Location: Citrus and Other Subtropical Products Research
2020 Annual Report
Objectives
1. Determine the critical factors that limit standard and novel citrus cultivars’
ability to thrive and become productive in Florida, where huanglongbing (HLB) and its vector are
endemic.
1a. Determine the effects of HLB on response to abiotic and biotic stresses.
1b. Determine the HLB susceptibility of various rootstock/scion combinations in
green house trials.
2. Protect and/or rescue valuable and unique ARS citrus germplasm from infection by HLB through appropriate methods, including, micrografting, cyrotherapy,
thermotherapy and antibiotics as necessary.
2a: Rescue high-value, novel germplasm that is threatened by HLB as well as other
maladies.
2b: Develop improved methods for elimination of CLas from infected citrus.
3. Conduct field trials to evaluate promising scion selections for tree health,
productivity and fruit quality.
3a: Determine HLB tolerance for various combinations of rootstocks and scions.
3b: Determine fruit quality attributes of advanced selections of Poncirus trifoliata hybrids that show tolerance to HLB.
Approach
Sustainability of the U.S. citrus industry, especially in Florida, is currently
threatened by Huanglongbing (HLB), a disease associated with the bacterium
Candidatus Liberibacter asiaticus (CLas) and its vector, the Asiatic citrus psyllid. HLB debilitates trees, reduces yield, ruins fruit quality and eventually kills trees. Young trees are especially susceptible to HLB, making the establishment of new orchards challenging. The foundation of sustainable citrus production in the face of HLB will be cultivars, both scion, rootstock and the appropriate combinations thereof, that are able to survive to bearing age and produce high quality fruit for fresh and juice markets. Therefore, determination of susceptibility of various rootstock/scion combinations will be done in greenhouse and later evaluated in field trials trials. Complicating the HLB impact are the effects of other stress which may increase the impact of HLB. Thus, abiotic (drought) and biotic (nematode, fungal, and insect) stresses will be compared for HLB and healthy grafted trees. Citrus breeding is a long-term endeavor that requires growing and evaluation of trees in the field before promising selections can be identified. Historically, first stage testing of new hybrids has been conducted using own-rooted trees that are planted into the field, grown to maturity and evaluated for horticultural characteristics. We have been conducting projects for the last several years to rescue and protect these elite lines, and the process is ongoing. Selections are grafted onto seedling rootstocks and maintained in the greenhouse. Trees that survive are tested for CLas and Citrus Tristeza virus (CTV), trees identified as negative are maintained in the greenhouse for subsequent testing. Trees that continue to test negative for CLas after one year are submitted to the FDACS DPI Budwood program for entry into the parent tree program. Another approach is to investigate the ability of various treatments for the elimination of CLas from citrus budwood. Most research on elimination of CLas from citrus has focused on either shoot-tip grafting or whole plant thermotherapy. Such approaches are laborious and require lengthy periods for evaluation. We will collect buds from known HLB-positive branches to be subjected to heat, cold, antibiotics and/or other treatments that may come available.
Progress Report
This serves as the final report for Project No. 6034-21000-016-00D. The long-term objective of this project was to develop new knowledge that will contribute to sustainability of citrus production in areas where Huanglongbing (HLB) disease is endemic. The potential for HLB to destroy the entire US citrus industry is real.
Research contributing to the viability of citrus production and marketing under the HLB threat is essential. This project focuses on: 1) field and greenhouse trials to determine how abiotic and biotic stresses impact trees affected by HLB; 2) methods to preserve and protect valuable citrus germplasm threatened by HLB; 3) field trials to determine how various combinations of scions and rootstocks perform under commercial citrus production practices in Florida; and 4) determining if HLB tolerant Poncirus trifoliata hybrids possess acceptable juice quality.
Each of the project objectives required accurate quantification of Candidatus Liberibacter asiaticus (CLas) in citrus so that incidence of infection as well as CLas titers could be assessed. Considerable effort was devoted in the first year of the project to estimate CLas titer in citrus based on the standard Animal and Plant Health Inspection Service (APHIS) work instruction. Based on that research it was determined that the detection of single copies of CLas target DNA is possible and that the standard “cutoff” used by APHIS to distinguish infected from non-infected was too stringent and could, in some cases, result in false negative diagnoses. Results were shared with APHIS and resulted a revision of the diagnostic standard. This has also become the diagnostic cutoff used by the California Department of Agriculture.
The results of this work also led to an APHIS funded project to determine the effects of temperature on detection of CLas in citrus nursery stock. It was demonstrated that a reduction of CLas titer occurs during and shortly after exposure of trees to temperatures of 40 degrees °C but that titer soon thereafter returned to levels seen before exposure to elevated temperatures. Based on reports of this work at scientific and industry meetings, the California Research Board requested work be conducted on detection of CLas in citrus and Asian citrus psyllid (ACP) nymphs. Results of this work demonstrated that greatest diagnostic reliability for CLas in the absence of HLB symptoms could be achieved by adopting a hierarchical sampling strategy at the tree level. If there is evidence of ACP nymphs, eggs adults or leaf notching (indicative of ACP feeding) those samples are the most likely to be infected. This data was shared with APHIS and a modified sampling protocol has been drafted. This protocol will provide more efficient diagnostics, especially in areas where CLas has recently been introduced into an area. During the course of these experiments, an elegant greenhouse system for controlled inoculations of citrus with CLas by ACP was developed. This system has been used during the project to compare susceptibility of various citrus types (oranges, grapefruit, mandarins, lemons and citrons) and to conduct studies on the impact of CLas infection on citrus physiology. Results demonstrate clearly that citrus trees infected with CLas begin to express HLB symptoms within four months of infection. This sheds new light on the concept of latency of HLB disease development, which was believed to be as long as years. It was also demonstrated that HLB symptom development is closely related to CLas titer and become apparent when CLas titer reaches ca. 1000 cells per mg of citrus petiole. However, there are differences among citrus genotypes in the severity of HLB symptoms, with grapefruit being the most sensitive, mandarins varying in sensitivity depending on cultivar and lemons and citrons, while developing clear HLB symptoms, continue to grow.
As members of the HLB research community became aware of the infrastructure developed in this project, numerous requests for collaborative projects with other researchers developed. This has resulted in USDA receiving three NIFA grants dealing with HLB detection, management and breeding and four citrus research board grants dealing with detection and management of HLB.
Extensive field trails were established with grower cooperators to screen novel germplasm for tolerance to CLas infection. Some elite lines have shown promise, however, some have Poncirus trifoliate in their background, a sexually compatible citrus relative imparting poor fruit quality, which is not always acceptable. Because flavor is such an important aspect of citrus fresh fruit and juice quality, research was conducted in this project to develop sensorial data. A panel was trained and used to establish standard descriptors of ponicirus types and their hybrids. This information will be critical as novel Poncirus hybrids with resistance to HLB are developed.
In addition, numerous unique citrus hybrids, currently only in the field in Florida and infected with CLas needed to be rescued. Experiments were conducted to determine if budlings of trees produced from field-grown material could be established free of CLas. During the course of this project over 50 hybrid selections have been rescued and are now free of infection.
Accomplishments
1. Improved diagnostic sampling protocol for detection of citrus Huanglongbing (HLB) pathogen, Candidatus Liberibacter asiaticus (CLas) infections in citrus trees. Detection of CLas infection triggers regulatory action and impacts management
decisions by citrus growers, thus early detection is a critical component of HLB
management. However, detection of CLas in citrus trees is challenging in the absence of HLB symptoms. Frequently, samples collected from CLas-infected trees test negative for infection when in fact the tree is infected (false negative). Research was conducted by ARS scientists in Ft. Pierce, Florida, to determine if a hierarchical sampling strategy, at the tree level, could reduce the probability of false negative diagnoses of CLas. The hierarchical sampling scheme is based on where CLas is most likely to be found in a tree. Results demonstrated that CLas infections can actually be detected within 24 hours after infection occurs. Based on these results, the official APHIS sampling protocol is now being revised.
Review Publications
Zuniga, C., Peacock, B., Liang, B., Mccollum, T.G., Irogoyen, S.C., Tec, D., Marotz, C., Weng, N., Zepeda, A., Vidalakis, G. 2020. An in vitro pipeline for screening and selection of citrus- associated microbiota with potential anti-“Candidatus Liberibacter asiaticus” properties. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.02883-19.
Kunta, M., Park, J., McCollum, T.G., Gonzalez, M., Vedasharan, P., Da Graca, J.V. 2018. Development of a sensitive real-time PCR detection method for citrus tatter leaf virus. Journal of Plant Pathology. 100:67. https://doi.org/10.1007/s42161-018-0029-7.
