Resistance assessment of pyraoxystrobin in Magnaporthe oryzae and the detection of a point mutation in cyt b that confers resistance

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.

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Detection of a Point Mutation (G143A) in Cyt b of Corynespora cassiicola That Confers Pyraclostrobin Resistance

Horticulturae ◽

10.3390/horticulturae7060155 ◽

2021 ◽

Vol 7 (6) ◽

pp. 155

Author(s):

Xiuhuan Li ◽

Chengcheng Li ◽

Guixiang Li ◽

Jiamei Zhu ◽

Feng Liu ◽

...

Keyword(s):

Point Mutation ◽

Phytopathogenic Fungi ◽

Shandong Province ◽

Lamp Method ◽

Corynespora Cassiicola ◽

Cyt B ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Target Spot ◽

Qoi Resistance

Point mutation G143A in the cytochrome b (Cyt b) protein commonly confers resistance to quinone outside inhibitor (QoI) fungicides in phytopathogenic fungi, including Corynespora cassiicola, which causes cucumber target spot disease. However, the effect of G143A on the binding between the QoI fungicide and the Cyt b protein, and the use of LAMP (loop-mediated isothermal amplification) to detect this point mutation had not been reported previously in C. cassiicola. In this study, the sensitivity of 131 C. cassiicola isolates—collected from Shandong province, China in 2019 and 2020—to pyraclostrobin was determined. The EC50 values ranged from 1.67 to 8.82 μg/mL, and sequencing results showed that all C. cassiicola isolates contained the G143A mutation. Molecular docking results suggested that G143A significantly alters the affinity of pyraclostrobin to the Cyt b protein. Following development of three LAMP primer pairs, the best reaction condition for LAMP analysis was 65 °C for 60 min, and the detection limit was 0.01 ng/μL of DNA containing the point mutation. In conclusion, the G143A mutation conferring pyraclostrobin resistance is widespread in C. cassiicola from Shandong province, and the LAMP method can be used to monitor QoI resistance in C. cassiicola caused by the G143A mutation in the field.

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Resistance to QoI Fungicides Is Widespread in Brazilian Populations of the Wheat Blast Pathogen Magnaporthe oryzae

Phytopathology ◽

10.1094/phyto-06-14-0184-r ◽

2015 ◽

Vol 105 (3) ◽

pp. 284-294 ◽

Cited By ~ 57

Author(s):

Vanina L. Castroagudín ◽

Paulo C. Ceresini ◽

Samanta C. de Oliveira ◽

Juliana T. A. Reges ◽

João L. N. Maciel ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Cyt B ◽

Southern Brazil ◽

Qoi Fungicides ◽

Wheat Blast ◽

Wheat Diseases ◽

Gene Sequence Analysis ◽

Cyt B Gene ◽

Important Disease ◽

Qoi Resistance

Wheat blast, caused by Magnaporthe oryzae, is an important disease across central and southern Brazil. Control has relied mainly on strobilurin fungicides (quinone-outside inhibitors [QoIs]). Here, we report the widespread distribution of QoI resistance in M. oryzae populations sampled from wheat fields and poaceous hosts across central and southern Brazil and the evolution of the cytochrome b (cyt b) gene. Sequence analysis of the cyt b gene distinguished nine haplotypes, with four haplotypes carrying the G143A mutation associated with QoI resistance and two haplotypes shared between isolates sampled from wheat and other poaceous hosts. The frequency of the G143A mutation in the wheat-infecting population increased from 36% in 2005 to 90% in 2012. The G143A mutation was found in many different nuclear genetic backgrounds of M. oryzae. Our findings indicate an urgent need to reexamine the use of strobilurins to manage fungal wheat diseases in Brazil.

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Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance

Frontiers in Microbiology ◽

10.3389/fmicb.2016.00615 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 34

Author(s):

Jianqiang Miao ◽

Meng Cai ◽

Xue Dong ◽

Li Liu ◽

Dong Lin ◽

...

Keyword(s):

Point Mutation ◽

Phytophthora Capsici ◽

Resistance Assessment

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Sensitivity of Botrytis cinerea From Nectarine/Cherry in China to Six Fungicides and Characterization of Resistant Isolates

Plant Disease ◽

10.1094/pdis-02-18-0244-re ◽

2018 ◽

Vol 102 (12) ◽

pp. 2578-2585 ◽

Cited By ~ 7

Author(s):

W. X. Yin ◽

M. Adnan ◽

Y. Shang ◽

Y. Lin ◽

C. X. Luo

Keyword(s):

Botrytis Cinerea ◽

Point Mutation ◽

Gray Mold ◽

Stone Fruits ◽

Cyt B ◽

Control Efficacy ◽

Sdhb Gene ◽

Cyt B Gene ◽

Fitness Analysis

Botrytis cinerea, the causal agent of gray mold, can result in considerable preharvest and postharvest losses in many economically valuable plant species. Fungicides were widely used to minimize such losses, but fungicide resistances were detected frequently. In the present study, we collected 164 isolates from nectarine and cherry in China and tested the sensitivity to six fungicides. Among the tested isolates, 71 (43.3%) were resistant to azoxystrobin, 14 (8.5%) to cyprodinil, 7 (4.3%) to boscalid, 4 (2.4%) to carbendazim, 1 (0.6%) to iprodione, and no isolates were found to be resistant to fludioxonil. The EC50 value and resistance factor (RF) of resistant isolates were determined. Fitness analysis showed that there were no significant differences between sensitive and resistant isolates for osmotic stress and pathogenicity, while more conidia production was observed for some resistant isolates. Control efficacy of fungicides showed that the resistant isolates could not be controlled efficiently by using corresponding fungicides. The point mutation G143A was detected in the Cyt b gene of the isolates resistant to azoxystrobin, while the point mutation H272R of SdhB gene was confirmed in boscalid-resistant isolates, and mutations E198V/A of TUB2 gene and mutation I365S of BcOs1 occurred in carbendazim-resistant and iprodione-resistant isolates, respectively. These results indicate that the occurrence of fungicide resistance greatly threatens the management of gray mold on stone fruits nectarine and cherry.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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High throughput phenotype screening pipeline for functional genomics in Magnaporthe oryzae

Protocol Exchange ◽

10.1038/nprot.2007.168 ◽

2007 ◽

Cited By ~ 1

Author(s):

Sook-Young Park ◽

Myoung-Hwan Chi ◽

Junhyun Jeon ◽

Yong-Hwan Lee

Keyword(s):

Functional Genomics ◽

High Throughput ◽

Magnaporthe Oryzae ◽

Phenotype Screening

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Paris I Dysfibrinogenemia: A Point Mutation in Intron 8 Results in Insertion of a 15 Amino Acid Sequence in the Fibrinogen γ-Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651583 ◽

1993 ◽

Vol 69 (03) ◽

pp. 217-220 ◽

Cited By ~ 12

Author(s):

Jonathan B Rosenberg ◽

Peter J Newman ◽

Michael W Mosesson ◽

Marie-Claude Guillin ◽

David L Amrani

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Point Mutation ◽

Genomic Dna ◽

Comparative Sequence Analysis ◽

Chain Gene ◽

Molecular Defect ◽

Nucleotide Position ◽

Normal Individuals ◽

Polymerase Chain

SummaryParis I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal γ-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the γ-chain region of the Paris I subject’s genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A→G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I γ-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature γparis I chain mRNA, and encodes a 15 amino acid insert after γ350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I γ-chain mRNA also results after translation into a substitution of S for G at position γ351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the γpariS I chain. We conclude that the insertion of this amino acid sequence leads to a conformationallyaltered, and dysfunctional γ-chain in Paris I fibrinogen.

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