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.
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.
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.
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
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.
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.
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.
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.
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
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
The first four HLB-symptomatic trees were discovered in the ACPS research block in August 2010, at which time the trees were in the ground for 19 months. The affected trees were removed in order to limit the acquisition and dispersal of Las by psyllids from within the block. In September 2011, a year later, the HLB survey uncovered 304 symptomatic trees scattered throughout the block, but particularly prevalent in the western boundary adjoining the residential road and properties with backyard citrus trees. Average infection incidence for the block was 8.5%, but on the western boundary was approaching 25%.
For the winter quarter (January ‘ March, 2012), the citrus agents have collectively conducted and/or assisted with 9 educational events to provide timely information regarding HLB and psyllid management. These programs have included the Florida Citrus Show (Ft. Pierce, 673 participants), citrus production schools (Arcadia, 110 participants) and local county programs. In addition to the above programs, the citrus agents made 76 grower consultations or grove visits. To educate urban audiences related to citrus production and HLB management, 10 programs were conducted for Master Gardener, urban and/or youth audiences. Five field days were conducted or assisted with that highlighted HLB management, including Lake Placid, Immokalee and Mid Florida Citrus Foundation near Winter Garden. Training for 523 citrus grove workers in the Indian River citrus area was conducted to educate grove workers on canker and HLB identification. Worker safety program for over 220 grove workers in Arcadia highlighting safety practices related to equipment operation, pesticide application and general farm safety which are all important in local farming operations. The agents are actively assisting with CHMA spray programs and/or meeting with area team captains to promote and foster the development of area wide spray applications.
The quarterly tree size measurements in the ridge ACPS experiment were collected on June 8, 2011. Average tree height was 21% greater in the drip fertigated treatments than in the conventional “grower” treatment. The average tree canopy volumes of drip fertigated trees were up to 43% greater than those in the conventional “grower” treatment. A slight visible yellowing of new spring leaf flush in April became very widespread in all the fertigated treatments and was quickly identified with leaf tissue sample analyses as manganese (Mn) deficiency and marginal zinc (Zn) deficiency. Leaf tissue concentrations of Mn were as low as 13 mg/kg and Zn was as low as 19 mg/kg. To rectify the problem, we injected additional supplementary chelated Mn and Zn fertilizer through the irrigation system and the trees responded very quickly by greening up. The conventional “grower” treatment did not develop any Mn or Zn deficiencies during this time, and that may be due to the much higher fertilization it received compared to the fertigation treatments, and also due to the higher growth rates experienced in the fertigation treatments. The drip fertigation treatment continued to demonstrate the highest nutrient use efficiencies as measured by the rate of canopy growth per unit of fertilizer applied. The drip fertigation treatment also measured the lowest soil solution nitrate-N concentrations below the root zone, indicating that there was negligible fertilizer wasted and that the risk of environmental contamination was reduced.
In addition to our primary mission of generating bioinformatic analyses of Liberibacter genomes, my group provides bioinformatic support for a range of projects related to the pathogen, plant, and vector interactions that contribute to citrus greening. A recent focus of attention has concerned the identification and characterization of bacterial endosymbionts in Diaphorina citri, the psyllid vector of L. asiaticus, using sequence data from the psyllid metagenome generated by the International Psyllid Genome Consortium. A set of reference genomes was assembled corresponding to those previously found in association with various psyllid species/isolates as determined by PCR amplification of ribosomal RNA genes. Reads from the D. citri metagenome were mapped to the set of reference genomes in order to determine the extent of support for their presence. These analyses are part of a larger effort directed toward development of computational pipelines for identification and taxonomic classification of resident endosymbionts and epiphytes in eukaryotic metagenome sequences that may affect vector efficacy or plant host susceptibility. Confirmation of the presence of several bacteria in the D. citri metagenome has been obtained, with the best sequence coverage apparent for the Wolbachia endosymbiont. Wolbachia is of particular interest given that its association with the psyllid appears to be relatively stable and that manipulation of resident Wolbachia strains has proven an effective strategy for reducing the fitness of other insect such as mosquito. A manuscript is currently in preparation that summarizes the endosymbiont survey and describes the draft genome assembly, annotation, and phylogenetic analysis of the Wolbachia strain from D. citri. We have recently joined ongoing efforts to complete the draft genome sequence of D. citri, assisting the International Psyllid Genome Consortium with characterization of repeat sequences in the current draft assembly. Additionally we have provided assistance to Dr. Angela Douglas (Cornell University) in identification of gut sucrase and aquaporin sequences in the psyllid genome for the purpose of biological characterization of these potential targets for insect control. Recent discussions with Hong-Lin (ARS Parlier) and Goutam Gupta (LANL) have focused on collaborative bioinformatic analyses of two or more new Liberibacter sequences, pending their anticipated completion by the end of this year. Analytical pipelines and comparative analyses established for the Liberibacter genomes currently available at the HLB Genome Resources Website (http://citrusgreening.org/) should be readily applicable to these new sequences.
Objective 1. Localization of Candidatus Liberibacter asiaticus (Las) in organs and tissues of the Asian citrus psyllid (ACP). This objective has been achieved and, as mentioned in our previous report, two papers have been published: Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104:526-533; and Ammar et al., 2011, J. of Phytopathology, 159:726-734. Using quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH), the proportion of Las-infected salivary glands was significantly lower than that of infected alimentary canals or other organs/tissues. Our results show the near systemic infection of ACP organs/tissues by Las and suggest that both the alimentary canal and salivary gland constitute infection and/or transmission barriers to Las in this vector. In the salivary glands both entry and exit barriers are proposed, whereas in the alimentary canal only an entry barrier is indicated. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We completed our study for developing 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). This new method (described briefly in Ammar et al. 2011, Proceedings of 2nd Inter. Res. Conference on HLB, Orlando, FL, Jan. 2011) is now being prepared for a full publication. In our study, 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 spp. 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 were also obtained with the following: a. higher densities of inoculating psyllids (5-10 adults/leaf), b. longer inoculation access periods, and c. incubation of the excised leaves for 1-wk post-inoculation before being processed for PCR. Furthermore, logistic regression analysis indicated a positive correlation between Las titer (as evidenced by Cq values) 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. Such a correlation can be used to predict the rate/proportion of ‘Las inoculative’ psyllids from that of ‘PCR-positive’ ones in field samples collected in various geographical regions or at various times of the year, which can provide timely and valuable information for predicting and/or combating HLB. [B] 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 and transmission as well as on Las replication in psyllids. In the first two experiments, using qPCR with specific primers to Las and to a psyllid gene, 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 analyzing the third experiment by qPCR to confirm these results. [C] New methods for studying hemipteran stylets, as well as their salivary sheaths in citrus plants, have been developed and used to study the feeding behavior of ACP nymphs and adults on young and old citrus leaves. [D] Transmission and scanning electron microscopy studies on the stylets of ACP nymphs and adults are continuing, which may reveal the negative and/or positive roles of the salivary and food canals of the maxillary stylets in Las transmission by ACP from the phloem of diseased plants.
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