Citrus sinensis seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg (NO₃)₂ for 16 weeks. Mg-deficiency-induced interveinal chlorosis, vein enlargement and corkiness, and alterations of gas exchange, pigments, chlorophyll a fluorescence (OJIP) transients and related parameters were observed in middle and lower leaves, especially in the latter, but not in upper leaves. Mg-deficiency might impair the whole photosynthetic electron transport, including structural damage to thylakoids, ungrouping of photosystem II (PSII), inactivation of oxygen-evolving complex (OEC) and reaction centers (RCs), increased reduction of primary quinone electron acceptor (Q_A) and plastoquinone pool at PSII acceptor side and oxidation of PSI end-electron acceptors, thus lowering energy transfer and absorption efficiency and the transfer of electrons to the dark reactions, hence, the rate of CO₂ assimilation in Mg-deficiency middle and lower leaves. Although potassium, Mg, manganese and zinc concentration in blades displayed a significant and positive relationship with the corresponding element concentration in veins, respectively, great differences existed in Mg-deficiency-induced alterations of nutrient concentrations between leaf blades and veins. For example, Mg-deficiency increased boron level in the blades of upper leaves, decreased boron level in the blades of lower leaves, but did not affect boron level in the blades of middle leaves and veins of upper, middle and lower leaves. To conclude, Mg-deficiency-induced interveinal chlorosis, vein enlargement, and corkiness, and alterations to photosynthesis and related parameters increased with increasing leaf age. Mg-deficiency-induced enlargement and corkiness of veins were not caused by Mg-deficiency-induced boron-starvation.

This present study examined excess copper (Cu) effects on seedling growth, leaf Cu concentration, gas exchange, and protein profiles identified by a two-dimensional electrophoresis (2-DE) based mass spectrometry (MS) approach after Citrus sinensis and Citrus grandis seedlings were treated for six months with 0.5 (control), 200, 300, or 400 μM CuCl₂. Forty-one and 37 differentially abundant protein (DAP) spots were identified in Cu-treated C. grandis and C. sinensis leaves, respectively, including some novel DAPs that were not reported in leaves and/or roots. Most of these DAPs were identified only in C. grandis or C. sinensis leaves. More DAPs increased in abundances than DAPs decreased in abundances were observed in Cu-treated C. grandis leaves, but the opposite was true in Cu-treated C. sinensis leaves. Over 50% of DAPs were associated with photosynthesis, carbohydrate, and energy metabolism. Cu-toxicity-induced reduction in leaf CO₂ assimilation might be caused by decreased abundances of proteins related to photosynthetic electron transport chain (PETC) and CO₂ assimilation. Cu-effects on PETC were more pronounced in C. sinensis leaves than in C. grandis leaves. DAPs related to antioxidation and detoxification, protein folding and assembly (viz., chaperones and folding catalysts), and signal transduction might be involved in Citrus Cu-toxicity and Cu-tolerance.

The plant rhizosphere microbiome is known to play a vital role in plant health by competing with pathogens or inducing plant resistance. This study aims to investigate rhizosphere microorganisms responsive to a devastating citrus disease caused by ‘Candidatus Liberibacter asiaticus’ (CLas) infection, by using 16S rRNA sequencing and metagenome technologies. The results show that 30 rhizosphere and 14 root bacterial genera were significantly affected by CLas infection, including 9 plant resistance-associated bacterial genera. Among these, Amycolatopsis, Sphingopyxis, Chryseobacterium, Flavobacterium, Ralstonia, Stenotrophomonas, Duganella, and Streptacidiphilus were considerably enriched in CLas-infected roots, while Rhizobium was significantly decreased. Metagenome analysis revealed that the abundance of genes involved in carbohydrate metabolism, such as glycolysis, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, was significantly reduced in the CLas-infected citrus rhizosphere microbial community. Likewise, the abundance of genes involved in phosphoinositide signaling and phosphoinositide metabolism, which play important roles in energy metabolism (such as carbohydrate metabolism and lipid metabolism), was also decreased in the CLas-infected samples. Taken together, our results indicate that CLas infection could affect the resistance potential and energy metabolism of the citrus rhizosphere microbial community, which may help us to understand the rhizosphere responses to plant disease and thus facilitate the development and application of antagonistic microorganism products in citrus industry.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of ‘Candidatus Liberibacter asiaticus’, a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of ‘Ca. L. asiaticus’ with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of ‘Ca. L. asiaticus’.

Background: Leaves are the main organs involved in photosynthesis. They capture light energy and promote gas exchange, and their size and shape affect yield. Identifying the regulatory networks and key genes that control citrus leaf size is essential for increasing citrus crop yield. Methods: In this study, transcriptome sequencing was performed on three leaf materials: the ‘Cuimi’ kumquat (Nor) variety and its leaf variants, larger-leaf (VarB) and smaller-leaf (VarS) varieties. Results: Correlation and principal component analyses revealed a relatively close correlation between Nor and VarS. A total of 7264 differentially expressed genes (DEGs), including 2374 transcription factors (TFs), were identified, and 254 DEGs were common among the three materials. GO and KEGG enrichment analyses revealed significant enrichment in glucose metabolism, cell wall composition, starch biosynthesis, and photosynthesis pathways. WGCNA identified three specific modules related to the different leaf sizes of these three citrus materials. Fifteen candidate genes related to leaf size, including three transcription factors, Fh5g30470 (MYB), Fh7g07360 (AP2/ERF), and Fh5g02470 (SAP), were identified on the basis of connectivity and functional annotations. Conclusions: These findings provide a theoretical foundation for a deeper understanding of the molecular mechanisms underlying citrus leaf size and offer new genetic resources for the study of citrus leaf size.