МИКРОБИОЛОГИЯ, 2013, том 82, № 1, с. 106-114
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
MICROWAVE IRRADIATION IS A USEFUL TOOL FOR IMPROVING ISOLATION OF ACTINOMYCETES FROM SOIL
© 2013 r. D. S. Wang", Q. H. Xue", W. J. Zhu", J. Zhao4, J. L. Duan", and G. H. Shenb
aCollege of Resource and Environment, Northwest A&F University, Yangling, Shaanxi, 712100 China bCollege of Life Science, Northwest A&F University, Yangling, Shaanxi, 712100 China
Received February 23, 2012
Abstract — Actinomycetes are an important source of novel, biologically active compounds. New methods need to be developed for isolating previously unknown actinomycetes from soil. The objective of this experiment was to study microwave irradiation of soil as a means for isolating previously unknown actinomycetes. Soil samples were collected at ten elevations between 800 and 3670 m on Taibai Mountain, Shaanxi Province, China. Moistened soil samples were irradiated at 120 W heating power (2450 MHz) for 3 min using a household microwave oven. Irradiation increased total actinomycete, streptomycete, and antagonistic actinomycete counts on three types of culture media. Irradiation also increased the number of culturable actinomycete isolates. Some actinomycete isolates were culturable only after the soil was irradiated, whereas other isolates could not be cultured after irradiation. Irradiation of soil from elevations >3000 m increased actinomycete counts significantly but had little effect on the number of culturable actinomycete isolates. In contrast, irradiation of samples from elevations <3000 m had relatively little effect on actinomycete counts, but significantly increased the number of culturable actinomycete isolates. We used 16S rDNA sequence analysis to identify 14 actinomycete isolates that were only culturable after irradiation. Microwave irradiation of soil was helpful for isolating Streptomyces spp., Nocardia spp., Streptosporangium spp., and Lentzea spp. Slightly more than 90% of the identified actinomycete species were biologically active. In conclusion, microwave irradiation is a useful tool for isolating biologically active actinomycetes from soil.
Keywords: Gause's synthetic medium, antagonistic actinomycetes, Taibai Mountain, 16S rDNA.
DOI: 10.7868/S0026365612060183
It is estimated that about 45% of all biologically active microbial metabolites are produced by actinomycetes [1]. Many actinomycete species that produce biologically active compounds have been isolated from soil. However, more than 90% of soil microorganisms can not be cultured using current isolation methods and techniques [2]. Scientists are searching for improved methods for isolating actinomycetes from soil, including new methods of treating the soil, new ways of inhibiting the growth of unwanted microorganisms, and new types of culture media [3—7]. As a result of these efforts, it has been reported that the addition of yeast extract to soil can activate actinomycetes spores [8]. One research group selectively isolated Micromonospora spp., Dactylosporangium spp., Micro-bispora spp., Microtetraspora spp., Actinomadura spp., and Streptosporangium spp. by adding 0.05% SDS and 6% yeast extract to a soil suspension and then shaking at 40 °C for 20 min [9]. Another research group found that treating soil with calcium carbonate increased the number of colony forming units (CFU) of Actinokine-ospora spp. [4]. In terms of physical treatments, heat-
1 Corresponding author; e-mail: xuequanhong@nwsuaf.edu.cn
ing the soil helped with the isolation of rare actino-mycetes [10] and ultrasonication increased the cultur-ability of some actinomycete species but decreased the culturability of others [11].
Microwaves are part of the electromagnetic spectrum, ranging in frequency from 300 million cycles per second (300 MHz, X = 1000 mm) to 300 billion cycles per second (300 GHz, X = 1 mm). Many studies have examined the use of microwaves for sterilization of soil. For example, Ferriss [12] reported that microwave irradiation of soil reduced total fungal and total prokaryote counts in soil extracts. The same author found that the effects of microwave irradiation increased with treatment time, decreased with the amount of soil, and decreased as the soil water content increased from 16 to 37% (w w-1). There are few reports about the effect of microwave irradiation on the culturability of microorganisms, and especially the culturability of actinomycetes [13-15]. Bulina et al. [13] reported that microwave irradiation significantly increased the number of culturable rare actinomycete taxa in soil, including Micromonospora, Micropolyspora, Norcardia, and Actinomadura. Yang et al. [14] re-
Table 1. Selected chemical properties of the soil samples
Sample Elevation (m) Soil type Organic matter (g kg 1) pH CaCO3(g kg-1)
1 800 Alpine cinnamonic soil 24.4 7.24 8.23
2 1200 Alpine cinnamonic soil 27.7 6.73 5.96
3 1850 Alpine brown soil 57.9 6.58 1.84
4 2270 Alpine brown soil 40.6 5.76 0.98
5 3490 Paramo soil 32.5 5.7 0.55
6 3530 Paramo soil 72.0 5.57 0.72
7 3600 Paramo soil 40.8 5.86 0.47
8 3640 Paramo soil 50.5 6.29 0.52
9 3660 Paramo soil 32.0 6.34 1.03
10 3670 Paramo soil 42.6 6.61 0.82
ported that short periods of microwave irradiation increased culturable actinomycete counts and the number of culturable actinomycete isolates in a sandy aeolian soil. The same author also found that irradiation increased the number of antagonistic actinomycete isolates as a percentage of the total number of cultural actinomycete isolates. Xue et al. [15] reported that microwave irradiation of a calcareous soil increased the total counts of culturable actinomycetes, Streptomyces sppv and Micromonospora spp. in soil extracts. The increases were greater when moist soil was irradiated, rather than dry soil. These studies demonstrated that microwave irradiation can increase the total counts and isolates number of culturable actino-mycetes in sandy aeolian or calcareous soils, however the novel actinomycete isolates isolated from microwave irradiated soil were not identified.
The objective of this research was to investigate the use of microwave irradiation as a possible means for isolating previously unknown actinomycete species from soil. We compared the actinomycetes population, isolates number, and the number of antagonistic actinomycete isolates in irradiated and non-irradiated soil samples. Isolates which were unique to irradiated samples were identified by 16S rDNA sequence analysis.
MATERIAL AND METHODS
Soil sampling. Soil samples (5—20 cm depth) were collected at ten elevations on the north side of Taibai Mountain, Shaanxi Province, China (33°57'~34°58' N, 107°45'~107°53' E) (Table 1). The samples were placed in sterile polyethylene bags, sealed, and stored in the dark at 4°C until use.
Soil chemical analyses. Soil organic matter was measured by the Walkley and Black method. Calcium carbonate was determined by the vacuum-gasometric method. Soil pH was measured with a pH meter (Leici PHS-3D, Shanghai, China) using a soil : water ratio of 1 : 2.5. Selected soil properties are shown in Table 1.
Soil sample pretreatment. Soil samples were air-dried for two weeks and then passed through a 1 mm sieve to remove gravel and large organic material. Soil samples (5.0 g) were put into 10 mL centrifuge tubes and then moistened with 2 mL sterile water to help absorb microwave irradiation. The tubes with moistened samples were put into a 1000 mL breaker with 900 mL room-temperature water to reduce heating. The breaker was placed in the center of a 2450 MHz microwave oven (Galanz P7021TP-6, Guangdong, China) and irradiated at 120 W power for 3 min. Change in the water temperature in the breaker was monitored with a mercury thermometer. The change was less that 1°C. These soil samples will be referred to as the microwave irradiated samples. Non-irradiated soil samples were the controls.
Isolation. We used the soil dilution plate technique to isolate actinomycetes from the soil samples. Serial dilutions were prepared by adding 5.0 g of irradiated or non-irradiated soil to 45.0 mL sterile distilled water (10-1), followed by shaking and further dilution to 10-5. Three agar media were tested: Gause's synthetic agar (G) (Gause et al., 1983), Gause's synthetic calcium agar (5 g CaCl2 added to 1000 mL Gause's synthetic agar, GCa), and Gause's synthetic nutrient-poor agar (Gause's synthetic agar at one tenth the recommended concentration, GP). All media were supplemented with potassium dichromate (80 mg L-1) to inhibit the
Table 2. Soil actinomycete population (x104 CFU g 1 dry soil) on Gause's synthetic agar
Soil Microbe type Control Irradiated samples Soil Microbe type Control Irradiated samples
Total 98.8 ± 27.4 147.0 ± 29.4 Total 3.4 ± 0.4 11.9 ± 1.5*
1 Strepa 48.1 ± 2.2 56.0 ± 21.6 6 Strep 1.4 ± 0.3 4.6 ± 0.6*
Other 50.7 ± 29.5 91.0 ± 29.5 Other 2.0 ± 0.4 7.4 ± 1.1*
Total 214.0 ± 38.1 259.0 ± 12.8 Total 5.4 ± 0.3 11.9 ± 1.5*
2 Strep 92.5 ± 24.7 105.0 ± 21.8 7 Strep 2.4 ± 0.2 5.1 ± 0.7*
Other 121.6 ± 53.2 154.0 ± 32.6 Other 3.0 ± 0.4 6.9 ± 1.1*
Total 277.4 ± 24.9 366.8 ± 35.7*b Total 16.1 ± 1.3 19.4 ± 1.8
3 Strep 73.5 ± 11.6 100.8 ± 25.5 8 Strep 8.3 ± 1.0 8.9 ± 1.1
Other 203.9 ± 36.5 266.0 ± 10.6* Other 7.8 ± 0.3 10.5 ± 1.3
Total 45.6 ± 23.1 134.4 ± 80.1 Total 1.3 ± 0.4 3.4 ± 0.4*
4 Strep 19.0 ± 65.8 64.4 ± 35.7 9 Strep 0.3 ± 0.1 0.6 ± 0.1
Other 26.6 ± 21.2 70.0 ± 46.5 Other 1.0 ± 0.3 2.8 ± 0.5*
Total 1.6 ± 0.4 2.1 ± 0.7 Total 82.3 ± 24.7 243.6 ± 70.7*
5 Strep 0.1 ± 0.02 0.3 ± 0.06* 10 Strep 27.9 ± 9.6 81.2 ± 25.7*
Other 1.5 ± 0.5 1.8 ± 0.6 Other 54.5 ± 22.3 162.4 ± 45.3*
a Total, total actinomycetes; Strep, streptomycetes; Other, total actinomycetes minus streptomycetes. b * Significant difference at P < 0.05.
growth of bacteria and fungi. All plates were incubated at 28°C for 15 days. Colonies were identified by their cultural and morphological characteristics, and by microscopi
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.