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.

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.

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.

Abutilon theophrasti's defense against the allelochemical benzoxazolin-2(3H)-one: support by Actinomucor elegans.

Abutilon theophrasti Medik., previously found to be rather insensitive to benzoxazinoid containing rye mulch and the allelochemical benzoxazolin-2(3H)-one (BOA), can be associated with the zygomycete Actinomucor elegans, whereby the fungus colonizes the root relatively superficially and mainly in the maturation zone. The fungus mitigates necrosis of the cotyledons when seedlings are incubated with 2 mM BOA, in contrast to those that lack the fungus. In liquid cultures of the fungus, tryptophan was identified. The accumulation of tryptophan is increased in presence of BOA. This amino acid seems to be important in protecting Abutilon against BOA and its derivatives since it suppressed the accumulation of BOA derived, highly toxic 2-aminophen-oxazin-3-one (APO) in the medium and on the root surface during BOA incubations of Abutilon seedlings. Although A. elegans is insensitive to BOA and APO, the fungus is not able to protect the plant against harmful effects of APO, when seedlings are treated with the compound. Abutilon can detoxify BOA via BOA-6-OH glucosylation probably by a cell wall associated glucosyltransferase, but only low amounts of the product accumulate. Low tryptophan concentrations can contribute to a degradation of the toxic intermediate BOA-6-OH by Fenton reactions, whereby the amino acid is oxidized. One of the oxidation products was identified as 4(1H)-quinolinone, which is the core substructure of the quorum sensing molecule 2-heptyl-3-hydroxy-4-quinolone. The mutualistic association of Abutilon theophrasti with Actinomucor elegans is considered as opportunistic and facultative. Such plant-fungus associations depend rather likely on environmental conditions, such as the mode of fertilization.

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.

Benzoxazinoids in rye allelopathy - from discovery to application in sustainable weed control and organic farming.

The allelopathic potency of rye (Secale cereale L.) is due mainly to the presence of phytotoxic benzoxazinones-compounds whose biosynthesis is developmentally regulated, with the highest accumulation in young tissue and a dependency on cultivar and environmental influences. Benzoxazinones can be released from residues of greenhouse-grown rye at levels between 12 and 20 kg/ha, with lower amounts exuded by living plants. In soil, benzoxazinones are subject to a cascade of transformation reactions, and levels in the range 0.5-5 kg/ha have been reported. Starting with the accumulation of less toxic benzoxazolinones, the transformation reactions in soil primarily lead to the production of phenoxazinones, acetamides, and malonamic acids. These reactions are associated with microbial activity in the soil. In addition to benzoxazinones, benzoxazolin-2(3H)-one (BOA) has been investigated for phytotoxic effects in weeds and crops. Exposure to BOA affects transcriptome, proteome, and metabolome patterns of the seedlings, inhibits germination and growth, and can induce death of sensitive species. Differences in the sensitivity of cultivars and ecotypes are due to different species-dependent strategies that have evolved to cope with BOA. These strategies include the rapid activation of detoxification reactions and extrusion of detoxified compounds. In contrast to sensitive ecotypes, tolerant ecotypes are less affected by exposure to BOA. Like the original compounds BOA and MBOA, all exuded detoxification products are converted to phenoxazinones, which can be degraded by several specialized fungi via the Fenton reaction. Because of their selectivity, specific activity, and presumably limited persistence in the soil, benzoxazinoids or rye residues are suitable means for weed control. In fact, rye is one of the best cool season cover crops and widely used because of its excellent weed suppressive potential. Breeding of benzoxazinoid resistant crops and of rye with high benzoxazinoid contents, as well as a better understanding of the soil persistence of phenoxazinones, of the weed resistance against benzoxazinoids, and of how allelopathic interactions are influenced by cultural practices, would provide the means to include allelopathic rye varieties in organic cropping systems for weed control.

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.

BOA detoxification of four summer weeds during germination and seedling growth.

A recent greenhouse study revealed a significant reduction of germination and growth of redroot pigweed (Amaranthus retroflexus) and common purslane (Portulaca oleracea) by rye mulch, whereas velvetleaf (Abutilon theophrasti) and common lambsquarters (Chenopodium album) were not suppressed. Since BOA detoxification by metabolic alteration may influence the relation between the benzoxazinoid content of the soil mulch and weed suppression, we tested the dynamics in BOA detoxification in different plant organs of three and 10-day-old seedlings of four warm season weeds incubated with five BOA concentrations (4, 20, 40, 80, and 200 µmol g(-1) fresh weight). In addition, germination and length of 3-day-old seedlings were measured after exposure to 0, 0.3, 1.5, 3, 6, and 15 µmol BOA. Finally, we tested the influence of the MDR translocator inhibitors verapamil, nifedipine, and the GST inhibitor ethycrynic acid on BOA accumulation and detoxification activity. Due to BOA-detoxification, all weeds were able to grow in environments with low BOA contents. At higher contents, Abutilon theophrasti and Chenopodium album had a better chance to survive because of highly active mechanisms that avoided the uptake of BOA (A. theophrasti) and of efficient detoxification activities in youngest seedlings (C. album). The interpretation of all of the data gave the following sequence of increasing sensitivity: A. theophrasti <<< C. album << P. oleracea ≤ A. retroflexus. The results were in agreement with recent findings of the suppression of these weeds by rye mulches and their benzoxazinoid contents. Our studies demonstrate for the first time that the detoxification of BOA influences the survival of certain weeds in environments enriched with this allelochemical. Therefore, detoxification processes affect the potential for weed suppression by soil allelochemicals in sustainable weed management.

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.

Pea Grain Protein Content Across Italian Environments: Genetic Relationship With Grain Yield, and Opportunities for Genome-Enabled Selection for Protein Yield.

Wider pea (Pisum sativum L.) cultivation has great interest for European agriculture, owing to its favorable environmental impact and provision of high-protein feedstuff. This work aimed to investigate the extent of genotype × environment interaction (GEI), genetically based trade-offs and polygenic control for crude protein content and grain yield of pea targeted to Italian environments, and to assess the efficiency of genomic selection (GS) as an alternative to phenotypic selection (PS) to increase protein yield per unit area. Some 306 genotypes belonging to three connected recombinant inbred line (RIL) populations derived from paired crosses between elite cultivars were genotyped through genotyping-by-sequencing and phenotyped for grain yield and protein content on a dry matter basis in three autumn-sown environments of northern or central Italy. Line variation for mean protein content ranged from 21.7 to 26.6%. Purely genetic effects, compared with GEI effects, were over two-fold larger for protein content, and over 2-fold smaller for grain and protein yield per unit area. Grain yield and protein content exhibited no inverse genetic correlation. A genome-wide association study revealed a definite polygenic control not only for grain yield but also for protein content, with small amounts of trait variation accounted for by individual loci. On average, the GS predictive ability for individual RIL populations based on the rrBLUP model (which was selected out of four tested models) using by turns two environments for selection and one for validation was moderately high for protein content (0.53) and moderate for grain yield (0.40) and protein yield (0.41). These values were about halved for inter-environment, inter-population predictions using one RIL population for model construction to predict data of the other populations. The comparison between GS and PS for protein yield based on predicted gains per unit time and similar evaluation costs indicated an advantage of GS for model construction including the target RIL population and, in case of multi-year PS, even for model training based on data of a non-target population. In conclusion, protein content is less challenging than grain yield for phenotypic or genome-enabled improvement, and GS is promising for the simultaneous improvement of both traits.

Molecular basis of rootstock-related tolerance to water deficit in Vitis vinifera L. cv. Sangiovese: A physiological and metabolomic combined approach.

The rootstock M4 (V. vinifera × V. berlandieri) × V. berlandieri cv. Resseguier n.1) is a recent selection reported to confer improved drought tolerance to grafted V. vinifera scions, a very desired feature in the era of global warming. Therefore, a short-term study was performed on a batch of 12 potted cv. Sangiovese vines grafted either on M4 or on the drought susceptible SO4 rootstock. Ecophysiological assessments as whole canopy net CO2 exchange rate (NCER), transpiration (Tc), and pre-dawn leaf water potential (Ψpd) and UHPLC-ESI/QTOF-MS metabolomics were then used to investigate the different vine responses during water limiting conditions. Water stress was induced by applying 50 % of estimated daily water use from days of year 184-208. M4 was able to deliver similar CO2, at a significantly reduced water use, compared to SO4 grafting. In turn, this resulted in enhanced canopy water use efficiency (NCER/Tc ratio) quantified as +15.1 % during water stress and +21.7 % at re-watering. Untargeted metabolomics showed a similar modulation of brassinosteroids and ABA between the two rootstocks, whereas the up accumulation of cytokinins and gibberellins under drought was peculiar of M4 grafted vines. The increase in gibberellins, together with a concurrent down accumulation of chlorophyll precursors and catabolites and an up accumulation of folates in M4 rootstock suggests that the capacity of limiting reactive-oxygen-species and redox imbalance under drought stress was improved. Finally, distinctive osmolyte accumulation patterns could be observed, with SO4 investing more on proline and glycine-betaine content and M4 primarily showing polyols accumulation.

Pigmented sorghum polyphenols as potential inhibitors of starch digestibility: An in vitro study combining starch digestion and untargeted metabolomics.

Polyphenols from five pigmented sorghum (PS) flours were in vitro evaluated as possible modulators of starch digestibility. White sorghum (WS) flour was used as control. Untargeted metabolomics depicted the phenolic composition of raw and cooked flours (obtained through heating at 100 °C for 30 min in water) highlighting differences in flavonoids and phenolic acids. Raw PS flours were characterized by greater tannin and kafirin contents when compared to WS, and, after cooking, PS flours had greater resistant starch (from 4.2 to 21.4 g /100 g dry matter), and lower starch hydrolysis index (HI) with respect to cooked WS. Multivariate statistics showed that flavonoids characterizing PS were the most discriminant compounds during the in vitro digestion. In addition, kafirin and total tannins content (on raw ingredients) along with the anthocyanin profiles (on cooked samples) were negative correlated with HI. Therefore, PS flours might be good candidates for the formulation of functional foods.

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.