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 objective 1, we have generated transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene. The transgenic plants are currently under canker resistance test. We will propagate these plants for citrus greening test. We have shown that overexpressing the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker, suggesting that the salicylic acid (SA) signaling pathway plays an important role in citrus disease resistance. We recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem with the support of a USDA special grant. Using the Arabidopsis-Xcc pathosystem, we found that mutants of the SA signaling pathway are more susceptible to Xcc. A manuscript about these results has been accepted by PLoS ONE. We are trying to generate citrus transgenic plants that accumulate high levels of SA. For objective 2, we are continuing the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Two quarts of seeds treated with gamma-ray irradiation at 50 Gy have been plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated were 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. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test. We are also testing whether a direct genetic screen would work for identifying citrus greening-resistant varieties. We germinated gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculated the seedlings with psyllids carrying greening bacteria. We are watching the development of greening symptoms on the seedlings.
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. Previous 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. However, 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. Therefore during the present quarter we focused on developing a system for 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. Adult psyllids are placed in the culture tubes and within 7 days most tomato shoots are qPCR positive for CLps. After two weeks Ct values are typically less than 25. 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 soon. The project received a no-cost extension to Jan 31, 2012 due to delays in initial funding, so there are 5 quarterly reports this year.
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. Previous work showed that the model plant Arabidopsis thaliana can be infected with CLps by potato psyllids. However, only plants growing in soil could be infected. We were not successful in inoculating plants grown in solid or liquid culture media as would be best for screening chemicals that may affect transmission or bacterial replication. Work during this quarter focused on testing methods to expose plants to chemicals before and after psyllid feeding. We tested growing plants in hydroponic culture but did not find CLps positives following psyllid inoculation. Additional testing is planned. We repeated screening of soil-grown plants of 10 Arabidopsis ecotypes for susceptibility or partial resistance using a larger number of Arabodopsis plants per ecotype to obtain better discrimination among ecotypes. qPCR assays to assess CLps levels in the plants have not yet been completed for all samples, but as in previous tests most plants are positive for CLps. Because of the difficulties we have experienced in inoculating Arabidopsis plants in culture systems that would be suitable for chemical screening, we will begin testing small tomato shoots in liquid culture.
In this project we are examining how the distribution of the HLB bacterium within infected trees affects the efficiency of HLB transmission by psyllids and what types of tree flushes provide better inoculum for psyllids to transmit the infection. During this period of funding we established and maintained a healthy-psyllid colony and an infected-psyllid colony for the use in our experiments. One portion of healthy psyllids is maintained on healthy Murraya paniculata plants in special insect cages and the other portion is exposed to HLB-infected citrus plants to generate HLB-infected psyllids that can be further used for inoculation of new citrus plants. Some of the transmission experiments were conducted using already available HLB-infected plants. In addition, we keep inoculating new sweet orange and grapefruit plants using the infected psyllids and graft-inoculation with HLB-containing buds to generate more plant material that can be used in our further experiments. One of the obstacles that we face in our research is that we have plenty of old symptomatic flushes on the infected plants, but much less often we have young, asymptomatic yet containing the bacteria tissue that grows on the infected plants. This can be explained by the fact that growth of HLB-infected plants (especially small greenhouse trees that we are working with) usually slows down. To overcome this obstacle, we try to generate more HLB-infected plants. For examination of psyllid acquisition of the bacterium from different types of flushes, 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. After 21 days traps were removed and 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. The numbers of plants that become infected upon inoculation with psyllids fed on different types of flushes will be analyzed and compared. Another observation: TEM examination of the material extracted from psyllids demonstrated that psyllids fed on different types of flushes contained different forms of Las: psyllids fed on the young flushes contained mostly rod shaped bacteria, while bacteria extracted from psyllids that fed on the old symptomatic flushes were mainly present in a round form. Our next step is to understand whether the HLB bacterium can have different forms or can be present at different stages in different types of tissues. Several approaches are being undertaken to characterize those forms. We are also examining what types of flushes are more susceptible to psyllid inoculation. In these experiments infected psyllids were placed on different types of flushes of healthy plants. Plants were grouped in two sets: first set in which only young flushes were exposed to psyllids and second set in which psyllids were placed on old flushes for a period of 21 days. The inoculated plants are being maintained and monitored for infection to analyze and compare a percentage of plants that become infected.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las is much shorter that we expected. In our population of psyllids in the containment room, the proportion of infected psyllids born on newly inserted healthy plants starts increasing after about 30 days suggesting that the receptor plants begin becoming donors at about that time. We are examining this process in more detail now. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and plants containing potential anti-psyllid genes for Kirsten Pelz-Stelinski’s lab and for Bob Shatters et al. lab in Fort Pierce. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or anti-psyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 50 new peptides that are now being screened. We are eliminating peptides that do not work and continuing to make and screen new ones. ‘ We have greatly improved our efficiency of screening. We are using small plants in order to screen faster. However, we have to balance psyllid damage with inoculation of HLB. We now are ‘pulse-inoculating’ plants by incubating them about 2-3 weeks with psyllids between intervals of no psyllids in the greenhouse. ‘ We have greatly improved the CTV vector to produce probably 100x more peptide. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
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. In another study, host response of Rangpur lime (Citrus . limonia Osbeck) which shows tolerance to the bacteria, to Las infection, was examined using suppression subtractive hybridization (SSH). Differential expression of selected genes of Ca. L. asiaticus in cultivars of citrus Our result indicate that the Ca. L. asiaticus genes involved in the survival and virulence in the plant are up-regulated in planta compared to the insect. We tested whether these potential virulence related genes would be differentially expressed in susceptible and tolerant varieties of citrus. Genes that were highly expressed in planta were selected, their expression were evaluated in selected cultivars of citrus. The selected genes included those involved in Heme biosynthesis, ABC transport of iron and Zinc, production of an RTX type toxin (CLIBASIA_01555: hemolysin) and a metalloprotease (CLIBASIA_01345: serralysin). The citrus cultivars selected ranged from those highly susceptible to Ca. L. asiaticus infection, to the ones that are tolerant. The expression of of these genes were significantly lower in the moderately tolerant and tolerant varieties of citrus, however, most these genes were expressed at very high levels in the sensitive varieties Riored, Kinkoji and Valencia. Thus, our results indicate that there is a significant difference in the expression of Ca. L. asiaticus genes depending on host reactivity to the disease.
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 have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. 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 Xac 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. A manuscript entitled ‘Overexpression of the Citrus CtNH1 Gene Confers Resistance to Canker Disease’ is in preparation. The CtNH1 plants have been propagated by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). No conclusive results can be reported at this time. Microarray experiments were conducted using the transgenic line CtNH1-1 and non-transgenic ‘Duncan’ grapefruit inoculated with Xac306. 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. A second manuscript describing these results is in preparation. 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. We will characterize these plants by Northern blot and propagate the lines with overexpressed CtNH1 for Las inoculation.
Huanglongbing (HLB) and Citrus Bacterial Canker present serious threats to citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters are used to generate numerous transformants of rootstock and scion genotypes. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events continue to grow better than all controls at 8 months after initiating the challenge, with 35% greater trunk-cross-sectional area increase than the overall experimental average and 64% greater growth than the mean of the controls, but do not show immunity to CLas development. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. Anthocyanin regulatory genes, give bright red shoots (UF Gray collaboration) and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Unfortunately, when antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight, presumably due to high levels of phenolic compounds. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 34 months and a repeated trial has been in the field for 22 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. Comparison of field-grown and greenhouse-grown valencia following graft-inoculation show much more rapid CLas development in greenhouse-grown trees. Several new collaborations are being explored to feed new HLB-suppressing transgenes and novel strategies into the citrus transformation pipeline.
A transgenic test site has numerous experiments in place at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than seventeen 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 transgenics produced by Erik Mirkov. Discussions have been ongoing with 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.
On May 8th, the research team met with growers at the University of Florida’s Citrus Research and Education Center in Lake Alfred. The project and the need for grower participation were discussed. At that time, one grower had provided data on the use of enhanced nutrient programs, soil and leaf analyses, and yield. Since then, other growers have begun providing data. As data are received, they are entered into Excel spreadsheets in preparation for analysis. Preliminary exploration of the data has begun.
In this fifth quarter, and based on the low number of trees that acquired HLB infection 5 months after the first inoculation with HLB infected buds, 200 plants of Valencia orange on Kuharske Carrizo rootstocks were re-infected with one HLB infected bud per plant during the last week of September and the first week of October, 2011 using the standard inverted ‘T’ budding technique as describe by Ferguson (2007). Each plant will be inoculated with one additional HLB infected bud on March of 2012. HLB infection will be verified monthly by quantitative real-time PCR (qRT-PCR) starting on February 2012 as described in the 9/30/11 quarterly report. In brief, tree leaves per each inoculated tree will be harvested to verify HLB infection. Petioles and midribs of the citrus leaf will be used (100 mg per sample) to extract total DNA using ‘Qiagen DNeasy Plant Mini Kit’. After DNA isolation the yield and purity of the DNA will be estimated by measuring OD260 and OD260/280, respectively, with a NanoDrop Spectrophotometer ND-1000. The qRT-PCR for Candidatus.Liberibacter species will be performed using 16S rDNA primer and probe sets as described by Li et al. (2006). While we are waiting for the plants at the greenhouse get HLB diseased, we are going ahead with an alternative plan using citrus diseased trees at Florida’s orchards, which will allow us to start applying the different therapeutic strategies in 2012 to evaluate if they enhance citrus response to the disease, prolog life of HLB-infected plants and reduce bacteria titer and counteract the detrimental effect on citrus production. Literature cited: Ferguson J. 2007. Your Florida dooryard citrus guide. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Document HS 884. Li W., Hartung J.S. and Levy L. 2006. Quantitative real time PCR for detection and identification of Candidatus Liberobacter species associated with citrus huanglongbing. J. Microbiol. Methods 66: 104-115.
Dr. Pena and his greenhouse manager traveled again to Florida last October 2011 to continue evaluating the project. They started evaluating the establishment of the citrus germplasm. They checked the cultivars to guarantee that they were true to type since passing through in vitro conditions for cleaning the germplasm can cause somaclonal variation. Plants that were not true to type were discarded. They also found that the germplasm was very clean but plants were shorter than they should be for their age. They suggested changing the photoperiod in the growth room to be able to manipulate the plant growth. The suggestion was discussed with the facility coordinator and steps towards a reprogramming process to have a better control of the program have been initiated but at this date the programmer was not able to start the job. The germplasm was organized in lots that will be the material of origin of the mature in vitro experiments in the laboratory. Mother plants were also selected to be transplanted next year. The inventory was organized and I was trained in how to establish a ‘production’ schedule with the current germplasm and how to plan and manage the lots in the future. The greenhouse personnel was again trained in grafting. The personnel is taking too long to master the technique, and they are not consistent in the quality of the job they do which does not help to establish a calendar. Training, checking, and keeping the personnel on task in the growth room has been extremely difficult. Dr. Pena and his greenhouse manager also dedicated some time to check the infrastructure and growth room protocols. We found that the humidifiers are still not working. As a result of many months of waiting the solution was to bypass the filters to get enough water in the humidifiers and to place the program in manual daily. It seems like the system was not designed properly and a new set of filters will replace the current system next year. The facility coordinator is still looking for somebody to do this job. Growth room protocols were checked. The use of coconut fiber as proposed initially has been postponed until we master the current situation without major problems. Use of coconut fiber requires more trained personnel that is not available at this moment in the growth room. Once we master the current situation we will be able to move forward on this. We started doing citrus mature transformation experiments in the laboratory. We used A. tumefaciens with the pCambia 2301containing the GUS gene as a marker. The first experiment was done the first week of November with a small batch of Valencia 1-14-10 and we were able to obtain some positive plants. They are currently micro-grafted in vitro and will be ready to transfer to soil by the end of January 2012. We will evaluate efficiency of transformation after a few experiments are performed with the different cultivars.
The following has been accepted for publication in the Proceedings of the Florida State Horticultural Society: Huanglongbing (HLB) was first discovered in Florida in 2005. In response, Florida citrus nurseries began treating rootstock seed trees located outdoors with insecticide applications to reduce risk of psyllid transmission of ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent. In 2008, a survey identified two ‘Carrizo’ citrange trees with symptoms of HLB. To assess the potential for seed transmission from HLB-affected seed source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 1557 seedlings germinated from ‘Pineapple’ sweet orange seeds from trees in Collier Co. were assayed by quantitative polymerase chain reaction (qPCR) using 16S rRNA gene primers. Of these seedlings, a single plant was positive for (Las+), although additional tests were negative. In 2009, no Las+ plants were detected among 332 ‘Murcott’ tangor seedlings from trees in Hendry Co. From nurseries in 2008, one Las+ seedling was detected in 290 seedlings from fruit located on symptomatic branches of two ‘Carrizo’ citrange trees, but it’s Las+ status was not confirmed after repeated testing. In 2009, a single Las+ result was obtained for one of 100 Cleopatra mandarin seedlings, whereas no Las+ seedlings were detected for 125 seedlings from seeds from two trees of ‘Swingle’ citrumelo, 649 seedlings from four trees of ‘Kuharske’ citrange, or 100 seedlings from one tree of ‘Shekwasha’ mandarin. Despite the occasional Las+ qPCR tests, no plants developed HLB symptoms. The most probable explanation for these results is transient transmission of Las from seed obtained from HLB-infected trees with no subsequent disease establishment.
We have continued our work to investigate the susceptibility of various Rutaceous plant species in Florida to Candidatus Liberibacter asiaticus (Las), and the psyllid transmission from these hosts to citrus. Also work has been done to determine the performance of the bacterium in these alternative hosts and to see if passage through them affects the biology (pathogenicity) of it. We have now developed improved methodology to quantitatively detect live bacteria inside HLB hosts. This technique will soon be reported in a published manuscript and will be available to other researchers. We have found that the population of live bacteria is different from the population of the total DNA detected in the different plants infected. The qPCR method has been correlated with direct counting of bacterial rods/field of view and this has been converted to rods/.L using parameters from the microscope. The logarithmic values of the rods/.L data were plotted against Cq values from their qPCR side and a standard curve by linear regression was developed using the data. We are using this with some of our different alternative hosts and with different citrus cultivars. No significant differences were found among the different citrus cultivars that were tested. More testing was done on various alternative hosts which included Severinia buxifolia, Calomondin, Xanthoxylum fagara, Citropsis gillentiana, Chiosya spp., Esenbeckia runyonii and Amyris texana. One hundred percent transmission was accomplished with 10 psyllids per plant from citrus to S. buxifolia but only a 46% transmission was accomplished from S. buxifolia to citrus. Positive transmissions were accomplished from citrus to Calomondin, X. fagara, C. gillentiana, Choisya spp. and to E. runyonii. Transmission results back to citrus are pending. Live bacterial population data and the live bacteria ratio (LBR) over a period of 12 months were obtained for both Severinia and sweet orange.
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