Silencing of δ-aminolevulinic acid dehydratase via virus induced gene silencing promotes callose deposition in plant phloem.

The δ-aminolevulinic acid dehydratase (ALAD) enzyme is an intermediate in the biosynthetic pathway of tetrapyrroles. It combines two δ-aminolevulinic acid (δ-ALA) molecules to form the pyrrole, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, light-sensing, and nutrient uptake. Our recent efforts showed that, in citrus, silencing of ALAD gene via Citrus tristeza virus-induced gene silencing, caused yellow spots and necrosis in leaves and in developing new shoots. Silencing of ALAD gene reduced leaf pigments and altered leaf metabolites. Moreover, total phenolic content, H2O2, and reactive oxygen species (ROS) increased, indicating that silencing of ALAD induced severe stress. Herein, we hypothesized that conditions including lower sucrose, elevated ROS, alteration of microRNA involved in RNAi regulatory protein Argonaute 1 (AGO1) and ROS lead to higher deposition of callose in phloem tissues. Using aniline blue staining and gene expression analysis of callose synthases, we showed significant deposition of callose in ALAD-silenced citrus.

Influence of Rootstock on the Leaf Volatile Organic Compounds of Citrus Scion Is More Pronounced after the Infestation with Diaphorina citri.

Nowadays, citrus greening or Huanglongbing is considered the most destructive disease in the citrus industry worldwide. In the Americas and Asia, the disease is caused by the putative pathogen, 'Candidatus Liberibacter asiaticus' and transmitted by the psyllid vector, Diaphorina citri. It has been shown that volatile organic compounds (VOC) that are released from citrus leaves attract the psyllid vector. Herein, we tested whether the rootstock influenced the stored VOC profile in the scion leaves and if these influences were altered after infestation with D. citri. The VOC profiles of the hexane-extracted leaves of the mandarin hybrid 'Sugar Belle' that were grafted on three different rootstocks (C-35, sour orange (SO), and US-897) with and without infestation with D. citri were studied. The GC-MS analysis showed that the scion VOC profiles of the non-infested control trees were similar to each other, and rootstock was not a strong influence. However, after one month of infestation with D. citri, clear differences in the scion VOC profiles appeared that were rootstock dependent. Although the total scion leaf VOC content did not differ between the three rootstocks, the infestation increased scion monoterpenes significantly on US-897 and C-35 rootstock, increased terpene alcohols on US-897 and SO rootstock, and increased sesquiterpenes on SO. Infestation with D. citri significantly reduced fatty acids and fatty acid esters across all of the rootstocks. Therefore, our results suggest that rootstock choice could influence scions with an inducible volatile defense by enhancing the amounts of VOCs that are available for repelling vectors or for signaling to their natural enemies or parasitoids. According to this study, US-897 may be the best choice among the three that were studied herein, due to its diverse and robust VOC defense response to infestation with D. citri.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus.

Recently in Florida, foliar treatments using products with the antibiotics oxytetracyclineand streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which iscaused by the putative bacterial pathogen 'Candidatus Liberibacter asiaticus'. Herein, we assessedthe levels of oxytetracycline and 'Ca. L. asiaticus' titers in citrus trees upon foliar applications withand without a variety of commercial penetrant adjuvants and upon trunk injection. The level ofoxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and 'Ca. L.asiaticus' titer was measured using quantitative PCR. Low levels of oxytetracycline were taken upby citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to theoxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level ofoxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliarapplications. The titer of 'Ca. L. asiaticus' in the midrib of leaves from trees receiving oxytetracyclineby foliar application was not affected after four days and thirty days of application, whereas thetiter was significantly reduced in oxytetracycline-injected trees thirty days after treatment.Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidizedcuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake ofoxytetracycline, decreasing the titer of 'Ca. L. asiaticus' in citrus leaves upon foliar application.Taken together, our findings indicate that trunk injection is more efficient than foliar spray evenafter the use of adjuvants. Our conclusion could help in setting useful recommendations for theapplication of oxytetracycline in citrus to improve tree health, minimize the amount of appliedantibiotic, reduce environmental exposure, and limit off-target effects.

All roads lead to Rome: Towards understanding different avenues of tolerance to huanglongbing in citrus cultivars.

Citrus tolerance to huanglongbing could result from tolerance to the pathogen Candidatus Liberibacter asiaticus (CLas) and/or to its vector Diaphorina citri. Field observations and greenhouse-controlled studies showed that some citrus cultivars were more tolerant than others. However, the mechanism(s) behind the tolerance has not been determined yet. Using GC-MS, we investigated the volatile organic compounds (VOCs) and the non-volatile metabolite profiles of two tolerant citrus cultivars- Australian finger lime, 'LB8-9' Sugar Belle® mandarin hybrid, and a recently released mandarin hybrid 'Bingo'. The three were grafted onto the rootstock, Carrizo citrange. Our findings showed that the metabolomic profiles of Australian finger lime were different from that of 'LB8-9'. Finger lime was high in many amino acids and tricarboxylic acid intermediates, whereas 'LB8-9' was high in several amino acids, sugars, and sugar alcohols. 'LB8-9' was high in thymol, which is known for its strong antimicrobial activity against a panel of pathogenic bacteria. The metabolomic profiles of 'Bingo' were intensely different from the other mandarin hybrid, 'LB8-9', including a reduced thymol biosynthetic pathway and low amounts of most of the amino acids and sugar alcohols. Remarkably, 1,8-cineole (eucalyptol) was only detected in 'Bingo', indicating that eucalyptol could have feeding and ovipositional repellency against D. citri. The metabolite profiles generated for HLB-tolerant citrus species will improve the ability of citrus breeders and will allow them to take more informed decisions. Metabolomic profiling of HLB-tolerant citrus species could identify tolerance specific markers that can be introduced to other commercial citrus cultivars to improve their tolerance to HLB disease.

Effect of different rootstocks on the leaf metabolite profile of 'Sugar Belle' mandarin hybrid.

Currently, citrus greening is threatening the citrus industry worldwide. So far, there is no effective cure for this destructive disease and management mainly depends on the control of Diaphorina citri vector using insecticides. Although the use of different rootstocks could increase citrus scions' tolerance to biotic and abiotic stresses, little work has been conducted to investigate the effect of rootstocks on citrus tolerance to citrus greening pathogen. In this study, we investigated the effect of rootstock on the metabolite profile of 'Sugar Belle' mandarin hybrid using gas-chromatography mass spectrometry (GC-MS). The principle component analysis showed that the metabolite profiles of the 'Sugar Belle' mandarin hybrid on the three selected rootstocks were different from each other. These results indicated that rootstocks could affect the primary and secondary metabolites of citrus scions, and consequently could affect scion tolerance to pathogens.

Metabolic alterations in the nymphal instars of Diaphorina citri induced by Candidatus Liberibacter asiaticus, the putative pathogen of huanglongbing.

Currently, huanglongbing is the most damaging disease of citrus causing huge economic losses. The disease is caused by the Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). The pathogen is transmitted in a persistent propagative circulative manner within its vector, the Asian citrus psyllid, Diaphorina citri. Exploring the metabolic alteration in the vector may lead to a better understanding of the nutritional needs of CLas and to designing an artificial medium for culturing the pathogen. It has been shown that the nymphal stages have a greater role in transmission mainly because they feed on plants more actively than adults. In this study, we carried out an untargeted comparative metabolomic analysis for healthy and CLas-infected 4th / 5th instar nymphs. The metabolic analysis was performed using trimethylsilylation and methyl chloroformate derivatization followed by Gas Chromatography-Mass Spectrometry (GC-MS). Overall, the changes in the nymph metabolism due to the infection with CLas were more pronounced than in adults, as we previously published. Nymphs reared on CLas-infected Valencia sweet orange were higher in many metabolites, mainly those of the TCA cycle, C16 and C18 fatty acids, glucose, sucrose, L-proline, L-serine, pyroglutamic acid, saccharic acid, threonic acid and myo-inositol than those reared on healthy plants. In contrast, CLas-infected nymphs were lower in putrescine, glycine, L -phenylalanine, L -tyrosine, L -valine, and chiro-inositol. The information provided from this study may contribute in acceleration of the availability of CLas in culture and consequent screening of antibacterial compounds to discover a definitive solution for huanglongbing.

Metabolically speaking: Possible reasons behind the tolerance of 'Sugar Belle' mandarin hybrid to huanglongbing.

Huanglongbing (HLB) is currently considered the most destructive disease of citrus. Since its spread to the Americas, HLB has killed millions of trees and caused a sharp decline in production in many citrus growing regions. With the continuous spread of HLB disease in Florida and worldwide, there is an urgent need for the development of commercial citrus cultivars with a strong tolerance to HLB. Interestingly, field observations showed that some of the recently released mandarin hybrids such as 'Sugar Belle' were tolerant to HLB. In this study, we investigated the volatile and non-volatile metabolites of greenhouse-grown 'Sugar Belle' mandarin and four of its ancestors in order to understand why 'Sugar Belle' mandarin is relatively tolerant to HLB. Leaf volatiles were directly extracted with hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Leaf polar metabolites were extracted with a mixture of methanol:water (1:1, v/v), derivatized to their trimethylsilyl ethers, and analyzed using GC-MS. Forty-seven volatile compounds and forty-two polar metabolites were detected in 'Sugar Belle' mandarin leaves and its ancestors. 'Sugar Belle' was high in several volatiles such as α-thujene, para-cymene, γ-terpinene, thymol, ß-elemene, and (E)-ß-caryophyllene. Some of these volatiles, especially thymol, ß-elemene, and (E)-ß-caryophyllene are known for their anti-microbial activity. In addition, 'Sugar Belle' mandarin was the highest in synephrine, benzoic acid, ferulic acid, caffeic acid, chiro-inositol, fructose, glucose, threonic acid, saccharic acid, and galactaric acid, and the second in threonine, malic acid, and myo-inositol compared to the ancestors. Phenolic compounds such as benzoic, ferulic, and caffeic acids may act as antibacterial agents, whereas others like sugar alcohols may protect 'Sugar Belle' mandarin from stress during pathogen attack. The tolerance of 'Sugar Belle' and other newly released mandarin hybrids should be further evaluated using greenhouse controlled studies. If tolerance of these hybrids is confirmed, they could be used to replace the traditionally susceptible cultivars.

Profiling of volatile organic compounds released from individual intact juvenile and mature citrus leaves.

Plants release volatiles to communicate with each other and to attract or repel insects. The methods used to collect volatiles are varied. Here, we describe a simple solvent-less, solid phase microextraction-based method to collect the volatiles released from intact citrus leaves. We were able to collect up to 39 volatiles from both juvenile and mature leaves. Our results indicated that juvenile leaves produced both monoterpenes and sesquiterpenes, and while mature leaves continued to produce a variety of monoterpenes, their release of sesquiterpenes decreased dramatically. The finding that juvenile leaves emitted higher levels of sesquiterpenes while mature leaves released mostly monoterpenes suggests that younger leaves of plants may be involved in a more complex chemical communication system.

An HPLC-MS characterization of the changes in sweet orange leaf metabolite profile following infection by the bacterial pathogen Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) presumably caused by Candidatus Liberibacter asiaticus (CLas) threatens the commercial U.S. citrus crop of an annual value of $3 billion. The earliest shift in metabolite profiles of leaves from greenhouse-grown sweet orange trees infected with Clas, and of healthy leaves, was characterized by HPLC-MS concurrently with PCR testing for the presence of Clas bacteria and observation of disease symptoms. Twenty, 8-month-old 'Valencia' and 'Hamlin' trees were grafted with budwood from PCR-positive HLB source trees. Five graft-inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Thirteen weeks after inoculation, Clas was detected in newly growing flushes in 33% and 55% of the inoculated 'Hamlin' and 'Valencia' trees, respectively. Inoculated trees remained asymptomatic in the first 20 weeks, but developed symptoms 30 weeks after grafting. No significant differences in the leaf metabolite profiles were detected in Clas-infected trees 23 weeks after inoculation. However, 27 weeks after inoculation, differences in metabolite profiles between control leaves and those of Clas-infected trees were evident. Affected compounds were identified with authentic standards or structurally classified by their UV and mass spectra. Included among these compounds are flavonoid glycosides, polymethoxylated flavones, and hydroxycinnamates. Four structurally related hydroxycinnamate compounds increased more than 10-fold in leaves from 'Hamlin' and 'Valencia' sweet orange trees in response to Clas infection. Possible roles of these hydroxycinnamates as plant defense compounds against the Clas infection are discussed.