Fusarium Graminearum Species Complex Research Articles

Phylogenetic Variation of Tri1 Gene and Development of PCR-RFLP Analysis for the Identification of NX Genotypes in Fusarium graminearum Species Complex.

NX toxins have been described as a novel group of type A trichothecenes produced by members of the Fusarium graminearum species complex (FGSC). Differences in structure between NX toxins and the common type B trichothecenes arise from functional variation in the trichothecene biosynthetic enzyme Tri1 in the FGSC. The identified highly conserved changes in the Tri1 gene can be used to develop specific PCR-based assays to identify the NX-producing strains. In this study, the sequences of the Tri1 gene from type B trichothecene- and NX-producing strains were analyzed to identify DNA polymorphisms between the two different kinds of trichothecene producers. Four sets of Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were successfully developed to distinguish the common type B trichothecene producers and NX producers within FGSC. These promising diagnostic methods can be used for high-throughput genotype detection of Fusarium strains as a step forward for crop disease management and mycotoxin control in agriculture. Additionally, it was found that the Tri1 gene phylogeny differs from the species phylogeny, which is consistent with the previous studies.

Genome Sequence Resource of the Head Blight Pathogens Fusarium asiaticum and F. graminearum Isolated from Cereal Crops and Gramineous Weeds in Korea and China

Head blight is a serious fungal disease that results in yield loss and mycotoxin contamination in cereal crops worldwide. Head blight is primarily caused by members of the Fusarium graminearum species complex (FGSC), which consists of at least 16 species, including Fusarium asiaticum and Fusarium graminearum. FGSC members produce trichothecene mycotoxins and can be divided into four groups (chemotypes) depending on the type of trichothecenes produced: NIV (production of nivalenol), 3ADON (production of 3-acetyldeoxynivalenol), 15ADON (production of 15-acetyldeoxynivalenol), and NX-2 (production of 3α-acetoxy, 7α, 15-dihydroxy-12, and 13-epoxytrichothe-9-ene). Whereas F. graminearum mainly causes head blight in wheat-producing areas, F. asiaticum is associated primarily with rice head blight in eastern Asia. To date, the NCBI database archives 116 assembled genome sequences of F. graminearum, but only four accessions correspond to F. asiaticum. Accessions for three F. graminearum strains (PH-1 = NRRL 31084, CS3005, and FG-12) are chromosome assemblies (i.e., include contigs/scaffolds consisting of full-length or nearly full-length chromosomes), but none of the F. asiaticum accessions is a chromosome assembly. This announcement reports the assembled genome sequences of nine F. asiaticum strains and seven F. graminearum strains isolated from rice, wheat, barley, and other gramineous plants in Korea and China. [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 .

First report of Rice panicle blight caused by Fusarium sporotrichioides in China.

Rice (Oryza sativa L.) panicle blight, caused by various fungal and bacterial pathogens, is an emerging threat to rice production, due to the impact on rice yield and quality. In the autumn of 2020, a survey was conducted to understand the etiology of the disease in Liaoning province, an important rice growing area in northeastern China. Rice seeds with typical reddish or brown spots on the glumes were collected from various rice fields. Symptomatic seeds were sterilized with 5% sodium hypochlorite and 75% ethanol, rinsed in sterile distilled water, and placed on potato dextrose agar (PDA) plates. After 3 to 5 days of incubation at 25°C, suspected Fusarium strains showing cream to salmon colonies on PDA were purified by the single-spore isolation method. The identification of these strains were performed based on morphological and molecular characteristics. Fusarium incarnatum-equiseti species complex was the most frequently, followed by the members of Fusarium graminearum species complex and Fusarium fujikuroi species complex. However, one strain was identified as F. sporotrichioides Sherb. based on the following results: (I) Colonies on PDA produced dense mycelia and typical red pigment on the undersurface. Macroconidia were usually 3- to 5-septate, moderately curved to straight, and 27.46 ± 7.16 × 3.78 ± 0.8 μm (n = 50). Microconidia were ellipsoid to fusoid and 9.77 ± 2.29 × 2.99 ± 0.65 μm (n = 50). (II) Genomic DNA was extracted by AxyPrep Multisource Genomic DNA Miniprep Kit according to the manufacturer's instructions; the sequence analysis of partial translation elongation factor-1α (TEF-1α) and RNA polymerase II second largest subunit (RPB2) were accomplished with the primers EF1/2 and RPB5F/7CR, respectively. BLAST searches of the obtained sequences had 99-100% homology with several F. sporotrichioides strains from GenBank. DNA sequences of partial TEF-1α and RPB2 were deposited in GenBank as OQ068267 and OQ068269, respectively. (III) This strain can synthesis T-2, HT-2, diacetoxyscirpenol (DAS), and Neosolaniol (NEO) toxin at the concentration of about 5000, 600, 700 and 8000 μg/kg in rice culture, respectively, according to the previous culture and analysis methods (1,2). (IV) Pathogenicity tests were conducted with the rice variety Nanjing 9108 by spraying spore suspension (106 conidia/ml) on rice spikes (5 mL per spike) at the flowering stage. Control spikes were inoculated with sterile distilled water. Three weeks after inoculation, the inoculated rice glumes showed similar symptoms with the original samples in the field. No symptoms were observed on controls. Besides, F. sporotrichioides was successfully recovered from the inoculated rice spikes but not from controls. To our knowledge, this is the first report of F. sporotrichioides causing rice panicle blight in China and this disease appears to be a serious risk to food safety and human health. Funding: This work was supported by Jiangsu Agriculture Science and Technology (CX(21)1005).

Fusarium Species and Mycotoxins Associated with Sorghum Grains in Uruguay.

Grain mold and stalk rot are among the fungal diseases that cause significant losses in sorghum worldwide and are caused by different Fusarium spp. The presence of Fusarium species in sorghum grains causes yield losses and mycotoxin contamination, which represents a risk to consumers. In this study, Fusarium graminearum species complex (FGSC) had a high incidence, followed by Fusarium fujikuroi species complex (FFSC) and F. incarnatum-equiseti species complex. Within FFSC, F. proliferatum, F. andiyazi, F. fujikuroi, F. thapsinum, F. verticillioides and F. subglutinans were identified, and this was the first report of F. fujikuroi in sorghum. The most frequent toxins found in sorghum samples were deoxynivalenol (DON) and zearalenone (ZEN). The presence of fumonisins and nivalenol (NIV) was detected at low levels. This study adds new knowledge about the occurrence of Fusarium species and mycotoxins in sorghum grains. Furthermore, this is the first report in Uruguay on fungicide sensitivity for Fusarium isolates from sorghum, which constitutes an important starting point for defining management practices to minimize fungal infection and mycotoxin contamination.

Genetics and Environmental Factors Associated with Resistance to Fusarium graminearum, the Causal Agent of Gibberella Ear Rot in Maize

Maize is one of the most important food and feed sources at the worldwide level. Due to this importance, all the pathogens that can infect this crop can harm both food safety and security. Fungi are the most important pathogens in cultivated maize, and Fusarium spp. are one of the most important families. Reduction in yield and production of dangerous mycotoxins are the main effects of Fusarium spp. infection. Fusarium graminearum (part of the Fusarium graminearum species complex) is one the most important fungi that infect maize, and it is the causative agent of Gibberella ear rot (GER). The main characteristics of this species include its ability to infect various species and its varying infection pressures across different years. This fungus produces various harmful mycotoxins, such as deoxynivalenol, zearalenone, butanolide, and culmorin. Infection can start from silk channels or from ear wounds. In the first case, the environmental conditions are the most important factors, but in the second, a key role is played by the feeding action of lepidopteran larvae (in Europe, Ostrinia nubilalis). All these factors need to be taken into account to develop a successful management strategy, starting from cropping methods that can reduce the source of inoculum to the direct control of the fungus with fungicide, as well as insect control to reduce ear wounds. But, the most important factor that can reduce the effects of this fungus is the use of resistant hybrids. Different studies have highlighted different defensive methods developed by the plant to reduce fungal infections, like fast drying of silk and kernels, chemical compounds produced by the plant after infection, and mechanical protection from insects’ wounds. The aim of this paper is to review the scientific evidence of the most important management strategies against GER in maize and to highlight the genetic basis which is behind hybrid resistance to this disease, with a focus on genes and QTLs found in studies conducted across the world and with different types of maize from tropical cultivars to European flint.

Relationship Between Mycotoxin Production and Gene Expression in Fusarium graminearum Species Complex Strains Under Various Environmental Conditions.

The Fusarium graminearum species complex (FGSC) can produce various mycotoxins and is a major concern for food quantity and quality worldwide. In this study, we determined the effects of water activity (aw), temperature, incubation time and their interactions on mycotoxin accumulation and the expression levels of biosynthetic genes in FGSC strains from maize samples in China. The highest deoxynivalenol (DON), 3-acetyldeoxynivalenol(3ADON) and 15-acetyldeoxynivalenol (15ADON) levels of the F. boothii and F. graminearum strains were observed at 0.98 aw/30°C or 0.99 aw/25°C. F. asiaticum and F. meridionale reached maximum nivalenol (NIV) and 4-acetylnivalenol (4ANIV) contents at 0.99 aw and 30°C. With the extension of the incubation time, the concentrations of DON and NIV gradually increased, while those of their derivatives decreased. F. boothii, F. meridionale and one F. asiaticum strain had the highest zearalenone (ZEN) values at 0.95 aw and 25°C, while the optimum conditions for the other F. asiaticum strain and F. graminearum were 0.99 aw and 30°C. Four genes associated with trichothecene and zearalenone synthesis were significantly induced under higher water stress in the early stage of production. The results indicated independence of mycotoxin production and gene expression, as maximum amounts of these toxic metabolites were observed at higher aw in most cases. This study provides useful information for the monitoring and prevention of such toxins entering the maize production chain.

A multivariate analysis of phenotypic traits of strains of Fusarium graminearum and F. meridionale supports structure by species

AbstractFusarium head blight (FHB) of wheat and Gibberella ear and stalk rot (GER and GSR) of maize are diseases of worldwide importance. Several phylogenetic species belonging to the Fusarium graminearum species complex (FGSC) cause FHB and GER/GSR in Brazil, but the dominant species varies depending on host. Fusarium graminearum is the most frequent cause of FHB, while F. meridionale is more often associated with GER/GSR. These two species are distinguished based on fixed sequence variations at a few genetic loci; however, relevance of these differences to reproduction, pathogenicity and toxigenicity has not been fully explored. A range of phenotypic traits were evaluated for a group of strains of F. graminearum and F. meridionale from wheat and maize. These included radial growth on agar plates, sexual and asexual reproduction, fungicide sensitivity, toxigenicity and pathogenicity on wheat and maize. Although intraspecies variation was observed for most traits, a multivariate analysis showed the strains were strongly structured by species, regardless of host origin. Increased sexual or asexual reproduction, deoxynivalenol production and aggressiveness on wheat heads were most important for distinguishing strains of F. graminearum, while faster mycelial growth in culture, nivalenol production and reduced colonization on detached maize silks were most important for distinguishing strains of F. meridionale. Thus, the phylogenetic distinction between F. graminearum and F. meridionale is supported by phenotypic differences that are relevant for pathogenicity (aggressiveness and mycotoxin production on wheat heads and maize silks, mycelial growth and sexual and asexual reproduction) and disease management (tebuconazole sensitivity).

Deoxynivalenol and zearalenone in Brazilian barley destined for brewing

ABSTRACT Barley is an important cereal worldwide. However, fungal contamination during pre and postharvest is a recurrent problem for barley production, causing a direct impact on the quality of the grains and their by-products due to spoilage and mycotoxin accumulation. The Fusarium graminearum species complex is the main contaminant during preharvest and some species can produce deoxynivalenol and zearalenone, important mycotoxins that represent a risk to human and animal health. This study evaluated the fungal diversity and the levels of DON and ZEN in barley grains produced in Brazil. The results showed high frequency (60%) of Fusariumcontamination in barley grains. Additionally, mycotoxin levels ranged from 46 to 2074 µg/kg for DON and from 74 to 556 µg/kg for ZEN. Co-occurrence of DON and ZEN was observed in 40% of the samples and 30% of barley samples had DON and ZEN levels higher than the maximum levels established by Brazilian and European legislations.

Quantitative PCR assays for the species-specific detection of Fusarium graminearum sensu stricto and Fusarium asiaticum in winter wheat growing regions in China

Fusarium graminearum species complex (FGSC) is one of the most devastating fungal plant pathogens of cereal crops worldwide, resulting in a corresponding mycotoxins contamination in cereal-based food. The detection of FGSC to study its population structure and species distribution is of great concern for the integrated control of mycotoxins contamination in grains entering food supply chains. In this study, real time quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR) methods were developed for the species-specific detection of Fusarium graminearum species complex in winter wheat growing regions in China. Primers and probes were designed basing the on the sequence of Fg-16 SCAR fragment (sequence characterized amplified regions analysis) and confirmed to make a distinguishment between the two prevailing species including Fusarium graminearum sensu stricto and Fusarium asiaticum. The assay specificity was tested against 24 isolates of target Fusarium species and several non-target Fusarium species that were frequently isolated from wheat in China. Consistent results could be obtained by the developed RT-qPCR and ddPCR assays, and both of them were sensitive enough for the detection of FGSC in these regions. Population structure and species distribution of FGSC in North China plain and Yangtze River plain by the developed qPCR assays accorded with previous results obtained by fungal isolation method. The newly developed qPCR assays are time-saving and will provide new insights during the routine surveillance of FGSC in winter wheat growing regions in China and possibly other countries.

Exploring and applying genes to enhance the resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) is a destructive disease in wheat worldwide. Fusarium graminearum species complex (FGSC) is the main causal pathogen causing severe damage to wheat with reduction in both grain yield and quality. Additionally, mycotoxins produced by the FHB pathogens are hazardous to the health of human and livestock. Large numbers of genes conferring FHB resistance to date have been characterized from wheat and its relatives, and some of them have been widely used in breeding and significantly improved the resistance to FHB in wheat. However, the disease spreads rapidly and has been severe due to the climate and cropping system changes in the last decade. It is an urgent necessity to explore and apply more genes related to FHB resistant for wheat breeding. In this review, we summarized the genes with FHB resistance and mycotoxin detoxication identified from common wheat and its relatives by using forward- and reverse-genetic approaches, and introduced the effects of such genes and the genes with FHB resistant from other plant species, and host-induced gene silencing (HIGS) in enhancing the resistance to FHB in wheat. We also outlined the molecular rationale of the resistance and the application of the cloned genes for FHB control. Finally, we discussed the future challenges and opportunities in this field.

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

Current species composition of Fusarium population affecting the main wheat-growing regions in Uruguay and evolution of their sensitivity to triazoles after long-term application

Fusarium head blight (FHB) is a destructive disease of cereal grains caused by several Fusarium species, of which Fusarium graminearum is considered the primary causal agent. In this work 586 pure cultures of Fusarium spp. were obtained from infected grains, of which 64.9% belonged to the Fusarium graminearum species complex. 96.4% of those isolates had 15-acetyldeoxynivalenol genotype and the rest exhibited Nivalenol genotype. The second most predominant species was F. poae (19.1%) followed by F. avenaceum (8.2%) and F. tricinctum (4.6%). An increase in the tolerance to tebuconazole of Uruguayan Fusarium spp. isolates was detected.

Fusarium species that cause corn stalk rot in the Ubaté valley of Cundinamarca, Colombia

In recent years, corn (Zea mays L.) crops in the Colombian cold tropics located in the Ubaté valley in Cundinamarca have been affected by stalk rot with incidences up to 40%. Despite the importance of this disease, accurate diagnosis has not been conducted. The objectives of the study were to determine the causal agents of corn lodging associated with stalk rot in this corn-producing region and describe the symptoms of the disease. Two plots with stalk rot reported in the municipality of Simijaca in July 2016 were sown with the regional varieties Simijaca and Sogamoso. Plants were randomly inspected on a monthly basis for describing disease symptoms and isolating the pathogen. The Fusarium species isolated were morphologically and molecularly identified and pathogenicity tests were conducted. The disease was detected at early plant developmental stages with the combination of chlorosis, leaf anthocyanosis, and dwarfism as the main symptoms in the two corn varieties evaluated. Crown and node necrosis in longitudinal sections of the stalk and purple colorations in the crown, nodes and internodes of plants were observed 90 d after sowing. Finally, lodging occurred at any phenological stage of the crop. Fusarium spp. were isolated in all stages of plant development. Fusarium species were identified as F. graminearum in the Fusarium graminearum species complex and F. subglutinans in the Fusarium fujikuroi species complex, which have cold-climate production zones as their ecological niche. Pathogenicity tests confirmed F. graminearum and F. subglutinans as the causal agents of stalk rot in the regional corn variety Simijaca in the Ubate valley in Cundinamarca.

Identification of Candidate Genes Associated with Trichothecene Biosynthesis in Fusarium graminearum Species Complex Combined with Transcriptomic and Proteomic Analysis.

The Fusarium graminearum species complex is the main causal agent of wheat head blight worldwide. Trichothecenes produced by the pathogen in infected grains have important food safety implications. Previously reported studies on trichothecene production have all focused on the conditions conducive to mycotoxin production, while the molecular mechanisms of trichothecene biosynthesis in Fusarium strains under normal or non-inducing conditions are still unclear. Here, a global analysis of the fungal gene expression of three strains using the Affymetrix Fusarium GeneChip under non-inducing conditions is reported. Differentially expressed genes were identified among strains with different trichothecene-production ability, and some novel genes associated with trichothecene biosynthesis were found by bioinformatics analysis. To verify the transcriptome results, proteomic analyses of the three strains were conducted under the same culture conditions. In total, 69 unique fungal proteins were identified in 77 protein spots. Combined with transcriptome and proteome analysis, 27 novel genes were predicted to be associated with trichothecene mycotoxin production. A protein, encoded by FGSG_01403, was found to be associated with trichothecene production via proteome analysis. Gene knock-out mutations of FGSG_01403 resulted in mutants with increased production of trichothecenes. Future functional analysis of the candidate genes identified in this study may reveal new insights into the negative regulation of trichothecene production in the Fusarium graminearum species complex.

Effectiveness of Species- and Trichothecene-Specific Primers in Monitoring Fusarium graminearum Species Complex in Small Grain–Pea Intercropping Systems

Tracking the distribution of Fusarium species and the detection of changes in toxin production provides epidemiological information that is essential for Fusarium head blight (FHB) management. Members of Fusarium graminearum species complex (FGSC) were characterized using species and trichothecene-specific primers. Associations between members of the FGSC, cereal crop species (wheat, rye, triticale, and oat), seeding time (winter and spring), type of cultivation (monocrop and intercrop) and chemotype grouping were investigated with multiple correspondence analysis and multiple regression modeling. We found that triticale and oat were more related to isolates classified into F. graminearum s. lato than with other isolates. In contrast, wheat and rye were more associated with F. graminearum s. stricto. Cereal crop species affected the frequencies of F. graminearum s. stricto (p = 0.003) and F. graminearum s. lato (p = 0.08) and unidentified isolates with morphological characteristics like those of FGSC members (p = 0.02). The effectiveness of species-specific primers was 60.3% (Fg16F/R) and 76.2% (FgrF/FgcR), and the effectiveness of primer sets for the trichothecene genotyping of the Tri5 and Tri3 genes was 100% and 90.6%, respectively. The decrease in Fusarium-damaged kernel values in the wheat–pea intercropping system indicated that intercropping systems have the potential to control FHB.

First Report of Fusarium meridionale Causing Stalk Rot of Ryegrass in China.

Members of the Fusarium graminearum species complex (FGSC) are the main causing agents of head blight, seedling blight, or stalk rot in wheat and other cereals worldwide. Surveys on species composition and mycotoxin production of FGSC populations have mainly focused on food crops such as wheat, maize, and barley, but little is known about the identity of FGSC pathogens present in pasture grass. In April 2021, a survey of grass diseases in the Hongya County (29.90661 N; 103.37313 E) in Sichuan Province was conducted to understand the etiology of stalk rot in perennial ryegrass (Lolium perenne). It was observed in several pastures that about 10% of yield loss in perennial ryegrass was caused by stalk rot. Affected plant stalks were brown to dark brown in colour and appeared soggy. As infections continued or under conditions of high humidity, some plant stalks also became flattened. Perennial ryegrass samples with symptoms of stalk rot or browning of the stem were collected. Symptomatic tissues were cut into short segments (approximately 5 mm), surface-sterilized in 3% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, air dried, plated onto potato dextrose agar (PDA), and then incubated in the dark at 28 °C. After 3 to 5 days, Fusarium-like fungal colonies with reddish-orange mycelium were collected and transferred to new PDA plates for further purification, and the purified cultures were obtained by single spore isolation. Four uniform isolates were obtained and their colonies on PDA resembled typical FGSC colonies (Leslie and Summerell 2006; O'Donnell et al. 2004). Colonies had an average radial growth rate of 8.5 to 11.0 mm/day at 28 °C in the dark on PDA. Conidial characteristics were studied on Spezieller Nährstoffarmer agar (SNA) as described by Wang et al. (2014). Macroconidia were falcate to almost straight, usually with parallel dorsal and ventral lines, 3- to 5-septate, 20.65 to 55.22 μm in length (average 39.16 μm), and 2.38 to 6.93 μm in width (average 4.42 μm) (n = 200). No microconidia were observed. The pathogenicity of the isolated Fusarium strains was then tested on healthy perennial ryegrass (variety Changjiang 1). Ryegrass plants grown for 2 months were inoculated by punching a hole in the stem using a sterile toothpick, followed by an injection of 20 μL macroconidia suspension at a concentration of 105 spores/mL. Ryegrass stems treated with water served as the control. Twenty plants were included in each treatment. After inoculation, the plants were grown in a growth chamber at 25 °C and 90% humidity for 24 h. Stalk tissues at the wound site turned brown after 3 days and the brown area then extended to regions above and below. No symptoms were observed in the water-treated controls. As well, the same pathogen was reisolated from the infected grass stems, but not from the controls. Thus, the isolated Fusarium spp. are a cause of stalk rot in perennial ryegrass based on the fulfillment of Koch's postulates. To identify the Fusarium spp. to species level, portions of the translation elongation factor 1-α (TEF) gene sequences from all four strains were amplified and sequenced as described by Wang et al. (2015). The obtained sequences were identical, and a sequence of isolate SC1 was submitted to GenBank (accession no. MZ964308). BLASTn searches were conducted on the TEF sequence (607 bp) in two databases, revealing it had 100% similarity to the sequence of Fusarium meridionale strain DS27 (accession no. MN629330) in NCBI and strain NRRL28723 from FUSARIUM-ID (http://isolate.fusariumdb.org/). A concatenated four-gene phylogeny (supplementary figure) resolved SC1 and the type specimens of F. meridionale (NRRL28723, 29010, and 28436) in a monophyletic clade with 100% bootstrap support, confirming that the strain SC1 belongs to F. meridionale. Finally, trichothecene productions of F. meridionale strains were evaluated using rice cultures kept at 28 °C in the dark for two weeks, as described by Desjardins and Proctor (2011). LC-MS/MS analysis indicated that the fungus could produce NIV and 4ANIV in rice cultures with average concentrations of 1400.44 and 3144.10 μg/kg, respectively. To the best of our knowledge, this is the first report of F. meridionale causing disease in perennial ryegrass in China. Further research will be necessary to determine its distribution, aggressiveness, and trichothecene production.

Trichothecene Genotype Profiling of Wheat Fusarium graminearum Species Complex in Paraguay

Paraguay is a non-traditional wheat-producing country in one of the warmest regions in South America. Fusarium Head Blight (FHB) is a critical disease affecting this crop, caused by the Fusarium graminearum species complex (FGSC). A variety of these species produce trichothecenes, including deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). This study characterized the phylogenetic relationships, and chemotype diversity of 28 strains within FGSC collected from wheat fields across different country regions. Phylogenetic analysis based on the sequence of elongation factor-1α gene (EF-1α) from 28 strains revealed the presence of four species in the FGSC: F. graminearum sensu stricto, F. asiaticum, F. meridionale and F. cortaderiae. Ten strains selected for further analysis revealed that all F. graminearum strains were 15-ADON chemotype, while the two strains of F. meridionale and one strain of F. asiaticum were NIV chemotype. Thus, the 15-ADON chemotype of F. graminearum sensu stricto was predominant within the Fusarium strains isolated in the country. This work is the first report of phylogenetic relationships and chemotype diversity among Fusarium strains which will help understand the population diversity of this pathogen in Paraguay.

An Efficient Strategy Combining Immunoassays and Molecular Identification for the Investigation of Fusarium Infections in Ear Rot of Maize in Guizhou Province, China

Fusarium is one of the most important phytopathogenic and mycotoxigenic fungi that caused huge losses worldwide due to the decline of crop yield and quality. To systematically investigate the infections of Fusarium species in ear rot of maize in the Guizhou Province of China and analyze its population structure, 175 samples of rotted maize ears from 76 counties were tested by combining immunoassays and molecular identification. Immunoassay based on single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein was first employed to analyze these samples. Fusarium pathogens were isolated and purified from Fusarium-infected samples. Molecular identification was performed using the partial internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) sequences. Specific primers were used to detect toxigenic chemotypes, and verification was performed by liquid chromatography tandem mass spectrometry (LC–MS/MS). One-hundred and sixty three samples were characterized to be positive, and the infection rate was 93.14%. Sixteen species of Fusarium belonging to six species complexes were detected and Fusarium meridionale belonging to the Fusarium graminearum species complex (FGSC) was the dominant species. Polymerase chain reaction (PCR) identification illustrated that 69 isolates (56.10%) were potential mycotoxin-producing Fusarium pathogens. The key synthetic genes of NIV, NIV + ZEN, DON + ZEN, and FBs were detected in 3, 35, 7, and 24 isolates, respectively. A total of 86.11% of F. meridionale isolates carried both NIV- and ZEN-specific segments, while Fusarium verticillioides isolates mainly represented FBs chemotype. All the isolates carrying DON-producing fragments were FGSC. These results showed that there are different degrees of Fusarium infections in Guizhou Province and their species and toxigenic genotypes display regional distribution patterns. Therefore, scFv-AP fusion-based immunoassays could be conducted to efficiently investigate Fusarium infections and more attention and measures should be taken for mycotoxin contamination in this region.

Mechanism of Fusarium graminearum Resistance to Ergosterol Biosynthesis Inhibitors: G443S Substitution of the Drug Target FgCYP51A.

Fusarium head blight (FHB), caused by the Fusarium graminearum species complex, is a devastating fungal disease resulting in substantial yield and quality losses. Ergosterol biosynthesis inhibitors (EBIs) are the most popular chemicals for controlling FHB. Recently, the resistance of F. graminearum to EBIs has emerged in the field, and an amino acid substitution (G443S) of the sterol 14α-demethylase FgCYP51A was detected in the field resistant strains. To further illustrate the resistance mechanism of F. graminearum to EBIs, site-directed mutants conferring the G443S substitution of FgCYP51A were generated from the progenitor strain PH-1 via genetic transformation with site-directed mutagenesis. We found that the FgCYP51A-G443S substitution significantly decreased the sensitivity of F. graminearum to EBIs with EC50 values ranging from 0.1190 to 0.2302 μg mL-1 and EC90 values ranging from 1.3420 to 9.1119 μg mL-1 for tebuconazole. Furthermore, the FgCYP51A-G443S substitution decreased sexual reproduction and virulence, which will reduce the initial infection source of pathogen populations in the field, while the increase of sporulation capability may enhance the frequencies of the disease cycle, thereby contributing to epidemics of FHB disease. Surprisingly, the FgCYP51A-G443S substitution accelerated DON biosynthesis by upregulating TRI5 expression and enhancing the fluorescence intensity of TRI1-GFP, the marker protein of Fusarium toxisomes. Thus, we concluded that the FgCYP51A-G443S substitution regulates EBI-fungicide resistance and DON biosynthesis, increasing the risk of fungicide resistance development in the field, thereby threatening the control efficacy of EBIs against FHB.

First report of Head blight of wheat caused by Fusarium vorosii in Serbia.

The cosmopolitan species Fusarium graminearum Schwabe directly reduces yield, as well as grain quality of cereals, due to its ability to synthesize mycotoxins. Previously it was considered to be one species occurring on all continents. However, phylogenetic analysis employing the GCPSR method (Genealogical Concordance Phylogenetic Species Recognition) revealed the existence of 15 phylogenetic species within what is now recognised as the Fusarium graminearum Species Complex (FGSC) (Sarver et al. 2011). During 1996-2008, a MRIZP collection of FGSC isolates was established and isolates originating from wheat (5), maize (3) and barely (2) were selected for further study. Morphological features including the appearance of colonies and macroconidia (average size 38.5-53.1 × 4.6-5.4 µm, No 50) of all 10 isolates on PDA were consistent with descriptions of F. graminearum (O'Donnell et al. 2004, Leslie and Summerell 2006). Total DNA was isolated from mycelium removed from 7-day old colonies of single-spore isolates grown on PDA using the DNeasy Plant Mini Kit (Qiagen, Hilden). Further identification was based on amplification and sequencing of elongation factor TEF-1α, histone H3 and β-tubulin in both directions, with primers ef1/ef2, H3-1a/H3-1b and T1/T22, respectively (Jacobs et al. 2010). The sequences were deposited in NCBI under accession numbers MF974399 - MF974408 (TEF-1α), MG063783 - MG063792 (β-tubulin) and MF999139 - MF999148 (histone H3). Sequence analysis was performed using BLAST while genetic similarity was calculated using MEGA 6.0 software. Isolate 1339 originating from wheat (collected at the locality of Kikinda in 2006), shared 100% nucleotide identity with TEF-1α (DQ459745), histone H3 (DQ459728) and β-tubulin (DQ459643) of F. vorosii isolate NRRL37605 (Starkey et al. 2007). The remaining nine isolates were identified as F. graminearum as they shared 99% to 100% nucleotide similarity with F. graminearum NRRL 28439 (O'Donnell et al. 2004). Pathogenicity was tested using artificial inoculations of spikes during wheat flowering (Mesterhazy et al. 1999). Thirty classes were inoculated with each isolate, in three replicates. Inoculum was prepared from 7-day colonies on PDA, and 30 ml of a conidia suspension (1x105 conidia/ml) was used. Control plants were inoculated with sterile water. Three weeks after inoculation, typical Fusarium head blight symptoms were visible on inoculated plants, from which all 10 isolates were successfully reisolated. Control spikes remained symptomless. Disease severity was estimated on the 1-7 scale (Blandino et al. 2012). Average pathogenicity of the F. vorosii isolate 1339 was 1.9, and 2.4 -5.1 of F. graminearum isolates. Toxin production was determined using gas chromatography-tandem mass spectrometry. Kernels inoculated with the 10 isolates were ground and tested for the presence of deoxynivalenol (DON) and its acetyl derivatives 3ADON, 15ADON and NIV. F. vorosii isolate 1339 possessed the 15ADON chemotype, as well as eight F. graminearum isolates, while only one F. graminearum isolate was 3ADON chemotype. To date, F. vorosii has only been detected in Hungary on wheat (Toth et al. 2005) and Korea on barley, corn and rice (Lee et al. 2016). This is the first report of F. vorosii in Serbia, which is of great importance, because it indicates the spread of this toxigenic species. Further studies should be focused on determining the distribution, aggressiveness and toxicological profile of F. vorosii.