Merfa e Silva, Marcus Vinicius

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) and ‘Candidatus Liberibacter americanus’ (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

The phloem-restricted, insect-transmitted bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. The inability to culture CLas impairs the understanding of its virulence mechanisms and the development of effective management strategies to control this incurable disease. Previously, our research group used commercial grapefruit juice (GJ) to prolong the viability of CLas in vitro. In the present study, GJ was amended with a wide range of compounds and incubated under different conditions to optimize CLas growth. Remarkably, results showed that CLas growth ratios were inversely proportional to the initial inoculum concentration. This correlation is probably regulated by a cell density-dependent mechanism, because diluting samples between subcultures allowed CLas to resume growth. Moreover, strategies to reduce the cell density of CLas, such as subculturing at short intervals and incubating samples under flow conditions, allowed this bacterium to multiply and reach maximum growth as early as 3 days after inoculation, although no sustained exponential growth was observed under any tested condition. Unfortunately, cultures were only transient, because CLas lost viability over time; nevertheless, we obtained populations of about 105 genome equivalents/ml repeatedly. Finally, we established an ex vivo system to grow CLas within periwinkle calli that could be used to propagate bacterial inoculum in the lab. In this study we determined the influence of a comprehensive set of conditions and compounds on CLas growth in culture. We hope our results will help guide future efforts toward the long-sought goal of culturing CLas axenically.

In recent decades, ‘Candidatus Liberibacter spp.’ have emerged as a versatile group of psyllid-vectored plant pathogens and endophytes capable of infecting a wide range of economically important plant hosts. The most notable example is ‘Candidatus Liberibacter asiaticus’ (CLas) associated with Huanglongbing (HLB) in several major citrus-producing areas of the world. CLas is a phloem-limited α-proteobacterium that is primarily vectored and transmitted among citrus species by the Asian citrus psyllid (ACP) Diaphorina citri. HLB was first detected in North America in Florida (USA) in 2005, following introduction of the ACP to the State in 1998. HLB rapidly spread to all citrus growing regions of Florida within three years, with severe economic consequences to growers and considerable expense to taxpayers of the state and nation. Inability to establish CLas in culture (except transiently) remains a significant scientific challenge toward effective HLB management. Lack of axenic cultures has restricted functional genomic analyses, transfer of CLas to either insect or plant hosts for fulfillment of Koch’s postulates, characterization of host-pathogen interactions and effective screening of antibacterial compounds. In the last decade, substantial progress has been made toward CLas culturing: (i) three reports of transient CLas cultures were published, (ii) a new species of Liberibacter was identified and axenically cultured from diseased mountain papaya (Liberibacter crescens strain BT-1), (iii) psyllid hemolymph and citrus phloem sap were biochemically characterized, (iv) CLas phages were identified and lytic genes possibly affecting CLas growth were described, and (v) genomic sequences of 15 CLas strains were made available. In addition, development of L. crescens as a surrogate host for functional analyses of CLas genes, has provided valuable insights into CLas pathogenesis and its physiological dependence on the host cell. In this review we summarize the conclusions from these important studies.