The pH plays a key role in the growth and colonization of plant pathogens as well as the onset and progression of the symptoms they cause within the host. Plants may quickly alter their apoplastic pH (pHapo) to protect themselves against infection. However, pathogens can also alter the pH of their ambient environment to promote their own growth. Citrus canker is a serious plant disease caused by Xanthomonas citri subsp. citri (Xcc). This Gram-negative aerobic rod is usually cultured in Luria–Bertani (LB) medium at pH 7. However, little is known about the changes in pH both in this medium as Xcc grows and in the leaf apoplast in response to Xcc infection and colonization. Moreover, the differences in leaf apoplast pH between Xcc-resistant and Xcc-susceptible citrus genotypes are also unknown. Here, Xcc grew well in liquid LB medium at initial pH 6–8 and the pathogen altered the medium pH to 6.8 ± 0.4. Xcc growth declined at pH 5 and was zero at pH 3, 4, 9, and 10. In susceptible sweet orange infected with Xcc inoculum, canker symptoms were inhibited at pH 3, 4, and 10 but did not differ in the range of pH 5–9. As expected, canker symptoms were absent at all inoculum pH in the resistant Citron C-05. For both genotypes, Xcc only grew well in the leaves exposed to pH 5–8 inoculums. At four days post-inoculation (4 dpi), the foliar pHapo of resistant Citron C-05 had rapidly declined from 5.6 to 4.4. At 2 dpi, the pHapo of susceptible sweet orange had rapidly increased from 5.6 to 6.7, Xcc grew quickly, and canker symptoms appeared. Plasma membrane (PM) H+-ATPase activation with fusicoccin (FC) acidified the apoplast and upregulated the pathogenesis-related genes (PRs) in the sweet orange leaves. Hence, Xcc colonization and canker development were inhibited. The results of this study revealed that apoplastic acidification is implicated in the resistance of Citron C-05 to Xcc infection and provided insight into the association between pHapo regulation and resistance to bacterial pathogen invasion in plants.
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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 is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), which can infect branches, leaves and fruits, and affects almost all major citrus varieties. The results of a previous survey of 39 citrus orchards in Hunan Province showed that soil acidification and exchange calcium deficiency in citrus orchards were serious and calcium deficiency existed in all leaves. Calcium is one of the elements required by plants in large quantities, and calcium deficiency causes nutritional imbalance, reduced growth potential and compromised plant immunity level. However, the effect of calcium element on the process of citrus infection with canker is not clear.
The objective of this study is to analyze the pathogenic differences after inoculation with Xcc in Poncirus trifoliata (sensitive to citrus canker) leaves under different calcium concentrations, and to explore the role of calcium in Xcc infection of P. trifoliata leaves.
The seedlings of P. trifoliata were sand cultured with calcium concentrations of 0, 0.75, 3, and 30 mmol·L-1. During the growth period of P. trifoliata, the biomass, chlorophyll a and b concentrations, and calcium content in roots and leaves were determined, as well as the formation of reactive oxygen species (ROS) in roots and callose deposition. The effects of Xcc inoculation on cell wall synthesis-related genes and immune-related genes in P. trifoliata leaves were investigated.
Compared with 3 mmol·L-1 calcium treatment, 0, 0.75 and 30 mmol·L-1 calcium treatments inhibited the growth and development of aboveground and underground parts of P. trifoliate, and chlorophyll a and b concentrations decreased. The calcium content in roots and leaves was proportional to the amount of exogenous calcium. ROS and callose deposition were generated in roots after treatment with different calcium concentrations, reaching the maximum at 3 mmol·L-1. After inoculation with Xcc, the leaf symptoms gradually decreased with the increase of calcium concentration, but the growth of Xcc had no significant difference. Compared with 3 mmol·L-1 treatment, PtCESA4, a gene involved in cell wall synthesis, was up-regulated and then down-regulated by Xcc under 0 mmol·L-1 treatment, and up-regulated by Xcc under 30 mmol·L-1 treatment. PtPME and PtFLA were down-regulated by Xcc under 0 mmol·L-1 treatment, and up-regulated by Xcc under 30 mmol·L-1 treatment. The expression levels of immune pathway related genes PtGSL, PtGST1 and PtWRKY22 induced by Xcc at 30 mmol·L-1 were higher than those at 0 and 3 mmol·L-1 after inoculation with Xcc at 0, 2, 4 and 6 dpi.
The growth and development of P. trifoliate is affected by calcium deficiency and excess, resulting in leaf chlorosis, and ROS production and callose deposition in roots decreased. The sensitive symptoms on the leaf surface caused by Xcc were greatly attenuated after calcium application, but the bacterial content was not significantly different from that of the control. Calcium may promote cell wall thickening by regulating genes related to cell wall synthesis, thereby inhibiting Xcc from breaking through leaf epidermis and forming typical symptoms.
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.
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