Improving Citrus tolerance to Huanglongbing through neutralizing soil acidity and exploring biocontrol resources

Huanglongbing (HLB), previously called citrus greening disease, is one of the most devastating diseases in citrus production. Since the causal pathogen (Candidatus Liberibacter asiaticus), which is mainly transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, of HLB has not been successfully cultured artificially, further researches on its pathogenic mechanism and the development of pathogen control technology are needed. Meanwhile, field observations showed that low pH values of soil and the prevalence of HLB disease in citrus orchards in South China generally occurred simultaneously. However, there is a lack of knowledge about the relationships between soil properties and the occurrence and development of HLB. The influence of soil acidify on citrus tolerance to HLB is also seldom taken into account to understand a suitable soil environment for plant growth and defense. Moreover, although it is well known that a stable plant-related microbial community is essential to plant health, most of current studies only focus on HLB interference with the inherent host-microbial interaction. There is no in-depth study on how to improve the resistance of citrus to HLB by remodeling root-related microorganisms.
In this work, the aim was to enlighten the proteome-level response of diseased citrus trees to C. L. asiaticus infection during the development of HLB symptoms and to reveal the role of soil environment and beneficial bacteria in contribution of enhanced citrus tolerance to HLB. This is based on the conceptual model of disease triangle, trying to provide a candidate scheme for the HLB control from the perspective of optimizing the environmental factors of its three causal components. HLB should be controlled and mitigated by different and comprehensive measures in order to maintain the quality and productivity of citrus fruits. In spite of this, the use of biological control approaches to control HLB and ACP is still in the initial stage of research and beneficial microbial resources are obviously insufficient. To this end, the exploration and development of biological control agents with insecticidal and antibacterial characteristics are worthy of more efforts. Then, the study was carried out in four research axes described in the following paragraphs.
As preliminaries, we detailed the changes in biological processes of host citrus during the establishment of HLB disease symptoms. Comparative proteomics analysis were performed to reveal the global protein accumulation profiles in leaf petiole of CLas-infected Citrus at the asymptomatic and symptomatic stages compared to healthy counterpart. We found that photosynthesis was suppressed throughout the whole CLas infection cycle. Also, lots of crucial starch biosynthesis and trivial immune defense associated proteins were induced after the symptom-free prodromal period. Moreover, the protein-protein interaction network analysis indicated that a F-type ATPase and an alpha-1,4 glucan phosphorylase were the core nodes in the interactions of differentially accumulated proteins. The analysis of endogenous hormones in different disease stages of citrus supported that the changes of SA and JA content were positively correlated with the abundance of phytohormone biosynthesis-related proteins.
In the second research axis, lime application (3000 kg/ha ploughed into topsoil) proved to be a manageable and practical method to optimize soil pH and enhance the endurance capacity of citrus against C. L. asiaticus, resulting in increased yield and fruit quality of citrus in liming-amended soils. These meaningful effects may be attributed to the improvement of acidic soil that can increase the root metabolic activity and up-regulate the expression of ion transporter-related genes in HLB-infected roots, as well as alleviate the physiological disorders of sieve tube blockage of HLB-infected leaves and improve the content of chlorophyll. Meanwhile, the immune response involved in SAR and activating the salicylic acid signal pathway of diseased citrus was strengthen after applying lime.
The third research axis focused on the relationship between liming practice the changes of soil properties and root-associated microbes of HLB-infected citrus. The extent of soil acidification and aluminum toxicity was mitigated after soil improvement, accompanied by the increase of root activity and the decrease of pathogen concentration in citrus roots. Likewise, lime application strongly altered root endophytic microbial community diversity and structure, which represented by the enrichment of beneficial microorganisms in disease roots. It also enriched functional categories involved in host-microbe interactions and nitrogen and sulfur metabolisms in the HLB-infected citrus rhizosphere. Then, we highlight the important role of root microbiota changes and ecological factors, such as soil acidity, in delaying and alleviating HLB disease.
In the last experimental chapter, a nematode-symbiotic bacterium Xenorhabdus budapestensis strain C72 was identified with broad-spectrum antimicrobial activity and high-efficiency insecticidal activity. In Bipolaris maydis - Maize pathosystem, the early colonization of pathogenic fungi was significantly impaired by C72 supernatant treatment. Symbiotic bacteria secrete extracellular enzymes and induce the expression of host defense-related genes may be the reasons for the antimicrobial potential of C72. In addition, a high-quality complete and annotated genome sequence was reported, showing that the genome contains multiple secondary metabolite biosynthetic gene clusters.
In conclusion, this study is among the first to focus on the interfer effects on the soil acidify and dynamic plant microbiota on the occurrence and progression of HLB. It provides interesting results on global protein accumulation profiles in response to CLas infection during the prodromal stage and advances the understanding of the association between soil improvement with the HLB endurance of diseased citrus.