Dynamics of 'Candidatus Liberibacter asiaticus' Growth, Concentrations of Reactive Oxygen Species, and Ion Leakage in Huanglongbing-Positive Sweet Orange.

Citrus Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

Challenging battles of plants with phloem-feeding insects and prokaryotic pathogens.

For the past 4 decades, intensive molecular studies of mostly leaf mesophyll cell-infecting pathogens and chewing insects have led to compelling models of plant-pathogen and plant-insect interactions. Yet, some of the most devastating pathogens and insect pests live in or feed on the phloem, a systemic tissue belonging to the plant vascular system. Phloem tissues are difficult to study, and phloem-inhabiting pathogens are often impossible to culture, thus limiting our understanding of phloem-insect/pathogen interactions at a molecular level. In this Perspective, we highlight recent literature that reports significant advances in the understanding of phloem interactions with insects and prokaryotic pathogens and attempt to identify critical questions that need attention for future research. It is clear that study of phloem-insect/pathogen interactions represents an exciting frontier of plant science, and influx of new scientific expertise and funding is crucial to achieve faster progress in this important area of research that is integral to global food security.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Zinkicide Is a ZnO-Based Nanoformulation with Bactericidal Activity against Liberibacter crescens in Batch Cultures and in Microfluidic Chambers Simulating Plant Vascular Systems.

Phloem-limited bacterial "Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (ZnO)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens, whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by "Candidatus Liberibacter" species and other plant vascular pathogens.IMPORTANCE "Candidatus Liberibacter" species are associated with incurable plant diseases that have caused billions of dollars of losses for United States and world agriculture. Chemical control of these pathogens is complicated, because their life cycle combines intracellular vascular stages in plant hosts with transmission by highly mobile insect vectors. To date, "Candidatus Liberibacter" species are mostly unculturable, except for Liberibacter crescens, a member of the genus that has been used as a model for in vitro assays. Here, we evaluated the potential of Zinkicide (ZnK) as an antimicrobial against "Candidatus Liberibacter" species in batch cultures and under flow conditions, using L. crescens as a biological model. ZnK displayed bactericidal activity against L. crescens in batch cultures and showed increased mobility and bactericidal activity in microfluidic devices resembling "Candidatus Liberibacter" species natural habitats. ZnK performance observed here against L. crescens makes this compound a promising candidate to control plant diseases caused by vascular pathogens.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface.

BACKGROUND: Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus. L. asiaticus, L. americanus, L. africanus cause citrus greening disease, while Ca. L. solanacearum causes potato Zebra chip disease. None of the pathogens grows in axenic culture. L. crescens grows in three media: a BM-7, a serum-free Hi® Grace's Insect Medium (Hi-GI), and a chemically-defined medium called M15. To date, no optimal growth parameters of the model species L. crescens have been reported. Studying the main growth parameters of L. crescens in axenic culture will give us insights into the lifestyle of the Ca. Liberibacter pathogens. RESULTS: The evaluation of the growth parameters-pH, aeration, temperature, and buffering capacity-reflects the optimal living conditions of L. crescens. These variables revealed that L. crescens is an aerobic, neutrophilic bacterium, that grows optimally in broth in a pH range of 5.8 to 6.8, in a fully oxygenated environment (250 rpm), at 28 °C, and with monosodium phosphate (10 mM or 11.69 mM) as the preferred buffer for growth. The increase of pH in the external media likely results from the deamination activity within the cell, with the concomitant over-production of ammonium in the external medium. CONCLUSION: L. crescens and the Ca. Liberibacter pathogens are metabolically similar and grow in similar environments-the phloem and the gut of their insect vectors. The evaluation of the growth parameters of L. crescens reveals the lifestyle of Liberibacter, elucidating ammonium and phosphate as essential molecules for colonization within the hosts. Ammonium is the main driver of pH modulation by active deamination of amino acids in the L. crescens amino acid rich media. In plants, excess ammonium induces ionic imbalances, oxidative stress, and pH disturbances across cell membranes, causing stunted root and shoot growth and chlorosis-the common symptoms of HLB-disease. Phosphate, which is also present in Ca. L. asiaticus hosts, is the preferred buffer for the growth of L. crescens. The interplay between ammonium, sucrose, potassium (K+), phosphate, nitrate (NO3-), light and other photosynthates might lead to develop better strategies for disease management.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Plant growth-promoting bacteria Kosakonia radicincitans mediate anti-herbivore defense in Arabidopsis thaliana.

MAIN CONCLUSION: This study demonstrates that the application of the PGPB strain, Kosakonia radicincitans enhances a plant's resistance against phloem-feeding and chewing insects in Arabidopsis thaliana. The plant growth-promoting bacterial strain K. radicincitans DSM 16656 applied to A. thaliana reduced the number of phloem-feeding insects of both the specialist Brevicoryne brassicae and the generalist Myzus persicae. While weight gain of the generalist chewing insect Spodoptera exigua was reduced by 30% on A. thaliana plants treated with K. radicincitans, growth of the specialist caterpillar Pieris brassicae was not affected when compared with caterpillars from control plants. Since generalist and specialist chewing insects responded differentially to PGPB application, the implication of signaling pathways in PGPB mediated changes in plant defense was studied using two signaling pathway mutants impaired in their salicylic acid (npr1-1 mutant) or jasmonic acid (coi1-1 mutant) pathway. We found that the jasmonic acid pathway is relevant for upregulation of aliphatic glucosinolates and suppression of the chewing generalist S. exigua larval growth. Chewing from generalist P. brassicae increased glucosinolate content in A. thaliana leaves mediated via both signaling pathways. However, only in the npr1-1 mutant, which contains the highest aliphatic glucosinolate content, the P. brassicae induced further enrichment of glucosinolates, resulting in a reduction of larval growth. Effects of K. radicincitans on plant resistance could not be explained by changes in glucosinolate contents or composition. Our results demonstrate the distinct role played by K. radicincitans in suppressing insect performance in A. thaliana.

Transgenic citrus expressing synthesized cecropin 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. 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.

Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

Combined microscopy and molecular analyses show phloem occlusions and cell wall modifications in tomato leaves in response to 'Candidatus Phytoplasma solani'.

Callose deposition, phloem-protein conformational changes and cell wall thickening are calcium-mediated occlusions occurring in the plant sieve elements in response to different biotic and abiotic stresses. However, the significance of these structures in plant-phytoplasma interactions requires in-depth investigations. We adopted a novel integrated approach, based on the combined use of microscopic and molecular analyses, to investigate the structural modifications induced in tomato leaf tissues in presence of phytoplasmas, focusing on vascular bundles and on the occlusion structures. Phloem hyperplasia and string-like arrangement of xylem vessels were found in infected vascular tissue. The diverse occlusion structures were differentially modulated in the phloem in response to phytoplasma infection. Callose amount was higher in midribs from infected plants than in healthy ones. Callose was observed at sieve plates but not at pore-plasmodesma units. A putative callose synthase gene encoding a protein with high similarity to Arabidopsis CalS7, responsible for callose deposition at sieve plates, was upregulated in symptomatic leaves, indicating a modulation in the response to stolbur infection. P-proteins showed configuration changes in infected sieve elements, exhibiting condensation of the filaments. The transcripts for a putative P-protein 2 and a sieve element occlusion-related protein were localized in the phloem but only the first one was modulated in the infected tissues.

Synthesis and bioactivities of amino acid ester conjugates of phenazine-1-carboxylic acid.

Phenazine-1-carboxylic acid (PCA) is a natural product that has been characterized by special chemical structures, interesting bioactivities and has been registered for fungicide against rice sheath blight in China. Phloem mobility is of great significance to long-distance transport of systemic pesticides in plants. In order to improve the phloem mobility and bioactivities of PCA, seventeen PCA-amino acid ester conjugates were designed and synthesized by conjugating PCA with different amino-acid esters. The conjugates were evaluated for their fungicidal activities against Rhizoctonia solani Kuhn and their phloem mobility was determined by HPLC. Results showed that conjugates a, b, c, d, e, l, m and p (EC50 values between 5.35 and 18.85µg/mL) were more active than PCA (25.66µg/mL). In particular, conjugates l and m exhibited the best fungicidal activities against Rhizoctonia solani Kuhn (EC50 values of them were 6.47µg/mL and 5.35µg/mL respectively). All these conjugates didn't have phloem mobility.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale.

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10% in the control to 30-50% in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.

Resistance to Cucurbit aphid-borne yellows virus in Melon Accession TGR-1551.

The genetic control of resistance to Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) in the TGR-1551 melon accession was studied through agroinoculation of a genetic family obtained from the cross between this accession and the susceptible Spanish cultivar 'Bola de Oro'. Segregation analyses were consistent with the hypothesis that one dominant gene and at least two more modifier genes confer resistance; one of these additional genes is likely present in the susceptible parent 'Bola de Oro'. Local and systemic accumulation of the virus was analyzed in a time course experiment, showing that TGR-1551 resistance was expressed systemically as a significant reduction of virus accumulation compared with susceptible controls, but not locally in agroinoculated cotyledons. In aphid transmission experiments, CABYV inoculation by aphids was significantly reduced in TGR-1551 plants, although the virus was acquired at a similar rate from TGR-1551 as from susceptible plants. Results of feeding behavior studies using the DC electrical penetration graph technique suggested that viruliferous aphids can salivate and feed from the phloem of TGR-1551 plants and that the observed reduction in virus transmission efficiency is not related to reduced salivation by Aphis gossypii in phloem sieve elements. Since the virus is able to accumulate to normal levels in agroinoculated tissues, our results suggest that resistance of TGR-1551 plants to CABYV is related to impairment of virus movement or translocation after it reaches the phloem sieve elements.

Genetic Diversity of the Indian Populations of 'Candidatus Liberibacter asiaticus' Based on the Tandem Repeat Variability in a Genomic Locus.

Citrus huanglongbing (HLB, citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, noncultured, gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being 'Candidatus Liberibacter asiaticus'. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen 'Ca. L. asiaticus' populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian 'Ca. L. asiaticus' based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat number (TRN); Class I (TRN≤5), Class II (TRN>5≤10), Class III (TRN>10≤15), and Class IV (TRN>15). The study revealed that the Indian population of 'Ca. L. asiaticus' is more diverse than reported for Florida and Guangdong populations, which showed less diversity. While Florida and Guangdong populations were dominated by a TRN5 and TRN7 genotype, respectively, the Indian 'Ca. L. asiaticus' populations with TRN copy numbers 9, 10, 11, 12, and 13 were widely distributed throughout the country. Additionally, TRN2 and TRN17 genotypes were also observed among the Indian 'Ca. L. asiaticus' populations. The predominant 'Ca. L. asiaticus' genotypes from the northeastern region of India were TRN6 and TRN7 (53.12%) and surprisingly similar to neighboring South China populations. Preliminary results showed absence of preference of citrus cultivars to any specific 'Ca. L. asiaticus' genotype.

The soybean mycorrhiza-inducible phosphate transporter gene, GmPT7, also shows localized expression at the tips of vein endings of senescent leaves.

GmPT7 was originally identified as an arbuscular mycorrhiza-inducible gene of soybean that encodes a member of subfamily I in the PHOSPHATE TRANSPORTER 1 family. In the present study, we established conditions under which a number of dwarf soybean plants complete their life cycles in a growth chamber. Using this system, we grew transgenic soybean with a GmPT7 promoter-ß-glucuronidase fusion gene and evaluated GmPT7 expression in detail. GmPT7 was highly expressed in mature, but not in collapsed, arbuscule-containing cortical cells, suggesting its importance in the absorption of fungus-derived phosphate and/or arbuscule development. GmPT7 was also expressed in the columella cells of root caps and in the lateral root primordia of non-mycorrhizal roots. The expression of GmPT7 occurred only in the late stage of phosphorus translocation from leaves to seeds, after water evaporation from the leaves ceased, and later than the expression of GmUPS1-2, GmNRT1.7a and GmNRT1.7b, which are possibly involved in nitrogen export. GmPT7 expression was localized in a pair of tracheid elements at the tips of vein endings of senescent leaves. Transmission electron microscopy revealed that the tip tracheid elements in yellow leaves were still viable and had intact plasma membranes. Thus, we think that GmPT7 on the plasma membranes transports phosphate from the apoplast into the tip elements. GmPT7 knockdown resulted in no significant effects, the function of GmPT7 remaining to be clarified. We propose a working model in which phosphate incorporated in vein endings moves to seeds via xylem to phloem transfer.

Metabolomic analysis reveals the potential metabolites and pathogenesis involved in mulberry yellow dwarf disease.

To analyse the molecular mechanisms of phytoplasma pathogenicity, the comprehensive metabolomic changes of mulberry leaf and phloem sap in response to phytoplasma infection were examined using gas chromatography-mass spectrometry. The metabolic profiles obtained revealed that the metabolite compositions of leaf and phloem sap were different, and phytoplasma infection has a greater impact on the metabolome of phloem sap than of leaf. Phytoplasma infection brought about the content changes in various metabolites, such as carbohydrates, amino acids, organic acids, etc. Meanwhile, the results of biochemical analysis showed that the degradation of starch was repressed, and the starch content was increased in the infected leaves. In addition, we found that phytoplasma infection changed the levels of abscisic acid and cytokinin and break phytohormone balance. Interestingly, our data showed that the contents of H2O2 and superoxide were increased in the infected leaves, but not in the phloem saps. Based on the results, the expression levels of the genes involved in the metabolism of some changed metabolites were examined, and the potential molecular mechanisms of these changes were discussed. It can be concluded that both the leaf and phloem saps have a complicated metabolic response to phytoplasma infection, but their response mechanisms were different.

Spatial imaging of Zn and other elements in Huanglongbing-affected grapefruit by synchrotron-based micro X-ray fluorescence investigation.

Huanglongbing (HLB) is a highly destructive, fast-spreading disease of citrus, causing substantial economic losses to the citrus industry worldwide. Nutrient levels and their cellular distribution patterns in stems and leaves of grapefruit were analysed after graft-inoculation with lemon scions containing 'Candidatus Liberibacter asiaticus' (Las), the heat-tolerant Asian type of the HLB bacterium. After 12 months, affected plants showed typical HLB symptoms and significantly reduced Zn concentrations in leaves. Micro-XRF imaging of Zn and other nutrients showed that preferential localization of Zn to phloem tissues was observed in the stems and leaves collected from healthy grapefruit plants, but was absent from HLB-affected samples. Quantitative analysis by using standard references revealed that Zn concentration in the phloem of veins in healthy leaves was more than 10 times higher than that in HLB-affected leaves. No significant variation was observed in the distribution patterns of other elements such as Ca in stems and leaves of grapefruit plants with or without graft-inoculation of infected lemon scions. These results suggest that reduced phloem transport of Zn is an important factor contributing to HLB-induced Zn deficiency in grapefruit. Our report provides the first in situ, cellular level visualization of elemental variations within the tissues of HLB-affected citrus.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter.

The Gram-stain-negative, rod-shaped bacterial isolate BT-1(T) is the closest relative to the genus 'Candidatus Liberibacter' cultured to date. BT-1(T) was recovered from the phloem sap of a defoliating mountain papaya in Puerto Rico. The BT-1(T) 16S rRNA gene sequence showed that strain BT-1(T) is most closely related to members of the genus 'Ca. Liberibacter' sharing 94.7% 16S rRNA gene sequence similarity with 'Ca. Liberibacter americanus' and 'Ca. Liberibacter asiaticus'. Additionally, average nucleotide identity, 16S rRNA gene sequences and conserved protein sequences supported inclusion of the previously described species of the genus 'Ca. Liberibacter' in a genus with BT-1(T). The prominent fatty acids of isolate BT-1(T) were C18 : 1ω7c (77.2%), C16 : 0 OH (4.8%), C18 : 0 (4.4%) and C16 : 0 (3.5%). Both physiological and genomic characteristics support the creation of the genus Liberibacter, as well as the novel species Liberibacter crescens gen. nov., sp. nov. with type strain BT-1(T) ( = ATCC BAA-2481(T) = DSM 26877(T)).