Abstracts of Presentations Development of the Hydro-Solar Thermotherapy (HSTT) for treatment of HLB infected citrus

G Aiken, T Gottwald, J Kainz, G Poole, J Truett
unpublished
The use of thermotherapy to treat HLB-infected citrus trees has been demonstrated by various systems over the past few years, which basically fall into two primary categoriespassive solar systems (tents), and active heated water/steam spray systems. The system envisioned here is a combination of the twoa solar-heated water system, that is active but requires no power or user input, or expendable materials that need to be replenished. Circulating heated water is used to transfer heat to the tree
more » ... by means of a thermal insolated jacket placed around the trees trunk, or on larger trees, around main scaffold branches. Multiple trees can be linked to the water source in series or in parallel subunits. Preliminary tests to determine the maximum temperature achievable along with feasibility of operation were done with a solar heating system, similar to the envisioned final design. Water temperatures in excess of 60C can be generated, and under bark temperatures reached the low to mid 40's. A pulsed system has also been tested which runs for only 5 hrs and produced higher temperatures at the trunk, but not in excess of 45C. Once the proof of concept was completed, tests beds were designed to determine the optimum temperature of operation. The first test bed was basic and only provided heated water at a constant temperature, for extended periods of time. Studies showed that the maximum temperature that a diseased tree could tolerate was 48-49C over 48hrs. The second test bed, known as the Mobile Thermotherapy Lab, or MTL, is more versatile and designed to provide heating and cooling of water to simulate what might be achieved through solar heating and supplemental cooling with a 40C differential in temperature. The goal of the MTL is to look more in depth at the parameters, and to test the effect of sequential heating and cooling on CLas bacteria survival in the phloem. Results indicate that a passive solar water heating system can produce higher temperatures under the bark of the trunk, as well as increased temperatures into the canopy. This heat generated is below the thermal tolerance level of the tree, but also may not be hot enough to significantly decrease the bacterial population in situ as determined by pre-and post-PCR assay. We will discuss the question, 'If thermotherapy does not decrease bacterial populations, why do treated trees display disease remission for multiple months?". Temperature of 49C would be the maximum usable temperature due to the thermal tolerance of citrus trees, however optimal duration at that temperature has not been determined. Genetic diversity of the locus CLIBASIA 05640-05650 in strains of Candidatus Liberibacter from Mexico The bacterium associated with huanglongbing (HLB) in Mexico, Candidatus Liberibacter asiaticus (CLas) is present in 23 citrus growing regions, where its dissemination is due to the presence of its vector, Diapohrina citri Kuwayama (Hemiptera: Liviidae). In Mexico, some reports have documented differences in the severity of symptoms in infected trees according to the type of citrus (sour or sweet); those differences could be associated with CLas genetic variation. In order to probe this hypothesis, we analyzed a hypervariable region of the CLas genome from infected citrus leaves and D. citri adult samples from the Yucatán peninsula, and the Southeast of Mexico, as well as, the coastal zone of the Pacific Ocean and Northeast Mexico. Analysis of the samples yielded at least seven electrophoretic profiles; one is widely distributed among the states of the coastal zone of the Pacific Ocean and Northeast Mexico, and the rest is in the Yucatán peninsula and the Southeast part of the country. Our results suggest the presence of two CLas populations in Mexico, one of them showed genetic diversity and characteristically restricted to the Yucatán peninsula and the Southeast; meanwhile, the other lacks such diversity, the records suggest wide distribution in the country, and is infecting the Mexican lime in the citrus region of the Pacific Ocean. Our data provide insights into the understanding of the epidemiologic development of HLB in Mexico, and for the improvement of the current management strategies. Micro-CT scanning of Asian citrus psyllid, Diaphorina citri, anatomy and feeding An international collaboration to establish an interactive Digital Video Library for a Systems Biology Approach to study Bacteria transmission by the Asian citrus Psyllid, linking psyllid genomic/proteomic interactions is demonstrated. Advances in micro-CT, digital computed tomography (CT) scan uses X-rays to make detailed pictures of structures inside of the body, can now visualize very small insects with great detail. Combining micro-CT imaging which permits digital section of an insect with Digital Video Library systems which links information from other databases enables a new visualization and interaction by the viewer. 'Interactive Information' systems are changing the way researchers, entomologist, and the public search for and use complex information. Diaphorina citri, spreads a plant-infecting bacterium linked to Citrus greening disease, a worldwide threat to citrus sustainability. The anatomy was elucidated using a high resolution Bruker Skyscan 1172 micro tomography system, micro-CT (www.Skyscan.be) Department of Zoology, University of Granada, Spain. Live psyllids were prepared by overnight fixation in 4% glutaraldehyde with 2.5% paraformaldehyde made with sodium cacodolate buffer pH 6.5. Samples were rinsed three times, 10 min each, with 30% ethanol, and dehydrated in an ethanol series, 30 min per step, 50%,70,80,90,95, three times at 100%. Samples were chemically dried by placing in 2 mL of 100% Hexamethyldisilazane (HDMDS) for 2 hrs, drying overnight at 35C. High resolution scans combined with computer software enabled rotation, 'digital sectioning', and coloring of specimens providing an innovative, interactive system to explore insect anatomy. Video http://www.youtube.com/watch?v=LLz5QWyNM98. In-depth explana-tions of sample preparations of leafhoppers and other insects, plus operation of the software for the Bruker SkyScan system at: A recent study investigated leaf metabolite profiles of six citrus rootstock cultivars grown as seedlings, with different responses to HLB using untargeted GC-TOF MS analysis. The study found that tolerance to HLB does not seem to be associated with an accumulation of larger amounts of protective metabolites in response to infection with Candidatus Liberibacter asiaticus (Las), but rather with different concentrations of specific metabolites independent of infection. The highest number (166) of metabolites responding to infection with Las at 12 months after graft inoculation (mai) was found for the susceptible rootstock cultivar Cleopatra mandarin (Citrus reticulata), but the majority of compounds (122) were of unknown chemical identity. The present study focuses on the chemically known metabolites and their involvement in the different pathways associated with primary metabolism. The study was conducted on greenhouse-grown rootstock seedlings at three different stages (8, 10 and 12 mai) of disease progression. Among the metabolites most strongly affected by Las in Cleopatra were those associated with arginine and proline metabolism, specifically the amino acids proline, hydroxyproline, ornithine, and citrulline. Numerous roles for proline have been suggested, and include osmoprotection, stress adaptation, reactive oxygen species (ROS) generation, and phytohormone-induced senescence. Other important functions of the amino acids related to this pathway are involvement in nitrogen metabolism and root architecture. Interestingly, whilst compounds of arginine and proline metabolism were up-regulated in response to Las at 8 and 10 mai, a significant down-regulation was observed at 12 mai, suggesting collapse of defense responses at this later stage of disease, and resulting in intensification of disease symptoms and plant decline. Also down-regulated at this stage of disease were metabolites associated with other sugar metabolism pathways, namely raffinose, galactinol, melibiose, sorbitol, glucose-1-phosphate, glucose, and fructose. Sugars are important components of plant signaling reactions and defense and provide important sources of energy for pathogens. Comparison of responses of Cleopatra with the more tolerant rootstock cultivar Carrizo citrange (C. sinensis x Poncirus trifoliata) showed a similar down regulation for several pathways at 12 mai, most notably pathways associated with arginine and proline metabolism, galactose metabolism, and propanoate metabolism. The similarity of metabolic responses of Cleopatra and Carrizo is surprising, but may be associated with the foliar disease symptoms which were similar in manifestation in both cultivars at this stage of infection. It is important to note that except for galactose metabolism, other pathways of carbohydrate metabolism as well as TCA cycle reactions were not affected in Carrizo, which may explain the better performance of this rootstock cultivar under HLB pressure. In contrast, the tolerant rootstocks US-897 and US-942 which did not show any disease symptoms at 12 mai responded to infection by changes in the amounts of only three metabolites. This suggests that different and rootstockspecific mechanisms are associated with tolerant responses to HLB. Citrus flowering and vegetative growth characteristics in relationship to Asian citrus psyllid control in HLB management LG ALBRIGO Due to concern for pollinating bees in citrus trees in the spring, spraying for psyllid control with harsher, but often more effective pesticides is suspended from when 5 to 10 % of the citrus flowers are open until 95 % petal fall is reached. After use of dormant sprays at least one spray on emerging flush is desired before bee-friendly sprays are applied during the 'flowering period'. Data was not available for how long a period existed from various stages of vegetative growth until 5 to 10 % open flowers is reached. Hamlin, Valencia, Sunburst and Murcott blocks were monitored during the 2015, 2016 and 2017 bloom periods to determine vegetative bud break, leaf feather stage and unfolded leaves as well as pinhead, popcorn, open and petal fall stages of flowering. In the first two years the average time period from bud break to 10 % open flowers was 39 and 45 days, respectively, and the days from 10 % leaf feathers until 10 % open flowers was 26 and 20 days. This provided 13 and 25 day intervals between bud break and feather leaves in the first two years. Round oranges and mandarins differed in time from bud break to full bloom in 2015, 72 versus 57 days, but were similar in 2016, 57 and 56 days. Petal fall (95 %) occurred 8 to 20 days after full bloom. Weather data will be evaluated to determine if any differences in time for vegetative development between the three years was due to daily temperatures, similar to the temperature response for flower development. More details including 2017 data will be discussed, particularly as pertaining to the best time to apply the first vegetative spray before the 10 % open flower stage is reached. The predicted date on which vegetative flush may be optimum for applying a psyllid spray could be obtained from the 'Citrus Flowering Monitor System', which is on-line: http://disc.ifas.ufl.edu/bloom. Another aspect of interaction between the citrus tree and HLB is the increase in off-season flowering. This is most pronounced in early-to mid-winter under Florida conditions and has consistently occurred over the last four years. This flowering reduces the available buds for normal spring flowering and probably contributes to carry over of fungal inoculum for postbloom fruit drop (PFD). The role of fall tree water stress on the induction of this off-season flowering, and the association of this tree water stress with debilitation from HLB will be discussed. Diaphorina citri samples from many different venues in peninsular Florida were tested for presence of Candidatus Liberibacter asiaticus (Las) between 2005 and 2008. The positive samples were geocoded and plotted on a map of the state. Independently, suspect plant samples also were collected from many different venues and tested for Las at the Florida Department of Agriculture and Consumer Services, Division of Plant Industry between 2005 and 2010. The positive samples also were geocoded. The distribution of positive plant samples was compared with the distribution of positive psyllid samples to determine whether the two sets of samples were correlated, and whether positive psyllids predict positive plants or vice versa.
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