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In vitro axillary shoot proliferation can be used to increase availability of citrus (Citrus) types in high demand, while limiting somaclonal variation. However, established protocols could be improved to increase efficiency. Therefore, this study investigated some factors [plant growth regulators (PGRs), basal media, and successive subculturing] which affect the in vitro axillary shoot proliferation of mature ‘Washington Navel’ orange (Citrus sinensis) and juvenile ‘Carrizo’ citrange (C. sinensis × Poncirus trifoliata). In ‘Washington Navel’ orange, maximum axillary shoot induction (66.9% explants producing axillary shoots with a mean of 2.45 shoots per explant) was obtained in Driver and Kuniyuki walnut (DKW) medium supplemented with 0.1 mg·L⁻¹ 6-benzylaminopurine (BA), 0.05 mg·L⁻¹ naphthalene acetic acid (NAA) along with 1 mg·L⁻¹ 6-furfurylaminopurine [kinetin (kin)], whereas in ‘Carrizo’ citrange, axillary shoot production was greatest (82.6% and 87.5% of explants producing axillary shoots with a mean of 4.3 and 4.1 shoots per explant) at 1.0 or 2.0 mg·L⁻¹ BA in DKW medium. The initial nodal propagules (with basal tissue remaining from removed shoots) were repeatedly subcultured for six times every 4 weeks onto DKW medium with the same levels of PGRs used for initial culturing. Woody plant medium (WPM), Murashige and Skoog medium (MS), and DKW were also compared for rooting at quarter to full strength for salt components, all amended with 2.0 mg·L⁻¹ indolebutyric acid (IBA) and 0.5 mg·L⁻¹ NAA. MS at full strength provided the highest rooting in ‘Carrizo’ citrange (93%) and longest root length (58 mm), whereas half-strength MS provided the highest rooting in ‘Washington Navel’ orange (60% to 61%) and the longest roots (26 mm). Addition of 1 μm spermidine to the rooting medium enhanced root length only for ‘Washington Navel’ orange on full-strength MS, but accelerated rooting for both cultivars on all media. The plantlets were successfully transferred to greenhouse conditions, exhibiting normal development, with high uniformity, and no evidence of somaclonal variation.

A test population consisting of progenies of 92 seed-source genotypes (hereafter called “parent genotypes”) of Citrus and Citrus relatives in the field in east–central Florida was assessed after natural freeze events in the winters of 2010 and 2011. Eight seedlings per parent genotype were planted in a randomized complete block design; however, as a result of mortality, the number of plants assessed in some genotype groups was reduced at some or all sampling dates. The citrus diseases huanglongbing and citrus canker were endemic in the planting and may have influenced tree response to cold temperatures. Unusually low temperatures (near –4 °C each winter) for east–central Florida were experienced during the trial period. Defoliation and dieback were significantly greater in the winter of 2011 than in the winter of 2010. The winter in 2011 was preceded by a period of extraordinarily low temperatures in mid-December with no period of cool temperatures to allow trees to acclimate. In 2010 the average defoliation was 53% ± 28% and less than 13% of the trees exhibited any noticeable dieback, whereas in 2011, the average defoliation and dieback were 93% ± 17% and 51% ± 35%, respectively. Within the genus Citrus, several progenies were identified that had 16% to 24% dieback in 2011 and these were from parent genotypes C. reticulata (CRC 2590) (23%), C. sinensis (CRC 3858) (24%), C. maxima (CRC 3945) (16%), C. hassaku (CRC 3907 and 3942) (16% and 17%), C. aurantium (CRC 628 and 2717) (18% and 7%), C. taiwanica (CRC 2588) (21%), and C. neo-aurantium (C. obovoidea + C. unshiu graft chimera) (CRC 3816) (23%). Within other genera in the Aurantiodeae, Poncirus trifoliata (CRCs 301, 3957, 3549, and 4007), Severinia buxifolia (CRC 1497), Bergera koenigii (CRC 3165), and Glycosmis pentaphylla (CRC 3285) had the least amount of dieback, all at less than 23%. The two species within the Toddalioideae subfamily of the Rutaceae (Casimiroa edulis and Zanthoxylum ailanthoides) had among the least amount of dieback (1% and 8%, respectively). When considered by groups, the Citrons and Australian natives had the greatest amount of dieback in 2011, 68% and 65%, respectively. The trifoliates (Poncirus and hybrids) had the least dieback, ranging from 4% to 40%. The information from this study may be useful in germplasm enhancement and Citrus breeding targeting greater cold tolerance.

The world market for citrus (Citrus spp.) products has undergone dramatic shifts over the last decade. These shifts are influencing development and planting of new citrus cultivars. Seedlessness and very easy peeling have become paramount in mandarin types (C. reticulata and hybrids), and new cultivars are being developed through plant breeding and selection of new sports. In both sweet orange (C. sinensis) and grapefruit (C. paradisi), essentially all important cultivars are derived from a single original hybrid of each fruit type, and plant improvement has focused on selection of sports with redder color and extended maturity. The existence of many active citrus breeding programs makes it likely that we will continue to see evolution of new citrus cultivars over the foreseeable future.

Bloom in individual citrus (Citrus) trees often continues for more than 1 month in south Florida, with even greater bloom duration within most orchard blocks because of variation in bloom timing between trees. Prolonged bloom contributes to variable fruit maturity as harvest approaches and increases severity of postbloom fruit drop (PFD) disease (caused by Colletotrichum acutatum). Hydrogen cyanamide (cyanamide) has been effective in accelerating bloom in various deciduous fruits, and its potential use in citrus was investigated in this preliminary study. Cyanamide was applied at a range of concentrations, from 0% to 1.0% a.i., to potted trees of six citrus types reflecting fairly broad diversity in commercial citrus that was readily available as seed [alemow (Citrus macrophylla), ‘Duncan’ grapefruit (Citrus paradisi), sour orange (Citrus aurantium), ‘Smooth Flat Seville’ sour orange hybrid (C. aurantium hybrid), ‘Swingle’ citrumelo (C. paradisi × Poncirus trifoliata), and ‘Sun Chu Sha’ mandarin (Citrus reticulata)] in Dec. 1999 while trees were quiescent. Phytotoxicity increased with cyanamide rate, with some damage at 0.125% cyanamide on most tested plants, and large variation among citrus types. All cyanamide rates hastened flushing. Airblast application of cyanamide (0, 0.025%, 0.05%, and 0.10%) was made to mature trees of ‘Valencia’ and ‘Navel’ sweet orange (Citrus sinensis) in Ft. Pierce, FL, on 27 Jan. 2000. On 15 Feb. and 28 Feb. additional trees received cyanamide at 0.05%. There was considerable defoliation, which increased linearly with cyanamide rate. Flushing and flowering were unaffected by cyanamide compared with controls except in February where cyanamide applied at 0.05% increased flowers per tree in ‘Valencia’ sweet orange, and in contrast, 0.1% cyanamide on 27 Jan. reduced ‘Navel’ sweet orange flowering. Cyanamide application to ‘Valencia’ sweet orange on 28 Feb., after initial flowering but 16 days before peak bloom, significantly reduced yield per tree but there were no other effects on cropping. In these trials, cyanamide was not an effective agent for hastening bloom in south Florida citrus with applications late January through February. Further work is needed to determine whether December applications of cyanamide to trees in the field may be more effective in concentrating subsequent flush and bloom.

Candidatus Liberibacter asiaticus (CLas), the bacteria responsible for citrus greening disease [huanglongbing (HLB)], has become a worldwide threat to citrus (Citrus sp.) production. HLB has proven difficult to study and treat because of the complex interactions between CLas, the citrus host, and insect vectors. We have selected for single chain fragment variable (scFv) antibodies from a specialized bacteriophage library for binding activity against CLas proteins InvA and TolC. Portions of each protein were chosen as antigens based on predicted binding availability and theorized necessary functions in pathogenicity. Binding affinity for individual scFv-expressing clones was confirmed by phage enzyme-linked immunosorbent assay (ELISA). The scFv sequences were stably transformed under the control of a tandem Cauliflower mosaic virus 35S (CaMV 2x35S) promoter by Agrobacterium tumefacien–mediated transformation into ‘Carrizo’ citrange (Citrus sinensis × Poncirus trifoliate), a citrus rootstock cultivar. Replicated plants of single transformations were inoculated by infestation with CLas positive asian citrus psyllid (Diaphorina citri), a CLas vector. Inoculation and disease progression was monitored through quantitative real-time polymerase chain reaction. Inoculated transgenic plants showed significantly reduced CLas titer compared with wild types. A subpopulation of transgenic plants displayed no measurable surviving bacteria after 12 months. Interestingly, individual replicated plants from the same transgenic events strongly segregated into two populations by resistance phenotype: a minority that were indistinguishable from wild-type plants and a majority that were highly resistant. Our results are the first step in developing a novel protection strategy for HLB.

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.

Clonal woody crop germplasm collections often originate and are grown in distinct geographical locations. Because the degree of cold-hardiness is known to be a factor in the successful use of dormant bud cryopreservation for Malus, it was suggested that material from relatively warmer climates would not respond to cryopreservation as well as material from colder environments. To test this hypothesis, the effect of growing provenance on cryosurvival of dormant buds from three Malus (apple) cultivars grown in three locations (Geneva, NY; Davis, CA; and Corvallis, OR) was tested in 3 consecutive years. Dormant winter buds were harvested at the three locations, cryopreserved, and bud viability was tested by grafting. The collective 3-year mean viability for cryopreserved dormant apple buds for the three locations ranged from 63% to 81% of the buds surviving with the highest survival from the Corvallis site; however, the Geneva twigs were exposed to the lowest preharvest temperature. These results suggest that the temperature at the growing location may not hinder application of the dormant bud cryopreservation method with Malus to the extent previously speculated.

Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus-growing regions worldwide. Current control of D. citri and HLB is inadequate, but the identification and incorporation of D. citri resistance traits from uncultivated Citrus spp. and Citrus relatives is seen as a potential disease management strategy. In this study, seedlings of 87 Rutaceae seed-source genotypes, primarily in the orange subfamily Aurantioideae, were assessed in the field for their propensity in a free-choice situation for infestations of natural south Florida populations of D. citri. The majority of test populations surveyed hosted all three life stages of D. citri; however, there were significant differences among the test populations in the mean ranks for D. citri eggs (F = 3.13, df = 86, P < 0.0001), nymphs (F = 9.01, df = 86, P < 0.0001), and adults (F = 4.21, df = 86, P < 0.0001). The only sampled test population that was completely avoided by all life stages of D. citri was seedlings of Casimiroa edulis Llave et Lex, commonly known as white sapote, which was one of the few plants included in the study belonging to the Rutaceae subfamily Toddalioideae. Although not completely avoided, very low levels of D. citri were found on two surveyed test populations of Poncirus trifoliata (L.) Raf, seedlings of ‘Simmon's trifoliate’ and ‘Little-Leaf’. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp., is commonly incorporated into rootstock varieties, and has been used in breeding-advanced scion material. The identification of partial resistance in P. trifoliata to D. citri could prove useful in future citrus breeding efforts aimed at reducing the incidence and spread of HLB. Determining if there is true resistance to D. citri in this and other germplasm sources with a low propensity for infestations in free-choice situations will require no-choice experiments.

Six trials were conducted to determine whether lower spray volumes or inclusion of different surfactants would permit adequate thinning of mandarin hybrids (Citrus reticulata hybrids) at a much lower cost per hectare. Sprays were applied using a commercial airblast orchard sprayer during physiological drop when fruitlets averaged 8 to 16 mm in diameter. Surfactant was always included at 0.05% v/v. NAA always reduced fruit per tree, increased fruit size, and decreased production of smallest size fruit. However, in only three experiments, contrast of all NAA treatments vs. controls indicated increased production of the largest (80–100 fruit per carton) and most valuable fruit. In four of five experiments, comparison of spray volumes of 600 (only examined in three of four experiments), 1200, or 2300 L·ha^–1 demonstrated significant fruit size enhancement from all NAA applications. Most individual NAA treatments resulted in fewer fruit per tree, but there were no statistically significant differences between NAA treatments at different spray volumes. In only one of the four experiments, there was a marked linear relationship between spray volume and fruit per tree, yield, mean fruit size, and production of largest fruit sizes. The effects of surfactants (Activator, a nonionic, Silwet L-77, and LI-700) on NAA thinning were tested in both `Murcott' and `Sunburst'. In comparisons between Silwet L-77 and Activator surfactant, one experiment with `Murcott' showed greater fruit per tree and yield reduction from using Silwet, but with a smaller increase in production of largest fruit sizes, whereas in another `Murcott' experiment, Silwet L-77 reduced numbers of smaller fruit size with no increase in production of larger fruit. Based on these findings, current recommendations for NAA thinning of Fla. mandarins are use of spray volume of ≈1100–1400 L·ha^–1 on mature trees with proportionally lower volume on smaller trees. These data appear to support use of a nonionic surfactant rather than other tested surfactants in NAA thinning of Florida mandarins. Because experience with NAA thinning of Florida citrus is limited, it is only recommended where the disadvantages of overcropping are perceived to substantially outweigh the potential losses from overthinning.

Asiatic huanglongbing (HLB) is a devastating disease of citrus associated in North America with the bacterium ‘Candidatus Liberibacter asiaticus’ (LAS) vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. ACP management is considered a vital component of a program aimed at reducing the incidence and spread of HLB. Considerable research has been published comparing the efficacy of different insecticides for ACP control as well as on seasonal strategies for applying ACP insecticides. However, published information was largely lacking for even the most intense insecticide programs on their effectiveness for keeping HLB out of a new citrus planting in Florida citrus. We therefore conducted two replicated (individual plots 0.2 to 0.4 ha in size) experiments on protecting young citrus from HLB using different ACP management programs. An intensive insecticide program was evaluated in each experiment: eight annual calendar applications of traditional insecticides (hereafter referred to as the “complete” program). In one experiment, citrus was either planted alone and subjected to the complete program or citrus was interplanted with orange jasmine, Murraya exotica L. (a favored ACP host plant) and subjected to a reduced insecticide program (four calendar sprays of traditional insecticides). There was one set of plots in which both jasmine and citrus were treated with insecticides and one set in which jasmine was not treated at all. In the second experiment, citrus was either subjected to the complete program or to one of two other programs: a reduced insecticide program consisting of five calendar applications of traditional insecticides or a mineral oil program (oil applications every 3 weeks plus one dormant insecticide spray). The results of the two experiments were similar. Relatively good ACP control was achieved under each ACP management program during the first year but, as the experiments progressed and trees increased in size, ACP outbreaks occurred regardless of the psyllid management program. Little HLB developed under any ACP management program during the first year, but thereafter HLB increased and large percentages of the trees in each experiment became LAS-infected in less than two to three years. The combined results of the experiments indicated that up to eight monthly pesticide treatments per year applied on a calendar schedule were ineffective for preventing young citrus from becoming diseased. Of important significance is that the orchard within which the two experiments were conducted was subjected to a minimal psyllid management program and contained many older trees known to be infected by the HLB pathogen, a challenging situation for getting young trees into production without contracting the disease. Thus, the ACP management programs we evaluated might have been more effective if ACP in the surrounding areas had been more aggressively controlled and diseased trees in the surrounding areas removed to reduce inoculum loads. Also, the programs might have been more effective for slowing the spread of HLB if our research plots had been larger.