Three-dimensional imaging reveals that positions of cyst nematode feeding sites relative to xylem vessels differ between susceptible and resistant wheat.

KEY MESSAGE: Resistance conferred by the Cre8 locus of wheat prevents cereal cyst nematode feeding sites from reaching and invading root metaxylem vessels. Cyst nematodes develop syncytial feeding sites within plant roots. The success of these sites is affected by host plant resistance. In wheat (Triticum aestivum L.), 'Cre' loci affect resistance against the cereal cyst nematode (CCN) Heterodera avenae. To investigate how one of these loci (Cre8, on chromosome 6B) confers resistance, CCN-infected root tissue from susceptible (-Cre8) and resistant (+Cre8) wheat plants was examined using confocal microscopy and laser ablation tomography. Confocal analysis of transverse sections showed that feeding sites in the roots of -Cre8 plants were always adjacent to metaxylem vessels, contained many intricate 'web-like' cell walls, and sometimes 'invaded' metaxylem vessels. In contrast, feeding sites in the roots of +Cre8 plants were usually not directly adjacent to metaxylem vessels, had few inner cell walls and did not 'invade' metaxylem vessels. Models based on data from laser ablation tomography confirmed these observations. Confocal analysis of longitudinal sections revealed that CCN-induced xylem modification that had previously been reported for susceptible (-Cre8) wheat plants is less extreme in resistant (+Cre8) plants. Application of a lignin-specific stain revealed that secondary thickening around xylem vessels in CCN-infected roots was greater in +Cre8 plants than in -Cre8 plants. Collectively, these results indicate that Cre8 resistance in wheat acts by preventing cyst nematode feeding sites from reaching and invading root metaxylem vessels.

EPG combined with micro-CT and video recording reveals new insights on the feeding behavior of Philaenus spumarius.

The meadow spittlebug Philaenus spumarius plays a key role in the transmission of the bacterium Xylella fastidiosa to olive in Apulia (South Italy). Currently, available data on P. spumarius feeding behavior is limited, and a real-time observation of the different steps involved in stylet insertion, exploratory probes, and ingestion, has never been carried out. Therefore, we performed an EPG-assisted characterization of P. spumarius female feeding behavior on olive, in order to detect and analyze the main EPG waveforms describing their amplitude, frequency, voltage level, and electrical origin of the traces during stylet penetration in plant tissues. Thereafter, each of the main waveforms was correlated with specific biological activities, through video recording and analysis of excretion by adults and excretion/secretion by nymphs. Furthermore, the specific stylet tips position within the plant tissues during each of the waveforms observed was assessed by microcomputer tomography (micro-CT). Additional EPG-recordings were carried out with males of P. spumarius on olive, in order to assess possible sex-related differences. P. spumarius feeding behavior can be described by five main distinct waveforms: C (pathway), Xc (xylem contact/pre-ingestion), Xi (xylem sap ingestion), R (resting), N (interruption within xylem phase). Compared to males, females require shorter time to begin the first probe, and their Xi phase is significantly longer. Furthermore, considering the single waveform events, males on olive exhibit longer np and R compared to females.

Pine wilt disease causes cavitation around the resin canals and irrecoverable xylem conduit dysfunction.

Physiological mechanisms of irreversible hydraulic dysfunction in seedlings infected with pine wilt disease (PWD) are still unclear. We employed cryo-scanning electron microscopy (cryo-SEM) to investigate the temporal and spatial changes in water distribution within the xylem of the main stem of 2-year-old Japanese black pine seedlings infested by pine wood nematodes (PWNs). Our experiment was specifically designed to compare the water relations among seedlings subjected to the following water treatment and PWN combinations: (i) well-watered versus prolonged drought (no PWNs); and (ii) well-watered with PWNs versus water-stressed with PWNs (four treatments in total). Cryo-SEM imaging observations chronicled the development of patchy cavitations in the xylem tracheids of the seedlings influenced by PWD. With the progression of drought, many pit membranes of bordered pits in the xylem of the main stem were aspirated with the decrease in water potential without xylem cavitation, indicating that hydraulic segmentation may exist between tracheids. This is the first study to demonstrate conclusively that explosive and irreversible cavitations occurred around the hydraulically vulnerable resin canals with the progression of PWD. Our findings provide a more comprehensive understanding of stressors on plant-water relations that may eventually better protect trees from PWD and assist with the breeding of trees more tolerant to PWD.

Phytocercomonas venanatans, a New Species of Cercozoa Associated with Chlorotic Streak of Sugarcane.

Chlorotic streak is a global disease of commercial sugarcane (Saccharum spp. hybrids). The disease is transmitted by wet soil, water, as well as in diseased planting material. Although first recognized almost 90 years ago and despite significant research effort, the identity of the causal agent has been elusive. Metagenomic high throughput sequencing (HTS) facilitated the discovery of novel protistan ribosomal and nuclear genes in chlorotic streak-infected sugarcane. These sequences suggest a possible causal agent belonging to the order Cercomonadida (Rhizaria, phylum Cercozoa). An organism with morphological features similar to cercomonads (=Cercomonadida) was isolated into pure axenic culture from internal stalk tissues of infected sugarcane. The isolated organism contained DNA sequences identical to those identified in infected plants by HTS. The DNA sequences and the morphology of the organism did not match any known species. Here we present a new genus and species, Phytocercomonas venanatans, which is associated with chlorotic streak of sugarcane. Amplicon sequencing also supports that P. venanatans is associated with this disease. This is the first reported member from Cercomonadida showing a probable pathogenic association with higher plants.

De novo transcriptome assemblies of four xylem sap-feeding insects.

Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced. Findings: Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91 384 for Cuerna arida to 106 998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species. Conclusions: These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization.

Contribution of gall microscopic structure to taxonomy of gallicolous aphids on Pistacia.

Aphids inducing galls on Pistacia plants belong to the tribe Fordini. According to the Heie & Wegierek classification, the genera are grouped into three subtribes. Previous microscopic studies showed that this taxonomy is not consistent with the histological characteristics of the galls. In this paper, galls induced by Aplonerura lentisci, Asiphonella cynodonti, Forda riccobonii, Slavun wertheimae and Smynthurodes betae were analyzed for the first time, as well as nine other galls previously described. Based on histological features three groups of galls can be establish: the first group comprises closed galls, induced by Baizongia pistaciae, Geoica utricularia, Rectinasus buxtoni and Slavun wertheimae; the second group includes two species of Geopemphigus (G. blackmani and G. torsus), and the third one is divided into two subgroups, the first comprises Aplonerura lentisci, Asiphonella cynodonti and Geopemphigus morral, and the second that includes Forda formicaria, F. marginata, F. riccobonii, Paracletus cimiciformis and Smynthurodes betae. An identification key of species based on microscopic features of galls is presented.

Poplar-Root Knot Nematode Interaction: A Model for Perennial Woody Species.

Plant root-knot nematode (RKN) interaction studies are performed on several host plant models. Though RKN interact with trees, no perennial woody model has been explored so far. Here, we show that poplar (Populus tremula × P. alba) grown in vitro is susceptible to Meloidogyne incognita, allowing this nematode to penetrate, to induce feeding sites, and to successfully complete its life cycle. Quantitative reverse transcription-polymerase chain reaction analysis was performed to study changes in poplar gene expression in galls compared with noninfected roots. Three genes (expansin A, histone 3.1, and asparagine synthase), selected as gall development marker genes, followed, during poplar-nematode interaction, a similar expression pattern to what was described for other plant hosts. Downregulation of four genes implicated in the monolignol biosynthesis pathway was evidenced in galls, suggesting a shift in the phenolic profile within galls developed on poplar roots. Raman microspectroscopy demonstrated that cell walls of giant cells were not lignified but mainly composed of pectin and cellulose. The data presented here suggest that RKN exercise conserved strategies to reproduce and to invade perennial plant species and that poplar is a suitable model host to study specific traits of tree-nematode interactions.

Rice sucrose transporter1 (OsSUT1) up-regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles.

The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3-week-old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1- to 10-day infestation time course, using a combination of gene expression and ß-glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide-field fluorescence microscopy was used to confirm the expression of OsSUT1-promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up-regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up-regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid-related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.

Histopathological response of Lens culinaris roots towards root-knot nematode, Meloidogyne incognito.

Lens culinaris (lentil) is an important pulse crop. The yield of the crop is reduced if grown in root-knot nematode (Meloidogyne incognita) infested field. Meloidogyne incognita caused infection in primary and the secondary roots leading to the anomalies in the affected part of the root. The study revealed that the second stage juveniles (J2) of Meloidogyne incognita entered the growing roots and their branches inter and intracellularly. The immediate response was hypertrophy and hyperplasia in the root tissue near the nematode head. In response to hypertrophy some cells became very large and contained dense and granular cytoplasm. Adjacent to the giant cells, the vascular tissue was found to be disturbed. Shape, size and orientation of the vascular elements was so much altered that it had become difficult to trace the normal course of vascular strands. In various sections vascular strands were found disrupted. The vessel elements had the shapes resembling the shapes of parenchyma cells. Similarly sieve tube elements of the phloem, near the giant cells were shorter and resembled with nearby parenchyma cells. Abnormalities in xylem and phloem favored transport water, minerals and metabolites towards the giant cells. From this study, it might be inferred that alteration in the cells of galled tissue was essential for the sustenance of giant cells and for the survival of the nematode.

Syncytia formed by adult female Heterodera schachtii in Arabidopsis thaliana roots have a distinct cell wall molecular architecture.

• Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. • Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. • Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. • This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.

Root-derived oxylipins promote green peach aphid performance on Arabidopsis foliage.

Oxylipins function as signaling molecules in plant growth and development and contribute to defense against stress. Here, we show that oxylipins also facilitate infestation of Arabidopsis thaliana shoots by the phloem sap-consuming green peach aphid (GPA; Myzus persicae), an agronomically important insect pest. GPAs had difficulty feeding from sieve elements and tapping into the xylem of lipoxygenase5 (lox5) mutant plants defective in LOX activity. These defects in GPA performance in the lox5 mutant were accompanied by reduced water content of GPAs and a smaller population size of GPAs in the mutant compared with the wild-type plant. LOX5 expression was rapidly induced in roots in response to infestation of shoots by GPAs. In parallel, levels of LOX5-derived oxylipins increased in roots and in petiole exudates of GPA-colonized plants. Application of 9-hydroxyoctadecadienoic acid (an oxylipin produced by the LOX5 enzyme) to roots restored water content and GPA population size in lox5 plants, thus confirming that a LOX5-derived oxylipin promotes infestation of the foliage by GPAs. Micrografting experiments demonstrated that GPA performance on foliage is influenced by the LOX5 genotype in roots, thus demonstrating the importance of root-derived oxylipins in colonization of aboveground organs by an insect.

Gall formation in clubroot-infected Arabidopsis results from an increase in existing meristematic activities of the host but is not essential for the completion of the pathogen life cycle.

Plasmodiophora brassicae (clubroot) infection leads to reprogramming of host development resulting in the formation of characteristic galls. In this work we explored the cellular events that underly gall formation in Arabidopsis thaliana with the help of molecular markers of cell division (CYCB1:GUS) and meristematic activity (ANT:GUS). Our results show that gall development involved the amplification of existing meristematic activities within the vascular cambium (VC) and phloem parenchyma (PP) cells in the region of the hypocotyl. Additionally we found that the increase in VC activity and prolonged maintenance of cambial-derived cells in a meristematic state was crucial for gall formation; disruption of the VC activity significantly decreased the gall size. Gall formation also perturbed vascular development with a significant reduction in xylem and increase in PP in infected plants. This situation was reflected in a decrease in transcripts of key factors promoting xylogenesis (VND6, VND7 and MYB46) and an increase in those promoting phloem formation and function (APL, SUC2). Finally we show, using the cell cycle inhibitor ICK1/KRP1 and a cle41 mutant with altered regulation of cambial stem cell maintenance and differentiation, that a decrease in gall formation did not prevent pathogen development. This finding demonstrates that although gall formation is a typical symptom of the disease and influences numbers of spores produced, it is not required for completion of the pathogen life cycle. Together, these results provide an insight into the relationship of the cellular events that accompany Plasmodiophora infection and their role in disease progression.

Quantitative comparison of stylet penetration behaviors of glassy-winged sharpshooter on selected hosts.

New Zealand is threatened by invasion of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), an important vector of Xylella fastidiosa, a gram-negative bacterium that causes Pierce's disease in grape (Vitis spp.) and scorch diseases in many other horticultural crops. Therefore, an understanding of the host acceptability, feeding behavior, and potential vector efficiency of glassy-winged sharpshooter on New Zealand crops is important. We tested host plant acceptance and feeding behaviors of glassy-winged sharpshooter on three common horticultural crops grown in New Zealand (apple [Malus spp.], grape, and citrus [Citrus spp.]), and a native plant (Metrosideros excelsa [=tomentosa] Richard, pohutukawa), using the electrical penetration graph (EPG) technique. Probing (stylet penetration) behaviors varied among the host plants, primarily due to differences in waveform event durations. Apple and grape were the most accepted host plants, on which glassy-winged sharpshooter spent the majority of its time on the plant probing and readily located and accepted a xylem cell for ingestion. This resulted in long durations of sustained xylem fluid ingestion. In contrast, pohutukawa was the least accepted host. On this plant, glassy-winged sharpshooter spent less time probing and engaged in longer and more frequent testing/searching and xylem-testing activities, rejected xylem cells frequently, and spent less time with stylets resting, before accepting a xylem cell and ultimately performing the same amount of sustained ingestion. Citrus plants contaminated with sublethal insecticide residues were intermediate between these extremes, with some acceptance of xylem, but less ingestion, probably due to presumed partial paralysis of the cibarial muscles. Implications of the results in terms of host plant acceptance and the development of a stylet penetration index are discussed.

A role for IAA in the infection of Arabidopsis thaliana by Orobanche aegyptiaca.

BACKGROUND: Vascular continuity is established between a host plant and the root parasite broomrape. It is generally accepted that the direction of vascular continuity results from polar flow of auxin. Our hypothesis was that chemical disruptions of auxin transport and activity could influence the infection of the host by the parasite. METHODS: A sterile system for the routine infection of Arabidopsis thaliana seedlings in Nunc cell culture plates by germinated seeds of Orobanche aegyptiaca was developed. This method permitted a quantitative assay of the rate of host infection. The three-dimensional structure of the vascular contacts was followed in cleared tissue. IAA (indole acetic acid) or substances that influence its activity and transport were applied locally to the host root. RESULTS: The orientation of the xylem contacts showed that broomrape grafts itself upon the host by acting hormonally as a root rather than a shoot. Local applications of IAA, PCIB (p-chlorophenoxyisobutyric acid) or NPA (naphthylphthalamic acid) all resulted in drastic reductions of Orobanche infection CONCLUSIONS: Broomrape manipulates the host by acting as a sink for auxin. Disruption of auxin action or auxin flow at the contact site could be a novel basis for controlling infection by Orobanche.