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. We have propagated the CtNH1 line by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). 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. Five transgenic lines have been obtained.
Funds for this project have not yet been received by Dr. McNellis. Penn State has assigned a fund number, but the Office of Sponsored Programs has not yet finalized a budget for the funds. Once funds are received, the development of the NodT antibody will be initiated immediately.
As proposed, a transgenic test site has been prepared 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 fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress 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 wihc have altered Ca metabolism to target canker, HLB and other diseases. Jude Grosser will be planting ~250 additional trees on the test site next week. 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.
One of the most recent challenges with deploying DMDS in the ISCA release device called SPLAT (specialized pheromone lure application technology) has been the phytotoxicity of the treatment to leaves and branches. A substantial amount of the DMDS active ingredient is needed in order to affect psyllid population densities. Unfortunately, we have found the DMDS active ingredient burns tree foliage and can even kill entire small tree branches, if applied directly to the wood surface. Therefore, we have been developing alternative release devices that would allow deploying the DMDS active ingredient within trees without touching tree surfaces. Our most recent prototype is a sachet that is hung in trees with a wire hanger. The sachet contains the SPLAT-DMDS, which is allowed to evaporate through a porous membrane. Therefore, the DMDS dispenser is deployed in trees; however, the active ingredient does not come in direct contact with tree branches or leaves. We are currently investigating whether these devices will be effective. The experiment was initiated in September and we are still analyzing data. The results of an earlier experiment conducted in August with a newer formulation of SPLAT-DMDS have been analyzed. The results of this test did not replicate the success we observed with the same formulation last fall. Psyllid populations were not significantly reduced by deployment of this formulation in this mid-summer test (as compared with control plots)as was observed last fall. We are unsure why we were unable to replicate the earlier success. We are trying to determine if the inconsistent results are because of differences in psyllid population densities between these tests or differences in environmental conditions when the different tests were conducted. It is possible that the formulation is not holding up to the intense temperatures and rainfall experienced in the summer as compared with cooler and dryer conditions in the fall. Finally, we have initiated testing of three new formulations of SPLAT that contain repellents other than DMDS that have proven effective against psyllids in laboratory tests. These experiments have only recently been initiated and we should have initial results in 3-4 months.
We have been investigating the mechanisms underlying why Asian citrus psyllid (ACP). We have found that ACP are attracted to common volatiles released by citrus such as .-ocimene and D-limonene, implicating these as general host selection cues. Both .-ocimene and D-limonene are predominant citrus volatiles released by citrus flush, foliage and fruits. However, the release of methyl salicylate (MeSA) was increased (presumably induced) in Las-infected plants as compared with uninfected plants. Correspondingly, this induced compound attracted ACP, suggesting that it may be a chemical cue that facilitates spread of pathogen. Insect herbivory can change the volatile profile of host plants. Insects that use a piercing/sucking mode of feeding have long-lasting interactions with plants cells and/or phloem. It may not be surprising that plant responses to phloem-feeding are distinct from that of chewing insects and that phloem-feeders often induce salicylate-dependent defense pathways commonly activated by bacterial, fungal, and viral pathogens. Such salicylate-dependent associations are implicated in the current investigation given detection of MeSA release from plants infected with the HLB causal pathogen. We hypothesized that psyllids (phloem-feeding insects) could also induce a plant response similar to that caused by the pathogen. Therefore, we investigated whether release of MeSA may be induced by ACP feeding and also used by psyllids as a cue for locating established conspecifics. Volatile collections from psyllid-infested plants detected induced release of MeSA, suggesting a coincidental convergence on a single cue that may simultaneously benefit the pathogen by deceptively attracting its vector, which also uses this cue to locate conspecifics. Movement of psyllids from infected to uninfected plants after initial selection of infected plants suggests that their initial response to olfactory cues may not be directly linked to the most beneficial host plant. Relatively more ACP responded to Las-infected plants under light than dark conditions. This suggests that visual cues may have also played role in attracting ACP to diseased plants. Yellowing of leaves is a typical initial symptom of Las infection on citrus plants. Furthermore, ACP adults are strongly attracted to yellow color. Las-infected plants were initially more attractive to ACP adults than uninfected plants; however, psyllids dispersed subsequently to uninfected plants to make them their final location of settling rather than diseased plants. The mechanisms underlying ACP host acceptance are currently under investigation. Specifically, we are trying to understand why leave infected plants after they initial prefer infected plants over uninfected plants. The duration of initial setting on infected plants was sufficient for ACP to acquire the Las pathogen. Thus, the pathogen is modifying the behavior of the vector by inducing changes in the attractiveness of the host plant through both olfactory and visual cues. This scenario suggests a mechanism for spread of the pathogen in the field because initial attraction and feeding of ACP on diseased host plants should facilitate acquisition of the pathogen while subsequent movement to uninfected plants should facilitate inoculation of uninfected plants. Overall, this behavioral manipulation of the vector by the action of the pathogen on the plant appears to favor spread of pathogen-induced disease. Our results indicate that MeSA may be the specific chemical cue mediating initial psyllid attraction to Las-infected plants. At low dosages, synthetic MeSA is an ACP attractant and at high dosages it is a repellent. We are investigating whether this can be used for possible management applications for ACP. To determine why ACP leave infected plants after after acquiring the HLB bacterium, we are more analyzing psyllid feeding preference between infected and uninfected plants as it relates to nutritional imbalances due to HLB infection.
In order to examine how Candidatus Liberibacter asiaticus (Las) infection affects gene expression in citrus tissues, transcriptional response of leaf, stem and root tissues of Valencia sweet orange (Citrus sinensis) to Las infection was compared using Affymetrix microarray. The analysis revealed that Las reprograms several metabolic and cellular processes in citrus, and identified genes whose expression is regulated in a tissue-specific manner. Tissue specific regulation was observed for several genes and gene groups including those encoding cell wall pectins, transcriptional factors with MADS box and PHOR1 domains, G-proteins, legume-lectin family proteins, pectatelyases, SUT4, SUC6, BAP12, protein kinase THESEUS1 and vacuolar invertase, which were regulated only in leaves; major intrinsic protein family proteins, crinkly 4-like protein, SUT1, ERF5, CPRD2, CNGC1, CSLD4, and FERONIA regulated in stems; and NAT12, GLR4, DDM1, SCL14, APS kinase, resistance protein RGC2, CCR4-associated factor 1-related protein and Arabidopsis response regulator 1 regulated in roots. 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). Genes encoding senescence-associated protein, dehydroascorbate reductase, stress enhanced protein 1, protein kinases, miraculin-like protein 2, actin related protein 2/3 complex, checkpoint-like protein CHK1, ACC oxidase, type I proteinase inhibitor-like protein, xyloglucan endotransglycosylase, protease inhibitor, and PR10-related Cas s 1 pollen allergen were up-regulated, while genes encoding photosystem II subunit R, DNA binding protein, a putative zinc finger protein, SLL1 protein, CCR4-associated factor, metallothionein-like protein, cysteine proteinase, cinnamyl alcohol dehydrogenase-like protein were down-regulated. Many of these genes were unaffected in the susceptible C. sinensis, suggesting genotype dependent response to Las. Sequence analysis and comparison of the expression of a set of pathogenesis-related (PR) proteins is being done using quantitative real-time reverse transcription PCR. To examine the molecular response of citrus to infection by Las under field conditions, Affymetrix microarray analysis was repeated using samples collected from Valencia sweet orange in citrus grove. The analysis is currently undergoing to compare the microarray results from the greenhouse, and the citrus grove (two different times). This research has significantly advanced our understanding of how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Importantly, the manipulation of host response especially plant defense response by Las provides valuable information regarding how to manage HLB.
Using Liberibacter from the alimentary canals of psyllids as inoculum, we have tested over 560 media formulations for growth of liberibacter. Some formulations support prolonged survival of Liberibacter in culture. After 2-3 weeks in culture, masses of Liberibacter have been observed in biofilms on the psyllid alimentary canals used as a source of inoculum and streaming from the canals into the media. We will to continue testing different formulations of the media to determine if they will support planktonic growth of Liberibacter. Information obtained from the genome sequence of Liberibacter and by comparing that genome with the genome of the closely related Babaco bacterium from culture is being used to devise new media formulations. Other possible growth factors, such as diffusible signal factors produced by the psyllids, are also being evaluated.
1. Data mining for ACP continues for annotated transcriptomes (phase I) of the whole adult and gut ACP to identify candidate genes for functional analysis, with an emphasis targets for siRNA-mediated silencing. These data have been used to design RNAi solutions for bioassay. 2. Illumina sequence data for ACP ESTs (from PAVE) was analyzed based on R-stat and normalized expression levels. Pairwise comparisons were done for the Adult,Nymph and infected adult and infected nymph libraries, revealing a number of genes differentially expressed in the four pairwise comparisons, based on transcript copy number. Not all genes that are differentially expressed in the Adult and Nymph were exclusively expressed in either one. The varying range of differential expression suggests that there are well defined developmental cues. Expression of genes in all pairs and the fold change ranges from very obvious to very subtle differences. 3. Potato psylllid ESTs.The Six adult psyllid libraries (adults, dissected salivary glands and alimentary canal) were used to construct mRNA enriched from total RNA extracts (Trizol). The paired Illumina libraries were prepared for sequencing, subjected to DNA sequencing, and are currently being assembled by NCGR. DNA sequences will be organized in PAVE, our data management system that constructs a database and has tools for mining transcripts, and will allow for subsequent RNASeq quantification (Soderlund). 4. FISH probe localization was highly successful for Liberibacter solanacearum localization in dissected guts using the 16S rRNA, outer membrane protein, and abc transporter genes probes when fixation conditions were modified and the new fluorescent camera was employed. Testing with the 16S rRNA for Carsonella ruddii (primary endosymbiont, potato psyllid) as an internal control, are underway to demonstrate specificity. Optimization is underway using the 16S rRNA probe for salivary glands and the oral box. FISH results for whole psyllids indicated decolorization will be essential and that this may confound the technique’s utility for intact adult psyllids, however, parameters are being tested in an attempt to overcome this limitation. 5. Feeding studies are underway to optimize an artificial feeding assay for potato psyllid using a system developed for whitefly. Psyllids were found to remain alive on sucrose feeding solutions tested for 3-7 days.
1. mtCOI haplotyping. More samples are being received for mtCOI haplotyping from the R. Lee lab (USDA, CA) to explore baseline diversities in different locations, in an attempt to relate populations from the US and elsewhere in the Western Hemisphere to haplotypes from a primary region of endemism. All sequences in hand have been edited. 2. Time-course and transmission studies continue bioassay to assess transmission frequency in relation to Rt-PCR detection of Ca. Liberibacter solanacearum (AZ) and in Ca. L. asiaticus (FL) in individual psyllids reared on infected plant material. Results using dot blot hybridization were found to be less sensitive than RT-PCR but were generally comparable with a few exceptions; nonetheless we will continue the work using qPCR even though it is the more expensive method, results are robust. Five psyllids reared on infected tomato, and given a range of inoculation access feeds on tomato seedlings from 30 min, and 1,2,4,8,12,and 24 hr transmitted Ca. L. solanacearum 5-20%, 35, 25-30,70, 80, 90, and 95% of the time (20 plants per rep). Plants were assayed using RT-PCR and were scored for symptom development. Only the former data were used to calculate transmission frequencies because psyllid feeding causes aberrations in symptom phenotype that disallow Liberibacter-incitedsymptoms to be differentiated consistently. This above result was corroborated in two replicated studies and a third replicate is underway. Results suggest that once acquired psyllids transmit at relatively high frequency. The goal is to select individuals for light microscopy, and SEM-TEM ultrastructural observations to pin point localization following a range of different AAP and IAPs. We initiated two AAP time points, starting with 8 and 24 hours using a 24 hr IAP. IAP studies with ACP have proven too difficult owing to the apparent uneven distribution of bacteria in plants, which confounds reliable detection and transmission bioassay to further the localization effort. The goal to produce time course feeding populations for both ACP and potato psyllid is overly ambitious given the problems experienced obtaining ACPs with consistent Liberibacter titer, despite season or host. Even so, extrapolations from the more tractable potato psyllid study system, (an approach approved by the project Director) will clearly help clarify AAP and IAP time-points in relation to organs that are key to the transmission pathway. 3. As noted we are using the potato psyllid as a surrogate for the Asian citrus psyllid since they are closely related, both have digestive systems so similar that we can’t find any differences using the methods and instruments available to us (Cicero et al 2010), and, thirdly, the potato psyllid is easy to rear on tomato in our Arizona laboratory. Thanks to timely publications (Cicero et al 2010, Ammar et al 2011a, b), collaborative efforts, and FPRAC quarterly/annual reporting by the teams involved, the general motif for the transmission cycle of Ca. Liberibacter has been worked out in the potato psyllid. We are in publication mode now. Further, we are now developing the toolbox to answer more detailed questions, such as the infra-instar changes in bacterial titers, and the role of the pharate instar in the movement of bacteria from the midgut, where the anatomically operational beginning of the cycle occurs, to the stylets, where the cycle finishes. We are also moving to apply the methods, used for the above breakthrough, on the Asian citrus psyllid as a check to be sure no significant differences occur in that species.
In this fourth quarter, 208 plants of Valencia orange on Kuharske Carrizo rootstocks, inoculated with HLB infected buds on the 3rd week of April of 2011 using a standard inverted ‘T’ budding technique as describe by Ferguson (2007), were individually tested for HLB infection monthly, starting on July 11, 2011. Tree leaves per each inoculated tree were harvested to verify HLB infection by quantitative real-time PCR (qRT-PCR). Petioles and midribs of the citrus leaf were 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 was estimated by measuring OD260 and OD260/280, respectively, with a NanoDrop Spectrophotometer ND-1000. The qRT-PCR for Candidatus.Liberibacter species was performed using 16S rDNA primer and probe sets as described by Li et al.(2006). The qRT-PCR consisted of 2 min incubation at 50 ‘C followed by 10 min incubation at 95 ‘C and 40 cycles at 95 ‘C for 15 s and 60 ‘C for 1.0 min. qRT-PCR data were analyzed using the Applied Biosystems software Version 1.4.0. The qRT-PCR results from July, August and September showed HLB infection on 0, 6, and 11 inoculated trees, respectively. Based on the low number of tree that acquired HLB infection 5 months after inoculation, starting on October, 2011 citrus trees will be re-budding with HLB infected buds. To obtain a base line of gene expression on leaves form HLB inoculated and HLB non-inoculated trees were sampled for RNA isolation on August 22, 2011. For control trees for each treatment, 10 mid-vein + petioles were pooled. For the HLB-infected trees, 18 were pooled (mid-vein + petiole only). A total of 448 frozen pooled samples were ground and immediately 1 g was weighed for RNA isolation and the rest was stored for back up as pre-ground RNA tissue. RNA was extracted from August 24 to August 30, 2011. The RNA extraction was done using the 3 day guanidine isothiocyanate (GITC) protocol and stored at -80 ‘C, gene expression will be determined by qRT-PCR. 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.
Since the recent release of the citrus genome sequences, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory. In some genes, however, we observed single nucleotide polymorphisms in our cloned genes, compared with the ones published in the citrus sequence database. We think that it might be due to the difference in the plants used in our lab from that used in the citrus genome sequencing. Nevertheless, we found that most published defense genes have full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of these genes is at various stages, some have been amplified from the cDNA library and cloned into pGEM vector while others have already been moved to the binary vector pBinARSplus. The genes newly cloned into the binary vector pBinARSplus include ctNHL1, ctMOD1, and ctJAR1. Since previously cloned ctEDS5 had many single nucleotide polymorphisms (SNP) from the published sequence, we recloned the ctEDS5, which now has fewer SNPs, into pBINARSplus. We have planted the corresponding Arabidopsis mutants plants and transformation will be conducted with the constructs, ctEDS5, ctNHL1, ctMOD1, and ctJAR1 soon. For ctNDR1 + ndr1-1 plants, we are continuously characterizing the defense phenotypes of the homozygous lines. So far we observed a range of defense phenotypes displayed by different individual plants, which is normal with independently transformed lines from one construct. Some plants with stronger disease resistance are also associated with minor cell death phenotypes. For ctPAD4 + pad4-1, ctEDS1 + eds1-2, and ctEDS5 + eds5-1 plants, we are trying to get more transgenic plants, obtain homozygous lines, and/or perform initial defense tests.
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. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. 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 Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
The main objective of this project is to examine how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development and how it correlates with the distribution of the bacterium within infected trees. During this period of funding we established a 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. Psyllid transmission experiments were initiated using already available HLB-infected plants. Additionally, we inoculated 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. The newly inoculated plants will be monitored for development of HLB infection and then used for transmission experiments. To examine acquisition of the bacterium from different types of flushes, healthy psyllids (as nymphs and adults) 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. However, TEM observations 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. We are now conducting experiments that will determine the viability of the bacteria acquired during psyllid feeding on different tissue sources. Another interesting observation was made during these experiments. Psyllids fed on the old symptomatic tissue exhibited phenotypical abnormalities. Our next step is to assess whether these developmental abnormalities are due to psyllid feeding on the symptomatic diseased tissue or similar phenotype can be obtained from incubation of psyllids on old flushes of healthy plants. During this period we also began experiments in which we are examining what types of flushes are more susceptible to psyllid inoculation. In these experiments infected psyllids are placed on different types of flushes of healthy plants. Plants are grouped in two sets: first set in which only young flushes are exposed to psyllids and second set in which psyllids are placed on old flushes. Psyllids are kept for a period of 21 days on those plants. The experiments are in progress. Percentage of plants that become infected will be analyzed.
The initial indexed mature material that was maintained in vitro for a long time did not adapt properly after planting in soil. The plants have already 3 months and they have very long leaves and are growing stunted, most of them are not behaving like normal plants. We are going to continue monitoring them the next couple of months to see if they recover and come out of this stage. The new batches of indexed mature material introduced from May to July 2011 are being grafted on Swingle citrumelo, Macrophylla and C. Volkameriana that were growing originally inside the laboratory. This material will also be used to establish mother plants and to establish the first batch of plants that will give us the material for mature transformation experiments. The rootstocks that we started planting when the growth room was finished are not ready for grafting yet. Only a few plants coming from this material were available to graft and the whole group will be ready in October 2011. The Growth Room is still under “modification”. It took almost 5 months to be able to have completely access to the computer program, however it is still an issue to get access for users that come and go which is the case with the personnel we currently have. The Citrus Research and Education Center does not have enough IT help to assist in a timely fashion with the several needs required for this project. The IT people usually respond within weeks instead of hours to any problem we may have. The humidifier in the small room is still not working properly. The company responsible for the job was not able to coordinate the different subcontractors to finalize the job. The warranty in this case will not work and we will need to pay for them to finish this task. Another problem that we are currently facing is related with filtrations among the different areas where the floor meets the walls but also window sealing. The water is moving between growth rooms and between the growth room and the office. The caulking applied to close the gaps and to provide a seal between the concrete floor and the panel walls is not working. The subcontractor came and applied a new coat of caulking but it was not enough to stop the filtration. It is still happening as of today. Water leaking through the window has not been addressed yet. We also experience problems with tripped breakers and bad electrical connections of some lamps. We are still monitoring the situation.
The causal agent of greening, Candidatus Liberibacter asiaticus ( L asiaticus) is a fastidious phloem-inhabiting gram-negative bacterium. It is transmitted by Asian citrus psyllid (ACP) during feeding. Transmission mechanisms of Las have been intensely investigated recently, yet key information gaps still existed. We investigated whether Las is transmitted between infected and uninfected ACP adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male ACP to uninfected females at a low rate (< 4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of seven days or more was required to detect the bacterium in recipient females. In addition, Las was detected in the unlaid eggs and offspring of infected females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy but were absent in ovaries from uninfected ACP females. The size of the rod shaped structures varied from 0.39 to 0.67 'm in length and 0.19 to 0.39 'm in width. The spherical structures measured from 0.61 to 0.80 'm in diameter. Although, the overall percentage of sexual transmission of Las from males to females appears small, it could be a highly significant contributing factor to pathogen spread given that thousands of psyllids colonize individual trees. Collectively our results demonstrate that Las is transmitted from male to female ACP at a low rate during mating. These results suggest that the presumed causal agent of HLB may spread within populations of the vector even in absence of infected host plants. These findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of ACP, even in the absence of infected host trees. Detection of Las in female psyllids reared on Las-free citrus plants after mating with infected males strongly suggests that this plant pathogenic bacterium was transferred sexually from male to female insects during mating. This was also confirmed with lack of transmission from females to males, or between same sex pairs. While this phenomenon has been demonstrated experimentally for endosymbiotic bacteria of insects, this is the first report of sexual transmission of plant pathogenic bacteria among insect vectors. The distribution of Las appears to be ubiquitous throughout the hemolymph and organs of infected psyllids, although the bacterial titer is highest in the alimentary canal and salivary glands. We observed a low bacterial titer (Cq values 37 to 39) in 2.3% and 8.9% of individual male and female genital samples, respectively, suggesting a low bacterial titer in the reproductive organs of ACP. Cq values could also be high because of the small amount of tissue that is processed during DNA extraction even though there may be a high bacterial load relative to the size of the organ examined. If the overall titer of bacteria in the infected psyllid donor is low, the likelihood of bacteria occurring in the reproductive organs should also be correspondingly low. Detection of Las in the unlaid eggs and offspring of infected females is congruent with previously reported transovarial transmission of Las by ACP. Sexual transmission of Las provides additional, albeit indirect, evidence that the relationship between the pathogen and ACP could be mutualistic rather than pathogenic.
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