Evaluation and Genome Mining of Bacillus stercoris Isolate B.PNR1 as Potential Agent for Fusarium Wilt Control and Growth Promotion of Tomato.

Recently, strategies for controlling Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of Fusarium wilt of tomato, focus on using effective biocontrol agents. In this study, an analysis of the biocontrol and plant growth promoting (PGP) attributes of 11 isolates of loamy soil Bacillus spp. has been conducted. Among them, the isolates B.PNR1 and B.PNR2 inhibited the mycelial growth of Fol by inducing abnormal fungal cell wall structures and cell wall collapse. Moreover, broad-spectrum activity against four other plant pathogenic fungi, F. oxysporum f. sp. cubense race 1 (Foc), Sclerotium rolfsii, Colletotrichum musae, and C. gloeosporioides were noted for these isolates. These two Bacillus isolates produced indole acetic acid, phosphate solubilization enzymes, and amylolytic and cellulolytic enzymes. In the pot experiment, the culture filtrate from B.PNR1 showed greater inhibition of the fungal pathogens and significantly promoted the growth of tomato plants more than those of the other treatments. Isolate B.PNR1, the best biocontrol and PGP, was identified as Bacillus stercoris by its 16S rRNA gene sequence and whole genome sequencing analysis (WGS). The WGS, through genome mining, confirmed that the B.PNR1 genome contained genes/gene cluster of a nonribosomal peptide synthetase/polyketide synthase, such as fengycin, surfactin, bacillaene, subtilosin A, bacilysin, and bacillibactin, which are involved in antagonistic and PGP activities. Therefore, our finding demonstrates the effectiveness of B. stercoris strain B.PNR1 as an antagonist and for plant growth promotion, highlighting the use of this microorganism as a biocontrol agent against the Fusarium wilt pathogen and PGP abilities in tomatoes.

Evaluation of Soil Streptomyces spp. for the Biological Control of Fusarium Wilt Disease and Growth Promotion in Tomato and Banana.

Fusarium oxysporum f. sp. lycopersici (Fol) and Fusarium oxysporum f. sp. cubense (Foc), are the causal agent of Fusarium wilt disease of tomato and banana, respectively, and cause significant yield losses worldwide. A cost-effective measure, such as biological control agents, was used as an alternative method to control these pathogens. Therefore, in this study, six isolates of the Streptomyces-like colony were isolated from soils and their antagonistic activity against phytopathogenic fungi and plant growth-promoting (PGP) activity were assessed. The results showed that these isolates could inhibit the mycelial growth of Fol and Foc. Among them, isolate STRM304 showed the highest percentage of mycelial growth reduction and broad-spectrum antagonistic activity against all tested fungi. In the pot experiment study, the culture filtrate of isolates STRM103 and STRM104 significantly decreased disease severity and symptoms in Fol inoculated plants. Similarly, the culture filtrate of the STRM304 isolate significantly reduced the severity of the disease and symptoms of the disease in Foc inoculated plants. The PGP activity test presents PGP activities, such as indole acetic acid production, phosphate solubilization, starch hydrolysis, lignin hydrolysis, and cellulase activity. Interestingly, the application of the culture filtrate from all isolates increased the percentage of tomato seed germination and stimulated the growth of tomato plants and banana seedlings, increasing the elongation of the shoot and the root and shoot and root weight compared to the control treatment. Therefore, the isolate STRM103 and STRM104, and STRM304 could be used as biocontrol and PGP agents for tomato and banana, respectively, in sustainable agriculture.

Enzyme Inhibition and Antimicrobial Activities of Fractionated Wild Grape (Ampelocissus martinii Planch.) Seed Extracts.

BACKGROUND AND OBJECTIVE: Study on medicinal plant extract is gradually interested and distributed, especially their biological activities. The present study aimed to determine the enzyme inhibition and antimicrobial activities of the fractionated extracts of wild grape (Ampelocissus martinii Planch.) seeds. MATERIALS AND METHODS: Wild grape seeds in different growth stages were extracted with methanol before fractionation by silica gel chromatography. The anti-glucosidase and anti-tyrosinase enzyme activities of the extracts were then tested by using UV-Vis spectrophotometry and antimicrobial activities were observed from MIC, MBC values and time killing assay. RESULTS: The sub-fraction of immature stage eluted by ethyl acetate/methanol at 75/25 (%v/v) has the highest enzyme inhibition activity and the most potent efficiency for time kills profiles. The MIC values of the potent immature, mature and ripe fractioned extracts were ranging from 1.25-50.00, 1.25-50.00 and 1.56-25.00 mg mL-1, respectively, while the MBC values ranged from 3.12-6.25, 3.12-25.00 and 3.12-25.00 mg mL-1, respectively. CONCLUSION: The wild grape seed composed of α-glucosidase and tyrosinase inhibition and antibacterial activities compounds. The wild grape seed extracts may be used as active ingredients sources of health-supporting products or cosmetics.

Comparative Study on the Effect of Aqueous and Ethanolic Mycelial Extracts from Polycephalomyces nipponicus (Ascomycetes) against Human Breast Cancer MCF-7 Cells.

Recently, the entomopathogenic fungus Polycephalomyces nipponicus has been determined to be a prolific producer of bioactive compounds that have both antibacterial and antimarial activities, but the anticancer effects of the mycelial extracts have not been well studied. The present study investigates the effects and mechanisms of action of P. nipponicus extracts that are responsible for cell death in the human breast cancer MCF-7 cell line. The results showed that the 50% ethanol extract had greater anticancer activity than the aqueous extract. The 50% ethanolic extract inhibited cell growth at concentrations of just 109.75 ± 3.54 µg mL-1 for 72 h. The aqueous extract's activity was > 400 µg mL-1 against MCF-7 cells. Similar results were obtained from the colony formation assay. Moreover, the 50% ethanolic extract caused a significant increase in the distribution of cells at the G1 phase in a dose-dependent manner. The aqueous extract induced MCF-7 cells to arrest at the G2/M phase in a dose-dependent manner. These were supported by the reduction in the cyclin-dependent kinase (cdk)2, cdk4, and cdk6 genes' expression levels along with an induction of the cyclin-dependent kinase inhibitor p21 in MCF-7 cells after treatment with the extracts. In conclusion, P. nipponicus may be useful for breast cancer prevention and treatment in the future.

Evaluation of Antibacterial and Anticancer Activities of the Medicinal Fungus Ophiocordyceps sobolifera (Ascomycetes) from Thailand.

Eleven strains of an entomopathogenic fungus, isolated and identified as Ophiocordyceps sobolifera, were screened for activity against 5 strains of Gram-negative and 5 strains of Gram-positive bacteria. Four of the isolates, Cod-KK1634, Cod-KK1643, Cod-KS1601, and Cod-SN1626, had activity against the test strains of Grampositive bacteria. Of these 4 extracts, the Cod-KK1643 extract had the lowest minimum inhibitory and bactericidal concentrations. The Cod-KK1643 extract exhibited both concentration- and time-dependent bactericidal activity. Moreover, the Cod-KK1643 extract induced morphological alterations in bacterial cells, including decreased cell size, a crushed appearance, and cell lysis. It is surprising to note that the extracts also inhibited MCF-7 cell proliferation, with a half-maximal inhibitory concentration of 47.09 ± 33.64 mg/mL after 72 hours. The extracts also inhibited MCF-7 cell migration, with the lowest percentage of relative closure achieved with Cod-KK1643. These findings represent what is to our knowledge the first information on the activities of O. sobolifera mycelial extracts against bacteria and the human breast cancer MCF-7 cell line.

Colony Characteristics, Nucleoside Analog Profiles, and Genetic Variations of Medicinal Fungus Polycephalomyces nipponicus (Ascomycetes) Isolates from Northeast Thailand.

The entomopathogenic fungus Polycephalomyces nipponicus is known to have activity against human pathogenic bacteria and the malaria pathogen; however, information about its genetic variation is limited. In this study, cicada nymphs infected with entomopathogenic fungi were collected from various locations in the northeast of Thailand. Internal transcribed spacer sequencing was used to identify the fungal pathogen P. nipponicus. A total of 36 isolates of P. nipponicus from 6 provinces were investigated for variations in fungal morphology, nucleoside analog content, and genetics. The results showed that colony morphology varied depending on the strain of the tested fungi, without influence from its geographic origin. A similar finding was observed with regard to the production of nucleoside analog content. Interestingly, the important bioactive compound adenosine was detected in the mycelial extract of all 36 isolates. This indicates that P. nipponicus could possibly be used as a source of potential therapeutic bioactive compounds. In addition, random amplified polymorphic DNA analysis, as supported by the Nei Index and Shannon Index values, showed high genetic variation within and between the populations. These findings represent what is, to our knowledge, the first information on the colony morphology, adenosine analog profile, and genetic variation of P. nipponicus.

Effects of Ethyl Acetate Extracts from the Polycephalomyces nipponicus Isolate Cod-MK1201 (Ascomycetes) against Human Pathogenic Bacteria and a Breast Cancer Cell Line.

This study aimed to identify a suitable organic solvent for extracting bioactive compounds from Polycephalomyces nipponicus and to evaluate the antibacterial and anticancer activities of the extracts obtained. Only extracts obtained with ethyl acetate exhibited antibacterial activity, so ethyl acetate was chosen for large-scale extraction. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Gram-positive and Gram-negative human pathogenic bacteria of the 3 ethyl acetate-derived extracts-ethyl acetate extract from P. nipponicus (PN-ME), ethyl acetate extract after defatting (PN-ME*), and ethyl acetate extract after refluxation (PN-ME')-were determined. PN-ME' exhibited the most potent activity, inhibiting 12 of the 18 test bacteria, especially Bacillus cereus ATCC 11778 and Vibrio cholera (O1) DMST 9700, with low MIC and MBC values. PN-ME* showed greater inhibitory activity than PN-ME. The effects of the extracts on bacterial cell morphology were also determined. After 120 minutes of treatment with PN-ME* or PN-ME', B. cereus ATCC 11778 exhibited an abnormal rod-shaped cell structure, with some cells elongated to multiple times their original size and others appearing collapsed. V cholera (O1) DMST 9700 cells showed shrinkage and the formed subsurface cavities. PN-ME* and PN-ME' also inhibited the growth of MCF-7 breast cancer cells. In conclusion, the fungal isolate P. nipponicus Cod-MK1201 represents a source of antibacterial and anti-breast cancer compounds.

Isolation of the Entomopathogenic Fungal Strain Cod-MK1201 from a Cicada Nymph and Assessment of Its Antibacterial Activities.

The entomopathogenic fungus Cod-MK1201 was isolated from a dead cicada nymph. Three regions of ribosomal nuclear DNA, the internal transcribed spacers of nuclear ribosomal DNA repeats (ITS), the partial small subunit of rDNA (nrSSU) , and the partial large subunit of rDNA (nrLSU), and two protein-coding regions, the elongation factor 1α (EF-1α), and the largest subunit of the RNA polymerase II (rpb1) gene, were sequenced and used for fungal identification. The phylogenetic analysis of the ITS and the combined data set of the five genes indicated that the fungal isolate Cod-MK1201 is a new strain of Cordyceps sp. that is closely related to Cordyceps nipponica and C. kanzashiana. Crude extracts of mycelium-cultured Cod-MK1201 were obtained using distilled water and 50% (v/v) ethanol, and the antibacterial activity of each was determined. Both extracts had activity against Gram-positive and Gram-negative bacteria, but the ethanol extract was the more potent of the two. The antibacterial activity of the protein fractions of these extracts was also determined. The protein fraction from the ethanol extract was more antibacterial than the protein fraction from the aqueous extract. Three antibacterial constituents including adenosine, the total phenolic content (TPC), and the total flavonoid content (TFC) was also determined. The results showed that the adenosine content, the TPC, and the TFC of the ethanol extract were more active than those of the aqueous extract. Moreover, synergism was detected between these antibacterial constituents. In conclusion, the entomopathogenic fungal isolate Cod-MK1201 represents a natural source of antibacterial agents.

Comparison of mosquito densoviruses: two clades of viruses isolated from indigenous mosquitoes.

We analyzed the phylogenetic tree of densoviruses isolated from indigenous mosquitoes and mosquito cell lines. Our findings suggest two distinct clades of densovirus. The viruses in the first clade were isolated from an indigenous mosquito which had the Aedes aegypti densovirus (AaeDNV) as a representative virus. The other clade of viruses was isolated from mosquito indigenous cell line which had the Aedes albopictus densovirus (AalDNV) as the representative virus. The origin of the two clades of DNVs is unclear but the phylogenetic trees were significantly different from each other. The two major densoviruses, AaeDNV and AalDNV, that infect mosquitoes that are known to carry viruses responsible for dengue hemorrhagic fever and yellow fever. Understanding the evolution of these two clades of densoviruses is important for studying the distribution of these viruses in mosquito cell lines and the information gained may be applied to understanding other viruses in various mosquito cell lines.

Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection.

The present study aimed to use enterobacterial repetitive intergenic consensus (ERIC) fingerprints to design SCAR primers for the detection of Escherichia coli. The E. coli strains were isolated from various water sources. The primary presumptive identification of E. coli was achieved using MacConkey agar. Nineteen isolates were selected and confirmed to be E. coli strains based on seven biochemical characteristics. ERIC-PCR with ERIC 1R and ERIC 2 primers were used to generate DNA fingerprints. ERIC-PCR DNA profiles showed variant DNA profiles among the tested E. coli strains and distinguished all E. coli strains from the other tested bacterial strains. A 350 bp band that predominated in five E. coli strains was used for the development of the species-specific SCAR primers EC-F1 and EC-R1. The primers showed good specificity for E. coli, with the exception of a single false positive reaction with Sh. flexneri DMST 4423. The primers were able to detect 50 pg and 10(0) CFU/ml of genomic DNA and cells of E. coli, respectively.

New genetic variation of Aedes albopictus densovirus isolated from mosquito C6/36 cell line.

Densovirus (DNV) is a small single-stranded DNA, non-enveloped virus belonging to the subfamily Densovirinae of the Parvoviridae family. This group of invertebrate viruses infects exclusively insects. Two of the major densoviruses, Aedes aegypti (AaeDNV) and Ae. albopictus (AalDNV), infect mosquitoes that carry viruses responsible for two important public health diseases, namely, dengue hemorrhagic fever and yellow fever. The present study describes cloning, sequencing and phylogenetic analysis of a new densovirus, AalDNV-4, from infected Ae. albopictus C6/36 cell line. The total nucleotide sequence (3.9 kb) of AalDNV-4 was obtained from sequencing of DNA fragments, and is 98% homologous to the initial AalDNV previously isolated, and distinguishable from other AalDNVs reported earlier. This full-length viral genome contains a 40-bp deletion at the left terminal region, 12 substitutions and 3 indels. Phylogenetic analysis of AalDNV-4 genome indicates that this virus is more closely related to the original AalDNV found in C6/36 cell line than to AaeDNV isolated from other mosquitoes. It was concluded that AalDNV-4 may have been derived from the original DNV found in the C6/36 cell line and has transferred worldwide from the exchange of this cell line among laboratories.

Heterologously type IV pilus expressed protein of Burkholderia pseudomallei is immunogenic but fails to induce protective immunity in mice.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe disease of humans and animals. At present, no effective vaccine against melioidosis exists. Bacterial type IV pilin proteins have been used successfully as subunit vaccines. In this study, we evaluated a heterologously expressed and purified type IV pilus protein (PilV) of B. pseudomallei strain K96243 as a candidate subunit vaccine. PilV protein was assessed for its ability to protect BALB/c mice against B. pseudomallei strain G207 infection. Mice subcutaneously immunized with purified PilV protein produced high titers of IgG antibodies, which were strongly biased towards IgG1, with lower levels of IgG2a. Even though the PilV protein was highly immunogenic, it could not induce protection against a lethal B. pseudomallei challenge. Possible mechanisms of this non-protection phenomenon are discussed.