Spatial prediction of caterpillar (<Emphasis Type="Italic">Ormiscodes</Emphasis>) defoliation in Patagonian <Emphasis Type="Italic">Nothofagus</Emphasis> forests

In the temperate forests of the southern Andes, southern beech species (Nothofagus), the dominant tree species of the region, experience severe defoliation caused by caterpillars of the Ormiscodes genus (Lepidoptera: Saturniidae). Despite the recent increase in defoliation frequency in some areas, there is no quantitative information on the spatial extent and dynamics of these outbreaks. This study examines the spatial patterns of O. amphimone outbreaks in relation to landscape heterogeneity. We mapped defoliation events caused by O. amphimone in northern (ca. 40–41°S) and southern Patagonian (ca. 49°S) Nothofagus forests from Landsat imagery and analyzed their spatial associations with vegetation cover type, topography (elevation, slope angle, aspect) and mean annual precipitation using overlay analyses. We used these data and relationships to develop a logistic regression model in order to generate maps of predicted susceptibility to defoliation by O. amphimone for each study area. Forests of N. pumilio are typically more susceptible to O. amphimone outbreaks than lower elevation forests of other Nothofagus species (N. dombeyi and N. antarctica). Stands located at intermediate elevations and on gentle slopes (<15°) are also more susceptible to defoliation than higher and lower elevation stands located on high angle slopes. Stands in areas with intermediate to high precipitation, relative to the distribution of Nothofagus along the precipitation gradient, are more susceptible to O. amphimone attack than are drier areas. Our study represents the first mapping and spatial analysis of insect defoliator outbreaks in Nothofagus forests in South America.     

Transient co-expression with three <Emphasis Type="Italic">O</Emphasis>-glycosylation enzymes allows production of GalNAc-<Emphasis Type="Italic">O</Emphasis>-glycosylated Granulocyte-Colony Stimulating Factor in <Emphasis Type="Italic">N. benthamiana</Emphasis>

Background

Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.

Results

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.

Conclusions

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

     

The Genus <Emphasis Type="Italic">Fragaria </Emphasis>in Croatia

Only three native species of genus Fragaria (F. moschata, F. vesca and F. viridis) are recorded in three regions of Croatia. These species as well as many of their hybrids, are, or once were, cultivated for their edible fruits. The majority of cultivated strawberries in Europe belong to garden strawberries F. x ananassa (hybrids of F. chiloensis and F. virginiana). The most expanded wild strawberry species in Croatia is a woodland strawberry (F. vesca) whose berries are gathered seasonally as wild edible fruits. They often contain higher amount of nutrients and bioactive compounds in comparison to cultivated varieties. The research on the genus Fragaria species distribution in Croatia has not been carried out, and so is the case with many others wild growing fruit species in Croatia. By summing up a number of individual citations and observations, it is possible to get a perspective regarding the current state of their distribution.     

Optimization of Factors Affecting <Emphasis Type="Italic">In vitro</Emphasis> Establishment, <Emphasis Type="Italic">Ex vitro</Emphasis> Rooting and Hardening for Commercial Scale Multiplication of Silk Banana (<Emphasis Type="Italic">Musa</Emphasis> AAB)

Factors concerning aseptic culture establishment and hardening were studied in detail in choicest Silk Banana. Effect of size of sucker (small, medium and large), carbon concentration (10, 20 and 30 g/l), season of initiation (wet and dry) and pre-treatments such as segmentation and incision to the explants were studied during initiation. Further, hardening related factors such as substrates used for primary and secondary hardening, nursery nutrition (source and frequency of application) and pre-treatments for ex vitro rooting were also studied. Results revealed that small suckers were most suited for initiations with the least contamination, maximum establishment and higher percentage of greening. Lower concentration of sucrose, though delayed greening, resulted in 100?% establishment of explants. Initiations performed during the drier period were completely free from the fungal contamination and showed less bacterial contamination than those performed during the rainy season. Segmentation of explant into four parts during first subculture supported maximum shoot proliferation by overcoming apical dominance. Coir pith was observed to be the most congenial substrate during primary hardening, whereas coir pith alone or sand: red earth: coir pith (1:1:1) supported superior performance of plantlets during secondary hardening. Single application of mono ammonium phosphate improved growth of plantlets during secondary hardening. Pre-treatment with mono ammonium phosphate gave the best response in terms of rooting and hardening of un-rooted micro-shoots. Thus, the discussed methodology could help the industries to take up commercial scale propagation of Silk Banana.     

Timing of breeding in <Emphasis Type="Italic">Ochrotomys nuttalli</Emphasis> and <Emphasis Type="Italic">Peromyscus leucopus</Emphasis> is related to a latitudinal isotherm

Previous comparative studies on patterns of reproduction in small-mammal species focus primarily on latitudinal differences in average litter size. Few studies compare reproductive patterns among northern and southern populations at the landscape scale. Our study compares differences in seasonal patterns of reproduction in northern and southern populations of the golden mouse, Ochrotomys nuttalli, and the white-footed mouse, Peromyscus leucopus. These are remarkably similar species with regard to bioenergetics, body mass, feeding behavior, home-range size, natural history, nest-site preference, and periods of activity. Both species also exhibit very similar intraspecific seasonal patterns of reproduction across their respective geographic ranges. We found that O. nuttalli and P. leucopus switch from a summer breeding season, extending from late spring through early autumn in the north to a winter breeding season extending from late autumn through early spring in the south, near the isotherm where mean annual temperature is 15.6°C (60°F), or approximately 35° N latitude. This latitudinal isotherm provides a geographic benchmark to address future changes in patterns of reproduction attributed to climate change. Findings also suggest that length of the breeding season and patterns of reproduction between species partially explain why P. leucopus is typically more abundant than O. nuttalli in similar habitat types.     

Study of Genetic Diversity of Pear Genotypes and Cultivars (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) Using Inter<Emphasis Type="Italic">-</Emphasis>Simple Sequence Repeat Markers (ISSR)

ISSR molecular marker was used to investigate genetic diversity of ‘Dare Gazi’ genotypes of Mashhad Esteghlal orchard and its relationship with other commercial and native cultivars of pear. Among ‘Dare Gazi’ genotypes of Mashhad Esteghlal orchard 23 genotypes were selected base on difference in tree vigor, leaf color, shape and color of fruit and also 33 other commercial and native pear cultivars from Esteghlal orchard and other Mashhad commercial orchards were studied. A total number of 230 DNA fragments were obtained using 11 primers of which 225 were polymorphic. On average, each primer produced 20.9 bands. Dice similarity coefficient ranged from 0.27 (between ‘Dom Kaj’ and Asian pear) to 1 (between ‘Dare Gazi’ 1 and 2 genotypes). Sample cluster dendrogram indicated that 56 genotypes were divided into 12 distinct clusters. The dendogram generated on the principle of Unweight Pair Wise Method using Arithmetic Average (UPGMA) was constructed by Dice coefficient and the genotypes were grouped into 12 clusters. ‘Dare Gazi’ genotypes did not show 100% similarity due to seed propagation or mutation, as ‘Dare Gazi’ 3 and 18 genotypes had the lowest similarity coefficient (0.64). Asian pears were placed in a separate group from European pears. And ‘Dare Gazi’ genotypes from different orchards were grouped separately, but all of them are called as ‘Dare Gazi’ pear for convenience. ISSR molecular marker can well identify the genetic variability among genotypes and cultivars and found suitable for grouping them.     

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the <Emphasis Type="Italic">Solanaceae</Emphasis> and <Emphasis Type="Italic">Rosaceae</Emphasis> families

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.     

High-level diterpene production by transient expression in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Background

Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.

Results

Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm².

Conclusion

With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

     

Freeze Injure and Antioxidant Enzyme Activity of Grapevine (<Emphasis Type="Italic">Vitis Vinifera</Emphasis>) Under Bio-Boron Fertilizer Applications

This study was conducted to determine the effects of plant growth promoting bacteria (PGPR) and boron (Bio-B) on the plant freeze injury, antioxidant enzyme activity and fruit yield of grapevine cultivar. The experimental plot was a completely randomized design with 5 replicates. As a trial model Bio-B and control were used as fertilizer agent. Bio-B fertilizer has been applied in different ways as soil?+?foliar, soil and foliar application methods to grapevine plants. Data through 2 years trials results showed that the use of Bio-B significantly decreased freeze injury and increased antioxidant enzyme activity of grapevine leaf. The highest damage rate at control group occurred with ?20?^°C and 94.89% ratio. In the same temperature degree at grape plant, the damage ratio of the Bio-B application from soil decreased by 21.55%; the application from leaf by 25.53% and with the application from soil?+?leaf by 26.24%. In addition to this, compared with control, in the CAT, POD and SOD enzyme activities increases occurred with the ratios as 28.57%, 22.05% and 39.25%, respectively. Generally, as results of this study under field conditions, Bio-B fertilizer application decreased freeze injure and increased antioxidant enzyme activity of the grapevine plant.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence,under half of the adaptive growth-irradiance,for high-throughput sensing of leaf-water deficit in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> accessions

Background

Non-invasive and high-throughput monitoring of drought in plants from its initiation to visible symptoms is essential to quest drought tolerant varieties. Among the existing methods, chlorophyll a fluorescence (ChlF) imaging has the potential to probe systematic changes in photosynthetic reactions; however, prerequisite of dark-adaptation limits its use for high-throughput screening.

Results

To improve the throughput monitoring of plants, we have exploited their light-adaptive strategy, and investigated possibilities of measuring ChlF transients under low ambient irradiance. We found that the ChlF transients and associated parameters of two contrasting Arabidopsis thaliana accessions, Rsch and Co, give almost similar information, when measured either after ~20 min dark-adaptation or in the presence of half of the adaptive growth-irradiance. The fluorescence parameters, effective quantum yield of PSII photochemistry (Φ_PSII) and fluorescence decrease ratio (R _FD) resulting from this approach enabled us to differentiate accessions that is often not possible by well-established dark-adapted fluorescence parameter maximum quantum efficiency of PSII photochemistry (F _V/F _M). Further, we screened ChlF transients in rosettes of well-watered and drought-stressed six A. thaliana accessions, under half of the adaptive growth-irradiance, without any prior dark-adaptation. Relative water content (RWC) in leaves was also assayed and compared to the ChlF parameters. As expected, the RWC was significantly different in drought-stressed from that in well-watered plants in all the six investigated accessions on day-10 of induced drought; the maximum reduction in the RWC was obtained for Rsch (16%), whereas the minimum reduction was for Co (~7%). Drought induced changes were reflected in several features of ChlF transients; combinatorial images obtained from pattern recognition algorithms, trained on pixels of image sequence, improved the contrast among drought-stressed accessions, and the derived images were well-correlated with their RWC.

Conclusions

We demonstrate here that ChlF transients and associated parameters measured even in the presence of low ambient irradiance preserved its features comparable to that of measured after dark-adaptation and discriminated the accessions having differential geographical origin; further, in combination with combinatorial image analysis tools, these data may be readily employed for early sensing and mapping effects of drought on plant’s physiology via easy and fully non-invasive means.

     

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis>)

Background

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.

Results

After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N₆ macronutrient/B₅ micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.

Conclusion

The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

     

Isolation of intact sub-dermal secretory cavities from <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.     

Shaken but not stirred: multiscale habitat suitability modeling of sympatric marten species (<Emphasis Type="Italic">Martes martes</Emphasis> and <Emphasis Type="Italic">Martes foina</Emphasis>) in the northern Iberian Peninsula

Context

Multispecies and multiscale habitat suitability models (HSM) are important to identify the environmental variables and scales influencing habitat selection and facilitate the comparison of closely related species with different ecological requirements.

Objectives

This study explores the multiscale relationships of habitat suitability for the pine (Martes martes) and stone marten (M. foina) in northern Spain to evaluate differences in habitat selection and scaling, and to determine if there is habitat niche displacement when both species coexist.

Methods

We combined bivariate scaling and maximum entropy modeling to compare the multiscale habitat selection of the two martens. To optimize the HSM, the performance of three sampling bias correction methods at four spatial scales was explored. HSMs were compared to explore niche differentiation between species through a niche identity test.

Results

The comparison among HSMs resulted in the detection of a significant niche divergence between species. The pine marten was positively associated with cooler mountainous areas, low levels of human disturbance, high proportion of natural forests and well-connected forestry plantations, and medium-extent agroforestry mosaics. The stone marten was positively related to the density of urban areas, the proportion and extensiveness of croplands, the existence of some scrub cover and semi-continuous grasslands.

Conclusions

This study outlines the influence of the spatial scale and the importance of the sampling bias corrections in HSM, and to our knowledge, it is the first comparing multiscale habitat selection and niche divergence of two related marten species. This study provides a useful methodological framework for multispecies and multiscale comparatives.

     

Gap Crossing Decisions by Reed Warblers (<Emphasis Type="Italic">Acrocephalus Scirpaceus</Emphasis>) in Agricultural Landscapes

To meet the need for research on the requirements for corridors for marshland birds, this study set out to quantify gap crossing decisions made by reed warblers moving through the landscape. In three experiments, reed warblers were released into landscape situations with different gap sizes and their movement towards reed patches fringing a watercourse were monitored. In all experiments, most birds flew over the smallest gap towards the nearest reed patch. In the experiment with two gap sizes, the probability of crossing a gap was a function of the ratio between distances to the reed patches. In the experiment with increasing gap sizes, most birds crossed the smaller gaps frequently. Near the bigger gaps, birds did not cross the gaps; instead, they only crossed the watercourse repeatedly. In the third experiment with more realistic landscape configurations, the birds preferred nearby non-reed landscape elements to more distant reed patches. It is concluded that reed warblers were reluctant to cross gaps wider than 50 m. The results suggest that the presence and size of gaps in reed patches affect reed warblers’ local gap-crossing decisions: when given a choice, the birds prefer to cross the smallest gap. Furthermore, reed warblers may be directed towards suitable marshlands by creating corridors of reed vegetation with gaps no wider than 50 m. The surrounding agricultural landscape and the presence of trees and ditches could decrease the reluctance to cross gaps in corridors.     

A plastome primer set for comprehensive quantitative real time RT-PCR analysis of <Emphasis Type="Italic">Zea mays</Emphasis>: a starter primer set for other <Emphasis Type="Italic">Poaceae</Emphasis> species

Background  

Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species.     

Analysis of Genetic Relationships Between Wild Roses (<Emphasis Type="Italic">Rosa</Emphasis> L. Spp.) Growing in Turkey

Rosa L. taxa are dominantly used for horticulture, food, medicinal, ornamental and aesthetic purposes. Rosa is represented with approximately 200 wild species in the world. The number of natural Rosa species in Turkey is reported as about 31. Rosa species are widely distributed in all regions in Turkey. In this study, RAPD and ISSR markers were applied to assess genetic diversity and genetic relationships among 27 species of Rosa. The UPGMA cluster was constructed using a combination of data from RAPD and ISSR markers. The dendrogram revealed two main clusters. Each cluster was divided into subgroups. This investigation showed that genetic distance was relatively significant among the species. As the results of the study close genetic relationships were found between evolutionary relations, morphplogical properties, and phytogeographical distributions of the Rosa species, The results also propose that RAPD and ISSR markers are useful tools for indicating genetic relationships among Rosa genotypes.     

Rapid metabolic profiling of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> defence responses against <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> using direct infrared laser desorption ionization mass spectrometry and principal component analysis

Background  

Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.     

Genome-wide identification of microsatellites in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) using FIASCO and phpSSRMiner

Background  

Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.     

Editing of the urease gene by CRISPR-Cas in the diatom <Emphasis Type="Italic">Thalassiosira pseudonana</Emphasis>

Background

CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results

A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.

Conclusions

CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

     

Fire severity and seed source influence lodgepole pine (<Emphasis Type="Italic">Pinus contorta</Emphasis> var. <Emphasis Type="Italic">murrayana</Emphasis>) regeneration in the southern cascades,Lassen volcanic National Park,California

Rocky Mountain lodgepole pine, (Pinus contorta var. latifolia) regenerates quickly after high severity fire because seeds from serotinous cones are released immediately post-fire. Sierra lodgepole pine (P. contorta var. murrayana) forests burn with variable intensity resulting in different levels of severity and because this variety of lodgepole pine does not have serotinous cones, little is known about what factors influence post-fire regeneration. This study quantifies tree regeneration in a low, moderate, and high severity burn patch in a Sierra lodgepole forest 24 years after fire. Regeneration was measured in ten plots in each severity type. In each plot, we quantified pre- and post-fire forest structure (basal area, density), counted and aged tree seedlings and saplings of all species, and measured distance to the nearest seed bearing tree. There was no difference in the density of seedlings and saplings among severity classes. Distance and direction to the nearest seed bearing lodgepole pine were the best predictors of lodgepole seedling and sapling density in high severity plots. In contrast to Rocky Mountain lodgepole pine, regeneration of Sierra lodgepole pine appears to rely on in-seeding from surviving trees in low or moderate severity burn patches or live trees next to high severity burn patches. Our data demonstrate that Sierra lodgepole pine follows stand development pathways hypothesized for non-serotinous stands of Rocky Mountain lodgepole pine.