This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 75 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We also continue to screen transgenic plants for other labs.
Function of individual X. citri transcription activator like effectors (TALEs): The activity and specificity of specific X. citri TALE proteins PthA1-4 have been tested using two different approaches. In one approach, activity was tested transiently using a reporter assay in Duncan grapefruit leaves. In these assays, plants were co-inoculated with a reporter construct consisting of a 14 TALE binding element (EBE) version of the Bs3 promoter driving the GUSi reporter gene together with Agrobacterium containing individual pthA genes or combinations of genes. Our results from these studies show that on its own Xc PthA4 is the most effective activator of gene expression, however co-inoculation with other individual proteins increases expression. We also used a second approach in which stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct were inoculated with individual citrus TALEs introduced via X. campestris pv. campestris. We then quantified activity using GUS leaf disc staining and fluorescence MUG assays. In both assays, we could observe that individual TALEs did trigger expression of the GUS gene, so long as the corresponding EBE was present. This system also permitted us to examine the activity of pthA genes from a range of strains, including the sequenced Brazilian A 306 strain, A44 from Argentina, a typical A strain from Miami, an unusual strain isolated from Etrog in Florida,and a C strain designated #93 Brazil. TALEs from these strains triggered expression of the construct when matching EBEs were present. These data show that the promoter constructs are functioning as designed, with specificity for individual TALEs from a wide range of strains. We also now have a number of assays to evaluate TALE-promoter interactions to evaluate the roles of TALEs individually and in combination. Transformation and production of stable citrus lines: We have experienced difficulty in recovering functional stable transgenic citrus with from our transformation efforts. We have obtained many transformants, but to date we have not identified a line with a functional, intact transgene construct. In response, we are pursuing several alternative approaches to obtain stable transgenics, including further examination and testing of original constructs, preparation of new constructs in alternative vectors, additional controls, optimization of the transformation protocol, and new methods of transformation. We continue to regularly set up transformation experiments and analyze transformants by PCR, sequencing and pathogen testing. Given the successful function of constructs in transient reporter and disease resistance assays and the success of stable transformation with other constructs, we expect to recover functioning stable transformants through continued optimization of the transformation process.
Thermal treatment has been regarded as an effort to prolong the productivity of citrus trees infected by the Huanglongbing disease (HLB). A commonly known method of thermotherapy application in the grove is to cover or tent a tree, allowing solar radiation to raise the air temperature under the cover which subsequently raises the temperature of the tree. Two specific objectives were addressed: 1) to develop a simple in-field system that collects solar energy to raise tree canopy temperature, and 2) to extend the productive life of HLB-infected trees using in-field solar thermal treatment. In accomplishing objective 1, two thermal treating systems were developed. The first system was in the form of a mobile enclosure which covers individual trees with greenhouse plastic mounted onto a metal frame. In this system, temperature rise under the cover occurs solely by solar radiation. The second system was an improvement on the first system and used additional supplemental heat to make the temperature rise faster as well as provide more consistency in maintaining the intended temperature. Supplemental heat was provided by infrared heaters attached to the lower parts of the cover s frame. In accomplishing objective 2, the second system was used in an experiment with nine different treatments on mature citrus trees. The treatments varied in their temperature and duration. The temperature and duration combinations used were 45 C at 60, 120, and 180 minutes, 50 C at 40, 80, and 120 minutes, and 55 C at 20, 40, 60 minutes. The parameters used to evaluate the effect of the treatments on the trees were yield, juice quality, physiological markers (leaf water potential, stomatal conductance, and phloem area), and tree health markers (chlorophyll fluorescence, fruit set, fruit diameter, leaf area, and PCR analysis). It was found that treatments at 55 C regardless of the tested duration gave the best results. For the trees treated at this temperature, there was an increase in phloem area 30 DAT, fruit set, fruit size, and leaf areas had the highest values, and the PCR analysis showed a decrease in the number of CLas copies on the leaves after treatment. One of the major challenges of this project was the lack of reliable technique for detecting live to death CLas in plant as a results of heat treatment. The high chance of reinfection of treated heat trees also added to uncertainty of the effects of the treatment towards the disease and the plant. Due to this, it is difficult to conclude how thermal treatment is actually influencing HLB-infected citrus trees. Future work will include further investigation in determining how the treatment actually affects the disease in the plant and the plant itself. Additional future work also includes continued use of supplemental heat, primarily steam, and development of systems to treat both the canopy and the roots of the tree.
A new, rugged, heating tunnel prototype that incorporates two infrared radiant heaters and four fans was built. The heating and air circulation system has been integrated with an adjustable temperature control switch for automatic control of the temperature. As mentioned in our early report there was a lack of repeatability of live bacteria PCR analyses due to both intrinsic variability in the leaves and experimental variability due to the large number of steps required for DNA extraction procedures. Therefore, to assess the effect of the 2012 heating treatments on tree health at the time of fruit harvest, i.e. April 2013, we assessed overall appearance of the trees, and determined fruit yield and juice quality. We also determined the number of new shoots, new leaves and the mean leaf surface area in selected portions of treated and control trees. The main findings are summarized below. Qualitative observations: The trees that were treated in 2012 were those that showed the most prominent symptoms of all the trees in this research project. The trees treated in July of 2012 reached a temperature of at least 45 ‘C for at least 4 h. In April 2013, the treated trees looked healthier than the surrounding, untreated trees that now show severe symptoms. This suggests that the thermal treatment was effective in mitigating the progress of the disease. Because of cold weather, trees treated in September of 2012 did not reach 45 ‘C for as long as those treated in July of 2012. Trees treated in September of 2012 did not appear to be as healthy as the trees treated in July, but appeared healthier than trees that were infected and were untreated (symptomatic control). Overall, asymptomatic (healthy control) trees looked healthier than treated or untreated trees. A large amount of fruit drop was observed for all trees. Quantitative results: Data from the April 2013 harvest showed that the average fruit yield for the healthy control trees was 217 lb/tree. Trees treated in July yielded an average of 200 lb/tree. Symptomatic control trees had a mean yield of 162 lb/tree. Although yield variability was high, (standard deviations in the order of 50 lb) these results suggest that treated trees had a yield close to that of healthy trees.Healthy control trees had the same mean soluble solids content (SSC) of 12.0 ‘Brix as trees that were treated in 2012. In contrast, untreated symptomatic control and trees that had a mild heat treatment had a mean SSC of 11.3 and 11.0 ‘Brix, respectively. Mean new leaf surface area of healthy trees (16.6 cm2) was slightly greater than that of treated trees (13.4 cm2). In contrast, the mean surface area of new leaves from untreated trees (7.6 cm2) was considerably smaller (about one half) than that of healthy or treated trees. This suggests that symptomatic trees suffering stress conditions might have slower canopy foliage development compared to healthy trees. Also, thermally treated trees have good health/vigor compared to symptomatic control trees. Primarily, it was observed that healthy trees have well-developed medium to dense foliage from past seasons; whereas, symptomatic control trees have thin foliage. The number of new shoots in a 0.5 x 0.5 m frame was on average 30 for healthy control trees, 56 for untreated symptomatic controls, and 42 for treated trees. In conclusion, results from 2012 experiments suggest that thermal treatment is effective in mitigating the effects of HLB. It is anticipated that the use of fans and electric heaters will result in more uniform and faster tree treatments.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ Finished the 2008-2012 fungicide sensitivity tests. During this trimester, 182 isolates were tested for sensitivity to azoxystrobin and pyraclostrobin using the resazurin-based microtiter assay. ‘ Out of 182 isolates tested, 55 were resistant while the remaining isolates were sensitive. ‘ In summary, from 2008 to 2012 survey, 817 isolates were tested for QoI fungicide sensitivity. The number of resistant isolates were 471 (57.6% of the total isolates) ‘ DNA extraction of 123 isolates was done in order to identify the point mutation that corresponds with the resistant phenotype. ‘ In total, 235 isolates were DNA extracted from the 2008-2012 survey, including eight isolates from our baseline collection. ‘ RFLP-PCR analyses were performed using the cytochrome b gene amplification of the 235 isolates to identify the G143A mutation in resistant isolates. ‘ In summary, 161 isolates were resistant and all of them carried the G143A mutation. In contrast, the remaining 74 isolates were sensitive and none of them had the G143A point mutation. ‘ We found two genotypes associated with the partial structure of the cytochrome b gene, named profile I and profile II. ‘ Two experiments were conducted using detached leaves from 4 cultivars using 10 isolates (5 R and 5 S). In vitro inoculations were performed to evaluate some fitness parameters related with incubation period and number of lesions per cm2 in individual detached leaves. ‘ In vitro fitness components were established using the same group of isolates described above (5 R and 5 S). Fitness parameters evaluated were: mycelium growth, sporulation and conidium germination. ‘ A second experiment under greenhouse conditions was conducted to test the ability to infect tangerine plants previously applied with a full rate (15 fl oz) of Abound (azoxystrobin). During this experiment 10 different isolates were used (the same isolates used in the experiments described above). After 5 days of incubation, the numbers of lesions were counted on individual leaves per treatment-isolate combination. A significant difference (p<0.001) was found between sensitive and resistant isolates, means that resistant isolates were able to infect plants treated with the full rate of fungicide. ‘ A second experiment using the spiroplate method was done to test boscalid sensitivity from 16 different isolates. ‘ The paper ‘Distribution of QoI resistance in population of tangerine-infecting Alternaria alternata in Florida’ was finished and ready to be submitted in Plant Disease Journal within the week.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 8 full-length genes in these categories in the entry vector pJET and two of the genes were further cloned to the binary vector pBIN19plusARS. Transformation of Arabidopsis and citrus plants will be simultaneously performed to obtained transgenic plants over-expressing the constructs for further analysis of plant disease resistance. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A manuscript describing the cloning and characterization of the citrus NDR1 ortholog was recently under revision in the journal Frontiers in Plant Science.
Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (Las) infection has emerged as a major threat to citrus production worldwide owing to the fact that it can take up to two years following infection before outward symptoms become apparent. The goal of this project was to find metabolic indicators for Las infection in citrus that is complementary to PCR, and may be useful in areas where Las remains an exotic threat such as California. Our work began looking at the effect of this pathogen on citrus fruit metabolism. Through measuring metabolites in healthy, asymptomatic, and symptomatic fruit, we were able to detect distinct differences that provided some potential information on the mechanism of Las attack. Specifically, we determined that the bacteria may be suppressing plant defense mechanisms, such as preventing generation of hydrogen peroxide, nitric oxide, and cinnamic acid, all compounds that can aid in killing potential pathogens. This work was published in the Journal of Proteome Research in 2012, and has provided valuable preliminary information for understanding the mechanism of Las infection. At the same time, we began to study the effect of Las infection on citrus leaves. Through collaboration with the USDA in Fort Pierce (Mark Hilf), we obtained leaf samples from a longitudinal study in which asian citrus psyllids (ACP) carrying Las were released into a controlled greenhouse and allowed to infect the citrus plants, including varieties of sweet orange. At the end of the experiment, none of the trees were symptomatic for Las infection, and approximately 8-9 months after infection, sweet orange varieties of citrus tested positive for Las. Our work indicated that we could detect obvious plant metabolic changes, related to stress and plant defense, 3-5 months prior to detection by PCR-based methods. These results provided the necessary information for us to propose extending our project by an additional 2 years. The work that we started this year continues from our first year of funding. We have obtained additional samples from other longitudinal experiments performed by Dr. Hilf that were started at different times to rule out any changes in plant metabolism caused by weather. These new experiments appear to confirm our previous findings. We have also coordinated to collect leaf samples from citrus infected with various pathogens such as citrus tristeza virus (CTV) or citrus canker. In collaboration with Dr. Cynthia LeVesque and Dr. MaryLou Polek, we have received citrus leaf samples that have been damaged by Las negative ACP. We have determined that the metabolic signature from insect damage is different than the signature from Las pathogen infection further confirming that our results in the longitudinal experiment were due to the pathogen rather than damage from the insect. We are currently in the process of writing the results from the Las negative ACP experiment for publication.
In the past few months, the most significant progress made on the FT project was the completion of the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter. This construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. In order to ensure that the cDNA was as effective as the genomic, this FMVcDNA27 construct will be compared to the original p27 construct which contains a genomic FT3 insert in the pCAMBIA2201 vector with the FMV promoter. Transformation of Carrizo and tobacco tissue is already underway in order to compare the action of these two constructs. Additionally, we have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we will begin development of the inducible promoter constructs.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 3,000 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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. This report marks the end of the third quarter of the project, during which we have began large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. A total of 2,124 transgenic plants have passed through the screening program. A total of 43,680 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Maintaining an open infestation of infected psyllids (phase 2 of the inoculation process) was challenging this past quarter because of surprise pest problems, notably western flower thrips which invaded the greenhouse initially attacking new flush and then first instar psyllids (facultative predation). Tamarixia radiata invaded the house during January, killing a majority of psyllid nymphs. Measures have been taken to limit these unwanted pests, but these measures are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects for the control of spider mites and thrips.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant gene(s). First group of samples for RNA-Seq were selected at Picos Farm at Fort Pierce, including three Jackson grapefruit plants (resistant/tolerant) and three Marsh grapefruit plants (susceptible). Five pioneer Illumina paired-end libraries were constructed and sequenced by Hiseq-2000 at BGI. Each library has about 10 M 2.90 bp paired-end reads with more than 10 G data. We maped the RNA-Seq data to reference genome, C. clementina using the computer program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24275 genes of original predicted genes had been found with expressions. And a total of 10539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to have an expression. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of EST varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component analysis (PCA) study on the expressions of five samples. The plot of first PC against second PC showed that R2017 and R20T18, were grouped to the one group (resistance group) and R19T23, R19T24 and R20T24 were grouped to another group (susceptible group). This result showed that the gene expressions were significantly different in resistant and susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and FDR threshold of 0.01. Among them, 306 genes are up-regulated expression genes and 515 are down-regulated in resistant citrus. Using program iAssembler, a total of 53981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, comparing with the citurs reference genome. To reveal the differences in resistance, we also identified the exon variations (SNP/INDEL). A total of 612618 SNP/INDELs were identified using mpileup method employed in samtools. We focused on two types of mutations that could contribute to the resistance difference. The first type mutation is the mutation in the genes of susceptible citrus leading to pseudogenes (Type1) and the other type of mutation is the mutations in resistant citrus genes that may gain a new function (Type2). Type1 mutation should have a homozygote mutation genotype in the susceptible citrus. We identified 146 candidate genes having Type1 mutations, which had high impact variations, such as frame shift, splice site acceptor, splice site donor, start lost, stop gain or stop lost and 3578 genes with Type2 mutations. We expect that with more libraries being sequencing, the candidates should be reduced to a reasonable number for further validation.
The main goal of this project is to assess how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. Electron microscopy examination of the sites on the leaves of citrus plants where HLB-positive psyllids fed for 7 days demonstrated that even at early stages of infection (starting from 14 days after the beginning of the experiment) the bacteria could be already visualized in the initial sites of introduction. To characterize inoculum sources of the bacterium available for psyllids within an infected tree, we are 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 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. We also conducted a similar experiment that was slightly modified in a way that psyllids fed on old and young leaves that were detached from plants and kept in 50 ml tubes (‘detached leaf experiment’). Some differences in the bacterium acquisition were obtained from these two experiment series. On average 48.33% of psyllids fed on old symptomatic flushes tested positive and 58.33% of psyllids fed on young pre-symptomatic flushes were positive. In the ‘detached leaf’ experiment, an average acquisition from young pre-symptomatic tissue was significantly higher than from old symptomatic flushes: with average of 64.26% and 23.9%, respectively. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected (52% of Duncan grapefruit plants and 53% of Madam Vinous sweet orange plants) and less proportion of receptor plants inoculated with psyllids that fed on old mature flushes got infected (19 and 33% of the same varieties, respectively). In order to assess what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria, we exposed sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes to HLB-infected psyllids (“no young flush” plants). According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. The next 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 were moved to greenhouse and monitored for the development of HLB infection. The first four varieties showed the highest infection rates (80-100% infection), while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. Overall, our results support the initial observation of young flushes being more likely crucial for the disease spread at both steps of the pathogen transmission, either acquisition and inoculation are higher when young flush are present. Nonetheless, transmission associated with old tissues, which occurs at a reduced level, should not be ignored also.
In this project we are examining ways to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. We are conducting initial experiments to examine the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We already prepared plant material that will be used in this project. Inoculum sources (different isolates of CTV propagated in the greenhouse as well as collected on the field) are also available. A series of experiments to assess the effect of preexisting CTV infections on multiplication of the superinfecting vector in inoculated citrus trees are ongoing. We first graft-inoculated sweet orange trees with the T36 or T30 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field. In different experiment sets we are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. For another objective, to select rootstock/scion combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector, we are preparing trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants are now growing and later will be used for the experiments similar to the experiments described above.
HLB incidence is approaching 100%, especially in young groves. The 2012-2013 season was dry before July and after September to the present. Fruit drop statewide has led to five reductions in the USDA crop estimate (unprecedented). The concensus among researchers and growers is that most of the drop is due to HLB. Fruit drop is greater than in past seasons due to increased HLB incidence and disease effects which we defined from our recent greenhouse and field studies on root health of HLB-affected trees,i.e. 1) Candidatus Liberibacter asiaticus (Las) moves to the roots after initial infection /transmission in the shoots; 2) Las infects structural and fibrous roots; 3) Las colonizes the roots before the shoots, phloem is not plugged; 3) the infection causes a rapid fibrous root loss of 27-40% before symptoms in the canopy; 4) Phytophthora interacts to further reduce root health but the majority of the root loss is due to HLB; 5) Phytophthora populations of HLB trees in groves initially increase and then the populations decline rapidly as roots are lost due to Las infection, . Statewide drop in Phytophthora counts in 2012 may indicate more HLB-induced root loss which accelerates fruit drop. This Phytophthora population dynamic was confirmed in potting soil at 2, 8 and 14 mpi for bud-inoculated trees (HLB+) and mock-inoculated (HLB-) trees. We measured a 27-40% reduction in root density for presymptomatic and recently symptomatic HLB trees. Root loss equates with the ‘ 30% yield losses on early symptomatic trees in Florida treated with good irrigation and nutritional management. Recent population survey of a mefenoxam treated block showed a significantly lower population of Phyopthtora per root mass than in the adjacent non-treated half of the block. Hence, we are recommending that, when the Phytophthora count is >10-20 prop/cm3, the fungus should be managed aggressively to sustain root health. We are furthermore recommending alternation of fungicides as follows: after spring shoot flush – Aliette/phosphites; 45 days later ‘ mefenoxam (injection); 45 days later – Aliette/phosphites; After fall shoot flush ‘ mefenoxam (injection). The costs of root health management should be balanced with other resources for HLB, i.e. psyllid control, best management practices for irrigation and nutrition, as well as, control of other pests and diseases.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Multiple assays to determine the ability of canker strains to move in response to chemical stimuli have been concluded. Differences among species and types of xanthomonads were found for motility, chemotaxis, bacterial growth and the profile of chemicals that act as chemotaxis inducers. The diversity of chemotaxis profiles was related to the patterns of methyl-accepting chemotaxis proteins (MCPs) that act as sensors. Cluster analysis of chemotaxis profiles and MCP sequences grouped narrow host range citrus bacterial canker (CBC) strains into the same clade. In addition an in silico study was performed to identify MCPs from complete genome sequence in databases. MCP sequences were clustered in twenty seven phylogenetic groups, fifteen of these groups included MCP sequences found in every strain and are considered conserved in Xanthomonas. Twelve groups are restricted to certain strains and of interest due to their possible link with the unique chemotactic profile for strains associated with host range. Moreover, differences among strains were detected mainly as amino acid modifications in the putative ligand binding domain.To confirm chemotaxis at an early stage of the infection and to determine possible differences due to host range, the bacterial strains were exposed to leaf fractions from different plant species. No response was elicited by leaf washings, revealing inability of absent or low concentrations compounds to stimulate a chemotaxis response on the intact leaf surface. In contrast, apoplastic fluids definitely produced chemoattractant and repellant responses, even more so than those produced by crude leaf extract. Therefore the natural chemotactic interaction is probably due to apoplastic fluids emanating from opened stomata or leaf damages. Moreover differences were shown between narrow and wide host range strains of CBC in their chemotaxis behavior. Objective 2 (To investigate bifofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads). Type IV pilus from Xanthomonas citri subsp. citri is being purified in order to obtain antibodies as a strategy to confirm such protein is a main component of the protein fraction of the biofilm matrix. In addition assays to detect cellulose and amyloid fiber production by CBC strains, using calcofluor and Congo red have been started. Differences in biofilm formation among the diverse CBC strains as compared to X. campestris and X. alfalfae subsp. citrumelonis were shown in minimal or nutritive culture media. In addition assays to evaluate presence of DNA in the biofilm matrix are under progress by varying DNAse concentration during the biofilm development or for biofilm removal.Finally, initial assays of gene expression revealed differences in the level of transcription between wide and narrow host range strains of CBC for genes related to biofilm and motility.
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. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, 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 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. Early in the next quarter Dr. Grosser is removing the unsuccessful trees from the first planting and planting additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
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