Antagonistic activity of glucanolytic bacteria Bacillus subtilis W3.15 against Fusarium oxysporum and its enzyme characterization

Putri RE, Mubarik NR, Ambarsari L, Wahyudi AT. 2021. Antagonistic activity of glucanolytic bacteria Bacillus subtilis W3.15 against Fusarium oxysporum and its enzyme characterization. Biodiversitas 22: 4067-4077. Biocontrol of Fusarium oxysporum, a phytopathogenic fungus that causes plant wilt can be approached with cell-wall degrading enzymes such as ?-glucanase. The aim of this study was to evaluate the prospective ability in glucanase production from several soil bacterial isolates and to characterize its ?-glucanase activity of ammonium sulfate precipitation, and to determine its antifungal activity against F. oxysporum in vitro. Twenty bacterial isolates were screened qualitatively and quantitatively as ?-glucanase producers. The results showed that the prospective isolate W3.15 can produce ?-glucanase on glucan agar as the selection medium. From 16S rRNA sequences identification, the isolate belongs to the genus Bacillus, closely related to Bacillus subtilis. The enzyme activity of the ammonium sulfate fraction of isolate W3.15 is optimum at a pH of 7 and temperature range of 60-80oC. B. subtilis W3.15 exhibits high inhibition against the mycelial growth of F. oxysporum and significantly reduced fungal biomass.

Gamma Radiation Effect on Agrobacterium tumefaciens-Mediated Gene Transfer in Potato (Solanum tuberosum L.)

Potato (Solanum tuberosum L.) is one of the major crops of the world. Significant improvements can be achieved in terms of yield and quality by the determination of efficient transformation methods. On the other hand, low transformation frequency seriously limits the application of molecular techniques in obtaining transgenic crops. In the present study, the effect of gamma radiation on Agrobacterium tumefaciens-mediated transformation to the potato was firstly investigated. Sterile seedlings of potato cv. ‘Marabel’, which was grown on Gamborg’s B5 medium in Magenta vessels, were irradiated with different gamma radiation doses (0-control, 40, 80, 120 Gy 60Co). Stem parts having axillary meristems were excised from irradiated seedlings and inoculated by A. tumefaciens (GV2260), which harbors the binary plasmid p35S GUS-INT contains and GUS (β-glucuronidase) gene controlled by 35S promoter (CaMV) and nptII (neomycin phosphotransferase II) gene driven by NOS (nopaline synthase) promoter). Inoculated stem parts having axillary meristems explants were then directly transported to a selection medium containing duocid (500 mg l−1), and kanamycin (100 mg l−1), 4 mg l−1 gibberellic acid, 1 mg l−1 BAP and 0.1 mg l−1 NAA. The adult transgenic plants were detected by polymerase chain reaction (PCR) analysis. According to the number of transgenic plants determined by PCR analysis, results obtained from explants treated with 40 Gy gamma gave the best results compared to the control (0 Gy) application. The doses over 40 Gy were also found statistically significant compared to the control (0 Gy). It is expected that the protocol described in this study make the transformation studies easier by skipping the stages of ‘co-cultivation’, ‘culturing explants on selection medium’ and ‘recovery of transgenic shoots on selection medium’ not only for potato but also for other crop plants. This study was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBİTAK) (Grant number 113O280 to Prof. Dr. Mustafa YILDIZ).

First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

Cell Suspension Culture and In Vitro Screening for Drought Tolerance in Soybean Using Poly-Ethylene Glycol

Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL−1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL−1 sucrose and 7.5 gL−1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

SCREENING OF ACIT SULPHATE SOILS FUNGI FROM SOUTH KALIMANTAN AS SOURCE OF EXTRACELULAR ENZYMES

Kalimantan acid sulphate land has the potential to be developed into productive land, with good land optimization. Utilization of rhizosphere microorganism diversity, especially mold can potentially provide a solution in optimizing agricultural land, namely the ability to produce extracellular enzymes. This study aims to determine the potential of mold originating from acid sulphate fields in producing extracellular enzymes (pectinase, chitinase, glucanase, cellulase, and phosphatase). The study was conducted in June-July 2019 at the Microbiology Laboratory, Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development. Screening of extracellular enzyme-producing fungi was carried out on selection media. The results obtained by some isolates have the ability to produce extracellular enzymes. Indications of the ability of mold to produce extracellular enzymes are the presence of clear zones in the selection medium. In pectinase, chitinase and glucanase testing all isolates showed negative results. Potential isolates in producing extracellular enzymes include Penicillium sp. Paddy 4.1 (cellulolytic index 2.43), Clonostachys sp. KRMT 17.9 and Penicillium singorense KLK 13.7 (proteolytic indices 3.97 and 3,00, respectively). The difference in index values ​​indicates the variation in the level of enzyme activity.

Epidemiological and Genomic Characterization of Campylobacter jejuni Isolates from a Foodborne Outbreak at Hangzhou, China

Background: Foodborne outbreaks caused by Campylobacter jejuni have become a significant public health problem worldwide. Applying genomic sequencing as a routine part of foodborne outbreak investigation remains in its infancy in China. We applied both traditional PFGE profiling and genomic investigation to understand the cause of a foodborne outbreak in Hangzhou in December 2018. Method: A total of 43 fecal samples, including 27 sick patients and 16 canteen employees from a high school in Hangzhou city in Zhejiang province, were recruited. Routine real-time fluorescent PCR assays were used for scanning the potential infectious agents, including viral pathogens (norovirus, rotavirus, adenovirus, and astrovirus), and bacterial pathogens (Salmonella, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus and Vibrio cholerae). Bacterial selection medium was used to isolate and identify the positive bacteria identified by molecular test. Pulsed field gel electrophoresis (PFGE), and next generation sequencing (NGS) were applied to fifteen recovered C. jejuni isolates to further understand the case linkage of this particular outbreak. Additionally, we retrieved reference genomes from the NCBI database and performed a comparative genomics analysis with the examined genomes produced in this study. Results: The analyzed samples were found to be negative for the queried viruses. Additionally, Salmonella, Shigella, Vibrio parahaemolyticus and Vibrio cholera were not detected. Fifteen C. jejuni strains were identified by the real-time PCR assay and bacterial selection medium. These C. jejuni strains were classified into two genetic profiles defined by the PFGE. Out of fifteen C. jejuni strains, fourteen have a unified consistent genotype belonging to ST2988, and the other strain belongs to ST8149, with a 66.7% similarity in comparison with the rest of the strains. Moreover, all fifteen strains harbored blaOXA-61 and tet(O), in addition to a chromosomal mutation in gyrA (T86I). The examined fourteen strains of ST2988 from CC354 clone group have very minimal genetic difference (3~66 SNPs), demonstrated by the phylogenomic investigation. Conclusion: Both genomic investigation and PFGE profiling confirmed that C. jejuni ST2988, a new derivative from CC354, was responsible for the foodborne outbreak Illustrated in this study.

Expression of Hemagglutinin-Neuraminidase (HN) and Fusion (F) Epitopes of Newcastle Disease Virus (NDV) in Chlamydomonas reinhardtii

Owing to their unique characteristics which combines the properties of both prokaryotes and eukaryotes, microalgae have emerged as an ideal platform for heterologous production of recombinant proteins including subunit vaccines. In an attempt to develop recombinant vaccine against Newcastle Disease, an agrobacterium-mediated genetic transformation was carried out to express a chimeric gene construct including Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) in Chlamydomonas reinhardtii. Four tandem repeat of HN epitope with 96bp length followed by three tandem repeat of F epitope of NDV with 153bp length were used. Microalgal cells (Chlamydomonas reinhardtii) were co-cultivated with Agrobacterium tumefaciens cells harboring foreign gene construct and then transferred to selection medium. Single colonies representing putative transformation events were screened in selection medium enriched with kanamycin. PCR assay confirmed integration of F-HN sequence in microalgal nuclei. RT-PCR assay showed that the F-HN sequence was expressed in transformed colonies. Finally, translation of the foreign gene was confirmed by protein dot blotting, western blot and Elisa assay. The results of this experiment may contain both research and practical implications.