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.     

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of ‘Hamlin’, ‘Pera’, and ‘Valencia’ sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08?% among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.     

Transgenic citrus expressing synthesized <Emphasis Type="Italic">cecropin</Emphasis> B genes in the phloem exhibits decreased susceptibility to Huanglongbing

Key message

Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing.

Abstract

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus (Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8, these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.

     

Stylet Morphometrics and Citrus Leaf Vein Structure in Relation to Feeding Behavior of the Asian Citrus Psyllid Diaphorina citri,Vector of Citrus Huanglongbing Bacterium

The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae), is the primary vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus (LAS) associated with huanglongbing (HLB, citrus greening), considered the world’s most serious disease of citrus. Stylet morphometrics of ACP nymphs and adults were studied in relation to citrus vein structure and to their putative (histologically verified) feeding sites on Valencia orange leaves. ACP nymphs preferred to settle and feed on the lower (abaxial) side of young leaves either on secondary veins or on the sides of the midrib, whereas adults preferred to settle and feed on the upper (adaxial) or lower secondary veins of young or old leaves. Early instar nymphs can reach and probe the phloem probably because the distance to the phloem is considerably shorter in younger than in mature leaves, and is shorter from the sides of the midrib compared to that from the center. Additionally, the thick-walled ‘fibrous ring’ (sclerenchyma) around the phloem, which may act as a barrier to ACP stylet penetration into the phloem, is more prominent in older than in younger leaves and in the center than on the sides of the midrib. The majority (80–90%) of the salivary sheath termini produced by ACP nymphs and adults that reached a vascular bundle were associated with the phloem, whereas only 10–20% were associated with xylem vessels. Ultrastructural studies on ACP stylets and LAS-infected leaves suggested that the width of the maxillary food canal in first instar nymphs is wide enough for LAS bacteria to traverse during food ingestion (and LAS acquisition). However, the width of the maxillary salivary canal in these nymphs may not be wide enough to accommodate LAS bacteria during salivation (and LAS inoculation) into host plants. This may explain the inability of early instar nymphs to transmit LAS/HLB in earlier reports.     

Fragile Sites of ‘Valencia’ Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in ‘Valencia’ C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of ‘Valencia’ C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid ‘Valencia’ C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in ‘Valencia’ sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in ‘Valencia’ sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.     

Evaluation of the Genetic Structure of Xylella fastidiosa Populations from Different Citrus sinensis Varieties

Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H_T) values of X. fastidiosa were similar for both sets of strains; however, H_TRAPD values were substantially lower than H_TVNTR values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.     

Etiology of three recent diseases of citrus in São Paulo State: sudden death, variegated chlorosis and huanglongbing

The state of S?o Paulo (SSP) is the first sweet orange growing region in the world. Yet, the SSP citrus industry has been, and still is, under constant attack from various diseases. In the 1940s, tristeza-quick decline (T-QD) was responsible for the death of 9 million trees in SSP. The causal agent was a new virus, citrus tristeza virus (CTV). The virus was efficiently spread by aphid vectors, and killed most of the trees grafted on sour orange rootstock. Control of the disease resided in replacing sour orange by alternative rootstocks giving tolerant combinations with scions such as sweet orange. Because of its drought resistance, Rangpur lime became the favourite alternative rootstock, and, by 1995, 85% of the SSP sweet orange trees were grafted on this rootstock. Therefore, when in 1999, many trees grafted on Rangpur lime started to decline and suddenly died, the spectre of T-QD seemed to hang over SSP again. By 2003, the total number of dead or affected trees was estimated to be over one million. The new disease, citrus sudden death (CSD), resembles T-QD in several aspects. The two diseases have almost the same symptoms, they spread in time and space in a manner strikingly similar, and the pathological anatomy of the bark at the bud union is alike. Transmission of the CSD agent by graft-inoculation has been obtained with budwood inoculum taken not only on CSD-affected trees (grafted on Rangpur lime), but also on symptomless trees (grafted on Cleopatra mandarin) from the same citrus block. This result shows that symptomless trees on Cleopatra mandarin are tolerant to the CSD agent. Trees on rootstocks such as Sunki mandarin or Swingle citrumelo are also tolerant. Thus, in the CSD-affected region, control consists in replacing Rangpur lime with compatible rootstocks, or in approach-grafting compatible rootstock seedlings to the scions of trees on Rangpur lime (inarching). More than 5 million trees have been inarched in this way. A new disease of sweet orange, citrus variegated chlorosis (CVC), was observed in 1987 in the Triangulo Mineiro of Minas Gerais State and the northern and north-eastern parts of SSP. By 2000, the disease affected already 34% of the 200 million sweet orange trees in SSP. By 2005, the percentage had increased to 43%, and CVC was present in all citrus growing regions of Brazil. Electron microscopy showed that xylem-limited bacteria were present in all symptomatic sweet orange leaves and fruit tissues tested, but not in similar materials from healthy, symptomless trees. Bacteria were consistently cultured from twigs of CVC-affected sweet orange trees but not from twigs of healthy trees. Serological analyses showed the CVC bacterium to be a strain of Xylella fastidiosa. The disease could be reproduced and Koch's postulates fulfilled, by mechanically inoculating a pure culture of X. fastidiosa isolate 8.1.b into sweet orange seedlings. The genome of a CVC strain of X. fastidiosa was sequenced in SSP in the frame of a project supported by FAPESP and Fundecitrus. X. fastidiosa is the first plant pathogenic bacterium, the genome of which has been sequenced. Until recently, America was free of huanglongbing (HLB), but in March 2004 and August 2005, symptoms of the disease were recognized, respectively in the State of S?o Paulo (SSP) and in Florida, USA. HLB was known in China since 1870 and in South Africa since 1928. Because of its destructiveness and its rapid spread by efficient psyllid insect-vectors, HLB is probably the most serious citrus disease. HLB is caused by a phloem sieve tube-restricted Gram negative bacterium, not yet available in culture. In the 1990s, the bacterium was characterized by molecular techniques as a member of the alpha proteobacteria designated Candidatus Liberibacter africanus for the disease in Africa, and Candidatus Liberibacter asiaticus for HLB in Asia. In SSP, Ca. L. asiaticus is also present, but most of the trees are infected with a new species, Candidatus Liberibacter americanus.     

Construction of Commercial Sweet Cherry Linkage Maps and QTL Analysis for Trunk Diameter

A cross between the sweet cherry (Prunus avium) cultivars ‘Wanhongzhu’ and ‘Lapins’ was performed to create a mapping population suitable for the construction of a linkage map. The specific-locus amplified fragment (SLAF) sequencing technique used as a single nucleotide polymorphism (SNP) discovery platform and generated 701 informative genotypic assays; these, along with 16 microsatellites (SSRs) and the incompatibility (S) gene, were used to build a map which comprised 8 linkage groups (LGs) and covered a genetic distance of 849.0 cM. The mean inter-marker distance was 1.18 cM and there were few gaps > 5 cM in length. Marker collinearity was maintained with the established peach genomic sequence. The map was used to show that trunk diameter (TD) is under the control of 4 loci, mapping to 3 different LGs. Different locus influenced TD at a varying stage of the tree’s development. The high density ‘W×L’ genetic linkage map has the potential to enable high-resolution identification of QTLs of agronomically relevant traits, and accelerate sweet cherry breeding.     

Mi-1-Mediated Nematode Resistance in Tomatoes is Broken by Short-Term Heat Stress but Recovers Over Time

Tomato (Solanum lycopersicum L.) is among the most valuable agricultural products, but Meloidogyne spp. (root-knot nematode) infestations result in serious crop losses. In tomato, resistance to root-knot nematodes is controlled by the gene Mi-1, but heat stress interferes with Mi-1-associated resistance. Inconsistent results in published field and greenhouse experiments led us to test the effect of short-term midday heat stress on tomato susceptibility to Meloidogyne incognita race 1. Under controlled day/night temperatures of 25°C/21°C, ‘Amelia’, which was verified as possessing the Mi-1 gene, was deemed resistant (4.1 ± 0.4 galls/plant) and Rutgers, which does not possess the Mi-1 gene, was susceptible (132 ± 9.9 galls/plant) to M. incognita infection. Exposure to a single 3 hr heat spike of 35°C was sufficient to increase the susceptibility of ‘Amelia’ but did not affect Rutgers. Despite this change in resistance, Mi-1 gene expression was not affected by heat treatment, or nematode infection. The heat-induced breakdown of Mi-1 resistance in ‘Amelia’ did recover with time regardless of additional heat exposures and M. incognita infection. These findings would aid in the development of management strategies to protect the tomato crop at times of heightened M. incognita susceptibility.     

Sugars,organic acids,and phenolic compounds of ancient grape cultivars (Vitis vinifera L.) from Igdir province of Eastern Turkey

Background

The Eurasian grapevine (Vitis vinifera L.) is the most widely cultivated and economically important horticultural crop in the world. As a one of the origin area, Anatolia played an important role in the diversification and spread of the cultivated form V. vinifera ssp. vinifera cultivars and also the wild form V. vinifera ssp. sylvestris ecotypes. Although several biodiversity studies have been conducted with local cultivars in different regions of Anatolia, no information has been reported so far on the biochemical (organic acids, sugars, phenolic acids, vitamin C) and antioxidant diversity of local historical table V. vinifera cultivars grown in Igdir province. In this work, we studied these traits in nine local table grape cultivars viz. ‘Beyaz Kismis’ (synonym name of Sultanina or Thompson seedless), ‘Askeri’, ‘El Hakki’, ‘Kirmizi Kismis’, ‘Inek Emcegi’, ‘Hacabas’, ‘Kerim Gandi’, ‘Yazen Dayi’, and ‘Miskali’ spread in the Igdir province of Eastern part of Turkey.

Results

Variability of all studied parameters is strongly influenced by cultivars (P < 0.01). Among the cultivars investigated, ‘Miskali’ showed the highest citric acid content (0.959 g/l) while ‘Kirmizi Kismis’ produced predominant contents in tartaric acid (12.71 g/l). The highest glucose (16.47 g/100 g) and fructose (15.55 g/100 g) contents were provided with ‘Beyaz Kismis’. ‘Kirmizi Kismis’ cultivar had also the highest quercetin (0.55 mg/l), o-coumaric acid (1.90 mg/l), and caffeic acid (2.73 mg/l) content. The highest ferulic acid (0.94 mg/l), and syringic acid (2.00 mg/l) contents were observed with ‘Beyaz Kismis’ cultivar. The highest antioxidant capacity was obtained as 9.09 μmol TE g^-1 from ‘Inek Emcegi’ in TEAC (Trolox equivalent Antioxidant Capacity) assay. ‘Hacabas’ cultivar had the highest vitamin C content of 35.74 mg/100 g.

Conclusions

Present results illustrated that the historical table grape cultivars grown in Igdir province of Eastern part of Turkey contained diverse and valuable sugars, organic acids, phenolic acids, Vitamin C values and demonstrated important antioxidant capacity for human health benefits. Further preservation and use of this gene pool will be helpful to avoid genetic erosion and to promote continued agriculture in the region.     

Effective Antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-Affected Citrus Plants Identified via the Graft-Based Evaluation

Citrus huanglongbing (HLB), caused by three species of fastidious, phloem-limited ‘Candidatus Liberibacter’, is one of the most destructive diseases of citrus worldwide. To date, there is no established cure for this century-old and yet, newly emerging disease. As a potential control strategy for citrus HLB, 31 antibiotics were screened for effectiveness and phytotoxicity using the optimized graft-based screening system with ‘Candidatus Liberibacter asiaticus’ (Las)-infected citrus scions. Actidione and Oxytetracycline were the most phytotoxic to citrus with less than 10% of scions surviving and growing; therefore, this data was not used in additional analyses. Results of principal component (PCA) and hierarchical clustering analyses (HCA) demonstrated that 29 antibiotics were clustered into 3 groups: highly effective, partly effective, and not effective. In spite of different modes of actions, a number of antibiotics such as, Ampicillin, Carbenicillin, Penicillin, Cefalexin, Rifampicin and Sulfadimethoxine were all highly effective in eliminating or suppressing Candidatus Liberibacter asiaticus indicated by both the lowest Las infection rate and titers of the treated scions and inoculated rootstock. The non-effective group, including 11 antibiotics alone with three controls, such as Amikacin, Cinoxacin, Gentamicin, Kasugamycin, Lincomycin, Neomycin, Polymixin B and Tobramycin, did not eliminate or suppress Las in the tested concentrations, resulting in plants with increased titers of Las. The other 12 antibiotics partly eliminated or suppressed Las in the treated and graft-inoculated plants. The effective and non-phytotoxic antibiotics could be potential candidates for control of citrus HLB, either for the rescue of infected citrus germplasm or for restricted field application.