Morphological and Genetic Characterization of Maize Landraces Adapted to Marginal Hills in North-West Italy.

The growing interest in maize landraces over the past two decades has led to the need to characterize the Italian maize germplasm. In Italy, hundreds of maize landraces have been developed, but only a few of them have been genetically characterized, and even fewer are currently employed in agriculture or for breeding purposes. In the present study, 13 maize landraces of the west Emilia-Romagna region were morphologically and genetically characterized. These accessions were sampled in 1954 from three provinces, Modena, Parma, and Piacenza, during the characterization project of Italian maize landraces. The morphological characterization of these 13 accessions was performed according to the UPOV protocol CPVO/TP2/3, examining 34 phenotypic traits. A total of 820 individuals were genotyped with 10 SSR markers. The genetic characterization revealed 74 different alleles, a FST mean value of 0.13, and a Nm mean of 1.73 over all loci. Moreover, AMOVA analysis disclosed a low degree of differentiation among accessions, with only 13% of genetic variability found between populations, supporting PCoA analysis results, where the first two coordinates explained only 16% of variability. Structure analysis, supported by PCoA, showed that only four accessions were clearly distinguished for both K = 4 and 6. Italian landraces can be useful resources to be employed in maize breeding programs for the development of new varieties, adapted to different environmental conditions, in order to increase crop resilience and expand the maize cultivation area.

Characterization and Valorization of Maize Landraces from Aosta Valley.

While there is a rich collection of maize germplasm from Italy, it lacks genetic resources from the Aosta Valley, an isolated mountain region where landraces have been preserved in the absence of modern germplasm introductions. These local materials, which are still cultivated mainly at household level, can have high importance from a genetic and historical point of view. In the present study, five landraces named, after the collecting sites, Arnad, Arnad-Crest, Châtillon, Entrebin and Perloz, were sampled in Aosta Valley and subjected to historic, morphologic and genetic characterization. This study provided evidence for the landraces' long presence in Aosta Valley, a significant genetic variability and differentiation among the investigated landraces. Globally, 67 different alleles were detected ranging from 4 for markers phi127 and p-bnlg176 to 10 for phi031, with a mean of 6.7 alleles per locus. Observed heterozygosity levels were comprised from 0.16 to 0.51 and are generalkly lower than expected heterozigosity supporting fixation at some loci. STRUCTURE analysis revealed clear separation between accessions revealing the presence of four ancestral populations. This may be explained by the long reproductive isolation experienced by these materials. Finally, morphological observations confirm the high diversity between landraces revealing that they generally have flint kernels, variable color from yellow to dark red (Châtillon) while Perloz showed kernels with an apical beak. The present work confirms the importance of mountain areas in conserving biodiversity and increases the rich Italian maize germplasm with materials well adapted to marginal areas. Such new genetic variability may be used to breed new materials for more resilient agriculture.

Diverse mycorrhizal maize inbred lines differentially modulate mycelial traits and the expression of plant and fungal phosphate transporters.

Food production is heavily dependent on soil phosphorus (P), a non-renewable mineral resource essential for plant growth and development. Alas, about 80% is unavailable for plant uptake. Arbuscular mycorrhizal fungi may promote soil P efficient use, although the mechanistic aspects are yet to be completely understood. In this study, plant and fungal variables involved in P acquisition were investigated in maize inbred lines, differing for mycorrhizal responsiveness and low-P tolerance, when inoculated with the symbiont Rhizoglomus irregulare (synonym Rhizophagus irregularis). The expression patterns of phosphate transporter (PT) genes in extraradical and intraradical mycelium (ERM/IRM) and in mycorrhizal and control maize roots were assessed, together with plant growth responses and ERM extent and structure. The diverse maize lines differed in plant and fungal accumulation patterns of PT transcripts, ERM phenotypic traits and plant performance. Mycorrhizal plants of the low-P tolerant maize line Mo17 displayed increased expression of roots and ERM PT genes, compared with the low-P susceptible line B73, which revealed larger ERM hyphal densities and interconnectedness. ERM structural traits showed significant correlations with plant/fungal expression levels of PT genes and mycorrhizal host benefit, suggesting that both structural and functional traits are differentially involved in the regulation of P foraging capacity in mycorrhizal networks.

Functional Study of Lipoxygenase-Mediated Resistance against Fusarium verticillioides and Aspergillus flavus Infection in Maize.

Mycotoxin contamination of maize kernels by fungal pathogens like Fusarium verticillioides and Aspergillus flavus is a chronic global challenge impacting food and feed security, health, and trade. Maize lipoxygenase genes (ZmLOXs) synthetize oxylipins that play defense roles and govern host-fungal interactions. The current study investigated the involvement of ZmLOXs in maize resistance against these two fungi. A considerable intraspecific genetic and transcript variability of the ZmLOX family was highlighted by in silico analysis comparing publicly available maize pan-genomes and pan-transcriptomes, respectively. Then, phenotyping and expression analysis of ZmLOX genes along with key genes involved in oxylipin biosynthesis were carried out in a maize mutant carrying a Mu transposon insertion in the ZmLOX4 gene (named UFMulox4) together with Tzi18, Mo17, and W22 inbred lines at 3- and 7-days post-inoculation with F. verticillioides and A. flavus. Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while UFMulox4 was highly susceptible to both pathogens with the most elevated mycotoxin content. F. verticillioides inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to A. flavus. Additionally, oxylipin analysis revealed prevalent linoleic (18:2) peroxidation by 9-LOXs, the accumulation of 10-oxo-11-phytoenoic acid (10-OPEA), and triglyceride peroxidation only in F. verticillioides inoculated kernels of resistant genotypes.

Cell death induced by mycotoxin fumonisin B1 is accompanied by oxidative stress and transcriptional modulation in Arabidopsis cell culture.

KEY MESSAGE: Fumonisin B1 induces rapid programmed cell death in Arabidopsis cells, oxidative and nitrosative bursts, and differentially modulates cell death responsive genes. Glutathione is the main antioxidant involved in the stress response. Fumonisin B1 (FB1) is a fungal toxin produced by Fusarium spp. able to exert pleiotropic toxicity in plants. FB1 is known to be a strong inducer of the programmed cell death (PCD); however, the exact mechanism underling the plant-toxin interactions and the molecular events that lead to PCD are still unclear. Therefore, in this work, we provided a comprehensive investigation of the response of the model organism Arabidopsis thaliana at the nuclear, transcriptional, and biochemical level after the treatment with FB1 at two different concentrations, namely 1 and 5 µM during a time-course of 96 h. FB1 induced oxidative and nitrosative bursts and a rapid cell death in Arabidopsis cell cultures, which resembled a HR-like PCD event. Different genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, pathogenesis, and sugar transport were upregulated, especially during the late treatment time and with higher FB1 concentration. Among the antioxidant enzymes and compounds studied, only glutathione appeared to be highly induced in both treatments, suggesting that it might be an important stress molecule induced during FB1 exposure. Collectively, these findings highlight the complexity of the signaling network of A. thaliana and provide information for the understanding of the physiological, molecular, and biochemical responses to counteract FB1-induced toxicity.

Fusarium verticillioides and Aspergillus flavus Co-Occurrence Influences Plant and Fungal Transcriptional Profiles in Maize Kernels and In Vitro.

Climate change will increase the co-occurrence of Fusarium verticillioides and Aspergillus flavus, along with their mycotoxins, in European maize. In this study, the expression profiles of two pathogenesis-related (PR) genes and four mycotoxin biosynthetic genes, FUM1 and FUM13, fumonisin pathway, and aflR and aflD, aflatoxin pathway, as well as mycotoxin production, were examined in kernels and in artificial medium after a single inoculation with F. verticillioides or A. flavus or with the two fungi in combination. Different temperature regimes (20, 25 and 30 °C) over a time-course of 21 days were also considered. In maize kernels, PR genes showed the strongest induction at 25 °C in the earlier days post inoculation (dpi)with both fungi inoculated singularly. A similar behaviour was maintained with fungi co-occurrence, but with enhanced defence response at 9 dpi under 20 °C. Regarding FUM genes, in the kernels inoculated with F. verticillioides the maximal transcript levels occurred at 6 dpi at 25 °C. At this temperature regime, expression values decreased with the co-occurrence of A. flavus, where the highest gene induction was detected at 20 °C. Similar results were observed in fungi grown in vitro, whilst A. flavus presence determined lower levels of expression along the entire time-course. As concerns afl genes, considering both A. flavus alone and in combination, the most elevated transcript accumulation occurred at 30 °C during all time-course both in infected kernels and in fungi grown in vitro. Regarding mycotoxin production, no significant differences were found among temperatures for kernel contamination, whereas in vitro the highest production was registered at 25 °C for aflatoxin B1 and at 20 °C for fumonisins in the case of single inoculation. In fungal co-occurrence, both mycotoxins resulted reduced at all the temperatures considered compared to the amount produced with single inoculation.

Fusarium verticillioides Induces Maize-Derived Ethylene to Promote Virulence by Engaging Fungal G-Protein Signaling.

Seed maceration and contamination with mycotoxin fumonisin inflicted by Fusarium verticillioides is a major disease concern for maize producers worldwide. Meta-analyses of quantitative trait loci for Fusarium ear rot resistance uncovered several ethylene (ET) biosynthesis and signaling genes within them, implicating ET in maize interactions with F. verticillioides. We tested this hypothesis using maize knockout mutants of the 1-aminocyclopropane-1-carboxylate (ACC) synthases ZmACS2 and ZmACS6. Infected wild-type seed emitted five-fold higher ET levels compared with controls, whereas ET was abolished in the acs2 and acs6 single and double mutants. The mutants supported reduced fungal biomass, conidia, and fumonisin content. Normal susceptibility was restored in the acs6 mutant with exogenous treatment of ET precursor ACC. Subsequently, we showed that fungal G-protein signaling is required for virulence via induction of maize-produced ET. F. verticillioides Gß subunit and two regulators of G-protein signaling mutants displayed reduced seed colonization and decreased ET levels. These defects were rescued by exogenous application of ACC. We concluded that pathogen-induced ET facilitates F. verticillioides colonization of seed, and, in turn, host ET production is manipulated via G-protein signaling of F. verticillioides to facilitate pathogenesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Loss of ZmLIPOXYGENASE4 Decreases Fusarium verticillioides Resistance in Maize Seedlings.

Fusarium verticillioides is one of the most relevant fungal species in maize responsible for ear, stalk and seedling rot, as well as the fumonisin contamination of kernels. Plant lipoxygenases (LOX) synthesize oxylipins that play a crucial role in the regulation of defense mechanisms against pathogens and influence the outcome of pathogenesis. To better uncover the role of these signaling molecules in maize resistance against F. verticillioides, the functional characterization of the 9-LOX gene, ZmLOX4, was carried out in this study by employing mutants carrying Mu insertions in this gene (named as UFMulox4). In this regard, the genotyping of five UFMulox4 identified the mutant UFMu10924 as the only one having an insertion in the coding region of the gene. The impact of ZmLOX4 mutagenesis on kernel defense against F. verticillioides and fumonisin accumulation were investigated, resulting in an increased fungal susceptibility compared to the inbred lines W22 and Tzi18. Moreover, the expression of most of the genes involved in the LOX, jasmonic acid (JA) and green leaf volatiles (GLV) pathways, as well as LOX enzymatic activity, decreased or were unaffected by fungal inoculation in the mutant UFMu10924. These results confirm the strategic role of ZmLOX4 in controlling defense against F. verticillioides and its influence on the expression of several LOX, JA and GLV genes.

Perspectives on Global Mycotoxin Issues and Management From the MycoKey Maize Working Group.

During the last decade, there have been many advances in research and technology that have greatly contributed to expanded capabilities and knowledge in detection and measurement, characterization, biosynthesis, and management of mycotoxins in maize. MycoKey, an EU-funded Horizon 2020 project, was established to advance knowledge and technology transfer around the globe to address mycotoxin impacts in key food and feed chains. MycoKey included several working groups comprising international experts in different fields of mycotoxicology. The MycoKey Maize Working Group recently convened to gather information and strategize for the development and implementation of solutions to the maize mycotoxin problem in light of current and emerging technologies. This feature summarizes the Maize WG discussion and recommendations for addressing mycotoxin problems in maize. Discussions focused on aflatoxins, deoxynivalenol, fumonisins, and zearalenone, which are the most widespread and persistently important mycotoxins in maize. Although regional differences were recognized, there was consensus about many of the priorities for research and effective management strategies. For preharvest management, genetic resistance and selecting adapted maize genotypes, along with insect management, were among the most fruitful strategies identified across the mycotoxin groups. For postharvest management, the most important practices included timely harvest, rapid grain drying, grain cleaning, and carefully managed storage conditions. Remediation practices such as optical sorting, density separation, milling, and chemical detoxification were also suggested. Future research and communication priorities included advanced breeding technologies, development of risk assessment tools, and the development and dissemination of regionally relevant management guidelines.

Application of an Integrated and Open Source Workflow for LC-HRMS Plant Metabolomics Studies. Case-Control Study: Metabolic Changes of Maize in Response to Fusarium verticillioides Infection.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) represents the most powerful metabolomics platform to investigate biological systems. Reproducible and standardized workflows allow obtaining a meaningful biological interpretation. The purpose of this study was to set up and apply an open-source workflow for LC-HRMS plant metabolomics studies. Key steps of the proposed workflow were as follows: (1) experimental design, (2) sample preparation, (3) LC-HRMS analysis, (4) data processing, (5) custom database search, (6) statistical analysis, (7) compound identification, and (8) biochemical interpretation. Its applicability was evaluated through the study of metabolomics changes of two maize recombinant inbred lines with contrasting phenotypes with respect to disease severity after Fusarium verticillioides infection of seedlings. Analysis of data from the case-control study revealed abundance change in metabolites belonging to different metabolic pathways, including two amino acids (L-tryptophan and tyrosine), five flavonoids, and three N-hydroxynnamic acid amides.

A Genome-Wide Association Study To Understand the Effect of Fusarium verticillioides Infection on Seedlings of a Maize Diversity Panel.

Fusarium verticillioides, which causes ear, kernel and stem rots, has been reported as the most prevalent species on maize worldwide. Kernel infection by F. verticillioides results in reduced seed yield and quality as well as fumonisin contamination, and may affect seedling traits like germination rate, entire plant seedling length and weight. Maize resistance to Fusarium is a quantitative and complex trait controlled by numerous genes with small effects. In the present work, a Genome Wide Association Study (GWAS) of traits related to Fusarium seedling rot was carried out in 230 lines of a maize association population using 226,446 SNP markers. Phenotypes were scored on artificially infected kernels applying the rolled towel assay screening method and three traits related to disease response were measured in inoculated and not-inoculated seedlings: plant seedling length (PL), plant seedling weight (PW) and germination rate (GERM). Overall, GWAS resulted in 42 SNPs significantly associated with the examined traits. Two and eleven SNPs were associated with PL in inoculated and not-inoculated samples, respectively. Additionally, six and one SNPs were associated with PW and GERM traits in not-inoculated kernels, and further nine and thirteen SNPs were associated to the same traits in inoculated kernels. Five genes containing the significant SNPs or physically closed to them were proposed for Fusarium resistance, and 18 out of 25 genes containing or adjacent to significant SNPs identified by GWAS in the current research co-localized within QTL regions previously reported for resistance to Fusarium seed rot, Fusarium ear rot and fumonisin accumulation. Furthermore, linkage disequilibrium analysis revealed an additional gene not directly observed by GWAS analysis. These findings could aid to better understand the complex interaction between maize and F. verticillioides.

Unravelling the genetic basis of Fusarium seedling rot resistance in the MAGIC maize population: novel targets for breeding.

Fungal infection by Fusarium verticillioides is cause of prevalent maize disease leading to substantial reductions in yield and grain quality worldwide. Maize resistance to the fungus may occur at different developmental stages, from seedling to maturity. The breeding of resistant maize genotypes may take advantage of the identification of quantitative trait loci (QTL) responsible for disease resistance already commenced at seedling level. The Multi-parent Advance Generation Intercross (MAGIC) population was used to conduct high-definition QTL mapping for Fusarium seedling rot (FSR) resistance using rolled towel assay. Infection severity level, seedling weight and length were measured on 401 MAGIC maize recombinant inbred lines (RILs). QTL mapping was performed on reconstructed RIL haplotypes. One-fifth of the MAGIC RILs were resistant to FSR and 10 QTL were identified. For FSR, two QTL were detected at 2.8 Mb and 241.8 Mb on chromosome 4, and one QTL at 169.6 Mb on chromosome 5. Transcriptomic and sequencing information generated on the MAGIC founder lines was used to guide the identification of eight candidate genes within the identified FSR QTL. We conclude that the rolled towel assay applied to the MAGIC maize population provides a fast and cost-effective method to identify QTL and candidate genes for early resistance to F. verticillioides in maize.

A Genome Wide Association Study Reveals Markers and Genes Associated with Resistance to Fusarium verticillioides Infection of Seedlings in a Maize Diversity Panel.

Fusarium verticillioides infects maize, causing ear rot, yield loss and contamination by fumonisin mycotoxins. The fungus can be transmitted via kernels and cause systemic infection in maize. Maize resistance to the fungus may occur at different developmental stages, from seedling to maturity. Resistance during kernel germination is part of the plant-pathogen interaction and so far this aspect has not been investigated. In the present study, a genome wide association study (GWAS) of resistance to Fusarium during the seedling developmental stage was conducted in a maize diversity panel using 226,446 SNP markers. Seedling germination and disease phenotypes were scored on artificially inoculated kernels using the rolled towel assay. GWAS identified 164 SNPs significantly associated with the traits examined. Four SNPs were associated with disease severity score after inoculation, 153 were associated with severity in asymptomatic kernels and 7 with the difference between the severity ratings in inoculated and non-inoculated seeds. A set of genes containing or physically near the significant SNPs were identified as candidates for Fusarium resistance at the seedling stage. Functional analysis revealed that many of these genes are directly involved in plant defense against pathogens and stress responses, including transcription factors, chitinase, cytochrome P450, and ubiquitination proteins. In addition, 25 genes were found in high linkage disequilibrium with the associated SNPs identified by GWAS and four of them directly involved in disease resistance. These findings contribute to understanding the complex system of maize-F. verticillioides and may improve genomic selection for Fusarium resistance at the seedling stage.

The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology.

Genome editing technologies have progressed rapidly and become one of the most important genetic tools in the implementation of pathogen resistance in plants. Recent years have witnessed the emergence of site directed modification methods using meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Recently, CRISPR/Cas9 has largely overtaken the other genome editing technologies due to the fact that it is easier to design and implement, has a higher success rate, and is more versatile and less expensive. This review focuses on the recent advances in plant protection using CRISPR/Cas9 technology in model plants and crops in response to viral, fungal and bacterial diseases. As regards the achievement of viral disease resistance, the main strategies employed in model species such as Arabidopsis and Nicotiana benthamiana, which include the integration of CRISPR-encoding sequences that target and interfere with the viral genome and the induction of a CRISPR-mediated targeted mutation in the host plant genome, will be discussed. Furthermore, as regards fungal and bacterial disease resistance, the strategies based on CRISPR/Cas9 targeted modification of susceptibility genes in crop species such as rice, tomato, wheat, and citrus will be reviewed. After spending years deciphering and reading genomes, researchers are now editing and rewriting them to develop crop plants resistant to specific pests and pathogens.

Phenolic Profile and Susceptibility to Fusarium Infection of Pigmented Maize Cultivars.

Maize is a staple food source in the world, whose ancient varieties or landraces are receiving a growing attention. In this work, two Italian maize cultivars with pigmented kernels and one inbred line were investigated for untargeted phenolic profile, in vitro antioxidant capacity and resistance to Fusariumverticillioides infection. "Rostrato Rosso" was the richest in anthocyanins whilst phenolic acids were the second class in abundance, with comparable values detected between cultivars. Tyrosol equivalents were also the highest in "Rostrato Rosso" (822.4 mg kg-1). Coherently, "Rostrato Rosso" was highly resistant to fungal penetration and diffusion. These preliminary findings might help in breeding programs, aiming to develop maize lines more resistant to infections and with improved nutraceutical value.

Oxylipins from both pathogen and host antagonize jasmonic acid-mediated defence via the 9-lipoxygenase pathway in Fusarium verticillioides infection of maize.

Oxylipins are a newly emerging group of signals that serve defence roles or promote virulence. To identify specific host and fungal genes and oxylipins governing the interactions between maize and Fusarium verticillioides, maize wild-type and lipoxygenase3 (lox3) mutant were inoculated with either F. verticillioides wild-type or linoleate-diol-synthase 1-deleted mutant (ΔFvlds1D). The results showed that lox3 mutants were more resistant to F. verticillioides. The reduced colonization on lox3 was associated with reduced fumonisin production and with a stronger and earlier induction of ZmLOX4, ZmLOX5 and ZmLOX12. In addition to the reported defence function of ZmLOX12, we showed that lox4 and lox5 mutants were more susceptible to F. verticillioides and possessed decreased jasmonate levels during infection, suggesting that these genes are essential for jasmonic acid (JA)-mediated defence. Oxylipin profiling revealed a dramatic reduction in fungal linoleate diol synthase 1 (LDS1)-derived oxylipins, especially 8-HpODE (8-hydroperoxyoctadecenoic acid), in infected lox3 kernels, indicating the importance of this molecule in virulence. Collectively, we make the following conclusions: (1) LOX3 is a major susceptibility factor induced by fungal LDS1-derived oxylipins to suppress JA-stimulating 9-LOXs; (2) LOX3-mediated signalling promotes the biosynthesis of virulence-promoting oxylipins in the fungus; and (3) both fungal LDS1- and host LOX3-produced oxylipins are essential for the normal infection and colonization processes of maize seed by F. verticillioides.

Molecular Basis of Resistance to Fusarium Ear Rot in Maize.

The impact of climate change has been identified as an emerging issue for food security and safety, and the increased incidence of mycotoxin contamination in maize over the last two decades is considered a potential emerging hazard. Disease control by chemical and agronomic approaches is often ineffective and increases the cost of production; for this reason the exploitation of genetic resistance is the most sustainable method for reducing contamination. The review focuses on the significant advances that have been made in the development of transcriptomic, genetic and genomic information for maize, Fusarium verticillioides molds, and their interactions, over recent years. Findings from transcriptomic studies have been used to outline a specific model for the intracellular signaling cascade occurring in maize cells against F. verticillioides infection. Several recognition receptors, such as receptor-like kinases and R genes, are involved in pathogen perception, and trigger down-stream signaling networks mediated by mitogen-associated protein kinases. These signals could be orchestrated primarily by hormones, including salicylic acid, auxin, abscisic acid, ethylene, and jasmonic acid, in association with calcium signaling, targeting multiple transcription factors that in turn promote the down-stream activation of defensive response genes, such as those related to detoxification processes, phenylpropanoid, and oxylipin metabolic pathways. At the genetic and genomic levels, several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium ear rot deriving from QTL mapping and genome-wide association studies are described, indicating the complexity of this polygenic trait. All these findings will contribute to identifying candidate genes for resistance and to applying genomic technologies for selecting resistant maize genotypes and speeding up a strategy of breeding to contrast disease, through plants resistant to mycotoxin-producing pathogens.

QTL mapping and candidate genes for resistance to Fusarium ear rot and fumonisin contamination in maize.

BACKGROUND: Fusarium verticillioides is a common maize pathogen causing ear rot (FER) and contamination of the grains with the fumonisin B1 (FB1) mycotoxin. Resistance to FER and FB1 contamination are quantitative traits, affected by environmental conditions, and completely resistant maize genotypes to the pathogen are so far unknown. In order to uncover genomic regions associated to reduced FER and FB1 contamination and identify molecular markers for assisted selection, an F2:3 population of 188 progenies was developed crossing CO441 (resistant) and CO354 (susceptible) genotypes. FER severity and FB1 contamination content were evaluated over 2 years and sowing dates (early and late) in ears artificially inoculated with F. verticillioides by the use of either side-needle or toothpick inoculation techniques. RESULTS: Weather conditions significantly changed in the two phenotyping seasons and FER and FB1 content distribution significantly differed in the F3 progenies according to the year and the sowing time. Significant positive correlations (P < 0.01) were detected between FER and FB1 contamination, ranging from 0.72 to 0.81. A low positive correlation was determined between FB1 contamination and silking time (DTS). A genetic map was generated for the cross, based on 41 microsatellite markers and 342 single nucleotide polymorphisms (SNPs) derived from Genotyping-by-Sequencing (GBS). QTL analyses revealed 15 QTLs for FER, 17 QTLs for FB1 contamination and nine QTLs for DTS. Eight QTLs located on linkage group (LG) 1, 2, 3, 6, 7 and 9 were in common between FER and FB1, making possible the selection of genotypes with both low disease severity and low fumonisin contamination. Moreover, five QTLs on LGs 1, 2, 4, 5 and 9 located close to previously reported QTLs for resistance to other mycotoxigenic fungi. Finally, 24 candidate genes for resistance to F. verticillioides are proposed combining previous transcriptomic data with QTL mapping. CONCLUSIONS: This study identified a set of QTLs and candidate genes that could accelerate breeding for resistance of maize lines showing reduced disease severity and low mycotoxin contamination determined by F. verticillioides.

Association of Effector Six6 with Vascular Wilt Symptoms Caused by Fusarium oxysporum on Soybean.

The Fusarium oxysporum species complex (FOSC) is a widely distributed group of fungi that includes both pathogenic and nonpathogenic isolates. In a previous study, isolates within the FOSC collected primarily from soybean were assessed for the presence of 12 fungal effector genes. Although none of the assayed genes was significantly associated with wilt symptoms on soybean, the secreted in xylem 6 (Six6) gene was present only in three isolates, which all produced high levels of vascular wilt on soybean. In the current study, a collection of F. oxysporum isolates from soybean roots and F. oxysporum f. sp. phaseoli isolates from common bean was screened for the presence of the Six6 gene. Interestingly, all isolates for which the Six6 amplicon was generated caused wilt symptoms on soybean, and two-thirds of the isolates showed high levels of aggressiveness, indicating a positive association between the presence of the effector gene Six6 and induction of wilt symptoms. The expression profile of the Six6 gene analyzed by quantitative reverse-transcription polymerase chain reaction revealed an enhanced expression for the isolates that caused more severe wilt symptoms on soybean, as established by the greenhouse assay. These findings suggest the suitability of the Six6 gene as a possible locus for pathogenicity-based molecular diagnostics across the various formae speciales.

Constitutive expression of pathogenesis-related proteins and antioxydant enzyme activities triggers maize resistance towards Fusarium verticillioides.

Fusarium verticillioides is a fungal pathogen of maize that causes ear rot and contaminates the grains with fumonisin mycotoxins. Breeding for resistance to Fusarium emerged as the most economic and environmentally safe strategy; therefore the discovery of resistant sources and effective molecular markers are a priority. Ears of resistant (CO441 and CO433) and susceptible (CO354 and CO389) maize lines were inoculated with F. verticillioides and the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes that protect from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) were evaluated in the kernels at 72h post inoculation. In addition, the oxidation level and the enzymatic activity of ascorbate-glutathione cycle, catalase, superoxide dismutase and cytosolic and wall peroxidases were investigated. The uninoculated kernels of the resistant lines showed higher gene expression and enzymatic activities, highlighting the key role of constitutive resistance in limiting pathogen attack. In contrast, the susceptible lines activated defensive genes only after pathogen inoculation, resulting in increased levels of H2O2 and lipid peroxidation, as well as lower enzymatic activities. The constitutive defenses observed in this study from seed could be profitably exploited to develop markers to speed up conventional breeding programs in the selection of resistant genotypes.