Regeneration of somatic embryos from sweet orange (C. sinensis) protoplasts using semi-permeable membranes

Sweet orange (C. sinensis L. Osbeck) protoplasts were isolated from nucellar-derived embryogenic callus, cultured in alginate beads for 5–30 days, and the resulting p-calli released by liquefaction and cultured on semi-permeable membranes overlaid on MT culture medium. Somatic embryos did not develop from 5- to 10-day-old p-calli but did develop from 15-, 20-, 25-, and 30-day-old p-calli. There were no significant differences in the numbers of embryos produced among the 15- to 30-day-old p-calli and no abnormal embryo morphologies were observed. The minimum size of p-calli to form embryos was 77.84 μm in diameter. Embryos were smaller from p-calli than those produced from embryogenic callus; p-calli-derived embryos ranged in size between 0.5 and 0.8 mm, while embryos derived from embryogenic callus ranged between 1 and 2 mm.     

Differentiation of Indian,East Timorese,Papuan and Floridian ‘Candidatus Liberibacter asiaticus’ isolates on the basis of simple sequence repeat and single nucleotide polymorphism profiles at 25 loci

Japanese isolates of ‘Candidatus Liberibacter asiaticus’ have been shown to be clearly differentiated by simple sequence repeat (SSR) profiles at four loci. In this study, 25 SSR loci, including these four loci, were selected from the whole‐genome sequence and were used to differentiate non‐Japanese samples of ‘Ca. Liberibacter asiaticus’ (13 Indian, 3 East Timorese, 1 Papuan and 8 Floridian samples). Out of the 25 SSR loci, 13 were polymorphic. Dendrogram analysis using SSR loci showed that the clusters were mostly consistent with the geographical origins of the isolates. When single nucleotide polymorphisms (SNPs) were searched around these 25 loci, only the upstream region of locus 091 exhibited polymorphism. Phylogenetic tree analysis of the SNPs in the upstream region of locus 091 showed that Floridian samples were clustered into one group as shown by dendrogram analysis using SSR loci. The differences in nucleotide sequences were not associated with differences in the citrus hosts (lime, mandarin, lemon and sour orange) from which the isolates were originally derived.     

Limonin biosynthesis from obacunone via obacunoate in Citrus limon

Radioactive tracer work showed that [¹⁴C]obacunone was converted to at least four metabolites in Citrus limon. Two were identified as obacunoate and limonin. When [¹⁴C]methyl obacunoate was fed, limonin was found to be one of the metabolites. Based on these results and data accumulated thus far, biosynthetic pathways of limonoids in citrus are proposed.     

Photosynthetic responses of citrus trees to soil flooding

Continuous soil flooding reduced leaf photosynthetic rate, stomatal conductance to water vapor, chlorophyll concentration and activity of ribulose bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39) of sweet orange [ Citrus sinensis (L.) Osbeck cv. Hamlin] trees, grafted onto rough lemon (RL; C. jambhiri Lush.) and sour orange (SO; C. aurantium L.) rootstocks. After 24 days of waterlogging, trees showed senescence, wilting and abscission of leaves, and these symptoms were more evident with flooded Hamlin/SO than Hamlin/RL. Reduction of leaf photosynthetic rate at day 24 was ca 94%, of stomatal conductance, 71%, of chlorophyll, 38% and of Rubisco, 62% for flooded Hamlin/SO, compared with 22, 5, 18 and 33%, respectively, for flooded Hamlin/RL. For both Hamlin/RL and Hamlin/SO, leaf photosynthetic rate and stomatal conductance to water vapor were closely correlated (r²= 0.87). Leaf internal CO₂ concentration of flooded trees, however, was not decreased by reduced stomatal conductance. Dark respiration rates of fibrous roots of flooded trees were greatly reduced, but not in leaf tissues. Total nonstructural carbohydrate concentrations were higher in leaves (50 and 80% increases for Hamlin/SO and Hamlin/RL, respectively), but drastically reduced in roots (60 and 45% reductions for Hamlin/SO and Hamlin/RL, respectively), as a result of flooding. The data indicate that Hamlin grafted onto RL rootstocks was more tolerant to soil flooding than Hamlin grafted onto SO rootstocks.     

A multi‐Gaussian model for predicting crawler occurrence of Unaspis yanonensis in citrus orchards

Arrowhead scale, Unaspis yanonensis (Kuwana) (Hemiptera: Diaspididae), feeds on the foliage, stems, and fruits of citrus trees and causes tree dieback when heavy infestations occur. Multi‐Gaussian models (three‐ and two‐peak models) were developed for better management of the arrowhead scale in citrus orchards and later validated against several field data sets. The oviposition activities observed in the laboratory were highly correlated with both models (r² = 0.88). The three peak oviposition times estimated by the three‐peak model were at 282, 500, and 694 degree‐days, based on a low threshold temperature of 13 °C. Also, the peak oviposition times of the two‐peak model were identical to the first and second peak times of the three‐peak model. Both models accurately predicted the first oviposition peak period of field populations. In the later peak period, both model outputs well predicted the actual crawler populations, except for the tail end of actual peak periods which were underestimated in the two‐peak model and overestimated in the three‐peak model. Overall, both models showed a strong robustness for correlation with actual data. The newly developed multi‐Gaussian models better described the actual population phenology of U. yanonensis than the previously published models, and either model would be useful for the management of U. yanonensis in the field.     

Comparative proteomic analysis between fifth‐instar nymphs and adults of Asian citrus psyllid Diaphorina citri

Asian citrus psyllid Diaphorina citri Kuwayama is extremely problematic worldwide, particularly where Huanglongbing (HLB) disease, the most serious and devastating of citrus diseases, is found. The threat is a result of its ability to transmit the causal agent of HLB, Candidatus Liberibacter asiaticus (CLas) bacterium. Improvements in proteomics, mass spectrometry, bioinformatics tools and gene ontology annotation facilitate the mapping and large‐scale identification and quantification of proteins. To date, only a few comparative proteomic studies report the developmental proteomic changes of hemimetabolous and plant–disease vector insects. Two‐dimensional gel electrophoresis analysis of D. citri total protein is able to detect qualitative and quantitative developmental differences. Liquid chromatography‐tandem mass spectrometry identifies 89 protein spots. Most proteins are metabolism and bioenergetics‐related. Nineteen protein spots are found to be implicated in stress/defence/immunity; 7 in development regulation; 9 in nervous system functions; 4 in the reproductive system; 23 in cytoskeleton and muscle organization; and 4 in movement, flight and other processes. Significant increases in the level of proteins related to structural constitution of the skeleton, stress/defence/immunity, reproduction system, muscles, locomotion and flight are found in adults, consistent with the fact that D. citri is a hemimetabolous insect, whereas proteins involved in developmental regulation are higher in the nymphal stage. The identification of these variably expressed proteins between the nymph and adult stages, linked with the basis of their physiological roles, will lead to a better understanding of the factors influencing development in D. citri and the regulation of some crucial metabolic pathways. It may also help to identify targets for genetic manipulation using RNA interference or other techniques to disrupt Asian citrus psyllid development, lifespan or its ability to transmit CLas.     

A prophage-encoded effector from “Candidatus Liberibacter asiaticus” targets ASCORBATE PEROXIDASE6 in citrus to facilitate bacterial infection

Citrus huanglongbing (HLB), associated with the unculturable phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of CLas remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the CLas genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis. The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H₂O₂ and O₂⁻) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri. This study reveals a novel mechanism underlying how CLas uses a prophage-encoded effector, AGH17488, to target a reactive oxygen species accumulation-related gene, APX6, in the host to facilitate its infection.     

Leaching Soluble Salts Increases Population Densities of Tylenchulus semipenetrans

The effect of salinity on population densities of Tylenchulus semipenetrans was measured on 3-month-old salt-tolerant Rangpur lime growing on either loamy sand, sand, or organic mix and on 4-month-old salt-sensitive Sweet lime in organic mix. Salinity treatments were initiated by watering daily with 25 mol/m³ NaCl + 3.3 mol/m³ CaCl₂ for 3 days and every other day with 50 mol/m³ NaC1 + 6.6 mol/m³ CaC1₂ for one week, with no salt (NS) treatments as controls. Salinity was discontinued in one treatment (DS) by leaching with tap water prior to inoculation with nematodes, whereas the continuous salinity (CS) treatment remained unchanged. Overall, in Rangpur lime organic soil supported the highest population densities of T. semipenetrans, followed by loamy sand and sand. The DS treatment resulted in the highest (P ≤ 0.05) mean population densities of T. semipenetrans in the three soil types. Similarly, the DS treatment in Sweet lime resulted in the highest (P ≤ 0.05) nematode populations. The DS treatment predisposed citrus to nematode infection through accumulated salt stress, whereas leaching soluble salt in soil solution offered nematodes a suitable nonosmotic habitat. Nematode females under the DS treatment also had the highest (P ≤ 0.05) fecundity.     

The effect of shoot and root genotype on phosphorus concentrations of shoots and roots

Genotypes of two morphologically different populations of white clover (Trifolium repens L.) were reciprocally and self-grafted. Ungrafted stolon tips were also grown as controls. Grafting per se had no significant effect on shoot size, root size, leaflet width or shoot and root % P. The scion genotype had a significant effect on shoot and root size, and leaflet width. Neither scion nor rootstock genotype had a significant effect on either shoot or root % P. However, there was a significant scion × rootstock × P level interaction for shoot % P. This along with other evidence suggests that conflicting results regarding effects of scion and rootstock on % P content of plants within species is probaby due to the interaction of scion and rootstock with environment.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.