Valencia fruit were harvested and juiced, with blends of symptomatic greening fruit (based on % weight of fruit) as previously described for Hamlin fruit. Blends include 0% greening (negative control), 2.5% greening, 5% greening, 10% greening, 20% greening, 50% greening, and 100% greening (positive control). Due to breakdown of the microthermics juice pasteurizer, all juice remains at -20’C until equipment is repaired. We evaluated impacts of HLB and girdling on oil compounds in ‘Valencia’. The girdle treatments were performed on trees as previously reported. Fruit affected by HLB and/or girdling were collected in April 2011. At least 8 trees for each treatment, and at least 4 fresh fruit from each tree (a biological replicate) were used for this study. The collected samples include healthy (H), asymptomatic (AS), symptomatic (SA), ungirdled (UG), half girdled (HG), and full girdled (FG) fruit. The cold-water press method was performed to extract total oil from fresh fruit flavedo. Results indicated H, AS, UG, and HG fruit had similar amount of oils (from 5.5 to 6.1 .l of oil/g fruit weight). Oil content was significantly reduced in SA and FG fruit (3.0 and 3.2 .l of oil/g fruit weight). GC-MS analysis was performed to compare the oil components in all of the treatments. Uneven pigmentation was observed in HLB and FG fruit peel. Thus, oil for GC-MS analysis was extracted from stem end, equator and blossom end separately to account for positional differences, if any. Here we report on oil extracted from the fruit equator. For each replicate, 0.1.l oil was directly injected into GC-MS. 37 compounds were identified by GC-MS analysis. Principal component analysis (PCA) will be performed to statistically analyze data from all treatments from different fruit positions when completed. When compared with UG fruit, the accumulation of 9 out of 37 volatiles was changed in FG, including octanal (0.2x), linalool (0.1x), trans-P-mentha-2 (0.5x), limonene oxide (3x), citronellal (53x), .-terpineol (0.4x), decanal (0.3x), neryl acetate (2.6x) and dodecanal (0.4x). Only one volatile (citronellal) was found altered in HG compared to UG fruit. When compared with H fruit, 10 compounds were shifted in SY fruit, including octanal (2x), .-terpinolene (0.5x), nonanal (0.4x), limonene oxide (2x), citronellal (41x), .-bergamotene (0.5x), dodecanal (0.5x), .-cubabene (0.5x), .-farnesene (3.1x) and .-sinensal (3.3x). Five volatiles were also changed in AS fruit when compared with H fruit. The preliminary results indicate that HLB and girdling altered the amount and components of citrus oil. Accumulation of 7 volatiles was affected by HLB but not girdling. Those include octanal, .-terpinolene, nonanal, .-bergamotene, .-cubabene, .-farnesene and .-sinensal.
Our study with Citrus sinensis ‘Valencia’ trees indicated fruit drop in HLB-infected trees was initiated at breaker stage. When compared with fruit in healthy trees,10-fold higher symptomatic fruit dropped to the ground and the detachment force of attached fruit (FDF) was reduced 75%. There was no difference of % abscission and FDF between asymptomatic fruit and healthy fruit. When compared with abscission zones of healthy fruit, the expression of abscission-induced genes such as 1-aminocyclopropane-1-carboxylate synthase (CsACS1), cellulose-a1 (CsCel-a1), polygalacturonase (CsPG), phospholipaseA2. (CssPLA2.) and PhospholipaseA2. (CsPLA2.) were 17-, 7-, 530-, 1.5-, and 2-fold higher in symptomatic fruit abscission zones respectively. The expression of CsACS1 and CsCel-a1 were 5-fold increased and CsPG was 150-fold increased in asymptomatic fruit abscission zones. In addition, ethylene production was 6- and 2-fold higher in the areas of symptomatic and asymptomatic fruit abscission zones respectively. HLB increased leaf abscission by 7% after 80 observation days. A 10% reduction of the detachment force and increased expression of abscission-induced genes were also found in symptomatic laminar and petiole abscission zones compared to healthy controls. We analyzed girdled tissues to study the impacts of carbohydrate deprivation on abscission and HLB-like symptom development. A bark full ring or half ring around the twig located 10-cm above a fruit was removed at immature stage. Leaves between the girdled region and fruit were removed. Girdling was also applied on a branch that had between 50 to 100 of leaves. The study showed symptomatic and full girdled fruit shared similar impacts on size, weight, and color. Lopsided symptoms were only found in symptomatic, but not girdled fruit. In most of the full girdled fruit, the flavedo and immediately underlying regions of albedo very close to fruit abscission zone was orange in color. Mottled chlorosis appeared on leaves subtending the full girdled areas. There was no visual differences between half-girdled compared to ungirdled tissues. Similar to symptomatic tissues, full girdled tissues showed the shift of starch and sucrose contents, premature fruit drop, leaf drop, and the increased expression of abscission-induced genes at abscission zones. However, the impacts of girdling were greater than HLB infection especially on tissue drop and carbohydrate metabolism.
During the final quarter of the year, the Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. During the 4th quarter, the CCTF continued to utilize the provided funding to process the orders for transgenic plants, mostly servicing CRDF funded researchers studying transgenes with potential to generate resistance to HLB. Orders processed during this quarter include: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. The impact of citrus diseases on Florida citriculture is rapidly growing, and our participation in this battle is growing with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. A number of plant derived insecticidal transgenes, each driven by a root specific promoter were incorporated into Carrizo citrange. As part of this project we cloned four insecticidal genes and two plant derived promoters. Constructs were initially tested on N. benthamiana for confirmation of transgene activity. Carrizo citrange was subsequently transformed utilizing the conventional Agrobacterium mediated transformation process. A total of 123 putative transgenic lines were generated. PCR screening identified 74 of these lines to be transgenic. 54 of these transgenic lines had a high level of transgene expression as determined by qPCR. We have also identified a number of putative root specific genes from Citrus clementina by data mining the phytozome database. Of them several putative sequences were selected for characterization using qPCR. RNA was extracted from mature and juvenile roots from non-transgenic Swingle citrange. Each of the identified sequences were characterized in these different tissues. In addition, transcript levels in the leaves were also measured. The Cic1867m gene was determined to be very root specific and a 1.2 kb fragment of its promoter was cloned from Clementine genomic DNA using PCR. Deletion analyses identified a 0.8 kb fragment from that promoter fragment to be sufficient for root specific activity and transgenic plants were produced using this promoter. Cuttings from all the better performing lines have been made and are being rooted in the mist bed. These clones will be sized up for Diaprepes feeding experiments. Clonally propagated plants will be force fed with Diaprepes neonates – when available and root damage / insect mortality evaluated.
This project had a single objective to assure un-interrupted production of transgenic citrus plants through the services of Citrus Transformation Facility (CTF). Continued operation of the facility was going to guarantee a foundation regarding this aspect of research for the scientific community involved in fight against huanglongbing (HLB) and citrus canker. By producing transgenic plants and bringing into life the ideas of scientists working in different laboratories, CTF would facilitate the process of search for candidate plants that would be tolerant/resistant to diseases and provide major relief for Florida Citrus Industry. The objective of this project was successfully accomplished. Throughout the funding period of three and-a-half years, CTF operated without major interruptions and provided service in the form of production of transgenic plants. The level of production stayed at expected, satisfactory level and resulted in creation of 781 citrus plants that represent independent transgenic events. These plants belong to eight different cultivars: Duncan grapefruit, Carrizo citrange, Pineapple sweet orange, Mexican lime, Valencia sweet orange, Swingle citrumelo, Kumquat, and Pomelo.During this project, CTF worked on 103 orders that were placed by 10 clients and this group included eight faculty members based at University of Florida, one faculty from University of California and one Foundation. The number of projects for which the plants were produced was higher than the number of clients. For example, Dr. Nian Wang was one of the clients but orders from his lab were the part of at least five different projects done by his post-doctoral associates.All produced transgenic plants were associated only with research that has to do with disease resistance. This is a clear indication of the role CTF plays in the efforts to overcome effects of HLB and citrus canker.CTF supported the projects from Dr. Nian Wang’s lab that resulted in successful application of CRISPR/Cas9-mediated genome editing technology in Citrus. Plants produced this way will be the most market-friendly as they only have minor modifications of their own gene(s) and in the near future will be free of any other inserted DNA. The work done in CTF lead to production of Duncan grapefruit plants that exhibit high degree of resistance to citrus canker. Other commercially important cultivars could be made with improved resistance to canker based on results of this project.As a part of another research project aimed at control of Asian Citrus Psyllid (ACP) in citrus groves, transgenic Indian curry leaf plants were also produced in CTF. There are six such plants that are being tested for the ability to kill ACP after they were allowed to feed on their leaves. All these successes were achieved even though CTF experienced high flux of employees, had to move to temporary location for six months, and was exposed to adverse effects of hurricane Irma.Future opportunities for the CTF are determined by the needs of Florida Citrus Industry. As those needs change from the search for the solution to HLB and canker to a development of specialty fruit with consumer-oriented traits in post-HLB era, CTF will be there to support all these efforts.
There were three significant accomplishments during the quarter. A visit to the citrus sector of Sao Paulo, Brazil was completed in May. The trip included visits to two large citrus growing farms and several smaller farms. FUNDECITRUS, the major citrus research entity in Brazil was also visited and researchers there gave an update on the greening situation in Sao Paulo. They have developed a model of greening spread at the grove level which may prove valuable in grove level decision-making. A main finding from the trip is that greening is widespread in the central-east portion of the Sao Paulo citrus growing area. A large number of trees in this area have been eradicated and more eradication will occur in the near future. While new planting to replace eradicated trees was accomplished at a high rate in 2006-08, new planting has declined with lower fruit prices which will have implications for future fruit production. A second accomplishment is that a relationship with researchers at the University of Sao Paulo has been established which will foster information exchange. Dr. Margarete Boteon will visit Florida in August to make a presentation at the Southwest Florida Citrus Expo. She has recently published a study which was two case studies that analyzed the cost of production for citrus in Sao Paulo. This study provides detail on production practices and costs not published previously. It will be of great assistance to analyze both future production prospects and the competitiveness of the citrus sector in Sao Paulo. An update of the economic impact of the citrus sector on the economy of Florida was begun late in the quarter. With the loss of both tree numbers and acres planted across all citrus varieties grown in Florida, it is important to establish a new benchmark on the economic impact of the industry. A similar approach to previous studies is being taken in which an input-output model of the Florida economy is modified to identify the economic contribution of the citrus industry.
Two major accomplishments were completed during this quarter. First, the world orange juice model was updated to reflect the 2008 Florida Citrus Tree Census. Second, Dr. Margarette Boteon, a collaborator on the project came to Florida in August to participate in the SW Florida Citrus Expo. She provided new data with respect to orange production in Sao Paulo which will be incorporated into the world orange juice model. A meeting of all project investigators (except Alan Hodges) was held in Ft. Myers and a plan was formulated to guide future work on the project. A M.S. thesis by Jordan Malugen in which an investment model for citrus was developed was completed. This model will be used in the grove level assessments to be made in the project.
We continued the work this grant focusing on the infection of various citrus and other rutaceous plants that might be alternative hosts for both the psyllid and the bacterium Candidatus Liberibacter species associated with HLB. One of the new hosts for the psyllid that we reported in the last quarter, Choisya ternata, was grafted with PCR positive budsticks of HLB infected citrus. The grafts were not successful and died in the plants. Plant materials were sent to both the USDA, ARS, Molecular Plant Pathology Lab, Beltsville Exotic Citrus Pathogen Collection and to the USDA, ARS Foreign Disease and Weed Science Research Lab, Frederick, MD for grafting and psyllid transmission of the Ca. Liberibacter americanus and the Ca. Liberibacter. africanus strains. Infectivity assays are pending. In psyllid inoculation tests with Ca. Las we were able to infect four of six Choisya ternata plants in 3 months. In field surveys for alternative hosts we sampled a group of 18 sour orange trees that were growing in a pine planting close to commercial citrus plantings. Of the 18 trees sampled one was found to be PCR positive for Ca. Liberibacter asiaticus. In other field work we sampled a swamp area close to where the initial infections of a grove were discovered. Of 35 plants samlee no infected plants were found including a number sweet orange and grapefruit seedlings. Additional sampling is planned to look for alternative hosts for HLB that could be reservoirs for infections. We continue to propagate and inoculate seedlings and grafted plants of IAPAR 73. As reported previously we were unable to infect some seedlings with HLB material. We presented these results at the Florida State Horticultural Society meeting in June and have submitted a manuscript to the proceedings. Publications: Damsteegt, V. D. E. N. Postnikova, A. L. Stone, M. Kuhlmann, A. Sechler, N. W. Schaad, R. H. Brlansky, and W. L. Schneider. 2010. The relevance of Murraya paniculata and related species as potential hosts and inoculum reservoirs of ‘Candidatus Liberibacter asiaticus’, the suspected causal agent of Huanglongbing. Plant Disease 94:528-533. (Editor’s paper of the month of May).
During the first year of this project, we evaluate the efficacy of different phytohormones in suppressing the production of new flush growth on potted citrus plants in the greenhouse. Two separate field trials were also conducted 1) to determine how new flush growth suppression by these phytohormones will relate to Asian citrus psyllid (ACP) population and 2) to elucidate the impacts of pruning date and fertilization level and timing on the population of ACP in a mature sweet orange block. In the greenhouse, potted lime trees were first pruned to stimulate flush production, and each phytohormone was immediately sprayed following the label recommended rate. Ten potted plants per treatment along with an untreated control that received tap water treatment were used. The numbers of new flush shoots on potted plants were recorded twice a week for the first two weeks and weekly during week 3 and 4. Three phytohormones, namely Apogee (0.7g/L), Sumagic PGR (100mL/L), and NAA-1-Naphthaleneacetic acid (0.5’L/L) significantly reduced the number and delayed the growth of new flush shoots produced by lime trees. The number of new flush shoots produced by plants treated with these 3 growth regulators was at least 2-fold lower than the untreated control. In field trials, rows of a mature ‘Valencia’ block were pruned and each of the 10 phytohormones was applied to a group of 2 contiguous trees. A randomized block design with 4 replications was used. Number of new flush shoots and densities of ACP were recorded weekly for 4 weeks. Out of the 10 phytohormones, only two (Apogee and NAA-1-Naphthaleneacetic acid) significantly reduced the number of new flush produced by trees, but the reduction was less than 20% relative to the untreated control. Due to the overall low numbers of psyllid recorded during the trial in October, no meaningful statistical inference could be made. In the second field trial two hedging dates (one early in February and one late in April) and two nitrogen fertilization regimes (one application of 100 lb/ac in February and two applications of 50 lb/ac each in February and in June) were tested in a factorial design in a mature sweet orange block. Weekly counts of new flush shoot growth and ACP densities were made. Both hedging dates and application of nitrogen significantly affected ACP infestation and densities. Hedging significantly altered the phenology and intensity of new flush shoot production on trees. Both the early and late hedging dates of trees stimulated profuse flush shoot production 2 to 3 weeks post-hedging. Significantly higher ACP infestation levels and densities were recorded in the late hedging date compared to the other treatments from May to October. Although more new flush shoots were produced in the early hedging treatment relative to the non-hedged treatment, ACP populations were comparable in these two treatments. These results clearly demonstrate that early hedging (February) should be encouraged as it prevents severe outbreaks of ACP populations, while providing the intended physiological benefit of the practice. By contrast, late pruning in spring will likely lead to ACP outbreaks in citrus orchards. Application of nitrogen also affected the abundance of new flush growth. Although no alteration of flush cycles resulted from N application, densities of new flush growth were higher in fertilized plots than in the non-fertilized control blocks. The effect was more dramatic in blocks where N was applied in a single dose. In the one-time N application treatment, significantly more new flush shoots were produced which resulted in higher densities of ACP eggs and nymphs for most of the sampling dates. ACP densities in the split application and non-fertilized control were similar throughout the sampling period. In summary, N management, and in particular, split N fertilization and early pruning of trees were associated with lower ACP population densities on sweet orange trees. These cultural practices can be used to manipulate the phenology and abundance of citrus flush shoots and consequently the population densities of ACP.
As a preliminary step to understand and characterize what metabolites are responsible for the bitter off-favor of Huanglongbing infected fruit, the thresholds of limonin, nomilin, and their combination in a sugar and acid matrix, as well as in healthy ‘Valencia’ orange juice were determined by taste panels. Food grade limonin and nomilin were added alone or in combination to a simple (sucrose and citric acid) or complex (sucrose, glucose, fructose, citric and malic acid) matrix, or were added directly into orange juice. Thresholds were determined by taste panels, composed of 16 to 23 trained panelists, using a three-alternative forced choice (3-AFC) method (ASTM: E-679). In the simple matrix, the threshold of limonin was lower than nomilin. The synergetic effect of limonin and nomilin was significant in decreasing their individual thresholds. Interestingly, the thresholds of limonin and nomilin were lower in orange juice compared to the thresholds measured in the complex matrix. Our current results show that the threshold concentrations of limonin and nomilin when added to healthy ‘Valencia’ orange juice are higher than the concentrations of those compounds measured in juice made with symptomatic HLB fruit, which was perceived bitter by a taste panel. Possibly, the lower sugar and higher acid content of HLB fruit decreased the threshold of those bitter compounds. Moreover, different concentrations of ‘Valencia’ and ‘Hamlin’ HLB infected juice were blended into healthy juice to determine the detection and recognition thresholds. Panelists were able to detect the symptomatic HLB juice at different levels depending on the variety. For both Hamlin and Valencia juices, however, panelists could detect a difference when blending normal juice with 25% HLB symptomatic juice and could describe the difference (bitter, metallic) when normal juice was blended with 50% HLB symptomatic juice. Nomilin was discovered to have a lingering metallic taste that was different from limonin, which was found to be just bitter. This study looked at flavor compounds in juice made from fruit harvested from 15+ trees symptomatic for HLB compared to healthy trees grown in the same area for multiple harvests of Hamlin (December/January, 2009) and Valencia (April/June, 2009). Fruit from HLB symptomatic trees were separated into asymptomatic (normal looking, HLBAS) and symptomatic (small, green and lopsided, HLBS) fruit for comparison to healthy (H) fruit prior to juicing using a JBT extractor/pasteurizer. For Valencia, there were no differences in Brix, but HLB juices tended to have higher titratable acidity (TA), lower ratio (April) and higher oil (June). For Hamlin, Brix was higher in H juice (December), TA higher in HLBS (December), ratio lower in HLBS and oil higher for HLBS juice (December). Healthy juice tended to have higher levels of sucrose and fructose, whereas glucose was variable, especially for HLBS juice. HLB juice had higher levels of citric acid (December Hamlin/April Valencia) but there were no differences for the other two harvests/cultivars. Malic acid tended to be lower in HLB juices. Limonin and nomilin were higher in HLB juices, especially HLBS, but were below reported thresholds. Gas chromatography-olfactometry (GC-O) research did not show differences between H and HLBAS juice, but observed some volatiles that were found either exclusively or at higher aroma intensity in either H or HLBS juices. There were more :green’/ ‘fatty’ aromas in HLBS while there were more ‘sweet’/’fruity’ components in H juices with respect to each other.
Objective 1 was to understand the cause of leaf chlorosis. In the leaf, starch packing of chloroplasts occurred upstream after phloem blockage and often after some necrosis was detected. Subsequently chloroplast grana were disorganized and disappeared and chlorosis of these affected cells in the leaf tissue occurred. Phloem plugging was elucidated as smooth (callose formation) and fibrillar (phloem protein 2 (PP2) ligand formation) materials in sieve elements accompanied by some phloem necrosis. The combination leads to phloem blockage and starch accumulation above blocked phloem. The cascade of blockage down the phloem system leads to root starvation and tree decline. Plugging by callose was about 3 times more prevalent than PP2 plugging in HLB affected trees in both the greenhouse and field. Objective 2 was to attempt to alter the primary plugging cause (callose formation) and determine how important callose plugging was to the overall disease symptomology of HLB. This was accomplished by over expressing the citrus beta-1, 3 glucanase gene in citrus plant phloem. The goal was to either accelerate breakdown of the callose ligand or block its breakdown depending on how metabolism reacted to the over expression. Plants were produced and are now under greenhouse screening to compare their development to non-transformed plants both exposed to the HLB causing bacteria C. Las and without the disease. Objective 3 was to determine if any virulent genes from C. Las could directly cause any of the symptoms described for HLB infected citrus plants. The known bacterial genes were evaluated for overexpression in HLB affected citrus and 44 putative virulent factors were identified. Two of the hypothetical genes that were overexpressed in planta were screened in Nicotiana benthamiana, and the tobacco plants showed disease symptoms, primarily general necrosis. Transgenic citrus plants (Duncan) expressing these two genes were constructed at the Citrus Transformation Facility. Another potential virulence factor overexpressed in planta was hemolysin. Results indicated that C. Liberibacter encodes a functional hemolysin protein which might be involved in pathogen and host interaction. Two papers and 2 abstracts were published related to Objective 1 and 2.
Transmission tests using endemic healthy Brevivalpus phoenicis mites from Florida were done in quarantine at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD. Cytoplasmic citrus leprosis infected samples were sent under permit from Colombia to the USDA, APHIS, PPQ quarantine facility at Beltsville, MD and then shipped under permit to the Ft. Detrick lab. The leprosis materials arrived in excellent condition as compared with previous samples that were detained in customs. Mite transmission tests were also done at the same time by our cooperator in Colombia. Results of both transmission tests are pending. The manuscript describing the new cytoplasmic citrus leprosis virus from Colombia (“A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis) was accepted for publication in Phytopathology) Contacts have been made with Mexico where another type of citrus leprosis has been detected.
Cytoplasmic citrus leprosis infected samples were sent from Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. Again they were negative in PCR and antibody tests for cytoplasmic citrus leprosis virus (CiLV-C). Samples were from different cultivars of citrus in order to see if the samples differed in the virus contained. Samples again were prepared for electron microscopy to verify the type of viral particle and particles similar to those previously published for cytoplasmic citrus leprosis were seen. Sequencing (funded on another project) was completed and the bioinformatics confirmed a new cytoplasmic citrus leprosis virus. New primers were produced for detection and samples were sent from Colombia for detection of both types of cytoplasmic citrus leprosis virus. Mr.Leon in Colombia as earlier reported continues to do successful mite transmission experiments with PCR negative isolates from Colombia. Samples were sent under permit to the USDA and using primers to the the new cytoplasmic citrus leprosis virus (CilV-C2) we have found that he has transmitted this new virus with Brevipalpus mites from Colombia. A manuscript is in preparation.
Healthy endemic Brevivalpus phoenicis mites from Florida were reared and acquired from Dr. Jorge Pena, University of Florida, Tropical Research and Education Center, Homestead, FL. The mites were shipped under permit to the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD. Cytoplasmic citrus leprosis infected samples were sent under permit from Colombia to the USDA, APHIS, PPQ quarantine facility at Beltsville, MD and then shipped under permit to the Ft. Detrick lab. Healthy citrus plants were shipped at the same time for transmission tests. Transmission tests were performed however mite survival was less than expected. We are awaiting results. Mite transmission tests were also done by Guillermo Leon in Colombia and the mites were shipped in alcohol for comparison with the Florida mites. The Florida mites and a sample of the mites were prepared for low temperature SEM by Dr. Ronald Ochoa and Dr. Gary Baucham of the USDA, ARS, Beltsville, MD. They reported and produced photos of the mites. The mites from Colombia and the Florida mites were both found to be B. phoenicis type II. All transmission experiments will be with the same mite species and type.
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