Citrus blight continues to be a major economic problem in citrus groves in Florida. Thousands of trees each year succumb to citrus blight, with estimated losses at over $60 million per year. The disease can occur on all common citrus cultivars, and Carrizo citrange are especially susceptible. Early symptoms are zinc deficiency in the leaves which may disappear, zinc accumulation in the phloem and eventually high zinc levels in the xylem. Blockage of xylem tissues with amorphous plugs follows with reduced water uptake. The causal agent of citrus blight is unknown. However, symptoms and all of the characteristics associated with citrus blight can be reproduced by root graft inoculations. Therefore in a project previously funded by CRDF we used NGS RNA sequencing protocols to look for novel viruses in roots of sweet orange with blight, but not present in roots of healthy trees, or trees affected by HLB. We identified several related endogenous pararetroviruses related to Petunia Vein Clearing Virus (PVCV) using a collection of 10 RNA libraries prepared from 10 different root samples collected from healthy trees or those with blight or HLB. In the quarter just ending we have expanded the correlation studies for the blight related pararetrovirus. At the end of the last quarter leaves and roots were collected from over 50 trees from five geographically distinct locations. The majority of these trees were identified as being blight affected by water uptake testing, but putatively healthy trees were also sampled. In some cases, bark tissue from trunks was also collected for testing. RNA extractions were completed for all samples from all trees, and the presence of active pararetrovirus was assessed using the two primer sets selected in the optimization study from the previous quarter. Every tree that showed diminished water take up using the syringe injection test was positive for citrus blight associated pararetrovirus DNA. When the RNA extractions were treated with DNAse to eliminate potential genomic DNA sources of citrus blight associated pararetrovirus, all but 1 tree tested positive for pararetroviral RNA. However, no reverse transcription negative controls suggest that some samples still had low levels of genomic DNA, and these results need to be confirmed. Still, in initial analysis, there is a very strong correlation between the reduced water uptake via the syringe test and the presence of active citrus blight associated pararetrovirus. In addition, progress is being made towards generation of a complete genome sequence for the blight associated pararetrovirus. A primer walking strategy is being implemented to extend known sequences in both directions to generate a full genome. Sequencing efforts are being complicated by the need for complete removal of genomic DNA from the template RNA. In addition, the screening of multiple trees has indicated that there may be more than one active citrus blight associated pararetrovirus. The active viruses are more closely related to each other than they are to the non-active endogenous pararetroviruses.
This project provides the infrastructure to support six CRB-funded projects that investigate multiple methods of detection of Candidatus Liberibacter asiaticus (CLas), the causal agent of huanglongbing (HLB), in asymptomatic plants or in psyllids. Early detection of CLas in asymptomatic plants uses a systems approach that includes transcriptomics, proteomics, and metabolomics. Detection of CLas using digital drop PCR uses preserved psyllids and CLas-infected plant material from colonies maintained in the Contained Research Facility. Colonies of both CLas-negative and CLas-positive Asian citrus psyllids and a culture of CLas (Hacienda Heights strain) are maintained for use in the above studies. The first set of studies on the early detection of CLas in asymptomatic plants using the systems approach was conducted using Washington navels, Lisbon lemons and Tango mandarins on Carrizo rootstock. The plants were assigned to one of the following treatments: Control-control (no manipulation); graft-control (grafted with clean scion with 3 grafts per plant); and HLB-graft (grafted with scion that tested positive by qPCR for CLas and grafted with 3 grafts per plant). The plants used to get CLas-infected plant material had the following Ct values: Washington navel ‘ 30.17; Lisbon lemon ‘ 24.00; and Tango mandarin ‘ 27.39. The clean scion used for the graft-control all had Ct values of 40. The plants were grafted on the following days ‘ February 25, 2014 ‘ navel graft-control and mandarin graft-control; February 26, 2014 ‘ lemon graft-control, and navel ‘ HLB-graft; and February 27, 2104 ‘ lemon HLB-graft, and mandarin HLB-graft. All plants were housed in the same greenhouse under the same environmental conditions (80’F, 16L: 8D). The plants were sampled regularly by each group, and samples for qPCR analysis were taken at approximately monthly intervals. The qPCR samples were taken such that they did not interfere with sampling by any of the research groups. The plant tissue was held in a -80 freezer until it was sent to the Citrus Research Board Laboratory for qPCR testing using USDA-APHIS standard methods. The number of plants becoming CLas-positive and the time to the first CLas-positive reading by qPCR varied with cultivar. Of the 22 lemons graft-inoculated with CLas, 10 plants became CLas positive after 319 days. For the navels, 15 plants out of 19 inoculated plants became positive after 320 days. Of the 16 mandarins graft inoculated with CLas, only 2 had at least one positive Ct value, and 2 had suspect Ct values. However, the sample immediately following the positive or suspect value gave a negative value. A small number of these plants are still being held and sampled in an attempt to get consistent positive Ct values. The time to the first Ct value that indicated the plant was CLas positive was the shortest for the lemons, followed by the navels, and then the mandarins. The mean days to the first positive Ct value for lemons was 121.1 days (‘17.424); for the navels, 160.93 days (‘ 14.36); and for the mandarins, 267 days (‘ 10.99). A second set of experimental plants has been generated and include 25 Washington navels, 42 Lisbon lemons, and 31 Tango mandarins, all on Carrizo rootstock. These plants will be subjected to one of the following treatments: control-control (no manipulation); CLas-negative psyllid feeding; and CLas-positive psyllid feeding. The psyllids will be caged on the plants and allowed to feed for 10 days, after which the psyllids will be removed. All plants will be treated with insecticides to insure removal of the psyllids. The insect inoculation is expected to occur in April 2015. A third set of experimental plants have been started, and there are 236 Carrizo plants waiting to be grafted with the appropriate scion in September 2015.
Dec 2014 The objectives of this project are to optimize Guignardia citricarpa ascospore extraction procedures and qPCR with automated extraction system, determine if prototype passive ascospore traps will capture a sufficient number of Guignardia citricarpa ascospores to be an effective monitoring tool and monitor for G. citricarpa ascospores in six locations around state. We have continued to deploy the slides in the traps but there are very few spores on the Burkhard and new traps to compare counts. Through out the fall there were nearly no spores with less than 10 per slide. There has been little reason to see if qPCR would work because the limit of detection would likely not be met.
For the time period of October 16, 2014 through January 15, 2015, the Southern Gardens Diagnostic Laboratory has run 6301 samples for a total of 18,410 samples for the funding period. For the period of October 16, 2014 through January 15, 2015, 2466 were grower samples, 188 were psyllid samples and the remaining 3647 samples were research samples from various sources. Cumulative over the funding cycle, of the 18,410 samples, 7500 were sent in as grower samples, 1024 were psyllid samples and the remaining 9886 were research samples from various sources. As has been mentioned previously, virtually all of the samples received by the laboratory are “research” samples of some sort. Very few samples are now diagnostic samples to determine if greening is present or not in a grove. The research samples come from various sources including University, federal, corporate and private suppliers/researchers as well from grove owners who are conducting their own trials.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We have propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild stain status was maintained after three consecutive propagation. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. We have developed a digital PCR (dPCR) system for early detection of HLB and tracking of lysogenic and lytic activities of the Las prophage/phage. We show that as few as 1 to 2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. The copy number measurement of the targeted DNA molecules can be statistically differentiated from the healthy sample and negative water controls. We are optimizing dPCR-based assay for differentiation of Las populations that carry different prophages/phages. New primers were designed to amplify the Las phage region iFP3. These new primers contained a thiophosphate modification between the last two nucleotides at the 3′ end to increase their stability during rolling circle amplification (RCA). Samples from a Las positive citrus plant, a Las positive periwinkle plant, and a Las negative control plant were used for RCA. After several attempts at amplification via commercially available RCA kits, a protocol that amplified the DNA approximately 7 fold was developed. The specificity of the product obtained with the designed primers is currently being tested. In addition, Las-infected plants were subjected to heat stress using the same parameters as those currently used in thermotherapy. Sampling at various durations of time while the plant is enduring this stress will allow for the effects of heat stress on phage-encoded genes to be investigated.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, December The lab has been in operation for more than 7 years, and as of December 2014, we have processed more than 2,800 grower samples. Additionally, more than 34,000 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers for more than 72,500 samples processed. Grower samples are typically processed and reports returned within a two to four week time period. Numbers specific to this report are 1950 samples received from growers. This number represents an increase from the previous two years. The increased number is likely due to the increased efforts to mitigate the HLB-associated tree stresses. Grower in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
The search for possible treatments of the citrus greening bacterium Liberibacter asiaticus continues with the antimicrobial screening of the closest culturable relative Liberibacter crescens. Since the last report, 317 additional compounds have been tested, bringing the total tested to date to 769. This quarter resulted with the following compounds being tested: 5 tetracycline derivatives, 15 essential oil microemulsion formulations, 13 alkyl polyamides, 26 essential oil extracts, 5 chemical formulations from Bayer Crop Science, and 253 microbial fermentation products. The essential oil extracts and emulsions were highly effective with almost all samples showing ‘90% inhibition. About half of the tetracyclines, alkyl polyamides, and Bayer compounds were highly effective. Only 86 out of the 253 fermentation products showed ‘90% inhibition. Work also continues with group from the UF Particle Engineering and Research Center. GC-MS headspace analysis was done on citrus and the oils to determine if it may be possible to detect the sprayed oil in the phloem of treated plants. Most of the method development was completed, but the experimental trial has yet to be conducted.
In the Triplett lab work on defining BM7, the growth medium used to cultivate Liberibacter crescens, continues. Another approach to eliminating unessential components of the media is underway. Since L. crescens is highly similar to L. asiaticus (75.5%), we assume that knowing the exact components will eventually contribute to the development of a medium formulation to sustain L. asiaticus growth. In order to achieve this, our approach seeks to break down the main constituents of the BM7 medium to determine which are essential for L. crescens growth. Keeping the basic elements such as buffering conditions and pH, which are already defined, we are simulating BM7 original conditions, but eliminating some of its components, including, mainly, those that are undefined. If L. crescens is able to grow without a specific ingredient, that component will be deemed inessential. Any essential, undefined component will be broken down further into its exact components, if possible, to attempt to understand which portion is truly indispensable. Once defined, these components will be added to the already formulated components established just before. The process will be repeated until we develop a defined medium that supports the growth of L. crescens. BM7 main ingredients include ACES buffer; TMN-FH, which is a Grace’s Insect Medium enriched with yeastolate and lactalbumin hydrolysate; and Fetal Bovine Serum (FBS), the last three being complex undefined nutrients. By now, we are working in evaluating the growth of L. crescens on the BM7 control, as well as removing the TMN-FH, or FBS or both from the BM7, in order to determine which of the components are the most essential for L. crescens growth. We are also breaking down these main components by replacing the TMN-FH component in the BM7 medium by the Grace’s Defined Insect medium with and without enrichment. We believe that breaking down the components of the BM7 one by one will lead us to define a medium for L. crescens. Using transcriptome and proteome data, work continues on uncovering what genes may be essential for growth of L. crescens in culture. This may give clues to why the uncultured Liberibacters remain elusive. We may also be able to use these genes as targets for antimicrobial applications. In the Davis lab, work continues as stated in the previous report. Medium BM752 was selected to test the effects of additional ingredients on the growth of Candidatus Liberibacter asiaticus. Addition of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, uridine monophosphate, CoA, adenine triphosphate, and riboflavin did not significantly enhance growth. Further supplementation with fresh yeast extract had no effect. Riboflavin and zinc sulfate seemed to have a positive effect on growth and further tests are underway. In the Hilf lab, ARS and Galaxy Diagnostics have negotiated a Material Transfer Agreement that will allow ARS to test their proprietary media as a possible growth medium for Liberibacter. Galaxy provides diagnostic capability for the detection of potential Bartonella spp. infections in humans and animals using their proprietary media. Bartonella is phylogenetically and taxonomically related to Liberibacter and historically species of Bartonella were difficult to culture or could not be cultured. This is an issue Galaxy appears to have solved to a certain extent. Galaxy approached ARS in fall 2014 to partner in testing their media formulations to see if any would support in vitro culture of Liberibacter and the two entities have arrived finally to a mutually acceptable agreement which allows this to occur. ARS should receive the first shipment
This research project aims to develop an alternative to Cu biocides using Quaternary Ammonium compound (Quat) as active ingredient. Despite superior antimicrobial properties, direct spray application of Quat for managing citrus canker is not possible due to severe phytotoxicity of Quat. In this project we have developed ‘Fixed-Quat’ nanoparticle/nanogel material which is completely non-phytotoxic but maintains its outstanding antimicrobial properties. In this reporting period, we have successfully synthesized Fixed-Quat Nanoparticles (Fixed-Quat A NPs and Fixed-Quat B NPs) using a modified sol-gel process. This time we used EPA approved Quat compounds for the synthesis. The resulting Fixed-Quat NPs exhibited exceptional colloidal stability with a shelf-life of at least nine months. With further optimization of synthesis protocol, formulation shelf-life can be extended up to two years. In order to confirm the safety of Fixed-Quat nanoparticles, phytotoxicity assays were carried out in a controlled environment using a Panasonic Environmental Test Chamber (Model MLR- 352H). This test chamber allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate natural summer weather conditions. Studies were conducted on Vinca sp, an ornamental plant and Tomato sp. as model plant systems. In a typical procedure, plants were spray-treated with Fixed-Quat materials and were allowed to dry-up. Treated plants were then transferred to the environmental chamber and monitored for 96 hrs. For both Vinca sp and Tomato sp, it was observed as received Quat compounds (Quat A and Quat B) caused phytotoxicity at 500 ppm. As expected, Fixed-Quat A and B NP formulations did not cause any damage when tested in a wide range up to 900 ppm. Previously we reported that Fixed-Quat NPs, A and B exhibited superior antimicrobial efficacy against Xanthamonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate; MIC as low as 1.1 ppm). Detailed understanding of structure-property relationship of Fixed-Quat is important which we will study in coming months. In future reports, we will discuss additional materials characterization results.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. The scFv have been modified by the addition of a four amino acid leader sequence (KDEL) and both Sma I and Spe I cloning sites. The KDEL sequence is expected to stabilize the concentration of scFv in phloem cells by facilitating proper folding of the protein in the microtubules and thereby protecting the ScFv from proteolytic digestion. Eleven scFv inserts have been sequenced to be sure that the expected sequences are correct, and five ScFv sequences have been successfully cloned into the recombinant vector pUSHRL-26 for transformation of citrus rootstocks. These inserts include three different scFv that bind to the protein InvA and two that bind to the protein TolC. The protein InvA is produced by CaLas and secreted into the host to prevent the infected host cells from entering into apoptosis, and the protein TolC targeted by the scFv, is in the external membrane and is essential for the removal of antimicrobial substances produced by the plant. The vector is designed to direct expression of the scFv into the phloem cells of citrus, where CaLas grows, and the vector encoding the scFv genes is being introduced into rootstock varieties by Agrobacterium mediated transformation. To date we have purified 19 scFv genes, and cloned them into the plant transformation vector pUSHRL-26 developed by Ed Stover at Fort Pierce. This includes 9 antibodies directed at InvA and 10 directed at the external opening of the TolC protein. Sequencing confirmed the clones were correct. The Stover laboratory has transformed the constructs into Agrobacterium and the Agrobacterium has been used to transform Carrizo seedlings. Nineteen transgenic lines have been established or are in development with between 150-400 epicotyl explants for each line. These explants include scFv for both TolC and InvA, and are being grown at Fort Pierce for subsequent evaluation by inoculation with psyllids infected with CaLas. There are hundreds of independent transformants containing these scFv constructs at Ft. Pierce (Stover). These transformants are being grafted on to high titer root stocks and will be incorporated into the separate NuPsyllid project to determine if they have adverse effect on acquisition or transmission of CaLas by the psyllid. We are making a second recombinant antibody library using ribosomal display technology. This technology is not expected to be encumbered by patent restrictions. Mice have been obtained and have been injected 3 times with psyllid extracts, following the previously successful immunization protocol used to make the scFc library.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. We have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. As described in earlier reports, the HisTag incorporated in the scFv vector leads to unacceptable cross reactions and will not be useful in detection assays. Wehave improved the basic tissue printing protocol, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. We have used the tissue print protocol with detection of the scFv via the FLAG epitope incorporated into the scFv. However the secondary monoclonal antibodies that bind to the FLAG epitope in the scFv vector also bound to HLB diseased tissue in the absence of the primary scFv and produced images that were not distinguishable from those produced with the scFv. This was completely unexpected and eliminates the FLAG epitope for detection of the scFv. Surprisingly, the secondary anti-FLAG monoclonal antibody also ‘detected’ CTV, Xylella fastidiosa and CCDV with tissue in the absence of primary scFv. Thus the anti FLAG secondary antibody reacts to a protein produced in response to infection. This is a subject of investigation. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and flagellar antigen FlgL and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. These polyclonal rabbit antibodies are very useful in a tissue print detection format. Cross reactions as described for the anti-FLAG or anti His-monoclonals are not a problem. The tissue print assay also preserves the anatomical distribution of CaLas in plant tissues. The sensitivity of the assay is comparable to qPCR but interestingly is more successful for detection of CaLas in asymptomatic plant tissues than is qPCR. This is because although the concentration of CaLas may be locally high in individual phloem cells, the overall concentration of CaLas in tissues can be low in these presymptomatic leaves. The ease and cost of the dot blot assay is also much less than qPCR. We have used the assay to follow the distribution of CaLas in infected trees. The assay shows the distribution of CaLas in roots, stems, leaves, peduncles and seed of citrus samples collected from HLB affected groves in Florida. The results of the tissue print assays have been correlated with results from qPCR. The rabbit polyclonals work as we hoped, and the tissue print assay for CaLas is faster better and cheaper than previous assays. One manuscript has been submitted and two more are in final stages of preparation documenting this work.
Sept 2015 The objectives of this project are to optimize Guignardia citricarpa ascospore extraction procedures and qPCR with automated extraction system, determine if prototype passive ascospore traps will capture a sufficient number of Guignardia citricarpa ascospores to be an effective monitoring tool and monitor for G. citricarpa ascospores in six locations around state. We have designed and built prototype spore traps. The first one was placed at CREC near our Burkhard trap so that spore counts could be compared. Once we were satisfied with the design and that we had made them so they would not break, further traps were constructed. Six traps were deployed at different heights (0.5, 1.0, and 1.5 m) near our Burkhard traps in the Immokalee area. We have continued to deploy the slides in the traps but there are very few spores on the Burkhard and new traps to compare counts. This may be time of year.
Extensive accumulation of Callose and Phloem protein 2 (PP2) have been observed in citrus plants infected with Huanglongbing (HLB) implicated in plugging sieve tubes interfering with source-sink transport of nutrients. Transcriptome analyses have indicated the over-expression/accumulation of the transcripts for callose and PP2 genes. During the course of our investigation we have tried to silence the endogenous citrus genes responsible for the accumulation of callose and PP2 in phloem (callose accumulation in phloem is mainly due to the activity of callose 7 gene) using silencing potential of the citrus tristeza virus vector. Towards this end, we engineered the truncated versions of Callose and PP2 genes into CTV vector. We have generated citrus macrophylla plants expressing double-stranded RNA (dsRNA) to regulate the expression of endogenous callose 7 in this case we used callose 7, so as to silence the phloem callose. We have observed reduced accumulation of callose 7 and PP2 in phloem as determined by real time quantitative PCR (RTqPCR). Presently we are exposing these plants to psyllids reared on HLB positive citrus plants (hot psyllids) to study changes in accumulation of callose 7 and PP2 with the progression of HLB disease.
Oct 23, 2014 As part of this project, we were able to complete the statewide survey of tangerine and tangerine hybrid groves in 2013 for strobilurin resistance. We found that 57.6% of our isolates had an EC50 (effective concentration to inhibit 50% growth) that were >5 ug/ml and 1 ug/ml for azoxystrobin and pyraclostrobin, respectively, indicating that they were functionally resistant. The resistance was not significantly related to field disease severity. We confirmed our results by molecular means. We sequenced a subset of the isolates and the known resistance conferring mutation G143A was present in resistant isolates and absent in sensitive isolates. Details can be found in: Vega, B., and Dewdney, M. M. 2014. Distribution of QoI resistance in populations of tangerine-infecting Alternaria alternata in Florida. Plant Dis. 98: 67-76. We then tested to determine if the resistance phenotype was stable. Resistance stability will determine whether the resistance will remain a problem or slowly disappear without the selection pressure. There was no change in the phenotype of the resistant fungi without the selection pressure. We also looked at sporulation rates, mycelial growth, spore germination, and spore production and there was no difference between sensitive and resistant isolates. We did find that resistant isolates were significantly more virulent than sensistive isolates on Dancy, Minneola, and Sunburst but not Murcott. When tested in the greenhouse, field rates of azoxystrobin did not control resistant isolates on Murcott. More details can be found in: Vega, B., and Dewdney, M. M. 2014. QoI-resistance stability in relation to pathogenic and saprophytic fitness components of Alternaria alternata from citrus. Plant Disease 98: 1371-1378. We also determined the baseline sensitivity to the newly introduced fungicide boscalid, an SDHI fungicide. We established the best method to measure sensitivity to boscalid by screening multiple methods and media. We determined that the resazurin reduction method with complete media was best. We did find that spore germination and mycelial growth were not suppressed at the highest concentration although it was possible to establish an EC50. We tested 419 isolates collected between 1996-2012 never exposed to boscalid. We found that all the isolates were sensitive with an EC50 range of 0.07-5.84 ug/ml. We also molecularly characterized the SDH genes of 15 isolates representing a range of EC50s. We found multiple mutations in these genes but none that are known to confer resistance. Currently there is low risk of SDHI resistance in the next few years but monitoring should continue. This is available in preprint at: Vega, B., and Dewdney, M. M. 2014. Sensitivity of Alternaria alternata from citrus to boscalid and polymorphism in the iron-sulfur and in the anchored membranes subunits of succinate dehydrogenase. Plant Disease. http://dx.doi.org/10.1094/PDIS-04-14-0374-RE. We also determined the baseline sensitivity to the other newly introduced fungicide, the DMI difenoconazole. We determined that the best way to measure the sensitivity to this fungicide via microtiter plate with growth measured via absorbance. No isolates were found to have any shifted sensitivity. We are preparing the manuscript and plan to submit it to Plant Disease.
In agriculture, Copper (Cu) has been and is continuing to be used extensively to protect multiple crop species from a wide variety of infectious diseases. While its low cost and potent protection make it useful, there are many downsides involved. Protracted usage of copper based biocides has led to an increased risk of copper toxicity in fertile soil from accumulation and the development of copper resistance among infectious organisms threatens its effectiveness. The goal of this project is to develop an attractive, environmentally safe, industrially feasible alternative to Cu biocides. The use of Quaternary Ammonium Compounds (Quat) as an antimicrobial agent is extensive within medical and industrial fields as a disinfectant. Quat compounds have not been used in agriculture due to their potential to cause plant tissue damage (Phytotoxicity). Combining Quat with a silica delivery system to produce Fixed-Quat can reduce its potential phytotoxicity and allow it to be used in agricultural applications. For the 2013-2014 period, we have successfully synthesized a variety of Fixed-Quat nanomaterials with Quat immobilized within a silica matrix either covalently or electrostatically. Fixed-Quat nanomaterials were synthesized using EPA approved compounds and followed modified Stober protocols by hydrolyzing a silica precursor under either acidic (nanogel) or basic (nanoparticle) conditions. The large series of nanomaterials were originally developed to determine the best Quat to Silica ratio for generating Fixed-Quat systems. The various Fixed-Quat nanomaterials were characterized for Quat presence, particle size and morphology using Fourier transform infrared spectroscopy (FTIR), Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Fixed-Quat nanoparticles conjugated covalently or electrostatically polydispersed exhibited sizes in the sub-micron range of 300-600nm. Fixed-Quat nanogels exhibited a large size distribution from sub-micron to micron. Fixed-Quat nanomaterials were found to be stable at concentrations exceeding 10,000 ppm Quat content, indicating potential large scale production. Potential plant tissue damage (Phytotoxicity) of Fixed-Quat nanomaterials was tested on a variety of plants, including Vinca sp (Ornamental plant highly susceptible to phytotoxicity), Hamlin Orange and Pink Grapefruit. Fixed-Quat Nanogels were found to be completely safe to plants at rates as high as 900ppm. Antibacterial studies have been conducted using in-vitro microplate alamar blue assay, bacterial viability expressed as colony forming units (CFU/mL), growth curves and the determination of the Minimum Inhibitory Concentration (MIC). Antimicrobial efficacy was tested using gram-negative model bacterial system, E. coli and X. alfalfae (a citrus canker surrogate), with Kocide 3000 (commercial product from DuPont) as a control. Studies indicated Fixed-Quat nanomaterials were effective at MICs as low as 2ppm. One of the Fixed-Quat nanogel materials has been delivered for 2014 field trials. Field efficacy will be evaluated at two different foliar spray rates. Trial data is being collected in October 2014 and will be analyzed and presented in the next progress report.
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