Mitigation of Huanglongbing: Implications of a Biologically Enhanced Nutritional Program on Yield, Pathogen Localization, and Host Gene Expression Profiles

Huanglongbing (HLB, citrus greening disease), the most destructive disease affecting citrus production, is primarily linked to the gram-negative, insect-vectored, phloem-inhabiting α-proteobacterium ' Liberibacter asiaticus' (CLas). With no effective treatment available, management strategies have largely focused on the use of insecticides in addition to the destruction of infected trees, which are environmentally hazardous and cost-prohibitive for growers, respectively. A major limitation to combating HLB is the inability to isolate CLas in axenic culture, which hinders studies and creates a need for robust CLas detection and visualization methods. The aim of this study was to investigate the efficacy of a nutritional program-based approach for HLB treatment, and to explore the effectiveness of an enhanced immunodetection method to detect CLas-infected tissues. To achieve this, four different biologically enhanced nutritional programs (bENPs; P1, P2, P3, and P4) were tested on CLas-infected citrus trees. Structured illumination microscopy preceded by a modified immunolabeling process and transmission electron microscopy were used to show treatment-dependent reduction of CLas cells in phloem tissues. No sieve pore plugging was seen in the leaves of P2 trees. This was accompanied by an 80% annual increase in fruit number per tree and 1,503 (611 upregulated and 892 downregulated) differentially expressed genes. These included an MLRQ subunit gene, UDP-glucose transferase, and genes associated with the alpha-amino linolenic acid metabolism pathway in P2 trees. Taken together, the results highlight a major role for bENPs as a viable, sustainable, and cost effective option for HLB management.