Cytoplasmic citrus leprosis infected samples were sent from our cooperator in Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. Five sets of samples were sent during this funding period and again samples did not arrive in very good condition due to delays in shipment. PCR was done on all samples and all samples were prepared for electron microscopy to verify the PCR results. Viral particles as previously published for cytoplasmic citrus leprosis were discovered in all the samples. However the PCR was not positive for cytoplasmic citrus leprosis. Since the samples were not positive samples for mite transmissions were not sent to the quarantine lab at the USDA, ARS, FDWSRU in Ft. Detrick for endemic mite transmission tests. Mr.Leon in Colombia as previously reported did transmission experiments with the mites and these PCR negative isolates from Colombia. Funding for Mr. Leon’s work was received and he continues to do transmission tests. PCR positive samples were successfully shipped from Mexico and Panama to quarantine in Maryland as controls for PCR tests. In January the first experiments were planned with with the endemic mites from Florida and PCR positive leprosis samples from Colombia. However none of the samples were PCR positive. We began sequencing funded by another grant to find out why the samples were not PCR positive for cytoplasmic leprosis compared to samples from Mexico and Panama.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For the transgenic approach proposed in objective 1, besides transforming the Arabidopsis MKK7 (AtMKK7) gene into citrus, we are making transgenic citrus plants overexpressing SA biosynthesis genes. We expect that citrus transgenic plants overproducing SA would have increased resistance to citrus canker. Although exogenous application of SA does not increase resistance to citrus greening, increasing endogenous SA levels may have different effect. Transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene are currently under canker resistance test. We have propagated these plants for citrus greening test. We are trying to generate citrus transgenic plants overexpressing several other Arabidopsis disease resistance genes including ELP3 and ELP4. The mutant screen proposed in objective 2 has been continued. More gamma ray-irradiated Ray Ruby grapefruit seeds have been gamma ray irradiated. Part of the seeds will be plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Some shoots formed on these gamma irradiated seeds have been screened again on the selective medium. Part of the seeds will be directly sown into soil, and seedlings from these seeds will be test for greening resistance. We would like to test whether a direct genetic screen could work for identifying citrus greening-resistant varieties. We will continue to germinate gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculate the seedlings with psyllids carrying greening bacteria. We have been watching the development of greening symptoms on the seedlings.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We searched the citrus genome database (http://www.phytozome.org/citrus.php) with BLAST and identified nine genes similar to AtNPR1 or its Arabidopsis homologs. Among them, CtNPR1 (also named CtNH1) is the most closely-related to AtNPR1 based on phylogenetic analysis, supporting an orthologous relationship. The Figwort mosaic virus (FMV) promoter was used to overexpress CtNH1 in citrus. Previous studies in soybean showed that the FMV promoter is significantly stronger than the Cauliflower mosaic virus (CaMV) 35S promoter for gene expression (MPMI 21: 1027). Three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts have been identified. When inoculated with X. citri subsp. citri (Xcc), they all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xcc in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. CtNH1 plants have been propagated by grafting and are inoculating with Candidatus Liberibacter asiaticus (Las) in two laboratories. A microarray experiment was conducted using CtNH1 and non-transgenic Duncan grapefruit inoculated with Xcc. A needleless syringe was used to infiltrate the leaves with the bacterial culture (OD600 to 0.3). Three time points were used for this study. For each time point, three replications were used. Data analysis indicates that at p value <0.01, a total of 451, 725, and 2144 genes were differentially expressed at 6, 48, and 120 hours post inoculation (HPI), respectively. Using the visualization tool Mapman 3.5.1, the differentially regulated genes (Log FC ' 1 and Log FC ' -1) were mapped to give an overview of the pathways affected. Interestingly, at 120 HPI, a large number of genes involved in protein degradation and post-translational modification were differentially regulated. Furthermore, numerous genes involved in signaling also showed differential expression at this time. The results indicate that a large number of genes involved in the regulation of transcription were up-regulated in the transgenic plants at 120 HPI, and also at 48 HPI, although to a lesser extent. The photosynthetic pathway was affected to a larger extent at 48 HPI, which is signified by a large number of genes involved in photosynthesis being up-regulated in the transgenic plant when compared to the non-transgenic citrus. We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into 'Duncan' grapefruit. To date, ten transgenic lines have been obtained. They are ready for Las inoculation.
We now have citrus transformants in the greenhouse at UF for all of our constructs. PCR confirmations for all plants have been completed and indicate that from 17 to 88% of the surviving plants are positive for the R gene constructs. The least successful was the snc1 constitutive mutant expressed using the AtSUC2 promoter (2 positives out of 12 total). In addition, we have 11 (out of 15) citrus transformants containing the GusPlus reporter expressed using the AtPAD4 promoter, which we have recently found to be inducible by psyllid feeding. Our working hypothesis is that restricting expression of the R proteins to the phloem will lessen the negative impact that these proteins may have on normal growth and development. Transgenic citrus plants expressing either SNC1 or SSI4 wild type proteins using the phloem-specific AtSUC2 promoter appeared to exhibit a normal growth and leaf phenotype. This result was predicted since the respective R proteins should not have been activated in the absence of pathogens, and hence, would not trigger the hypersensitive response. In contrast, however, expression of the constitutively-active ssi4 protein in a phloem-specific manner resulted in approximately 60% of the plants with an obvious negative phenotype consisting of yellowish leaves, wavy-edged leaves, reduced internodal lengths, general stunting and leaf drop. This stunted phenotype resulted in death for 6 out of the 15 original transformants (40%). Although phloem-specific expression of the ssi4 mutant protein often (60% of the plants) resulted in either death or a negative growth phenotype, phloem expression of the snc1 mutant protein produced no abnormal phenotype. However, only 2 out of 12 plants were confirmed transgenics (AtSUC2/snc1). A similar tendency for the ssi4 mutant protein constructs to show an abnormal phenotype was also seen when expressed using the wound-inducible AtPAD4 promoter. The AtPAD4 promoter-snc1 construct showed no unusual phenotype. There are two questions that remain unresolved: 1) Why are some transformants showing a negative growth phenotype and others not, and 2) Do any of the R protein constructs confer resistance to Liberibacter? We are planning experiments that will evaluate the survival of Liberibacter in our transformed citrus lines. In addition, psyllid feeding tracks are being characterized histochemically to determine the relationship between AtPAD4/GusPlus reporter expression and the location of stylet sheaths and callose induction.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. It required serial dilutions of genomic templates to reduce interference of the inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with the presence of this specific construct. It is possible that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. These were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. Citrus leaves were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials were in the range of 40 mg. The primers used for PCR detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in specific standard curves in the real-time PCR reactions to determine copy numbers of respective genes. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las, 14 copies of Cox and Wg. Also, lower concentrations of PCR primers were more optimal. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our overall goal of the early Liberibacter detection in citrus plants.
New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. Trials of the new TMR wafers have now moved to evaluations in typical citrus growing areas of California. Mallet TMR dispensers are being weathered inside Jackson traps in citrus trees in Tulare, Ventura, Riverside, Kern, and Orange Counties of California. Two series of trials are being conducted in conditions representative of summer (August-September) and winter (January-March) conditions in California. Each week weathered wafers are sent to Kauai Island, HI or Hawaii Island, HI (USDA-ARS-PBARC) for fruit fly bioassays and to Farma Tech International, North Bend, Washington for chemical analysis using conventional gas liquid chromatography methods. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
Transcriptional analyses of citrus stem, root, and leaf responses to Candidatus Liberibacter asiaticus infection Candidatus Liberibacter asiaticus is known to cause Huanglongbing disease, which is currently the most destructive disease that affects citrus plants. Previous studies indicate that Ca. L. asiaticus is distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. The leaves, stems, and roots play distinct roles in the photosynthesis and transportation of water, nutrients, etc. However, the effects of Ca. L. asiaticus on gene expression in the stems and roots remain to be elucidated despite the recent progress that has been made toward understanding the transcriptome of leaves that are infected with Ca. L. asiaticus. Dramatic differences were observed in the transcriptional responses in the citrus leaves, stems, and roots to Ca. L. asiaticus infection. Overall, 1909, 884, and 111 genes were regulated in leaves, stems, and roots, respectively, by Ca. L. asiaticus infection. Only 2 genes overlapped in the leaves, stems and roots, whereas 289 genes were regulated in both the leaves and stems, 16 in the leaves and roots, and 6 in the roots and stems. The low similarities among the leaf, stem and root expression profiles indicate that Ca. L. asiaticus affects them all differently. Further analyses showed that Ca. L. asiaticus reprograms multiple cellular and metabolic processes in citrus and identified genes whose expression are regulated in organ-specific manners. Broad variations in expression levels were detected for genes that are involved in carbohydrate metabolism, cell wall biogenesis, lipid metabolism, hormone signaling, secondary metabolism, transportation, amino acid metabolism, and signaling and transcriptional regulation. Our analysis has revealed that Ca. L. asiaticus reprograms multiple metabolic and cellular processes in citrus and has identified genes whose expression are regulated in an organ-specific manner. Most of the genes were regulated in the leaves, followed by the stems and the least were observed in the roots. The genes that showed significantly altered expression between the organs were very different in each of the three organs, indicating organ specialization in response to Ca. L. asiaticus, which affects their distinct functions. Microscopy analysis has been conducted to compare the lignin, and anatomic changes in the stems and roots of HLB positive trees and healthy trees. In order to understand how seasonal variation affects the molecular response of citrus to infection by Ca. L. asiaticus, we conducted transcriptional analyses of infected sweet orange in Florida in June where temperatures are high (Summer) and in November where temperatures are relatively lower (Fall). With P value ‘0.05 and log2 fold change (LFC) of >1.0 or < -1.0 as cutoff thresholds, significantly altered gene expression was recorded for 2012 genes in Fall, and 1399 in summer. Of these, 1031 and 751 were up-regulated, while 981 and 648 were down-regulated in Fall and summer samples, respectively. A total of 330 genes were regulated under both growth conditions, out of which 214 were up-regulated, and 116 were repressed in the Fall, while 78 were up-regulated, and 252 were repressed in summer. At least 1682 genes were regulated only in Fall, while 1069 were affected in the summer. Ca. L. asiaticus regulated citrus genes were mapped into 29 out of 34 pathways/processes represented in the MapMan system. In both environmental conditions, Ca. L. asiaticus infection most significantly regulated genes corresponding to the following gene groups including hormone metabolism and response, transportation, signaling, photosynthesis, secondary metabolism, biotic and abiotic stress, development, lipid metabolism, carbohydrate metabolism, redox, and amino acid metabolism.
A series of transgenics, produced in the last several years, continue to move forward in the testing pipeline. Currently, it appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Several D4E1 lines appear to grow substantially better than controls even when infected, but do show the presence of CLas. Initial hopes of quickly identifying truly immune transgenics have matured to current efforts focusing on identifying significant resistance. For a disease that is devastating many Florida citrus groves, it is surprisingly difficult to get a consistent high-level of HLB disease even using known susceptible plants and inoculum sources with high levels of CLas. APHIS funded a grant, written by Gloria Moore, Ed Stover and Bob Shatters, focusing on identifying methods for highly-efficient and more standardized resistance screening, including exploration of strain x genotype effects. At the request of CRDF most FL researchers conducting research examining HLB-resistance met and shared observations and information. These data were summarized and used to finalize a set of experiments which are now underway. Jude Grosser and Ron Brlansky are playing key roles in addition to the grant authors. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support 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 more than twenty-one months. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place: Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M defensin transgenics produced by Erik Mirkov and his trees expressing the snow-drop Lectin (to suppress ACP) are now on the Stover permit. Information has been provided to complete the permit application by Eliezer Louzada of Texas A&M to plant his transgenics which have altered Ca metabolism to target canker, HLB and other diseases. 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 will be 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. Additional plantings are welcome from the research community.
The first objective of this proposal is to examine initiation of HLB infection after psyllid inoculation to investigate how introduction of the pathogenic bacterium into different types of flushes of a tree affects establishment of infection. For this purpose, we have been setting a number of trials using sweet orange plants that have young growing flushes and plants that have only matured flushes. These plants have been exposed to HLB-infected psyllids. Leaves on which psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. As a result in this experiment, we were able to detect presence of the bacterium fairly early after the initial exposure. Plants exposed to infected psyllids have been transferred to greenhouse and further monitored for the development of infection. Currently we are in a process of analyzing and comparing infection rates of plants with young flushes versus plants with only matured flushes. Our next objective is to characterize potential inoculum sources of the bacterium available for psyllids within an infected tree by examining presence of viable bacterium in different types of flushes that are produced during the development of the disease after psyllid-mediated inoculation of a tree and evaluating the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes during infection development and their ability to transmit infection to new trees. We conducted several trials in which healthy psyllids were placed on either a young growing flush or an older symptomatic flush of an infected tree. Psyllids were secured on those flushes by using small traps made up of mesh material and after 21 days psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. These plants are being monitored for the development of infection. Currently we are analyzing numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes. Another goal is to understand whether the HLB bacterium can have different forms or can be present at different stages in different types of tissues. One of the approaches that are being undertaken is to compare levels of the HLB bacterium genes expression in old symptomatic and young pre-symptomatic flushes. This work is ongoing. The third objective is to examine psyllid transmission rates from and to citrus varieties that are highly tolerant to HLB. We have propagated 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. Currently these plants are being exposed to HLB-infected psyllids. After 1-month exposure, plants will be moved to greenhouse and monitored for the development of HLB infection. Infection rates for these varieties will be analyzed and compared. Later infected plants will be used as inoculum donors to examine psyllid transmission to new plants.
The goal of this project is to obtain knowledge of the genetic and biological diversity of CTV isolates in CA and to identify and put in place mild isolates of the virus that can provide sustained protection against severe SP isolates. We are conducting molecular characterization of CTV isolates (Folimonova lab) collected from various regions in California. This approach will provide important information regarding genetic diversity of CTV populations in this region required for further selection of mild protective isolates. We have developed approaches for molecular characterization of CTV isolates by designing several sets of oligonucleotide primers that specifically amplify sequences of particular CTV genotypes (strains), including T30, T36, T3, VT, T68 and RB (Poncirus resistance braking strain) genotypes and further tested and optimized protocols for conventional reverse transcription PCR and quantitative real-time reverse transcription PCR amplification using the selected primers coupled with further sequencing analysis. We have analyzed the genotype composition of nearly 90 CTV isolates that have been collected from main citrus growing regions in California and then propagated in CTV isolates collections (CCTEA, CCPP). Most of the isolates had a mixture of the T30 plus VT genotypes. Few isolates have T3 or T36 genotypes. We also got amplification of PCR products with primers specific to the T68 and RB genotypes for a few isolates. The latter genotypes are not very well characterized, so follow up sequencing will be needed to determine whether some of the tested isolates contain those two genotypes. As the next step, we are setting up cross-protection trials using selected mild isolates along with isolates that cause severe stem pitting phenotype. Our first trial set has been already set up using T519 (mild isolate) and SY 568 (severe isolate) in Ray Yokomi lab at the USDA facility in Parlier. Currently plants that were inoculated first with the mild isolate and sequentially challenged with the severe one are being monitored for symptoms development. From the results of the molecular characterization of a large number of CTV isolates, we made a list of isolates (mild and severe) that will be used for additional cross-protection trials. We are now in a process of setting up those trials in Ray Yokomi and Georgios Vidalakis labs. In order to further understand how CTV cross-protection works in the field, we studied the cross-protection system developed by Klaus Bederski in Peru. This system represents an example of successful in-field selection of mild protecting isolates that were able to provide sustained protection of sweet oranges against severe stem pitting isolates that were introduced into the country. Molecular characterization of the Peruvian protecting isolates and isolates that caused severe stem pitting disease demonstrated that both protecting and severe isolates shared the same genotype composition: they all contained the VT genotype. This result very well correlates with the data that we obtained from our studies on CTV cross-protection mechanism and confirms that sustained protection could be provided only by mild isolates that have the same genotype as that of the severe isolate.
Eight experiments to estimate the viability of LAS over time, each conducted using a separate LAS-infected pomelo, have recently been completed. Data analyses from these experiments are ongoing. These are expected to be the final experiments of this type, and data from these experiments will be part of a publication. Time-series experiments to monitor LAS viability were conducted in four different types of media. These were: K (1/3 King’s B), J50 (50% juice from the infected pomelo), G50 (50% store-bought grapefruit juice), and G ( 100% storebought grapefruit juice). Replicate culture flasks for each media type were inoculated with LAS inoculum from seeds of the infected pomelo fruit. Starting immediately after inoculation and continuing every other day for a total of 10 time points (18 days), samples were collected from the culture flasks. Triplicate samples were frozen unmodified from the culture flasks, while another triplicate set was treated with ethidium monoazide (EMA) and then frozen. Additional samples of the media before and after each experiment were collected for later analyses, and pH and salinity of these samples were measured. Also during the course of the time-series, samples were collected and cultured on both 1/3 KB solid media and G50 solid media to see if any colonies would develop. As 1/3 KB media served as the control media, and previous experiments showed a quick loss of LAS viability in this medium, growth on these plates served as an estimate of contamination. Experiments with significant contamination were excluded from further analyses. No growth of any kind was seen on G50 solid media. Currently, data analysis of the frozen samples is being conducted. DNA extractions are being performed, and the DNA is being analyzed by qPCR to determine both total LAS DNA and viable LAS DNA (EMA-treated samples). So far, results agree with previous findings showing that LAS viability is maintained longer in media containing fruit juice. Also, plans are being made to analyze media sample nutrient concentrations before and after the experiments via inductively coupled plasma-optical emission spectroscopy. This will allow media composition comparisons and may help identify nutrients that LAS requires for growth. While conducting these experiments, it was noticed that a white biofilm forms at the air-liquid interface of many of the culture flasks. This biofilm was more frequent and clearly larger in flasks containing juice in the media. In an effort to reproduce this biofilm for further study, 50 ml conicals containing a glass slide have recently been inoculated with LAS to monitor biofilm formation. Any growth will be observed under a microscope, with the intention of gaining more information on the conditions needed to produce this biofilm in microfluidic chambers in the future. We would like to determine how this biofilm forms and if it contains any LAS cells or not.
The first objective of the project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. Unfortunately, the greenhouse was unable to be constructed due to miscalculations in the planning budget in the original proposal. Instead a growth room was constructed adding a clean head house structure that could be used as support of the growing area. It took approximately 7 months to construct the growth room. Up to date, almost a year after finishing the construction, we are still correcting a few problems with humidity, computer sensors and the water filtration system. Also, a generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and plant the rootstocks at the beginning of April 2011. At the same time three (3) sweet orange varieties were indexed in vitro and micrografted; the cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA are adjusting to our specific environmental conditions and clone specificities. In our conditions, mature Valencia was very responsive to organogenic regeneration. We obtained positive plants. Hamlin was also transformed but was less responsive to organogenic regeneration, we have a few positive plants, but we are still adjusting the protocol to improve the efficiency. Our control cultivar, Pineapple, has some quality problems with the starting material and new introductions are needed in the future, however since we don’t have too much space in the growth room, we are introducing another important cultivar for Florida. The Valencia positive plants are already growing in the growth room to be tested by PCR and Southern Blot.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. During year 1, most work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. We tested 19 diverse Arabidopsis lines for susceptibility to infection by psyllids and found significant differences among lines in the percentage of plants that became infected, and in the amount of bacteria present in tissue samples from each plant as judged from the Ct value of qPCR detection. No lines were completely resistant, the most resistant having only 1 of 5 plants infected. We did not detect any visible disease symptoms on leaves or stems of infected plants, and later experiments with a susceptible line showed no differences in root weight, number of fruits, number of seeds or other traits. However, seeds produced by most inoculated plants had much lower germination than those of non-inoculated plants. Potato psyllid nymphs can mature into adults on Arabidopsis, but the these adults soon die and do not lay eggs. We repeated screening of soil-grown plants of 10 Arabidopsis ecotypes for susceptibility or partial resistance using a larger number of Arabidopsis plants per ecotype to obtain better discrimination among ecotypes. Results of these experiments have not yet been analyzed. For chemical screening experiments, we must be able to grow plants in a system that facilitates introduction of small volumes of chemicals through the root system, and it must be possible to inoculate such plants with potato psyllids raised on infected tomato plants. Repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. This does not appear related to the size of the plant, but the culture condition may induce stress responses that make the plants immune to infection and/or reduce psyllid feeding. Late in the project year we began to focus on developing a system for chemical genomics assays in tomato. Small side shoots from tomato plants were placed in 50 ml culture tubes with the cut stem end immersed in water in a microfuge tube. This design was adapted from one shown on a poster by Ammar et al. at the Citrus Health Research Forum in Denver in October 2011. Adult psyllids are placed in the culture tubes and within 7 days most tomato shoots were qPCR positive for CLps. After two weeks Ct values ranged from 22 to 29. This system appears promising since chemicals can be introduced into the water for plant uptake. We have not yet demonstrated chemical uptake, but the system has all of the other essential characteristics necessary for a chemical genomics experiment and we plan to initiate these experiments early in year 2. The project received a no-cost extension to January 31, 2012 due to delays in initial funding, so there are 5 quarterly reports this year.
An Excel model that uses the income approach to asset valuation as the methodology has previously been developed by UF/IFAS to evaluate citrus investments in Florida. This model was modified to develop an economic model to evaluate emerging solutions to citrus greening. The model can be divided into three components: grove characteristics, disease characteristics, and economic variables. Each component enables the user to enter values for a number of variables that, when combined with variables from the other components, represent the planting and operation of a new grove, or the operation of an existing grove, as a business unit. Each variable can be changed to determine its effect on costs, production, revenues and cash flows. An economic evaluation of the tradeoffs among current greening management alternatives was then done that included the following: (1) removing infected trees and spraying to control psyllids; (2) not managing greening; (3) resetting removed trees; (4) switching to an enhanced foliar nutrient program instead of removing diseased trees and (5) replanting the entire grove when cumulative tree losses make it economically unproductive, and replanting to traditional tree densities versus higher tree densities. The results of this analysis were published on EDIS and in a citrus trade magazine, as well as presented at two industry citrus conferences, at two International Research Conferences on HLB, and at three grower meetings. An economic model was also developed that evaluates the grove replanting decision. The general principles of asset replacement were applied to orange groves, where the existing typical grove (defender) was compared to self-replacement with another typical grove and to replacement with a grove planted at high tree densities (advanced production systems, or APS). This was called the challenger. Two types of groves were analyzed: A traditional grove (defender) planted with 145 trees per acre and a grove planted with 270 trees per acre (challenger). The results showed that once the grove was replanted with an APS grove, if trees lost were reset, the grove never needed replanting. If trees lost were not reset, the grove had an economic life of 28 years, far beyond the typical planning horizon of 15-20 years. Thus, groves planted at tree densities similar to APS groves have either infinite or much longer economic lives. Data for both groves were from commercial groves in Florida planted at these densities. When data from an APS grove become available from this project, this model will be used to see if APS groves potentially have infinite economic lives. If they do, that suggests that APS technology offers a way for the Florida citrus industry to survive greening until a cure is discovered. This optimal grove replanting analysis was published in a leading Florida citrus trade magazine, and presented at both a scientific conference and at grower meetings. It is also in review for publication in the Journal of Agricultural and Applied Economics as ‘Optimal Asset Replacement under Conditions of Technological Change: The Use of Advanced Citrus Production Systems to Mitigate Endemic Citrus Greening.’
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