In-vitro assessment of a novel plant rhizobacterium, Citrobacter freundii, for degrading and biocontrol of food mycotoxin deoxynivalenol.

Deoxynivalenol (DON) is one of the most harmful and well-known toxins present in food and animal feed throughout the world. Citrobacter freundii (C. freundii-ON077584), a novel DON-degrading strain, was isolated from rice root-linked soil samples. The degrading properties, including DON concentrations, incubation pH, incubation temperatures, bacterial concentrations, and acid treatment effect on degradation, were evaluated. At pH 7 and an incubation temperature of 37 °C, C. freundii demonstrated the capability to degrade more than 90% of DON. The degraded products of DON were identified as 3-keto-DON and DOM-1, which were confirmed by High Performance Liquid Chromatography (HPLC) and Ultra-Performance Liquid Chromatography hyphenated with Tandem Mass Spectrometry (UPLC-MS/MS) analyses. The mechanism of DON degradation into 3-keto-DON and DOM-1 by this bacterial strain will be further explored to identify and purify novel degrading enzymes that can be cloned to the microorganism and added to the animal feed to degrade the DON in the digestion tract.

Biological Control Potential of Endophytic Fungi with Amelioration of Systemic Resistance in Sunflower and GC-MS Metabolic Profiling of Talaromyces assiutensis.

Endophytic fungi live inside plant tissues but do not cause any disease. Several reports have now revealed that they have great influence on host. In this study, the beneficial role of endophytic fungi is highlighted and explored. Endophytic fungi isolated from healthy plants were identified as Aspergillus terreus, Curvularia lunata, C. hawaiiensis, Macrophomina phaseolina, Fusarium solani, Talaromyces assiutensis, and T. trachyspermus using 18S rRNA gene sequencing. In vitro, fungi evaluated for antimicrobial activity, showed significant activity. These fungi were tested in field application by exploring their broad spectrum. Talaromyces assiutensis and T. trachyspermus were applied in pots and field plot experiments using sunflower as test plants, along with endophytic Cephalosporium sp., and Chaetomium sp. Endophytic fungi showed significant activity against root rot pathogens affecting sunflower and improved plant biomass. They also improved production of plant defense biochemical markers (polyphenolic content and salicylic acid) with improvement in antioxidant potential. These fungi are used as biological control agents, so their culture filtrates are used to check the presence of metabolites by GC-MS. Several new compounds were isolated from T. assiutensis. The major bioactive compounds are Coumarin, 3,4-dihydro-6-methoxy-4,4-dimethyl, 1-Monolinoleoylglycerol trimethylsilyl ether, 1,2-Propanediol, 3-(octadecyloxy), Ethyl iso-allocholate, and 1H-Pyrazole, which possess antioxidant, antitumor, antibacterial, anticancer, and antimicrobial properties. These findings will lead to further in-depth research toward the potential use of these endophytic fungi for their possible use in agriculture and drug formation.

LC-MS/MS based appraisal of multi-mycotoxin co-occurrence in poultry feeds from different regions of Punjab, Pakistan.

Mycotoxins, being a threat to animal and human health, contribute significantly towards economic losses in the poultry sector. A liquid chromatography-mass spectrometry-based study was conducted on poultry feed samples collected from Punjab, Pakistan to evaluate the prevalence, contamination levels, and co-occurrence of multi-mycotoxins across different processed forms of the feed, types of utilities and sampling regions. All samples were found to be contaminated with aflatoxin B1 (AFB1) and fumonisin B1 (FB1). The European Commission (EC) maximum level for AFB1 in complete feedingstuffs in poultry and guidance values for FB1 and zearalenone (ZEN) were exceeded in 73%, 2%, and 14% of the contaminated samples, respectively. The corresponding median values were 39.9 µg/kg, 205 µg/kg, and 34.5 µg/kg. In addition to exceeding contamination levels, a varying co-occurrence of three to fourteen mycotoxins was observed in each of the feed samples that calls for mitigation measures to safeguard the feed and its ingredients.

The Psychological Impact of COVID-19 Among Pakistani Adults in Lahore.

Background: In the wake of the worldwide spread of the novel coronavirus and the resultant restrictive measures, mental health has become a crucial issue. Physical health is not the only aspect of humans that is at risk. Globally, the rates and severity of mental illness are being significantly impacted by this pandemic. Two scales have been validated to measure the impact of coronavirus disease 2019 (COVID-19) on the levels of anxiety and obsessional thinking in clinical and non-clinical populations. The present study was designed to investigate the levels of anxiety and obsessions related to COVID-19 in the general public of Lahore, Pakistan. Materials and Methods: Data were collected via snowball sampling from May 9 to May 19. An online survey consisting of a demographic profile and two scales, Coronavirus Anxiety Scale (CAS) and Obsession with COVID-19 Scale (OCS), was sent through email, WhatsApp, and Facebook groups to adults (18 years and above) of Lahore, Pakistan. Results: A total of 240 individuals (20% men and 80% women) recorded their responses. The majority belonged to a nuclear family system (60%), and their education level ranged from high school to Ph.D. The cut-off score for probable dysfunctional coronavirus anxiety and obsession levels was not met within this sample (CAS, M = 3.24, SD = 4.21; OCS, M = 4.14, SD = 3.15), suggesting that the general population of Lahore, Pakistan is not suffering from dysfunctional anxiety or obsessions related to COVID-19. Forty-seven participants' score on OCS and 35 participants' scores on CAS were above the cut-off, i.e., ≥7 and ≥9, respectively. The results of the correlation analysis showed a significant positive relationship (**p < 0.619) between anxiety and obsessions related to COVID-19. Conclusion: One important, yet surprising, conclusion of this study is that the average adult in Lahore does not show much anxiety or obsessions related to COVID-19. Other studies around the world using these measurement tools have indicated significantly high levels of both anxiety and obsessions related to COVID-19. These findings may demonstrate the resilience of Pakistanis or perhaps the lack of understanding of the seriousness of the situation.

LC-MS/MS-based determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in poultry meat from the Punjab-Pakistan.

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry-based confirmatory method was redeveloped and validated for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscles. The analytes were extracted from minced chicken muscle with acetonitrile and ammoniated water mixture. A second extraction with ethyl acetate was followed by evaporation and dissolution of the residue in ammoniated methanol before defatting with n-hexane. Finally, the extract was further cleaned up by dispersive solid phase extraction using C-18 end-capped dispersive material. The validation protocol was adapted from the European Commission Decision 2002/657/EC and all the performance characteristics were successfully satisfied. The recoveries of all the analytes were found to be in the range of 86.4-108.1% and the precision values, within day and between days, ranged from 2.7% to 11% and 4.4% to 16.3%, respectively. The method was tested in various incurred samples and applied to analyse a wide range of random poultry market samples (n = 120) collected from three cities of the Punjab, Pakistan. Chloramphenicol and florfenicol residues were detected at low levels in less than 11% of the samples. Chloramphenicol was detected only in 4 samples with the concentration range of 0.17-0.477 µg kg-1, whereas the levels of florfenicol/florfenicol amine residues detected in 9 samples ranged from 8.7 to 32.8 µg kg-1. Moreover, most of the florfenicol residues were identified as tissue bound, extractable only after strong acid hydrolysis.

Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease.

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management.

Multiple Mycotoxins in Rice: Occurrence and Health Risk Assessment in Children and Adults of Punjab, Pakistan.

Mycotoxin contamination in rice can create a health risk for the consumers. In this study, the measurement of 23 mycotoxins in rice samples (n = 180) was performed using a validated LC-MS/MS method. A food frequency questionnaire was used to get rice consumption data for the assessment of mycotoxin dietary exposure, before calculating the health risk in adults and children of north and south regions of the Pakistani Punjab province. The prevalence of aflatoxin B1 (56%), aflatoxin B2 (48%), nivalenol (28%), diacetoxyscirpenol (23%), fumonisin B1 (42%), zearalenone (15%), HT-2 toxin (10%), deoxynivalenol (8%), and ochratoxin A (6%) was estimated in samples with a mean concentration range between 0.61 and 22.98 µg/kg. Aflatoxin degradation by traditional Pakistani cooking recipes was evaluated and observed to be 41-63%. The dietary exposure to aflatoxins exceeded the tolerable daily intake at all levels, and ochratoxin A and zearalenone posed health risk at high contamination and high consumption levels. The margin of aflatoxin B1 exposure ranged between 10 and 69 in adults and 10 and 62 in children. The mean cancer risk by aflatoxin B1 exposure was 0.070 (adults) and 0.071 (children) cases/year/100,000 people in South Punjab population, and 0.122 (adults) and 0.127 (children) cases/year/100,000 people in North Punjab population. This study will provide new insights for the planning and management of mycotoxins in Pakistan.

Analysis of Nutritional Traits and Aflatoxin Contamination in Selected Maize Varieties from Pakistan.

Recently, cultivation of high-yielding hybrid maize varieties has revolutionized maize production in Pakistan. Analyses of nutritional traits and aflatoxin (AF) contamination in these varieties can aid in the identification of susceptible and resistant varieties, particularly for cultivation in the Pakistani agro-climatic environment. Five spring maize varieties-Pioneer, Neelam, DK-919, Desi, and Hi-maize-were selected for analyses of their nutritional, tocopherol, and AF contents. Protein, carbohydrate, oil, ash, fiber, and moisture contents ranged between 8.7 and 10.8%, 68 and 71%, 3.72 and 5.56%, 1.09 and 1.81%, 1.1 and 3.1%, and 11.7 and 14.2%, respectively. Tocopherol levels in selected varieties were in the range of 461 to 1,430 µg/g. Hi-maize exhibited significantly higher protein and tocopherol contents than the other varieties, indicating its better suitability for feed and silage applications. The highest mean level of total AFs, 14.5 ± 0.12 µg/kg, was found in Desi, and results showed that the most dominant AF found in the maize varieties was AFB1. Furthermore, the results showed that the higher the level of tocopherol, the lower the concentration of total AFs and vice versa in maize varieties. The results can be used to investigate additional susceptible maize varieties that are resistant to fungal attack.