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RNAi-based insecticides for crop protection have witnessed rapid improvement over the years. However, their potential to efficiently control maize stem borer (Chilo partellus) pests has remained underexplored. In this study, double-stranded C. partellus chitinase (dsCHI) toxicity was investigated in C. partellus larvae. Furthermore, we developed transgenic maize lines expressing dsRNA targeted against C. partellus chitinase transcripts and performed detached leaf insect feeding bioassays. Our results revealed that C. partellus chitinase transcript expression was significantly downregulated by 57% and 82% in the larvae. Larvae exhibited various phenotypic distortion levels across developmental stages, and 53% mortality occurred in transgenic fed larvae compared to those fed on nontransgenic leaves. In conclusion, we have identified the C. partellus chitinase gene as a potential target for RNAi-mediated control and demonstrated that oral delivery via bacteria and plant-mediated delivery are viable means of achieving C. partellus RNAi-mediated control. [ABSTRACT FROM AUTHOR]

Coat protein gene of potato virus X is a structural protein that plays a vital role in viral transmission and pathogenesis. RNA interference or post transcriptional gene silencing is a regulatory conserved mechanism that uses small interfering RNAs to control the expression of desired gene by inhibiting mRNA transcription. The present study aims to investigate the potential role of different siRNAs in down-regulating the mRNA expression of potato virus X in cell culture assays. Three specific siRNAs against CP-PVX gene were designed in which siRNA 3 significantly reduced the CP-PVX mRNA expression in HepG 2 cells upto 91.91% (12.5 fold) as compared to siRNA 1 and siRNA 2 where knockdown percentages were only 20 and 73.04%, respectively as revealed by realtime PCR. Maximum knockdown values were obtained at 100 nM concentration of siRNA 1 and 2, while for siRNA 3 promising knockdown was obtained at 50 nM concentration. For long lasting effect of siRNAs, short hairpin RNA corresponding to siRNA3 was designed and tested against CP-PVX which resulted in a similar pattern of inhibition on RNA expression of target gene as synthetic siRNA3. It was found that mRNA expression of targeted gene was reduced to 91% depicting strong RNAi response generated by shRNA. Our findings support the possibility of using consensus siRNA and shRNA-based approach as a promising strategy in effective inhibition of PVX in in-vitro assay. Down regulation of potato virus X through coat protein gene targeted siRNA is a potential way of inhibiting invading virus and to effectively stop its systemic spread. [ABSTRACT FROM AUTHOR]

Copyright of Genetika (0534-0012) is the property of Serbian Genetics Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Fungi have been used in modern scientific research due to their high potential for different enzymes production based on genomic features. The great proportion of soil mycoflora represented by saprobic fungi plays an important role in decomposition, thus contribute to the global carbon cycle. Sordaria fimicola strains (n= 61) collected from different environments were evaluated for catalase enzyme activity at first stage. Among all 61 isolates of S. fimicola, five strains viz. S1, S2, N7, N6 and SF13 were found to be most efficient in catalase enzyme activity. The complete catalase gene including exons and introns was amplified and sequenced from the most efficient strains of S. fimicola and then submitted in the NCBI data base under accession numbers KM282183, KM282184, KM282186, KM282185 and KM282182 for strains S1, S2, N7, N6 and SF13 respectively. The significant differences in the genes sequences and theoretically translated proteins were observed for all five strains of S. fimicola. As regards catalase enzyme activity, S. fimicola strains were found comparable to the Aspergillus niger strains, therefore being a saprophytic fungus with short life cycle S. fimicola can become a fungus of choice to produce catalase enzyme at large scale. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum L.), an important food crop in the world, is susceptible to many fungal pathogens including Alternaria solani and Fusarium oxysporum causing Fusarium wilt and early blight diseases. Mycoparasitic fungi like Trichoderma encode chitinases, cell wall degrading enzymes, with high antifungal activity against a wide range of phytopathogenic fungi. In this study, a binary vector harboring endochitinase gene of ~1,000 bp was constructed and used to transform potato nodes through Agrobacterium-mediated transformation. Out of several primary transformants, two transgenic potato lines were verified for transgene insertion and integration by Southern blot. In a pot experiment for Fusarium resistance, the transgenic potato lines didn't show any symptoms of disease, instead they remained healthy post infection. The transgenic potato lines exhibited 1.5 fold higher mRNA expression of endochitinase at 7 days as compared to 0 day post fungus inoculation. It was evident that the mRNA expression decreased over days of inoculation but was still higher than at 0 day and remained stable upto 30 days post inoculation. Similarly, for A. solani infection assay, the mRNA expression of the endochitinase gene was 3 fold higher 7 days post inoculation compared to expression at 0 day. Although the expression decreased by1.2 fold during subsequent days post infection, it remained stable for 30 days, suggesting that protection in transgenic potato plants against fungal pathogens was achieved through an increase in endochitinase transcript. [ABSTRACT FROM AUTHOR]

We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours. [ABSTRACT FROM AUTHOR]

The future of food security in Africa is being severely threatened due to an exponential increase in population, which is almost three times the increase of food production. Maize production is constrained by stem borers which cause significant yield losses. Yield losses can be further compounded by higher temperatures due to climate changes, which are expected to increase the population of maize stem borers. While several methods have been employed in stem borer management, there is still significant damage caused by maize stem borers. This necessitates better control methods including the adoption of recent biotechnological advancement in RNA interference (RNAi) technology. This review highlights evidence of an increase in the stem borer population as well as the foreseen decline in maize production worldwide due to the effects of climatic changes. Furthermore, we have drawn attention to improved methods that have been used to control stem borers in maize production as well as a reluctant acceptance of traditional biotechnology in Africa. Finally, we suggest the application of alternative RNA interference techniques to breed maize for efficient pest control in order to achieve food security, improve nutrition and promote sustainable maize production. [ABSTRACT FROM AUTHOR]

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the bC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vb, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants. [ABSTRACT FROM AUTHOR]

Phosphate solubilizing bacteria (PSB) have the ability to solubilize insoluble phosphorus to make it available for plant roots to be absorbed. In this study, isolation of rhizobacteria and screening of their ability for phosphate solubilization, production of indole acetic acid (IAA), antagonistic activity against fungal pathogen and intrinsic antibiotic resistance was studied. In total, 15 isolates of PSB were found gram negative with rod shaped cells. Different levels of antibiotic resistance was seen against four antibiotics (Ampicillin, Kanamycin, Tetracycline and Streptomycin 25, 30, 30 and 10 μg/ml respectively) by Rhizobacterial isolates (Seven isolates were found to show antifungal activity against Fusarium oxysporum ). All PSB isolates were found to solubilize insoluble phosphorous Ca 3 (PO 4 ) and produce IAA. Two PSB isolates sequences were found novel and were submitted in NCBI database. Conclusively, combine application of rhizobacterial isolates, Sugarcane (SC-22) in addition with antifungal agent can prove to be an excellent combination to solubilize insoluble phosphorus and to act as antifungal remedy. Further this combination can be eco-friendly and prove to be cost effective strategy to improve crop production. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Slovenica is the property of Biotechnical Faculty of the University of Ljubljana and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Saprobic fungi play an important role in decomposition and thus contributing to the global carbon cycle. Sordaria fimicola strains collected from diverse environment were evaluated for their laccase enzyme activity, while Aspergillus niger was used as control fungus. In laccase assay, S. fimicola strain N6 collected from the station 6 located at the Northern Facing Slope (NFS) of the Evolution Canyon 1 (EC 1), showed the maximum laccase enzyme activity (1.1 ΔA/min). In the next step, complete laccase gene (1.917 Kb) was amplified and sequenced from the biochemically efficient isolates of S. fimicola viz. S1, S2, SF13, N6 and N7 and on comparison with reported sequence of Neurospora crassa (Accession no. M18334.1) 245 point mutations, 65 amino acid and post-translational modifications (PTMs) changes were detected in the in silico translation proteins. In phylogenetic analysis, S1, S2, SF13, N6 and N7 strains of S. fimicola were found in clade-I alone, while reference sequence of N. crassa (Accession no. M18334.1) was present in clade-II. The laccase gene sequences were submitted to NCBI data base under accession numbers KM282173, KM282174, KM282172, KM282175 and KM282176 for S. fimicola strains S1, S2, SF13, N6 and N7, respectively. The biochemical as well as molecular data of the study depicted that environmental stresses affected the specific genes by bringing in mutations, which may result in genomic diversity among the organisms and their frontier molecules such as proteins. The observed laccase enzyme activity of S. fimicola-a non-pathogenic fungus, was found even better and comparable to A. niger, a pathogenic fungus. Therefore, being a saprophytic with short life cycle S. fimicola can become a fungus of choice to produce laccase enzyme at large scale. [ABSTRACT FROM AUTHOR]

Soil, air and water pollution have become a global problem due to unprocessed emission of toxic heavy metals into the environment. The uncontrolled increase in release of hazardous heavy metals such as arsenic into the soil and water is mainly due to the untreated industrial waste. The accumulation of heavy metals causes damage not only to soil and water flora but also have deleterious effects on human health. The conventional methods such as thermal treatment, excavation and land fill, electro-reclamation and acid leaching used for degradation were time and cost consuming with release of toxic products. In nature, there are microorganisms residing in soil, which are capable of degrading toxic metals through phytoremediation. In phytoremediation, rhizobacteria play an important role. Rhizobacteria not only prevent contaminated soil's fertility but also enhance the growth of plants by release of special plant growth hormones. Phytohormones secreted by plant growth-promoting rhizobacteria (PGPR) are major chemicals involved in metal uptake. This in situ and environment friendly method of bioremediation is cost effective and efficient. The advance techniques such as genetic engineering have been introduced to increase the spectrum and degrading capacity of rhizobacteria. [ABSTRACT FROM AUTHOR]

Copyright of Zemdirbyste-Agriculture is the property of Lithuanian Research Centre for Agriculture & Forestry and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Introduction of multiple traits in crop plants for desirable character is an essential tool of Biotechnological application. To overcome problem of insect pests and weeds cotton variety FBS-37 was genetically modified by transformation of CEMB Cry1Ac+Cry2A and GTGene cloned in different plant expression vectors under CaMV35S Promoter and nopaline synthase Nos Terminator. CEMB optimized protocol of cotton gene transformation i.e. Agrobacterium mediated shoot apex cut method was applied for transformation of these genes constructs in cotton. Overall transformation efficiency was found to be 1.05%. Putative transgenic cotton plants were shifted to soil pots and acclimatized in the green house conditions. PCR with gene specific primers amplified 190bp for EPSPS (Glyphosate GTG) and 1000bp for each of Cry1Ac and Cry2A. Quantification of Cry1Ac, Cry2A and GTG protein through ELISA determined maximum of 0.8μg/g, 1μg/g and 0.9μg/g of tissue respectively. 100% mortality was obtained in 2nd instar larvae of Heliothus armigera when compared with non-transgenic control cotton plants where maximum damaged was seen in leaf bioassay. Moreover, transgenic cotton plants successfully survived when sprayed with 1600ml/acre of glyphosate as compared to control where 100% mortality and necrosis occur after 7 days of spray assay in control conditions. [ABSTRACT FROM AUTHOR]

RNA interference is a defense response against virus multiplication. The goal of this paper was to test if expressing virus-specific shRNAs against potato virus Y (PVY) could confer resistance against PVY in potatoes. The recombinant binary vectors consisted of short hairpin RNA (shRNA) transgenes, expressed under the CaMV35S promoter, directed against the conserved portion of the potato virus Y (PVY) coat protein gene. To provide the shRNA a particular shape and stability in vivo , stem and loop sequences from a microRNA (miR403; an active regulatory microRNA in potato) were included with the siRNA sequences. The shRNA constructs were delivered into the “Cardinal” potato variety using Agrobacterium , and transgenic status was confirmed by polymerase chain reaction (PCR) and Southern blot. Compared to wild-type plants inoculated with PVY, expression of PVY coat protein mRNA, as assayed by reverse transcriptase (RT)-PCR, was reduced to 0.05% in shRNA1-transgenic potato lines and 22% in shRNA4-transgenic lines. In addition, via northern blots, the maximum accumulation of the transgene-specific siRNA was observed in shRNA1 transgenic plants, less was observed in shRNA4-transgenic lines, and the least amount was detected in control plants, which correlates with silencing of the PVY coat protein gene. Conclusively, by simultaneous RNA silencing, shRNA1-transgenic plants were found to be almost immune to PVY infection compared with shRNA4-potato plants. Therefore, the expression of virus-specific shRNAs is a novel, effective, and predictable approach to engineer resistance against PVY. [ABSTRACT FROM AUTHOR]

Biological control is a novel approach in crop protection. Bacteria, such as Bacillus spp. and Pseudomonas spp., are reported for this purpose and some of their products are already commercially available. In this study, the rhizosphere and phyllosphere of healthy cotton plants were used as a source of bacterial isolates with properties of potential biocontrol agents. The isolates were screened for phosphate solubilization activity, indole acetic acid (IAA) production and antifungal activity. Two isolates, S1HL3 and S1HL4, showed phosphate solubilization and IAA production simultaneously, while another two, JS2HR4 and JS3HR2, demonstrated potential to inhibit fungal pathogens. These bacteria were identified as Pseudomonasaeruginosa (S1HL3), Burkholderia sp. (S1HL4) and Bacillus sp. (JS2HR4 and JS3HR2) based on biochemical and molecular characteristics. The isolates were tested against Cotton leaf curl virus (CLCuV) in greenhouse conditions, both as individual bacterial isolates and consortia. Treated plants were healthy as compared to control plants, where up to 74% of the plants were symptomatic for CLCuV infection. Maximum inhibition of CLCuV was observed in the plants treated with a mixture of bacterial isolates: the viral load in the treated plants was only 0.4% vs. up to 74% in controls. This treatment consortium included P.aeruginosa S1HL3, Burkholderia sp. S1HL4 and Bacillus spp. isolates, JS2HR4 and JS3HR2. The principal-component biplot showed a highly significant correlation between the viral load percentage and the disease incidence. [ABSTRACT FROM AUTHOR]

Unnecessary weed growth in sugarcane fields forces plants to compete for nutrients and sunlight for survival, which most often leads to significant yield losses. As chemical herbicides cannot differentiate between crop plants and weeds, the development of herbicide-tolerant crops is anticipated. Four sugarcane varieties, CPF-234, CPF-213, HSF-240, and CPF-246, were used to develop glyphosate herbicide tolerance. A glyphosate-tolerant gene of 1368 bp cloned directionally under the 35S promoter with the β-glucuronidase (GUS) reporter gene was used as the transgene. Through the biolistic transformation of sugarcane, calli of all cultivars were transformed with glyphosate-tolerant gene constructs. Efficient regeneration conditions were optimized on 1.5-2.5 mg/L kinetin, 1.5-2.5 mg/L 6-benzylaminopurine (BAP), and 1 mg/L gibberellic acid (GA3). The transformants exhibited the best shoot regeneration on a medium containing 2 mg/L kinetin, 2 mg/L BAP, 2 mg/L GA3, and 1 mg/L indole-3-acetic acid. Based on initial screenings through GUS assay, the transformation efficiency was 22%, 32%, 17%, and 13% for cultivars 246, 234, 213, and 240, respectively. In transformed sugarcane plants, the transgene of 1368 bp was amplified. ELISA with gene-specific coated monoclonal IgG confirmed the transgene protein expression. It was revealed that all acclimatized transgenic sugarcane plants survived the glyphosate spray application of 900 mL/0.404 ha, except for the control nontransformed plants. However, at spray application of 1100 mL/0.404 ha, transgenic plants having the transgene protein OD of 0.2 to 1.0 did not survive, while those that had a transgene protein OD range of between 1.0 and 2.0 did. In addition, weeds growing alongside transgenic sugarcane plants turned brown and subsequently died at glyphosate spray applications of both 900 and 1100 mL/0.404 ha. [ABSTRACT FROM AUTHOR]

Ten local Aspergillus niger isolates were subjected to RAPD analysis and out of 20 random oligonucleotide primers, primer P2 (5'-ACGGCGTATG-3') was optimized to screen all ten isolates of A. niger. Results showed amplifications of different sizes in range of 150-1500 bp and isolates 658 and 880 showed 100% genomic similarity while isolate 764 showed least genomic homology (22.54%). Based on RAPD analysis representative isolates from each cluster were short listed for further downstream molecular and biochemical analysis. These isolates included 880, 764, 506, 1005 and 744. In ribotyping hyper variable region V4 of 18S rRNA gene was amplified in five isolates which were found to be genomically diverse in RAPD analysis. Results showed that amplified sequence of 18S rRNA gene was highly conserved and 100% similar with each other and to the already reported sequence (Accession No JX393054.1). In catalase test on all isolates of A. niger indicated that isolates 744, 506, 764 and 880 were the most efficient in the production of catalase enzyme, while remaining isolates except 1109 showed intermediate catalase activity. In laccase assay isolate 744 was found to be most efficient and isolate 840 was found to be least efficient, while the remaining isolates were found to be at same level in laccase assay. Results suggested that isolate 744 can be exploited further for the production of catalase and laccase enzyme at industrial scale. [ABSTRACT FROM AUTHOR]

Cormels pieces of four Fusarium susceptible Gladiolus cultivars (Friendship, Peter Pears, Victor Borge and Novalux) formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85–95% germination after breaking dormancy of 8 weeks at 4 °C. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6 cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum. [ABSTRACT FROM AUTHOR]

Four tomato F1 hybrid cultivars (Roma, Maya, Rio Grande and T-55) were used to develop haploid lines during 2005-08. Anthers were cytogenetically analyzed for anther size and meiotic stage. Anthers of varying sizes and meiotic stages were collected at different flowering stages. These were subjected to various pretreatments like low temperature (4, 7 and 10°C) and high sucrose concentration (5, 7, 10, and 12%) for enhancement in androgenic response. Anthers were cultured on MS basal media containing different concentrations of cytokinins, auxins, vitamins and adjuvant. Plants regenerated from androgenic calli were analyzed for their ploidy level under microscope and 9-15 chromosomes could be visualized per haploid cell. [ABSTRACT FROM AUTHOR]

Fungi have been used in modern scientific research due to their high potential for different enzymes production based on genomic features. The great proportion of soil mycoflora represented by saprobic fungi plays an important role in decomposition, thus contribute to the global carbon cycle. Sordaria fimicola strains (n= 61) collected from different environments were evaluated for catalase enzyme activity at first stage. Among all 61 isolates of S. fimicola, five strains viz. S1, S2, N7, N6 and SF13 were found to be most efficient in catalase enzyme activity. The complete catalase gene including exons and introns was amplified and sequenced from the most efficient strains of S. fimicola and then submitted in the NCBI data base under accession numbers KM282183, KM282184, KM282186, KM282185 and KM282182 for strains S1, S2, N7, N6 and SF13 respectively. The significant differences in the genes sequences and theoretically translated proteins were observed for all five strains of S. fimicola. As regards catalase enzyme activity, S. fimicola strains were found comparable to the Aspergillus niger strains, therefore being a saprophytic fungus with short life cycle S. fimicola can become a fungus of choice to produce catalase enzyme at large scale.

Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35kDa exhibited highest expression at 0.5mM concentration of IPTG. Expressed recombinant protein of 35kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80μg and 200μg., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)