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Asiatic citrus canker (ACC) foliar lesions were evaluated on progenies of 84 seed-source genotypes (“parent genotypes”) from the Citrus Variety Collection (CVC) of the University of California at Riverside (UCR) of Citrus trifoliata and hybrids between C. trifoliata and other Citrus species and hybrids. Trees were planted Aug. 2013 in a completely randomized design at the Fort Pierce U.S. Department of Agriculture (USDA) grove. Plants were assessed visually Aug. 2017, Sept. 2019, and Sept. 2020 for distinctive ACC lesion incidence and severity. Progeny were compared by parent genotypes using nonparametric analysis. Incidence of ACC [percentage of leaves displaying symptoms, verified by quantitative polymerase chain reaction (qPCR) to be associated with Xanthomonas citri pv. citri] across parent genotypes ranged from 8% to 80% (mean, 49%) of leaves affected in 2017, from 4% to 58% (mean, 29%) in 2019, and 8% to 46% (mean, 25%) in 2020. In 2017, of 49 C. trifoliata parent genotypes, only four separated from the two highest ACC-incidence statistical categories [Citrus Research Center (CRC) 3345, 3484, 3888, and 4017]. whereas 29 of the 35 C. trifoliata hybrids displayed lower ACC incidence, which separated from the two highest statistical categories. In 2019, of the C. trifoliata, only six separated from the highest ACC-incidence statistical category (CRC 3330, 3484, 3547, 3549, 3876, and 3888), whereas all 35 C. trifoliata hybrids displayed lower ACC incidence and separated from the highest statistical category, and 26 hybrids separated from 18 of the C. trifoliata. In 2020, only three C. trifoliata separated from the highest ACC-incidence statistical category (CRC 2861, 3549, and 3888) and 20 hybrids separated from 18 of the C. trifoliata. By parent genotype, ACC incidence correlated substantially between each pair of the 3 years, with r 2 values of 0.39, 0.57, and 0.65. Of 34 hybrids validated, similar numbers had C. trifoliata, grapefruit (C. ×aurantium var. racemosa), and sweet orange (C. ×aurantium var. sinensis) chloroplasts. Chloroplast type affected ACC incidence and severity, but not in a consistent manner. Near-isogenic groups within C. trifoliata, as determined by DNA markers, were associated with some statistically different ACC sensitivity. Overall, hybrids of C. trifoliata with other citrus types displayed markedly reduced ACC sensitivity compared with C. trifoliata, indicating that this trait is readily overcome through breeding.
Rootstock significantly affected the development of stem-end rind breakdown (SERB) on `Valencia' and navel oranges (Citrus sinensis), but not `Ray Ruby' grapefruit (C. paradisi) or `Oroblanco' (C. grandis × C. paradisi), and affected postharvest decay on navel orange, `Ray Ruby' grapefruit, `Oroblanco' and one of two seasons (2002) on `Valencia' orange. In `Valencia' and navel oranges, fruit from trees grown on Gou Tou (unidentified Citrus hybrid) consistently developed low SERB. `Valencia' oranges on US-952 [(C. paradisi × C. reticulata) × Poncirus trifoliata] developed high levels of SERB in both years tested. Relative SERB of fruit from other rootstocks was more variable. Navel oranges, `Ray Ruby' grapefruit, and `Oroblanco' fruit from trees on Cleopatra mandarin (C. reticulata) rootstock consistently developed relatively low levels of decay, and in navel this level was significantly lower than observed from trees on all other rootstocks. In three of five trials we observed significant differences between widely used commercial rootstocks in their effects on postharvest SERB and/or decay. Given the expanding importance of sales to distant markets, it is suggested that evaluations of quality retention during storage be included when developing citrus rootstocks and scion varieties for the fresh market.
In a Fort Pierce, FL, field planting, plant growth, and Huanglongbing (HLB) severity were assessed as indicators of HLB tolerance on progenies of 83 seed-source accessions of Citrus and Citrus relatives mainly from the Riverside, CA, genebank. The HLB-associated pathogen [Candidatus Liberibacter asiaticus (CLas)] and vector [asian citrus psyllid (ACP), Diaphorina citri] were abundant, and trees were naturally challenged for 6 years before metrics (leaf mottle, percent canopy mottle, overall health, canopy density, canopy width, canopy height, and trunk diameter) were collected in Oct. and Nov. 2015. The healthiest trees with low or no HLB symptoms were distant citrus relatives: Balsamocitrus dawei, Bergera koenigii, Casimiroa edulis, Clausena excavata, Murraya paniculata, and one accession of Severinia buxifolia. Within Citrus, most of the healthiest trees with densest canopies, little leaf loss, and greater growth were those with pedigrees that included Citrus medica (citron). These included progenies of Citrus hybrid (‘Limon Real’), Citrus limetta, Citrus limettioides, Citrus limonia, C. medica, Citrus volkameriana, and some Citrus limon accessions. Trees in this category exhibited distinct leaf-mottle characteristic of HLB and substantial pathogen titers, but maintained dense canopies and exhibited good growth. Trees from seed-source accessions in the genus Citrus without citron in their background were generally among the least healthy overall with less dense canopies. The exceptions were progenies of two Citrus aurantium accessions, which were markedly healthier than progenies of other Citrus seed-source accessions not derived from citron. Linear regression analysis, between metrics collected and pedigree of seed parent, indicated that percentage of citron in the pedigree significantly correlated with measures of tolerance. Although no commercial Citrus genotypes yielded progenies with strong HLB resistance, in this field experiment several progenies maintained dense canopies and good growth, and may be useful for breeding HLB tolerant cultivars.
Xanthomonas citri ssp. citri (Xcc) is the causal agent of Asiatic citrus canker (ACC), a commercially important disease in Florida citrus as well as in many other regions. In this study we evaluated occurrence of foliar lesions from ACC on progenies of 94 seed-source genotypes (hereafter called “parent genotypes”) of Citrus and Citrus relatives in the field in east–central Florida to identify the relative susceptibility to ACC. Eight seedlings per parent genotype were planted in a randomized complete block planting, but the number of plants assessed in some genotype groups was reduced by mortality at some or all sampling dates. Plants experienced ambient exposure to high Xcc inoculum pressure and plants were assessed in Sept. 2010, July 2011, Oct. 2011, May 2012, and Sept. 2012. The incidence and severity of ACC lesions were assessed and evaluated using non-parametric analyses to compare progeny from the 94 parent genotypes. Progeny of 14 parent genotypes did not exhibit ACC symptoms at any date. All were in genera other than Citrus with only Microcitrus and Eremocitrus being cross-compatible with Citrus. The kumquat hybrid C. halimii, two accessions of C. reticulata, C. nobilis, and C. sunki were the only Citrus species in the group that had a low severity (percent total leaf area showing symptoms) on each date of assessment. The aforementioned accessions had an average incidence and severity of ACC lesions of less than 4% in 2011 and 2012, but 26% to 38% in 2010 when no chemical control for ACC was applied in the adjoining citrus groves at our field site. Fourteen of the 16 progeny of C. reticulata and related parent genotypes were in the group with the lowest incidence and severity of ACC on two or more assessment dates. However, for analysis of only the most symptomatic leaves on each plant, 10 C. reticulata parent genotype progenies were in the most resistant category on the Sept. 2012 assessment despite having a low incidence and severity of ACC symptoms overall. Progeny of Poncirus and its hybrids as well as those of C. maxima, C. limon, and related species were the most severely diseased at all assessment dates. There were few instances in which progeny of different accessions of the same species had markedly different responses to ACC: progeny of C. reticulata ‘Fremont’ displayed more severe ACC compared with several other C. reticulata groups and C. aurantium ‘Zhuluan’ displayed much lower incidence and severity of ACC compared with several other accessions designated C. aurantium. Information on ACC susceptibility in diverse Citrus and Citrus relatives may prove useful for breeding programs aimed at reducing ACC susceptibility and will be of value to researchers interested in mechanisms of ACC resistance and susceptibility.
`McIntosh' apples (Malus ×domestica Borkh.) display a rapid increase in ethylene production as they ripen, resulting in more preharvest drop and accelerated softening compared with other major cultivars. Economic considerations often dictate a choice between delaying harvest to achieve color development or harvesting earlier to avoid excessive fruit softening and drop. We have evaluated the effects of plant growth regulators (PGRs) and summer pruning on this balance. Treatments were applied to trees in the Mid-Hudson region in New York state in 1995 and 1996, and a subset of treatments was applied in the Champlain Valley region in 1996. NAA, applied at 10 mg·L-1 in 1995 and 20 mg·L-1 in 1996, reduced drop on only one sample date in only one of the three trials. Ethephon at 150 mg·L-1 plus 10 mg·L-1 NAA, accelerated ripening and permitted harvest before substantial drop occurred. However, earlier harvest resulted in smaller fruit size, and if ethephon-treated fruit were not picked within a narrow window, rapid drop ensued, and fruit developed a high senescent breakdown incidence during storage. ReTain, containing AVG, at 124 g·ha-1 a.i. delayed drop in all three trials, but its use resulted in firmer fruit after storage in only two of seven comparisons. Use of ethephon on AVG-treated trees enhanced red color but accelerated drop, although it was reduced less than when ethephon was used alone. Severe late summer pruning accelerated red color development, drop and ripening in both years of the study. AVG was more effective for management of `McIntosh' harvest in the cooler Champlain Valley region than in the Mid-Hudson Valley region. Chemical names used: naphthalene acetic acid (NAA); 2-chloroethylphosphonic acid (ethephon); aminoethoxyvinylglycine (AVG).
The citrus disease huanglongbing (HLB) has become endemic in Florida, with estimates that greater than 80% of citrus trees are currently infected. Although there are no commercial citrus varieties with strong HLB resistance, some field tolerance has been observed in trees exposed to the disease after they were mature. There is great urgency to identify citrus which may permit economic citrus production where HLB is endemic. Therefore, the objective of this study was to assess field tolerance to HLB. To expedite the trial due to urgency, nursery trees were purchased on rootstock varieties as available. The trial included the following unbalanced scion/rootstock combinations: ‘Hamlin/Kinkoji’, ‘Hamlin/Cleopatra’, ‘Temple/Cleopatra’, ‘Fallglo/Kinkoji’, ‘Sugar Belle/Sour Orange’, ‘Tango/Kuharske’, and ‘Ruby Red/Kinkoji’, with most comparisons based solely on scion/rootstock combinations. A randomized complete block experiment was established at Fort Pierce, FL, in Sept. 2010. All trees exhibited symptoms of HLB and tested positive for the Candidatus Liberibacter asiaticus (CLas) bacterium by Oct. 2012, with similar titers [directly assessed as cycle threshold (Ct) using quantitative polymerase chain reaction (qPCR)] measured for all scion/rootstocks at most sample dates, but early titer development in ‘Ruby Red/Kinkoji’ was significantly lower than several other scion/rootstocks. Across all time-points, ‘Fallglo/Kinkoji’ had the lowest rating of distinctive HLB mottling and ‘Ruby Red/Kinkoji’ had the highest rating, but ‘SugarBelle/Sour Orange’ had the highest percentage of leaves affected. After 5 years, ‘SugarBelle/Sour Orange’ and ‘Tango/Kuharske’ had the greatest overall increase in trunk diameter, and were among the healthiest in overall appearance. In Oct. 2015, ‘SugarBelle/Sour Orange’ and ‘Tango/Kuharske’ trees had significantly greater fruit load (80–88 fruit/tree) followed by ‘Temple/Cleopatra’ and ‘Fallglo/Kinkoji’ (31–35 fruit/tree) while ‘Hamlin/Kinkoji’, ‘Hamlin/Cleopatra’, and ‘Ruby Red/Kinkoji’ produced less than 20 fruit per tree. Despite becoming infected by CLas in less than 2 years after planting, the trees continued to grow and all scion/rootstocks displayed increasing fruit production, although very low in ‘Hamlin/Kinkoji’, ‘Hamlin/Cleopatra’, and ‘Ruby Red/Kinkoji’. Growth and fruit production in the highest performing scion/rootstocks were likely less than would be expected for healthy trees, but these results are promising, with markedly better response of some scion/rootstocks with mandarin hybrid scions compared with trees with sweet orange or grapefruit scions. Larger fully replicated trials are underway. It is noteworthy that the most pronounced HLB symptoms and higher early pathogen titer, which are the two criteria most widely used in assessing HLB resistance, were not associated with the lowest growth and cropping, and focus on early symptomatic traits when screening for resistance may obscure important disease tolerance.
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.
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.
Huanglongbing {HLB [associated with Candidatus Liberibacter sp. (CLas)]} and asiatic citrus canker {ACC [causal organism Xanthomonas citri ssp. citri (XCC)]} are bacterial diseases that seriously threaten sustainability of the Florida citrus (Citrus sp.) industry. Sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) are highly susceptible to ACC and improvement through conventional breeding is a long-term process, making transgenic solutions attractive. No strong HLB resistance has been identified within cultivated citrus scion types: creation of transgenic citrus that would permit economic citrus production where HLB is endemic is a high priority. Little is known about the HLB pathosystem and thus broad-spectrum antimicrobial peptides (AMPs) have been the focus for current work, and identification of safe and effective transgenes is essential to our efforts. In vitro assessment of minimum inhibitory concentration (MIC) for 44 AMPs was conducted using Sinorhizobium meliloti and Agrobacterium tumefaciens as surrogates for the unculturable CLas because they are closely related alpha proteobacteria (class Alphaproteobacteria). XCC is also a gram-negative bacterium and was included in these analyses in anticipation that HLB and ACC resistance can be achieved with the same AMP transgene if expressed using non-tissue-specific promoters. Twenty AMPs from diverse sources were initially tested. AMPs with the lowest MICs included tachyplesin I from horseshoe crab (Tachypleus tridentatus), SMAP-29 from sheep (Ovis aries), D4E1 and D2A21 (which are synthetic AMPs derived through evaluation of critical amino acid residues in AMPs, overall peptide structure, and AMP effectiveness), the human (Homo sapiens) LL-37, and the honeybee (Apis mellifera) venom AMP melittin. These AMPs inhibited growth of all three test bacterial species at 1 μM or less. An additional 20 synthetic AMPs were designed based on structures of the most effective AMPs and seven of these showed effectiveness at 1 μM or less across all three test bacteria. Most AMPs were comparable in effectiveness across the three bacterial species, but some species × AMP interactions were observed. Hemolytic activity was assessed by exposure of porcine erythrocytes (from Sus scrofa) to the AMPs. Hemolysis from most AMPs was not significantly different from water, whereas melittin was highly hemolytic.
The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a key pest in most citrus-growing regions worldwide. Adult citrus leafminers oviposit primarily on young elongating flush of Citrus as well as other Rutaceae and some ornamental plants. Larvae feed on the epidermal cell layer of developing leaves and injury to leaves provides a pathway for infection by the bacterium Xanthomonas citri subsp. citri (Hasse), the causal agent of Asiatic citrus canker. In this study, we quantified abundance of citrus leafminer larvae on progeny of 87 seed parent genotypes of Citrus and Citrus relatives (family Rutaceae) in the field in East–central Florida to identify those that have low abundance of leafminers. Progeny from the 87 parent genotypes varied in abundance of the leafminer. Progeny of 15 parent genotypes had a high mean abundance of more than six leafminers per flush shoot. All but one of these genotypes were in the Citrus genus. Progeny of 16 parent genotypes had zero, or nearly zero, leafminers, but none were from the Citrus genus. However, many of these 16 genotypes were from genera closely related to true citrus (subtribe Citrinae) and are sexually compatible with Citrus. Progeny of two parent genotypes in the subfamily Toddalioideae and Glycosmis pentaphylla (Retz.) Corr. also had a low abundance of leafminer. Glycosmis pentaphylla also is a poor host for the Asian citrus psyllid, Diaphorina citri Kuwayama, and has biochemical resistance to the citrus weevil, Diaprepes abbreviatus (L.), so this genotype as well as others identified as poor hosts for the leafminer may prove useful in breeding programs aimed at reducing the abundance of multiple insect pests on citrus.