scholarly journals Molecular and Biochemical Characterization of Laboratory and Field Mutants of Botrytis cinerea Resistant to Fludioxonil

Plant Disease ◽  
2016 ◽  
Vol 100 (7) ◽  
pp. 1414-1423 ◽  
Author(s):  
Weichao Ren ◽  
Wenyong Shao ◽  
Xu Han ◽  
Mingguo Zhou ◽  
Changjun Chen
Keyword(s):  
Botrytis Cinerea ◽  
Biochemical Characterization ◽  
Resistance Mechanism ◽  
Gray Mold ◽  
Terminal Region ◽  
Excellent Activity ◽  
Resistant Mutants ◽  
First Time

Botrytis cinerea is a filamentous phytopathogen with a high risk of developing resistance to fungicides. The phenylpyrrole fungicide fludioxonil has been reported to have excellent activity against B. cinerea and increasingly has been applied to control gray mold in China. In this study, molecular and biochemical characteristics of laboratory and field mutants of B. cinerea resistant to fludioxonil has been investigated. During 2012 to 2014, B. cinerea isolates collected from Jiangsu and Shandong Provinces in China were tested in vitro for sensitivity to fungicides commonly used to suppress gray mold of cucumber and tomato. Among the 75 isolates collected from cucumber in 2013, two were highly resistant (HR) to fludioxonil. Of the 308 isolates collected from tomato in 2014, four were fludioxonil-HR. This was the first time that B. cinerea isolates HR to fludioxonil had been detected in the field. Six fludioxonil-resistant mutants were obtained in the laboratory by selection on fungicide-amended media. These mutants exhibited stable resistance to fludioxonil, as indicated by resistance factor values that ranged from 34.38 to >10,000. In comparison with fludioxonil-sensitive isolates of B. cinerea, all field and laboratory mutants showed reduced fitness, as defined by mycelial growth, sporulation, virulence, and sensitivity to osmotic stress. When treated with fludioxonil at 1 μg/ml, sensitive isolates showed increased glycerol contents in mycelium and expression levels of Bchog1, while levels in field and laboratory HR mutants increased only slightly. Sequences of the Bos1 gene of field and laboratory fludioxonil-HR mutants showed that mutations in field mutants were located in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) domains of the N-terminal region, whereas mutations in the laboratory mutants were distributed in HAMP domains or in the HATPase_c domain of the C-terminal region. These results will enhance our understanding of the resistance mechanism of B. cinerea to fludioxonil.

scholarly journals Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates From Commercially Stored Apple Fruit

2017 ◽  
Vol 107 (3) ◽  
pp. 362-368 ◽  
Author(s):  
Wayne M. Jurick ◽  
Otilia Macarisin ◽  
Verneta L. Gaskins ◽  
Eunhee Park ◽  
Jiujiang Yu ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Gray Mold ◽  
Apple Fruit ◽  
Sublethal Dose ◽  
Long Term Storage ◽  
Pose Control ◽  
First Time

Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruit, vegetables, and ornamentals. Fludioxonil-sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had values for effective concentrations for inhibiting 50% of mycelial growth of 0.0004 to 0.0038 μg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sublethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multiresistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multiresistant B. cinerea strain caused decay on apple fruit treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multipostharvest fungicide-resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanisms to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil, which may pose control challenges in the packinghouse environment and during long-term storage.

scholarly journals The Relaxase of the Rhizobium etli Symbiotic Plasmid Shows nic Site cis-Acting Preference

2006 ◽  
Vol 188 (21) ◽  
pp. 7488-7499 ◽  
Author(s):  
Daniel Pérez-Mendoza ◽  
María Lucas ◽  
Socorro Muñoz ◽  
José A. Herrera-Cervera ◽  
José Olivares ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Rhizobium Etli ◽  
Cis Acting ◽  
Cleavage Activity ◽  
Symbiotic Plasmid ◽  
The Family ◽  
Nick Site ◽  
First Time

ABSTRACT Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1

2001 ◽  
Vol 356 (1) ◽  
pp. 257-267 ◽  
Author(s):  
Jeffrey A. SKINNER ◽  
Alan R. SALTIEL
Keyword(s):  
Molecular Mass ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Terminal Region ◽  
Protein Phosphatase 1 ◽  
Chromosome 15 ◽  
Glutathione S Transferase ◽  
Interacting Protein

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns > 99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1–46.5cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related ∼ 110–130kDa MYPT subunits by its molecular mass of 58kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)–MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST–MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

Characterization of Botrytis cinerea and B. prunorum from Healthy Floral Structures and Decayed ‘Hayward’ Kiwifruit During Post-Harvest Storage

Plant Disease ◽  
2020 ◽  
Author(s):  
Danae Riquelme ◽  
Zdenka Aravena ◽  
Hector Valdes ◽  
Bernardo Antonio Latorre ◽  
Gonzalo A Díaz ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Central Valley ◽  
Gray Mold ◽  
Heat Shock Protein 60 ◽  
Post Harvest ◽  
Diseased Fruit ◽  
Apparently Healthy

Gray mold is the primary post-harvest disease of ‘Hayward’ kiwifruit (Actinidia deliciosa) in Chile, with a prevalence of 33.1% in 2016 and 7.1% in 2017. Gray mold develops during postharvest storage, which is characterized by a soft, light to brown watery decay that is caused by Botrytis cinerea and B. prunorum. However, there is no information related to the role of B. prunorum during the development and storage of kiwifruit in Chile. For this purpose, asymptomatic flowers and receptacles were collected throughout fruit development and harvest from five orchards over two seasons in the Central Valley of Chile. Additionally, diseased kiwifruits were selected after storage for 100 days at 0°C plus 2 days at 20° C. High (HCP) and low conidial production (LCP) colonies of Botrytis sp. were consistently obtained from apparently healthy petals, sepals, receptacles, styles, and diseased kiwifruit. Morphological and phylogenetic analysis using three partial gene sequences encoding glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) were able to identify and separate B. cinerea and B. prunorum species. Consistently, B. cinerea was predominantly isolated from all floral parts and fruit in apparently healthy tissue and diseased kiwifruit. During full bloom, the highest colonization by B. cinerea and B. prunorum was obtained from petals followed by sepals. In storage, both Botrytis species were isolated from the diseased fruit (n=644), of which 6.8% (n=44) were identified as B. prunorum. All Botrytis isolates grew from 0°C to 30°C in vitro and were pathogenic on kiwifruit leaves and fruit. Notably, B. cinerea isolates were always more virulent than B. prunorum isolates. This study confirms the presence of B. cinerea and B. prunorum colonizing apparently healthy flowers and floral parts in fruit and causing gray mold during kiwifruit storage in Chile. Therefore, B. prunorum plays a secondary role in the epidemiology of gray mold developing in kiwifruit during cold storage.

scholarly journals Purification and characterization of embryo-specific soy urease (Glycine max) and its antifungal potential against Paracoccidioides brasiliensis

2021 ◽  
Vol 46 (1SI) ◽  
pp. 41-52
Author(s):  
Elisângela Andrade Ângelo ◽  
Tainá Michelle Da Cruz ◽  
José Renato Pattaro Júnior ◽  
Daniele Maria Zanzarin ◽  
Franciele Abigail Vilugron Rodrigues ◽  
...  
Keyword(s):  
Glycine Max ◽  
Biochemical Characterization ◽  
Paracoccidioides Brasiliensis ◽  
Maximum Velocity ◽  
Enzymatic Function ◽  
Potential Applications ◽  
First Time

Ureases are amidohydrolases that catalyze the hydrolysis of urea to ammonia and carbamate. In addition to the enzymatic function, ureases have fungitoxic and insecticidal function, which are independent of their catalytic activity. Soy (Glycine max) has two main urease isoforms: ubiquitous and embryo-specific, the latter is present in beans. In view of the potential applications of ureases, this work aimed to extract, purify, characterize the structure, activity and fungitoxic activity of soy urease against Paracoccidioides brasiliensis. The biochemical characterization was performed, in terms of optimal pH and temperature, as well as the determination of the Michaelis�Menten constant (KM) and maximum velocity (Vmax). The protein sequence was identified by mass spectrometry and used in computational modeling of the biological structure. The optimum pH and temperature of the enzyme were 6.5 and 65 �C, respectively, KM 526 mmol L-1 and Vmax 7.4 mmol L-1NH3׵gurease-1�s-1 and biological unity as a trimer. The antifungal activity assays (in vitro) were promising, showing a fungicidal profile of the urease, with a minimum inhibitory concentration of 10 �g�mL-1. This work demonstrated, for the first time, the fungitoxic activity of embryo-specific soy urease against the Pb18 strain of P. brasiliensis.

Characterization of sparfloxacin-resistant mutants of Staphylococcus aureus obtained in vitro

2001 ◽  
Vol 18 (2) ◽  
pp. 107-112 ◽  
Author(s):  
Joaquim Ruiz ◽  
Josep M. Sierra ◽  
M.Teresa Jiménez De Anta ◽  
Jordi Vila
Keyword(s):  
Staphylococcus Aureus ◽  
Resistant Mutants

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

1985 ◽  
Vol 101 (2) ◽  
pp. 427-440 ◽  
Author(s):  
E Bartnik ◽  
M Osborn ◽  
K Weber
Keyword(s):  
Positive Reaction ◽  
Intermediate Filaments ◽  
Biochemical Characterization ◽  
Helix Pomatia ◽  
Molar Ratio ◽  
Negative Stain ◽  
Epithelial Morphology ◽  
Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

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