Isolation and Identification of Lipopeptide-Producing Bacillus velezensis Strains from Wheat Phyllosphere with Antifungal Activity against the Wheat Pathogen Zymoseptoria tritici

Septoria tritici blotch, caused by the fungal pathogen Zymoseptoria tritici, is a highly significant disease on wheat crops worldwide. The objective of the present study was to find out new bacterial strains with bio-antimicrobial activity against Z. tritici. Two phyllospheric bacteria (S1 and S6) were isolated from wheat ears and identified as Bacillus velezensis strains according to 16S rRNA Sanger sequencing. Antagonistic assays performed with either living strains or cell-free culture filtrates showed significant in vitro antifungal activities against Z. tritici. For the culture filtrates, the half-maximal inhibitory dilution and the minimal inhibitory dilution were 1.4% and 3.7% for the strain S1, and 7.4% and 15% for the strain S6, respectively. MALDI—ToF analysis revealed that both strains synthesize cyclic lipopeptides but from different families. Interestingly, only strain S1 produces putative bacillomycin D. Such differential lipopeptide production patterns might explain the difference observed between the antifungal activity of the culture filtrates of the two strains. This study allows the identification of new lipopeptide-producing strains of B. velezensis with a high potential of application for the biocontrol of Z. tritici.

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Characterisation of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici

10.1101/130393 ◽

2017 ◽

Cited By ~ 1

Author(s):

Graeme J. Kettles ◽

Carlos Bayon ◽

Caroline A. Sparks ◽

Gail Canning ◽

Kostya Kanyuka ◽

...

Keyword(s):

Expression Pattern ◽

Effector Proteins ◽

Yeast Cells ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

List Type ◽

Expression Systems ◽

Zymoseptoria Tritici ◽

Loop Region

Abstract-The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Z. tritici secretes many functionally uncharacterised effector proteins during infection. Here we characterised a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern.-Transient expression systems were used to characterise Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi.-We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino acid N-terminal “loop” region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells but not to Z. tritici itself.-Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggests that whilst it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.

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Prevalence of QoI resistance and mtDNA diversity in the Irish Zymoseptoria tritici population

Irish Journal of Agricultural and Food Research ◽

10.2478/ijafr-2019-0004 ◽

2019 ◽

Vol 58 (1) ◽

pp. 27-33

Author(s):

S. Kildea ◽

D.E. Bucar ◽

F. Hutton ◽

S. de la Rosa ◽

T.E. Welch ◽

...

Keyword(s):

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Entire Group ◽

Zymoseptoria Tritici ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Strain Collection ◽

Increased Sensitivity ◽

Mtdna Diversity

Abstract The emergence and spread of Quinone outside Inhibitor (QoI) fungicide resistance in the Irish Zymoseptoria tritici population in the early 2000s had immediate impacts on the efficacy of the entire group of fungicides for the control of septoria tritici blotch. As a result, a dramatic reduction in the quantities applied to winter wheat occurred in the following seasons. Even in the absence of these fungicides, the frequency of the resistance allele, G143A in the pathogens mtDNA has remained exceptionally high (>97%), and as such, it can be anticipated that continued poor efficacy of current QoI fungicides will be observed. Amongst the isolates with G143A, differences in sensitivity to the QoI pyraclostrobin were observed in vitro. The addition of the alternative oxidase (AOX) inhibitor salicylhydroxamic acid increased sensitivity in these isolates, suggesting some continued impairment of respiration by the QoI fungicides, albeit weak. Interestingly, amongst those tested, the strains from a site with a high frequency of inserts in the MFS1 transporter gene known to enhance QoI efflux did not exhibit this increase in sensitivity. A total of 19 mtDNA haplotypes were detected amongst the 2017 strain collection. Phylogenetic analysis confirmed the suggestion of a common ancestry of all the haplotypes, even though three of the haplotypes contained at least one sensitive strain.

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A Microbial Fermentation Mixture Reduces Fusarium Head Blight and Promotes Grain Weight but does not impact Septoria tritici blotch

European Journal of Plant Pathology ◽

10.1007/s10658-021-02396-4 ◽

2021 ◽

Author(s):

Tony Twamley ◽

Mark Gaffney ◽

Angela Feechan

Keyword(s):

Fusarium Head Blight ◽

Fungal Pathogens ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Microbial Fermentation ◽

In Planta ◽

Head Blight ◽

Zymoseptoria Tritici ◽

The Impact

AbstractFusarium graminearum and Zymoseptoria tritici cause economically important diseases of wheat. F. graminearum is one of the primary causal agents of Fusarium head blight (FHB) and Z. tritici is the causal agent of Septoria tritici blotch (STB). Alternative control methods are required in the face of fungicide resistance and EU legislation which seek to cut pesticide use by 2030. Both fungal pathogens have been described as either hemibiotrophs or necrotrophs. A microbial fermentation-based product (MFP) was previously demonstrated to control the biotrophic pathogen powdery mildew, on wheat. Here we investigated if MFP would be effective against the non-biotrophic fungal pathogens of wheat, F. graminearum and Z. tritici. We assessed the impact of MFP on fungal growth, disease control and also evaluated the individual constituent parts of MFP. Antifungal activity towards both pathogens was found in vitro but MFP only significantly decreased disease symptoms of FHB in planta. In addition, MFP was found to improve the grain number and weight, of uninfected and F. graminearum infected wheat heads.

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Will Triazoles Still Be of Importance in Disease Control of Zymoseptoria tritici in the Future?

Agronomy ◽

10.3390/agronomy11050933 ◽

2021 ◽

Vol 11 (5) ◽

pp. 933

Author(s):

Holger Klink ◽

Joseph-Alexander Verreet ◽

Mario Hasler ◽

Tim Birr

Keyword(s):

Control Strategies ◽

Fungal Growth ◽

Current Control ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Significant Shift ◽

Active Ingredients ◽

Zymoseptoria Tritici ◽

Wheat Diseases

Septoria tritici blotch (STB), caused by Zymoseptoria tritici, is one of the most important foliar wheat diseases worldwide. Current control strategies of STB rely mainly on fungicides, whereby triazoles (demethylation inhibitors; DMIs) have been the backbone in the control of Z. tritici in the last decades. However, in recent years a gradual loss of sensitivity of Z. tritici to several active ingredients of the triazole group has been reported in several European wheat-growing areas. Nevertheless, a new triazole fungicide, namely, mefentrifluconazole, has recently become available in disease management of STB, which belongs to a completely new triazole subclass, the so-called isopropanol triazoles. In this study, the trend in sensitivity development of Z. tritici towards older triazoles (tebuconazole, prothioconazole, and propiconazole) and the new isopropanol triazole mefentrifluconazole was determined in microtiter assays using Z. tritici field populations isolated in 1999, 2009, 2014, and 2020 in a high-disease-pressure and high-fungicide-input area in northern Germany in order to investigate whether the loss of sensitivity of Z. tritici to older triazoles also applies to mefentrifluconazole. For the three triazole fungicides tebuconazole, prothioconazole and propiconazole, a significant shift towards decreasing sensitivity of Z. tritici field populations was observed from 1999 to 2020, whereas the efficacy of mefentrifluconazole in reducing the in vitro fungal growth by 50% (EC50) remained unchanged over the investigated period, demonstrating a stable sensitivity of Z. tritici towards mefentrifluconazole. Although older triazoles are suffering from a loss of sensitivity of Z. tritici field populations due to the selection and spread of less triazole sensitive strains within the Z. tritici population, the efficacy of the new triazole mefentrifluconazole with its unique isopropanol unit was not affected by these changes within the Z. tritici population. Thus, the introduction of such new molecular units could also represent an important contribution for older groups of active ingredients, which previously suffered from a loss of sensitivity.

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Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat

Plant Disease ◽

10.1094/pdis-02-20-0318-sc ◽

2020 ◽

pp. PDIS-02-20-0318

Author(s):

Adam Kuzdraliński ◽

Justyna Leśniowska-Nowak ◽

Michał Nowak ◽

Magdalena Kawęcka ◽

Anna Kot ◽

...

Keyword(s):

High Specificity ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

In Vitro Tests ◽

Zymoseptoria Tritici ◽

Specificity And Sensitivity ◽

Wheat Leaf ◽

Primer Sets ◽

Species Specific

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

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Linear Correlation Analysis of Zymoseptoria tritici Aggressiveness with In Vitro Growth Rate

Phytopathology ◽

10.1094/phyto-12-15-0338-r ◽

2016 ◽

Vol 106 (11) ◽

pp. 1255-1261 ◽

Cited By ~ 5

Author(s):

Fangfang Zhan ◽

Yiekun Xie ◽

Wen Zhu ◽

Danli Sun ◽

Bruce A. McDonald ◽

...

Keyword(s):

Growth Rate ◽

Pathogenic Fungus ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

In Vitro Growth ◽

Zymoseptoria Tritici ◽

Average Growth ◽

Positive Correlation ◽

Linear Correlation Analysis

Zymoseptoria tritici is a globally distributed plant-pathogenic fungus causing Septoria tritici blotch of wheat. In this study, the in vitro growth rates and aggressiveness of 141 genetically distinct isolates sampled from four wheat fields on three continents were assessed to determine the association of these two ecological parameters. Aggressiveness was assessed on two spring wheat cultivars (‘Toronit’ and ‘Greina’) in a greenhouse using percentages of leaf area covered by lesions and pycnidia. We found a positive correlation between aggressiveness of pathogen strains on the two cultivars, consistent with a quantitative and host-nonspecific interaction in this pathosystem. We also found a positive correlation between aggressiveness and average growth rate at two temperatures, suggesting that in vitro pathogen growth rate may make a significant contribution to pathogen aggressiveness.

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The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogenZymoseptoria tritici

10.1101/595454 ◽

2019 ◽

Author(s):

Michael Habig ◽

Sharon Marie Bahena-Garrido ◽

Friederike Barkmann ◽

Janine Haueisen ◽

Eva Holtgrewe Stukenbrock

Keyword(s):

Transcription Factor ◽

Reverse Genetics ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Zymoseptoria Tritici ◽

Fungal Cell ◽

Narrow Host Range ◽

Host Interaction ◽

Putative Transcription Factor

SummaryZymoseptoria triticiis a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections the fungus shows a markedly different expression pattern. However, the underlying mechanisms causing differential gene expression during host and non-host interaction are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection program in compatible hosts. In the rice blast pathogenMagnaporthe oryzae, MoCOD1, a member of the fungal Zn(II)2Cys6transcription factor family, has been shown to directly affect pathogenicity. Here, we analyse the role of the putative transcription factor Zt107320, a homolog of MoCOD1, during infection of compatible and incompatible hosts byZ. tritici. We show for the first time thatZt107320is differentially expressed in host versus non-host infections and that lower expression corresponds to an incompatible infection of non-hosts. Applying reverse genetics approaches we further show that Zt107320 regulates the dimorphic switch as well as the growth rate ofZ. triticiand affects fungal cell wall compositionin vitro. Moreover, ΔZt107320mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 regulates growth processes and pathogenicity during infection. Our results suggest that this putative transcription factor is involved in discriminating compatible and non-compatible infections.

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Evaluation of λ-Carrageenan, CpG-ODN, Glycine Betaine, Spirulina platensis, and Ergosterol as Elicitors for Control of Zymoseptoria tritici in Wheat

Phytopathology ◽

10.1094/phyto-11-17-0367-r ◽

2019 ◽

Vol 109 (3) ◽

pp. 409-417 ◽

Cited By ~ 2

Author(s):

Geraldine Le Mire ◽

Ali Siah ◽

Brice Marolleau ◽

Matthieu Gaucher ◽

Claude Maumené ◽

...

Keyword(s):

Glycine Betaine ◽

Spirulina Platensis ◽

Crop Protection ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

In Vitro Tests ◽

Cpg Odn ◽

Zymoseptoria Tritici

Wheat crops are constantly challenged by the pathogen Zymoseptoria tritici, responsible for Septoria tritici Blotch (STB) disease. The present study reports the evaluation of five elicitor compounds (λ-carrageenan, cytosine-phosphate-guanine oligodesoxynucleotide motifs [CpG ODN], glycine betaine, Spirulina platensis, and ergosterol) for the protection of wheat against STB in order to offer new alternative tools to farmers for sustainable crop protection. Screening of elicitors of wheat defenses was carried out through a succession of experiments: biocidal in vitro tests enabled checking for any fungicidal activities, glasshouse experiments allowed determination of the efficacy of a given compound in protecting wheat against STB, and quantitative reverse-transcription polymerase chain reaction biomolecular tests investigated the relative expression of 23 defense genes in treated versus untreated plants. Therefore, we demonstrated that λ-carrageenan, CpG-ODN, glycine betaine, S. platensis, and ergosterol are potential elicitors of wheat defenses. Foliar treatment with these compounds conferred protection of wheat by up to approximately 70% against Z. tritici under semicontrolled conditions and induced both salicylic acid- and jasmonic acid-dependent signaling pathways in the plant. These findings contribute to extending the narrow list of potential elicitors of wheat defenses against Z. tritici.

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Importance of the C12 Carbon Chain in the Biological Activity of Rhamnolipids Conferring Protection in Wheat against Zymoseptoria tritici

Molecules ◽

10.3390/molecules26010040 ◽

2020 ◽

Vol 26 (1) ◽

pp. 40

Author(s):

Rémi Platel ◽

Ludovic Chaveriat ◽

Sarah Le Guenic ◽

Rutger Pipeleers ◽

Maryline Magnin-Robert ◽

...

Keyword(s):

Plasma Membranes ◽

Biological Activities ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Multi Drug Resistance ◽

Activity Levels ◽

In Planta ◽

Zymoseptoria Tritici ◽

Relationship Analysis

The hemibiotrophic fungus Zymoseptoria tritici, responsible for Septoria tritici blotch, is currently the most devastating foliar disease on wheat crops worldwide. Here, we explored, for the first time, the ability of rhamnolipids (RLs) to control this pathogen, using a total of 19 RLs, including a natural RL mixture produced by Pseudomonas aeruginosa and 18 bioinspired RLs synthesized using green chemistry, as well as two related compounds (lauric acid and dodecanol). These compounds were assessed for in vitro antifungal effect, in planta defence elicitation (peroxidase and catalase enzyme activities), and protection efficacy on the wheat-Z. tritici pathosystem. Interestingly, a structure-activity relationship analysis revealed that synthetic RLs with a 12 carbon fatty acid tail were the most effective for all examined biological activities. This highlights the importance of the C12 chain in the bioactivity of RLs, likely by acting on the plasma membranes of both wheat and Z. tritici cells. The efficacy of the most active compound Rh-Est-C12 was 20-fold lower in planta than in vitro; an optimization of the formulation is thus required to increase its effectiveness. No Z. tritici strain-dependent activity was scored for Rh-Est-C12 that exhibited similar antifungal activity levels towards strains differing in their resistance patterns to demethylation inhibitor fungicides, including multi-drug resistance strains. This study reports new insights into the use of bio-inspired RLs to control Z. tritici.

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Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692845 ◽

2021 ◽

Vol 12 ◽

Author(s):

Berit Samils ◽

Björn Andersson ◽

Eva Edin ◽

Malin Elfstrand ◽

Tilman Rönneburg ◽

...

Keyword(s):

High Throughput ◽

Fungicide Resistance ◽

Housekeeping Genes ◽

Target Site ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Zymoseptoria Tritici ◽

Multiplex Amplification ◽

Long Read

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.

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