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Abstract [Objectives] This study was conducted to investigate the effects of artificially cultured Eisenia foetida and wild earthworms （Pheretima sp.） on the changes of microbial populations in forest soil in different seasons.
[Methods]Artificially cultured E. foetida and wild earthworms were introduced to forest soil， and changes in the number of microbial populations under earthworm microbe interaction were investigated in different seasons by tracking observation. The effects of earthworm activities on the number of different microbial populations were discussed also.
[Results] Different microbial populations all increased in the soil to different degrees after the introduction of earthworms. The effects of the wild earthworms on the changes in bacteria， actinomycetes and fungi in the soil were different from those of artificially cultivated earthworms to certain degrees.
[Conclusions] This study provides a theoretical basis for organic agriculture that relies on the decomposition of organic matter to release nutrients.
Key words Earthworm; Forest land; Microorganisms; Season
Earthworms play an important role in soil ecosystems. They can participate in the decomposition of organic matter， mineralization of effective nutrients， and the formation and maintenance of stable soil aggregates and pore structures， thereby influencing plant growth， which has been confirmed by a large number of studies[1-2]. In the study of the number of soil microbial populations under earthworm microbe interaction， as reported in Parle in 1963， the number of bacteria and actinomycetes increased significantly in the soil after passing through the intestinal canals of earthworms， and the number of fungi remained unchanged[3]. There are also various discussions and controversies in the conclusion that the effects of earthworms on soil microbes have great significance[4]. Although there are many factors affecting the composition of microorganisms in the soil and the changes in the number of microorganisms in the soil， activities of other animals in the soil are also a cause of the changes in the number and composition of microorganisms in the soil. However， in the organisms present in the soil， earthworms are a typical animal that can affect the changes of microbial populations in the soil. The main reason is that， through the digestive system of earthworms， under the action of protease， lipase， cellulase and amylase， the organic matter in the soil can be fully decomposed and transformed to materials that are directly and indirectly used by themselves or other organisms， and the manure organic fertilizer produced by the organisms own activities can promote the formation of soil aggregate structure， resulting in improved oil permeability， water retaining property and fertility retaining capacity， which is conducive to the reproduction and increase of microorganisms， and helps to improve the capacity of the soil to absorb nutrients and store nutrients[5]. Because the composition of microorganisms in the soil and the number of microorganisms are related to soil fertility[6]， soil types and environmental conditions have a greater impact on the growth and reproduction of soil microorganisms. Different soil types and different land cultivation methods cause certain differences in soil microorganism populations and number[7]. The fresh earthworm manure contains high microbial biomass. The interaction between earthworms and microorganisms can regulate the composition of soil microflora and change the function of soil ecosystem. The activities of earthworms can change the composition and quantity of microorganisms. Microbes have a fundamental role in the transformation of soil nitrogen， thereby promoting the mineralization of nitrogen[8]. Therefore， the study of the variation of soil microbial populations under earthworm microbe interaction not only can analyze the physical and chemical properties of soil and the effects on microbial populations in the soil， but also can provide a basis for the improvement of soil nutrients and the restoration of soil ecological environment. At the same time， it has a more realistic significance in the improvement of soil fertility. At present， there are few studies on the changes of soil microbial populations under earthworm microbe interaction， especially under long term interaction. Many studies have carried out research on soil microbial biomass under relatively short term interaction[9]. In addition， the differences in soil texture and cultivation and farming methods also lead to certain differences in the number of soil microbial populations; and the amounts and types of applied fertilizers and soil temperature in different cultivating periods may also cause changes in soil microbial populations and number. Due to the lack of sufficient research on the relationship between the activities of earthworms and the changes of soil microbial populations and quantities， in this study， artificial earthworms and wild earthworms were introduced into forest soil， respectively， followed by the observation of the changes in the number of microbial populations in three seasons， and the effects of the artificially cultivated earthworms and the wild earthworms on the numbers of microbial populations in the forest soil through earthworm microbe interaction were analyzed in different seasons， respectively. Materials and Methods
Tested materials
In this experiment， the tracking observation on the changes in the number of microbial populations in the soil was carried out in the Wanchun Plantation in Wenjiang District， Chengdu. The used earthworms were wild earthworms （Pheretima sp.） collected near the plantation and cultured Eisenia foetida provided by the Chengdu Earthworm Farm.
Methods
Experimental design
The exposed soil layer （0-25 cm） was collected in a multi point manner and mixed uniformly[10-11]. In the open soil of the plantation， a plastic film was used to form a 1.1 m ×1.0 m×0.5 m earthworm culture tank （bottomless）， and 40 cm thick open soil was filled in the tanks. Six individuals of each of the two varieties with basically the same size and weight were selected for culture. The reason for culturing six earthworms is that it was found from the investigation of the earthworm distribution in the plantation that the average number of earthworms in the volume of 1.1 m×1.0 m×0.5 m was 6， though the earthworms differ in body shape and weight. In order to prevent earthworms from escaping， plastic spacers 30 cm high were buried around each culture bank. Moreover， in order to prevent the culture tanks from being wetting by rainwater， a corresponding anti proof canopy was built over each culture tank.
Into culture tank I and culture tank II， Pheretima sp. and E. foetida were input， respectively. After 15 d of culture， the soils were collected and mixed in the laboratory， and analyzed repeatedly for 3 times according to the above sample microbiological analysis method.
Sample collection
The experiment was conducted from April 2018 to October 2018. In culture tanks I and II， the soils were collected in the multi point manner and mixed. After mixing， about 1.2 kg of the samples were weighed， and further analyzed and identified in the laboratory[14].
Sample analysis
The soil microbial quantity analysis was carried out by the plate dilution method， using beef extract peptone medium for culturing bacteria， Gauzes synthetic medium No.1 for culturing actinomycetes， and Martins medium for culturing fungi[12]. Soil nutrients were determined according to conventional analytical methods[13].
According to Table 3 and Table 4， the effects of artificially cultivated E. foetida and the wild earthworms on the number of soil bacteria， actinomycetes and fungi were compared， and the number of microbial populations differed to certain degrees in the soil in different seasons. From the respect of the effect on the number of bacteria in the soil， in April （spring）， July （summer） and October （autumn）， the activities of artificially raised E. foetida increased the number of bacteria in the soil by 26.5%， 24.5% and 18.7%， respectively; the number of actinomycetes in the soil increased by 19.7%， 26.1% and 13.6%， respectively; and the number of fungi in the soil increased by 20.6%， 18.9% and 19.6% ， respectively. In the soil cultured with the wild earthworms， the number of bacteria in the soil increased by 20.7%， 25.0% and 14.2%， respectively; the number of actinomycetes in the soil increased by 20.8%， 18.0% and 7.5%， respectively; and the number of fungi in the soil increased by 22.3%， 20.2% and 22.6%. This suggests that the effects of artificially introduced wild earthworms on the changes in bacteria， actinomycetes and fungi in the soil are different from those of artificially cultivated earthworms. Specifically， in the soil introduced with the artificially cultivated E. foetida， the number of bacteria and fungi increased significantly in the spring soil; the number of actinomycetes increased significantly in the summer soil; and fungi increased significantly in the autumn soil. In the soil introduced with wild earthworms， the quantity of fungi increased significantly in the spring soil; in summer， the number of soil bacteria and actinomycetes increased significantly; and fungi increased significantly in the autumn soil. However， there were some significant differences in the number of soil microbial populations cultured with artificially cultivated E. foetida and wild earthworms. Yingsen HE et al. Effects of Pheretima sp. and Eisenia foetida on Changes in Microbial Populations of Forest Soil in Different Seasons
Discussion
Earthworms are particularly important for the promotion of soil microbial activity and the improvement of the decomposition and utilization of organic matter and crop growth， especially for organic agriculture that relies on the decomposition of organic matter to release nutrients[15]. The effect of earthworms on the soil is mainly manifested by the interaction with microorganisms， because earthworms could influence the number， activity and composition of soil microbial populations by improving microhabitats （defecation， home building， agitation）， increasing the surface area of organic matter， feeding directly and carrying microorganisms. Therefore， earthworm microbe interaction may be a relatively important issue in soil ecology research. The experiment on the effects of earthworm activities on the number of soil microbial populations has preliminarily proved that the number of bacteria， actinomycetes and fungi in the soil after the introduction of earthworms increased significantly， and the increases in different seasons were different. Three systems that can significantly increase the number of bacteria ， actinomycetes and fungi in the soil may be responsible for the increases in the number of bacteria， actinomycetes and fungi in different seasons. Firstly， the channels formed by earthworm themselves and the granulated manure improve the ventilation， water retention and drainage conditions of the soil， making the organic matter more easily decomposed by microorganisms， thereby promoting the growth and reproduction of microorganisms. Secondly， the secretions on the surface of the earthworm body are rich in amino acids， polysaccharides and biological enzymes， which constitute the nutrient source of soil microorganisms， which promotes the growth and reproduction of soil microorganisms to some extent. Thirdly， the digestive tract of earthworms is also a source of certain microorganisms， and the feces of earthworms are rich in microorganisms such as bacteria， actinomycetes and fungi. Due to the presence of these three systems， the number of microorganisms can be significantly increased. However， how these three systems of earthworm microbe interaction work together still needs further study.
References
[1] BRUSSAARD L. On the mechanisms of interactions between earthworms and plants[J]. Pedobiologia， 1999， 43： 880-885. [2] BROWN GG， BARIOS I， LAVELLE P. Regulation of soil organic matter dynamics and microbial activity in the drilosphere and the role of interactions with other edaphic functional domains[J]. European Journal of Soil Biology， 2000， 36： 177-198.
[3] PARLR J. A microbiological study of earthworm casts[J]. J. General Mirobiol.， 1963， 31： 13-22.
[4] ZHANG BG. Interaction between earthworms and microorganisms[J]. Acta Ecologica Sinica， 1997， 17（5）： 556-560. （in Chinese）
[5] LI HX， HU F， SHEN QR， et al. Effect of earthworm inoculation on soil carbon and nitrogen dynamics and on crop yield with application of corn residues[J]. Chinese Journal of Applied Ecology？，2002，13（12）： 1636-1641. （in Chinese）
[6] HUANG FZ， ZHANG YZ， YANG FR. Soil improvement and comprehensive utilization by earthworms[M]. Chongqing： Chongqing Publishing House， 1984： 28-36. （in Chinese）
[7] ZHANG JE， LIU WG， HU G， et al. The relationship between quantity index of soil microorganisms and soil fertility of different land use systems[J]. Soil and Environment，2002，11（2）：140-143. （in Chinese）
[8] ARAUJO Y， LUIZAO F， BARROS E. Effects of earthworm addition on soil nitrogen availability，microbial biomass and litter decomposition in mesocosms[J]. Biology and Ferlility of soils， 2004， 39： 146-152.
[9] ZHANG BG， LI GT， SHEN TS. Influence of the earthworm Pheretima guillelmi on soil microbial biomass and activity[J]. Acta Ecologica Sinica， 2000， 20（1）：168-172. （in Chinese）
[10] CHEN WX. Soil and environmental microbiology[M]. Beijing： Beijing Agricultural University Press， 1990. （in Chinese）
[11] LI FD. Agricultural microbiology experimental techniques[M]. Beijing： Agriculture Press， 1996. （in Chinese）
[12] Microbiology Department， Institute of Soil Science， Chinese Academy of Sciences. Soil microbial research[M]. Beijing： Science Press， 1985. （in Chinese）
[13] LU RK. Soil agricultural chemical analysis methods[M]. Beijing： China Agricultural Science and Technology Press， 1999. （in Chinese）
[14] XIANG WS， WU JS， XIAO HA. Advances in extraction and purification of soil microorganism[J]. Chinese Journal of Applied Ecology， 2003， 14（3）： 453-456. （in Chinese）
[15] WOLTERS V. Invertebrate control of soil organic matter stability[J]. Biology and Fertility of Soils， 2000， 31： 1.
[Methods]Artificially cultured E. foetida and wild earthworms were introduced to forest soil， and changes in the number of microbial populations under earthworm microbe interaction were investigated in different seasons by tracking observation. The effects of earthworm activities on the number of different microbial populations were discussed also.
[Results] Different microbial populations all increased in the soil to different degrees after the introduction of earthworms. The effects of the wild earthworms on the changes in bacteria， actinomycetes and fungi in the soil were different from those of artificially cultivated earthworms to certain degrees.
[Conclusions] This study provides a theoretical basis for organic agriculture that relies on the decomposition of organic matter to release nutrients.
Key words Earthworm; Forest land; Microorganisms; Season
Earthworms play an important role in soil ecosystems. They can participate in the decomposition of organic matter， mineralization of effective nutrients， and the formation and maintenance of stable soil aggregates and pore structures， thereby influencing plant growth， which has been confirmed by a large number of studies[1-2]. In the study of the number of soil microbial populations under earthworm microbe interaction， as reported in Parle in 1963， the number of bacteria and actinomycetes increased significantly in the soil after passing through the intestinal canals of earthworms， and the number of fungi remained unchanged[3]. There are also various discussions and controversies in the conclusion that the effects of earthworms on soil microbes have great significance[4]. Although there are many factors affecting the composition of microorganisms in the soil and the changes in the number of microorganisms in the soil， activities of other animals in the soil are also a cause of the changes in the number and composition of microorganisms in the soil. However， in the organisms present in the soil， earthworms are a typical animal that can affect the changes of microbial populations in the soil. The main reason is that， through the digestive system of earthworms， under the action of protease， lipase， cellulase and amylase， the organic matter in the soil can be fully decomposed and transformed to materials that are directly and indirectly used by themselves or other organisms， and the manure organic fertilizer produced by the organisms own activities can promote the formation of soil aggregate structure， resulting in improved oil permeability， water retaining property and fertility retaining capacity， which is conducive to the reproduction and increase of microorganisms， and helps to improve the capacity of the soil to absorb nutrients and store nutrients[5]. Because the composition of microorganisms in the soil and the number of microorganisms are related to soil fertility[6]， soil types and environmental conditions have a greater impact on the growth and reproduction of soil microorganisms. Different soil types and different land cultivation methods cause certain differences in soil microorganism populations and number[7]. The fresh earthworm manure contains high microbial biomass. The interaction between earthworms and microorganisms can regulate the composition of soil microflora and change the function of soil ecosystem. The activities of earthworms can change the composition and quantity of microorganisms. Microbes have a fundamental role in the transformation of soil nitrogen， thereby promoting the mineralization of nitrogen[8]. Therefore， the study of the variation of soil microbial populations under earthworm microbe interaction not only can analyze the physical and chemical properties of soil and the effects on microbial populations in the soil， but also can provide a basis for the improvement of soil nutrients and the restoration of soil ecological environment. At the same time， it has a more realistic significance in the improvement of soil fertility. At present， there are few studies on the changes of soil microbial populations under earthworm microbe interaction， especially under long term interaction. Many studies have carried out research on soil microbial biomass under relatively short term interaction[9]. In addition， the differences in soil texture and cultivation and farming methods also lead to certain differences in the number of soil microbial populations; and the amounts and types of applied fertilizers and soil temperature in different cultivating periods may also cause changes in soil microbial populations and number. Due to the lack of sufficient research on the relationship between the activities of earthworms and the changes of soil microbial populations and quantities， in this study， artificial earthworms and wild earthworms were introduced into forest soil， respectively， followed by the observation of the changes in the number of microbial populations in three seasons， and the effects of the artificially cultivated earthworms and the wild earthworms on the numbers of microbial populations in the forest soil through earthworm microbe interaction were analyzed in different seasons， respectively. Materials and Methods
Tested materials
In this experiment， the tracking observation on the changes in the number of microbial populations in the soil was carried out in the Wanchun Plantation in Wenjiang District， Chengdu. The used earthworms were wild earthworms （Pheretima sp.） collected near the plantation and cultured Eisenia foetida provided by the Chengdu Earthworm Farm.
Methods
Experimental design
The exposed soil layer （0-25 cm） was collected in a multi point manner and mixed uniformly[10-11]. In the open soil of the plantation， a plastic film was used to form a 1.1 m ×1.0 m×0.5 m earthworm culture tank （bottomless）， and 40 cm thick open soil was filled in the tanks. Six individuals of each of the two varieties with basically the same size and weight were selected for culture. The reason for culturing six earthworms is that it was found from the investigation of the earthworm distribution in the plantation that the average number of earthworms in the volume of 1.1 m×1.0 m×0.5 m was 6， though the earthworms differ in body shape and weight. In order to prevent earthworms from escaping， plastic spacers 30 cm high were buried around each culture bank. Moreover， in order to prevent the culture tanks from being wetting by rainwater， a corresponding anti proof canopy was built over each culture tank.
Into culture tank I and culture tank II， Pheretima sp. and E. foetida were input， respectively. After 15 d of culture， the soils were collected and mixed in the laboratory， and analyzed repeatedly for 3 times according to the above sample microbiological analysis method.
Sample collection
The experiment was conducted from April 2018 to October 2018. In culture tanks I and II， the soils were collected in the multi point manner and mixed. After mixing， about 1.2 kg of the samples were weighed， and further analyzed and identified in the laboratory[14].
Sample analysis
The soil microbial quantity analysis was carried out by the plate dilution method， using beef extract peptone medium for culturing bacteria， Gauzes synthetic medium No.1 for culturing actinomycetes， and Martins medium for culturing fungi[12]. Soil nutrients were determined according to conventional analytical methods[13].
According to Table 3 and Table 4， the effects of artificially cultivated E. foetida and the wild earthworms on the number of soil bacteria， actinomycetes and fungi were compared， and the number of microbial populations differed to certain degrees in the soil in different seasons. From the respect of the effect on the number of bacteria in the soil， in April （spring）， July （summer） and October （autumn）， the activities of artificially raised E. foetida increased the number of bacteria in the soil by 26.5%， 24.5% and 18.7%， respectively; the number of actinomycetes in the soil increased by 19.7%， 26.1% and 13.6%， respectively; and the number of fungi in the soil increased by 20.6%， 18.9% and 19.6% ， respectively. In the soil cultured with the wild earthworms， the number of bacteria in the soil increased by 20.7%， 25.0% and 14.2%， respectively; the number of actinomycetes in the soil increased by 20.8%， 18.0% and 7.5%， respectively; and the number of fungi in the soil increased by 22.3%， 20.2% and 22.6%. This suggests that the effects of artificially introduced wild earthworms on the changes in bacteria， actinomycetes and fungi in the soil are different from those of artificially cultivated earthworms. Specifically， in the soil introduced with the artificially cultivated E. foetida， the number of bacteria and fungi increased significantly in the spring soil; the number of actinomycetes increased significantly in the summer soil; and fungi increased significantly in the autumn soil. In the soil introduced with wild earthworms， the quantity of fungi increased significantly in the spring soil; in summer， the number of soil bacteria and actinomycetes increased significantly; and fungi increased significantly in the autumn soil. However， there were some significant differences in the number of soil microbial populations cultured with artificially cultivated E. foetida and wild earthworms. Yingsen HE et al. Effects of Pheretima sp. and Eisenia foetida on Changes in Microbial Populations of Forest Soil in Different Seasons
Discussion
Earthworms are particularly important for the promotion of soil microbial activity and the improvement of the decomposition and utilization of organic matter and crop growth， especially for organic agriculture that relies on the decomposition of organic matter to release nutrients[15]. The effect of earthworms on the soil is mainly manifested by the interaction with microorganisms， because earthworms could influence the number， activity and composition of soil microbial populations by improving microhabitats （defecation， home building， agitation）， increasing the surface area of organic matter， feeding directly and carrying microorganisms. Therefore， earthworm microbe interaction may be a relatively important issue in soil ecology research. The experiment on the effects of earthworm activities on the number of soil microbial populations has preliminarily proved that the number of bacteria， actinomycetes and fungi in the soil after the introduction of earthworms increased significantly， and the increases in different seasons were different. Three systems that can significantly increase the number of bacteria ， actinomycetes and fungi in the soil may be responsible for the increases in the number of bacteria， actinomycetes and fungi in different seasons. Firstly， the channels formed by earthworm themselves and the granulated manure improve the ventilation， water retention and drainage conditions of the soil， making the organic matter more easily decomposed by microorganisms， thereby promoting the growth and reproduction of microorganisms. Secondly， the secretions on the surface of the earthworm body are rich in amino acids， polysaccharides and biological enzymes， which constitute the nutrient source of soil microorganisms， which promotes the growth and reproduction of soil microorganisms to some extent. Thirdly， the digestive tract of earthworms is also a source of certain microorganisms， and the feces of earthworms are rich in microorganisms such as bacteria， actinomycetes and fungi. Due to the presence of these three systems， the number of microorganisms can be significantly increased. However， how these three systems of earthworm microbe interaction work together still needs further study.
References
[1] BRUSSAARD L. On the mechanisms of interactions between earthworms and plants[J]. Pedobiologia， 1999， 43： 880-885. [2] BROWN GG， BARIOS I， LAVELLE P. Regulation of soil organic matter dynamics and microbial activity in the drilosphere and the role of interactions with other edaphic functional domains[J]. European Journal of Soil Biology， 2000， 36： 177-198.
[3] PARLR J. A microbiological study of earthworm casts[J]. J. General Mirobiol.， 1963， 31： 13-22.
[4] ZHANG BG. Interaction between earthworms and microorganisms[J]. Acta Ecologica Sinica， 1997， 17（5）： 556-560. （in Chinese）
[5] LI HX， HU F， SHEN QR， et al. Effect of earthworm inoculation on soil carbon and nitrogen dynamics and on crop yield with application of corn residues[J]. Chinese Journal of Applied Ecology？，2002，13（12）： 1636-1641. （in Chinese）
[6] HUANG FZ， ZHANG YZ， YANG FR. Soil improvement and comprehensive utilization by earthworms[M]. Chongqing： Chongqing Publishing House， 1984： 28-36. （in Chinese）
[7] ZHANG JE， LIU WG， HU G， et al. The relationship between quantity index of soil microorganisms and soil fertility of different land use systems[J]. Soil and Environment，2002，11（2）：140-143. （in Chinese）
[8] ARAUJO Y， LUIZAO F， BARROS E. Effects of earthworm addition on soil nitrogen availability，microbial biomass and litter decomposition in mesocosms[J]. Biology and Ferlility of soils， 2004， 39： 146-152.
[9] ZHANG BG， LI GT， SHEN TS. Influence of the earthworm Pheretima guillelmi on soil microbial biomass and activity[J]. Acta Ecologica Sinica， 2000， 20（1）：168-172. （in Chinese）
[10] CHEN WX. Soil and environmental microbiology[M]. Beijing： Beijing Agricultural University Press， 1990. （in Chinese）
[11] LI FD. Agricultural microbiology experimental techniques[M]. Beijing： Agriculture Press， 1996. （in Chinese）
[12] Microbiology Department， Institute of Soil Science， Chinese Academy of Sciences. Soil microbial research[M]. Beijing： Science Press， 1985. （in Chinese）
[13] LU RK. Soil agricultural chemical analysis methods[M]. Beijing： China Agricultural Science and Technology Press， 1999. （in Chinese）
[14] XIANG WS， WU JS， XIAO HA. Advances in extraction and purification of soil microorganism[J]. Chinese Journal of Applied Ecology， 2003， 14（3）： 453-456. （in Chinese）
[15] WOLTERS V. Invertebrate control of soil organic matter stability[J]. Biology and Fertility of Soils， 2000， 31： 1.