PCR-RFLP for detection of Fusarium graminearum genotypes with resistance to phenamacril

Plant Disease ◽  
2020 ◽  
Author(s):  
Jiao-Sheng Li ◽  
Luo-Yu Wu ◽  
Hui Zhang ◽  
Xiu-Shi Song ◽  
Jian-Xin Wang ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Conventional Method ◽  
Resistance Mutations ◽  
Head Blight ◽  
Fusarium Asiaticum ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Resistant Strains ◽  
Codon Mutation

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and Fusarium asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in vitro lab experiments. In this study, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217, and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L) or 1649 bp (for mutation of E420K) in myosin-5 gene were amplified respectively by appropriate primer pairs. Restriction enzyme KpnⅠ, TasⅠ or DraⅠ was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L and E420K, respectively. KpnⅠ digested the 841 bp PCR products of phenamacri-resistant strains with codon mutation A135T into two fragments of 256 bp and 585 bp. In contrast, KpnⅠ did not digest the PCR products of sensitive strains. TasⅠ digested the 802 bp PCR products of phenamacril-strains with codon mutation S217L into three fragments of 461 bp, 287bp and 54 bp. In contrast, TasⅠ digestion of the 802 bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515bp and 287bp. DraⅠ digested the 1649 bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 bp and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three resistant mutation genotypes of F. graminearum to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

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

Plant Disease ◽  
2018 ◽  
Vol 102 (9) ◽  
pp. 1759-1765 ◽  
Author(s):  
J. B. Qiu ◽  
M. Z. Yu ◽  
Q. Yin ◽  
J. H. Xu ◽  
J. R. Shi
Keyword(s):  
Fungal Pathogens ◽  
Toxin Production ◽  
Resistance Mutations ◽  
Head Blight ◽  
Fusarium Asiaticum ◽  
Seedborne Disease ◽  
Induced Mutants ◽  
Fitness Parameters ◽  
Resistant Strains ◽  
Kinase Signaling Pathway

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.

Impact of the biofungicide tetramycin on the development of Fusarium head blight, grain yield and deoxynivalenol accumulation in wheat

2020 ◽  
Vol 13 (2) ◽  
pp. 235-246
Author(s):  
W.Q. Shi ◽  
L.B. Xiang ◽  
D.Z. Yu ◽  
S.J. Gong ◽  
L.J. Yang
Keyword(s):  
Fusarium Head Blight ◽  
Fusarium Graminearum ◽  
Spore Germination ◽  
Field Experiments ◽  
Synergistic Effects ◽  
Field Trials ◽  
Head Blight ◽  
Mycelium Growth ◽  
Key Aspects

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease that leads to extensive yield and quality loss in wheat and barley production. Integrated pest management (IPM) is required to control this disease and biofungicides, such as tetramycin, could be a novel addition to IPM strategies. The current study investigated in vitro tetramycin toxicity in Fusarium graminearum and evaluated its effectiveness for the control of Fusarium head blight FHB. Tetramycin was shown to affect three key aspects of Fusarium pathogenicity: spore germination, mycelium growth and deoxynivalenol (DON) production. The in vitro results indicated that tetramycin had strong inhibitory activity on the mycelial growth and spore germination. Field trials indicated that tetramycin treatment resulted in a significant reduction in both the FHB disease index and the level of DON accumulation. The reduced DON content in harvested grain was correlated with the amount of Tri5 mRNA determined by qRT-PCR. Synergistic effects between tetramycin and metconazole, in both the in vitro and field experiments were found. Tetramycin could provide an alternative option to control FHB.

Chemotype and aggressiveness evaluation of Fusarium graminearum and Fusarium culmorum isolates from wheat fields in Wisconsin

Plant Disease ◽  
2021 ◽  
Author(s):  
Brian Mueller ◽  
Carol Groves ◽  
Damon L. Smith
Keyword(s):  
Growth Rate ◽  
Fusarium Head Blight ◽  
Fusarium Graminearum ◽  
Fusarium Culmorum ◽  
Head Blight ◽  
Growing Seasons ◽  
Progress Curve ◽  
Sporulation Capacity ◽  
Derivatives Of

Fusarium graminearum commonly causes Fusarium head blight (FHB) on wheat, barley, rice, and oats. Fusarium graminearum produces nivalenol and deoxynivalenol (DON) and forms derivatives of DON based on its acetylation sites. The fungus is profiled into chemotypes based on DON derivative chemotypes (3 acetyldeoxynivalenol (3ADON) chemotype; 15 acetyldeoxynivalenol (15ADON) chemotype) and/or the nivalenol (NIV) chemotype. The current study assessed the Fusarium population found on wheat and the chemotype profile of the isolates collected from 2016 and 2017 in Wisconsin. Fusarium graminearum was isolated from all locations sampled in both 2016 and 2017. Fusarium culmorum was isolated only from Door County in 2016. Over both growing seasons, 91% of isolates were identified as the 15ADON chemotype while 9% of isolates were identified as the 3ADON chemotype. Aggressiveness was quantified by area under disease progress curve (AUDPC). The isolates with the highest AUDPC values were from the highest wheat producing cropping districts in the state. Deoxynivalenol production in grain and sporulation and growth rate in vitro were compared to aggressiveness in the greenhouse. Our results showed that 3ADON isolates in Wisconsin were among the highest in sporulation capacity, growth rate, and DON production in grain. However, there were no significant differences in aggressiveness between the 3ADON and 15ADON isolates. The results of this research detail the baseline frequency and distribution of 3ADON and 15ADON chemotypes observed in Wisconsin. Chemotype distributions within populations of F. graminearum in Wisconsin should continue to be monitored in the future.

scholarly journals One Small RNA of Fusarium graminearum Targets and Silences CEBiP Gene in Common Wheat

2019 ◽  
Vol 7 (10) ◽  
pp. 425 ◽  
Author(s):  
Jiao Jian ◽  
Xu Liang
Keyword(s):  
Fusarium Graminearum ◽  
Small Rna ◽  
Pathogenic Fungus ◽  
Effective Means ◽  
Transcriptional Level ◽  
Head Blight ◽  
Protein Coding ◽  
Pathogen Invasion

The pathogenic fungus Fusarium graminearum (F. graminearum), causing Fusarium head blight (FHB) or scab, is one of the most important cereal killers worldwide, exerting great economic and agronomic losses on global grain production. To repress pathogen invasion, plants have evolved a sophisticated innate immunity system for pathogen recognition and defense activation. Simultaneously, pathogens continue to evolve more effective means of invasion to conquer plant resistance systems. In the process of co-evolution of plants and pathogens, several small RNAs (sRNAs) have been proved in regulating plant immune response and plant-microbial interaction. In this study, we report that a F. graminearum sRNA (Fg-sRNA1) can suppress wheat defense response by targeting and silencing a resistance-related gene, which codes a Chitin Elicitor Binding Protein (TaCEBiP). Transcriptional level evidence indicates that Fg-sRNA1 can target TaCEBiP mRNA and trigger silencing of TaCEBiP in vivo, and in Nicotiana benthamiana (N. benthamiana) plants, Western blotting experiments and YFP Fluorescence observation proofs show that Fg-sRNA1 can suppress the accumulation of protein coding by TaCEBiP gene in vitro. F. graminearum PH-1 strain displays a weakening ability to invasion when Barley stripe mosaic virus (BSMV) vector induces effective silencing Fg-sRNA1 in PH-1 infected wheat plants. Taken together, our results suggest that a small RNA from F. graminearum can target and silence the wheat TaCEBiP gene to enhance invasion of F. graminearum.

scholarly journals Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by qPCR

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 560
Author(s):  
Elena Maria Colombo ◽  
Andrea Kunova ◽  
Claudio Gardana ◽  
Cristina Pizzatti ◽  
Paolo Simonetti ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Plant Pathogens ◽  
Fungal Growth ◽  
Toxin Production ◽  
Fungal Mycelium ◽  
Head Blight ◽  
Wheat Grains ◽  
Streptomyces Spp ◽  
Dual Plate

Streptomyces spp. can be exploited as biocontrol agents (BCAs) against plant pathogens such as Fusarium graminearum, the main causal agent of Fusarium head blight (FHB) and against the contamination of grains with deoxynivalenol (DON). In the present research, four Streptomyces strains active against F. graminearum in dual plate assays were characterized for their ability to colonize detached wheat grains in the presence of F. graminearum and to limit DON production. The pathogen and BCA abundance were assessed by a quantitative real-time PCR, while DON production was assessed by HPLC quantification and compared to ergosterol to correlate the toxin production to the amount of fungal mycelium. Fungal growth and mycotoxin production were assessed with both co-inoculation and late inoculation of the BCAs in vitro (three days post-Fusarium inoculation) to test the interaction between the fungus and the bacteria. The level of inhibition of the pathogen and the toxin production were strain-specific. Overall, a higher level of DON inhibition (up to 99%) and a strong reduction in fungal biomass (up to 71%) were achieved when streptomycetes were co-inoculated with the fungus. This research enabled studying the antifungal efficacy of the four Streptomyces strains and monitoring their development in DON-inducing conditions.

scholarly journals Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Dimitrios Evangelopoulos ◽  
Gareth A. Prosser ◽  
Angela Rodgers ◽  
Belinda M. Dagg ◽  
Bhagwati Khatri ◽  
...  
Keyword(s):  
Biological Factor ◽  
Resistance Mutations ◽  
Compensatory Mechanisms ◽  
Medical Problems ◽  
Antibiotic Resistant ◽  
Clinical Resistance ◽  
Resistant Strains ◽  
Rate Of Emergence ◽  
Fitness Analysis

Abstract Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations.

scholarly journals Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Stephen Baker ◽  
Pham Thanh Duy ◽  
Tran Vu Thieu Nga ◽  
Tran Thi Ngoc Dung ◽  
Voong Vinh Phat ◽  
...  
Keyword(s):  
Target Genes ◽  
Systemic Infection ◽  
Critical Factor ◽  
Selective Advantage ◽  
Salmonella Typhi ◽  
Antimicrobial Treatment ◽  
Resistance Mutations ◽  
Resistant Strains ◽  
And Control

Fluoroquinolones (FQ) are the recommended antimicrobial treatment for typhoid, a severe systemic infection caused by the bacterium Salmonella enterica serovar Typhi. FQ-resistance mutations in S. Typhi have become common, hindering treatment and control efforts. Using in vitro competition experiments, we assayed the fitness of eleven isogenic S. Typhi strains with resistance mutations in the FQ target genes, gyrA and parC. In the absence of antimicrobial pressure, 6 out of 11 mutants carried a selective advantage over the antimicrobial-sensitive parent strain, indicating that FQ resistance in S. Typhi is not typically associated with fitness costs. Double-mutants exhibited higher than expected fitness as a result of synergistic epistasis, signifying that epistasis may be a critical factor in the evolution and molecular epidemiology of S. Typhi. Our findings have important implications for the management of drug-resistant S. Typhi, suggesting that FQ-resistant strains would be naturally maintained even if fluoroquinolone use were reduced.

scholarly journals Variation Among Isolates of Fusarium graminearum Associated with Fusarium Head Blight in North Carolina

Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 404-410 ◽  
Author(s):  
Scott L. Walker ◽  
Steven Leath ◽  
Winston M. Hagler ◽  
J. Paul Murphy
Keyword(s):  
North Carolina ◽  
Fusarium Head Blight ◽  
Fusarium Graminearum ◽  
Phenotypic Diversity ◽  
In Vitro Growth ◽  
Head Blight ◽  
Potential Threat ◽  
Previous Crop

Fusarium head blight (FHB) can reduce yield of wheat and decrease the value of harvested grain by accumulation of detrimental toxins. Understanding the variability of the fungal population associated with infection could improve disease control strategies. Sixty-six isolates of Fusarium graminearum associated with FHB were collected in North Carolina and tested for in vitro growth rate, in vitro production of deoxynivalenol (DON) and zearalenone, and pathogenicity on three cultivars of soft red winter wheat. Significant differences among isolates were found for all three traits. Randomly Amplified Polymorphic DNA (RAPD) analysis revealed high levels of genotypic diversity among isolates. Isolates of F. graminearum, F. culmorum, and F. avenaceum acquired from the Pennsylvania State University Fusarium Center were included for comparison in all tests. In vivo levels of DON were measured for the five isolates associated with the highest levels of disease and the five isolates associated with the lowest levels of disease, and no significant differences were found. However, all ten isolates produced detectable levels of DON in vivo. Mean disease ratings ranged from 3.4 to 96.4%, in vitro (DON) levels ranged from 0 to 7176.2 ppm, and zearalenone ranged from 0 to 354.7 ppm, among isolates. A multiple regression model using in vitro growth, in vitro DON, and zearalenone production, collection location, wheat cultivar of isolate origin, plot, tillage conditions, and previous crop as independent variables and percent blighted tissue as the dependent variable was developed. The cumulative R2 value for the model equaled 0.27 with in vitro rate of growth making the largest contribution. Analysis of phenotype and genotype among isolates demonstrated diversity in a single plot, in a single location, and in North Carolina. Genotypic and phenotypic diversity were significant under both conventional and reduced tillage conditions, and diversity was high regardless of whether the previous crop had been a host or non-host for F. graminearum. These data indicate a variable pathogen population of F. graminearum exists in North Carolina, and members of this population can be both highly pathogenic on wheat and produce high levels of detrimental toxins, indicating a potential threat for problems with FHB within the state.

Endophytic fungi as a promising biocontrol agent to protect wheat from Fusarium graminearum head blight

Plant Disease ◽  
2021 ◽  
Author(s):  
Zachary Albert Noel ◽  
Ludmilla Roze ◽  
Mikaela Breunig ◽  
Frances Trail
Keyword(s):  
Microbial Community ◽  
Fusarium Graminearum ◽  
Endophytic Fungi ◽  
Seed Weight ◽  
Biocontrol Agent ◽  
Fungal Endophytes ◽  
In Planta ◽  
Head Blight ◽  
Species Definitions

The search for beneficial endophytes that can be part of a constructed microbial community has increased in recent years. We characterized three endophytic fungi previously isolated from wheat for their in vitro and in planta antagonism toward the Fusarium head blight pathogen, Fusarium graminearum. The endophytes were phylogenetically characterized and shown to be Alternaria destruens, Fusarium commune, and Fusarium oxysporum. Individual fungal endophytes significantly increased seed weight and lowered the accumulation of the mycotoxin deoxynivalenol compared to F. graminearum infected wheat heads without endophyte pretreatment. Investigation into the mechanism of competition in vitro showed that endophytes competitively excluded F. graminearum by pre-emptive colonization and possible inhibition over a distance. Investigations on the use of these endophytes in the field are in progress. Identification of these three endophytes highlights a common quandary in searching for beneficial microbes to use in agriculture: species definitions often do not separate individual isolates’ lifestyles. A greater understanding of the risks in using intraspecies variants for biocontrol is needed and should be examined in the context of the ecology of the individuals being investigated.

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