Kernel Colonization Research Articles

Investigation of nutritional content and aflatoxin B1 in landraces and varieties of Ethiopian groundnut (Arachis hypogaea L.)

Aflatoxins, a group of toxic compounds produced by certain Aspergillus species, pose significant health risks due to their strong carcinogenic properties. Groundnut (Arachis hypogaea L.) is susceptible to aflatoxin contamination both pre- and post-harvest. This study aimed to investigate the nutritional content and aflatoxin B1 (AFB1) levels in various landraces and varieties of Ethiopian groundnut. Groundnut germplasm was collected from three key regions: Gamo zone, Haramaya University (Haramaya), and Werer Agricultural Research Center (Werer). Key parameters, including individual seed weight (ISW), kernel colonization by Aspergillus flavus, total carbohydrates, protein, total free amino acids, and AFB1 content, were analyzed. The results revealed significant variation (P < 0.05) among the landraces and varieties. The groundnut varieties from Haramaya exhibited significantly higher mean ISW. All five varieties from Haramaya showed zero percent kernel colonization by A. flavus, indicating certain level of resistance. Landraces from Gamo zone were found to be rich in total carbohydrates and total free amino acids. AFB1 levels, determined using indirect competitive ELISA, ranged from 1.84 ppb to 6.89 ppb. While four landraces from Gamo, two varieties from Haramaya, and five varieties from Werer met the European Union’s AFB1 safety threshold (2 ppb), the remaining germplasm exceeded this limit, posing health and trade concerns. No relationship was found between nutritional content and AFB1 levels; however, a significant correlation was observed between kernel colonization and AFB1 content, suggesting kernel colonization could serve as a cost-effective proxy for estimating AFB1 contamination in groundnuts.

Factors influencing aflatoxin B1 levels in the groundnut (Arachis hypogaea L.) germplasm of Ethiopia

As there was no maximum permissible limit prescription for aflatoxin B1 (AFB1) in Ethiopia, this study has been conducted to generate data on AFB1 levels in Ethiopian groundnut accessions/landraces. Besides, an attempt was made to find out if there is any relationship between AFB1 and other parameters such as altitude of cultivation, individual seed weight, kernel colonization by Aspergillus flavus, total carbohydrates, protein and total free amino acids. Out of the 28 accessions studied, merely six accessions registered ≤2 ppb AFB1 and thus, they comply with maximum permissible limit set by European Union. Altitude of cultivation had no relationship with AFB1 levels. Interestingly, total carbohydrates in the seeds as well as kernel colonization by A. flavus showed statistically significant (p < 0.01) positive relationships with AFB1 levels. It is suggested to use kernel colonization measurement as an alternative to the expensive ELISA based AFB1 measurement. Besides, suitable pre- and post-harvest aflatoxin management strategies should be developed to alleviate the AFB1 levels in Ethiopian groundnut.

Relationship of altitude, individual seed weight, and kernel colonization by Aspergillus flavus with biochemical parameters of various Ethiopian groundnut (Arachis hypogaea L.) accessions

Groundnut is one of the five extensively grown oil crops of Ethiopia. Groundnut kernels contain 40-50% fat, 20-50% protein, and 10-20% carbohydrate and are rich in vitamin E, niacin, riboflavin, thiamine, falacin, calcium, phosphorus, magnesium, zinc, iron, and potassium. This study aimed to determine individual seed weight, kernel colonization by Aspergillus flavus, and biochemical parameters in groundnut seeds collected from different areas of Ethiopia. Groundnut germplasm in the form of seeds was collected from the Ethiopian Biodiversity Institute (EBI), further information about each accession location and altitude was also collected from the institute. Parameters such as individual seed weight (ISW), kernel colonization by A. flavus, and biochemical parameters (i.e. total carbohydrate, protein, and total free amino acids) were measured in the groundnut seeds. A negative relationship between ISW and altitude (p&lt;0.01) was observed in this study which indicates that an increase in altitude would result in a decrease in seed weight. A positive relationship between altitude with total carbohydrate (p&lt;0.01) and with total free amino acids (p&lt;0.05) was also observed. Low-ISW accessions exhibited significantly higher total carbohydrates while high-ISW accessions exhibited significantly higher total free amino acids in their seeds. Groundnut seeds of EBI accessions with 0% kernel colonization contained less total carbohydrate, and the accessions with 100% kernel colonization contained the highest total carbohydrates. Besides, total carbohydrates correlated positively with kernel colonization (p&lt;0.01). So, it is postulated that high total carbohydrate makes groundnut seeds susceptible to A. flavus colonization.

Resistance to Aspergillus flavus and Aspergillus parasiticus in Almond Advanced Selections and Cultivars and Its Interaction with the Aflatoxin Biocontrol Strategy.

Aflatoxin contamination of almond kernels, caused by Aspergillus flavus and A. parasiticus, is a severe concern for growers because of its high toxicity. In California, the global leader of almond production, aflatoxin can be managed by applying the biological control strain AF36 of A. flavus and selecting resistant cultivars. Here, we classified the almond genotypes by K-Means cluster analysis into three groups (susceptible [S], moderately susceptible [MS], or resistant [R]) based on aflatoxin content of inoculated kernels. The protective effects of the shell and seedcoat in preventing aflatoxin contamination were also examined. The presence of intact shells reduced aflatoxin contamination >100-fold. The seedcoat provided a layer of protection but not complete protection. In kernel inoculation assays, none of the studied almond genotypes showed a total resistance to the pathogen. However, nine traditional cultivars and four advanced selections were classified as R. Because these advanced selections contained germplasm derived from peach, we compared the kernel resistance of three peach cultivars to that shown by kernels of an R (Sonora) and an S (Carmel) almond cultivar and five pistachio cultivars. Overall, peach kernels were significantly more resistant to the pathogen than almond kernels, which were more resistant than pistachio kernels. Finally, we studied the combined effect of the cultivar resistance and the biocontrol strain AF36 in limiting aflatoxin contamination. For this, we coinoculated almond kernels of R Sonora and S Carmel with AF36 72 h before or 48 h after inoculating with an aflatoxin-producing strain of A. flavus. The percentage of aflatoxin reduction by AF36 strain was greater in kernels of Carmel (98%) than in those of Sonora (83%). Cultivar resistance also affected the kernel colonization by the biological control strain. AF36 strain limited aflatoxin contamination in almond kernels even when applied 48 h after the aflatoxin-producing strain. Our results show that biocontrol combined with the use of cultivars with resistance to aflatoxin contamination can result in a more robust protection strategy than the use of either practice in isolation.

Performance of Winter Wheat Cultivars Grown Organically and Conventionally with Focus on Fusarium Head Blight and Fusarium Trichothecene Toxins.

Growing acreage and changing consumer preferences cause increasing interest in the cereal products originating from organic farming. Lack of results of objective test, however, does not allow drawing conclusions about the effects of cultivation in the organic system and comparison to currently preferred conventional system. Field experiment was conducted in organic and conventional fields. Thirty modern cultivars of winter wheat were sown. They were characterized for disease infection including Fusarium head blight, seed sowing value, the amount of DNA of the six species of Fusarium fungi as well as concentration of ergosterol and trichothecenes in grain. The intensity Fusarium head blight was at a similar level in both systems. However, Fusarium colonization of kernels expressed as ergosterol level or DNA concentration was higher for the organic system. It did not reflect in an increased accumulation of trichothecenes in grain, which was similar in both systems, but sowing value of organically produced seeds was lower. Significant differences between analyzed cropping systems and experimental variants were found. The selection of the individual cultivars for organic growing in terms of resistance to diseases and contamination of grain with Fusarium toxins was possible. Effects of organic growing differ significantly from the conventional and grain obtained such way can be recommended to consumers. There are indications for use of particular cultivars bred for conventional agriculture in the case of organic farming, and the growing organic decreases plant stress resulting from intense fertilization and chemical plant protection.

Set3 Is Required for Asexual Development, Aflatoxin Biosynthesis, and Fungal Virulence in Aspergillus flavus.

Aspergillus flavus is an opportunistic pathogenic fungus for both plant and animal that produces carcinogenic toxins termed aflatoxins (AFs). To identify possible genetic targets to reduce AF contamination, in this study, we have characterized a novel A. flavus Set3, and it shares sequence homology with the yeast protein Set3. The set3 deletion mutants present no difference in growth rate but alterations in asexual development and secondary metabolite production when compared to the A. flavus wild type. Specifically, deletion of set3 gene decreases conidiophore formation and conidial production through downregulating expression of brlA and abaA genes. In addition, normal levels of set3 are required for sclerotial development and expression of sclerotia-related genes nsdC and sclR. Further analyses demonstrated that Set3 negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. Importantly, our results also display that A. flavus Set3 is involved in crop kernel colonization. Taking together, these results reveal that a novel Set3 plays crucial roles in morphological development, secondary metabolism, and fungal virulence in A. flavus.

Infection incidence, kernel colonisation, and mycotoxin accumulation in durum wheat inoculated with Fusarium sporotrichioides, F. langsethiae or F. poae at different growth stages

A 2-year field experiment was conducted to determine the effects of Fusarium sporotrichioides, F. langsethiae, or F. poae on durum wheat plants artificially inoculated at different growth stages. The percentage of symptomatic kernels was similar among the three species, but incidence of infected kernels was lower for F. langsethiae. Kernel colonization was higher when plants were inoculated before and during anthesis for F. sporotrichioides and F. poae, but unaffected by timing of inoculation for F. langsethiae. Production of T-2/HT-2 toxins was higher for F. sporotrichioides than for F. langsethiae. Significant accumulations of nivalenol were detected for F. poae. Across all three species, there was a weak correlation (r = 0.16; P = 0.031) between the incidences of symptomatic and infected kernels, but a stronger correlation (r = 0.53; P 0.58; P < 0.007) with infection incidence or fungal DNA in kernels, but only for F. sporotrichioides and F. poae.

The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus

AbstractA kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.

Evaluation of maize inbred lines and topcross progeny for resistance to pre-harvest aflatoxin contamination

Pre-harvest aflatoxin contamination occurs in maize following kernel colonization by Aspergillus flavus. Aflatoxin contamination resistance is a highly desired trait in maize breeding programs. The identification of novel sources of resistance to pre-harvest aflatoxin contamination is a major focus in germplasm screening efforts. Here, we performed a field evaluation of 64 inbred lines over two years for pre-harvest aflatoxin contamination. Topcrosses were also performed with two testers, B73 and Mo17, to generate 128 F1 hybrids which were also evaluated over two years. Hybrid performance was used to calculate both general combining ability (GCA) of the inbreds, and observed heterosis for aflatoxin resistance. Over both years of the study, aflatoxin concentrations ranged from 80 ± 47 to 17,617 ± 8816 μg kg−1 for inbreds, and from 58 ± 39 to 2771 ± 780 μg kg−1 for hybrids with significant variation between years and lines. The inbred lines CML52, CML69, CML247, GT-603, GEMS-0005, Hi63, Hp301, and M37 W showed <1000 μg kg−1 of aflatoxin accumulation in both years of the study and less than the resistant check, Mp313E, in at least one season. Among these, CML52, GT-603, and Hi63 also showed significant GCA with the testers in hybrid progeny. CML52, GT-603, and M37 W also showed heterotic effects of −13.64%, −12.47%, and − 24.50%, respectively, with B73 resulting in reduced aflatoxin contamination. GT-603 also showed a similar heterotic effect for aflatoxin contamination, −13.11%, with Mo17 indicating that this line may serve as a versatile source of aflatoxin contamination resistance in breeding programs.

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus.

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus. Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus, were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points.

Grain colonization by fumonisin-producing Fusarium spp. and fumonisin synthesis in South African commercial maize in relation to prevailing weather conditions

Fumonisins are mycotoxins produced primarily by Fusarium verticillioides and F. proliferatum on maize. These mycotoxins are secondary, carcinogenic metabolites with a worldwide distribution. This study was performed to evaluate the relationship between weather variables, the colonization of grain by F. verticillioides and resultant fumonisin contamination. Grain colonization by Fusarium spp. was determined using quantitative real-time PCR (qPCR) and contamination with fumonisins using HPLC analysis. Results indicated high natural infection by fumonisin-producing Fusarium spp. and fumonisin concentrations in warmer production areas such as Northern Cape, North West and Free State Provinces. Site-specific weather data, including temperature, radiation, humidity, rainfall and evapo-transpiration were provided by the ARC-Institute for Soil Water and Climate's meteorology office. Stepwise multiple regression analysis selected the variables mean maximum temperature and mean minimum humidity (days 1–14 post-silking) as having significant relationships with colonization of maize kernels by fumonisin-producing Fusarium spp.. Further relationships were calculated using the non-linear, 3-dimensional Lorentzian equation (Sigmaplot 10.0). The optimum temperature for colonization of maize by Fusarium spp. were 28.97, 32.14 and 30.40 °C and the optimum minimum humidities were calculated at 27.29, 31.86 and 29.74% respectively over the three recorded seasons. The application of correlation analysis and stepdown multiple regression analysis on sequential means of weather data and the inclusion of observed fungal biomass as a variable, suggested two phases in the development of fumonisins in maize kernels i.e. colonization of maize tissues during the early post-silking stage, followed by fumonisin production during the dough stage of grain fill. Based on this statistical model it would appear that fumonisin production by a specific biomass of fumonisin producing Fusarium spp. on grain was influenced largely by temperature and less so by rainfall. Optimum fumonisin concentrations were obtained with fungal biomasses of 39.94 pg during 2007, 47.65 pg during 2008 and 90.69 pg during 2009. Optimum temperatures for fumonisin production were 30.33, 31.12 and 29.80 °C for 2007–2009, respectively.Although these statistical models were not consistent over seasons with regard to actual prediction values, they were consistent regarding time of fungal infection and fumonisin production. It is evident that additional driving variables need to be identified.

Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production

Effects of Aspergillus flavus colonization of maize kernels under different water activities (aw; 0.99 and 0.91) and temperatures (30, 37°C) on (a) aflatoxin B1 (AFB1) production and (b) the transcriptome using RNAseq were examined. There was no significant difference (p=0.05) in AFB1 production at 30 and 37°C and 0.99 aw. However, there was a significant (p=0.05) increase in AFB1 at 0.91 aw at 37°C when compared with 30°C/0.99 aw. Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 aw showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30°C and 702 genes at 37°C. Increasing temperature from 30 to 37°C at both aw levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing aw at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both aw and temperature. An interaction effect for 4 of the 25 AFB1 genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by aw stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed.

The novel fungal-specific gene FUG1 has a role in pathogenicity and fumonisin biosynthesis in Fusarium verticillioides.

Fusarium verticillioides is a globally important pathogen of maize, capable of causing severe yield reductions and economic losses. In addition, F. verticillioides produces toxic secondary metabolites during kernel colonization that pose significant threats to human and animal health. Fusarium verticillioides and other plant-pathogenic fungi possess a large number of genes with no known or predicted function, some of which could encode novel virulence factors or antifungal targets. In this study, we identified and characterized the novel gene FUG1 (Fungal Unknown Gene 1) in F. verticillioides through functional genetics. Deletion of FUG1 impaired maize kernel colonization and fumonisin biosynthesis. In addition, deletion of FUG1 increased sensitivity to the antimicrobial compound 2-benzoxazolinone and to hydrogen peroxide, which indicates that FUG1 may play a role in mitigating stresses associated with host defence. Transcriptional profiling via RNA-sequencing (RNA-seq) identified numerous fungal genes that were differentially expressed in the kernel environment following the deletion of FUG1, including genes involved in secondary metabolism and mycelial development. Sequence analysis of the Fug1 protein provided evidence for nuclear localization, DNA binding and a domain of unknown function associated with previously characterized transcriptional regulators. This information, combined with the observed transcriptional reprogramming in the deletion mutant, suggests that FUG1 represents a novel class of fungal transcription factors or genes otherwise involved in signal transduction.

Involvement of FST1 from Fusarium verticillioides in virulence and transport of inositol.

Fumonisin B1 (FB1), a polyketide mycotoxin produced by Fusarium verticillioides during the colonization of maize kernels, is detrimental to human and animal health. FST1 encodes a putative protein with 12 transmembrane domains; however, its function remains unknown. The FST1 gene is highly expressed by the fungus in the endosperm of maize kernels compared with the levels of expression in germ tissues. Previous research has shown that FST1 affects FB1 production, virulence, hydrogen peroxide resistance, hydrophobicity and macroconidia production. Here, we examine the phylogeny of FST1, its expression in a Saccharomyces cerevisiae strain lacking a functional myo-inositol transporter (ITR1) and the effect of amino acid changes in the central loop and C-terminus regions of FST1 on functionality. The results indicate that expression of FST1 in an ITR1 mutant strain restores growth on myo-inositol medium to wild-type levels and restores the inhibitory effects of FB1, suggesting that FST1 can transport both myo-inositol and FB1 into yeast cells. Our results with engineered FST1 also indicate that amino acids in the central loop and C-terminus regions are important for FST1 functionality in both S. cerevisiae and F. verticillioides. Overall, this research has established the first characterized inositol transporter in filamentous fungi and has advanced our knowledge about the global regulatory functions of FST1.

Variations in grain lipophilic phytochemicals, proteins and resistance to Fusarium spp. growth during grain storage as affected by biological plant protection with Aureobasidium pullulans (de Bary)

Modern agriculture relies on an integrated approach, where chemical treatment is reduced to a minimum and replaced by biological control that involves the use of active microorganisms. The effect of the antagonistic yeast-like fungus Aureobasidium pullulans on proteins and bioactive compounds (alkylresorcinols, sterols, tocols and carotenoids) in winter wheat grain and on the colonization of wheat kernels by fungal microbiota, mainly Fusarium spp. pathogens, was investigated. Biological treatment contributed to a slight increase contents of tocols, alkylresorcinols and sterols in grain. At the same time, the variation of wheat grain proteins was low and not significant. Application of A. pullulans enhanced the natural yeast colonization after six months of grain storage and inhibited growth of F. culmorum pathogens penetrating wheat kernel. This study demonstrated that an integrated approach of wheat grain protection with the use of the yeast-like fungus A. pullulans reduced kernel colonization by Fusarium spp. pathogens and increased the content of nutritionally beneficial phytochemicals in wheat grain without a loss of gluten proteins responsible for baking value.

Impact of Crop Residue and Corn-soybean Rotation on the Survival of Fusarium virguliforme a Causal Agent of Sudden Death Syndrome of Soybean

Soybean sudden death syndrome (SDS), caused by Fusarium virguliforme, is an economically important soilborne disease and is a major risk to many soybean [Glycine max, (L.) Merr.] production regions worldwide. Two-year studies were conducted in a greenhouse and in fields to examine survival of the fungus in corn-soybean residues. Corn kernels consistently showed significantly (P<0.05) higher F. virguliforme colony-forming units per gram of dry soil (CFU/g) in a greenhouse and in field micro-plots compared to no additional residue added treatment (control). None of the soil samples from commercial fields in Iowa showed significant (P<0.05) difference in F. virguliforme CFU/g within year of sampling, but between years there were numerical differences but not statistically different in samples if the previous crop had been corn, compared with winter wheat or soybean. In Fusarium spp. not causing SDS, CFU/g were significantly (P<0.05) higher in micro-plots amended with six different corn-soybean residue treatments compared with the control in 2008, while in 2009, only corn stock spread on soil showed significant (P<0.05) difference over the control. Our results suggest that a clean corn harvest could reduce SDS risk by reducing colonization of corn kernels that supports survival of F. virguliforme, while a considerable corn loss during harvest could increase SDS risk.

The Novel Monocot-Specific 9-Lipoxygenase ZmLOX12 Is Required to Mount an Effective Jasmonate-Mediated Defense Against Fusarium verticillioides in Maize

Fusarium verticillioides is a major limiting factor for maize production due to ear and stalk rot and the contamination of seed with the carcinogenic mycotoxin fumonisin. While lipoxygenase (LOX)-derived oxylipins have been implicated in defense against diverse pathogens, their function in maize resistance against F. verticillioides is poorly understood. Here, we functionally characterized a novel maize 9-LOX gene, ZmLOX12. This gene is distantly related to known dicot LOX genes, with closest homologs found exclusively in other monocot species. ZmLOX12 is predominantly expressed in mesocotyls in which it is strongly induced in response to F. verticillioides infection. The Mutator transposon-insertional lox12-1 mutant is more susceptible to F. verticillioides colonization of mesocotyls, stalks, and kernels. The infected mutant kernels accumulate a significantly greater amount of the mycotoxin fumonisin. Reduced resistance to the pathogen is accompanied by diminished levels of the jasmonic acid (JA) precursor 12-oxo phytodienoic acid, JA-isoleucine, and expression of jasmonate-biosynthetic genes. Supporting the strong defense role of jasmonates, the JA-deficient opr7 opr8 double mutant displayed complete lack of immunity to F. verticillioides. Unexpectedly, the more susceptible lox12 mutant accumulated higher levels of kauralexins, suggesting that F. verticillioides is tolerant to this group of antimicrobial phytoalexins. This study demonstrates that this unique monocot-specific 9-LOX plays a key role in defense against F. verticillioides in diverse maize tissues and provides genetic evidence that JA is the major defense hormone against this pathogen.

Trichoderma harzianum seed treatment controls Fusarium verticillioides colonization and fumonisin contamination in maize under field conditions

In northern Italy, the most frequently occurring class of mycotoxins in maize are fumonisins, mainly those produced by Fusarium verticillioides. Currently, good agricultural practices (GAPs) represent the best line of defense for controlling the contamination of maize by Fusarium-toxins. Annual fluctuations in weather conditions can strongly reduce the advantages conferred by GAPs, and thus integration with biological control strategies can be a sustainable way to achieve reliable control of Fusarium colonization and toxin contamination. Trichoderma harzianum is a good biocontrol agent against a wide range of plant pathogens, and previous studies have reported its ability to reduce F. verticillioides colonization under greenhouse conditions. Field trials were conducted in two locations to assess the effect of seed treatment with T. harzianum strain T22 on F. verticillioides kernel colonization and on fumonisin contamination under various natural conditions. An average reduction of 58% in fungal infestation and 53% in mycotoxin contamination was observed during our three-year experiments. This research suggests that seed biopriming with T. harzianum T22 can be a promising and environmentally friendly way to control F. verticillioides kernel colonization and fumonisin accumulation.

Effect of fungal colonization of wheat grains with Fusarium spp. on food choice, weight gain and mortality of meal beetle larvae (Tenebrio molitor).

Species of Fusarium have significant agro-economical and human health-related impact by infecting diverse crop plants and synthesizing diverse mycotoxins. Here, we investigated interactions of grain-feeding Tenebrio molitor larvae with four grain-colonizing Fusarium species on wheat kernels. Since numerous metabolites produced by Fusarium spp. are toxic to insects, we tested the hypothesis that the insect senses and avoids Fusarium-colonized grains. We found that only kernels colonized with F. avenaceum or Beauveria bassiana (an insect-pathogenic fungal control) were avoided by the larvae as expected. Kernels colonized with F. proliferatum, F. poae or F. culmorum attracted T. molitor larvae significantly more than control kernels. The avoidance/preference correlated with larval feeding behaviors and weight gain. Interestingly, larvae that had consumed F. proliferatum- or F. poae-colonized kernels had similar survival rates as control. Larvae fed on F. culmorum-, F. avenaceum- or B. bassiana-colonized kernels had elevated mortality rates. HPLC analyses confirmed the following mycotoxins produced by the fungal strains on the kernels: fumonisins, enniatins and beauvericin by F. proliferatum, enniatins and beauvericin by F. poae, enniatins by F. avenaceum, and deoxynivalenol and zearalenone by F. culmorum. Our results indicate that T. molitor larvae have the ability to sense potential survival threats of kernels colonized with F. avenaceum or B. bassiana, but not with F. culmorum. Volatiles potentially along with gustatory cues produced by these fungi may represent survival threat signals for the larvae resulting in their avoidance. Although F. proliferatum or F. poae produced fumonisins, enniatins and beauvericin during kernel colonization, the larvae were able to use those kernels as diet without exhibiting increased mortality. Consumption of F. avenaceum-colonized kernels, however, increased larval mortality; these kernels had higher enniatin levels than F. proliferatum or F. poae-colonized ones suggesting that T. molitor can tolerate or metabolize those toxins.

UBL1 of Fusarium verticillioides links the N‐end rule pathway to extracellular sensing and plant pathogenesis

Fusarium verticillioides produces fumonisin mycotoxins during colonization of maize. Currently, molecular mechanisms underlying responsiveness of F.verticillioides to extracellular cues during pathogenesis are poorly understood. In this study, insertional mutants were created and screened to identify genes involved in responses to extracellular starch. In one mutant, the restriction enzyme-mediated integration cassette disrupted a gene (UBL1) encoding a UBR-Box/RING domain E3 ubiquitin ligase involved in the N-end rule pathway. Disruption of UBL1 in F.verticillioides (Δubl1) influenced conidiation, hyphal morphology, pigmentation and amylolysis. Disruption of UBL1 also impaired kernel colonization, but the ratio of fumonisin B1 per unit growth was not significantly reduced. The inability of a Δubl1 mutant to recognize an N-end rule degron confirmed involvement of UBL1 in the N-end rule pathway. Additionally, Ubl1 physically interacted with two G protein α subunits of F.verticillioides, thus implicating UBL1 in G protein-mediated sensing of the external environment. Furthermore, deletion of the UBL1 orthologue in F.graminearum reduced virulence on wheat and maize, thus indicating that UBL1 has a broader role in virulence among Fusarium species. This study provides the first linkage between the N-end rule pathway and fungal pathogenesis, and illustrates a new mechanism through which fungi respond to the external environment.