An Improved Method to Track Changes of Candidatus Liberibacter asiaticus Titer in HLB-affected Citrus Trees

Etxeberria, Ed; González, Pedro; Singerman, Ariel; Ebert, Timothy A.

doi:10.21273/hortsci14025-19

Cited by 5 publications

(4 citation statements)

References 12 publications

(16 reference statements)

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“…Samples of two leaves from each tree were taken at time 0 and 6 weeks after the treatment, for qPCR analysis of ‘ Ca . Liberibacter asiaticus’ using Applied Biosystems 7500 PCR instrument (Life Technologies, Carlsbad, CA, USA) as described earlier [ 42 ].…”

Section: Methodsmentioning

confidence: 99%

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

et al. 2020

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Recently in Florida, foliar treatments using products with the antibiotics oxytetracycline and streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which is caused by the putative bacterial pathogen ‘Candidatus Liberibacter asiaticus’. Herein, we assessed the levels of oxytetracycline and ‘Ca. L. asiaticus’ titers in citrus trees upon foliar applications with and without a variety of commercial penetrant adjuvants and upon trunk injection. The level of oxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and ‘Ca. L. asiaticus’ titer was measured using quantitative PCR. Low levels of oxytetracycline were taken up by citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to the oxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level of oxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliar applications. The titer of ‘Ca. L. asiaticus’ in the midrib of leaves from trees receiving oxytetracycline by foliar application was not affected after four days and thirty days of application, whereas the titer was significantly reduced in oxytetracycline-injected trees thirty days after treatment. Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidized cuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake of oxytetracycline, decreasing the titer of ‘Ca. L. asiaticus’ in citrus leaves upon foliar application. Taken together, our findings indicate that trunk injection is more efficient than foliar spray even after the use of adjuvants. Our conclusion could help in setting useful recommendations for the application of oxytetracycline in citrus to improve tree health, minimize the amount of applied antibiotic, reduce environmental exposure, and limit off-target effects.

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Section: Methodsmentioning

confidence: 99%

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

et al. 2020

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“…Here, lack of homogeneity impedes effective evaluations of treatments (especially those with low efficacy). Nonetheless, it has been reported that the use of multiple disks taken from the same leaf significantly reduces the random variability associated with C Las [ 57 ], and thus, this hindrance was addressed in our assay. Low initial infection rates are another hurdle in analyzing the efficacy of treatments, particularly due to the minimized effect potential.…”

Section: Discussionmentioning

confidence: 99%

“…Two hundred μL of each plant extract treatment (at 1%) and 0.5 mM streptomycin were added to their respective wells (7 replicates of each treatment per 96-well plate). To reduce variability associated with C Las, this assay used a cross-comparison of disks taken from the same leaf as a blocking factor [ 57 ]. Using a sterile biopsy punch, 4 mm leaf disks were punched along the midrib of each leaf, beginning from the base of the leaf and moving out.…”

Section: Methodsmentioning

confidence: 99%

Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing

et al. 2023

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Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide–based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB.

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“…Plant tissues were sampled monthly from the same 12 mature leaves on each replicate tree. Samples were collected by harvesting discs from these leaves beginning at the leaf tip and progressing down the petiole on each sampled leaf as described in [ 37 , 38 ]. Plant tissue samples were immediately flash frozen in liquid nitrogen, ground with a tissue lyser, and stored at − 80 °C for further analyses.…”

Section: Methodsmentioning

confidence: 99%

Salicylic acid mediated immune response of Citrus sinensis to varying frequencies of herbivory and pathogen inoculation

et al. 2022

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Background Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. Results A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) ‘pulses’ of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). Conclusions Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease.

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An Improved Method to Track Changes of Candidatus Liberibacter asiaticus Titer in HLB-affected Citrus Trees

Cited by 5 publications

References 12 publications

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing

Salicylic acid mediated immune response of Citrus sinensis to varying frequencies of herbivory and pathogen inoculation

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