All 4 goals were completed. These were: 1) cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter in E. coli; 2) cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter constructs in E. coli, and 3) cloning and expression of all 4 Las and the one possible Lam anti-repressors, and determining responsiveness of the reporter and clones from Milestone 2. The primary goal was Goal 4: development of a assay for Las phage late gene expression. We used quantitative, real time PCR (qRT-PCR) assays to determine levels of expression of the Las SC1 late genes in both citrus and periwinkle. In an effort to functionally confirm that those genes annotated as being lytic cycle genes were in fact lytic cycle (late) genes, we functionally characterized one of them, SC1_gp110, annotated as a holin). Heterologous expression of SC1_gp110 in E. coli revealed bacteriostatic activity and functionally confirmed the annotation of the expressed, predicted protein product as a holin. We used RT-qPCR to monitor the expression of this holin and 4 other putative phage lytic cycle genes in Las- infected periwinkle and citrus. Surprisingly, holin mRNA levels increased significantly in citrus 12 to 24 hours after leaves were detached, indicating a potential lysis triggering mechanism in citrus. In citrus, expression was monitored before and after heat treatment at 42’C for two days, a level demonstrated to cure plants of Las infection. The relative expression of four of the five putative lytic cycle genes examined, SC1_gp025, SC1_gp110, SC2_gp095, and SC2_gp100 were much more highly expressed in periwinkle than citrus. However, heat treatment of citrus leaves failed to increase expression of any of these genes, indicating that lytic cycle induction of these prophages is not tied into a presumed heat stress response of Las in citrus. The possibility of activation by biochemical inducer(s) specific to periwinkles is currently being examined. Conclusions from this year’s funding are: 1) Functional holin activity of the predicted SC1_gp110 gene confirms the annotation. 2) Expression analyses revealed much higher levels of expression of phage late genes, particularly the holin, in periwinkle as compared to citrus, possibly indicating a phage response specific to periwinkle. 3) The significant increase in holin mRNA levels in citrus, 12 to 24 hours after being detached, regardless of temperature, indicates a potential lysis triggering signals in citrus. The holin promoter region is being developed into a reporter gene construct that may be useful for monitoring lytic cycle activation by potential inducers in citrus. 4) The mode of action of thermal therapy (heat curing of Las) in infected citrus does not seem to be connected to phage induction.
Liberibacter crescens BT-1 (Lcr, isolated from mountain papaya), has been cultured and sequenced (GenBank CP003789, Leonard et al., 2012) and has the potential to be developed into a model Liberibacter system. for functional analyses of Las, Lam and Lso genes. To become a model system, BT-1 needs to be 1) reliably and stably transformed with a shuttle vector so that different genes can be added in; 2) gene knockouts in BT-1 must be reliably made, and 3) the strain needs to be introduced into a plant and be shown capable of growth in planta. The first two of these 3 goals have already been met. To determine if BT-1 might be tractable for functional genomics studies, the minimum inhibitory concentrations (MICs) of several antibiotics commonly used for plasmid selection were determined. BT-1 was found to be quite sensitive to: chloramphenicol, < 4 mg L-1; gentamycin, < 1 mg L-1; kanamycin, < 2.5 mg L-1; and tetracycline, < 0.3 mg L-1. BT-1 cells were readily transformed at high frequencies with two different wide host range replicons tested: pUFR071 (RepW; Al-Saadi et al., 2007) and pUFJ05 (Bordatella replicon; Reddy et al., 2007). The transformation frequencies were estimated to be 3000 transformants / mg of DNA for pUFJ05 and ~ 20. greater for pUFR071. Stability of pUFR071 was evaluated; this plasmid was >95% stable, without selection, when grown in BM7 medium for over 20 generations. pUFR071 was extracted from BT-1, retransformed into E. coli and appeared from restriction analysis to be unchanged. BT-1 cells transformed with pUFR071 showed only a marginal loss of plasmid, even when grown for more than 70 generations in the absence of antibiotic selection. The results are in line with the expected stability of RepW origin in the presence of stabilizing partition locus parA (De Feyter et al. 1990). A gene knockout in BT-1 was successfully demonstrated, following the strategy outlined in Casta’eda et al. (2005). Briefly, a partial internal fragment of the BT-1 gene encoding the Type I restriction-modification system restriction subunit R (GenBank B488_07050) was PCR-amplified and cloned into TOPO’ 2.1 (Invitrogen, Carlsbad, CA). The resulting plasmid was used for electroporation of BT-1 cells. A single homologous recombination event at the target site duplicated the cloned region and integrated the vector between incomplete copies of the target gene. The antibiotic resistant colonies were analyzed by PCR to confirm interruption of the target gene and integration of the plasmid backbone. Attempts to artificially inoculate marked BT-1 strains into tobacco, citrus and periwinkle are currently in progress.
The main objective of this proposal is the down regulatin of genes of phloem specific Callose synthase andd phloem proteins involved in phloem plugging in citrus infected citrus greening pathogen. Differential gene expression following infection of citrus by Candidatus Liberibacter asiaticus (CLas) bacterium has been documented by microarray and deep transcriptome profiling. CLas infection elicits over-accumulation of callose, and phloem-specific lectin PP2-like protein in the phloem of infected plants, in addition to number of transcripts of starch and sucrose metabolism, hormone biosynthesis, transcription factors etc. Over-accumulation of callose and PP-2-like proteins presumably causes phloem-plugging and consequent impairment in photoassimilate transport and excessive starch accumulation in leaf chloroplasts which results in disease manifestation. Down-regulation of over-expressed genes responsible for phloem-plugging would potentially negate the CLas disease manifestation. Towards this end, we have identified Callose7 as phloem specific callose among many calloses in plants based on their homology to Arabidopsis thaliana. At the same time we have also identified phloem protein B8 and B14 as phloem specific. These have been amplified from the total RNA isolated from healthy citrus tissues by reverse transcription and have been cloned into pGEMT easy vector which will be eventually engineered into the Citrus trizteza virus vector as outlined in the proposal and the details will be presented as they become available in the quarterly report. I am in the process of identifying a suitable post-doctoral candidate to continue with this research.
Understanding foamy bark rot of Fukumoto navel: Preliminary results support the hypothesis that foamy bark rot is initiated by scion-rootstock incompatibility, resulting in nutritional and/or physiological abnormalities, which provide opportunity for infection by certain pathogen(s) and foam production is possibly due to yeast activities. Inoculations with fungi (Fusarium solani, Fusarium sp. and Neofusicoccum mediterraneum) obtained from declining Fukumoto show that most of these fungi are pathogenic on Fukumoto. Bacterial inoculations produced no obvious symptoms on Fukumoto. Analysis of soil samples indicated no significant correlation between foamy bark rot and pH, total N, C, Olsen-P, K, Na, Ca, Mg, and lime. Results from in vitro fungicide screens have identified a number of products currently available that have the potential for use as pruning protectants. Results from this study have been published and can be found at http://www.citrusresearch.org/nov-dec_citrograph Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: In this survey across six counties, 11 different species of Botryosphaeriaceae, comprising five genera (Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum and Neoscytalidium), were found associated with cankers of multiple citrus varieties and different ages. All isolates tested were found pathogenic on citrus and further tests on one-year old citrus plants showed the ability of most isolates to produce symptoms of gumming within one to two weeks post inoculation. Results from an in vitro fungicide screen show that a number of commercially available products currently on the market have promise for use as pruning protectants. Results from this study have been published and can be found at http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-05-13-0492-RE. The alliance between Fusarium solani and other factors in citrus dry root rot: Results from this study indicate that in addition to F. solani and two species of Phytophthora (P. citrophthora P. nicotiana), F. oxysporum and a new Fusarium sp. are major organisms associated with DRR. Although there was no correlation between season and isolation of Fusarium spp., there was correlation between seasons and the isolation of Phytophthora spp. No correlation between nutrient content and DRR was found. A rootstock susceptibility screen is underway to determine the tolerances of 33 rootstocks to infection by F. solani as well as other Fusaria identified from this study. Results from this study have been published and can be found at http://www.citrusresearch.org/nov-dec_citrograph Investigating Branch Canker and Dieback in Lemon and Grapefruit in the California Desert Region: Surveys conducted across Riverside, Imperial and San Diego counties in Southern California show the presence of Neoscytalidium dimidiatum, Eutypella citricola, E. microtheca and Eutypella sp. associated with citrus branch canker and dieback. Pathogenicity tests on detached shoots show all three species of Eutypella are pathogenic on citrus, however these fungi appear to have a low to moderate virulence. Preliminary results from an in vitro fungicide screen show there are some products currently available that have the potential for use as pruning protectants. Results from this study have been published and can be found at http://www.citrusresearch.org/current-citrograph
The major aim of this research was to develop a method for in vitro culture of ‘Candidatus Liberibacter asiaticus’ (LAS). Specifically, it was hypothesized that microfluidic chambers, by mimicking plant phloem vessels to which LAS is restricted, could be used to facilitate LAS culture. However, liquid culture medium optimization was required prior to successful study of LAS in microfluidic chambers. Therefore, research was focused on systematically quantifying LAS viability over time in different culture media to identify the characteristics of a suitable culture medium for LAS. In the first year of the project, experiments were conducted to compare LAS viability characteristics among different media types over time via EMA-qPCR. In the second year of this project, all data was analyzed from the first year’s experiments. Additional experiments were conducted to analyze the bacterial community composition of the biofilm formed in the media treatments as well as the chemical characteristics of the media tested. The overall finding was that LAS culture media containing citrus juice from different sources promoted cell viability. A peer-reviewed publication of these results has been produced: ‘Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium’ (2013, Phytopathology). In this publication, we indicate many features of our experiments that will be informative in future design of a successful culture medium for LAS. This publication represents the first significant published contribution to finding a solution for the culture of LAS since the publications from Davis et al. (2008) and Sechler et al. (2009). Future work will focus on refining this liquid medium and testing potential new media formulations both in batch culture as well as in flow conditions in microfluidic chambers.
The main goal of this project is developing of a new method for rapid and efficient inoculation of plants with HLB based on a Pulse Micro Dose Injection System (PMDIS). We are conducting experiments in order to 1) identify what types of tissue within an infected citrus plant can serve as a good resource of the HLB bacteria for preparation of the inoculum by comparing extracts from stems, leaves and seed coats as inoculum sources; 2) examine whether HLB-infected psyllids can be utilized for preparation of the inoculum suspension; 3) optimize the composition of the extraction buffer used for preparation of the bacterial suspension and the extraction conditions, so they would support high efficiency of the PMDIS-mediated transmission of the pathogen; 4) optimize the parameters of injection. We are also evaluating how age of receptor plants, types of citrus varieties used as HLB bacterium donors as well as for plants being inoculated, types of flushes being inoculated affect efficiency of inoculation. Several sets of plants have been already injected using PMDIS. Those are being maintained in the greenhouse and monitored for the disease development. Some successful infections of citrus plants using PMDIS were achieved, however infection rates were less than those seen upon graft-inoculation of plants with HLB-containing tissue. Currently we are working on improvement of PMDIS-based inoculation procedure. We are trying to improve our project by using two strategies. First, we are building a collaboration with Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University. Dr. Gonzalez uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. We would like to learn more about their injection system and the injection protocol, so we can transfer the obtained knowledge into our project on the development of a system to inoculate citrus with HLB. Second, we have obtained a culture of Liberibacter crescens and are using this culture as a model system to develop and improve our injection protocol. The culture is being used for pulse injection-mediated inoculation of both citrus and papaya (the original host of L. crescens, here is used as a model host).
The objective of this project is to provide an alternative to traditional Cu fungicide/bactericide for protection against citrus canker. Extensive Cu use for plant protection has led to Cu accumulation in fertile soil. It is desirable to develop an alternative to Cu fungicide/bactericide for preventing citrus canker. In this project a new non-phytotoxic quaternary ammonium compounds based nanoformulations (Fixed-Quat) is being developed for canker protection. We have successfully synthesized a few batches of Fixed-Quat formulas using EPA approved quaternary ammonium compounds. Material characterization using HRTEM, SEM, DLS, FTIR etc. is in progress. Antimicrobial efficacy, phytotoxicity and film forming ability of Fixed-Quat formula is being evaluated on Hamlin orange, grapefruit citrus varieties as well as on vinca ornamental species. Future reports will include results from materials characterization results, antimicrobial studies, film-forming capacity and phytotoxicity.
The objective of this project is to improve the bioavailability of copper loaded silica nanogel (CuSiNG) material. Further studies of mixed-valence Cu within a CuSiNG system have been performed. Two different mixed-valence Cu systems have been created using different reducing agents to lower the oxidation state of Cu within the Copper silica system. Increases in Cu (I) content have been confirmed using XPS, HRTEM and UV-Vis with the Cu(I) specific chelator neocuproine. HRTEM revealed the presence of cupric oxide, cuprous oxide and metallic Cu crystallites within the mixed valence Cu system. Antimicrobial tests have shown an increase in efficacy over previous CuSiNG materials, along with Kocide 3000, Cu-sulfate and Cu(I)-chloride controls. The presence and action of hydroxyl radicals in the mechanism of action of CuSiNG was indirectly studied using the hydroxyl radical scavengers D-Mannitol and Dimethylthiourea. Phytotoxicity testing was carried out on Hamlin Orange and Vinca sp. Results indicated that Hamlin Orange was resistant to phytotoxicity at all levels tested while Vinca sp was susceptible to plant tissue damage at higher Cu level. No damage was observed at levels used for canker protection in field trials.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, while Lam and Las have both been found in Texas. In March 2013, twelve different isolates from plants identified as being naturally infected with Ca. Liberibacter species but which would test negative for Las, Lam, and Laf, were inoculated into receptor plants in a greenhouse at Ft. Pierce. We are currently testing these inoculated plants to verify the establishment of the Ca. Liberibacter species in the receptor plants. Once this has been verified, the ‘cross protection’ trials will be established. DNA extractions from the isolates has been used for macro array qPCR to identify genomic regions which differ from Las, and selected isolates are being prepared for further sequencing to verify their relationship with Las and Lam and other reported Ca. Liberibacter species.
Based on the PCR results, five classes were defined for the Valencia variety dataset. They included 20 healthy, 20 HLB symptomatic, 20 magnesium (Mg) deficient, 6 zinc (Zn) deficient, and 30 zinc deficient and HLB symptomatic. As explained in the previous report, 30 textural features were extracted from the images. A principal component analysis (PCA) was performed on all 30 features to have a visual characteristic of the samples. The samples were plotted in a two dimensional coordinate system of the first and second principal components (PCs) and a step by step classification model was designed based on this plot. Based on the model, all the samples were classified into two classes of Healthy-Mg deficient class and HLB-Zn deficient class in the first step. Then, the healthy and Mg deficient classes were individually recognized from the Healthy-Mg deficient class. Also, the HLB and Zn deficient classes were identified individually, and the HLB infection within the Zn deficient class was identified as the last step of the model. The best set of features and classifiers was identified for each step by comparing the results achieved using different sets and a 5-fold cross validation model was employed to confirm the results. A programming code for the classification model was prepared and all the dataset was divided randomly into two equal sets. Then the classification model was performed on the first half, while the second half was used as a training set. The classification model was repeated on the second half, and this time the first half was used as a training set. Based on the classification results, all the healthy and HLB symptomatic samples were classified correctly with zero error. Also most of the magnesium deficient samples were classified correctly with a 95% accuracy and only one magnesium deficient sample was misclassified in the HLB symptomatic class. However, the classification accuracies within the zinc deficient class were comparatively lower than other classes because either the zinc deficiency symptoms buried the HLB symptoms or their HLB statuses were questionable. A conference paper was prepared based on these results and submitted for the 2013 ASABE international meeting. The paper was selected from sixty submissions to receive the IET Select Paper Award. An imaging system for the on-the-go HLB detection method was assembled and tested successfully in the lab. A DGPS receiver was added to the system to acquire coordinates of where the images were taken. Software was developed using LabView programing to control the camera, take the images, and log the coordinates. The on-the-go imaging system was tested on July 2nd, 2013 in CREC grove after sunset (9:00 PM to 2:00 AM). A total of 486 images from both sides of 243 trees in two blocks (10A and 2C) were captured and their coordinates were logged. The HLB statuses of these blocks were updated in April, 2013 by a ground inspection crew in CREC who has monitored the grove continually. The imaging system was mounted at the height of 1.6 m on a gator utility vehicle. The vehicle was stopped in front of each tree, the illumination was turned on, tree images and coordinate were stored, and the illumination was turned off. Now the dataset is being analyzed to determine its capability to identify the HLB infection.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, March 2013. The lab has been in operation for more than 5 years, and as of March 2012, we have processed more than 40,000 grower samples. Additionally, more than 30,000 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers. Numbers specific to this report are 941 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, March 2013. The lab has been in operation for more than 5 years, and as of March 2012, we have processed more than 40,500 grower samples. Additionally, more than 32,000 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers. Numbers specific to this report are 605 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
To date, we have tested more than 824 culture medium formulations for their ability to support growth of Candidatus Liberibacter asiaticus. Several formulations have been developed which support initial growth of the bacterium. This is indicated by an increase in the numbers of bacterial cells observed in samples of inoculated medium by epifluorescent microscopy after staining the cells with the DNA-binding fluorochrome SYTO 13. The population of cells reaches a peak after 1-2 weeks. After addition of fresh medium, the population usually diminishes until they no longer can be detected. However, occasionally the population peaks again and then declines to undetectable levels. Our efforts are now aimed at identifying those conditions that will support continuous cultivation of the bacterium.
Nutrient application for maintaining the productivity of HLB-infected orchards is becoming a common practice in Florida, especially with increasing rate of infection. Although the foliar nutritional application program does not remove/reduce the inoculum, it offers the growers an opportunity to maintain productivity and profitability. One of the major challenges in nutrient application is deciding on the sufficient amount or proportion of nutrients to be applied in the field to maintain citrus yield. In this project, we proposed to evalute the potential of a new sensing technology based on laser-induced breakdown spectroscopy (LIBS) for rapid and real-time measurment of foliar nutrient quantity. During the first stage of the project, citrus anomalies with abiotic (iron, zinc, manganese, magnesium) and biotic (HLB, canker) stressed leaf samples were tested with LIBS system. Qualitative analysis using a statistical algorithm (bagged decision tree) indicated that classification of citrus anomalies can be achieved with an accuracy of about 94%. Based on these results, in the second phase of the project, we evaluated the LIBS system for quantification of leaf nutrients. Two sets of data, dataset 1 and dataset 2, were collected during different seasons (fall and spring). The partial least square regression (PLS-R) model with a training dataset was developed to establish a calibration model. Once developed, the calibration model was tested with an independent dataset. In dataset 1, the PLS-R model showed a good correlation between the LIBS spectra and nutrient concentrations during calibration, although the model could not be validated with an independent dataset. However, in dataset 2, amongst the nutrients that were estimated, iron, magnesium, potassium, boron, and calcium showed R2 greater than 0.70. Calcium showed a maximum R2 of 0.80. One of the major challenges during this work was the low variability of foliar nutrient concentrations that limited the robustness of the calibration model. Better results could have been acheived if the concentration of nutrients had had a wider range of variability.
Early detection is critical to delaying and limiting the spread of HLB into California. While commercial growers are inspecting regularly for HLB, approximately half of the citrus trees in California are located in plantings other than commercial groves; many in the yards of homeowners, where regular and comprehensive inspection presents challenges. If HLB infection gains a foothold in non-commercial plantings it could spread widely before being detected in commercial groves. The objective of this task is to complete a proof-of-concept demonstration of the use of airborne surveys with the Airborne Visible and Infrared Imaging Spectrometer (AVIRIS), a high-performance sensor developed by NASA, to improve early detection of huanglongbing (HLB) by mapping the locations of citrus trees in urban and suburban areas as an aid to extension of effective survey coverage to include non-commercial plantings. Since the sensor observes from an aircraft, this approach could regularly and comprehensively survey wide areas to map non-commercial citrus and detect HLB-infected trees. The following milestones have been completed as of the date of this report: 1. A Space Act Agreement for this experiment was drafted, approved, negotiated, and signed by the National Aeronautics and Space Administration (NASA) and the California Citrus Research Board. 2. A Task Plan was written by JPL and approved by NASA. 3. A Task Order was issued by NASA for the Jet Propulsion Laboratory to begin work on the task. 4. A Flight Request was approved by NASA for the use of the AVIRIS Classic instrument in support of this task. 5. AVIRIS flights in support of this task have been tentatively scheduled for late July 2013. 6. The JPL team and CRB representatives met to discuss flight planning, particularly to define the areas of interest for airborne data collection and relative priorities among these areas. 7. Analysis has begun on archival data collected by an instrument similar to AVIRIS over an area that included the main Citrus Variety Collection (CVC) at UC Riverside.
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