The Chitinolytic Activity of Bacillus Cohn Bacteria Antagonistic to Phytopathogenic Fungi

Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, -1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r _{x , y} = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.     

The study of mycolytic properties of aerobic spore-forming bacteria producing extracellular chitinases

The mycolytic activity of 27 strains of antagonistic bacilli belonging to two taxonomic groups (18 strains of Bacillus subtilis and 9 strains of Paenibacillus ehimensis) capable of induced synthesis of chitinolytic enzymes was studied. Most of the B. subtilis strains neither displayed visible mycolytic effects on the phytopathogenic fungus Bipolaris sorokiniana in vitro, nor produced chitinases in the presence of an autoclaved mycelium. On the contrary, P. ehimensis strains grown under conditions favorable for induction of chitinases and other hydrolases exhibited a pronounced lytic effect on B. sorokiniana and actively grew by utilizing mycelium as the sole source of carbon and nitrogen. Comparison of the mycolytic activities of extracellular hydrolases in the studied strains demonstrated low correlation between chitinase production and the ability of the strains to degrade the cell walls of B. sorokiniana. Characterization of enzyme profiles in the studied strains revealed that β-1,3-glucanase was a more significant factor than chitinase for determining the mycolytic potential of bacteria and their ability to utilize the mycelium of phytopathogenic fungi as a growth substrate.     

Bt几丁质酶的基础表达及诱导合成的多态现象

多数微生物可以产生几丁质酶。一般认为几丁质酶基因表达受几丁质的诱导和葡萄糖抑制。但是苏云金芽胞杆菌Bacillus thuringiensis(简称Bt)几丁质酶的诱导表达方式是否与其他微生物相同,至今尚无定论。采用DNS法检测77株Bt在有或无诱导物培养基中的几丁质酶活力。研究了葡萄糖对4株不同表达类型菌株酶活力的影响,以及葡萄糖抑制与几丁质诱导之间的关系。研究发现在无几丁质诱导条件下,全部试验菌株都可以产生几丁质酶,保持一定量的基础表达,说明Bt能组成型合成几丁质酶,不需要诱导。添加诱导物之后,31株菌的酶活力没有任何变化,44株菌有不同程度的提高,但其中绝大部分诱导特性并不典型,酶活力提高不显著。许多Bt菌株几丁质酶表达兼具组成型和诱导型的特点。葡萄糖能够抑制几丁质的诱导作用,但是不能完全抑制Bt菌株几丁质酶的基础表达。比较组成型和诱导型菌株的几丁质酶基因chiA、chiB调节区域核苷酸序列,发现仅存在个别碱基的差异。     

Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification

The purpose of this study was to explore the microbial potential of a semi-arid sandy soil from south-central Algeria in order to isolate new chitinolytic actinobacteria. This soil is subjected to high temperatures (up to 43 °C) and has low nutrient content. Strains were isolated by plating soil suspensions on Bennett and Colloidal Chitin (CCM) medium. An initial clustering of isolates was made through BOX-PCR genetic profiling. Next, a 16S rRNA gene sequencing of representative isolates was realized. We also identified optimum physicochemical conditions for chitinolytic activity. A rapid in vitro assay based on glucose catabolic repression was developed to select isolates having a chitinase-dependent antifungal activity against two phytopathogenic fungi. Gene identification of glycosyl hydrolase family 18 (GH18) permitted us to assess the divergence of chitinase genes. Forty isolates were obtained from the semi-arid sandy soil. The molecular identification permitted us to assign them to Streptomyces or Micromonospora genera with seven possibly new bacterial species. For chitinolytic activity, 100% of isolates were able to grow and degrade colloidal chitin at pH 7 and at a temperature ranging from 30 to 40 °C. We also observed that Micromonospora strains had atypical activity patterns, with a strong chitinase activity maintained at high temperature. Finally, three strains presented an interesting chitinolytic potential to reduce fungal growth with new GH18 sequences. This study presents a new rapid method to detect antifungal chitinase-dependent activity that allowed to identify potentially new species of actinobacteria and new GH18 gene sequences.     

Chitinolytic activity of endophytic Streptomyces and potential for biocontrol

Aims: Biological sources for the control of plant pathogenic fungi remain an important objective for sustainable agricultural practices. Actinomycetes are used extensively in the pharmaceutical industry and agriculture owing to their great diversity in enzyme production. In the present study, therefore, we evaluated chitinase production by endophytic actinomycetes and the potential of this for control of phytopathogenic fungi. Methods and Results: Endophytic Streptomyces were grown on minimum medium supplemented with chitin, and chitinase production was quantified. The strains were screened for any activity towards phytopathogenic fungi and oomycetes by a dual‐culture in vitro assay. The correlation between chitinase production and pathogen inhibition was calculated and further confirmed on Colletotrichum sublineolum cell walls by scanning electron microscopy. Conclusions: This paper reports a genetic correlation between chitinase production and the biocontrol potential of endophytic actinomycetes in an antagonistic interaction with different phytopathogens, suggesting that this control could occur inside the host plant. Significance and Impact of the Study: A genetic correlation between chitinase production and pathogen inhibition was demonstrated. Our results provide an enhanced understanding of endophytic Streptomyces and its potential as a biocontrol agent. The implications and applications of these data for biocontrol are discussed.     

Rhizosphere bacteria of maize with chitinolytic activity and its potential in the control of phytopathogenic fungi

The chitinase enzyme was identified in isolated bacteria of maize rhizosphere as well as its potential for the biological control of fungi associated at seeds of the same plant. The production of chitinase enzyme was found in the genera identified as Acinetobacter, Bacterium, Burkholderia, Paenibacillus, Pseudomonas, Rhizobium, Shewanella, Sphingomonas and Stenotrophomonas. Bacterial isolates with ability to degrade fungal mycelium from maize fungi as Fusarium and Alternaria among others, were detected. Bacterial chitinase activity and the presence of the chiA gene were determined. The inoculation of chitinolytic bacteria showed a positive effect in the control of fungi in maize seeds. The results support the potential use of chitinase enzyme producing bacteria on the control of phytopathogenic fungi.     

Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae

AIMS: The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. METHODS AND RESULTS: Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. CONCLUSIONS: Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.     

Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus

The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed.     

Molecular screening of Streptomyces isolates for antifungal activity and family 19 chitinase enzymes

Thirty soil-isolates of Streptomyces were analyzed to determine their antagonism against plant-pathogenic fungi including Fusarium oxysporum, Pythium aristosporum, Colletotrichum gossypii, and Rhizoctonia solani. Seven isolates showed antifungal activity against one or more strain of the tested fungi. Based on the 16S rDNA sequence analysis, these isolates were identified as Streptomyces tendae (YH3), S. griseus (YH8), S. variabilis (YH21), S. endus (YH24), S. violaceusniger (YH27A), S. endus (YH27B), and S. griseus (YH27C). The identity percentages ranged from 98 to 100%. Although some isolates belonged to the same species, there were many differences in their cultural and morphological characteristics. Six isolates out of seven showed chitinase activity according to a chitinolytic activity test and on colloidal chitin agar plates. Based on the conserved regions among the family 19 chitinase genes of Streptomyces sp. two primers were used for detection of the chitinase (chiC) gene in the six isolates. A DNA fragment of 1.4 kb was observed only for the isolates YH8, YH27A, and YH27C. In conclusion, six Streptomyces strains with potential chitinolytic activity were identified from the local environment in Taif City, Saudi Arabia. Of these isolates, three belong to family 19 chitinases. To our knowledge, this is the first reported presence of a chiC gene in S. violaceusniger YH27A.     

Isolation of novel chitinolytic bacteria and production optimization of extracellular chitinase

Chitin is one of the most abundant biopolymers widely distributed in the marine and terrestrial environments. Chitinase enzyme has received increased attention due to its wide range of biotechnological applications, especially in agriculture for biocontrol of phytopathogenic fungi and harmful insects. In the present study, 58 bacterial isolates were screened for chitinolytic activity and on the basis of chitin hydrolysis zone 6 isolates were selected for chitinase production in broth media. Based on enzyme production, two most potent isolates identified as Aeromonas hydrophila HS4 and Aeromonas punctata HS6 were selected for further study. The effects of media composition and various fermentation conditions for optimization of chitinase production were studied. The maximum chitinase production was obtained at 37 °C and pH 8.0 after 24–48 h of incubation by HS4; and at 37 °C and pH 7 after 48 h incubation by HS6. Among the substrates colloidal chitin was the best for both the strains. Regarding carbon sources, starch (1%) was the best for both strains; while malt and yeast extract (1%) was found as the best nitrogen source for HS4 and HS6, respectively. Out of metal ions Mn²⁺ and Cu²⁺ enhanced enzyme production in the case of HS6. However, Co²⁺ was the most appropriate for HS4.     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Biodegradation of shrimp processing bio-waste and concomitant production of chitinase enzyme and N-acetyl-D-glucosamine by marine bacteria: production and process optimization

A total of 250 chitinolytic bacteria from 68 different marine samples were screened employing enrichment method that utilized native chitin as the sole carbon source. After thorough screening, five bacteria were selected as potential cultures and identified as; Stenotrophomonas sp. (CFR221?M), Vibrio sp. (CFR173?M), Phyllobacteriaceae sp. (CFR16?M), Bacillus badius (CFR198?M) and Bacillus sp. (CFR188?M). All five strains produced extracellular chitinase and GlcNAc in SSF using shrimp bio-waste. Scanning electron microscopy confirmed the ability of these marine bacteria to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. HPLC analysis of the SSF extract also confirmed presence of 36-65?% GlcNAc as a product of the degradation. The concomitant production of chitinase and GlcNAc by all five strains under SSF using shrimp bio-waste as the solid substrate was optimized by 'one factor at a time' approach. Among the strains, Vibrio sp. CFR173?M produced significantly higher yields of chitinase (4.8 U/g initial dry substrate) and GlcNAc (4.7?μmol/g initial dry substrate) as compared to other cultures tested. A statistically designed experiment was applied to evaluate the interaction of variables in the biodegradation of shrimp bio-waste and concomitant production of chitinase and GlcNAc by Vibrio sp. CFR173?M. Statistical optimization resulted in a twofold increase of chitinase, and a 9.1 fold increase of GlcNAc production. These results indicated the potential of chitinolytic marine bacteria for the reclamation of shrimp bio-waste, as well as the potential for economic production of chitinase and GlcNAc employing SSF using shrimp bio-waste as an ideal substrate.     

Methodological aspects of assessing chitin utilization by soil microorganisms

A computational method for estimating specific activity of chitin decomposition by microorganisms is proposed. Spectrophotometric and gas chromatographic methods have been used to determine the rates of chitinase production, biomass accumulation, and carbon dioxide emission by pure cultures of microorganisms grown on a chitin-containing medium. Among dominants of the chitinolytic community of chernozem (Trichoderma viride, Stretomyces albolongus, Alcaligenes, and Arthrobacter), the highest chitinolytic activity is characteristic of prokaryotes. In brown desert-steppe soil, the main destructor prokaryotes are actinomycetes (S. roseolilacinus). The biomass of the fungus T. viride growth on the chitin-containing medium markedly exceeds that of prokaryotes, but the specific activity of respiration and chitinase production in actinomycetes S. roseolilacinus and S. albolongus is an order of magnitude higher than in T. viride.     

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.     

Glycogen in Methanolobus and Methanococcus

Abstract Ultraviolet light and nitrosoguanidine were used to mutagenize a red pigmented culture of Serratia marcescens , strain EB415, which produced chitinase. After mutagenesis, a stable, non-pigmented mutant designated BL40 was isolated which produced larger colonies and zones of clearing on solid medium containing colloidal chitin.
In liquid medium with colloidal chitin as the sole carbon source both strains grew similarly but BL40 produced 160 units/ml of chitinase compared with 60 units/ml for EB 415, an increase of 167%. When chitin concentration was increased in the medium, chitinase production also increased. Chitinase appeared to be extracellular, since the supernatant from washed, sonicated cells for both strains showed no detectable amount of chitinolytic activity.     

Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi

A chitinolytic enzyme from Bacillus thuringiensis subsp. aizawai has been purified and its molecular mass was estimated ca. 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was able to hydrolyze chitin to chitobiosides but not carboxymethylcellulose, cellulose, pullulan, and laminarin. Optimal pH and temperature were detected at 6 and 50 degrees C, respectively. Stability, in the absence of substrate, was observed at temperatures less than 60 degrees C and pH between 5 and 8. Enzyme activity was significantly inhibited by K+ and EDTA and completely inhibited by Hg2+. Purified chitinase showed lytic activity against cell walls from six phytopathogenic fungi and inhibited the mycelial growth of both Fusarium sp. and Sclerotium rolfsii. The biocontrol efficacy of the enzyme was tested in the protection of bean seeds infested with six phytopathogenic fungi.     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

Improvement of Chitinase Production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> NM101-19 for Antifungal Biocontrol through Physical Mutation

Chitinase is one of the most important enzymes due to its diversity and a variety of potential uses. This study is an attempt to enhance chitinase production for antifungal biocontrol by subjecting Bacillus thuringiensis NM101-19 strain grown on shrimp shell wastes to various doses of gamma irradiation. Six mutants (BM-4, BM-6, BM-8, BM-12, BM-15, and BM-17) obtained at gamma ray doses of 40, 60, 80, 100, 120 and 140 Gy, respectively produced higher levels of chitinolytic activities in comparision to the wild-type strain. The BM-15 mutant strain showed the highest chitinase production (65.41 U/mL) which was 2.60 times more than the wild type (25.11 U/mL). Biocontrol efficacy of the mutants was statistically superior to the wild-type strain against all tested phytopathogens. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) PCR techniques, with five primers for each, were used in order to detect the variation in DNA profile between the mutant and wild-type strains in response to gamma-radiation treatments. RAPD and ISSR analysis indicated the appearance and disappearance of DNA polymorphic bands at different gamma ray doses. The results confirmed that the mutagenesis technique is a potent strategy to enhance the chitinase activity for industrial and agricultural purposes.     

Production of an extracellular chitinolytic system byTalaromyces emersonii CBS 814.70.

Summary The thermophilic fungusTalaromyces emersonii CBS 814.70 produces a thermostable extracellular chitinolytic system when cultured on chitin containing media. The chitinolytic system consists of chitinase (EC 3.2.1.14) and N-acetylglucosaminidase (EC 3.2.1.30). Using fluorescent substrate analogues, in zymogram staining of polyacrylamide gradient and isoelectric focusing gels on which the chitinase system was electrophoresed and focused, respectively, it was found that a number of bands could be resolved. Using isoelectric focusing it was observed that at least 4 extracellular forms of chitinase activity are produced.