Extremozymes and compatible solute production potential of halophilic and halotolerant bacteria isolated from crop rhizospheric soils of Southwest Saurashtra Gujarat.

Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.

Chitinase-functionalized UiO-66 framework nanoparticles active against multidrug-resistant Candida Auris.

Candida auris (C. auris) is a yeast that has caused several outbreaks in the last decade. Cell wall chitin plays a primary role in the antifungal resistance of C. auris. Herein, we investigated the potential of chitinase immobilized with UiO-66 to act as a potent antifungal agent against C. auris. Chitinase was produced from Talaromyces varians SSW3 in a yield of 8.97 U/g dry substrate (ds). The yield was statistically enhanced to 120.41 U/g ds by using Plackett-Burman and Box-Behnken design. We synthesized a UiO-66 framework that was characterized by SEM, TEM, XRD, FTIR, a particle size analyzer, and a zeta sizer. The produced framework had a size of 70.42 ± 8.43 nm with a uniform cubic shape and smooth surface. The produced chitinase was immobilized on UiO-66 with an immobilization yield of 65% achieved after a 6 h loading period. The immobilization of UiO-66 increased the enzyme activity and stability, as indicated by the obtained Kd and T1/2 values. Furthermore, the hydrolytic activity of chitinase was enhanced after immobilization on UiO-66, with an increase in the Vmax and a decrease in the Km of 2- and 38-fold, respectively. Interestingly, the antifungal activity of the produced chitinase was boosted against C. auris by loading the enzyme on UiO-66, with an MIC50 of 0.89 ± 0.056 U/mL, compared to 5.582 ± 0.57 U/mL for the free enzyme. This study offers a novel promising alternative approach to combat the new emerging pathogen C. auris.

Three-Step Enzymatic Remodeling of Chitin into Bioactive Chitooligomers.

Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of ß-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.

Biocontrol potential and growth-promoting effect of endophytic fungus Talaromyces muroii SD1-4 against potato leaf spot disease caused by Alternaria alternata.

BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.

Production of a new chitinase from Nocardiopsis halophila TN-X8 utilizing bio-waste from the blue swimming crab: enzyme characterization and immobilization.

In accordance with the framework of the Circular Blue Bioeconomy in the Mediterranean region, the objective of this study was to evaluate the biotransformation of blue swimming crab (Portunus segnis) residues obtained from the port of Sfax by an extracellular chitinase produced by Nocardiopsis halophila strain TN-X8 isolated from Chott El Jerid (Tozeur, Tunisia). From the analysis of multiple extremophilic Actinomycetota, it was determined that strain TN-X8 exclusively utilized 60 g/L of raw blue swimming crab as its carbon and energy source, achieving a chitinase activity of approximately 950 U/mL following a 6-day incubation period at 40 °C. Pure chitinase, designated as ChiA-Nh30, was obtained after heat treatment, followed by ammonium sulfate fractionation and Sephacryl® S-200 column chromatography. The maximum ChiA-Nh30 activity was observed at pH 3 and 75 °C. Interestingly, compared with cyclohexamidine, ChiA-Nh30 showed a good antifungal effect against four pathogenic fungi. Furthermore, when using colloidal chitin as substrate, ChiA-Nh30 demonstrated a higher degree of catalytic efficiency than the commercially available Chitodextrinase®. In addition, ChiA-Nh30 could be immobilized by applying encapsulation and encapsulation-adsorption techniques. The kaolin and charcoal used acted as excellent binders, resulting in improved ChiA-Nh30 stability. For the immobilized ChiA-Nh30, the yield of N-acetyl-D-glucosamine monomers released from 20% (w/v) blue swimming crab residues increased by 3.1 (kaolin) and 2.65 (charcoal) times, respectively.

Combined analysis of transcriptomics with metabolomics provides insights into the resistance mechanism in winter jujube using L-Methionine.

Black rots lead to great economic losses in winter jujube industry. The objective of this research was to delve into the underlying mechanisms of enhanced resistance of winter jujube fruit to black rot by L-Methionine (Met) treatment. The findings revealed that the application of Met significantly curtailed lesion diameter and decay incidence in winter jujube fruit. The peroxidase (POD) activity in the Met-treated jujubes was 3.06-fold that in the control jujubes after 4 d of treatment. By day 8, the activities of phenylalanine ammonia-lyase (PAL), chitinase (CHI) and ß-1,3-glucanase (GLU) in the Met-treated jujubes had surged to their zenith, being 1.39, 1.22, and 1.52 times in the control group, respectively. At the end of storage, the flavonoid and total phenol content remained 1.58 and 1.06 times than that of the control group. Based on metabolomics and transcriptomics analysis, Met treatment upregulated 6 key differentially expressed metabolites (DEMs) (succinic acid, trans-ferulic acid, salicylic acid, delphinium pigments, (S)-abscisic acid, and hesperidin-7-neohesperidin), 12 key differentially expressed genes (DEGs) (PAL, CYP73A, COMT, 4CL, CAD, POD, UGT72E, ANS, CHS, IAA, TCH4 and PR1), which were involved in phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway and plant hormone signal transduction pathway. Further analysis revealed that the most of the enzymes, DEMs and DEGs in this study were associated with both antioxidant and disease resistance. Consequently, Met treatment enhanced disease resistance of winter jujube fruit by elevating antioxidant capacity and triggering defense response. This study might provide theoretical support for utilizing Met in the management and prevention of post-harvest black rot in winter jujube.

Active Discovery of the Allosteric Inhibitor Targeting Botrytis cinerea Chitinase Based on Neural Relational Inference for Food Preservation.

Currently, allosteric inhibitors have emerged as an effective strategy in the development of preservatives against the drug-resistant Botrytis cinerea (B. cinerea). However, their passively driven development efficiency has proven challenging to meet the practical demands. Here, leveraging the deep learning Neural Relational Inference (NRI) framework, we actively identified an allosteric inhibitor targeting B. cinerea Chitinase, namely, 2-acetonaphthone. 2-Acetonaphthone binds to the crucial domain of Chitinase, forming the strong interaction with the allosteric sites. Throughout the interaction process, 2-acetonaphthone diminished the overall connectivity of the protein, inducing conformational changes. These findings align with the results obtained from Chitinase activity experiments, revealing an IC50 value of 67.6 µg/mL. Moreover, 2-acetonaphthone exhibited outstanding anti-B. cinerea activity by inhibiting Chitinase. In the gray mold infection model, 2-acetonaphthone significantly extended the preservation time of cherry tomatoes, positioning it as a promising preservative for fruit storage.

Marine Bacillus haynesii chitinase: Purification, characterization and antifungal potential for sustainable chitin bioconversion.

The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0-9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL-1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL-1 and Vmax of 5.75 mmol min-1. Kcat and catalytic efficiency were measured at 1.91 s-1 and 191 mL mg-1 s-1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent.

Assessment of Fungal Lytic Enzymatic Extracts Produced Under Submerged Fermentation as Enhancers of Entomopathogens' Biological Activity.

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of - 80, - 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml-1, chitinase activity of 0.28 ± 0.01 U ml-1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml-1, lipase activity of 0.32 U ml-1, and protease activity of 0.24 U ml-1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents' performance.

Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste.

Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

The chitinase genes TuCht4 and TuCht10 are indispensable for molting and survival of Tetranychus urticae.

Insect chitinases (Chts) play a crucial role in the molting process, enabling continuous growth through sequential developmental stages. Based on their high homology to insect Chts, TuCht1 (group II), TuCht4 (group I) and TuCht10 (group IV) were identified, and their roles during molting process were investigated. TuCht1 was mainly expressed in the deutonymphal stage, while TuCht4 was mainly expressed in the nymphal stage and the highest expression level of TuCht10 was observed in the larvae. Feeding RNAi assays have shown that group I TuCht4 and group Ⅳ TuCht10 are involved in mite molting. Suppression of TuCht4 or TuCht10 resulted in high mortality, molting abnormalities and the absence of distinct electron dense layers of chitinous horizontal laminae in the cuticle, as demonstrated by scanning electron microscopy and transmission electron microscopy. The nanocarrier mediated RNAi had significantly higher RNAi efficiency and caused higher mortality. The results of the present study suggest that chitinase genes TuCht4 and TuCht10 are potential targets for dietary RNAi, and demonstrates a nanocarrier-mediated delivery system to enhance the bioactivity of dsRNA, providing a potential technology for green pest management.

Toxoplasma gondii chitinase-like protein TgCLP1 regulates the parasite cyst burden.

Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis.

Chitinase Producing Gut-Associated Bacteria Affected the Survivability of the Insect Spodoptera frugiperda.

BACKGROUND: Fall armyworm (Spodoptera frugiperda) is a highly destructive maize pest that significantly threatens agricultural productivity. Existing control methods, such as chemical insecticides and entomopathogens, lack effectiveness, necessitating alternative approaches. METHODS: Gut-associated bacteria were isolated from the gut samples of fall armyworm and screened based on their chitinase and protease-producing ability before characterization through 16S rRNA gene sequence analysis. The efficient chitinase-producing Bacillus licheniformis FGE4 and Enterobacter cloacae FGE18 were chosen to test the biocontrol efficacy. As their respective cell suspensions and extracted crude chitinase enzyme, these two isolates were applied topically on the larvae, supplemented with their feed, and analyzed for their quantitative food use efficiency and survivability. RESULTS: Twenty-one high chitinase and protease-producing bacterial isolates were chosen. Five genera were identified by 16S rRNA gene sequencing: Enterobacter, Enterococcus, Bacillus, Pantoea, and Kocuria. In the biocontrol efficacy test, the consumption index and relative growth rate were lowered in larvae treated with Enterobacter cloacae FGE18 by topical application and feed supplementation. Similarly, topical treatment of Bacillus licheniformis FGE4 to larvae decreased consumption index, relative growth rate, conversion efficiency of ingested food, and digested food values. CONCLUSION: The presence of gut bacteria with high chitinase activity negatively affects insect health. Utilizing gut-derived bacterial isolates with specific insecticidal traits offers a promising avenue to control fall armyworms. This research suggests a potential strategy for future pest management.

Analysis of chitinase gene family in barley and function study of HvChi22 involved in drought tolerance.

BACKGROUND: Chitinase (Chi) is a pathogenesis-related protein, also reported to play an important role in plant responses to abiotic stress. However, its role in response to abiotic stress in barley is still unclear. RESULTS: In this study, a total of 61 Chi gene family members were identified from the whole genome of wild barley EC_S1. Phylogenetic analysis suggested that these family genes were divided into five groups. Among these genes, four pairs of collinearity genes were discovered. Besides, abundant cis-regulatory elements, including drought response element and abscisic acid response element were identified in the promoter regions of HvChi gene family members. The expression profiles revealed that most HvChi family members were significantly up-regulated under drought stress, which was also validated by RT-qPCR measurements. To further explore the role of Chi under drought stress, HvChi22 was overexpressed in Arabidopsis. Compared to wild-type plants, overexpression of HvChi22 enhanced drought tolerance by increasing the activity of oxidative protective enzymes, which caused less MDA accumulation. CONCLUSION: Our study improved the understanding of the Chi gene family under drought stress in barley, and provided a theoretical basis for crop improvement strategies to address the challenges posed by changing environmental conditions.

Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

Heterologous Expression and Characterization of a pH-Stable Chitinase from Micromonospora aurantiaca with a Potential Application in Chitin Degradation.

To promote the bioconversion of marine chitin waste into value-added products, we expressed a novel pH-stable Micromonospora aurantiaca-derived chitinase, MaChi1, in Escherichia coli and subsequently purified, characterized, and evaluated it for its chitin-converting capacity. Our results indicated that MaChi1 is of the glycoside hydrolase (GH) family 18 with a molecular weight of approximately 57 kDa, consisting of a GH18 catalytic domain and a cellulose-binding domain. We recorded its optimal activity at pH 5.0 and 55 °C. It exhibited excellent stability in a wide pH range of 3.0-10.0. Mg2+ (5 mM), and dithiothreitol (10 mM) significantly promoted MaChi1 activity. MaChi1 exhibited broad substrate specificity and hydrolyzed chitin, chitosan, cellulose, soluble starch, and N-acetyl chitooligosaccharides with polymerization degrees ranging from three to six. Moreover, MaChi1 exhibited an endo-type cleavage pattern, and it could efficiently convert colloidal chitin into N-acetyl-D-glucosamine (GlcNAc) and (GlcNAc)2 with yields of 227.2 and 505.9 mg/g chitin, respectively. Its high chitin-degrading capacity and exceptional pH tolerance makes it a promising tool with potential applications in chitin waste treatment and bioactive oligosaccharide production.

BpAFP, a Broussonetia papyrifera latex chitinase, exhibits a dual role in resisting to both Verticillium wilt disease and lepidopterous pests, Plutella xylostella and Prodenia litura.

Paper mulberry (Broussonetia papyrifera) is a fast-growing tree known for its tolerance to diverse biotic and abiotic stresses. To explore genes combating Verticillium wilt, a devasting and formidable disease damage to cotton and many economically significant crops, we purified an antifungal protein, named BpAFP, from the latex of paper mulberry. Based on peptide fingerprint, we cloned the full cDNA sequence of BpAFP and revealed that BpAFP belongs to Class I chitinases, sharing 74 % identity with B. papyrifera leaf chitinase, PMAPII. We further introduced BpAFP into Arabidopsis, tobacco, and cotton. Transgenic plants exhibited significant resistance to Verticillium wilt. Importantly, BpAFP also demonstrated insecticidal activity against herbivorous pests, Plutella xylostella, and Prodenia litura, when feeding the larvae with transgenic leaves. Our finding unveils a dual role of BpAFP in conferring resistance to both plant diseases and lepidopterous pests.

Co-overexpression of chitinase and ß-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium.

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and ß-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and ß-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 µg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.