Monoclonal-Antibody-Based Immunoassays for the Mycotoxins NX-2 and NX-3 in Wheat.

The fungal infestation of crops can cause major economic losses. Toxins produced by the causative fungi (mycotoxins) represent a potential safety hazard to people and livestock consuming them. One such mycotoxin is deoxynivalenol (DON, also known as vomitoxin), a trichothecene associated with Fusarium Head Blight of wheat. DON is commonly found in cereal crops worldwide. A group of trichothecene mycotoxins closely related to DON, the NX toxins, have been reported to occur in the northeastern United States and southern Canada. While many commercial immunoassays are available to detect DON, there are no rapid screening assays for the NX toxins. We describe the development and isolation of three monoclonal antibodies (mAbs) specific towards two NX toxins: NX-2 and NX-3. The mAbs did not recognize DON or several other closely related trichothecenes. One of the mAbs was selected for development of an enzyme-linked immunosorbent assay (ELISA) for NX-2 and NX-3 in wheat. The dynamic ranges for the assay were 7.7 to 127 µg/kg for NX-2 and 59 µg/kg to 1540 µg/kg for NX-3 in wheat. Recoveries from spiked wheat averaged 84.4% for NX-2 and 99.3% for NX-3, with RSDs of 10.4% and 11.3%, respectively (n = 24). The results suggest that this assay can be used to screen for NX toxins in wheat at levels relevant to human food and animal feed safety.

Dynamic geospatial modeling of mycotoxin contamination of corn in Illinois: unveiling critical factors and predictive insights with machine learning.

Mycotoxin contamination of corn is a pervasive problem that negatively impacts human and animal health and causes economic losses to the agricultural industry worldwide. Historical aflatoxin (AFL) and fumonisin (FUM) mycotoxin contamination data of corn, daily weather data, satellite data, dynamic geospatial soil properties, and land usage parameters were modeled to identify factors significantly contributing to the outbreaks of mycotoxin contamination of corn grown in Illinois (IL), AFL >20 ppb, and FUM >5 ppm. Two methods were used: a gradient boosting machine (GBM) and a neural network (NN). Both the GBM and NN models were dynamic at a state-county geospatial level because they used GPS coordinates of the counties linked to soil properties. GBM identified temperature and precipitation prior to sowing as significant influential factors contributing to high AFL and FUM contamination. AFL-GBM showed that a higher aflatoxin risk index (ARI) in January, March, July, and November led to higher AFL contamination in the southern regions of IL. Higher values of corn-specific normalized difference vegetation index (NDVI) in July led to lower AFL contamination in Central and Southern IL, while higher wheat-specific NDVI values in February led to higher AFL. FUM-GBM showed that temperature in July and October, precipitation in February, and NDVI values in March are positively correlated with high contamination throughout IL. Furthermore, the dynamic geospatial models showed that soil characteristics were correlated with AFL and FUM contamination. Greater calcium carbonate content in soil was negatively correlated with AFL contamination, which was noticeable in Southern IL. Greater soil moisture and available water-holding capacity throughout Southern IL were positively correlated with high FUM contamination. The higher clay percentage in the northeastern areas of IL negatively correlated with FUM contamination. NN models showed high class-specific performance for 1-year predictive validation for AFL (73%) and FUM (85%), highlighting their accuracy for annual mycotoxin prediction. Our models revealed that soil, NDVI, year-specific weekly average precipitation, and temperature were the most important factors that correlated with mycotoxin contamination. These findings serve as reliable guidelines for future modeling efforts to identify novel data inputs for the prediction of AFL and FUM outbreaks and potential farm-level management practices.

Manipulating atmospheric CO2 concentration induces shifts in wheat leaf and spike microbiomes and in Fusarium pathogen communities.

Changing atmospheric composition represents a source of uncertainty in our assessment of future disease risks, particularly in the context of mycotoxin producing fungal pathogens which are predicted to be more problematic with climate change. To address this uncertainty, we profiled microbiomes associated with wheat plants grown under ambient vs. elevated atmospheric carbon dioxide concentration [CO2] in a field setting over 2 years. We also compared the dynamics of naturally infecting versus artificially introduced Fusarium spp. We found that the well-known temporal dynamics of plant-associated microbiomes were affected by [CO2]. The abundances of many amplicon sequence variants significantly differed in response to [CO2], often in an interactive manner with date of sample collection or with tissue type. In addition, we found evidence that two strains within Fusarium - an important group of mycotoxin producing fungal pathogens of plants - responded to changes in [CO2]. The two sequence variants mapped to different phylogenetic subgroups within the genus Fusarium, and had differential [CO2] responses. This work informs our understanding of how plant-associated microbiomes and pathogens may respond to changing atmospheric compositions.

Elevated CO2 Can Worsen Fusarium Head Blight Disease Severity in Wheat but the Fhb1 QTL Provides Reliable Disease Resistance.

Fusarium head blight (FHB) is a destructive fungal disease of wheat that causes significant economic loss due to lower yields and the contamination of grain with fungal toxins (mycotoxins), particularly deoxynivalenol (DON). FHB disease spread and mycotoxin contamination has been shown to worsen at elevated CO2, therefore, it is important to identify climate-resilient FHB resistance. This work evaluates whether wheat with the Fhb1 quantitative trait locus (QTL), the most widely deployed FHB resistance locus in wheat breeding programs, provides reliable disease resistance at elevated CO2. Near-isogenic wheat lines (NILs) derived from either a highly FHB susceptible or a more FHB resistant genetic background, with or without the Fhb1 QTL, were grown in growth chambers at ambient (400 ppm) and elevated (1000 ppm) CO2 conditions. Wheat was inoculated with Fusarium graminearum and evaluated for FHB severity. At elevated CO2, the NILs derived from more FHB-resistant wheat had increased disease spread, greater pathogen biomass and mycotoxin contamination, and lower rates of DON detoxification; this was not observed in wheat from a FHB susceptible genetic background. The Fhb1 QTL was not associated with increased disease severity in wheat grown at elevated CO2 and provided reliable disease resistance.

Insights into the Aggressiveness of the Emerging North American Population 3 (NA3) of Fusarium graminearum.

In the United States and Canada, Fusarium graminearum (Fg) is the predominant etiological agent of Fusarium head blight (FHB), an economically devastating fungal disease of wheat and other small grains. Besides yield losses, FHB leads to grain contamination with trichothecene mycotoxins that are harmful to plant, human, and livestock health. Three genetic North American populations of Fg, differing in their predominant trichothecene chemotype (i.e., NA1/15ADON, NA2/3ADON, and NA3/NX-2), have been identified. To improve our understanding of the newly discovered population NA3 and how population-level diversity influences FHB outcomes, we inoculated heads of the moderately resistant wheat cultivar Alsen with 15 representative strains from each population and evaluated disease progression, mycotoxin accumulation, and mycotoxin production per unit Fg biomass. Additionally, we evaluated population-specific differences in induced host defense responses. The NA3 population was significantly less aggressive than the NA1 and NA2 populations but posed a similar mycotoxigenic potential. Multiomics analyses revealed patterns in mycotoxin production per unit Fg biomass, expression of Fg aggressiveness-associated genes, and host defense responses that did not always correlate with the NA3-specific severity difference. Our comparative disease assay of NA3/NX-2 and admixed NA1/NX-2 strains indicated that the reduced NA3 aggressiveness is not due solely to the NX-2 chemotype. Notably, the NA1 and NA2 populations did not show a significant advantage over NA3 in perithecia production, a fitness-related trait. Together, our data highlight that the disease outcomes were not due to mycotoxin production or host defense alone, indicating that other virulence factors and/or host defense mechanisms are likely involved.

NX Trichothecenes Are Required for Fusarium graminearum Infection of Wheat.

Fusarium graminearum causes Fusarium head blight (FHB) on wheat and barley and contaminates grains with various mycotoxins that are toxic to humans and animals. Deoxynivalenol (DON), a type B trichothecene, is an essential virulence factor that is required for F. graminearum to spread within a wheat head. Recently, novel type A trichothecenes NX-2 and NX-3 (NX) have been found in F. graminearum. NX trichothecenes lack a keto group at the C8 position. To determine if NX trichothecenes play a role similar to that of DON during F. graminearum infection, deletion mutants of TRI5, the first gene for trichothecene biosynthesis, were generated from strains PH-1, NRRL46422, and NRRL44211 (hereafter 44211) representing the 15-acetyl-DON, 3-acetyl-DON, and NX chemotypes. No trichothecene production was detected in any of the Δtri5 mutants in cultures or inoculated wheat heads. FHB symptoms were restricted to the inoculated wheat spikelets when point-inoculated with the Δtri5 mutants, confirming the necessity of NX and DON for FHB spread. Furthermore, whole-head dip inoculations revealed significant reductions in disease and fungal biomass in wheat heads inoculated with 44211Δtri5 compared with 44211. Introduction of the native 44211 TRI5 and a Trichoderma arundinaceum TRI5 ortholog in the 44211Δtri5 mutant complemented trichothecene production in vitro; however, introducing both TRI5 partially restored wild-type levels of NX in infected heads. Our results demonstrate that NX trichothecenes play an important role in Fusarium graminearum initial infection as well as FHB spread. Thus, TRI5 may serve as an ideal target to control plant infection, FHB spread, and mycotoxin production simultaneously. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fhb1 disease resistance QTL does not exacerbate wheat grain protein loss at elevated CO2.

Fusarium head blight, a devastating cereal crop disease, can cause significant yield losses and contaminate grain with hazardous fungal toxins. Concerningly, recent evidence indicates that substantial grain protein content loss is likely to occur in wheat that is moderately resistant to head blight when it is grown at elevated CO2. Although wheat breeders in North America utilize a number of resistance sources and genes to reduce pathogen damage, the Fhb1 gene is widely deployed. To determine whether Fhb1 is associated with the protein content loss at elevated CO2, twelve near-isogenic spring wheat lines from either a susceptible or moderately susceptible genetic background, and with, or without the Fhb1 QTL, were grown at ambient and elevated CO2 conditions. The near-isogenic lines were evaluated for differences in physiology, productivity, and grain protein content. Our results showed that the Fhb1 QTL did not have any significant effect on plant growth, development, yield, or grain protein content at ambient or elevated CO2. Therefore, other factors in the moderately susceptible wheat genetic background are likely responsible for the more severe grain protein loss at elevated CO2.

Gradient boosting and bayesian network machine learning models predict aflatoxin and fumonisin contamination of maize in Illinois - First USA case study.

Mycotoxin contamination of corn results in significant agroeconomic losses and poses serious health issues worldwide. This paper presents the first report utilizing machine learning and historical aflatoxin and fumonisin contamination levels in-order-to develop models that can confidently predict mycotoxin contamination of corn in Illinois, a major corn producing state in the USA. Historical monthly meteorological data from a 14-year period combined with corresponding aflatoxin and fumonisin contamination data from the State of Illinois were used to engineer input features that link weather, fungal growth, and aflatoxin production in combination with gradient boosting (GBM) and bayesian network (BN) modeling. The GBM and BN models developed can predict mycotoxin contamination with overall 94% accuracy. Analyses for aflatoxin and fumonisin with GBM showed that meteorological and satellite-acquired vegetative index data during March significantly influenced grain contamination at the end of the corn growing season. Prediction of high aflatoxin contamination levels was linked to high aflatoxin risk index in March/June/July, high vegetative index in March and low vegetative index in July. Correspondingly, high levels of fumonisin contamination were linked to high precipitation levels in February/March/September and high vegetative index in March. During corn flowering time in June, higher temperatures range increased prediction of high levels of fumonisin contamination, while high aflatoxin contamination levels were linked to high aflatoxin risk index. Meteorological events prior to corn planting in the field have high influence on predicting aflatoxin and fumonisin contamination levels at the end of the year. These early-year events detected by the models can directly assist farmers and stakeholders to make informed decisions to prevent mycotoxin contamination of Illinois grown corn.

Measurement of Fumonisins in Maize Using a Portable Mass Spectrometer.

Fumonisins are a group of mycotoxins that routinely contaminate maize. Their presence is monitored at multiple stages from harvest to final product. Immunoassays are routinely used to screen commodities in the field while laboratory-based methods, such as mass spectrometry (MS), are used for confirmation. The use of a portable mass spectrometer unlocks the potential to conduct confirmatory analyses outside of traditional laboratories. Herein, a portable mass spectrometer was used to measure fumonisins in maize. Samples were extracted with aqueous methanol, cleaned up on an immunoaffinity column, and tested with the portable MS. The limits of detection were 0.15, 0.19, and 0.28 mg/kg maize for fumonisins B1 (FB1), FB2/FB3, and total fumonisins, respectively. The corresponding limits of quantitation in maize were 0.33, 0.59, and 0.74 mg/kg. Recoveries ranged from 93.6% to 108.6%. However, RSDs ranged from 12.0 to 29.8%. The method was applied to the detection of fumonisins in 64 samples of maize collected as part of the Illinois Department of Agriculture's monitoring program. Good correlations were observed between the portable MS and a laboratory-based LC-MS method (r2 from 0.9132 to 0.9481). Results suggest the portable MS can be applied to the measurement of fumonisins in maize at levels relevant to international regulations.

Fusarium head blight resistance exacerbates nutritional loss of wheat grain at elevated CO2.

The nutritional integrity of wheat is jeopardized by rapidly rising atmospheric carbon dioxide (CO2) and the associated emergence and enhanced virulence of plant pathogens. To evaluate how disease resistance traits may impact wheat climate resilience, 15 wheat cultivars with varying levels of resistance to Fusarium Head Blight (FHB) were grown at ambient and elevated CO2. Although all wheat cultivars had increased yield when grown at elevated CO2, the nutritional contents of FHB moderately resistant (MR) cultivars were impacted more than susceptible cultivars. At elevated CO2, the MR cultivars had more significant differences in plant growth, grain protein, starch, fructan, and macro and micro-nutrient content compared with susceptible wheat. Furthermore, changes in protein, starch, phosphorus, and magnesium content were correlated with the cultivar FHB resistance rating, with more FHB resistant cultivars having greater changes in nutrient content. This is the first report of a correlation between the degree of plant pathogen resistance and grain nutritional content loss in response to elevated CO2. Our results demonstrate the importance of identifying wheat cultivars that can maintain nutritional integrity and FHB resistance in future atmospheric CO2 conditions.

Use of the volatile trichodiene to reduce Fusarium head blight and trichothecene contamination in wheat.

Fusarium graminearum is the primary cause of Fusarium head blight (FHB), one of the most economically important diseases of wheat worldwide. FHB reduces yield and contaminates grain with the trichothecene mycotoxin deoxynivalenol (DON), which poses a risk to plant, human and animal health. The first committed step in trichothecene biosynthesis is formation of trichodiene (TD). The volatile nature of TD suggests that it could be a useful intra or interspecies signalling molecule, but little is known about the potential signalling role of TD during F. graminearum-wheat interactions. Previous work using a transgenic Trichoderma harzianum strain engineered to emit TD (Th + TRI5) indicated that TD can function as a signal that can modulate pathogen virulence and host plant resistance. Herein, we demonstrate that Th + TRI5 has enhanced biocontrol activity against F. graminearum and reduced DON contamination by 66% and 70% in a moderately resistant and a susceptible cultivar, respectively. While Th + TRI5 volatiles significantly influenced the expression of the pathogenesis-related 1 (PR1) gene, the effect was dependent on cultivar. Th + TRI5 volatiles strongly reduced DON production in F. graminearum plate cultures and downregulated the expression of TRI genes. Finally, we confirm that TD fumigation reduced DON accumulation in a detached wheat head assay.

Weeds Harbor Fusarium Species that Cause Malformation Disease of Economically Important Trees in Western Mexico.

Mango malformation disease (MMD) caused by Fusarium spp. is an important limiting factor in most production areas worldwide. Fusarium mexicanum and F. pseudocircinatum have been reported as causing MMD in Mexico. These two pathogens also cause a similar disease in Swietenia macrophylla (big-leaf mahogany malformation disease) in central western Mexico, and F. pseudocircinatum was recently reported as causing malformation disease in Tabebuia rosea (rosy trumpet) in the same region. These studies suggest that additional plant species, including weeds, might be hosts of these pathogens. The role that weed hosts might have in the disease cycle is unknown. The objectives of this work were to recover Fusarium isolates from understory vegetation in mango orchards with MMD, identify the Fusarium isolates through DNA sequence data, and determine whether F. mexicanum is capable of inducing disease in the weedy legume Senna uniflora (oneleaf senna). Additional objectives in this work were to compare Fusarium isolates recovered from weeds and mango trees in the same orchards by characterizing their phylogenetic relationships, assessing in vitro production of mycotoxins, and identifying their mating type idiomorph. A total of 59 Fusarium isolates from five species complexes were recovered from apical and lateral buds from four weed species. Two of the species within the F. fujikuroi species complex are known to cause MMD in Mexico. Trichothecene production was detected in five isolates, including F. sulawense and F. irregulare in the F. incarnatum-equiseti species complex and F. boothii in the F. sambucinum species complex. Both mating types were present among mango and weed isolates. This is the first report of herbaceous hosts harboring Fusarium species that cause mango malformation in Mexico. The information provided should prove valuable for further study of the epidemiological role of weeds in MMD and help manage the disease.

A Novel Trichothecene Toxin Phenotype Associated with Horizontal Gene Transfer and a Change in Gene Function in Fusarium.

Fusarium trichothecenes are among the mycotoxins of most concern to food and feed safety. Production of these mycotoxins and presence of the trichothecene biosynthetic gene (TRI) cluster have been confirmed in only two multispecies lineages of Fusarium: the Fusarium incarnatum-equiseti (Incarnatum) and F. sambucinum (Sambucinum) species complexes. Here, we identified and characterized a TRI cluster in a species that has not been formally described and is represented by Fusarium sp. NRRL 66739. This fungus is reported to be a member of a third Fusarium lineage: the F. buharicum species complex. Cultures of NRRL 66739 accumulated only two trichothecenes, 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol. Although these are not novel trichothecenes, the production profile of NRRL 66739 is novel, because in previous reports 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol were components of mixtures of 6-8 trichothecenes produced by several Fusarium species in Sambucinum. Heterologous expression analysis indicated that the TRI13 gene in NRRL 66739 confers trichothecene 7-hydroxylation. This contrasts the trichothecene 4-hydroxylation function of TRI13 in other Fusarium species. Phylogenetic analyses suggest that NRRL 66739 acquired the TRI cluster via horizontal gene transfer from a close relative of Incarnatum and Sambucinum. These findings provide insights into evolutionary processes that have shaped the distribution of trichothecene production among Fusarium species and the structural diversity of the toxins.

Distribution, Function, and Evolution of a Gene Essential for Trichothecene Toxin Biosynthesis in Trichoderma.

Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.

Effects of Atmospheric CO2 and Temperature on Wheat and Corn Susceptibility to Fusarium graminearum and Deoxynivalenol Contamination.

This work details the impact of atmospheric CO2 and temperature conditions on two strains of Fusarium graminearum, their disease damage, pathogen growth, mycotoxin accumulation, and production per unit fungal biomass in wheat and corn. An elevated atmospheric CO2 concentration, 1000 ppm CO2, significantly increased the accumulation of deoxynivalenol in infected plants. Furthermore, growth in cool growing conditions, 20 °C/18 °C, day and night, respectively, resulted in the highest amounts of pathogen biomass and toxin accumulation in both inoculated wheat and corn. Warm temperatures, 25 °C/23 °C, day and night, respectively, suppressed pathogen growth and toxin accumulation, with reductions as great as 99% in corn. In wheat, despite reduced pathogen biomass and toxin accumulation at warm temperatures, the fungal pathogen was more aggressive with greater disease damage and toxin production per unit biomass. Disease outcomes were also pathogen strain specific, with complex interactions between host, strain, and growth conditions. However, we found that atmospheric CO2 and temperature had essentially no significant interactions, except for greatly increased deoxynivalenol accumulation in corn at cool temperatures and elevated CO2. Plants were most susceptible to disease damage at warm and cold temperatures for wheat and corn, respectively. This work helps elucidate the complex interaction between the abiotic stresses and biotic susceptibility of wheat and corn to Fusarium graminearum infection to better understand the potential impact global climate change poses to future food security.

Malformation Disease in Tabebuia rosea (Rosy Trumpet) Caused by Fusarium pseudocircinatum in Mexico.

Tabebuia rosea (rosy trumpet) is an economically important neotropical tree in Mexico that is highly valued for the quality of its wood, which is used for furniture, crafts, and packing, and for its use as an ornamental and shade tree in parks and gardens. During surveys conducted in the lower Balsas River Basin region in the states of Guerrero and Michoacán, symptoms of floral malformation were detected in T. rosea trees. The main objectives of this study were to describe this new disease, to determine its causal agent, and to identify it using DNA sequence data. A second set of objectives was to analyze the phylogenetic relationship of the causal agent to Fusarium spp. associated with Swietenia macrophylla trees with malformation surveyed in the same region and to compare mycotoxin production and the mating type idiomorphs of fusaria recovered from T. rosea and S. macrophylla. Tabebuia rosea showed malformed inflorescences with multiple tightly curled shoots and shortened internodes. A total of 31 Fusarium isolates recovered from symptomatic T. rosea (n = 20) and S. macrophylla (n = 11) trees were identified by molecular analysis as Fusarium pseudocircinatum. Pathogenicity tests showed that isolates of F. pseudocircinatum recovered from T. rosea induced malformation in inoculated T. rosea seedlings. Eighteen F. pseudocircinatum isolates were tested for their ability to produce mycotoxins and other secondary metabolites. Moniliformin, fusaric acid, bikaverin, beauvericin, aurofusarin. and 8-O-methylbostrycoidin were produced by at least one strain of the 18 isolates tested. A multiplex PCR assay for mating type idiomorph revealed that 22 F. pseudocircinatum isolates were MAT1-1 and that 9 were MAT1-2. Here, we report a new disease of T. rosea in Mexico caused by F. pseudocircinatum.

Correction: Phylogenetic diversity, trichothecene potential, and pathogenicity within Fusarium sambucinum species complex.

[This corrects the article DOI: 10.1371/journal.pone.0245037.].

Effects of Double-Stranded RNAs Targeting Fusarium graminearum TRI6 on Fusarium Head Blight and Mycotoxins.

Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and contaminates grains with poisonous trichothecene mycotoxins, including deoxynivalenol (DON). DON functions as an important virulence factor that promotes FHB spread in wheat; therefore, reducing DON production will decrease yield losses to FHB and increase food safety. Recent progress in the topical application of double-stranded RNA (dsRNA) to reduce F. graminearum infection has provided encouraging results. In this study, we designed and synthesized dsRNA targeting the transcription factor TRI6 (TRI6-dsRNA), which is a key regulator of DON biosynthesis. The expression of F. graminearum TRI6 was significantly lower in detached wheat heads treated with TRI6-dsRNA solution compared with the controls. Furthermore, TRI6-dsRNA treatments reduced disease and DON accumulation in inoculated detached wheat heads. Therefore, topical applications of TRI6-dsRNA on wheat heads of intact plants were assessed for their ability to reduce FHB and DON under growth chamber and greenhouse conditions. When wheat heads were treated with TRI6-dsRNA solution in growth chamber conditions, TRI6-dsRNA treatments failed to prevent FHB spread. However, when wheat heads were treated with TRI6-dsRNA solution under greenhouse conditions, FHB and DON were significantly reduced, and infection was restricted to the inoculated floret. In addition, addition of TRI6-dsRNA to toxin induction liquid media had no effect on F. graminearum 15-ADON production. Our study demonstrates that the efficacy of dsRNA applications is strongly dependent on application methods and environmental conditions.

Phylogenetic diversity, trichothecene potential, and pathogenicity within Fusarium sambucinum species complex.

The Fusarium sambucinum species complex (FSAMSC) is one of the most taxonomically challenging groups of fusaria, comprising prominent mycotoxigenic plant pathogens and other species with various lifestyles. Among toxins produced by members of the FSAMSC, trichothecenes pose the most significant threat to public health. Herein a global collection of 171 strains, originating from diverse hosts or substrates, were selected to represent FSAMSC diversity. This strain collection was used to assess their species diversity, evaluate their potential to produce trichothecenes, and cause disease on wheat. Maximum likelihood and Bayesian analyses of a combined 3-gene dataset used to infer evolutionary relationships revealed that the 171 strains originally received as 48 species represent 74 genealogically exclusive phylogenetically distinct species distributed among six strongly supported clades: Brachygibbosum, Graminearum, Longipes, Novel, Sambucinum, and Sporotrichioides. Most of the strains produced trichothecenes in vitro but varied in type, indicating that the six clades correspond to type A, type B, or both types of trichothecene-producing lineages. Furthermore, five strains representing two putative novel species within the Sambucinum Clade produced two newly discovered type A trichothecenes, 15-keto NX-2 and 15-keto NX-3. Strains of the two putatively novel species together with members of the Graminearum Clade were aggressive toward wheat when tested for pathogenicity on heads of the susceptible cultivar Apogee. In planta, the Graminearum Clade strains produced nivalenol or deoxynivalenol and the aggressive Sambucinum Clade strains synthesized NX-3 and 15-keto NX-3. Other strains within the Brachygibbosum, Longipes, Novel, Sambucinum, and Sporotrichioides Clades were nonpathogenic or could infect the inoculated floret without spreading within the head. Moreover, most of these strains did not produce any toxin in the inoculated spikelets. These data highlight aggressiveness toward wheat appears to be influenced by the type of toxin produced and that it is not limited to members of the Graminearum Clade.