rDNA PCR-RFLP Research Articles

A historical and integrative taxonomic account of mangrove oyster species native to the Atlantic American coast: A re-evaluation of Brazilian Crassostrea species

The identification of native mangrove oyster species of the genus Crassostrea in Brazil is a recurring issue for specialists. Studies have recognized the presence of two species in the littoral zone: C. rhizophorae and C. gasar. However, other names may be used, such as C. mangle for C. rhizophorae and C. brasiliana or C. tulipa for C.gasar. As in most oyster taxonomic studies, their identification requires genetic evaluation for the precise determination of taxa. Moreover, previous revisions have proposed some morphological traits by which each species could be precisely identified, leading to a series of debates about the best model for species identity. The aim of this study was to apply an integrative taxonomic approach to native Crassostrea mangrove oyster species on the Brazilian coast to assert the correct species names, delimitations, and concise descriptions. Molecular tracing of cultivated specimens, along with wild specimens of Crassostrea collected from the Brazilian coast, was used to confirm the morphological identity of the taxa. The wild specimens were tentatively identified as either C. gasar or C. rhizophorae based on previous morphological revisions. PCR-RFLP and DNA analyses of the mitochondrial 16S gene mitochondrial rDNA from wild specimens were used to confirm taxa and re-evaluate morphological boundaries and descriptions of the species. We confirmed two patterns in the RFLP and sequence analyses: C. gasar and C. rhizophorae. Conchological and anatomical evaluations of 1006 identified Crassostrea specimens confirmed the inherent polymorphism between the native species, preventing reliable identification of the species by morphology alone. A clear exception, however, is found for specimens whose shell height is greater than 110 mm with internal view of white-colored valves and without an umbonal cavity, fitting the description of C. paraibanensis, which belongs to C. gasar. C. brasiliana and C. mangle are synonyms of C. gasar and C. rhizophorae, respectively. C. tulipa represents a species disconnected from any described mangrove oyster species from Atlantic seas. Conchological and anatomical descriptions of both species were improved to include the variability encountered.

Indigenous Yeasts for the Biocontrol of Botrytis cinerea on Table Grapes in Chile.

One hundred twenty-five yeast strains isolated from table grapes and apples were evaluated for the control Botrytis cinerea of in vitro and in vivo. Ten strains were selected for their ability to inhibit mycelial growth of B. cinerea in vitro. In the in vivo assays, these yeasts were tested at 20 °C on 'Thompson Seedless' berries for 7 days; only three were selected (m11, me99 and ca80) because they significantly reduced the incidence of gray mold. These three yeast strains were then evaluated at different concentrations (1 × 107, 1 × 108 and 1 × 109 cells mL-1) on 'Thompson Seedless' grape berries at 20 °C. The strains m11, me99 and ca80 reduced the incidence of B. cinerea to 11.9, 26.1 and 32.1%, respectively, when the berries were submerged in a yeast suspension at a concentration of 1 × 109 cells mL-1 24 h before inoculation with B. cinerea. The most favorable pH for antifungal activity was 4.6 in the three isolates. The three yeast strains secreted the hydrolytic enzymes chitinase and β-1-glucanase, and two strains (me99 and ca80) produced siderophores. The three yeast strains exhibited low oxidative stress tolerance and only strain m11 had the ability to produce biofilms. The strains were identified using 5.8S-ITS rDNA PCR-RFLP and correspond to the Meyerozyma guilliermondii (m11) and Aureobasidium pullulans (me99 and ca80) species.

Liver Fluke Species Identification Isolated From Humans and Animals Using PCR-RFLP and DNA Sequencing

Fasciola species are a member of flatworms belonging to the trematodes (flukes), commonly known as liver fluke, they are extremely pathogenic parasites that affect the liver of humans and animals, nowadays, most laboratories and research facilities use molecular-based techniques for identifying and describing Fasciola species. The molecular diagnostic markers such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-RFLP methods are accurate and more specific than the immunological and microscopical methods. The identification of the species of liver flukes will give a new clue for the treatment and control of fascioliasis. The aim, of this study, is to find the molecular characterization of Fasciola spp. isolated from humans and animals in Sulaimani city. The flukes were isolated from humans using endoscopic techniques and from slaughtered livestock at the new slaughterhouse of Sulaimani, 48 liver flukes were collected from different hosts; human (n = 3), cattle (n = 20), sheep (n = 20), and goats (n = 5) from October 2021 to April 2022. The uinversal primers ribosomal Deoxy Ribo Nuclic Acid (rDNA) were used, then the PCR products were subjected to restriction fragment polymorphism (RFLP) assay and The PCR Product was digested with restriction enzymes DraII, also the DNA sequencing was used for the PCR product of the primer Cytochrome Oxidase subuint 1 (COX1). The results of the PCR-RFLP of the 28s rDNA show the genetic polymorphisms among the flukes and two patterns of RFLP were observed F. hepatica, and F. gigantica, also the sequence analysis of the partial gene of the COX1 showed the isolated flukes belonged to F. hepatica and F. gigantica with some genetic variation, and the result of the sequences was deposited in the Gene Bank under the following Accession numbers; F. gigantica (OP718780 and OP718781) and F. hepatica (OP718782, OP718783, and OP718784). The present study concludes thatF. hepatica and F. gigantica are both responsible for human and animal Fasciolasis in Kurdistan-Iraq, Therefore, RFLP techniques and DNA sequencing are a reliable, and differential method for species and genotyping identification of liver fluke.

Potential role of rhizobia to enhance chickpea-growth and yield in low fertility-soils of Tunisia.

Plant growth-promoting rhizobacteria are bacteria that improve plant growth and reduce plant pathogen damages. In this study, 100 nodule bacteria were isolated from chickpea, screened for their plant growth-promoting (PGP) traits and then characterised by PCR-RFLP of 16S rDNA. Results showed that most of the slow-growing isolates fixed nitrogen but those exhibiting fast-growth did not. Fourteen isolates solubilized inorganic phosphorus, 16 strains produced siderophores, and 17 strains produced indole acetic acid. Co-culture experiments identified three strains having an inhibitory effect against Fusarium oxysporum, the primary pathogenic fungus for chickpea in Tunisia. Rhizobia with PGP traits were assigned to Mesorhizobium ciceri, Mesorhizobium mediterraneum, Sinorhizobium meliloti and Agrobacterium tumefaciens. We noted that PGP activities were differentially distributed between M. ciceri and M. mediterraneum. The region of Mateur in northern Tunisia, with clay-silty soil, was the origin of 53% of PGP isolates. Interestingly, we found that S. meliloti and A. tumefaciens strains did not behave as parasitic nodule-bacteria but as PGP rhizobacteria useful for chickpea nutrition and health. In fact, S. meliloti strains could solubilize phosphorus, produce siderophore and auxin. The A. tumefaciens strains could perform the previous PGP traits and inhibit pathogen growth also. Finally, one candidate strain of M. ciceri (LL10)-selected for its highest symbiotic nitrogen fixation and phosphorus solubilization-was used for field experiment. The LL10 inoculation increased grain yield more than three-fold. These finding showed the potential role of rhizobia to be used as biofertilizers and biopesticides, representing low-cost and environment-friendly inputs for sustainable agriculture.

Epidemiology of Pityriasis versicolor in Tunisia: Clinical features and characterization of Malassezia species

Malassezia (M.) genus includes commensal yeasts of increasing medical importance, as they result in many diseases, ranging from pityriasis versicolor (PV) to systemic infections. Previous studies reported geographical variations in distribution of Malassezia species in PV lesions. The aims of the current study were to define the clinico-demographic features of PV in Tunisia, to characterize Malassezia isolates using phenotypic and molecular techniques and to find out any association between species and clinico-demographic parameters.In total, 120 PV patients were enrolled in this study. Skin scrapings were collected and inoculated on Sabouraud agar and modified Dixon medium. Malassezia species were identified using conventional phenotypic methods and 26 s rDNA PCR-RFLP. The highest prevalence of PV was observed among young adults’ group. The most affected body areas were the back and neck. In overall, 50.8% and 35% of PV cases had pruritus and history of recurrence respectively. The overall concordance between phenotypic and molecular methods was high (80.95%). The discordant results are rather due to the presence of multiple species in a single culture than true misidentification. Using PCR-RFLP, M. furfur was the most isolated species (38.7%) followed by M. globosa (37.7%), M. restricta and M. sympodialis. No statistically significant association was noted between Malassezia spp. and clinico-demographic characteristics. Unlike many reports from temperate climate countries, M. furfur and M. globosa along together were the most frequently isolated species in Tunisian PV patients. Although phenotypic methods remain simple and cost-effective, molecular techniques are considered as fast and accurate methods for diagnosis purposes.

OENOLOGICAL CHARACTERISTICS AND VINIFICATION RESULTS OF THE YEAST OF MALAGOUSIA GRAPE ISOLATED IN GREECE

Malagousia grapes, selected from 5 different PGI Greek zones, in 3 different growing seasons (2018-2019, 2019-2020, 2020-2021) were collected in the stage of technological maturity. Quantity of 25 Kg grapes fermented spontaneously in 30 l thermo regulated stain steel tanks, produced white wine. The indigenous yeast flora, isolated at three phases of the alcoholic fermentations, was studied. Different yeast species were isolated, purified and characterized. The restriction fragment length polymorphism of PCR-amplified fragments from the rDNA gene cluster (PCR RFLP of rDNA) has been used for the differentiation of yeast species. The standard identification procedure has been performed on representative strains that shared identical RFLP profiles showed great diversity of the yeast population. Including grape berries, must and fermented must, the following yeast species were identified: a number of Pichia and Candida species, Kloeckera apiculata, Cryptococcus curvatus, Metschnikowia pulcherrima, Kluyveromyces, as well as Saccharomyces cerevisiae and Saccharomyces stranieri. We performed microscopic, macroscopic and carbon assimilation tests by API 32C standardized system, biotechnological testing and hydrolase profiling obtained by the API-ZYM system. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were bigger diversity of different species of Non- Saccharomyces in organic grapes and almost complete absence of non-Saccharomyces species, at least at grapes and at the beginning of the alcoholic fermentations at conventionally cultivated grapes, from all the examined PGI zones in all the three years of the study. The use and enhancement of indigenous yeasts is an increasing trend. Rapid identification of the yeast population is necessary for successful monitoring of the fermentation process and for obtaining a good wine quality as well as contributing to the optimization of Greek wine production.

The Screening and Identification of Heterotrophic Nitrified Aerobic Denitrifier in the Penaeus Vannamei Boone Aquaculture System without Water Renew

The experiments respectively take the artificial substratum (mesh) form the indoor aquaculture systems without water renew of Penaeus vannamei Boone as well as the mature bio-films. Through the complex inoculation on BTB ammonia, nitrite and nitrate medium, screened and obtained the library of the heterotrophic nitrified aerobic denitrifier. Selected 25 single colonies from the bacteria library of two membrane systems to conduct analysis based on the 16s rDNA PCR-RFLP and the results indicated that 50 colonies can be divided into 5 spectral types, in which 2 for the mesh, 3 for the coral sand. After sequencing, the blast comparison shows that all the heterotrophic nitrified aerobic denitrifiers are from the γ-proteobacteria (Gammaproteobacteria), where the bacteria on the mesh belong to the Acinetobacter sp.and Klebsiella sp.; while the bacteria on the coral sand belong to the Pseudomonas sp., Shewanella sp. and Klebsiella sp. According to the analysis on the phylogenetic tree, we know its evolutionary position is basically consistent with the traditional classification. Gram stain analysis indicates that all bacteria are red and belong to the Gram-negative bacteria. The research is of great significance for the selection of the heterotrophic nitrified aerobic denitrifier in the prawn aquaculture systems.

Identification and Characterization of Verticillium longisporum Lineage A1/D1 from Brassica Crops in Manitoba, Canada.

Verticillium stripe in canola (Brassica napus L.) caused by Verticillium longisporum was first reported in Manitoba in 2014. In this study, Brassica crops including canola, mustard (Brassica juncea) and radish (Raphanus sativus) with visible symptoms of Verticillium stripe were collected from Portage La Prairie, Manitoba, and the pathogens were isolated. Isolates from canola and radish were identified to V. longisporum, which produced longer conidia (7.92–12.00 µm) than Verticillium dahliae (4.32–7.04 µm). An isolate derived from mustard was characterized as V. dahliae. Molecular diagnostics with 18S rDNA, 5.8S rDNA and mating-type marker primers were used to confirm the identification of Verticillium isolates. PCR-RFLP of the mitochondrial small subunit rDNA and the cytochrome b gene were also employed to distinguish V. longisporum isolates from V. dahliae. The multi-gene characterization approach allowed for lineage determination, and V. longisporum isolates from canola and radish were in the A1/D1 group. Isolates of Verticillium longisporum from canola inoculated onto the canola cultivar ‘Westar’ caused symptoms of stem striping, stunting and short plants. Re-isolated fungal strains from infected stems were again inoculated onto canola plants, in order to confirm that V. longisporum was the causal agent of Verticillium stripe disease in the pathogenicity test.

Phylogenetic diversity and cross-inoculation of indigenous isolated Bradyrhizobium from nodules of peanut in Liaoning province of China.

Arachis hypogaea. L is a legume of economic importance, which is nodulated by Bradyrhizobium, a slow-growing bacteria. However there is no well characterization of this rhizobia in many areas of China. In the present study, cross-inoculation experiments were performed in cowpea and soybean. The isolated bacteria strains were characterized physiologyically, biochemically and identified through 16S rDNA sequence analysis showing that it belongs to Bradyrhizobium japonicum. The genetic diversity of the seventeen isolated strains were assessed through PCR-RFLP of 16S rDNA and 16S-23S rDNA IGS region. Cross inoculation test indicated that isolates could nodulate cowpea but not soybean. The cluster analysis based on physiological and biochemical characteristics showed the lower correlation between isolates and sites. The isolates were grouped into four clusters based on 16S rDNA gene sequence analysis. Thirteen polymorphisms were variable across all observations in 16S rDNA RFLP and six different IGS types from isolates. The results implies that there was some association between geographical factor and phylogenetic diversity of indigenous Bradyrhizobium isolates.

Identification of predominant epiphytic and endophytic bacterial isolates in rice seeds effective for enhancement of seed germination and plant growth

ABSTRACT-During rice growing season, the symptomless rice seeds from different paddy fields in Guilan province, Iran, were collected. After isolation of epiphytic and endophytic bacteria, 39 isolates including 19 epiphytes and 20 endophytes were selected based on the predominant characteristics. Five Operational Taxonomic Units (OTUs) were obtained based on PCR-RFLP of 16S r-DNA in these isolates. According to biochemical tests and partial sequencing of 16S-rDNA, in both epiphyte and endophyte bacteria of rice seeds, the most populated OTUs (V and II) were identified as Pantoea ananatis and Pseudomonas oryzihabitans, respectively. Six representative isolates from these two OTUs were selected to be evaluated for their abilities for rice seed germination and growth enhancement. Among them, P. oryzihabitans was not beneficially effective. However, JpB1 isolate of P. ananatis was considered to be the most effective plant growth promoting isolate, since it showed stable beneficial effects on most surveyed characteristics in both rice seed germination and growth enhancement experiments. Although, OpB3 isolate of P. ananatis produced IAA in higher amount and solubilized phosphate more than the other isolates followed by JpB1 and P. ananatis L3pB3, it was not beneficially effective on rice seed germination.

Endophytes isolated from ginger rhizome exhibit growth promoting potential for Zea mays

ABSTRACTPlant growth promoting bacteria (PGPB) are known to colonize ginger rhizome. A total of 57 strains were isolated and identified by 16S rDNA PCR-RFLP fingerprinting, and were grouped into genera Serratia, Enterobacter, Acinetobacter, Pseudomonas, Stenotrophomonas, Agrobacterium, Ochrobactrum, Bacillus and Tetrathiobacter. Out of 34 representative strains, 14 were positive for indole-3-acetic acid (IAA) production (1.02–49.66 µg ml–1), 16 were able to solubilize phosphate (9.3–233.05 µg ml–1) and 18 showed siderophore activities (9.47–70.66%). Six representative strains with plant growth promoting (PGP) activities boosted the growth of Zea mays, where not only the plant height, leaf area, and biomass yield were significantly improved, plant N, P and K uptakes were also increased. Plants co-inoculated with both PGPB and chemical fertilizer also showed an increased yield. Our study thus indicates that there is an abundance of endophytic bacteria in ginger rhizome, some of which possess promising capabilities for enhancing the growth of Z. mays and have the potential to serve as bio-fertilizers for crops.

Phytoplasmas of sesame and Orosius orientalis are genetically diverse based on 16S rDNA sequencing and PCR-RFLP in Turkey

Sesame phyllody causes significant yield losses mainly in Mediterranean region of Turkey. To detect and identify phytoplasmas of sesame and the cicadellid vector Orosius orientalis, samples were collected during 2011–2013 in Antalya. Nested PCR amplification of the 16S rDNA gave an amplification band of approximately 1246 bp corresponding to the 16Sr F2nR2 region. Identification of the phytoplasmas based on sequence homology revealed presence of the 16Sr groups II (Peanut witches’-broom, PnWB), VI (Clover proliferation, CP) and IX (Pigeon pea witches’-broom, PPWB) in sesame plants. PnWB and CP were detected in O. orientalis. Subgroups were determined by sequencing and PCR-RFLP of the F2nR2 region. 16Sr subgroups II-D, VI-A and IX-C were identified from sesame and II-D and VI-A subgroups from the insect vector. This study shows that phytoplasmas in sesame and O. orientalis are genetically diverse and to the knowledge, VI-A group phytoplasmas from sesame and O. orientalis were characterised molecularly for the first time in Turkey.

Diversity, phylogeny and pathogenicity of Botryosphaeriaceae on non-native Eucalyptus grown in an urban environment: A case study

The Botryosphaeriaceae are opportunistic pathogens mostly of woody plants, including Eucalyptus. These fungi can cause cankers and die-back diseases on non-native Eucalyptus trees in South African plantations. Botryosphaeriaceae were isolated from diseased and asymptomatic twigs and leaves from 20 Eucalyptus spp. grown in a Pretoria, South Africa arboretum and its surroundings. The isolates were initially grouped based on conidial morphology and Internal Transcribed Spacer (ITS) rDNA PCR-RFLP profiles. They were further identified using DNA sequence data for the ITS rDNA and translation elongation factor 1-α (TEF-1α) gene regions and tested for pathogenicity. Five species were identified including Botryosphaeria dothidea and four Neofusicoccum species namely Neofusicoccum parvum; N. cryptoaustrale and N. ursorum that were recently described from plant tissues collected as a part of the current study; and Neofusicoccum eucalypti (Winter) Maleme, Pavlic & Slippers comb. nov. The latter species is recorded for the first time on Eucalyptus in South Africa. Most of the identified species were collected from the leaves of 17 different Eucalyptus spp. Neofusicoccum parvum was most commonly isolated (72% of all isolates) followed by B. dothidea species complex (17%). With exception of N. parvum which was isolated from majority of Eucalyptus spp. the other species were isolated from limited number of Eucalyptus species indicating host-preferences. All the isolated Botryosphaeriaceae species produced lesions on inoculated Eucalyptus grandis plants that were significantly larger than those associated with the controls.

Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains

Novel cry genes are potential candidates for resistance management strategies, due to their different structures and modes of action. Therefore, it is desirable to clone and express novel cry genes from several new isolates of Bacillus thuringiensis (Bt). In the present study, 28 Bt strains were characterized at morphological and molecular level. All these strains are Gram positive, endospore forming and had shown different crystal morphologies when viewed under the microscope. The ARDRA (16S rDNA PCR-RFLP technique) with AluI, HaeIII, HinfI and TaqI produced unique and distinguishable restriction patterns used for the molecular characterization of these isolates. Based on UPGMA clustering analysis, Bt strains showed significant molecular diversity and the dendrogram obtained differentiated 28 Bt strains into 1 major cluster at a similarity coefficient 0.56. PCR analysis demonstrated that the Bt strains showed diverse cry gene profiles with several genes per strain. The Bt strain G3C1 showed the presence of maximum cry-type genes by PCR. The toxicological characterization of these cry genes will have huge importance in transgenic technology and will be useful in transgenesis of crop plants for better resistance management.

Microbiological quality of selected ready-to-eat leaf vegetables, sprouts and non-pasteurized fresh fruit-vegetable juices including the presence of Cronobacter spp.

Bacteria of the genus Cronobacter are emerging food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or adults with suppressed immunity. This study was aimed at determining the microbiological quality of ready-to-eat (RTE) plant-origin food products available on the Polish market with special emphasis on the prevalence of Cronobacter genus bacteria.Analyses were carried out on 60 samples of commercial RTE type plant-origin food products, including: leaf vegetables (20 samples), sprouts (20 samples) and non-pasteurized vegetable, fruit and fruit-vegetable juices (20 samples). All samples were determined for the total count of aerobic mesophilic bacteria (TAMB) and for the presence of Cronobacter spp. The isolates of Cronobacter spp. were subjected to genetic identification and differentiation by 16S rDNA sequencing, PCR-RFLP analysis and RAPD-PCR and evaluation of antibiotic susceptibility by the disk diffusion assay.The TAMB count in samples of lettuces, sprouts and non-pasteurized fruit, vegetable and fruit-vegetable juices was in the range of 5.6–7.6, 6.7–8.4 and 2.9–7.7 log CFU g−1, respectively. The presence of Cronobacter spp. was detected in 21 (35%) samples of the products, including in 6 (30%) samples of leaf vegetables (rucola, lamb’s lettuce, endive escarola and leaf vegetables mix) and in 15 (75%) samples of sprouts (alfalfa, broccoli, small radish, lentil, sunflower, leek and sprout mix). No presence of Cronobacter spp. was detected in the analyzed samples of non-pasteurized fruit, vegetable and fruit-vegetable juices. The 21 strains of Cronobacter spp. isolated from leaf vegetable and sprouts included: 13 strains of C. sakazakii, 4 strains of C. muytjensii, 2 strains of C. turicensis, one strain of C. malonaticus and one strain of C. condimenti. All isolated C. sakazakii, C. muytjensii, C. turicensis and C. malonaticus strains were sensitive to ampicillin, cefepime, chloramphenicol, gentamycin, streptomycin, tetracycline, ciprofloxacin and cotrimoxazol, whereas the C. condimenti isolate showed intermediate resistance to streptomycin and cotrimoxazole.

16S-23S ITS rDNA PCR-RFLP APPROACH AS A TOOL FOR IDENTIFICATION AND DIFFERENTIATION OF BACTERIAL SPOT- CAUSING XANTHOMONAS

Bacterial spot of pepper and tomato plants is one of the most important constraints limiting crops yield in Bulgaria and Macedonia. Therefore, early identification of the pathogen is necessary for the control and prevention of the disease. In order to explore the strength of 16S-23S ITS rDNA PCR-RFLP as an approach for identification and differentiation of the causative agents of bacterial spot 262 Bulgarian and Macedonian strains pathogenic of pepper and tomato plants were used. The strains were previously identified as Xanthomonas euvesicatoria (132 strains), Xanthomonas vesicatoria (115 strains), and Xanthomonas gardneri (15 strains). Each restriction analysis resulted in two profiles that grouped the used strains. The restriction with AluI endonuclease differentiated X. vesicatoria isolates from the other three species. The enzyme HpaII separated the X. euvesicatoria strains. A combination of three restriction endonucleases (AluI, MboI, HpaII) successfully differentiated the four species described as causative agents of bacterial spot.

Characterization of Rhizobial Bacteria Nodulating Astragalus corrugatus and Hippocrepis areolata in Tunisian Arid Soils.

Fifty seven bacterial isolates from root nodules of two spontaneous legumes (Astragalus corrugatus and Hippocrepis areolata) growing in the arid areas of Tunisia were characterized by phenotypic features, 16S rDNA PCR-RFLP and 16S rRNA gene sequencing. Phenotypically, our results indicate that A. corrugatus and H. areolata isolates showed heterogenic responses to the different phenotypic features. All isolates were acid producers, fast growers and all of them used different compounds as sole carbon and nitrogen source. The majority of isolate grew at pHs between 6 and 9, at temperatures up to 40°C and tolerated 3% NaCl concentrations. Phylogenetically, the new isolates were affiliated to four genera Sinorhizobium, Rhizobium, Mesorhizobium and Agrobacterium. About 73% of the isolates were species within the genera Sinorhizobium and Rhizobium. The isolates which failed to nodulate their host plants of origin were associated to Agrobacterium genus (three isolates).

Symbiotic effectiveness and phylogeny of rhizobia isolated from faba bean (Vicia faba L.) in Sichuan hilly areas, China

A total of 54 rhizobial strains were isolated from faba bean root nodules in 21 counties of Sichuan hilly areas in China, and their symbiotic effectiveness, genetic diversity and phylogeny were assessed. Only six strains increased the shoot dry mass of the host plant significantly (P≤0.05). Based on the cluster analysis of combined 16S rDNA and intergenic spacer region (IGS) PCR-RFLP, the strains were divided into 31 genotypes in 11 groups, indicating a high degree of genetic diversity among the strains. The sequence analysis of three housekeeping genes (atpD, glnII and recA) and 16S rDNA indicated that the strains represented two R. leguminosarum, two Rhizobium spp., R. mesosinicum, Agrobacterium sp. and A. tumefaciens. The strains representing four Rhizobium species were divided into two distinct nodC and nifH genotypes. However, the phylogeny of housekeeping genes and symbiotic genes was not congruent, implying that the strains had been shaped by vertical evolution of the housekeeping genes and lateral evolution of the symbiotic genes.

Bacterial Groups Concerned with Maturing Process in Manure Production Analyzed by a Method Based on Restriction Fragment Length Polymorphism Analysis

Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was no reliable ground. In order to find out shortening method of the maturing phase, the microorganisms concerned with a progress of the maturing was determined by using the most probable number method (MPN) and PCR-RFLP of the 16S rDNA, which was found effective to provide numbers and taxonomy of polymyxin B resistant bacterial groups in the former paper [1]. Compared to the numbers after thermophilic phase, those of Actinobacteria, δ-proteobacteria, and the other gram negative bacteria increased to 50 times, 20 times, and 105 times, respectively, after maturing phase, while those of Bacillus spp., and α and β-proteobacteria decreased to 1/10, and 1/105 after maturing phase. Numbers of the other Fumicutes, and γ- proteobacteria remained in the same revel. Actinobacteria, δ-proteobacteria, and the other gram negative bacteria might be concerned with a progress of the maturing phase, because these bacterial groups were detected and enumerated due to their proliferation ability. Although number of Acitinobacteria might be underestimated because of a PCR bias, the method was found effective for the purpose to monitor bacteria actively proliferated in culture medium.

Confronting the constraints of morphological taxonomy in the Botryosphaeriales.

Identification of fungi and the International Code of Nomenclature underpinning this process, rests strongly on the characterisation of morphological structures. Yet, the value of these characters to define species in many groups has become questionable or even superfluous. This has emerged as DNA-based techniques have increasingly revealed cryptic species and species complexes. This problem is vividly illustrated in the present study where 105 isolates of the Botryosphaeriales were recovered from both healthy and diseased woody tissues of native Acacia spp. in Namibia and South Africa. Thirteen phylogenetically distinct groups were identified based on Internal Transcribed Spacer (ITS) rDNA PCR-RFLP and translation elongation factor 1-α (TEF1-α) sequence data, two loci that are known to be reliable markers to distinguish species in the Botryosphaeriales. Four of these groups could be linked reliably to sequence data for formerly described species, including Botryosphaeria dothidea, Dothiorella dulcispinae, Lasiodiplodia pseudotheobromae and Spencermartinsia viticola. Nine groups, however, could not be linked to any other species known from culture and for which sequence data are available. These groups are, therefore, described as Aplosporella africana, A. papillata, Botryosphaeria auasmontanum, Dothiorella capri-amissi, Do. oblonga, Lasiodiplodia pyriformis, Spencermartinsia rosulata, Sphaeropsis variabilis and an undescribed Neofusicoccum sp. The species described here could not be reliably compared with the thousands of taxa described in these genera from other hosts and regions, where only morphological data are available. Such comparison would be possible only if all previously described taxa are epitypified, which is not a viable objective for the two families, Botryosphaeriaceae and Aplosporellaceae, in the Botryosphaeriales identified here. The extent of diversity of the Botryosphaeriales revealed in this and other recent studies is expected to reflect that of other undersampled regions and hosts, and illustrates the urgency to find more effective ways to describe species in this, and indeed other, groups of fungi.