Low Aflatoxin Levels in Aspergillus flavus-Resistant Maize Are Correlated With Increased Corn Earworm Damage and Enhanced Seed Fumonisin.
ABSTRACT: Preharvest mycotoxin contamination of field-grown crops is influenced not only by the host genotype, but also by inoculum load, insect pressure and their confounding interactions with seasonal weather. In two different field trials, we observed a preference in the natural infestation of corn earworm (CEW; Helicoverpa zea Boddie) to specific maize (Zea mays L.) genotypes and investigated this observation. The field trials involved four maize lines with contrasting levels of resistance to Aspergillus flavus. The resistant lines had 7 to 14-fold greater infested ears than the susceptible lines. Seed aflatoxin B1 (AF) levels, in mock- and A. flavus-inoculated ears were consistent with genotype resistance to A. flavus, in that the resistant lines showed low levels of AF (<30 ppb), whereas the susceptible lines had up to 500 ppb. On the other hand, CEW infestation showed a positive correlation with seed fumonisins (FUM) contamination by native Fusarium verticillioides strains. We inferred that the inverse trend in the correlation of AF and FUM with H. zea infestation may be due to a differential sensitivity of CEW to the two mycotoxins. This hypothesis was tested by toxin-feeding studies. H. zea larvae showed decreasing mass with increasing AF in the diet and incurred >30% lethality at 250 ppb. In contrast, CEW was tolerant to fumonisin with no significant loss in larval mass even at 100 ppm, implicating the low seed aflatoxin content as a predominant factor for the prevalence of CEW infestation and the associated fumonisin contamination in A. flavus resistant maize lines. Further, delayed flowering of the two resistant maize lines might have contributed to the pervasive H. zea damage of these lines by providing young silk for egg-laying. These results highlight the need for integrated strategies targeting mycotoxigenic fungi as well as their insect vectors for enhanced food safety.
Project description:The emergence of super-toxigenic strains by recombination is a risk from an intensive use of intraspecific aflatoxin (AF) biocontrol agents (BCAs). Periodical alternation with interspecific-BCAs will be safer since they preclude recombination. We are developing an AF-biocontrol system using rice-associated Bacilli reported previously (RABs). More than 50% of RABs inhibited the growth of multiple A. flavus strains, with RAB4R being the most inhibitory and RAB1 among the least. The fungistatic activity of RAB4R is associated with the lysis of A. flavus hyphal tips. In field trails with the top five fungistatic RABs, RAB4R consistently inhibited AF contamination of maize by Tox4, a highly toxigenic A. flavus strain from Louisiana corn fields. RAB1 did not suppress A. flavus growth, but strongly inhibited AF production. Total and HPLC-fractionated lipopeptides (LPs) isolated from culture filtrates of RAB1 and RAB4R also inhibited AF accumulation. LPs were stable in vitro with little loss of activity even after autoclaving, indicating their potential field efficacy as a tank-mix application. A. flavus colonization and AF were suppressed in RAB1- or RAB4R-coated maize seeds. Since RAB4R provided both fungistatic and strong anti-mycotoxigenic activities in the laboratory and field, it can be a potent alternative to atoxigenic A. flavus strains. On the other hand, RAB1 may serve as an environmentally safe helper BCA with atoxigenic A. flavus strains, due its lack of strong fungistatic and hemolytic activities.
Project description:Aspergillus flavus, the main aflatoxin B? producing fungal species, Fusarium graminearum, a deoxynivalenol producer, and the fumonisin-producing species F. proliferatum and F. verticillioides are the main toxigenic fungi (TF) that colonize maize. Several strategies are available to control TF and related mycotoxins, such as chemical control. However, there is poor knowledge on the efficacy of fungicides on maize plants since few molecules are registered. The sensitivity of F. graminearum, F. proliferatum, F. verticillioides, and A. flavus to eleven fungicides, selected based on their different modes of action, was evaluated in both in vitro assays and, after selection, in the field. In vitro, demethylation inhibitors (DMI) showed excellent performances, followed by thiophanate-methyl and folpet. Among the succinate dehydrogenase inhibitors (SDHI), isopyrazam showed a higher effectiveness against Fusarium species than boscalid, which was ineffective against Fusarium, like the phenyl-pyrrole fludioxonil. Furthermore, both SDHIs and fludioxonil were more active against A. flavus than Fusarium species. In field trials, prothioconazole and thiophanate-methyl were confirmed to be effective to reduce F. graminearum (52% and 48%) and F. proliferatum contamination (44% and 27%). On the other hand, prothioconazole and boscalid could reduce A. flavus contamination at values of 75% and 56%, respectively.
Project description:Fusarium fujikuroi, a member of the Fusarium fujikuroi species complex, stands out as a rice bakanae disease pathogen with a high production of gibberellic acid. Not all, but some F. fujikuroi strains are known to produce a carcinogenic mycotoxin fumonisin. Fumonisin biosynthesis is dependent on the FUM cluster composed of 16 FUM genes. The FUM cluster was detected not only from a fumonisin producing strain, but also from a fumonisin nonproducing strain that does not produce a detectable level of fumonisin. Genetic mapping indicated the causative mutation(s) of fumonisin nonproduction is present in the FUM cluster of the fumonisin nonproducing strain. Comparative analyses of FUM genes between the fumonisin producing and the nonproducing strains and gene complementation indicated that causative mutation of fumonisin nonproduction is not a single occurrence and the mutations are distributed in FUM21 and FUM7. Our research revealed a natural variation in the FUM cluster involving fumonisin production difference in F. fujikuroi.
Project description:Several are the inputs which are able to modulate mycotoxin synthesis. In particular, when a fungus receives an external stimulus reacts by activating, through a quite well-defined signal cascade, an evident switch in its lifestyle. This profound change is also due to the activation of global gene regulators and, in particular, of transcription factors able to switch on the mycotoxin gene clusters expression. Aflatoxins (AF) are harmful carcinogenic compounds produced mainly by Aspergillus flavus and A. parasiticus. AF are produced during the contamination of maize kernels into the field, even if their role in phyto-toxicity is not yet assessed. Nevertheless, AF biosynthesis is tightly regulated by host-derived signals. Recently, the nature of some of these signals have been elucidated. In particular, a role in susceptibility and resistance of maize to A. flavus contamination has been assigned to some plant oxylipins. These findings draw a scenario in which a complex interplay is under way between A. flavus and maize. For uncovering all the implications of this cross-talk we decide to follow a holistic approach. In particular, we designed experimental conditions aimed to mimic the different phases of A. flavus infection cycle on maize and then by performing a microarray analysis on the harvested mycelia. The microarray data set has been processed for performing the differential expression analysis of almost 14000 gene probes, the pathway analysis based on the gene ontology and inter pro annotations, the secondary metabolites cluster co-expression analysis and an identification of groups of co-expressed neighbor genes, possibly associated with production of secondary metabolites. Overall design: Analysis of 12 microarrays monitoring gene expression of Aspergillus flavus over various growth conditions
Project description:Maize (Zea mays L.) is a globally important staple food crop prone to contamination by aflatoxin, a carcinogenic secondary metabolite produced by the fungus Aspergillus flavus. An efficient approach to reduce accumulation of aflatoxin is the development of germplasm resistant to colonization and toxin production by A. flavus. Lipoxygenases (LOXs) are a group of non-heme iron containing dioxygenase enzymes that catalyze oxygenation of polyunsaturated fatty acids (PUFAs). LOX derived oxylipins play critical roles in plant defense against pathogens including A. flavus. The objectives of this study were to summarize sequence diversity and expression patterns for all LOX genes in the maize genome, and map their effect on aflatoxin accumulation via linkage and association mapping. In total, 13 LOX genes were identified, characterized, and mapped. The sequence of one gene, ZmLOX10, is reported from 5 inbred lines. Genes ZmLOX1/2, 5, 8, 9, 10 and 12 (GRMZM2G156861, or V4 numbers ZM00001D042541 and Zm00001D042540, GRMZM2G102760, GRMZM2G104843, GRMZM2G017616, GRMZM2G015419, and GRMZM2G106748, respectively) fell under previously published QTL in one or more mapping populations and are linked to a measurable reduction of aflatoxin in maize grains. Association mapping results found 28 of the 726 SNPs tested were associated with reduced aflatoxin levels at p ? 9.71 x 10-4 according to association statistics. These fell within or near nine of the ZmLOX genes. This work confirms the importance of some lipoxygenases for resistance to aflatoxin accumulation and may be used to direct future genetic selection in maize.
Project description:There has been significant interest in the development of formulations of non-toxigenic strains of Aspergillus flavus for control of toxigenic strains to reduce the aflatoxin B1 (AFB1) contamination of maize. In the future, climate change (CC) abiotic conditions of temperature (+2-4°C), CO2 (existing levels of 400 vs. 800-1,200 ppb), and drought stress will impact on the agronomy and control of pests and diseases. This study has examined (1) the effect of two-way interacting factors of water activity × temperature on colonization and AFB1 contamination of maize cobs of different ripening ages; (2) the effect of non-toxigenic strains of A. flavus (50:50 inoculum ratio) on relative control of toxigenic A. flavus and AFB1 contamination of ripening cobs; (3) post-harvest control of AFB1 by non-toxigenic strains of A. flavus in non-GM and isogenic GM maize cultivars using the same inoculum ratio; and (4) the impact of three-way interacting CC factors on relative control of AFB1 in maize cobs pre-harvest and in stored non-GM/GM cultivars. Pre-harvest colonization and AFB1 production by a toxigenic A. flavus strain was conserved at 37°C when compared with 30°C, at the three ripening stages of cob development examined: milk ripe (R3), dough (R4), and dent (R5). However, pre-harvest biocontrol with a non-toxigenic strain was only effective at the R3 and R4 stages and not at the R5 stage. This was supported by relative expression of the aflR regulatory biosynthetic gene in the different treatments. When exposed to three-way interacting CC factors for control of AFB1 pre-harvest, the non-toxigenic A. flavus strain was effective at R3 and £4 stages but not at the R5 stage. Post-harvest storage of non-GM and GM cultivars showed that control was achievable at 30°C, with slightly better control in GM-cultivars in terms of the overall inhibition of AFB1 production. However, in stored maize, the non-toxigenic strains of A. flavus had conserved biocontrol of AFB1 contamination, especially in the GM-maize cultivars under three-way interacting CC conditions (37°C × 1,000 ppm CO2 and drought stress). This was supported by the relative expression of the aflR gene in these treatments. This study suggests that the choice of the biocontrol strains, for pre- or post-harvest control, needs to take into account their resilience in CC-related abiotic conditions to ensure that control of AFB1 contamination can be conserved.
Project description:Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ? 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses.
Project description:Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related (PR) proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance in plants. Among these, production of the PR maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Downregulation of ZmPRms in transgenic kernels resulted in a ?250-350% increase in A. flavus infection accompanied by a ?4.5-7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms-RNAi lines demonstrated downregulation (vs. control) of a majority of these ZmPRms-regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.
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:Several are the inputs which are able to modulate mycotoxin synthesis. In particular, when a fungus receives an external stimulus reacts by activating, through a quite well-defined signal cascade, an evident switch in its lifestyle. This profound change is also due to the activation of global gene regulators and, in particular, of transcription factors able to switch on the mycotoxin gene clusters expression. Aflatoxins (AF) are harmful carcinogenic compounds produced mainly by Aspergillus flavus and A. parasiticus. AF are produced during the contamination of maize kernels into the field, even if their role in phyto-toxicity is not yet assessed. Nevertheless, AF biosynthesis is tightly regulated by host-derived signals. Recently, the nature of some of these signals have been elucidated. In particular, a role in susceptibility and resistance of maize to A. flavus contamination has been assigned to some plant oxylipins. These findings draw a scenario in which a complex interplay is under way between A. flavus and maize. For uncovering all the implications of this cross-talk we decide to follow a holistic approach. In particular, we designed experimental conditions aimed to mimic the different phases of A. flavus infection cycle on maize and then by performing a microarray analysis on the harvested mycelia. The microarray data set has been processed for performing the differential expression analysis of almost 14000 gene probes, the pathway analysis based on the gene ontology and inter pro annotations, the secondary metabolites cluster co-expression analysis and an identification of groups of co-expressed neighbor genes, possibly associated with production of secondary metabolites. Analysis of 12 microarrays monitoring gene expression of Aspergillus flavus over various growth conditions