Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

Grazing effects on plant diversity in the endemic Erica mackayana heathland community of north-west Spain

Background: Large areas of heathland landscapes in Galicia, north-west Spain, have traditionally been extensively grazed by free-ranging cattle and wild ponies. Recently, a large reduction in the number of these larger herbivores has been observed, with unknown consequences for the habitat.

Aims: To evaluate the effects of grazing and herbivore density on plant diversity, community composition and vegetation structure of the endemic wet heathlands dominated by Erica mackayana in Galicia.

Methods: Field sampling of vascular plants, generalised linear models (GLMs), non-metric multidimensional distance scaling (NMDS).

Results: Grazed sites had significantly higher total and rare species richness and diversity than ungrazed sites. Higher densities of cattle resulted in lower numbers of rare species, while wild pony density had no effect on rare species richness. In grazed sites, vegetation was lower with greater variation in height, resulting in greater heterogeneity of the habitat. Precipitation and summer temperatures were related to plant diversity, mainly beta diversity. Soil organic matter negatively correlated with rare species.

Discussion: Grazing, mostly by wild ponies, was demonstrated to be positively related to plant diversity and vegetation structure. Lack of grazing or high cattle densities resulted in a negative effect on total and rare species richness and diversity. Future climate change may negatively affect heathland plant diversity. Galician wild ponies represent a unique case of sustainable management of a wild species and an invaluable cultural heritage. Moreover, they have a significant role in maintaining the endemic E. mackayana heathlands, what would justify specific conservation actions for these large herbivores.     

华南野牡丹科野生花卉种质资源的收集和繁殖

野牡丹科植物主要分布于热带亚热带地区,其中不乏许多有观赏价值的野生花卉.为了发掘其观赏方面的价值,本项目在华南地区调查和引种的基础上,进行了初步地繁殖和示范栽培工作.并从中选出了几个有较大观赏价值的种类供今后开发利用.     

Lectin-like binding and antibiotic sensitivity of enterococci from wild herbivores

Fifty eight enterococcal isolates from wild herbivores were tested for their antibiotic sensitivity pattern and lectin-like binding of extracellular matrix (ECM) and serum proteins. Kanamycin resistance was very frequent; many multiresistant strains were also isolated. All isolates were sensitive to rifampicin. Resistance to gentamicin, novobiocin, and tetracycline was widely distributed in the microflora of wild herbivores breeded in zoological garden in Košice.

No autoaggregating strains were detected among these 58 enterococcal isolates. Various degrees of binding of mucins, fetuin, heparin, fibrinogen, and fibronectin were observed in individual strains. However, bovine lactoferrin binding by enterococci from deers and chamoises was either negative (0) or strongly positive (3).

With regard to influence of growth media, TH agar was found to be better for the expression of lectin-like binding than blood agar, TH broth and Nutrient broth.

A significant effect (P < 0.001 or P < 0.05) of proteolytic treatment was observed in six selected strains. However, there is a difference between the effect of trypsin and pronase P.Pronase treatment more effectively decreased binding of some strains (1H, 6A, EF 1111, EC 1292), while trypsin treatment decreased more binding of other enterococcal strains (EF953 and 1E). Significant (P < 0.001) influence of metaperiodate, which cleaves the C-C bond between vicinal groups of sugars, on collagen I binding by three selected strains (1E, 1H, 6A) and bovine lactoferrin binding (by EF 1111, EC 1292, EF 953) was also observed. However, its influence was very different. In two strains (1H and EC 1292), ECM binding was decreased, while in four other strains (1E, 6A, EF 1111, EF 953) it was increased.     

贵州少数民族地区作物稀有种质资源和野生近缘植物

"贵州农业生物资源调查"项目于2012-2014年已对贵州省21个县(市)进行了系统调查。系统调查的21个县(市)均有少数民族集居,各民族有不同的传统文化和生活习俗,在世代相传中赋予了当地作物种质资源丰富的民族文化内涵。并且,当地的地形地貌复杂,气候类型多样。正因为这种多样的自然条件和各异的民族文化,造就了这里丰富的作物稀有种质资源和野生近缘植物。本次调查获得的作物稀有种质资源和野生近缘植物,对作物的分类研究和新品种选育以及商业开发,将会有一定的利用价值。     

野生动物分子水平研究中的取样方法进展

对野生动物进行分子水平研究时 ,在取材上经常存在野外找不到动物或得到的材料提取不到足够的DNA等困难。但随着PCR技术的产生和DNA提取技术的进步 ,研究材料也不再局限于新鲜的组织样本 ,从而取样的范围扩大到微量的血液、单根的毛发、羽毛、指甲、唾液、粪便、骨骼 ,甚至古代化石等样品 ,研究范围随之也不断扩大。     

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E−. Plants infected by N. lolii exhibited higher production of phenolics relative to the E− plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, β-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P < 0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P < 0.05) and β-caryophyllene (P < 0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants’ protection.     

Rice genetic resources: history,conservation, investigative characterization and use in Japan

Rice has been grown in Japan for about 3000 years. Although both japonica and indica varieties have been grown in Japan, now japonica rices are grown. Japanese rice breeding has used an ecological breeding approach. While emphasis in rice breeding in the 1940's and 1950's focussed on yield in recent decades quality has been of major importance. Consumer preference and name recognition of high quality varieties, such as Koshihikari, has resulted in slow acceptance of new varieties.Rice germplasm was systematically collected throughout Japan between 1962 and 1963. Subsequent acquisition and collecting, in Japan and other countries, has resulted in 28,000 accessions being conserved in the National Genebank, based at the National institute of Agrobiological Resources (NIAR).Research on genetic diversity of rice using a range of techniques, for example esterase isozymes, has revealed clinal variation in rice radiating from the center of diversity of rice in and around southwest China. Newly found genes in traditional rice germplasm, such as genes for non-elongating mesocotyl, are now routinely identified on the rice genome. Pioneering studies on eco-genetic differentiation of species in the genus Oryza in Japan has revealed much about the complex genepool for which rice evolved.Pest and disease resistance sources, particularly to blast, bacterial blight and brown plant hopper, from many countries have been incorporated into Japanese varieties. Cold tolerance at the booting stage was found in the Indonesian variety Silewah. In the future in characterisation of rice germplasm and interaction between rice germplasm specialists and rice molecular scientists, both in Japan and internationally, will be corner stones to securing rice genetic diversity and rice improvement in the next century.     

Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum

Genetic diversity was studied in six subpopulations (a total of 60 individuals) of wild barley, Hordeum spontaneum , the progenitor of cultivated barley, sampled from six stations located along a transect of 300 m across the two opposing slopes of 'Evolution Canyon', a Mediterranean microsite at Lower Nahal Oren, Mt Carmel. The two opposing slopes are separated by between 100 and 400 m and designated SFS (South-Facing Slope) and NFS (North-Facing Slope) with each having three equidistant test stations. The SFS, which receives up to 300% more solar radiation, is drier, ecologically more heterogeneous, fluctuating, and more stressful than the NFS. Analysis of 12 RAPD primers, representing a total of 51 putative loci, revealed a significant inter- and intraslope variation in RAPD band polymorphism. A significantly higher proportion of polymorphic RAPD loci was found amongst the subpopulations on the SFS (mean P = 0.909) than on the NFS (mean P = 0.682), on the basis of the presence and absence of 22 strong bands. Polymorphism generally increased upwards from the bottom to the top of the SFS (0.636, 0.773, 0.955) and NFS (0.409, 0.500, 0.545), respectively. Gametic phase disequilibria estimates, D, revealed SFS and NFS unique predominant combinations which sharply differentiated the two slopes and indicated that there is differential interslope selection favouring slope-specific multilocus combinations of alleles, or blocks of genes over tens to hundreds of meters. This suggests that selection overrides migration. RAPD polymorphism appears to parallel allozyme diversity which is climatically adaptive and driven by natural selection in the same subpopulations at the microsite.     

Distribution of Competition Potential Between Native Ungulates and Free-Roaming Equids on Western Rangelands

Free-roaming equids (i.e., feral horses [Equus caballus] and burros [Equus asinus]) are widely distributed and locally abundant across the rangelands of the western United States. The 1971 Wild Free Roaming Horse and Burro Act (WFRHBA) gave the Bureau of Land Management (BLM) and United States Forest Service (USFS) the legal authority to manage these animals on designated public lands. To fulfill this responsibility, federal agencies established an Appropriate Management Level (AML), defined as the number of horses or burros that can be sustained on a given management unit under prevailing environmental conditions and land uses. Although the WFRHBA specifies that feral equids must be managed in ecological balance with other land uses, including conservation of native wildlife, population control measures such as gathers, contraception, and adoptions have failed to keep pace with intrinsic growth rates. Over 80% of federally managed herds currently exceed prescribed population levels, making the potential for competition between native ungulates and feral equids a growing concern among state wildlife agencies. Mule deer (Odocoileus hemionus), pronghorn (Antilocapra americana), elk (Cervus canadensis), and bighorn sheep (Ovis canadensis) are of ecological and economic value to the states where they occur, and all exhibit some degree of distributional, habitat, or dietary overlap with horses or burros. Notwithstanding the scale of the problem, to date there have been no range-wide assessments of competition potential among native and feral ungulates for space, forage, or water. To address this need, we compiled demographic, jurisdictional, and species occurrence data collected from 2010–2019 by federal and state agencies. We used these data to map the distributions of 4 native ungulate species across federal equid management units (FEMUS) in 10 western states (n = 174). We then made within-state rankings of the 50 units that were ≥2 times over AML and encompassed ≥3 native ungulates. Collectively, FEMUs covered approximately 225,000 km², representing 18% of all BLM and USFS lands in affected states. Each FEMU supported ≥1 native ungulate and 14% contained all 4. The degree of overlap between native and feral species varied by state, ranging from <1% for mule deer in Montana, to 40% for bighorn sheep in Nevada. Oregon had the largest proportion of units that supported all 4 native ungulates (58%), whereas Montana and New Mexico had the fewest equids, but all populations were over target densities. Despite the perception that the problem of equid abundance is limited to the Great Basin states, high intrinsic growth rates and social constraints on management practices suggest all affected states should monitor range conditions and native ungulate demography in areas where forage and water resources are limited and expanding equid populations are a concern. © 2021 The Wildlife Society.