Genome-wide association mapping of quantitative traits in a breeding population of sugarcane.

BACKGROUND: Molecular markers associated with relevant agronomic traits could significantly reduce the time and cost involved in developing new sugarcane varieties. Previous sugarcane genome-wide association analyses (GWAS) have found few molecular markers associated with relevant traits at plant-cane stage. The aim of this study was to establish an appropriate GWAS to find molecular markers associated with yield related traits consistent across harvesting seasons in a breeding population. Sugarcane clones were genotyped with DArT (Diversity Array Technology) and TRAP (Target Region Amplified Polymorphism) markers, and evaluated for cane yield (CY) and sugar content (SC) at two locations during three successive crop cycles. GWAS mapping was applied within a novel mixed-model framework accounting for population structure with Principal Component Analysis scores as random component. RESULTS: A total of 43 markers significantly associated with CY in plant-cane, 42 in first ratoon, and 41 in second ratoon were detected. Out of these markers, 20 were associated with CY in 2 years. Additionally, 38 significant associations for SC were detected in plant-cane, 34 in first ratoon, and 47 in second ratoon. For SC, one marker-trait association was found significant for the 3 years of the study, while twelve markers presented association for 2 years. In the multi-QTL model several markers with large allelic substitution effect were found. Sequences of four DArT markers showed high similitude and e-value with coding sequences of Sorghum bicolor, confirming the high gene microlinearity between sorghum and sugarcane. CONCLUSIONS: In contrast with other sugarcane GWAS studies reported earlier, the novel methodology to analyze multi-QTLs through successive crop cycles used in the present study allowed us to find several markers associated with relevant traits. Combining existing phenotypic trial data and genotypic DArT and TRAP marker characterizations within a GWAS approach including population structure as random covariates may prove to be highly successful. Moreover, sequences of DArT marker associated with the traits of interest were aligned in chromosomal regions where sorghum QTLs has previously been reported. This approach could be a valuable tool to assist the improvement of sugarcane and better supply sugarcane demand that has been projected for the upcoming decades.

Evaluation of PSP1 biostimulant on Fusarium graminearum-wheat pathosystem: impact on disease parameters, grain yield, and grain quality.

BACKGROUND: Plant defense elicitors are valuable tools in sustainable agriculture, providing an environmentally friendly and effective means of enhancing plant defense and promoting plant health. Fusarium head blight (FHB) is one of the most important fungal diseases of cereal crops worldwide. The PSP1 is a novel biopesticide formulated based on an elicitor, the extracellular protein AsES, from the fungus Sarocladium strictum. The present work aimed to evaluate the effectiveness of PSP1 in controlling FHB under field conditions. Experiments were conducted during three consecutive growing seasons (2019, 2020, and 2021). Three biostimulant treatments were tested in different physiological stages (from late tillering to heading stage), and FHB inoculations were performed at anthesis. Disease parameters, seed parameters, grain yield, and grain quality parameters were evaluated. RESULTS: Depending on the year and the genotype, reductions in disease incidence (up to 11%) and disease severity (up to 5%) were reported, although these differences could not be attributed to the use of the PSP1 biostimulant. Occasional improvements in seed parameters and grain quality were observed, suggesting that early treatments could work better than late treatments, probably due to early activation/priming of defense response mechanisms. However, more studies are deemed necessary. CONCLUSION: The use of PSP1 biostimulant in commercial wheat crops could be a biological alternative or complement to traditional chemical fungicides to manage FHB. The reduced environmental impact and the potential benefits in grain yield and quality are other reasons that can generate new adherents of this technology in worldwide agriculture systems in the coming years. © 2024 Society of Chemical Industry.

Pathogen-induced accumulation of an ellagitannin elicits plant defense response.

In an incompatible interaction between Colletotrichum fragariae and strawberry plants, the accumulation of phenolic compounds in plant leaves was observed. A particularly abundant penta-esterified ellagitannin that accumulated in response to pathogen attack was identified as 1-0-galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-ß-d-glucopyranose (HeT) by mass spectroscopy and nuclear magnetic resonance. Foliar application of purified HeT prior to inoculation with a virulent pathogen was shown to increase resistance toward C. acutatum in strawberry plants and to Xanthomonas citri subsp. citri in lemon plants. The induced resistance in strawberry was associated with a rapid oxidative burst, callose deposition, a transient increase of salicylic acid in phloem, and induction of gene expression responsive to salicylic acid. Results obtained suggested that HeT could be a common plant defense response molecule capable of inducing pathogen resistance in different plant species.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

Selecting putative drought-tolerance markers in two contrasting soybeans.

Identifying high-yield genotypes under low water availability is essential for soybean climate-smart breeding. However, a major bottleneck lies in phenotyping, particularly in selecting cost-efficient markers associated with stress tolerance and yield stabilization. Here, we conducted in-depth phenotyping experiments in two soybean genotypes with contrasting drought tolerance, MUNASQA (tolerant) and TJ2049 (susceptible), to better understand soybean stress physiology and identify/statistically validate drought-tolerance and yield-stabilization traits as potential breeding markers. Firstly, at the critical reproductive stage (R5), the molecular differences between the genotype's responses to mild water deficit were explored through massive analysis of cDNA ends (MACE)-transcriptomic and gene ontology. MUNASQA transcriptional profile, compared to TJ2049, revealed significant differences when responding to drought. Next, both genotypes were phenotyped under mild water deficit, imposed in vegetative (V3) and R5 stages, by evaluating 22 stress-response, growth, and water-use markers, which were subsequently correlated between phenological stages and with yield. Several markers showed high consistency, independent of the phenological stage, demonstrating the effectiveness of the phenotyping methodology and its possible use for early selection. Finally, these markers were classified and selected according to their cost-feasibility, statistical weight, and correlation with yield. Here, pubescence, stomatal density, and canopy temperature depression emerged as promising breeding markers for the early selection of drought-tolerant soybeans.

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains.

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.

Genetic Transformation of Sugarcane, Current Status and Future Prospects.

Sugarcane (Saccharum spp.) is a tropical and sub-tropical, vegetative-propagated crop that contributes to approximately 80% of the sugar and 40% of the world's biofuel production. Modern sugarcane cultivars are highly polyploid and aneuploid hybrids with extremely large genomes (>10 Gigabases), that have originated from artificial crosses between the two species, Saccharum officinarum and S. spontaneum. The genetic complexity and low fertility of sugarcane under natural growing conditions make traditional breeding improvement extremely laborious, costly and time-consuming. This, together with its vegetative propagation, which allows for stable transfer and multiplication of transgenes, make sugarcane a good candidate for crop improvement through genetic engineering. Genetic transformation has the potential to improve economically important properties in sugarcane as well as diversify sugarcane beyond traditional applications, such as sucrose production. Traits such as herbicide, disease and insect resistance, improved tolerance to cold, salt and drought and accumulation of sugar and biomass have been some of the areas of interest as far as the application of transgenic sugarcane is concerned. Although there have been much interest in developing transgenic sugarcane there are only three officially approved varieties for commercialization, all of them expressing insect-resistance and recently released in Brazil. Since the early 1990's, different genetic transformation systems have been successfully developed in sugarcane, including electroporation, Agrobacterium tumefaciens and biobalistics. However, genetic transformation of sugarcane is a very laborious process, which relies heavily on intensive and sophisticated tissue culture and plant generation procedures that must be optimized for each new genotype to be transformed. Therefore, it remains a great technical challenge to develop an efficient transformation protocol for any sugarcane variety that has not been previously transformed. Additionally, once a transgenic event is obtained, molecular studies required for a commercial release by regulatory authorities, which include transgene insertion site, number of transgenes and gene expression levels, are all hindered by the genomic complexity and the lack of a complete sequenced reference genome for this crop. The objective of this review is to summarize current techniques and state of the art in sugarcane transformation and provide information on existing and future sugarcane improvement by genetic engineering.

Strawberry fatty acyl glycosides enhance disease protection, have antibiotic activity and stimulate plant growth.

An increasing interest in the development of products of natural origin for crop disease and pest control has emerged in the last decade. Here we introduce a new family of strawberry acyl glycosides (SAGs) formed by a trisaccharide (GalNAc-GalNAc-Glc) and a monounsaturated fatty acid of 6 to 12 carbon atoms linked to the glucose unit. Application of SAGs to Arabidopsis thaliana (hereafter Arabidopsis) plants triggered a transient oxidative burst, callose deposition and defense gene expression, accompanied by increased protection against two phytopathogens, Pseudomonas viridiflava and Botrytis cinerea. SAGs-induced disease protection was also demonstrated in soybean infected with the causal agent of target spot, Corynespora cassiicola. SAGs were shown to exhibit important antimicrobial activity against a wide-range of bacterial and fungal phytopathogens, most probably through membrane destabilization, and the potential use of SAGs as a biofungicide for postharvest disease protection was demonstrated on lemon fruits infected with Penicillium digitatum. Plant growth promotion by application of SAGs was shown by augmented primary root elongation, secondary roots development and increased siliques formation in Arabidopsis, whereas a significant increment in number of seed pods was demonstrated in soybean. Stimulation of radicle development and the induction of an auxin-responsive reporter system (DR5::GUS) in transgenic Arabidopsis plants, suggested that SAGs-stimulated growth at least partly acts through the auxin response pathway. These results indicate that strawberry fatty acid glycosides are promising candidates for the development of environmental-friendly products for disease management in soybean and lemon.

The Endophytic Strain Klebsiella michiganensis Kd70 Lacks Pathogenic Island-Like Regions in Its Genome and Is Incapable of Infecting the Urinary Tract in Mice.

Klebsiella spp. have been isolated from many different environmental habitats but have mainly been associated with nosocomial acquired diseases in humans. Although there are many recently published sequenced genomes of members of this genus, there are very few studies on whole genome comparisons between clinical and non-clinical isolates, and it is therefore still an open question if a strain found in nature is capable of infecting humans/animals. Klebsiella michiganensis Kd70 was isolated from the intestine of larvae of Diatraea saccharalis but genome analysis revealed multiple genes associated with colonization and growth promotion in plants suggesting an endophytic lifestyle. Kd70 cells labeled with gfp confirmed capability of root colonization and soil application of Kd70 promoted growth in greenhouse grown sugarcane. Further genomic analysis showed that the Kd70 genome harbored fewer mammalian virulence factors and no pathogen island-like regions when compared to clinical isolates of this species, suggesting attenuated animal/human pathogenicity. This postulation was corroborated by in vivo experiments in which it was demonstrated that Kd70 was unable to infect the mouse urinary tract. This is to the best of our knowledge the first experimental example of a member of a pathogenic Klebsiella spp. unable to infect a mammalian organism. A proteomic comparison deduced from the genomic sequence between Kd70 and several other K. michiganensis strains showed a high similarity with isolates from many different environments including clinical strains, and demonstrated the existence of conserved genetic lineages within this species harboring members from different ecological niches and geographical locations. Furthermore, most genetic differences were found to be associated with genomic islands of clinical isolates, suggesting that evolutionary adaptation of animal pathogenicity to a large extent has depended on horizontal gene transfer. In conclusion our results demonstrate the importance of conducting thorough in vivo pathogenicity studies before presupposing animal/human virulence of non-clinical bacterial isolates.

Elicitor-Based Biostimulant PSP1 Protects Soybean Against Late Season Diseases in Field Trials.

Currently, fungicide application in soybean production accounts for an important amount of global pesticide use, and it is therefore most desirable to find new healthier and more environmental friendly alternatives for the phytosanitary management in this crop. In this study, we present convincing evidence for effective induction of disease protection by the agricultural biostimulant PSP1, a formulation based on the plant-defense eliciting activity of the fungal protease AsES (Acremonium strictum elicitor subtilisin), in multiple field trials in Argentina. PSP1 was shown to combine well with commercial spray adjuvants, an insecticide, a herbicide and fungicides used in Argentinian soybean production without losing any defense-inducing activity, indicating an easy and efficient adaptability to conventional soybean production and disease management in the region. Results from multiple soybean field trials conducted with different elite genotypes at several locations during two consecutive growing seasons, showed that PSP1 is able to induce an enhanced pathogen defense which effectively reduced late season disease (LSD) development in field-grown soybean. This defense response seems to be broad-range as disease development was clearly reduced for at least three different fungi causing LSDs in soybean (Septoria glycines, Cercospora kikuchii and Cercospora sojina). It was noteworthy that application of PSP1 in soybean alone gave a similar protection against fungal diseases as compared to the commercial fungicides included in the field trials and that PSP1 applied together with a fungicide at reproductive stages enhanced disease protection and significantly increased grain yields. PSP1 is the first example of an elicitor-based strategy in order to efficiently control multiple fungal diseases under field conditions in the soybean crop. These results show the feasibility of using induced resistance products as complements or even full-good replacements to currently used chemical pesticides, fulfilling a role as important components of a more sustainable crop disease management system.

Development of PSP1, a Biostimulant Based on the Elicitor AsES for Disease Management in Monocot and Dicot Crops.

In this work, we present a novel biostimulant for sustainable crop disease management, PSP1, based on the plant defense-elicitor AsES, an extracellular protease produced by the strawberry fungal pathogen Acremonium strictum. Fungal fermentation conditions and downstream processing were determined to maximize extracellular protein production, product stability and a high plant defense-eliciting activity, as monitored by anthracnose resistance in supernatant-treated strawberry plants subsequently infected with a virulent strain of Colletotrichum acutatum. Fermentation batches were shown to reduce anthracnose development by 30-60% as compared to infected non-treated plants. Product formulation was shown to be stable for 6 months when stored at temperatures up to 45°C and toxicological tests showed that PSP1 was harmless to beneficial organisms and non-toxic to mammalian species at concentrations 50 times higher than those used in plant experiments. Furthermore, disease protection studies using dilutions of PSP1 indicated that there is a minimum threshold protease activity needed to induce pathogen defense in strawberry and that this induction effect is dose-independent. A significant characteristic of PSP1 is its broad-range protection against different diseases in various crop species. In soybean, PSP1 reduced the symptomatology by 70% of Corynespora cassiicola, etiological agent of the target spot. This protection effect was similar to the commercial inducer BION 500 WG based on BTH, and both products were shown to induce an oxidative burst and up-regulated PR1-gene expression in soybean. Furthermore, a double PSP1-treatment on greenhouse-grown sugarcane plants provided protection against bacterial red stripe disease caused by Acidovorax avenae and a double foliar application of PSP1 on field-grown wheat plants significantly increased resistance against Fusarium graminearum, causal agent of head blight disease, manifested mainly in an increased seed germination rate. In summary, these disease protection studies demonstrated an effective control against both bacterial and fungal pathogens in both monocot and dicot crop species, which together with its low production cost, effectiveness at low concentrations, long shelf-life, tolerance to high temperatures, harmlessness to non-target organisms and simple handling and application, make PSP1 a very promising candidate for effective and sustainable disease management in many crop species.

Isolation of cellulolytic bacteria from the intestine of Diatraea saccharalis larvae and evaluation of their capacity to degrade sugarcane biomass.

As a strategy to find efficient lignocellulose degrading enzymes/microorganisms for sugarcane biomass pretreatment purposes, 118 culturable bacterial strains were isolated from intestines of sugarcane-fed larvae of the moth Diatraea saccharalis. All strains were tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays or by growing bacteria on sugarcane biomass as sole carbon sources. Out of the 118 strains isolated thirty eight were found to possess cellulose degrading activity and phylogenetic studies of the 16S rDNA sequence revealed that all cellulolytic strains belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. Within the three phyla, species belonging to five different genera were identified (Klebsiella, Stenotrophomonas, Microbacterium, Bacillus and Enterococcus). Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella. Good cellulolytic activity correlated with high extracellular protein concentrations. In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates. The results of this study indicate the possibility to find efficient cellulose degrading enzymes and microorganisms from intestines of insect larvae feeding on sugarcane and their possible application in industrial processing of sugarcane biomass such as second generation biofuel production.

Use of AFLP markers to estimate molecular diversity of Phakopsora pachyrhizi

Background Asian soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. & Syd., is one of the main diseases affecting soybean and has been reported as one of the most economically important fungal pathogens worldwide. Knowledge of the genetic diversity of this fungus should be considered when developing resistance breeding strategies. We aimed to analyze the genetic diversity of P. pachyrhizi combining simple sampling with a powerful and reproducible molecular technique. Results We employed Amplified Fragment Length Polymorphism (AFLP) technique for the amplification of P. pachyrhizi DNA extracted from naturally SBR-infected plants from 23 production fields. From a total of 1919 markers obtained, 77% were polymorphic. The high percentage of polymorphism and the Nei's genetic diversity coefficient (0.22) indicated high pathogen diversity. Analysis of molecular variance showed higher genetic variation within countries than among them. Temporal analysis showed a higher genetic variation within a year than between years. Cluster, phylogenetic and principal co-ordinate analysis showed that samples group by year of collection and then by country sampled. Conclusions The study proposed combining a simple collection of urediniospore with a subsequent analysis by AFLP was useful to examine the molecular polymorphism of samples of P. pachyrhizi collected and might have a significant contribution to the knowledge of its genetic diversity. Also, AFLP analysis is an important and potent molecular tool for the study of genetic diversity and could be useful to carry out wider genetic diversity studies.

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose.

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.

A dehydrin gene in Physcomitrella patens is required for salt and osmotic stress tolerance.

We isolated a dehydrin-like (DHN-like) gene fragment, PpDHNA, from the moss Physcomitrella patens by PCR amplification using degenerate primers directed against conserved amino acid segments of DHNs of higher plants. The full-length cDNA was found to encode a 59.2-kDa glycine-rich protein, DHNA, with typical characteristics of DHNs, including the presence of several Y repeats and one conserved K segment. DHNA had a high sequence similarity with a protein from Tortula ruralis, Tr288, which is thought to be involved in cellular dehydration tolerance/repair in this moss. Northern and Western analysis showed that PpDHNA is upregulated upon treatment of plants with abscisic acid, NaCl or mannitol, indicating a similar expression pattern to DHNs from higher plants. To analyze the contribution of DHNA to osmotic stress tolerance, we generated a knockout mutant (dhnA) by disruption of the gene using homologous recombination. Growth and stress response studies of the mutant showed that dhnA was severely impaired in its capacity to resume growth after salt and osmotic-stress treatments. We provide direct genetic evidence in any plant species for a DHN exerting a protective role during cellular dehydration allowing recovery when returned to optimal growth conditions.