New! NTSYS Pc 2.2 Free
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New! NTSYS Pc 2.2 Free
Save the ntinst22.exe file to disk into a temporary directory and run it by double clicking on it. This will start the installation process once you enter a password (the unlocking code that came with your CD). The install program will also request a registration serial number that was also provided with the CD. The 'Updates' topic in the help file describes the changes that have been made since the programs\'s initial release. Check the Frequently Asked Questions file for answers to common questions about NTSYSpc. See item 19 about the change in the location of the ntsys.ini starting with release 2.20w. The ntnew22 page lists the features of the current version. For more details, the current help file can be download here (1.4 MB).
This file is not a free upgrade from version 2.0 or earlier versions. You will need a password and an NTSYSpc version 2.1 registration serial number in order to run this program. Save the file (ntinst21.exe) to disk into a temporary directory and run it by double clicking on it. This will start the installation process once you enter a password (the unlocking code that came with your CD). The install program will also request a registration serial number that was also provided with the CD. The 'Updates' topic in the help file describes the changes that were made in this version. Download (3.4MB). Check the Frequently Asked Questions file for answers to common questions about NTSYSpc.
NTSYSpc 2.2 was available to download from the developer's website when we last checked. We cannot confirm if there is a free download of this software available. The software lies within Development Tools, more precisely Database Tools.
This PC software can be installed on 32-bit versions of Windows XP/XP Professional/Vista/7/8/10/11. This tool was originally developed by Exeter Software. The most popular versions of the tool 2.2, 2.1 and 2.0. The NTSYSpc installer is commonly called ntedit.exe or ntsys.exe etc. We recommend checking the downloaded files with any free antivirus.
With the purpose of isolating and characterizing free nitrogen fixing bacteria (FNFB) of the genus Azotobacter, soil samples were collected randomly from different vegetable organic cultures with neutral pH in different zones of Boyacá-Colombia. Isolations were done in selective free nitrogen Ashby-Sucrose agar obtaining a recovery of 40%. Twenty four isolates were evaluated for colony and cellular morphology, pigment production and metabolic activities. Molecular characterization was carried out using amplified ribosomal DNA restriction analysis (ARDRA). After digestion of 16S rDNA Y1-Y3 PCR products (1487pb) with AluI, HpaII and RsaI endonucleases, a polymorphism of 16% was obtained. Cluster analysis showed three main groups based on DNA fingerprints. Comparison between ribotypes generated by isolates and in silico restriction of 16S rDNA partial sequences with same restriction enzymes was done with Gen Workbench v.2.2.4 software. Nevertheless, Y1-Y2 PCR products were analysed using BLASTn. Isolate C5T from tomato (Lycopersicon esculentum) grown soils presented the same in silico restriction patterns with A. chroococcum (AY353708) and 99% of similarity with the same sequence. Isolate C5CO from cauliflower (Brassica oleracea var. botrytis) grown soils showed black pigmentation in Ashby-Benzoate agar and high similarity (91%) with A. nigricans (AB175651) sequence. In this work we demonstrated the utility of molecular techniques and bioinformatics tools as a support to conventional techniques in characterization of the genus Azotobacter from vegetable-grown soils.
With the purpose of isolating and characterizing free nitrogen fixing bacteria (FNFB) of the genus Azotobacter, soil samples were collected randomly from different vegetable organic cultures with neutral pH in different zones of Boyacá-Colombia. Isolations were done in selective free nitrogen Ashby-Sucrose agar obtaining a recovery of 40%. Twenty four isolates were evaluated for colony and cellular morphology, pigment production and metabolic activities. Molecular characterization was carried out using amplified ribosomal DNA restriction analysis (ARDRA). After digestion of 16S rDNA Y1-Y3 PCR products (1487pb) with AluI, HpaII and RsaI endonucleases, a polymorphism of 16% was obtained. Cluster analysis showed three main groups based on DNA fingerprints. Comparison between ribotypes generated by isolates and in silico restriction of 16S rDNA partial sequences with same restriction enzymes was done with Gen Workbench v.2.2.4 software. Nevertheless, Y1-Y2 PCR products were analysed using BLASTn. Isolate C5T from tomato (Lycopersicon esculentum) grown soils presented the same in silico restriction patterns with A. chroococcum (AY353708) and 99% of similarity with the same sequence. Isolate C5CO from cauliflower (Brassica oleracea var. botrytis) grown soils showed black pigmentation in Ashby-Benzoate agar and high similarity (91%) with A. nigricans (AB175651) sequence. In this work we demonstrated the utility of molecular techniques and bioinformatics tools as a support to conventional techniques in characterization of the genus Azotobacter from vegetable-grown soils.
Soil samples from organic broccoli (Brassica oleracea var. italica), zucchini (Cucurbita pepo), cauliflower (B. oleracea var. botrytis), spinach (Spinacia oleracea), tomato (Lycopersicon esculentum) and carrot (Daucus carota) grown soils were collected during October 2006 in Sogamoso (5º43'N, 72º55'W) and Tibasosa (5º44'N, 73º00'W) (Boyacá-Colombia) (Table 1). Five random samples each of 500 g were withdrawn from 10-15 cm depth and sieved through a 4.75 mm-mesh sieve. Soil pH was measured according to Van Lierop (38), analyzing samples fully suspended in distilled water 1:1 (v/v). The primary isolation was made using the grain soil technique (Figure 1A) according to the methodology previously described by Aquilanti et al. (1) and Becking (4) in Ashby-Sucrose agar (Agar 1.5%, Sucrose 0.5%, CaCO3 0.5%, MgSO4 0.02%, NaCl 0.02%, KH2PO4 0.02%, FeSO4 0.0005%). Plates were incubated at 28ºC for 7 days until observing sticky and glistening colonies around grains (Figure 1F). Counts were made for each cultivated soil (% Recovery = Number of grains with sticky and glistening colonies/total x 100). Isolates were purified by streaking on free nitrogen Ashby-Sucrose agar and morphological observations were done and subsequently stored at - 80ºC in nutrient broth (Scharlau, Barcelona, Spain) containing 50% glycerol. Pigment production was observed by growing the cultures in Ashby agar with 0.5% (w/v) of Benzoate (4, 22) and incubation at 28ºC for 7 days. A. vinelandii ATCC12518 and Azotobacter sp. strain (CCT) previously isolated were used for biochemical identification and molecular characterization. Biochemical identification was carried out in triplicate using different sources of carbon: glucose (GLU), maltose (MAL), mannitol (MAN), ramnose (RAM) 1% (w/v) and benzoate (BNZ) 0.5% (w/v) in phenol red broth (Merck, Germany). Oxidase (OXI), catalase (CAT) and nitrate reduction tests were done (34) and biochemical sets were incubated at 28ºC and evaluated after 24 h.
Azotobacter species are found in agricultural soils playing different beneficial roles: atmospheric nitrogen fixation, production of phytohormones, degradation of toxic compounds (7, 16) and driving the ecological balance in agro-ecosystems. For the isolation and molecular characterization of FNFB of the genus Azotobacter we sampled soils from different organic vegetable cultures located in Boyacá-Colombia, fertilized with considerable amounts of organic matter and phosphates that benefit the proliferation of FNFB as reported by Gordon (13) and Tsain et al. (37) and without nitrogen fertilizers in the rotation scheme that could inhibit the growth of Azotobacter species (3). FNFB were recovered in free nitrogen Ashby-Sucrose agar (4) and A. chrococcum in Ashby agar with mannitol or benzoate (31). Benzoate is used as a carbon source by A. chrococcum, which uses the β-ketoadipate pathway to convert it into succinate. Succinate is introduced into the respiratory chain by means of the flavine-dependent system and the production of melanines, which are related to the colony pigmentation (4, 7). In addition, some important microelements such as iron and molybdenum are necessary for nitrogen fixation (18). Carrot-grown soils presented the lowest percentage of FNFB recovery (31.3%), possibly due to its low pH (6.25), while for other soils the recovery was between 35% and 45%. Although most isolates displayed the characteristic colony morphology of the genus Azotobacter, some presented transparent, glistening, shining, 2-5mm in diameter colonies, similar to the described ones for other FNFB such as Beijerinckia and Azospirillum (Figure 1G) (17, 34), which renders colony identification based on morphology a difficult task sometimes producing ambiguous results. Isolates C5CA and C1Z formed the characteristic cysts of Azotobacter (Figure 1C). According to Hitchins and Sadoff (15) cysts can be formed by the action of the calcium ion present in the Ashby agar after the exponential phase of growth or under adverse conditions due to the lack of nutrients. These structures are characteristic of Beijerinckia although unlike as in Azotobacter, in this case they do not appear forming pairs. An important characteristic of Azotobacter is the Gram-negative bacillary morphology, with cells between 2 µm and 4 µm in diameter (Figure 1D). Some isolates presented this morphology, while others consisted of small Gram-negative bacilli but with a morphology very similar to that displayed by Azospirillum, Beijerinckia, Herbaspirillum and Derxia (Figure 1E). However, it is known that Azotobacter is a pleiomorphic microorganism (4). In general, morphological identification and characterization are not necessarily useful in defining the genus as it was the case of isolates C3BR and C5BR, which exhibited morphological characteristics similar to those reported for Azotobacter besides growing in nitrogen-free culture media but the molecular identification revealed that they do not belong to that genus. It has been reported that Azotobacter has the capacity to produce soluble pigments (1, 4), this can be a useful tool in the characterization of some Azotobacter species. Brown or black pigmentation in the Ashby-Benzoate agar allowed in this study the differentiation between A. chroococcum and A. nigricans (22). Isolates C1CO, C5E, C1T, C3T and C5T exhibited morphological traits and similar pigmentation to those displayed by A. chroococcum and A. nigricans (Figure 1H and Table 1). On the another side, isolates C3CA and C4E presented colonies with brown-yellow pigmentation, Gram-negative bacilli, and catalase and oxidase negative tests, suggesting that they can be aerobic FNFB that do not belong to the genus Azotobacter. In the biochemical identification, tests allowing the differentiation between genera and species were used. An example is that A. vinelandii is the only species capable of assimilating ramnose and using it as carbon source. In addition, all Azotobacter species have the capacity to produce oxidases and catalases for the protection of their nitrogenase. Universal primers (Y1-Y3) have been used in FNFB 16S rDNA amplification (2, 17, 20) although not specific for the genus Azotobacter. FNFB characterization can be done based on several different genes coding for many functions, as is the case of the gene nif that codifies the nitrogenase enzyme which drives the atmospheric nitrogen fixation (21, 26), in this work a partial sequence of nifH gene was amplified in several isolates including in the tree groups of ARDRA (data not shown). Nevertheless, both the 16S rDNA characteristics and its vertical transmission render this gene as an excellent molecular marker (23, 39). ARDRA is a molecular technique that allows establishing phylogenetic relationships between individuals. The enzymes used must generate high levels of polymorphism for specific groups to facilitate the detection of intra or inter-specific significant differences (14). The comparison between ARDRA and in silico analysis revealed that ARDRA is highly useful in establishing phylogenetic relationships between isolates and in clarifying the taxonomy. In general, the percentages of polymorphism of enzymes used in this work were relatively high, 16.5% on average, and similar to those reported by Aquilanti et al. (2) who obtained polymorphisms of 22%, 23% and 16% for AluI, HpaII and RsaI, respectively. A directly proportional relationship was demonstrated between the percentages of polymorphism and the ribotypes produced. UPGMA showed three main groups of similarity (Figure 3). We noted that group I isolates are found only in crop-grown soils located in Sogamoso, whereas group II and III isolates occur in crop-grown soils in both localities. Based solely on soil characteristics and the grown crops, clustering may differ from that one obtained with ARDRA. On the other hand, there was no relationship between the groups and the pH of crop-grown soils, although most isolates of group II were recovered in pH with values between 7.06 and 7.44. C5T and C1Z always showed irregular patterns for each enzyme and isolate C5E displayed a unique band with RsaI (Figu re 2); these unique bands are particularly important since they can be useful for designing specific primers. When comparing the patterns generated in ARDRA with those obtained from the in silico restriction, a great consistency between number of bands and their corresponding molecular weights was observed in some cases. In other cases, this consistency results only for one or two enzymes. Inconsistencies could be due to the fact that the lengths of some of the GenBank 16S rDNA sequences do not fully match with the lengths of the sequences of our isolates, probably because the primers used differed from ours. By this reason the molecular weights of the bands could differ by a few base pairs, especially in fragments of the flanking regions of the gene, considering as similar those bands that differed by 15-25 bp. On the other hand, in some isolates the presence of bands of greater molecular weights was observed after the digestion, suggesting that some copies of the gene were not completely digested. Isolates of group I of the ARDRA (Figure 3) showed restriction patterns similar to those obtained from the sequences of Azotobacter species used in the in silico restriction, whereas the majority of isolates of group II did not show this similarity. With respect to isolates of group III, a special finding was isolate C5T that produced patterns identical to A. chroococcum (accession number AY353708). In general, the BLASTn analysis carried out with partial sequences of the 16S rDNA gene obtained from every isolate revealed for group I similarity values between 74% and 84% with sequences of A. vinelandii; isolate C5CA showed a similarity of 78% (EF620439). Strain C5CO showed a similarity of 91% with A. nigricans, confirming its preliminary identification. Regarding isolates of group III from ARDRA, isolate C5T showed a similarity of 99% with A. chroococcum (AY353708). In conclusion, from the 15 isolates obtained from vegetable-grown fields, 14 exhibited phenotypical similarity with species of the genus Azotobacter and 13 showed restriction patterns similar to the ones obtained with the sequences of Azotobacter in the in silico restriction. Isolate C5T obtained from a tomato-grown field (Lycopersicon esculentum) could be identified as A. chroococcum. It is important to note that the short length of the sequences analyzed with BLASTn did not suffice to obtain a clarifying view of the taxonomic position of the majority of isolates. However, ARDRA proved to be a molecular tool that allows the identification of interspecific ribotypes of FNFB using a few polymorphic enzymes. The analysis of diversity is an important factor in the measurement of quality of agricultural soils. On the other hand, it is necessary to design species-specific primers for the genus Azotobacter because of its large importance as atmospheric nitrogen fixing and plant growth promoting rhizobacteria. 153554b96e
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