Occurrence of Fusarium Crown Rot Caused by Fusarium culmorum on Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Wheat (Triticum aestivum L.) is the predominant grain crop and plays a pivotal role in grain production in Xinjiang Uygur Autonomous Region (XUAR), China. Its cultivated area constitutes approximately half of the total sown area of grain crops in XUAR, with 1.14 million hectares in 2021. Fusarium crown rot (FCR) of wheat, caused by Fusarium culmorum (W.G. Smith) Sacc., is one of the most devastating soil-borne diseases known to seriously reduce grain yield (Ma et al. 2024; Saad et al. 2023). In 2016, FCR of wheat, caused by F. culmorum, was firstly identified in Henan Province, China (Li et al. 2016). In June 2023, during the investigation of FCR of wheat in Aksu Prefecture, XUAR, FCR on winter wheat (cv. Xindong 20) was found (82.761349°E, 41.612202°N). The grain-filling period for winter wheat in early June coincided with a period of high temperatures and water demand in Aksu Prefecture. Approximately 8% of the Xindong 20 wheat plants exhibited symptoms of white heads and browning at the stem base, with the disease present in 82% of the wheat fields surveyed. To identify the pathogens, 20 samples of diseased stem basal tissue, each 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, followed by three rinses with sterile water. These samples were then plated onto potato dextrose agar (PDA) medium at 25°C for 5 days. A total of 17 isolates with consistent morphological characteristics were obtained using single-spore technique, with an isolation rate of 85%. The isolated strains exhibited rapid growth on PDA, producing fluffy, pale-yellow hyphae, and accumulating a pale-yellow to dark red pigment on the bottom of the medium. On carnation leaf agar (CLA), these strains formed orange colonies due to the aggregation of a large number of macroconidia. The macroconidia were short and thick, with three to four septa and rounded apical cell, averaging 31.94 to 40.96 × 5.62 to 6.71 µm (Magnification of ×400). Microconidia were not observed. These morphological characters were consistent with those of F. culmorum (Leslie and Summerell. 2006). Two isolates (D-9 and D-11) were selected for molecular identification. The EF-1α gene fragment was amplified using primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') as previously described by O'Donnell et al. (1998). The two 665 bp PCR products were sequenced and submitted to GenBank (GenBank Accession No: PP763247 and PP763248) with 99. 7% identity to the published F. culmorum sequences (e.g., OP985478, OP985477, MG195126, KX702638). The molecular identification was further confirmed by F. culmorum species-specific PCR primers FcOIF/FcOIR (Nicholson et al. 1998). The expected PCR products of 553 bp were produced only in F. culmorum. Strains D-9 and D-11 were used to conduct the pathogenicity experiment on 7-day-old winter wheat (cv. Xindong 20) using drip in the lower stem inoculation method with a 10-µl of 106 macroconidia ml-1 suspension, and the control 7-day-old winter wheat were treated with sterile water (Xu et al. 2017). The experiments were replicated five times in a greenhouse at temperatures ranging from 20℃ to 25℃. After 4 weeks, all inoculated wheat seedlings showed stem base browning or even death. No symptoms were observed on the control plants. The fungus was reisolated from all inoculated wheat plants by the method described above and identified by morphological and PCR amplification using F. culmorum species-specific primers FcOIF/FcOIR. No F. culmorum was isolated from the control wheat plants, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F.culmorum causing FCR on winter wheat in XUAR, China. Considering wheat is the predominant grain crop and plays a pivotal role in grain production in China, necessary measures should be taken to prevent the spread of F. culmorum to other regions.

Synthesis of nanoparticles using Trichoderma Harzianum, characterization, antifungal activity and impact on Plant Growth promoting Bacteria.

Globally cultivated cereals are frequently threatened by various plant pathogenic agents such as Fusarium fungi. To combat these pathogens, researchers have made nanoparticles as potential agricultural pesticides. In this study, selenium and titanium dioxide NPs were synthesized using Trichoderma harzianum metabolites. Characterization of the NPs indicated varying size and shapes of both NPs and functional groups existence to constitute both NPs. The evaluation of antifungal activity of NPs against plant pathogenic fungi, Fusarium culmorum, indicated both NPs maximum antifungal activity at concentration of 100 mg/L. The impacts of nanoparticles on some beneficial plant growth promoting bacteria (PGPB) were evaluated and showed their inhibition effect on optical density of PGPB at a concentration of 100 mg/L but they did not have any impact on nitrogen fixation by bacteria. Existence of TiO2NPs reduced the intensity of color change to pink compared to the control indicating auxin production. Both NPs demonstrated different impact on phosphate solubilization index. This study suggests that the synthesized nanoparticles have the potential to serve as antifungal compounds at special concentration against plant diseases without significantly reducing the potential of PGPB at low concentrations.

A novel and efficient strategy for the biodegradation of di(2-ethylhexyl) phthalate by Fusarium culmorum.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is used worldwide and raises concerns because of its prevalence in the environment and potential toxicity. Herein, the capability of Fusarium culmorum to degrade a high concentration (3 g/L) of DEHP as the sole carbon and energy source in solid-state fermentation (SSF) was studied. Cultures grown on glucose were used as controls. The biodegradation of DEHP by F. culmorum reached 96.9% within 312 h. This fungus produced a 3-fold higher esterase activity in DEHP-supplemented cultures than in control cultures (1288.9 and 443.2 U/L, respectively). In DEHP-supplemented cultures, nine bands with esterase activity (24.6, 31.2, 34.2, 39.5, 42.8, 62.1, 74.5, 134.5, and 214.5 kDa) were observed by zymography, which were different from those in control cultures and from those previously reported for cultures grown in submerged fermentation. This is the first study to report the DEHP biodegradation pathway by a microorganism grown in SSF. The study findings uncovered a novel biodegradation strategy by which high concentrations of DEHP could be biodegraded using two alternative pathways simultaneously. F. culmorum has an outstanding capability to efficiently degrade DEHP by inducing esterase production, representing an ecologically promising alternative for the development of environmental biotechnologies, which might help mitigate the negative impacts of environmental contamination by this phthalate. KEY POINTS: • F. culmorum has potential to tolerate and remove di(2-ethylhexyl) phthalate (DEHP) • Solid-state fermentation is an efficient system for DEHP degradation by F. culmorum • High concentrations of DEHP induce high levels of esterase production by F. culmorum.

Phytoconstituents and Ergosterol Biosynthesis-Targeting Antimicrobial Activity of Nutmeg (Myristica fragans Houtt.) against Phytopathogens.

In recent years, nutmeg (Myristica fragans Houtt.) has attracted considerable attention in the field of phytochemistry due to its diverse array of bioactive compounds. However, the potential application of nutmeg as a biorational for crop protection has been insufficiently explored. This study investigated the constituents of a nutmeg hydroethanolic extract via gas chromatography-mass spectrometry and vibrational spectroscopy. The research explored the extract's activity against phytopathogenic fungi and oomycetes, elucidating its mechanism of action. The phytochemical profile revealed fatty acids (including tetradecanoic acid, 9-octadecenoic acid, n-hexadecanoic acid, dodecanoic acid, and octadecanoic acid), methoxyeugenol, and elemicin as the main constituents. Previously unreported phytochemicals included veratone, gelsevirine, and montanine. Significant radial growth inhibition of mycelia was observed against Botrytis cinerea, Colletotrichum acutatum, Diplodia corticola, Phytophthora cinnamomi, and especially against Fusarium culmorum. Mode of action investigation, involving Saccharomyces cerevisiae labeled positively with propidium iodide, and a mutant strain affected in ERG6, encoding sterol C-24 methyltransferase, suggested that the extract induces a necrotic type of death and targets ergosterol biosynthesis. The evidence presented underscores the potential of nutmeg as a source of new antimicrobial agents, showing particular promise against F. culmorum.

First report of Fusarium culmorum (W.G. Sm.) Sacc causing crown rot on wheat in Jordan.

Fusarium crown rot (FCR) is a disease caused by numerous Fusarium species, primarily F. culmorum (W. G. Sm.) Sacc., F. pseudograminearum (O'Donnell & T. Aoki), and F. graminearum Schwabe (Paulitz et al., 200). FCR on wheat is a worldwide distributed disease that causes significant yield losses. In the Middle East, FCR was reported in Iraq (Motallebi et al., 2015; Matny et al., 2019) and Syria (Motallebi et al., 2015). In Jordan, Fusarium occurrence on wheat was documented in a checklist publication in 1984 (Mamluk et al., 1984) without further identification of the causative species and its pathogenicity level. There have been no other reports of Fusarium on wheat in Jordan since then. Symptoms of Fusarium crown rot were observed in 2016-2022 (Alananbeh et al., 2018) across Jordan through annual surveys of wheat diseases. The disease severity was higher in the dry seasons such as that of 2017 and 2021. Very severe symptoms were noted on wheat planted at the University of Jordan experimental wheat plots (n=4) in 2016-2022. A total of 40 symptomatic plants were randomly collected from these plots. Roots and stems of the 40 plants were then cut into small sections, disinfected in 0.5% hypochlorite for 5 minutes, 70% ethanol for one minute, and finally rinsed in sterile distilled water three times. The sections were dried under the laminar flow, plated on potato dextrose agar (PDA), and incubated for 10 -14 days at 25 ℃. The fungal cultures were purified by hyphal tipping. At least one pure isolate exhibited a typical morphology of F. culmorum was recovered from each plant. The colonies of pure cultures grew rapidly on PDA with fluffy floccose aerial mycelium and dark red to reddish brown pigment diffused in the agar. The isolates produced monophialidic conidiogenous cells. The formed marcoconidia were slightly curved, with pointed apical and foot cells, 3-5 septated, on average 28.5 - 46.5 X 4.5-7.0 µm, indication the cultures as Fusarium spp. (Figure 1). Chlamydospores were intercalary in hyphae and microconidia were absent. Two representative isolates (Iso-1 and Iso-2) identified putatively as F. culmorum, based on their morphological features, were sent to Macrogen Inc., South Korea to Sanger sequence a portion of the translation elongation factor 1-α gene using the EF1/EF2 primers (Geiser et al. 2004). Raw sequences were used to create consensus sequences using the BioEdit sequence alignment editor. The consensus sequences for the two representatives isolates were used to conduct BLASTn queries of NCBI (https://www.ncbi.nlm.nih.gov) which revealed they are 99.67% and 100% identical to MW233082.1, a TEF11-α sequence of the ex-epitype of F. culmorum (NRRL 25475, Crous et al. 2021). The two sequences generated herein were accessioned in GenBank (accession numbers: OQ785278 and OQ785279). Combined with the morphological and molecular analysis, the Iso-1 and Iso-2 were identified as Fusarium culmorum. The pathogenicity of the isolates was tested on two wheat cultivars using two methods: in vitro on seeds grown in sterile dishes and on seedlings. A 4 X 104 macroconidia suspension was prepared from 10 day-old culture of the isolate grown on PDA at 28 ºC. Seeds of two wheat cultivars, Hourani and Norsi were surface sterilized in 1% (v/v) bleach and rinsed in sterile distilled water three times. For the first method, seeds were soaked in the F. culmorum conidia suspension for 15 min and then dried using filter paper. The seeds were plated onto sterile paper towels in sterile plastic boxes and placed in a growth chamber. Three replicates with 10 seeds/replicate were used. Control Mock treatments used seeds treated with sterile distilled water. The germination percentage, coleoptile length, radicle length, longest seminal root length, and number of seminal roots were measured after 5 days. For the seedling-based pathogenicity test, seeds were planted in seedlings trays filled with sterilized 1:1:1 peat moss: sand: soil. 5 mL conidia suspension was drenched following seedling emergence. Ten replicates with one seed/replicate were used. Plants were watered when necessary to maintain appropriate soil growth conditions. The control seedlings were drenched with sterile distilled water. Disease symptoms were rated by the disease severity index (CRI) described by Mitter et al. (2006) after 35 days of inoculation. The in vitro test showed a reduction of germination and other seeds measurements in the presence of F. culmorum as compared to the control (Table 1 and Table 2, Figure 2). Similarly, the seedling's height, length of discoloration, disease score, disease severity index and germination percentage were all reduced in F. culmorum treated seedlings compared to the control. The two experiments showed that Cv. Norsi was more susceptible to FCR than Hourani (Table 1, Figure 2). F. culmorum was re-isolated from the roots of inoculated plants of both cultivars. The present study is the first report of the crown rot pathogen, F. culmorum on Jordanian wheat. Fusarium culmorum can cause significant economic losses and current research is ongoing to survey FCR-associated Fusarium spp. in Jordan, their genetic diversity, and QTL mapping for resistance genes in wheat landraces.

Determining the biocontrol capacities of Trichoderma spp. originating from Turkey on Fusarium culmorum by transcriptional and antagonistic analyses.

In this study aiming to investigate potential fungal biocontrol agents for Fusarium culmorum, several isolates of Trichoderma spp. were evaluated for their antagonistic effects by means of transcriptional analyses. At first, 21 monosporic Trichoderma spp. isolates were obtained from natural wood debris and wood area soils in Manisa, Turkey. Trichoderma spp. Isolates were identified as belonging to four different species (T. atroviride, T. harzianum, T. koningii, and T. brevicompactum) by tef1-α sequencing. Then, the linear growth rate (LGR) of each species was calculated and determined to be in a range between 13.22 ± 0.71 mm/day (T. atroviride TR2) and 25.06 ± 1.45 mm/day (T. harzianum K30). Inter-simple sequence repeat (ISSR) genotyping validated the tef1-α sequencing results by presenting two sub-clusters in the dendrogram. We determined the genetically most similar (TR1 & TR2; 97.77%) and dissimilar (K9 & K17; 40.40%) individuals belonging to the same and different species, respectively. Dual sandwich culture tests (which are useful for antagonism studies) revealed that T. harzianum K21 (the least suppressive) and T. brevicompactum K26 (the most suppressive) isolates suppressed F. culmorum with growth rates of 3% and 46%, respectively. Expressions of genes previously associated with mycoparasitism-plant protection-secondary metabolism (nag1, tgf-1, and tmk-1) were tested by quantitative real-time polymerase chain reaction (qRT-PCR) in both those isolates. While there were no significant differences (p>0.05) in expression that were present in the K21 isolate, those three genes were upregulated with fold change values of 2.69 ± 0.26 (p<0.001), 2.23 ± 0.16 (p<0.001), and 5.38 ± 2.01 (p<0.05) in K26, meaning that the presence of significant alteration in the physiological processes of the fungus. Also, its mycoparasitism potential was tested on Triticum aestivum L. cv Basribey in planta, which was infected with the F. culmorum FcUK99 strain. Results of the trials, including specific plant growth parameters (weight or length of plantlets), confirmed the mycoparasitic potential of the isolate. It can be concluded that (i) nag1, tgf-1, and tmk-1 genes could be approved as reliable markers for evaluation of BCA capacities of Trichoderma spp. and (ii) the T. brevicompactum K26 strain can be suggested as a promising candidate for combating in F. culmorum diseases following the necessary procedures to ensure it is non-hazardous and safe.

Assessment of Tunisian Trichoderma Isolates on Wheat Seed Germination, Seedling Growth and Fusarium Seedling Blight Suppression.

Beneficial microorganisms, including members of the Trichoderma genus, are known for their ability to promote plant growth and disease resistance, as well as being alternatives to synthetic inputs in agriculture. In this study, 111 Trichoderma strains were isolated from the rhizospheric soil of Florence Aurore, an ancient wheat variety that was cultivated in an organic farming system in Tunisia. A preliminary ITS analysis allowed us to cluster these 111 isolates into three main groups, T. harzianum (74 isolates), T. lixii (16 isolates) and T. sp. (21 isolates), represented by six different species. Their multi-locus analysis (tef1, translation elongation factor 1; rpb2, RNA polymerase B) identified three T. afroharzianum, one T. lixii, one T. atrobrunneum and one T. lentinulae species. These six new strains were selected to determine their suitability as plant growth promoters (PGP) and biocontrol agents (BCA) against Fusarium seedling blight disease (FSB) in wheat caused by Fusarium culmorum. All of the strains exhibited PGP abilities correlated to ammonia and indole-like compound production. In terms of biocontrol activity, all of the strains inhibited the development of F. culmorum in vitro, which is linked to the production of lytic enzymes, as well as diffusible and volatile organic compounds. An in planta assay was carried out on the seeds of a Tunisian modern wheat variety (Khiar) by coating them with Trichoderma. A significant increase in biomass was observed, which is associated with increased chlorophyll and nitrogen. An FSB bioprotective effect was confirmed for all strains (with Th01 being the most effective) by suppressing morbid symptoms in germinated seeds and seedlings, as well as by limiting F. culmorum aggressiveness on overall plant growth. Plant transcriptome analysis revealed that the isolates triggered several SA- and JA-dependent defense-encoding genes involved in F. culmorum resistance in the roots and leaves of three-week-old seedlings. This finding makes these strains very promising in promoting growth and controlling FSB disease in modern wheat varieties.

Weapons against Themselves: Identification and Use of Quorum Sensing Volatile Molecules to Control Plant Pathogenic Fungi Growth.

Quorum sensing (QS) is often defined as a mechanism of microbial communication that can regulate microbial behaviors in accordance with population density. Much is known about QS mechanisms in bacteria, but fungal QS research is still in its infancy. In this study, the molecules constituting the volatolomes of the plant pathogenic fungi Fusarium culmorum and Cochliobolus sativus have been identified during culture conditions involving low and high spore concentrations, with the high concentration imitating overpopulation conditions (for QS stimulation). We determined that volatolomes emitted by these species in conditions of overpopulation have a negative impact on their mycelial growth, with some of the emitted molecules possibly acting as QSM. Candidate VOCs related to QS have then been identified by testing the effect of individual volatile organic compounds (VOCs) on mycelial growth of their emitting species. The antifungal effect observed for the volatolome of F. culmorum in the overpopulation condition could be attributed to ethyl acetate, 2-methylpropan-1-ol, 3-methylbutyl ethanoate, 3-methylbutan-1-ol, and pentan-1-ol, while it could be attributed to longifolene, 3-methylbutan-1-ol, 2-methylpropan-1-ol, and ethyl acetate for C. sativus in the overpopulation condition. This work could pave the way to a sustainable alternative to chemical fungicides.

Evaluation of Fusarium Head Blight Resistance Effects by Haplotype-Based Genome-Wide Association Study in Winter Wheat Lines Derived by Marker Backcrossing Approach.

Fusarium head blight (FHB) of wheat caused by Fusarium species is a destructive disease, causing grain yield and quality losses. Developing FHB-resistant cultivars is crucial to minimize the extent of the disease. The first objective of this study was incorporation of Fhb1 from a resistant donor into five Polish wheat breeding lines with good agronomical traits and different origins. We also performed a haplotype-based GWAS to identify chromosome regions in derived wheat families associated with Fusarium head blight resistance. As a result of marker-assisted backcrossing (MABC), five wheat combinations were obtained. Fungal inoculation and disease assessment were conducted for two years, 2019 and 2020. In 2019 the average phenotypic response of type II resistance was 2.2, whereas in 2020 it was 2.1. A haploblock-based GWAS performed on 10 phenotypic traits (related to type of resistance, year of experiment and FHB index) revealed nine marker-trait associations (MTA), among which six belong to chromosome 2D, two to 3B and one to 7D. Phenotypic variation (R2) explained by the identified haplotypes in haploblocks ranged from 6% to 49%. Additionally, an association weight matrix (AWM) was created, giving the partial correlation-information theory (PCIT) pipeline of 171 edges and 19 nodes. The resultant data and high level of explained phenotypic variance of MTA create the opportunity for data utilization in MAS.

Fungal Endophytes Isolated from Elymus repens, a Wild Relative of Barley, Have Potential for Biological Control of Fusarium culmorum and Pyrenophora teres in Barley.

Twenty-four fungal endophytes, isolated from a wild relative of barley, Elymus repens, were screened in barley against an isolate of Fusarium culmorum and an isolate of Pyrenophora teres under controlled conditions. In all experiments, the endophytes were applied individually as seed dressings. Five endophytes could significantly reduce symptoms of Fusarium culmorum (Periconia macrospinosa E1 and E2, Epicoccum nigrum E4, Leptodontidium sp. E7 and Slopeiomyces cylindrosporus E18). In particular, treatment with Periconia macrospinosa E1 significantly reduced Fusarium symptoms on roots by 29-63% in two out of four experiments. Using, a gfp transformed isolate of P. macrospinosa E1, it was possible to show that the fungus was present on roots 14 days after sowing, coinciding with the disease scoring. To test for a potential systemic effect of the seed treatment, eight endophyte isolates were tested against the leaf pathogen Pyrenophora teres. Three isolates could significantly reduce symptoms of P. teres (Lasiosphaeriaceae sp. E10, Lindgomycetaceae sp. E13 and Leptodontidium sp. E16). Seed treatment with Lasiosphaeriaceae sp. E10 reduced net blotch leaf lesion coverage by 89%, in one out of three experiments. In conclusion, specific endophyte isolates exerted varying degrees of protection in the different experiments. Nevertheless, data suggest that endophytic strains from E. repens in a few cases are antagonistic against F. culmorum and P. teres, but otherwise remain neutral when introduced to a barley host in a controlled environment.

Effect of Compactin on the Mycotoxin Production and Expression of Related Biosynthetic and Regulatory Genes in Toxigenic Fusarium culmorum.

Zearalenone (ZEN) and deoxynivalenol (DON) are mycotoxins produced by various species of Fusarium fungi. They contaminate agricultural products and negatively influence human and animal health, thus representing a serious problem of the agricultural industry. Earlier we showed that compactin, a secondary metabolite of Penicillium citrinum, is able to completely suppress the aflatoxin B1 biosynthesis by Aspergillus flavus. Using the F. culmorum strain FC-19 able to produce DON and ZEN, we demonstrated that compactin also significantly suppressed both DON (99.3%) and ZEN (100%) biosynthesis. The possible mechanisms of this suppression were elucidated by qPCR-based analysis of expression levels of 48 biosynthetic and regulatory genes. Expression of eight of 13 TRI genes, including TRI4, TRI5, and TRI101, was completely suppressed. A significant down-regulation was revealed for the TRI10, TRI9, and TRI14 genes. TRI15 was the only up-regulated gene from the TRI cluster. In the case of the ZEN cluster, almost complete suppression was observed for PKS4, PKS13, and ZEB1 genes, and the balance between two ZEB2 isoforms was altered. Among regulatory genes, an increased expression of GPA1 and GPA2 genes encoding α- and ß-subunits of a G-protein was shown, whereas eight genes were down-regulated. The obtained results suggest that the main pathway for a compactin-related inhibition of the DON and ZEN biosynthesis affects the transcription of genes involved in the G-protein-cAMP-PKA signaling pathway. The revealed gene expression data may provide a better understanding of genetic mechanisms underlying mycotoxin production and its regulation.

Fusarium culmorum Produces NX-2 Toxin Simultaneously with Deoxynivalenol and 3-Acetyl-Deoxynivalenol or Nivalenol

Fusarium culmorum is a major pathogen of grain crops. Infected plants accumulate deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), or nivalenol (NIV), which are mycotoxins of the trichothecene B group. These toxins are also produced by F. graminearum species complex. New trichothecenes structurally similar to trichothecenes B but lacking the carbonyl group on C-8, designated NX toxins, were recently discovered in atypical isolates of F. graminearum from North America. Only these isolates and a few strains of a yet to be characterized Fusarium species from South Africa are known to produce NX-2 and other NX toxins. Here, we report that among 20 F. culmorum strains isolated from maize, wheat, and oat in Europe and Asia over a period of 70 years, 18 strains produced NX-2 simultaneously with 3-ADON and DON or NIV. Rice cultures of strains producing 3-ADON accumulated NX-2 in amounts corresponding to 2−8% of 3-ADON (1.2−36 mg/kg). A strain producing NIV accumulated NX-2 and NIV at comparable amounts (13.6 and 10.3 mg/kg, respectively). In F. graminearum, producers of NX-2 possess a special variant of cytochrome P450 monooxygenase encoded by TRI1 that is unable to oxidize C-8. In F. culmorum, producers and nonproducers of NX-2 possess identical TRI1; the reason for the production of NX-2 is unknown. Our results indicate that the production of NX-2 simultaneously with trichothecenes B is a common feature of F. culmorum.

Abscisic Acid-Defensive Player in Flax Response to Fusarium culmorum Infection.

Fusarium culmorum is a ubiquitous soil pathogen with a wide host range. In flax (Linum ussitatissimum), it causes foot and root rot and accumulation of mycotoxins in flax products. Fungal infections lead to huge losses in the flax industry. Moreover, due to mycotoxin accumulation, flax products constitute a potential threat to the consumers. We discovered that the defense against this pathogen in flax is based on early oxidative burst among others. In flax plants infected with F. culmorum, the most affected genes are connected with ROS production and processing, callose synthesis and ABA production. We hypothesize that ABA triggers defense mechanism in flax and is a significant player in a successful response to infection.

Computational Strategy for Minimizing Mycotoxins in Cereal Crops: Assessment of the Biological Activity of Compounds Resulting from Virtual Screening.

Cereal crops are frequently affected by toxigenic Fusarium species, among which the most common and worrying in Europe are Fusarium graminearum and Fusarium culmorum. These species are the causal agents of grain contamination with type B trichothecene (TCTB) mycotoxins. To help reduce the use of synthetic fungicides while guaranteeing low mycotoxin levels, there is an urgent need to develop new, efficient and environmentally-friendly plant protection solutions. Previously, F. graminearum proteins that could serve as putative targets to block the fungal spread and toxin production were identified and a virtual screening undertaken. Here, two selected compounds, M1 and M2, predicted, respectively, as the top compounds acting on the trichodiene synthase, a key enzyme of TCTB biosynthesis, and the 24-sterol-C-methyltransferase, a protein involved in ergosterol biosynthesis, were submitted for biological tests. Corroborating in silico predictions, M1 was shown to significantly inhibit TCTB yield by a panel of strains. Results were less obvious with M2 that induced only a slight reduction in fungal biomass. To go further, seven M1 analogs were assessed, which allowed evidencing of the physicochemical properties crucial for the anti-mycotoxin activity. Altogether, our results provide the first evidence of the promising potential of computational approaches to discover new anti-mycotoxin solutions.

Reduction of the Fusarium Mycotoxins: Deoxynivalenol, Nivalenol and Zearalenone by Selected Non-Conventional Yeast Strains in Wheat Grains and Bread.

Mycotoxins, toxic secondary metabolites produced by fungi, are important contaminants in food and agricultural industries around the world. These toxins have a multidirectional toxic effect on living organisms, causing damage to the kidneys and liver, and disrupting the functions of the digestive tract and the immune system. In recent years, much attention has been paid to the biological control of pathogens and the mycotoxins they produce. In this study, selected yeasts were used to reduce the occurrence of deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA) produced by Fusarium culmorum, F. graminearum, and F. poae on wheat grain and bread. In a laboratory experiment, an effective reduction in the content of DON, NIV, and ZEA was observed in bread prepared by baking with the addition of an inoculum of the test yeast, ranging from 16.4% to 33.4%, 18.5% to 36.2% and 14.3% to 35.4%, respectively. These results indicate that the selected yeast isolates can be used in practice as efficient mycotoxin decontamination agents in the food industry.

Quantitative High-Throughput Screening Methods Designed for Identification of Bacterial Biocontrol Strains with Antifungal Properties.

Large screens of bacterial strain collections to identify potential biocontrol agents often are time-consuming and costly and fail to provide quantitative results. In this study, we present two quantitative and high-throughput methods to assess the inhibitory capacity of bacterial biocontrol candidates against fungal phytopathogens. One method measures the inhibitory effect of bacterial culture supernatant components on the fungal growth, while the other accounts for direct interaction between growing bacteria and the fungus by cocultivating the two organisms. The antagonistic supernatant method quantifies the culture components' antifungal activity by calculating the cumulative impact of supernatant addition relative to the growth of a nontreated fungal control, while the antagonistic cocultivation method identifies the minimal bacterial cell concentration required to inhibit fungal growth by coinoculating fungal spores with bacterial culture dilution series. Thereby, both methods provide quantitative measures of biocontrol efficiency and allow prominent fungal inhibitors to be distinguished from less effective strains. The combination of the two methods sheds light on the types of inhibition mechanisms and provides the basis for further mode-of-action studies. We demonstrate the efficacy of the methods using Bacillus spp. with different levels of antifungal activities as model antagonists and quantify their inhibitory potencies against classic plant pathogens. IMPORTANCE Fungal phytopathogens are responsible for tremendous agricultural losses on an annual basis. While microbial biocontrol agents represent a promising solution to the problem, there is a growing need for high-throughput methods to evaluate and quantify inhibitory properties of new potential biocontrol agents for agricultural application. In this study, we present two high-throughput and quantitative fungal inhibition methods that are suitable for commercial biocontrol screening.

Evaluation of Durum Wheat Genotypes for Resistance against Root Rot Disease Caused by Moroccan Fusarium culmorum Isolates.

Fusarium culmorum is one of the most important causal agents of root rot of wheat. In this study, 10 F. culmorum isolates were collected from farms located in five agro-ecological regions of Morocco. These were used to challenge 20 durum wheat genotypes via artificial inoculation of plant roots under controlled conditions. The isolate virulence was determined by three traits (roots browning index, stem browning index, and severity of root rot). An alpha-lattice design with three replicates was used, and the resulting ANOVA revealed a significant (P < 0.01) effect of isolate (I), genotype (G), and G × I interaction. A total of four response types were observed (R, MR, MS, and S) revealing that different genes in both the pathogen and the host were activated in 53% of interactions. Most genotypes were susceptible to eight or more isolates, while the Moroccan cultivar Marouan was reported resistant to three isolates and moderately resistant to three others. Similarly, the Australian breeding line SSD1479-117 was reported resistant to two isolates and moderately resistant to four others. The ICARDA elites Icaverve, Berghisyr, Berghisyr2, Amina, and Icaverve2 were identified as moderately resistant. Principal component analysis based on the genotypes responses defined two major clusters and two sub-clusters for the 10 F. culmorum isolates. Isolate Fc9 collected in Khemis Zemamra was the most virulent while isolate Fc3 collected in Haj-Kaddour was the least virulent. This work provides initial results for the discovery of differential reactions between the durum lines and isolates and the identification of novel sources of resistance.

Biological Control of Fusarium culmorum, Fusarium graminearum and Fusarium poae by Antagonistic Yeasts.

The genus Fusarium is considered to be one of the most pathogenic, phytotoxic and toxin-producing group of microorganisms in the world. Plants infected by these fungi are characterized by a reduced consumer and commercial value, mainly due to the contamination of crops with mycotoxins. Therefore, effective methods of reducing fungi of the genus Fusarium must be implemented already in the field before harvesting, especially with alternative methods to pesticides such as biocontrol. In this study we identified yeasts that inhibit the growth of the pathogenic fungi Fusarium culmorum, F. graminearum and F. poae. Tested yeasts came from different culture collections, or were obtained from organic and conventional cereals. The greater number of yeast isolates from organic cereals showed antagonistic activity against fungi of the genus Fusarium compared to isolates from the conventional cultivation system. Cryptococcus carnescens (E22) isolated from organic wheat was the only isolate that limited the mycelial growth of all three tested fungi and was the best antagonist against F. poae. Selected yeasts showed various mechanisms of action against fungi, including competition for nutrients and space, production of volatile metabolites, reduction of spore germination, production of siderophores or production of extracellular lytic enzymes: chitinase and ß-1,3-glucanase. Of all the investigated mechanisms of yeast antagonism against Fusarium, competition for nutrients and the ability to inhibit spore germination prevailed.

Potential of Trichoderma harzianum and Its Metabolites to Protect Wheat Seedlings against Fusarium culmorum and 2,4-D.

Wheat is a critically important crop. The application of fungi, such as Trichoderma harzianum, to protect and improve crop yields could become an alternative solution to synthetic chemicals. However, the interaction between the fungus and wheat in the presence of stress factors at the molecular level has not been fully elucidated. In the present work, we exposed germinating seeds of wheat (Triticum aestivum) to the plant pathogen Fusarium culmorum and the popular herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of T. harzianum or its extracellular metabolites. Then, the harvested roots and shoots were analyzed using spectrometry, 2D-PAGE, and MALDI-TOF/MS techniques. Although F. culmorum and 2,4-D were found to disturb seed germination and the chlorophyll content, T. harzianum partly alleviated these negative effects and reduced the synthesis of zearalenone by F. culmorum. Moreover, T. harzianum decreased the activity of oxidoreduction enzymes (CAT and SOD) and the contents of the oxylipins 9-Hode, 13-Hode, and 13-Hotre induced by stress factors. Under the influence of various growth conditions, changes were observed in over 40 proteins from the wheat roots. Higher volumes of proteins and enzymes performing oxidoreductive functions, such as catalase, ascorbate peroxidase, cytochrome C peroxidase, and Cu/Zn superoxide dismutase, were found in the Fusarium-inoculated and 2,4-D-treated wheat roots. Additionally, observation of the level of 12-oxo-phytodienoic acid reductase involved in the oxylipin signaling pathway in wheat showed an increase. Trichoderma and its metabolites present in the system leveled out the mentioned proteins to the control volumes. Among the 30 proteins examined in the shoots, the expression of the proteins involved in photosynthesis and oxidative stress response was found to be induced in the presence of the herbicide and the pathogen. In summary, these proteomic and metabolomic studies confirmed that the presence of T. harzianum results in the alleviation of oxidative stress in wheat induced by 2,4-D or F. culmorum.