Fleites, Laura

Abstract Delivery of therapeutic biomolecules into plant vascular tissues remains a challenge in management of vector-borne plant pathogens. The symbiont concept uses reprogrammed Agrobacterium tumefaciens galls (called symbionts) to produce biomolecules while remaining connected to host vasculature. We evaluated whether symbionts expressing antimicrobial peptides (AMPs) suppress ‘ Candidatus Liberibacter solanacearum’ (CLso), the causal agent of potato zebra chip disease. Symbionts were engineered to express a Streptococcus mutans bacteriocin associated with bacterial membrane disruption (Blp-Sm), or an AMP isolated from finger lime and associated with resistance to citrus greening disease (MaSAMP). Effects of AMP-producing symbionts on CLso titers, infection incidence, pathogen movement, and disease symptoms were evaluated in tomato and potato. In tomato, neither AMP significantly reduced CLso titers or infection incidence. However, in potato, AMP-producing symbionts reduced CLso accumulation and movement from CLso-inoculated source shoots into non-inoculated sink shoots connected through underground tubers. Blp-Sm produced the strongest reduction in CLso accumulation and infection incidence in sink tissues. In separate assays where symbionts were established directly on potato seed tubers, MaSAMP significantly reduced CLso titers in stems and tubers and reduced zebra chip symptoms in tubers, despite no reduction of CLso titers in terminal leaves. These findings demonstrate that AMP-producing symbionts suppress vascular pathogen accumulation and movement within plants and highlight the symbiont concept as a potential platform for managing diseases caused by vascular-restricted pathogens. Further, they show the potato-CLso system is a promising infection model to both refine and improve symbiont technology, and to test additional AMPs for potency against related pathogens.

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by ‘Ca. L. asiaticus’, the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the ‘Ca. L. asiaticus’ bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the ‘Ca. L. asiaticus’ bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of ‘Ca. L. asiaticus’, while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate ‘Ca. L. asiaticus’ bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of ‘Ca. Liberibacter’-infected plants in nursery and greenhouse settings.