Screening For Resistance Research Articles

Evaluation of Wheat Cultivars and Germplasm Lines for Resistance to Pratylenchus neglectus Populations Collected in North Dakota.

Root-lesion nematode (RLN; Pratylenchus neglectus) is a migratory endoparasite and a major soilborne pathogen that affects wheat (Triticum spp.) production worldwide. Genetic resistance is one of the most economical and effective ways to manage P. neglectus in wheat. This study evaluated 37 local cultivars and germplasm lines in seven greenhouse experiments, including 26 hexaploid wheat, six durum wheat, two synthetic hexaploid wheat, one emmer wheat, and two triticale for P. neglectus resistance from 2016 to 2020. North Dakota field soils infested with two RLN populations (350 to 1,125 nematodes per kilogram of soil) were used for resistance screening under controlled greenhouse conditions. The final nematode population density for each cultivar and line was counted under the microscope to categorize the resistance ranking of these entries as resistant, moderately resistant, moderately susceptible, and susceptible. Out of the 37 cultivars and lines, one was classified as resistant (Brennan); 18 were moderately resistant (Divide, Carpio, Prosper, Advance, Alkabo, SY Soren, Barlow, Bolles, Select, Faller, Briggs, WB Mayville, SY Ingmar, W7984, PI 626573, Ben, Grandin, and Villax St. Jose); 11 were moderately susceptible; and seven were susceptible to P. neglectus. The resistant to moderately resistant lines identified in this study could be used in breeding programs after the resistance genes or loci are further elucidated. This research provides valuable information about P. neglectus resistance among wheat and triticale cultivars used in the Upper Midwest region of the United States.

Genome-wide CRISPR/Cas9 screening for drug resistance in tumors

Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) screening is a simple screening method for locating loci under specific conditions, and it has been utilized in tumor drug resistance research for finding potential drug resistance-associated genes. This screening strategy has significant implications for further treatment of malignancies with acquired drug resistance. In recent years, studies involving genome-wide CRISPR/Cas9 screening have gradually increased. Here we review the recent application of genome-wide CRISPR/Cas9 screening for drug resistance, involving mitogen-activated protein kinase (MAPK) pathway inhibitors, poly (ADP-ribose) polymerase inhibitors (PARPi), alkylating agents, mitotic inhibitors, antimetabolites, immune checkpoint inhibitors (ICIs), and cyclin-dependent kinase inhibitors (CDKI). We summarize drug resistance pathways such as the KEAP1/Nrf2 pathway MAPK pathway, and NF-κB pathway. Also, we analyze the limitations and conditions for the application of genome-wide CRISPR/Cas9 screening techniques.

Polymorphism of Drug Resistance Genes dhfr and dhps in Plasmodium falciparum Isolates among Chinese Migrant Workers Who Returned from Ghana in 2013.

In 2013, an epidemic of falciparum malaria involving over 820 persons unexpectedly broke out in Shanglin County, Guangxi Zhuang Autonomous Region, China, after a large number of migrant workers returned from Ghana, where they worked as gold miners. Herein, we selected 146 isolates randomly collected from these patients to investigate the resistance characteristics of the parasite to sulfadoxine-pyrimethamine (SP) by screening mutations in the dhfr and dhps genes. All 146 isolates were successfully genotyped for dhps, and only 137 samples were successfully genotyped for dhfr. In the dhfr gene, point mutations occurred at three codons: 51 (83.2%, 114/137), 59 (94.9%, 130/137), and 108 (96.4%, 132/137). In the dhps gene, mutations occurred at four codons: 436 (36.3%, 53/146 for S436A, 0.7%, 1/146 for S436Y), 437 (95.2%, 139/146), 540 (3.4%, 5/146), and 613 (2.7%, 4/146). All 146 isolates had mutations in at least one codon, either within dhfr or dhps. Quadruple mutation I51R59N108/G437 (41.1%, 60/146) of partial or low resistance level was the most prevalent haplotype combination. Quintuple I51R59N108/G437E540 accounted for 2.1% (3/146). Sextuple I51R59N108/A436G437S613 was also found and accounted for 1.4% (2/146). A chronological assay incorporating two sets of resistance data from the studies of Duah and Amenga-Etego provided an overview of the resistance trend from 2003 to 2018. During this period, the results we obtained generally coincided with the total development tendency of SP resistance. It can be concluded that Plasmodium falciparum samples collected from Chinese migrant workers from Ghana presented prevalent but relatively partial or low resistance to SP. A chronological assay incorporating two sets of data around 2013 indicates that our results possibly reflect the SP resistance level of Ghana in 2013 and that the possibility of increased resistance exists. Therefore, reasonable drug use and management should be strengthened while also maintaining a continuous screening of resistance to SP. These findings also underscore the need to strengthen the prevention of malaria importation from overseas and focus on preventing its reintroduction and transmission in China.

Identification of novel associations of candidate marker genes with resistance to onion-fusarium basal rot interaction pathosystem

We evaluated the selected populations of eighteen open-pollinated short-day onion genotypes for FBR-Fusarium basal rot (Fusarium oxysporum f. sp. cepae-FOC) susceptibility; population genetic variation via ISSR marker; and transcriptome analysis using qRT-PCR with six novel selected marker genes: R1, R5, RGA29, lectin, LOX, and Osmotin, at tree time post inoculation (wpi). Screening for resistance showed the average severity between 4.7 and 88.9%; of which, the lowest one was in ‘Saba’ and ‘Saba – HS (6.7 and 4.7%, respectively). ISSR analysis recorded 226 amplified bands, of which 160 bands showed polymorphism, of which ISSR1 and ISSR10 primers showed the best performance. We also found that following inoculation with FOC could regulate defense-related marker genes; R1, PR5, Lectin, LOX, Osmotin, and RGA29 in resistant onion “Saba” and ‘Saba’-HS in comparison to susceptible and controls, non-inoculated ones ranging from 1.23 to 6.99 -fold significantly. Surprisingly, marker genes; Lectin, LOX, and Osmotin were also expressed to FOC simultaneous, though basically are resistance to other biotic and abiotic stress: Lectin to Rhizoctonia solani, Aphid, and major sap-sucking pests; LOX to root-knot and cyst nematode, Heterodera glycines; Osmotin to drought stredd, and oxidative burst in plants. This indicates the double, and or multiple roles of our selected marker genes covering two or more functions at a time. The findings introduce newly resistant onion genotypes, and also can be used in management programs to reduce damages caused by FOC disease. Cumulatively, the transcriptome-data provide novel-insights into the response of onions for improving onion-breeding to FOC.

High oral carriage of multidrug resistant Gram-negative bacilli in adolescents: the SOPKARD-Junior study.

The colonization of the oral cavity by potentially pathogenic antimicrobial-resistant bacteria in adolescents and its consequences is very poorly understood. The present study focused on the occurrence of oral colonization by Gram-negative bacilli (GNB) and their multidrug resistance, including the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases, among healthy adolescents and risk factors associated with GNB colonization. This study was conducted as part of "A program for the early detection of risk factors for lifestyle diseases SOPKARD-Junior" (SOPKARD-Junior). Oral samples were collected from 182 adolescents from four public elementary schools in Sopot, Poland, aged 13-14 years. Bacterial strains were identified by the MALDI-TOF MS method. Screening of antimicrobial resistance was performed using a disk diffusion method. The NG-Test® CARBA-5 was used to detect and differentiate the five most widely distributed carbapenemases. Demographic and clinical data were collected and statistical analysis of risk factors was performed. A total of 68 out of 182 (37.4%) healthy adolescents was documented oral colonization with Gram-negative bacilli, including 50/182 (27.5%) multidrug resistant (MDR-GNB) strains. Over 60% of oral carriage concerned three main genera Enterobacter spp., Pseudomonas spp., and Serratia spp., which were detected in 22.1%, 19.1%, and 19.1% of participants, respectively. Citrobacter spp., Escherichia coli, Klebsiella spp., Hafnia spp., Aeromonas spp., Acinetobacter spp., and Stenotrophomonas spp. were also isolated. The antimicrobial resistance to ampicillin (100%), ceftazidime (69.1%), meropenem (60.3%), gentamycin (60.3%), piperacillin/tazobactam (52.9%), and piperacillin (45.6%) were the most common. Among 73.5% GNB strains multidrug resistance was observed, including all Pseudomonas spp. strains. Among MDR-GNB, 30.4% were resistant to four groups of antibiotics, half of the MDR Pseudomonas spp. strains were resistant to 10 groups of antibiotics. Extended-spectrum β-lactamases were produced by Enterobacter cloacae, Klebsiella spp., and Serratia spp. (7.4%). Colonization by ESBLs-positive GNB strains was significantly associated with recurrent respiratory infections, nasal congestion, and bronchitis (p<0.05). Our study revealed high oral carriage of multi-drug resistant Gram-negative bacilli in healthy adolescents and the association of ESBL-producing strains with respiratory infections. Further studies on oral colonization with GNB are necessary due to the possibility of distinct infections and the acquisition of antibiotic resistance by resident microbiota.

Assessing O.K.N.V.I. RESIST‑5 performance for post‑mortem biological samples: A prospective pilot study.

In recent years, the emergence of carbapenem-resistant strains has been increasing worldwide, including in Romania. Rapid tests for post-mortem examinations have been researched and currently have several applications. In the present study, we aimed to test the performance of O.K.N.V.I. RESIST-5 tests on impure post-mortem biological samples compared with a standard of pure cultures. When a death occurs during hospitalization and the issue of malpractice arises, the medico-legal practice would benefit from rapid tests applicable to post-mortem samples. Thus, detection and differentiation of the five targeted carbapenemases, namely oxacilinase-48, Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase, Verona integron-encoded metallo-β-lactamase and imipenemase, could be useful in guiding sampling for third-party microbiological assessment and could also be an asset from an epidemiological standpoint. The present prospective and observational pilot study included medico-legal autopsy cases performed at Mina Minovici National Institute of Legal Medicine (Romania) between June and July 2022. A total of two sets of O.K.N.V.I. RESIST-5 tests were performed: Test I, which was performed on-site from biological samples obtained during autopsy; and Test II, which was performed on pure cultures after sample inoculation and incubation. Total of 39 O.K.N.V.I. RESIST-5 rapid tests were performed on 19 biological samples, at least one sample per case. The O.K.N.V.I. RESIST-5 tests performed on-site showed an overall sensitivity of 92.3% with a 100% specificity. The results obtained through rapid tests using post-mortem impure samples were comparable to the results obtained from sample cultures with good sensitivity and specificity. Through post-mortem screening for carbapenem resistance, it would be possible to narrow down the number of cases that require further bacteriological assessment.

Epidemiologic case investigation on the zoonotic transmission of Methicillin-resistant Staphylococcus pseudintermedius among dogs and their owners

Dogs often carry methicillin-resistant Staphylococci asymptomatically. These bacteria are frequently linked to conditions such as canine pyoderma and otitis. Close interaction between dogs and humans can facilitate the exchange of resistant strains, particularly Methicillin-resistant Staphylococcus pseudintermedius (MRSP). This represents a public health issue, since these strains, in addition to occasionally causing infections in humans, can also serve as a source of resistance and virulence genes for strains of greater importance in human medicine, such as Staphylococcus aureus. Furthermore, MRSP strains are often multidrug resistant, which ends up compromising the treatment of infections. This study aimed to assess the potential transmission of Staphylococcus pseudintermedius among dogs and their owners. We examined a total of one hundred canine samples collected from cases of pyoderma and otitis to detect the presence of staphylococci. Simultaneously, we conducted evaluations on all dog owners. Staphylococci strains were identified using MALDI-TOF MS and PCR targeting the nuc gene. Methicillin resistance screening was also performed by detecting the mecA gene using PCR. Among the sampled dogs, 64 carried S. pseudintermedius. Nine were identified as MRSP. In six instances, dogs and their owners exhibited S. pseudintermedius. These samples underwent genome sequencing and were screened for antimicrobial resistance genes, SCCmec typing, MLST characterization, and Single Nucleotide Polymorphisms (SNP) analyses. The results of the phylogenetic analysis revealed that in three cases, dogs and owners had closely related isolates, suggesting interspecies transmission. Two of these cases involved MRSP and one MSSP. Moreover, in the two MRSP cases, the same SCCmec type (type V) was detected. Additionally, the sequence type was consistent across all three cases involving dogs and owners (MSSP ST2277, MRSP ST2282, and ST2286). These findings strongly indicate a transmission event. Since Staphylococcus pseudintermedius is primarily isolated from canine samples, it is plausible that dogs may have acted as a potential source. In the remaining three cases, despite identifying the same species in both samples, they had notable phylogenetic differences.

Improvement of micro-perforated panel absorbers sound performance using resistive screens and sensitivity analysis of the composite absorber models

The sound attenuation performance of a composite absorber constituted by a micro- perforated panel, a resistive screen that is inserted into the air cavity and a rigid back plate, is studied. Using a nonlinear acoustic impedance model, the surface impedance of the absorber is determined by transfer matrix method, and the models are validated by comparison with experimental measurements performed at different sound pressure levels up to 150 dB. The contribution of the resistive screens in improving the acoustic performances of micro-perforated panel absorbers is presented. It is shown that the resistive screen increases the resistance of the absorber resulting in broadening of the absorption frequency band and improvement of the absorption coefficient when an appropriate perforation ratio of the panel is used. Sensitivity analysis is performed at higher pressure excitations, and the dimensionless input parameters are the perforation ratio of the panel, the ratio of the perforation diameter by the thickness of the panel, the ratio of the cavity depth by the thickness, the orifice Mach number and the resistance per unit area of the screen. The analysis shows the impact of the input parameters on the normalized surface impedance and the sound absorption coefficient of the absorber. It is demonstrated that the resistance per unit area of the screen influences strongly the acoustic properties of the absorber in the linear regime, while the perforation ratio of the panel and the orifice Mach number are the dominant parameters which control the acoustic behavior of the absorber in the nonlinear regime.

Molecular Detection of Key Virulence-associated Genes and Phenotypic Analysis of Virulence Traits of Klebsiella pneumoniae Clinical Isolates from Kenya

Klebsiella pneumoniae is an opportunistic pathogen and a major cause of nosocomial infections. Phenotypic analysis of virulence and molecular typing of virulence-associated genes are powerful approaches to understanding Klebsiella pneumoniae infection biology. This study subjected 102 clinical Klebsiella pneumoniae isolates to virulence gene screening and phenotypic analysis of serum resistance, biofilm formation, and hypermucoviscosity. The virulence genes mrkD, ybtS, wcaJ, entD, and rmpA had a prevalence of 95.1%, 30.4%, 27.5%, 22.5%, and 0.98%, respectively. 54.9%, 36.3%, and 8.8% were serum resistant, intermediate, and susceptible, respectively. There was no significant correlation between the presence of mrkD, ybtS, wcaJ, entD, and rmpA genes and serum non-susceptibility. 53.9%, 22.5%, 6.9%, and 16.7% were strong, moderate, weak, and non-biofilm formers, respectively. The biofilm-forming phenotype was significantly correlated with mrkD (P= 0.000098) and ybtS (P=0.032) gene presence. In addition, 11.8 % of the isolates had the hypermucoviscous phenotype indicating hypervirulence. All of these hypervirulent isolates were positive for the mrkD gene and were significantly associated with the presence of the wcaJ gene (P = 0.000085). These results indicate a positive association between virulence genes with biofilm formation and hypervirulence. In conclusion, Klebsiella pneumoniae isolates circulating in Kenya are predominantly serum non-susceptible and biofilm formers. mrkD, ybtS, and wcaJ genes were identified as key genes influencing biofilm formation and hypervirulence and would be good targets for vaccine development to reduce the severity of Klebsiella pneumoniae infections in Kenya.

Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10.

Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.

Herbicide resistance in Alopecurus myosuroides: The value of routine testing of seed samples submitted by farmers since 1985

AbstractIn 35 years of routine screening for herbicide resistance, a total of 3758 seed sample/herbicide combinations were assayed. Resistance testing was done in either pots in glasshouses (chlortoluron, fenoxaprop and mesosulfuron‐methyl + iodosulfuron‐methyl sodium) or Petri‐dishes in incubators (sethoxydim, cycloxydim and pendimethalin). With all herbicides, the relationship between herbicide efficacy and year of sampling was linear, with the slope representing the annual loss of efficacy. This was higher for the ALS inhibitors mesosulfuron + iodosulfuron (3.73% year−1) and ACCase inhibitors sethoxydim/cycloxydim (1.92% year−1) and fenoxaprop (1.36% year−1) than for the substituted urea chlorotoluron (0.69% year−1) and the dinitroaniline pendimethalin (1.10% year−1). These results are consistent with other studies on the relative resistance risk associated with these different modes of action. There was also a surprisingly good correlation between results for random and non‐random resistance testing, which has also been found in studies with other weed species in Canada and Australia. This indicates that routine testing of submitted samples can replace, at least partly, the need for random surveys which tend to be both labour intensive and expensive. These results, compiled over 35 years, show the value of routine resistance screening, not only for detecting resistance at the individual field level (‘micro’ scale), but also the distribution, evolution and impact of resistance country‐wide (‘macro’ scale). However, it is important that standardised testing methods, including appropriate reference populations, are used by different testing centres to ensure consistent results.

Development of Glycine max Germplasm Highly Resistant to Sclerotinia sclerotiorum

Sclerotinia stem rot (SSR) of soybean caused by Sclerotinia sclerotiorum is a devastating disease of soybean, especially in the Upper Midwest region of the United States. To mitigate yield losses due to this disease, many control methods are available for producers, including cultural control practices, chemical control, and cultivars with quantitative resistance. However, due to there being few commercial cultivars with high levels of resistance, producers are often limited in their seed selection. The aim of this study was to develop novel conventional soybean cultivars with high levels of resistance to SSR, favorable agronomic traits, and resistance to additional economically important diseases. Initial crosses were conducted in 2016 with two different sources of SSR resistance. Across multiple generations of screening for resistance to SSR, three highly resistant soybean lines were identified as the elite lines. These elite lines were demonstrated to be highly resistant across multiple years in both greenhouse and field trials, including high levels of resistance to multiple diverse S. sclerotiorum isolates. The three selected elite lines also resulted in moderately high yields and favorable agronomic traits, such as low lodging and moderate branching, indicating their viability to be released for production. In addition to SSR resistance, these three elite lines demonstrated resistance to other economically important soybean diseases, such as frogeye leaf spot, anthracnose, Cercospora leaf blight, and brown stem rot. Overall, this work has led to three SSR-resistant soybean lines that could be useful for future breeding efforts or commercial soybean production. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genome-wide association mapping in hairy vetch (Vicia villosa) discovers a large effect locus controlling seed dormancy.

Hairy vetch (Vicia villosa Roth), a winter-hardy annual legume, is a promising cover crop. To fully leverage its potential, seed production and field performance of V. villosa must be improved to facilitate producer adoption. Two classic domestication traits, seed dormancy (hard seed) and dehiscence (pod shatter), are selection targets in an ongoing breeding program. This study reports a genome-wide association study of 1,019 V. villosa individuals evaluated at two sites (Knox City, Texas and Corvallis, Oregon) for the proportion of dormant seed, visual pod dehiscence scores, and two dehiscence surrogate measures (force to dehiscence and pod spiraling score). Trait performance varied between sites, but reliability (related to heritability) across sites was strong (dormant seed proportion: 0.68; dehiscence score: 0.61; spiraling score: 0.42; force to dehiscence: 0.41). A major locus controlling seed dormancy was found (q-value: 1.29 × 10-5; chromosome 1: position: 63611165), which can be used by breeding programs to rapidly reduce dormancy in breeding populations. No significant dehiscence score QTL was found, primarily due to the high dehiscence rates in Corvallis, Oregon. Since Oregon is a potentially major V. villosa seed production region, further dehiscence resistance screening is necessary.

Insights into the microbiological and virulence characteristics of bacteria in orthopaedic implant infections: A study from Pakistan.

The exponential increase in the prevalence of multidrug resistant bacteria has resulted in limiting surgical treatment options globally, potentially causing biofilm-related complications, implant failure, and severe consequences. This study aims to isolate and characterize bacteria from post-surgical orthopaedic implant infections and screening for multiple antibiotic resistance. A cross-sectional study was conducted, involving isolation of forty-four dominant pathogenic bacterial isolates from 16 infected implant samples from across Islamabad and Rawalpindi. Out of forty-four, 38% cocci and 61% bacilli were obtained. Approximately 90% of isolates showed multiple antibiotic resistance (MAR) index of more than 0.2. Eleven strains were identified via 16S rRNA gene sequencing as Pseudomonas aeruginosa, Bacillus spp., Planococcus chinensis, Staphylococcus, Escherichia coli and Enterobacter cloacae. The bacterial strain E. coli MB641 showed sensitivity to Polymyxin only, and was resistant to all other antibiotics used. Maximum biofilm forming ability 0.532 ± 0.06, 0.55 ± 0.01 and 0.557 ± 0.07 was observed in Pseudomonas aeruginosa MB663, Pseudomonas aeruginosa MB664 and Bacillus spp. MB647 respectively after 24 hours of incubation. EPS production of bacterial strains was assessed, the polysaccharides and protein content of EPS were found to be in the range of 11-32 μg/ml and 2-10 μg/ml, respectively. Fourier transform infrared spectroscopic analysis of EPS showed the presence of carbohydrates, proteins, alkyl halides, and nucleic acids. X-ray diffraction analysis revealed crystalline structure of EPS extracted from biofilm forming bacteria. These findings suggest a high prevalence of antibiotic-resistant bacteria in orthopaedic implant-associated surgeries, highlighting the urgent need for ongoing monitoring and microorganism testing in infected implants.

Brown rust resistance screening and molecular characterization of wheat cultivars by molecular markers

Brown rust resistance screening and molecular characterization of wheat cultivars by molecular markers

Physical Screening of Resistance on Selected Rice Accessions against Rice Leaf Folder – Cnaphalocrocis medinalis Guenee (Pyralidae: Lepidoptera) under Screen House Condition

The experiment was carried out at screen house III, Department of Entomology, Faculty of Agriculture, Annamalai University, Chidambaram. India. Total 13 numbers of rice accessions were concerned along with one susceptible check (TN-1) to picked up to quantify the most prominent resistant accessions on different selected rice genotypes by showing their resistance characters against the rice leaf folder, Cnaphalocrocis medinalis during Kharif 2022. Among which, based on the morphological traits viz., plant height, length and width of flag leaves, productive tiller numbers, peduncle length, panicle length and trichome density, only one accession (BA-207) was recorded as highly resistant and five (BA-18, BA-31, BA-187, BA-193 and BA-266) of them were reported as resistant accessions. The observations were made at 60 days after transplanting. All the results are given based on the standard evaluation system (SES) build up by International Rice Research Institute. Three of accessions exhibit moderate resistance viz., BA-30, BA-116 and BA-243. Three numbers of accessions were noted as moderately susceptible, which are BA-5, BA-80 and BA-91. At the same time, TN-1 was noticed as highly susceptible. A detailed report given in this research article is based on 13 rice accessions that were tested under screen house conditions. In the farmers point of view, susceptible varieties should replaced with resistant accessions to decrease the pest incidence and enhance the grain productions.

Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3'5'H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3'5'H. In this study, PgF3'5'H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3'5'H showed a higher expression level in all PgF3'5'H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3'5'H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3'5'H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3'5'H products have F3'5'H enzyme activity. In addition, PgF3'5'H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3'5'H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.

Automating high-throughput screening for anthracnose resistance in common bean using allele specific PCR

BackgroundCommon beans (Phaseolus vulgaris L.) provide important protein and calories globally. Anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889) is a major disease in common bean and causes significant yield losses in bean production areas. Screening for markers linked to known disease resistance genes provides useful information for plant breeders to develop improved common bean varieties. The Kompetitive Allele Specific PCR (KASP) assay is an affordable genetic screening technique that can be used to accelerate breeding programs, but manual DNA extraction and KASP assay preparation are time-consuming. Several KASP markers have been developed for genes involved in resistance to bean anthracnose, which can reduce yield by up to 100%, but their usefulness is hindered by the labor required to screen a significant number of bean lines. Our research objective was to develop publicly available protocols for DNA extraction and KASP assaying using a liquid handling robot (LHR) which would facilitate high-throughput genetic screening with less active human time required. Anthracnose resistance markers were used to compare manual and automated results.ResultsThe 12 bean anthracnose differential cultivars were screened for four anthracnose KASP markers linked to the resistance genes Co-1, Co-3 and Co-42 both by hand and with the use of an LHR. A protocol was written for DNA extraction and KASP assay thermocycling to implement the LHR. The LHR protocol reduced the active human screening time of 24 samples from 3h44 to 1h23. KASP calls were consistent across replicates but not always accurate for their known linked resistance genes, suggesting more specific markers still need to be developed. Using an LHR, information from KASP assays can be accumulated with little active human time.ConclusionResults suggest that LHRs can be used to expedite time-consuming and tedious lab work such as DNA extraction or PCR plate filling. Notably, LHRs can be used to prepare KASP assays for large sample sizes, facilitating higher throughput use of genetic marker screening tools.

An updated review of Alternaria blight of pigeonpea (Cajanus cajan (L.) Mill sp.)

Pigeonpea (Cajanus cajan (L.) Mill sp.) is an important food legume grown in semi-arid tropical and sub-tropical farming systems under diverse agroecological environments. Alternaria blight (Alternaria tenuissima (Kunze ex Pers.) Wiltshire) is a minor disease when the pigeonpea is sown in June-July but becomes the most serious disease in crops that are sown in mid-September in north-eastern India. Alternaria blight disease has been reported from 13.67 to 49.33% disease intensity in different climatic conditions. This disease is found on leaves, stems, and pods, and dark spots on the leaves and pods reduce the photosynthesis capacity and immature ripening, reducing the amount of quality seeds. The recurrence of the disease depends upon weather conditions, genotypes, age of plants, and virulence of the pathogen, and crop growing areas prioritized the research for developing an extensive range of Alternaria blight. Earlier workers on pathological, physiological, and biochemical characteristics of pathogen the nature of the infection process, and the genetic basis of the pathogen in variability could not clearly be reported. It is required to investigate Alternaria blight including signs, symptoms, biology, status, epidemiology, pathogenic, biochemical, and genetic variability, refinement of the resistance screening techniques, and develop integrated disease management technology of the pathogen to understand the change of disease scenario in the context of climate change. Available information on pathogen symptomatology, status, variability, source of resistance, different management options, and genetic basis of resistance have been updated and discussed with the identification of future research priorities.

Synergistic action of endophytic fungi secondary metabolites against multidrug-resistant strains of Staphylococcus aureus

The global threat to treatment of infections associated with multidrug-resistant microorganisms as well as side effects associated with combination therapy calls for the urgent development of new medicines to address these challenges. Endophytic fungi are efficient bio synthesizers and offer a wide array of these reliable bioactive compounds. The present study is aimed at evaluating the effect of combined extracts of endophytic fungi isolated from the leaves of Musa paradisiaca against multidrug-resistant Staphylococcus aureus (MRSA) strains. Screening for multidrug resistance was carried out using the Kirby-Bauer disc diffusion assay followed by a preliminary assay of endophytic fungal extracts bioactivity using agar diffusion assay against selected MRSA. A synergistic study using checkerboard assay technique was performed, then extracts were subjected to quantitative evaluation of secondary metabolites using Gas Chromatography with Flame Ionization Detector. Axenic cultures of five endophytic fungi from M. paradisiaca (EMp1-Emp5) were isolated from the sampled leaves. All the endophytic fungal extracts inhibited the growth of all the multi-drug resistant Staphylococcus aureus at 0.5 mg/mL. Varying inhibition zones that were concentration-dependent were observed. The combined effects of EMp3 and EMp5 extracts at 9:1; 8:2; 4:6; 3:7; and 2:8 combination ratios produced synergistic effects against S. aureus (S18) with very low Fractional inhibitory Concentrations of 0.03:0.01, 0.01:0.01, 0.006:0.02, 0.01:0.04 and 0.01:0.1 mg/mL respectively. Several bioactive secondary metabolites such as anthocyanin, (-) epicatechin, naringenin, resveratrol, catechin, rutin, phenol were produced by the fungal endophytes. Our findings further affirm fungal endophytes associated with M. paradisiaca leaves as producers of natural antibacterial agents with synergistic potentials.