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Abstract The current reports on phytopathogens multidrug resistance have become a significant issue for plant health and global food security. Antimicrobial peptides (AMPs) have recently gained generous attention as potential alternatives to prevent plant disease resistance because of their potent, multifarious antimicrobial activity. AMPs are low-weight protein molecules. Living organisms secrete a wide range of AMPs, with some synthesised by canonical gene expression, known as ribosomal AMPs, and non-ribosomal AMPs, synthesised by modular enzyme-generating systems. Plants produce an array of AMPs, yet they are still unknown to many infection processes of causal agents. Plant-derived AMPs have a wide range of structures and functions, and they induce an innate immune system in plants. The biologically active AMPs in plants mainly depend on direct and indirect interactions with membrane lipids. Transgenic plants have expressed several AMPs, the basis for the model of new synthetic analogues, to provide support against diseases. These peptides have shown significant ability to manage plant diseases and can provide an eco-friendly alternative to hazardous conventional methods. Here, we have a comprehensive study on AMPs to identify their role in plant pathogen stress suppression activities and their mode of action. This would surely facilitate a bottomless insight into AMPs' mode of action against pathogen infections. An improved understanding of the mechanism will facilitate the development of the next generation of antimicrobial peptides, potentially employing a multitargeted approach.

Bakanae, caused by Fusarium fujikuroi, is a serious seed-borne disease affecting rice production worldwide. Herein, we investigated the potential of propolis, a natural honeybee product, collected from three geographic locations in Egypt: North (Menofia), Middle (Cairo), and South (Sohag), and extracted with four different solvents (ethanol, olive oil, hexane, and water) as a sustainable eco-friendly alternative to control the Bakanae disease in rice. In vitro experiments showed that propolis extracts exhibited antifungal activity against F. fujikuroi and significantly suppressed its mycelial growth and sporulation. The antifungal activity of propolis extracts was associated with its chemical composition which varied depending on geographical origin, and the extraction solvents. Moreover, SEM-based analysis revealed that ethanolic extract of northern propolis prominently altered the microconidia morphology of F fujikuroi, which shrunken and seemed to lose their viability. These findings were further confirmed in vivo under greenhouse conditions. Soaking F fujikuroi-inoculated rice seeds in 1000 ppm of propolis extract significantly reduced the bakanae disease incidence, and disease severity index compared to mock-treated controls. Although infection with F fujikuroi induced the accumulation of H2O2, the application of propolis extracts alleviated oxidative stress and significantly reduced the H2O2 levels within infected rice seedlings. Moreover, propolis extracts enhanced the profile of enzymatic antioxidants (guaiacol-dependent peroxidase [POX] and polyphenol oxidase [PPO]) in F. fujikuroi-infected rice seedlings. Finally, Propolis extracts-treated rice seedlings exhibited higher transcript levels of three PTI-marker genes including nonexpressor of pathogenesis-related genes 1 (OsNPR1), WRKY transcription factor 21(OsWRKY21), and phenylalanine ammonia-lyase (OsPAL1), which are associated with systemic acquired resistance (SAR) and crucial for the plant's defense response against pathogens. Collectively, these findings suggest that propolis might be a promising sustainable, eco-friendly alternative to control bakanae disease and other fungal seed-borne phytopathogens due to its antifungal properties and ability to induce a complex multilayered defense system within infected plants.

White mold, caused by the necrotrophic fungus Sclerotinia sclerotiorum, is a challenging disease to common bean cultivation worldwide. In the current study, two non-proteinogenic amino acids (NPAAs), γ-aminobutyric acid (GABA) and ß-alanine, were suggested as innovative environmentally acceptable alternatives for more sustainable management of white mold disease. In vitro, GABA and ß-alanine individually demonstrated potent dose-dependent fungistatic activity and effectively impeded the radial growth and development of S. sclerotiorum mycelium. Moreover, the application of GABA or ß-alanine as a seed treatment followed by three root drench applications efficiently decreased the disease severity, stimulated plant growth, and boosted the content of photosynthetic pigments of treated S. sclerotiorum-infected plants. Furthermore, although higher levels of hydrogen peroxide (H2O2), superoxide anion (O2•−), and malondialdehyde (MDA) indicated that S. sclerotiorum infection had markedly triggered oxidative stress in infected bean plants, the exogenous application of both NPAAs significantly reduced the levels of the three studied oxidative stress indicators. Additionally, the application of GABA and ß-alanine increased the levels of both non-enzymatic (total soluble phenolics and flavonoids), as well as enzymatic (catalase [CAT], peroxidases [POX], and polyphenol oxidase [PPO]) antioxidants in the leaves of S. sclerotiorum-infected plants and improved their scavenging activity and antioxidant efficiency. Applications of GABA and ß-alanine also raised the proline and total amino acid content of infected bean plants. Lastly, the application of both NPAAs upregulated the three antioxidant-related genes PvCAT1, PvCuZnSOD1, and PvGR. Collectively, the fungistatic activity of NPAAs, coupled with their ability to alleviate oxidative stress, enhance antioxidant defenses, and stimulate plant growth, establishes them as promising eco-friendly alternatives for white mold disease management for sustainable bean production.

Gray mold, caused by Botrytis cinerea Pers. Fr., is one of the most vital plant diseases, causing extensive pre- and post-harvest losses in apple fruits. In the current study, we isolated and identified two potential endophytic bioagents, Bacillus subtilis and Streptomyces endus. Both bioagents exhibited a potent fungistatic effect against B. cinerea under both in vitro and in planta conditions. Moreover, two experiments were carried out; (i) the first experiment was conducted at room temperature after artificial inoculation with B. cinerea to monitor the progression of the infection and the corresponding biochemical responses of the apples. Our in vivo findings showed that the treated B. cinerea-infected apple fruits with the cell-free bacterial filtrate of B. subtilis and S. endus (dipping or wrapping) significantly reduced the rotten area of the treated apple at room temperature. Additionally, B. subtilis and S. endus enhanced the enzymatic (POX and PPO) and non-enzymatic (phenolics and flavonoids) antioxidant defense machinery in treated apples. (ii) The second experiment focused on the preventive effects of both bioagents over a 90-day storage period at 1 °C of healthy apples (no artificial inoculation). The application of both bacterial filtrates prolonged the storage period, reduced the relative weight loss, and maintained high-quality parameters including titratable acidity, firmness, and total soluble solids of apple fruits under cold storage at 1 °C. The Kaplan–Meier analysis of rotten apples over 90 days during cold storage showed that the treated apples lasted longer than the non-treated apples. Moreover, the lifespan of apple fruits dipped in the culture filtrate of B. subtilis, or a fungicide, was increased, with no significant differences, compared with the non-treated apples. The current results showed the possibility of using both bioagents as a safe and eco-friendly alternative to chemical fungicides to control gray mold disease in apples.

IntroductionBacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastative diseases that threatens rice plants worldwide. Biosynthesized nanoparticle (NP) composite compounds have attracted attention as environmentally safe materials that possess antibacterial activity that could be used in managing plant diseases.MethodsDuring this study, a nanocomposite of two important elements, nickel and silicon, was biosynthesized using extraction of saffron stigmas (Crocus sativus L.). Characterization of obtained nickel-silicon dioxide (Ni-SiO2) nanocomposite was investigated using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Transmission/Scanning electron microscopy (TEM/SEM), and energy-dispersive spectrum (EDS). Antibacterial activities of the biosynthesized Ni-SiO2 nanocomposite against Xoo were tested by measuring bacterial growth, biofilm formation, and dead Xoo cells.Results and discussionsThe bacterial growth (OD600) and biofilm formation (OD570) of Xoo treated with distilled water (control) was found to be 1.21 and 1.11, respectively. Treatment with Ni-SiO2 NPs composite, respectively, reduced the growth and biofilm formation by 89.07% and 80.40% at 200 μg/ml. The impact of obtained Ni-SiO2 nanocomposite at a concentration of 200 μg/ml was assayed on infected rice plants. Treatment of rice seedlings with Ni-SiO2 NPs composite only had a plant height of 64.8 cm while seedlings treated with distilled water reached a height of 45.20 cm. Notably, Xoo-infected seedlings treated with Ni-SiO2 NPs composite had a plant height of 57.10 cm. Furthermore, Ni-SiO2 NPs composite sprayed on inoculated seedlings had a decrease in disease leaf area from 43.83% in non-treated infected seedlings to 13.06% in treated seedlings. The FTIR spectra of biosynthesized Ni-SiO2 nanocomposite using saffron stigma extract showed different bands at 3,406, 1,643, 1,103, 600, and 470 cm−1. No impurities were found in the synthesized composite. Spherically shaped NPs were observed by using TEM and SEM. EDS revealed that Ni-SiO2 nanoparticles (NPs) have 13.26% Ni, 29.62% Si, and 57.11% O. Xoo treated with 200 µg/ml of Ni-SiO2 NPs composite drastically increased the apoptosis of bacterial cells to 99.61% in comparison with 2.23% recorded for the control.ConclusionsThe application of Ni-SiO2 NPs significantly improved the vitality of rice plants and reduced the severity of BLB.

Secondary metabolites in plants have been recognized as a novel basis of potential bio-pesticides, paving the way for their use in sustainable agriculture. Plant secondary metabolites have pivotal roles in plant-pathogen interactions. Some important secondary metabolites of plants are terpenoids, flavanols, flavones, etc., are stress-inducible phytochemicals playing an important role in plant immune response development. Pathogen enters into host cell, multiply and utilise the biological mechanism of plants, causing a hazard to world food assembly. Under stressed circumstances, plants evolve a powerful and intricate system of growth and defensive action. On the other hand, transcription factors (TFs) assist host plants to counter adverse environments by acting as mediators of stress signal and regulating the stress-responsive gene expression. The transcriptional and post-transcriptional manipulation of transcriptional factors is capable of aid in molecular breeding and genetic modification meant for improved secondary metabolite synthesis. Although the presence of numerous secondary metabolites has been established in plant life, very slight is known about their interaction with pathogens and the specific mechanisms involved in leading to plant immunity. Chemical pesticides are wreaking havoc on our environment. As a consequence, environmental-friendly alternatives to disease management, like plant-based metabolites, should be explored. In this appraisal, we have reviewed plant secondary metabolites in relation to pathogens, their contribution to innate immunity, mechanism of action, and regulation of TFs in response to combating plant infections in an eco-friendly approach.

Although the mitochondria retain all required enzymes for an intact tricarboxylic acid (TCA) cycle, plants might shift the cyclic flux from the TCA cycle to an alternative noncyclic pathway via γ-aminobutyric acid (GABA) shunt under specific physiological conditions. We hypothesize that several genes may ease this noncyclic flux and contribute to the citrus response to the phytopathogenic bacterium ‘Candidatus Liberibacter asiaticus’, the causal agent of Huanglongbing in citrus. To test this hypothesis, we used multiomics techniques (metabolomics, fluxomics, and transcriptomics) to investigate the potential roles of putative gab homologies from Valencia sweet orange (Citrus sinensis). Our findings showed that ‘Ca. L. asiaticus’ significantly increased the endogenous GABA and succinate content but decreased ketoglutarate in infected citrus plants. Citrus genome harbors three putative gab genes, including amino-acid permease (also known as GABA permease; CsgabP), GABA transaminase (CsgabT), and succinate-semialdehyde dehydrogenase (also known as GABA dehydrogenase; CsgabD). The transcript levels of CsgabP, CsgabT, and CsgabD were upregulated in citrus leaves upon the infection with ‘Ca. L. asiaticus’ and after the exogenous application of GABA or deuterium-labeled GABA isotope (GABA-D6). Moreover, our finding showed that exogenously applied GABA is quickly converted to succinate and fed into the TCA cycle. Likewise, the fluxomics study showed that GABA-D6 is rapidly metabolized to succinate-D4. Our work proved that GABA shunt and three predicated gab genes from citrus, support the upstream noncyclic flux toward succinate rather than an intact TCA cycle and contribute to citrus defense responses to ‘Ca. L. asiaticus’.[Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Huanglongbing (HLB; also known as citrus greening) is the most destructive bacterial disease of citrus worldwide with no known sustainable cure yet. Herein, we used non-targeted metabolomics and transcriptomics to prove that γ-aminobutyric acid (GABA) accumulation might influence the homeostasis of several metabolic pathways, as well as antioxidant defense machinery, and their metabolism-related genes. Overall, 41 metabolites were detected in ‘Valencia’ sweet orange (Citrus sinensis) leaf extract including 19 proteinogenic amino acids (PAA), 10 organic acids, 5 fatty acids, and 9 other amines (four phenolic amines and three non-PAA). Exogenous GABA application increased most PAA in healthy (except L-threonine, L-glutamine, L-glutamic acid, and L-methionine) and ‘Candidatus L. asiaticus’-infected citrus plants (with no exception). Moreover, GABA accumulation significantly induced L-tryptophan, L-phenylalanine, and α-linolenic acid, the main precursors of auxins, salicylic acid (SA), and jasmonic acid (JA), respectively. Furthermore, GABA supplementation upregulated most, if not all, of amino acids, phenolic amines, phytohormone metabolism-related, and GABA shunt-associated genes in both healthy and ‘Ca. L. asiaticus’-infected leaves. Moreover, although ‘Ca. L. asiaticus’ induced the accumulation of H2O2 and O2•− and generated strong oxidative stress in infected leaves, GABA possibly stimulates the activation of a multilayered antioxidative system to neutralize the deleterious effect of reactive oxygen species (ROS) and maintain redox status within infected leaves. This complex system comprises two major components: (i) the enzymatic antioxidant defense machinery (six POXs, four SODs, and CAT) that serves as the front line in antioxidant defenses, and (ii) the non-enzymatic antioxidant defense machinery (phenolic acids and phenolic amines) that works as a second defense line against ‘Ca. L. asiaticus’-induced ROS in citrus infected leaves. Collectively, our findings suggest that GABA might be a promising alternative eco-friendly strategy that helps citrus trees battle HLB particularly, and other diseases in general.

The devastating citrus disease, Huanglongbing (HLB), is associated with ‘Candidatus Liberibacter sp.’ and transmitted by citrus psyllids. Unfortunately, HLB has no known sustainable cure yet. Herein, we proposed γ-aminobutyric acid (GABA) as a potential eco-friendly therapeutic solution to HLB. Herein, we used GC/MS-based targeted metabolomics combined with gene expression to investigate the role of GABA in citrus response against HLB and to better understand its relationship(s) with different phytohormones. GABA supplementation via root drench boosts the accumulation of endogenous GABA in the leaves of both healthy and ‘Ca. L. asiaticus’-infected trees. GABA accumulation benefits the activation of a multi-layered defensive system via modulating the phytohormone levels and regulating the expression of their biosynthesis genes and some pathogenesis-related proteins (PRs) in both healthy and ‘Ca. L. asiaticus’-infected plants. Moreover, our findings showed that GABA application stimulates auxin biosynthesis in ‘Ca. L. asiaticus’-infected plants via the activation of the indole-3-pyruvate (I3PA) pathway, not via the tryptamine (TAM)-dependent pathway, to enhance the growth of HLB-affected trees. Likewise, GABA accumulation was associated with the upregulation of SA biosynthesis genes, particularly the PAL-dependent route, resulting in higher SA levels that activated CsPR1, CsPR2, CsPR5, and CsWRKY70, which are prominent to activation of the SA-mediated pathway. Additionally, higher GABA levels were correlated with an enhanced JA profile and linked with both CsPR3 and CsPR4, which activates the JA-mediated pathway. Collectively, our findings suggest that exogenous GABA application might be a promising alternative and eco-friendly strategy that helps citrus trees battle HLB.

Drought stress is one of the main environmental challenges that dramatically reduce global rice production within several agricultural ecosystems. Breeding drought-tolerant rice genotypes is an important sustainable strategy to overcome this constraint. In this work, drought tolerance levels were assessed according to biochemical, anatomical, and molecular aspects, which led to selecting three promising crosses (Sakha 107 × Sakha super 300, Sakha 107 × M206, and Sakha 107 × Sakha 108) that were compared with their parents as controls. The antioxidant capabilities of the chosen potential crosses, such as the ascorbate peroxidase activity (APX), superoxide dismutase activity (SOD), catalase activity (CAT), and total phenolics, were significantly higher compared with their parents under drought stress. Moreover, the promising selected crosses could accumulate greater proline and chlorophyll contents. The potential superiority of the three selected rice crosses was anatomically represented throughout cross-sections of roots, stems, and leaves, which recorded higher values of cross-section diameter, epidermal thickness, cortex thickness, mesophyll thickness, and bundle sheath thickness as well as a broader range of xylem vessel diameters than their parents under a water deficit. The observed superiority of the antioxidant activities in the overall drought-tolerance mechanisms and anatomical characteristics reflected their protective role in the adaptation process under water stress. Molecular analyses using inter-simple sequence repeat (ISSR) markers suggested two promising crosses (Sakha 107 × Sakha super 300 and Sakha 107 × M206) to be the most suitable crosses for saving water. They had the highest similarity values and were grouped in a distinct cluster. The relative gene expression of OsACS2, OsCML31, OsCYP94C2a, and OsSRO1c was significantly elevated in the two selected drought-tolerant rice genotypes (Sakha 107 × Sakha super 300 and Sakha 107 × M206).

White rot, caused by Stromatinia cepivora (Anamorph: Sclerotium cepivorum Berk), is a serious soil-borne disease of the onion that restricts its cultivation and production worldwide. Herein, we isolated and characterized a plant growth-promoting rhizobacterium Stenotrophomonas maltophilia from healthy onion roots and an endophytic bacterium Serratia liquefaciens from healthy bean leaves. Both isolates showed strong fungistatic activity against S. cepivora using the dual culture and culture filtrate methods. This effect might be due to the presence of several volatile compounds, especially menthol in both culture filtrates as shown with a GC-MS analysis. Additionally, the root drench application of cell-free culture filtrates of S. maltophilia and S. liquefaciens significantly reduced the incidence and severity of white rot disease on treated onion plants, which was associated with the activation of both enzymatic (POX and PPO) and non-enzymatic (phenolics and flavonoids) antioxidant defense machineries of S. cepivora-infected onion plants. Moreover, the culture filtrates of both bacterial bioagents remarkably enhanced the growth (as expressed by root length, plant height, and number of leaves) and yield parameters (as indicated by bulb circumference, fresh weight of the bulb, and bulb yield per plot) of treated onion plants under field conditions during two successive seasons (2020/2021 and 2021/2022). This might be because of a reduced disease severity and/or the accumulation of the main auxin, indole-3-acetic acid (IAA), and its precursor, the amino acid tryptophan. Our findings suggest that both bioagents might be utilized as eco-friendly alternative control measures to reduce the utilization of chemical fungicides entirely or partially for the safer production of onion in S. cepivora-infested soils.

Ras cheese is one of the main hard cheeses in Egypt and is well-known worldwide. Herein, we investigated the potential effects of different coating techniques on the physico-chemical characteristics, sensory properties, and aroma-related volatile organic compounds (VOCs) of Ras cheese over a six-month ripening period. Four coating techniques were tested, including (I) uncoated Ras cheese (the benchmark control), (II) Ras cheese coated with paraffin wax (T1), (III) Ras cheese coated with a plastic film under a vacuum (PFUV; T2), and (IV) Ras cheese coated with a plastic film treated with natamycin (T3). Although none of the treatments significantly affected the salt content, Ras cheese coated with a plastic film treated with natamycin (T3) slightly reduced the moisture content over the ripening period. Moreover, our findings revealed that while T3 had the highest ash content, it showed the same positive correlation profiles of fat content, total nitrogen, and acidity % as the control cheese sample, indicating no significant effect on the physico-chemical characteristics of the coated cheese. Furthermore, there were significant differences in the composition of VOCs among all tested treatments. The control cheese sample had the lowest percentage of other VOCs. T1 cheese, coated with paraffin wax, had the highest percentage of other volatile compounds. T2 and T3 were quite similar in their VOC profiles. According to our GC-MS findings, thirty-five VOCs were identified in Ras cheese treatments after six months of ripening, including twenty-three fatty acids, six esters, three alcohols, and three other compounds identified in most treatments. T2 cheese had the highest fatty acid % and T3 cheese had the highest ester %. The development of volatile compounds was affected by the coating material and the ripening period of the cheeses, which played a major role in the quantity and quality of volatile compounds.

Ras cheese is the main Egyptian hard cheese that is well-known worldwide. Herein, we investigated how different salting techniques affect the physio-chemical properties, sensory properties, and volatile compounds of Ras cheese over a six-month ripening period. Five Ras cheese treatments were made from pasteurized cow’s milk using various salting techniques: traditional salting of Ras cheese, salting by applying all of the salt to the curd after the entire whey drainage, salting by applying all of the salt to the curd after half to two-thirds of the whey drainage, salting in a brine solution for 24 h without dry salting, and salting in a brine solution for 12 h and then dry salting. The obtained results by GC-MS recorded that thirty-eight volatile compounds were identified in Ras cheese treatments after six months of ripening, and the development of volatile compounds was affected by the salting technique as well as the ripening period of the cheeses, which played a major role in the type and concentration of volatile compounds. Results revealed that there are six esters, 15 fatty acids, five ketones, two aldehydes, four alcohols, and eight other compounds identified in most treatments. Some physio-chemical characteristics and sensory properties were found to have high correlations with the storage period, while some others have low correlations during the ripening period.

The important vegetable crop, tomato, is challenged with numerous abiotic and biotic stressors, particularly the newly emerged fungicide-resistant strains of phytopathogenic fungi such as Alternaria alternata, the causal agent of early blight disease. The current study investigated the potential antifungal activity of four cinnamate derivatives including cinnamic acid, ρ-coumaric acid, caffeic acid, and ferulic acid against A. alternata. Our in vitro findings showed that all tested compounds exhibited dose-dependent fungistatic action against A. alternata when their concentrations were increased from 0.1, 0.3, 0.5, and 0.7, to 0.9 mM, respectively. The high concentration of ferulic acid (0.9 mM) completely inhibited the radial mycelial growth of A. alternata and it was comparable to the positive control (difenoconazole fungicide). Additionally, under greenhouse conditions, foliar application of the four tested cinnamates significantly reduced the severity of early blight disease without any phytotoxicity on treated tomato plants. Moreover, it significantly improved the growth traits (plant height, total leaf area, number of leaves per plant, and shoot fresh weight), total chlorophyll, and yield components (number of flowers per plant, number of fruits per plant, and fruit yield) of treated A. alternata-infected plants. Collectively, our findings suggest that cinnamate derivatives could be good candidates as eco-friendly alternatives to reduce the use of chemical fungicides against A. alternata.

Soil and water salinity are among the most fatal environmental challenges that threaten agricultural production worldwide. This study investigated the potential impact(s) of soil amendment using composted bagasse and/or foliar application of cyanobacteria-based bio-stimulants (Arthrospira platensis, also known as Spirulina platensis) to combat the harmful effect(s) of using saline water to irrigate barley plants grown in salt-affected soils during 2020/2021 and 2021/2022. Briefly, the dual application of composted bagasse and cyanobacteria-based bio-stimulants significantly improved the soil properties, buffered the exchangeable sodium percentage (ESP), and enhanced the activity of soil enzymes (urease and dehydrogenase). Moreover, both treatments and their combination notably augmented the water relations of barley plants under salinity stress. All treatments significantly decreased stomatal conductance (gs) and relative water content (RWC) but increased the electrolyte leakage (EL) and balanced the contents of Na+ and K+, and their ratio (K+/Na+) of barley leaves under salinity stress compared with those irrigated with fresh water during the 2020/2021 and 2021/2022 seasons. Additionally, composted bagasse and cyanobacteria-based bio-stimulants diminished the oxidative stress in barley plants under salinity stress by improving the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). Consequently, the combination of composted bagasse and cyanobacteria extract resulted in superior yield-related traits such as spike length, number of grains per spike, 1000-grain weight, grain yield, straw yield, and harvest index. Collectively, our findings suggest that the integrative application of composted bagasse and cyanobacteria is promising as a sustainable environmental strategiy that can be used to improve soil properties, plant growth, and productivity of not only barley plants but also maybe other cereal crops irrigated with saline water in salt-affected soil.

In the present work, the quality of sweet basil leaves volatile oil dried by indirect mode forced convection solar dryer (IMFCSD) integrated with thermal storage material was investigated and compared with the traditional air-drying method. It is found that the indirect solar dryer is capable of drying the basil leaves for 31 h (including the night hours) when the phase change material was used, while it is required 60 h to dry the basil leaves in natural air. In order to ensure that the proposed solar drying method preserves the basil valuable components, an untargeted gas chromatography-mass spectrometry (GC/MS)-based metabolomic technique was used to investigate the impact of the indirect solar drying process on the Volatile organic compounds (VOCs) profile of the basil essential oil. GC/MS analysis revealed that 62 different VOCs were detected in either natural-air dried, indirect solar-dried samples or both together. The usage of indirect solar dryer resulted in higher abundances of 31 detected VOCs. There was no significant difference in the abundance of methyl eugenol, however, there were significant differences in the abundance of most minor components for the favor of IMFCSD. Collectively, these findings indicate that the IMFCSD is much better than natural air drying which might be referred to the controlled drying environment and the stability of drying temperature.

Sweet pepper (Capsicum annuum L.), also known as bell pepper, is one of the most widely grown vegetable crops worldwide. It is attacked by numerous phytopathogenic fungi, such as Fusarium equiseti, the causal agent of Fusarium wilt disease. In the current study, we proposed two benzimidazole derivatives, including 2-(2-hydroxyphenyl)-1-H benzimidazole (HPBI) and its aluminum complex (Al−HPBI complex), as potential control alternatives to F. equiseti. Our findings showed that both compounds demonstrated dose-dependent antifungal activity against F. equiseti in vitro and significantly suppressed disease development in pepper plants under greenhouse conditions. According to in silico analysis, the F. equiseti genome possesses a predicted Sterol 24-C-methyltransferase (FeEGR6) protein that shares a high degree of homology with EGR6 from F. oxysporum (FoEGR6). It is worth mentioning that molecular docking analysis confirmed that both compounds can interact with FeEGR6 from F. equiseti as well as FoEGR6 from F. oxysporum. Moreover, root application of HPBI and its aluminum complex significantly enhanced the enzymatic activities of guaiacol-dependent peroxidases (POX), polyphenol oxidase (PPO), and upregulated four antioxidant-related enzymes, including superoxide dismutase [Cu-Zn] (CaSOD-Cu), L-ascorbate peroxidase 1, cytosolic (CaAPX), glutathione reductase, chloroplastic (CaGR), and monodehydroascorbate reductase (CaMDHAR). Additionally, both benzimidazole derivatives induced the accumulation of total soluble phenolics and total soluble flavonoids. Collectively, these findings suggest that the application of HPBI and Al−HPBI complex induce both enzymatic and nonenzymatic antioxidant defense machinery.

Powdery mildew disease, caused by Erysiphe betae, is one of the most threatening diseases on sugar beet plants worldwide. It causes a great loss in the root yield, sugar percentage, and quality of produced sugar. In the current study, we aimed to evaluate the susceptibility of 25 sugar beet cultivars to infection with powdery mildew disease under Egyptian conditions. Moreover, we evaluated the impacts of three eco-friendly materials, including potassium bicarbonate (KHCO3; at 5 and 10 g L−1), Moringa oleifera seed extract (25 and 50 g L−1), and the biocontrol agent, Bacillus subtilis (108 cell suspension) against E. betae in two successive seasons 2020 and 2021. Our findings showed that there were significant differences between these 25 cultivars in their susceptibility to the disease under study. Using the detached leaves technique in vitro, B. subtilis showed strong antifungal activity against E. betae. Moreover, both concentrations of KHCO3 and moringa seed extract significantly reduced the disease severity. Under field conditions, tested treatments significantly reduced the severity of powdery mildew disease and prevented E. betae from producing its conidiophores and conidia. Scanning electron microscope examination of treated leaves demonstrated the presence of the decomposition of fungal hyphae, conidiophores, conidia, and the occurrence of plasmolysis to fungal cells and spores on the surface of the leaves. Furthermore, these treatments greatly improved the percent of sucrose and soluble solids content, as well as the enzymatic activity of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase. It is noteworthy that treatment with moringa seed extract gave the best results, followed by potassium bicarbonate, then B. subtilis cell suspension. Generally, it is recommended to use the substances used in this research to combat powdery mildew to minimize or prevent the use of chemical fungicides harmful to public health and the environment.

Combined stressors (high soil salinity and saline water irrigation) severely reduce plant growth and sugar beet yield. Seed inoculation with plant growth-promoting rhizobacteria (PGPR) and/or foliar spraying with silica nanoparticles (Si-NP) is deemed one of the most promising new strategies that have the potential to inhibit abiotic stress. Herein, sugar beet (Beta vulgaris) plants were treated with two PGPR (Pseudomonas koreensis MG209738 and Bacillus coagulans NCAIM B.01123) and/or Si-NP, during two successive seasons 2019/2020 and 2020/2021 to examine the vital role of PGPR, Si-NP, and their combination in improving growth characteristics, and production in sugar beet plants exposed to two watering treatments (fresh water and saline water) in salt-affected soil. The results revealed that combined stressors (high soil salinity and saline water irrigation) increased ion imbalance (K+/Na+ ratio; from 1.54 ± 0.11 to 1.00 ± 0.15) and declined the relative water content (RWC; from 86.76 ± 4.70 to 74.30 ± 3.20%), relative membrane stability index (RMSI), stomatal conductance (gs), and chlorophyll content, which negatively affected on the crop productivity. Nevertheless, the application of combined PGPR and Si-NP decreased oxidative stress indicators (hydrogen peroxide and lipid peroxidation) and sodium ions while increasing activities of superoxide dismutase (SOD; up to 1.9-folds), catalase (CAT; up to 1.4-folds), and peroxidase (POX; up to 2.5-folds) enzymes, and potassium ions resulting in physiological processes, root yield, and sugar yield compared to non-treated controls under combined stressors (high soil salinity and saline water irrigation). It is worth mentioning that the singular application of PGPR improved root length, diameter, and yield greater than Si-NP alone and it was comparable to the combined treatment (PGPR+Si-NP). It was concluded that the combined application of PGPR and Si-NP has valuable impacts on the growth and yield of sugar beet growing under combined stressors of high soil salinity and saline water irrigation.

Wheat is one of the most important crops in the world and was considered the major grain crop grown in Egypt. Nowadays, Egypt is the largest wheat importer in the world and consumes an extensive amount of it. To decrease the gap between production and consumption and increase the yield, we need to control wheat diseases, especially stripe rust, due to its major damage to wheat. Further, we need to advise farmers as early as we can to control and treat them. The paper proposed an interactive intelligent system to monitor, predict and give the correct advice at the right time to farmers. This system is called MISSR (Mentoring Interactive System for Stripe Rust). The system is considered an important means to effectively prevent risks in agricultural production. It also plays an important role in guiding farmers and decision-makers to plan and implement suitable practices to increase yield and mitigate stripe-rust disease. On the other hand, it can acquire relevant and timely information in the areas where this information or data is unavailable. To build this model for the wheat crop in Egypt, we used wheat experts’ knowledge and climate data API. MISSR is available as a mobile application to provide access for more farmers and increase its availability.

Wheat stem rust caused by Puccinia graminis f. sp. tritici is one of the most destructive wheat diseases worldwide. Identifying stem rust races in general, Ug99 lineage particularly, and determining resistance genes are critical goals for disease assessment. Thirty wheat varieties and monogenic lines with major stem rust resistance genes (Sr) were examined here over the course of three succeeding seasons from 2020 to 2022. Fourteen stem rust races have been identified in ten African countries, as well as Central and West Asia and North Africa (CWANA) and ten European countries. The Ug99 group (Clade I) included four races (TTKSK, TTKST, TTKTK, and TTKTT) and was reported in five African countries (Egypt, Kenya, Rwanda, Tanzania, and Uganda) and Iran, but none of the European countries. On the other hand, none of the races in Clade III-B (TTRTF) and Clade IV-B (TKTTF and TTTTF) were found in Egypt. Furthermore, Egyptian races were clustered separately from races identified from other countries, and six races were found only in Egypt, including PKSTC, RKTTH, TKTTC, TTTSK, TCKTC, and TKTTH. Races from Kenya, Tanzania, Uganda, Rwanda, and Iran were all closely associated with one another, according to correlation analysis. However, most races identified from other investigated regions, including Eritrea, Spain, Ethiopia, Morocco, Italy, Poland, Kenya, Tanzania, and Uganda, were adversely linked with Egyptian races. The diagnostic 350 bp long PCR fragment linked with virulence to Sr31, Clement (Sr31), and Brigardier (Sr31) was used to identify the TTKSK (Ug99) race. The identification of the regional associations and genetic diversity of newly emerged races within the Ug99 lineage of P. graminis tritici in Africa, Asia, and Europe is one of the key goals of this study. It will help plant breeders to develop new resistant lines against the virulent races, especially TTKSK (Ug99) and TTTSK. This helps in ensuring global food security in the context of climate change.

Sweet pepper (Capsicum annuum L.) is one of the most widely produced vegetable plants in the world. Fusarium wilt of pepper is one of the most dangerous soil-borne fungal diseases worldwide. Herein, we investigated the antifungal activities and the potential application of two chalcone derivatives against the phytopathogenic fungus, Fusarium equiseti, the causal agent of Fusarium wilt disease in vitro and in vivo. The tested compounds included 3-(4-dimethyl amino-phenyl)-1-{6-[3-(4 dimethyl amino-phenyl)-a cryloyl]-pyridin-2-yl}-propanone (DMAPAPP) and its metal complex with ruthenium III (Ru-DMAPAPP). Both compounds had potent fungistatic activity against F. equiseti and considerably decreased disease progression. The tested compounds enhanced the vegetative growth of pepper plants, indicating there was no phytotoxicity on pepper plants in greenhouse conditions. DMAPAPP and Ru-DMAPAPP also activated antioxidant defense mechanisms that are enzymatic, including peroxidase, polyphenole oxidase, and catalase, and non-enzymatic, such as total soluble phenolics and total soluble flavonoids. DMAPAPP and Ru-DMAPAPP also promoted the overexpression of CaCu-SOD and CaAPX genes. However, CaGR and CaMDHAR were downregulated. These results demonstrate how DMAPAPP and Ru-DMAPAPP could be employed as a long-term alternative control approach for Fusarium wilt disease as well as the physiological and biochemical mechanisms that protect plants.

The genus Aspergillus comprises several species that play pivotal roles in agriculture. Herein, we morphologically and physiologically characterized four genetically distinct Aspergillus spp., namely A. japonicus, A. niger, A. flavus, and A. pseudoelegans, and examined their ability to suppress the white mold disease of bean caused by Sclerotinia sclerotiorum in vitro and under greenhouse conditions. Seriation type of Aspergillus spp. correlates with conidiospores discharge as detected on the Petri glass lid. Members of Nigri section cover their conidial heads with hard shells after prolonged incubation. In addition, sporulation of the tested Aspergillus isolates is temperature sensitive as it becomes inhibited at low temperatures and the colonies become white. Examined Aspergillus spp. were neither infectious to legumes nor aflatoxigenic as confirmed by HPLC except for A. flavus and A. pseudoelegans which, secreted 5 and 1 ppm of aflatoxin B1, respectively. Co-inoculations of Sclerotinia’s mycelium or sclerotia with a spore suspension of Aspergillus spp. inhibited their germination on PDA at 18 °C and 28 °C, and halted disease onset on detached common bean and soybean leaves. Similarly, plants treated with A. japonicus and A. niger showed the highest survival rates compared to untreated plants. In conclusion, black Aspergillus spp. are efficient biocides and safe alternatives for the management of plant diseases, particularly in organic farms.

Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector.

Citrus is grown globally throughout the subtropics and semi-arid to humid tropics. Abiotic stresses such as soil water deficit negatively affect plant growth, physiology, biochemistry, and anatomy. Herein, we investigated the effect(s) of three water regimes (control, moderate drought, and severe drought) on the physiological and anatomical structure of 10 different citrus rootstocks with different degrees of tolerance to drought stress. Brazilian sour orange and Gadha dahi performed well by avoiding desiccation and maintaining plant growth, plant water status, and biochemical characters, while Rangpur Poona nucellar (C. limonia) and Sunki × bentake were the most sensitive rootstocks at all stress conditions. At severe water stress, the highest root length (24.33 ± 0.58), shoot length (17.00 ± 1.00), root moisture content (57.67 ± 1.53), shoot moisture content (64.59 ± 1.71), and plant water potential (−1.57 ± 0.03) was observed in tolerant genotype, Brazilian sour orange. Likewise, chlorophyll a (2.70 ± 0.06), chlorophyll b (0.87 ± 0.06) and carotenoids (0.69 ± 0.08) were higher in the same genotype. The lowest H2O2 content (77.00 ± 1.00) and highest proline content (0.51 ± 0.06) were also recorded by Brazilian sour orange. The tolerance mechanism of tolerant genotypes was elucidated by modification in anatomical structures. Stem anatomy at severe drought, 27.5% increase in epidermal cell thickness, 25.4% in vascular bundle length, 30.5% in xylem thickness, 27.7% in the phloem cell area, 8% in the pith cell area, and 43.4% in cortical thickness were also observed in tolerant genotypes. Likewise, leaf anatomy showed an increase of 27.9% in epidermal cell thickness, 11.4% in vascular bundle length, 21% in xylem thickness, and 15% in phloem cell area in tolerant genotypes compared with sensitive ones. These modifications in tolerant genotypes enabled them to maintain steady nutrient transport while reducing the risk of embolisms, increasing water-flow resistance, and constant transport of nutrients across.

Plant viral infection is one of the most severe issues in food security globally, resulting in considerable crop production losses. Chitosan is a well-known biocontrol agent against a variety of plant infections. However, research on combatting viral infections is still in its early stages. The current study investigated the antiviral activities (protective, curative, and inactivation) of the prepared chitosan/dextran nanoparticles (CDNPs, 100 µg mL−1) on Nicotiana glutinosa plants. Scanning electron microscope (SEM) and dynamic light scattering analysis revealed that the synthesized CDNPs had a uniform, regular sphere shapes ranging from 20 to 160 nm in diameter, with an average diameter of 91.68 nm. The inactivation treatment was the most effective treatment, which resulted in a 100% reduction in the alfalfa mosaic virus (AMV, Acc# OK413670) accumulation level. On the other hand, the foliar application of CDNPs decreased disease severity and significantly reduced viral accumulation levels by 70.43% and 61.65% in protective and curative treatments, respectively, under greenhouse conditions. Additionally, the induction of systemic acquired resistance, increasing total carbohydrates and total phenolic contents, as well as triggering the transcriptional levels of peroxidase, pathogen-related protein-1, and phenylalanine ammonia-lyase were observed. In light of the results, we propose that the potential application of CDNPs could be an eco-friendly approach to enhance yield and a more effective therapeutic elicitor for disease management in plants upon induction of defense systems.

The phytopathogenic basidiomycetous fungus, Rhizoctonia solani, has a wide range of host plants including members of the family Poaceae, causing damping-off and root rot diseases. In this study, we biosynthesized spherical-shaped silicon dioxide nanoparticles (SiO2 NPs; sized between 9.92 and 19.8 nm) using saffron extract and introduced them as a potential alternative therapeutic solution to protect wheat seedlings against R. solani. SiO2 NPs showed strong dose-dependent fungistatic activity on R. solani, and significantly reduced mycelial radial growth (up to 100% growth reduction), mycelium fresh and dry weight, and pre-, post-emergence damping-off, and root rot severities. Moreover, the impact of SiO2 NPs on the growth of wheat seedlings and their potential mechanism (s) for disease suppression was deciphered. SiO2 NPs application also improved the germination, vegetative growth, and vigor indexes of infected wheat seedlings which indicates no phytotoxicity on treated wheat seedlings. Moreover, SiO2 NPs enhanced the content of the photosynthetic pigments (chlorophylls and carotenoids), induced the accumulation of defense-related compounds (particularly salicylic acid), and alleviated the oxidative stress via stimulation of both enzymatic (POD, SOD, APX, CAT, and PPO) and non-enzymatic (phenolics and flavonoids) antioxidant defense machinery. Collectively, our findings demonstrated the potential therapeutic role of SiO2 NPs against R. solani infection via the simultaneous activation of a multilayered defense system to suppress the pathogen, neutralize the destructive effect of ROS, lipid peroxidation, and methylglyoxal, and maintain their homeostasis within R. solani-infected plants.

Water scarcity, due to physical shortage or inadequate access, is a major global challenge that severely affects agricultural productivity and sustainability. Deficit irrigation is a promising strategy to overcome water scarcity, particularly in arid and semiarid regions with limited freshwater resources. However, precise application of deficit irrigation requires a better understanding of the plant response to water/drought stress. In the current study, we investigated the potential impacts of biochar-based soil amendment and foliar potassium-humate application (separately or their combination) on the growth, productivity, and nutritional value of onion (Allium cepa L.) under deficient irrigation conditions in two separate field trials during the 2018/2019 and 2019/2020 seasons. Our findings showed that deficit irrigation negatively affected onion resilience to drought stress. However, these harmful effects were diminished after soil amendment using biochar, K-humate foliar application, or their combination. Briefly, integrated biochar and K-humate application increased onion growth, boosted the content of the photosynthetic pigments, enhanced the water relations, and increased the yield traits of deficient irrigation onion plants. Moreover, it improved the biochemical response, enhanced the activities of antioxidant enzymes, and enriched the nutrient value of deficiently irrigated onion plants. Collectively, these findings highlight the potential utilization of biochar and K-humate as sustainable eco-friendly strategies to improve onion resilience to deficit irrigation.

Adult plant resistance in wheat is an achievement of the breeding objective because of its durability in comparison with race-specific resistance. Partial resistance to wheat stripe rust disease was evaluated under greenhouse and field conditions during the period from 2016 to 2021. Misr 3, Sakha 95, and Giza 171 were the highest effective wheat genotypes against Puccinia striiformis f. sp. tritici races. Under greenhouse genotypes, Sakha 94, Giza 168, and Shandaweel1 were moderately susceptible, had the longest latent period and lowest values of the length of stripes and infection frequency at the adult stage. Partial resistance levels under field conditions were assessed, genotypes Sakha 94, Giza 168, and Shandaweel1 exhibited partial resistance against the disease. Leaf tip necrosis (LTN) was noted positively in three genotypes Sakha 94, Sakha 95, and Shandaweel1. Molecular analyses of Yr18 were performed for csLV34, cssfr1, and cssfr2 markers. Only Sakha 94 and Shandaweel1 proved to carry the Yr18 resistance allele at both phenotypic and genotypic levels. Scanning electron microscopy (SEM) observed that the susceptible genotypes were colonized extensively on leaves, but on the slow-rusting genotype, the pustules were much less in number, diminutive, and poorly sporulation, which is similar to the pustule of NIL Jupateco73 ‘R’.

Characterization of the genetic structure and the physiological races of Puccinia triticina is a growing necessity to apply host genetic resistance against wheat leaf rust as a successful control strategy. Herein, we collected and identified about 130 isolates of P. triticina from 16 Egyptian commercial wheat cultivars grown at different locations, over two seasons (2019/2020 and 2020/2021). The 130 isolates of P. triticina were segregated into 17 different physiological races. TTTST and TTTKS were the most common virulent races, whereas TTTST and MTTGT were the most frequent races. The races were classified into three groups, based on their distinct DNA band sizes (150 bp, 200 bp, and 300 bp) after RAPD analysis. The new wheat cultivars (Sakha-94, Sakha-95, and Shandweel-1) infected with the most virulent race (TTTST), Gemmeiza-12, and Misr-3 were resistant to all physiological races. The resistance of these cultivars was mostly due to the presence of Lr19- and Lr28-resistant genes. Our results serve as a warning about emerging aggressive races capable of supplanting resistance to leaf rust, and help in the understanding of the pathotype–cultivar–location association and its role in the susceptibility/resistance of new wheat cultivars to P. triticina.

Osmotic stress is a major physiologic dysfunction that alters the water movement across the cell membrane. Soil salinity and water stress are major causal factors of osmotic stress that severely affect agricultural productivity and sustainability. Herein, we suggested and evaluated the impact of integrated biochar-based soil amendment and exogenous glycine betaine application on the growth, physiology, productivity, grain quality, and osmotic stress tolerance of rice (Oryza sativa L., cv. Sakha 105) grown in salt-affected soil under three irrigation intervals (6, 9, or 12 days), as well as soil properties and nutrient uptake under field conditions during the 2019 and 2020 seasons. Our findings showed that dual application of biochar and glycine betaine (biochar + glycine betaine) reduced the soil pH, electrical conductivity, and exchangeable sodium percentage. However, it enhanced the K+ uptake which increased in the leaves of treated-rice plants. Additionally, biochar and glycine betaine supplementation enhanced the photosynthetic pigments (chlorophyll a, b, and carotenoids) and physiological attributes (net photosynthetic rate, stomatal conductance, relative water content, and electrolyte leakage) of osmotic-stressed rice plants. Biochar + glycine betaine altered the activity of antioxidant-related enzymes (catalase, ascorbate peroxide, and peroxidase). Moreover, it improved the yield components, biological yield, and harvest index, as well as the nutrient value of rice grains of osmotic-stressed rice plants. Collectively, these findings underline the potential application of biochar and glycine betaine as a sustainable eco-friendly strategy to improve plant resilience, not only rice, but other plant species in general and other cereal crops in particular, to abiotic stress, particularly those growing in salt-affected soil.

The utilization of low-quality water or slightly saline water in sodic-saline soil is a major global conundrum that severely impacts agricultural productivity and sustainability, particularly in arid and semiarid regions with limited freshwater resources. Herein, we proposed an integrated amendment strategy for sodic-saline soil using biochar and/or plant growth-promoting rhizobacteria (PGPR; Azotobacter chroococcum SARS 10 and Pseudomonas koreensis MG209738) to alleviate the adverse impacts of saline water on the growth, physiology, and productivity of maize (Zea mays L.), as well as the soil properties and nutrient uptake during two successive seasons (2018 and 2019). Our field experiments revealed that the combined application of PGPR and biochar (PGPR + biochar) significantly improved the soil ecosystem and physicochemical properties and K+, Ca2+, and Mg2+ contents but reduced the soil exchangeable sodium percentage and Na+ content. Likewise, it significantly increased the activity of soil urease (158.14 ± 2.37 and 165.51 ± 3.05 mg NH4+ g−1 dry soil d−1) and dehydrogenase (117.89 ± 1.86 and 121.44 ± 1.00 mg TPF g−1 dry soil d−1) in 2018 and 2019, respectively, upon irrigation with saline water compared with non-treated control. PGPR + biochar supplementation mitigated the hazardous impacts of saline water on maize plants grown in sodic-saline soil better than biochar or PGPR individually (PGPR + biochar > biochar > PGPR). The highest values of leaf area index, total chlorophyll, carotenoids, total soluble sugar (TSS), relative water content, K+ and K+/Na+ of maize plants corresponded to PGPR + biochar treatment. These findings could be guidelines for cultivating not only maize but other cereal crops particularly in salt-affected soil and sodic-saline soil.

Tomato early blight, caused by Alternaria solani, is a destructive foliar fungal disease. Herein, the potential defensive roles of benzoic acid (BA) and two of its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA), and protocatechuic acid (PCA) against A. solani were investigated. All tested compounds showed strong dose-dependent fungistatic activity against A. solani and significantly reduced the disease development. Benzoic acid, and its hydroxylated derivatives, enhanced vegetative growth and yield traits. Moreover, BA and its derivatives induce the activation of enzymatic (POX, PPO, CAT, SlAPXs, and SlSODs) and non-enzymatic (phenolics, flavonoids, and carotenoids) antioxidant defense machinery to maintain reactive oxygen species (ROS) homeostasis within infected leaves. Additionally, BA and its hydroxylated derivatives induce the accumulation of salicylic acid (SA) and its biosynthetic genes including isochorismate synthase (SlICS), aldehyde oxidases (SlAO1 and SlAO2), and phenylalanine ammonia-lyases (SlPAL1, SlPAL2, SlPAL3, SlPAL5, and SlPAL6). Higher SA levels were associated with upregulation of pathogenesis-related proteins (SlPR-1, SlPR1a2, SlPRB1-2, SlPR4, SlPR5, SlPR6), nonexpressor of pathogenesis-related protein 1 (SlNPR1), and salicylic acid-binding protein (SlSABP2). These findings outline the potential application of BA and its hydroxylated derivatives as a sustainable alternative control strategy for early blight disease and also deciphering the physiological and biochemical mechanisms behind their protective role.

Oxytetracycline (OTC) has been used for the control of several plant diseases and was recently approved for the control of Huanglongbing, the citrus greening disease. Huanglongbing is caused by the phloem limited ‘Candidatus Liberibacter asiaticus’. Determination of OTC in the xylem and phloem of citrus plants is of great interest as they are the main routes of translocation in citrus. In addition, the determination of the level of OTC in the phloem sap is necessary for the control of the ‘Ca. L. asiaticus’ pathogen, which resides in the phloem. Herein, we demonstrated that the level of OTC in the citrus phloem and xylem saps obtained using the centrifugation method can be successfully measured using the europium (Eu) method directly or with cleanup by solid-phase extraction (SPE). Recovery of OTC from spiked sap samples purified by solid-phase extraction (SPE) was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. The ‘Ca. L. asiaticus’-infected leaf and phloem sap samples showed higher inhibition of the fluorescence intensity of the OTC standard compared to non-infected control leaf and phloem samples. In agreement with this finding, the levels of phenols and flavonoids in ‘Ca. L. asiaticus’-infected leaves were higher than those controls and were shown to interfere with the Eu method. Therefore, the SPE cleanup step only improved OTC recovery from leaf samples containing the interfering compounds. The Eu method was then used to determine OTC levels in the phloem and xylem sap of OTC-treated plants, and the results were similar whether measured directly or after SPE. Visualization under ultraviolet light (400 nm) showed the presence of OTC in citrus xylem and phloem saps with and without the use of SPE.

Huanglongbing, a destructive disease of citrus, is caused by the fastidious bacterium ‘Candidatus Liberibacter asiaticus’ and transmitted by Asian citrus psyllid, Diaphorina citri. The impact of ‘Ca. L. asiaticus’ infection or D. citri infestation on Valencia sweet orange (Citrus sinensis) leaf metabolites was investigated using gas chromatography mass spectrometry, followed by gene expression analysis for 37 genes involved in jasmonic acid (JA), salicylic acid (SA), and proline-glutamine pathways. The total amino acid abundance increased after ‘Ca. L. asiaticus’ infection, while the total fatty acids increased dramatically after infestation with D. citri, compared with control plants. Seven amino acids (glycine, l-isoleucine, l-phenylalanine, l-proline, l-serine, l-threonine, and l-tryptophan) and five organic acids (benzoic acid, citric acid, fumaric acid, SA, and succinic acid) increased in ‘Ca. L. asiaticus’-infected plants. On the other hand, the abundance of trans-JA and its precursor α-linolenic increased in D. citri-infested plants. Surprisingly, the double attack of both D. citri infestation and ‘Ca. L. asiaticus’ infection moderated the metabolic changes in all chemical classes studied. In addition, the gene expression analysis supported these results. Based on these findings, we suggest that, although amino acids such as phenylalanine are involved in citrus defense against ‘Ca. L. asiaticus’ infection through the activation of an SA-mediated pathway, fatty acids, especially α-linolenic acid, are involved in defense against D. citri infestation via the induction of a JA-mediated pathway.

Huanglongbing (HLB) is currently the largest threat to global citrus production. We examined the effect of HLB pathogen ‘Candidatus Liberibacter asiaticus’ infection or infestation by its vector, Diaphorina citri, on ‘Valencia’ sweet orange leaf pigments using high-performance liquid chromatography, followed by gene expression analysis for 46 involved genes in carotenoid and chlorophyll biosynthesis pathways. Both ‘Ca. L. asiaticus’ and D. citri alter the total citrus leaf pigment balance with a greater impact by ‘Ca. L. asiaticus’. Although zeaxanthin was accumulated in ‘Ca. L. asiaticus’-infected leaves, chlorophyllide a was increased in D. citri-infested plants. Our findings support the idea that both ‘Ca. L. asiaticus’ and D. citri affect the citrus pigments and promote symptom development but using two different mechanisms. ‘Ca. L. asiaticus’ promotes chlorophyll degradation but accelerates the biosynthesis of carotenoid pigments, resulting in accumulation of abscisic acid and its precursor, zeaxanthin. Zeaxanthin also has a photoprotective role. By contrast, D. citri induced the degradation of most carotenoids and accelerated chlorophyll biosynthesis, leading to chlorophyllide a accumulation. Chlorophyllide a might have an antiherbivory role. Accordingly, we suggest that citrus plants try to defend themselves against ‘Ca. L. asiaticus’ or D. citri using multifaceted defense systems, based on the stressor type. These findings will help in better understanding the tritrophic interactions among plant, pathogen, and vector.

Oxytetracycline (OTC) and streptomycin have been used for the control of several plant diseases and were recently permitted for the control of citrus greening disease, Huanglongbing. Consequently, sensitive and reliable methods are highly needed for the detection of OTC in citrus tissues. Herein, we studied the replacement of cetyltrimethylammonium chloride (CTAC) by citrate (Cit) as a sensitizing agent for the analysis of OTC in citrus tissues using the recently established europium (Eu) method. In addition, we determined the optimal conditions for the formation of the Eu-OTC-Cit ternary complex in tris buffer. Our results showed that the plant matrix significantly decreased the fluorescence intensity of the Eu-OTC-Cit complex even after the replacement of CTAC. Our investigations showed that phenols such as gallic acid degrade slowly at high pH and their degradation was enhanced in the presence of the (Eu+3) cation. To reduce the plant matrix interference, the sample extract was cleaned using solid-phase extraction (SPE). The OTC recoveries from spiked healthy and Candidatus Liberibacter asiaticus (CLas)-infected trees were 91.4 ± 7.8% and 82.4 ± 3.9%, respectively. We also used the citrate method to determine the level of OTC in trunk-injected trees. The level of OTC as measured using the Eu-OTC-Cit complex (117.5 ± 20.3 µg g−1 fresh weight “FWT”) was similar to that measured using Eu-OTC-CTAC complex (97.5 ± 14 µg g−1 FWT). In addition, we were able to visualize the OTC in citrus leaf extract, under ultraviolet light (400 nm), after it was cleaned with the SPE. Our study showed that the citrate can be successfully used to replace the harmful CTAC surfactant, which could also react with phenols.

Melatonin is synthesized from the amino acid L-tryptophan via the shikimic acid pathway and ubiquitously distributed in both prokaryotes and eukaryotes. Although most of melatonin biosynthesis genes were characterized in several plants and animal species including the insect model, Drosophila melanogaster, none of these enzymes have been identified from the Asian citrus psyllid, Diaphorina citri. We used comprehensive in silico analysis and gene expression techniques to identify the melatonin biosynthesis-related genes of D. citri and to evaluate the expression patterns of these genes within the adults of D. citri with gradient infection rates (0, 28, 34, 50, 58, and 70%) of the phytopathogenic bacterium Candidatus Liberibacter asiaticus and after the treatment with exogenous melatonin. We showed that the D. citri genome possesses six putative melatonin biosynthesis-related genes including two putative tryptophan 5-hydroxylase (DcT5H-1 and DcT5H-2), a putative aromatic amino acid decarboxylase (DcAADC), two putative arylalkylamine N-acetyltransferase (DcAANAT-1 and DcAANAT-2), and putative N-acetylserotonin O-methyltransferase (DcASMT). The infection with Ca. L. asiaticus decreased the transcript levels of all predicted genes in the adults of D. citri. Moreover, melatonin supplementation induced their expression levels in both healthy and Ca. L. asiaticus-infected psyllids. These findings confirm the association of these genes with the melatonin biosynthesis pathway.

Antimicrobial compounds have been successfully used to control many plant and animal diseases. Recently, oxytetracycline (OTC) and streptomycin have been approved for the treatment of Huanglongbing in citrus. Since the application of OTC is under strict regulations, several methods have been developed to determine and monitor its levels in the environment including high-performance liquid chromatography, ELISA, colorimetric, and fluorometric assays. In this study, we developed a fluorometric method for the determination of OTC in plant tissues based on its complexation with europium. Our preliminary trials showed that phenols and flavonoids interfere with the europium assay by reacting with the sensitizing reagent, cetyltrimethylammonium chloride. Consequently, we used the 60 mg hydrophilic–lipophilic balanced (HLB) cartridges to purify the OTC from the plant matrix. The recovery of OTC from spiked leaf samples was 75 ± 7.6%. Using the 500 mg HLB, we were able to detect 0.3 ppm OTC in the final sample extract, which corresponds to 3 µg g−1 fresh weight (FWT). The developed method was successfully used to measure the level of OTC in leaves obtained from trunk-injected trees. The results obtained by the europium method were similar to those obtained using the ELISA assay. We also tested the cross-reactivity of OTC metabolites with the europium method. The 4-epi-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-apo-OTC and β-apo-OTC showed small cross-reactivity. We showed that the europium-sensitized fluorescence-based method can be successfully used to assess OTC in citrus plant tissues after a cleanup step. Our results showed that this method was sensitive, reproducible, and can be used to analyze many samples simultaneously.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen “Candidatus Liberibacter asiaticus” and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Antibiotics have been successfully used to control plant diseases for more than fifty years. Recently, oxytetracycline and streptomycin have been approved for the treatment of Huanglongbing, which is threatening the citrus industry in many regions. Because the efficiency of antibiotics in planta is highly affected by their movement and distribution, understanding the mechanism of antibiotics’ uptake and distribution could lead to a better control of plant pathogens. Herein, we investigated the movement of oxytetracycline within citrus plants. Oxytetracycline was applied by root drenching to both girdled and non-girdled citrus seedlings. In addition, oxytetracycline was applied by trunk injection to girdled and non-girdled citrus trees. After the exposure time (24 h), citrus seedlings were dissected and the levels of oxytetracycline in the different tissues were measured using an oxytetracycline ELISA kit. Upon root application (laboratory experiment), oxytetracycline was detected in the inner part of the stem (xylem-associated tissue), cortex (phloem-associated tissue), and leaves above and below the girdled area. Likewise, oxytetracycline was also detected in leaves of trunk-injected field trees (girdled and non-girdled) three days post treatment. Interestingly, cortex girdling did not affect the distribution and translocation of oxytetracycline, indicating that the xylem is the main path for oxytetracycline translocation. Taken together, our results indicate that oxytetracycline translocation mainly occurs via xylem vessels, and that movement into the phloem occurs subsequent to xylem translocation. Our findings also clearly demonstrated that upon trunk injection, only trace levels of oxytetracycline reached the roots, minimizing its therapeutic value there. Thus, our recommendation is to time tree injections to coincide with the flushing periods when the bacteria are moving into new shoots to maximize the efficiency of oxytetracycline.

Huanglongbing (HLB), formerly known as citrus greening disease, is one of the most devastating bacterial diseases in citrus worldwide. HLB is caused by ‘Candidatus Liberibacter asiaticus’ bacterium and transmitted by Diaphorina citri. Both ‘Ca. L. asiaticus’ and its vector manipulate the host metabolism to fulfill their nutritional needs and/or to neutralize the host defense responses. Herein, we discuss the history of HLB and the complexity of its pathosystem as well as the geographical distribution of its pathogens and vectors. Recently, our recognition of physiological events associated with ‘Ca. L. asiaticus’ infection and/or D. citri-infestation has greatly improved. However, the roles of citrus metabolites in the development of HLB symptoms are still unclear. We believe that symptom development of HLB disease is a complicated process and relies on a multilayered metabolic network which is mainly regulated by phytohormones. Citrus metabolites play vital roles in the development of HLB symptoms through the modulation of carbohydrate metabolism, phytohormone homeostasis, antioxidant pathways, or via the interaction with other metabolic pathways, particularly involving amino acids, leaf pigments, and polyamines. Understanding how ‘Ca. L. asiaticus’ and its vector, D. citri, affect the metabolic pathways of their host is critical for developing novel, sustainable strategies for HLB management.

Tomato (Solanum lycopersicum L.) is among the most important vegetable crops worldwide. Early blight disease, caused by Alternaria solani, is a destructive foliar disease of tomato and other Solanaceae species. Herein, we investigated the in vitro antifungal properties of gallic acid and two of its derivatives (syringic and pyrogallic acids) against A. solani during 2019 and 2020 seasons. The physiological and biochemical effects of these compounds on infected tomato plants were also investigated using the whole plant bioassay. The in vitro investigation showed that all tested compounds showed fungistatic action and inhibited the mycelial radial growth of A. solani in a dose-dependent manner. In two separate pot-experiments, those compounds efficiently suppressed the development of the disease symptoms and area under disease progress curve (AUDPC), without any phytotoxic effects on the treated tomato plants. Additionally, all tested compounds positively enhanced the biochemical traits of treated plants including the chlorophyll content, the total soluble phenolics, the total soluble flavonoids, and the enzymatic activities of catalase, peroxidase, and polyphenol oxidase during 2019 and 2020 seasons. Moreover, the treatment with gallic acid and its derivatives significantly increased all yield components of A. solani-infected tomato plants such as the total number of flowers and fruits, and the fruit yield for each tomato plant in both experiments. Considering the fungitoxicity of phenolic acids against A. solani with no phytotoxicity on treated tomato plants, we believe that gallic acid and its derivatives might be a sustainable eco-friendly control strategy to reduce the usage of chemical fungicides partially or entirely against A. solani particularly, and fungal diseases in general.

Polyamines (PAs) are ubiquitous biogenic amines found in all living organisms from bacteria to Archaea, and Eukaryotes including plants and animals. Since the first description of putrescine conjugate, feruloyl-putrescine (originally called subaphylline), from grapefruit leaves and juice, many research studies have highlighted the importance of PAs in growth, development, and other physiological processes in citrus plants. PAs appear to be involved in a wide range of physiological processes in citrus plants; however, their exact roles are not fully understood. Accordingly, in the present review, we discuss the biosynthesis of PAs in citrus plants, with an emphasis on the recent advances in identifying and characterizing PAs-biosynthetic genes and other upstream regulatory genes involved in transcriptional regulation of PAs metabolism. In addition, we will discuss the recent metabolic, genetic, and molecular evidence illustrating the roles of PAs metabolism in citrus physiology including somatic embryogenesis; root system formation, morphology, and architecture; plant growth and shoot system architecture; inflorescence, flowering, and flowering-associated events; fruit set, development, and quality; stomatal closure and gas-exchange; and chlorophyll fluorescence and photosynthesis. We believe that the molecular and biochemical understanding of PAs metabolism and their physiological roles in citrus plants will help citrus breeding programs to enhance tolerance to biotic and abiotic stresses and provide bases for further research into potential applications.

Abstract δ‐aminolevulinic acid dehydratase (ALAD) is an important enzyme in tetrapyrrole synthesis. ALAD combines two δ‐aminolevulinic acid (δ‐ALA) molecules to form the pyrrole molecule, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, and nutrient uptake. In this study, we investigated the effects of silencing of ALAD gene on citrus leaf pigments and metabolites. The ALAD enzyme was inhibited using virus‐induced gene silencing (VIGS) technology using citrus tristeza virus (CTV). δ‐ALA accumulated in citrus plants inoculated with the recombinant virus (CTV‐tALAD) to silence ALAD and resulted in discrete yellow spots (yellow islands) and necrosis in leaves and stems. The levels of chlorophylls, starch, sucrose, trans‐ and cis‐violaxanthin, and α‐ and β‐cryptoxanthin were reduced in CTV‐tALAD plants, whereas zeaxanthin was increased. The increase in zeaxanthin and the decrease in its precursors indicated that the reduction in chlorophylls resulted in light damage. Salicylic acid and jasmonic acid levels, as well as emission of (E)‐α‐bergamotene and (E)‐β‐farnesene, increased in CTV‐tALAD plants indicating these plants were under stress. Our results showed that silencing of ALAD induces stress in plants and that VIGS using mild CTV strains is a promising technique to study biological function of citrus genes.

Huanglongbing (HLB), or citrus greening, is the most destructive disease to the citrus industry. In Florida, it is caused by the bacterium, Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllid, Diaphorina citri. Recent studies suggested that antibiotics could inhibit the growth of the CLas pathogen in planta. In the current study, we investigated the uptake and translocation of oxytetracycline and streptomycin in citrus seedlings. Oxytetracycline and streptomycin were delivered via root and stem and their level in various tissues was monitored using enzyme-linked immunosorbent assay (ELISA). Oxytetracycline and streptomycin were detected in the leaves, xylem, phloem, and root after root drench and stem delivery. High levels of antibiotics were detected in the roots after root drench, whereas high levels of antibiotics were detected in the canopy after stem delivery. The level of oxytetracycline detected in the phloem, xylem, and leaves after root drench was higher than that of streptomycin. Whereas the level of streptomycin in root was higher than that of oxytetracycline, indicating that streptomycin was bound to the xylem tissues. Oxytetracycline and streptomycin were detected in the phloem, xylem, leaves, and root tissues thirty-five days after the root incubation in 200 µg·mL−1 solution. These results demonstrated that oxytetracycline and streptomycin were relatively stable and could inhibit CLas growth for a couple of months in citrus trees. Observations reported in this study regarding the distribution and stability of oxytetracycline and streptomycin in citrus plants could be useful for designing an effective program for the control of HLB disease using antibiotics.