Molecular Characterization of Laboratory Mutants of Fusarium oxysporum f. sp. niveum Resistant to Prothioconazole, a Demethylation Inhibitor (DMI) Fungicide

Fusarium oxysporum f. sp. niveum (FON) is the causal agent of Fusarium wilt in watermelon, an international growth-limiting pathogen of watermelon cultivation. A single demethylation inhibitor (DMI) fungicide, prothioconazole, is registered to control this pathogen, so the risk of resistance arising in the field is high. To determine and predict the mechanism by which FON could develop resistance to prothioconazole, FON isolates were mutagenized using UV irradiation and subsequent fungicide exposure to create artificially resistant mutants. Isolates were then put into three groups based on the EC50 values: sensitive, intermediately resistant, and highly resistant. The mean EC50 values were 4.98 µg/mL for the sensitive, 31.77 µg/mL for the intermediately resistant, and 108.33 µg/mL for the highly resistant isolates. Isolates were then sequenced and analyzed for differences in both the coding and promoter regions. Two mutations were found that conferred amino acid changes in the target gene, CYP51A, in both intermediately and highly resistant mutants. An expression analysis for the gene CYP51A also showed a significant increase in the expression of the highly resistant mutants compared to the sensitive controls. In this study, we were able to identify two potential mechanisms of resistance to the DMI fungicide prothioconazole in FON isolates: gene overexpression and multiple point mutations. This research should expedite growers’ and researchers’ ability to detect and manage fungicide-resistant phytopathogens.

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Characterization of Maize Genotypes (Zea mays L.) for Resistance to Striga asiatica and S. hermonthica and Compatibility with Fusarium oxysporum f. sp. strigae (FOS) in Tanzania

Agronomy ◽

10.3390/agronomy11051004 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1004

Author(s):

John Lobulu ◽

Hussein Shimelis ◽

Mark D. Laing ◽

Arnold Angelo Mushongi ◽

Admire Isaac Tichafa Shayanowako

Keyword(s):

Grain Yield ◽

Fusarium Oxysporum ◽

Biocontrol Agent ◽

Zea Mays L ◽

Control Treatment ◽

Yield Reduction ◽

Maize Genotypes ◽

Significant Yield ◽

The Mean

Striga species cause significant yield loss in maize varying from 20 to 100%. The aim of the present study was to screen and identify maize genotypes with partial resistance to S. hermonthica (Sh) and S. asiatica (Sa) and compatible with Fusarium oxysporum f. sp. strigae (FOS), a biocontrol agent. Fifty-six maize genotypes were evaluated for resistance to Sh and Sa, and FOS compatibility. Results showed that FOS treatment significantly (p < 0.001) enhanced Striga management compared to the untreated control under both Sh and Sa infestations. The mean grain yield was reduced by 19.13% in FOS-untreated genotypes compared with a loss of 13.94% in the same genotypes treated with FOS under Sh infestation. Likewise, under Sa infestation, FOS-treated genotypes had a mean grain yield reduction of 18% while untreated genotypes had a mean loss of 21.4% compared to the control treatment. Overall, based on Striga emergence count, Striga host damage rating, grain yield and FOS compatibility, under Sh and Sa infestations, 23 maize genotypes carrying farmer preferred traits were identified. The genotypes are useful genetic materials in the development of Striga-resistant cultivars in Tanzania and related agro-ecologies.

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Characterization of Spontaneous, In Vitro-Selected, Rifampin-Resistant Mutants of Mycobacterium tuberculosisStrain H37Rv

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.12.3298-3301.2000 ◽

2000 ◽

Vol 44 (12) ◽

pp. 3298-3301 ◽

Cited By ~ 47

Author(s):

Glenn P. Morlock ◽

Bonnie B. Plikaytis ◽

Jack T. Crawford

Keyword(s):

Point Mutations ◽

Laboratory Strain ◽

Resistant Mutant ◽

Small Region ◽

Clinical Isolates ◽

Gene Coding ◽

Resistant Mutants ◽

Strain H37rv

ABSTRACT Resistance to rifampin in Mycobacterium tuberculosisresults from mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). At least 95% of rifampin-resistant isolates have mutations in rpoB, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified, but point mutations in two codons, those coding for Ser531 and His526, are seen in about 70% of rifampin-resistant clinical isolates, with Ser531-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized M. tuberculosislaboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the rpoBgene from one colony per plate revealed that 39 (60.9%) were Ser531 to Leu. We conclude that, for unknown reasons, the associated rpoB mutation occurs at a substantially higher rate than other rpoB mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.

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Detection and Molecular Characterization of Resistance to the Dicarboximide and Benzamide Fungicides in Botrytis cinerea From Tomato in Hubei Province, China

Plant Disease ◽

10.1094/pdis-10-17-1531-re ◽

2018 ◽

Vol 102 (7) ◽

pp. 1299-1306 ◽

Cited By ~ 8

Author(s):

M. Adnan ◽

M. S. Hamada ◽

G. Q. Li ◽

C. X. Luo

Keyword(s):

Botrytis Cinerea ◽

Management Strategies ◽

Resistance Management ◽

Point Mutations ◽

Hubei Province ◽

Cross Resistance ◽

Resistance Development ◽

Fitness Parameters ◽

The Mean

Altogether, 192 Botrytis cinerea isolates collected from tomato greenhouses at different locations in Hubei Province were evaluated for their sensitivity to fungicides procymidone and zoxamide. The mean effective concentration to cause 50% growth inhibition (EC50) values of procymidone for sensitive and resistant isolates were 0.25 and 3.60 μg/ml, respectively. The frequency of procymidone-resistant (ProR) isolates was 18%, and the highest frequency was recorded in Jingmen. Positive cross-resistance was observed for ProR isolates to other dicarboximide fungicides but not to phenylpyrroles. Significant differences were observed for fitness parameters (i.e., mycelial growth, osmotic sensitivity, and virulence between sensitive and resistant isolates). Amino acid sequence of the Bos1 gene revealed that ProR isolates carried either point mutations at codon 365 (I365S) or a pair of point mutations at codons 369 (Q369P) and 373 (N373S). For zoxamide, the mean EC50 values for sensitive and resistant isolates were 0.22 and 5.32 μg/ml, respectively. Approximately 14% of the isolates were found to be resistant to zoxamide, and the highest frequency of resistance was also observed in Jingmen. There was positive cross-resistance for zoxamide-resistant (ZoxR) isolates to carbendazim. No significant differences were observed for fitness parameters between zoxamide-sensitive and ZoxR isolates. Sequence analysis of the β-tubulin gene of Botrytis cinerea revealed two previously reported point mutations (E198A and E198K) and one new point mutation (T351I). This new mutation was detected in only those isolates which possessed the E198K but not E198A substitution. This study allows for a better understanding of the resistance development profile in Hubei Province. Results will be useful for the improvement of fungicide resistance management strategies.

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Characterization of a new TEM-derived beta-lactamase produced in a Serratia marcescens strain.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.11.2374 ◽

1997 ◽

Vol 41 (11) ◽

pp. 2374-2382 ◽

Cited By ~ 25

Author(s):

M Perilli ◽

A Felici ◽

N Franceschini ◽

A De Santis ◽

L Pagani ◽

...

Keyword(s):

Serratia Marcescens ◽

Three Dimensional ◽

Point Mutations ◽

Catalytic Efficiency ◽

Point Of View ◽

Multiple Point ◽

Crystallographic Structure ◽

Beta Lactamase

A natural TEM variant beta-lactamase was isolated from an epidemic strain of Serratia marcescens. Nucleotide gene sequencing revealed multiple point mutations located in the 42-to-44 tripeptide and positions 145 to 146, 178, and 238. In addition, a glutamic acid 212 deletion was also found. The purified enzyme was studied from a kinetic point of view, revealing the highest catalytic efficiency (k[cat]/Km) values for ceftazidime and aztreonam compared with the TEM-1 prototype enzyme. The in vitro resistance correlated with kinetic parameters, and the enzyme also mediated resistance to some penicillins and an ampicillin-clavulanic acid combination. The mutational and kinetic changes are discussed in relation to the three-dimensional crystallographic structure of the wild-type TEM-1 enzyme.

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Caracterização da Peneira Média em Clones de Coffea canephora

REVISTA FIMCA ◽

10.37157/fimca.v5i2.58 ◽

2018 ◽

Vol 5 (2) ◽

pp. 28-31

Author(s):

Darlan Darlan Sanches Barbosa Alves ◽

Victor Mouzinho Spinelli ◽

Marcos Santana Moraes ◽

Carolina Augusto De Souza ◽

Rodrigo da Silva Ribeiro ◽

...

Keyword(s):

Genetic Improvement ◽

Block Design ◽

Coffea Canephora ◽

Coffee Production ◽

Randomized Block Design ◽

Significant Difference ◽

The Mean ◽

Four Blocks ◽

Selection Of

Introdução: O estado de Rondônia se destaca como tradicional produtor de café, sendo o segundo maior produtor brasileiro de C. canephora. No melhoramento genético de C. canephora, a seleção de plantas de elevada peneira média está associada à bebida de qualidade superior. Objetivos: O objetivo desse estudo foi avaliar a variabilidade genética de clones de C. canephora para o tamanho dos grãos, mensurado a partir da avaliação da peneira média (PM). Materiais e Métodos: Para isso, foi conduzido ao longo de dois anos agrícolas experimento no campo experimental da Embrapa no município de Ouro Preto do Oeste-RO, para a avaliação da peneira média de 130 genótipos (clones) com características das variedades botânicas Conilon, Robusta e híbridos intervarietais. O delineamento experimental utilizado foi de blocos ao acaso, com quatro repetições de quatro plantas por parcela. Resultados: Não houve resultados significativos para a interação clones X anos, indicando uma maior consistência no comportamento das plantas ao longo do tempo. Porém foram observadas diferenças significativas para o tamanho dos grãos entre os genótipos avaliados, possibilitando selecionar genótipos superiores. Conclusão: Os genótipos agruparam-se em cinco classes de acordo com o teste de média, subsidiando a caracterização de um gradiente de variabilidade da característica avaliada ABSTRACTIntroduction: Coffea canephora accounts for approximately 35% of the world's coffee production. The state of Rondônia stands out as a traditional coffee producer, being the second largest Brazilian producer of C. canephora. In the classical genetic improvement of C. anephora, the selection of plants of high average sieve is associated with a drink of superior quality. Objectives: The objective of this udy was to evaluate the genetic variability of Coffea canephora clones for the agronomic medium sieve (PM). Materials and Methods: The experiment was conducted in the experimental field of Embrapa, municipality of OuroPreto do Oeste-RO, located at coordinates 10º44'53 "S and 62º12'57". One hundred thirty genotypes (clones) of botanical characteristics Conilon, Robusta and intervarietal hybrids were evaluated in the agricultural years 2013-2014 and 2014-2015. The experimental design was a randomized block design with four blocks and four plants per plot, spacing 3.5 x 1.5 meters between plants. Results: Significant difference was found for the grain size. According to the F test, at 5% probability, the genotypes were grouped into five classes according to the mean test. Conclusion: The results obtained subsidized the characterization of a variability gradient of the evaluated trait.

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Optimization of the Expression of 1,3-1,4-β-glucanase Gene from Rhizomucor miehei in the Komagataella kurtzmanii yeast

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-5-3-11 ◽

2019 ◽

Vol 35 (5) ◽

pp. 3-11 ◽

Cited By ~ 1

Author(s):

I.I. Gubaidullin ◽

A.S. Fedorov ◽

D.G. Kozlov

Keyword(s):

Target Gene ◽

Rhizomucor Miehei ◽

Leader Peptide ◽

Promoter Regions ◽

Functional Elements ◽

Resource Center ◽

The Status ◽

Pichia Pastoris Yeast ◽

The Difference ◽

Genetic Constructs

Key functional elements of the vector (promoter, leader and terminator regions) that provide the expression of a target l,3-l,4-(3-glucanase gene from Rhizomucor miehei in the Komagataella kurtzmanii yeast have been optimized. It was shown that the promoter regions of the gene AOX1 from the Pichia pastoris yeast currently reclassified as Komagataella phaffti and from К. kurtzmanii yeast as parts of a vector provided equal levels of expression of the target gene in the cells of the recipient strain К. kurtzmanii Y727his4, i.e. they were completely interchangeable. This means that genetic constructs that were previously developed for the biosynthesis of recombinant proteins in К. phajfii are able to provide an effective expression in the К kurtzmanii yeast. The leader peptide MF4I (used as a variant of mif4I containing one amino acid substitution) and the leader peptide maxHH (containing the double proregion of the Hspl50 protein from Saccharomyces cerevisiae) confirmed the status of the most powerful elements among the five leader sequences analyzed. Their efficiency was 1.7 times higher than that of the standard leader from the yeast alpha-factor, and by 20% higher than the characteristics of the second group of artificial leaders. At the same time, it was found that, the choice of the terminator region had the strongest influence on the expression of the target gene among all of the vector functional elements. The best terminator elements were variants derived from the transcription termination region of the AOX1 gene, and the difference in the expression level of the target gene using different terminators was approximately 4.5 times. Based on the analysis of the obtained data, the optimal composition of the key functional elements of the expression vector was determined ; it included the promoter and terminator regions of the AOX1 yeast gene and one of the artificial leaders, mif4I or maxHH. β-glucanase, Komagataella kurtzmanii, yeast, secretion, strain producer The work was financially supported by the Ministry of Science and Higher education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) using the Unique Scientific Facility of the National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

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Pathogenicity and race characterization of Fusarium oxysporum f. sp. phaseoli isolates from Spain and Greece

Plant Pathology ◽

10.1046/j.1365-3059.2002.00745.x ◽

2002 ◽

Vol 51 (5) ◽

pp. 605-611 ◽

Cited By ~ 38

Author(s):

F. M. Alves-Santos ◽

L. Cordeiro-Rodrigues ◽

J. M. Sayagués ◽

R. Martín-Domínguez ◽

P. García-Benavides ◽

...

Keyword(s):

Fusarium Oxysporum

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The Banana Root Endophytome: Differences between Mother Plants and Suckers and Evaluation of Selected Bacteria to Control Fusarium oxysporum f.sp. cubense

Journal of Fungi ◽

10.3390/jof7030194 ◽

2021 ◽

Vol 7 (3) ◽

pp. 194

Author(s):

Carmen Gómez-Lama Cabanás ◽

Antonio J. Fernández-González ◽

Martina Cardoni ◽

Antonio Valverde-Corredor ◽

Javier López-Cepero ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Canary Islands ◽

High Throughput Sequencing ◽

Fungal Communities ◽

Phenological Stage ◽

Mother Plants ◽

Fusarium Wilt Of Banana ◽

Identification And Characterization

This study aimed to disentangle the structure, composition, and co-occurrence relationships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root endosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phenological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7).

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

International Journal of Antimicrobial Agents ◽

10.1016/s0924-8579(01)00366-1 ◽

2001 ◽

Vol 18 (2) ◽

pp. 107-112 ◽

Cited By ~ 7

Author(s):

Joaquim Ruiz ◽

Josep M. Sierra ◽

M.Teresa Jiménez De Anta ◽

Jordi Vila

Keyword(s):

Staphylococcus Aureus ◽

Resistant Mutants

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Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages

Viruses ◽

10.3390/v13030459 ◽

2021 ◽

Vol 13 (3) ◽

pp. 459

Author(s):

Casey Stamereilers ◽

Simon Wong ◽

Philippos K. Tsourkas

Keyword(s):

Comparative Studies ◽

Point Mutations ◽

Paenibacillus Larvae ◽

Bacterial Disease ◽

American Foulbrood ◽

Antibiotic Resistant ◽

Future Studies ◽

Complete Genomes ◽

Point Of Reference

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage–host systems.

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