Heteroplasmy and Response Against Azoxystrobin in Cercospora

Heteroplasmy and Response Against Azoxystrobin in genus CercosporaIntroductionThe quinone immaterial inhibitor (QoI) or Strobilurin is one of the most important antifungals use to control fungous and some Oomycetes pathogens in agricultural crops. This class of fungicide was commencement exercise isolated from a wood-rotting fungus called Strobilurus tenacellus. Several chemically modified derivatives of natural fungicide, Strobilurin A, atomic number 18 available which ar more stable, efficacious, less harmful to human and environment. These fungicides are commercially available with different names and active ingredients azoxystrobin (Syngenta), fenamidone (Bayer), fluoxastrobin (Arysta), kresoxim methyl (Cheminova), pyraclostrobin (BASF) and trifloxystrobin (Bayer) (bartlett pear et al., 2002 Vincelli, 2012).QoI fungicides confront both translaminar (across leaf blade) and weak systemic movement within the plant. all QoI fungicides pee-pee the same mode of action which dis rupt mitochondrial respiration and maintain energy production inside fungal cells (Vincelli 2012). The disruption of ATP coevals occurs because of binding of strobilurin at Qo site of cytochrome b hence preventing electron shipping from cytochrome b to cytochrome c1 (Bartlett et al., 2002).QoI fungicides are applied to control a broad wrap of plant pathogens including fungi, water molds, downy forms, powdery mildews and rusts (Vincelli, 2012). They are mainly employ as protective and healthful fungicides because of effective action against spore germination and shrewdness (Balba, 2007). The eradicative property has also been reported by preventing sporulation of fungal pathogen (Anesiadis et al., 2003). more(prenominal) than 50 species of plant pathogens repellent to QoI fungicides has been reported and there is a full(prenominal) risk of selecting insusceptible isolates in the field (Fungicide Resistant Action Committee, 2013). ternion different point vicissitude in m itochondrial cytochrome b gene has been associated with foul mechanics against QoI fungicide. The primary mechanism of foeman is by amino vitriolic substitution from genus Glycine to alanine at 143rd codon (G143A) (Bartlett et al., 2002). Other two point athletics at cytochrome b gene is the substitution of phenylalanine with leucine at position 129 (F129L) and glycine with arginine at position 137 (G137R) which confer QoI resistance (Fernndez-Ortuo et al. 2010). Another mechanism has also been identified that can bypass the blockage of electron transfer. alternative oxidase (AOX) is a strobilurin-insensitive terminal oxidase which can bypass electron transfer in Complex III and Salicylhydroxamic acid (SHAM) is an active inhibitor of AOX (Wood and Hollomon, 2003).Resistant mechanism of C. sojina against QoI fungicides is associated with a mitochondrial genome which is present in multiple copies within a angiotensin-converting enzyme cell. The coexistence of speculative and mu tated alleles in QoI resistant/sensitive locus has been reported in several(prenominal) other fungal pathogens such as Corynespora cassiicola, Collectotrichumgloeosporioides, Venturia inequalis and Mycovellosiella nattrassii (Ishii et al., 2007 Villani and cox, 2014). The proportion of wild and mutant allele in the mitochondrial genome has a major role for quantitative resistance (Villani and Cox, 2014). Protective efficacy of the full dose of azoxystrobin against powdery and downy mildew has been found to decrease as populations contained 10% resistant isolates (Ishii et al., 2007). There have been reports of loss of resistance stability in the absence of selection crush and vice versa (Fraaije et al., 2002 Ishii et al., 2007). The main objectives of this study are to i) list heteroplasmy in Cercospora sojina ii) monitor the proportion of resistant and sensitive allele in the aim of selection pressure in the laboratory and, iii) study the aesthesia of C. sojina against azoxys trobin.Materials and Methods sequester selection and development of bingle spore culturesIsolates of C. sojina were screened for resistant and sensitive allele development Taqman assay. After screening, leash isolates each having resistant and sensitive alleles were chosen for single spore cultures. Isolates were transferred to V8-RA media and grown in dark cabinet to enhance sporulation. After three weeks, casingd were flooded with water and filtered with muslin filter cloth. Water was observed under dissecting microscope to identify single spores. Sterilized needed were used to pick single spore and transferred to refreshful V8-RA plates. Culture was left at room temperature, mycelium harvested, lyophilized and DNA was extracted. radial tire development studyA nitty-gritty of two isolates 158-1 (resistant) and 312-1 (sensitive) were selected for fungicide esthesia and radial appendage study. Four different compactnesss of azoxystrobin including control were used to culture both isolates in two replications. Technical grade formulation of azoxystrobin (0.104 gm) (96% a.i. Syngenta play Protection) was used to make 100,000 g a.i./ml stock in 1 ml acetone. Serial dilution was done to make four different concentration stocks 10,000, 1000, 100 and 100 g a.i./ml. V8 media was prepared with four different concentrations (10, 1, 0.1, 0.01 g a.i./ml) by adding 1ml of respective fungicide stock in 1 lambert of media. All four media along with control was amended with salicylhydroxamic acid (SHAM) at 60 g a.i./ml.Two straight line at 90o were draw at the center of the plate. For resistant and sensitive isolates, a 5 mm mycelium disc was taken and placed at the center of amended plates in two replications. For each plate, diameters of growth were measured at the interval of 11, 21 and 30 days. Mycelium disc from amended plates was again transferred to the newly amended plate after 10 days. Diameters were measured similarly for three contemporariess.Taqman as say and Sanger sequencingThe G/C point mutation in cytochrome b gene go out be discriminated by Taqman assay consisting of two dyes. VIC can detect resistant allele C and FAM can detect sensitive allele G. verge cycle or Ct of two dyes provide be used in detecting the presence of two alleles in a single spore culture. Ct value is the cycle number at which the fluorescence generated crosses the threshold fluorescence and is inversely proportional to the amount of nucleic acid. Lower Ct indicates higher copies in the sample.Sanger sequencing testament be done to confirm the presence of both alleles in a single spore. Two primers pairs (Forward 5 CTCATTAAATTAGTAATAACTGTGGC 3 and Reverse 5 TAATACAGCTTCAGCATTTTTCTTCT 3) get out be used to amplify a part of cytochrome b gene. PCR reaction will be done in a total volume of 25 l consisting of 1.25 l (10 M) of each primer, 12.5 l of 2x Veriseq PCR intermix (Enzymatics Inc.), 1.25 l DNA and 8.5 l water and run in future(a) settings ini tial denaturation at 94 C for 2 min followed by 29 cycles of denaturation at 94 C for 20 s, annealing at 55 C for 25 s, extension at 72 C for 1 min and final extension at 72 C for 10 min.Data analysisSequences derived from Sanger sequencing will be aligned to in public available cytochrome b gene of C. sojina. The QoI resistant/sensitive point mutation locus will be observed for Heterozygosity. The proportions of resistant and sensitive alleles will be calculated based on Ct values and statistical analysis will be performed to compare among different generations.The percent growth inhibition will be calculated as (colony diameter on control media 5 mm colony diameter on fungicide amended media 5 mm) / (colony diameter on control media 5 mm) x 100. Further, radial growth of the same isolate among three generations and four different treatments will be compared statistically.Expected resultsThis study will help to explore if heteroplasmy exists in C. sojina as in other Cercospora species. The proportion of resistant and sensitive isolates determines the limit of disease, so it is important to know this ratio. In vitro assay to check the sensitivity of isolates against azoxystrobin at different concentration in a different generation will help to understand the effect of selection pressure. Further measuring stick of resistant and sensitive proportion with qPCR would help to determine the change occurred in following generations. Genetic study after fungicide treatment will also contribute in identifying changes due to selection pressure.ReferencesAnesiadis T, Karaoglanidis G and TzavellaKlonari K. 2003. Protective, curative and eradicant activity of the strobilurin fungicide azoxystrobin against Cercospora beticola and Erysiphe betae. Journal of Phytopathology 151(1112)647-651.Balba H. 2007. Review of strobilurin fungicide chemicals. Journal of Environmental Science and Health Part B 42(4)441-451.Bartlett DW, Clough JM, Godwin JR, Hall AA, Hamer M and Par rDobrzanski B. 2002. The strobilurin fungicides. Pest management lore 58(7)649-662.Fernndez-Ortuo D, Tors JA, De Vicente A and Prez-Garca A. 2010. Mechanisms of resistance to QoI fungicides in phytopathogenic fungi. International Microbiology 11(1)1-9.Fraaije B, Butters J, Coelho J, Jones D and Hollomon D. 2002. Following the dynamics of strobilurin resistance in Blumeria graminis f. sp. tritici using quantitative allelespecific realtime PCR measurements with the fluorescent dye SYBR Green I. lay down pathology 51(1)45-54.Fungicide Resistant Action Committee. 2013. List of plant pathogenic organisms resistant to disease control agents. http//www.frac.info/docs/default-source/publications/list-of-resistant-plant-pathogens/list-of-resistant-plant-pathogenic-organismsfebruary-2013.pdf?sfvrsn=4.Ishii H, Yano K, Date H, Furuta A, Sagehashi Y, Yamaguchi T, Sugiyama T, Nishimura K and Hasama W. 2007. Molecular characterization and diagnosing of QoI resistance in cucumber and eggplant fun gal pathogens. Phytopathology 97(11)1458-1466.Villani SM and Cox KD. 2014. Heteroplasmy of the cytochrome b gene in Venturia inaequalis and its involvement in quantitative and mulish resistance to trifloxystrobin. Phytopathology 104(9)945-953.Vincelli P. 2012. QoI (Strobilurin) Fungicides Benefits and Risks. The Plant Health Instructor. DOI 10.1094/PHI-I-2002-0809-0.Wood PM and Hollomon DW. 2003. A critical evaluation of the role of alternative oxidase in the performance of strobilurin and associate fungicides acting at the Qo site of complex III. Pest management science 59(5)499-511.