Root rot is a prevalent soil-borne fungal disease in citrus. Citron C-05 (Citrus medica) stands out as a germplasm within Citrus spp. due to its complete resistance to citrus canker and favorable characteristics such as single embryo and easy rooting. However, Citron C-05 was found to be highly susceptible to root rot during cultivation, with the specific pathogens previously unknown. In this study, four candidate fungal species were isolated from Citron C-05 roots. Sequence analysis of ITS, EF-1α, RPB1, and RPB2 identified two Fusarium solani strains, Rr-2 and Rr-4, as the candidates causing root rot in Citron C-05. Resistance tests showed these two pathogens increased root damage rate from 10.30% to 35.69% in Citron C-05, sour orange (Citrus aurantium), sweet orange (Citrus sinensis) and pummelo (Citrus grandis). F. solani exhibited the weak pathogenicity towards trifoliate orange (Poncirus trifoliata). DAB staining revealed none of reddish-brown precipitation in the four susceptible citrus germplasm after infection with F. solani, while trifoliate orange exhibited significant H₂O₂ accumulation. Trypan blue staining indicated increased cell death in the four susceptible citrus germplasm following infection with these two pathogens but not in trifoliate orange. These findings provide a comprehensive understanding of citrus root rot and support future research on the mechanisms of root rot resistance in citrus.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a globally quarantine disease infecting nearly all Citrus cultivars. Citron C-05 has been identified with complete and active resistance to Xcc. However, the mechanism underlying Citron C-05's resistance to Xcc remains elusive. We identified a gene cluster on chromosome 8 of the citrus genome comprising five pathogenesis-related 4-like genes. PR4A was upregulated in Citron C-05 leaves under Xcc infection, exhibiting the highest expression among these PR4-like genes. In addition, PR4A expression was higher in leaves of disease-resistant genotypes than susceptible genotypes under Xcc invasion. Bimolecular fluorescence complementation (BiFC) and Split-Luc assays indicated that CmWRKY75, a positive regulator of PR4A, interacted with pthA4 and upregulated expression of PR4A in Citron C-05 leaves. Regulatory function for the expression of CmPR4A was localized to a 516-nucleotide region upstream of the translational start site, which was designated ProCmPR4A-P516. Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange, and three transgenic lines of OE-CmPR4A exhibited resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis. Furthermore, CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait, BiFC and Split-Luc assays further verified their interaction. Transient overexpression of CmSMU2 in sweet orange increased resistance to Xcc. Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A, indicating that they may synergistically enhance resistance to Xcc in citrus. These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.