Two methods for isolation of endophytic and edaphic Bacillus spp. from sugarcane fields / Dois métodos para isolamento de Bacillus spp. endofíticos e edáficos de canaviais

Bacillus has been widely studied and used for the control of pests and diseases. The adapted protocol proposed by POLANCZYK (2004) proved to be more efficient than the one by the World Health Organization (WHO, 1985) to isolate edaphic strains of Bacillus. However, it has not been assessed for isolation of endophytic strains, which are much less abundant in the nature and more difficult to be isolated. This study aimed to compare two methodological procedures for isolation of Bacillus, established by the WHO (1985) and by POLANCZYK (2004), regarding their efficiency for isolation of endophytics and edaphics Bacillus strains from inside the root tissue of sugarcane, as well as from the associated soil sample, collected from 11 locations; and to compare the density of bacteria in both environments. Endophytic and edaphic strains of Bacillus were isolated by both procedures. However, the isolation protocol performed by POLANCZYK (2004) made more efficient by having a greater number of colony forming units (CFU) per gram of soil and root indicating that this procedure is more useful, especially for isolation of endophytic strains of Bacillus, which are much less abundant in the nature than edaphic strains, being therefore more difficult to be isolated. Using the Polanczyk protocol (2004), Bacillus strains were recovered from all roots (endophytic) and soil (edaphic) samples of all the 11 fields, suggesting that the plant root may be another important source for isolation of Bacillus besides the soil. Higher densities of Bacillus were isolated from the edaphic environment compared with the endophytic environment, with significant differences when isolated by Polanczyk method (2004).(AU)

Bacillus tem sido amplamente estudado e usado para o controle de pragas e doenças. O protocolo adaptado proposto por POLANCZYK (2004) mostrou-se mais eficiente do que o da Organização Mundial de Saúde (WHO, 1985) para isolar cepas edáficas de Bacillus. No entanto, não foi avaliado quanto ao isolamento de estirpes endofíticas, que são muito menos abundantes na natureza e mais difíceis de isolar. Este estudo teve como objetivos comparar dois procedimentos metodológicos para o isolamento de Bacillus, o estabelecido pela OMS (WHO, 1985) e o de POLANCZYK (2004), quanto a sua eficiência para o isolamento de estirpes endofitas e edáficas de Bacillus originárias do interior do tecido radicular de cana-de-açúcar, bem como de amostras de solos associados, coletada de 11 locais; e comparar a densidade de bactérias em ambos os ambientes. As cepas endofíticas e edáficas de Bacillus foram isoladas por ambos os procedimentos. No entanto, o protocolo de isolamento realizado por POLANCZYK (2004) demonstrou-se mais eficiente por gerar maior número de unidades de formação de colônias (CFU) por grama de solo e raiz, indicando que esse procedimento é mais útil, especialmente para isolamento de estirpes endofíticas de Bacillus, que são muito menos abundantes na natureza do que as cepas edáficas, sendo, portanto, mais difíceis de serem isoladas. Usando o protocolo de POLANCZYK (2004), as cepas de Bacillus foram isoladas de todas as amostras de raízes (endofíticas) e de solo (edáficas) de todos os 11 campos, sugerindo que a raiz da planta pode ser outra fonte importante de isolamento de Bacillus além do solo. As densidades mais altas de Bacillus foram isoladas do ambiente edáfico em comparação com o ambiente endofítico, com diferenças significativas quando isoladas pelo método de POLANCZYK (2004).(AU)

Two methods for isolation of endophytic and edaphic Bacillus spp. from sugarcane fields / Dois métodos para isolamento de Bacillus spp. endofíticos e edáficos de canaviais

Bacillus has been widely studied and used for the control of pests and diseases. The adapted protocol proposed by POLANCZYK (2004) proved to be more efficient than the one by the World Health Organization (WHO, 1985) to isolate edaphic strains of Bacillus. However, it has not been assessed for isolation of endophytic strains, which are much less abundant in the nature and more difficult to be isolated. This study aimed to compare two methodological procedures for isolation of Bacillus, established by the WHO (1985) and by POLANCZYK (2004), regarding their efficiency for isolation of endophytics and edaphics Bacillus strains from inside the root tissue of sugarcane, as well as from the associated soil sample, collected from 11 locations; and to compare the density of bacteria in both environments. Endophytic and edaphic strains of Bacillus were isolated by both procedures. However, the isolation protocol performed by POLANCZYK (2004) made more efficient by having a greater number of colony forming units (CFU) per gram of soil and root indicating that this procedure is more useful, especially for isolation of endophytic strains of Bacillus, which are much less abundant in the nature than edaphic strains, being therefore more difficult to be isolated. Using the Polanczyk protocol (2004), Bacillus strains were recovered from all roots (endophytic) and soil (edaphic) samples of all the 11 fields, suggesting that the plant root may be another important source for isolation of Bacillus besides the soil. Higher densities of Bacillus were isolated from the edaphic environment compared with the endophytic environment, with significant differences when isolated by Polanczyk method (2004).

Bacillus tem sido amplamente estudado e usado para o controle de pragas e doenças. O protocolo adaptado proposto por POLANCZYK (2004) mostrou-se mais eficiente do que o da Organização Mundial de Saúde (WHO, 1985) para isolar cepas edáficas de Bacillus. No entanto, não foi avaliado quanto ao isolamento de estirpes endofíticas, que são muito menos abundantes na natureza e mais difíceis de isolar. Este estudo teve como objetivos comparar dois procedimentos metodológicos para o isolamento de Bacillus, o estabelecido pela OMS (WHO, 1985) e o de POLANCZYK (2004), quanto a sua eficiência para o isolamento de estirpes endofitas e edáficas de Bacillus originárias do interior do tecido radicular de cana-de-açúcar, bem como de amostras de solos associados, coletada de 11 locais; e comparar a densidade de bactérias em ambos os ambientes. As cepas endofíticas e edáficas de Bacillus foram isoladas por ambos os procedimentos. No entanto, o protocolo de isolamento realizado por POLANCZYK (2004) demonstrou-se mais eficiente por gerar maior número de unidades de formação de colônias (CFU) por grama de solo e raiz, indicando que esse procedimento é mais útil, especialmente para isolamento de estirpes endofíticas de Bacillus, que são muito menos abundantes na natureza do que as cepas edáficas, sendo, portanto, mais difíceis de serem isoladas. Usando o protocolo de POLANCZYK (2004), as cepas de Bacillus foram isoladas de todas as amostras de raízes (endofíticas) e de solo (edáficas) de todos os 11 campos, sugerindo que a raiz da planta pode ser outra fonte importante de isolamento de Bacillus além do solo. As densidades mais altas de Bacillus foram isoladas do ambiente edáfico em comparação com o ambiente endofítico, com diferenças significativas quando isoladas pelo método de POLANCZYK (2004).

Two methods for isolation of endophytic and edaphic Bacillus spp. from sugarcane fields / Dois métodos para isolamento de Bacillus spp. endofíticos e edáficos de canaviais

Bacillus has been widely studied and used for the control of pests and diseases. The adapted protocol proposed by POLANCZYK (2004) proved to be more efficient than the one by the World Health Organization (WHO, 1985) to isolate edaphic strains of Bacillus. However, it has not been assessed for isolation of endophytic strains, which are much less abundant in the nature and more difficult to be isolated. This study aimed to compare two methodological procedures for isolation of Bacillus, established by the WHO (1985) and by POLANCZYK (2004), regarding their efficiency for isolation of endophytics and edaphics Bacillus strains from inside the root tissue of sugarcane, as well as from the associated soil sample, collected from 11 locations; and to compare the density of bacteria in both environments. Endophytic and edaphic strains of Bacillus were isolated by both procedures. However, the isolation protocol performed by POLANCZYK (2004) made more efficient by having a greater number of colony forming units (CFU) per gram of soil and root indicating that this procedure is more useful, especially for isolation of endophytic strains of Bacillus, which are much less abundant in the nature than edaphic strains, being therefore more difficult to be isolated. Using the Polanczyk protocol (2004), Bacillus strains were recovered from all roots (endophytic) and soil (edaphic) samples of all the 11 fields, suggesting that the plant root may be another important source for isolation of Bacillus besides the soil. Higher densities of Bacillus were isolated from the edaphic environment compared with the endophytic environment, with significant differences when isolated by Polanczyk method (2004).(AU)

Bacillus tem sido amplamente estudado e usado para o controle de pragas e doenças. O protocolo adaptado proposto por POLANCZYK (2004) mostrou-se mais eficiente do que o da Organização Mundial de Saúde (WHO, 1985) para isolar cepas edáficas de Bacillus. No entanto, não foi avaliado quanto ao isolamento de estirpes endofíticas, que são muito menos abundantes na natureza e mais difíceis de isolar. Este estudo teve como objetivos comparar dois procedimentos metodológicos para o isolamento de Bacillus, o estabelecido pela OMS (WHO, 1985) e o de POLANCZYK (2004), quanto a sua eficiência para o isolamento de estirpes endofitas e edáficas de Bacillus originárias do interior do tecido radicular de cana-de-açúcar, bem como de amostras de solos associados, coletada de 11 locais; e comparar a densidade de bactérias em ambos os ambientes. As cepas endofíticas e edáficas de Bacillus foram isoladas por ambos os procedimentos. No entanto, o protocolo de isolamento realizado por POLANCZYK (2004) demonstrou-se mais eficiente por gerar maior número de unidades de formação de colônias (CFU) por grama de solo e raiz, indicando que esse procedimento é mais útil, especialmente para isolamento de estirpes endofíticas de Bacillus, que são muito menos abundantes na natureza do que as cepas edáficas, sendo, portanto, mais difíceis de serem isoladas. Usando o protocolo de POLANCZYK (2004), as cepas de Bacillus foram isoladas de todas as amostras de raízes (endofíticas) e de solo (edáficas) de todos os 11 campos, sugerindo que a raiz da planta pode ser outra fonte importante de isolamento de Bacillus além do solo. As densidades mais altas de Bacillus foram isoladas do ambiente edáfico em comparação com o ambiente endofítico, com diferenças significativas quando isoladas pelo método de POLANCZYK (2004).(AU)

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.(AU)

The genetic diversity of genus Bacillus and the related genera revealed by 16S rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.