Insights into the molecular mechanism of Trichoderma stimulating plant growth and immunity against phytopathogens.

Trichoderma species have received significant interest as beneficial fungi for boosting plant growth and immunity against phytopathogens. By establishing a mutualistic relationship with plants, Trichoderma causes a series of intricate signaling events that eventually promote plant growth and improve disease resistance. The mechanisms contain the indirect or direct involvement of Trichoderma in enhancing plant growth by modulating phytohormones signaling pathways, improving uptake and accumulation of nutrients, and increasing soil bioavailability of nutrients. They contribute to plant resistance by stimulating systemic acquired resistance through salicylic acid, jasmonic acid, and ethylene signaling. A cascade of signal transduction processes initiated by the interaction of Trichoderma and plants regulate the expression of defense-related genes, resulting in the synthesis of defense hormones and pathogenesis-related proteins (PRPs), which collectively improve plant resistance. Additionally, advancements in omics technologies has led to the identification of key pathways, their regulating genes, and molecular interactions in the plant defense and growth promotion responses induced by Trichoderma. Deciphering the molecular mechanism behind Trichoderma's induction of plant defense and immunity is essential for harnessing the full plant beneficial potential of Trichoderma. This review article sheds light on the molecular mechanisms that underlie the positive effects of Trichoderma-induced plant immunity and growth and opens new opportunities for developing environmentally friendly and innovative approaches to improve plant immunity and growth.

Methylorubrum rhodesianum M520 as a biocontrol agent against Meloidogyne incognita (Tylenchida: Heteroderidae) J2s infecting cucumber roots.

AIMS: Root-knot nematodes (RKNs) are plant pathogens that cause huge economic losses worldwide. The biological management of RKNs may be a sustainable alternative to chemical control methods. Here, the biocontrol potential of Methylorubrum rhodesianum M520 against the RKN Meloidogyne incognita was investigated to theoretically support its application as a biocontrol agent in field production. METHODS AND RESULTS: In-vitro assays showed 91.9% mortality of M. incognita second-stage juveniles in the presence of strain M520 and that the hatching rate of M. incognita eggs was 21.7% lower than that of eggs treated with sterile water. In pot experiments, the M520 treatment caused 70.8% reduction in root-knots and increased plant shoot length and stem and root fresh weights, compared to control plant values. In split-root experiments, cucumber roots treated with M520 showed 25.6% decrease in root gall number, compared to that in control roots. CONCLUSION: M520 has multiple mechanisms against RKNs and might be used as a biocontrol agent against M. incognita in cucumber, laying a foundation for further studying M520 biocontrol against RKNs.

Enhanced suppression of soil-borne phytopathogenic bacteria Ralstonia solanacearum in soil and promotion of tomato plant growth by synergetic effect of green synthesized nanoparticles and plant extract.

AIMS: Because of severe economic losses and food security concerns caused by plant pathogenic bacteria, Ralstonia solanacearum, there is a need to develop novel control methods. This study was aimed to green synthesize the zinc oxide nanoparticles (ZnO NPs) through Withania coagulans leaf extracts and checked their antibacterial potential alone or in combination with W. coagulans leaf extract for the management of R. solanacearum causing bacterial wilt disease in tomato. METHODS AND RESULTS: ZnO NPs were synthesized through an eco-friendly approach using leaves extract of W. coagulans and characterized through various spectroscopic approaches, that is Fourier transform infrared spectroscopic, UV-visible spectroscopy and energy dispersive spectroscopy. The antibacterial effect of W. coagulans leaf extract and ZnO NPs alone and in combination was tested in vitro and in vivo against bacterial wilt pathogen in tomato plants. The results showed that combine application of leaf extract and ZnO NPs inhibited in vitro growth of R. solanacearum more than applying alone. Three application times (0, 6 and 12 days before transplantation) of leaf extract, ZnONPs and their combine application were tested in vivo. The combine treatment and longest application time (12 days before transplantation) were more effective in suppressing soil population of R. solanacearum, reducing disease severity and enhancing plant growth than applying alone and smaller application time. CONCLUSION: It is concluded that W. coagulans leaf extract and ZnO NPs have strong antibacterial potential against R. solanacearum in vitro and in vivo. SIGNIFICANCE AND IMPACT OF STUDY: The results of this study suggest the potential application of leaf extract and ZnO nanoparticles for controlling R. solanacearum as safe, eco-friendly and less expensive integrated disease management strategy in tomato crop.

Rapid and mass production of biopesticide Trichoderma Brev T069 from cassava peels using newly established solid-state fermentation bioreactor system.

Converting agricultural waste into value-added biopesticides to replace chemical pesticides for plant protection is a good alternative for environmental sustainability and resource recycling. In this study, five tropical wastes (cassava peels, banana pseudostem, coconut shell, sugarcane bagasse, and pineapple peels) were screened as substrates for the rapid production of biopesticide Trichoderma Brev T069. Five single tests and a Box-Behnken design (BBD) with response surface methodology were used to optimize the culture conditions to improve the spore yield. The results showed that cassava peel was the optimal solid fermentation substrate, and the optimization enabled a spore yield of 9.31 × 109 spores/g at 3rd day, which was equal to 93.19% of spore yield obtained at 5th day (9.99 × 109 spores/g). A newly packed-bed bioreactor with agitation and ventilation system was developed and used to expand the production that 250 kg of biopesticide (2.89 × 109 spores/g) could be available on the 3rd day. A pot experiment indicated that the biopesticide T. Brev T069 obtained under this production system, when applied at 1 × 107 spores/g of soil had a 64.65% biocontrol efficiency on banana fusarium wilt. This study provides a practical solution for turning a tropical waste into an effective biopesticide which can prevent banana wilt disease, thereby helping to reduce disease management cost and overcome environmental hazards caused by synthetic pesticides.

Phenolic Contents, Organic Acids, and the Antioxidant and Bio Activity of Wild Medicinal Berberis Plants- as Sustainable Sources of Functional Food.

Wild fruits have increasingly been investigated as part of recent searches for food products with a high antioxidant activity. In this study, wild edible berberis Berberis vulgaris collected from three different provinces (Jilin, Heilongjiang, and Liaoning) were investigated for their phenolic contents, organic acid contents, mineral contents, antioxidant activity as well as their antimicrobial potential against a range of common food borne pathogens. In addition, a physiochemical and mineral analysis of the fruits was also performed. The methanol extracts of berberis fruit collected from Jilin province were highly active against all the studied food borne bacterial pathogens, i.e., S. aureus and L. monocytogenes, E. coli, P. fluorescens, V. parahaemolyticus, and A. caviae while the berberis extracts from Heilongjiang and Liaoning showed activity only against Gram-negative bacteria. The phenolic content and antioxidant activity were determined by the HPLC separation method and ß-carotene bleaching methods, respectively. Four organic acids such as malic acid, citric acid, tartaric acid, and succinic acid were identified while a variety of phenolic compounds were detected among which catechin, chlorogenic acid, and gallic acid were found to be the predominant phenolic compounds in all three of berberis fruit samples. The berberis fruit from Jilin was found to be superior to the Heilongjiang and Liaoning fruit regarding desired physiochemical analysis; however, there were no significant differences in the mineral contents among the three samples. Overall, the berberis fruit from Jilin was ranked as the best in term of the nutritional, physiochemical, antimicrobial, and antioxidant properties. This study confirms the various useful characteristics and features of berberis at a molecular level that can be used as a sustainable source for their potential nutritional applications for making functional foods in different food industries.

Draft Genome Resource of Fusarium oxysporum f. sp. capsici, the Infectious Agent of Pepper Fusarium Wilt.

Fusarium oxysporum f. sp. capsici is the specific pathogen of pepper Fusarium wilt and causes a significant reduction in pepper yield. Its narrow host specificity has led to the concept of formae speciales. This interesting phenomenon has great potential and needs to be analyzed at the molecular level. In this study, we obtained the draft genome sequence of F. oxysporum f. sp. capsici, using the Oxford Nanopore sequencing technology. The long read-based assembly consisted of 34 contigs, with a total length of 54,516,562 bp. The contig N50 was 4,962,668 bp and the GC content was 47.6%. Our genome assembly of F. oxysporum f. sp. capsici provides a valuable resource for the study of pepper Fusarium wilt, and the comparative genomic study of F. oxysporum.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Volatile Organic Compounds of Bacillus cereus Strain Bc-cm103 Exhibit Fumigation Activity against Meloidogyne incognita.

Bacillus cereus strain Bc-cm103 shows nematicidal activity and, therefore, has been used as a biological control agent to control the root-knot nematode Meloidogyne incognita. However, it remains unknown whether volatile organic compounds (VOCs) produced by B. cereus strain Bc-cm103 are effective in biocontrol against M. incognita. Therefore, in this study, we investigated the activity of Bc-cm103 VOCs against M. incognita. The B. cereus strain Bc-cm103 significantly repelled the second-stage juveniles (J2s) of M. incognita. In vitro evaluation of VOCs produced by the fermentation of Bc-cm103 in a three-compartment Petri dish revealed the mortality rates of M. incognita J2s as 90.8% at 24 h and 97.2% at 48 h. Additionally, evaluation of the ability of Bc-cm103 VOCs to suppress M. incognita infection in a double-layered pot test showed that root galls on cucumber roots decreased by 46.1%. Furthermore, 21 VOCs were identified from strain Bc-cm103 by solid-phase microextraction gas chromatography-mass spectrometry, including alkanes, alkenes, esters, and sulfides. Among them, dimethyl disulfide (30.63%) and S-methyl ester butanethioic acid (30.29%) were reported to have strong nematicidal activity. Together, these results suggest that B. cereus strain Bc-cm103 exhibits fumigation activity against M. incognita.

Management of Ralstonia solanacearum in Tomato Using ZnO Nanoparticles Synthesized Through Matricaria chamomilla.

Matricaria chamomilla flower extract was used as a biocompatible material for synthesis of zinc oxide nanoparticles (ZnONPs). The synthesized NPs were evaluated for their antibacterial potential in vitro and in vivo against the Gram-negative bacterium Ralstonia solanacearum, which causes devastating bacterial wilt disease in tomato and other crops. Synthesized ZnONPs were further analyzed by UV-visible spectroscopy, Fourier transform infrared spectroscopy, x-ray diffraction, transmission electron microscopy, and scanning electron microscopy with energy-dispersive spectroscopy. The synthesized polydisperse ZnONPs were found to be in the size range of 8.9 to 32.6 nm, and at 18.0 µg ml-1 exhibited maximum in vitro growth inhibition of the pathogen R. solanacearum. Scanning electron microscopy analysis of affected bacterial cells showed morphological deformation such as disruption of the cell membrane and wall, and the leakage of cell contents. Results of in vivo studies also showed that application of ZnONPs to the artificially inoculated tomato plants with the pathogen R. solanacearum significantly enhanced the plant growth by reducing bacterial soil population and disease severity as compared with the untreated control. Biosynthesized ZnONPs could be an effective approach to control the bacterium R. solanacearum.

Discovery of a new antifungal lipopeptaibol from Purpureocillium lilacinum using MALDI-TOF-IMS.

Fungi are considered to be rich in biologically active natural products for agricultural and medicinal purposes. The discovery and accurate identification of the bioactive fungal natural products is important for their efficient utilization. During the course of our continuing search for the new natural products from the fungal agents, we found the well-known bio-control fungus Purpureocillium lilacinum showed in vitro activity against Botrytis cinerea, an airborne plant pathogenic fungus causing gray mold disease in many vegetables and fruits. The co-culture of two fungi on agar plate showed that P. lilacinum inhibited the growth of B. cinerea which means P. lilacinum has potential to produce some bioactive secondary metabolites against B. cinerea. In this study, we applied matrix-assisted laser desorption ionization-time of flight mass spectrometry imaging mass spectrometry (MALDI-TOF-IMS), as a fast identification tool, for the discovery of a new antifungal lipopeptaibol (leucinostatin Z) from P. lilacinum against B. cinerea. The planar structure of leucinostatin Z was further established by using the LC-HRESI-MS-MS analysis. MALDI-TOF-IMS is becoming a new approach that allows us to observe the bioactive natural products directly on growth media between the colonies of two fungi, which is faster and more effective than the traditional techniques to discover new bioactive compounds in fungi.

Eco-friendly Management of Bacterial Wilt in Tomato Using Dried Powder of the Wild Arid-Land Medicinal Shrub Rhazya stricta.

Bacterial wilt (BW) disease caused by Ralstonia solanacearum species complex is a devastating plant disease that inflicts heavy losses to the large number of economic host plants it infects. In this study, the potential of dried powder of the arid-land medicinal shrub Rhazya stricta to control BW of tomato was explored. Both, in vitro and in planta studies were conducted, using different concentrations of dried powder of plant parts, and applied (surface mulched or mixed) to infested soil at 0, 10, and 20 days before transplanting (DBT). Aqueous extract of leaves (16% w/v) was found to be as effective as streptomycin (100 ppm) in inhibiting the in vitro growth of R. solanacearum. As evident from the scanning electron micrograph, 16% aqueous extract of leaves produced severe morphological changes, such as rupture of the bacterial cell walls. Results from the greenhouse experiments demonstrated that the higher powder dose (succulent shoot), namely, 30 g/kg of soil mixed with infested soil 20 DBT, was found to be the most effective in controlling BW. It increased root length (cm), shoot length (cm), and plant fresh biomass (g) by 55, 42, and 40%, respectively, over control plants. Mixing of plant powder with the artificially infested (35 ml of 108 CFU/ml per kilogram of soil) pot soil was better than surface mulching. The 30 g/kg of soil dose mixed with soil increased root length (cm), shoot length (cm), and plant fresh biomass (g) of treated plants by 67, 36, and 46%, respectively, over control plants. A 37% decrease in disease severity over the control was observed with drench application of 30 g of powder per kilogram of soil applied once at 20 DBT. Our results indicated that the dried powder (30 g/kg of soil) of leaves or succulent shoots of R. stricta, thoroughly mixed with soil, 20 DBT, could act as an effective control method against BW.

Genetic Susceptibility to Chronic Liver Disease in Individuals from Pakistan.

Chronic liver disease, with viral or non-viral etiology, is endemic in many countries and is a growing burden in Asia. Among the Asian countries, Pakistan has the highest prevalence of chronic liver disease. Despite this, the genetic susceptibility to chronic liver disease in this country has not been investigated. We performed a comprehensive analysis of the most robustly associated common genetic variants influencing chronic liver disease in a cohort of individuals from Pakistan. A total of 587 subjects with chronic liver disease and 68 healthy control individuals were genotyped for the HSD17B13 rs7261356, MBOAT7 rs641738, GCKR rs1260326, PNPLA3 rs738409, TM6SF2 rs58542926 and PPP1R3B rs4841132 variants. The variants distribution between case and control group and their association with chronic liver disease were tested by chi-square and binary logistic analysis, respectively. We report for the first time that HSD17B13 variant results in a 50% reduced risk for chronic liver disease; while MBOAT7; GCKR and PNPLA3 variants increase this risk by more than 35% in Pakistani individuals. Our genetic analysis extends the protective role of the HSD17B13 variant against chronic liver disease and disease risk conferred by the MBOAT7; GCKR and PNPLA3 variants in the Pakistani population.

Whole-Exome sequencing identifies GYS2 biallelic variants in individuals with suspected epilepsy.

BACKGROUND: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2. METHODS: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families. RESULTS: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM_021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM_021957.3: c.343G>A; p.Val115Met and NM_021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency. CONCLUSIONS: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene.

Nematicidal glycosylated resorcylic acid lactones from the fungus Pochonia chlamydosporia PC-170 and their key biosynthetic genes.

Chemical study of the nematicidal biocontrol fungus Pochonia chlamydosporia PC-170 led to discovery of six resorcylic acid lactones (RALs), including three nematicidal glycosylated RALs, monocillin VI glycoside (1), colletogloeolactone A (2) and monocillin II glycoside (3), and three antibacterial non-glycosylated RALs, monocillin VI (4), monocillin IV (5) and monocillin II (6). The planar structure of the new compound monocillin VI glycoside (1) was elucidated using HRESIMS and NMR data, and its monosaccharide configuration was further determined through sugar hydrolysis experiment and GC-MS analysis method. Furthermore, their two biosynthetic-related PKS genes, pchE and pchI, were identified through the gene knockout experiment. The glycosylated RALs 1-3 exhibited nematicidal activity against Meloidogyne incognita, with LC50 values of 94, 152 and 64 µg/mL, respectively, and thus had great potential in the development of new nematicidal natural products to control M. incognita in the future.

Deciphering the role of rhizosphere microbiota in modulating disease resistance in cabbage varieties.

BACKGROUND: Cabbage Fusarium wilt (CFW) is a devastating disease caused by the soil-borne fungus Fusarium oxysporum f. sp. conglutinans (Foc). One of the optimal measures for managing CFW is the employment of tolerant/resistant cabbage varieties. However, the interplay between plant genotypes and the pathogen Foc in shaping the rhizosphere microbial community, and the consequent influence of these microbial assemblages on biological resistance, remains inadequately understood. RESULTS: Based on amplicon metabarcoding data, we observed distinct differences in the fungal alpha diversity index (Shannon index) and beta diversity index (unweighted Bray-Curtis dissimilarity) within the rhizosphere of the YR (resistant to Foc) and ZG (susceptible to Foc) cabbage varieties, irrespective of Foc inoculation. Notably, the Shannon diversity shifts in the resistant YR variety were more pronounced following Foc inoculation. Disease-resistant plant variety demonstrate a higher propensity for harboring beneficial microorganisms, such as Pseudomonas, and exhibit superior capabilities in evading harmful microorganisms, in contrast to their disease-susceptible counterparts. Furthermore, the network analysis was performed on rhizosphere-associated microorganisms, including both bacteria and fungi. The networks of association recovered from YR exhibited greater complexity, robustness, and density, regardless of Foc inoculation. Following Foc infection in the YR rhizosphere, there was a notable increase in the dominant bacterium NA13, which is also a hub taxon in the microbial network. Reintroducing NA13 into the soil significantly improved disease resistance in the susceptible ZG variety, by directly inhibiting Foc and triggering defense mechanisms in the roots. CONCLUSIONS: The rhizosphere microbial communities of these two cabbage varieties are markedly distinct, with the introduction of the pathogen eliciting significant alterations in their microbial networks which is correlated with susceptibility or resistance to soil-borne pathogens. Furthermore, we identified a rhizobacteria species that significantly boosts disease resistance in susceptible cabbages. Our results indicated that the induction of resistance genes leading to varied responses in microbial communities to pathogens may partly explain the differing susceptibilities of the cabbage varieties tested to CFW. Video Abstract.

Rice false smut pathogen: implications for mycotoxin contamination, current status, and future perspectives.

Rice serves as a staple food across various continents worldwide. The rice plant faces significant threats from a range of fungal, bacterial, and viral pathogens. Among these, rice false smut disease (RFS) caused by Villosiclava virens is one of the devastating diseases in rice fields. This disease is widespread in major rice-growing regions such as China, Pakistan, Bangladesh, India, and others, leading to significant losses in rice plantations. Various toxins are produced during the infection of this disease in rice plants, impacting the fertilization process as well. This review paper lightens the disease cycle, plant immunity, and infection process during RFS. Mycotoxin production in RFS affects rice plants in multiple ways, although the exact phenomena are still unknown.

A Salt-Tolerant Strain of Trichoderma longibrachiatum HL167 Is Effective in Alleviating Salt Stress, Promoting Plant Growth, and Managing Fusarium Wilt Disease in Cowpea.

Salt stress is a constraint factor in agricultural production and restricts crops yield and quality. In this study, a salt-tolerant strain of Trichoderma longibrachiatum HL167 was obtained from 64 isolates showing significant salt tolerance and antagonistic activity to Fusarium oxysporum. T. longibrachiatum HL167 inhibited F. oxysporum at a rate of 68.08% in 200 mM NaCl, penetrated F. oxysporum under 200 mM NaCl, and eventually induced F. oxysporum hyphae breaking, according to electron microscope observations. In the pot experiment, pretreatment of cowpea seedlings with T. longibrachiatum HL167 reduced the accumulation level of ROS in tissues and the damage caused by salt stress. Furthermore, in the field experiment, it was discovered that treating cowpea with T. longibrachiatum HL167 before root inoculation with F. oxysporum can successfully prevent and control the development of cowpea Fusarium wilt, with the best control effect reaching 61.54%. Moreover, the application of HL 167 also improved the K+/Na+ ratio of cowpea, alleviated the ion toxicity of salt stress on cowpea, and HL167 was found to effectively colonize the cowpea roots. T. longibrachiatum HL167, which normally survives in saline-alkali environments and has the functions of disease prevention and plant growth promotion capabilities, has important research implications for improving the saline-alkali soil environment and for the sustainable development of green agriculture.

6-Pentyl-2H-pyran-2-one from Trichoderma erinaceum Is Fungicidal against Litchi Downy Blight Pathogen Peronophythora litchii and Preservation of Litchi.

The postharvest losses of litchi caused by litchi downy blight are considerably high. We identified a natural antifungal volatile pyrone, 6-pentyl-2H-pyran-2-one (6PP), synthesized by Trichoderma erinaceum LS019-2 and investigated as biocontrol for litchi downy blight and preservation. 6PP significantly inhibited the growth and sporangial germination of Peronophythora litchii, the causal agent of litchi downy blight, and caused severe cellular and intracellular destructions, as evidenced by electron microscopic analysis. Furthermore, in the treatment, the fruit kept better color, higher weight, and antioxidant activity, so it can maintain freshness and prolong shelf life. Metabolome analysis confirmed the decline of lipids and the accumulation of organic acids in litchi fruits in response to 6PP treatment. These effects from 6PP could alleviate disease effects and prolong the shelf life of litchi fruits. These findings suggested that 6PP could be a useful natural product to control downy blight disease and a new preservative of litchi fruits.

Mitigating Cd and bacterial wilt stress in tomato plants through trico-synthesized silicon nanoparticles and Trichoderma metabolites.

There has been a growing apprehension in recent years about the harmful effects of environmental pollutants on agricultural output, encompassing both living organisms and non-living factors that cause stress. In this study, the soil application of bulk silicon (Si), silicon nanoparticles (SiNPs) and Trichoderma metabolites (TM) were investigated alone or in combination for the management of an important abiotic stress i.e. Cd toxicity and biotic stress i.e. bacterial wilt (BW) in tomato plants. SiNPs were synthesized by Trichoderma and confirmed through XRD, FTIR, and Ranman spectrum analysis. Results showed that Si, SiNPs and TM were all effective treatments. The combine treatment of SiNPs and TM followed by SiNPs alone were superior over other treatments in mitigating Cd toxicity and reducing BW disease on tomato plants. The soil application of these treatments reduced the Cd toxicity by enhancing Cd-tolerance index, decreasing bioavailability of soil Cd, reducing Cd contents and translocation in plants, improving gaseous exchange, photosynthesis, and increasing the antioxidant enzyme activities and their transcriptions. These treatments significantly suppressed BW pathogen leading to the significant decrease in disease index and severity on plants. In vitro evaluation and scanning electron microscopic (SEM) analysis revealed that SiNPs and TM significantly disrupted the cellular morphology of BW pathogen Ralstonia solanacearum. Findings of this study proposes the possible use of SiNPs and TM in mitigating the Cd and BW stress in tomato plants and possibly in other crops.

9p21 Locus Polymorphism Is A Strong Predictor of Metabolic Syndrome and Cardiometabolic Risk Phenotypes Regardless of Coronary Heart Disease.

The world population is genetically predisposed to metabolic syndrome (MetS) and its components, also known as cardiometabolic risk phenotypes, which can cause severe health complications including coronary heart disease (CHD). Genetic variants in the 9p21 locus have been associated with CHD in a number of populations including Pakistan. However, the role of the 9p21 locus in MetS and cardiometabolic risk phenotypes (such as obesity, hypertension, hyperglycemia, and dyslipidemia) in populations with CHD or no established CHD has not been explored. Therefore, the present study was designed to explore the association of the minor/risk allele (C) of 9p21 locus SNP rs1333049 with MetS or its risk phenotypes regardless of an established CHD, in Pakistani subjects. Genotyping of rs1333049 (G/C) was performed on subjects under a case-control study design; healthy controls and cases, MetS with CHD (MetS-CHD+) and MetS with no CHD (MetS-CHD-), respectively. Genotype and allele frequencies were calculated in all study groups. Anthropometric and clinical variables (Means ± SD) were compared among study groups (i.e., controls, MetS + CHD and MetS-CHD) and minor/risk C allele carriers (GC + CC) vs. non-carriers (Normal GG genotype). Associations of the risk allele of rs1333049 SNP with disease and individual metabolic risk components were explored using adjusted multivariate logistic regression models (OR at 95% CI) with a threshold p-value of ≤0.05. Our results have shown that the minor allele frequency (MAF) was significantly high in the MAF cases (combined = 0.63, MetS-CHD+ = 0.57 and MetS-CHD- = 0.57) compared with controls (MAF = 0.39). The rs1333049 SNP significantly increased the risk of MetS, irrespective of CHD (MetS-CHD+ OR = 2.36, p < 0.05 and MetS-CHD- OR = 4.04, p < 0.05), and cardiometabolic risk phenotypes; general obesity, central obesity, hypertension, and dyslipidemia (OR = 1.56-3.25, p < 0.05) except hyperglycemia, which lacked any significant association (OR = 0.19, p = 0.29) in the present study group. The 9p21 genetic locus/rs1333049 SNP is strongly associated with, and can be a genetic predictor of, MetS and cardiometabolic risks, irrespective of cardiovascular diseases in the Pakistani population.

Risk variants of obesity associated genes demonstrate BMI raising effect in a large cohort.

Obesity is highly polygenic disease where several genetic variants have been reportedly associated with obesity in different ethnicities of the world. In the current study, we identified the obesity risk or protective association and BMI raising effect of the minor allele of adiponectin, C1Q and collagen domain containing (ADIPOQ), cholesteryl ester transfer protein (CEPT), FTO alpha-ketoglutarate dependent dioxygenase (FTO), leptin (LEP), and leptin receptor (LEPR) genes in a large cohort stratified into four BMI-based body weight categories i.e., normal weight, lean, over-weight, and obese. Based on selected candidate genetic markers, the genotyping of all study subjects was performed by PCR assays, and genotypes and allele frequencies were calculated. The minor allele frequencies (MAFs) of all genetic markers were computed for total and BMI-based body weight categories and compared with MAFs of global and South Asian (SAS) populations. Genetic associations of variants with obesity risk were calculated and BMI raising effect per copy of the minor allele were estimated. The genetic variants with higher MAFs in obese BMI group were; rs2241766 (G = 0.43), rs17817449 (G = 0.54), rs9939609 (A = 0.51), rs1421085 (C = 0.53), rs1558902 (A = 0.63), and rs1137101 (G = 0.64) respectively. All these variants were significantly associated with obesity (OR = 1.03-4.42) and showed a high BMI raising effect (ß = 0.239-0.31 Kg/m2) per copy of the risk allele. In contrast, the MAFs of three variants were higher in lean-normal BMI groups; rs3764261 A = 0.38, rs9941349 T = 0.43, and rs7799039 G = 0.40-0.43). These variants showed obesity protective associations (OR = 0.68-0.76), and a BMI lowering effect per copy of the protective allele (ß = -0.103-0.155 Kg/m2). The rs3764261 variant also showed significant and positive association with lean body mass (OR = 2.38, CI = 1.30-4.34). Overall, we report six genetic variants of ADIPOQ, FTO and LEPR genes as obesity-risk markers and a CETP gene variant as lean mass/obesity protective marker in studied Pakistani cohort.