A weighing method to identify the microbial growth phases in solid-state fermentation tests

A general equation correlating the variation rate of the fermenting medium weight (dry matter) and the cell biomass growth rate in solid-state fermentation tests is proposed with the main purpose of identifying the microbial growth phases. This revised version was published online in July 2006 with corrections to the Cover Date.     

One-year survey of a single Micronesian reef reveals extraordinarily rich diversity of <Emphasis Type="Italic">Symbiodinium</Emphasis> types in soritid foraminifera

Recent molecular studies of symbiotic dinoflagellates (genus Symbiodinium) from a wide array of invertebrate hosts have revealed exceptional fine-scale symbiont diversity whose distribution among hosts, regions and environments exhibits significant biogeographic, ecological and evolutionary patterns. Here, similar molecular approaches using the internal transcribed spacer-2 (ITS-2) region were applied to investigate cryptic diversity in Symbiodinium inhabiting soritid foraminifera. Approximately 1,000 soritid specimens were collected and examined during a 12-month period over a 40 m depth gradient from a single reef in Guam, Micronesia. Out of 61 ITS-2 types distinguished, 46 were novel. Most types found are specific for soritid hosts, except for three types (C1, C15 and C19) that are common in metazoan hosts. The distribution of these symbionts was compared with the phylotype of their foraminiferal hosts, based on soritid small subunit ribosomal DNA sequences, and three new phylotypes of soritid hosts were identified based on these sequences. Phylogenetic analyses of 645 host-symbiont pairings revealed that most Symbiodinium types associated specifically with a particular foraminiferal host genus or species, and that the genetic diversity of these symbiont types was positively correlated with the genetic diversity found within each of the three host genera. Compared to previous molecular studies of Symbiodinium from other locations worldwide, the diversity reported here is exceptional and suggests that Micronesian coral reefs are home to a remarkably large Symbiodinium assemblage.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Employment of 16S rDNA gene sequencing techniques for improved identification of difficult-to-identify bacterial veterinary pathogens

To employ 16S rDNA PCR and automated sequencing techniques to identify a collection of bacterial veterinary pathogens from avian, equine, canine and ovine sources, that have proven difficult to identify, employing conventional cultural techniques. Universal or “broad-range” eubacterial PCR was performed on a collection of 46 difficult-to-identify bacterial isolates originating from clinical veterinary specimens. 16S rDNA PCR was performed using two sets of universal primers to successfully generate a composite amplicon of 1,068 bp, which was sequenced to obtain each isolate’s identity. Sequence analysis was able to identify all isolates examined with relative ease. Where the use of molecular identification methods is justified, such as in outbreak control or bioterrorism in animal health, employment of partial 16S rDNA PCR and sequencing employing universal or “broad-range” 16S rDNA, provides a valuable and reliable method of identification of such pathogens.     

Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 10²–10⁵ colony forming units g⁻¹ fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR^+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 10⁵ – 4.0 × 10⁷ colony forming units g⁻¹ tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.     

Alleviation of deleterious effects of protein mutation through inactivation of molecular chaperones

Molecular chaperones recognize and bind destabilized proteins. This can be especially important for proteins whose stability is reduced by mutations. We focused our study on a major chaperone system, RAC-Ssb, which assists folding of newly synthesized polypeptides in the yeast cytosol. A sensitive phenotypic assay, the red color of Ade2 mutants, was used to screen for variants with metabolic activity dependent on RAC-Ssb. None of the Ade2 mutants were found to exhibit lower metabolic activity after inactivation of RAC-Ssb. In order to explicitly test the relationship between protein instability and activity of chaperones, a series of temperature sensitive Ade2 mutants were tested in the presence or absence of RAC-Ssb. The growth of Ade2(ts) mutants at elevated temperatures was enhanced if chaperones were missing. Similar pattern was found for thermally sensitive mutants of several other genes. Because RAC-Ssb normally supports the folding of proteins, it appears paradoxical that catabolic activity of mutants is reduced when these chaperones are present. We suggest that under non-stressful conditions, molecular chaperones are tuned to support folding of native proteins, but not that of mutated ones.     

Phylogeny of Snub-Nosed Monkeys Inferred from Mitochondrial DNA,Cytochrome B,and 12S rRNA Sequences

Snub-nosed monkeys (Rhinopithecus spp.) are confined to isolated mountainous regions in China and North Vietnam. Their systematic classification and phylogenetic relationship has been controversial. The structures of mitochondrial DNA cytochrome b and 12S rRNA show that the 4 species of Rhinopithecus are quite different from other colobines. It is reasonable to regard them as an independent genus, as determined by external features, morphometric characters and behavior. However, whether or not there should be a subdivision between the Vietnamese and Chinese species at the subgeneric level remains to be clarified; more evidence from a large range of Asian colobine species is needed. The Guizhou species, Rhinopithecus brelichi, is a valid species, which is more closely related to Pygathrix than the other species ( R. roxellana, R. bieti and R. avunculus) are. Results also indicate that 3 species—Rhinopithecus roxellana, R. bieti and R. avunculus—might have diverged from R. brelichi, but the phylogenetic relationship of R. avunculus is not clear.     

Molecular evidence for the taxonomic status of three species of the <Emphasis Type="Italic">Sphyraena obtusata</Emphasis> group (Perciformes: Sphyraenidae) from East Asia

Genetic differences among the three species of Sphyraena with two gill rakers from East Asia (S. iburiensis, S. obtusata, and S. pinguis, defined recently as the S. obtusata group), were investigated using 799-bp sequences of the mitochondrial cytochrome b gene. Pairwise sequence differences within each of the three species were 0.0–0.4% (S. iburiensis), 0.0–0.4% (S. obtusata), and 0.0–0.6% (S. pinguis), respectively, pairwise sequence differences among the three species being 18.3–18.6% (S. iburiensis vs. S. obtusata), 14.9–15.4% (S. iburiensis vs. S. pinguis), and 17.6–18.3% (S. obtusata vs. S. pinguis), respectively. The extent of the latter were comparable to those among four other congeneric species, S. africana, S. forsteri, S. helleri, and S. japonica (16.0–24.5%). Phylogenetic analysis using the maximum-likelihood method indicated reciprocal monophyly of the three species, each clade being supported by a bootstrap value of 100%. These findings fully supported the taxonomic status of these species, recently elucidated by detailed morphological comparisons. In addition, the S. obtusata group formed a strongly supported clade against the four other congeneric species.     

Antimicrobial activity in cultures of endophytic fungi isolated from Garcinia species

The aim of the present study was to screen for antimicrobial activity in endophytic fungi isolated from surface sterilized leaves and branches of five Garcinia plants, G. atroviridis, G. dulcis, G. mangostana, G. nigrolineata and G. scortechinii, found in southern Thailand. Fermentation broths from 377 isolated fungi were tested for antimicrobial activity by the agar diffusion method. Minimum inhibitory concentrations (MICs) were obtained for crude ethyl acetate extracts. Seventy isolates (18.6%) displayed antimicrobial activity against at least one pathogenic microorganism, such as Staphylococcus aureus, a clinical isolate of methicillin-resistant S. aureus, Candida albicans and Cryptococcus neoformans. The results revealed that 6-10%, 1-2% and 18% of the crude ethyl acetate extracts inhibited both strains of S. aureus (MIC 32-512 microg mL(-1)), Ca. albicans and Cr. neoformans (MIC 64-200 microg mL(-1)), and Microsporum gypseum (MIC 2-64 microg mL(-1)), respectively. Isolates D15 and M76 displayed the strongest antibacterial activity against both strains of S. aureus. Isolates M76 and N24 displayed strong antifungal activity against M. gypseum. Fungal molecular identification based on internal transcribed spacer rRNA gene sequence analysis demonstrated that isolates D15 (DQ480353), M76 (DQ480360) and N24 (DQ480361) represented Phomopsis sp., Botryosphaeria sp. and an unidentified fungal endophyte, respectively. These results indicate that some endophytic fungi from Garcinia plants are a potential source of antimicrobial agents.