Leaf Tissue Samples Research Articles

Genomic DNA isolation of Acrocomia aculeata (Arecaceae) from leaf and stipe tissue samples for PCR analysis

Macaw palm, Acrocomia aculeata is an oleaginous species of the Arecaceae family; it has been identified as one of the most promising plants for sustainable production of renewable energy, especially biodiesel. We developed an efficient protocol of genomic DNA extraction for A. aculeata using leaf and stipe tissues, based on the cationic hexadecyltrimethylammonium bromide method, and we evaluated the quantity, purity, and integrity of the resultant DNA. We also determined whether these procedures interfere with PCR amplification using SSR molecular markers. The lowest concentration of DNA was obtained from stipe tissues (135 ng/μL), while fresh leaf tissues provided the highest concentration of DNA (650 ng/μL). Good quality DNA was obtained from fresh leaf, lyophilized leaf, and stipe tissues (relative purity, 1.79-1.89 nm). Differences in quantity and quality of DNA extracted from different tissues did not interfere with general patterns of PCR amplification based on SSR markers.

Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae

Peroxiredoxins (Prxs), also termed thioredoxin peroxidases (TPXs), are a family of thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative chloroplastic 2-Cys thioredoxin peroxidase (OsTPX) was identified by proteome analysis from leaf tissue samples of rice (Oryza sativa) seedlings exposed to 0.1 M NaCl for 3 days. To investigate the relationship between the OsTPX gene and the stress response, OsTPX was cloned into the yeast expression vector p426GPD under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD1) promoter, and the construct was transformed into Saccharomyces cerevisiae cells. OsTPX expression was confirmed by semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. OsTPX contained two highly conserved cysteine residues (Cys114 and Cys236) and an active site region (FTFVCPT), and it is structurally very similar to human 2-Cys Prx. Heterologous OsTPX expression increased the ability of the transgenic yeast cells to adapt and recover from reactive oxygen species (ROS)-induced oxidative stresses, such as a reduction of cellular hydroperoxide levels in the presence of hydrogen peroxide and menadione, by improving redox homeostasis. OsTPX expression also conferred enhanced tolerance to tert-butylhydroperoxide, heat shock, and high ethanol concentrations. Furthermore, high OsTPX expression improved the fermentation capacity of the yeast during glucose-based batch fermentation at a high temperature (40 °C) and at the general cultivation temperature (30 °C). The alcohol yield in OsTPX-expressing transgenic yeast increased by approximately 29 % (0.14 g g(-1)) and 21 % (0.12 g g(-1)) during fermentation at 40 and 30 °C, respectively, compared to the wild-type yeast. Accordingly, OsTPX-expressing transgenic yeast showed prolonged cell survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsTPX expression increases acquired tolerance to ROS-induced oxidative stress by improving cellular redox homeostasis and improves fermentation capacity due to improved cell survival during fermentation, especially at a high temperature.

Boron accumulation and tolerance of hybrid poplars grown on a B-laden mixed paper mill waste landfill

Paper mill wastes are a mixture of by-products from pulp production and on-site energy production, consisting of paper mill sludge, ash and cinders. Landfilling of these highly boron (B) and heavy metal laden waste products carries environmental risks. Poplars have been successfully employed in the phytomanagement and hydraulic control of B contaminated sites. Here, we assess the performance of hybrid poplars on a paper-mill waste landfill, investigate the accumulation of B by the trees and explore the relationship between local-scale root growth and substrate properties. Leaf and root tissue samples were collected on three plots and analyzed for their chemical properties and root traits. Additionally, we sampled four soil cores in the vicinity of each of the trees and determined chemical and physical properties. Using a principal component analysis followed by a cluster analysis, we identified three substrate types. This method delineated the soil effects on tree survival and growth, although correlations with individual soil element concentrations were weak. Despite signs of B toxicity in some leaves, B was not the key limiting factor for poplar growth. Instead, Ca deficiency caused by a Mg:Ca imbalance was the primary reason for the poor performance of some trees. Root growth was not limited by toxicity effects of soil contaminants. Our results show that hybrid poplars perform well under the harsh growing conditions on a multi-contaminated, B-laden substrate in a hemiboreal climate. Exploiting the differences in the performance of the four clones in relation to the soil types, could increase the success of revegetation on this and other landfills.

Calcium isotope fractionation in alpine plants

In order to develop Ca isotopes as a tracer for biogeochemical Ca cycling in terrestrial environments and for Ca utilisation in plants, stable calcium isotope ratios were measured in various species of alpine plants, including woody species, grasses and herbs. Analysis of plant parts (root, stem, leaf and flower samples) provided information on Ca isotope fractionation within plants and seasonal sampling of leaves revealed temporal variation in leaf Ca isotopic composition. There was significant Ca isotope fractionation between soil and root tissue \(\Updelta^{44/42}\hbox{Ca}_{\rm root-soil} \approx -0.40\,\permille\) in all investigated species, whereas Ca isotope fractionation between roots and leaves was species dependent. Samples of leaf tissue collected throughout the growing season also highlighted species differences: Ca isotope ratios increased with leaf age in woody species but remained constant in herbs and grasses. The Ca isotope fractionation between roots and soils can be explained by a preferential binding of light Ca isotopes to root adsorption sites. The observed differences in whole plant Ca isotopic compositions both within and between species may be attributed to several potential factors including root cation exchange capacity, the presence of a woody stem, the presence of Ca oxalate, and the levels of mycorrhizal infection. Thus, the impact of plants on the Ca biogeochemical cycle in soils, and ultimately the Ca isotope signature of the weathering flux from terrestrial environments, will depend on the species present and the stage of vegetation succession.

Development of a Model System to Identify Differences in Spring and Winter Oat

Our long-term goal is to develop a Swedish winter oat (Avena sativa). To identify molecular differences that correlate with winter hardiness, a winter oat model comprising of both non-hardy spring lines and winter hardy lines is needed. To achieve this, we selected 294 oat breeding lines, originating from various Russian, German, and American winter oat breeding programs and tested them in the field in south- and western Sweden. By assaying for winter survival and agricultural properties during four consecutive seasons, we identified 14 breeding lines of different origins that not only survived the winter but also were agronomically better than the rest. Laboratory tests including electrolytic leakage, controlled crown freezing assay, expression analysis of the AsVrn1 gene and monitoring of flowering time suggested that the American lines had the highest freezing tolerance, although the German lines performed better in the field. Finally, six lines constituting the two most freezing tolerant lines, two intermediate lines and two spring cultivars were chosen to build a winter oat model system. Metabolic profiling of non-acclimated and cold acclimated leaf tissue samples isolated from the six selected lines revealed differential expression patterns of 245 metabolites including several sugars, amino acids, organic acids and 181 hitherto unknown metabolites. The expression patterns of 107 metabolites showed significant interactions with either a cultivar or a time-point. Further identification, characterisation and validation of these metabolites will lead to an increased understanding of the cold acclimation process in oats. Furthermore, by using the winter oat model system, differential sequencing of crown mRNA populations would lead to identification of various biomarkers to facilitate winter oat breeding.

First Report of Quinoa Downy Mildew Caused by Peronospora variabilis in the United States.

Quinoa, Chenopodium quinoa Willd., is an Andean crop prized for its high nutritional value and adaptability to harsh environments. Quinoa is plagued by downy mildew caused by Peronospora variabilis Gäum (formerly Peronospora farinosa f. sp. chenopodii Byford) (1). Quinoa production has spread beyond native Andean ranges and quinoa downy mildew has been reported in India, Canada, and Denmark (1). During the summer of 2011, quinoa trials were established to determine the ability of quinoa to grow under Mid-Atlantic conditions and monitor for regional disease problems. In July, after cool, rainy conditions, downy mildew-like symptoms were observed on quinoa at research plots in Centre and Lancaster counties of Pennsylvania. Symptoms and signs consisted of irregularly shaped areas of foliar chlorosis or pink discoloration accompanied by dense, gray sporulation on both leaf surfaces. Sporangia were tan to gray-brown, semi-ovoid, often with a pedicel, mean length of 31 μm, and mean width of 23 μm. Sporangiophores branched dichotomously, and the terminal branchlets curved and tapered to a point. Orange oospores were present in field samples of leaf tissue. DNA was extracted from infected foliar tissue and sporangial suspensions. A seminested PCR protocol (2) was used to obtain partial internal transcribed spacer (ITS) sequences of six Peronospora isolates. The sequences shared 99% maximum identity to a known P. variabilis accession (FM863721.2) in GenBank. A voucher specimen was deposited into the U.S. National Fungus Collections (BPI 882064). Pathogenicity of each of two strains of P. variabilis was confirmed by inoculating quinoa with sporangia (4). Sporangia were shaken from leaves in sterile distilled water and the suspension was filtered through cheesecloth. A 0.01% Tween solution was added and the suspension diluted to 103 sporangia/ml. With an atomizer, a 10-ml sporangial suspension (or sterile water for noninoculated control plants) was sprayed onto one flat of 18 2-week-old quinoa plants, and relative humidity was increased to saturation using a humidity dome for 24 h. After 1 week, chlorosis and pink discoloration were noted on leaves of inoculated quinoa, and after 18 h of subsequent increased humidity (>95% relative humidity), dense gray sporulation was observed. No symptoms were noted on noninoculated control plants. Sporangia and sporangiophores were examined morphologically and confirmed to be P. variabilis, confirming Koch's postulates. For culture maintenance, 2-week-old quinoa leaves were placed onto a sporangial suspension on top of 1% water agar and maintained in a growth chamber at 20°C with 16 h of light per day. Quinoa downy mildew is seedborne (3) and initial infections may have occurred from oospores in the pericarp, despite intensive processing of consumable quinoa seeds to remove saponins. To our knowledge, this is the first report of quinoa downy mildew in the United States and also the first report of P. variabilis in the United States.

First Report of Alfalfa mosaic virus Infection in Viburnum tinus in Chile.

Viburnum tinus L., commonly known as laurustinus, is an ornamental shrub that is widely used as a garden plant and flower crop. V. tinus is popular because of its desirable characteristics such as evergreen foliage, tolerance to pruning, winter blooms, and its adaptation to cold temperate zones. It is also relatively easy to grow and is commonly used as a windbreak. Infection of this ornamental species by Alfalfa mosaic virus (AMV) has been associated with yellow mottling or variegated leaf coloring, including light green and white, and has been referred to as the "Viburnum Calico" (1,4). In April 2011, at the onset of winter in the Southern Hemisphere, intense yellow spotting and mottling was observed on V. tinus leaves in the San Joaquin Campus at Pontificia Universidad Catolica de Chile. Presence of Aphis spiraecola Patch was observed on the shrubs, however, in the area it is also common to find other aphid species such as Toxoptera aurantii (Boyer de Fonscolombe) and A. fabae Scopoli. Leaf tissue samples from 10 asymptomatic and 10 symptomatic plants were examined for the presence of AMV by tissue-blot immunoassay with a commercially available polyclonal antibody (Agdia Inc., Elkhart, IN) along with the Goat affinity purified anti-rabbit IgG conjugated (Whole Molecule) (Molecular Probes, Invitrogen Corp., Carlsbad, CA). First-strand cDNA synthesis and PCR were performed with specific primers CP-AMV1 and CP-AMV2 (3). AMV was detected in all symptomatic leaves and also in two of the asymptomatic tissue analyzed by tissue blot assay. Reverse transcription (RT)-PCR produced 753-bp amplicons in all samples that were positive to AMV by tissue printing. No amplification product was observed when water control or seronegative samples were used as templates in the RT-PCR assays. Two amplicons were directly sequenced in both directions to confirm the identification of AMV in the leaf samples. The sequences obtained were homologous and BLASTN analysis of the submitted sequence (GenBank Accession No. JN040542) showed 99% nucleotide sequence identity to an AMV isolate described from Nicotiana tabacum L. (GenBank Accession No. FJ527749). These results demonstrate the presence of AMV in V. tinus in Chile. This pathogen has also been described to be affecting V. tinus in France (1) and V. lucidum Mill. in Spain (2). In Chile, V. tinum is increasingly grown as an ornamental plant. Therefore, care should be taken to ensure that the propagative materials of V. tinum are devoid of AMV infection to prevent further spread of this virus.

Fertilizer Regime and Weed Pressure Minimally Influence Leaf Tissue Nutrient Levels during Cranberry Vine Establishment

Cranberry leaf tissue samples were analyzed annually over a 6-year period. Vines received varied nitrogen (N) fertilizer rates and were subjected to low or high weed pressure. With few exceptions, levels of N, phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), manganese (Mn), iron (Fe), and boron (B) were sufficient during the 6-year period. Calcium and B concentrations in cranberry vines treated with low, medium, or high rates of N were greater in areas with high weed pressure than in areas of low weed pressure. Levels of Zn and Mn were higher and K was lower in tissue collected from high weed areas in 4 out of 6 years. Calcium, Mg, Zn, Mn, B, and Fe levels were negatively correlated with increasing N rate. Overall, application of various N rates did not adversely affect cranberry leaf tissue nutrient content during the first 6 years.

BIOCHEMICAL MARKERS: A USEFUL TOOL FOR ASSESSING GENETIC DIVERSITY IN JACKFRUIT (ARTOCARPUS HETEROPHYLLUS LAM.)

Using polyacrylamide gel electrophoresis, peroxidase and esterase isozymes were investigated from leaf tissue samples of 44 selected (superior) jackfruit genotypes. These selected genotypes were collected following jackfruit descriptor 2000, after surveying 1500 trees in the districts of Nadia, 24 Parganas (N) and Coochbehar in West Bengal. The selected genotypes were analyzed for isozyme variation for peroxidase and esterase. Polymorphism was observed in both the enzyme systems studied, where several bands were found showing similarities and dissimilarities among the selected types. In the peroxidase assay, 7 loci were identified among the 44 genotypes. The relative mobility (R m ) values of the loci ranged between 0.29 and 0.50. Whereas in the esterase assay, 6 loci were identified and the relative mobility (R m ) values of the loci ranged between 0.22 and 0.65. Moreover, it was observed that genotypes collected from nearby locations did not show similar banding patterns, indicating that forces other than geographical origin, such as exchange of genetic stocks, genetic drift, spontaneous variation, natural and artificial selection, cross pollination and seedling origin of the plant are responsible for the genetic diversity.

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

Variation in Oxalic Acid Content among Commercial Table Beet Cultivars and Related Crops

Oxalic acid (C2O42–) is a compound of interest as a result of its relationship with kidney stone formation and antinutritive properties. Because table beet [Beta vulgaris ssp. vulgaris (garden beet group)] is considered a high oxalate food, breeding to decrease oxalic acid levels is an area of interest. In this study, a field trial was conducted over 2 years for 24 members of the Chenopodiaceae using two different planting dates to determine if variation exists for both total and soluble oxalic acid levels in roots and leaves. Total and soluble oxalic acid was extracted from homogenized root core and leaf tissue samples and a colorimetric enzymatic assay was used to determine total and soluble oxalic acid levels. Mean values ranged from 722 to 1909 mg/100 g leaf tissue and 553 to 1679 mg/100 g leaf tissue for total and soluble oxalate levels, respectively. Beet cultivar Forono and swiss chard [B. vulgaris ssp. vulgaris (leaf beet group)] cultivar Burpee's Fordhook Giant Chard produced the respective highest and lowest soluble and total oxalic acid leaf levels. Swiss chard cultivars produced 38% less total oxalate compared with table beet cultivars based on overall means. Root soluble oxalate values ranged from 103 to 171 mg/100 g root tissue and total values ranged from 95 to 142 mg/100 g root tissue. Significant variation for both total and soluble oxalic acid levels were detected, indicating progress could be made toward breeding for lower oxalic acid levels in table beet. However, gains in oxalic acid nutritional quality may be limited because it would take a substantial decrease in levels for table beet to be reclassified as a low oxalate food.

Inter Simple Sequence Repeats (ISSR) Reveal Genetic Variation Among Mid-Atlantic Populations of Threatened Amaranthus pumilus and Phylogenetic Relationships

Abstract Amaranthus (Amaranthaceae) is a globally distributed plant genus composed of both weedy and cultivated species. While there have been previous attempts to resolve phylogenetic relationships within the genus, little attention has been placed on systematic relationships of the federally threatened coastal species Amaranthus pumilus Raf., endemic to eastern United States barrier islands, nor on genetic variability within the genus. In the present study, single primer ISSRs were employed to measure both genetic diversity and the phylogenetic position of A. pumilus. Leaf tissue samples were taken from wild populations on Fenwick Island, Delaware and from wild and propagated populations on Assateague Island National Seashore, Maryland. Genetic variation was detected among and within A. pumilus populations, though variability was low. Fenwick populations exhibited the highest genetic variability (h = 0.1016), while on Assateague the wild A. pumilus population had higher variability (0.0340) than the p...

MARCADORES RAPDs ASOCIADOS A LA EXPRESIÓN DEL SEXO EN Ceratozamia mexicana Brongniart (Zamiaceae)

Ceratozamia mexicana Brongniart is a strictly dioecious species with a long juvenile stage. In order to detect molecular markers associated with sex, it was analyzed by the technique of random amplified polymorphic DNA (RAPD) and masal segregant analysis (BSA) samples of leaf tissue of leaf tissue of sexually differentiated individuals of the population of this species located at Coacoatzintla, Veracruz, México. 5 primers were used to amplify three bulk DNA samples of five known individuals from each sex. A total of 37 bands were produced of which 63% were polymorphic. The OPB08 and OPK10 primers generated distinctive bands to male sex in C. mexicana. With the OPK10 primer a band of 500 bp was observed in two of the three male bulk samples tested, while the OPB08 primer detected two bands (1400 and 1100 bp) in the male samples tested. Although it is necessary to validate this observation, these results could open a new alternative to early determination of sex in C. mexicana.

INDEXING TISSUE CULTURE BANANA (MUSA SPP.) SEEDLINGS FOR BANANA STREAK VIRUS WITH THE AIM OF ESTABLISHING A CLEAN MOTHER BLOCK

A total of 1137 tissue culture banana (Musa spp.) seedlings were collected between May and July 2007 from nurseries of the Thika National Horticulture Research Centre of the Kenyan Agricultural Research Institute (KARI), Jomo Kenyatta University of Applied Technology and Agro-Genetic Technologies. The seedlings were approximately 3 months old, since potting. Leaf samples from the seedlings were indexed for Banana streak virus (BSV) at KARI-Kabete laboratory. Immuno-capture PCR specific primers to BSV-GF and BSV-Cav were used. Of the 1137 seedlings indexed, 120, 175, 112, 200, 130, 150, 100, 75 and 75 were of cultivar 'Dwarf Chinese' (AAA genome), 'FHIA 18' (AAAB genome), 'Solio' (AAA-EA genome), 'FHIA 17' (AAAA genome), 'Nusu Ng'ombe' (AAA-EA genome), 'Grand Naine' (AAA genome), 'Williams' (AAA genome), 'Uganda Green' (AAA genome) and 'Chinese Cavendish' (AAA genome), respectively. A total of 265 samples from the nine cultivars tested positive for BSV. 'Dwarf Chinese', 'Grand Naine' and 'Williams' cultivars tested positive both for BSV-Cav and BSV-GF. The PCR protocol for detection of BSV was validated. Validation tests were conducted, utilizing fresh banana leaf tissue samples from cultivars 'Lisulya' and 'Khabusi Mboki' (AAA-EA genome), greenhouse tissue cultured plantlets of 'Dwarf Chinese' and 'Ex-Rongai' (AAA genome), a positive control, consisting of freeze-dried banana leaves from Queensland University of Technology (QUT), and a PCR positive control plasmid DNA sample, also from QUT. Extraction of DNA was done using Qiagen DNeasy plant mini kit to generate a DNA template for PCR amplification.

Molecular Genetic Characterization of Olpidium virulentus Isolates Associated with Big-Vein Diseased Lettuce Plants.

Lettuce plants showing symptoms of lettuce big-vein disease were collected from fields in the Perth Metropolitan region of southwest Australia. When root extracts from each plant were tested by polymerase chain reaction (PCR) using primers specific to the rDNA internal transcribed spacer (ITS) region of Olpidium brassicae and O. virulentus, only O. virulentus was detected in each of them. The nucleotide sequences of the complete rDNA ITS regions of isolates from five of the root samples and 10 isolates of O. virulentus from Europe and Japan showed 97.9 to 100% identities. However, with the six O. brassicae isolates, their identities were only 76.9 to 79.4%. On phylogenetic analysis of the complete rDNA-ITS region sequences of the five Australian isolates and 10 others, the Australian isolates fitted within two clades of O. virulentus (I and II), and within clade I into two of its four subclades (Ia and Id). Japanese isolates had greatest sequence diversity fitting into both clades and into all of clade I subclades except Ib, while European isolates were restricted to subclades Ib and Id. When the partial rDNA-ITS region sequences of two additional southwest Australian isolates, four from Europe, and four from the Americas were included in the analyses, the Australian isolates were within O. virulentus subclades Ia and Id, the European isolates within subclade Ic, and the American isolates within subclades Ia and Ib. These findings suggest that there may have been at least three separate introductions of O. virulentus into the isolated Australian continent since plant cultivation was introduced following its colonization by Europeans. They also have implications regarding numbers of different introductions to other isolated regions. Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus were both detected when symptomatic lettuce leaf tissue samples corresponding to the root samples from southwest Australia were tested using virus-specific primers in reverse transcription-PCR, so presence of both viruses was associated with O. virulentus occurrence.

An efficient high‐throughput flow cytometric method for estimating DNA ploidy level in plants

We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating DNA ploidy using a plate loader on a BD FACS-Canto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone sample handling. In two 96-well plates of samples, the histogram peaks of DNA content from flow cytometry were wellresolved in 189 of 192 samples tested (98.4%), with CV values ranging from 2.98% to 6.20% with an average CV of 4.35% (SD = 0.68%). This new method is useful in doubled haploid plant breeding programs where early discrimination of haploid and doubled haploid (i.e., diploid) plantlets can confer significantly improved operational efficiencies. We discuss how this method could be further refined including adapting the method to robotic sample processing.

Insufficient evidence for the discovery of transgenes in Mexican landraces

In the recently published article ‘Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations’ by Pineyro-Nelson et al. (2009), the authors report the finding of transgenes in Oaxacan maize landraces and make several claims to which we feel compelled to respond. The authors maintain that testing of leaf tissues in general, and in particular at Genetic ID (GID), is inclined to false negative results and that the presence of metabolites in landraces could lead to the reporting of false negatives. Much of the data reported by Pineyro-Nelson et al. is from end point PCR analyses of leaf material from single plants. We suggest and provide evidence that, for a study of such importance, conclusions must be drawn from real time qPCR analyses. Genetically any given plant should be either non-transgenic or transgenic, therefore for leaf tissue of a single transgenic plant, a GMO level close to 100% is expected. In their study, the authors chose to classify leaf samples as transgenic despite GMO levels of 0.1%. We contend that results such as these are incorrectly interpreted as positive and are more likely to be indicative of contamination in the laboratory. The authors claim that analysis of lyophilized leaf tissue can lead to reporting of false negative results. Real time PCR analysis of dried leaf tissue samples from three transgenic plants (MON810, NK603, Bt11) verified that transgenic corn can be detected in such samples. Dried leaves were stored at GID for 3 years at room temperature prior to testing. DNA was extracted from 10 mg of leaf material, and the transgenic events were successfully detected using endpoint PCR. Subsequent analysis of the DNA by real time qPCR confirmed the expected GMO value of 100% for all three of the transgenic plants for the 35S promotor, NOS terminator and the specific event. Identical results were obtained from a MON810 dried leaf tissue positive control which the authors sent to us in a blinded fashion in 2005 (i.e. 100% GMO value for the 35S promoter target gene and correct identification of the MON810 event).

First Report of a New Phytoplasma Subgroup, 16SrIII-T, Associated with Decline Disease Affecting Sweet and Sour Cherry Trees in Lithuania

During July 2007, sweet (Prunus avium) and sour cherry (P. cerasus) trees exhibiting disease symptoms suggestive of possible phytoplasma infection were observed in a large orchard in the Kaunas Region of Lithuania. Samples of leaf tissue were collected from 13 sweet cherry trees that were affected by a decline disease (designated cherry decline, ChD) characterized by symptoms that included leaf reddening and premature leaf drop and two sour cherry trees exhibiting proliferation of branches and nonseasonal flowering. To assess the diseased trees for phytoplasma infection, DNA was extracted with a Genomic DNA Purification Kit (Fermentas, Vilnius, Lithuania) and used as template in nested PCRs, primed by phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 for amplification of 16S ribosomal (r) DNA sequences (1,2). The 1.2-kbp DNA sequences amplified from all 15 trees were subjected to restriction fragment length polymorphism (RFLP) analyses with AluI, MseI, KpnI, HhaI, HaeIII, HpaII, RsaI, HinfI, TaqI, Sau3AI, and BfaI. The collective profiles indicated that DNAs were derived from two different phytoplasmas. One of them, designated ChD phytoplasma, was found in 11 sweet cherry trees and two sour cherry trees and tentatively classified as a member of new subgroup designated 16SrIII-T in 16S rDNA RFLP group 16SrIII (X-disease phytoplasma group). It was observed that the ChD phytoplasma caused different symptoms in sweet and sour cherry. The amplified ChD phytoplasma 16S rDNA was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank database (Accession No. FJ231728). The ChD phytoplasma 16S rDNA shared 98.4 and 98.6% sequence identity with the 16S rDNAs from stone fruit-infecting phytoplasmas associated with western X-disease (GenBank Accession No. L04682) and Canada X-disease (GenBank Accession No. L33733), respectively, indicating that the three strains are closely related. Interestingly, the ChD phytoplasma 16S rDNA shared 99.8% sequence identity with 16S rDNA from one operon (rrnB, GenBank Accession No. AF370120) from a phytoplasma previously found to be associated with dandelion virescence (DanVir) disease in Lithuania. The operon rrnA (GenBank Accession No. AF370119) shared 99.3% sequence identity (2). The high similarity of the ChD 16S rRNA gene sequence to that of DanVir rrnB suggests the possibility that ChD and DanVir may belong to a single phytoplasma species and that dandelion is possibly an alternate host of ChD phytoplasma. The other phytoplasma, found in two sweet cherry trees, was classified in subgroup 16SrI-B of 16S rDNA RFLP group 16SrI ('Candidatus Phytoplasma asteris' and related strains) and was designated cherry proliferation phytoplasma (GenBank Accession No. FJ231729). Thus, in Europe, cherry may be affected by diseases associated with phytoplasmas belonging to groups 16SrI, 16SrIII, 16SrX, and 16SrXII (3,4). The infections by diverse phytoplasma strains and species underscore the need for production of phytoplasma-free planting stock and for intensified research to reduce ecological and economic impacts of these phytoplasmas.

Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

Incidence, Distribution, and Association of Spongospora subterranea and Potato mop-top virus in Costa Rica.

A survey was conducted in 39 potato (Solanum tuberosum) fields in Costa Rica to determine incidence and association of Spongospora subterranea f. sp. subterranea and Potato mop-top pomovirus (PMTV). The fields were located in Costa Rica's two major potato-production regions and were further characterized by their altitude. In all, 633 paired samples of leaf tissue and corresponding tubers were collected, assessed visually for disease, and subsequently assayed by enzyme-linked immunosorbent assay (ELISA). S. subterranea presence in tuber tissue was tested by double-antibody sandwich (DAS)-ELISA and PMTV presence in leaf and tuber tissues was tested by triple-antibody sandwich (TAS)-ELISA. Moreover, soil samples were collected from 10 fields surveyed and were evaluated for both pathogens via ELISA and bioassay. The incidence of both diseases ranged from 0 to 100% within individual fields, with incidences lower than 40% occurring in more than 70% of the fields. Higher incidences were found in fields located at higher altitudes. Of the 633 paired samples, 179 and 146 were positive for PMTV and S. subterranea, respectively, according to ELISA in either the foliage or tubers. A low correlation was found for PMTV visual symptoms and ELISA test results. Only 14 of the 81 foliar samples testing positive for PMTV had visual symptoms; the remaining 67 samples were asymptomatic. Conversely, comparison of visual evaluation with detection of S. subterranea by ELISA on tubers showed that 70% of the results were coincident. S. subterranea was detected in 4 of 10 soil samples tested by ELISA. Soilborne PMTV was detected by ELISA in roots of bait plants sown in these soil samples. Co-occurrence of both pathogens was detected in 64 samples. A significant but low degree of association for vector and virus was determined, and data suggests that S. subterranea is participating in the transmission of PMTV in Costa Rica in low frequency.