Effects of inoculation of corn silage with Saccharomyces cerevisiae on silage fermentation characteristics, nutrient digestibility, mycoflora and aflatoxin production.

In the present study, fresh whole corn plants along with half milk kernels line were treated with live and hydrolysed yeast with different concentrations before ensiling and kept in airtight bags and then into mini silos in order to achieve anaerobic conditions for proper fermentation. The buckets were opened after different time intervals to characterise the material, quick acidification, dry matter recovery, and aerobic stability of silage respectively. Moreover, mycoflora and aflatoxin contamination were also analysed. The overall result reported that the silage quality was improved by the application of live and hydrolysed yeast. The best result was reported by the application of live yeast (T2: 10 g/kg) which significantly improved the fermentative, proximate, and digestibility parameters and reduced the mycoflora and aflatoxin contamination. Our results present promising new options for the use of natural compounds that may help to improve silage quality and reduce aflatoxin contamination.

Phytomediated Silver Nanoparticles (AgNPs) Embellish Antioxidant Defense System, Ameliorating HLB-Diseased 'Kinnow' Mandarin Plants.

Citrus production is harmed worldwide by yellow dragon disease, also known as Huanglongbing (HLB), or citrus greening. As a result, it has negative effects and a significant impact on the agro-industrial sector. There is still no viable biocompatible treatment for Huanglongbing, despite enormous efforts to combat this disease and decrease its detrimental effects on citrus production. Nowadays, green-synthesized nanoparticles are gaining attention for their use in controlling various crop diseases. This research is the first scientific approach to examine the potential of phylogenic silver nanoparticles (AgNPs) to restore the health of Huanglongbing-diseased 'Kinnow' mandarin plants in a biocompatible manner. AgNPs were synthesized using Moringa oleifera as a reducing, capping, and stabilizing agent and characterized using different characterization techniques, i.e., UV-visible spectroscopy with a maximum average peak at 418 nm, scanning electron microscopy (SEM) with a size of 74 nm, and energy-dispersive spectroscopy (EDX), which confirmed the presence of silver ions along with different elements, and Fourier transform infrared spectroscopy served to confirm different functional groups of elements. Exogenously, AgNPs at various concentrations, i.e., 25, 50, 75, and 100 mgL-1, were applied against Huanglongbing-diseased plants to evaluate the physiological, biochemical, and fruit parameters. The findings of the current study revealed that 75 mgL-1 AgNPs were most effective in boosting the plants' physiological profiles, i.e., chl a, chl b, total chl, carotenoid content, MSI, and RWC up to 92.87%, 93.36%, 66.72%, 80.95%, 59.61%, and 79.55%, respectively; biochemical parameters, i.e., 75 mgL-1 concentration decreased the proline content by up to 40.98%, and increased the SSC, SOD, POD, CAT, TPC, and TFC content by 74.75%, 72.86%, 93.76%, 76.41%, 73.98%, and 92.85%, respectively; and fruit parameters, i.e., 75 mgL-1 concentration increased the average fruit weight, peel diameter, peel weight, juice weight, rag weight, juice pH, total soluble solids, and total sugarby up to 90.78%, 8.65%, 68.06%, 84.74%, 74.66%, 52.58%, 72.94%, and 69.69%, respectively. These findings enable us to develop the AgNP formulation as a potential citrus Huanglongbing disease management method.

Evaluation of Selenium Nanoparticles in Inducing Disease Resistance against Spot Blotch Disease and Promoting Growth in Wheat under Biotic Stress.

In the present study, SeNPs were synthesized using Melia azedarach leaf extracts and investigated for growth promotion in wheat under the biotic stress of spot blotch disease. The phytosynthesized SeNPs were characterized using UV-visible spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and Fourier-transformed infrared spectroscopy (FTIR). The in vitro efficacy of different concentrations of phytosynthesized SeNPs (i.e., 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, and 300 µg/mL) was evaluated using the well diffusion method, which reported that 300 µg/mL showed maximum fungus growth inhibition. For in vivo study, different concentrations (10, 20, 30, and 40 mg/L) of SeNPs were applied exogenously to evaluate the morphological, physiological, and biochemical parameters under control conditions and determine when infection was induced. Among all treatments, 30 mg/L of SeNPs performed well and increased the plant height by 2.34% compared to the control and 30.7% more than fungus-inoculated wheat. Similarly, fresh plant weight and dry weight increased by 17.35% and 13.43% over the control and 20.34% and 52.48% over the fungus-treated wheat, respectively. In leaf surface area and root length, our findings were 50.11% and 10.37% higher than the control and 40% and 71% higher than diseased wheat, respectively. Plant physiological parameters i.e., chlorophyll a, chlorophyll b, and total chlorophyll content, were increased 14, 133, and 16.1 times over the control and 157, 253, and 42 times over the pathogen-inoculated wheat, respectively. Our findings regarding carotenoid content, relative water content, and the membrane stability index were 29-, 49-, and 81-fold higher than the control and 187-, 63-, and 48-fold higher than the negative control, respectively. In the case of plant biochemical parameters, proline, sugar, flavonoids, and phenolic contents were recorded at 6, 287, 11, and 34 times higher than the control and 32, 107, 33, and 4 times more than fungus-inoculated wheat, respectively. This study is considered the first biocompatible approach to evaluate the potential of green-synthesized SeNPs as growth-promoting substances in wheat under the spot blotch stress and effective management strategy to inhibit fungal growth.

Antifungal activity of green synthesized selenium nanoparticles and their effect on physiological, biochemical, and antioxidant defense system of mango under mango malformation disease.

Plant extract-based green synthesis of nanoparticles is an emerging class of nanotechnology that has revolutionized the entire field of biological sciences. Green synthesized nanoparticles are used as super-growth promoters and antifungal agents. In this study, selenium nanoparticles (SeNPs) were synthesized using Melia azedarach leaves extract as the main reducing and stabilizing agent and characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray (EDX), and fourier transform infrared spectrometer (FTIR). The green synthesized SeNPs were exogenously applied on Mangifera indica infected with mango malformation disease. The SeNPs at a concentration of 30 µg/mL were found to be the best concentration which enhanced the physiological (chlorophyll and membrane stability index), and biochemical (proline and soluble sugar) parameters. The antioxidant defense system was also explored, and it was reported that green synthesized SeNPs significantly reduced the biotic stress by enhancing enzymatic and non-enzymatic activities. In vitro antifungal activity of SeNPs reported that 300 µg/mL concentration inhibited the Fusarium mangiferae the most. This study is considered the first biocompatible approach to evaluate the potential of green synthesized SeNPs to improve the health of mango malformation-infected plants and effective management strategy to inhibit the growth of F. mangifera.

Phytomediated Selenium Nanoparticles Improved Physio-morphological, Antioxidant, and Oil Bioactive Compounds of Sesame under Induced Biotic Stress.

Vegetable oil consumption is expected to reach almost 200 billion kilograms by 2030 in the world and almost 2.97 million tons in Pakistan. A large quantity of edible oil is imported annually from other countries to fill the gap between local production and consumption. Compared to other edible oil crops such as soybean, rapeseed, peanut and olive, sesame has innately higher (55%) oil content, which makes it an excellent candidate to be considered to meet local edible oil production. Oil seed crops, especially sesame, are affected by various pathogens, which results in decreased oil production with low quality oil. Selenium nanoparticles (SeNPs) work synergistically, as it has antifungal activity along with improving plant growth. Different concentrations of SeNPs were used, on three different varieties of sesame (TS-5, TH-6, and Till-18). Plant growth and development were accelerated by SeNPs, which ultimately led to an increase in crop yield. Morphological parameters revealed that SeNPs resulted in a growth increase of 55.7% in root length, 48% increase in leaf number/plant, and 38% in stem diameter. Out of three sesame varieties, TS-5 seedlings treated with 40 mg/L SeNPs showed 96.7% germination and 53% SVI at 40 mg/L. Sesame varieties dramatically increased antioxidant capability using SeNPs, resulting in 147% increase in SOD and 140% increase in POD enzyme units in TH-6 and 76% elevation in CAT enzymes in TS-5 (mean ± S.E). GCMS analysis revealed that bioactive compound I, sesamin, sesamol, and tocopherol contents were increased along with enhanced production of different unsaturated fatty acids. Kegg pathway analysis and MSEA revealed that these compounds were mainly involved in biosynthesis of unsaturated fatty acids, suggesting that SeNPs have elicited the biosynthesis of unsaturated fatty acids such as oleic acid, linoleic acid, and α-linoleic acid. This study concluded that SeNPs (40 mg/L) have an excellent capability to be used for crop improvement along with better oil quality.

Proximate composition, fungal isolation and contamination of aflatoxin B1 in chickpea seeds from the Punjab, Pakistan.

Chickpea, Cicer arietinum L., is a nutrient rich crop that is widely cultivated and consumed in Pakistan. However, chickpea is highly prone to fungal growth leading to contamination with aflatoxins, the most potent carcinogen found in nature. In this study, fifty chickpea seed samples were collected from the local markets of the Punjab, Pakistan, to evaluate their nutritional quality, fungal and AFB1 contamination. Proximate analysis suggested that chickpea seeds contained 5.5-6.93% moisture, 62.24-63.24% carbohydrates, 22.75-23.44% protein, 4.99-5.4% fat, 5.62-5.84% fiber and 2.92-3.16% ash. Morphological identification techniques revealed fourteen fungal species belonging to six fungal genera from which Aspergillus flavus was the leading contaminant. AFB1 analysis revealed that sixty-two percent samples were contaminated with AFB1. All the AFB1 positive samples contained AFB1 level more than 2 ppb and 12.9% samples contain AFB1 level more than 20 ppb, exceeded the maximum limit (ML) assigned by EU and USA (FDA & FAO) respectively. The results of the present studies reported that chickpea is a highly contaminated commodity in terms of fungi and AFB1 that's why further investigations and monitoring are required to reduce the fungal and AFB1 contamination. These baseline data are an initial step in the effort to deal with this significant food safety issue.

Effect of Phytosynthesized Selenium and Cerium Oxide Nanoparticles on Wheat (Triticum aestivum L.) against Stripe Rust Disease.

In this study, selenium nanoparticles (SeNPs) and cerium oxide nanoparticles (CeONPs) were synthesized by using the extract of Melia azedarach leaves, and Acorus calamusas rhizomes, respectively, and investigated for the biological and sustainable control of yellow, or stripe rust, disease in wheat. The green synthesized NPs were characterized by UV-Visible spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and X-ray diffraction (XRD). The SeNPs and CeONPs, with different concentrations (i.e., 10, 20, 30, and 40 mg/L), were exogenously applied to wheat infected with Puccinia striformis. SeNPs and CeONPs, at a concentration of 30 mg/L, were found to be the most suitable concentrations, which reduced the disease severity and enhanced the morphological (plant height, root length, shoot length, leaf length, and ear length), physiological (chlorophyll and membrane stability index), biochemical (proline, phenolics and flavonoids) and antioxidant (SOD and POD) parameters. The antioxidant activity of SeNPs and CeONPs was also measured. For this purpose, different concentrations (50, 100, 150, 200 and 400 ppm) of both SeNPs and CeONPs were used. The concentration of 400 ppm most promoted the DPPH, ABTS and reducing power activity of both SeNPs and CeONPs. This study is considered the first biocompatible approach to evaluate the potential of green synthesized SeNPs and CeONPs to improve the health of yellow, or stripe rust, infected wheat plants and to provide an effective management strategy to inhibit the growth of Puccinia striformis.

Comprehensive Review of Aflatoxin Contamination, Impact on Health and Food Security, and Management Strategies in Pakistan.

Aflatoxins (AFs) are the most important toxic, mutagenic, and carcinogenic fungal toxins that routinely contaminate food and feed. While more than 20 AFs have been identified to date, aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), and M1 (AFM1) are the most common. Over 25 species of Aspergillus have been shown to produce AFs, with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being the most important and well-known AF-producing fungi. These ubiquitous molds can propagate on agricultural commodities to produce AFs in fields and during harvesting, processing, transportation, and storage. Countries with warmer climates and that produce foods susceptible to AF contamination shoulder a substantial portion of the global AF burden. Pakistan's warm climate promotes the growth of toxigenic fungi, resulting in frequent AF contamination of human foods and animal feeds. The potential for contamination in Pakistan is exacerbated by improper storage conditions and a lack of regulatory limits and enforcement mechanisms. High levels of AFs in common commodities produced in Pakistan are a major food safety problem, posing serious health risks to the population. Furthermore, aflatoxin contamination contributes to economic losses by limiting exports of these commodities. In this review, recent information regarding the fungal producers of AFs, prevalence of AF contamination of foods and feed, current regulations, and AF prevention and removal strategies are summarized, with a major focus on Pakistan.

Light and scanning electron microscopic characterization of aflatoxins producing Aspergillus flavus in the maize crop.

Maize (Zea mays L.) is considered as one of the main cereals, used as a source of food, forage, and processed products. The loss of maize productivity is reported due to effect on roots, stalks, ears, and kernels mainly caused by many fungi. Among these fungal pathogens of maize, Aspergillus flavus (A. flavus) are the most prevalent that produces highly toxigenic aflatoxins that are highly carcinogenic to the consumers. The present study is confined to isolate and characterize the A. flavus from maize seeds for accurate identification that can be helpful for determination and management of aflatoxins in maize crop. Eighty stored seed samples of maize were collected from warehouses where seeds are stored for food and feeding purposes. For the isolation of A. flavus, Potato Dextrose Agar was used. Isolated fungi were identified macro and microscopically using light microscope and scanning electron microscope. A total of 212 Aspergillus isolates were identified based on macro-morphological and micro-morphological characteristics. The results showed that A. flavus colonies were granular, flat with yellow-green to deep yellow-green colony color having a white border and compact, spherical spore heads. Rapid rate of growth was observed maturing in about 3-5 days. In microscopic features, A. flavus have apically swollen conidiophores with various conidia bearing cells in long and dry chains. Spherical conidial heads were split into several columns ranging 300-400 µm in diameter. This will be helpful for farmers, researchers and traders in future for correct identification of sources of aflatoxins. RESEARCH HIGHLIGHTS: Maize seed samples were collected from Pothohar region of Pakistan. The fungi were isolated on PDA. Aspergillus flavus was identified macro-morphologically by observing growth rate, colony color and texture. Furthermore, these fungi were identified micro-morphologically by using light and scanning electron microscope. The 212 Aspergillus flavus strains were isolated and identified.

Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan.

Sesame Sesamum indicum L. is a major oil-based seed crop that has been widely cultivated and consumed in Pakistan. Unfortunately, sesame is highly prone to Aspergillus fungal growth in the field, and under inappropriate storage conditions can become contaminated with aflatoxins, the most potent carcinogen found in nature. Here, we have isolated a high number of Aspergillus isolates from sesame seeds in fresh and stored conditions obtained from rainfed and irrigated zones of Punjab, Pakistan, and characterized them for aflatoxigenic potentials. Using morphological identification techniques, 260 isolates were grouped as potential Aspergillus section Flavi, with 126 and 134 originating from the rainfed and irrigated zones, respectively. Out of 260 in total, 188 isolates were confirmed to produce aflatoxins. There were no significant differences in potential aflatoxigenic isolates with respect to the rainfed and irrigated zones. However, the number of potential aflatoxigenic isolates was significantly higher (p < 0.05) in stored samples than that of those from fresh sesame seeds in the rainfed and irrigated zone. Whole genome sequencing and comparative analyses of 12 select isolates have revealed that one of the A. flavus isolates, which produced very low aflatoxins (AFP10), has an elevated missense variant rate, numerous high impact mutations, and a 600 base pair deletion in the norB gene. In summary, our study provides insights into aflatoxigenic potential and the associated genetic diversity of indigenous Aspergillus section Flavi isolates and potential management strategies for reducing aflatoxin contamination levels in a major crop consumed in Punjab, Pakistan.

Mycobiota Isolation and Aflatoxin B1 Contamination in Fresh and Stored Sesame Seeds from Rainfed and Irrigated Zones of Punjab, Pakistan.

ABSTRACT: This study was carried out to evaluate the prevalence of mycobiota and aflatoxin (AF) B1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. For this purpose, 100 sesame seed samples were collected directly from the fields of major sesame-producing areas in rainfed and irrigated zones. The agar plate method was used for isolation of mycobiota, and thin-layer chromatography was used to determine AFB1 concentrations. Seed samples were then stored for 12 months. After 12 months, the seeds were again analyzed for mycobiota and AFB1 for comparison. All samples were positive for fungal growth under fresh and stored conditions. Twenty-one fungal species of 10 genera were isolated. Aspergillus flavus was the most prevalent contaminant found in fresh and stored sesame seeds from rainfed and irrigated zones, followed by Aspergillus niger, Alternaria alternata, and Fusarium oxysporum. The least prevalent fungi were Aspergillus ochraceus and Cladosporium oxysporum. Analysis revealed that 92% of fresh and 99% of stored seed samples were contaminated with AFB1. In the rainfed zone, 88% of fresh and 100% of stored seed samples were contaminated with AFB1, with mean concentrations of 15.74 and 33.8 ppb, respectively. In the irrigated zone, 96% of fresh and 98% of stored seed samples were contaminated with AFB1, with mean concentrations of 20.5 and 27.56 ppb, respectively. AFB1 concentrations >20 ppb were found in 20% of fresh and 100% of stored seeds samples from the rainfed zone and in 28% of fresh and 60% of stored samples from the irrigated zone and thus were not fit for human consumption as per the maximum limit (20 ppb) assigned by the U.S. Food and Drug Administration and the Food and Agriculture Organization of the United Nations. This report is the first on the mycobiota and AFB1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. These baseline data are an initial step in the effort to deal with this significant food safety issue.

Titanium dioxide nanoparticles elicited agro-morphological and physicochemical modifications in wheat plants to control Bipolaris sorokiniana.

The current study involves the biogenesis of titanium dioxide nanoparticles (TiO2 NPs) by using Moringa oleifera Lam. aqueous leaf extract for the reduction of titanium dioxide salt into TiO2 nanoparticles. The biosynthesized TiO2 nanoparticles were observed by using the UV-visible spectrophotometry, SEM, EDX and XRD analytical methods. It was confirmed that the nanoparticles are crystalline and exist in the size range of 10-100 nm. The FTIR analysis confirmed the presence of O-H (hydrogen bonding), N-H (amide), C-C (alkanes) and C-I (Iodo-stretch) functional groups responsible for the stabilization of nanoparticles. Various concentrations (20, 40, 60 and 80 mg/L) of TiO2 NPs were applied exogenously on wheat plants infected with a fungus Bipolaris sorokiniana responsible to cause spot blotch disease at different time intervals. The measurement of disease incidence and percent disease index showed the time-dependent response and 40 mg/L was reported a stable concentration of TiO2 NPs to reduce the disease severity. The effects of biosynthesized TiO2 NPs were also evaluated for agro-morphological (leaf and root surface area, plant fresh and dry weight and yield parameters), physiological (relative water content, membrane stability index and chlorophyll content) and non-enzymatic metabolites (soluble sugar, protein, soluble phenol and flavonoid content) in wheat plants under biotic stress and 40 mg/L concentration of TiO2 NPs was found to be effective to elicit modifications to reduce biotic stress. The current study highlights the significant role of biosynthesized TiO2 NPs in controlling fungal diseases of wheat plants and thus ultimately improving the quality and yield of wheat plants.

The Incidence of Alternaria Species Associated with Infected Sesamum indicum L. Seeds from Fields of the Punjab, Pakistan.

Sesame (Sesamum indicum) is an important oil seed crop of Asia. Yields can be negatively impacted by various factors, including disease, particularly those caused by fungi which create problems in both production and storage. Foliar diseases of sesame such as Alternaria leaf blight may cause significant yield losses, with reductions in plant health and seed quality. The work reported here determined the incidence of Alternaria species infecting sesame seeds grown in the Punjab, Pakistan. A total of 428 Alternaria isolates were obtained from 105 seed samples and grouped into 36 distinct taxonomic groups based on growth pattern and morphological characters. Isolation frequency and relative density of surface sterilized and non-surface sterilized seeds showed that three isolates (A13, A47 and A215) were the most common morphological groups present. These isolates were further identified using sequencing of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA) and the Alternaria major allergen gene (Alt a 1). Whilst ITS of rDNA did not resolve the isolates into Alternaria species, the Alt a 1 sequences exhibited > 99% homology with Alternaria alternata (KP123850.1) in GenBank accessions. The pathogenicity and virulence of these isolates of Alternaria alternata was confirmed in inoculations of sesame plants resulting in typical symptoms of leaf blight disease. This work confirms the identity of a major source of sesame leaf blight in Pakistan which will aid in formulating effective disease management strategies.

Pre-harvest aflatoxins and Aspergillus flavus contamination in variable germplasms of red chillies from Kunri, Pakistan.

Various cultivars of red chilli were collected from a small town named Kunri, located in the province Sindh, Pakistan. This town is a hub of red chilli production in Asia. A total of 69 samples belonging to 6 cultivars were obtained and analysed for the occurrence of aflatoxins and Aspergillus flavus, to explore the potential of resistant and susceptible germplasm. Aflatoxins were detected by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), while A. flavus was isolated and identified using agar plate, blotter paper, deep freezing and dilution techniques. Molecular characterization using internal transcribed spacer (ITS) 1/4 and A. flavus specific FL1-F/R primers confirmed the identity of A. flavus. The data revealed that 67 and 75% samples contaminated with aflatoxin B1 (AFB1) and with A. flavus, respectively. A highly susceptible chilli cultivar was 'Nagina', showing 78.8% frequency of total aflatoxins (1.2-600 µg/kg) and a mean of 87.7 µg/kg for AFB1 and 121.9 µg/kg for total aflatoxins. A. flavus was detected with 93% frequency and 2.14 × 104 colony forming units. In contrast, cultivars 'Kunri' and 'Drooping Type' were found to be resistant, with low levels of aflatoxins and fungal counts. The study was conducted for the first time to explore two potential cultivars that were less susceptible towards A. flavus and aflatoxin contamination. These cultivars could be preferably cultivated and thereby boost Pakistan's chilli production.

Evaluation of antioxidant potential and HPLC based identification of phenolics in Polygonum amplexicaule extract and its fractions.

There is a growing interest for the plant-based medicines in pharmaceutical industry. Plant derived Antioxidants have gained huge importance regarding their medicinal value. The present study was designed to establish pharmaceutical value of Polygonum amplexicaule for their antioxidant activity using shoot, leaf and rhizome crude methanolic extract along with their n-butanolic, ethanolic, ethyl acetate and aqueous fractions. DPPH assay was used to assess antioxidants, which shows the maximum activity by crude methanolic extract of leaves (CMEL) having IC(50) 1.03 µg/ml where all other fractions showed IC(50) in a range of 1.03-58.2 µg/mL. The DNA plasmid protection assay showed that 10 ppm and 100 ppm concentrations of crude methanolic extracts (rhizome and leaf), aqueous fractions (shoot and leaf extract), n-butanolic fractions (shoot and leaf extract) and ethanolic fraction (rhizome extract) have DNA protection properties. TLC and HPLC based Identification of different antioxidants present in shoot, leaf and rhizome crude extracts and their fractions showed the presence of gallic acid, quercetin, catechin, caffeic acid, rutin, myricetin and kaempferol. This study suggested that this plant have high content of antioxidants, which needs to be investigated further for their medicinal and/cosmaceutical applications.