The results of the analysis that estimates the annual HLB infection rate that switching from the traditional HLB management program to an enhanced foliar nutrient program maximizes profits were presented at the 2nd International Research Conference on HLB in Orlando, Florida on January 12, 2011. Results of the grove replacement model were published in Citrus Industry Magazine in April of 2011. This model is in the process of being developed into a more user-friendly version that will enable growers and other users to determine when groves with declining fruit yields should be replanted. The Asian Citrus Psyllid (ACP) and HLB have spread rapidly throughout all citrus growing counties in Florida. At this time, control of ACP through intense use of pesticides remains the only viable means of managing HLB. Evolution of pesticide resistance in ACP has already been documented in Florida. Stakeholders are alarmed that the best defense against ACP and HLB may be eroding. At the recommendation of the National Academy of Sciences ‘Citrus Health Management Areas (CHEMA)’ were created in Florida. These contiguously-managed areas of groves improve the effectiveness of psyllid spray programs Florida. The costs of ACP management programs will be monitored in several CHEMAs each year of this five year project. The first two years will provide a benchmark of these costs for the standard management protocol, while the remaining three years will provide a comparison of the increased costs and hopefully reduced ACP populations and resulting reduction in tree mortality from the implementation of these programs. Costs will be based on recommended application rates, frequency of application, and material and application costs. Estimation of the returns associated with key rotation spray modules and binary mixtures will involve estimation of production not lost assuming ACP control reduces the likelihood of resistance development and spread of HLB in a particular block. We will utilize a block-level HLB Gompetz function spread model developed in Brazil (Gottwald et al. 2008) to depict the spread of HLB as a function of tree age at time of first infection, years elapsed since first infection, ACP control, and the level of external pressure of surrounding infections. Yield will be predicted as HLB progresses through a block. The value associated with varying levels of premature fruit drop and tree mortality will also be estimated. Costs and returns computed for key rotation spray modules and binary mixtures will be compared to those associated with conventional HLB cultural management and with the benchmark costs obtained in the first two years. Costs in CHEMAs are expected to be reduced from increased aerial pesticide applications and greater economies of scale in ground applications. Costs are expected to be increased from the use of more expensive pesticides in the pesticide resistance rotation program. Economic benefits are expected from lower tree loss and greater fruit production as a result of reduced ACP populations and infection rates.
When dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. The oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid. Comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and than that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
1-The first objective of the second year was to build and start the operation of a plant growth room at the Citrus Research and Education Center in Florida (CREC). The growth room construction started on October 22nd 2010 and the projected finish date was February 11th 2011. There was a delay of a few weeks and the main contractor will finalize on March 25th, but the computer system contractor is still finishing the programming that will control the environmental conditions. We checked the growth room and it is working as expected however disposal of the waste stream will be a concern when the growth room is in full operation since the water that we will dispose needs to be collected in an external tank and test by the county to guarantee that we are not disposing contaminants that can affect the environment. We already started furnishing and placing equipment inside the building. Because of the delay in the construction, the growth room is not in fully operation yet. We believe this will take at least one additional month. The cost of the construction was higher than the original budget plan; the extra funding was provided by CREC and IFAS facilities as agreed initially. 2- A full time technician with nursery experience was hired after several months of searching. The process of hiring was slow. A first candidate was hired and the offer of employment was rejected due to a low salary offer. A second candidate was found but he quit two months after hiring. We hired a third technician with limited experience and he will start the first week of April. It seems like salary will be an important issue in the future to recruit and retain personal. 3- Training of the manager Dr. Zapata was completed at the IVIA under the supervision of Dr. Pena. It was emphasized during the training the improvement of transformation methods for more recalcitrant types, molecular analysis of the regenerants and plant material preparation at the greenhouse/growth room, including micrografting, phytosanitary treatments, fertilization and pruning. An annual schedule for completion of planting, transplanting, grafting and obtaining budsticks to transform was developed. 4- The Mature Transformation Laboratory was established, using an existing laboratory located at CREC. Two technicians with limited experience were hired and are currently being trained in the first tissue culture techniques, including culture media preparation, grafting, micrografting, explant preparation, culture and regeneration, etc. 5-Our selected varieties Hamlin 1-4-1, Pineapple S-F-60-3 and Valencia S-SPB-1-14-19 were subjected to cleaning through shoot tip-grafting at the Florida Department of Agriculture and Consumer Services (DOACS) with the help of Dr. Peggy Sieburth. They are kept at out lab and ready to be grafted on rootstocks when the growth room is fully operative. 6-Dr. Leandro Pena and his greenhouse and growth room manager Josep Peris made a visit of one week in March 2011 to supervise the last steps of the growth room construction before been finalized and suggested minor details to make the facility more reliable and helpful for operators. They also short-trained the tissue culture technicians in horticultural practices. They checked substrate, seed stock and nutrition issues with the manager Dr. Zapata.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Here are the major achievements: 1. We are currently assessing citrus genes modulated by Las infection in 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing), 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway),3) Comparison of different citrus varieties that are different in tolerance and susceptibility (in progress). 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing The alteration of gene expressions by Las in leaf, stem and root tissues of Valencia sweet orange was investigated using Affymetrix microarray analysis. Out of 30,279 probe sets, a total of 8667, 2795 and 1142 showed significantly altered (p< 0.05) expression in leaves, stems and roots, respectively. Using 2 fold change as cut-off value, 1008, 580 and 58 transcripts were significantly up-regulated in leaf, stem and root tissues, respectively, whereas, 1109, 350 and 58 were correspondingly down-regulated in Las infected plants. Differences were observed for genes involved in cell wall synthesis and remodeling, lipid metabolism, photosynthesis, secondary metabolism, and starch and sucrose metabolism. Biotic stress induced signaling and transcription factors, PR-proteins, heat shock proteins, hormones and genes involved in protein modification, redox reactions and secondary metabolism were affected more in leaves and stems than in roots. PR genes were mainly repressed in roots but showed both patterns in leaves and stems; JA genes were up-regulated in stems, down-regulated in roots but up- and down-regulated in leaves; Calvin cycle genes were mainly altered in roots; SA and heat shock proteins were not significantly altered in roots. Transcriptional factors with WRKY, AP2/EREBP, MYB, bZIP, bHLH and Zinc finger domains were differentially regulated in all tissues, but least in roots. Differences were shown by C2C2(Zn) DOF zinc finger family proteins, affected in leaves and stems only; homologs of MEE47, nuclear factor PBF-2 and ATRR1 were regulated in roots only while MADS box transcription factor family and several unclassified transcriptional factors were down-regulated only in leaf tissues. We are further analyzing the data to understand how Las affects leaves, stems, and roots since they have distinct roles and function and how they contribute to the HLB disease development. 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway using multiple tools including MapMan. 3) Comparison of different citrus varieties that are different in tolerance and susceptibility. This work is in progress.
The goal of this proposal has been to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to better understand the mechanisms by which disease is spread in field. We carried out a series of experiments to evaluate if Ca. Las is transmitted from male to female psyllids during routine mating. Our preliminary investigations indicated that Ca. Las may be transmitted from male to female psyllids but not from females to males or among psyllids of the same sex. Pairs of Ca. Las infected male and healthy female, Ca. Las infected female and healthy male, Ca. Las infected female and healthy female, Ca. Las infected male and healthy male, Ca. Las infected female and Ca. Las infected male (positive control) healthy female and healthy male (negative control) adult psyllids were introduced separately in Petri dishes filled with agar medium. The insects were allowed to mate for 72 hrs. After 72 hrs, the insects were transferred to Ca. Las resistant Murraya koenigii plants for 12-14 days for multiplication of bacteria in recipient psyllids. DNA was prepared from each of the female and male psyllids separately and analyzed for Ca. Las presence utilizing a real time PCR assays. Our subsequent investigations confirmed that Ca. Las is sexually transmitted from Ca. Las-infected male psyllids to healthy females but not from infected females to healthy males or among psyllids of the same sex. Ca. Las was transmitted from Ca. Las-infected male psyllids to roughly 3% of healthy females. Ca. Las was not detected in the recipient sex immediately after mating but required a minimum incubation period of 2 weeks in psyllid bodies for PCR detection. These results also suggested multiplication of bacteria within psyllid bodies. No Ca. Las was detected in recipient psyllids when the recipient psyllids were maintained on HLB-resistant (Murraya koenigii) plants for longer than 4 weeks suggesting that psyllids may lose infectivity if they continuously live on HLB-resistant plants. We were able to detect Ca. Las bacteria in ACP ovaries of recipient females with PCR. However, we were unable to detect the presence of bacteria in genital parts of male and female psyllids with scanning and transmission electron microscopy perhaps due to washing of bacteria during sample preparation procedures. Ca. Las was also not detected in psyllid salivary glands using electron microscopy. More precise and accurate procedures such as in situ hybridization may be required to detect the presence of bacteria in psyllids. Also, we were able to detect Ca. Las in eggs of recipient females with PCR but not with electron microscopy. PCR detection of Ca. Las in psyllid ovaries suggested transovarial transmission of bacteria. Transovarial transmission was also confirmed in F2 generations of Ca. Las-recipient females which were produced on M. koenigii plants. We continue to evaluate if the Ca. Las-recipient females are capable of infecting new citrus plants. The experimental procedures for this have been completed; however, in some cases we are still awaiting to collect the results because a minimum 10 week period is required for detection of HLB in newly infected plants. In some cases this period may be longer; therefore, we have asked for a short extension of this 1-year project. These results show a new mechanism for C. Las transmission in addition to the known mechanisms (through plant feeding, and transovarial transmission). Although the level of this type of transmission is low; it is present. These results further underscore the importance of effective psyllid control for HLB management.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac; (3) Inoculation of the transgenic plants with the HLB pathogen, and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. We have identified three transgenic lines overexpressing CtNPR1. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants. We also conducted growth curve analyses. Nineteen days after inoculation, the bacterial population in one of the NPR1 overexpression lines is 10,000 fold lower than that in the control plants. These results demonstrate that overexpression of CtNPR1 confers resistance to canker disease. We also graft-inoculated the NPR1 overexpression lines with greening to determine whether NPR1 is functional in greening resistance. We are in the process of monitoring Candidatus Liberibacter asiaticus populations in the inoculated plants using quantitative PCR. Five transgenic lines containing the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene, have been generated. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. In a few weeks when the plants produce enough leaf tissue, we will perform Northern blot analyses to monitor the levels of CtNPR1 transcripts. The transgenic plants with high levels of CtNPR1 will be propagated by grafting and inoculated with greening. Finally, we have initiated microarray analyses of the CtNPR1 plants in response to Xac inoculations. The results will be reported soon. To continue our research, we request funds for the third year to achieve the following goals as originally proposed: (1) Characterization of the CtNPR1 transgenic plants inoculated with the HLB pathogen; (2) Molecular characterization of the SUC2::CtNPR1 plants; (3) inoculation of the SUC2::CtNPR1 plants with the HLB pathogen; (4) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac; (5) Plant maintenance.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. Based on these observations a collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus tristeza virus (CTV) expression vector to produce TMOF and CPI in the in the citrus phloem. Clones containing the sequences of mosquito TMOF and CPI (cathepsin protease inhibitor) (both shown to be effective against psyllids) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us with the plants to evaluate the effect on psyllids while feeding on these plants. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Using this information, we have initiated experiments to produce citrus plants that express these dsRNAs in citrus phloem cells. A collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus Tristeza Virus (CTV) expression vector to produce the psyllid dsRNAs in the citrus phloem. Clones containing the sequences of a psyllid Vacuolar ATPase and Cathepsin protease (both shown to be effective dsRNA targets) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us the plants to evaluate the effect on psyllids while feeding on these plants. We have also designed expression vectors for the production of transgenic plants expressing the psyllid dsRNAs through Agrobacterium-mediated plant transformation. These will be used to initiate the production of transgenic citrus plants that constitutively produce these dsRNAs in the phloem.
Cross protection has been used as a control strategy against virulent stem pitting strains of Citrus tristeza virus. Although it has been shown that CTV cross protection only works against CTV isolates of the same genotype, its underlying mechanism remains unknown. Cross protection can break down over time, typically as a consequence of challenge by a new virulent strain of CTV. This study of cross protection was undertaken with a model system where a parental CTV isolate called Dekopon was a mixture of three genetically different CTV strains. The Dekopon isolate protected sour orange seedlings from expression of seedling yellows (SY) symptoms which is significant because SY is associated with virulence of CTV. The parental Dekopon mixture was separated by aphid transmission and the resultant sub-isolates induced different symptoms, as expected based on individual genotypes; namely, the VT- and T3- genotypes induced strong SY in seedlings of sour orange and Duncan grapefruit; whereas the NS genotype was very mild. Various combinations of these sub-isolates failed to show cross protection or at least at the level expressed by the parental strain. Thus, the Dekopon isolate contained the essential component(s) of symptom amelioration. Deep sequencing using the Illumina platform was undertaken to analyze small interfering (si) RNAs profiles in sour orange severely affected by SY and in cross protected sour orange. An accumulation of virus-derived siRNAs mapping to the 3′-end of the CTV genome was found to be common in all three strains. Contigs from these datasets identified sequences homologous to the 282 Kb Ctv resistance locus of Poncirus trifoliata, thus, providing evidence that this region is involved in siRNA pathways. A total of 50 micro (mi) RNA (regulators of gene expression) families had significant frequency of which 8 miRNA families showed differential expression after CTV infection. Analysis of putative mRNA (chemical blueprint resulting in a protein product) targets in the EST database revealed 433 were common to sour orange plants showing protection or strong SY symptoms. Other mRNA targets were specific to cross protected plants (873) or sour orange showing severe SY (753). The different mRNA targets identified suggested that different CTV strains induced different miRNA-mRNA interactions. The experimental procedures undertaken in this research were designed to explore the siRNA pathway involved in the Citrus-CTV interactions and to identify the siRNAs and miRNAs associated with host response. The understanding gained should provide a new insights on mechanisms of CTV disease expression and cross protection.
During October ‘ December 2010 the citrus agents collectively provided 22 educational events and 61 grove visits. These activities were designed to enhance knowledge and practice adoption to combat HLB in all commercial citrus producing areas of Florida. In an effort to meet the recommendation of the National Academy of Science to develop citrus health management areas (CHMAs), the agents along with state faculty have been actively working with citrus growers to aid in the formation of the recommended pest management areas. Throughout the state, approximately 32 CHMAs are actively functioning or in various stages of development. CHMAs are being implemented in all citrus producing areas and agents are continuing to schedule meetings and other educational events to promote to pest management areas. In October of 2010, the citrus agents conducted their annual fall grower meeting series. The program was titled ‘Citrus Management Strategies in a New Disease Era’. The program series was conducted in 6 locations (Arcadia, Bartow, Ft. Pierce, Immokalee, Sebring, and Tavares) and attended by 356 growers. Program presentations were conducted and presented by the citrus agents. Presented topics included: citrus black spot; psyllid management; growing young citrus trees in the greening era; and foliar feeding and SAR for citrus trees. These presentations are currently available on the citrus agent’s web site (www.citrusagents.ifas.ufl.edu) and can be viewed upon demand. The citrus agents individually conducted or participated in 17 additional programs where HLB or topics to minimize the impact the disease has on citrus at various locations around Florida. Attendance at these programs exceeded 350 growers. The agents are documenting conditions of citrus trees that are PCR positive with HLB a photo series. This web based series is updated monthly with photos from the same selected trees to document tree conditions over time. All citrus production areas are being represented in the series. These photos can be viewed from the citrus agents web page.
Early signs of boron deficiency symptoms have begun to develop on the plants subjected to low boron fertilization. HLB infected budwood for graft inoculation is being tested for Las titers and graft inoculation will be performed as soon as the budwood status has been confirmed. Las titer and microscopy samples will be taken monthly after inoculation. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
This project aims to provide comprehensive knowledge of the genetic and biological diversity of CTV isolates in CA and to identify and put in place mild isolates of the virus that can provide sustained protection against severe SP isolates. As the first step we are conducting molecular characterization of CTV isolates (Folimonova lab) collected from various regions in California. This approach will provide important information regarding genetic diversity of CTV populations in this region required for further selection of mild protective isolates. To this date, 53 samples received from Vidalakis lab in 2010 (as nucleic acid extracts from isolates of CCCP collection) were assayed using conventional reverse transcription PCR with CTV genotype-specific primers designed to specifically amplify sequences of the T30 or VT strains. Majority of those isolates demonstrated presence of the VT genotype, while some contained the T30 strain. Thirteen isolates appeared to contain mixed populations of both genotypes. Further analysis of those samples with primers specific to other CTV genotypes (T68, T3, T36) is in progress. The results of molecular characterization will be then evaluated along with information obtained from biological indexing of those isolates on indicator hosts in order to select mild non-stem pitting isolates as potential protective candidates against severe stem pitting isolates which share the same genotype composition. In addition to conventional reverse transcription PCR technique, we are developing a CTV genotype-specific quantitative reverse transcription PCR method that will allow us not only to determine what are the components of each isolate but also to quantify the amount of each genotype component present in isolates that contain several different isolates. Also this technique will speed up our work by allowing us to process more samples in a less amount of time. Recently we received 45 additional samples from CCTEA collection. These samples are being prepared for further characterization using the approaches described above. In order to obtain an understanding what CTV genotypes are currently present in the southern part of California where no previous eradication occurred, we are planning to conduct additional sampling of the new material from this region (Vidalakis lab). Those samples will be then used for inoculation of greenhouse trees for bioindexing characterization (Vidalakis lab) as well as sent (as nucleic acid extracts) to Folimonova lab for further molecular characterization.
The long-term goal of this project is to identify early diagnosis markers and to understand the mechanisms of plant defense responses against bacterium Candidatus Liberibacter (Ca. L.), which causes one of the most devastating citrus diseases – Citrus greening or ‘Huanglongbing’ (HLB). By examining the responses of 30 different genotypes of citrus to Florida isolates of Ca. L. asiaticus under controlled conditions in the green house, tolerant genotypes as well as sensitive genotypes of citrus have been identified (Folimonova et al., 2009). This project aims to identify important small RNAs and mRNAs that encoding signaling genes that contribute to HLB defense responses, by profiling small RNAs and mRNAs from HLB-infected tolerant and sensitive genotypes. We expect that different set of small RNAs and mRNAs will be identified from the tolerant and sensitive genotypes. HLB-induced small RNAs may target negative regulators of plant defense and HLB-suppressed small RNAs should target positive regulators of defense pathways in tolerant genotypes. mRNA profiling will help identify and validate these targets. Similarly, HLB-regulated small RNAs and mRNAs may help identify important components in pathogenicity of the greening. We chose Nules clementine as a sensitive variety and Sun Chu Cha mandarin as a tolerant one for deep sequencing analysis because both of them are mandarins and the genome sequence of a mandarin was just released which will ensure the success of our project. Currently, Dr. Folimonova’s lab is helping preparing and collecting the plant material. We will construct and sequence small RNA libraries from grafting- and Psyllid-infected Nules clementine and Sun Chu Cha mandarin use Illumina high throughput deep sequencer. Currently, we are in the processing of preparing and collecting the material to validate our small RNA results from previous studies using HLB plants that were infected by psyllid transmission. Our previous studies used only the grafting infected HLB plants from Dr. Kim Bowman’s lab at USDA lab Florida. Madam Vinous sweet orange plants (represent sensitive group) and Citrus macrophylla plants (tolerant group) that were already infected with HLB via psyllid transmission, they are at early stages of infection (about 3 months ago they we put in the psyllid room and still remain there). In addition, bacteria-regulated small RNAs often target important regulatory components in plant defense pathways. In comparison with mRNA profiling data, we will identify a series of plant regulatory genes that may contribute to HLB-tolerance and resistance responses. We propose to use high-throughput deep-sequencing approach to profile the mRNA populations of these two distinct tolerant and sensitive varieties. The newly released genome sequence of a mandarin will be essential for the data analysis. This study not only leads us to the discovery of HLB-induced small RNAs for early diagnosis marker development, but also helps us understand the pathogenicity of citrus greening, and helps us identify important components in natural defense of HLB disease.
The purpose of this California Citrus Research Board-supported project is to develop and implement methods to extend the shipping and shelf-life of fresh citrus fruits. The postharvest life of citrus fruit is often ended by fungal decay pathogens; in arid production regions of the California and Arizona, the preponderance of losses are caused by green mold and blue mold, caused by Penicillium digitatum and P. italicum, respectively. Phosphites (also termed phosphonates) are salts of phosphorous acid (H3PO3), nutritionally ineffective forms of phosphorus with an oxidation state of +5, as opposed to phosphorus compounds of oxidation state +3 that are used in fertilizers and utilized by plants. Known to control diseases caused by Oomycetes, such as brown rot of citrus, they inhibit other fungi but their activity can be modest (Guest and Grant, 1991). They merit thorough evaluation because of their relatively benign environmental aspects. Most formulations are neutral in pH and, in soil, they oxidize to phosphate. This facilitates disposal of used solutions from packinghouse tanks and drenchers, compared to solutions that contain fungicides, sodium salts, or are alkaline. Phosphites are exempt from residue tolerances for many applications. In postharvest applications to fruit inoculated the prior day, brief immersion (15-60 sec) in 1 to 3% phosphite solutions controlled both green and blue molds significantly, and were particularly effective on mandarin cultivars where another fungicide alternative treatment, sodium bicarbonate, often performs poorly. For example, a 1 min immersion at 25oC in 2% calcium or sodium phosphite reduced green mold from 63.0% among untreated ‘Murrcott’ mandarins to 12.2 and 14.5%, respectively, while 30.5% decayed after 2% sodium bicarbonate treatment. Similar treatments at 50oC with calcium or sodium phosphite further reduced green mold to 6.5 and 6.9%, respectively, while 14.3% decayed after sodium bicarbonate treatment. In most tests, heating the phosphite solution consistently improved its performance. Mixing phosphites with imazalil, thiabendazole, fludioxonil/azoxystrobin, or pyrimethanil improved the effectiveness of these fungicides. Synergy with imazalil occurred with solutions at neutral pH and not at low pH. Applications of 1 to 3% (wt/vol) solutions of calcium or potassium phosphite to run-off one week before harvest to ‘Atwood’ navel oranges reduced postharvest decay from natural or artificial infections by approximately 50%, although these very high phosphite rates, greatly above those recommended when phosphites are applied to trees for other reasons, caused some defoliation. At lower rates (less than 1%), preharvest applications of phosphite did not control postharvest Penicillium decay. Tests to control preharvest brown rot, caused by Phytopthora spp., and in progress. Brown rot is usually minor in California in most seasons, but severe outbreaks are occurring this year because of the prolonged wet conditions in groves caused by exceptionally abundant rainfall. Phosphites were effective as a postharvest treatment, but their preharvest use for postharvest decay control was not promising. Amiri and Bompeix (2011) reported similar promising results with postharvest phosphite use on pomes. Postharvest registration of potassium phosphite was granted in 2010 in the USA. Amiri, A.; Bompeix, G., 2011. Control of Penicillium expansum with potassium phosphite and heat treatment. Crop Protect. 30:222-227. Guest, D.I.; Grant, B. 1991. The complex action of phosphonates as antifungal agents. Biol. Rev. 66: 159-187.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. A journal manuscript was submitted on research showing that use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime; experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publications supported by this grant:1. Dutt, M., D.H. Lee and J.W. Grosser. 2010. Bifunctional selection-reporter systems for genetic transformation of citrus: mannose and kanamycin based systems. In Vitro Cellular & Developmental Biology-Plant 46:467-476; 2.Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266 Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and a commercial harvesting company (w/ Frank Rogers), a plan to collect meaningful yield and fruit quality from the St. Helena project was developed – with harvest expected later this month. Approximately 10 acres of trees planted 2.8 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The majority of trees have a significant yield and the trial is showing significant rootstock affects on precocious bearing and early fruit quality – the best selections from this trial will be ideal candidates for testing with juvenile transgenics. Also of interest is the cultural program being used at the St. Helena project that mimics OHS principals but with reduced input. The trees have been grown with a UF research slow-release fertilizer mix (in cooperation with Harrell’s Fertilizer) and daily irrigation. Two trees were confirmed with HLB the first year; but even with bad neighbors, there has been no detected additional spread of HLB during the past year. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL sweet oranges (a group of clones with Rhode Red quality that show high solids in young trees) were regenerated and successfully micrografted for further study of early flowering and transgene expression. Approximately 25 transgenic sweet orange trees from OLL selections were produced containing four different gene constructs. Progress was also made transforming OLL clones with the alternative embryogenic culture transformation system, as numerous transformed somatic embryos have been recovered. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
Objective 1 (confirm biofilm formation by X. citri subsp. citri (Xcc) in comparison with other bacteria that are well known to form biofilms). When grapefruit, Citrus macrophylla, Swingle citrumelo, and Carrizo were spray inoculated and kept under greenhouse conditions, aggregate formation was most prevalent on Swingle and Carrizo, reduced on C. macrophylla, and greatly reduced on grapefruit. Conversely, lesions were most prevalent on grapefruit, moderate on C. macrophylla, and infrequent on Swingle and Carrizo, although all four hosts developed lesions after injection-infiltration. This suggests a host interaction between biofilm formation and Xcc infection. Expression of genes, gumD, cheZ and rpf, involved directly or indirectly in biofilm formation is under evaluation. Detection of a gumD RNA fragment previously demonstrated to be a viability marker was higher in aggregated than in planktonic bacteria which is correlated with higher survival after 3 or 7 days in aggregates compared to suspension bacteria. Expression of cheZ involved in signal transduction for cell motility was initally higher at the onset of aggregate formation (3 dpi) but decreased once the biofilm was formed. This implies a role for bacterial motility at initial stage of biofilm formation. Expression of rfp involved in quorum sensing is lower in planktonic than in aggregating cells and in biofilm after 7days. This preliminary result confirms the importance of quorum sensing in the aggregation of Xcc bacteria which may serve as a target for disruption of the Xcc infection process. Objective 2. New disinfectants were evaluated for their ability to inhibit biofilm formation or disrupt bacterial aggregates. Zixvirox and Aquazix are used as surface disinfectants for food and have reported activity against biofilms. Aquazix is a mixture of hydrogen peroxide and silver as active ingredients. Zixvirox is a mixture of hydrogen peroxide and peracetic acid. Different concentrations of these products were tested against planktonic Xcc and as inhibitors of biofilm formation. To assess the effect on planktonic bacteria, culturability on LB medium plates and viability by membrane integrity (live-dead kit) assay were measured. Zixvirox at 0.03% and Aquazix at 2% were 100% effective for killing bacteria in suspension and complete disruption of cell integrity was confirmed by live dead assay. To evaluate the effect of these products on biofilm, aggregation was observed on borosilicate plates using a non stable Gfp strain and confocal laser scanning microscopy. At sublethal concentrations, Zixvirox was not completely effective for inhibition of biofilm formation because some bacterial aggregates formed. Aquafix at sublethal concentration prevented aggregation whereas bacteria were still viable in culture suspension. Although Aquazix and Zixvirox show promise as disinfectants, further assays are needed to confirm their activity as biofilm inhibitors. The effect of peroxide, CuSO4, SOPP, NaCl and NaClO at different concentrations was evaluated after 30 minute exposure of preformed biofilms on slides. Efficacy for disruption of bacterial aggregates varied with the compound. Treatments with SOPP, NaCl and NaClO disrupted the biofilm. CuSO4 or peroxide did not disrupt and may have stimulated biofilm formation based on the observed higher aggregation. Currently, assays are underway to evaluate the viability of bacteria in aggregates after bactericide treatment of the preformed biofilms. These assays are based on respiratory activity of the aggregate and fluorescence detection of non stable Gfp expressing strains.
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