Nested PCR Assays for Detection of Monilinia fructicola in Stone Fruit Orchards and Botryosphaeria dothidea from Pistachios in California

Nested polymerase chain reaction (PCR) assays were developed based on microsatellite regions for detection of Monilinia fructicola, the causal agent of brown rot of stone fruits, and Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio. The nested PCR primers specific to M. fructicola were developed based upon the sequence of a species‐specific DNA fragment amplified by microsatellite primer M13. The external and internal primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571‐ and a 468‐bp fragment, respectively, from M. fructicola, but not from any other fungal species present in stone fruit orchards. The nested PCR primer pairs specific to B. dothidea were developed based upon the sequence of a species‐specific 1330‐bp DNA fragment amplified by microsatellite primer T3B. The external and internal primer pairs EBdF + EBdR and IBdF + IBdR amplified a 701‐ and a 627‐bp fragment, respectively, from B. dothidea, but not from any other fungal species associated with pistachio. The nested PCR assays were sensitive enough to detect the specific fragments in 1 fg of M. fructicola or B. dothidea DNA or in the DNA from only two conidia of M. fructicola or B. dothidea. The nested PCR assays could detect small numbers of M. fructicola conidia caught on spore‐trap tapes and detect visible infections of B. dothidea in pistachio tissues. Microsatellite regions with high numbers of copies are widely dispersed in eukaryotic genomes. The results of this study indicate that microsatellite regions could be useful in developing highly sensitive PCR detection systems for phytopathogenic fungi.     

Mutations of the β‐tubulin gene fragments from carbendazim‐resistant isolates of Pestalotiopsis sp. causing strawberry leaf blight in Chiang Mai,Thailand

Screening of field isolates of Pestalotiopsis sp. from strawberry leaf blight in Thailand identified 56 carbendazim‐resistant isolates. Of 56 isolates, 39 highly resistant (HR) phenotypes grew well on PDA amended with carbendazim even at ≥500 mg/L. Isolates with carbendazim‐resistant phenotype had a conspicuous mutation at particular sites in the beta‐tubulin (β‐tubulin) gene sequence. A β‐tubulin encoding gene from this pathogen was cloned and sequenced. Analysis of the β‐tubulin gene in highly resistant (HR) isolates showed a substitution at codon 79 and 102 from serine (AGT) to lysine (AAA) and valine (GTA) to alanine (GCA), respectively. The detection of such point mutations in the β‐tubulin gene allows the rapid screening to detect carbendazim‐resistant isolates in the field.     

Development and application of a simple,rapid and sensitive method for detecting moderately carbendazim‐resistant isolates in Botrytis cinerea

Sustainable disease management depends on the ability to monitor the development of fungicide resistance in pathogen populations. A point mutation resulting in an alteration (F200Y) at codon 200 of the target protein β‐tubulin leads to a moderate level of resistance to carbendazim in Botrytis cinerea. Although traditional methods remain a cornerstone in detection of fungicide resistance, molecular methods that do not require the isolation of pathogens, can detect the presence of resistance alleles at low frequencies, and require less time and labour than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting the moderately carbendazim‐resistant isolates in B. cinerea based on loop‐mediated isothermal amplification (LAMP). By using specific LAMP primers, we detected the resistance‐conferring mutation underlying β‐tubulin F200Y. The concentrations of LAMP components and LAMP parameters were optimised, resulting in reaction temperatures and times of 61–65°C and 45 min, respectively. The feasibility of the LAMP assay was verified by assaying the diseased samples with artificial inoculation in the different hosts. The LAMP assay developed in the current study was specific, stable, repeatable and sensitive, and was successfully applied for detection of moderately carbendazim‐resistant isolates of B. cinerea in plant samples.     

Detection of Aflatoxin in Aspergillus Species Isolated from Pistachio in Iran

To estimate the incidence contamination of fresh pistachio nuts by aflatoxigenic fungi in Iran, nut samples were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan regions. Out of the 200 Aspergillus isolates obtained, 11 species were identified as A. alliaceous, A. candidus, A. flavus, A. niger, A. niveus, A. ochraceus, A. parasiticus, A. tamari, A. terreus, A. unguis and A. wentii. For detection of aflatoxin production ability of the isolates, three target genes, namely aflR, aflJ, and omtB, used in PCR amplification. In all the examined cases, the degenerate primer designed for amplification of omtB gene, named omtBII, was able to amplify an expected 611 bp fragment in aflatoxigenic isolates in this study and yielded the same result as those obtained from TLC analysis and fluorescence ability by application of methylated β‐cyclodextrin in culture media. Using this procedure the significant incidence of aflatoxin‐producing aspergilli was confirmed in pistachio nuts produced in different regions of Iran. The results indicated that PCR method described here, in combination with fluorescence assay, is a reliable and simple confirmatory test for monitoring pistachio nuts contaminated with aflatoxinogenic aspergilli.     

Detection,Identification and Phylogenetic Analysis of Indian Ralstonia solanacearum Isolates by Comparison of Partial hrpB Gene Sequences

A species‐specific Polymerase Chain Reaction (sPCR) method was developed to identify and detect isolates of Ralstonia solanacearum, the cause of bacterial wilt disease in chilli. PCR primers for R. solanacearum were identified by alignment of hrpB gene sequences and selection of sequences specific for R. solanacearum at their 3′ ends. The primers were shown to be specific for R. solanacearum, as no PCR product was obtained when genomic DNA from other bacterial species including closely related Ralstonia species, were used as test species. Lone pair of primers (RshrpBF and RshrpBR) was designed using hrpB gene sequence, unique to R. solanacearum which amplified a predicted PCR product of 810 bp from 20 different isolates. Phylogenetic analysis was also attempted to understand the evolutionary divergence of Indian R. solanacearum isolates. Based on phylogenetic analysis, Indian isolates showed homology with the standard reference isolates from other countries but, interestingly, one new isolate showed complete evolutionary divergence by forming an out‐group.     

Antibiotic resistance in faecal bacteria (Escherichia coli,Enterococcus spp.) in feral pigeons

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli and Enterococcus spp. in feral pigeons (Columba livia forma domestica) in the Czech Republic. Methods and Results: Cloacal swabs of feral pigeons collected in the city of Brno in 2006 were cultivated for antibiotic‐resistant E. coli. Resistance genes, class 1 and 2 integrons, and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). The samples were also cultivated for enterococci. Species status of enterococci isolates was determined using repetitive extragenic palindromic‐PCR. Resistance genes were detected in resistant enterococci by PCR. E. coli isolates were found in 203 of 247 pigeon samples. Antibiotic resistance was recorded in three (1·5%, n_E. coli = 203) isolates. Using agar containing ciprofloxacin, 12 (5%, n_samples = 247) E. coli strains resistant to ciprofloxacin were isolated. No ESBL‐producing E. coli isolates were detected. A total of 143 enterococci were isolated: Ent. faecalis (36 isolates), Ent. faecium (27), Ent. durans (19), Ent. hirae (17), Ent. mundtii (17), Ent. gallinarum (12), Ent. casseliflavus (12) and Ent. columbae (3). Resistance to one to four antibiotics was detected in 45 (31%) isolates. Resistances were determined by tetK, tetL, tetM, tetO, aac(6′)aph(2′′), ant(4′)‐Ia, aph(3′)‐IIIa, ermB, pbp5, vanA and vanC1 genes. Conclusions: Antibiotic‐resistant E. coli and Enterococcus spp. occurred in feral pigeons in various prevalences. Significance and Impact of the Study: Feral pigeon should be considered a risk species for spreading in the environment antimicrobial resistant E. coli and enterococci.     

At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc₁ complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

Effect of azoxystrobin and kresoxim‐methyl on rice blast and rice grain yield in China

Sensitivity to azoxystrobin and kresoxim‐methyl of 80 single‐spore isolates of Magnaporthe oryzae was determined. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting mycelial growth of the 80 M. oryzae isolates were 0.006–0.056 and 0.024–0.287 µg mL^?1, respectively. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting conidial germination of the M. oryzae populations were 0.004–0.051 and 0.012–0.105 µg mL^?1, respectively. There was significant difference in sensitivity to azoxystrobin or kresoxim‐methyl between the tested isolates representing differential sensitivity to carbendazim (MBC) and kitazin P (IBP); however, there was no correlation between this difference in sensitivity to azoxystrobin or kresoxim‐methyl and sensitivity to MBC or IBP, indicating that there was no cross‐resistance between azoxystrobin or kresoxim‐methyl and MBC or IBP. In the protective and curative experiments, kresoxim‐methyl exhibited higher protective and curative activity than azoxystrobin when applied at 150 and 250 µg mL^?1 accordingly, while azoxystrobin exhibited stronger inhibitory activity against M. oryzae isolates than that of kresoxim‐methyl in the in vitro test. The results of field experiments also suggested that both azoxystrobin and kresoxim‐methyl at 187.5 g.a.i. ha^?1 gave over 73% control efficacy in both sites, exhibiting excellent activity against rice blast. Taken together, azoxystrobin and kresoxim‐methyl could be a good substitute for MBC or IBP for controlling rice blast in China, but should be carefully used as they were both at‐risk.     

Species‐specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Inteins (internal proteins) are self‐splicing transportable genetic elements present in conserved regions of housekeeping genes. The study highlights the importance of intein as a potential diagnostic marker for species‐specific identification of Candida tropicalis, a rapidly emerging opportunistic human pathogen. Initial steps of primer validation, sequence alignment, phylogenetic tree analysis, gel electrophoresis and real‐time polymerase chain reaction (PCR) assays were performed to confirm the specificity of the designed primers. The primers were selective for C. tropicalis with 100% inclusivity and showed no cross‐species or cross‐genera matches. The established technique is a prototype for developing multifaceted PCR assays and for point‐of‐care testing in near future.

Significance and Impact of the Study

Development of molecular markers for specific detection of microbial pathogens using real‐time polymerase chain reaction (PCR) is an appealing and challenging technique. A real‐time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species‐specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost‐effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species‐specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point‐of‐care testing in near future.     

Activity of fungicide mixtures against Botrytis cinerea isolates resistant to benzimidazoles,strobilurins and dicarboximides

Botrytis cinerea, the causal agent of grey mould in a broad range of crops, is considered a high‐risk plant pathogen for fungicide resistance development. The use of fungicide mixtures, particularly combinations with synergistic activity, can be a useful tactic to counteract resistance build‐up in pathogen populations. The present study aimed to investigate the effects of different ratios of two‐way mixtures of carbendazim, iprodione, kresoxim‐methyl, tebuconazole and penconazole on four B. cinerea isolates that were sensitive or resistant to benzimidazoles, dicarboximides and strobilurins. The isolates that were resistant to benzimidazoles and strobilurins had E198A and G143A mutations in β‐tubulin and cytochrome b genes, respectively. The mixtures had different effects on each of the isolates in vitro but, in 13 combinations, the synergistic effect was observed against all or three isolates. In greenhouse experiments, 11 fungicide combinations used in decreased (EC₇₅) concentrations showed the maximum control efficiency. The two follow‐up greenhouse experiments using six selected combinations revealed they were highly effective against additional isolates with various fungicide resistance profiles. The identified mixtures‐ratios have potential for use in grey mould management programs in the greenhouse.     

A premature stop codon in the TYRP1 gene is associated with brown coat colour in the European rabbit (Oryctolagus cuniculus)

Classical genetic studies in European rabbits (Oryctolagus cuniculus) suggested the presence of two alleles at the brown coat colour locus: a wild‐type B allele that gives dense black pigment throughout the coat and a recessive b allele that in the homozygous condition (b/b genotype) produces brown rabbits that are unable to develop black pigmentation. In several other species, this locus is determined by mutations in the tyrosinase‐related protein 1 (TYRP1) gene, encoding a melanocyte enzyme needed for the production of dark eumelanin. In this study, we investigated the rabbit TYRP1 gene as a strong candidate for the rabbit brown coat colour locus. A total of 3846 bp of the TYRP1 gene were sequenced in eight rabbits of different breeds and identified 23 single nucleotide polymorphisms (SNPs; 12 in intronic regions, five in exons and six in the 3′‐untranslated region) and an insertion/deletion of 13 bp, in the 3′‐untranslated region, organised in a few haplotypes. A mutation in exon 2 (g.41360196G>A) leads to a premature stop codon at position 190 of the deduced amino acid sequence (p.Trp190ter). Therefore, translation predicts a truncated TYRP1 protein lacking almost completely the tyrosinase domain. Genotyping 203 rabbits of 32 different breeds identified this mutation only in brown Havana rabbits. Its potential functional relevance in disrupting the TYRP1 protein and its presence only in brown animals strongly argue for this non‐sense mutation being a causative mutation for the recessive b allele at the brown locus in Oryctolagus cuniculus.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

CAPS and DHPLC Analysis of a Single Nucleotide Polymorphism in the Cytochrome b Gene Conferring Resistance to Strobilurins in Field Isolates of Blumeria graminis f. sp. hordei

A single nucleotide polymorphism in the wheat powdery mildew (Blumeria graminis f. sp. tritici) cytochrome b gene is responsible for resistance to inhibitors of the quinol outer binding site of the cytochrome bc₁ complex (QoI) fungicides. Analysis of a partial sequence of the cytochrome b gene from field isolates resistant and sensitive to QoI fungicides revealed the same point mutation in barley powdery mildew (B. graminis f. sp. hordei). Analysis of 118 and 40 barley powdery mildew isolates using a cleaved amplified polymorphic sequence assay and denaturing high performance liquid chromatography, respectively, confirmed that this single nucleotide polymorphism also confers resistance to QoI fungicides in barley powdery mildew.     

Dissemination of ceftazidime-resistant Acinetobacter baumannii clonal complex 92 in Korea

Aims: This study was performed to describe the epidemiological traits of ceftazidime‐resistant Acinetobacter baumannii clinical isolates from Korea. Methods and Results: Antimicrobial susceptibilities were determined by disk diffusion assay. PCR experiments were performed to detect genes encoding extended‐spectrum β‐lactamases and metallo‐β‐lactamases. Detection of ISAba1 upstream of the bla_ADC gene was also performed by PCR amplification. The genetic organization of the bla_PER‐1 gene was investigated by PCR mapping and sequencing of the regions surrounding the gene. Multilocus sequence typing was performed using seven housekeeping genes. A. baumannii isolates of clonal complex (CC) 92 exhibited a higher resistance rate (286/289, 99%) against ceftazidime compared to A. baumannii isolates of non‐CC92 (7/87, 8%). Amongst 286 ceftazidime‐resistant isolates of CC92, 100 (35%) isolates carried the bla_PER‐1 gene, while none of the 87 isolates of non‐CC92 carried the gene. The bla_ADC gene associated with an ISAba1 element was detected in 98% (281/286) of ceftazidime‐resistant isolates of CC92 and in all seven ceftazidime‐resistant isolates of non‐CC92. The bla_PER‐1 gene was located on a transposon, Tn1213 (ISPa12‐bla_PER‐1‐Δgst‐ISPa13), in 95 isolates and on a complex class 1 integron (orf513‐bla_PER‐1‐putative ABC transporter gene) in five isolates. Southern blot experiments confirmed the chromosomal location of the bla_PER‐1 gene. Conclusions: Acinetobacter baumannii CC92 which has acquired ceftazidime resistance by the production of PER‐1 extended‐spectrum β‐lactamases and/or the overproduction of Acinetobacter‐derived cephalosporinase is widely disseminated in Korea. Significance and Impact of the Study: This study shows the mechanisms of acquiring ceftazidime resistance in A. baumannii and the epidemiological traits of ceftazidime‐resistant A. baumannii isolates from Korea.     

Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop‐mediated isothermal amplification assay

Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug‐resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing‐specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time‐consuming and technically demanding. In the present study, a loop‐mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain‐identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c‐targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207‐PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c‐multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.     

Characterization and pathogenicity of Fusarium proliferatum causing stem rot of Hylocereus polyrhizus in Malaysia

During a series of sampling in 2008 and 2009, stem rot disease was detected in Hylocereus polyrhizus plantations in Malaysia, with symptom appeared as circular, brown sunken lesion with orange sporodochia and white mycelium formation on the lesion surface. Eighty‐three isolates of Fusarium were isolated from 20 plantations and were morphologically identified as F. proliferatum based on the variability of colony appearance, pigmentation, growth rate, length of chains, production of bluish sclerotia, concentric ring aerial mycelium and sporodochia. Three species‐specific primers, namely ITS1/proITS‐R, PRO1/2 and Fp3‐F/4‐R successfully produced PCR products and confirmed that the isolates from stem rot of H. polyrhizus were F. proliferatum isolates. From BLAST search of translation elongation factor 1‐alpha (TEF1‐α) sequences, the isolates showed 99–100% similarity with F. proliferatum deposited in GenBank which further confirmed that the isolates were F. proliferatum. The results from amplification of MAT‐allele specific primers indicated that 14.5% of F. proliferatum isolates carried MAT‐1 allele and 85.5% carried MAT‐2. Crossing results showed that all 83 F. proliferatum isolates were male fertile showing positive crosses with the tester strains of MATD‐1 and MATD‐2. Perithecia oozing ascospore were produced. Forty isolates as representative were evaluated for pathogenicity test, produced rot symptoms similar to those observed in the fields which confirmed the isolates as the causal agent of stem rot of H. polyrhizus. To our knowledge, this is the first report of stem rot of H. polyrhizus caused by F. proliferatum in Malaysia.     

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

Mimicking natural polymorphism in eIF4E by CRISPR‐Cas9 base editing is associated with resistance to potyviruses

In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus‐resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss‐of‐function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR‐nCas9‐cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single‐point mutation through C‐to‐G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome‐editing technologies offers a feasible solution for engineering transgene‐free genetic resistance in plants, even across species barriers.