Through both the CRISPR/Cas may provide a powerful tool for development of HLB resistant citrus vsarieties. The most wide-used applications of CRISPR utilize stable integration of Cas9/sgRNA genes into host plant genomes and subsequently their expression in plant cells produces targeted mutations. For sexually-propagated annual crop plants, the Cas9/sgRNA transgenes can be eliminated from plant genomes following sexual reproduction. However, this strategy is not feasible for vegetatively propagated perennial plants, such as citrus, requiring years to reach sexual maturity or being heterozygous in genotype. To circumvent that problem, we have used Agrobacterium-mediated transient expression of Cas9/sgRNA genes to produce targeted mutations with no stable integration of transgenes into the host plant genome. We have also developed a highly efficient method based on DNA sequencing and high resolution DNA melt analyses to identify shoots with desirable mutations. The method we developed may be useful in developing nontransgenic HLB tolerant cultivars of citrus. Huanglongbing , first reported in China in 1919, has been the most devastating citrus disease. Asian citrus psyllid was proved to be the vector of HLB in China in 1977. Today, ACP and HLB are widely distributed in 11 of the 18 citrus plantation provinces in China. Ponkan tangerine is one of the most HLB susceptible cultivars and over 80% of Ponkan orchards in Chinese Yongchun County have been ruined since HLB broke out in 2000. To develop a new system for the citrus sustainable plantation in HLB-infected areas becomes very important for citrus industry in China, as well as in the world.

Here, a promising plantation pattern was developed for Ponkan tangerine in Yongchun County,hydroponic dutch buckets which was summarized as following. The orchard should be segregated by mountains or forest belt . On the other hand, it is better to plant ACP disliked trees, such as China fir around each planting plot. Before replanting or planting citrus disease-free seedlings, all HLB positive or negative trees and other ACP host species should be removed thoroughly. Prior to removal, all trees should be treated with a foliar insecticide to kill all adult psyllids feeding on that tree. The insect pest controlling system, such as insecticide mechanical spraying system should be installed in the orchard to ensure suppressing the ACP in a short time. It is compulsory to use disease-free seedlings. Moreover, we recommend using big container-grown seedlings and the planting density of 1.5~2 m×3~4m to accelerate the orchard producing profit. The main task in the second year after planting is thinning flower in the spring, promoting tree growth in the summer and the early autumn, and stretching erected branches in the late autumn. To clean the orchard in winter, suppress summer shoot and intensively promote autumn shoot plus spraying insecticide are the three key steps to control ACP spread. Establish dynamic removal mechanism for HLB infected trees. Scouting for HLB infected trees should be done routinely so that infected trees can be removed in time and replaced with healthy trees . By adopting this pattern, the 4-year-old tree has the height of 2.0~3.0 m and the average yield of 30 kg per tree; the percentage of the HLB infected tree is below 3%. Moreover, over one thousand hectares of Ponkan tangerine groves with the present pattern were added in Yongchun County. This plantation pattern shows a promising prospect for sustainable citrus plantation in HLB-infected region. During the last two years, the Mexican monitoring program of Diaphorina citri, the vector of the pathogenic bacteria associated with huanglongbing, has shown a marked reduction in presence of the insect in most of the citrus growing areas of the country.

As management of the vector in Mexico, exhibits marked differences in respect to the performed in Brazil, or Florida, U.S.A., where there is an intensive use of insecticides, there are concerns that probably the official monitoring program is underestimating pest densities or that the traps have been failing to capture the insect, yielding optimistic results about abundance of D. citri. In order to contribute to discard the possible use of inefficient traps, the present study was conducted to evaluate these tools that have been used in the monitoring, as well as to obtain a better trap to be used under possibly scarce populations of the vector in Mexico, as a resultant future scenario from an effective pest control program. In this study, sticky traps in different designs, colors and color patterns were evaluated to determine the capacity to capture adults of the vector. During 2015- 2016, we carried out 13 experiments in Nuevo León state, and two regional simultaneous tests in Michoacán, Nayarit, Nuevo León, Sinaloa, Tamaulipas, and Veracruz states. Several traps captured a noticeable number of D. citri adults. Notably, when the glue was standardized for all the traps by removing the own and applying one commercial, generic glue, the capture of insects in the specific ACP American trap was lower or similar than most commercial traps evaluated; while, in some of them such change in glue produced higher D. citri capture, suggesting effects from quality and quantity of glue used in its manufacture. Compared with the 48 officially recognized CHMAs in FL, the Treasure Coast area exhibited very poor control of ACP in 2014 where multiple ACP ‘hot spots’ were recorded. The number of abandoned groves and uncoordinated spray patterns can lead to increasing ACP pressure. Without CHMA-wide control, HLB will spread quickly and the implications for new tree plantings may not be viable for production. A series discussion among Indian River Citrus League growers was held to assist in the development of CHMAs for Treasure Coast Citrus that integrate ACP control into new planting plans.

Based on our risk models, many risk factors are considered in the Treasure Coast CHMA optimization including: ACP population dynamics, residential risk , abandoned groves, distance to transportation corridors, citrus nurseries, farmers markets, packing houses and green waste facilities. Other logistical concerns are also considered, such as commercial planting density, maximum/optimum number of growers per management area and elevation difference for movement of equipment. Following thousands of simulations and optimization, seven CHMAs are proposed for the Treasure Coast area. They are defined as Indian River North, Indian River South, 95 East, Okeechobee, St. Lucie West, East and South. The generated CHMA maps have been shared with growers via UF/IFAS extension to monitor ACP control performance locally and statewide. Ever since first detection in late 2005, HLB is now widely spread throughout most commercial citrus groves in Florida and caused a rapid decline of trees, as the disease continues to tighten its grip on Florida’s citrus industry. However, there are limited studies that quantify the impact of HLB on citrus reduction rate over large spatial scales. Aerial photographs are most commonly used to measure historical changes since the advancement and availability of high-resolution, large-scale vertical aerial photography. This study presents a semi-automatic statistical classification method for annual citrus tree decline rate calculation and pattern recognition/analysis. High-resolution photography of Florida from 2006 to 2015 can be obtained from LABINS ,bato bucket where citrus field imagery is converted to digital format and filtered out using user-defined citrus polygons in GIS. Subjected to selection bias, a number of commercial citrus fields are used to fully represent the spatial variability of the HLB situation. In order to estimate active citrus coverage accurately, we specified multiple training areas as the color-spectral properties of citrus canopy photography are apt to change for different times and locations. Also, efforts have been made to remove tree shadows by identifying neighboring points with similar color intensity. Transition from healthy to serious infected citrus grove results in potential significant changes in tree canopy size . We compared annual tree coverage reduction rates across different landscapes, identified major patterns of tree loss within grove , and quantified the relationship with HLB progress in Florida. Incorporating both spatio-temporal analysis and aerial photography has the benefit in determining the economic impact of HLB on actual citrus landscape. This proposed technique could be used as an enhancement tool in future HLB survey design and modeling. Spatially interpolated Asian citrus psyllid density population estimates were calculated from the Florida multi-pest survey . ACP density is summarized at 2km grid resolution across all commercial citrus plantations in Florida to provide an overview of ACP population change, i.e., dynamics. By investigating ACP changes through all cycles from 2011 to 2016, a new rating system has been designed for bench marking and analysis of performance characteristics of each individual Citrus Health Management Area .

A total rating value for each CHMA is calculated to rank its comparative efficacy of ACP control. Also, various relevant factors, such as grower participant rate, urban population size, abandoned grove acreage, ratio between commercial citrus and residential area, are analyzed to quantify their influence on CHMA performance. The relationship between citrus production and CHMA performance is investigated, and this will help us understand the benefit of CHMA from an economic perspective. Accurate estimation of ACP population is also critical for future cost-effective pest management at the CHMA level. Related with CHMA performance scores, there are a number of CHMAs with high ACP uncertainty or ‘vague’ values. The amount of uncertainty is mainly influenced by the local variation of ACP observation in the field and the spatial pattern of ACP distribution within the CHMA. Bootstrap-based analysis has been implemented to address the suitable sample size for each CHMA, justifying realistically that the sample variance of the ACP population is not constant over time. The method is particularly useful for the MPS survey design as the project budget constrains the total number of citrus blocks that can be surveyed during a yearly time frame. Citrus Huanglongbing , spread by the Asian citrus psyllid vector, is a devastating disease threatening nearly every citrus producing area worldwide. Given the recent findings of ACP in California’s Central Valley, an increase in ACP prevalence is inevitable which heightens the risk of HLB introduction into commercial citrus. This poses a major threat to the viability of the citrus industry emphasizing the urgent need for proactive regulatory intervention and disease control. Understanding the spread of ACP across different landscapes is crucial in designing optimal survey methodologies and control strategies to mitigate the impact of HLB infection. We utilize an agent-based model approach to investigate the spread of ACP and HLB in a real-world setting. Instead of using ‘toy’ data, a 16 mile2 area of Porterville, CA with residential and commercial citrus present is used as a case study for the simulation. The mathematical model incorporates ACP population dynamics as well as ACP and HLB spread by considering factors such as psyllid life span and mortality, net reproduction rate in the natural environment, psyllid dispersal distance, citrus host type and density, flush availability through new flush production or infected tree decline, and effects of different management schemes including biological control , large scale or infield survey, and chemical spray. We also conduct a costbenefit analysis by assessing the productivity after different control methods are applied. This simulation model can quantify the influence of input epidemiological parameters on ACP/HLB development, and be utilized as a tool for HLB control planning. The development of symptoms of HLB in citrus plants is associated with the process of colonization by the bacteria Candidatus Liberibacter asiaticus and the increase in the production of reactive oxygen compounds . The formation of free radicals increases under conditions of high temperature and radiation, aggravating symptoms of HLB. The temperature of leaves of plants with symptoms is higher, probably due to lower rate of transpiration. Evidence of the benefit of mitigating the symptoms with use of sunscreens was previously observed. The objective of this work was to verify the expression of symptoms of HLB in citrus growing in an agroforestry system, where solar radiation, temperature and humidity are modified. For this purpose, both the severity of symptoms and incidence of diseased plants were evaluated in a 4 years old orchard under organic cultivation of Valencia orange tree on the Rangpur lemon root stock. The measurements were taken in the plants without shading and under two situations of shading, shadow of Gliricidia and Eucalyptus + Teak .