Other


Pathogen-Vector Relations between Asian Citrus Psyllid and Liberibacter asiaticus

Report Date: 07/13/2012   Project: Hall-93

Pathogen-Vector Relations between Asian Citrus Psyllid and Liberibacter asiaticus

Report Date: 07/13/2012
Project: Hall-93
Category: Other
Author: David Hall
Sponsor: Citrus Research and Development Foundation

This grant started in May 2009. Three peer-reviewed journal articles have been published and three others submitted for publication so far. Following are some of the key findings in this project on the interactions of the Asian citrus psyllid (ACP) and the HLB-associated bacterium, Candidatus Liberibacter asiaticus (Las): (1) Transmission barriers and localization of Las in organs and tissues of ACP: Our results show the near systemic infection of ACP organs and tissues by Las. Additionally, the proportion of Las-infected salivary glands was significantly lower than that of infected alimentary canals or other organs suggesting that the salivary gland constitutes a major barrier to Las transmission by ACP (Ammar et al. 2011, Ann. Entomol. Soc. of Amer. 104:526-533; Ammar et al. 2011, J. Phytopath. 159:726-734). Furthermore, ultrastructural studies indicated that the food and/or salivary canals in the maxillary stylets of ACP may be also a barrier to Las transmission especially in younger nymphs (unpublished results). (2) We developed a new ‘excised-leaf assay’ method that potentially speeds up Las-inoculativity tests on ACP from the current 6-12 months, when using whole citrus seedlings for inoculation, to only 2-3 weeks when using excised citrus leaves (abstract: Ammar et al. 2011, Proc. of 2nd Inter. Res. Conf. on HLB, Orlando, FL, Jan. 2011). We demonstrated that excised citrus leaves can be successfully used to assay the rate of inoculativity in ACP adults, and that such assays can save considerable time, material and greenhouse space, and may hopefully enhance vector-relation studies on Las and probably other Liberibacter species associated with HLB. The percentage of Las-positive leaves, when single ACP adults were tested per leaf, ranged from 2-6% using the more conventional HLBaspr primers, and 10-20% using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves, however, were obtained with the following: a. increasing the number of inoculating psyllids (to 5-10 adults) per leaf, b. longer inoculation access periods, and c. incubation of the excised leaves for 1-wk post-inoculation before being processed for qPCR. Furthermore, logistic regression analysis indicated a positive correlation between Las titer in the ACP adults tested singly and the probability of detecting Las in the inoculated excised leaves, which can be very useful in epidemiological studies, e.g. to predict the rate/proportion of ‘Las inoculative’ psyllids from that of ‘PCR-positive’ ones in field samples collected in various locations or at various times of the year, which can provide timely and valuable information for monitoring and/or combating HLB (full paper submitted for publication). (3) Las replication in ACP nymphs and adults: We conducted three large experiments to study the effects of various acquisition access periods (AAP) on Las-infected citrus plants by ACP nymphs and adults on Las acquisition, transmission and replication in psyllids. In the first two experiments, using qPCR, the relative Las titer was generally higher in ACP nymphs than in adults and higher with longer AAP in both life stages. We are currently finishing and analyzing the third experiment by qPCR to confirm these results. (4) Development of new techniques: In addition to the ‘excised-leaf assay method’ mentioned above, we developed new and simple methods for rearing ACP (Ammar & Hall 2011, Florida Entomol., 94:340-342), and for studying the stylets, salivary sheaths and bacteriomes (organs containing symbiotic bacteria) of ACP and other Hemiptera, which can enhance various studies on vector relations of HLB and other hemipteran-borne pathogens (submitted for publication).



Grower educational programs to enhance adoption of psyllid and HLB control

Report Date: 07/13/2012  

Grower educational programs to enhance adoption of psyllid and HLB control

Report Date: 07/13/2012
Category: Other
Author: Stephen Futch
Sponsor: Citrus Research and Development Foundation

In April, the citrus agents conducted the annual Citrus Growers Institute in Avon Park with over 300 participating in the event. The program consisted of 12 presentations with a major focus related to HLB and management of psyllids. Presentations from the 2012 event (as well as the 2008, 09, 10 and 11) are posted to the citrus agents website at: http://citrusagents.ifas.ufl.edu/events/GrowersInstitute2012/GrowersInstitute2012.htm . Presentations as well as oral recordings can be viewed via internet connections from any location worldwide. These recorded programs serve as an archive of progress within the industry to manage HLB and psyllids. Citrus agents have also been assisting growers with 34 field visits to discuss citrus production issues and offer possible solutions. Grove visits are an essential tool in increasing knowledge and participation, especially with smaller growers. Citrus agents are providing CHMA assistance to aid in adoption of area wide spray events targeting psyllids over a regional effort instead of individual efforts. These regional efforts appear to be providing greater psyllid control as compared to areas where regional sprays are not being conducted. Seven citrus grower meetings were conducted around the state to address educational needs and enhance participation in various production related issues including HLB and psyllid management. The citrus agents are also involved in: hosting international visitors; assisting youth with 4-H citrus tree projects; conducting master gardener trainings; and making presentations at Florida State Horticultural Society. In addition to the above activities, a new foliar nutritional trial has been established in 2 plantings (Immokalee and Arcadia) where young citrus trees (3 years old and recently planted trees) are being grown. Trees are sprayed on a monthly basis (during the growing season) to determine the impact of applying foliar nutritional materials on the growth of young trees in areas where HLB is endemic. The study consists of 9 nutritional treatments that are similar to those being studied in mature trees that are HLB positive. The 3-year old block is virtually 100% HLB infected.



Characterization of critical genes involved in spread of citrus canker pathogen Xanthomonas axonopodis pv. citri

Report Date: 07/12/2012   Project: 445

Characterization of critical genes involved in spread of citrus canker pathogen Xanthomonas axonopodis pv. citri

Report Date: 07/12/2012
Project: 445
Category: Other
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. How bacteria escape from infected plants is underexplored. Understanding the molecular determinants of lesion rupture, how Xcc survives in the intercellular spaces, and how Xcc releases from lesions of host plants will provide many fundamental and practical benefits. Despite the tremendous effort to eradicate citrus canker, the pathogen has spread to most citrus production areas in Florida and continues to spread. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes. Currently, complementation analysis of the mutants is underway. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. Three mutants were tested for affect in release from citrus canker lesions (dispersal assay) on grapefruit for 5, 7, 10 and 14 days as compared to wild type strain. The preliminary assay showed differences in growth rate (total count) and dispersal rates (surface count). The dispersal will thus be calculated according to percentage point difference to negate any effect due to growth changes. Pectate lyase assay, proteinase assay and motility tests (both swimming and swarming) have been completed for all the mutants. Further characterization of the mutants using EPS, LPS, capsule and biofilm assays, is in progress.



Development of a central genome resources website for Ca. Liberibacter asiaticus

Report Date: 07/12/2012   Project: 123

Development of a central genome resources website for Ca. Liberibacter asiaticus

Report Date: 07/12/2012
Project: 123
Category: Other
Author: Magdalen Lindeberg
Sponsor: Citrus Research and Development Foundation

Recent activities have involved a multi-pronged effort focused on bioinformatic characterization of genome sequence data for the D. citri insect vector, bacterial endosymbionts in the insect, and Liberibacter asiaticus, the pathogenic agent of citrus greening. Using data provided by Wayne Hunter and other members of the International Psyllid Genome Consortium, sequences derived from bacterial endosymbionts in the currently available D. citri sequence data have been characterized and a draft genome sequence for the Wolbachia endosymbiont generated. Wolbachia is of particular interest owing to reports of successful control of other diseases through Wolbachia-based manipulation of the biology of the insect vector. In addition to supporting the presence of Wolbachia, the D. citri genome data provides support for the presence of an enteric bacterium most closely related to Salmonella. Phylogenetic analysis of several Wolbachia gene sequences from the Florida D. citri isolate supports an origin of the insect distinct from China and consistent with a Southwest Asian origin. The genome has been manually curated and closely analyzed for features potentially linked to interaction with the host insect including ankyrin proteins, secretion pathways, and potential sources of nutritional provisioning of the insect. Comparison with sequenced Wolbachia isolates from other insects have also been generated. We are additionally involved in the effort to characterize features in the D. citri sequence itself, with particular focus on identification of repeats that can interfere with correct assembly of the genome. Strategies developed for analysis of the Florida D. citri isolate and its component endosymbionts are readily applicable to sequence data for other isolates as more data becomes available. Comparison of Liberibacter strains is ongoing, information on which will be presented by collaborators at the upcoming Citrus Health Research Forum in Fort Collins, CO.



Control of Citrus Huanglongbing by disruption of the transmission of citrus greening pathogen by psyllids

Report Date: 07/11/2012   Project: 337

Control of Citrus Huanglongbing by disruption of the transmission of citrus greening pathogen by psyllids

Report Date: 07/11/2012
Project: 337
Category: Other
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Ca. L. asiaticus in planta and to reduce the innoculum for psyllid transmission. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. In our recent study, we further expanded our previous study in identifying lead antimicrobial compounds with higher activities by targeting SecA using various computational techniques like homology modeling, virtual screening, molecular docking & minimization. Due to the uncultivable nature of Ca. L. asiaticus, we tested the potential inhibitory effect of the selected compounds against Agrobacterium tumefaciens, which is phylogenetically related to Ca. L. asiaticus. Twenty compounds were selected for biological activity study against SecA of Ca. L. asiaticus and A. tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 ug/ml to 256 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Ca. L. asiaticus. Those findings were published in the article entitled: Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design. on Eur J Med Chem (http://dx.doi.org/10.1016/j.ejmech.2012.05.035). To test the application potential of those compounds on plants, the phytotoxicity studies were performed on the five compounds against citrus. At higher concentrations (0.25mg/ml), all five compounds showed phytotoxicity. When the concentrations were diluted to 0.025mg/ml, the toxicity went down to mild to low. Currently, we are testing the inhibitory effects of the five compounds again Ca. L. asiaticus in planta.



Characterize the effect of application of beneficial bacteria (Microbe Program) on management of Huanglongbing

Report Date: 07/11/2012   Project: 8

Characterize the effect of application of beneficial bacteria (Microbe Program) on management of Huanglongbing

Report Date: 07/11/2012
Project: 8
Category: Other
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

Pathogenic microorganisms affecting plant health are major and chronic threat to food production and ecosystem stability worldwide. As agricultural production intensified over past few decades, producers became more and more dependent on agrochemicals as a relatively reliable method of crop production helping with economic stability of their operations. However, increasing use of chemical inputs causes several negative effects, e.g. development of pathogen resistance to the applied agents and their non target environmental impacts. Furthermore the growing cost of pesticides and consumer demand for pesticide free food has led to the search of substitute of these products. In addition, there are a number of fastidious diseases for which chemical solutions are few, ineffective or nonexistence. Biological control is thus being considered as an alternative or a supplemental way of reducing the use of chemicals for agriculture and to manage fastidious diseases. Citrus Huanglongbing (HLB, greening) is one of the most devastating diseases and presents an unprecedented threat to Florida citrus industry. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las) (Duan et al. 2009). Thus, alternative management approaches to manage HLB are necessary. The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. We have developed a culture collection of approximately 400 bacteria initially isolated from the root and rhizosphere of citrus. These bacterial isolates have been screened for various beneficial traits related to P solubilization, siderophore production, nitrogen fixation, indole acetic acid (IAA) synthesis, production of antibiotic and lytic enzymes, induction of systemic resistance, production of ACC deaminase1-amino-cyclopropane-1-carboxylate deaminase and production of quorum sensing [N-acyl homoserine lactones] signals. We are also evaluating the antagonistic activity of these bacterial strains against some well-known plant pathogenic fungi. Especially we have screened a bacterial isolate designated as 43A which possess multiple plant growth promoting activity and is also able to antagonize different fungi. We are also testing the plant growth promoting activity of 24 isolates using seed germination pouch in greenhouse. We have also selected several Bacillus spp. possessing multiple beneficial traits to develop bacterial consortium which can be further developed as carrier based bioformulation.



Control of citrus Huanglongbing by exploiting the virulence mechanisms of Candidatus Liberibacter asiaticus and inducing plant defense

Report Date: 07/11/2012   Project: 609

Control of citrus Huanglongbing by exploiting the virulence mechanisms of Candidatus Liberibacter asiaticus and inducing plant defense

Report Date: 07/11/2012
Project: 609
Category: Other
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. As requested by CRDF and SAB, we have revised project and will focus on the following two objectives: to characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors; to test different compounds in controlling HLB and characterize their mechanisms in controlling HLB. Recently, we compared the gene expression of PR1 and PR5 in healthy trees and Las infected citrus plants. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed once with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB.



Improve the management of citrus canker by protecting citrus fruits through interfering with biofilm formation and quorum sensing of Xanthomonas citri ssp. citri

Report Date: 07/11/2012   Project: 610

Improve the management of citrus canker by protecting citrus fruits through interfering with biofilm formation and quorum sensing of Xanthomonas citri ssp. citri

Report Date: 07/11/2012
Project: 610
Category: Other
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. The hypotheses of the proposed research are (1) Biofilm formation and QS play important roles in X. citri ssp. citri infection of citrus fruit; (2) Control of citrus canker could be improved by interfering with biofilm formation and QS of X. citri ssp. citri. The hypotheses are based on previous studies and our preliminary studies. The specific objectives of this proposed research are as follows: Objective I: To understand the role of biofilm formation and QS in X. citri ssp. citri infection of citrus fruit. Objective II: To prevent X. citri ssp. citri infection of fruit by combining copper with inhibitors of biofilm formation and QS. Recently, we compared the attachment of the QS mutants on the citrus fruit surface. Compared with wild type stain Xac 306, the quorum sensing mutant ‘rpfF showed significantly reduced attachment to the fruit surface as revealed by CLSM (confocal laser scanning microscopy) observation with the GFP-labeled bacterial strains. We also evaluated the effect of nine compounds on Xac biofilm formation on abiotic surfaces using the crystal violet staining method. The data obtained showed that three compounds were active in inhibiting Xac biofilm formation in NB liquid medium at. We are currently testing the effect of those compounds in inhibiting biofilm formation in planta using grapefruit plants in the greenhouse.



Characterize the causal agent of citrus blight through metagenomic approaches

Report Date: 07/11/2012   Project: 611

Characterize the causal agent of citrus blight through metagenomic approaches

Report Date: 07/11/2012
Project: 611
Category: Other
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

Per the requirements from CRDF, we have revised our goal of this proposal to focus it on the identification of the causal agent of citrus blight. Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. Recently, we have survey three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. Once we finalize the trees, we will begin to characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches



Expand Research Plots and Maintain Existing Areas at Mid Florida Citrus Foundation

Report Date: 07/02/2012  

Expand Research Plots and Maintain Existing Areas at Mid Florida Citrus Foundation

Report Date: 07/02/2012
Category: Other
Author: Gary England
Sponsor: Citrus Research and Development Foundation

Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (April 1 to June 30, 2012), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Harvest of Valencias concluded the 2011-12 harvest season. ‘ The Sugar Belle’ area was toped to reduce crop load and improve fruit size and quality. ‘ Dr. Singh is making evaluations of: o A pre-mix product containing Oryzalin and Glyphosate for efficacy in young citrus plantings. o Comparison of Alion and Chateau on non-bearing fresh fruit varieties. o Middles management trial ‘ Dr. Futch established an herbicide trial evaluating Alion and Chateau on non-bearing Valencia. ‘ Applications continued in the ‘commercial scale’ nutritional trial. ‘ BASF has o Made additional applications in their Headline trial o Initiated an herbicide trial ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB. ‘ The Plant Improvement Group has: o Topped and made applications of foliar nutritionals to enhance tree health o Planted 500 trees in a rootstock evaluation ‘ Additional small plantings have been made: o Approximately 1 acre for Dr. Futch for herbicide studies o Additional selections for the New Varieties Development and Management Corporation ‘ Agri Quest initiated a Citrus Root Health Improvement Evaluation in young Valencia.



Improving the innate immune response of citrus to HLB

Report Date: 06/30/2012   Project: CATP09-305

Improving the innate immune response of citrus to HLB

Report Date: 06/30/2012
Project: CATP09-305
Category: Other
Author: Abhaya Dandekar
Sponsor: Citrus Research and Development Foundation

In this quarter, we conducted three experiments using therapeutic compounds. The treatments were applied on 27 healthy, 3 years old, Valencia trees grafted on Cuban rootstocks grown in 5 gallon plastic nursery containers. The therapeutic compounds used for the different foliar treatment were 1) L- Arginine, to stimulate the plant innate immune response; 2) Gibberellin in combination with 6-benzyl adenine, to improve innate immunity, to inhibit pathogen mediated source’sink relationship in favor of the plant and to improve nutrition especially phosphate; and 3) Sucrose plus the herbicide atrazine, to improve the photosynthetic capacity and stress responses of the leaf. All treatments were sprayed on the citrus foliage; the volume sprayed per tree was enough to wet both the upper and lower leaf surfaces just to the point of runoff. Since some of the compounds we are using in the different treatment have soil activity, the soil surface was cover before spraying to eliminate that variable. After the sprayed solution dried, the soil cover was removed. For the first experiment we used 4 different concentrations of L-arginine dissolved in water with a surfactant to improve penetration. We use 3 healthy trees (1 tree = 1 biological replicate) for each L-arginine concentration. Also 3 healthy trees were treated with water containing just the surfactant and used as controls. This will provide us 5 treatments x 3 trees (biological replicates) X 5 sampling points = 75 samples for analysis (RNA) For the second experiment we used 2 different concentrations gibberellic acid in combination with a single 6-Benzyladenine concentration, the solutions were prepared as explained above. We use 3 healthy trees for each combination. This experiments will provide 2 treatments x 3 trees (biological replicates) X 5 sampling points = 30 samples for analysis(RNA). For the third experiment we used 2 different concentrations of atrazine in combination with one concentration of sucrose, solutions were prepared as explained above. We use 3 healthy trees for each combination. This experiments will provide us with 2 treatments x 3 trees (biological replicates) X 5 sampling points = 30 samples for further analysis (RNA). Three leaves were collected from each individual treated tree and processed individually. Leaf samples were collected temporally at 0 time (right before treatment), 6h, 24h, 3 days and 6 days after treatment. Leaves were immediately frozen in liquid nitrogen and keep at -80’C until extracted. Total RNA will be extracted using the 3 day guanidine isothiocyanate (GITC) protocol and stored at -80’C. Expression levels of biomarker genes for each treatment will be determined by qRT-PCR. In the mean time our 200 plants of Valencia orange on Kuharske Carrizo rootstocks that were re-infected with HLB infected buds appear to display ~90% infection that needs to be confirmed by quantitative real-time PCR (qRT-PCR) before we can initiation of our spray treatments.



Identification of small molecules that disrupt pathogenicity determinants of Liberibacter asiaticus

Report Date: 06/26/2012   Project: 80214

Identification of small molecules that disrupt pathogenicity determinants of Liberibacter asiaticus

Report Date: 06/26/2012
Project: 80214
Category: Other
Author: Claudio Fabricio Gonzalez
Sponsor: Citrus Research and Development Foundation

Due to its reduced genome, Candidatus Liberibacter asiaticus (CLas) has only a few transcriptional regulators. We hypothesized that natural and/or synthetic chemicals can interact with transcription factors to interfere with its regulatory activities. The screening of these molecules involves the use of pure proteins form CLas. While three out of five transcription factors were expressed and purified in Escherichia coli, CLIBASIA_03370 and CLIBASIA_00835 were non-soluble. The specific goals of this quarter were to clone, express and purify CLIBASIA_03370 and CLIBASIA_00835 using Sinorhizobium meliloti as an expression host. The rationale is that by using a host that is phylogenetically related to CLas the solubility of the proteins would be improved, compared to the low expression and solubility previously obtained in Escherichia coli. The genes were cloned and the proteins purified using nickel affinity chromatography. The results obtained indicate that the CLas genes can be expressed in a soluble form in S. meliloti. Current efforts are directed to improve the purity of the protein obtained. The validation of small molecules-transcription factor interactions is the next step in the identification of new therapeutics against CLas. The inability to culture CLas in a laboratory setting constitutes a big challenge towards the in vivo studies. In this sense our approach is the use of S. meliloti as a surrogate host. We constructed several gene fusions to the gusA reporter gene. These transcriptional fusions are aimed to analyze the effect of the small molecules on the activity of the CLIBASIA_01180 and CLIBASIA_01510 transcription factors. Current experiments are being performed to analyze the effects under different stress conditions.



Expand Research Plots and Maintain Existing Areas at Mid Florida Citrus Foundation

Report Date: 06/19/2012   Project: 238

Expand Research Plots and Maintain Existing Areas at Mid Florida Citrus Foundation

Report Date: 06/19/2012
Project: 238
Category: Other
Author: Gary England
Sponsor: Citrus Research and Development Foundation

Mid Florida Citrus Foundation (MFCF) a 501c3 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care cost tends to be higher than a commercial grove due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. In 2011 MFCF expansion projects include an economic study of high density citrus rooted cuttings for early production of citrus in the presence of greening, remedial and preventive tests for HLB infection, new herbicide trials, variety evaluation with HLB tolerance in Florida and ACP studies. A total of 31 acres has been prepared for planting with 25 acres planted this May and the remaining acreage to be planted this year. This is in addition to the 17 acres that were planted last year. This large planting of young trees also requires added young tree care expenses and an increase in grove care costs without any fruit to support continuing operations. Additionally private companies with a vested interest in the citrus industry have made considerable donations of materials and irrigation equipment for these new projects. This is a leverage of research dollars that does not get recognized. Between private donations and EQIP grant monies, the CRDF is getting approximately 2.00+ dollars towards research projects for every 1.00 dollar in grant monies awarded.



Intensively managed citrus production systems for early high yields and vegetative flush control in the presence of greening and canker diseases

Report Date: 06/09/2012   Project: 72357

Intensively managed citrus production systems for early high yields and vegetative flush control in the presence of greening and canker diseases

Report Date: 06/09/2012
Project: 72357
Category: Other
Author: Arnold Schumann
Sponsor: Citrus Research and Development Foundation

In January 2011, an additional 41 HLB-symptomatic trees were identified in the 14-acre ACPS block near Auburndale, bringing the average confirmed HLB incidence to 9.6%. Because the source of Las-carrying adult psyllids across the road from the research block cannot be controlled, and the already high HLB incidence in the block indicates an exponential increase phase, we decided that alternative management strategies should be developed and tested to allow the infected trees to continue producing profitable yields and more importantly, for the block to break even and produce pure profit in not too many years. The treatment structure of the Auburndale experiment is ideal for studying the interaction of HLB with the fertilizer source (fertigation versus granular), rootstock (C-35 versus swingle), and irrigation (drip versus microsprinkler), specifically in young trees. A basal prophylactic spray program consisting of potassium phosphite and major- and micro-nutrients was initiated in December 2011, with the objective to help mitigate the severity of the HLB symptoms on infected trees and to make them economically productive. Phosphite with DKP is being applied every 8 weeks and the other nutrients are being applied on the main leaf flushes when they are about 2/3 expanded. The 28 plots in the ACPS research experiment were sampled for fruit yield and quality in December 2011, followed by commercial harvesting of the entire 14-acre block. The third year fruit yield in boxes per acre by treatment are listed below: STD-Sw (218*) 91.1 MS-OH-Sw (218) 93.1 Drip-OH-Sw (218) 115 Drip-OH-Sw (303) 159.7 Drip-OH-C35 (303) 200.9 Drip-OH-C35 (363) 222.2 *Values in parentheses are the tree densities in trees/acre; Sw, C-35 are the rootstocks. STD-Sw is the conventional granular fertilized treatment, MS indicates microsprinkler fertigation, Drip indicates drip fertigation, OH indicates open hydroponics. See “Designing Advanced Citrus Production Systems To Maximize Early Production”. FLORIDA CITRUS SHOW, 25 January, 2012, Ft. Pierce, FL http://128.227.177.113/Powerpoint/ACPS-FL%20citrus%20show%20Jan%202012/index.html



Intensively managed citrus production systems for early high yields and vegetative flush control in the presence of greening and canker diseases

Report Date: 06/04/2012   Project: 72357

Intensively managed citrus production systems for early high yields and vegetative flush control in the presence of greening and canker diseases

Report Date: 06/04/2012
Project: 72357
Category: Other
Author: Arnold Schumann
Sponsor: Citrus Research and Development Foundation

A CREC field day attracted about 170 visitors on the 15th November 2011, and half of the day’s events were located in the ACPS experiment near Auburndale. Visitors were taken on a guided 4-stop tour of the 14-acre ‘Hamlin’ block to view the different treatments, including the comparison of Swingle and C-35 rootstocks, drip versus microsprinkler computerized fertigation versus standard irrigation practice and granular fertilizer, and a comparison of three planting densities (10×20′[218 trees/acre], 8×18′[303 trees/acre] and 8×15′[363 trees/acre]). A static demonstration of a 48-inch wide tractor and sprayer was displayed inside the 363 trees/acre plot to illustrate the appropriate matching of specialized farm machinery with the highly productive open hydroponics concept. A highly compact, dense root ball which had developed under a drip emitter in the drip ACPS treatment was uncovered for visitors to observe and the merits of drip versus microsprinkler ACPS were discussed. See photos at http://128.227.177.113/ACPS/Photos.html After the field day the visitors enjoyed a sponsored lunch at the CREC, followed by an expert panel discussion about the ACPS research and in particular the increasing problem of adequately protecting young trees from psyllids and early HLB infections. Photographs of the field day and the informative handout are available at http://128.227.177.113/ACPS/Index.html