Chitinase Production Research Articles

Extraction of RNA from the fungus Moniliophthora Perniciosa (Stahel) Aime and Phillips-Mora cultivated in induction medium for characterization of chitininases

Chitin is considered the second most important biopolymer in nature, both for its physiological functions in different organisms and for its high availability in the biosphere. However, its natural accumulation does not occur in the environment, this demonstrates that there are natural mechanisms responsible for its degradation. The chitinolytic enzymes found in living beings are targets of great biotechnological interest because they degrade chitin into useful derivatives for various sectors of the economy. Given this, this study aimed to extract RNA for subsequent molecular characterization of chitinases from the fungus Moniliophthora perniciosa. To this end, the fungus was cultivated in WY medium, remaining in the greenhouse for 10-14 days. After growth, RNA was extracted using the traditional Trizol method and the Direct-zol commercial kit. The resulting samples were analyzed using the spectrophotometric technique and verified by photodocumentation after agarose electrophoresis. It was observed that the culture medium was considered satisfactory for RNA extraction and the ideal growth time was 8-10 days for the strain to present sufficient mycelial mass for extraction. Furthermore, the commercial kit used was more favorable than the traditional Trizol method, the product had a low level of contaminants and electrophoresis demonstrated high RNA quality due to the presence of intact rRNA bands. Therefore, as it was extracted from cultivation in a medium that induces chitinase production, the RNA extraction described becomes the most suitable method for the molecular characterization of chitinase production.

Production of chitinase in elicited tomato cell suspensions and its application as a biopesticide and fungicide against soil-borne pests and fungi

Soil-borne fungi and pests present significant challenges to crop production, causing diseases and reducing yields. This study explores the potential of producing chitinase from tomato cell suspension cultures and its application in controlling soil-borne fungi and pests. Tomato cell suspension cultures were established from seeds, and different elicitors—including chitin, fungal debris, and fungal metabolites—were used to optimize chitinase production. The enzyme was purified, and its physicochemical properties were characterized. Its antifungal activity was tested against various soil-borne fungi, while termiticidal activity was evaluated using four bioassay methods. Additionally, the nematicidal activity of chitinase was assessed against different stages of root-knot nematodes. Results indicated that chitin media induced the highest chitinase production and cell growth compared to other elicitors. The purified chitinase had a molecular weight of 34 kDa, with a maximum velocity (Vmax) of 0.940 μg NAG/min and a Michaelis constant of 1.236 mg colloidal chitin/mL. The turnover number (Kcat) was 1.598 μg NAG/min/mol chitinase. The enzyme exhibited a broad range of activity and stability across varying pH levels and temperatures, as well as remarkable stability under different storage times and in the presence of metal ions. Tomato-derived chitinase demonstrated superior antifungal, nematicidal, and termiticidal activities compared to common chemical fungicides and pesticides. Chitinase was successfully produced from tomato cell suspension culture in this study, highlighting its potential as a biocontrol agent for sustainable pest and disease management in agriculture.

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

Genomics, Proteomics, and Antifungal Activity of Chitinase from the Antarctic Marine Bacterium Curtobacterium sp. CBMAI 2942.

This study aimed to evaluate the genomic profile of the Antarctic marine Curtobacterium sp. CBMAI 2942, as well as to optimize the conditions for chitinase production and antifungal potential for biological control. Assembly and annotation of the genome confirmed the genomic potential for chitinase synthesis, revealing two ChBDs of chitin binding (Chi C). The optimization enzyme production using an experimental design resulted in a 3.7-fold increase in chitinase production. The chitinase enzyme was identified by SDS-PAGE and confirmed through mass spectrometry analysis. The enzymatic extract obtained using acetone showed antifungal activity against the phytopathogenic fungus Aspergillus sp. series Nigri CBMAI 1846. The genetic capability of Curtobacterium sp. CBMAI 2942 for chitin degradation was confirmed through genomic analysis. The basal culture medium was adjusted, and the chitinase produced by this isolate from Antarctica showed significant inhibition against Aspergillus sp. Nigri series CBMAI 1846, which is a tomato phytopathogenic fungus. This suggests that this marine bacterium could potentially be used as a biological control of agricultural pests.

Sustainable quick quantitative screening for the physiology of chitinases production by Bacillus thuriengiensis gallariae and detection of Aedes aegypti biopesticide activity

Background Chitin is considered the world’s first and most abundant amino-carbon substrate. Objective The proposed method aims to speed up the detection and measuring of microbial chitinase production. Materials and methods Solid-state fermentation, plates, viscosity, and spectrophotometric methods were used to detect microbial chitinase activity. Analysis of variance was used for statistical analysis. Results and conclusion The trailed Bacillus thuriengiensis gallariae strain was differentiated from other tested strains of Bacillus thuriengiensis in terms of mosquito resistance, besides being superior to other studied Bacillus sphaericus. As anticipated, results from the clear zone plates method were not clear compared with the viscosity method where the strain exhibited the best. Worthy, the same result was reached in the color methods. The superlative incubation period for the physiological properties of chitinase production from Bacillus thuriengiensis gallariae was attained after 5 days. Results indicated that the best size of chitin particles was 1 mm, the best percentage of humidity of the growth was 67%, the best inoculum volume was 10 ml, and the best carbon source for the production of crude chitinases was glucose. The optimal concentration for the production of chitinases was 20% of the weight of the growth medium, yet 5% of the weight of the growth medium was the optimal percentage of glucose as a carbon source for the production of other protein metabolites. The proposed colorimetric method seemed to be an effective and rapid method to survey the aptitude of huge quantities of microorganisms to produce chitinases.

Evaluation and Characterization of Rhizobacteria and Endophytes for Antagonistic and Growth Promotional Traits Against Collar Rot (Sclerotium rolfsii Sacc.) of Soybean

Background: Sclerotium rolfsii Sacc. is one of the destructive soil-borne plant pathogens due to its significant loss in terms of yield and quality in several crop plants including soybean. Use of biocontrol agents in the management of disease is an ecofriendly strategy. Therefore, the study aimed to assess in vitro antagonisticactivity of rhizobacteria and endophytes against Sclerotium rolfsii following dual culture technique. Methods: A total of 75 rhizobacterial isolates, 3 endophytes along with reference cultures were evaluated for the antagonistic activity against Sclerotium rolfsii following dual culture technique. The effective rhizobacterial isolates were tested for the various plat growth promoting rhizobacteria traits viz., indole acetic acid production, siderophore production, Phosphate solubilization, production of ammonia, hydrogen cyanide production and chitinase test following the standard procedure. Result: The two promising rhizobacterial strains i.e., AUUB 209 (Streptomyces enissocaesilis) and Streptomyces racemochromogenes (AUDT 626) were found effective with 76.79 and 64.75 percent mycelial inhibition of Sclerotium rolfsii under in vitro. The antagonistic activity of these two rhizobacteria is mainly attributed to the positive confirmation of various PGPR activities viz., production of Indole acetic acid, siderophore, phosphate solubilization and chitinase production.

Genome assembly, comparative genomics, and identification of genes/pathways underlying plant growth-promoting traits of an actinobacterial strain, Amycolatopsis sp. (BCA-696)

The draft genome sequence of an agriculturally important actinobacterial species Amycolatopsis sp. BCA-696 was developed and characterized in this study. Amycolatopsis BCA-696 is known for its biocontrol properties against charcoal rot and also for plant growth-promotion (PGP) in several crop species. The next-generation sequencing (NGS)-based draft genome of Amycolatopsis sp. BCA-696 comprised of ~ 9.05 Mb linear chromosome with 68.75% GC content. In total, 8716 protein-coding sequences and 61 RNA-coding sequences were predicted in the genome. This newly developed genome sequence has been also characterized for biosynthetic gene clusters (BGCs) and biosynthetic pathways. Furthermore, we have also reported that the Amycolatopsis sp. BCA-696 produces the glycopeptide antibiotic vancomycin that inhibits the growth of pathogenic gram-positive bacteria. A comparative analysis of the BCA-696 genome with publicly available closely related genomes of 14 strains of Amycolatopsis has also been conducted. The comparative analysis has identified a total of 4733 core and 466 unique orthologous genes present in the BCA-696 genome The unique genes present in BCA-696 was enriched with antibiotic biosynthesis and resistance functions. Genome assembly of the BCA-696 has also provided genes involved in key pathways related to PGP and biocontrol traits such as siderophores, chitinase, and cellulase production.

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 60g/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.

Fungal endo and exochitinase production, characterization, and application for Candida biofilm removal.

Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.

Exploring the potential of Bacillus for crop productivity and sustainable solution for combating rice false smut disease.

Rice false smut, which is caused by the soil-borne fungal pathogen Ustilaginoidea virens (U. virens), is one of the most threatening diseases in most of the rice-growing countries including India that causes 0.5-75% yield loss, low seed germination, and a reduction in seed quality. The assessment of yield loss helps to understand the relevance of disease severity and facilitates the implementation of appropriate management strategies. This study aimed to mitigate biotic stress in rice by employing a rhizobacterial-based bioformulation, which possesses diverse capabilities as both a plant growth promoter and a biocontrol agent against U. virens. Rhizobacteria were isolated from the soil of the rice rhizospheres from the healthy plant of the false smut affected zone. Furthermore, they were identified as Bacillus strains: B. subtilis (BR_4), B. licheniformis (BU_7), B. licheniformis (BU_8), and B. vallismortis (KU_7) via sequencing. Isolates were screened for their biocontrol potential against U. virens under in vitro conditions. The antagonistic study revealed that B. vallismortis (KU_7) inhibited U. virens the most (44.6%), followed by B. subtilis BR_4 (41.4%), B. licheniformis BU_7 (39.8%), and B. licheniformis BU_8 (43.5%). Various biochemical and plant growth promoting attributes, such as phosphate and Zn solubilization, IAA, ammonium, siderophore, and chitinase production, were also investigated for all the selected isolates. Furthermore, the potential of the isolates was tested in both in vitro and field conditions by employing talc-based bioformulation through bio-priming and root treatment. The application of bioformulation revealed a 20% decrease in disease incidence in plants treated with B. vallismortis (KU_7), a 60.5% increase in the biological yield, and a 45% increase in the grain yield. This eco-friendly approach not only controlled the disease but also improved the grain quality and reduced the chaffiness.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation

BackgroundHydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by response surface methodology (RSM) which delvs into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers.ResultsIn this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts.ConclusionsThe recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Impact of microbial consortium of Rhizobium tropici and Rhizobium mayense on the growth of Phaseolus vulgaris L.

The main objectives of the present investigation was to characterize and recognize potential indigenous Rhizobium spp. associated with red clover nodules, as well as to evaluate the influence of prospective rhizobia inoculation (individual and consortia) on common bean plant growth. Total of 8 rhizobia like bacteria were isolated and were investigated for their specific PGP traits, production of hydrolytic enzymes. Results revealed, four isolates synthesize IAA (22.3 ± 1.45 to 88.6 ± 1.45 μg mL−1), three isolates solubilized phosphate (40.69 ± 1.25 to 216.3 ± 1.31 μg mL−1), three isolates solubilized potassium, produced siderophore, and showed chitinase production, two isolates possess ACC deaminase activity, one isolate was able to solubilize zinc, 5 isolates produced β- 1, 3-glucanase (0.38 ± 0.005 to 2.82 ± 0.011 % units) and four isolates produced cellulase. Two bacterial isolates found to exhibit most of the PGP activities, were selected and identified on the basis of phenotypic, biochemical tests and 16S rRNA sequencing, as Rhizobium tropici IHTF-1 and Rhizobium mayense IHTF-2. Interaction of these potential rhizobium strains individually and consortia with common bean plants under greenhouse conditions boosted root and shoot length, fresh and dry biomass compared to un-inoculated control plants. SEM study revealed that both the strains, IHTF-1 and IHTF-2 colonized common bean roots. These results imply that Rhizobium spp., identified in this work, can be used as bio-inoculants to increase common bean plant production in a sustainable way.

Response surface optimization, purification, characterization and short-chain chitooligosaccharides production from an acidic, thermostable chitinase from Thermomyces dupontii

Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul−1 h−1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol−1, 100.59 kJ mol−1, 102.64 kJ mol−1 and 5.95 J mol−1 K−1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2–4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2–4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.

Enzyme production by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and their application in the control of nematodes (Haemonchus spp. and Meloidogyne incognita) in vitro

Brazil is affected by the infection of gastrointestinal nematodes (Haemonchus spp.) because it causes subclinical diseases in small ruminants that directly affect weight and milk production and, in turn, generate a health risk for the animals. In the same way root-knot nematodes (Meloidogyne incognita) is a serious disease, which parasitize the roots of tomato plants causing damage such as poor nutrient absorption leading to significant yield losses. In order to minimize the economic impact of these nematodes, it is important to establish new control strategies. Beauveria bassiana and Metarhizium anisopliae are two main mycoinsecticides used to control many orders of insects, such as Lepidoptera and Hemiptera. These fungi have the ability to produce extracellular enzymes, which play an important role to control in the pest infection process. The aim of this study was to evaluate the production of proteases and chitinases by the entomopathogenic fungi B. bassiana and M. anisopliae on different solid and liquid culture media and their application in vitro to the control of Haemonchus spp and Meloidogyne incognita. To achieve this goal, enzymes were produced under solid and liquid fermentation conditions to determine the highest chitinolytic and proteolytic activity of commercial two strain of B. bassiana (IBCB 66 and ESALQ PL63) and M. anisopliae (IBCB 425 and ESALQ E9) in liquid media: SDY broth, YPG culture medium, synthetic, and soluble starch with yeast extract. The solid media tested were: rice supplemented with whey and chrysalis flour mixed with rice. In addition, the nematicidal action of their extracts (containing concentrated enzymes and without fungal cells) was measured. The results showed that the best culture medium (p< 0.01) for protease was solid serum rice medium, with a value for M. anisopliae IBCB 425 of 52 U/mg, and for B. bassiana ESALQ PL63 the value was 36 U/mg, compared to all the tested media. On the other hand, for chitinase of M. anisopliae strain IBCB 425, the activity value was close to 0.60 U/mg. In contrast, for B. bassiana isolate ESALQ PL63, SDY medium was the best inducer for chitinase production, with a value of 0.90 U/mg. Regarding the nematicidal activity of the crude extracts, the reduction percentages were 58% for ESALQ PL63 and 100% for IBCB 425 in the case of animal parasites. For plant parasites, the reduction percentages were 19% for ESALQ PL63 and 71% for IBCB 425. Thus, the use of enzyme-rich crude extracts presents promising control options for pest control.

Rhizospheric Fungal spp. of Tomato (Solanum lycopersicum L.) and Their Effect on Tomato Plant Growth and on Alternaria solani, the Causative Agent of Early Blight of Tomato

Tomato (Solanum lycopersicum L.) is a widely consumed crop throughout the world including Sri Lanka. Use of agrochemicals for enhanced yields and managing diseases associated with tomato results in deleterious impacts on soil microflora, fauna and leads to human health problems. A possible alternative is the use of rhizospheric microorganisms, as they are reported to improve productivity and protect various crops. Hence this study aimed to isolate rhizospheric fungal assemblages of tomato plants and evaluate their ability to solubilize phosphate while, controlling Alternaria solani, the causative agent of Early Blight of tomatoes. Rhizospheric fungi were isolated from commercial tomato cultivations in two agro-ecological zones (WM3b and IM2b), using soil dilution plate technique. Composite samples of soil attached to the root systems of five healthy plants, uprooted from each field were prepared and 10 g of each sample was used for the isolations. Twenty-seven fungal genera were isolated and Trichoderma and Penicillium were the frequently isolated genera. Phosphate solubilization ability of each fungal species was evaluated using Pikovskaya's agar and liquid medium, and four isolates (Eupenicillium sp., Talaromyces purpureogenus, Paecilomyces sp., and Purpureocillium lilacinum) with the highest phosphate solubilizing abilities were evaluated on tomato plant growth under greenhouse condition. The inocula of each fungal sp. were prepared by adding fifteen, 6mm diameter mycelial discs from each 5-day-old culture into polypropylene bags containing 90 g of sterile rice husk medium and incubated for 02 weeks at room temperature. Each pot contained a 4.6 kg layer of non- sieved sterilized potting medium (Sand: topsoil: Compost at 2:1:1 ratio) and a 2.5 kg layer of sieved medium on top, which was mixed with 15 g of inoculum. Three-week old, healthy tomato seedlings (variety-Bathiya) were planted (with 3 replicates) and allowed to grow with regular watering. Control pots contained potting medium with uninoculated sterile rice husk. Growth parameters such as Root and shoot lengths, fresh and dry weights of roots, shoots, and leaves were evaluated at 02-week intervals for 02 months and the results were analysed using ANOVA with Tukey‘s pairwise comparison. Plants treated with T. purpureogenus and P. lilacinum showed significant increases (p≤0.05) in all growth parameters in every evaluation. Dual culture plate assay on the Potato Dextrose Agar was used to test the effect of rhizospheric fungal spp. on the radial colony growth of A. solani, and Eupenicillium sp. showed the highest percentage inhibition (85.09±5.68%). Microscopic observations of inhibition zones between two colonies showed the presence of coils, loops, knobs, and haustoria, formed by inhibitory rhizospheric fungal species. Assays for Chitinase and Glucanase enzyme production showed that Mortierella sp.-1, P. lilacinum, Trichoderma sp.-1, and Acremonium sp. were the highest chitinase producers, whereas P. lilacinum was the highest glucanase producer. These results indicate the ability of some rhizospheric fungal species of tomato to increase the growth of tomato plants significantly by phosphate solubilization and ability to inhibit the growth of foliar pathogen A. solani under in vitro conditions. &#x0D; Keywords: Rhizospheric fungi, Phosphate solubilization, Dual culture plate, Plant growth parameters

Characterization of endophytic bacteria growth-promoting in potato plants (Solanum tuberosum)

Objective/Background. The purpose of this research was to evaluate the in vitro plant growth-promoting activity of endophytic bacteria isolated in tissue from Atlantic variety potato plants from the municipality of Guasave, Sinaloa, Mexico. Materials and Methods. The bacterial population was isolated in Lb agar culture medium; two bacterial isolates were obtained from the root and two from the stem, all four Gram positive. The bacterial population of the tissue samples was expressed as (CFU/g-1). The phosphate solubilization capacity, production of chitinases and siderophores were qualitatively evaluated. Results. Partial sequencing of the 16S rDNA gene was performed, allowing the identification of associated bacterial species within the Firmicutes. 100% of the strains were identified as Bacillus sp. with identities greater than 97%: B. cereus, B. tropicus, B. thuringiensis, B. fungorum. The B. thuringiensis and B. cereus strains showed positive activity in promoting plant growth in vitro through phosphate solubilization, production of chitinases and siderophores. B. cereus and B. tropicus presented inhibitory capacity greater than 50% for Sclerothium rolfsii. Conclusion. It is relevant to continue research carried out in the laboratory, in order to determine its potential in the field, improving the production of potato crops.

Impact of chitin-derived β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine on chitinase upregulation in Shewanella baltica.

The first steps in chitin degradation in marine bacteria involve chitinase, which produces N,N'-diacetylchitobiose (GlcNAc)2 from chitin. Moreover, in Vibrio bacteria, chitinase activity is enhanced by heterodisaccharide β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) produced from (GlcNAc)2 by chitin oligosaccharide deacetylase (COD). However, the role of COD in other marine bacteria, such as Shewanella, remains unexplored. This study investigates GlcNAc-GlcN's impact on chitinase gene expression and enzyme production in S. baltica ATCC BAA-1091, drawing parallels with Vibrio parahaemolyticus RIMD2210633. Using real-time quantitative PCR, the study assesses the upregulation of chitinase gene expression in S. baltica in response to GlcNAc-GlcN, informed by COD's known ability to produce GlcNAc-GlcN from (GlcNAc)2. In Vibrio, GlcNAc-GlcN considerably upregulates chitinase gene expression. This study posits a similar regulatory mechanism in S. baltica, with preliminary investigations indicating COD's capacity to produce GlcNAc-GlcN. This study highlights the importance of exploring GlcNAc-GlcN's regulatory role in chitin metabolism across diverse marine bacteria. The potential induction of chitinase production in S. baltica suggests broader ecological implications. Further research is crucial for a comprehensive understanding of chitin utilization and regulatory pathways in marine bacterial genera.

Identification of the Csr global regulatory system mediated by small RNA decay in Aeromonas salmonicida.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Chitinase production ability of Trichoderma harzianum isolates by Turbidity method

Chitinase production ability of Trichoderma harzianum isolates by Turbidity method