Meida continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from the haemolymph of the Asian citrus psyllid to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including heptadecanoic acid, n-methyl-L-proline, methyl-maleic acid, homoserine, pyroglutamic acid, arabinopyranose, D-glucopyranoside, fucose, azaleic acid, arabinofuranose, iso-citric acid, n-acetylglucosamine, alpha-D galactoside, inositol-2-phosphate , trehalose, citrulline, ornithine, AMP, CMP, NADP, GMO, IMP, and FAD. In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. During this period, we continued to follow-up on our discovery that citrate is a preferred carbon source of L. crescens. We discovered that the optimal level of citrate required for growth was 2.5 g/l or 13mM. We also found that the pH of the medium increases dramatically during growth from 5.9 to 7.6. This increase in pH is expected from the metabolism of citric acid. We are now testing medium with more buffering capacity to maintain pH near 6.0 during culture growth. This is being tested with higher levels of phosphate buffer and ACES buffer. During this period we have considered ideas on whether CLas can be starved of citrate by modifying the nutrition of citrus saplings. The first experiment tested whether changes in nitrogen nutrition (potassium nitrate, sodium nitrate, or ammonium nitrate) can alter the levels of citrate in phloem. These nutritional changes are expected to alter the ionic balance in the plant. Sodium nitrate was expected to reduce the level of citrate in phloem. However, after 60 days of treatment in the greenhouse, we saw no difference in citrate phloem levels between these nutritional treatments. We are now testing the notion that adeqaute phosphate nutrition is required to reduce citrate levels in phloem. Under conditions of phosphorous deficiency, plants load citric acid into the phloem in order to exude citric acid from roots (Gaume et al. 2001. Plant and Soil 228:253-264). It is also known that the exuded citric acid can then solubilize rock phosphate (P2O5) or other forms of bound phosphate for use by plants (Chen and Liao 2016. J. Genetics & Genomics 43:631-638). Zhao et al. (2013, Molec. Plant 6:301-310) reported that certain small RNAs made in response to HLB infection were involved in P nutrition. They also found a 35% reduction of P in CLas-positive citrus trees compared to healthy trees. Applying phosphate to HLB-positive sweet orange trees in southwest Florida reduced HLB symptom severity and significantly improved fruit production in a 3-year field trial. So our hypothesis is that the severity of citrus greening disease can be greatly reduced with application of phosphate (not rock phosphate as is sometimes recommended). Added phosphate will improve the nutrition of the tree and lower citrate loading into the phloem. The reduced citrate levels in the phloem is expected to starve Liberibacter. These ideas will be proposed in a grant proposal to the USDA SCRI program with Killiny, Wang, and Vincent from the CREC as co-PIs with Triplett.
During the past quarter, we have optimized dsRNA targets and have conducted immune priming assays to investigate the specificity of immune priming in ACP following exposure to dsRNA. The yield of redesigned dsRNA using MEGAscript RNAi Kit transcription was initially low, possibly because the QG buffer in the Zymoclean Gel DNA Recovery Kit may have decreased transcription. In order to avoid using QG buffer in gel purification and increased PCR concentration,gel purification method was modified. PCR products were analyzed on 1% agarose gel and the band was excised, and transferred to a filter tip and centrifuged at 5000 rpm for 10 min. Three l of purified gel product were run on 1% agarose gel which showed a high concentration (based on the intensity of the band) of the product. The remaining purified gel product was precipitated and resuspended in 30 l of RNase-DNase free water, and 2 l of the product were analyzed on agarose gel and directly amplified. PCR products obtained from the modified purification protocol for dsRNA synthesis yielded significantly higher dsRNA concentrations to compared to the kit. Bioassays were conducted with adult uninfected ACP. After 3 hrs of starvation, insects were fed on dsRNA for 24hrs and 5 days. After 24 hrs, insects were starved for 3 hrs and then transferred to a lethal dose of S. marcescens for 4 days. For each dsRNA concentration, two cages were exposed to dsRNA; then either fed on S. marcescens in diet (20% sucrose, and 0.5 green food coloring dye) or diet only (control). Diet containing the pGEM-T easy vector without a T7 tail, a blank sucrose + buffer diet, and container without food were also included as controls in the feeding experiment. The stability of different concentrations of dsRNA on diet solution were confirmed with agarose gel electrophoresis at the end of the 24hr and 5 d feeding periods. DsRNA of 1000 ng/ l and 100 ng/ l were present in agarose gel. Also the survival of S. marcescens after 4 days feeding was checked on nutrient agar plate. S. marcescens from feeding cage was streaked on nutrient agar and kept O/N at 30 C. Result confirmed that bacteria were viable during the feeding assay. Following exposure to bacteria, insect were placed on Citrus macrophylla in the greenhouse, and mortality was recorded daily. After 14 d, live insects from the 24hr and 5 d feeding treatments were collected and laterally bisected. Half of the insects were examined for the presence of S. marcescens, and the remaining insects were used for RNA extraction. To quantify S. marcescens, single insects were smashed in tube with 20 l of nuclease-free water and plated on nutrient agar, and kept at 30 C O/N. Colonies were obtained from most of the insects on nutrient agar. No colonies were obtained from control insects. Conventional PCR confirmed the presence S. marcescens in insects. To study the effect of dsRNA on mRNA levels, total RNA was extracted from single ACP using TRIzol (Invitrogen) and cDNA was prepared. The survival rate of insects after feeding on S. marcescens and from 14 days on C. macrophylla will be analyzed and presented when all replicates are completed; however, initial results indicate thigh mortality after feeding on S. marcescens as compared with those feeding on sucrose, suggesting that dsRNA exposure does not modulate ACP immune responses to S. marcenscens e.g. does not prime ACP to tolerate or mitigate lethal doses of pathogenic bacteria, although it must still be determined whether this response is similar following exposure to sublethal doses of bacteria. Whether this is responses is specific or is generalizable to other Gram negative bacteria, such as Liberibacter, will be determined in the next quarter.
Obj. 4. The purpose of this objective is to determine whether prior pathogen or dsRNA exposure inhibits Las acquisition by psyllids. We investigated if D. citri exhibits immune priming and produces a different response to secondary infections and the specificity of that protection. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Surviving D. citri were moved to a second sachet containing 1e6 cells/mL live S. marcescens where they remained until all D. citri were dead. Mortality was recorded once daily. Transgenerational immune priming bioassays were conducted with psyllids placed on artificial diets containing heat inactivated E. coli, M. luteus, or no bacteria. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Data presented here are preliminary, as additional replicates were collected during February and March 2017. These insects are currently being processed and analyzed via QPCR. Offspring of female D. citri that were fed a diet containing M. luteus (n = 17, 2.60E7 1.35E7) or E. coli (n = 18, 2.09E6 8.40E5) had higher CLas titers than offspring of non-primed females (n = 8, 1.22E5 6.20E4). The Ct of plant material was correlated with the titer of CLas in D. citri (b = -0.140, F1,41 = 4.115, p = 0.050, R2 = 0.09). Previously, we demonstrated that the D. citri immune response is induced due to recognition of Gram-positive bacteria, such as M. luteus. Given that M. luteus protected D. citri from S. marcescens, and CLas is also a Gram-negative bacterium, it was expected that CLas titers would be lower in offspring of M. luteus primed adults. In fact, the opposite was observed. Females fed a diet containing M. luteus prior to mating had higher CLas titers and produced offspring that acquired CLas at a much higher rate than those from control or E. coli fed females. This suggests that immune priming with M. luteus infection may facilitate CLas infection.
A paper was submitted during this period (Cruz-Munoz M, Cohn A, Lai K.-K, Dia, R, Rusoff K, Conrad R, Triplett EW 2016. A chemically defined medium suggests a-ketoglutarate and malate as primary carbon sources for Liberibacter crescens BT-1 growth. Submitted to Applied Microbiology). We continued to improve the defined medium for L. crescens and now use a medium called M12. In addition, we obtained more phloem metabolomics data from Nabil Killiny which was used to create more media formations for CLas. We have also learned that the typical primers used to assess CLas growth in culture give misleading results because they were created to amplify the bacteriophage sequence and not those of the bacterial genome. A new set of primers was designed that sigificatly improve our ability to assess growth. . The metabolomics data for the culture of L. crescens in defined and undefined media are encouraging. The strain grows much faster in BM-7 than in M11n so we are identifying those components that are limiting for growth in the defined medium (M11n). When identified, their concentration will be increased in the CLas medium. We have had difficulty maintaining an infected psyllid colony in Gainesville which has prevented us from studying the heterogeneity of CLas strains
An important breakthrough came for us during this quarter that was quite accidental. We are designing defined media for L. crescens. Our first such medium was made about 9 months earlier and we continue to improve upon it. We want to develop the best defiend medium we can that can then be easily adapted to a CLas medium based on the metabolomics of the psyllid hemolymph and citrus phloem (data from the Killiny lab). The design of these medium is largely based on trying to reproduce the constituents of BM-7, the undefined medium typcailly used to culture L. crescens. One of those constituents is an insect cell culture medium called Grace’s medium. The recipe for Grace’s is well known and the medium is available commercially from at least three sources. We have made Grace’s medium from scratch ourselves and it doesn’t do as well as the commerically obtained Grace’s medium for culturing L. crescens. So there is something awry in the recipes that are published. Furthermore, one of the sources of Grace’s medium we obtained commercially provides excellent growth of L. crescens. This was the case with one batch from this company and wasn’t reproducible in other batches. So we are in the process of performing metabolomics of this purchased medium to learn how it might be doing so well in the culturing of L. crrescens. Once we know that, we should be able to design better media for L. crescens. We are also using this great batch of Grace’s medium in various media to culture CLas.
In this quarter, we continue to focus on the metabolomics of culturing CLas and have identified many mass spectrometry ions that either disappear or appear with growth over time. Those that disappear with growth over time may be limiting the growth of L. creascens in culture, and, by extension, may be limiting in the growth of CLas. Those compounds that accumulate with time in these cultures may be toxic for the growth of L. crescens, and, by extension, may be toxic to CLas as well. One those compounds are identified we can re-design the media accordingly. We also continue to modify media for CLas according to the metabolomics or citrus phloem. In this quarter, we obtain data from Dr. Killiny on the metabolome of the insect hemolymph. We now have media based on the hemolymph and citrus phloem. We are also using a better primer set to assess the growth of these cultures. We aso started a new nutrition experiment in December 2016 that is based on our discovery that citric acid is a preferred carbon substract for Liberibacter in culture. Our hypothesis is that if we can limit the amount of citric acid in phloem, we should be able to limit the survivablity of CLas in citrus phloem. Our first experiment on this is designed to determine whether we can alter the ionic balance in phloem to prevent citric acid loading into the phloem. We are testing this by varying the source of nitrogen provided to citrus saplings in the greenhouse. In addition to the traditional ingredients of a citrus nutrient solution, four sets of 20 plants are receiving wither no N, sodium nitrate, ammonium nitrate, or potassium nitrate. We will continue this experiment through a period of flushing since new growth may be required. We expect to harvest this experiment in February or March of 2017. We will extract phloem from these plants and mearsure citruc acid with the help of Nabil Killiny. We expect citric acid levels to be the highest in the postassium nitrate teated plants and lowest in the sodum nitrate treated plants.
This project is a continuation of the funding that has been provided to Southern Gardens Citrus (SGC) to provide growers, researchers and private companies with a laboratory to detect the pathogen that causes huanglongbing (syn. citrus greening). For the second quarter of the second year of funding (October 1-December 31, 2016), 6768 samples were analyzed. Of these, 92% were plant samples and 8% were psyllid samples. This compares with 8,449 for the same period for year 1 and 8,407 for the first quarter of year 2. Total for year 2, 15,175 samples have been analyzed compared to 15,400 for the same period of year 1. Cumulative for the grant period, 48,124 samples have been run. If the sample submission rate continues, this will result in an approximate 50% increase in sample load compared to what was budgeted.
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 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. In order to define the mechanisms that phage are employing to overcome abiotic stress, we designed and optimized specific primer sets for quantitative reverse transcription PCR (qRT-PCR) for genes within the phage region that were likely regulated by heat treatment and other stress. Because this analyses are based on mRNA transcript level and not on genomic DNA, the upregulation of phage genes reflects the relative level of transcript of active living cells. In order to ensure adequate generation of cDNA from Las, we found it necessary to use individual primers specific for the targeted region instead of generalized primers such as random hexamers. The cDNA that was generated in this fashion was then used as the template for qRT-PCR. The present analysis included three biological samples for each condition and three technical replicates for each sample for statistical purposes. Particular genes that were found to be upregulated included: CLIBASIA_5590 encoding an unknown protein, CLIBASIA_5610 encoding a putative phage terminase (large subunit), CLIBASIA_5665 encoding an unknown protein, CLIBASIA_5390, which has the conserved sodium: dicarboxilate symporter family domain, CLIBASIA_5525 encoding a guanylate kinase that catalyzes the reaction ATP + GMP <->ADP + GDP. Phage genes found to be downregulated included: CLIBASIA_00005 encoding an unknown protein, CLIBASIA_00010, which has an NTPase domain of typical DNA-packaging enzyme, CLIBASIA_00030 encoding a putative DNA polymerase of bacteriophage origin, CLIBASIA_5565, which has the conserved domain TolA protein and is thought to be required for the translocation of the phage DNA. This data correlated well with what was seen via our previous RNA-Seq analysis and helps reveal the transcriptional response of the phage to abiotic stress factors. Given that previous studies on thermotherapy showed an overall reduction in Las titer in citrus affected by HLB post heat treatment, harnessing the ability to control these particular genes may allow us to lower the bacterium s ability to handle stress. Based on the variations of Las prophages/phages, we recognized certain molecular mechanisms behind the symptom variations and their association with “mild strains” of Las bacteria and host tolerance/resistance. The titration dynamics between 16S DNA-based and phage gene-based results revealed the association of host tolerance with the dynamics. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermo-therapy. To investigate the effects of stress on the genes involved in the phage lytic cycle, we identified several phage genes that were over-expressed in citrus plant after heat treatment. These results indicated that thermotherapy has a direct effect on Las bacteria by actively regulating specific phage genes. To further evaluate genes related to stages of the phage lytic cycle, we further compared Las genes expression profile from two distinct insect vectors, psyllids, and mealybugs. Mealybugs were found to contain much higher titers of Type D when compared with psyllids, indicating that the phage may be more active in the mealybugs than in the psyllids. Using an enrichment method to acquire RNAseq data, we compared the Las transcriptomes between the two insect vectors, and revealed that psyllids samples contained more than four times reads of the Las 16s rRNA than those of mealybugs samples, indicating much higher bacterial titers in psyllids than in mealybugs. However, mealybug s transcriptome profiling showed much higher expression level of prophage genes than those in psyllids, where expression level of prophage genes was completely absent or extremely low. Interestingly, more than 2/3 of the highly expressed genes in mealybugs were identified as prophage/phage genes. Interestingly, eight genes with the highest level of expression in mealybugs were identified as the highly expressed ones in Las-infected citrus after heat treatment. These results indicates that both stresses caused by thermotherapy and in mealybugs environment triggered similar signaling pathways, and result in the expression of prophage genes that may induce lytic cycle and eventually reduce Las titer in citrus plant treated with heat stress, and maintain low titer in mealybugs.
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. The objectives of this work included the development of an assay specific for active pararetroviruses in Florida citrus, and to use this assay to assess correlation between the active pararetrovirus and blight affected trees. An assay was initially developed using sequence inromation from the original work. Then 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. A third objective was to generate complete genome sequences for any and all active blight associated pararetroviruses and developing a active virus specific assay comprehensive enough to detect all blight associated pararetroviruses. As of the last quarter we had successfully generated a complete genome sequence for a blight associated active pararetrovirus, we are prepared to hand the sequence data to CRDF. In addition to the original genome sequence we have continued sequencing other pararetrovirus isolates from additional trees and geographic locations to determine levels of diversity. Six other complete genome sequences are underway. Also in our last quarter we had tested multiple assays for the active pararetrovirus on a large sample set to determine which of the assays was specific only to active pararetrovirus while still being inclusive enough to detect all active pararetroviruses. A single assay was developed that was very effective in detecting all active pararetrovirus samples in the 2015 sampling, this will meet the objective of the project. As a final check, an additional set of samples was taken in the summer of 2016 for testing and validating the new assay. This assay is ready for delivery to CRDF. .
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. The objectives of the proposed work are the following: 1. Generate a complete genome sequence for CBAPRV. This has been completed. We would like to transmit the complete sequence to CRDF. 2. Develop a highly specific RT-PCR assay that can determine when CBAPRV is active. This has been completed. The primers are ready to be transferred to CRDF. 3. Use this assay to screen a large number of trees from blight affected areas in Florida. This has been completed. The correlation between presence of the active viral RNA and blight is very high (96%). In addition, active viral RNA has never been found in healthy trees. 4. Transmission tests to determine if CBAPRV is the causal agent of citrus blight. This has been attempted, without success using multiple approaches. In the quarter just ending we have focused our efforts on the final objective. Previous sampling of 100 blight affected trees and 20 healthy trees from 5 blight affected areas (two sequential years) in Florida demonstrated a strong but not 100% correlation between the presence of CBAPRV RNA (the hallmark of active exogenous virus) and the onset of blight. 96% of the trees that were identified as affected by citrus blight have active viral CBAPRV RNA in leaves and roots, and none of the non-blighted trees have evidence of CBAPRV RNA. This is a strong correlation, but the 4% of trees that have been identified as blighted without the presence of CBAPRV suggests two possibilities: 1) the presence of the active virus is associated with the stresses of blighted trees, but the virus is not the causal agent of the disease, or 2) the method of sampling for CBAPRV RNA is not 100% effective. The final objective attempts to address which of these possible explanations is correct. Attempts to transmit the virus by aphids and psyllids were unsuccessful, in agreement with previous litereature. We have grafted CBAPRV onto a variety of rootstocks and scions, and these trees tested positive for viral DNA but not RNA. We will continue to monitor these trees, as well as applying a number of stresses (drought, cold, heat, etc…) in an attempt to induce blight. This work will continue beyond the scope of the project. In addition, attempts at purification of viral particles identified some likely particles, but these particles were only found in roots of blighted trees. Thus the efficiency of purification was very low. We may attempt to do further purifications and subsequent mechanical inoculations if enough particles can be generated, but that will occur beyond the scope of this project.
This project is a continuation of the funding that has been provided to Southern Gardens Citrus (SGC) to provide growers, researchers and private companies a laboratory to detect the pathogen that causes huanglongbing (syn. citrus greening). For the first quarter of the second year of funding (July 1, 2016 – September 30, 2016), 8,407 samples were processed. This compares to 6,951 for the first quarter of 2015. Of the samples processed for the first quarter of year 2 funding, 7,749 of the samples were plant samples and 658 samples were psyllid samples. Cumulative for the funding provided to date (5 quarters) a total of 41,356 samples have been run. This represents a 65% increase in sample load compared to what was budgeted for the period. During year 2 of the funding, we plan on adapting a PMA protocol that will allow the differentiation of live versus dead cells in the qPCR determination. This protocol will be implemented on a limited basis for specific trials. Once the procedure is validated, it may be made available on a limited basis to a wider group of researchers.
Under field conditions where latent Huanglongbing infections may be present, but undetectable by PCR, it is not known whether psyllids transmit low-level infections among trees. New HLB management strategies, such as heat-treating trees (thermal therapy), may reduce pathogen titers in infected trees to low or undetectable levels (Esahni et al.). Heat treatment methods currently under development include enclosing trees underneath tented structure, or applying steam heat directly to tree trunks, for a specific time period such that temperatures rise to approximately 55oC. The objective of this study was to determine how thermal treatment of CLas-infected citrus trees in influenced acquisition of a systemic, circulative infection CLas by the Asian citrus psyllid. Leaf CLas titers were not significantly reduced in infected plants receiving thermal treatment as compared to untreated plants, although numerical reductions in the CLas titers of infected plants occurred during the first sample period following thermal treatment. Similarly, CLas acquisition from infected, thermal-treated trees was not significantly different as compared to acquisition from infected, untreated trees in the field study. Mean adult and nymph acquisition rates were positively correlated with the mean Las titer in leaves. These results suggests that treating the above-ground portion of infected citrus trees at 55 C for 30 seconds is not adequate to reduce acquisition of CLas by psyllids. In a separate greenhouse study, whole tree application of thermal therapy to potted citrus tree resulted in no acquisition from thermal-treated trees compared with 75% of psyllids acquiring CLas from untreated, CLas-positive trees. The replication in this trial was low, and further studies are needed to confirm the efficacy of whole tree thermal therapy on psyllid acquisition; however, the data indicate that whole tree thermal therapy reduces CLas acquisition.
In Year 3, our major focus was to further optimize the best performing Fixed-Quat material to obtain a commercially-viable product formulation. This included stability (shelf-life) and in vitro efficacy evaluation of the product that was formulated with agriculture-grade chemicals. In Year 1 and 2, the source of silica for the preparation of Fixed-Quat nanoparticle and nanogel materials was tetraethylorthosilicate (TEOS; product labeled as Fixed-Quat A gel). Previously, Fixed-Quat A was used in the 2014 Canker field trial on “Ruby Ray” grapefruit in Vero Beach, FL and was found to reduce the incidence of infection from 63% to 15% at 200 ppm spray rate, matching Kocide 3000 (0.9 lb/acre Cu) which reduced the infection to 16%. This result was promising as this is for the first time we have shown that a non-metal based non-traditional disinfectant material could be potentially used as an alternative to traditional Cu bactericide/fungicide for crop protection. The results from the 2015 canker field trial reported that the untreated control displayed an incidence of infection of 60% while our Fixed-Quat A-II nanogel reduced the infection to 26% at 200 ppm which matched Magna-Bon (0.14 lb/acre Cu) commercial product which had a reduced infection of 25%. In the Fixed-Quat A-II gel, EPA approved Quat was used at the 13,500 ppm level or higher with no issues observed with formulation stability. Although the TEOS is a well-studied chemical (in particular for proof of concept studies for making colloidal silica particles), due to its chemical reactivity and other limitations, it is not approved by the US EPA for the use as an inert at this time. Therefore, instead of using TEOS, we have used EPA approved (for food and non-food use) silica source such as Sodium Silicate which resulted in newer version of products formulations, Fixed-Quat A-II, Fixed-Quat AP-II and Fixed-Quat E. The formulation composition and interactions between the components (silica and Quat) was confirmed using Fourier Transform Infrared Spectroscopy (FTIR) in particularly looking at the Si-O stretching and SiO-H stretching frequencies of silica. The Si-O stretching frequency changed from 1039 cm (-1) to 1031 cm (-1) whereas the SiO-H frequency changed from 3391 cm (-1) to 3388 cm (-1), suggesting interaction of positively charged Quat with the silica gel. The Quat C-H bending frequency of 2923 cm (-1) was found in both controls and formulations, confirming the presence of Quat. This change also supports the interaction of Quat with silica gel. The Fixed-Quat AP-II and E nanogel s plant safety (phytotoxicity studies) was carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions. Studies conducted on Sour orange, a common citrus variety and Roma Tomato sp, an ornamental plant revealed no sign of plant injury when tested with Quat concentration as high as 1000 ppm. The morphology of Fixed-Quat E nanogel was examined using Scanning Electron Microscopy (SEM), displaying sub-micron to micron composites with irregular shapes in multiple layers. X-ray Diffraction (XRD) revealed that the material is primarily amorphous in nature. Antimicrobial studies of optimized Fixed-Quat E nanogel was conducted against Xanthomonas alfalfae subsp. citrumelonis (citrus canker surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Minimum Inhibitory Concentration (MIC) was determined and compared against Kocide 3000 and copper sulfate controls. MICs of Fixed-Quat A-II and E were found to be = 1.0 g/mL for X. alfalfae, = 1.0 g/mL for P. syringae and = 1.0 g/mL for C. michiganensis. MIC results indicate no loss in efficacy with new source of silica. 2016 Citrus canker trials on “Ruby Ray” grapefruit includes both Fixed-Quat A-II and Fixed-Quat E nanogel materials. This trial results are expected sometime in January, 2017.
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. The objectives of the proposed work are the following: 1. Generate a complete genome sequence for CBAPRV. This has been completed. 2. Develop a highly specific RT-PCR assay that can determine when CBAPRV is active. This has been completed. 3. Use this assay to screen a large number of trees from blight affected areas in Florida. This is largely completed. 4. Transmission tests to determine if CBAPRV is the causal agent of citrus blight. This is underway. In the quarter just ending we have focused our efforts on the remaining objectives. During this quarter we have been validating the CBAPRV assay that was developed, and further sampling blight affected areas in Florida to assess the correlation between CBAPRV and blight affected trees. At this point, there is a strong but not 100% correlation between the presence of CBAPRV RNA (the hallmark of active exogenous virus) and the onset of blight. 94% of the trees that were identified as affected by citrus blight have active viral CBAPRV RNA in leaves and roots, and none of the non-blighted trees have evidence of CBAPRV RNA. This is a strong correlation, but the 6% of trees that have been identified as blighted without the presence of CBAPRV suggests two possibilities: 1) the presence of the active virus is associated with the stresses of blighted trees, but the virus is not the causal agent of the disease, or 2) the method of sampling for CBAPRV RNA is not 100% effective. To assess which of these possible explanations is correct we are moving on to attempted transmissions of the virus. There is no evidence that the virus is insect transmitted, so we have begun to work in two directions: 1) grafting of virus present tissue onto a variety of rootstocks and scions, and 2) attempts at identification and purification of viral particles that could be used to establish modified Koch’s postulates. The final quarter of work will focus exclusively on these efforts. Some additional testing of field materials collected in August of 2016 will also be completed.
We have proposed targeting specific regulators of key phage encoded virulence genes (such as the Las peroxidase) as well as the peroxidase itself and key regulators of the (lethal) phage lytic cycle. Objective 1 is control of HLB using the putative Las chromosomally encoded LexA-like repressor protein LC1 (Clibasia_01645), potentially a key phage lytic cycle regulator. We had previously shown that LC1 binds specifically to its own promoter as well as to a key lytic phage SC1 promoter region midway between the divergent late and early gene promoter regions. Bioinformatics revealed several putative sites for LC1 binding. Electrophoretic mobility shift assays demonstrated that LC1 binds to its own bidirectional promoter and to another DNA region between SC1_gp125 and SC1_gp130 genes on SC1. Moreover, SC1_gp125 is predicted to encode a C2-like repressor (LC2), therefore, if functionally confirmed as a repressor, LC2 would constitute a second potential target implicated in lytic cycle activation. To evaluate the potential activity of the LC1 and LC2 repressors, promoter::GFP reporter constructs were transformed into Liberibacter crescens (Lcr) and E. coli. SC1_gp125 (LC2) promoter activity was significantly higher than SC1_gp130 and even the strong LacZ promoter. These promoter::GFP reporters were cotransformed with vectors expressing the full-length LC1, truncated LC1 and LC2 repressors (all driven by LacZ promoter) in Lcr. Not only did LC2 self-repress its own promoter, but both full length and truncated LC1 repressors also repressed the LC2 promoter. Inactivation of either repressor would likely lead to Las cell lysis. Objective 2 is control of HLB using a repressor protein of unknown identity from psyllids as target. We previously reported functional repression of a Las phage lytic cycle holin promoter by a predicted Wolbachia repressor protein found in aqueous psyllid extracts. The in vitro translated Wolbachia protein partially repressed the holin::GUS reporter in a dose-independent manner, as compared to the aqueous extract from the psyllid, thus indicating that complete suppression of holin promoter requires an additional partner. Heat-inactivated psyllid extract was unable to enhance the Wolbachia repressor- induced suppression of holin promoter activity, confirming the heat labile proteinaceous nature of the additional psyllid-sourced partner. Including Objective 1 results, we have identified 3 protein repressors and target promoters that are being used for high-throughput screening of combinatorial libraries for chemicals that might be used to activate the phage lytic cycle in Las, both in Las-infected psyllids and citrus. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. Bacteria use a variety of enzymes, including peroxidase, peroxiredoxin, catalase, and bifunctional catalase/peroxidases, to degrade Reactive Oxygen Species (ROS). In addition to the previously identified secreted peroxidase, we recently identified and cloned two chromosomally encoded peroxiredoxins, one secreted. Lcr transformed with the BCP-like peroxiredoxin showed a significant (200 fold) increase in tolerance to 100 M hydrogen peroxide, as compared to a marginal 4-5 fold tolerance provided by either the PR2 peroxiredoxin or SC2 peroxidase. Most importantly, BCP-like peroxiredoxin augmented Lcr tolerance to 50 M tert-butyl hydroperoxide (tBOOH; an organic oxidizing agent) by nearly 1000 fold. Las BCP-like peroxiredoxin may represent a second critical secreted effector, this one conserved across all pathogenic Liberibacter species (even in the phageless Las strains), that functions to suppress host symptoms.
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