The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. Members of Burkholderia and Bradyrhizobium have been known to benefit plants. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. Salicylic acid (SA)-mediated ISR is an important benefit of beneficial bacteria to the plant host. We determined the expression of three SA mediated ISR marker genes, SAM, PR1 and PR2, of the inoculated trees. Plants treated with strain A53 exhibited a significant upregulation of PR2 gene at 3 dpi compared with negative control plants. A63 induced expression of the SAM gene at 5 dpi and the PR1 gene at 7 dpi. Similarly, Actigard induced the PR1 and SAM gene expression at 5 and 7 dpi. Large scale experiment is ongoing. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. Shoots from more survival trees are being grafted. We are also characterizing the potential mechanism why some branches are Las free. We are testing the effect of application of isolates on plant defenses and attractiveness to psyllids. One manuscript entitled: “Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome” has been published by Microbiome.
June 2017 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. During this time, the focus was on extraction of fungal DNA from plant tissues that had previous been enriched and then stored at -20 C. 1,056 extractions were carried out using the Qiagen MoBio PowerSoil kit. Another 576 samples were taken from the field biweekly, 384 of these samples were processed for incubation. After incubation 288 of these samples were excised of their bark and stored for the DNA extraction process. Salts have been selected and will be used in the future for the RH temperature treatments. Experiments were started to look at the effect of temperature on the level of sporulation of P. citricarpa. It can be quite difficult to get consistent sporulation even under controlled conditions. The temperatures that are being tested 15, 20, 24, 28, 32, and 36C. After incubation in complete darkness to avoid the confounding effects of light, it was found for 5 isolates that 24C was the best temperature for sporulation (P < 0.05) followed by 28C. The repetition of the experiment is not yet completed. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. The disinfectants have been preliminarily evaluated in the presence of finely ground plant debris (twigs and leaves as would be found on mowers or hedgers). Citrus debris itself had no significant effect on conidia germination but there was a significant effect on the efficacy of the disinfectants. Disinfectant treatment in the presence of citrus tissue debris has a much lower efficacy than determined from previous experiments lacking citrus tissue debris. This loss of efficacy can be attributed to two factors. The first is a reduction in potency due to the presence of tissue debris within the liquid treatment. The second and more profound factor is the availability of disinfectant as a free liquid. Testing of quaternary ammonium was completed during this quarter. Results showed that at the lowest ratio of disinfectant to debris, which is 100 l of disinfectant, 500 ppm a.i. was required to reduce the percentage spore germination to zero. This is still well below the recommended rate of 2000 ppm. At the highest volume (1500 l), only 20-50 ppm a.i. was required to kill 100% of the spores. In the absence of debris, only 20 ppm a.i. of quaternary ammonium was required to reduce spore germination to zero. Results of our work demonstrate that the presence of debris significantly reduces the efficacy of disinfectant, but this can be mitigated by using a large volume, relative to the amount of debris. Therefore, the results of this study illustrate that when decontaminating equipment with disinfectant, it should be applied to the point of run-off, rather than lightly sprayed on.
March 2017 There were three main objectives for this project: 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. Progress was made on each objective. In objective 1, we determined that there was little observable effect on the number of Phyllosticta spp. structures in the leaf litter per se between the untreated control, urea, and soil-set. But there were significant differences in the incidence of fruit with black spot symptoms with the soil-set treatment having the lowest in 2015 and 2016 (P < 0.05) but equivalent incidence in 2017. However, the severity was lowest with the soil-set treatment for all three years. It was found that the high volume fungicide application practices used in Australia does slow the decomposition of leaf litter. In the small plot trials used from Australia, urea was not a preferred treatment choice as it did not improve decomposition but organic mulch like bagasse was excellent. Bagasse was also found to be an excellent mulch choice in small experiments in Florida. In objective 2, sporulation and structure formation was followed over three years in Florida and Australia. In Florida, leaf litter was collected all year where as in Australia it was collected during the fruit susceptibility period. We found that pycnidia formation preceded pseudothecia formation in both Australia and Florida. In Australia, the majority of the fungal structures observed were P. citricarpa but in Florida, the majority of structures were P. capitalensis. From 2014 to 2017, the level of P. citricarpa increased to nearly equivalent levels of P. capitalensis. In Florida, pycnidia and conidia were observed all year but pseudothecia and ascspores tended to be present in cycles and always at lower levels. It is hypothesized that the ascospores were all from P. capitalensis. We also demonstrated that P. citricarpa has two mating types required for ascospore formation and only one is present in Florida, unlike the rest of the world. This means that only conidia are present in Florida. Our team was able to demonstrate that the two mating types were needed for ascospore formation and were able to get them to form in vitro. Again a first. In objective 3, the Florida team refined and used a method to inoculate potentially resistant 'Chinotto' hybrid leaves. There were different levels of spore formation on the leaves but it is unknown how this relates to fruit susceptibility. In Australia, fruit inoculations were done in a citrus collections of many breeding lines. Sour orange hybrids were differentially susceptible but the most promising results were from pomelo lines where inoculated fruit had no disease expression.
Huanglongbing (HLB, also known as citrus greening) is a severe threat to the sustainability of the US citrus industry particularly in Florida. HLB is caused by the bacterial species, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the vector, Asian Citrus Psyllid (ACP) during feeding. Altering or disrupting the feeding behavior of ACPs has potential to minimize disease progression. The goal of this project is to change the feeding behavior of ACPs by using a film barrier material, called hereafter Organo-Silica based Composite Film (OSCF) . The OSCF is a foliar product formulation that has polymer blended with silica based particles and gels. We have used metal ions (plant micronutrient) such as Ca, Mg, Zn as ionic cross-linker to stabilize polymer silica gel structure. A series of OSCF materials (total 17) were prepared, characterized and evaluated for phytotoxicity, rainfastness and heat-trapping potential. The size and morphology of OSCF materials was observed using Scanning Electron Microscopy. All OSCF were found to be composite materials with multiple micron sizes with some versions displaying layered structures and others being more particulate. The interactions between silica, polymer and metal cross-linkers were confirmed using Fourier transform infrared spectroscopy and UV-Vis spectroscopy. Plant safety of OSCF materials was evaluated by observing phytotoxicity and heat-trapping potential on plant surface after foliar application. All OSCF materials developed over three years were found to be safe on citrus species. Rainfastness studies conducted on citrus seedlings treated with OSCF materials revealed that most OSCF materials retained at least 50% of their content after 3 simulated rainfalls. Efficacy of OSCF materials in minimizing HLB infection was evaluated in field conditions. ACP citrus infection trials conducted with Valencia orange plants indicated with earlier versions of OSCF product formulations. Results demonstrated good potential for lowering infection incidence of HLB. Subsequent ACP trials were unable to confirm the effectiveness of later versions of OSCF product formulations due to low infection among untreated controls.
Strigolactone (SL) applications on HLB-affected trees consistently resulted in vegetative and reproductive flush following foliar treatment. Fruit set and retention were both enhanced by SL treatments. No effect was noted when SL were applied to healthy HLB- trees. SL-enhanced vegetative growth was accompanied by substantial increases in vascular tissue as evidenced by the growth in thickness and health of phloem elements. In addition, SL can regulate root architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Accompanying each burst in vegetative growth, there is a substantial decline in starch content, and to a lesser extent soluble sugars. The decline in starch likely fuels the establishment of new phloem and corresponding flush. Thus, foliar and/or soil drench applications of SL may induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restored tree health. Presently SL are relatively expensive, yet costs have declined substantially (reduced in half) during the experimental period. Although effective in improving health to HLB-affected trees, the cost remains prohibited at the moment. Further decline in costs may offer an opportunity to integrate SL with present grove care practices.
Typically, citrus trees in Florida groves under low volume microsprinkler irrigation concentrate fibrous roots in the wetted zone. In recent decades, soils under citrus tree canopies statewide have increased in pH and bicarbonate concentrations because of irrigation with alkaline water from deep wells extending into Florida s limestone aquifers. As soils become more alkaline some nutrients become more available (e.g. N and Mg) for uptake by plants and others (e.g. Fe, Mn, Zn and B) become less available for uptake. Elevated soil pH is not known to cause decline in healthy trees. However, declines in tree vigor and productivity above that caused by Huanglongbing (HLB) alone have been documented in trees growing in soils impacted by alkaline irrigation water. To verify the relationship between soil alkalinity and growth of trees affected with HLB, a greenhouse experiment was initiated to maintain HLB positive or negative trees by exclusion of the vector. Water uptake by trees receiving water supplemented with calcium bicarbonate was significantly reduced (10-15%) compared with health trees but substantially greater reduction for HLB affected trees (>20%). Tree heights were similar for HLB affected and healthy trees irrigated with calcium carbonate but significantly smaller than healthy trees not receiving modified irrigation water. Mean leaf Ca, Mg, Mn, Zn and B concentrations were not significantly different during the study period (April 2014 to April 2017). However, all nutrients were significantly different among treatments with the exception of Ca during the last year of the study. The cause of reduced water and nutrient uptake appears to be correlated with reductions in root density and increases in soil pH in soil irrigated with higher concentrations of calcium carbonate resulting in reduced leaf area and tree height. Two field studies (one grove with trees >10 years old, and one grove with trees < 5 year old) were conducted to determine the effect of soil pH on tree health. Leaf Ca, Mg, Mn, Zn and B concentrations in leaves were greater at both sites with reduced water and/or soil pH than non-treated controls at both the young tree and mature tree groves. Leaf Ca, Mg, Mn, and Zn concentrations were significantly different among treatments in the mature tree grove but only significantly different for Ca, Mn, and Zn at the young tree grove when averaged over the entire study period. However, Ca, Mg, Mn, Zn and B were significantly different among treatments when only the last year of data was considered. Root density samples taken in February to June indicate a significantly greater root length density with lower soil pH. These results indicate a positive correlation between root density and reduction in soil pH from greater than 7.0 to less than 5.0. Leaf Ca, Mg, Mn, and Zn in November samples were greater for trees treated with both irrigation water acidification and sulfur application compared with irrigation water acidification only. These results verify previous finding that leaf nutrient status is negatively correlated with soil pH.
During the past quarter, we have optimized dsRNA targets and have conducted immune priming assays to investigate the specificity of immune priming in ACP following exposure to dsRNA. The yield of redesigned dsRNA using MEGAscript RNAi Kit transcription was initially low, possibly because the QG buffer in the Zymoclean Gel DNA Recovery Kit may have decreased transcription. In order to avoid using QG buffer in gel purification and increased PCR concentration,gel purification method was modified. PCR products were analyzed on 1% agarose gel and the band was excised, and transferred to a filter tip and centrifuged at 5000 rpm for 10 min. Three l of purified gel product were run on 1% agarose gel which showed a high concentration (based on the intensity of the band) of the product. The remaining purified gel product was precipitated and resuspended in 30 l of RNase-DNase free water, and 2 l of the product were analyzed on agarose gel and directly amplified. PCR products obtained from the modified purification protocol for dsRNA synthesis yielded significantly higher dsRNA concentrations to compared to the kit. Bioassays were conducted with adult uninfected ACP. After 3 hrs of starvation, insects were fed on dsRNA for 24hrs and 5 days. After 24 hrs, insects were starved for 3 hrs and then transferred to a lethal dose of S. marcescens for 4 days. For each dsRNA concentration, two cages were exposed to dsRNA; then either fed on S. marcescens in diet (20% sucrose, and 0.5 green food coloring dye) or diet only (control). Diet containing the pGEM-T easy vector without a T7 tail, a blank sucrose + buffer diet, and container without food were also included as controls in the feeding experiment. The stability of different concentrations of dsRNA on diet solution were confirmed with agarose gel electrophoresis at the end of the 24hr and 5 d feeding periods. DsRNA of 1000 ng/ l and 100 ng/ l were present in agarose gel. Also the survival of S. marcescens after 4 days feeding was checked on nutrient agar plate. S. marcescens from feeding cage was streaked on nutrient agar and kept O/N at 30 C. Result confirmed that bacteria were viable during the feeding assay. Following exposure to bacteria, insect were placed on Citrus macrophylla in the greenhouse, and mortality was recorded daily. After 14 d, live insects from the 24hr and 5 d feeding treatments were collected and laterally bisected. Half of the insects were examined for the presence of S. marcescens, and the remaining insects were used for RNA extraction. To quantify S. marcescens, single insects were smashed in tube with 20 l of nuclease-free water and plated on nutrient agar, and kept at 30 C O/N. Colonies were obtained from most of the insects on nutrient agar. No colonies were obtained from control insects. Conventional PCR confirmed the presence S. marcescens in insects. To study the effect of dsRNA on mRNA levels, total RNA was extracted from single ACP using TRIzol (Invitrogen) and cDNA was prepared. The survival rate of insects after feeding on S. marcescens and from 14 days on C. macrophylla will be analyzed and presented when all replicates are completed; however, initial results indicate thigh mortality after feeding on S. marcescens as compared with those feeding on sucrose, suggesting that dsRNA exposure does not modulate ACP immune responses to S. marcenscens e.g. does not prime ACP to tolerate or mitigate lethal doses of pathogenic bacteria, although it must still be determined whether this response is similar following exposure to sublethal doses of bacteria. Whether this is responses is specific or is generalizable to other Gram negative bacteria, such as Liberibacter, will be determined in the next quarter.
The overall objectives of these studies were to provide predictive knowledge of the time of spring leaf flush and flowering for spring flush ACP control and the time for optimum bee foraging in. Weather, flowering and leaf flush data were collected and used to improve the Citrus Flowering Monitor System. Particular goals were to improve near-bloom temperature responses of the model and add leaf flushing time to the flowering time in the monitor system. The 10 % open flower and petal fall dates as the start and ending times of bee activity were determined. Overall the on-line Citrus Flowering Monitor System was improved to allow easy movement to different FAWN sites for flowering evaluations. Further, it now provides dates of initiation of growth and hours of induction to those dates, plus full bloom dates along with the graphic data. Visible budbreak, spring leaf development and flower bud development stages were monitored for three years. Data were collected in order to determine time of budbreak, leaf feather flush stage as well as the intended 10 % open flowers and 95 % petal fall. The peak flowering period was monitored to test it for the required period for minimal bee toxic ACP sprays. We discussed with growers and beekeepers the spray scheduling feasibility with concern of the grower for adequate ACP control and beekeepers for adequate safe access to citrus flowers for their bees. The active bee period was identified, but scheduling issues exist. About 55 days were required from bud break until full bloom, while 10 to 16 days were required from 10 % open flowers to full bloom and another 8 to 20 days from full bloom to 95 % petal fall, roughly a 24 to 30 day period in each flower bud cohort s development for bee activity. Two examples, normal bloom dates at Lake Alfred in 2015 and 2016 were 2/11 and 3/3 in 2015 (20 days apart) and 3/30 and 4/10 in 2016 (11 days apart). This increased the flowering period 11 or 20 days increasing the bee active bloom period to at least 35 to 50 days. Discussions with the CREC Entomologists suggests that a better psyllid control strategy may be to control (spray for) adult psyllids at budbreak and try to get maximum control into the bloom period (about 55 days until full bloom) and then use bee friendly pesticides for psyllid control until the bloom period is over. With two growers in the Ft. Meade area, using 4 blocks, psyllids were monitored after spraying at budbreak. Spray control of psyllids from one spray at budbreak lasted over 30 days. A second spray then continued control into the bloom period. Monitoring budbreak of the first summer flush also appears to be a good second window for effective psyllid control of the second synchronized flush each year. An aerial spray at spring and first summer budbreak followed by second sprays could provide good protection of the 2 major flushes with just four sprays.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being arranged. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors are being tested in vitro. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and are testing whether they can be degraded by SA hydroxylase. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Around 10 Gb clean reads data was generated per metagenome sample. Totally around 120 billion bp (120 Gb) were pulled together for assembly.The final assembly was composed of 17,676,569 contigs longer than 200 bp, totally 10.8 Gb, the longest contig length was 536,098 bp, average length was 613 bp and the N50 was 651 bp. The statistics indicated the quality of the assembly was good. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. Members of Burkholderia and Bradyrhizobium have been known to benefit plants. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. Salicylic acid (SA)-mediated ISR is an important benefit of beneficial bacteria to the plant host. We determined the expression of three SA mediated ISR marker genes, SAM, PR1 and PR2, of the inoculated trees. Plants treated with strain A53 exhibited a significant upregulation of PR2 gene at 3 dpi compared with negative control plants. A63 induced expression of the SAM gene at 5 dpi and the PR1 gene at 7 dpi. Similarly, Actigard induced the PR1 and SAM gene expression at 5 and 7 dpi. Large scale experiment is ongoing. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. The work is ongoing. We are testing the effect of application of isolates on plant defenses and attractiveness to psyllids. Two manuscripts have been accepted for publication.
December 2016 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. Samples collection for inoculum potential continue to be gathered biweekly. Bark is being removed from the twigs and DNA extracted from that. Since determining the best way to extract DNA from the field twigs took longer than expected, processing previously collected twigs continues and qPCR has not been conducted in a systematic manner yet. To get a larger range on temperatures and relative humidities, preliminary studies on the stability of RH measurements for different super saturated salt solutions at a temperature range has been conducted and a statistical analysis to determine whether the variability in RH measurements is excessive for the various salts. The selection will be based on the statistical outcome Experiments were started to look at the effect of temperature on the level of sporulation of P. citricarpa. It can be quite difficult to get consistent sporulation even under controlled conditions. The temperatures that are being tested 15, 20, 24, 28, 32, and 36C. After incubation in complete darkness to avoid the confounding effects of light, it was found for 5 isolates that 24C was the best temperature for sporulation (P < 0.05) followed by 28C. The repetition of the experiment is not yet completed. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. The disinfectants have been preliminarily evaluated in the presence of finely ground plant debris (twigs and leaves as would be found on mowers or hedgers). Citrus debris itself had no significant effect on conidia germination but there was a significant effect on the efficacy of the disinfectants. Disinfectant treatment in the presence of citrus tissue debris has a much lower efficacy than determined from previous experiments lacking citrus tissue debris. This loss of efficacy can be attributed to two factors. The first is a reduction in potency due to the presence of tissue debris within the liquid treatment. The second and more profound factor is the availability of disinfectant as a free liquid. When disinfectant is applied at low volumes and entirely or mostly absorbed by the tissue debris, the ai concentration must be raised greater than 200 fold to provide comparable efficacy observed in the presence of free liquid. Confirmatory experiments are being conducted.
Predicting the emergence and arrival of insect pests is paramount for integrated pest management. To achieve this goal, it is important to understand how abiotic factors influence pest dispersal behavior. We investigated the effects of abiotic conditions on flight initiation by the Asian citrus psyllid, Diaphorina citri Kuwayama. We first explored the effect of barometric pressure changes on flight initiation. We used a custom-made barometric chamber and observed the activity of D. citri as measured by the number of psyllids captured on yellow cardboard panels coated with adhesive. We found that psyllid flight initiation changed in response to variations in barometric pressure rather than to differences in stable pressures. D. citri were equally active at 1009 mbar and 1022 mbar. However, D. citri dispersed more as barometric pressure increased, and less when barometric pressure decreased. In a subsequent experiment, we manipulated temperature and relative humidity and observed how D. citri dispersed between citrus plants. Psyllids dispersal increased linearly with temperature. Changes in humidity did not affect dispersal of D. citri. Less than 1% of psyllids dispersed at 15 C, compared to 7.7% at 21 C and 27% at 25 C. The minimal threshold for D. citri to initiate flight is estimated to be 16.5 C. Collectively, our results provide an initial step toward developing predictive models of D. citri movement as influenced by abiotic factors. Densities of an herbivorous pest may be impacted by landscape and orchard architecture. We present two orchard experiments where the densities of the Asian citrus psyllid (Diaphorina citri) were compared depending on: (1) the presence or absence of a windbreak and (2) if the orchards consisted of a solid set re-planting or an orchard with a mixture of mature and reset-replacement trees. (1) Psyllid abundance was measured on the edges of five orchards. The factor investigated was the presence or absence of a windbreak. We observed significantly fewer psyllids on the edges of orchards with windbreaks as compared to those without windbreaks. We found no significant difference in the number of natural enemies between the edges with or without windbreaks, suggesting that windbreaks do not affect densities of psyllid natural enemies. (2) During two consecutive years, we compared the densities of psyllids on young trees less than 3 years of age in a solid set re-planting versus on resets (trees planted in replacement of dead or huanglongbing-infected trees) present randomly within mature orchards. This was conducted in four orchards and among three citrus varieties. More psyllids were found in the solid set re-plantings as compared with on the resets within mature orchards. To our knowledge, this is the first report to demonstrate that the planting strategy of new trees in orchards may impact the populations of a horticultural pest. Overall our data suggest that establishment and conservation of windbreaks might be beneficial to protect orchards from D. citri. The data also suggest that D. citri populations increase more within uniform landscapes of seedling trees as compared with mature orchards with randomly interspersed young seedlings.
The goals of this project were to 1) identify toxins derived from Bacillus thuringiensis (Bt) with toxicity against Asian citrus psyllid (ACP), 2) isolate a peptide that binds to the gut of ACP, and 3) modify the selected Bt toxin with the gut binding peptide to provide an artificial anchor for the toxin resulting in enhanced toxicity. We screened toxin mixtures derived from 35 strains of Bt with diverse insect toxicities and toxin profiles for toxicity against ACP. The bioassay protocol was optimized to avoid toxin precipitation, but control mortality was high in some of the bioassays. Of the 35 strains screened, statistical analysis could only be conducted on data generated for 18 strains. Of these, toxin mixtures derived from a total of six Bt strains showed toxicity to adult ACP at 500 g/mL of proteolytically activated toxin. Individual toxins from one of these strains were identified by LC-MS/MS, cloned, expressed, and tested against adult ACP. Of four individual toxins tested, one showed significant toxicity against adult ACP at 500 ug / ml. Given the smaller size of nymphs and large volumes of phloem ingested, it is expected that toxin efficacy will be greater against nymphs than against adult ACP. ACP mortality was associated with severe disruption of the midgut epithelium for the individual toxin and toxin mixture. The microvilli that line the gut epithelium were disorganized or lost, consistent with the mode of action of Bt-derived toxins. Toxicity also correlated with a drop in honeydew secretion indicative of reduced feeding which is also consistent with Bt toxin effects. We screened a phage display library to identify short amino acid peptide sequences that bind to the gut epithelium of ACP. This screen resulted in identification of four candidate peptides. Of these, two were shown to bind to the ACP gut when fused to the reporter enzyme, mCherry but only one, peptide 15, was found to bind specifically to the gut in competition assays. Peptide 15 was shown to bind to a 50 kDa gut protein. Having identified specific insertion sites in the ACP-active Bt toxin, we constructed a set of modified toxins by addition to- or replacement of- amino acid sequences at four different sites in the toxin, with peptide 15. Some of the modified toxins did not express well using standard expression in E. coli. Expression of these modified toxins using different expression strategies is now underway. Once the expression protocol has been optimized, modified toxins will be purified and tested against ACP. It is expected that peptide 15 will provide a peptide anchor for increased toxin binding to the ACP gut epithelium, resulting in enhanced toxicity against ACP.
Obj. 4. The purpose of this objective is to determine whether prior pathogen or dsRNA exposure inhibits Las acquisition by psyllids. We investigated if D. citri exhibits immune priming and produces a different response to secondary infections and the specificity of that protection. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Surviving D. citri were moved to a second sachet containing 1e6 cells/mL live S. marcescens where they remained until all D. citri were dead. Mortality was recorded once daily. Transgenerational immune priming bioassays were conducted with psyllids placed on artificial diets containing heat inactivated E. coli, M. luteus, or no bacteria. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Data presented here are preliminary, as additional replicates were collected during February and March 2017. These insects are currently being processed and analyzed via QPCR. Offspring of female D. citri that were fed a diet containing M. luteus (n = 17, 2.60E7 1.35E7) or E. coli (n = 18, 2.09E6 8.40E5) had higher CLas titers than offspring of non-primed females (n = 8, 1.22E5 6.20E4). The Ct of plant material was correlated with the titer of CLas in D. citri (b = -0.140, F1,41 = 4.115, p = 0.050, R2 = 0.09). Previously, we demonstrated that the D. citri immune response is induced due to recognition of Gram-positive bacteria, such as M. luteus. Given that M. luteus protected D. citri from S. marcescens, and CLas is also a Gram-negative bacterium, it was expected that CLas titers would be lower in offspring of M. luteus primed adults. In fact, the opposite was observed. Females fed a diet containing M. luteus prior to mating had higher CLas titers and produced offspring that acquired CLas at a much higher rate than those from control or E. coli fed females. This suggests that immune priming with M. luteus infection may facilitate CLas infection.
This project has been reported under ‘Epidemiology and Cultural Control’, but it seems more appropriate to be under ‘Vector Management’, see the category above. Trees and branches to monitor for vegetative and reproductive bud development were selected in the test blocks and initial ratings were established. The Flowering Monitor System provided a first wave flowering full bloom date of February 11 to 20 depending on the location within the Florida citrus production regions, but actually occurred a day earlier. A second wave of flowering was projected to occur from March 8 to 11 depending on location, but this wave was a week early. Warmer than normal weather caused the advanced development and initiation of earlier full bloom. Data collection was started with bud break estimated to occur in early January. It appeared that bud break may be more reliably estimated from initiation of differentiation than from the estimation of full bloom date. Some preliminary plotting showed that in several years mean temperature the first 2 or 3 weeks after initiation of growth was important to determining how many weeks were required to reach full bloom. Dr. L. Stelinski agreed to cooperate in evaluating psyllid control when a block is sprayed at the beginning of spring budbreak rather than later after feather leaves are present. Two properties near Fort Meade were evaluated from early January until present. At one property, 2 blocks were sprayed January 3rd and at the other location two blocks were sprayed January 17th. No adult psyllids were detected by tap counts in 30 days where the spray was applied at budbreak in early January. However adults psyllids were detected at the location where the spray was applied later even though this was a shorter control time. The idea of spraying at budbreak but before feather flush appears to have some merit. We had information for growers on full bloom and 10 % open flowers but did not work out a way to make this information available to bee keepers. We need to develop an extension plan for bee keeper information. We had some success in an associated project in reducing off-season winter bloom by applying gibberellic acid monthly in the fall. We will monitor these trials to see if yields were improved. It did appear however that fewer flowers were present in the first flowering wave also. We will monitor the five locations that growers sprayed and one we sprayed to determine yield of the next crop.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
