda and ‘Huapi’ kumquat) are resistant, whereas eight citrus germplasms (‘Chongyi’ wild mandarin, ‘Mangshan’ wild mandarin, ‘Ledong’ kumquat, ‘Dali’ citron, ‘Yiliang’ citron, ‘Longyan’ kumquat, ‘Bawang’ kumquat, and ‘Daoxian’ wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to increase the disease resistance of commercial citrus varieties via biotechnological approaches.

Alternaria brown spot disease is caused by the Alternaria alternata tangerine pathotype, which relies on ACT-toxin for infection. At present, all identified ACT-toxin biosynthesis-related genes are multi-copy genes. In this study, we summarized the advances in important host-specific toxins (HSTs), and listed key genes required for the pathogenicity of the A. alternata tangerine pathotype. Toxin virulence test results revealed that different citrus species displayed distinctly different tolerances to ACT-toxin. The extraction method of ACT-toxin crude extract was described in schematic form to make the method easier to understand. In addition, target gene disruption of two copies of ACTT5 (∆∆ACTT5) displayed significantly reduced virulence, indicating that ACTT5 is essential for the pathogenicity of the A. alternata tangerine pathotype.

Field Deployable Candidatus Liberibacter Asiaticus Detection Using