Pseudomonas synxantha 2-79 Transformed with Pyrrolnitrin Biosynthesis Genes Has Improved Biocontrol Activity Against Soilborne Pathogens of Wheat and Canola.

A four-gene operon (prnABCD) from Pseudomonas protegens Pf-5 encoding the biosynthesis of the antibiotic pyrronitrin was introduced into P. synxantha (formerly P. fluorescens) 2-79, an aggressive root colonizer of both dryland and irrigated wheat roots that naturally produces the antibiotic phenazine-1-carboxylic acid and suppresses both take-all and Rhizoctonia root rot of wheat. Recombinant strains ZHW15 and ZHW25 produced both antibiotics and maintained population sizes in the rhizosphere of wheat that were comparable to those of strain 2-79. The recombinant strains inhibited in vitro the wheat pathogens Rhizoctonia solani anastomosis group 8 (AG-8) and AG-2-1, Gaeumannomyces graminis var. tritici, Sclerotinia sclerotiorum, Fusarium culmorum, and F. pseudograminearum significantly more than did strain 2-79. Both the wild-type and recombinant strains were equally inhibitory of Pythium ultimum. When applied as a seed treatment, the recombinant strains suppressed take-all, Rhizoctonia root rot of wheat, and Rhizoctonia root and stem rot of canola significantly better than did wild-type strain 2-79.

The Highly Conserved Barley Powdery Mildew Effector BEC1019 Confers Susceptibility to Biotrophic and Necrotrophic Pathogens in Wheat.

Effector proteins secreted by plant pathogens play important roles in promoting colonization. Blumeria effector candidate (BEC) 1019, a highly conserved metalloprotease of Blumeria graminis f. sp. hordei (Bgh), is essential for fungal haustorium formation, and silencing BEC1019 significantly reduces Bgh virulence. In this study, we found that BEC1019 homologs in B. graminis f. sp. tritici (Bgt) and Gaeumannomyces graminis var. tritici (Ggt) have complete sequence identity with those in Bgh, prompting us to investigate their functions. Transcript levels of BEC1019 were abundantly induced concomitant with haustorium formation in Bgt and necrosis development in Ggt-infected plants. BEC1019 overexpression considerably increased wheat susceptibility to Bgt and Ggt, whereas silencing this gene using host-induced gene silencing significantly enhanced wheat resistance to Bgt and Ggt, which was associated with hydrogen peroxide accumulation, cell death, and pathogenesis-related gene expression. Additionally, we found that the full and partial sequences of BEC1019 can trigger cell death in Nicotiana benthamiana leaves. These results indicate that Bgt and Ggt can utilize BEC1019 as a virulence effector to promote plant colonization, and thus these genes represent promising new targets in breeding wheat cultivars with broad-spectrum resistance.

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici.

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/µg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

The effect of carabrone on mitochondrial respiratory chain complexes in Gaeumannomyces graminis.

AIMS: To evaluate the effect of carabrone on mitochondrial respiratory chain complexes in Gaeumannomyces graminis to identify carabrone targets. METHODS AND RESULTS: The influence of carabrone on mitochondria in G. graminis mycelia was determined, and the activity of complexes I-V, citrate synthase, and respiratory chain complexes I+III and II+III was evaluated following treatment with 30% effective concentration (EC30 ) as well as EC50 and EC70 of carabrone for 0, 6, 12 and 24 h. Quantitative real-time PCR analysis of the relative expression of genes encoding mitochondrial respiratory chain complexes was then conducted. Carabrone treatment influenced the activity of mitochondrial respiratory chain complexes III, I+III, and II+III in G. graminis in a dose- and time-dependent manner with a marked decrease (c. 50% compared to the control) in mitochondrial respiratory chain complex III activity following treatment with carabrone at the EC50 . The GgCyc1 was downregulated following carabrone treatment at the EC50 , and the other genes were upregulated with respect to the control. CONCLUSIONS: This study showed that mitochondrial respiratory chain complex III in G. graminis is highly sensitive to carabrone and is a potential target of this botanical fungicidal agent. SIGNIFICANCE AND IMPACT OF THE STUDY: Carabrone is a promising botanical fungicidal agent that is environmentally friendly and can control plant diseases and reduce chemical fungicides in agricultural production.

Novel screening strategy reveals a potent Bacillus antagonist capable of mitigating wheat take-all disease caused by Gaeumannomyces graminis var. tritici.

Take-all is a severe root disease of wheat worldwide that is caused by the soilborne fungal pathogen Gaeumannomyces graminis var. tritici (Ggt). In this study, 272 Bacillus isolates were screened for their antifungal activity in vitro to Ggt. Of the 128 strains that demonstrated an antagonistic action, 24 of these exhibited at least three of the four plant growth promotion parameters (i.e. indole acetic acid and siderophore production, inorganic phosphorus solubilization and organic phosphorus solubilization) that were tested in wheat plants. The most effective strain found was Bacillus subtilis Pnf-12; its disease reduction effect reached 69%. Pnf-12 also caused a significant improvement (P < 0·05) in the root and shoot weights of wheat plants, though their root length and shoot height were similar to the noninoculated treatment (P > 0·05). The mechanism for this disease control may be linked to the production of the antifungal lipopeptides surfactin, iturin and fengycin production, all of which were detected in the cell-free supernatant of Pnf-12. SIGNIFICANCE AND IMPACT OF THE STUDY: Take-all, which is caused by the soilborne fungal pathogen Gaeumannomyces graminis var. tritici (Ggt), is one of the most widespread and devastating root diseases of wheat plants. This study focuses on a novel screening strategy of Bacillus isolates to evaluate their potential biological control capacity for suppressing wheat take-all. The joint assessment of antifungal activities, growth promotion factors and variety of antibiotic synthesis genes, in addition to greenhouse experiments, allowed for the identification and demonstration of the Bacillus isolate Pnf-12 as an effective disease control agent.

Take-all or nothing.

Take-all disease of Poaceae is caused by Gaeumannomyces graminis (Magnaporthaceae). Four varieties are recognised in G. graminis based on ascospore size, hyphopodial morphology and host preference. The aim of the present study was to clarify boundaries among species and varieties in Gaeumannomyces by combining morphology and multi-locus phylogenetic analyses based on partial gene sequences of ITS, LSU, tef1 and rpb1. Two new genera, Falciphoriella and Gaeumannomycella were subsequently introduced in Magnaporthaceae. The resulting phylogeny revealed several cryptic species previously overlooked within Gaeumannomyces. Isolates of Gaeumannomyces were distributed in four main clades, from which 19 species could be delimited, 12 of which were new to science. Our results show that the former varieties Gaeumannomyces graminis var. avenae and Gaeumannomyces graminis var. tritici represent species phylogenetically distinct from G. graminis, for which the new combinations G. avenae and G. tritici are introduced. Based on molecular data, morphology and host preferences, Gaeumannomyces graminis var. maydis is proposed as a synonym of G. radicicola. Furthermore, an epitype for Gaeumannomyces graminis var. avenae was designated to help stabilise the application of that name.

1,2,4-Triazole benzamide derivative TPB against Gaeumannomyces graminis var. tritici as a novel dual-target fungicide inhibiting ergosterol synthesis and adenine nucleotide transferase function.

BACKGROUND: Isopropyl 4-(2-chloro-6-(1H-1,2,4-triazol-1-yl)benzamido)benzoate (TPB) was a 1,2,4-triazole benzoyl arylamine derivative with excellent antifungal activity, especially against Gaeumannomyces graminis var. tritici (Ggt). Its mechanism of action was investigated by transmission electron microscopy (TEM) observation, assays of sterol composition, cell membrane permeability, intracellular ATP and mitochondrial membrane potential, and mPTP permeability, ROS measurement, RNA sequencing (RNA-seq) analysis. RESULTS: TPB interfered with ergosterol synthesis, reducing ergosterol content, increasing toxic intermediates, and finally causing biomembrane disruption such as increasing cell membrane permeability and content leakage, and destruction of organelle membranes such as coarse endoplasmic reticulum and vacuole. Moreover, TPB destroyed the function of adenine nucleotide transferase (ANT), leading to ATP transport obstruction in mitochondria, inhibiting mPTP opening, inducing intracellular ROS accumulation and mitochondrial membrane potential loss, finally resulting in mitochondrial damage including mitochondria swelled, mitochondrial membrane dissolved, and cristae destroyed and reduced. RNA-seq analyses showed that TPB increased the expression of ERG11, ERG24, ERG6, ERG5, ERG3 and ERG2 genes in ergosterol synthesis pathway, interfered with the expression of genes (NDUFS5, ATPeV0E, NCA2 and Pam17) related to mitochondrial structure, and inhibited the expression of genes (WrbA and GST) related to anti-oxidative stress. CONCLUSIONS: TPB exhibited excellent antifungal activity against Ggt by inhibiting ergosterol synthesis and destroying ANT function. So, TPB was a novel compound with dual-target mechanism of action and can be considered a promising novel fungicide for the control of wheat Take-all. The results provided new guides for the structural design of active compounds and powerful tools for pathogen resistance management. © 2023 Society of Chemical Industry.

Strain-specific variation in a soilborne phytopathogenic fungus for the expression of genes involved in pH signal transduction pathway, pathogenesis and saprophytic survival in response to environmental pH changes.

The soilborne fungus Gaeumannomyces graminis var. tritici (Ggt) causes take-all, a wheat root disease. In an original strain-specific way, a previous study indicates that inside the Ggt species, some strains grow preferentially at acidic pH and other strains at neutral/alkaline pH. The most important mechanism for a fungal response to the environmental pH is the Pal pathway which integrates the products of the six pal genes and the transcription factor PacC. To evaluate whether the Ggt strain-specific growth in function of the ambient pH is mediated via the Pal pathway, a transcriptional study of the genes encoding this pathway was carried out. This study provided the first evidence that the pH signalling pathway similar to those described in other fungi operated in Ggt. The pacC gene was induced at neutral pH whatever the strain. In an original way, the expression of Ggt genes coding for the different Pal proteins depended on the strain and on the ambient pH. In the strain growing better at acidic pH, few pal genes were pH-regulated, and some were overexpressed at neutral pH when regulated. In the strain growing better at neutral pH, underexpression of most of the pal genes at neutral pH occurred. The strains displayed higher gene expression in the ambient pH that unfavoured their growth as if it was a compensation system. All pH taken together, a globally weaker Pal transcript level occurred in the strains that were less sensitive to acidic pH, and on the contrary, the strain growing better on neutral pH showed higher Pal mRNA levels. The expression of genes involved in pathogenesis and saprophytic growth was also regulated by the ambient pH and the strain: each gene displayed a specific pH-regulation that was similar between strains. But all pH taken together, the global transcript levels of four out of six genes were higher in the strain growing better on neutral pH. Altogether, for the first time, the results show that inside a species, conditions affecting environmental pH modulate the expression of genes in an original strain-specific way.

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

Magnaporthiopsis, a new genus in Magnaporthaceae (Ascomycota).

The phylogenetic relationships among taxa in the Magnaporthaceae are investigated based on DNA sequences of multiple genes including SSU, ITS, LSU, MCM7, RPB1 and TEF1. The genera Magnaporthe and Gaeumannomyces are shown to be polyphyletic and their members are divided into four major groups based on the phylogenetic analyses. Considering morphological, biological and molecular data, we establish a new genus, Magnaporthiopsis. It is characterized by black and globose perithecia with a cylindrical neck, two-layered perithecial wall, clavate asci with a refractive apical ring, fusiform to fusoid and septate ascospores, simple hyphopodia, and Phialophora-like anamorph. Species in this genus are necrotrophic parasites infecting roots of grasses. Three new combinations, Magnaporthiopsis poae, M. rhizophila and M. incrustans, are proposed accordingly. Pyricularia is suggested as the generic name for the rice blast fungus over Magnaporthe, following Article 59.1 of the International Code of Nomenclature for algae, fungi and plants. A new combination, Nakataea oryzae, is proposed for the rice stem rot fungus.

Ammonium fertilization increases the susceptibility to fungal leaf and root pathogens in winter wheat.

Nitrogen (N) fertilization is indispensable for high yields in agriculture due to its central role in plant growth and fitness. Different N forms affect plant defense against foliar pathogens and may alter soil-plant-microbe interactions. To date, however, the complex relationships between N forms and host defense are poorly understood. For this purpose, nitrate, ammonium, and cyanamide were compared in greenhouse pot trials with the aim to suppress two important fungal wheat pathogens Blumeria graminis f. sp. tritici (Bgt) and Gaeumannomyces graminis f. sp. tritici (Ggt). Wheat inoculated with the foliar pathogen Bgt was comparatively up to 80% less infested when fertilized with nitrate or cyanamide than with ammonium. Likewise, soil inoculation with the fungal pathogen Ggt revealed a 38% higher percentage of take-all infected roots in ammonium-fertilized plants. The bacterial rhizosphere microbiome was little affected by the N form, whereas the fungal community composition and structure were shaped by the different N fertilization, as revealed from metabarcoding data. Importantly, we observed a higher abundance of fungal pathogenic taxa in the ammonium-fertilized treatment compared to the other N treatments. Taken together, our findings demonstrated the critical role of fertilized N forms for host-pathogen interactions and wheat rhizosphere microbiome assemblage, which are relevant for plant fitness and performance.

Structure-Activity Studies of N-Heterocyclic Benzoyl Arylamine Derivatives Led to a Highly Fungicidal Candidate against Gaeumannomyces graminis var. tritici and Four Fusarium Wheat Pathogens.

Wheat root diseases can seriously reduce yields and quality of wheat. 1,2,4-Triazole benzoyl arylamine derivatives previously showed good activities against some wheat root fungal pathogens. To further systematically disclose the structure-activity relationship, a series of benzoyl arylamines were designed and prepared. Their structures were characterized and fungicidal activities against Gaeumannomyces graminis var. tritici and Fusarium graminearum were evaluated. The results indicated that the structure of the N-heterocyclic group and the substituted group and their position on the benzamide scaffold had an important influence on the activities, as predicted. Finally, compound 18f was found to show excellent activities against G. graminis var. tritici, F. graminearum, Fusarium culmorum, Fusarium pseudograminearum, and Fusarium moniliforme with half-maximum effective concentrations of 0.002, 0.093, 0.011, 0.881, and 0.287 µg/mL, respectively. These results proposed that compound 18f deserved serious consideration as a novel fungicide candidate for the control of wheat root diseases.

Influence of Bacillus subtilis strain Z-14 on microbial communities of wheat rhizospheric soil infested with Gaeumannomyces graminis var. tritici.

Wheat take-all disease caused by Gaeumannomyces graminis var. tritici (Ggt) spreads rapidly and is highly destructive, causing severe reductions in wheat yield. Bacillus subtilis strain Z-14 that significantly controlled wheat take-all disease effectively colonized the roots of wheat seedlings. Z-14 increased the metabolic activity and carbon source utilization of rhizospheric microorganisms, thus elevating average well-color development (AWCD) values and functional diversity indexes of soil microbial communities. Z-14 increased the abundance of Bacillus in the rhizosphere, which was positively correlated with AWCD and functional diversity indexes. The Z-14-treated samples acquired more linkages and relative connections between bacterial communities according to co-occurrence network analyses. After the application of Ggt, the number of linkages between fungal communities increased but later decreased, whereas Z-14 increased such interactions. Whole-genome sequencing uncovered 113 functional genes related to Z-14's colonization ability and 10 secondary metabolite gene clusters in the strain, of which nine substances have antimicrobial activity. This study clarifies how bacterial agents like Z-14 act against phytopathogenic fungi and lays a foundation for the effective application of biocontrol agents.

Complexity of Gaeumannomyces species causing take-all root rot of St. Augustinegrass in Texas.

Gaeumannomyces graminis var. graminis (Ggg) has been the etiological agent of take-all root rot (TARR) in St. Augustinegrass (Stenotaphrum secundatum) and root decline of the other warm-season turfgrasses. Seventy-five Ggg isolates were obtained from St. Augustinegrass in central and east Texas. Evaluation of colony morphologies on potato dextrose agar (PDA) within 2 wk and follow-up multilocus phylogenic analyses revealed three phenotypic groups associated with different Gaeumannomyces species: (i) G. floridanus, highly melanized with round colony formation; (ii) G. arxii, none to slightly melanized with round colony formation; and (iii) G. graminicola, highly melanized with irregular colony formation. Further examination with representative isolates from each group revealed that their phenotypic characterizations supported the distinctive genetic groups within Ggg associated with St. Augustinegrass TARR. Gaeumannomyces floridanus isolates grew faster at warmer temperature (30 C) than G. arxii or G. graminicola. Pathogenicity assays using rice seedlings indicated that G. floridanus was more aggressive in disease symptom development than G. arxii or G. graminicola. A multilocus phylogeny reconstruction supported that most of Gaeumannomyces isolates tested in this study were separated into three phylogenetically distinct groups: G. floridanus, G. arxii, and G. graminicola. The resolution of intravarietal complexities of causal fungi of TARR is important for proper diagnostics and management strategies for TARR in St. Augustinegrass and other root-decline diseases in warm-season turfgrasses.

Take-All Disease: New Insights into an Important Wheat Root Pathogen.

Take-all disease, caused by the fungal root pathogen Gaeumannomyces tritici, is considered to be the most important root disease of wheat worldwide. Here we review the advances in take-all research over the last 15 years, focusing on the identification of new sources of genetic resistance in wheat relatives and the role of the microbiome in disease development. We also highlight recent breakthroughs in the molecular interactions between G. tritici and wheat, including genome and transcriptome analyses. These new findings will aid the development of novel control strategies against take-all disease. In light of this growing understanding, the G. tritici-wheat interaction could provide a model study system for root-infecting fungal pathogens of cereals.

Biological Control of Take-All and Growth Promotion in Wheat by Pseudomonas chlororaphis YB-10.

Wheat is a worldwide staple food crop, and take-all caused by Gaeumannomyces graminis var. tritici can lead to a tremendous decrease in wheat yield and quality. In this study, strain YB-10 was isolated from wheat rhizospheric soil and identified as Pseudomonas chlororaphis by morphology and 16S rRNA gene sequencing. Pseudomonas chlororaphis YB-10 had extracellular protease and cellulase activities and strongly inhibited the mycelium growth of Gaeumannomyces graminis var. tritici in dual cultures. Up to 87% efficacy of Pseudomonas chlororaphis YB-10 in controlling the take-all of seedlings was observed in pot experiments when wheat seed was coated with the bacterium. Pseudomonas chlororaphis YB-10 was also positive for indole acetic acid (IAA) and siderophore production, and coating wheat seed with the bacterium significantly promoted the growth of seedlings at 107 and 108 CFU/mL. Furthermore, treatment with Pseudomonas chlororaphis YB-10 increased activities of the wheat defense-related enzymes POD, SOD, CAT, PAL and PPO in seedlings, indicating induced resistance against pathogens. Overall, Pseudomonas chlororaphis YB-10 is a promising new seed-coating agent to both promote wheat growth and suppress take-all.

Discovery and preliminary mechanism of 1-carbamoyl ß-carbolines as new antifungal candidates.

Natural ß-carboline alkaloids are ideal models for the discovery of pharmaceutically important entities. Various 1-substituted ß-carbolines were synthesized from commercially inexpensive tryptophan and demonstrated significant in vitro antifungal activity against G. graminis. Significantly, compound 4m (EC50 = 0.45 µM) with carboxamide at 1-position displayed the best efficacy and nearly 20 folds enhancement in antifungal potential compared to Silthiopham (EC50 = 8.95 µM). Moreover, compounds 6, 7, and 4i exhibited excellent in vitro antifungal activities and in vivo protective and curative activities against B. cinerea and F. graminearum. Preliminary mechanism studies revealed that compound 4m caused reactive oxygen species accumulation, cell membrane destruction, and deregulation of histone acetylation. These findings indicated that 1-carbamoyl ß-carboline can be selected as a promising model for the discovery of novel and broad-spectrum fungicide candidates.

Novel Fungicide 4-Chlorocinnamaldehyde Thiosemicarbazide (PMDD) Inhibits Laccase and Controls the Causal Agent of Take-All Disease in Wheat, Gaeumannomyces graminis var. tritici.

Our aim was to investigate the bioactivity and mode of action of a novel fungicide 4-chlorocinnamaldehyde thiosemicarbazide (PMDD). As a result of its efficacy against various plant pathogens, its protective fungicidal activity, and systemic transport after root treatment, PMDD could be a promising fungicide to control wheat root diseases. In a field assay, PMDD showed good control efficacy on wheat take-all disease. A biochemical study indicated that PMDD acts as a laccase inhibitor, a to date unique mode of fungicidal action. Moreover, a total of seven stable PMDD-resistant Gaeumannomyces graminis var. tritici (Ggt) mutants were generated and demonstrated no cross-resistance with any commercial fungicides used for take-all disease control, and the gene expression profile further confirmed that laccase could be the target of PMDD. Taken together, we conclude that PMDD is a laccase inhibitor and could be used on wheat to control take-all diseases. The current study could strongly benefit the registration and application of PMDD.

A functional analysis of mitochondrial respiratory chain cytochrome bc1 complex in Gaeumannomyces tritici by RNA silencing as a possible target of carabrone.

Gaeumannomyces tritici, an ascomycete soilborne fungus, causes a devastating root disease in wheat. Carabrone, a botanical bicyclic sesquiterpenic lactone, is a promising fungicidal agent that can effectively control G. tritici. However, the mechanism of action of carabrone against G. tritici remains largely unclear. Here, we used immunogold for subcellular localization of carabrone and the results showed that carabrone is subcellularly localized in the mitochondria of G. tritici. We then explored the functional analysis of genes GtCytc1 , GtCytb, and GtIsp of the mitochondrial respiratory chain cytochrome bc1 complex in G. tritici by RNA silencing as a possible target of carabrone. The results showed that the silenced mutant ∆GtIsp is less sensitive to carabrone compared to ∆GtCytc1 and ∆GtCytb. Compared with the control, the activities of complex III in all the strains, except ∆GtIsp and carabrone-resistant isolate 24-HN-1, were significantly decreased following treatment with carabrone at EC20 and EC80 in vitro (40%-50% and 70%-80%, respectively). The activities of mitochondrial respiratory chain complex III and the mitochondrial respiration oxygen consumption rates in all the strains, except ∆GtIsp and 24-HN-1, were higher with respect to the control when treated with carabrone at EC20 in vivo. The rates of mitochondrial respiration of all strains, except ∆GtIsp, were significantly inhibited following treatment with carabrone at EC80 (ranging from 57% to 81%). This study reveals that the targeting of the iron-sulphur protein encoded by GtIsp is highly sensitive to carabrone and provides a direction for the research of carabrone's target.