First report of tomato chlorotic dwarf viroid infecting litchi tomato (Solanum sisymbriifolium).

Litchi tomato (LT) (Solanum sisymbriifolium) is a solanaceous weed that is considered a biological control tool to manage potato cyst nematode (PCN) in Europe and is being explored for use in Idaho. Two Several LT lines were clonally maintained as stocks in the university greenhouse since 2013 and were also established in tissue culture at the same time. In 2018, tomato (Solanum lycopersicum cv. Alisa Craig) scions were grafted onto two LT rootstocks originating either from healthy-looking greenhouse stocks or from tissue culture-maintained plants. Unexpectedly, tomatoes grafted onto the greenhouse-maintained rootstocks of LT displayed severe symptoms of stunting, foliar deformation, and chlorosis, while grafts onto the same LT lines from tissue culture produced healthy-looking tomato plants. Tests for the presence of several viruses known to infect solanaceous plants were conducted on symptomatic tomato scion tissues using ImmunoStrips (Agdia, Elkhard, IN) and RT-PCR (Elwan et al. 2017) but yielded negative results. High throughput sequencing (HTS) was then used to identify possible pathogens that could have been responsible for the symptoms observed in tomato scions. Samples from two symptomatic tomato scions, two asymptomatic scions grafted onto the tissue culture-derived plants, and two greenhouse-maintained rootstocks were subjected to HTS. Total RNA from the four tomato and two LT samples was depleted of ribosomal RNA and subjected to HTS on an Illumina MiSeq platform producing 300-bp paired-end reads and raw reads were adapter and quality cleaned. For the tomato samples, the clean reads were mapped against the S. lycopersicum L. reference genome, and unmapped paired reads were assembled producing between 4,368 and 8,645 contigs. For the LT samples, all clean reads were directly assembled, producing 13,982 and 18,595 contigs. In the symptomatic tomato scions and the two LT rootstock samples, a 487-nt contig was found, comprising an ~1.35 tomato chlorotic dwarf viroid (TCDVd) genome and exhibiting 99.7% identity with it (GenBank accession AF162131; Singh et al. 1999). No other virus-related or viroid contigs were identified. RT-PCR analysis using a pospiviroid primer set Pospi1-FW/RE (Verhoeven et al. 2004), and a TCDVd-specific primer set TCDVd-Fw/TCDVd-Rev (Olmedo-Velarde et al. 2019) produced 198-nt and 218-nt bands, respectively, thus confirming the presence of TCDVd in tomato and LT samples. These PCR products were Sanger sequenced and confirmed to be TCDVd-specific; the complete sequence of the Idaho isolate of TCDVd was deposited in GenBank under the accession number OQ679776. Presence of TCDVd in LT plant tissue was confirmed by the APHIS PPQ Laboratory in Laurel, MD. Asymptomatic tomatoes and LT plants from tissue culture were found negative for TCDVd. Previously, TCDVd was reported to affect greenhouse tomatoes in Arizona and Hawaii (Ling et al. et al. 2009; Olmedo-Velarde et al. 2019), however, this is the first report of TCDVd infecting litchi tomato (S. sisymbriifolium). Five additional greenhouse-maintained LT lines were found TCDVd-positive using RT-PCR and Sanger sequencing. Given the very mild or asymptomatic infection of TCDVd in this host, molecular diagnostic methods should be used to screen LT lines for the presence of this viroid to avoid inadvertent spread of TCDVd. Another viroid, potato spindle tuber viroid, was reported to be transmitted through LT seed (Fowkes et al. 2021), and transmission of TCDVd through LT seed may also be responsible for this TCDVd outbreak in the university greenhouse, although no direct evidence was collected. To the best of our knowledge, this is the first report of TCDVd infection in S. sisymbriifolium and also the first report of the TCDVd occurrence in Idaho.

Control of Globodera pallida Using Brassica juncea Seed Meal Extract and the Trap Crop Solanum sisymbriifolium.

Globodera pallida, the pale cyst nematode, is a regulated potato pest which is economically detrimental. Restrictions on use of the soil fumigant methyl bromide and lack of resistant russet type varieties for U.S. markets have led to investigations of alternative strategies to control this pest. The efficacy of Brassica juncea seed meal extract (SME; 0, 0.14, 0.28, 0.56, 1.12, and 2.24 t/ha) was studied, either alone or in combination with the trap crop Solanum sisymbriifolium under greenhouse and field conditions. The impact of the application of SME pre- or postplanting of S. sisymbriifolium was also determined. S. sisymbriifolium only induced hatch of G. pallida and significantly fewer (up to 57 and 55% in pre- and postplant experiments, respectively) encysted eggs remained at termination of the experiment compared with the untreated control. However, when SME was applied preplant, the encysted eggs remained unchanged, which may indicate that SME inhibited egg hatch in the presence of S. sisymbriifolium. When applied individually, S. sisymbriifolium in all experiments, or SME at all rates tested in the greenhouse or 0.56 t/ha or higher rates of SME in the field, significantly reduced the viability, hatch, and reproduction of G. pallida. Combined treatment with S. sisymbriifolium and SME at lower rates (0.14 t/ha for preplant or 0.56 t/ha or less for the greenhouse postplant experiment) reduced G. pallida egg hatch further than each strategy alone. In the field, a combination of S. sisymbriifolium and SME at 1.12 t/ha or less reduced G. pallida more effectively than SME alone. SME alone applied at higher rates (0.56 and 1.12 t/ha) in preplant greenhouse trials, whether or not combined with S. sisymbriifolium, eliminated G. pallida reproduction. Under field conditions, SME applied at a rate of 1.12 t/ha highly reduced G. pallida reproduction compared with the untreated control by 97 and 61% in 2019 and 2020, respectively. Furthermore, reproduction of G. pallida was eliminated when SME was combined with S. sisymbriifolium. Our results indicated that a combination of SME and S. sisymbriifolium reduces the amount of SME needed to control G. pallida and further decreases the potential reserve of the viable population remaining after individual treatment with each strategy.

A Novel Rhabdovirus Associated with the Idaho Population of Potato Cyst Nematode Globodera pallida.

Globodera pallida, a potato cyst nematode (PCN), is a quarantine endoparasitic pest of potato (Solanum tuberosum) in the US due to its effects on yield and quality of potato tubers. A new rhabdovirus, named potato cyst nematode rhabdovirus (PcRV), was revealed and characterized in the G. pallida populations collected in Idaho through use of high-throughput sequencing (HTS) and RT-PCR and found to be most closely related to soybean cyst nematode rhabdovirus (ScRV). PcRV has a 13,604 bp long, single-stranded RNA genome encoding five open reading frames, including four rhabdovirus-specific genes, N, P, G, and L, and one unknown gene. PcRV was found present in eggs, invasive second-stage juveniles, and parasitic females of G. pallida, implying a vertical transmission mode. RT-PCR and partial sequencing of PcRV in laboratory-reared G. pallida populations maintained over five years suggested that the virus is highly persistent and genetically stable. Two other Globodera spp. reproducing on potato and reported in the US, G. rostochiensis and G. ellingtonae, tested negative for PcRV presence. To the best of our knowledge, PcRV is the first virus experimentally found infecting G. pallida. Based on their similar genome organizations, the phylogeny of their RNA-dependent RNA polymerase domains (L gene), and relatively high identity levels in their protein products, PcRV and ScRV are proposed to form a new genus, provisionally named "Gammanemrhavirus", within the family Rhabdoviridae.

Resisting Potato Cyst Nematodes With Resistance.

Potato cyst nematodes (PCN) are economically important pests with a worldwide distribution in all temperate regions where potatoes are grown. Because above ground symptoms are non-specific, and detection of cysts in the soil is determined by the intensity of sampling, infestations are frequently spread before they are recognised. PCN cysts are resilient and persistent; their cargo of eggs can remain viable for over two decades, and thus once introduced PCN are very difficult to eradicate. Various control methods have been proposed, with resistant varieties being a key environmentally friendly and effective component of an integrated management programme. Wild and landrace relatives of cultivated potato have provided a source of PCN resistance genes that have been used in breeding programmes with varying levels of success. Producing a PCN resistant variety requires concerted effort over many years before it reaches what can be the biggest hurdle-commercial acceptance. Recent advances in potato genomics have provided tools to rapidly map resistance genes and to develop molecular markers to aid selection during breeding. This review will focus on the translation of these opportunities into durably PCN resistant varieties.

Genomic Analyses of Globodera pallida, A Quarantine Agricultural Pathogen in Idaho.

Globodera pallida is among the most significant plant-parasitic nematodes worldwide, causing major damage to potato production. Since it was discovered in Idaho in 2006, eradication efforts have aimed to contain and eradicate G. pallida through phytosanitary action and soil fumigation. In this study, we investigated genome-wide patterns of G. pallida genetic variation across Idaho fields to evaluate whether the infestation resulted from a single or multiple introduction(s) and to investigate potential evolutionary responses since the time of infestation. A total of 53 G. pallida samples (~1,042,000 individuals) were collected and analyzed, representing five different fields in Idaho, a greenhouse population, and a field in Scotland that was used for external comparison. According to genome-wide allele frequency and fixation index (Fst) analyses, most of the genetic variation was shared among the G. pallida populations in Idaho fields pre-fumigation, indicating that the infestation likely resulted from a single introduction. Temporal patterns of genome-wide polymorphisms involving (1) pre-fumigation field samples collected in 2007 and 2014 and (2) pre- and post-fumigation samples revealed nucleotide variants (SNPs, single-nucleotide polymorphisms) with significantly differentiated allele frequencies indicating genetic differentiation. This study provides insights into the genetic origins and adaptive potential of G. pallida invading new environments.

Characterization of Superoxide Dismutase from the Potato Cyst Nematode, Globodera pallida.

Potato cyst nematodes (PCNs), such as Globodera pallida and Globodera rostochiensis, are some of the most agriculturally and economically important pests of potato. Upon nematode infection, a principal component of plant defense is the generation of the reactive oxygen species (ROSs). ROSs are highly toxic molecules that cause damage to pathogens and host alike. To infect the plant, nematodes protect themselves from ROSs by activating their own antioxidant processes and ROS scavenging enzymes. One of these enzymes is a superoxide dismutase (SOD; EC 1.15.1.1), which prevents cellular damage by catalyzing conversion of the superoxide radical (O2-·) to hydrogen peroxide (H2O2) and molecular oxygen (O2). We have isolated a putatively secreted isoform of a Cu-Zn SOD (SOD-3) from G. pallida and localized the expression of this gene in the posterior region of the nematode. Furthermore, we studied the expression of the SOD-3 gene during early parasitic stages of infection (24 to 72 h) in the susceptible potato cultivar Desiree, the resistant potato cultivar Innovator, and an immune host, Solanum sisymbriifolium. The SOD-3 gene was significantly upregulated, regardless of the host type; however, the expression pattern differed between the susceptible and the resistant or immune hosts. This finding suggests that SOD-3 gene is responding to infection in plant roots differently depending on whether the nematode is experiencing a compatible or an incompatible interaction.

Effect of Steroidal Glycoalkaloids on Hatch and Reproduction of the Potato Cyst Nematode Globodera pallida.

Steroidal glycoalkaloids (SGAs) are phytoanticipins found in solanaceous crops that act as the first line of chemical defense against pathogen attacks. Solanum sisymbriifolium, a trap crop for potato cyst nematodes, has been shown to effectively reduce populations of Globodera pallida. S. sisymbriifolium contains α-solamargine and other solasodine-type glycoalkaloids that may contribute to plant defenses. This study evaluated the influence of solanaceous SGAs on G. pallida hatch, development, and reproduction. Exposure to α-solamargine and α-solamarine reduced G. pallida hatch by 65 and 87%, respectively. Exposure of G. pallida cysts with the glycoalkaloids α-solamargine and solasodine significantly reduced infection in susceptible potato 'Russet Burbank' by 98 and 94% compared with the control. Exposure of cysts to either solasodine or solamargine significantly reduced reproduction of G. pallida on 'Russet Burbank' by 99% compared with the control. The study demonstrated the deleterious effect of SGAs on G. pallida hatch, infection, and reproduction.

Potato Cyst Nematode Egg Viability Assessment and Preparasitic Juvenile Screening Using a Large Particle Flow Cytometer and Sorter.

Potato cyst nematode (PCN) cysts consist of heterogenous populations of eggs, juveniles, and eggshells that make manual sorting of individual life stages cumbersome. The number of viable PCN eggs is a major determinant of crop damage. An accurate high-throughput PCN egg viability assay is useful for developing effective management and eradication plans. In this study, we present a method for rapid and precise enumeration and sorting of PCN eggs and juveniles, along with an egg viability assessment by staining eggs with the fluorescent stain, acridine orange, and sorting with the Complex Object Parametric Analyzer and Sorter (COPAS) system, a large particle flow cytometer. Both size sorting and fluorescent sorting capabilities of the COPAS were explored. By using the COPAS, sorting efficiency for eggs and preparasitic second-stage juveniles (J2s) was 97.6 and 97.2%, respectively, with 99% recovery at a flow rate of 15 events/s. Purity of sorted live and dead eggs was 95.5 and 94.1%, respectively. Sorting of J2s by size indicated that 15 to 16.4% of Globodera ellingtonae or G. pallida had an average body length of 436.1 ± 3.4 µm compared with an average size of 512.9 ± 4.4 µm for the majority of the J2 population for both species. A red autofluorescing J2 population was also identified through sorting. Sorting of eggs by flow cytometry did not significantly affect hatching (55.1 ± 1.2 and 53.9 ± 1.6%, respectively, for sorted or nonsorted eggs) or juvenile motility (91.3 ± 1.0 or 90.1 ± 1.1%, respectively), thus confirming that the method does not impair the biological activity of the nematode.

Belowground Chemical Interactions: An Insight Into Host-Specific Behavior of Globodera spp. Hatched in Root Exudates From Potato and Its Wild Relative, Solanum sisymbriifolium.

Understanding belowground chemical interactions between plant roots and plant-parasitic nematodes is immensely important for sustainable crop production and soilborne pest management. Due to metabolic diversity and ever-changing dynamics of root exudate composition, the impact of only certain molecules, such as nematode hatching factors, repellents, and attractants, has been examined in detail. Root exudates are a rich source of biologically active compounds, which plants use to shape their ecological interactions. However, the impact of these compounds on nematode parasitic behavior is poorly understood. In this study, we specifically address this knowledge gap in two cyst nematodes, Globodera pallida, a potato cyst nematode and the newly described species, Globodera ellingtonae. Globodera pallida is a devastating pest of potato (Solanum tuberosum) worldwide, whereas potato is a host for G. ellingtonae, but its pathogenicity remains to be determined. We compared the behavior of juveniles (J2s) hatched in response to root exudates from a susceptible potato cv. Desirée, a resistant potato cv. Innovator, and an immune trap crop Solanum sisymbriifolium (litchi tomato - a wild potato relative). Root secretions from S. sisymbriifolium greatly reduced the infection rate on a susceptible host for both Globodera spp. Juvenile motility was also significantly influenced in a host-dependent manner. However, reproduction on a susceptible host from juveniles hatched in S. sisymbriifolium root exudates was not affected, nor was the number of encysted eggs from progeny cysts. Transcriptome analysis by using RNA-sequencing (RNA-seq) revealed the molecular basis of root exudate-mediated modulation of nematode behavior. Differentially expressed genes are grouped into two major categories: genes showing characteristics of effectors and genes involved in stress responses and xenobiotic metabolism. To our knowledge, this is the first study that shows genome-wide root exudate-specific transcriptional changes in hatched preparasitic juveniles of plant-parasitic nematodes. This research provides a better understanding of the correlation between exudates from different plants and their impact on nematode behavior prior to the root invasion and supports the hypothesis that root exudates play an important role in plant-nematode interactions.

The Globodera pallida Effector GpPDI1 Is a Functional Thioredoxin and Triggers Defense-Related Cell Death Independent of Its Enzymatic Activity.

The plant-parasitic nematode Globodera pallida is an obligate biotroph that only reproduces on select species in the Solanum family. The establishment of the feeding site, the syncytium, involves secretion of effectors into the plant cell to combat the plant defense response and facilitate transformation of root cells into the syncytium. Despite the important predicted roles of effectors in the plant-pathogen interactions, the functionality of G. pallida effectors is largely unknown. In this study, we identified and characterized a G. pallida effector protein disulfide isomerase (GpPDI1). GpPDI1 contains two thioredoxin domains that function together to reduce disulfide bonds, as manifested by the nullification of enzymatic activity when either domain is absent. The transcript of GpPDI1 is localized in the dorsal gland of the nematode during the J2 stage. In addition, GpPDI1 can trigger defense-related cell death in Nicotiana benthamiana and tomato (Solanum lycopersicum) leaf tissue and localizes in the plant host cell's cytoplasm and nucleus when transiently expressed in plant cells. Significantly, the ability of elicitation of cell death is not dependent on the enzymatic activity of GpPDI1 or correlated with the subcellular distribution of GpPDI1, suggesting that a nondisulfide reducing function or structural feature of GpPDI1 is responsible for the recognition by the host immune system to elicit cell death.

Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium.

A transcriptome analysis of G. pallida juveniles collected from S. tuberosum or S. sisymbriifolium 24 h post infestation was performed to provide insights into the parasitic process of this nematode. A total of 41 G. pallida genes were found to be significantly differentially expressed when parasitizing the two plant species. Among this set, 12 were overexpressed when G. pallida was parasitizing S. tuberosum and 29 were overexpressed when parasitizing S. sisymbriifolium. Out of the 12 genes, three code for secretory proteins; one is homologous to effector gene Rbp-4, the second is an uncharacterized protein with a signal peptide sequence, and the third is an ortholog of a Globodera rostochiensis effector belonging to the 1106 effector family. Other overexpressed genes from G. pallida when parasitizing S. tuberosum were either unknown, associated with a stress or defense response, or associated with sex differentiation. Effector genes namely Eng-1, Cathepsin S-like cysteine protease, cellulase, and two unknown genes with secretory characteristics were over expressed when G. pallida was parasitizing S. sisymbriifolium relative to expression from S. tuberosum. Our findings provide insight into gene regulation of G. pallida while infecting either the trap crop S. sisymbriifolium or the susceptible host, S. tuberosum.

Current Status of Potato Cyst Nematodes in North America.

The potato cyst nematodes (PCNs) Globodera rostochiensis and Globodera pallida are internationally recognized quarantine pests. Although not widely distributed in either the United States or Canada, both are present and are regulated by the national plant protection organizations (NPPOs) of each country. G. rostochiensis was first discovered in New York in the 1940s, and G. pallida was first detected in a limited area of Idaho in 2006. In Canada, G. rostochiensis and G. pallida were first detected in Newfoundland in 1962 and 1977, respectively, and further detections of G. rostochiensis occurred in British Columbia and Québec, most recently in 2006. Adherence to a stringent NPPO-agreed-upon phytosanitary program has prevented the spread of PCNs to other potato-growing areas in both countries. The successful research and regulatory PCN programs in both countries rely on a network of state, federal, university, and private industry cooperatorspursuing a common goal of containment, management/eradication, and regulation. The regulatory and research efforts of these collaborative groups spanning from the 1940s to the present are highlighted in this review.

The potato cyst nematode effector RHA1B is a ubiquitin ligase and uses two distinct mechanisms to suppress plant immune signaling.

Plant pathogens, such as bacteria, fungi, oomycetes and nematodes, rely on wide range of virulent effectors delivered into host cells to suppress plant immunity. Although phytobacterial effectors have been intensively investigated, little is known about the function of effectors of plant-parasitic nematodes, such as Globodera pallida, a cyst nematode responsible for vast losses in the potato and tomato industries. Here, we demonstrate using in vivo and in vitro ubiquitination assays the potato cyst nematode (Globodera pallida) effector RHA1B is an E3 ubiquitin ligase that employs multiple host plant E2 ubiquitin conjugation enzymes to catalyze ubiquitination. RHA1B was able to suppress effector-triggered immunity (ETI), as manifested by suppression of hypersensitive response (HR) mediated by a broad range of nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors, presumably via E3-dependent degradation of the NB-LRR receptors. RHA1B also blocked the flg22-triggered expression of Acre31 and WRKY22, marker genes of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), but this did not require the E3 activity of RHA1B. Moreover, transgenic potato overexpressing the RHA1B transgene exhibited enhanced susceptibility to G. pallida. Thus, our data suggest RHA1B facilitates nematode parasitism not only by triggering degradation of NB-LRR immune receptors to block ETI signaling but also by suppressing PTI signaling via an as yet unknown E3-independent mechanism.

In situ Hybridization of Plant-parasitic Nematode Globodera pallida Juveniles to Detect Gene Expression.

In this study, we describe a standard whole mount in situ hybridization method which is used to determine the spatial-temporal expression pattern of genes from Globodera spp. Unlike more invasive radioactive labeling approaches, this technique is based on a safe, highly specific enzyme-linked immunoassay where a Digoxigenin (DIG)-tagged anti-sense probe hybridized to a target transcript is detected by anti-DIG antibodies conjugated with alkaline phosphatase enzyme (AP) (anti-DIG-AP). The hybrid molecules are visualized through an AP-catalyzed color reaction using as the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT). This method can be applied to both free-living pre-parasitic juveniles and early endoparasitic stages of cyst nematodes.

An Improved Technique for Sorting Developmental Stages and Assessing Egg Viability of Globodera pallida using High-Throughput Complex Object Parametric Analyzer and Sorter.

The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer designed for analyzing, sorting, and dispensing objects of varying sizes. We explored the potential of using this instrument to analyze and sort various developmental stages and egg viability of Globodera pallida. Cysts were successfully examined and sorted from debris by optimizing side-scatter and red-fluorescence parameters on the COPAS. We were able to separate eggs and second-stage juveniles from samples of mixed population using extinction and time of flight. Separation of live and dead eggs was examined following staining eggs with SYTOX Green and application of time of flight and green peak height. Data were compared with a commonly used viability assay by which eggs were stained with Meldola's Blue and examined by a microscope. COPAS proved to be effective in assessing viability by detecting two separate gates: live eggs having green fluorescence peaks <190 and dead eggs with the peaks >190. The application of COPAS in combination with SYTOX Green detected a greater number of live eggs than the Meldola's assay, suggesting that SYTOX Green provided an overestimate of live eggs. COPAS noticeably increased the accuracy and reduced the time required for screening and analyzing nematode populations.

Resistance of Potato Breeding Clones and Cultivars to Three Species of Potato Cyst Nematode.

In the United States, potato cyst nematodes Globodera rostochiensis and G. pallida are quarantined pests. A new cyst nematode species, Globodera ellingtonae, discovered in Oregon and Idaho, reproduces well on potato but is not currently a quarantine pest. Identifying resistance to all three Globodera spp. would provide a valuable management tool. Thirteen breeding clones and nine cultivars were evaluated in Oregon, Idaho, and New York laboratories where the nematode populations are maintained. Minitubers or tissue culture plants were planted into pots and inoculated with eggs in replicated experiments. Results indicated that five entries were partially resistant or resistant to all three species, while another five were resistant or partially resistant to G. rostochiensis and G. ellingtonae. Resistance to G. rostochiensis pathotypes Ro1 and Ro4 is controlled by the H1 gene and this study suggests that H1 may confer resistance to G. ellingtonae as well. Observed resistance to G. pallida was lower relative to the levels of resistance observed for G. rostochiensis and G. ellingtonae. Germplasm with G. pallida or G. ellingtonae resistance will be used in hybridizations to develop russet-skinned cultivars with long tubers which represent the predominant market class in western U.S. production, and to further explore the basis of potato resistance to Globodera spp.

Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium.

Solanum sisymbriifolium, also known as "Litchi Tomato" or "Sticky Nightshade," is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did.

Microaspiration of Solanum tuberosum root cells at early stages of infection by Globodera pallida.

BACKGROUND: Sedentary endoparasitic cyst nematodes form a feeding structure in plant roots, called a syncytium. Syncytium formation involves extensive transcriptional modifications, which leads to cell modifications such as increased cytoplasmic streaming, enlarged nuclei, increased numbers of organelles, and replacement of a central vacuole by many small vacuoles. When whole root RNA is isolated and analyzed, transcript changes manifested in the infected plant cells are overshadowed by gene expression from cells of the entire root system. Use of microaspiration allows isolation of the content of nematode infected cells from a heterogeneous cell population. However, one challenge with this method is identifying the nematode infected cells under the microscope at early stages of infection. This problem was addressed by staining nematode juveniles with a fluorescent dye prior to infection so that the infected cells could be located and microaspirated. RESULTS: In the present study, we used the fluorescent vital stain PKH26 coupled with a micro-rhizosphere chamber to locate the infected nematode Globodera pallida in Solanum tuberosum root cells. This enabled microaspiration of nematode-infected root cells during the early stages of parasitism. To study the transcriptional events occurring in these cells, an RNA isolation method from microaspirated samples was optimized, and subsequently the RNA was purified using magnetic beads. With this method, we obtained an RNA quality number of 7.8. For transcriptome studies, cDNA was synthesized from the isolated RNA and assessed by successfully amplifying several pathogenesis related protein coding genes. CONCLUSION: The use of PKH26 stained nematode juveniles enabled early detection of nematode infected cells for microaspiration. To investigate transcriptional changes in low yielding RNA samples, bead-based RNA extraction procedures minimized RNA degradation and provided high quality RNA. This protocol provides a robust procedure to analyze gene expression in nematode-infected cells.

Prospecting fungal parasites of the potato cyst nematode Globodera pallida using a rapid screening technique.

Seven filamentous fungal species were isolated from individual eggs of Globodera pallida cysts collected from infested fields in Shelley Idaho, USA and identified as Chaetomium globosum, Fusarium oxysporum, Fusarium solani, Fusarium tricinctum, Microdochium bolleyi, Purpureocillium lilacinum, and Plectosphaerella cucumerina. Their ability to reduce infection by G. pallida in planta were assessed in simple, reproducible micro-rhizosphere chambers (micro-ROCs). All fungi reduced G. pallida infection in potato, but greatest reduction was observed with C. globosum at an average reduction of 76%. Further non-destructive methods were developed to rapidly assess biological control potential of putative fungal strains by staining the infectious second stage juveniles of G. pallida with the live fluorescent stain PKH26. In comparisons between the standard, invasive acid fuchsin method and use of the live stain PKH26, no significant difference in infection level of G. pallida was observed whether roots were stained with PKH26 or acid fuchsin. For both methods, a similar reduction (77% for acid fuchsin, and 78% for PKH26 stain) in invasion of infectious stage of G. pallida was observed when potato plants were inoculated with C. globosum compared to non-inoculated potato.

Control of Globodera spp. Using Brassica juncea Seed Meal and Seed Meal Extract.

The eradication program for the potato cyst nematode (PCN), Globodera pallida, in the Northwest of the United States revolves around the use of soil fumigation. Alternative, integrated strategies are needed to continue to battle this invasive nematode. Laboratory, greenhouse, and field experiments were conducted with G. pallida and another cyst nematode found in the United States, Globodera ellingtonae, to evaluate the efficacy of a new formulated Brassica juncea seed meal extract, as well as a traditional B. juncea seed meal, as alternate eradication strategies. This is the first report on the efficacy of B. juncea seed meal extract against plant-parasitic nematodes. Rates of B. juncea seed meal greater than 2.2 t/ha and 4.5 t/ha for G. pallida and G. ellingtonae, respectively, were required for egg hatch suppression, as determined by a potato root diffusate (PRD) bioassay. Reproduction of G. pallida on potato after exposure to B. juncea seed meal at a rate of 2.2 t/ha was also significantly reduced. In the field, 8.9 t/ha B. juncea seed meal almost eliminated egg hatch of G. ellingtonae. Rates needed for Globodera spp. suppression were greatly reduced when using the B. juncea seed meal extract. When compared side-by-side, half as much B. juncea seed meal extract, 1.1 t/ha, was required to suppress G. ellingtonae egg hatch to the same extent as B. juncea seed meal. Exposure of G. pallida to B. juncea seed meal extract at 4.5 t/ha reduced egg hatch by 90% compared with a nonamended control. The ability to reduce the amount of material being applied to soil by using an extract has the potential for integration into a G. pallida eradication program.