Aflatoxins in Wheat Grains: Detection and Detoxification through Chemical, Physical, and Biological Means.

Wheat (Triticum aestivum L.) is an essential food crop in terms of consumption as well as production. Aflatoxin exposure has a widespread public health impact in economically developing nations, so there is a need to establish preventive techniques for these high-risk populations. Pre-harvest and post-harvest practices are the two strategies used to control aflatoxin contamination, which include the use of genetically modified crops that show resistance against Aspergillus infection, the use of pesticides, changing the planting and harvesting time of crops, and physical, chemical, and biological methods. In this research, aflatoxin detection and quantification were performed in different wheat varieties to determine quantitative differences in comparison to the European Commission's limit of 4 ppb aflatoxins in wheat. TLC for qualitative and the ELISA kit method for quantitative analysis of aflatoxins were used. Out of 56 samples, 35 were found contaminated with aflatoxins, while the remaining 21 samples did not show any presence of aflatoxins. Out of the 35 contaminated samples, 20 samples showed aflatoxin contamination within the permissible limit, while the remaining 15 samples showed aflatoxin concentration beyond the permissible level, ranging from 0.49 to 20.56 ppb. After quantification, the nine highly contaminated wheat samples were detoxified using physical, chemical, and biological methods. The efficiency of these methods was assessed, and they showed a significant reduction in aflatoxins of 53-72%, 79-88%, and 80-88%, respectively. In conclusion, the difference in aflatoxin concentration in different wheat varieties could be due to genetic variations. Furthermore, biological treatment could be the method of choice for detoxification of aflatoxins in wheat as it greatly reduced the aflatoxin concentration with no harmful effect on the quality of the grains.

Multi-Locus Phylogenetic Analysis Revealed the Association of Six Colletotrichum Species with Anthracnose Disease of Coffee (Coffea arabica L.) in Saudi Arabia.

Several Colletotrichum species are able to cause anthracnose disease in coffee (Coffea arabica L.) and occur in all coffee production areas worldwide. A planned investigation of coffee plantations was carried out in Southwest Saudi Arabia in October, November, and December 2022. Various patterns of symptoms were observed in all 23 surveyed coffee plantations due to unknown causal agents. Isolation from symptomatic fresh samples was performed on a PDA medium supplemented with streptomycin sulfate (300 mg L-1) and copper hydroxide (42.5 mg L-1). Twenty-seven pure isolates of Colletotrichum-like fungi were obtained using a spore suspension method. The taxonomic placements of Colletotrichum-like fungi were performed based on the sequence dataset of multi-loci of internal transcribed spacer region rDNA (ITS), chitin synthase I (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB2), and partial mating type (Mat1-2) (ApMat) genes. The novel species are described in detail, including comprehensive morphological characteristics and colored illustrations. The pathogenicity of the isolated Colletotrichum species was assessed on detached coffee leaves as well as green and red fruit under laboratory conditions. The multi-locus phylogenetic analyses of the six-loci, ITS, ACT, CHS-1, TUB2, GAPDH and ApMat, revealed that 25 isolates were allocated within the C. gloeosporioides complex, while the remaining two isolates were assigned to the C. boninense complex. Six species were recognized, four of them, C. aeschynomenes, C. siamense, C. phyllanthi, and C. karstii, had been previously described. Based on molecular analyses and morphological examination comparisons, C. saudianum and C. coffeae-arabicae represent novel members within the C. gloeosporioides complex. Pathogenicity investigation confirmed that the Colletotrichum species could induce disease in coffee leaves as well as green and red fruits with variations. Based on the available literature and research, this is the first documentation for C. aeschynomenes, C. siamense, C. karstii, C. phyllanthi, C. saudianum, and C. coffeae-arabicae to cause anthracnose on coffee in Saudi Arabia.

Antiviral Activity of Chitosan Nanoparticles and Chitosan Silver Nanocomposites against Alfalfa Mosaic Virus.

Plant viruses are a global concern for sustainable crop production. Among the currently available antiviral approaches, nanotechnology has been overwhelmingly playing an effective role in circumventing plant viruses. Alfalfa mosaic virus (AMV) was isolated and identified from symptomatic pepper plants in Egypt using symptomatology, serological tests using the direct ELISA technique, differential hosts and electron microscopy. The virus was biologically purified from a single local lesion that developed on Chenopodium amaranticolor. The AMV infection was further confirmed using an AMV coat protein-specific primer RT-PCR. We further evaluated the antiviral potential of chitosan nanoparticles (CS-NPs) and chitosan silver nanocomposites (CS-Ag NC) in different concentrations against AMV infections in pepper plants. All tested concentrations of CS-NPs and CS-Ag NC induced the inhibition of AMV systemically infected pepper plants when applied 24 h after virus inoculation. The foliar application of 400 ppm CS-NPs or 200 ppm CS-Ag NC produced the highest AMV inhibitory effect (90 and 91%) when applied 24 h after virus inoculation. Treatment with CS-NPs and CS-Ag NC considerably increased the phenol, proline and capsaicin contents compared to the infected plants. Moreover, the agronomic metrics (plant height, fresh and dry pod weights and number of pods per plant) were also significantly improved. According to our results, the potential applications of CS-NPs and CS-Ag NC may provide an effective therapeutic measure for better AMV and other related plant virus management.

Chitosan-Decorated Copper Oxide Nanocomposite: Investigation of Its Antifungal Activity against Tomato Gray Mold Caused by Botrytis cinerea.

Owing to the remarkable antimicrobial potential of these materials, research into the possible use of nanomaterials as alternatives to fungicides in sustainable agriculture is increasingly progressing. Here, we investigated the potential antifungal properties of chitosan-decorated copper oxide nanocomposite (CH@CuO NPs) to control gray mold diseases of tomato caused by Botrytis cinerea throughout in vitro and in vivo trials. The nanocomposite CH@CuO NPs were chemically prepared, and size and shape were determined using Transmission Electron Microscope (TEM). The chemical functional groups responsible for the interaction of the CH NPs with the CuO NPs were detected using the Fourier Transform Infrared (FTIR) spectrophotometry. The TEM images confirmed that CH NPs have a thin and semitransparent network shape, while CuO NPs were spherically shaped. Furthermore, the nanocomposite CH@CuO NPs ex-habited an irregular shape. The size of CH NPs, CuO NPs and CH@CuO NPs as measured through TEM, were approximately 18.28 ± 2.4 nm, 19.34 ± 2.1 nm, and 32.74 ± 2.3 nm, respectively. The antifungal activity of CH@CuO NPs was tested at three concentrations of 50, 100 and 250 mg/L and the fungicide Teldor 50% SC was applied at recommended dose 1.5 mL/L. In vitro experiments revealed that CH@CuO NPs at different concentrations significantly inhibited the reproductive growth process of B. cinerea by suppressing the development of hyphae, spore germination and formation of sclerotia. Interestingly, a significant control efficacy of CH@CuO NPs against tomato gray mold was observed particularly at concentrations 100 and 250 mg/L on both detached leaves (100%) as well as the whole tomato plants (100%) when compared to the conventional chemical fungicide Teldor 50% SC (97%). In addition, the tested concentration 100 mg/L improved to be sufficient to guarantee a complete reduction in the disease's severity (100%) to tomato fruits from gray mold without any morphological toxicity. In comparison, tomato plants treated with the recommended dose 1.5 mL/L of Teldor 50% SC ensured disease reduction up to 80%. Conclusively, this research enhances the concept of agro-nanotechnology by presenting how a nano materials-based fungicide could be used to protect tomato plants from gray mold under greenhouse conditions and during the postharvest stage.

Lignin-Loaded Carbon Nanoparticles as a Promising Control Agent against Fusarium verticillioides in Maize: Physiological and Biochemical Analyses.

Lignin, a naturally occurring biopolymer, is produced primarily as a waste product by the pulp and paper industries and burned to produce electricity. Lignin-based nano- and microcarriers found in plants are promising biodegradable drug delivery platforms. Here, we highlight a few characteristics of a potential antifungal nanocomposite consisting of carbon nanoparticles (C-NPs) with a defined size and shape containing lignin nanoparticles (L-NPs). Spectroscopic and microscopic studies verified that the lignin-loaded carbon nanoparticles (L-CNPs) were successfully prepared. Under in vitro and in vivo conditions, the antifungal activity of L-CNPs at various doses was effectively tested against a wild strain of F. verticillioides that causes maize stalk rot disease. In comparison to the commercial fungicide, Ridomil Gold SL (2%), L-CNPs introduced beneficial effects in the earliest stages of maize development (seed germination and radicle length). Additionally, L-CNP treatments promoted positive effects on maize seedlings, with a significant increment in the level of carotenoid, anthocyanin, and chlorophyll pigments for particular treatments. Finally, the soluble protein content displayed a favorable trend in response to particular dosages. Most importantly, treatments with L-CNPs at 100 and 500 mg/L significantly reduced stalk rot disease by 86% and 81%, respectively, compared to treatments with the chemical fungicide, which reduced the disease by 79%. These consequences are substantial considering the essential cellular function carried out by these special natural-based compounds. Finally, the intravenous L-CNPs treatments in both male and female mice that affected the clinical applications and toxicological assessments are explained. The results of this study suggest that L-CNPs are of high interest as biodegradable delivery vehicles and can be used to stimulate favorable biological responses in maize when administered in the recommended dosages, contributing to the idea of agro-nanotechnology by demonstrating their unique qualities as a cost-effective alternative compared to conventional commercial fungicides and environmentally benign nanopesticides for long-term plant protection.

Mycotoxins from Tomato Pathogenic Alternaria alternata and Their Combined Cytotoxic Effects on Human Cell Lines and Male Albino Rats.

The Alternaria species are considered to produce a plethora of several mycotoxins constituting a risk factor for both human and animal health. This work aimed mainly to explore the cytotoxicity of a combined mixture of altenuene (ALT), alternariol (AOH), tenuazonic acid (TeA), and altenuisol (AS) toxins produced by pathogenic A. alternata toward human oral epithelial cells (PCS-200-014), lung fibroblast cells (WI-38), and male albino rats. The sequencing of the multi-locus, RNA polymerase second largest subunit (rpb2), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and Alternaria major allergen gene (Alt a 1) was performed to infer relationships among isolated Alternaria species. The phylogenetic analysis of gapdh, rpb2, and Alt-a 1 sequence data indicated that all isolates resided in A. alternata. The pathogenic potentiality of A. alternata was investigated on tomato plants cv. super strain B under greenhouse conditions, and all isolates were pathogenic to tomato plants, with significant (p < 0.05) variations. The ability of A. alternata isolates to produce mycotoxins was also explored using high-performance liquid chromatography (HPLC). All tested isolates were able to produce at least one of the assessed mycotoxins-ALT, AOH, TeA, and AS-and ALT was reported as the dominant mycotoxin, produced by 80% of A. alternata isolates. The cytotoxic properties of the combined mixture of ALT, AOH, TeA, and AS at concentrations of 31.25, 62.50, 125, 250, and 500 µg/mL were assessed via the MTT assay method after exposure for 24 h versus the control. The treatment of both cell lines with combined mixtures of ALT, AOH, TeA, and AS showed a dose-dependent decrease in cell viability. The highest concentrations tested at 62.50, 125, 250, and 500 µg/mL significantly decreased cell viability and caused cell damage compared to the lowest concentration of 31.25 µg/mL and the control. The cytotoxicity and genotoxicity of the combined mixtures of ALT, AOH, TeA, and AS on male albino rats were also investigated via the gene expression of (TNF-α) and using hematological (CBC), chemical (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea and creatinine), and histopathological analyses. A marked increase was observed in the levels of ALT, AST, urea and creatinine, TNF-α gene expression, red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), and packed cell volume % (PCV) after 28 days of exposure relative to the untreated control. Pathological alterations were also observed in the liver and kidney tissues of rats. Conclusively, this work provides a new understanding on the cytotoxicity and genotoxicity of mycotoxins of pathogenic A. alternata from tomatoes.

Population Dynamics of Phytophthora infestans in Egypt Reveals Clonal Dominance of 23_A1 and Displacement of 13_A2 Clonal Lineage.

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are the most economically important vegetable crops in Egypt and worldwide. The winter crop in Egypt is particularly prone to late blight caused by Phytophthora infestans. A total of 152 P. infestans isolates were isolated from the 2013, 2014, 2016 and 2018 winter crops with 82 isolates from potato, 69 from tomato and one isolate from eggplant (S. melongena L.). All isolates belonged to the A1 mating type with no evidence of A2 or self-fertile strains. The majority of isolates (53%) were sensitive to metalaxyl, 32% were intermediate and 15% were resistant. Variation in aggressiveness between three P. infestans isolates EG-005 (13_A2) and EG-276 (23_A1) from potato, and EG-237 (23_A1) from eggplant was determined on tuber slices and leaflets of 10 potato cultivars. The eggplant isolate EG-237 showed higher sporulation capacity compared with the other tested isolates and was able to infect potato (Lady Rosetta cv) and tomato (Super Strain B cv). The simple sequence repeat (SSR) genotyping data showed that in contrast to our previous work (3-year period 2010-12) in which the proportion of 13_A2 lineage was 35%, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. The data have been submitted into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. The AVR2 and AVR2-like RXLR effector genes were amplified and sequenced. In the avirulent AVR2 gene, only one heterozygous SNP was detected at position 31 in the N terminus in six isolates out of eleven, whereas two heterozygous SNPs were detected at position 29 in the N-terminus and ninety-two in the C- terminus of the AVR2-like gene. This suggests that changes in the previously reported virulence profile of 23_A1 are not related to commercial cultivars carrying the R2 gene. In addition, this is the first report of P. infestans on eggplant in Egypt.

Diversity among Lasiodiplodia Species Causing Dieback, Root Rot and Leaf Spot on Fruit Trees in Egypt, and a Description of Lasiodiplodia newvalleyensis sp. nov.

Lasiodiplodia (family Botryosphaeriaceae) is a widely distributed fungal genus that causes a variety of diseases in tropical and subtropical regions. During 2020−2021, a routine survey of fruit tree plants was conducted in five Egyptian Governorates, and fresh samples exhibiting dieback, decline, leaf spot and root rot symptoms were collected. Collection from eight different symptomatic leaves, twigs, branches and roots of fruit trees yielded 18 Lasiodiplodia-like isolates. The sequencing data from the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (tef1-a) and ß-tubulin (tub2) were used to infer phylogenetic relationships with known Lasiodiplodia species. Two isolates obtained from black necrotic lesions on Phoenix dactylifera leaves were identified as a putative novel species, L. newvalleyensis sp. nov., and were thus subjected to further morphological characterization. The results of isolation and molecular characterization revealed that L. theobromae (n = 9) was the most common species on Mangifera indica, Citrus reticulata, C. sinensis, Ficus carica, Prunus persica, Prunus armeniaca and Pyrus communis trees. Lasiodiplodia pseudotheobromae (n = 5) was isolated from M. indica, Prunus persica and C. sinensis. Lasiodiplodia laeliocattleyae (n = 2) was isolated from C. reticulata. Pathogenicity test results suggested that all Lasiodiplodia species were pathogenic to their hosts. The present study is considered the first to characterize and decipher the diversity of Lasiodiplodia species associated with fruit trees in Egypt, using the multi-locus ITS, tef1-a and tub2 sequence data, along with morphological and pathogenic trials. To our knowledge, this is the first report of L. newvalleyensis on Phoenix dactylifera and L. laeliocattleya on C. reticulata in Egypt and worldwide.

First report of seedling stem blight of mango caused by Neofusicoccum parvum in Italy.

Seedling blight of mango (Mangifera indica L.) was observed in a commercial nursery located in Messina province (eastern Sicily, Italy) during winter of 2021. More than 30% of 3,000 seedlings, about three to six months old, of mango cv. Gomera 3 showed symptoms of basal stem blight. The symptoms started from seed, led to the decline and subsequent death of the plants. Necrotic lesions appeared at crown level two months after sowing. The stem tissues of ten symptomatic plants were cut, surface sterilized, dipped in 1.5% sodium hypochlorite for 1 min and transferred onto potato dextrose agar medium (PDA) and incubated at 25°C for four days. Approximately 60% of stem tissues developed very similar fungal colonies, resembling to Botryosphaeriaceae. A total of four representative isolates were collected through single hyphal-tip and stored at 4 °C. The internal transcriber spacer region (ITS) was amplified with primers, ITS5/ITS4 (White et al., 1990), and EF1-728F and EF1-986R (Carbone and Kohn, 1999) were used to amplify part of the translation elongation factor 1alpha gene (tef1-α), and primers Bt2a/Bt2b (Glass and Donaldson, 1995) were used for the partial ß-tubulin (tub2). The obtained sequences were deposited in GenBank with accession numbers: ON911292-95 for the ITS, ON933621-24 for tef1-α and ON933625-28 for tub2.To compare the results, 50 additional sequences were selected and inserted in the alignment according to the recent literature on the Botryosphaeriaceae (Bezerra et al., 2021; Zhang et al., 2021). Maximum parsimony analysis (MP) of concatenated dataset (ITS + tef1-α + tub2) was performed in PAUP v.4.0a. Clade support was assessed by 1,000 bootstrap replicates and Botryosphaeria dothidea was used as an outgroup. Our isolates clustered within the group of Neofusicoccum parvum (71% bootstrap value) (ex-type CMW9081). Based on these results, and morphological data (50 conidia length × width average: 18.1 × 6.6, respectively) our isolates (named MC) were identified and confirmed as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Pathogenicity tests were also conducted on 18 mango cv. Gomera 3 seedlings. The crown roots of each seedling were mechanically wounded and a mycelial plug of the isolate MC14 was placed onto them and covered with soil. Controls (three seedlings) were inoculated with sterile PDA only. Seedlings were maintained in a growth chamber with a 12 hrs photoperiod at 25°C ± 1°C and watered regularly. After five days, stem lesions appeared externally (1.6 cm) and one month after the inoculation, all the inoculated seedlings died. However, controls did not show any obvious symptoms. Re-isolations were conducted as described above and fulfilled Koch's postulates confirming pathogenicity. Among the diseases affecting mango plants, Botryosphaeriaceae represent a serious threat in Sicily as reported by Aiello et al., 2022. The endophytic behaviour of Botryosphaeriaceae is well known, making them latent pathogens (Slippers and Wingfield, 2007). In Italy, N. parvum was detected in mango orchards since 2013 (Ismail et al. 2013), but symptoms of seedlings stem blight have never been reported in the nursery. In Sicily, an increase of Botryosphaeriaceae infection has been observed recently, especially in nurseries, where N. parvum has been identified as a most destructive pathogen (Aiello et al., 2020; Gusella et al., 2021). To our knowledge, this is the first report worldwide of N. parvum causing mango seedling blight. The high incidence of infected seedlings detected in this study highlights the potential risk during propagation in the nursery, representing a significant source of inoculum for the field.

Humic Acid-Coated Fe3O4 Nanoparticles Confer Resistance to Acremonium Wilt Disease and Improve Physiological and Morphological Attributes of Grain Sorghum.

Acremonium wilt disease affects grain quality and reduces sorghum yield around the globe. The present study aimed to assess the efficacy of humic acid (HA)-coated Fe3O4 (Fe3O4/HA) nanoparticles (NPs) in controlling acremonium wilt disease and improving sorghum growth and yields. During the season 2019, twenty-one sorghum genotypes were screened to assess their response to Acremonium striticum via artificial infection under field conditions and each genotype was assigned to one of six groups, ranging from highly susceptible to highly resistant. Subsequently, over the two successive seasons 2020 and 2021, three different concentrations of 10, 40 and 80 mg L-1 of Fe3O4/HA NPs were tested against A. striticum. The concentrations of 40 and 80 mg L-1 were found to be highly effective in controlling acremonium wilt disease on different sorghum genotypes: LG1 (highly susceptible), Giza-3 (susceptible), and Local 119 (resistant) genotypes. After harvest, the physiological (growth and yield) and biochemical (peroxidase, catalase, and gibberellic acid) attributes of sorghum plants were determined, and the results demonstrated that concentrations of 40 and 80 mg L-1 increased peroxidase and catalase activities in healthy (uninoculated) sorghum genotypes compared to inoculated sorghum genotypes. Additionally, the toxicity of Fe3O4/HA NPs on male albino rats was investigated via hematological (CBC), chemical (ALT and AST) and histopathological analyses. The concentration 80 mg L-1 of Fe3O4/HA NPs caused a marked increase in ALT and creatinine level after 51 days of feeding. Severe pathological alterations were also observed in liver and kidney tissues of rats administered with grain sorghums treated with 80 mg L-1. In comparison with the untreated control plants, a concentration of 40 mg L-1 significantly increased the growth, yield and gibberellic acid levels (p ≤ 0.05) and was found to be safe in male albino rats. Conclusively, a concentration of 40 mg L-1 of Fe3O4/HA NPs showed promising results in curtailing A. striticum infections in sorghum, indicating its great potential to substitute harmful fertilizers and fungicides as a smart agriculture strategy.