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.

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.

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.

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.

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 Putative Fungal Effectors in Fusarium oxysporum with Wilt Symptoms in Soybean.

Fungi within the Fusarium oxysporum species complex can cause root rot, seedling blight, and wilt of soybean. Isolates recovered from soybean vary in aggressiveness and also the type of symptoms they produce. The aim of this study was to identify genetic markers to detect aggressive soybean wilt isolates. Eighty isolates collected primarily from soybean were tested in the greenhouse for their ability to produce wilt symptoms using susceptible 'Jack' soybean. The same 80 isolates were assessed for the presence of fungal effector genes Fmk1, Fow1, Pda1, PelA, PelD, Pep1, Prt1, Rho1, Sge1, Six1, Six6, and Snf1. All polymerase chain reaction amplicons were sequenced, phylogenies were inferred, and analysis of molecular variance (AMOVA) was performed for 10 of the 12 genes. High incidence of vascular discoloration of roots or stems was observed with 3 isolates, while moderate to low levels of incidence were observed for 25 isolates. Fungal effector genes Fmk1, Fow1, PelA, Rho1, Sge1, and Snf1 were present in all isolates screened, while Pda1, PelD, Pep1, Prt1, Six1, and Six6 were dispersed among isolates. The Bayesian and AMOVA analyses found that the genes Fmk1, Fow1, Pda1, PelA, Rho1, Sge1, and Snf1 corresponded to previously designated clades based on tef1α and mitochondrial small subunit sequences. None of the genes had a significant association with wilt symptoms on soybean. Interestingly, the Six6 gene was only present in three previously known wilt isolates from soybean, common bean, and tomato; of these, the soybean and common bean isolates produced high levels of vascular wilt in our study.

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.

Defense Responses to Mycotoxin-Producing Fungi Fusarium proliferatum, F. subglutinans, and Aspergillus flavus in Kernels of Susceptible and Resistant Maize Genotypes.

Developing kernels of resistant and susceptible maize genotypes were inoculated with Fusarium proliferatum, F. subglutinans, and Aspergillus flavus. Selected defense systems were investigated using real-time reverse transcription-polymerase chain reaction to monitor the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes protective from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) at 72 h postinoculation. The study was also extended to the analysis of the ascorbate-glutathione cycle and catalase, superoxide dismutase, and cytosolic and wall peroxidases enzymes. Furthermore, the hydrogen peroxide and malondialdehyde contents were studied to evaluate the oxidation level. Higher gene expression and enzymatic activities were observed in uninoculated kernels of resistant line, conferring a major readiness to the pathogen attack. Moreover expression values of PR genes remained higher in the resistant line after inoculation, demonstrating a potentiated response to the pathogen invasions. In contrast, reactive oxygen species-scavenging genes were strongly induced in the susceptible line only after pathogen inoculation, although their enzymatic activity was higher in the resistant line. Our data provide an important basis for further investigation of defense gene functions in developing kernels in order to improve resistance to fungal pathogens. Maize genotypes with overexpressed resistance traits could be profitably utilized in breeding programs focused on resistance to pathogens and grain safety.

Transcriptome profiling of soybean (Glycine max) roots challenged with pathogenic and non-pathogenic isolates of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is one of the most common fungal pathogens causing soybean root rot and seedling blight in U.S.A. In a recent study, significant variation in aggressiveness was observed among isolates of F. oxysporum collected from roots in Iowa, ranging from highly pathogenic to weakly or non-pathogenic isolates. RESULTS: We used RNA-seq analysis to investigate the molecular aspects of the interactions of a partially resistant soybean genotype with non-pathogenic/pathogenic isolates of F. oxysporum at 72 and 96 h post inoculation (hpi). Markedly different gene expression profiles were observed in response to the two isolates. A peak of highly differentially expressed genes (HDEGs) was triggered at 72 hpi in soybean roots and the number of HDEGs was about eight times higher in response to the pathogenic isolate compared to the non-pathogenic one (1,659 vs. 203 HDEGs, respectively). Furthermore, the magnitude of induction was much greater in response to the pathogenic isolate. This response included a stronger activation of defense-related genes, transcription factors, and genes involved in ethylene biosynthesis, secondary and sugar metabolism. CONCLUSIONS: The obtained data provide an important insight into the transcriptional responses of soybean-F. oxysporum interactions and illustrate the more drastic changes in the host transcriptome in response to the pathogenic isolate. These results may be useful in the developing new methods of broadening resistance of soybean to F. oxysporum, including the over-expression of key soybean genes.

Functional genomic analysis of constitutive and inducible defense responses to Fusarium verticillioides infection in maize genotypes with contrasting ear rot resistance.

BACKGROUND: Fusarium verticillioides causes ear rot in maize (Zea mays L.) and accumulation of mycotoxins, that affect human and animal health. Currently, chemical and agronomic measures to control Fusarium ear rot are not very effective and selection of more resistant genotypes is a desirable strategy to reduce contaminations. A deeper knowledge of molecular events and genetic basis underlying Fusarium ear rot is necessary to speed up progress in breeding for resistance. RESULTS: A next-generation RNA-sequencing approach was used for the first time to study transcriptional changes associated with F. verticillioides inoculation in resistant CO441 and susceptible CO354 maize genotypes at 72 hours post inoculation. More than 100 million sequence reads were generated for inoculated and uninoculated control plants and analyzed to measure gene expression levels. Comparison of expression levels between inoculated vs. uninoculated and resistant vs. susceptible transcriptomes revealed a total number of 6,951 differentially expressed genes. Differences in basal gene expression were observed in the uninoculated samples. CO441 genotype showed a higher level of expression of genes distributed over all functional classes, in particular those related to secondary metabolism category. After F. verticillioides inoculation, a similar response was observed in both genotypes, although the magnitude of induction was much greater in the resistant genotype. This response included higher activation of genes involved in pathogen perception, signaling and defense, including WRKY transcription factors and jasmonate/ethylene mediated defense responses. Interestingly, strong differences in expression between the two genotypes were observed in secondary metabolism category: pathways related to shikimate, lignin, flavonoid and terpenoid biosynthesis were strongly represented and induced in the CO441 genotype, indicating that selection to enhance these traits is an additional strategy for improving resistance against F. verticillioides infection. CONCLUSIONS: The work demonstrates that the global transcriptional analysis provided an exhaustive view of genes involved in pathogen recognition and signaling, and controlling activities of different TFs, phytohormones and secondary metabolites, that contribute to host resistance against F. verticillioides. This work provides an important source of markers for development of disease resistance maize genotypes and may have relevance to study other pathosystems involving mycotoxin-producing fungi.

Public perception of new plant breeding techniques and the psychosocial determinants of acceptance: A systematic review.

Advancements in New Plant Breeding Techniques have emerged as promising tools for enhancing crop productivity, quality, and resilience in the face of global challenges, such as climate change and food security. However, the successful implementation of these techniques relies also on public acceptance of this innovation. Understanding what shapes public perception and acceptance of New Plant Breeding Techniques is crucial for effective science communication, policymaking, and the sustainable adoption of these innovations. The objective of this systematic review was to synthesize existing research on the public perception of New Plant Breeding Techniques applied to food crops and explore the psychosocial determinants that influence acceptance. Twenty papers published between 2015 and 2023 were included on various New Plant Breeding Techniques and their reception by the general public. Determinants affecting the acceptance of food crops derived from New Plant Breeding Techniques were categorized into six areas: sociodemographic factors, perceived benefits and risks, attitudes toward science, communication strategies, personal values, and product characteristics.

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.

Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

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.

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.

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.

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.

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.

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.