HLB control with bactericides - J. H. Graham 5
Novel bactericides and application methods to control Huanglongbing Disease of Citrus
Jim Graham
University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850 USA
Summary
Huanglongbing (HLB) caused by systemic Candidatus Liberibacter asiaticus (CLas) is unlikely to be controlled by topical applications of bactericides to leaves because the pathogen is phloem-limited and systemic throughout the tree. Traditional film-forming bactericides such as copper (Cu) or large molecular weight antibiotics applied to the leaf surface are not formulated to move directly into plant tissues and become systemic in the tree. Therefore, the objective is to evaluate soluble copper sulfate pentahydrate chelate (Magna-Bon LLC, M-B) which may be more systemic in plant tissue. Trunk injections of M-B were somewhat effective for HLB but caused phytotoxicity and unsuitable for young trees. Alternatively, soil drenches of M-B were evaluated for root uptake and movement of Cu to leaves. Because CLas is non-culturable, bactericidalleaves from treated trees were assayed with, Xanthomonas citri subsp. citri (Xcc), to detect locally systemic bactericidal activity. qPCR was used to monitor CLas titer. Field trials with uninfected or recently infected asymptomatic trees are being conducted to evaluate soil, trunk and foliar applications for providing systemic bactericidal activity. If systemic activity of bactericides prevents or slows CLas replication in the tree, the expectation is that pathogen-induced damage to the phloem tissue will be reduced and risk of inoculum spread mitigated. Hence, the goal is to develop a practical and economical chemotherapeutic tool for HLB management.
Current situation with HLB management in Florida
Foliar nutritional sprays of trees in Florida groves have been reported by some growers and researchers to suppress symptoms and maintain productivity of HLB-affected trees (Spann and Schumann 2012). In a recent 2 year study in Florida (Gottwald et al. 2012), foliar nutritional sprays failed to sustain tree health, yield, or fruit quality. More importantly, no significant difference in CLas titer was detected compared to the untreated control. Because the nutritional supplements had no effect on CLas titer, this management approach likely promoted area-wide CLas inoculum build-up and spread by insect psyllid vector, Diaphorina citri.
Historically, trunk injections of antibiotics such as oxytetracycline (OTC) were demonstrated to effectively control symptom expression of HLB in Taiwan (Su and Chang, 1976) and citrus greening in South Africa caused by Candidatus Liberibacter africanus (Schwarz and Van Vuuren, 1971). Tree injection worked best for trees in the early stage of disease development. The efficacy of the antibiotic therapy was improved by use of gravity fed infiltration of the antibiotic solution with an extended duration of application (Van Vuuren et al., 1977). The drawbacks were that multiple applications of OTC were required to control disease symptoms and were associated with phytotoxicity such as mild vein necrosis, slender leaves and defoliation.
Experimental approaches with novel bactericides and application methods
CLas inoculum suppression is the major goal of area-wide HLB management in Brazil and elsewhere (Bassanezi et al. 2013). Effective delivery of chemicals with bactericidal activity is needed to not only suppress HLB symptoms and sustain tree productivity, but also to control bacterial titer and reduce inoculum spread. Magna-Bon (M-B, Magna-Bon LLC) is a chelated copper pentahydrate which provides bactericidal control of canker on fruit with 30% or less metallic Cu per application compared to film-forming Cu formulations (Graham et al., 2010). In a field trial utilizing 3-5 ml trunk injections, M-B had a significant effect on Las titer and HLB symptom development. M-B injections at 500-8000 ppm were applied for 2 years at 1 and 3 month intervals to 5-yr old Hamlin orange that trees that at the beginning of the trial were healthy (PCR-), asymptomatic (PCR+ without HLB symptoms) and PCR+ with mild decline (Graham et al. 2011). Trees were assayed by PCR and HLB canopy symptoms visually assessed at 6 month intervals after injections began in April 2009. All M-B treatments produced changes in canopy ratings that reflected positive response compared to no change for untreated trees. Ct values for the untreated trees indicated CLas positive status, whereas Ct’s for M-B treated trees were higher and indicated CLas negative or threshold infection. After 2 years of injections, the trees displayed iron chlorosis in the tree canopy, a visual symptom of copper phytotoxicity, and Fe deficiency in leaves.
Trunk injections no matter how well they work are operationally difficult and expensive to apply on a grove scale, hence, more feasible and sustainable methods for systemic application of bactericides to the tree are being sought (Zhang et al. 2011). For canker control, the SAR-inducing neonicotinoids and acibenzolar-S-methyl (Actigard, Bion, Syngenta Crop Protection) are effective as a soil drench or trunk spray (Graham and Myers, 2011, 2013). The advantage of trunk application is that the chemical is easy to apply and does not reach the soil which alleviates risk of leaching, immobilization and/or breakdown in the soil.
We evaluated trunk applications of M-B mixed with Pentra-Bark (Quest Products; www.quest products.us) a siloxane surfactant to enhance uptake of Cu through the bark. In a greenhouse trial, trunks of Hamlin orange trees (1-yr old) were painted with a range of concentrations of M-B with 0.1% Pentra-Bark or left untreated. Cu concentration in leaves was measured one month after application. Systemic bactericidal activity in the trees was assayed by detaching immature leaves and inoculating them with Xanthomonas citri subsp. citri (Xcc) as a Gram negative bacterial surrogate for non-culturable CLas. Canker disease control effect was measured as the number and size of the canker lesions as previously described for the in vitro assay (Francis et al. 2010). Cu levels in leaves were inconsistently elevated and canker lesions on detached leaves from M-B treated trees were not reduced compared with the untreated trees indicating that the Cu applied to the trunk did not move systemically to the leaf mesophyll in sufficient concentration to produce bactericidal activity.
After the failure of trunk treatments to produce systemic movement of Cu, soil drenches of M-B were conducted on potted trees and trees in groves on trees up to 3 years old. Soil drenches consistently increased Cu levels in the recently expanded leaves. To evaluate bactericidal activity of soil treatments with soluble M-B, field trials of 2 and 3 yr-old Valencia trees without CLas were drenched in the spring and fall in two successive seasons. The solid young tree plantings were surrounded by adult trees with 100% incidence of HLB. After two seasons, leaf samples were collected to assess HLB status of trees in each trial in early 2014. In a 4-yr old block of Valencia trees on Swingle citrumelo rootstock, CLas positive trees across all treatments ranged from approximately 40 to 80% incidence and in a 3-yr old block of Valencia on Volkamer lemon rootstock ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB drenches was not significantly different from untreated trees. After two seasons visual tree health ratings were higher for treated trees than the untreated check indicating these treatments were having a phytotoxic effect rather than achieving reduction in bacterial infection and/or HLB symptom expression.
ZinkicideTM is a novel therapeutic technology designed to release Zn in a sustained manner. Objectives for evaluation of ZinkicideTM are (i) maximizing antimicrobial activity, (ii) optimizing residual bactericidal effect in planta, and (iii) promoting systemic mobility with minimum phytotoxicity. ZinkicideTM as a nano-particle has been designed to move through the leaf cuticle and be absorbed into the mesophyll where it can be delivered to the phloem. Nevertheless, soil drench and trunk spray are being compared with foliar spray for effective systemic delivery of ZinkicideTM into phloem tissue.
Field testing of ZinkicideTM for control of citrus canker in comparison with Nordox 75G (CuO, Nordox AS) and Nordox 30/30 (ZnO/CuO) and an untreated check is under evaluation in a 5-yr-old Red grapefruit trial. Bactericides are applied with a handgun every 21 days. The sprays commence when the fruit reached 2.0 cm diameter and continue until full expansion for a total of 10-11 sprays per season. Disease evaluations include foliar disease incidence on the last summer flush and incidence of fruit with canker lesions, the age of the lesions and severity of lesions.
Field trial of ZinkicideTM against HLB is under evaluation in 2-yr-old Valencia trees with mild to moderate symptoms of HLB. Foliar sprays, trunk sprays or soil drenches are every 30 days during the growing season for a total of 6-7 applications. Disease evaluations include monthly evaluation of tree canopy symptoms, collection of leaves for CLas titer and leaf analysis for Zn. Tree health is assessed by visual estimation of HLB symptoms. To assess root health, fibrous roots are collected for fibrous root density determination as previously described (Johnson et al. 2014).
Literature cited
Bassanezi, R.B., Montesino, L.H., Gimenes-Fernandes, N., Yamamoto, P.T., Gottwald, T.R., Amorim, L., and Bergamin-Filho, A. 2013. Efficacy of area-wide inoculum reduction and vector control on temporal progress of huanglongbing in young sweet orange plantings. Plant Dis. 97:789-796.
Francis, M. I., Peña, A. and Graham, J. H. 2010. Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot. Eur. J. of Plant Path. 127:571–578.
Gottwald, T. R., Graham, J. H., Irey, M. S., McCollum, T. G., and Wood, B. W. 2012. Inconsequential effect of nutritional treatments on Huanglongbing control, fruit quality, bacterial titer and disease progress. Crop Protection 36:73-82.
Graham, J. H., Dewdney, M. M. and Myers, M. E. 2010. Streptomycin and copper formulations for control of citrus canker on grapefruit. Proc. Fla. State Hort. Soc. 123:92–99.
Graham, J.H., Irey, M.S., Miele, F. 2011. Trunk injection of copper sulfate pentahydrate (Magna-Bon) affects expression of HLB. Intern. Res. Conf on HLB Proceedings Orlando, FL, Jan. 2011: www.plantmanagementnetwork.org P. 174.
Graham, J. H. and M. E. Myers. 2013. Trunk and soil applications of imidacloprid, thiamethoxam and acibenzolar-S-methyl for SAR control of citrus canker on young fruiting
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Graham, J. H. and Myers, M. E. 2011. Soil drenches of imidacloprid, thiamethoxam and acibenzolar-S-methyl for induction of SAR to control citrus canker in young citrus trees. Plant Dis. 95:725-728.
Johnson, E. G., Wu, J., Bright, D. B., Graham, J. H. 2014. Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-affected trees prior to appearance of foliar symptoms. Plant Pathology 63: 290–298.
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Su, H. J. and S. C. Chang. 1976. The response of the Likubin pathogen to antibiotics and heat therapy. pp. 27-34. In E. C. Calavan, (ed). Proc. 7th Conference of IOCV, University of California, Riverside.
Van Vuuren, S. P., Moll, J. N., and da Graca, J. V. 1977. Preliminary report of extended treatment of citrus greening with tetracycline hydrochloride by trunk injection. Plant Dis. Reptr. 61: 358-359.
Zhang, M. Q., Powell, C. A., Zhou, L. J., He, Z. L., Stover, E., and Duan,Y. P. 2011. Chemical compounds effective against the citrus Huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ in planta. Phytopathology 101:1097-1103.