Fusarium Head Blight In China Research Articles

Effect of cleaning, milling, and cooking on the reduction of deoxynivalenol in freshly harvested rice

Abstract Fusarium mycotoxins were frequently present in freshly harvested rice samples caused by Fusarium head blight in China recently. Due to the importance of rice and the risks associated with mycotoxins, the fate of Fusarium mycotoxins in rice was evaluated by physical methods. In the present study, a total of 157 freshly harvested rice samples were collected from fields in Jiangsu Province. Mycotoxin analysis showed that only Fusarium mycotoxins were found in these samples, with deoxynivalenol (DON) being the major mycotoxin. Twenty-three freshly harvested rice samples containing concentrations of DON higher than 200 μg/kg were selected for cleaning and milling. Compared with air or gravity separation, the combined air and gravity separation was the most effective cleaning method to decrease the content of DON. After milling, brown and white rice intended for human consumption presented lower level of DON than freshly harvested rice, husks, and bran. Three types of brown and white rice with different contents of DON were chosen for cooking. Compared with other treatments, preboiled was the most effective method to reduce DON in both brown and white rice. The results of this study improve our understanding of the transfer ratios of DON and the development of effective control measures during the postharvest stage.

Rapid and Sensitive Detection of Toxigenic Fusarium asiaticum Integrating Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Techniques.

Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. Both Fusarium graminearum and F. asiaticum are the causal agents of FHB in China. F. asiaticum is the predominant species in the Middle-Lower Reaches of the Yangtze River (MLRYR) and southwest China. Therefore, detecting F. asiaticum in a timely manner is crucial for controlling the disease and preventing mycotoxins from entering the food chain. Here, we combined rapid genomic DNA extraction, recombinase polymerase amplification, Cas12a cleavage, and lateral flow detection techniques to develop a method for the rapid detection of F. asiaticum. The reaction conditions were optimized to provide a rapid, sensitive, and cost-effective method for F. asiaticum detection. The optimized method demonstrated exceptional specificity in detecting F. asiaticum while not detecting any of the 14 other Fusarium strains and 3 non-Fusarium species. Additionally, it could detect F. asiaticum DNA at concentrations as low as 20 ag/μL, allowing for the diagnosis of F. asiaticum infection in maize and wheat kernels even after 3 days of inoculation. The developed assay will provide an efficient and robust detection platform to accelerate plant pathogen detection.

Biological and molecular characterizations of field fludioxonil-resistant isolates of Fusarium graminearum

Fusarium head blight (FHB) predominately caused by F. graminearum, is an economical devastating disease for grain cereal crops especially on wheat. The phenylpyrrole fungicide fludioxonil exhibits excellent activity against F. graminearum and has been registered to control FHB in China. In this study, 6 fludioxonil-resistant (FludR) isolates of F. graminearum were identified from 2910 isolates collected from wheat cultivated field in Jiang Su, An Hui and Henan province of China in 2020. The sensitivity assay showed that resistance factor (RF) of FludR isolates ranges from 170.73 to >1000. In comparison with fludioxonil-sensitive (FludS) isolates, all of FludR isolates showed fitness defects in terms of mycelial growth, conidiation and virulence. Under fludioxonil treatment condition, the glycerol accumulation was obviously increased in FludS isolates, but was slightly increased in FludR isolates. Four FludR isolates exhibited increased sensitivity to osmotic stresses. Moreover, there is no positive cross-resistance between fludioxonil and other fungicides including phenamacril, carbendazim and tebuconazole. When treated with fludioxonil, the phosphorylation level of Hog1 was significantly decreased in the four FludR isolates, which was in contrast to the observation in the FludS and two FludR isolates where phosphorylation level of Hog1 was increased. Sequencing assay showed that the mutations were identified in different domains in FgOS1, FgOS2 or FgOS4 in FludR isolates. This was first reported that biological and molecular characterizations of field isolates of F. graminearum resistant to fludioxonil. The results can provide scientific directions for controlling FHB using fludioxonil.

Fusarium graminearum FgSdhC1 point mutation A78V confers resistance to the succinate dehydrogenase inhibitor pydiflumetofen.

Fusarium head blight (FHB) caused by Fusarium graminearum complex (Fg) is a devastating disease of cereal crops worldwide. The succinate dehydrogenase inhibitor, pydiflumetofen, was registered for management of FHB in China in 2019. Previously, laboratory-induced pydiflumetofen-resistant (PyR) mutants of Fg have been characterized. However, resistance situation of Fg to pydiflumetofen in the field remains largely unknown. After screening 6468 isolates of Fg from various regions of China, six PyR isolates were identified. All six resistant isolates exhibited no fitness penalties based on mycelial growth, conidiation and virulence. However, no cross-resistance between pydiflumetofen and azoxystrobin, tebuconazole or fludioxonil in Fg was detected. Genome-sequencing revealed that all six PyR isolates contained a point mutation A78V in FgSdhC1 (FgSdhC1A78V ). Genetic replacement assay further confirmed that FgSdhC1A78V conferred resistance of Fg to pydiflumetofen. Based on this, a mismatch allele-specific polymerase chain reaction was developed for rapidly detecting the PyR isolates containing the FgSdhC1A78V mutation in Fg. This is the first time that resistance of Fg to pydiflumetofen in the field was reported and point mutation FgSdhC1A78V conferring resistance of Fg to pydiflumetofen was confirmed. This study provides critical information for monitoring and managing pydiflumetofen resistance in Fg.

Biological Characteristics and Molecular Mechanisms of Fludioxonil Resistance in Fusarium graminearum in China.

Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB) of wheat. The phenylpyrrole fungicide fludioxonil is not currently registered for the management of FHB in China. The current study assessed the fludioxonil sensitivity of a total of 53 F. graminearum isolates collected from the six most important wheat-growing provinces of China during 2018 and 2019. The baseline fludioxonil sensitivity distribution indicated that all of the isolates were sensitive, exhibiting a unimodal cure with a mean effective concentration for 50% inhibition value of 0.13 ± 0.12 μg/ml (standard deviation). Five fludioxonil-resistant mutants were subsequently induced by exposure to fludioxonil under laboratory conditions. Ten successive rounds of subculture in the absence of the selection pressure indicated that the mutation was stably inherited. However, the fludioxonil-resistant mutants were found to have reduced pathogenicity, higher glycerol accumulation, and higher osmotic sensitivity than the parental wild-type isolates, indicating that there was a fitness cost associated with fludioxonil resistance. In addition, the study also found a positive cross resistance between fludioxonil, procymidone, and iprodione, but not with other fungicides such as boscalid, carbendazim, tebuconazole, and fluazinam. Sequence analysis of four candidate target genes (FgOs1, FgOs2, FgOs4, and FgOs5) revealed that the HBXT2R mutant contained two point mutations that resulted in amino acid changes at K223T and K415R in its FgOs1 protein, and one point mutation at residue 520 of its FgOs5 protein that resulted in a premature stop codon. Similarly, the three other mutants contained point mutations that resulted in changes at the K192R, K293R, and K411R residues of the FgOs5 protein but none in the FgOs2 and FgOs4 genes. However, it is important to point out that the FgOs2 and FgOs4 expression of all the fludioxonil-resistant mutants was significantly (P < 0.05) downregulated compared with the sensitive isolates (except for the SQ1-2 isolate). It was also found that one of the resistant mutants did not have changes in any of the sequenced target genes, indicating that an alternative mechanism could also lead to fludioxonil resistance.

Baseline sensitivity and control efficacy of epoxiconazole against Fusarium graminearum in Henan Province, China

Fusarium head blight (FHB), caused by Fusarium graminearum species complex (FGSC) is one of the most destructive wheat diseases worldwide. F. graminearum is the dominant pathogen of FHB in Henan Province. Epoxiconazole is a demethylation inhibitor (DMI) fungicide, and is the main fungicide for controlling FHB in China. In this study, 118 F. graminearum isolates were collected from different regions of Henan Province in 2016 and 2017. The sensitivity of these F. graminearum isolates to epoxiconazole was determined by the method of mycelial growth inhibition. The results showed that the EC50 values range of epoxiconazole to the tested F. graminearum isolates was 0.0196 to 0.5708 μg ml−1, and the mean EC50 value was 0.2178 ± 0.1178 μg ml−1 (mean ± SD). The sensitivity frequency distribution of these F. graminearum isolates to epoxiconazole was a continuous single peak curve, and no isolates resistant to epoxiconazole were found in Henan Province, China. The field control efficacy of epoxiconazole against F. graminearum was evaluated and the results showed that epoxiconazole had a significantly higher control efficacy on FHB, compared to the control fungicide carbendazim. The thousand grain weight of all the fungicide treatments was significantly higher than water control (without fungicide). This study showed that the DMI fungicide epoxiconazole has high activity and field control efficacy on F. graminearum, and can effectively control the prevalence of FHB.

Analysis of Fusarium graminearum Species Complex from Freshly Harvested Rice in Jiangsu Province (China).

Members of Fusarium graminearum species complex (FGSC) are the major pathogens that cause Fusarium head blight (FHB) in cereals worldwide. Symptoms of FHB on rice, including dark staining or browning of rice glumes, were recently observed in Jiangsu Province, China. To improve our understanding of the pathogens involved, 201 FGSC isolates were obtained from freshly harvested rice samples and identified by phylogenetic analyses. Among the 201 FGSC isolates, 196 were F. asiaticum and the remaining 5 were F. graminearum. Trichothecene chemotype and chemical analyses showed that 68.4% of the F. asiaticum isolates were the 3-acetyldeoxynivalenol (3ADON) chemotype and the remainder were the nivalenol (NIV) chemotype. All of the F. graminearum isolates were the 15-acetyldeoxynivalenol chemotype. Pathogenicity assays showed that both the 3ADON and NIV chemotypes of F. asiaticum could infect wheat and rice spikes. FHB severity and trichothecene toxin analysis revealed that F. asiaticum with the NIV chemotype was less aggressive than that with the 3ADON chemotype in wheat, while the NIV-producing strains were more virulent than the 3ADON-producing strains in rice. F. asiaticum isolates with different chemotypes did not show significant differences in mycelial growth, sporulation, conidial dimensions, or perithecial production. These findings would provide useful information for developing management strategies for the control of FHB in China.

A Victorivirus from Fusarium asiaticum, the pathogen of Fusarium head blight in China.

A Victorivirus was detected in isolate F16176 of the fungus Fusarium asiaticum, the causal agent of Fusarium head blight in China. The full genome sequence of the virus was sequenced and characterized. The complete cDNA sequence is 5,281 nucleotides long with 64.2% G + C content and contains two open reading frames (ORFs) that overlap at the pentanucleotide UAAUG. The two ORFs are predicted to encode coat protein (CP) and RNA-dependent RNA polymerase (RdRp), which are conserved among the dsRNA mycoviruses of the genus Victorivirus. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that this dsRNA mycovirus is a new virus belonging to the species Rosellinia necatrix victorivirus 1 in the family Totiviridae. This study is the first to report a full-length genomic sequence of a putative member of the genus Victorivirus in F. asiaticum.

Molecular and biological characteristics of laboratory metconazole-resistant mutants in Fusarium graminearum

The Fusarium graminearum species complex (FGSC), the causal agents of Fusarium head blight (FHB) in wheat, has the different geographically distributed species. Our previous study suggested that a DMI fungicide metconazole exhibits a strong fungicidal activity in mycelial growth of Chinese FHB pathogens and metconazole is currently a most effective compound of commercial fungicides for controlling FHB in China. In the current study, metconazole-resistant F. graminearum mutants were induced by chemical taming and their molecular and biological characteristics were determined. Compared to the corresponding parental strains, three mutation genotypes (two single mutations G443S and D243N, and a combined mutation E103Q&V157 L) were observed in the FgCYP51A of metconazole-resistant mutants. In addition to FgCYP51A mutation, all the mutants had no change on sequences of FgCYP51B and FgCYP51C and promotor sequences of FgCYP51s, but expression patterns of FgCYP51s were different. Compared to the corresponding parental strains, overexpression of FgCYP51A, FgCYP51B and FgCYP51C was observed in the mutant conferring D243N mutation, overexpression of FgCYP51A and FgCYP51B was observed in the mutant conferring E103Q&V157L mutations, and overexpression of FgCYP51A was observed in the mutant conferring G443S mutation. Biological fitness of the mutants conferring D243N mutation or E103Q&V157 L mutations significantly decreased in comparison to the corresponding parental strains, suggesting a fitness penalty. The mutants conferring G443S mutation had no change in biological fitness as compared with the parental strain, indicating that the G443S mutation may emerge in field resistant populations of F. graminearum in the future. In addition, a positive cross resistance between metconazole and other tested DMI fungicides was observed in the mutants conferring D243N mutation or E103Q&V157L mutations, but no cross resistance between metconazole and ipconazole or prochloraz was observed in the mutants conferring G443S mutation. Therefore, we concluded that the mutation genotype of FgCYP51A may cause the differences of biological fitness, cross-resistance and FgCYP51s overexpression patterns. Such information will increase our understanding of resistance mechanism of F. graminearum to DMIs and could provide new reference data for the management of FHB.

Molecular Characterization, Fitness, and Mycotoxin Production of Fusarium asiaticum Strains Resistant to Fludioxonil.

Fludioxonil is used in seedborne disease management of various fungal pathogens, including Fusarium asiaticum, the predominant causal agent of Fusarium head blight in China. In this study, we screened resistant strains from a large number of F. asiaticum strains collected from 2012 to 2016 and found that 4 of 1,000 field strains were highly resistant to fludioxonil. The 50% effective concentration values of the resistant strains and induced mutants ranged from 80 to >400 μg/ml. Compared with field-sensitive strains, all field-collected and laboratory-induced resistant strains exhibited fitness defects in traits including mycelial growth, conidial production, pathogenicity, and sensitivity to osmotic conditions. In the presence of fludioxonil, significantly higher glycerol accumulation was found in sensitive strains but not in resistant individuals. The fludioxonil-resistant strains produced lower amounts of glycerol in liquid culture and lower amounts of trichothecene mycotoxins in rice culture and inoculated wheat spikelets than the fludioxonil-sensitive strains. Sequence analyses of the key genes of the two-component histidine kinase signaling pathway showed various amino acid substitutions in the Os1, Os4, and Os5 genes between field-sensitive and resistant strains or mutants. The results of this study suggest a potential risk of fludioxonil resistance development and a possible influence of resistance mutations on fitness parameters and toxin production in F. asiaticum.

A single-nucleotide-polymorphism-based genotyping assay for simultaneous detection of different carbendazim-resistant genotypes in the Fusarium graminearum species complex.

The occurrence resistance to methyl benzimidazole carbamates (MBC)-fungicides in the Fusarium graminearum species complex (FGSC) is becoming a serious problem in the control of Fusarium head blight in China. The resistance is caused by point mutations in the β2-tubulingene. So far, five resistant genotypes (F167Y, E198Q, E198L, E198K and F200Y) have been reported in the field. To establish a high-throughput method for rapid detection of all the five mutations simultaneously, an efficient single-nucleotide-polymorphism-based genotyping method was developed based on the Luminex xMAP system. One pair of amplification primers and five allele specific primer extension probes were designed and optimized to specially distinguish the different genotypes within one single reaction. This method has good extensibility and can be combined with previous reported probes to form a highly integrated tool for species, trichothecene chemotype and MBC resistance detection. Using this method, carbendazim resistant FGSC isolates from Jiangsu, Anhui and Sichuan Province in China were identified. High and moderate frequencies of resistance were observed in Jiangsu and Anhui Province, respectively. Carbendazim resistance in F. asiaticum is only observed in the 3ADON genotype. Overall, our method proved to be useful for early detection of MBC resistance in the field and the result aids in the choice of fungicide type.

Myosins FaMyo2B and Famyo2 Affect Asexual and Sexual Development, Reduces Pathogenicity, and FaMyo2B Acts Jointly with the Myosin Passenger Protein FaSmy1 to Affect Resistance to Phenamacril in Fusarium asiaticum.

We previously reported that mutations occurred in the gene myosin5 were responsible for resistance to the fungicide phenamacril in Fusarium graminearum. Here, we determined whether there is a functional link between phenamacril resistance and the myosin proteins FaMyo2B and Famyo2 in Fusarium asiaticum, which is the major causal agent of Fusarium head blight in China. We found that FaMyo2B acts jointly with FaSmy1 to affect resistance to phenamacril in F. asiaticum. We also found that FaMyo2B disruption mutant and Famyo2 deletion mutant were defective in hyphal branching, conidiation, and sexual reproduction. ΔFamyo2 also had an enhanced sensitivity to cell wall damaging agents and an abnormal distribution of septa and nuclei. In addition, the FaMyo2B and Famyo2 mutants had reduced pathogenicity on wheat coleoptiles and flowering wheat heads. Taken together, these results reveal that FaMyo2B and Famyo2 are required for several F. asiaticum developmental processes and activities, which help us better understand the resistance mechanism and find the most effective approach to control FHB.

Identification of a novel phenamacril-resistance-related gene by the cDNA-RAPD method in Fusarium asiaticum.

Fusarium asiaticum, a dominant pathogen of Fusarium head blight (FHB) in East Asia, causes huge economic losses. Phenamacril, a novel cyanoacrylate fungicide, has been increasingly applied to control FHB in China, especially where resistance of F. asiaticum against carbendazim is severe. It is important to clarify the resistance-related mechanisms of F. asiaticum to phenamacril so as to avoid control failures, and to sustain the usefulness of the new product. A novel phenamacril-resistance-related gene Famfs1 was obtained by employing the cDNA random amplified polymorphic DNA (cDNA-RAPD) technique, and was validated by genetic and biochemical assays. Compared with the corresponding progenitors, deletion of Famfs1 in phenamacril-sensitive or highly phenamacril-resistant strains caused a significant decrease in effective concentrations inhibiting radial growth by 50% (EC50 value). Additionally, the biological fitness parameters (including mycelial growth under different stresses, conidiation, perithecium formation and virulence) of the deletion mutants attenuated significantly. Famfs1 not only was involved in the resistance of F. asiaticum to phenamacril but also played an important role in adaptation of F. asiaticum to the environment. Moreover, our data suggest that the cDNA-RAPD method can be a candidate technique to clone resistance-related genes in fungi. © 2015 Society of Chemical Industry.

Evaluation of tebuconazole for the management of Fusarium head blight in China

Fusarium head blight (FHB) of wheat is caused by fungal populations belonging to the Fusarium graminearum species complex (FGSC). In this study, tebuconazole (a sterol demethylation inhibitor fungicide, DMI) was evaluated for FHB management in China using in vitro tests and field trials. Although tebuconazole has not been widely used in China, another DMI, triadimefon, has been constantly used to control wheat powdery mildew for more than 20 years. The sensitivity of 56 FGSC isolates collected from 2000 to 2002 and 107 FGSC isolates collected from 2012 to 2013 to tebuconazole were compared. The half-maximal effective concentration (EC50) values of the two groups of isolates ranged from 0.028 to 0.262 mg L−1 and from 0.045 to 0.497 mg L−1, respectively, and statistically significant differences (P < 0.05) were found between them. During the 13 years, the EC50 values for tebuconazole increased by a factor of 1.53. In 2012 and 2013 field trials, the control efficacy of tebuconazole was evaluated in fields where triadimefon had been constantly used for over 20 years. Tebuconazole at 125.00 g a.i. ha−1 decreased disease severity by 81.80 and 70.54 %, respectively, reduced the deoxynivalenol (DON) contents by 78.85 and 38.43 %, respectively, and the yield losses were much lower in both years. Therefore, despite the significant decrease in the sensitivity of in vitro tests, tebuconazole was still very effective in inhibiting FHB and DON, and accordingly in reducing yield losses. It can be used as an effective fungicide for the control of FHB of wheat in China.

Involvement of the anucleate primary sterigmata protein FgApsB in vegetative differentiation, asexual development, nuclear migration, and virulence inFusarium graminearum

The protein ApsB has been shown to play critical roles in the migration and positioning of nuclei and in the development of conidiophores in Aspergillus nidulans. The functions of ApsB in Fusarium graminearum, a causal agent of Fusarium head blight in China, are largely unknown. In this study, we used the blastp program at the Broad Institute to identify FgApsB, an F.graminearum homolog of A.nidulansApsB. The functions of FgApsB were evaluated by constructing a deletion mutant of FgApsB, designated ΔFgApsB-28. Conidiation and mycelial growth rate are reduced in ΔFgApsB-28. The hyphae of ΔFgApsB-28 are thinner than those of the wild type and have a different branching angle. ΔFgApsB-28 exhibited reduced aerial hyphae formation, but increased production of rubrofusarin. Whereas nuclei are evenly distributed in germ tubes and hyphae of the wild type, they are clustered and irregularly distributed in ΔFgApsB-28. The mutant exhibited increased resistance to cell wall-damaging agents, but reduced virulence on flowering wheat heads, which is consistent with its reduced production of the toxin deoxynivalenol. All of the defects in ΔFgApsB-28 were restored by genetic complementation with the parental FgApsB gene. Taken together, the results indicate that FgApsB is important for vegetative differentiation, asexual development, nuclear migration, and virulence in F.graminearum.

The transcription cofactor FgSwi6 plays a role in growth and development, carbendazim sensitivity, cellulose utilization, lithium tolerance, deoxynivalenol production and virulence in the filamentous fungus Fusarium graminearum

Fusarium head blight (FHB) is a worldwide devastating disease of wheat, barley and other small grain cereals and caused primarily by Fusarium graminearum. Carbendazim (MBC) is one of the fungicides widely used to control FHB in China. In the present study, we have identified the F. graminearum locus FGSG_04220 as the sequence homolog for Saccharomyces cerevisiae ScSWI6, named FgSWI6 hereafter. Deletion of FgSWI6 causes mycelium of F. graminearum become sensitive to MBC in liquid medium. In addition, deletion of FgSWI6 reduces mycelial growth as well as production and development of conidia. F. graminearum cells lacking FgSWI6 show reduced production efficiency and sizes of perithecia as well as a defect in the production of ascus and ascospore. FgSWI6 is required for the cellulose utilization, lithium tolerance and deoxynivalenol (DON) production of this pathogen. Furthermore, deletion of FgSWI6 significantly attenuates the virulence of F. graminearum on wheat. Therefore, FgSwi6p plays an important role in growth and development of the economically important fungal pathogen F. graminearum as well as its resistance to MBC.

Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.

Phylogenetic, Carbendazim Sensitivity and Mycotoxin Genotype Analyses of Fusarium graminearum Complex Species Isolated from Wheat Fusarium Head Blight in China

AbstractFusarium head blight (FHB) is a worldwide devastating disease of wheat, caused primarily by species in the Fusarium graminearum (Fg) complex. In this study, we obtained 55 Fusarium isolates from wheat with FHB collected from seven provinces along the north of the Yangtze River. One additional phylogenetic species of Fg complex, Fusarium meridionale, was identified for the first time from China in addition to two known ones, Fusarium asiaticum and F. graminearum. In addition, Fusarium acuminatum, distantly related to Fg complex, was for the first time identified in Northern China. Sensitivities of these isolates to carbendazim were examined and appeared to vary both within and between species. Mycotoxin genotype analyses indicated that F. asiaticum isolates were potential 3‐AcDON and NIV mycotoxin producers, while all F. graminearum isolates might be 15‐AcDON producers. These findings would provide useful information for developing management strategies for the control of FHB in Northern China.

Characterization of Fusarium graminearum Isolates Resistant to Both Carbendazim and a New Fungicide JS399-19

Fusarium head blight (FHB) of wheat and other cereals, caused mainly by Fusarium graminearum, is one of the most economically important diseases worldwide, especially in the United States and China. The benzimidazole fungicides, particularly carbendazim (MBC), have been consistently used during the period of wheat heading and flowering in areas with warm and moist weather to control FHB in China for over 30 years. The effectiveness of MBC, however, has been threatened by the emergence of resistant pathogen populations in the field. JS399-19 (experimental number; a.i. 2-cyano-3-amino-3-phenylancryic acetate) is a novel cyanoacrylate fungicide discovered and patented by the Jiangsu Branch of National Pesticide Research & Development South Center of China. To evaluate the potential risk of resistance development in MBC-resistant F. graminearum isolates to this new fungicide JS399-19, five isolates each of MBC-resistant or -sensitive, which were classified into three different sensitivity phenotypes, such as sensitive (S), moderately resistant (MR), and highly resistant (HR) to MBC, were selected to induce JS399-19-resistant mutants by selecting resistance on potato sucrose agar (PSA) plates amended with JS399-19 at 10 microg/ml. In this way, a total of 24 JS399-19-resistant mutants were obtained from all tested MBC-resistant or -sensitive isolates. All 50 single-spore progenies of each of the resistant mutants could grow normally on PSA plates amended with JS399-19 at 10 microg/ml, indicating stability of resistance to this fungicide. Also, all of the resistant mutants maintained their resistance to JS399-19 and/or MBC through eight transfers on PSA plates for 40 days and when stored on PSA slants at 4 degrees C for 60 days. The mycelial growth and conidial production capacity were decreased in 52.4% of the resistant mutants, indicating that a fitness cost was associated with JS399-19-resistant phenotypes of F. graminearum isolates. However, most of the mutants resistant to both MBC and JS399-19 exhibited high sexual reproduction capacity and pathogenicity as their parental isolates. Nevertheless, the majority of these mutants possessed fitness levels comparable to their parents. The results on the efficacy of the two fungicides for controlling FHB incited by the fungicide-resistant mutants were generally consistent with those of the in vitro sensitivity tests. JS399-19 was effective in controlling FHB caused by MBC-resistant isolates under field conditions, while it was not effective in controlling FHB caused by isolates resistant to JS399-19 or those that were resistant to both MBC and JS399-19. Moreover, the efficacy of the mixture of MBC and JS399-19 was also significantly lower in controlling FHB caused by the isolates resistant to both MBC and JS399-19 than the efficacy against the disease caused by the sensitive isolates, the MBC-resistant isolates, or the JS399-19-resistant isolates. The results suggest that JS399-19 possessed a high risk in development of resistance in MBC-resistant and -sensitive F. graminearum isolates, and this double resistance to both of these fungicides could presumable emerge and create a major problem since both these fungicides are extensively used in China. Therefore, careful use of JS399-19 should be followed to delay resistance development in natural populations of F. graminearum, avoid unexpected control failures, and sustain the usefulness of MBC and the new product JS399-19.

Engineering Fusarium Head Blight Resistance in Wheat by Expression of a Fusion Protein Containing a Fusarium-Specific Antibody and an Antifungal Peptide

Fusarium head blight (FHB) or scab of wheat is a devastating disease in warm and humid regions at wheat-flowering periods worldwide. Natural resistance against FHB pathogens is inadequate and the development of FHB-resistant wheat cultivars has been a challenge. Expression of pathogen-specific antibodies in plants has been proposed as a strategy for crop protection. In this study, an antibody fusion protein comprising a Fusarium-specific recombinant antibody derived from chicken and an antifungal peptide from Aspergillus giganteus was expressed in wheat as a method for protecting plants against FHB pathogens. Plants expressing the antibody fusion displayed a very significantly enhanced resistance in T2 and T3 generations upon single-floret inoculation with the macroconidia of Fusarium asiaticum, the predominant species causing FHB in China, indicating a type II resistance. Spraying inoculation further revealed an enhanced type I resistance in the transgenic wheat plants. Remarkably, more grains were produced in the transgenic plants than the nontransgenic controls. Our results demonstrated that the antibody fusion protein may be used as an effective tool for the protection of crops against FHB pathogens.