A bioassay that yields quantifiable symptoms of cucurbit yellow vine disease caused by Serratia marcescens.

Cucurbit yellow vine disease (CYVD), which is caused by the gram-negative bacterium Serratia marcescens and transmitted by squash bugs (Anasa tristis DeGeer), is a devastating disease of cucurbit crops that is emerging rapidly in the eastern half of the U.S. The lack of a robust pathogenicity assay for CYVD in the laboratory has hampered functional tests using genomic sequences to investigate the biology of this phytopathogen. In this study we developed and validated a bioassay that yielded consistent and quantifiable CYVD symptoms on squash in the lab. We compared inoculation by wounding with a multipronged floral pin frog to inoculation by injection in which a needle was moved in and out of the stem multiple times in each of multiple piercings to mimic the feeding behavior of squash bugs. We found that inoculation by needle injection of ≥108 CFU/ml of S. marcescens into the stem of squash (Cucurbita pepo) plants at the cotyledon growth stage reproducibly induced CYVD symptoms, whereas injecting 106 or 107 CFU/ml did not. Additionally, we found that S. marcescens induced symptoms on all of the squash cultivars tested, and induced symptoms that have not been previously reported, including stem elongation and leaf cupping. In short, through our injection approach of mimicking the natural process of S. marcescens transmission by squash bug feeding, we obtained robust and quantifiable CYVD symptoms. This laboratory bioassay provides a crucial tool for investigating the biology and pathology of this emerging pathogen and for plant breeding screens aimed at combatting CYVD.

Optimizing 'red Fuji' apple quality: Auxin-mediated calcium distribution via fruit-stalk in bagging practices.

In apples, a bottleneck effect in calcium (Ca) transport within fruit stalk has been observed. To elucidate that how auxin affects Ca forms and distribution in the apple fruit stalk, we investigated the effects of different concentrations of auxin treatment (0, 10, 20, and 30 mg·L-1) on Ca content, forms, distribution, and fruit quality during later stages of fruit expansion. The results showed that auxin treatment led to a dramatic reduction in total Ca content in stalk, while an approximately 30 % increase in fruit. Furthermore, auxin treatment effectively enhanced the functionality of xylem vessels in vascular bundles of the stalk in bagged apples. Finally, TOPSIS method was used to assess fruit quality, with treatments ranked as follows: IAA20 > NAA20 > IAA30 > IAA10 > CK > NPA. The findings lay a foundation for further studies on the bottleneck in Ca transport within stalk, uneven distribution of Ca in fruit, and provide insights into Ca utilization efficiency in bagged apples.

Adaptation of feeding behaviors on two Brassica species by colonizing and noncolonizing Bemisia tabaci (Hemiptera: Aleyrodidae) NW whiteflies.

Bemisia tabaci New World (NW) (Gennadius) (Hemiptera: Aleyrodidae), a whitefly in the B. tabaci species complex, is polyphagous on many plant species. Yet, it has been displaced, albeit not entirely, by other whitefly species. Potential causes could include issues with adaptation, feeding, and the colonization of new-hosts; however, insights that would help clarify these possibilities are lacking. Here, we sought to address these gaps by performing electropenetrography (EPG) recordings of NW whiteflies, designated "Napus" and "Rapa," reared on 2 colony hosts, Brassica napus and B. rapa, respectively. Analysis of 17 probing and pathway (pw) phase-related EPG variables revealed that the whiteflies exhibited unique probing behaviors on their respective colony hosts, with some deterrence being encountered on B. rapa. Upon switching to B. rapa and B. napus, the probing patterns of Napus and Rapa whiteflies, respectively, adapted quickly to these new-hosts to resemble that of whiteflies feeding on their colony hosts. Results for 3 of the EPG variables suggested that B. rapa's deterrence against Napus whitefly was significant prior to the phloem phase. This also suggested that adaptation by Rapa whitefly improved its pw probing on B. rapa. Based on analysis of 24 phloem phase-related EPG variables, Napus and Rapa whiteflies performed equally well once they entered phloem phase and exhibited comparable phloem acceptability on both the colony- and new-hosts. These findings demonstrate that NW whiteflies reared on a colony host are highly adaptable to feeding on a new host despite encountering some deterrence during the nonphloem phases in B. rapa plant.

Niche construction and niche choice by aphids infesting wheat ears.

The niche of aphids is largely defined by their consumption of plant phloem sap and its composition, including nutrients and specialized metabolites. Niche construction is the change of the environment by organisms, which may influence the fitness of these organisms and their offspring. To better understand interactions between plants and aphids, it is necessary to investigate whether aphids modify the chemical composition of the phloem sap of their host plants and whether conspecifics are affected by previous infestation. In the current study, ears of wheat (Triticum aestivum) plants were infested with clonal lineages of the English grain aphid (Sitobion avenae) or were left uninfested. The metabolic composition of ear phloem sap exudates was analyzed through amino acid profiling and metabolic fingerprinting. Aphids of the clonal lineages were either put on previously aphid-infested or on uninfested ears and their colony sizes followed over time. Furthermore, it was investigated whether aphids choose one treatment group over another. Sitobion avenae infestation affected the relative concentrations of some metabolites in the phloem exudates of the ears. Compared to uninfested plants, the relative concentration of asparagine was higher after aphid infestation. Colonies grew significantly larger on previously aphid-infested ears, which the aphids also clearly chose in the choice experiment. The pronounced positive effect of previous infestation on aphid colonies indicates niche construction, while the choice of these constructed niches reveals niche choice by S. avenae on wheat. The interplay between these different niche realization processes highlights the complexity of interactions between aphids and their hosts.

Salt tolerance in wheat is associated with the maintenance of shoot biomass, stomatal conductance, and sucrose in the phloem.

Wheat (Triticum aestivum L.) is a mega-staple for millions of the world's populations and its yield potential is impacted by soil salinization. This study investigated genotypic variation in salt tolerance among six wheat genotypes, Gladius, Drysdale, GD0014, GD0120, GD0180, and GD0185. The study also characterized shoot traits, photosynthetic traits, leaf Na and K concentrations, and phloem sucrose. The plants were grown under controlled growth room conditions at 0 mM NaCl (Control) and 100 mM NaCl. The results showed that the salt tolerance index (STISFW, SFW: shoot fresh weight) varied from 0.52 for GD0120 to 0.69 for GD0180. Based on the STISFW, salt tolerance for the wheat genotypes was in the order, GD0180 > Gladius > GD0185 > Drysdale > GD0014 > GD0120. Projected shoot area (PSA) at all growth stages, 14, 20, 27, 34, and 40 DAS were strongly correlated with SFW at 45 DAS. Salt treatment significantly increased phloem sucrose level in the salt intolerant, Drysdale, while having no effect on this parameter in Gladius. Gladius showed greater maintenance of stomatal conductance than Drysdale. The relative ratio of K/Na between treatment and control was strongly correlated with the relative ratio of SFW (r = .85). The correlation between PSA at 14 DAS and SFW at 45 DAS and the correlation between the relative ratio of K/Na between treatment and control with STISFW identify these parameters to be potential traits for screening salt tolerance in wheat. Higher salt tolerance in Gladius would be associated with higher maintenance of stomatal conductance and enhanced phloem sucrose transport.

Identification and characterization of transition metal-binding proteins and metabolites in the phloem sap of Brassica napus.

Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography (SEC) coupled online to sector-field ICP-MS. Our data identified known TM binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10-15 kD containing Cu, Fe, S and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

OsHAK4 functions in retrieving sodium from the phloem at the reproductive stage of rice.

Soil salinity significantly limits rice productivity, but it is poorly understood how excess sodium (Na+) is delivered to the grains at the reproductive stage. Here, we functionally characterized OsHAK4, a member of the clade IV HAK/KUP/KT transporter subfamily in rice. OsHAK4 was localized to the plasma membrane and exhibited influx transport activity for Na+, but not for K+. Analysis of organ- and growth stage-dependent expression patterns showed that very low expression levels of OsHAK4 were detected at the vegetative growth stage, but its high expression in uppermost node I, peduncle, and rachis was found at the reproductive stage. Immunostaining indicated OsHAK4 localization in the phloem region of node I, peduncle, and rachis. Knockout of OsHAK4 did not affect the growth and Na+ accumulation at the vegetative stage. However, at the reproductive stage, the hak4 mutants accumulated higher Na+ in the peduncle, rachis, husk, and brown rice compared to the wild-type rice. Element imaging revealed higher Na+ accumulation at the phloem region of the peduncle in the mutants. These results indicate that OsHAK4 plays a crucial role in retrieving Na+ from the phloem in the upper nodes, peduncle, and rachis, thereby preventing Na+ distribution to the grains at the reproductive stage of rice.

Vascular tissue - boon or bane? How pathogens usurp long-distance transport in plants and the defence mechanisms deployed to counteract them.

Evolutionary emergence of specialised vascular tissues has enabled plants to coordinate their growth and adjust to unfavourable external conditions. Whilst holding a pivotal role in long-distance transport, both xylem and phloem can be encroached on by various biotic factors for systemic invasion and hijacking of nutrients. Therefore, a complete understanding of the strategies deployed by plants against such pathogens to restrict their entry and establishment within plant tissues, is of key importance for the future development of disease-tolerant crops. In this review, we aim to describe how microorganisms exploit the plant vascular system as a route for gaining access and control of different host tissues and metabolic pathways. Highlighting several biological examples, we detail the wide range of host responses triggered to prevent or hinder vascular colonisation and effectively minimise damage upon biotic invasions.

Adaptive responses to elevated CO2 in fruit species with different phloem loading mechanisms.

Introduction: It has been suggested that the mechanism of phloem loading, that is apoplastic or symplastic loading, may affect a plant's ability to adapt to elevated CO2 levels. Strawberry (Fragaria × ananassa) and tomato (Solanum lycopersicum) are two fruit crops that use different mechanisms to load sugars into the phloem - the former symplastically and the latter apoplastically - yet both species can increase their yields when grown in a CO2-enriched environment. In this study, we subjected strawberry and tomato plants to long-term CO2 enrichment to determine the morphological and physiological adaptations that enable them to increase their yields in response to higher CO2 levels. Methods: Transplanted tomato and strawberry plants were subjected to ambient (400 ppm) and elevated (800 ppm) CO2 for three months. We examined various parameters associated with growth, yield, photosynthesis, and carbon allocation by means of phenotyping, gas exchange analysis, and 13C labelling combined with isotope ratio mass spectrometry. Results: We found that CO2 enrichment promoted growth and reproductive development in both species, resulting in more flowers per plant (tomato and strawberry), larger crown (strawberry), and, eventually, higher yields. Gas exchange analysis and A/c i curves revealed that elevated CO2 increased carbon assimilation rate in strawberry, but not in tomato - the latter being limited by Rubisco's carboxylation efficiency. Finally, whereas both species prioritized fruit development over the development of other sink organs, they were both limited by carbon export at elevated CO2, since new photoassimilates were equally distributed to various sinks between CO2 treatments. Discussion: The findings suggest that both species will benefit from future increases in CO2 levels and support current glasshouse practices entailing CO2 enrichment. Those benefits probably stem from an enhanced performance of both species at early developmental stages, as differences in carbon assimilation rate (tomato) and carbon allocation between treatments at late developmental stages were absent. Moreover, crop adaptation to elevated CO2 seems to depend on the ability of each species to respond to elevated CO2, rather than on the phloem loading mechanism per se.

Visualizing and quantifying 33P uptake and translocation by maize plants grown in soil.

Phosphorus (P) availability severely limits plant growth due to its immobility and inaccessibility in soils. Yet, visualization and measurements of P uptake from different root types or regions in soil are methodologically challenging. Here, we explored the potential of phosphor imaging combined with local injection of radioactive 33P to quantitatively visualize P uptake and translocation along roots of maize grown in soils. Rhizoboxes (20 × 40 × 1 cm) were filled with sandy field soil or quartz sand, with one maize plant per box. Soil compartments were created using a gravel layer to restrict P transfer. After 2 weeks, a compartment with the tip region of a seminal root was labeled with a NaH2 33PO4 solution containing 12 MBq of 33P. Phosphor imaging captured root P distribution at 45 min, 90 min, 135 min, 180 min, and 24 h post-labeling. After harvest, 33P levels in roots and shoots were quantified. 33P uptake exhibited a 50% increase in quartz sand compared to sandy soil, likely attributed to higher P adsorption to the sandy soil matrix than to quartz sand. Notably, only 60% of the absorbed 33P was translocated to the shoot, with the remaining 40% directed to growing root tips of lateral or seminal roots. Phosphor imaging unveiled a continuous rise in 33P signal in the labeled seminal root from immediate post-labeling until 24 h after labeling. The highest 33P activities were concentrated just above the labeled compartment, diminishing in locations farther away. Emerging laterals from the labeled root served as strong sinks for 33P, while a portion was also transported to other seminal roots. Our study quantitatively visualized 33P uptake and translocation dynamics, facilitating future investigations into diverse root regions/types and varying plant growth conditions. This improves our understanding of the significance of different P sources for plant nutrition and potentially enhances models of plant P uptake.

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.

SYNAPTOTAGMIN 4 is expressed mainly in the phloem and participates in abiotic stress tolerance in Arabidopsis.

Plant synaptotagmins structurally resemble animal synaptotagmins and extended-synaptotagmins. Animal synaptotagmins are well-characterized calcium sensors in membrane trafficking, and extended-synaptotagmins mediate lipid transfer at the endoplasmic reticulum-plasma membrane contact sites. Here, we characterize SYNAPTOTAGMIN 4 (SYT4), which belongs to the six-member family in Arabidopsis. Fluorometric GUS assay showed that the SYT4 promoter was strongest in roots and the least active in rosettes and cauline leaves, which was confirmed by qPCR. In seedlings, promoter activity was influenced by several factors, such as plant growth regulators, mannitol, sucrose, polyethylene glycol and cold. GUS histochemistry revealed SYT4 promoter activity in the phloem of all organs and even almost exclusively in sieve element precursors and differentiating sieve elements. Accordingly, the SYT-GFP fusion protein also accumulated in these cells with maximal abundance in sieve element precursors. The protein formed a network in the cytoplasm, but during sieve tube differentiation, it deposited at the cell periphery and disappeared from mature tubes. Using photoconvertible fluorescence technology, we showed that a high abundance of SYT4 protein in meristematic protophloem cells was due to its extensive synthesis. SYT4 protein synthesis was interrupted in differentiating sieve elements, but protein degradation was also reduced. In addition to phloem, the fusion protein was detected in shoot and root stem cell niche as early as the late heart stage of the embryo. We isolated and molecularly and biologically characterized five syt4 T-DNA insertion alleles and subjected them to phenotype analysis. The allele with the C2B domain interrupted by an T-DNA insertion exhibits increased sensitivity to factors such as auxins, osmotics, salicylic acid, sodium chloride, and the absence of sucrose in the root growth test.

Harnessing Viral Promoters for Targeted RNAi: Engineering Phloem-Specific Resistance Against Tomato Yellow Leaf Curl Thailand Virus.

Phloem-specific promoter efficiently triggers graft-transmissible RNA interference (gtRNAi). We leveraged a phloem-specific promoter derived from the Rice tungro bacilliform virus, optimizing the RNAi mechanism's efficiency and specificity. Here, we detail the construction of phloem-specific promoter-based gtRNAi system and its application through grafting experiments, demonstrating its effectiveness in inducing tomato yellow leaf curl Thailand virus (TYLCHTV) resistance in non-transgenic scions. This strategy presents a practical application for protecting crops against viruses without genetically modifying the entire plant.

Comparative Anatomical Analysis of Bark Structure in 10 Quercus Species.

Detailed anatomical features of bark are used and interpreted in plant taxonomy, phylogenetics, and other areas of plant science. However, the delicate nature of bark cells, combined with the difficulty of obtaining high-quality sections and reliable data, limits the potential for utilizing and processing bark. In this study, the anatomical structure of the bark of 10 Quercus species growing in Yunnan Province, China, was characterized in detail. The results indicate that the anatomical features of the barks of 10 Quercus spp. show a certain degree of consistency. Specifically, sieve tubes are distributed in solitary elements or in small groups, mostly as compound sieve plates containing 2-8 sieve areas, suggesting that Quercus spp. may occupy a conservative evolutionary position. Additionally, for the first time, this study reports the presence of simple sieve plates in the sieve tube elements of Quercus phloem. Each sieve tube element has a companion cell on one side. The companion cell strands contain 2-7 cells. Axial parenchyma is diffuse, with parenchyma strands typically consisting of 4-7 cells; druses are present within chambered crystalliferous cells. Phloem rays are of two distinct sizes and often exhibit dilatation and sclerification, and the ray composition consists of procumbent cells. Sclerenchyma is composed of fibers and sclereids, both of which contain prismatic crystals. Most of the fibers are gelatinous fibers, which are distributed in discontinuous tangential bands of about five cells in width. Sclereids appear in clusters. The presence of sclerenchyma provides mechanical support to the bark, reducing the collapse of the phloem. Periderm usually consists of around 10-30 layers of phellem, and Quercus acutissima and Q. variabilis can reach dozens or hundreds layers. The phelloderm typically consists of from two to five layers, with Q. variabilis having up to ten or more layers. The filling tissue of lenticels in all Quercus species is nonstratified (homogeneous) and largely nonsuberized. Overall, this study enriches our comprehension of Quercus bark anatomy, elucidating evolutionary patterns, functional adaptations, and ecological ramifications within this significant botanical genus.

Estimation of phloem conductance at tree level in young, middle-aged and old-aged Scots pine trees growing in different climatic conditions in boreal forests.

In forests, a significant proportion of the carbon fixed by trees during photosynthesis is transported belowground along the conducting phloem, so variations in phloem anatomy can lead to variations in transport capacity. Phloem conductance at tree level (Ktree) is also affected by tree height. Both the phloem anatomy and the tree size change during ontogeny, and also differ under different environmental conditions. The goal of our work was to identify the main drivers of variation in Ktree in Scots pine trees growing in natural boreal forests. We conducted a phloem anatomical study and calculated Ktree in trees of three age groups growing in different climatic conditions along a latitudinal gradient from south to north. We found that Ktree was maintained at the same level in actively growing pine trees (25-80-years-old) but increased in old-aged trees (180-190-years-old), possibly reflecting the shift in source-sink relationships of aboveground and belowground parts of trees. Trees of the same age group growing in different climatic conditions demonstrated similar values of Ktree due to coordinated changes in the phloem anatomy and the tree height. In general, the negative influence of tree height on Ktree is offset by the positive influence of phloem width (or trunk diameter) and sieve cell diameter. The exception was young trees growing in the transition zone of the northern taiga subzone to the tundra, where Ktree was the highest in its age group and even exceeded Ktree of middle-aged trees.

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.

Novel program for automatic calculation of EPG variables.

The electrical penetration graph (EPG) technique is the most powerful tool for studying the feeding behavior of pierce-sucking insects. However, calculating EPG variables is often very time-consuming, and consequently, several software programs have been developed for the automatic calculation of EPG variables. Here we present a new user-friendly Excel Workbook that uses a standardized list of EPG variables and follows expert guidelines for calculating them. The program developed in Visual Basic for Applications (VBA) is a step up from the existing software and allows easy data analysis and interpretation. It also includes a novel option for dealing with the common problem of "truncated"-waveforms artificially terminated by the end of recording. The only requirement to run the program is Microsoft Excel software running under a PC environment. The Workbook was validated by calculating variables from EPG recordings of aphids and psyllids and the results obtained were compared with those of existing software such as the Sarria Workbook. Our EPG Workbook provides researchers with a reliable and standardized tool for the automatic calculation of up to 127 EPG variables from phloem-sap-sucking insects.

Potato spindle tuber viroid (PSTVd) loop 27 mutants promote cell-to-cell movement and phloem unloading of the wild type: Insights into RNA-based viroid interactions.

Variations in infection progression with concurrent or prior infections by different viruses, viroids, or their strains are evident, but detailed investigations into viroid variant interactions are lacking. We studied potato spindle tuber viroid intermediate strain (PSTVd-I) to explore variant interactions. Two mutants, U177A/A182U (AU, replication- and trafficking-competent) and U178G/U179G (GG, replication-competent but trafficking-defective) on loop 27 increased cell-to-cell movement of wild-type (WT) PSTVd without affecting replication. In mixed infection assays, both mutants accelerated WT phloem unloading, while only AU promoted it in separate leaf assays, suggesting that enhancement of WT infection is not due to systemic signals. The mutants likely enhance WT infection due to their loop-specific functions, as evidenced by the lack of impact on WT infection seen with the distantly located G347U (UU) mutant. This study provides the first comprehensive analysis of viroid variant interactions, highlighting the prolonged phloem unloading process as a significant barrier to systemic spread.

Vascular cambium stem cells: past, present and future.

Secondary xylem and phloem originate from a lateral meristem called the vascular cambium that consists of one to several layers of meristematic cells. Recent lineage tracing studies have shown that only one of the cambial cells in each radial cell file functions as the stem cell, capable of producing both secondary xylem and phloem. Here, we first review how phytohormones and signalling peptides regulate vascular cambium formation and activity. We then propose how the stem cell concept, familiar from apical meristems, could be applied to cambium studies. Finally, we discuss how this concept could set the basis for future research.