Chitinase-encoding Gene Research Articles

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.

The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases.

Atlantic salmon (Salmo salar) possesses a genome containing 10 genes encoding chitinases, yet their functional roles remain poorly understood. In other fish species, chitinases have been primarily linked to digestion, but also to other functions, as chitinase-encoding genes are transcribed in a variety of non-digestive organs. In this study, we investigated the properties of two chitinases belonging to the family 18 glycoside hydrolase group, namely Chia.3 and Chia.4, both isolated from the stomach mucosa. Chia.3 and Chia.4, exhibiting 95% sequence identity, proved inseparable using conventional chromatographic methods, necessitating their purification as a chitinase pair. Biochemical analysis revealed sustained chitinolytic activity against β-chitin for up to 24 h, spanning a pH range of 2 to 6. Moreover, subsequent in vitro investigations established that this chitinase pair efficiently degrades diverse chitin-containing substrates into chitobiose, highlighting the potential of Atlantic salmon to utilize novel chitin-containing feed sources. Analysis of the gastric matrix proteome demonstrates that the chitinases are secreted and rank among the most abundant proteins in the gastric matrix. This finding correlates well with the previously observed high transcription of the corresponding chitinase genes in Atlantic salmon stomach tissue. By shedding light on the secreted chitinases in the Atlantic salmon's stomach mucosa and elucidating their functional characteristics, this study enhances our understanding of chitinase biology in this species. Moreover, the observed capacity to effectively degrade chitin-containing materials implies the potential utilization of alternative feed sources rich in chitin, offering promising prospects for sustainable aquaculture practices.

PbrChiA: a key chitinase of pear in response to Botryosphaeria dothidea infection by interacting with PbrLYK1b2 and down-regulating ROS accumulation.

Pear ring rot, caused by the pathogenic fungi Botryosphaeria dothidea, seriously affects pear production. While the infection-induced reactive oxygen species (ROS) burst of infected plants limits the proliferation of B. dothidea during the early infection stage, high ROS levels can also contribute to their growth during the later necrotrophic infection stage. Therefore, it is important to understand how plants balance ROS levels and resistance to pathogenic B. dothidea during the later stage. In this study, we identified PbrChiA, a glycosyl hydrolases 18 (GH18) chitinase-encoding gene with high infection-induced expression, through a comparative transcriptome analysis. Artificial substitution, stable overexpression, and virus induced gene silencing (VIGS) experiments demonstrated that PbrChiA can positively regulate pear resistance as a secreted chitinase to break down B. dothidea mycelium in vitro and that overexpression of PbrChiA suppressed infection-induced ROS accumulation. Further analysis revealed that PbrChiA can bind to the ectodomain of PbrLYK1b2, and this interaction suppressed PbrLYK1b2-mediated chitin-induced ROS accumulation. Collectively, we propose that the combination of higher antifungal activity from abundant PbrChiA and lower ROS levels during later necrotrophic infection stage confer resistance of pear against B. dothidea.

Cloning and biochemical characterization of a recombinant chitinase encoded by the CHT4 gene from Candida albicans

As one of the major components in the fungal cell wall, chitin is a polymer of β-1,4-linked N-acetylglucosamine. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. The human fungal pathogen Candida albicans has three chitinase-encoding genes, CaCHT1, CaCHT2 and CaCHT3. The CaCHT4 gene encodes a protein with the glycoside hydrolase family GH18 domain, Glyco_18, which suggests that CaCht4 might be a chitinase. In the present study, we have cloned, expressed and purified the N-terminally His6-tagged CaCht4 protein from bacterial cells. Further biochemical characterization has shown that this recombinant CaCht4 protein shows both exochitinase (chitobiosidase) and endochitinase activities, but has no N-acetylglucosaminase activity. The optimal temperature for the exochitinase activity of CaCht4 is 55 °C. Taken together, these data support that the CaCHT4 gene encodes a chitinase. Our finding provides a basis for us to understand the biological functions of the CaCHT4 gene in C. albicans.

Transcriptomic Response of Clonostachys rosea Mycoparasitizing Rhizoctonia solani.

Clonostachys rosea is an important mycoparasitism biocontrol agent that exhibits excellent control efficacy against numerous fungal plant pathogens. Transcriptomic sequencing may be used to preliminarily screen mycoparasitism-related genes of C. rosea against fungal pathogens. The present study sequenced and analyzed the transcriptome of C. rosea mycoparasitizing a Basidiomycota (phylum) fungal pathogen, Rhizoctonia solani, under three touch stages: the pre-touch stage, touch stage and after-touch stage. The results showed that a number of genes were differentially expressed during C. rosea mycoparasitization of R. solani. At the pre-touch stage, 154 and 315 genes were up- and down-regulated, respectively. At the touch stage, the numbers of up- and down-regulated differentially expressed genes (DEGs) were 163 and 188, respectively. The after-touch stage obtained the highest number of DEGs, with 412 and 326 DEGs being up- and down-regulated, respectively. Among these DEGs, ABC transporter-, glucanase- and chitinase-encoding genes were selected as potential mycoparasitic genes according to a phylogenetic analysis. A comparative transcriptomic analysis between C. rosea mycoparasitizing R. solani and Sclerotinia sclerotiorum showed that several DEGs, including the tartrate transporter, SDR family oxidoreductase, metallophosphoesterase, gluconate 5-dehydrogenase and pyruvate carboxylase, were uniquely expressed in C. rosea mycoparasitizing R. solani. These results significantly expand our knowledge of mycoparasitism-related genes in C. rosea and elucidate the mycoparasitism mechanism of C. rosea.

Cloning, expression, purification and biochemical characterization of the recombinant chitinase enzyme encoded by CTS2 in the budding yeast

Chitin is a polymer of β-1,4-linked N-acetylglucosamine (GlcNAc) and plays a central role in the assembly of the fungal cell wall. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. Chitinases are classified into three categories, endochitinases, exochitinases and N-acetylglucosaminases, according to the manner in which the enzyme cleaves the chitin polymer. Saccharomyces cerevisiae has two chitinase-encoding genes, CTS1 and CTS2. However, whether Cts2p shows a chitinase activity remains unknown. In this study, we have cloned, expressed and purified the recombinant Cts2p protein from bacterial cells. We have demonstrated that Cts2p has a higher chitobiosidase (exochitinase) activity than endochitinase activity, but no N-acetylglucosaminase activity. The optimal temperature for the chitobiosidase activity of Cts2p is 37 °C.

Comparative genomics of Bacillus cereus sensu lato spp. biocontrol strains in correlation to in-vitro phenotypes and plant pathogen antagonistic capacity.

Bacillus cereus sensu lato (Bcsl) strains are widely explored due to their capacity to antagonize a broad range of plant pathogens. These include B. cereus sp. UW85, whose antagonistic capacity is attributed to the secondary metabolite Zwittermicin A (ZwA). We recently isolated four soil and root-associated Bcsl strains (MO2, S-10, S-25, LSTW-24) that displayed different growth profiles and in-vitro antagonistic effects against three soilborne plant pathogens models: Pythium aphanidermatum (oomycete) Rhizoctonia solani (basidiomycete), and Fusarium oxysporum (ascomycete). To identify genetic mechanisms potentially responsible for the differences in growth and antagonistic phenotypes of these Bcsl strains, we sequenced and compared their genomes, and that of strain UW85 using a hybrid sequencing pipeline. Despite similarities, specific Bcsl strains had unique secondary metabolite and chitinase-encoding genes that could potentially explain observed differences in in-vitro chitinolytic potential and anti-fungal activity. Strains UW85, S-10 and S-25 contained a (~500 Kbp) mega-plasmid that harbored the ZwA biosynthetic gene cluster. The UW85 mega-plasmid contained more ABC transporters than the other two strains, whereas the S-25 mega-plasmid carried a unique cluster containing cellulose and chitin degrading genes. Collectively, comparative genomics revealed several mechanisms that can potentially explain differences in in-vitro antagonism of Bcsl strains toward fungal plant pathogens.

Effects of trichothecene production by Trichoderma arundinaceum isolates from bean-field soils on the defense response, growth and development of bean plants (Phaseolus vulgaris).

The trichothecene toxin-producing fungus Trichoderma arundinaceum has potential as a biological control agent. However, most biocontrol studies have focused only on one strain, IBT 40837. In the current study, three Trichoderma isolates recovered from bean-field soils produced the trichothecene harzianum A (HA) and trichodermol, the latter being an intermediate in the HA biosynthesis. Based on phylogenetic analysis, the three isolates were assigned to the species T. arundinaceum. Their genome sequences had a high degree of similarity to the reference IBT 40837 strain, in terms of total genome size, number of predicted genes, and diversity of putative secondary metabolite biosynthetic gene clusters. HA production by these bean-field isolates conferred significant in vitro antifungal activity against Rhizoctonia solani and Sclerotinia sclerotiorum, which are some of the most important bean pathogens. Furthermore, the bean-field isolates stimulated germination of bean seeds and subsequent growth of above ground parts of the bean plant. Transcriptomic analysis of bean plants inoculated with these T. arundinaceum bean-field soil isolates indicated that HA production significantly affected expression of plant defense-related genes; this effect was particularly significant in the expression of chitinase-encoding genes. Together, these results indicate that Trichoderma species producing non-phytotoxic trichothecenes can induce defenses in plants without negatively affecting germination and development.

Overexpression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea)

ABSTRACT In tropical and subtropical areas, peanuts are a very important legume crop that is widely cultivated for food and cooking oil. They are, however, extremely susceptible to a wide range of phytopathogens, particularly soil-borne fungi, which result in low yields and poor seed quality. This study aimed to express three Trichoderma asperellum SH16 42 kDa chitinase-encoding genes in peanut to improve their resistance to some soil-borne fungi. Chi42 is a synthetic, intronless, wild-type gene, whereas syncodChi42-1 and syncodChi42-2 are peanut codon-optimized synthetic genes. These genes were introduced into a local peanut strain through Agrobacterium-mediated transformation. Analysis of the transgenic peanut lines showed that chitinase-specific activities from the syncodChi42-1 and syncodChi42-2 genes were approximately 1.2 and 1.4 times higher than those of the wild-type gene, respectively. The engineered peanut plants also exhibited antifungal activity against the soil-borne pathogenic fungus Sclerotium rolfsii. The transgenic peanut lines transformed with the two synthetic genes have stronger antifungal activities than those transformed with the wild-type version, suggesting that they could be used as novel peanut lines to combat phytopathogenic fungi.

Evolutive History of Leishmania Genus and Differential Diagnosis of Clinical Important Species Based on a Unique Kinetoplastida Chitinase

Introduction: Leishmaniasis, a neglected infectious disease affecting humans, domestic, and wild animals, is caused by 20 from 53 Leishmania genus species and transmitted by sandflies. Leishmania genus, belonging to the Trypanosomatidae family and Kinetoplastida order, are grouped into five subgroups according to the biogeographic and evolutive history of parasites and hosts, leading to incongruences and paraphyly. The GH18 Leishmania chitinase, which is encoded by a species-specific single-copy gene, conserved in the basal groups of trypanosomatids, and absent in the genus Trypanosoma, was evaluated as a phylogenetic marker and a diagnostic target. Methods: Primers were designed to detect Leishmania in its host biological samples and obtain the chitinase sequence of species that are unavailable in public databanks. The GH18 chitinase gene and its genomic context were evaluated phylogenetically. A protocol was developed to discriminate Leishmania subgenera by adopting polymerase chain reaction (PCR) and restriction fragment length polymorphism and using in silico tools. The adopted PCR method for detecting a partial 953 bp GH18 chitinase-encoding gene represented high sensibility and specificity on DNA of isolated parasites and was used as negative controls, Trypanosoma cruzi, and DNA from Leishmania hosts. Results: Preservation of the chitinase locus in the aquatic free-living protozoan Bodo saltans disclosed a primitive common origin. Based on the comparative analysis, the amino acid sequence of GH18 trypanosomatidae chitinase demonstrated its high similarity to that of chitinase from marine prokaryotes and protozoans. Phylogenetic reconstruction based on chitinase corroborated the Supercontinent Origins Theory for Leishmania. Conclusion: The chitinase-encoding gene was effectively detected in biological samples and thus could be considered for differential molecular diagnosis among Leishmania clinical important species worldwide.

Foliar Application of Chitosan Increases Tomato Growth and Influences Mycorrhization and Expression of Endochitinase-Encoding Genes.

Nowadays, applying bio-organic fertilizer (e.g., chitosan, Ch) or integrating beneficial microorganisms (e.g., arbuscular mycorrhizal fungi, AMF) are among the successful strategies to promote plant growth. Here, the effect of two application modes of Ch (foliar spray or root treatment) and Ch-derived nanoparticles (NPs) on tomato plants colonized with the AMF Rhizophagus irregularis were analyzed, thereby focusing on plant biomass, flowering and mycorrhization. An increase of shoot biomass and flower number was observed in arbuscular mycorrhizal (AM) plants sprayed with Ch. The interaction with AMF, however, was reduced as shown by decreased mycorrhization rates and AM-specific gene expression. To get insights into Ch effect on mycorrhization, levels of sugars, jasmonates, abscisic acid, and the expression of two chitinase-encoding genes were determined in mycorrhizal roots. Ch had no effect on sugar and phytohormone levels, but the reduced mycorrhization was correlated with down- and upregulated expression of Chi3 and Chi9, respectively. In contrast, application of NPs to leaves and Ch applied to the soil did not show any effect, neither on mycorrhization rate nor on growth of mycorrhizal plants. Concluding, Ch application to leaves enhanced plant growth and flowering and reduced interaction with AMF, whereas root treatment did not affect these parameters.

IMPROVING THE CHITINOLYTIC ACTIVITY OF STREPTOMYCES GRISEORUBENS E44G BY MUTAGENESIS

Genetic improvement trials of the chitinolytic activity of Streptomyces griseorubens E44G were made by using physical, chemical and site-directed mutagenesis. Although the UV radiation, as a physical mutagen, was shed on the tested bacteria for different durations (5, 10, and 15 min), no change in the chitinolytic activity was observed when compared with the wild type. To induce the chemical mutagenesis, S. griseorubens E44G was treated with ethylmethane sulfonate for varied durations (20, 40, and 60 min). The chitinolytic activity decreased with the increment in the exposure period. Four different sets of primers were designed based on the DNA sequence of the wild type of S. griseorubens E44G.Overexpressionof chitinase-encoding genes was observed as three of the amplified mutated genes comparing with the wild-type gene. The chitinolytic activity of the recombinant gene P2 increased by 1.39-fold comparing with the wild-type gene. The molecular weight of the chitinase protein produced by the mutated gene was determined by SDS-PAGE. In conclusion, these results demonstrated that the recombinant gene of S. griseorubens E44G possess a higher level of chitinolytic activity than that of the wild-type. Genetic improvement of the chitinolytic activity of S. griseorubens E44G may enhance their biocontrol potential against phytopathogenic fungi.

In silico analysis of onion chitinases using transcriptome data.

Chitinases are glycoside hydrolase (GH) family of proteins having multifaceted roles in plants. It is of interest to identify and characterize chitinase-encoding genes from the popular bulbous plant onion (Allium cepa L.). We have used the EST sequences for onion chitinases to elucidate its functional features using sequence, structure and functional analysis. These contigs belong to the GH19 chitinases family according to domain architecture analysis. They have highly conserved chitinase motifs including motifs exclusive to plant chitinases as implied using the MEME based structural characterization. Estimation of biochemical properties suggested that these proteins have features to form stable and hydrophilic proteins capable of localizing extracellular and in vacuoles. Further, they have multiple cellular processes including defense role as inferred by DeepGO function prediction. Phylogenetic analysis grouped them as class I and class VII plant chitinase, with possible abundance of class I chitinase in onion. These observations help in the isolation and functional validation of onion chitinases.

Fusion of a chitin-binding domain to an antibacterial peptide to enhance resistance to Fusarium solani in tobacco (Nicotiana tabacum).

An antibacterial peptide-encoding gene from alfalfa seeds, alfAFP, was fused to the C-terminal part of chitin-binding domain (CBD) of the rice chitinase-encoding gene (CBD-alfAFP) and introduced to tobacco by Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) technique was used to confirm the integration of the recombinant CBD-alfAFP encoding gene in transgenic tobacco plants. A number of transgenic lines and a non-transgenic control plant were selected for further molecular analyses. The result of analyzing the transgenic plants by semi-quantitative RT-PCR showed that the recombinant gene is expressed in transgenic plants and there is a difference between the transgenic plants in terms of the level of CBD-alfAFP expression. The total protein was extracted from a few selected transgenic plants and used to evaluate the antibacterial/antifungal of recombinant protein activity against some important plant and human pathogens. The results of this experiment showed that the total protein extract obtained from transgenic lines significantly (P < 0.05) inhibited the growth of various bacteria and fungi compared to the non-transgenic plants. Transgenic lines showed a significant (P < 0.01) difference considering their ability to inhibit bacterial and fungal pathogens growth. The recombinant CBD-alfAFP protein significantly (P < 0.01) increased the resistance of the transgenic plants against Fusarium solani. Transgenic lines showed no significant wilting symptoms and obvious wilting symptoms were not observed even 30 days post-inoculation (dpi) with Fusarium solani spores. These results suggest that transgenic tobacco plants are resistant to Fusarium solani wilt and fusion of CBD to the alfAFP antimicrobial peptide is an efficient approach to control fungal diseases.

Direct Regulation of Extracellular Chitinase Production by the Transcription Factor LeClp in Lysobacter enzymogenes OH11

Lysobacter enzymogenes is a gram-negative bacterial biological control agent that produces abundant extracellular enzymes capable of degrading the cell walls of fungal pathogens. In strain OH11, an isolate from China, the global regulator LeClp controls the production of extracellular chitinase by regulating the transcription of the chitinase-encoding gene chiA. Using a combination of bioinformatic, genetic, and biochemical methods, we show that LeClp regulates chiA transcription by directly binding to the chiA promoter region. Although LeClp appears to be important in this role, it is not the sole regulator of chiA transcription. Furthermore, the sequence analysis of putative LeClp binding sites indicated that the LeClp homolog could be involved in the regulation of extracellular chitinase production in diverse Lysobacter spp. by a mechanism similar to that in L. enzymogenes. Our findings present new insights into the molecular mechanism of LeClp in controlling extracellular chitinase activity, providing a fundamental road to elucidate how LeClp regulates the production of other extracellular lytic enzymes in L. enzymogenes.

Isolation screening and characterisation of local beneficial rhizobacteria based upon their ability to suppress the growth of Fusarium oxysporum f. sp. radicis-lycopersici and tomato foot and root rot

Local beneficial rhizobacteria were selected based upon their ability to control the fungus Fusarium oxysporum f. sp. radicis-lycopersici which causes crown and root rot of tomato. Seven out of 384 strains prevailed in multiple and dual cultures and were identified as Pseudomonas chlororaphis (one strain), Bacillus cereus (one strain), Serratia marcescens (three strains) and Serratia rubidaea (two strains), by sequencing the 16S rRNA or the 16S and 23S rRNA inter-spacer region. The seven selected rhizobacteria were tested for their biocontrol and growth-promoting effects in planta, and their antifungal properties in vitro. All strains significantly reduced disease severity under controlled conditions, in a gnotobiotic system and in pots. Moreover, one P. chlororaphis and one S. marcescens strain significantly decreased disease severity to the level of the healthy control under natural conditions in pots experiments. The inhibitory activity of bacterial liquid cultures' metabolites on the fungus was demonstrated for all strains in vitro, using filter paper, thin layer chromatography and microtiter bioassays. Genes encoding phenazines were tentatively detected by PCR in the P. chlororaphis strain and chitinase-encoding genes were detected in one S. rubidaea and all three S. marcescens strains. Production of phenazine-1-carboxamide and hydrogen cyanide was evidenced for the P. chlororaphis strain while protease activity and production of siderophore-like compounds was confirmed in all bacterial strains. Possible use of these strains as biological control agents and their impact on natural biocontrol of pathogens in soils is discussed.

Retraction note to: Preliminary comparing the toxicities of the hybrid cry1Acs fused with different heterogenous genes provided guidance for the fusion expression of Cry proteins. World J Microbiol Biotechnol (2012 Jan; 28(1):397-400).

In order to provide guidance for selecting suitable heterogenous gene that can efficiently enhance toxicity or broaden insecticidal spectrum of Cry1Ac through fusion expression, two hybrid cry1Acs fused with chitinase-encoding gene tchiB and neurotoxin gene hwtx-1 respectively were constructed and their toxicities were compared. A Bacillus thuringiensis strain harboring the cry1Ac gene in vector pHT315 was used as control. Bioassay revealed that LC50 (after 72 h) of Cry1Ac protoxin was 41.01 μg mL−1, while the hybrid cry1Acs fused with tchiB and hwtx-1 were 4.89 and 23.14 μg mL−1, which were 8.23- and 1.77-fold higher than Cry1Ac protoxin in terms of relative toxicity respectively. Both fusion crystals had a higher toxicity than the original Cry1Ac protein and the toxicity of hybrid cry1Acs fused with hwtx-1 experienced a more significant increase than that fused with tchiB.

Construction of Streptomyces lydicus A01 transformant with the chit33 gene from Trichoderma harzianum CECT2413 and its biocontrol effect on Fusaria

Streptomyces lydicus A01 resists many plant pathogens (including Fusarium spp.) by producing the antifungal agent natamycin, which binds to the ergosterol of fungal cell membranes and inhibits the growth of pathogens. Trichoderma harzianum CECT2413 is a widely-distributed soil fungus that antagonizes several plant fungal pathogens (including Fusarium spp.) by producing chitinase and degrading chitin, a major component of the fungal cell wall. This study attempted to enhance the biocontrol effect of S. lydicus A01 on Fusarium spp. by transforming the chitinase gene of Trichoderma. Chitinase and natamycin could act synergistically on both the cell walls and cell membranes of pathogens. The 33-kD chitinase-encoding gene (chit33) was cloned and conjugal-transformed from T. harzianum CECT2413 to S. lydicus A01, and then confirmed via polymerase chain reaction (PCR) assays. Subsequent analyses using the 3,5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry showed that compared with its wild type strain (WT), the S. lydicus A01 conjugal transformant (CT) with chit33 gene exhibited substantially higher chitinase activity and natamycin production. The resistance of S. lydicus A01-chit33 CT and WT to four Fusaria in crops and vegetables was tested via the cup-plate method. Compared with the WT, the conjugal transformant of S. lydicus A01 with chit33 gene from T. harzianum CECT2413 showed greatly increased biocontrol effect on fusarium disease. This study would be beneficial to the development of high-quality antifungal bio-agents for agricultural applications via the synergy between the previously non-existent and pre-existing functions achieved through heterogeneous gene transformation.

Biodegradation of chitinous substances and chitinase production by the soil actinomycete Streptomyces rimosus

A chitinolytic actinomycete was isolated from agricultural soil in the centre of Poland. It was identified as Streptomyces rimosus. Identification was based on standard biochemical tests and on 16S rRNA gene sequence analysis. The degradation of chitinous substances (shrimp shell waste, crab chitin powder and chitosan) was examined on the basis of oxygen consumption by applying OxiTop (WTW). The enzyme catalyzed the hydrolysis of the disaccharide 4-MU-(GlcNAc)3 most efficiently and was therefore classified as an endochitinase. The chitinase was purified from culture medium by fractionation with ammonium sulfate and affinity chromatography. The purified proteins were subjected to identification by mass spectrometry. Based on analysis of the resultant fragments of the amino acid sequence of chitinase produced by the examined bacteria, degenerate primers for PCR were designed. The results of our research prove that the substances which are metabolised by S. rimosus the most efficiently are chitosan and the shrimp shell waste. Chitinolytic activity examination showed that shrimp shell waste was the finest inducer of chitinase synthesis. The molecular mass of the purified enzyme was 63 kDa. The highest activity of chitinase was obtained at the temperature of 40°–45 °C. Optimal pH for chitinase activity was 7.0. The activity of the purified chitinase was stabilized by Mg2+ ions. The activity of the enzyme was inhibited by Hg2+ and Pb2+ ions. Chitinase inhibited the growth of the fungal phytopathogens Fusarium solani and Alternaria alternata. The nucleotide sequence of the amplified gene fragment proved to be similar to the sequence of chitinase-encoding genes of the glycoside hydrolases family 18.

Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium

Fusarium head blight (FHB) is a re-emerging wheat disease that causes extensive damage through direct losses in yield and quality due to the presence of damaged Fusarium kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Biological control, including the treatment of crop residues with antagonists, in order to reduce pathogen inoculum of FHB, holds considerable promise. Ten Trichoderma isolates, previously selected for their ability to grow in the presence of DON, were preliminarily investigated as potential antagonists against Fusarium culmorum and F. graminearum mycotoxigenic strains in plate confrontation assays. The three Trichoderma isolates showing antibiosis and mycoparasitism were evaluated for their capacity to inhibit DON production by F. graminearum and F. culmorum on two natural substrates. The expression of some chitinase-encoding genes by the two best resulting Trichoderma strains, during interaction with F. culmorum and F. graminearum, was monitored. All investigated genes from chitinase subgroups A, B and the new subgroup C responded to mycoparasitic conditions and were upregulated before contact and/or when in contact with the host. T. gamsii 6085, the best antagonist, was finally used in a competition test against F. culmorum and F. graminearum on natural substrates, using a qPCR approach to evaluate its effect on the pathogen's growth and DON production in haulms and rice. This test confirmed the ability of T. gamsii 6085 to antagonize the pathogens on rice. On wheat haulms, an extreme oligotrophic environment, T. gamsii 6085 seemed to develop very poorly and the growth of both the pathogens was unaffected by the presence of the antagonist.