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M.G. DeWald, G.A. Moore and W.B. Sherman

Genetically characterized isozyme loci are useful for taxonomic studies. In an initial study a few Ananas genotypes were used to determine which enzyme systems would give well-resolved banding patterns on starch gels. The enzyme-staining systems that resulted in well-resolved banding patterns were used to survey more Ananas genotypes to identify and characterize isozyme polymorphism. Genetic studies were performed using seedling populations to determine the basis of variability observed among genotypes. Two peroxidase loci and three phosphoglucomutase loci were identified and characterized. Information from these studies, was used to formulate a system by which species and plant introductions could be identified and distinguished.

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C.A. Weber, W.B. Sherman and G.A. Moore

Segregating F2 peach populations in the Univ. of Florida breeding program were analyzed to determine linkage relationships among five qualitative traits: flower type, Sh/sh, flesh type, M/m; flesh color, Y/y; leaf gland type, E/e; and pubescence, G/g. Independent segregation was confirmed between flesh color and leaf gland type, between pubescence and flesh color, and between flower type and pubescence. Previously undocumented independent segregation was found between leaf gland type and flesh type and between pubescence and leaf gland type in our populations. The relationship between these latter characteristics should be investigated in other breeding populations. No correlation was found between fruit development period and flesh type. Also, no correlation was found between chilling requirement and flesh type.

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Pan-chi Liou, Fred G. Gmitter Jr. and Gloria A. Moore

Citrus genetic studies and cultivar improvement have been difficult with conventional techniques. Alternative approaches are needed to enhance efficiency of such studies. Our objectives were to characterize the Citrus genome and to initiate development of a linkage map using RFLP and isozyme analysis. Methods of Citrus DNA extraction were developed to allow the isolation of chromosomal DNA of acceptable quality for recombinant' DNA manipulations. A PstI Citrus genomic library was constructed to create DNA clones for the RFLP survey. A rapid, reliable procedure was developed to facilitate screening of the library for useful clones. The methods used and strategy followed minimized contamination with organelle DNA, increased the frequency of single copy clones, and allowed rapid screening of the newly–constructed library. Linkage relationships of 49. markers, including 36 RFLP and 6 isozyme loci, were analyzed and a map comprised of 8 linkage groups was constructed. Insertions or deletions were responsible for at least 30% of the RFLPs identified. A hypothesis of transposon activity in Citrus was proposed based on our observations.

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Ed Stover, David G. Hall, Robert G. Shatters Jr. and Gloria A. Moore

Assessments of the resistance of citrus germplasm to huanglongbing (HLB) can be expedited by inoculating plants under laboratory or greenhouse settings with the HLB bacterium, Candidatus Liberibacter asiaticus (CLas). Consistent rapid screening is critical to efficiently assess disease resistance among plant materials; however, a number of factors may govern the efficacy of such inoculations. Despite the rapidity at which HLB can spread in a grove, it often takes 8 to 10 months for high levels of CLas and HLB symptoms to develop even in highly susceptible sweet orange. Therefore, two experiments were conducted to assess factors that might influence efficiency in screening for HLB resistance. In one experiment, three test citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, HLB susceptible) were bud grafted using CLas-infected buds from four different source genotypes. All bud source genotypes had similar levels of CLas titer, but citron, rough lemon, and Volkamer lemon were hypothesized to be better bud inoculum sources as they are more tolerant of HLB than ‘Valencia’. Among the three test genotypes over all sources of infected buds, inoculations of ‘Kuharske’ resulted in lower CLas titers and fewer HLB symptoms than inoculations of rough lemon or ‘Valencia’. Inoculations of rough lemon resulted in higher CLas titers and more pronounced HLB symptoms when it was inoculated using infected buds from rough lemon or ‘Valencia’. Grafting ‘Valencia’ with infected buds from Volkamer lemon resulted in less disease than when ‘Valencia’ was grafted with infected citron, rough lemon, or ‘Valencia’ buds. Overall, these results suggest that the source of CLas-infected buds used to graft-inoculate some genotypes will influence disease development. Trunk cross-sectional area increase for the year following infection was 3× higher in ‘Kuharske’ and rough lemon, compared with ‘Valencia’. ‘Kuharske’ had very low levels of CLas (30 CLas/µg DNA), whereas ‘Valencia’ (43,000 CLas/µg DNA) and rough lemon (6700 CLas/ µg DNA) had relatively high levels. As an alternative to graft-inoculating plants with CLas-infected buds, plants can be subjected to infestations of CLas-infected Asian citrus psyllid (ACP) as occurs naturally. Of interest is if transmission rates of CLas and the development of HLB in a genotype are greater when the ACP have been feeding on the same host genotype. An experiment was therefore conducted to assess transmission of CLas by ACP reared on CLas-infected rough lemon to five different genotypes (‘Carrizo’, ‘Flame’ grapefruit, rough lemon, ‘Temple’, and ‘Valencia’). These assessments were made using a detached leaf assay recognized as a faster method of gauging transmission rates of CLas than using whole plants. Higher percentages of ACP died when they were transferred from infected rough lemon to healthy ‘Carrizo’, and lower percentages died when they were transferred to rough lemon or ‘Flame’. However, CLas transmission by infected ACP occurred to at least some leaves of each genotype in each of the five different assays, with 70% or more leaves of each genotype becoming infected in at least one assay. Over all assays, there was relatively little variation among genotypes in the percentage of leaves becoming CLas infected and in the titer of CLas developing in infected leaves. However, there were relatively large differences in transmission rates among individual assays unrelated to differences among test genotypes. Because of the rapidity of the detached leaf assay, efforts are merited to improve consistency of this inoculation method.

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D.J. Gray, J.A. Mortensen, CM. Benton, R.E. Durham and G.A. Moore

Ovules of seedless bunch grapes (Vitis spp.) fertilized by controlled pollination increased in size during berry development. More ovules cultured 10 days or 60 to 70 days after pollination became brown compared to those cultured at 20 to 40 days. Cultured ovules developed with and without endosperm. Globular to torpedo stage embryos were recovered. More embryos and plants were recovered from ovules cultured at 40 or 60 days than at 10 or 20 days after pollination. Pollen parent significantly affected both embryo and plant recovery at certain sampling times. BA incorporated into medium significantly increased embryo germination percentage. Electrophoretic analysis of glucosephosphate isomerase in progeny showed that 67% to 88% were hybrids of controlled crosses. Of four vines that fruited thus far, two were seedless. Seedless progeny had smaller seed traces than either parent. Chemical name used: N-(phenylmethyl) -1H-purin-6-amine (BA).

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Courtney A. Weber, Gloria A. Moore, Zhanao Deng and Fred G. Gmitter Jr.

Mapping quantitative trait loci (QTL) associated with freeze tolerance was accomplished using a Citrus grandis (L.) Osb. × Poncirus trifoliata (L.) Raf. F1 pseudo-testcross population. A progeny population of 442 plants was acclimated and exposed to temperatures of -9 °C and -15 °C in two separate freeze tests. A subpopulation of 99 progeny was genotyped for random amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR), and sequence tagged site (STS) markers to produce a linkage map for each parent. Potential QTL were identified by interval mapping, and their validity was corroborated with results from means comparison (t test), one-way analysis of variance (F test), and bulked segregant analysis (BSA). Multiple analytical methods provided evidence supporting putative QTL and decreased the probability of missing significant QTL associated with freeze tolerance. QTL with a large effect on freeze tolerance were located on both the Citrus and Poncirus linkage maps. In addition, clusters of markers with significantly different means between marker present and absent classes indicating minor QTL that contribute smaller effects on the level of tolerance were found on the linkage maps of both species.

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Ed Stover, David G. Hall, Jude Grosser, Barrett Gruber and Gloria A. Moore

The primary objective of this experiment was to determine if the selection of rootstock (Citrus and hybrids) could enhance the development of huanglongbing (HLB)-related symptoms associated with the pathogen Candidatus Liberibacter asiaticus (CLas) in sweet orange (Citrus sinensis). If so, then it may permit more rapid identification of HLB-susceptible compared to HLB-resistant scion types. The secondary objective was to assess the impact of different rootstocks on plant growth parameters and health to determine if trees on any rootstocks displayed reduced sensitivity to HLB-influenced growth restriction. ‘Valencia’ sweet orange was budded on each of the following eight genotypes: Carrizo (C. sinensis × Poncirus trifoliata); Cleopatra (C. reshni); Green-7 {a complex allotetraploid from somatic hybrids [C. clementina × (C. paradisi × C. reticulata) + C. grandis] × [(C. aurantium + (C. sinensis × P. trifoliata)]}; UFR-2 (a complex allotetraploid from somatic hybrids {[C. clementina × (C. paradisi × C. reticulata)] + C. grandis} × (C. reticulata + P. trifoliata)); UFR-4 (same pedigree as UFR-2); rough lemon (C. jambhiri); sour orange (C. aurantium); and US-897 (C. reticulata × P. trifoliata). Half of the trees on each rootstock were bud-inoculated with CLas and half were inoculated with the asian citrus psyllid [ACP (Diaphorina citri)], which is the CLas vector. During both experiments, no rootstock conferred significantly greater HLB symptom severity compared to trees on Carrizo; however, trees on several rootstocks had reduced HLB severity compared to those on Carrizo. Regarding the bud-inoculated trees after 3 years, trees on UFR-4 displayed greater overall health than trees on Carrizo, Green-7, sour orange, and US897, and trees on UFR-4 had a higher percentage of plants with leaf cycle threshold (Ct) values >36 compared with trees on Cleopatra and rough lemon (62 vs. 26-29 respectively). Regarding the ACP-inoculated trees after 3 years, trees on UFR-4 had better overall health than trees on Carrizo, rough lemon, and US-897, and trees on sour orange had a higher percentage of plants with leaf Ct values greater than 36 only compared to Cleopatra and US-897. The percentage increase in the trunk diameter per month over the course of each entire experiment was significantly greater for UFR-2 in both trials than all rootstocks except UFR-4. Only root CLas titers were sometimes significantly higher for trees on other rootstocks compared to those on Carrizo. Although no rootstock provided acceleration of HLB symptom development compared with Carrizo, some rootstocks conferred significantly greater health compared to Carrizo. However, it is uncertain whether the modest differences in health and growth observed in these greenhouse trials would translate to economic benefits in the field.

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Kimberly A. Moore, Amy L. Shober, Gitta Hasing, Christine Wiese and Nancy G. West

Previous research indicated that acceptable quality annual and perennial plant species can be grown in the landscape with low nitrogen (N) inputs. However, information on the impact of soil conditions and N use by ornamental plants grown in central Florida is lacking in the literature. Our objective was to evaluate plant growth and quality response of eight warm-season annuals, seven cool-season annuals, and four herbaceous perennial species to a range of N fertilizer rates when plants were grown in landscape beds containing native field soil or subsoil fill. A slow-release N source (42N–0P–0K) was applied every 12 weeks at annual N rates of 3, 5, or 7 lb/1000 ft2 for a period of 18 weeks (annual species) or 1, 3, or 5 lb/1000 ft2 for a period of 54 weeks (perennial species). Plants were evaluated for aesthetic quality every 6 weeks and shoot dry weight was measured at completion of the experiment. Dry weight production and aesthetic quality of most species evaluated was unaffected by N rate. For several species, shoot dry weight was higher when planted in the field plots containing native soil [alyssum (Lobularia maritima) ‘Bada Bing White’ wax begonia (Begonia ×semperflorens-cultorum), dahlberg daisy (Thymophylla tenuiloba), ‘Survivor Hot Pink’ geranium (Pelargonium ×hortorum), gomphrena (Gomphrena globosa), ‘Blue Puffs Improved’ (‘Blue Danube’) ageratum (Ageratum houstonianum), blanket flower (Gaillardia pulchella), goldenrod (Solidago chapmanii), ‘Mystic Spires’ salvia (Salvia longispicata ×farinacea)]. Quality response to soil condition was mixed over the course of the study. Several species performed as well (or better) in the field as when planted in the subsoil fill soils. These results illustrate that some landscape plant species are able to survive and thrive under various soil and fertility conditions. These “tougher” species may be good choices for installation in landscapes with marginal native soils or disturbed urban landscape soils.

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Ed Stover, Robert G. Shatters Jr., Barrett Gruber, Prem Kumar and Gloria A. Moore

Plants inoculated with the huanglongbing (HLB)-associated bacterium, Candidatus Liberibacter asiaticus (CLas) typically must be monitored for 8–10 months to identify differences in susceptibility between genotypes. Continuous light is reported to accelerate development of HLB symptoms and field observations suggest that trees girdled by tags or tree ties showed greater symptoms. Therefore, an experiment was conducted assessing HLB susceptibility as influenced by light/dark periods of 12 hours: 12 hours and 24 hours: 0 hours, in combination with scoring tree trunks to disrupt phloem. Sixty trees of each of three citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, highly HLB susceptible) were bud grafted using two CLas-infected buds (rough lemon and citron) per tree on 26 Mar. 2012, and were placed in controlled growth rooms (one 12 hour light: 12 hour dark and one constant light) on 4 June 2012. Ten trees of each genotype in each growth room were scored 10 cm above the soil (cutting through the bark but not the wood) with a knife on 18 July 2012 and the scoring was repeated at the same scoring wounds on 30 Aug. 2012. Trees were removed from growth rooms on 12 Dec. 2012 and subsequently maintained in a greenhouse. At two to three month intervals between June 2012 and May 2013, HLB symptoms and stem diameter at 5 cm above the soil were assessed, and three leaves per tree were collected for quantitative polymerase chain reaction (qPCR) determination of CLas titer. Six months after inoculation and 3 months following imposition of treatments, the ‘Valencia’ scored in the 12 hour light: 12 hour dark regime, the ‘Valencia’ non scored trees in 24 hours of light and the ‘Kuharske’ scored trees in 24 hours of light displayed higher CLas titers than most other trees. After an additional two months, both scored and non-scored trees of all three genotypes in 24 hours of light had significantly elevated CLas titers compared with trees in 12 hour light: 12 hour dark regime, but within most treatments all three genotypes had titers which were not statistically different from each other. Growth of ‘Kuharske’ and rough lemon was enhanced; whereas ‘Valencia’ growth was reduced when graft-inoculated plants were maintained in continuous light. Scoring enhanced early CLas development in ‘Kuharske’ when combined with continuous light, had no effect in rough lemon, and showed inconsistent effects in ‘Valencia’. Although continuous lighting enhanced disease progression, it did not reveal differences in HLB susceptibility.

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Kim D. Bowman, Frederick G. Gmitter Jr., Gloria A. Moore and Russell L. Rouseff

Citrus fruit with sector chimeras were collected in commercial packinghouses and from the field. Chimeric fruit from eight cultivars of sweet oranges [Citrus sinensis (L.) Osbeck], grapefruit (C. paradisi Macf.), tangelo (C. paradisi × C. reticulate Blanco), and tangors (C. reticulate × c. sinensis) were found at a frequency of 0.009% to 0.271%. Tetraploid plants obtained from one type of sector mutant (termed gigas) and albino plants obtained from another type of sector mutant confirmed that some genetic mutations observed in fruit rind can be recovered in nucellar seedlings. The gigas chimeras were identified as a source of citrus tetraploids. Several types of potentially useful sector mutants with altered rind color were observed, and plants were produced from some mutant sectors by developed seed or culture of aborted ovules. HPLC analysis of rind tissues from sectors of one chimeric fruit revealed substantial quantitative and qualitative differences in pigment composition. Propagation of plants from mutant sectors may yield cultivars with improved fruit color, altered maturation date, and reduced disease or mite susceptibility and may eventually lead to breeding of seedless triploid hybrids.