Discovery and Characterization of Four Aphelenchid Species from Cultivated Regions of Southern Alberta, Canada.

The nematode family Aphelenchoididiae is considered fungal-feeding, predatory, or root hair feeders. Some members of this family are universally present in agricultural landscapes and are an integral part of soil health and conservation studies. In the present soil nematode biodiversity survey, we detected four species of the genera Aphelenchus, Aphelenchoides, and Robustodorus. Because fungal-feeding nematodes from southern Alberta have not previously been reported, we conducted a detailed morphological and molecular investigation, identifying these species as Aphelenchus avenae, Aphelenchoides limberi, Aphelenchoides prairiensis n. sp. and Robustodorus paramegadorus n. sp. The first two species we document as new records from southern Alberta, whereas A. prairiensis n. sp. and R. paramegadorus n. sp. we describe in detail as new taxa. Briefly, A. prairiensis n. sp. is an amphimictic species having 4 lateral lines; hemispherical anteriorly flattened lip region; delicate stylet and swelling-like stylet knobs; excretory pore at the posterior edge of nerve ring. Female tail conical, gradually tapering towards a truncated end with single mucro. Spicule 23.0 (20.0-25.0) µm long having elongated rounded condylus, small, blunt conical rostrum, and lamina that gradually tapers towards the rounded distal end; three pairs of caudal papillae were present on the male tail. Robustodorus paramegadorus n. sp., is a parthenogenetic species with 3 lines in the lateral fields; lip region rounded, anteriorly flattened; stylet robust, with knobs rounded to bean-shaped; excretory pore located posterior to nerve ring; reproductive components were quite indiscernible with a short 24.0 (18.0-27.0) µm post-vulval uterine sac; tail conical, ending with pointed to wedge-shaped tip. We performed molecular characterizations for each species and constructed phylogenetic trees to study the phylogenetic relationship of these aphelenchid species. The discovery of A. prairiensis n. sp. and R. paramegadorus n. sp. indicates that soil nematode diversity is relatively unexplored in southern Alberta. The findings of this study will significantly enhance the identification processes and may contribute towards future soil health and biodiversity efforts.

Morpho-Anatomy, In Vitro Culture, and Phylogenetic Studies of Two Helicotylenchus Species from Southern Alberta, Canada.

Spiral nematodes (Helicotylenchus spp.) are polyphagous parasitic species exhibiting a broad host range and geographical distribution. However, their diversity in the cultivated regions of southern Alberta remains understudied. Hence, we conducted a comprehensive survey of the region's arable lands for the presence of spiral nematodes and revealed two Helicotylenchus species, H. crassatus and H. oscephalus. H. crassatus consisted of two distinct morphotypes: one morphotype had a conoid tail with slight ventral projection on the distal end, whereas the other had a broadly rounded tail. This study presents the first documentation of H. crassatus and H. oscephalus from southern Alberta, Canada. Molecular characterization was based on the partial 18S rRNA, the D2-D3 of 28S rRNA, ITS rRNA, and COI gene sequences, complemented by detailed morphological studies using scanning electron microscopy. In this work, Helicotylenchus species were often co-detected with root lesion nematodes, which made the evaluation of their role in crop damage more difficult. To meet the requirements for threshold and pathogenicity assessments, we introduced both spiral nematode species to sterile carrot disks and evaluated the feasibility of their multiplication and mass production in vitro. The present findings expand the taxonomic records of Helicotylenchus spp. and improve diagnostics of these morphologically similar species. Furthermore, our in vitro culture technique will provide a reliable source of the initial inoculum for future plant-nematode interaction studies.

New Records of Ditylenchus Species from Southern Alberta, Canada.

The presence of plant-parasitic nematodes (PPNs) in cultivated areas is a limiting factor in achieving marketable crop yield. To control and alleviate the effects of these nematodes and determine appropriate management strategies, species-level identification is crucial. Therefore, we conducted a nematode diversity survey, which resulted in the detection of four Ditylenchus species in cultivated areas of southern Alberta, Canada. The recovered species had six lines in the lateral field, delicate stylets (>10 µm long), distinct postvulval uterine sacs, and pointed to rounded tail tips. The morphological and molecular characterization of these nematodes revealed their identity as D. anchilisposomus, D. clarus, D. tenuidens and D. valveus, all of which are members of the D. triformis group. All of the identified species were found to be new records in Canada except for D. valveus. Accurate Ditylenchus species identification is crucial because false-positive identification can result in the implementation of quarantine measures over the detected area. Our current study not only documented the presence of Ditylenchus species from southern Alberta, but also described their morpho-molecular characteristics and subsequent phylogenetic relationships with related species. The results of our study will aid in the decision on whether these species should become a part of nematode management programs since nontarget species can become pests due to changes in cropping patterns or climate.

Overexpression of Douglas-Fir LEAFY COTYLEDON1 (PmLEC1) in Arabidopsis Induces Embryonic Programs and Embryo-like Structures in the lec1-1 Mutant but Not in Wild Type Plants.

Somatic embryogenesis (SE) is the most promising method for the quick propagation of desirable plant genotypes. However, application of SE to conifers remains challenging due to our limited knowledge about the genes involved in embryogenesis and the processes that lead to somatic embryo formation. Douglas-fir, an economically important lumber species, possesses a homolog of the angiosperm embryo-regulatory LEC1 gene. In the present study, we analyzed the potential of Douglas-fir PmLEC1 to induce embryonic programs in the vegetative cells of a heterologous host, Arabidopsis thaliana. PmLEC1 complemented the Arabidopsis lec1-1 null mutant and led to a variety of phenotypes ranging from normal morphology to developmental arrest at various stages in the T1 generation. PmLEC1 did not affect the morphology of wild type Arabidopsis T1 plants. More profound results occurred in T2 generations. PmLEC1 expression induced formation of recurrent somatic embryo-like structures in vegetative tissues of the rescued lec1-1 mutant but loss of apical dominance (bushy phenotype) in wild type plants. The activation of embryonic programs in the lec1-1PmLEC1 T2 plants was confirmed by the presence of the embryo-specific transcripts, OLEOSIN and CRUCIFERIN. In contrast, no embryo-like structures, and no OLEOSIN or CRUCIFERIN were observed in PmLEC1-expressing bushy wild type T2 plants.

Douglas-fir LEAFY COTYLEDON1 (PmLEC1) is an active transcription factor during zygotic and somatic embryogenesis.

Douglas-fir (Pseudotsuga menziesii) is one of the world's premier lumber species and somatic embryogenesis (SE) is the most promising method for rapid propagation of superior tree genotypes. The development and optimization of SE protocols in conifers is hindered by a lack of knowledge of the molecular basis of embryogenesis and limited sequence data. In Arabidopsis, the LEAFY COTYLEDON1 (AtLEC1) gene is a master regulator of embryogenesis that induces SE when expressed ectopically. We isolated the LEC1 homologue from Douglas-fir, designated as PmLEC1. PmLEC1 expression in somatic embryos and developing seeds demonstrated a unique, alternating pattern of expression with the highest levels during early stages of embryogenesis. PmLEC1 protein accumulation during seed development correlated with its transcriptional levels during early embryogenesis; however, substantial protein levels persisted until 2 weeks on germination medium. Treatment of mature, stratified seeds with 2,4-epibrassinolide, sorbitol, mannitol, or NaCl upregulated PmLEC1 expression, which may provide strategies to induce SE from mature tissues. Sequence analysis of the PmLEC1 gene revealed a 5' UTR intron containing binding sites for transcription factors (TFs), such as ABI3, LEC2, FUS3, and AGL15, which are critical regulators of embryogenesis in angiosperms. Regulatory elements for these and other seed-specific TFs and biotic and abiotic signals were identified within the PmLEC1 locus. Most importantly, functional analysis of PmLEC1 showed that it rescued the Arabidopsis lec1-1 null mutant and, in the T2 generation, led to the development of embryo-like structures, indicating a key role of PmLEC1 in the regulation of embryogenesis.

Overview of the Genus Boleodorus and First Reports of Boleodorus thylactus and B. volutus from Southern Alberta, Canada.

The present study provides the morphological and molecular characterization of Boleodorus thylactus and B. volutus populations, recovered from agricultural fields of southern Alberta. Despite a significant abundance of this group of nematodes, none of the Boleodorus species were previously reported in Canada. Therefore, representative adult specimens of each population were photographed and examined morphometrically. Phylogenetic trees were reconstructed using partial D2-D3 expansion segments of the 28S and 18S rDNA sequences to understand the relationships of Boleodorus species with Tylenchidae-related genera. Boleodorus species are relevant to soil ecological studies and therefore we summarized the important morphological and morphometric characters in tabular form for easy and efficient species identification. Moreover, we discuss the associated hosts and the distribution of all described Boleodorus species. This study will serve as a guide and basic framework for species diagnostics in the genus Boleodorus and will aid in filling the gaps in our knowledge of the species present in our cultivated lands.

Integrative taxonomy, distribution, and host associations of Geocenamus brevidens and Quinisulcius capitatus from southern Alberta, Canada.

Two stunt nematode species, Geocenamus brevidens and Quinisulcius capitatus, were recovered from the potato growing regions of southern Alberta, described and characterized based on integrative taxonomy. Morphometrics, distribution, and host associations of both species are discussed. The Canadian populations of both species displayed minor variations in morphometrical characteristics (viz., slightly longer bodies and tails) from the original descriptions. The populations of G. brevidens and Q. capitatus species examined in this study are proposed as standard and reference populations for each respective species until topotype specimens become available and molecularly characterized. Phylogenetic analyses, based on partial 18S, 28S, and ITS sequences, placed both species with related stunt nematode species. The present study updates the taxonomic records of G. brevidens and Q. capitatus from a new location, southern Alberta, Canada, and will aid in the decision whether these stunt nematodes should be included in nematode management programs.

Species Diversity of Pin Nematodes (Paratylenchus spp.) from Potato Growing Regions of Southern Alberta, Canada.

Pin nematodes ( Paratylenchus spp.) are polyphagous parasitic species with a wide host range and geographical distribution; their diversity is unknown in the potato growing region of Alberta, Canada. The present study aims to provide morphological and molecular characterization of three pin nematode species, namely P. neoprojectus , P. tateae , and a new species, Paratylenchus enigmaticus sp. nov. All of them were recovered from the potato growing region of southern Alberta. The nematodes were isolated using the sieving and flotation-centrifugation method, and their morphology was assessed by light microscopy. Molecular characterization was performed using partial 18S, D2-D3 expansion domains of the 28S and ITS ribosomal genes. This study is the first report of molecular characterization of P. tateaeand P. neoprojectus , being new records from southern Alberta, and two Spanish populations of P. tateae comprising the first report of this species in Europe. The phylogenetic analysis of the 18S, D2-D3 expansion domains of the 28S and ITS ribosomal DNA regions underscores the importance of using molecular data for accurate species identification and clarifies the status of P. nanus type B and P. sheri . Moreover, our findings will be useful to determine the impact of pin nematodes on potato production in future field research.

Agrobacterium rhizogenes-mediated transformation enhances the antioxidant potential of Artemisia tilesii Ledeb.

Plants belonging to the genus Artemisia L. have been used for medicinal purposes since ancient times. These aromatic plants produce and accumulate a wide range of potent secondary metabolites, many of which have shown antioxidant, antiparasitic, antimicrobial, anti-inflammatory, and even anticancer activities. Enhanced biosynthesis of these compounds is a prerequisite for comprehensive studies of their therapeutic properties and cost-efficient use. Transformation of plants with Agrobacterium rhizogenes native root locus (rol) genes is a promising approach to increase the biosynthesis of plant secondary metabolites. The aim of the present study was to evaluate the effects of A. rhizogenes-mediated transformation on the flavonoid contents in hairy roots of medicinal herb A. tilesii Ledeb. Transgenic A. tilesii hairy root lines were analyzed for stable integration of the rolB and rolC transgenes into the plant genome, total flavonoid contents, antioxidant activities of extracts, and the spatiotemporal expression of two flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL) and chalcone synthase (CHS). The flavonoid contents of A. tilesii directly correlated with the antiradical activity and reducing power of their respective lines, with the greatest antioxidant activity found in the plants with the highest level of total flavonoids. Furthermore, all hairy root lines demonstrated altered expression of plant native PAL and CHS genes. Most importantly, A. rhizogenes-mediated transformation enhanced the biosynthesis of natural antioxidants in A. tilesii, producing almost twice the amount of flavonoids than controls. These findings provide an opportunity for the identification of the bioactive molecules in A. tilesii extracts and their potential health benefits.

Simple sequence repeat analysis of new potato varieties developed in Alberta, Canada.

The worldwide demand for potato production requires the constant development of new potato varieties with improved yield, quality, disease resistance, and abiotic tolerance. However, cultivar registration is preceded by a long process to morphologically and physiologically characterize the plants. Notably, this process can be expedited by DNA marker analysis. Simple sequence repeats (SSRs), also known as microsatellites, are the most common reliable DNA markers used to discriminate between genotypes. In this study, 20 potato varieties, including five new genotypes developed in Alberta, Canada, were fingerprinted using 10 SSR markers selected for their high discriminatory power. Different SSRs were amplified from potato DNA using specific primers, and the DNA fragment sizes were analyzed by denaturing polyacrylamide gel electrophoresis. The number of alleles per locus ranged from two for the SSR marker STPoAc58 to six for STM0030 and STM0037 with an average of 4.4. In addition, a total of 77 unique patterns were observed for the 10 SSR markers. The polymorphic information content ranged from 0.477 to 0.802 with an average of 0.675 per locus. In this study, STM0037, STM1016, and STM1104 were found to be the best SSR markers to detect genetic differences between potato varieties. A minimum of two markers was required to distinguish between all 20 genotypes. Most importantly, this highly informative molecular tool confirmed that the developed potato varieties were genetically different from their respective maternal lines and potentially constituted new cultivars.

Enhancing disease resistance in poplar through modification of its natural defense pathway.

KEY MESSAGE: Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.

Correction to: Spatiotemporal activities of Douglas-fir BiP Pro1 promoter in transgenic potato.

In Table 1, the unit of measure provided for GUS activity in unstressed leaves was not given correctly in the original publication. It should read.

Spatiotemporal activities of Douglas-fir BiP Pro1 promoter in transgenic potato.

MAIN CONCLUSION: The PmBiPPro1 promoter of the luminal binding protein (BiP) from Douglas-fir is fully functional in transgenic potato, responsive to wounding, and has high transcriptional activity in tubers. A predefined pattern and level of transgene expression targeted to specific tissues or organs and at a particular developmental stage is a pre-requisite for the successful development of plants with desired traits. Here, we evaluated the transcriptional activity of the PmBiPPro1 promoter of the luminal binding protein (BiP) from Douglas-fir, by expressing reporter ß-D-glucuronidase (GUS) gene constructs containing three different PmBiPPro1 promoter versions (2258 bp, 1259 bp, and 278 bp) in transgenic potato. In conifers, this promoter regulates the endoplasmic reticulum (ER) molecular chaperon of the HSP70 stress-related protein family and is essential for proper functioning of the ER. Stable expression analysis demonstrated that two of three PmBiPPro1 promoter versions (PmBiPPro1-1 and PmBiPPro1-3) were fully functional in the heterologous host, exhibited high transcriptional activities in the leaves of unstressed potatoes, and were responsive to wounding. Deletion analysis showed that the positive cis-active regulatory elements necessary for higher level expression resided within the - 1243 to - 261 region, whereas negative cis-active elements encompassed nucleotides - 2242 to - 1243. Histochemical staining revealed high level of GUS activities in tissues associated with a high rate of cell division and secretory activities. Most importantly, the PmBiPPro1 promoters, especially the full-length version, had activity in microtubers at a level that was much higher than in any other potato organ or tissue. The - 2242 to - 1243 bp region likely contains important cis element(s) that interact with tuber-specific transcription factors required for promoter activation in the storage organs. The organ-specific activity of the PmBiPPro1 promoters may be useful for targeted expression of heterologous molecules in potato tubers.

Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. x P. maximowiczii A. Henry.

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. x P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the beta-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4-10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1-2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra x P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.

Comparison of pathogen-induced expression and efficacy of two amphibian antimicrobial peptides, MsrA2 and temporin A, for engineering wide-spectrum disease resistance in tobacco.

The rapid accumulation of defensive transgene products in plants only on pathogen invasion has clear advantages over their constitutive synthesis. In this study, two antimicrobial peptides from the skin secretions of frogs, MsrA2 (N-methionine-dermaseptin B1) and temporin A, were evaluated for engineering pathogen-induced disease resistance in plants. Both peptides inhibited plant-specific pathogens in vitro at micromolar concentrations that were not toxic to plant protoplasts. The plant-optimized nucleotide sequences encoding MsrA2 and temporin A were transcriptionally fused to the inducible win3.12T poplar promoter, which had strong systemic activity in response to fungal infection, and introduced into tobacco (Nicotiana tabacum L. cv. Xanthi). Transgene expression was very low in the leaves of unstressed plants; however, it was strongly increased after pathogen challenge or wounding. The pathogen responsiveness of the win3.12T promoter was found to be universal rather than species specific, with high activity in response to all pathogens tested. On induction, the amount of MsrA2 was up to 6-7 microg per gram of fresh leaf tissue. Most importantly, the induced accumulation of MsrA2 and temporin A in transgenic tobacco was sufficient to confer resistance to a variety of phytopathogenic fungi, such as Fusarium solani, F. oxysporum, Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum, the oomycete Pythium aphanidermatum and the bacterium Pectobacterium carotovorum. The accumulation of these peptides in transgenic plants did not alter the normal phenotype of tobacco. Thus, the expression of MsrA2 and temporin A in a pathogen-inducible manner enables the development of tobacco, and possibly other plant species, with wide-spectrum disease resistance, which can reduce the use of pesticides and the associated environmental risks.

Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco.

Expression of defensive genes from a promoter that is specifically activated in response to pathogen invasion is highly desirable for engineering disease-resistant plants. A plant transformation vector was constructed with transcriptional fusion between the pathogen-responsive win3.12T promoter from poplar and the gene encoding the novel cecropin A-melittin hybrid peptide (CEMA) with strong antimicrobial activity. This promoter-transgene combination was evaluated in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) for enhanced plant resistance against a highly virulent pathogenic fungus Fusarium solani. Transgene expression in leaves was strongly increased after fungal infection or mechanical wounding, and the accumulation of CEMA transcripts was found to be systemic and positively correlated with the number of transgene insertions. A simple and efficient in vitro regeneration bioassay for preliminary screening of transgenic lines against pathogenic fungi was developed. CEMA had strong antifungal activity in vitro, inhibiting conidia germination at concentrations that were non-toxic to tobacco protoplasts. Most importantly, the expression level of the CEMA peptide in vivo, regulated by the win3.12T promoter, was sufficient to confer resistance against F. solani in transgenic tobacco. The antifungal resistance of plants with high CEMA expression was strong and reproducible. In addition, leaf tissue extracts from transgenic plants significantly reduced the number of fungal colonies arising from germinated conidia. Accumulation of CEMA peptide in transgenic tobacco had no deleterious effect on plant growth and development. This is the first report showing the application of a heterologous pathogen-inducible promoter to direct the expression of an antimicrobial peptide in plants, and the feasibility of this approach to provide disease resistance in tobacco and, possibly, other crops.

Calcium/calmodulin activation of two divergent glutamate decarboxylases from tobacco.

Glutamate decarboxylase (GAD, EC 4.1.1.15) catalyses the alpha-decarboxylation of glutamate to produce gamma-aminobutyrate (GABA). The nucleotide sequences of two divergent GADs (designated GAD1 and GAD3) were isolated from a Nicotiana tabacum L. cv. Samsun NN leaf cDNA library. Open reading frames indicated that GAD1 encodes a polypeptide of 496 amino acids and has greater than 99% identity with known tobacco GADs, whereas GAD3 encodes a polypeptide of 491 amino acids and has about 14% divergence from known tobacco GADs. Genomic DNA analysis suggested that there are at least four tobacco GAD genes, existing in pairs of highly identical genes. An in vitro assay at pH 7.3 revealed that activities of the recombinant proteins, after isolation from Escherichia coli and partial purification by nickel-affinity chromatography, are 57-133 times the control levels in the presence of 0.5 mM calcium and 0.2 micro M bovine calmodulin.