Current Distribution of Huanglongbing (citrus greening disease) in India as Diagnosed by Real‐Time PCR

The widespread occurrence of Huanglongbing (HLB) was recorded in sixteen citrus growing states of India using the real‐time quantitative PCR and the derived threshold cycle (Ct) value. All the commercially important citrus varieties of mandarin, sweet orange, lime and lemon, pummelo and Satkara were infected with ‘Candidatus Liberibacter asiaticus’, the bacterium associated with HLB. Ct values positive for HLB were found in all the states except Arunachal Pradesh. The primer–probe combination HLBas‐HLBr‐HLBp was found specific to Ca. L. asiaticus and do not exhibit any cross‐reactivity with other pathogenic residents of citrus.     

Competitive adsorption of Pb, Cd and Zn ions onto Eichhornia crassipes in binary and ternary systems

A batch sorption technique was used to study the biosorption of Pb²⁺, Cd²⁺ and Zn²⁺ ions onto the vastly abundant water hyacinth weed, Eichhornia crassipes biomass in binary and ternary systems at a temperature of 30 °C and pH 4.84. Mutual interference effects were probed using equilibrium adsorption capacity ratios, , where the prime indicates the presence of one or two other metal ions. The combined action of the metals was found to be antagonistic, and the metal sorption followed the order Pb²⁺  Cd²⁺  Zn²⁺. The behaviour of competitive biosorption for Pb–Cd and Pb–Zn combinations were successfully described by the Langmuir Competitive Model (CLM), whilst the model showed poor fitting to the Cd–Zn data. In conclusion, Pb²⁺ ions could still be effectively removed from aqueous solution in the presence of both Cd²⁺ and Zn²⁺ ions, but removal of the Cd²⁺ and Zn²⁺ ions would be suppressed in the presence of Pb²⁺.     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Characterization of two novel tritiated radioligands for labelling Neuropeptide FF (NPFF1 and NPFF2) receptors

The binding characteristics of [³H]-NPVF and [³H]-EYF, the two first tritiated probes for the respective labelling of NPFF₁ and NPFF₂ receptors, are presented. In membranes from CHO cells transfected with the human NPFF₁ receptor, [³H]-NPVF labelled one class of binding sites with a high affinity (Bmax = 4 pmol/mg protein, Kd = 2.65 nM). In membranes from CHO cells transfected with the human NPFF₂ receptor, [³H]-EYF labelled one class of binding sites with a high affinity (Bmax = 16 pmol/mg protein, Kd = 0.54 nM). Both radioligands exhibited time-dependent binding, low (10–20%) non-specific binding and poor cross-reactivity towards the related receptor subtype. The potency of different NPFF ligands to displace [³H]-NPVF and [³H]-EYF binding profiles was in good agreement with the profile previously measured by using ¹²⁵I-probes (NPFF₁ receptor: NPVF ≥ 1DMe = SPA-NPFF > NPFF = SQA-NPFF = QFW-NPSF > NPSF > RF9; NPFF₂ receptor: SPA-NPFF > > SQA-NPFF = QFW-NPSF = 1DMe = NPFF  NPSF = NPVF > RF9). Therefore, [³H]-NPVF and [³H]-EYF are new valuable tools for performing binding on NPFF receptors.     

Essential oils of citrus fruit stimulate oviposition in the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae)

Female Mediterranean fruit flies (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) perceive both qualitative and quantitative aspects of citrus fruit chemistry. However, the behavioural and biological adjustments of this response remain largely unknown. In the present study, the ovipositional responses of gravid female medflies to essential oils (i.e. the most critical resistance factor to medfly infestation) of various citrus fruits are investigated. In dual‐choice (treatment versus distilled water control) experiments, females lay significantly more eggs into pre‐punctured hollow oviposition hemispheres (domes) provided with 1 µL of citrus peel oil from sweet orange, satsuma mandarin, bitter orange, grapefruit and lemon compared with odourless domes. No‐choice tests show a weak effect of lemon essential oils in stimulating oviposition. The female ovipositional response to sweet orange oil (the most active in eliciting oviposition) is dose‐dependent. Additionally, limonene, the most abundant chemical in all citrus oils, stimulates oviposition, whereas linalool, a representative compound of immature citrus fruit associated with high toxicity against immature stages of fruit flies, has a significant deterrent effect. In further no‐choice tests, females lay approximately 23% fewer eggs in limonene (93%) (amount found in orange oil) and 60% fewer eggs in limonene 93% plus linalool 3% (approximately 10‐fold the amount found in orange oil) mixtures, relative to sweet orange oil. The results suggest that the limonene content accounts largely (but not completely) for the ovipositional responses observed in sweet orange oil, whereas high linalool proportions are capable of significantly masking and/or disrupting its stimulatory effects in citrus oils. The importance and practical implications of these findings with respect to understanding how citrus fruit chemistry influences the ovipositional responses of medfly is discussed.     

Evidence for two endocytic transport pathways in plant cells

Intracellular trafficking of endocytic vesicles in eukaryotes varies with the nature of the cargo molecules and the targeted organelle, and proceeds through an intricate network of internal endosomal compartments. However, the path for fluid-phase endocytosis (FPE), the internalization of external solutes from the apoplast via plasmalemma generated vesicles, remains unresolved despite some indication of a direct transport route to the vacuole. To test this hypothesis, we made use of the membrane-impermeable Na-dependent fluorescent marker Coro-Na in combination with the fluorescent membrane marker FM 4-64 and confocal laser scanning microscopy. When protoplasts from sweet lime juice cells were incubated in Na-free solution, FM 4-64, Coro-Na, and 200 mM sucrose, two distinct types of labeled vesicles were evident. A set of vesicles (1 μm in diameter) was intensely labeled with Coro-Na and to a lesser extent with FM 4-64, whereas the second type of 1–7 μm structures appeared exclusively labeled with FM 4-64. These data demonstrate the parallel functioning of two endocytic pathways in plant cells. In one system, a set of small endocytic vesicles merge with the endosome, whereas a separate set of vesicles fuse to form larger vesicles independent from the endosome. Although it is likely that both vesicle systems eventually contribute to solutes reaching the vacuole, given their size (1–7 μm), and based on previous observations of endocytic vesicle formation protruding from the plasmalemma and merging with the vacuole, we conclude that these latter vesicles constitute the primary FPE vesicle system.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

不同柑桔品种上桔全爪螨的生长和种群动态差异

通过田间调查、室内盆栽苗接螨、离体叶片饲养等方法,考察了14个柑桔品种上桔全爪螨种群动态,组建了其实验种群生命表,结果表明：该螨在各柑桔品种上的种群密度有明显差异,平均密度以北碚447、台湾晚白柚和垫江白柚上较高,柑新生系最低。雌成螨寿命在台湾晚白柚上最长,垫江白柚次之,柑新生系最短;产卵量以取食台湾晚白柚时最多,垫江白柚、五步红心柚次之,以柑新生系最少;在垫江白柚上的内禀增长率（r_m）最大,沙田柚、台湾晚白柚次之,金弹最小。根据桔全爪螨实验种群的参数判断,金弹和柑新生系属抗螨品种,台湾晚白柚、垫江白柚和北碚447是感螨品种。从品种归类上来看,柚类品种和甜橙中的北碚447感螨程度较高,金柑属的金弹和宽皮柑桔类的柑新生系感螨程度较低。甜橙类与宽皮柑桔类比较,前者感螨程度较高,后者相对较低,但宽皮柑桔类的早津感螨程度相对较高。     

Attractiveness of binary blends of floral odorant compounds to moths in Florida, USA

Evaluation of combinations of flower odor compounds in the field revealed several compounds that were attractive or co-attractive with phenylacetaldehyde (PAA) to pest noctuid and pyralid moths. A number of moth species responded positively to the key floral odorant PAA. The floral odorants cis -jasmone, linalool, benzyl acetate, limonene, β-myrcene, methyl salicylate, and methyl 2-methoxybenzoate all increased captures of some moths when added to traps with PAA, but responses varied among the moth species that were trapped. For example, soybean looper moths, Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae), most strongly responded to PAA + β-myrcene, but benzyl acetate, cis -jasmone, and limonene also increased captures of these moths when these compounds were used in traps along with PAA. Velvetbean caterpillar moths, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), responded most strongly to PAA + linalool, but β-myrcene, cis -jasmone, and limonene also increased captures of these moths in traps over numbers trapped with PAA. Positive responses to floral compound blends were also noted for golden looper [ Argyrogramma verruca (F.)], grass looper [ Mocis disseverans (Walker)], tobacco budworm [ Heliothis virescens (F.)], southern armyworm [ Spodoptera eridania (Stoll) (all Lepidoptera: Noctuidae)], and melonworm [ Diaphania hyalinata (L.) (Lepidoptera: Pyralidae)]. Overall, PAA + β-myrcene appeared to be the strongest floral compound combination for pest species trapped, compared to PAA or other compound blends.     

Investigation of the pronounced synergism between prostaglandin E2 and other constrictor agents on rat femoral artery

This study investigates the pronounced synergism between the weak contractile action of prostaglandin E₂ (PGE₂) and strong actions of phenylephrine, U-46619 and K⁺ on rat isolated femoral artery. The potency ranking for synergism was SC-46275 (prostanoid receptor agonist selectivity: EP₃EP₁)=sulprostone (EP₃>EP₁)>17-phenyl PGE₂ (EP₁>EP₃). The novel EP₃ antagonist L-798106 (0.2–1 μM) blocked the enhanced action of sulprostone (pA₂=7.35–8.10), while the EP₁ antagonist SC-51322 (1 μM) did not (pA₂<6.0). Matching responses to priming agent and priming agent/sulprostone were similarly suppressed by nifedipine (300 nM) and the selective Rho-kinase inhibitors H-1152 (0.1–1 μM) and Y-27632 (1–10 μM). Our findings implicate an EP₃ receptor in the prostanoid component of contractile synergism. While the synergism predominantly operates through a Ca²⁺ influx–Rho-kinase pathway, the EP₃ receptor does not necessarily transduce via Rho-kinase.     

Volatile components of banana pseudostem of a cultivar susceptible to the banana weevil

Volatiles from banana (‘githumo’'cultivar) pseudostem were trapped using porapak S. The trapped volatiles were desorbed from porapak columns by elution with dichloromethane and analysed using GC and GC-MS. The volatile compounds identified included -pinene, β-pinene, β-myrcene, limonene, -cubebene, -copaene, -cedrene, β-caryophyllene and -humulene.     

Genetic relationships between some Tunisian Citrus species based on their leaf volatile oil constituents

Volatile oil constituents of Tunisian sweet orange (Citrus sinensis Osbeck) cv. Meski (MES), Valencia Late (VAL), Thomson Navel (THN) and Maltaise Blanc (MAB); mandarin (Citrus reticulata Blanco); sour orange (Citrus aurantium L. cv. Amara (AM)) and pummelo (Citrus grandis Osbeck) were obtained by hydrodistillation and analysed by gas chromatography (GC) combined with a flame ionisation detector (FID) and mass spectrometry (MS). A total of 41 components accounting for more than 95% of the total essential oils were identified, and oxygenated monoterpenes (69.5–99.9%) were found as the most prominent fraction in all oil samples. The main constituents were linalool (3.1–73%), isoborneol (0–55.8%) and tepinen-4-ol (1.1–19.2%). A high degree of inter-and intraspecific chemical variability between species and cultivars was found to be genetically determined, and a set of distinctive traits (chemical markers) in the essential oils profile was established. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of all identified components grouped the oils into two main chemotypes (linalool/isoborneol and isoborneol/linalool).     

Monoterpene biotransformation by <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Objective

To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.

Results

C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l^?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.

Conclusions

This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

     

Chemical variability of peel and leaf essential oils of 15 species of mandarins

Peel and leaf oils of 58 mandarin cultivars, belonging to 15 different species were obtained from fruits and leaves collected on mandarin-trees submitted to the same pedoclimatic and cultural conditions. Their chemical composition was investigated by capillary GC, GC/MS and 13C NMR and the results were submitted to a cluster analysis and a discriminant analysis. Three major chemotypes, limonene, limonene/gamma-terpinene and linalyl acetate/limonene, were distinguished for peel oils while three other chemotypes, sabinene/linalool, gamma-terpinene/linalool and methyl N-methylanthranilate, were observed for leaf oils.     

Toxicity of basil and orange essential oils and their components against two coleopteran stored products insect pests

Two commercialized essential oils and their constituent compounds were investigated for fumigant and contact activities against two grain storage insects, adults of the maize weevil (Sitophilus zeamais) and the red flour beetle (Tribolium castaneum). The two commercialized basil and orange oils showed strong fumigant and contact activities against S. zeamais and T. castaneum. The constituents of the basil oil were linalool (21.83%), estragole (74.29%), and α-humulene (2.17%), and those of the orange oil were α-pinene (0.54%), sabinene (0.38%), β-myrcene (1.98%), limonene (96.5%), and linalool (0.6%). As a toxic fumigant, the basil oil was more effective (24-h LC₅₀ = 0.014 and 0.020 mg cm^− 3) than the orange oil (24-h LC₅₀ = 0.106 and 0.130 mg cm^− 3) against S. zeamais and T. castaneum adults, respectively. Among the constituents of the two essential oils, the toxicity of estragole was the highest (0.004 and 0.013), followed by linalool (0.016 and 0.023), limonene (0.122 and 0.171), α-pinene (0.264 and 0.273), and β-myrcene (0.274 and 0.275) based on 24-h LC₅₀ values (mg cm^− 3). Similar results were obtained in a contact toxicity test. The contact activity of basil oil was more toxic than orange oil, and estragole and linalool showed pronounced contact toxicity against S. zeamais and T. castaneum adults. Alpha-humulene had no activity as a fumigant at the tested doses, but it did have an effect as a contact poison, having 24-h LD₅₀ values of 0.040 and 0.045 mg adult^− 1 to S. zeamais and T. castaneum, respectively. Although basil oil, orange oil, and their components displayed both contact and fumigant toxicities, their effects were mainly exerted by fumigant action via the vapor phase. Thus, basil oil, orange oil, and their components could be potential candidates as new fumigants for the control of S. zeamais and T. castaneum adults.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Transferability of the EST-SSRs developed on Nules clementine (<Emphasis Type="Italic">Citrus clementina</Emphasis> Hort ex Tan) to other <Emphasis Type="Italic">Citrus</Emphasis> species and their effectiveness for genetic mapping

Background

During the last decade, numerous microsatellite markers were developed for genotyping and to identify closely related plant genotypes. In citrus, previously developed microsatellite markers were arisen from genomic libraries and more often located in non coding DNA sequences. To optimize the use of these EST-SSRs as genetic markers in genome mapping programs and citrus systematic analysis, we have investigated their polymorphism related to the type (di or trinucleotide) or their position in the coding sequences.

Results

Among 11000 unigenes from a Clementine EST library, we have found at least one microsatellite sequence (repeated units size ranged from 2 to 6 nucleotides) in 1500 unigenes (13.6%). More than 95% of these SSRs were di or trinucleotides. If trinucleotide microsatellites were encountered trough all part of EST sequences, dinucleotide microsatellites were preferentially (50%) concentrated in the 5' 100th nucleotides. We assessed the polymorphism of 41 EST-SSR, by PCR amplification droved with flanking primers among ten Citrus species plus 3 from other genera. More than 90% of EST-SSR markers were polymorphic. Furthermore, dinucleotide microsatellite markers were more polymorphic than trinucleotide ones, probably related to their distribution that was more often located in the 5' UnTranslated Region (UTR). We obtained a good agreement of diversity relationships between the citrus species and relatives assessed with EST-SSR markers with the established taxonomy and phylogeny. To end, the heterozygosity of each genotype and all dual combinations were studied to evaluate the percentage of mappable markers. Higher values (> 45%) were observed for putative Citrus inter-specific hybrids (lime lemon, or sour orange) than for Citrus basic true species (mandarin, pummelo and citron) (<30%). Most favorable combinations for genome mapping were observed in those involving interspecific hybrid genotypes. Those gave higher levels of mappable markers (>70%) with a significant proportion suitable for synteny analysis.

Conclusion

Fourty one new EST-SSR markers were produced and were available for citrus genetic studies. Whatever the position of the SSR in the ESTs the EST-SSR markers we developed are powerful to investigate genetic diversity and genome mapping in citrus.