The purpose of this project was to collect additional data from growers so that the efficacy of the enhanced nutrient programs could be assessed more fully after some promising results were obtained from the limited data collected last year. After extensive effort, some data were collected from three growers. However, none of the data sets was complete enough for analysis, and efforts to obtain the additional information failed. Consequently, we will not be able to complete the goals of this project. Because all involved (growers and researchers) were optimistic at the start of the study, some funds were spent so that graduate students would be prepared to conduct the analyses. However, when it became evident that data would not be forthcoming, expenditures stopped. We believe that the growers had full intentions of supplying the data. However, recent events, such as extensive premature fruit drop, have been devastating to the industry, and it is understandable that the growers no longer have the time or energy to help with this project.
Overall goal: To engineer Liberibacter asiaticus gene regulation in Sinorhizobium meliloti. Premise behind the work done thus far: ‘ We want test one Liberibacter asiaticus regulator as a pilot experiment, and will troubleshoot and make necessary adjustments before working with the other La regulators on our list. ‘ The sigma factor La.rpoH was chosen as a pilot experiment, given extensive data on the S. meliloti rpoH1 homolog, which can serve as a control. ‘ With synthetic La rpoH and Sm rpoH1 constructs, activity will be assessed on fusions to known S. meliloti RpoH1 targets. Goals: optimize induction time and IPTG (inducer) concentrations and measure reporter fusion activity. Conditions will be used for later global transcription analysis. Cloning synthetic allele of the Liberibacter asiaticus rpoH gene: ‘ A synthetic La rpoH gene was designed with optimized codon usage and ribosome binding site for expression in Sinorhizobium meliloti ‘ The complete DNA for La rpoH was synthesized using Invitrogen’s GeneArt gene synthesis. ‘ The synthetic DNA was cloned into expression plasmid, pSRK-Tc. This plasmid has an inducible lac promoter with low basal activity (Khan 2008, AEM 74:5053). The Tc plasmid succeeded as cloning vector, but failed to function in S. meliloti (see below) and we subsequently turned to two other versions with alternate resistance markers (Km, Gm). Cloning S. meliloti rpoH1 gene: ‘ rpoH1 was amplified from our rpoH1 clone using primers that introduced the same ribosome binding site and restriction sites as the La rpoH clone. ‘ After sequencing to confirm correct sequence, the rpoH1 gene was cloned into pSRK-Tc ‘ After vector failure, a second cloning placed the DNA into pSRK-Km and pSRK-Gm Conjugation of pSRK constructs into S. meliloti strains CL150 and RFF157 (.rpoH1): ‘ The initial pSRK-Tc constructs did not work, nor did the empty vector. Despite published reports of pSRK-Tc being used successfully in S. meliloti, in our hands the trans-conjugants do not grow to singles on M9 sucrose with Tc-10 or Tc-5. A positive control (rpoH1 in vector pRK290) was done at the same time and this yielded many Tcr transconjugants. ‘ The clonings were repeated with the two other versions of the plasmid (Gmr and Kmr). These constructs do appear to work in S. meliloti; they now must be confirmed by PCR, etc. Summary of accomplishments: A Liberibacter asiaticus gene, encoding the RNA polymerase sigma factor RpoH, was engineered for optimum expression in S. meliloti, synthesized, and successfully cloned into three different expression vectors. Conjugation of pSRK-Tc into S. meliloti did not work, due to apparent problems with the vector; conjugations with two alternate constructs were initiated and each gave promising results with abundant transconjugants.
Overall goal: To engineer Liberibacter asiaticus gene regulation in Sinorhizobium meliloti. Previous results: As a pilot experiment for study of Liberibacter asiaticus regulators, we synthesized the sigma factor La.rpoH using codons tailored for opimum expression in Sinorhizobium meliloti. We cloned this, and the comparable S. meliloti homolog, into Gm-resistant and Km resistant variants of vector pSRK. These vectors have an inducible lac promoter for varying the expression level of the sigma factor. These cloned genes and the empty vector control plasmid were conjugated into S. meliloti. New results: ‘ To assess function, we tested these constructs in RFF157 (.rpoH1) compared to wild type CL150, to see if they allow growth on LB at 37 degrees. Specifically it is known previously that WT grows at 37, but RFF157 cannot grow at the higher (restrictive) temperature o The La rpoH and Sm rpoH1 constructs allow growth at 37 degrees. pSRK empty vectors do not. From this, we conclude that the Liberibacter RpoH (La RpoH) protein is successfully expressed from pSRK, and functions in S. meliloti to replace the endogenous Sm RpoH. o We asked whether expression from pSRK is tightly dependent on lac induction. We observed that La rpoH and Sm rpoH1 strains grow better on media with IPTG (inducing the lac promoter), but difference compared to the non-induced control is not large. This suggests that lac promoter may have leaky activity, and that the sigma factor clones are being expressed to some extent even without inducer. This evidence for likely background noise has implications for our eventual experiments in which we assay transcription profiles. o We also tested plasmids expressing La rpoH in RFF231 (mutated with deletions of both rpoH1 and rpoH2). The cloned La rpoH does allow growth at 37 degrees in the double mutant. ‘ The next step of functional assay is to test expression of specific genes. We have chosen to make beta-glucuronidase (uidA) and fluorescent protein (FP) gene fusions to endogenous S. meliloti genes with responsiveness to Sm RpoH. Conjugation of pSRK constructs into S. meliloti strains CL150 and RFF157 (.rpoH1): o This will provide a quantitative way to test activity of constructs ‘ 0.5 mM IPTG o We have chosen to construct fusions to five different promoters: trxB, groES5, hslV, clpB, ibpA o To date, we have both uidA and mCherry (a red FP) fusions, as single crossover of constructs into CL150 genome. o We have now conjugated La rpoH and Sm rpoH1 constructs, plus the plasmid with no cloned insert (empty vector control) into each of the fusion strains Summary of accomplishments: we showed that the Ca. Liberibacter asiaticus rpoH sigma factor gene can complement a growth defect in a S. meliloti rpoH mutant. We chose 5 genes whose promoters are likely to respond to RpoH protein, and we have introduced our newly cloned Liberibacter gene into strains bearing fusions to those promoters. Our next step is on assay gene expression effects of La RpoH.
Fruit harvests have now been completed for this study (comprising 2 seasons: 2012-2013 and 2013-2014, two harvest Hamlin and two Valencia/season), but sensory, biochemical and chemical analyses on frozen samples continue. All analyses have been completed for the first year (2012-2013 season) including trained and consumer sensory panels, chemical analyses of sugars, acids, aroma volatiles, vitamin C, limonoids, flavonoids, electronic tongue and of bacterial titer of the juice using qPCR. Sugar, acid and aroma volatile analyses have now been completed for the 2013-2014 season, but peel oil, limonoid and flavonoid as well as sensory analysis is still under way. Liberibacter titer in the juice was determined by qPCR for the Hamlin harvests but not Valencia as yet. So far physical fruit measurements show that fruit from HLB-infected trees for last season are smaller and greener than fruit from healthy trees, regardless of nutritional treatments of which 3 were investigated so far for the earlier harvests. Sugars (especially sucrose), acids and ratio were generally lower in HLB juice, and acids were sometimes higher in juice from symptomatic fruit, regardless of treatment, except for one nutritional treatment for Valencia in March, 2013. Bitter limonoids along with many other flavonoids were higher in HLB juice for the first season, regardless of nutritional treatment and especially in symptomatic fruit or fruit from severely infected trees, although this is more the case in early season harvests. So far, the nutritional treatments have not shown a consistent effect, but there are sporadic positive effects on flavor chemicals. The nutritional treatments did not show an effect on reduction of Liberbacter titer in the juice as evidenced by qPCR analysis so far for the first season, nor in the Hamlin harvests from the 2013-2014 season. Several times, however one nutritional treatment has shown the ability to make the juice taste sweeter although no major effect on sugars was found. Now for the second season, the same nutritional treatment has shown little to no reduction of sugars due to HLB for the two Hamlin harvests (12/13 and 1/14) and Valencia (5/14) while all other treatments showed sugar reduction due to HLB. One of the new nutritional treatments added for the Valencia 5/14 harvest also showed less reduction of sugars due to HLB. The electronic tongue (etongue) and nose could discriminate between juices from healthy, asymptomatic-HLB and symptomatic-HLB fruit, with the etongue being much more effective. The etongue could also discriminate the different nutritional treatments within a harvest, but was confounded by seasonal changes across harvests. The etongue was more effective for Hamlin than Valencia, reflecting the more severe HLB-induced flavor effects for Hamlin in the first season samples, but the spring, 2013, Valencia samples did show separation indicating that the disease is becoming more severe for Valencia. Etongue has not yet been run for the 2013-2014 season samples. Trained panel showed differences in perception of orange and grapefruit, fruity, green and stale flavors and sweet, sour, bitter, metallic, tingling, astingent and umami (salty) tastes. Consumer difference-from-control panels showed that the panelists detect differences that correlate to lower ratio and/or higher limonoids in HLB compared to healthy juice. The differences is greatest when there is both low ratio and high limonoids. Aroma volatile analysis, now completed through 2014 show that some tope notes, especially esters, are generally lower in HLB juice. Samples were taken in recent harvests for analysis of peel oil and were processed and now run on GC-O (GC olfectometry) and GS-MS, but data not yet tabulated and limonene was analyzed with not differences noted.
Putative transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP was obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107CFU/ml. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. Several transgenic Carrizo with thionin increased canker resistance remarkably with infiltration test at the concentration 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Two experiments were undertaken to evaluate the effectiveness of reflective mulch to repel ACP, reduce incidence of HLB, and improve growth of newly planted citrus compared to standard practices. The first trial planted 5 May 2013 at SWFREC consists of 24, 250 ft. rows of ‘Ray Ruby’ grapefruit on ‘smooth flat Seville’ divided into 8 main plots, half receiving organic amendments since 1993, most recently with 12 tons/ac composted horticultural waste applied in a 6 ft swath to the plant drill. The plot had been underlain with drain tile and was flat except for a 6 in beds 36 inches wide on 18 ft centers covered with polyethylene film mulch with irrigation provided through two drip tape lines. Each 3-row plot is divided into 2 subplots: whiteface or metalized mulch. One of 160 leaf samples collected 28 Jan 2014 tested positive for HLB came white mulch compost plot. A second round of HLB leaf samples were collected on June 25 which are now in the process of being analyzed. Of 258 ACP found on sticky cards, 133 were from white mulch+compost, 78 white mulch no compost, 30 in metallized mulch compost and 17 in metalized mulch no compost. Only 13 infested flush were found in trees on metalized mulch compared to 30 infested on white mulch no compost and 81 on white mulch with compost (N=17,638). Trees in in compost plots had 22% more flush shoots than trees without compost, with no effect of mulch type on flush. Two growth measurements indicated no significant differences between mulch types but larger trees in composted plots compared to no compost. Insecticide drenches are applied as needed as well as other standard grove maintenance sprays. The second trial was initiated at the Florida Research Center for Agricultural Sustainability in Vero Beach: Land prep was synchronized with tree planting and reflective mulch installation on a 15 acre site. Beds were initially mowed and new weed growth was treated with a systemic herbicide application followed by a contact herbicide. Fill sand was applied to low areas within the tree line to prevent standing water. Finally, beds were subjected to 2 passes with a rotovator and rolled twice to pack and stabilize the soil. A randomized complete design was used with 4 replicates and 3 treatments: beds covered with metalized polyethylene mulch, beds covered with urban plant debris (UPD), and bare ground. Mulch (6 ft. wide) was installed centered on the surveyed tree line using a plastic laying machine that buried the sides of the plastic leaving 5 ft. exposed. (‘Ray Ruby’ on ‘sour orange’ trees were planted at 12 ft. x 25 ft. tree spacing (145 trees/Ac) on 17 March 2014. Containerized trees (pretreated at the nursery with a neonicotinoid) were ‘mudded in’ according to commercial practices. After planting, the irrigation system was installed with two drippers (2 g/hr each) per tree arranged on each side within 6 in. of the tree trunk. The compost treatment was then applied as a 5 ft. wide by 3.5 in. deep layer UPD. Fertigation with a liquid 6-0-8 (with minors) was included all irrigation events. Imidacloprid was applied by soil drench for ACP control within 2 weeks of planting. Typical commercial young tree caretaking was continued including weed control, fertigation, and canopy sprays of insecticides, fungicides and foliar fertilizers. A second neonicotinoid (Belay 2.13 SC) trunk drench application was made for ACP control 65 days after planting. Field data collected included volumetric water content (%VMC) of soil and trunk caliper measurements for each tree.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July 2012 on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. To date, 2125 psyllids have been found on sticky cards of which greater than 70% are in no-mulch plots while only 11% have been found in plots that receive insecticides. Only 2% of the ACP found on sticky cards come from plots with both mulch and insecticides. Thus far over 61,000 flush have been observed of which 7,934 were infested with ACP and 1685 were infested with Aphids. ACP and aphid infested flush are predominately in no-mulch plots with more than 65% of the total. Very few infested flush have been found in plots receiving insecticides (513 ACP and 558 aphid infested flush) while very few infested flush (100 ACP and 178 aphid infested flush) have been found in plots with both mulch and insecticide applications. Leaf samples for HLB testing were collected mid-January 2014 of which there were 20 positive samples none of which were from the mulched treatments while 12 were from the no mulch foliar nutrition without insecticide plots. All other non-mulch treatments were similar. The most recent HLB leaf sample was collected on June 16, 2014 but results are not yet available. Growth measurements made 2014 by measuring trunk diameter showed results of which are different from previous reports. Trees on mulch and not receiving soil drenches of systemic insecticide are 20% larger than on bare ground whether or not they are receiving foliar nutrition. However, mulch did not improve growth of trees treated with insecticide; actually the contrary. This may be due to failure of the irrigation system during the month of May resulted in worse drought stress in trees on mulch compared to bare ground trees that profited more from rainfall. Normal grove care operations continued which included one herbicide application in June of glyphosate, Kocide is sprayed monthly for control of canker, and one application of AgriMek to reduce ACP and other citrus pest populations to more realistic levels in all treatments. Verimark was applied as a drench in all insecticide plots late in June as well as a Nurpid application in May.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. ‘Hamlin’ orange on ‘Carrizo’ citrange rootstock was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.643 degrees S. -81.455 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 August. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. 2,655 ACP have been found on sticky cards of which greater than 70% are in no-mulch plots while only 14% have been found in plots that receive insecticide drenches. Only 3% of the ACP found on sticky cards come from plots with both mulch and insecticides. Thus far over 72,270 flush shoots have been observed of which 8,386 were infested with ACP and 1,737 were infested with aphids. Greater than 70% of ACP and aphid infested flush were found in no-mulch plots. Only 599 ACP and 559 aphid infested shoots were found in plots receiving insecticides with 122 ACP and 191 aphid infested shoots found in trees on both mulch treated with insecticide. Leaf samples for HLB testing were collected July 2014 of which there were 55 positive samples 12 of which were trees on mulch,10 from trees treated with insecticide with only 1 positive tree on mulch and drenched with insecticide. The most recent HLB leaf sample for nutrient analysis was collected on 16 June 2014 but results are not yet available. Growth measurements were made 1 July 2014 by measuring trunk area cross section. Trees on mulch and receiving foliar nutrition are now 20% larger than the no mulch no foliar nutrient control. However, trees receiving insecticide drench both with or without foliar nutrition but without mulch were larger than with mulch. The reason may be due to an irrigation system failure during the month of May which impacted trees on mulch more than those not on mulch which made better use of rainfall. Treatments applied: July 9: Foliar nutrition went out monthly Griffin Green 1gpa, August 5: 1 lb Fortress and 1 gal N-Sure in a 50 gallon volume, August 21: the entire field was sprayed by Duda with foliar nutrition 6-0-8 10 gpa, September 3: 1 lb Fortress and 1 gal N-Sure August in a 50 gallon volume. Normal grove care operations continued which included one herbicide application in September of glyphosate, Kocide sprayed monthly for canker control, and one application of Intrepid for leafminer control.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of July 7, 2014, a total of 5,824 transgenic plants have passed through inoculation process. A total of 115,175 bacteriliferous psyllids have been used in no-choice inoculations.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of July 7, 2014, a total of 5,824 transgenic plants have passed through inoculation process. A total of 115,175 bacteriliferous psyllids have been used in no-choice inoculations.
The Core Citrus Transformation Facility (CCTF) continued to function as a platform for testing of different genes that could potentially render Citrus plants tolerant to greening disease. Almost all of the efforts in the facility are directed towards a single goal-producing valuable transgenic plants that may survive greening bacteria-induced infection. Due to a present way of functioning, in some periods of time the CCTF becomes, to a certain degree, an extended arm of research labs. As such, we follow developments taking place in those labs. Recently, five vectors that were previously supplied to the CCTF by one lab were modified and are being used in the latest co-incubation experiments. These five vectors could be considered as new orders. There were no other new orders. Within the last three months, CCTF serviced old orders but the work was also done on the recent order placed by the CRDF. The progress was made for all the orders. For the existing and new orders, additional co-incubation experiments were performed with the appropriate plant material and Agrobacterium strains. Selection of putatively transgenic shoots based on the PCR screen continued without interruption. Production of rootstock plants carrying the NPR1 gene requested by the CRDF is continuing as planned. High number of shoots (~800) was tested in the primary PCR and about 120 of them were positive. Because of our effort to accelerate the production of this material, shoots harvested from explants were placed on medium with gibberellic acid (GA3) to promote the elongation prior to primary PCR screen. Elongated shoots are much easier to graft. The treatment with GA3 turned out to be detrimental for many of these shoots and about 30 of them that were positive in the primary PCR screen were lost as their elongated stems did not sustain grafting well. Of those shoots that were positive in the primary screen and survived the grafting, 27 were moved from in vitro environment to pots. Four of those plants were negative in the secondary PCR while 23 were positive. Those 23 plants are growing well on the light bench in the lab. For the last three months, CCTF produced plants for the following orders: pX4- 18 plants, pNah-three plants, pX11- two plants, pHGJ2- two plants, pHGJ10+ pHGJ11-five plants, pNPR1-23 plants, pNPR1-G-three plants, pELP3-G-one plant, pELP4-G- one plant, pMG105- two plants, and pTMN1-seven plants. Within this group of transgenic plants, two were C. macrophylla, three were Valencia orange, four were Swingle citrumelo, 17 were Carrizo citrange, and 41 were Duncan grapefruit.
The overall goal of this project is to 1) determine the overall effects of ACPS/open hydroponics growing systems on drought susceptibility and 2) the efficacy of plant growth regulators on mitigating the effects of preharvest fruit drop resulting from HLB. During the past quarter field data was collected for trees growing under traditional grower irrigation systems compared to ACPS “open hydroponics”. Additional data on the vulnerability to drought symptoms was also collected during this quarter on the two different experimental groups. The data are now being analyzed for a formal report/publication. Data collection will continue through the summer with support by the Horticultural Sciences Ph.D. student funded by this project. Preharvest fruit drop data from the 4-acre plant growth regulator trial in Lake Alfred are in the final stages of analysis. The anatomical data from citrus trees in the different experimental blocks are also in the final stages of sectioning and analysis. As part of an extension of this project a greenhouse study has been initiated to test the efficacy of 2,4-D and other plant growth regulators on root health in small trees with and without HLB. The plants have been potted, graft inoculated, and their initial root mass measured prior to the first applications of the PGR treatments. These trees will be evaluated regularly over the next six months for changes in growth and physiology in response to the PGRs and HLB. Additional field sites were selected in the Ft. Pierce area for expansion of the project to evaluate the efficacy of 2,4-D on preharvest fruit drop in grapefruit cultivars. The field experiment will begin this summer and preharvest fruit drop counts will begin starting in August or September.
A genetic construct from Dr. Mou has been transformed into mature scion of Valencia, Hamlin, Ray Ruby, Pineapple, and mature rootstock of Swingle and Carrizo. A number of PCR positive, putatively transgenic shoots have been micro-grafted onto immature rootstock. There are numerous PCR positive shoots in all varieties, which will be verified by additional molecular methods once the plants are larger. PCR positive Pineapple sweet orange shoots have been secondarily micro-grafted in the growth room and are in soil. We have been able to promote rooting of immature tissues in one week with IBA but not NAA. After clonal propagation of desired transgenics, budding in additional combinations will commence. The capacity of our PCR screening methods has been increased using a direct tissue PCR method, which does not involve performing DNA extractions. Putative PCR positives can also been determined by fluorescence of the PCR reaction in the tube on a UV light, without the need to run a gel, although gel electrophoresis is still mandatory. Additional constructs obtained from other UF scientist(s) have been transformed into the appropriate scion and/or rootstock. Shoots have been regenerated and micro-grafted onto immature rootstock. For some constructs, sequence and maps have still not been obtained and therefore work has not commenced with these constructs. A second-hand laminar flow bench was procured for the growth room. This will become a dedicated micro-grafting station. The numbers of transgenic shoots that survive appear to be greater if micro-grafting is performed first and the shoots are screened later. Since none of our constructs have reporter genes, earlier detection by GFP florescence or GUS staining is not possible. Southern blots of plants transformed with marker genes are underway. The nonradioactive DIG labeling kit is being used, which required a few adjustments (e.g. different membrane, different probe generation) compared to the standard protocol using radioactive labeling. Copy number information and expression data for these transgenics are being compiled for a poster presentation at the annual American Horticultural Society (AHS) meeting in Orlando at the end of July, 2014. Once copy number and gene integration has been demonstrated, it will be possible to determine the number of false positives. The gene gun is operational and is being tested on mature and immature explants of both rootstock and scion. We have been able to obtain calli suitable for biolistics by indirect embryogenesis of immature citrus explants, and plant regeneration should be relatively facile. Mature scion will also be used in biolistics experiments and it is anticipated that plant regeneration will also be improved, without the inhibitory effects of the antibiotics used to prevent Agrobacterium overgrowth after transformation.
In this quarter over 12 tetracycline derivatives were designed and synthesized and examined for antimicrobial activity against the causative agent of HLB (citrus greening), as measured against the surrogate laboratory strain Liberibacter crescens. Synthetic modification of the starting tetracycline(s) have yielded separate series of compounds that are more potent (3 orders of magnitude) than EPA registered oxytetracycline, and semisynthesis has yielded extremely potent compounds following specific structure-versus-activity relationships. In one series, molecular descriptors describing increased activity against HLB have emerged, and further chemical changes will result in the most potent derivatives against HLB while remaining inactive against human microbial pathogens and displaying “non-antibiotic” activity. These in vitro studies against the surrogate strain have delineated superior compounds that are currently the subject of greenhouse studies of Liberibacter infection in young and infected citrus trees. Currently, three compounds are being evaluated in controlled studies of Liberibacter infection at the USDA, in the Shatters and Powell laboratories, whereby young trees will be treated with the test compounds and the bacterial titer of the trees determined after dosing using different conditions and using differing formulations. It is expected that bacterial infection levels would be reduced and readily measured by quantitative PCR over time, and the dosage modes and concentrations adjusted in further studies to optimize anti-HLB activity. In other studies being conducted in the Gonzalez laboratory (UofF) three compounds, including the most active recently determined from in vitro studies, have been synthetically scaled up for further testing in greenhouse studies complementing those ongoing in greenhouse field trials. As further improvements to the compounds and their activity against HLB occur, other compounds are planned for synthesis to further increase their potency specifically against HLB. The most potent compound will continue to progress through greenhouse assays and field studies as planned to determine the most potent and ideal agricultural agent to treat HLB and to begin field studies for EPA compound registration.
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