First Report of Diaporthe foeniculina Associated with Grapevine Trunk Diseases on Vitis vinifera in Cyprus

In June 2017, three vineyards were surveyed in the regions of Droushia (30-year-old, cv Mavro), Ineia (50-year-old, cv Xynisteri), and Lemona (15-year-old, cv Carignan) at the province of Paphos, Cyprus, with dieback incidence of 22%, 32%, and 14%, respectively. More specifically, affected grapevines exhibited severe dieback symptoms in spur and cordon positions, related to perennial cankers and internal brown discoloration. Thirty symptomatic samples, were surface-sterilized (95% ethanol) and wood chips were plated on potato dextrose agar (PDA), amended with streptomycin (500 μg/ml) at 25 °C for 3-5 days. Based on colony morphology (white to creamy color, with sparse aerial mycelium) and conidia production, nine Diaporthe-like isolates were obtained. For species identification, the internal transcribed spacer (ITS) region and β-tubulin (BT) genes were amplified using the primer pairs ITS1/ITS4 and Bt2a/Bt2b, respectively (Úrbez-Torres et al. 2008). Sequences of the isolates P101b, P114c, and P289a revealed >99.8% homology to NCBI voucher specimens of Diaporthe foeniculina (Sacc.) Udayanga & Castl. (ITS: CBS111553, MH050434; ΒΤ: KY511368, KF778966), and were deposited in the GeneBank (ITS: MT735646, MT737289, MT737287; BT: MT903969, MT903970, MT903971). Thus, 8.3% of the collected isolates (3 of 36) were identified as D. foeniculina, while the rest Diaporthe-like isolates were identified as D. ampelina. D. foeniculina isolates were also transferred on 2% water agar with sterile pine needles under a 12h/12h near-ultraviolet, light/darkness regime, at 25 °C, to induce sporulation (Guarnaccia and Crous 2017). Two weeks later, microscopic observations revealed dark brown to black, globose to sub-globose, ostiolate pycnidia (n = 30) 291 to 897 μm (595 ± 173) x 192 to 655 μm (364 ± 113) containing hyaline, unbranched conidiophores, bearing alpha‐ and beta‐conidia in the form of yellowish cirri. Alpha-conidia were aseptate, hyaline, ovate to ellipsoidal, ranging (n=100) from 5.6 to 9.9 μm (7.5 ± 0.8) x 1.9 to 3.3 μm (2.7 ± 0.3). Beta-conidia were abundant, aseptate, hyaline, filiform, slightly curved (n = 100) from 22.4 to 35.3 μm (28.1 ± 2.5) x 1.2 to 2.3 μm (1.6 ± 0.2) (Udayanga et al. 2014). Pathogenicity tests were conducted with isolates P101b and P289a under greenhouse conditions (24-32 ⁰C, 70% RH). Ten 1-year-old rooted canes cv Mavro were inoculated with 4 mm mycelium plugs from actively growing cultures into wounds made by drilling between two internodes at the middle of the trunk. The same number of cuttings were inoculated with sterile PDA plugs, sealed with Vaseline, and wrapped with parafilm, serving as controls. Seven months later, all inoculated cuttings developed brownish wood discolorations (average 39 ± 13 mm), similar to naturally infected plants. No symptoms were observed in the controls. Successful re-isolations were made only from the inoculated cuttings and confirmed by colony morphology. Previously, D. foeniculina (as D. neotheicola) has been reported as grapevine wood saprophyte (Úrbez-Torres et al. 2014). It has also been reported to cause shoot canker and dieback in numerous hosts, including almond, avocado, citrus, and sweet chestnut (Annesi et al. 2016; Guarnaccia and Crous 2017; Diogo et al. 2010; Mathioudakis et al. 2020). This is the first record of D. foeniculina associated with grapevine trunk diseases (GTDs) in Cyprus. However, its relative importance as the causal agent of GTDs remains to be further investigated.

Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster

In Drosophila, expression of eyeless (ey) gene is restricted to the developing eyes and central nervous system. However, the flanking genes, myoglianin (myo), and bent (bt) have different temporal and spatial expression patterns as compared to the ey. How distinct regulation of ey is maintained is mostly unknown. Earlier, we have identified a boundary element intervening myo and ey genes (ME boundary) that prevents the crosstalk between the cis-regulatory elements of myo and ey genes. In the present study, we further searched for the cis-elements that define the domain of ey and maintain its expression pattern. We identify another boundary element between ey and bt, the EB boundary. The EB boundary separates the regulatory landscapes of ey and bt genes. The two boundaries, ME and EB, show a long-range interaction as well as interact with the nuclear architecture. This suggests functional autonomy of the ey locus and its insulation from differentially regulated flanking regions. We also identify a new Polycomb Response Element, the ey-PRE, within the ey domain. The expression state of the ey gene, once established during early development is likely to be maintained with the help of ey-PRE. Our study proposes a general regulatory mechanism by which a gene can be maintained in a functionally independent chromatin domain in gene-rich euchromatin.

Bt cotton seed purity in Burkina Faso: Status and lessons learnt

BackgroundSince the commercial release of Bt cotton the issue of seed purity in producers' fields has been little addressed and in an unbalanced way when it was. It is well documented that the loss of purity in conventional seeds has endangered the continuation of organic cotton production. However, studies are rare on the purity of Bt-cotton seeds despite its implications on the effectiveness and sustainability of their use.This paper compensates for the mentioned lack of literature by analyzing data collected in 2015 in Burkina Faso, namely results of ELISA tests on samples of seeds from 646 fields grown with conventional or Bt varieties. ResultsAccording to the conservative criteria retained to declare the presence of Bt gene (more than 10% and 90% of controlled seeds for conventional and Bt variety, respectively), seed purity was very questionable for both types of varieties. For the conventional variety, the presence of Bt gene was observed on 63.6 and 59.3% of samples for Cry1Ac and Cry2Ab, respectively. Only 29.3% of samples corresponded to pure conventional seeds while 52.2% were double Bt seeds. Conversely, for the Bt variety, the presence of Bt gene was observed on 59.6 and 53.6% of samples for Cry1Ac and Cry2Ab, respectively. Actually BG2 seeds with both Bt genes were found in 40.4% of samples against 27.2% of samples of actually conventional seeds while the remaining of 32.4% of samples corresponded to single Bt gene seeds.Two factors affected the severe lack of seed purity. As regard to conventional seeds, it clearly resulted from a phenomenon of contamination, indicative of a failure in adjusting the seed production scheme to the use of Bt-cotton. With regard to the Bt variety, the lack of purity of the original seeds provided to Burkina Faso accounted and should even be the major factor.The observed lack of seed purity is a threat to the initiative of organic cotton production, albeit a very minor production mode in the country. It also calls upon the effectiveness and furthermore the sustainability of Bt cotton to control target pests. ConclusionOur results show the extent of purity loss when no especial attention is paid to the preservation of seed purity. Pure conventional seeds could totally vanish while Bt seeds become a combination of seeds of various types encompassing or not the expected Bt genes.Any country willing to embark the use of Bt cotton, or to resume this use like Burkina Faso, must previously adjust its seed production scheme and enforce its operation. This is a condition to preserve pure seeds both to enable the launch or the continuation of identity-cotton production and to ensure a sustainable effectiveness of Bt-cotton. The mentioned condition implies that seed purity must be checked and the related information shared.

Agronomic parameters and morpho-agronomic characteristics of genetically modified maize hybrids compared to conventional maize hybrids

The objectives were to study the behavior of fifteen pre-commercial upland maize hybrids, analyze their agronomic performance regarding grain yield, and evaluate productivity components, as well as morpho-agronomic characteristics, in the Midwest Region of Brazil. Two experiments were conducted in the municipalities of Formosa-GO and Planaltina-DF, 2016/17 crop year. Both consisted of five pre-commercial maize hybrid platforms (HPA252, HPB262, HPB621, HPB646, and HPD354). Each platform consisted of three different versions: conventional, transgenic with a Bt gene that expresses the protein Cry1F, and transgenic with two Bt genes that express the proteins Cry1F and Cry1AB. The experiment was randomized blocks with four replications. The experimental plot was four lines five meters long considering the two central lines as useful. The lines were spaced 0.75 meters apart, and the final density was five plants per linear meter. To estimate grain yield, the plots were harvested, and the weight was extrapolated to kg.ha-1. The moisture was standardized at 14%. Data were submitted to analysis of variance, and the means were compared by Tukey test at 5% probability using the Sisvar software. Grain yield between treatments ranged from 8,381 to 12,908 kg.ha-1, and the average yield was 11,234 kg.ha-1. The parameters evaluated were divided into two groups. The first group contained parameters determining grain yield: grain depth, thousand grains weight, number of rows of grains, number of grains per row, and grain yield. The second group contained morpho-agronomic parameters that directly interfere with resistance to lodging and plant breaking: plant height, ear insertion height, and mean stem diameter. There was no direct effect of the transgenes on the evaluated hybrids since the classification of productivity was not divided into conventional and transgenic classes. It is suggested that the HPA252YH, HP621H and HP646H versions be discarded because grain yield performance was unsatisfactory compared to their respective conventional and transgenic counterparts. The parameters GD and W1000 are more effective for grain yield estimation than NR and NG. It was observed for HP621H that, in addition to a lower grain yield, there was a significant reduction in stem diameter, indicating a possible reduction in lodging tolerance and/or plant breaking when exposed to adverse climatic conditions such as windstorms. For the HPD354H version, the release of the transgenic counterpart HPD354YH is preferentially indicated since the H version had a significant reduction in stem diameter and a possible greater tolerance to lodging and/or plant breaking under the same conditions.

Comprehensive genomic analysis and expression profiling of the BTB and TAZ (BT) genes in cucumber (Cucumis sativus L.)

BTB-TAZ (BT) proteins are plant-specific transcription factors containing a BTB domain and a TAZ domain. They play vital roles in various biological processes and stress responses. In this study, a total of three BT genes (CsBT1–3) were identified from cucumber genome, and they were unevenly distributed in two of the seven chromosomes. Phylogenetic analysis of the BT proteins from cucumber, Arabidopsis, apple, tomato, and rice revealed that these proteins could be distinctly divided into two groups in accordance with their motif distributions. We also determined the structures of BT genes from cucumber, Arabidopsis, and rice to demonstrate their differences. The quantitative real-time PCR (qRT-PCR) results showed that the CsBT genes displayed differential expression patterns in cucumber tissues, and their expression was regulated by cold, salt, and drought stresses. These findings suggest that CsBT genes may participate in cucumber development and responses to various abiotic stresses.

Effects of Cry toxins on non-target soil bacteria during a 2-year follow up study

Genetically modified (GM) plants with insecticidal Bacillus thuringiensis (Bt) genes are widely accepted but their commercial utilization highlights the biosafety issues worldwide. The risk assessment of GM crops demonstrates their impact on the ecosystem as well as non-target organisms (NTOs). Among the NTOs, plant growth promoting rhizobacteria (PGPR) demand more critical experimental studies as they play a significant role in plant growth. A comparative study of Bt with non-Bt cotton rhizosphere was conducted, on selected bacterial strains. During the course of the study, biochemical characterization, auxin biosynthesis and molecular characterization was done to assess the effect of Bt toxins (Cry1Ac and Cry2A) on non-target PGPR strains. A significant decrease (p<0.05) in phosphatase activity was recorded in some of the experimental bacterial strains as compared to those of control strains. However, no significant differences (p>0.05) were observed in other parameters like bacterial population, colony morphologies as well as biochemical activities. Thus, our study demonstrates the safe plantation of Bt crops with respect to soil bacteria.