Survey of Philippine coffee beans for the presence of ochratoxigenic fungi

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1 % sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17 %, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70 % of A. ochraceus produced OTA as high as 16238 ng g^?1 while 40 % of A. westerdijkiae produced maximum OTA of 36561 ng g^?1 in solid agar. Within section Nigri, 16.76 % of A. niger produced OTA at the highest 18439 ng g^?1, 10 % of A. japonicus at maximum level of 174 ng g^?1, and 21.21 % of A. carbonarius yielded maximum OTA of 1900 ng g^?1. Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g^?1.     

The taxonomic composition,characteristics, and neurotoxic activities of ribbon worm-associated bacteria from the Sea of Japan

The taxonomic composition of bacteria associated with two species of tetrodotoxin-bearing (TTX-bearing) (Hubrechtella juliae and Lineus alborostratus) and two species of non-TTX-bearing (Quasitetrastemma stimpsoni and Malacobdella grossa) ribbon worms collected from the Peter the Great Bay of Sea of Japan was studied. Bacterial isolates were identified using 16S rRNA gene sequencing and phenotypic characteristics. Thirty-eight strains of heterotrophic bacteria from the eight genera: Pseudoalteromonas, Shewanella, Ruegeria, Pseudomonas, Defluviicoccus, Vibrio, Alteromonas, and Bacillus, were isolated and characterized. γ-Proteobacteria dominated among the associated microflora of nemerteans (76.3% of the total number of isolates). Sensitivity analysis of 38 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all of the studied isolates. The 15 bacterial strains isolated in the study exhibited antimicrobial activities against at least one of five indicator microorganisms, most of which corresponded to the Pseudoalteromonas genus. Screening of the TTX-producing bacteria was performed using confocal laserscanning microscopy and polyclonal antibodies. A TTX-producing strain, Pseudoalteromonas sp., was found in the nemertean H. juliae. A correlation between the presence of TTX-positive microflora and the toxicity of nemerteans was determined.     

Genomic characterisation,detection of genes encoding virulence factors and evaluation of antibiotic resistance of <Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from cattle with clinical metritis

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.     

Antibiotic activity of bacterial endobionts of basidiomycete fruit bodies

Bacterial strains (93 isolates) capable of growth on full-strength nutrient media were isolated from 86 fungal fruit bodies collected in the Moscow region. Antimicrobial activity of the endobiont isolates against 12 bacterial and fungal test strains (including drug-resistant ones) was studied in submerged cultures. Most of the strains (84.9%) were found to produce antibiotic compounds with different antimicrobial properties, including antifungal activity in 18.3% of the strains. Morphological characteristics and analysis of the 16S rRNA gene sequences were used to determine the taxonomic position of 16 bacterial strains of the following 10 species: Bacillus subtilis, Ewingella americana, Pseudomonas sp., Stenotrophomonas maltophilia, as well as Achromobacter spanius, B. licheniformis, Hafnia paralvei, Micrococcus terreus, Nocardia coeliaca, and St. rhizophila, which have not been previously known to be endobionts of basidiomycete fruit bodies. Antimicrobial activity of A. spanius, E. americana, H. paralvei, M. terreus, N. coeliaca, and St. rhizophila has not been reported previously. Complex mechanisms of symbiotic relations between fungi and bacteria, including those associated with antibiotic formation, probably developed in the course of co-evolution.     

Strain differentiation of airborne opportunistic microorganisms within a municipal landfill area as assessed by PCR MP method

A municipal landfill is the site where occurs differentiation of microorganisms inclusive of several hazardous to human health. The aim of this study was to evaluate by a PCR melting profile (PCR MP) technique the level of genetic intraspecies relatedness of strains, representing several opportunistic bacteria and fungi commonly found in bioaerosol in the landfill site. In total, 27 strains representing four bacterial species (i.e. Escherichia coli, Proteus mirabilis, Staphylococcus sciurii, S. xylosus) and 36 fungal strains belonging to Aspergillus fumigatus and A. flavus were isolated from air samples collected by an Anderson impactor within the landfill area. The PCR melting profile approach clearly indicated that except E. coli, represented by one genotype, other microbial species underwent significant genetic variability in the active sector and surrounding the landfill forest and field areas. Although the genetic relatedness of some strains could testify to distribution of microbes from the active sector to the surroundings in the past, the bacterial and fungal isolates indicated site-specific genetic fingerprints. This is the first report on the distribution of airborne opportunistic microbial species within the landfill area, performing a comparison of their genotypes and evaluation of genetic relatedness between the isolates using the PCR MP method.     

Variation of cassiicolin genes among Chinese isolates of <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Corynespora cassiicola is a species of fungus that is a plant pathogen of many agricultural crop plants, including severe target spot disease on cucumber. Cassiicolin is an important effector of pathogenicity of this fungus. In this study, we collected 141 Corynespora isolates from eighteen hosts, and the casscolin gene was detected in 82 C. cassiicola strains. The deduced protein sequences revealed that 72 isolates contained the Cas2 gene, two strains from Gynura bicolor harboured the Cas2.2 gene, and 59 isolates without a cassiicolin gene were classified as Cas0. Phylogenetic analyses was performed for the 141 isolates using four loci (ITS, ga4, caa5, and act1) and revealed two genetic clusters. Cluster A is composed of four subclades: subcluster A1 includes all Cas2 isolates plus 18 Cas0 strains, subcluster A2 includes the eight Cas5 isolates and one Cas0 isolate, and subclusters A3 and A4 contain Cas0 strains. Cluster B consists of 21 Cas0 isolates. Twenty-two C. cassiicola strains from different toxin classes showed varying degrees of virulence against cucumber. Cas0 or Cas2 strains induced diverse responses on cucumber, from no symptoms to symptoms of moderate or severe infection, but all Cas5 isolates exhibited avirulence on cucumber.     

Diversity of Oil-Degrading Microorganisms in the Gulf of Finland (Baltic Sea) in Spring and in Summer

Diversity of the oil-degrading microbial strains isolated from the water and sediments of the Gulf of Finland (Baltic Sea) in winter and in summer was studied. Substrate specificity of the isolates for aliphatic and aromatic hydrocarbons was studied. The isolates belonged to 32 genera of the types Proteobacteria (alpha-, beta-, and gammaproteobacteria), Actinobacteria,Firmicutes, and Bacteroidetes. Seasonal variations of the oil-degrading microbial communities was revealed. The presence of the known genes responsible for the degradation of oil aliphatic and aromatic hydrocarbons was determined. The alkB sequence of the alkane hydroxylase gene was found in ~16% of the studied strains. The sequence of the phnAc phenanthrene 3,4- dioxygenase was found in Sphingobacterium sp. and Arthrobacter sp. isolates retrieved in winter and summer. In five Pseudomonas sp. strains from winter samples, the classical operons of naphthalene degradation (nah) were localized in catabolic plasmids, of which three belonged to IncР-9, one, to IncР-7, and two to an unidentified incompatibility group. Burkholderia and Delftia strains contained the operons for naphthalene degradation via salicylate and gentisate (nag). The presence of nag genes has not been previously reported for Delftia spp. strains. The sequences of the nagG salicylate 5-hydroxylase gene were also found in Achromobacter, Sphingobacterium, and Stenotrophomonas strains.     

Probiotic Potential of Autochthonous Bacteria Isolated from the Gastrointestinal Tract of Four Freshwater Teleosts

In this study, a total of 121 bacterial strains were isolated from the gastrointestinal tract of four teleostean species, namely striped snakehead (Channa striatus), striped dwarf catfish (Mystus vittatus), orangefin labeo (Labeo calbasu) and mrigal carp (Cirrhinus mrigala), among which 8 isolates showed promising antibacterial activity against four potential fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas sobria and Pseudomonas fluorescens and were non-hemolytic. The isolates were further screened in response to fish bile tolerance and extracellular digestive enzyme activity. Two bacterial strains MVF1 and MVH7 showed highest tolerance and extracellular enzymes activities, and selected for further studies. Antagonistic activity of these two isolates was further confirmed by in vitro growth inhibition assay against four selected fish pathogens in liquid medium. Finally, these two bacterial strains MVF1 and MVH7 were selected as potential probiotic candidates and thus identification by partial 16S rRNA gene sequence analysis. The bacterial isolates MVF1 and MVH7 were identified as two strains of Bacillus sp.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

Korean indigenous bacterial species with valid names belonging to the phylum <Emphasis Type="Italic">Actinobacteria</Emphasis>

To understand the isolation and classification state of actinobacterial species with valid names for Korean indigenous isolates, isolation source, regional origin, and taxonomic affiliation of the isolates were studied. At the time of this writing, the phylum Actinobacteria consisted of only one class, Actinobacteria, including five subclasses, 10 orders, 56 families, and 330 genera. Moreover, new taxa of this phylum continue to be discovered. Korean actinobacterial species with a valid name has been reported from 1995 as Tsukamurella inchonensis isolated from a clinical specimen. In 1997, Streptomyces seoulensis was validated with the isolate from the natural Korean environment. Until Feb. 2016, 256 actinobacterial species with valid names originated from Korean territory were listed on LPSN. The species were affiliated with three subclasses (Acidimicrobidae, Actinobacteridae, and Rubrobacteridae), four orders (Acidimicrobiales, Actinomycetales, Bifidobacteriales, and Solirubrobacterales), 12 suborders, 36 families, and 93 genera. Most of the species belonged to the subclass Actinobacteridae, and almost of the members of this subclass were affiliated with the order Actinomycetales. A number of novel isolates belonged to the families Nocardioidaceae, Microbacteriaceae, Intrasporangiaceae, and Streptomycetaceae as well as the genera Nocardioides, Streptomyces, and Microbacterium. Twenty-six novel genera and one novel family, Motilibacteraceae, were created first with Korean indigenous isolates. Most of the Korean indigenous actionobacterial species were isolated from natural environments such as soil, seawater, tidal flat sediment, and fresh-water. A considerable number of species were isolated from artificial resources such as fermented foods, wastewater, compost, biofilm, and water-cooling systems or clinical specimens. Korean indigenous actinobacterial species were isolated from whole territory of Korea, and especially a large number of species were from Jeju, Gyeonggi, Jeonnam, Daejeon, and Chungnam. A large number of novel actinobacterial species continue to be discovered since the Korean government is encouraging the search for new bacterial species and researchers are endeavoring to find out novel strains from extreme or untapped environments.     

First report of an atypical new <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> isolates with nucleotide insertion in <Emphasis Type="Italic">aflR</Emphasis> gene resembling to <Emphasis Type="Italic">A. sojae</Emphasis>

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B₁, B₂, G₁, and G₂ and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.     

Diversity and enzyme activity of <Emphasis Type="Italic">Penicillium</Emphasis> species associated with macroalgae in Jeju Island

A total of 28 strains of 19 Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β-tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The commonly isolated species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Among 19 Penicillium species, three species–P. kongii, P. olsonii, and P. viticola–have not been previously recorded in Korea.     

Relation to enterocins of variable <Emphasis Type="Italic">Aeromonas</Emphasis> species isolated from trouts of Slovakian aquatic sources and detected by MALDI-TOF mass spectrometry

Aeromonads represent bacteria thought to be primarily mostly autochthonous to aquatic environments. This study was focused on the relation with antibiotics and enterocins of identified Aeromonas species isolated from the intestine of trouts living in Slovakian aquatic sources. Intestinal samples from 50 trouts (3 Salmo trutta and 47 Salmo gairdnerii) were collected in April of years 2007, 2010, and 2015 from trouts of different water sources in Slovakia (pond Bukovec near Ko?ice, river ?ierny Váh). Due to the MALDI-TOF mass spectrometry evaluation, 25 strains were proposed to the genus Aeromonas involving nine different species (Aeromonas bestiarum—nine strains, Aer. salmonicida—four strains, Aer. encheleia, Aer. eucrenophila, Aer. molluscorum, Aer. media, Aer. sobria, Aer. popoffii, Aer. veronii). Phenotypic evaluation of individual strains confirmed their species identification. Twenty-five strains of different Aeromonas species were sensitive to azithromycin, amikacin, mecillinam, mezlocillin, piperacillin, gentamicin, chloramphenicol, and tetracycline. On the other side, they were resistant to carbenicillin and ticarcillin. The growth of Aer. bestiarum R41/1 was inhibited by treatment with Ent M and Ent 2019 (inhibition activity 100 AU/mL). Aer. bestiarum R47/3 was inhibited by eight enterocins (100 AU/mL). It is the first study testing enterocins to inhibit the growth of Aeromonas species from trouts.     

Yeasts from peat in a tropical peat swamp forest in Thailand and their ability to produce ethanol,indole-3-acetic acid and extracellular enzymes

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens, Debaryomyces fabryi, Meyerozyma guilliermondii, Saturnispora diversa and Schwanniomyces polymorphus var. africanus, and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides, Papiliotrema flavescens, Papiliotrema laurentii and Rhodotorula mucilaginosa. Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus, Cy. subsufficiens and D. fabryi. None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.     

Diversity and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> isolates obtained from rice rhizosphere soils in Taiwan

Azotobacter species, free-living nitrogen-fixing bacteria, have been used as biofertilizers to improve the productivity of non-leguminous crops, including rice, due to their various plant growth-promoting traits. The purposes of this study were to characterize Azotobacter species isolated from rice rhizospheres in Taiwan and to determine the relationship between the species diversity of Azotobacter and soil properties. A total of 98 Azotobacter isolates were isolated from 27 paddy fields, and 16S rRNA gene sequences were used to identify Azotobacter species. The characteristics of these Azotobacter strains were analyzed including carbon source utilization and plant growth-promoting traits such as nitrogen fixation activity, indole acetic acid production, phosphate-solubilizing ability, and siderophore secretion. Of the 98 strains isolated in this study, 12 were selected to evaluate their effects on rice growth. Four species of Azotobacter were identified within these 98 strains, including A. beijerinckii, A. chroococcum, A. tropicalis, and A. vinelandii. Of these four species, A. chroococcum was predominant (51.0%) but A. beijerinckii had the highest level of nucleotide diversity. Strains within individual Azotobacter species showed diverse profiles in carbon source utilization. In addition, the species diversity of Azotobacter was significantly related to soil pH, Mn, and Zn. Members of the same Azotobacter species showed diverse plant growth-promoting traits, suggesting that the 98 strains isolated in this study may not equally effective in promoting rice growth. Of the 12 strains evaluated, A. beijerinckii CHB 461, A. chroococcum CHB 846, and A. chroococcum CHB 869 may be used to develop biofertilizers for rice cultivation because they significantly promoted rice growth. This study contributes to the selection of suitable Azotobacter strains for developing biofertilizer formulations and soil management strategies of Azotobacter for paddy fields.     

Screening for potential probiotic from spontaneously fermented non-dairy foods based on in vitro probiotic and safety properties

The aim of this study was to screen potential probiotic lactic acid bacteria from Chinese spontaneously fermented non-dairy foods by evaluating their probiotic and safety properties. All lactic acid bacteria (LAB) strains were identified by 16S rRNA gene sequencing. The in vitro probiotic tests included survival under low pH and bile salts, cell surface hydrophobicity, auto-aggregation, co-aggregation, antibacterial activity, and adherence ability to cells. The safety properties were evaluated based on hemolytic activity and antibiotic resistance profile. The salt tolerance, growth in litmus milk, and acidification ability were examined on selected potential probiotic LAB strains to investigate their potential use in food fermentation. A total of 122 strains were isolated and identified at the species level by 16S rRNA gene sequencing and included 62 Lactobacillus plantarum, 40 Weissella cibaria, 12 Lactobacillus brevis, 6 Weissella confusa, and 2 Lactobacillus sakei strains. One W. cibaria and nine L. plantarum isolates were selected based on their tolerance to low pH and bile salts. The hydrophobicity, auto-aggregation, co-aggregation, and antagonistic activities of these isolates varied greatly. All of the 10 selected strains showed multiple antibiotic resistance phenotypes and no hemolytic activity. The highest adhesion capacity to SW480 cells was observed with L. plantarum SK1. The isolates L. plantarum SK1, CB9, and CB10 were the most similar strains to Lactobacillus rhamnosus GG and selected for their high salt tolerance and acidifying activity. The results revealed strain-specific probiotic properties were and potential probiotics that can be used in the food industry.