Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 25% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 25% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1130 - Entomology and acarology | 25% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 3030 - Information and communication | 15% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 2020 - Engineering | 10% |
Citrus is a very important fruit crop in the US. Huanglongbing (HLB) is a serious disease that has destroyed about half of Florida citrus and is now threatening the citrus industries in Texas and California. The pathogen is a bacterium known as Liberibacter and the disease is transmitted by an insect vector, Asian citrus psyllid. There is no known cure for HLB. Early detection and proper management done in a timely fashion are essential for disease management. Three species of the HLB pathogen are known. We have recently discovered a fourth species from Colombia. Proper identification of all forms of the pathogen is crucial for disease management. We propose to characterize pathogen variants and generate information that will be used for development of a testing system capable of detecting all species. Since the pathogen can be detected in the insect vectors several years before it is detectable in plants, we will focus mainly on testing the insect vectors of HLB. There is a need to trap the psyllids efficiently so that reliable testing can be conducted. The SmartTraps that we propose to develop will chiefly trap the insect vector of HLB and will preserve the psyllids such that they can be used efficiently for pathogen detection tests. We will use the genetic information developed for pathogen variants and design assays to detect all forms of the pathogen. In addition we will develop detection technologies using a very simple SmartDART unit that can be operated by growers and used for testing. Expensive equipment or scientific training will not be needed to utilize this system. The most important aspect of our project is extension. We will train a total of 30 growers in two states, California and Texas, in using these new technologies. SmartDART units, SmartTraps, and reagent kits will be provided to the participants. We believe that if the growers are capable of detecting HLB the surveillance system will be stronger and disease management becomes easier. Our goal is to engage a large number of growers and extension personnel in early detection of HLB so that the disease can be better managed in areas where it is already present and prevent and suppress the disease in other areas.
Overall goal is development of methodologies for early detection of all disease-associated forms ofLiberibacter (in psyllids), monitoring of the pathogen using field based diagnostic tools and development of integrated management strategies.HLB has resulted in significant losses to citrus industries worldwide. Reliable identification of all disease-associated Liberibacter pathogens may be crucial for taking necessary steps for disease management. Development of information required for designing generic testing capabilities for all Liberibacters may be an integral part of disease management. Secondly, if we make these technologies available to the interested parties including citrus growers and extension personnel, it will facilitate large-scale testing in future and will make the surveillance system stronger.We have four major goals:a) to develop sequence information on diverse Liberibacters; b) to develop methodologies for field detection of all Liberibacters associated with huanglongbing (HLB) in psyllids and plants; c) to develop improved methodologies collection and storage of psyllid vectors, and, d) to make the technologyavailable to citrus growers and extension workers. The four objectives are outlined below:
Objective one: Develop genomic sequence information of Liberibacter variants found in California, Texas and other citrus-growing regions1.1 Develop universal Liberibacter detection system. Using alignments of available genomic sequences of Rhizobiaceae members, several targets will be selected and several universal Liberibacter detection systems will be designed and used to screen existing collection of psyllid DNAs to find out sequence variants.1.2. Selection of samples: We will analyze the Liberibacter strain from California (samples from CRF in Davis), include samples from Texas, citrus relative samples showing HLB symptoms from FL, Central and South American countries; primarily using single psyllid extractions.1.3. 16S metagenome analysis. We will conduct this using services of Mr. DNA Company. Analysis of the 16S metagenome data will be helpful in understanding the nature of bacteria present in the sample. The methodology helped us to confirm a new species of Liberibacter in Colombia.1.4. 384 primer array. We have designed and used this array in our Riverside labs. Will use selected samples to do the array and study populations.1.5. Agilent Sureselect hybridization. We have already designed this panel. We will use this approach for select samples only.1.6. PACBIO and Illumina NextGen sequencing and Bioinformatic analysis. Isolates carefully selected by several of the above methods will be used for full genome sequencing. This aspect will be conducted at the core facilities of UCR and UC Irvine/Univ. of Florida.1.7. Selection of sequences conserved in Liberibacters. We will refine our universal Liberibacter detection system as needed when new sequences become available.Objective two: Develop a LAMP-based field testing system capable of detecting all HLB-associated Liberibacter populations from both psyllids and plants2.1. Develop LAMP capable of detecting all relevant Liberibacters: Identify suitable regions conserved in Liberibacters, absent in other Rhizobiaceae, design LAMP primers (Jenkins et al. 2011) and screen against multiple plant/psyllid extractions collected from various hosts and geographical locations (extractions available in the collection of the Riverside labs). We will design assimilating probes for promising primer sets and multiplex with internal controls.2.2. Standardize and optimize LAMP assays using a qPCR machine: Development of LAMP primer sets and assimilating probes for all HLB-associated Liberibacters will be done in year 1.2.3. Prepare and evaluate a lyophilized, ready-to-use, diagnostic kit for HLB: We will develop formulations which can be freeze-dried, to achieve stability at room temperature over long durations of time (> 6 months). Coordinated by Kubota, and Jenkins at the Univ. of Hawaii, Manoa.2.4. Develop Assimilating Probe-based detection systems for Liberibacter and psyllids: To enable use of CLas- and ACP-specific internal control probes in duplex assays for validation.2.5. Develop an assay for detection of Liberibacters in plants: Psyllid assay is fairly easy to handle without cross-contaminations. LAMP works well with purified plant DNAs (Keremane et al., 2014). Will develop a crude extraction method for plant samples. Instead of the COX (multicopy), we will use single copy gene as internal control (Ramadugu et al. 2013).2.6. Customization of Smart-DARTTM software tools/ interface: Existing Smart-DARTTM software has automated classification algorithms to reliably identify and report outcomes of tests. As new tests are developed with internal controls, we will customize the user interface for citrus industry stakeholders to simplify and standardize interpretation of results (i.e. sample is infected or not, or test results are inconclusive/invalid based on the presence of inhibitors or inappropriate use of sample or sample preparation).Objective three: Improve methods for psyllid collection, shipping and preservation3.1. Purchase two new 3-D printers to make variations to the shape and design of SmartTraps. Modify previous designs by adding texture, more appropriate lighting (different colors and LED lights. Placement, color and deployment of light are key elements of research in the second year.3.2 Compare SmartTraps on citrus with those on separate poles. Improve lighting parameters. Provide additional light at dawn and dusk only.3.3. Collect psyllids and the low level of non-psyllids from traps. Psyllids will be sent to Riverside lab for analysis. Document non-psyllid samples and preserve in DPI.Objective 3 will be conducted in FL by Smith and Halbert with help from Jenkins for improving the LED aspect.Objective four: Promote the use of LAMP assay and train growers, nurserymen, pest control agents, extension workers and other stakeholders for widespread testing of Liberibacters as a component of an overall integrated pest management (IPM) program.4.1 Train citrus growers (selected through advisory Board; 10 in year 1, 30 in years 3, 4 and 5; total no. is 30) in using SmartTrap, SmartDART TM and LAMP, sampling procedures, discuss anticipated project outcomes and expectations. Participants will collect psyllids from SmartTraps at biweekly intervals, conduct SmartDARTTM testing, initially for CLas and then for all Liberibacters (as soon a method is available; see objective 1). Each participant will periodically test pooled (1-10) psyllids for CLas using SmartDART TM and the remaining cohorts of trapped psyllids and crude extracts will be provided to Alabi in Texas and Keremane in Riverside for test validation. All data will be maintained in databases along with details of extension activities and test results. HLB finds in new locations will be subjected to standard reporting requirements with the local, state and federal regulatory agencies while maintaining grower confidentiality. As modifications and/or updates become available, we will provide participants with the improved units. Similarly, updated kits and protocols will be provided when the universal Liberibacter detection kit becomes available. All reagent kits will be prepared at Riverside and shipped to individual participants in coordination with project collaborators. We will work on room temperature stable reagent kits (objective 2), and the improved kits will be supplied to all participants when they become available. Detection systems for plant samples using Smart-DARTâ„¢ will also be provided when they become available (objective 2).
Target Audience
Citrus growers, regulatory agencies, officials overseeing trade in agricultural and biological materials, pest control agents, entomologists, plant pathologists, bio-engineers, students, home owners, agricultural producers and processors, experimental biologists, farmers and agricultural diagnostic service providers.
Changes / Problems
Nothing Reported
Training & Professional Development
We have provided opportunities for students (undergraduate, graduate, post-graduate levels) and laboratory technicians to have rich experiences while conducting various aspects of the project work. While performing trap work, there was an opportunity for learning design and printing of traps using a 3D printer. When we deployed traps in citrus groves, the extension work involved traveling to citrus groves, coordinating activities with grove managers, etc. During this period, the students and technicians had an opportunity to interact with members of the citrus industry involved in conducting pest management, grove managers, and other personnel directly responsible for the management of the citrus trees in a real-world situation. The project team realized the relevance and the importance of the research work conducted in the laboratory. They understand the need to have a positive interaction with the industry. Usually, these students and technicians are confined to a university lab and do not associate with the farm team. The lab team involved in conducting loop-mediated amplification experiments obtained extensive training in conducting biochemical assays. They learned the correct procedures for handling reagents, preparing enzyme mixes, and performing diagnostic tests in the lab and field situations. Analysis of the results of LAMP experiments taught the participants to troubleshoot data and find solutions to common problems. Improvement of the Bioranger testing units provided opportunities to learn various aspects of system fabrication and programming the software. Elementary statistics and hypothesis testing were taught. They practiced sterile technique, prepared and conducted isothermal LAMP reactions. In the process, the team obtained experience in lyophilization of biologically active reagents. In the Liberibacter characterization aspect of the project, the postdoctoral fellow and the technician involved obtained training in preparing high molecular weight DNA from plant and insect samples, generated sequencing libraries, and had an opportunity to learn principles of next generation sequencing methodologies. There was also an opportunity to learn bioinformatics, conduct an analysis of the genome sequences, compare sequences with related organisms, generate phylogenetic trees, and study implications of the sequence comparison of different Liberibacters, and correlate pathogenicity with sequence variations. Interaction of students, technicians, and scientists with the grower community was very valuable for the project personnel. It helped them to understand principles of diversity, inclusion, and responsibility. The Spanish speaking students were involved in direct communication with the growers and pest control agents. Some of them did not speak any English. Brochures prepared in English were translated to Spanish by the Spanish speaking students. When possible, the project personnel were encouraged to attend grower meetings as well as scientific conferences held in Florida, Texas, and California. This provided them with an opportunity to mix with other scientists and exchange ideas.
Dissemination Streams
We have conducted multiple demonstrations in Florida and California to show the field tools developed to various growers, pest control agents, and homeowners throughout the project. When requested, one-on-one demonstrations were also conducted. We have traveled to multiple regions in California with our tools to have presentations for interested groups. In California, we have routinely exhibited the tools developed in grower meetings like UCR citrus day and other events. When we had the opportunity, we have made oral presentations to grower groups to educate them about the tools available. We also keep in touch with many of the growers so that when they find psyllid populations in their areas, they will be able to contact us for help. We have encouraged growers to contact us for training and evaluation of tools developed. The Florida team has presented our work on traps in at least seven grower meetings held in different parts of Florida in the past year. Brochures were prepared in English and Spanish and distributed as needed. The team in Hawaii has helped to address issues related to technology implementation and practicality. The design of trap lights and experimental results were presented at professional meetings and invitations of various Universities and other state agencies. Some of the work has been published in peer-reviewed journals. We have about four manuscripts that are almost ready for publication in scientific journals. In Texas, the traps were displayed at the Annual citrus Winter Texan festival held yearly. Information was shared with growers at the bimonthly meetings of the Texas citrus pest and disease management corporation by co-PI from Texas. The project team has attended many meetings in California and Florida and presented results in scientific conferences. This provides an opportunity to interact with peers and also to advertise about the tools developed.
Next Reporting Steps
The project has now ended. But we plan to provide support to CDFA (California Department of Food and Agriculture) that is considering using the traps developed in this project for monitoring psyllids in Southern California. Similarly, we have established contacts with many growers, pest control agents and other industry personnel in southern California. We have been monitoring psyllids in Temecula to ensure that the citrus industry personnel in Temecula will be aware of the HLB status of their groves. We will continue to provide the support that we can offer since it will be useful for them to know about HLB situation in this critical time when the disease has started spreading in Southern California. If some growers want to test the psyllids and use the tools developed by us, we will be willing to provide support and help them. Several other citrus industries outside the country are interested in using the tools that we have developed. We have been approached by scientists and industry personnel from various sources. To the best of our ability, we will provide information, tools and expertise to provide assistance required using discretionary funds that are available from other sources.
Target Audience
Our target audience consists of citrus growers, regulatory agencies, officials overseeing trade in agricultural and biological materials, agricultural producers and processors, experimental biologists, analytical chemists, farmers and agricultural diagnostic service provides. Also, entomologists, pest control agents, plant pathologists, bio-engineers, students and home owners.
Changes / Problems
The genome sequence of Ca. L. caribbeanus was significantly different from other reported citrus and psyllid-associated Liberibacter genomes. Since the associated phenotypes in the citrus plant are vastly different with this Liberibacter variant that does not cause disease in citrus, analysis of the genome may provide new insights on the pathogen biology. The information generated may be useful in devising new approaches for disease control. We are in the process of completing the De novo sequence assembly and annotation of the novel Liberibacter genome. The newly designed traps have been performing better than the ones we reported in previous years. To cut costs involved, we used inexpensive materials for the prototypes. While this is desirable for use in controlled environments, for use in the field situation, a sturdier model is preferable. Since we want the new traps to be distributed to the growers, we are not in a position to distribute the current prototypes because of durability issues. This has caused some delay in distributing these to the grower participants. We are trying to find better alternatives for a custom molded plastic trap that can be manufactured for a fraction of the cost and that will be strong enough for use in the field situation. Increasing the psyllid capture efficiency by altering the shape of the trap and altering other parameters worked well in our hands. Using lures increased the capture rate only marginally. Lighted traps did not increase capture rates as we expected. The problem may be due to technical reasons and we will try to address this issue by altering the lighting parameters in the units and re-evaluating them.
Training & Professional Development
In the current project we have graduate students (both Ph.D. and Masters students) that have contributed significantly to different aspects of the project work. We have a total of six undergraduate students that have been involved in conducing the field, laboratory and extension work. Their involvement in the project provides them with a unique opportunity to interact with citrus growers. This valuable training makes the students realize that the research project that they are involved in is designed to address real life problems faced by the citrus growers. Also, when certain processes do not work as expected, the students involved understand the importance of conducting experiments that can be repeated by anyone who is interested in using the tools developed. We are also involved in doing some sequencing and sequence analysis type of experiments. This provides the project personnel an opportunity to learn new skills and understand the importance of using recent technological advances for problem solving. Professional development of the project participants is beneficial for the students, technicians and other employees involved. Depending on their situation, many project members are encouraged to attend scientific meetings, make presentations to grower communities, and participate in discussions leading to improvement of the techniques developed. We have presented progress of this project to citrus growers, pest control agents, citrus industry personnel and also home owners. This exercise provides an ideal atmosphere for professional growth and for development of scientific curiosity and interest in students and other workers. Many of the brochures prepared are in both English and Spanish so that we can reach a larger audience. The Spanish speaking students/technicians are involved in doing this translation and it helps them to understand principles of diversity, inclusion and developing helpful attitudes that are an integral part of achieving progress in any community.
Dissemination Streams
We have disseminated information regarding project progress through peer-reviewed scientific publications, semi-technical articles published in grower magazines, presentations at International, National, and Regional conferences, grower meetings, talks delivered for interested public groups, seminars in university settings, etc. (a list of some of the publications, talks, conference presentations etc. is included in the section on 'products'). In addition, we have also communicated with growers in the area and engaged them by including their groves in our evaluation studies and by conducting outreach and extension activities when possible. Most of this activity has taken place either in California or in Florida, to some extent in Hawaii. Many growers/pest control agents require one-on-one training. We are able to provide this service to only a few growers. We have realized that public interest in this project - especially in California where the disease is just starting- is tremendous. We are doing our best with the resources available to us to meet the needs of the local citrus industry.
Next Reporting Steps
During the next reporting period, we plan to do the following: Complete sequence assembly and annotation of the Liberibacter variant - Ca. L. caribbeanus. Deposit sequences of the CLas variant from Colombia and of the novel Liberibacter - Ca. L. caribbeanus in a publicly accessible sequence database and publish a manuscript in a peer-reviewed journal. Complete evaluation of the new cylinder traps in psyllid rearing facilities and also in the field situation in CA and in FL. Publish trap design and evaluation work in peer-reviewed journals. Re-configure trap lighting units to conduct more evaluations of lighted traps. Conduct more extension in California where the growers are interested in adopting new technologies that may help in disease mitigation. <br><br>
<br>What was accomplished under these goals? Impact of the project: Citrus huanglongbing has resulted in significant financial losses in Florida due to decrease in yield and fruit quality, increased grove maintenance costs required for management of the disease and death of severely infected trees. In California, in the past six months' period (June 2018- January 2019), the number of HLB positive tree finds increased from about 600 to about 1100. However, in CA, the disease is currently restricted to backyard citrus and has not yet affected commercial citrus. Proper management of the groves and increased surveillance may result in postponing the disease epidemic in CA. Hopefully, HLB resistant cultivars will be made available in the near future. Our project aims to develop field tools required for routine surveillance of groves that will facilitate timely management practices. These will be a part of the integrated pest management strategies required to manage this deadly disease. Testing psyllids for the presence of the HLB pathogen has been shown to be a reliable method to detect the pathogen early. Yellow sticky traps utilized routinely to monitor psyllid populations are useful for planning pesticide sprays but the psyllids captured on the sticky traps are not suitable for DNA testing. Hence psyllids are captured by using aspirators- an ineffective method of capture that is laborious and expensive. We have developed plastic 3D traps printed with a 3D printer for capture of psyllids. The designs are based on understanding psyllid behavior and biology and the prototypes are evaluated, tweaked to produce better performing traps. If these are used along with an inexpensive testing device that was developed in the project, it will be possible to conduct large scale testing by many interested parties. Increased surveillance will lead to early detection of the pathogen in a grove and can result in management decisions that will mitigate HLB. The project also aims to study pathogen variants that may become important during an epidemic and for understanding the HLB pathosystem. The progress made in the project is regularly disseminated to interested members of the citrus industries. The first objective is to characterize Liberibacter variants that are known to exist in nature. We have been working on characterization of two Liberibacter variants from Colombia, South America. The first isolate is a strain of Ca. Liberibacter asiaticus from Colombia. Since the organisms cannot be cultured, the method used will involve metagenomics approaches for obtaining sequence information. Psyllid populations collected from citrus plants that have huanglongbing symptoms were extracted by research collaborators from Instituto Colombiano Agropecuario (ICA) located in Bogota and the DNA obtained was shipped to the laboratory in Riverside, CA for further analysis. DNA extractions that were positive by qPCR for CLas were utilized for constructing sequencing libraries for Illumina platform. The sequences generated were filtered using bioinformatics tools to remove other sequences like the psyllid Diaphorina citri, endosymbionts like Wolbachia, Carsonella, Proftella etc. CLas Colombia sequences were assembled De novo and also aligned with other CLas sequences and variant regions identified. We now have a high quality draft assembly of CLas strain from Colombia. Similar procedures were utilized for Candidatus Liberibacter caribbeanus (CLca) samples. Psyllid extracts were tested by qPCR to identify samples that were positive only for CLca using primers specific for CLca genome. Selected samples were used for whole genome amplification, library construction and Illumina shotgun sequencing. De novo assembly of the genomic sequence of the novel Liberibacter is in progress. Comparison with CLas psy 62, Ca. L. americanus, Ca. L. africanus, Ca. L. solanacearum and L. crescens showed several blocks of microsyntenous and orthologous genes in CLca. Many genomic regions in CLca appear to have multiple sites of recombination. Studying the genome structure and biology of a psyllid-associated Liberibacter like CLca with a novel host-associated phenotype will be helpful in understanding the pathosystem. We have developed qPCR assays for detection of all citrus and psyllid-associated Liberibacters. The Taqman probe-based real time PCR assay will not amplify non-specific targets like Rhizobium, Agrobacterium or Sinorhizobium. Similarly, no reaction was observed with DNA extractions from healthy citrus or Liberibacter-free ACP and tomato psyllids harboring Ca. L. solanacearum. This assay for detection of all citrus-associated Liberibacters would be very useful in routine surveys. Objective two is to utilize sequence information developed to design inexpensive, sensitive, field deployable kits using LAMP technology and a field-adaptable detection device. Our original detection unit, 'Smart Dart' is now replaced with another unit 'BioRanger' that has several technical improvements over the original gadget. We have replaced all the old units of Smart Dart with the newer version. The improvements include: a). enclosure of power switch and indicator lights under an integral translucent lid; b). Additional hard ware for thermal shut off is incorporated. The thermal fuses that are now a part of the testing device improve safety; c). Optical components were reoriented to eliminate the need for additional mineral oil so that sample evaporation issues are minimized; d). Optional lysis step, ability to enter user defined thresholds for sample reaction classification and amplicon melting analysis incorporated. Objective three is to develop better traps for capture of psyllids. We have developed 'cylinder' traps that outperform the previous 'stem' traps. The design of the cylinder traps is simpler and the traps are most cost effective. Modifications were made to the design to increase retention of the psyllids that enter the traps. The capture rate increased significantly [from an average of 20 psyllids per trap in our previous model (no. 1015) to about 60 psyllids per trap in the cylinder traps]. Several iterations of the cylinder traps were printed and are under further evaluation. We have updated the design of the programmable lighting device that was reported in the previous years to include much more efficient energy regulation and low power usage extending the operating life of the device. The six LEDs incorporated into the traps (UV, Blue, green, Yellow, Amber or Red) can now be turned on in various combinations for programmable durations (before sunrise, after sunset, etc). The lighted traps are currently being evaluated for determining the capture rate. We have evaluated three different types of lures - commercial formulation purchased from Alphascents company, custom lures formulated by Dr. Joseph Patt (Collaborator from Florida) that are derived from Eureka lemon and from Orange jasmine. The traps used to conduct the evaluations were fitted with a holder that will accommodate a small plastic tube containing the lures. We have observed a modest increase in capture rate by the addition of lures. There was no significant difference between the different types of lures tested. We have also evaluated the effect of acetic acid on capture rate - based on a recent publication. In our experiments, we did not observe any improvement in capture rate due to acetic acid. Objective four is to conduct extension with citrus growers. We have given several presentations in California and in Florida to explain the new technologies developed to citrus growers, pest control agents and other members of the citrus industry (please see the list of talks delivered in the publications section). We have also conducted demonstrations to interested growers. It may be possible to make sufficient number of traps available to growers if they are made by a commercial partner that will utilize custom molding techniques. <br><br><b>Publications</b><br>
Target Audience
Our target audience consists of entomologists, plant pathologists, bio-engineers, pest control advicers, citrus growers, students, home owners, California Department of Food and Agriculture emloyees, Wonderful Citrus employeesinterested in using field detection units, advisory board membersand other collaborating scientists.
Changes / Problems
We had some delay in obtaining samples with Ca. L. caribbeanus from Colombia. The sequence that we have generated now appears to be different from published sequences and will need some more bioinformatic analysis before we can completely annotate the genome. The BioRanger unit had some technical issues resulting in variability in results. At this time we believe that we have fixed these problems. The capture rate of traps is probably sufficient for testing limited number of psyllids. The lures that we have tried to date have not signifciantly increased the capture rate. We will try to improve this some more.
Training & Professional Development
The current project involves some basic research like development of sequence information for variants of the HLB pathogen, bioinformatics component, identification of genomic regions suitable for development of pathogen detection assays, etc. In addition, there is a strong extension component associated with this project. This provides the participants an opportunity to be involved in conducting scientific research that can be passed on to the end user. In this project, the students, technicians and scientists involved have been in constant communication with growers, pest control agents, citrus industry personnel and homeowners. We have presented the project progress in several grower meetings, citrus day events, etc. We are constantly interacting with the industry. This provides an excellent opportunity for training and professional development of the project personnel involved. Since we tweak our systems based on the feedback obtained, there is constant interaction with the industry resulting in awareness of the practical problems and efforts to develop tools that will meet the needs of the citrus industry. The students involved in the projects have the rare opportunity of talking to citrus growers and disseminating the information needed.
Dissemination Streams
We have presented our work to citrus industry personnel in California, Florida, Texas and Hawaii. The grower meetings where our results are presented include Citrus day events held at Riverside, CA, California citrus conference organized by the citrus research board in Visalia, CA, Citrus nursery society events in California, entomological society meetings in Denver, presentations at the Florida state fair where the Laboratory of 3D printing was one of the featured events, at the AmCon Design and Contract manufacturing expo in Orlando, Florida department of Agriculture and Consumer services form scientific advisory workgroup, etc. Our group had five presentations at the International Research conference on Huanglongbing held in Orlando, Florida, during 2017. At this meeting, in addition to scientists and growers from the US, we had the opportunity to interact with many industry persons from central and south America. There was an immense amount of interest in the tools we are developing for the field detection of HLB pathogens. The practical utility of products developed in this project was evident and this was very encouraging to our project team.
Next Reporting Steps
For objective one, we plan to complete annotation of the CLas variant from Colombia. We will conduct detailed analysis of the metagenomic sequence obtained from Illumina sequencing of Candidatus Liberibacter caribbeanus from Asian citrus psyllidsample obtained fromColombia. We will design LAMP assay that can be used to test CLca as well as the CLas variants being characterized. For objective two, we will improve BioRanger based on the feedback received fromcitrus growers participating in our project. We will develop a stable, lyophilized, ready to use test kit (just add sample) that can be used for field testing. We have been working on a visual LAMP assay that will be very inexpensive. The assay is now working well under laboratory conditions. The assay is not field ready at this time. We will try to standardize conditionsand make this visual LAMP assay more reliable under field conditions. For objective three, we will evaluate the newer versions of the traps in the field in California and also in Florida to generate statistically signficant data that can be published. We will evaluate more lures and develop LED lighting capabilities. A combination of LED lights and lures may be useful for increasing capture of psyllids. We will include acetic acid in our trap evaluations. Acetic acid, a degradation product of a pheromone has been recently reported to increase capture rate. We will conduct extension work in California and in Texas (with Wonderful citrus) to popularize our tools and to train more citrus growers, pest control agents and home owners interested in monitoring their groves actively for HLB pathogen. <br><br>
<br>What was accomplished under these goals? Our first objective is to characterize Liberibacter variants that are known to exist and use the information to develop methods for detecting all HLB-associated Liberibacters. In California, Liberibacter crescens has been reported occasionally from citrus by CDFA. Several "Early Detection Technologies" (EDT) have been finding Liberibacter positives over the last few years from different regions of California, but most of them have not been confirmed by direct detection of the bacterium by either qPCR or conventional PCR. In view of the above, we would like to test if either another species of Liberibacter, or a closely related member of the family Rhizobiaceae is causing interference with EDTs. We have designed two sets of primers and probes for detection of all known Liberibacters (confirmed by testing against Ca. L. asiaticus, africanus, americanus, and solanacearum). We are trying to obtain DNA of other Liberibacters for this study. Similarly, we have also designed primers and probes for detection of members of Rhizobiaceae. We have detected Sinorhizobium from some leaf samples in a survey of 100 field trees in California, but no Liberibacter yet. We will be analyzing a large number of samples from Florida using thesenew primers and probes in the next year. In 2014, we found a new species, Candidatus Liberibacter caribbeanus (CLca) in Colombia mostly from psyllids. Since then we have monitored the movement of this bacterium through the years by testing psyllids. In 2016, we found Ca. L. asiaticus, (CLas) and associated HLB symptoms in another region close to Venezuela. It appears that the CLas is moving at a much faster rate than CLca. To characterize the CLas variants, we have we have obtainedpsyllid and citrus DNA from Colombia through collaborators, conducted whole genome amplifications using DNA extracted from single psyllids. This product was used to construct a library using Illumina Nextera library kit, and sequenced using Illumina NextSeq platform. After removal of sequences of Diaphorina citri, the sequences were aligned to the genome of Ca. L. asiaticus strain, Psy62. There was 99.71% coverage at 1X, and 96.87% coverage at 30X. The annotation of this CLas variant is now in progress. The gap regions of the genome with very low coverage or no coverage are being filled by PCR amplification of genomic fragments followed by Sanger sequencing. We have also developed sequence information for a different species of psyllid-associated Liberibacter from Colombia. Ca. L. caribbeanus reaches very high titers in psyllids (can reach Ct value of 15 in qPCR assays). We have not yet conclusively detected this species in citrus plants. In the past, we have used PACBIO platform to sequence the metagenome of Ca. L. caribbeanus, and obtained about 200 kb of sequence. Using the same approach as above, we have now sequenced the metagenome of Ca. L. caribbeanus from a psyllid. After filtering the sequence to remove non-target reads, the sequence will be assembled de novo. This assembly appears to be more challenging since the genome appears to be extremely diverse from other known Liberibacters. We have also conducted endosymbiont analysis of several psyllids, and foundsignificant reduction of other endosymbionts in Liberibacter containing psylldis. This reduction appeared to be very drastic in CLca containing psyllids from Colombia. We have also used plant DNA from Aegle and Afraegle (citrus relative genera thatcan harbor Liberibacter asiaticus and other related Liberibacters) from Florida, and conducted sequencing using Illumina platform. We are currently analyzing the sequence. Objective two is to utilize sequence information to design inexpensive, sensitive, field deployable kits using LAMP technology and a field adaptable detection device. Our earlier detection unit "Smart Dart" is now replaced with another unit, "BioRanger". The new devise is useful for conducting isothermal amplification assays for HLB by LAMP either in the field or in laboratories. Many improvements have been made in the most recent version of the Bioranger unit. The device is now packaged with a more professionally finished and commercially appealing translucent housing. Other improvements that have resulted in improved performance or additional functionality include: 1. Enclosure of power switch (and indicator lights) under an integral translucent lid. 2. Incorporation of additional hardware thermal shutoffs, in addition to thermal fuses, to improve the safety of the device. 3. Redesign of the orientation of optical components to eliminate the need for additional mineral oil to prevent sample evaporation. Addition of mineral oil is cumbersome and introduces undesirable opportunity for sample contamination. 4. Additional software features are now introduced - including optional lysis step (for integral sample preparation using new thermally stable strand displacing polymerases), incorporation of user defined thresholds for sample/reaction classification, and amplicon melting analysis to confirm amplification of the target sequence. The reconfiguration enumerated under item 3 listed above resulted in some additional background noise in the analysis due to undesired light scattering within the device. Hence an additional component was redesigned to incorporate into the BioRanger design and all released versions of the device have been serviced to include the new hardware, resulting in very significant improvements in signal to noise ratio. Objective three is to develop better traps for capture of Asian citrus psyllids. Six new trap designs were developed focused primarily on shape, color and phtometrics (Figure 2). Rhinoceros 5 modeling software was used for designing. Models were exported as stereo lithography files and printed on various 3D printers including MakerBot Replicator 2, MakerBot Z-18, Lulzbot Taz 5 & 6, German Reprap X 400. Manufacturer recommended software was used to prepare STL files for final printing: MakerBot Desktop, Cura and Simplify 3D. This software acted as a printer interface allowing for the manipulation of STL files and printer settings directly. All trap components were printed using polylactic filaments. Evaluations were conducted in the laboratory using cage trials as described before. The cages varied in size from 0.115 m3 to 0.192 m3. Traps having the highest success rates were used for field trials conducted at the location of an organic grove in DeLand, Florida. Trap 1015 performed better than other traps and also performed better than the sticky traps in certain evaluation trials. Further evaluations are in progress in California. We are in the process of evaluating several lures that are commercially available or provided by collaborating scientists to increase the capture rate of the traps. In California the trap evaluations are done in Pomona in the psyllid rearing facility and also in organic groves in Riverside county and Ventura county. Objective 4 is to conduct extension with citrus growers. In this reporting period we have conducted many workshops for interested growers. We have distributed educational material regarding the proper use of psyllid traps and testing methods using BioRanger devices. These were done in Santa Paula, Ojai, Moorpark, Camarillo and Fillmore areas of Southern California. We have also conducted demonstrations in Texas and trained field personnel working for Wonderful Citrus in McAllen, Texas. <br><br><b>Publications</b><br>
Target Audience
The target audience includes scientists (Plant pathologists, entomologists, extension scientists), home owners, citrus growers, pest control agents, bioengineers, California Department of Food and Agriculture employees, APHIS personnel, project collaborators, advisory board members, students and technicians.
Changes / Problems
Our efforts to characterize the new Liberibacter variant, Ca. L. caribbeanus was delayed due to insufficient coordination with the various cooperators in Colombia. The bacterium appears to be spreading in this region and at this time we do have material that can be used for whole genome amplification of the Liberibacter from the psyllids and can be used for genomics work. One of the problems we faced with the field evaluation of Smart traps is that the population of psyllids in Southern California was varying in grower's orchards. Now we are collecting the basic evaluation data in the psyllid rearing facility in Pomona, CA (maintained by Citrus research Board and APHIS). Some of the Smart Dart units have a lot of noise owing to the presence of interfering signals. The updated version is assumed to be free from this technical glitch and should be useful. California has many regulations regarding movement of putative infected samples with in the state. Hence we are conducting our trap evaluations and psyllid testing with selected growers, pest control agents and testing laboratories in Southern California.
Training & Professional Development
We have provided opportunities for many undergraduate students to be involved in this project. The California group has made many visits to citrus groves and this provides the project personnel to have direct contact with the growers. The students and technicians involved in the project appreciate the research being done since they realize the importance of the laboratory study in making a difference in the real world. For the growers, the capability to conduct their own testing is very empowering. In the process, they understand the science behind this deadly disease and get the feeling that they can keep track of the disease status in their groves - to some extent. The knowledge gained will make them feel responsible and also make them accountable to try and maintain the grove in good condiiton. The project is conducted in Universities, State departments and USDA labs in four different states.
Dissemination Streams
We have published research results in journals and presented in scientific meetings. The section on products has a list of the publications and research presentations. We have talked to growers in the area and presented our work in citrus workshops and other grower related activities that take place in California, Florida and in Texas. We have also prepared instructional material suitable for growers and have distributed these to people interested in trying our technologies. We have continuous conversations with some grower participants and have been improving our tools to suit the needs of the community.
Next Reporting Steps
During the next reporting period, we plan to generate the entire sequence of Ca. L. caribbeanus and have it annotated. We will develop more LAMP assays so that the most sensitive test can be used in our kits. We will provide the improved detection devices to growers. We hope to have alternate methods like visual LAMP working efficiently in the field situation so that the growers have different options and will be able to select the technology that works best under their circumstances. We will distribute more Smart traps to growers and have more growerinvolvement in field testing. Approval of the technologies by CDFA will facilitate large scale use of our methods in CA. <br><br>
<br>What was accomplished under these goals? Goal 1: To develop genomic sequence information for Liberibacter variants. A. Leaf samples containing Liberibacter variants have been collected from citrus relatives. These include Aegle marmelos, Aeglopsis chevalieri, Afraegle paniculata and Balsamocitrus daweii. In a separate project conducted inFL we have detected the presence of Las strain common in FL in many citrus relatives and have reported that these hosts can harbor HLB pathogens (Ramadugu et al., 2016). Based on studies conducted using 384-primer arrays, we have determined that many field trees of citrus relative genera in Southern Florida carry variants of Las. We have analyzed the Liberibacter genome from these sources and sequenced selected regions of the genome B. We have reported another variant of Las - Candidatus Liberibacter caribbeanus from Colombia, South America. To characterize this strain of Liberibacter, we have screened many psyllid samples (collaborators from Colombia) and generated information on many genomic fragments. Samples with sufficient Liberibacter titers were used for whole genome amplification and the samples are now utilized for generating genomic sequence information. Goal 2: To design loop-mediated amplification technology (LAMP) assays for developing field-deployable kits and detection devices. A. LAMP-based procedures can be utilized for devising field tests for detecting HLB pathogens. Since LAMP can be conducted at ambient temperatures, simple instrumentation will be adequate to conduct the amplification and to visualize the results (Keremane et al., 2015). At this time we are evaluating additional targets for developing new LAMP tests. The nrdB gene in Liberibacters codes for the ribonucleotide reductase enzyme and is a multicopy gene with five copies in the bacterial genome. This region has been described by Zheng et al. 2016 (Sci. Rep. 6:39020) as suitable for detection of the pathogen in asymptomatic HLB positive plants and in insect vectors. We are designing LAMPtest for the nrdB gene to develop a field assay for Liberibacters. B. The detection unit developed by us for visualizing the LAMP products (Smart Dart) costs about $3000. Development of more cost effective methods will be beneficial for large-scale use of the field test . In case of LAMP method that we have described earlier, a fluorescent molecule generated by the reaction is detected by the Smart Dart unit. It is also possible to generate products that will enable visual discrimination of the samples that are positive or negative for the HLB pathogen. The Visual LAMP method was developed for HLB detection and evaluated by us last year. At this time, we are improving the technique so that the results are consistent and the technique is field adaptable. We have been collaborating with New England Biolabs company and developing required reagents that are suitable for this inexpensive method for field testing of HLB. C. Several grower cooperators have tried the detection units that were made available last year. Based on the feedback obtained about the performance of the Smart Dart detection units, our team has now designed the next version of the Smart Dart, the 'Bioranger' to address all the technical problems of Smart-Dart. 'Bioranger' will have many improvements to the hardware and the software. It has less inter-device variability and is more reliable in areas of high wireless traffic. It includes a heater shutoff system to prevent device overheating, enables smaller assay volumes, and can be operated without the addition of mineral oil that is generally included to prevent evaporation/condensation. The new unit is packaged with a fully integrated lid with a gentle snap hook to lock in the closed position. The android app interface includes improvements like options for classifying reaction units based on manually set thresholds, interactive controls on the charts for better data visualization, and automatic reestablishment of lost Bluetooth connections. A melt curve analysis is included for confirmation of specific amplicons. D. As an additional detection method, we are also working on development of an antibody-based lateral flow detection methodology to detect Liberibacters from psyllid samples. Polyclonal and monoclonal antibodies raised to Liberibacter surface antigens will be utilized in simple to use dipsticks. Goal 3: Design Smart traps for psyllid capture and for testing captured psyllids for HLB pathogens. A. Smart traps printed using Rhinoceros-5 modeling software on 3D printers can be utilized for capture of psyllids in the fields. Twenty one models were designed and printed by our team members from Division of Plant Industry, Gainesville, FL. These were first evaluated in the laboratory in psyllid chambers in a pairwise comparison study. Selected traps were also tested in the field conditions in CA and in TX. At this time we are testing 100 traps of a specific model in the fieldor in psyllid cages (12'X12'X12') maintained by Citrus Research Board and APHIS in Pomona, CA (psyllid rearing facility). The optimal parameters required for capture of the psyllids was determined. B. Conducted experiments to evaluate the quality of DNA extracted from the psyllids captured in the low-tox preservative and exposed to the weather conditions for six weeks. Experimentsdone during winter, spring and summer seasons under field conditions (David Hall, FL). The conclusion was that the psyllids could be stored for up to six weeks under field conditions without any significant negative effect in the quality of bacterial DNA. Psyllids captured in the Smart traps in low-tox preservative can be used for testing for about 6 weeks. C. We are evaluating different lures (provided by collaborators) to see if the psyllid capture can be improved. At present, using Smart traps, we can capture about 20-30% of the psyllids trapped by yellow sticky traps placed in the general area. Use of sticky traps will be beneficial to determine the population dynamics of psyllids and to determine the occurrence of psyllids in certain locations in the grove (depending on environmental factors). Placement of Smart traps in areas determined to have sufficient psyllids will be helpful in capturing a portion of the psyllids in the preservative. These psyllids can be used tested for Las using field deployable units that we have developed. D. We have developed Smart traps with LED lighting inside them. These are currently under evaluation in Florida. E. Traps are now evaluated by many growers in CA. CDFA will evaluate the traps containing lures during this spring. Goal 4: Extension A. Smart traps: We have distributed Smart traps to interested growers in CA. We have demonstrated the use of these and distributed educational materials for proper usage of the traps. We have collected feedback from growers regarding the performance of the traps and passed these comments on to our collaborators in Gainesville, FL. The design of the traps was altered many times to incorporate the changes suggested. Some older versions of traps were also evaluated by CDFA in Southern CA. We plan to provide the current models of traps (with lures) to CDFA this spring for field evaluations. B. We have conducted several training sessions for citrus growers in CA to familiarize them with Smart Dart detection units. Certain growers have obtained training in our laboratory. Again, we have obtained feedback from the growers and have improved the detection units significantly. The current model, Bioranger (manufactured by Diagenetix, HI) incorporates the improvements needed for optimal use of this unit. C. We have distributed simple instructions and conducted many demonstrations in the grower's orchards. The intent is to make growers familiar with the technologies developed so that large scale testing can be done in the near future. <br><br><b>Publications</b><br>
Target Audience
Target audience: For the current reporting period (Feb 1, 2015 to Jan 31, 2016), the target audience include scientists (plant pathologists, entomologists, extension scientists), home owners, citrus growers, pest control agents, bioengineers, California Department of Food and Agriculture employees, APHIS personnel, project collaborators, project advisory board committee, students and technicians. Efforts: Science-based knowledge imparted to participating growers, students and extension personnel through informal educational programs. Hands-on training was provided to certain growers and pest control agents in conducting LAMP assays and subsequent detection of the HLB pathogen using SmartDart platform.
Changes / Problems
In California, the second find of HLB has caused a lot of alarm among citrus growers. Several small growers approached us and are interested in using our technologies for pathogen testing. In order to accommodate this need expressed by the industry, we tried to develop cheaper technologies. We have experimented with LAMP methods and detection devices and we have now developed two promising methods that can be used instead of SmartDart (after proper validation). This will reduce the initial expenditure from $3000 (for SmartDart) to $300 (for calcein or visual PCR methods). SmartDart method is still better since it will be more sensitive than the cheaper technologies. We hope that in addition to SmartDart, we can provide materials required for the newly developed methods to interested small growers. The SmartDart units did not perform well initially since there was discrepancy among the units. With the help of our Hawaiian collaborators, we have now improved the hardware and software of the SmartDart units and enforced rigorous quality control for all the units that are used in the project. The SmartTraps are better than the standard yellow sticky traps since they capture and preserve the psyllids. Unlike psyllids captured in sticky traps, the ones from SmartTraps can be tested for the pathogen DNA. However, the current models of SmartTraps are not catching sufficient number of psyllids. Hence we are now printing many more traps using the 3D printers and the 3D technology and we ae currently evaluating the various models. This caused delay in supplying the traps to the growers. We should be able to provide the technology that is currently available to the participants in 2016.
Training & Professional Development
For growers, the capability to conduct their own testing is very empowering. In the process, they understand the science behind the deadly disease and the feeling that they can keep track of the disease status in their groves - to some extent. The knowledge gained will make them feel responsible and also make them accountable to try and maintain the grove in a good condition. A group of growers in Los Angeles area of California have contacted us for training and we are in constant communication with this group. They are small growers and want to get technical support for their whole community and have the capability to conduct HLB testing. Our inspiration to develop cheaper methods of detection was because of such grower interactions. If we can provide tools to all of them without too much expenditure, that would serve the purpose of our project. We are training a middle school student to conduct a science project using LAMP technology and SmartDart unit. The student developed enormous interest in pursuing science and has learnt many scientific concepts because of her science project. The project is conducted in Universities, State departments and USDA labs in four different states. Our part time technicians and have the opportunity to visualize science in action. Periodically we have growers walk into the facility and discuss their concerns regarding management of HLB with the scientists. Working on a problem that is relevant in everyday life of a citrus grower is very powerful in influencing the thinking of someone who is new to research. It makes them understand that research can be very meaningful for solving real-life agricultural problems.
Dissemination Streams
We have published the LAMP technology in Crop science Journal. As an open source article this publication has been accessed by several researchers as shown by statistics. The detection technologies included in the project are based on LAMP (loop-mediated amplification) methodology. Interested scientists and growers can easily access the research article and understand the methodology. Keremane presented results of our psyllid testing projects to scientists, researchers, students, industry personnel and citrus growers in American Phytopathological Society meeting held in Pasadena, CA and also in the HLB meeting conducted in Davis, CA. Interesting discussions followed these presentations and several useful contacts were made. We are constantly in touch with many local growers in California (through Ramadugu and Keremane) and in Texas (through Alabi). We had two team meetings with the participating scientists of this project in this year. The first meeting was held in Orlando during the HLB International Conference in February 2015. This was a planning session and many of the participating scientists were involved in discussions about the project. A second team meeting was held in Pasadena, CA in August of 2015. All the participating scientists in this project who attended American Phytopathology Society meeting in Pasadena were involved in this discussion. Many issues and concerns were raised and we have been addressing these through e mail communications and phone meetings. In addition, project personnel travelled from Riverside, CA to Gainesville, FL in November of 2015 to discuss about SmartTrap experiments. Personnel also travelled from Riverside, CA to McAllen, Texas for discussions with the scientists, for training the scientists as well as some growers in Texas. Project personnel travelled to Davis, CA to present the project work to fellow scientists and local growers. Presentations in Davis and Pasadena (Keremane). The team from Hawaii visited Riverside two times to validate the new Smart Dart units that were purchased from Diagenetix and to conduct discussions on design of inexpensive detection units for calcein based methods. We are constantly reevaluating our methods and develop technologies to improve our current protocols and devices. The interactions between scientists from Riverside and Hawaii groups have been very successful in achieving these goals. We are constantly discussing with our advisory committee consisting of Dr. Richard Lee (retired Research Leader, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA), Dr. MaryLou Polek (Past Vice-president of research from Citrus Research Board, CA) and Mr. Alan Washburn (Pest Control Advisor, Southern California). Their input and broad perspective of the needs of the citrus industry are useful in guiding our project goals.
Next Reporting Steps
In the next reporting period, we will conduct sequencing of five isolates of Liberibacters. At present our list includes: Liberibacter isolates from San Gabriel HLB find site in California, Liberibacter variants from citrus relative genera, Aegle and Afraegle, Ca. Liberibacter caribbeanus from Colombia, Las isolates from Colombia/Venezuela. The sequence variations between these isolates will be useful in devising methods that can detect all Liberibacter variants associated with citrus. We will try to make the LAMP assay more user friendly by lyophilization of the mastermix. At present these trials are being conducted in Riverside and Hawaii. Easy storage of the reaction mix will be convenient for storage of the reagents required for the detection methods. We will improve the calcein-based methods that require an investment of about $300 for the initial set up. The lower prices will encourage more growers to adopt these technologies. The detection device that we have already designed needs to be further validated. As needed, we will improve the detection devices to make them safe, user-friendly and affordable. The visual PCR method is very promising as it is simple to use and fairly sensitive in detecting Liberibacters. We will evaluate this method further in the lab and also under field conditions. We will provide training to growers that includes visual PCR and calcein-based methods in addition to SmartDart devices. We will evaluate the programmable lighting that our team has designed. If there is improvement in psyllid capture, we will design appropriate lighting set up that can be easily incorporated into the best models of SmartTraps. Using available technologies, we will conduct training programs in California and also in Texas and increase grower participation. Certain growers in Florida are interested in participation since they would like to have the ability to monitor their nurseries for presence of psyllids that may have Liberibacters. Because of this recent development, we will provide our detection units and reaction reagents to the interested parties in Florida. We will conduct more trap evaluation experiments in backyard situations. Most growers conduct sprays as soon as psyllids arrive and it is difficult to complete the trials when pesticides are used. Conducting some of the trap trials in organic groves and backyards may provide better data for analysis and evaluation. <br><br>
<br>What was accomplished under these goals? Objective 1. The only way to save the citrus industry from citrus HLB is by prevention, exclusion and suppression of both the vector and the disease. Early on-site detection using 'easy to use', simple and reliable technologies would facilitate such an effort. In CA, there have been two reports of HLB positive citrus so far. Genomic sequence analyses reported by Keremane in collaboration with other scientists have shown sequence differences in the genomes. Two separate introductions are likely and the HLB find in 2012 probably did not spread to other regions; the disease containment efforts appear to be working. Information about strain differences will be useful in assessment of disease management strategies and understanding HLB epidemiology. We are conducting molecular characterization of Liberibacters of other HLB positive plant samples from San Gabriel HLB find site to test if all the San Gabriel isolates are genetically similar. We are characterizing Liberibacter isolates from citrus relative genera - Aegle and Afraegle from FL to understand sequence variability. From Colombia, South America, we have described a new species of citrus associated Liberibacter, Ca. Liberibacter caribbeanus. We have generated 250 Kb sequence of this strain by using the PAC BIO sequencing platform and using Illumina platform we aim to get the complete genome sequence. We have identified Las in another part of Colombia bordering Venezuela (first report of Las from Colombia). We will obtain citrus DNA from Venezuela and characterize the Liberibacter isolates. Objective 2. Using available genomic sequences of Liberibacters we have developed qPCR primers for universal amplification of all citrus Liberibacters. These primers have been validated with standard DNA preparations; evaluation with psyllid and plant samples is on-going. We will then design LAMP primers and develop assays for detection. We are improving the LAMP reaction mix by lyophilization of the mix to enable easy storage at room temperature. SmartDart unit works well to conduct field testing. It is suitable for large groves due to increased sensitivity and rapid assay time. Considering the current HLB situation in CA, there is an urgent need to develop less expensive technology so that a larger number of people can do testing. We are now developing a calcein-based fluorescence method to detect Las from psyllids. In this test, HLB positive samples will show fluorescence while negative samples will not. Once the method is developed, it would cost only $300 to obtain the necessary reagents and equipment to do testing (as compared to $3000 for SmartDart and the associated software). A low-cost, end-point version of the SmartDart system was developed with temperature controlled heating block and integral UV LEDs for visualizing LAMP reactions conducted with calcein indicator. As an alternate method affordable for small growers and home owners, we have developed a "visual PCR assay" that can be conducted in about an hour under field conditions. The test requires initial investment of about $250 only. Samples are scored after the simple test as positive or negative, visually, based on color. The calcein method and visual PCR will not be as sensitive as SmartDart. But, because of the decreased costs, many more growers/homeowners will be able to utilize the technology for HLB testing. Even though, our initial objective was to develop an universal detection system, we have focused presently on Las alone for field detection since there is an urgent need to conduct large scale testing in CA. We have made several improvements to SmartDart software and hardware. Objective 3. Standard sticky traps are useful for monitoring psyllid populations but not for HLB testing. To enable capture of psyllids that can be tested for Liberibacters, we developed 'SmartTraps'. The advantages are twofold: unlike sticky traps, psyllids captured in the preservative used in the SmartTraps will be testable for the presence of the HLB pathogen. Secondly, use of a trap that can preserve psyllids well will enable a 'dynamic' method of testing. For example, if psyllids are captured using aspirators, the testing method is 'static' and reflects the situation on a single day. With the traps that can preserve psyllid DNA, samples collected after two weeks will reflect the situation in the two week period. The Las titer in psyllids changes dramatically over time and hence dynamic sampling is better. When manual collections are conducted, most locations are visited once or twice a year; a SmartTrap placed in a certain location can be used to collect samples throughout the year. To improve psyllid capture in SmartTraps, we have adjusted many parameters including color of the plastic used to print the trap, external and internal shape of the barrel, pore size, presence of awnings, smooth versus textured surfaces, addition of a slippery 'Fluon' coating on the interior, etc. Psyllid behavior is taken into consideration for the designs. Different combinations of these variables are incorporated into each trial model and the psyllid capturing ability is evaluated in closed chambers and field situation. Improvements made resulted in avoiding larger insects and in preventing rain water from entering the trap. Presence of stem like structures and rough texture on the exterior was more attractive to the psyllid than the original smooth surfaces. Addition of the 'Fluon' coating has prevented escape of the captured psyllids from the traps. A combination of alterations are now being evaluated. Addition of a light source to the existing traps may be useful in increasing the trapping efficiency of the SmartTraps. We have designed a Bluetooth programmable lighting system with multiple wavelength high intensity LEDs to test different lighting conditions (UV, blue, green, yellow, amber and red). We will alter the light intensity and test various combinations of lights. Rechargeable batteries and solar panel capability are integrated into the system. Depending on the preliminary results, we will incorporate the most effective type of lighting into the trap design. Objective 4. The key to finding HLB quickly is - a) testing psyllids and b) large scale testing of psyllids. Towards this goal, we require traps capable of capturing psyllids and preserving them so that they are suitable for testing and secondly, we need testing systems that are easy to use, economical and efficient. At this time we have made significant improvements in both these areas. In CA, we have worked in cooperation with home owners, organic growers, extension agents, Pest Control Advisor (PCA; Alan Washburn), researchers and owners of small citrus groves. Evaluation studies were conducted in a citrus park, psyllid rearing and bio-control facilities. We discussed the trap evaluation project with entomologists, CDFA personnel and APHIS employees. Provided training to pest control agents and small growers in conducting LAMP assays using Smart Dart. In TX, we have trained scientists and some growers. Technical problems with the units were observed by Diagenetix Inc. personnel and corrected later. We revisited the hardware, software and reagent components of the detection technology in a systematic fashion and brought about several improvements. Strict quality control is now in place. Two alternate technologies - a) calcein based LAMP and b) visual PCR were pursued. We designed detection components for the calcein method and constructed 50 units at a cost of about $150 per unit. The calcein based method is still under development. The visual PCR method is at present very promising. After a few more validations in the lab setting, we will be able to launch this method as an alternate, more affordable technique. We will distribute the SmartTraps, SmartDart units and visual PCR kits to growers this spring as soon as the psyllid populations build up. <br><br><b>Publications</b><br>