Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Geographical variations, prevalence, and molecular dynamics of fastidious phloem-limited pathogens infecting sugar beet across Central Europe.

In Europe, two fastidious phloem-limited pathogens, 'Candidatus Phytoplasma solani' (16SrXII-A) and 'Candidatus Arsenophonus phytopathogenicus', are associated with rubbery taproot disease (RTD) and syndrome basses richesses (SBR) of sugar beet, respectively. Both diseases can significantly reduce yield, especially when accompanied by root rot fungi. This study investigates the presence, geographic distribution and genetic traits of fastidious pathogens and the accompanying fungus, Macrophomina phaseolina, found on sugar beet across four geographically separated plains spanning seven countries in Central Europe. The survey revealed variable incidences of symptoms linked to these fastidious pathogens in the Pannonian and Wallachian Plains, sporadic occurrence in the North European Plain, and no symptomatic sugar beet in the Bohemian Plain. Molecular analyses unveiled the occurrence of both 'Ca. P. solani' and 'Ca. A. phytopathogenicus' throughout Central Europe, with a predominance of the phytoplasma. These fastidious pathogens were detected in all six countries surveyed within the Pannonian and Wallachian Plains, with only a limited presence of various phytoplasmas was found in the North European Plain, while no fastidious pathogens were detected in Bohemia, aligning with observed symptoms. While 16S rDNA sequences of 'Ca. P. solani' remained highly conserved, multi-locus characterization of two more variable loci (tuf and stamp) unveiled distinct variability patterns across the plains. Notably, the surprising lack of variability of tuf and stamp loci within Central Europe, particularly the Pannonian Plain, contrasted their high variability in Eastern and Western Europe, corresponding to epidemic and sporadic occurrence, respectively. The current study provides valuable insights into the genetic dynamics of 'Ca. P. solani' in Central Europe, and novel findings of the presence of 'Ca. A. phytopathogenicus' in five countries (Slovakia, Czech Republic, Austria, Serbia, and Romania) and M. phaseolina in sugar beet in Slovakia. These findings emphasize the need for further investigation of vector-pathogen(s)-plant host interactions and ecological drivers of disease outbreaks.

Distinct metabolites affect the phloem fungal communities in ash trees (Fraxinus spp.) native and nonnative to the highly invasive emerald ash borer (AGRILUS PLANIPENNIS).

Emerald ash borer (EAB, Agrilus planipennis) is an invasive killer of ash trees (Fraxinus spp.) in North America and Europe. Ash species co-evolved with EAB in their native range in Asia are mostly resistant, although the precise mechanism(s) remain unclear. Very little is also known about EAB or ash tree microbiomes. We performed the first joint comparison of phloem mycobiome and metabolites between a native and a nonnative ash species, infested and uninfested with EAB, in conjunction with investigation of larval mycobiome. Phloem mycobiome communities differed between the tree species, but both were unaffected by EAB infestation. Several indicator taxa in the larval gut shared a similarly high relative abundance only with the native host trees. Widely targeted metabolomics revealed 24 distinct metabolites in native trees and 53 metabolites in nonnative trees, respectively, that differed in relative content between infested and uninfested trees only in one species. Interestingly, four metabolites shared a strong relationship with the phloem mycobiomes, majority of which affected only the native trees. Collectively, our results demonstrate a complex interplay between host tree chemistry and mycobiome, and suggest the shared relationships between the mycobiomes of the native host tree and EAB may reflect their shared co-evolution.

A Transmission Assay of 'Candidatus Liberibacter asiaticus' Using Citrus Phloem Sap and Topical Feeding to Its Insect Vector, Diaphorina citri.

'Candidatus Liberibacter asiaticus', the putative causal agent of citrus greening disease, is transmitted by the Asian citrus psyllid, Diaphorina citri, in a propagative, circulative, and persistent manner. Unfortunately, 'Ca. L. asiaticus' is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (wt/vol) sucrose and 0.1% ascorbic acid (wt/vol) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (10 nymphs per seedling) for an inoculation period of 2 weeks. A transmission rate of up to 80% was recorded at 6 months postinoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of 'Ca. L. asiaticus' becomes available.

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.

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.

Distinct microbial communities among different tissues of citrus tree Citrus reticulata cv. Chachiensis.

Plant microbiota colonize all organs of a plant and play crucial roles including supplying nutrients to plants, stimulating seed germination, promoting plant growth, and defending plants against biotic and abiotic stress. Because of the economic importance, interactions between citrus and microbes have been studied relatively extensively, especially citrus-pathogen interactions. However, the spatial distribution of microbial taxa in citrus trees remains under-studied. In this study, Citrus reticulata cv. Chachiensis was examined for the spatial distribution of microbes by sequencing 16S rRNA genes. More than 2.5 million sequences were obtained from 60 samples collected from soil, roots, leaves, and phloem. The dominant microbial phyla from all samples were Proteobacteria, Actinobacteria and Acidobacteria. The composition and structure of microbial communities in different samples were analyzed by PCoA, CAP, Anosim and MRPP methods. Variation in microbial species between samples were analyzed and the indicator microbes of each sample group were identified. Our results suggested that the microbial communities from different tissues varied significantly and the microenvironments of tree tissues could affect the composition of its microbial community.

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.

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.

Fourier-transform infrared (FT-IR) spectroscopy analysis discriminates asymptomatic and symptomatic Norway spruce trees.

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 - 912 cm-1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 - 994 cm-1 and 1104 - 994 cm-1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

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.

Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.

Carbon starvation is the current leading hypothesis of plant mortality mechanisms under drought stress; recently, it is also used to explain tree die-off in plant diseases. However, the molecular biology of the carbon starvation pathway is unclear. Here, using a punch inoculation system, we conducted transcriptome and physiological assays to investigate pathogen response in poplar stems at the early stages of Botryosphaeria and Valsa canker diseases. Transcriptome assays showed that the majority of differentially expressed genes (DEGs) in stem phloem and xylem, such as genes involved in carbon metabolism and transportation, aquaporin genes (in xylem) and genes related to the biosynthesis of secondary metabolites and the phenylpropanoid pathway (related to lignin synthesis), were downregulated at 7 days after inoculation (DAI). Results also showed that the expression of the majority of disease-resistance genes upregulated in poplar stems, which may be connected with the downregulation expression of the majority of WRKY family genes. Physiological assays showed that transpiration rate decreased but WUE (water use efficiency) increased the 3 and 7 DAI, while the net photosynthetic rate decreased at 11 DAI in Botryosphaeria infected poplars (ANOVA, P < 0.05). The NSC (non-structural carbohydrates) content assays showed that the soluble sugar content of stem phloem samples increased at 3, 7, and 11 DAI that might due to the impede of pathogen infection. However, soluble sugar content of stem xylem and root samples decreased at 11 DAI; in contrast, the starch content unchanged. Therefore, results revealed a chronological order of carbon related molecular and physiological performance: declination of genes involved in carbon and starch metabolism first (at least at 7 DAI), declination of assimilation and carbon reserve (at 11 DAI) second. Results implied a potential mechanism that affects the host carbon reserve, by directly inhibiting the expression of genes involved in carbon metabolism and transport.

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.

Phytoplasmas: An Introduction.

Phytoplasmas are among the most recently discovered plant pathogens. They are wall-less prokaryotes restricted to phloem tissue, associated with diseases affecting several hundred plant species. The impact of phytoplasma diseases on agriculture is impressive and, at the present day, no effective curative strategy has been developed. The availability of rapid and sensitive techniques for phytoplasma detection as well as the possibility to study their relationship with the host plants is a prerequisite for the management of phytoplasma-associated diseases.

Comparison of Different Procedures for DNA Extraction for Routine Diagnosis of Phytoplasmas.

This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.

Real-Time PCR Protocol for Phytoplasma Detection and Quantification.

Phytoplasmas are mollicutes restricted to plant phloem tissue and are normally present at very low concentrations. Real-time polymerase chain reaction (qPCR) offers several advantages over conventional PCR. It is a fast, sensitive, and reliable detection technique amenable to high throughput. Two fluorescent chemistries are available, intercalating dyes or hybridization probes. Intercalating dyes are relatively less expensive than TaqMan® hybridization probes but the latter chemistry is the most commonly used for phytoplasma detection. qPCR may be designed for universal detection of phytoplasma, group or subgroup specific detection, or for simultaneous detection of up to three or four phytoplasmas (multiplexing). qPCR may be used for relative or absolute quantification in host plants and in insect vectors. Therefore, qPCR plays an important role in phytoplasma detection as well as in host-pathogen interaction and in epidemiological studies. This chapter outlines the protocols followed in qPCR assay for phytoplasma detection and quantification, focusing mainly on the use of TaqMan® probes.

Sieve Elements: The Favourite Habitat of Phytoplasmas.

The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the translocation of photoassimilates and systemic signals and for survival of the enucleate sieve elements. Of paramount importance is the Ca2+-dependent gating of plasmodesmata by callose and proteins. Hence, some of the complex, regulatory mechanisms to maintain Ca2+ homoeostasis in sieve elements are presented. Finally, the peculiarities of the chemical and physical sieve-element environment offered to phytoplasmas are discussed.

Laser Microdissection of Phytoplasma-Infected Grapevine Leaf Phloem Tissue for Gene Expression Study.

Phytoplasmas have been found confined mainly in leaf phloem sieve elements. In spite of this, few researches have been focused on the infected phloem tissue, whereas the plant response at the infection site could be quite different compared to distal parts and almost completely masked when whole organs are considered. Herein, we provide a protocol for the isolation of leaf phloem from paraffin-embedded samples by Laser Microdissection, followed by RNA purification and RNA amplification to generate cDNA libraries. Our protocol, which has been set up for phytoplasma-infected field-grown grapevine and successfully used for gene expression profiling, can be modified according to different plant species.

DAPI and Confocal Laser-Scanning Microscopy for In Vivo Imaging of Phytoplasmas.

As phytoplasmas are located inside the phloem tissue, always surrounded by numerous layers of other cells, they can result difficult candidates for microscopical investigations. Moreover, the necessity to kill the plant tissues for microscopy observations causes instantaneous and irreversible modifications in the sieve elements, leading to misleading information and erroneous interpretations. Phytoplasmas were here investigated in intact Vicia faba host plants using DAPI as fluorescent probe and confocal laser scanning microscopy. The described nondestructive technique may be applied for the imaging of phytoplasmas and of different pathogen-related responses in planta.

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.