Screening Molecules for Control of Citrus Huanglongbing Using an Optimized Regeneration System for ‘Candidatus Liberibacter asiaticus’-Infected Periwinkle (Catharanthus roseus) Cuttings

Abstract

Citrus huanglongbing is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of 'Candidatus Liberibacter', of which 'Ca. L. asiaticus' is the most widely distributed. An optimized system using 'Ca. L. asiaticus'-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 microg/ml) and indole-3-butyric acid (4 microg/ml). This system allowed a plant regeneration rate of 60.6% for 'Ca. L. asiaticus'-infected cuttings in contrast to the <1% regeneration rate with water alone. Two chemical agents, penicillin G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA), were found to be effective at eliminating or suppressing the 'Ca. L. asiaticus' bacterium in this periwinkle regeneration system. When treated with penicillin G sodium at 50 microg/ml, all plants regenerated from 'Ca. L. asiaticus'-infected cuttings were 'Ca. L. asiaticus' negative as determined by both nested polymerase chain reaction (PCR) and quantitative real-time PCR. In addition, DBNPA was also able to significantly reduce the percentage of 'Ca. L. asiaticus'-positive plants and the titer of the 'Ca. L. asiaticus' bacterium at 200 microl/liter.

Effective Antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-Affected Citrus Plants Identified via the Graft-Based Evaluation

“…However, less than 10 antibiotics have been used commercially and, of those, only streptomycin and tetracycline have been knowingly applied to fruit trees (43). Although effective chemical treatments for commercial application have yet to be developed for the control of citrus HLB, we have demonstrated some chemical compounds that reduce Las titer in infected trees [9], [30], [31]. Earlier experiments, using the graft-based chemotherapy screening method, determined a mixture of penicillin and streptomycin could suppress or eliminate Las [9], [31].…”

“…Cheema et al (1986) also reported that penicillin combined with carbendazim eliminated Las [33]. We previously reported that penicillin G potassium [9], [30] or ampicillin [31] at 1,000 mg/L eliminated ‘ Ca. L. asiaticus’ in infected periwinkle and citrus [29], [30].…”

Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats

“…Liberibacter asiaticus"-infected periwinkles was obtained either by dodder transmission (56) or by graft transmission from "Ca. Liberibacter asiaticus"-infected periwinkle to healthy periwinkle (53). The "Ca.…”

Diverse DNA extraction methods and PCR primers for detection of Huanglongbing-associated bacteria from roots of ‘Valencia’ sweet orange on sour orange rootstock

“…Several HLB disease management and control strategies were reported such as antibiotics, rootstocks/interstocks, heat, and a combination of chemical compounds (Belasque et al, 2010;Hoffman et al, 2012;Shokrollah et al, 2011;Zhang et al, 2010). However, early detection and initiation of rapid disease management by controlling psyllid populations and removal of infected trees are essential in preventing further spread of the disease (Gottwald, 2010).…”