Effect of herbivore stress on transgene behaviour in maize crosses with different genetic backgrounds: cry1Ab transgene transcription, insecticidal protein expression and bioactivity against insect pests.

Background: Decades after their first commercial release, many theoretical assumptions are still taken for granted in the deployment of genetically modified (GM) crops. Theoretically, in the case of maize, active transcription of the cry1Ab transgene would result in dose-dependent production of the insecticidal Cry1Ab protein, which would in turn induce dose-dependent mortality on lepidopteran pests. We produced data to realistically approach this question by using a model that includes two genetic background contexts from two geographical provenances in Brazil and South Africa, and two lepidopteran pests (Helicoverpa armigera and Spodoptera littoralis). However, in this study, the effect of insect herbivory was superimposed to investigate possible stress-induced effects in transgene expression at three levels: mRNA, protein and bioactivity. Results: Overall, we found that herbivore damage by H. armigera was reflected only at the translational level, with a higher level of Cry1Ab protein measured in the Brazilian crosses under herbivore stress. On the other hand, compared to non-stress growing conditions, the herbivore damage by S. littoralis was not directly reflected in mRNA, protein or bioactivity in the South African crosses. Conclusions: The differences between South African and Brazilian genetic backgrounds, and between the stressor effect of the two herbivores used, highlight the complexity of transgene expression at the agroecological level. Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-023-00815-3.

Transgene behavior in Zea mays L. crosses across different genetic backgrounds: Segregation patterns, cry1Ab transgene expression, insecticidal protein concentration and bioactivity against insect pests.

Brazil and South Africa, countries with economies in transition, are characterized by a dual agrarian structure co-occurring, sometimes, alongside in the same region. Large-scale commercial farming produces crops for export to global markets on the one hand, and small-scale farming, on the other hand, provides for subsistence and local markets. In both systems, maize (Zea mays) is a key crop for these two countries. For the commercial system, maize is a commodity crop while for the small-scale system it is the prime staple crop. In commercial systems, farmers predominantly grow genetically modified (GM) hybrid maize. In small-scale systems, farmers rely on open pollinated varieties (OPVs) and/or landraces and are largely dependent on seed saving systems. The aim of this study was to understand the relationship between transgene expression rates, the resulting concentrations of the transgene product (Bt protein) and its bioactivity in insect pests following transgene flow from GM hybrid maize into non-genetically modified, non-GM near-isogenic maize hybrid (ISO) and OPVs. We modeled segregation patterns and measured cry1Ab transgene expression (mRNA quantification), Cry1Ab protein concentration and pest survival. Two groups of F1, F2 crosses and backcrosses with GM, ISO and OPV maize varieties from Brazil and South Africa were used. Bioassays with the larvae of two lepidopteran maize pest species, Helicoverpa armigera and Spodoptera littoralis, were carried out. Overall, the cry1Ab transgene outcrossed effectively into the genetic backgrounds tested. The cry1Ab transgene was stably expressed in both ISO and OPV genetic backgrounds. Transgene introgression led to consistent, though highly variable, concentrations of Cry1Ab toxins that were similar to those observed in GM parental maize. Most crosses, but not all, suggested the expected Mendelian segregation pattern. Transgene expression rates were significantly higher than expected from purely Mendelian segregation in the South African crosses. In South African materials, ISO and OPV crosses produced significantly lower Cry1Ab concentrations compared to the GM parental maize. The Cry1Ab toxins from crosses were bioactive and induced mortality rates of ≥92.19% in H. armigera and ≥40.63% in S. littoralis after a period of only 4 days. However, no correlations were observed between the quantitation of mRNA for cry1Ab and the corresponding Cry1Ab protein concentrations, nor between the Cry1Ab concentrations and insect mortality rates across different genetic backgrounds. We therefore suggest that while transcription of the cry1Ab transgene reliably determines the presence of Cry1Ab protein, mRNA levels do not reflect, by themselves, the end Cry1Ab protein concentrations found in the plant. Because predictably high Cry1Ab concentrations are a key prerequisite for effective insect resistance management (IRM) programs, this observation raises questions about the effectiveness of such programs in scenarios with complex crop genetic backgrounds. On the other hand, confirmed bioactivity in all crosses should be expected to impact small farmer's selection behavior, unknowingly favoring the insecticidal trait. This may lead to a fixation of the trait in the wider population, and may influence the genetic diversity of varieties maintained by small-scale farmers.

Teosinte in Europe - Searching for the Origin of a Novel Weed.

A novel weed has recently emerged, causing serious agronomic damage in one of the most important maize-growing regions of Western Europe, the Northern Provinces of Spain. The weed has morphological similarities to a wild relative of maize and has generally been referred to as teosinte. However, the identity, origin or genetic composition of 'Spanish teosinte' was unknown. Here, we present a genome-wide analysis of single-nucleotide polymorphism (SNP) data for Spanish teosinte, sympatric populations of cultivated maize and samples of reference teosinte taxa. Our data are complemented with previously published SNP datasets of cultivated maize and two Mexican teosinte subspecies. Our analyses reveal that Spanish teosinte does not group with any of the currently recognized teosinte taxa. Based on Bayesian clustering analysis and hybridization simulations, we infer that Spanish teosinte is of admixed origin, most likely involving Zea mays ssp. mexicana as one parental taxon, and an unidentified cultivated maize variety as the other. Analyses of plants grown from seeds collected in Spanish maize fields and experimental crosses under controlled conditions reveal that hybridization does occur between Spanish teosinte and cultivated maize in Spain, and that current hybridization is asymmetric, favouring the introgression of Spanish teosinte into cultivated maize, rather than vice versa.

Loop-mediated isothermal amplification of single pollen grains.

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

Phylogeography and evolution in matsutake and close allies inferred by analyses of ITS sequences and AFLPs.

Matsutake are commercially important ectomycorrhizal basidiomycetes in the genus Tricholoma. Despite their importance, the systematics of this species complex have remained elusive and little is known about their origin and biogeography. Using DNA analyses on a worldwide sample of matsutake, we present here the first comprehensive definition of natural groupings in this species complex. We infer patterns of migration and propose Eocene origins for the group in western North America by a transfer from an angiosperm-associated ancestor to an increasingly specialized conifer symbiont. From these origins, matsutake appear to have followed migratory routes parallel to those of coniferous hosts. Patterns of vicariance between eastern North America and eastern Asia are resolved and their origins are suggested to stem from migration through Beringia. Using an analysis of genetic dissimilarity and geographical distance, we reject both the possibility that migration into Europe and Asia occurred through Atlantic bridges and the connection between matsutake populations in the Mahgrebi Mountains and those from Europe. Instead, African and European matsutake appear to be the most recent ends of a westward expansion of the domain of these fungi from North America.

Isotopic fractionation during ammonium assimilation by basidiomycete fungi and its implications for natural nitrogen isotope patterns.

• 15 N natural abundance was examined to determine the fractionation associated with ammonium assimilation into fungi. The importance of fungi to the movement of nitrogen (N) within soils and into plants highlights the need to consider such fractionations in the interpretation of ecosystem 15 N distributions. • To separate the influence of physiological fractionations from N source preferences on fungal δ15 N, both ectomycorrhizal (EM) and saprotrophic (SAP) fungi were grown in liquid culture containing (NH4 )2 HPO4 of known δ15 N and harvested after 35 d. Time series experiments were employed for two taxa to determine how growth stage affects fractionation. • Mycelium δ15 N was positively associated with several measures of growth and no distinction was observed between EM and SAP fungi. Kinetic isotope exchange models explain the decrease in discrimination against 15 N at larger biomasses in terms of the amount of N utilized. The models indicate a fractionation of approx. -20‰, and suggest that assimilated N is excreted back into the medium during growth. • The observed fractionations at high and low N concentrations provide insight into the mechanisms that could drive fungal, plant and soil δ15 N. We suggest that detailed information on fungal life histories, territorial behaviours, and sink-source relationships in the fungus-plant-soil continuum could aid accurate ecological interpretations of δ15 N.

Phylogenetics of Lophodermium from pine.

Lophodermium comprises ascomycetous fungi that are both needle-cast pathogens and asymptomatic endophytes on a diversity of plant hosts. It is distinguished from other genera in the family Rhytismataceae by its filiform ascospores and ascocarps that open by a longitudinal slit. Nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships within Lophodermium. Twenty-nine sequences from approximately 11 species of Lophodermium were analyzed together with eight sequences from isolates thought to represent six other genera of Rhytismataceae: Elytroderma, Lirula, Meloderma, Terriera, Tryblidiopsis and Colpoma. Two putative Meloderma desmazieresii isolates occurred within the Lophodermium clade but separate from one another, one grouped with L. indianum and the other with L. nitens. An isolate of Elytroderma deformans also occurred within the Lophodermium clade but on a solitary branch. The occurrence of these genera within the Lophodermium clade might be due to problems in generic concepts in Rhytismataceae, such as emphasis on spore morphology to delimit genera, to difficulty of isolating Rhytismataceae needle pathogens from material that also is colonized by Lophodermium or to a combination of both factors. We also evaluated the congruence of host distribution and several morphological characters on the ITS phylogeny. Lophodermium species from pine hosts formed a monophyletic sister group to Lophodermium species from more distant hosts from the southern hemisphere, but not to L. piceae from Picea. The ITS topology indicated that Lophodermium does not show strict cospeciation with pines at deeper branches, although several closely related isolates have closely related hosts. Pathogenic species occupy derived positions in the pine clade, suggesting that pathogenicity has evolved from endophytism. A new combination is proposed, Terriera minor (Tehon) P.R. Johnst.

Growth-dependent stable carbon isotope fractionation by basidiomycete fungi: delta(13)C pattern and physiological process.

We grew 11 basidiomycetes in axenic culture to characterize their physiological capacities to fractionate stable C isotopes. Generally, delta(13)C values of the fungal biomass were (i) enriched in (13)C relative to the growth medium, (ii) variable among the isolates, and (iii) dependent on the growth rate and growth stage of the fungi. We found a multiphasic dynamic of fractionation for Cryptoporus volvatus and Marasmius androsaceus during various growth stages. The first phase, P1, corresponded to the exponential growth stage and was characterized by an increasing enrichment in (13)C content of the fungal biomass relative to the growth medium ranging between 4.6 and 6.9 per thousand. The second phase, P2, exhibited a continual depletion in (13)C of the fungal biomass, with the delta(13)C values of the fungal biomass asymptotically returning to the delta(13)C value of the growth medium at inoculation. The expression of the various fractionation phases was dependent on the amount of low-concentration micronutrients and growth factors added to the growth medium. The onset of P2 occurred at reduced concentrations of these elements. All of the sugars in the growth medium (sucrose, maltose, and glucose) were utilized for growth, indicating that the observed fractionation was not an artifact derived from the preferential use of (13)C-rich maltose, which was found at low concentrations in the growth medium. In this study, we establish a framework with which to explore the impact of physiological fractionations by fungal interfaces on natural distributions of stable C isotopes.

Ecophysiology of 13C and 15N isotopic fractionation in forest fungi and the roots of the saprotrophic-mycorrhizal divide.

To quantify and characterize N and C isotopic fractionation effects due to fungal transformation of organic substrates in forest ecosystems, we performed a field study in California and a meta-analysis of three additional studies conducted by others across the Northern Hemisphere. Basidiomycete fungal biomass was consistently enriched for the heavier isotope for C relative to substrate and either enriched or depleted for N relative to atmospheric N. Extent and pattern of fractionation was very variable, but the distinction between ectomycorrhizal and saprotrophic basidiomycetes was strongly supported, particularly when dual isotope analyses were performed. This differentiation, which we call the "EM-SAP Divide" holds for studies within a restricted ecosystem, but becomes less distinct over larger geographical regions, removing the rationale for using direct isotopic values from single specimens as diagnostic of ecophysiological role. For C, the EM-SAP Divide seems to reflect substrate effects, potentially due to differential access to recently synthesized versus recycled organic compounds, rather than distinct physiological pathways. Once substrate and ecophysiological role effects are removed, our meta-analysis suggests the existence of more than one mechanism causing C fractionations in fungi which is found equally in ectomycorrhizal and saprotrophic fungi. Similarly, a multimodal distribution of δ15N values suggests that physiological effects may play a much stronger influence on N natural isotopic distributions in fungi. Our meta-analysis provides a firm statistical base to evaluate fungal ecological statements based on natural isotopic distributions of C and N. We call into question the current practice of using direct isotopic measurements to make statements about trophic relationships of fungi in the absence of other supporting evidence.

La bioprospección en la era de la información: un análisis crítico de las iniciativas de conservación asociadas con el descubrimiento de nuevos fármacos / Bioprospecting in the information age: a critical analysis of conservation-linked pharmaceutical searches through biodiversity

I. Conexiones emergentes entre la biodiversidad y la biotecnología: modelos de prospección química

Resumen, conclusiones y opciones de política / Summary, conclusiones y opciones de política

VI: Epílogo

Comentario: llevará la prospección de la diversidad biológica para obtener fármacos al descubrimiento del "oro verde"? / Commentary: will biodiversity prospecting for pharmaceuticals lead to the discovery of "green gold"

VI: Epílogo

Bioprospecting in the information age: a critical analysis of conservation-linked pharmaceutical searches through biodiversity

I. Emerging connections between biodiversity and biotechnology: bioprospecting models

Summary, conclusiones y opciones de política

VI. Epilogue

Commentary: will biodiversity prospecting for pharmaceuticals lead to the discovery of "green gold"

VI. Epilogue