Molecular Insights into Defense Responses of Vietnamese Maize Varieties to Fusarium verticillioides Isolates.
ABSTRACT: Fusarium ear rot (FER) caused by Fusarium verticillioides is one of the main fungal diseases in maize worldwide. To develop a pathogen-tailored FER resistant maize line for local implementation, insights into the virulence variability of a residing F. verticillioides population are crucial for developing customized maize varieties, but remain unexplored. Moreover, little information is currently available on the involvement of the archetypal defense pathways in the F. verticillioides-maize interaction using local isolates and germplasm, respectively. Therefore, this study aims to fill these knowledge gaps. We used a collection of 12 F. verticillioides isolates randomly gathered from diseased maize fields in the Vietnamese central highlands. To assess the plant's defense responses against the pathogens, two of the most important maize hybrid genotypes grown in this agro-ecological zone, lines CP888 and Bt/GT NK7328, were used. Based on two assays, a germination and an in-planta assay, we found that line CP888 was more susceptible to the F. verticillioides isolates when compared to line Bt/GT NK7328. Using the most aggressive isolate, we monitored disease severity and gene expression profiles related to biosynthesis pathways of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), benzoxazinoids (BXs), and pathogenesis-related proteins (PRs). As a result, a stronger induction of SA, JA, ABA, BXs, and PRs synthesizing genes might be linked to the higher resistance of line Bt/GT NK7328 compared to the susceptible line CP888. All these findings could supply valuable knowledge in the selection of suitable FER resistant lines against the local F. verticllioides population and in the development of new FER resistant germplasms.
Project description:Fusarium ear rot (FER) caused by Fusarium verticillioides is one of the most common diseases affecting maize production worldwide. FER results in severe yield losses and grain contamination with health-threatening mycotoxins. Although most studies to date have focused on comprehensive analysis of gene regulation in maize during defense responses against F. verticillioides infection, less is known about the role of microRNAs (miRNAs) in this process. We used deep sequencing to compare small RNA libraries from the maize kernels of susceptible (N6) or resistant (BT-1) inbred lines from uninfected plants and upon F. verticillioides infection. We found that pathogen exposure was accompanied by dynamic alterations in expression levels of multiple miRNAs, including new members of previously annotated miRNA families. A combination of transcriptomic, degradomic, and bioinformatics analyses revealed that F. verticillioides-responsive miRNAs and their potential target genes displayed opposite expression patterns in the susceptible and resistant genotypes. Functional category analysis uncovered preferential enrichment of the pathogen-responsive miRNAs and their targets in the phenylpropanoid metabolic processes, plant-pathogen interactions, and plant phytohormone signal transduction pathways. Furthermore, transgenic maize plants overexpressing miR408b exhibited reduced resistance to F. verticillioides infection in a susceptible maize line. These findings provide new insights into the regulatory roles of miRNAs in maize immunity against FER and new resources for breeding disease resistance into maize.
Project description:The fungus Fusarium verticillioides can infect maize ears, causing Fusarium ear rot (FER) and contaminating the grain with fumonisins (FUM), which are harmful to humans and animals. Breeding for resistance to FER and FUM and post-harvest sorting of grain are two strategies for reducing FUM in the food system. Kernel and cob tissues have been previously associated with differential FER and FUM. Four recombinant inbred line families from the maize nested associated mapping population were grown and inoculated with F. verticillioides across four environments, and we evaluated the kernels for external and internal infection severity as well as FUM contamination. We also employed publicly available phenotypes on innate ear morphology to explore genetic relationships between ear architecture and resistance to FER and FUM. The four families revealed wide variation in external symptomatology at the phenotypic level. Kernel bulk density under inoculation was an accurate indicator of FUM levels. Genotypes with lower kernel density-under both inoculated and uninoculated conditions-and larger cobs were more susceptible to infection and FUM contamination. Quantitative trait locus (QTL) intervals could be classified as putatively resistance-specific and putatively shared for ear and resistance traits. Both types of QTL mapped in this study had substantial overlap with previously reported loci for resistance to FER and FUM. Ear morphology may be a component of resistance to F. verticillioides infection and FUM accumulation.
Project description:Fusarium ear rot (FER) is a common fungal disease in maize (Zea mays L.) caused by Fusarium verticillioides. Resistant germplasm resources for FER are rare in cultivated maize; however, teosintes (Z. mays ssp. parviglumis and Z. mays ssp. diploperennis), which are wild-type species of maize, have the potential to offer a novel source of resistance alleles to enhance pathogen resistance in modern maize. Therefore, the aim of this study was to identify favorable alleles that confer significant levels of resistance toward FER. Three populations of BC2F8 recombinant inbred lines (RILs) were developed by crossing two different teosintes, Z. diploperennis and Z. parviglumis, with maize inbred lines B73 and Zheng58, and were screened for FER resistance. We found that Z. diploperennis and Z. parviglumis had higher resistance toward F. verticillioides in the leaves than B73 and Zheng58. However, the resistance toward F. verticillioides in the leaf and ear was unrelated among RILs. FER resistance was positively correlated with grain yield in the B73 × diploperennis (BD) and Zheng58 × parviglumis (ZP) populations, partly because the quantitative trait loci (QTLs) of FER resistance and yield traits were located close together. Four coincident QTLs (qFERbd5.177, qFERbd10.140, qFERzp4.066, and qFERzp5.116) and two highly reliable resistance-yield synergistic QTLs (qFERbd10.140 and qFERzp4.066) were identified in the BD and ZP populations, opening up the possibility of breeding for FER resistance without reducing yield.
Project description:Driven by climate change, Fusarium ear rot (FER) caused by Fusarium verticillioides occurs frequently in maize worldwide. In parallel, legislative regulations and increasing environmental awareness have spurred research on alternative FER biocontrol strategies. A promising group of bacterial control agents is Streptomyces species due to their metabolic versatility. However, insights into the molecular modes of action of these biocontrol agents are often lacking. This study aims at unraveling the biocontrol efficacy of Streptomyces rhizobacterial strains against F. verticillioides. We first assessed the direct antagonism of four Streptomyces strains ST02, ST03, ST07, and ST08. Then, a profile of 16 genes associated with intrinsic plant defense signaling was assessed in maize plants. Both in vitro and in vivo data showed that the biocontrol strain ST03 perfectly suppressed the growth of F. verticillioides. High inhibition efficacy was also observed for extracellular compounds in the supernatant secreted by this strain. Especially, for maize cobs, the biocontrol strain ST03 not only inhibited the proliferation of F. verticillioides but also significantly repressed fungal fumonisin production 7 days after inoculation. On maize plants, the direct antagonism was confirmed by a significant reduction of the fungal DNA level in soils when co-applied with F. verticillioides and strain ST03. In terms of its action on plants, strain ST03 induced downregulation of auxin responsive genes (AUX1, ARF1, and ARF2) and gibberellic acid (GA)-related gene AN1 even in the absence of F. verticillioides at early time points. In leaves, the biocontrol strain induced the expression of genes related to salicylic acid (SA), and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-mediated pathways, and pathogenesis-related proteins in the presence or absence of the pathogen. Interestingly, the biocontrol strain significantly promoted plant growth even in the presence of F. verticillioides. All of which demonstrated that the Streptomyces strain ST03 is a promising FER biocontrol and a growth-promoting candidate.
Project description: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.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.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.
Project description:Fusarium ear rot (FER) incited by Fusarium verticillioides is a major disease of maize that reduces grain quality globally. Host resistance is the most suitable strategy for managing the disease. We report the results of genome-wide association study (GWAS) to detect alleles associated with increased resistance to FER in a set of 818 tropical maize inbred lines evaluated in three environments. Association tests performed using 43,424 single-nucleotide polymorphic (SNPs) markers identified 45 SNPs and 15 haplotypes that were significantly associated with FER resistance. Each associated SNP locus had relatively small additive effects on disease resistance and accounted for 1-4% of trait variation. These SNPs and haplotypes were located within or adjacent to 38 candidate genes, 21 of which were candidate genes associated with plant tolerance to stresses, including disease resistance. Linkage mapping in four biparental populations to validate GWAS results identified 15 quantitative trait loci (QTL) associated with F. verticillioides resistance. Integration of GWAS and QTL to the maize physical map showed eight colocated loci on chromosomes 2, 3, 4, 5, 9, and 10. QTL on chromosomes 2 and 9 are new. These results reveal that FER resistance is a complex trait that is conditioned by multiple genes with minor effects. The value of selection on identified markers for improving FER resistance is limited; rather, selection to combine small effect resistance alleles combined with genomic selection for polygenic background for both the target and general adaptation traits might be fruitful for increasing FER resistance in maize.
Project description:Maize expressing Cry1Ab insecticidal toxin (Bt maize) is an effective method to control <i>Sesamia nonagrioides</i> and <i>Ostrinia nubilalis</i>, the most damaging corn borers of southern Europe. In this area, maize is prone to <i>Fusarium</i> infections, which can produce mycotoxins that pose a serious risk to human and animal health, causing significant economic losses in the agrifood industry. To investigate the influence of corn borer damage on the presence of <i>Fusarium</i> species and their mycotoxins, Bt maize ears and insect-damaged ears of non-Bt maize were collected from commercial fields in three Bt maize growing areas in Spain, and differences in contamination were assessed. Additionally, larvae of both borer species were collected to evaluate their role as vectors of these molds. Non-Bt maize ears showed significantly higher presence of <i>F. verticillioides</i>, <i>F. proliferatum</i>, and <i>F. subglutinans</i> than Bt maize ears. For the first time, <i>Fusarium</i> species have been isolated from larvae of the two species. The most frequently found mycotoxins in ears were fumonisins, with non-Bt ears being significantly more contaminated than those of Bt maize. High levels of fumonisins were shown to correlate with the occurrence of corn borers in the ear and the presence of <i>F. verticillioides</i> and <i>F. proliferatum</i>.
Project description:Fusarium verticillioides is the most commonly reported fungal species responsible for ear rot of maize which substantially reduces grain yield. It also results in a substantial accumulation of mycotoxins that give rise to toxic response when ingested by animals and humans. For inefficient control by chemical and agronomic measures, it thus becomes more desirable to select more resistant varieties. However, the molecular mechanisms underlying the infection process remain poorly understood, which hampers the application of quantitative resistance in breeding programs. Here, we reveal the disease-resistance mechanism of the maize inbred line of BT-1 which displays high resistance to ear rot using RNA high throughput sequencing. By analyzing RNA-seq data from the BT-1 kernels before and after F. verticillioides inoculation, we found that transcript levels of genes associated with key pathways are dramatically changed compared with the control treatment. Differential gene expression in ear rot resistant and susceptible maize was confirmed by RNA microarray and qRT-PCR analyses. Further investigation suggests that the small heat shock protein family, some secondary metabolites, and the signaling pathways of abscisic acid, jasmonic acid, or salicylic acids (SA) may be involved in the pathogen-associated molecular pattern-triggered immunity against F. verticillioides. These data will not only provide new insights into the molecular resistant mechanisms against fungi invading, but may also result in the identification of key molecular factors associated with ear rot resistance in maize.
Project description:The promotion and application of transgenic <i>Bt</i> crops provides an approach for the prevention and control of target lepidopteran pests and effectively relieves the environmental pressure caused by the massive usage of chemical pesticides in fields. However, studies have shown that <i>Bt</i> crops will face a new risk due to a decrease in exogenous toxin content under elevated carbon dioxide (CO<sub>2</sub>) concentration, thus negatively affecting the ecological sustainability of <i>Bt</i> crops. Arbuscular mycorrhizal fungi (AMF) are important beneficial microorganisms that can effectively improve the nutrient status of host plants and are expected to relieve the ecological risk of <i>Bt</i> crops under increasing CO<sub>2</sub> due to global climate change. In this study, the <i>Bt</i> maize and its parental line of non-transgenic <i>Bt</i> maize were selected and inoculated with a species of AMF (<i>Funneliformis caledonium</i>, synonyms: <i>Glomus caledonium</i>), in order to study the secondary defensive chemicals and yield of maize, and to explore the effects of <i>F. caledonium</i> inoculation on the growth, development, and reproduction of the pest <i>Mythimna separata</i> fed on <i>Bt</i> maize and non-<i>Bt</i> maize under ambient carbon dioxide concentration (<i>a</i>CO<sub>2</sub>) and elevated carbon dioxide concentration (<i>e</i>CO<sub>2</sub>). The results showed that <i>e</i>CO<sub>2</sub> increased the AM fungal colonization, maize yield, and foliar contents of jasmonic acid (JA) and salicylic acid (SA), but decreased foliar Bt toxin content and <i>Bt</i> gene expression in <i>Bt</i> maize leaves. <i>F. caledonium</i> inoculation increased maize yield, foliar JA, SA contents, <i>Bt</i> toxin contents, and <i>Bt</i> gene expression in <i>Bt</i> maize leaves, and positively improved the growth, development, reproduction, and food utilization of the <i>M. separata</i> fed on non-<i>Bt</i> maize. However, <i>F. caledonium</i> inoculation was unfavorable for the fitness of <i>M. separata</i> fed on <i>Bt</i> maize, and the effect was intensified when combined with <i>e</i>CO<sub>2</sub>. It is indicated that <i>F. caledonium</i> inoculation had adverse effects on the production of non-<i>Bt</i> maize due to the high potential risk of population occurrence of <i>M. separata</i>, while it was just the opposite for <i>Bt</i> maize. Therefore, this study confirms that the AMF can increase the yield and promote the expression levels of its endogenous (JA, SA) and exogenous (Bt toxin) secondary defense substances of <i>Bt</i> maize under <i>e</i>CO<sub>2</sub>, and finally can enhance the insect resistance capacity of <i>Bt</i> crops, which will help ensure the sustainable utilization and safety of <i>Bt</i> crops under climate change.
Project description:In order to control lepidopteran and coleopteran insects, the genes expressing <i>Bacillus thuringiensis</i> (<i>Bt</i>) insecticidal proteins have been transferred into crops. Ecological risk assessments of the transgenic plants have included impacts on non-target entomophagous insects, such as parasitoid wasps. Herbivore-induced plant volatiles are considered to be important defensive traits of plants because these compounds play as an important role in recruitment of natural enemies. Here, we evaluated induced volatile emissions of maize seedlings of two <i>Bt</i> cultivars (5422Bt1, event Bt11 and 5422CBCL, event Mon810), and their nearly isogenic non-<i>Bt</i> line 5422. We damaged plants mechanically and then applied with the regurgitant of <i>Spodoptera litura</i> (F.) caterpillars (Lepidoptera: Noctuidae), or treated the plants with the plant hormone jasmonic acid (JA), to trigger similar defensive responses of plants. Compared to the non-<i>Bt</i> isoline 5422 and the <i>Bt</i> maize 5422CBCL, the other <i>Bt</i> maize 5422Bt1 released more (3<i>E</i>)-4,8-dimethyl-1,3,7-nonatriene (DMNT) when they were all treated by artificial wounds and caterpillar regurgitant; and released more linalool, DMNT and (<i>E</i>)-?-farnesene when applied with JA solution. As a result, the total volatile emission of the 5422Bt1 was highest. However, the difference in volatile emission did not affect the attractiveness of the <i>Bt</i> maize plants to the egg parasitoid <i>Trichogramma ostriniae</i> Pang et Chen (Hymenoptera: Trichogrammatidae) compared to the nearly isogenic non-<i>Bt</i> plants. The variability of induced volatiles of maize cultivars derived from conventional breeding programs and transgenic methods are discussed.