Exploring the biochemical and molecular mechanisms that contribute to Huanglongbing (HLB) tolerance in Citrus australis hybrids.

Huanglongbing (HLB), also known as citrus greening, is a devastating disease that affects citrus crops. Citrus australis (Australian round lime), a wild citrus species, has been reported to exhibit some resistance to HLB. In this study, we investigated the biochemical and molecular responses to HLB by several F1 hybrids between the Marisol clementine crossed with the Australian round lime (MRL), with a focus on identifying the mechanisms underlying HLB tolerance. A selected HLB-tolerant hybrid (MRL2-12) showed fewer HLB symptoms and maintained a healthy canopy, whereas the other C. australis hybrids exhibited typical HLB symptoms. The MRL2-12 hybrid exhibited the highest chlorophyll content and the least starch accumulation, both of which are important markers for HLB tolerance. Based on the Candidatus Liberibacter asiaticus (CaLas) titer and biochemical analysis, the gene expression patterns of a selected susceptible hybrid (MRL2-11) and the tolerant MRL2-12 hybrid were further analyzed using RNA-seq data to investigate plant defense responses in the context of HLB. The transcriptomic data from the MRL2-11 and MRL2-12 hybrids revealed different responses to HLB, with a set of differentially expressed genes between the tolerant C. australis hybrid and the susceptible hybrid, which were both grown under the same field conditions. These results revealed that the expression of genes related to cellular defense and pathogenesis-related defense mechanisms was significantly upregulated in the tolerant MRL2-12 hybrid compared with the MRL2-11 hybrid. MRL2-12 showed upregulated expression of pattern recognition receptors (PRRs), receptor-like kinases (RLKs), calcium-dependent protein kinases (CDPKs), and cysteine protease proteins, indicating effective defense mechanisms. Comparative genomic analysis identified significant polymorphic variants in MRL hybrids, indicating a genetically diverse background. These findings suggest that early, coordinated activation of immune signaling and physical defense mechanisms, such as cell wall fortification, plays a critical role in HLB tolerance in the C. australis citrus hybrids.