CARD-FISH研究食细菌线虫对氨氧化细菌(AOB)数量的影响

Effects of bacterial-feeding nematodes on the amount of ammonia oxidizing bacteria colony in soils using CARD-FISH

土壤动物与微生物的取食与反馈之间的关系是土壤生态学研究的核心内容之一。通过接种原位的食细菌线虫和微生物群落模拟土壤真实环境,采用CARD-FISH方法来观察食细菌线虫的不同取食密度下,氨氧化细菌(ammonia oxidizing bacteria)数量的动态变化,以揭示土壤食细菌线虫对AOB数量的影响及AOB的反馈强度。结果表明:与单独接种细菌的处理(SB)相比,接种食细菌线虫显著地增加了土壤中AOB的数量,3个不同线虫接种密度处理中AOB数量表现为接种20条g-1干土的处理(SBN20)>接种10条g-1干土的处理(SBN10)>接种40条g-1干土的处理(SBN40)。由于过度取食,SBN40处理中AOB的数量在培养了14d后低于SB处理,且在第28天时显著低于SB处理。接种食细菌线虫显著增加了土壤中NH4+-N和NO3-N的含量,表明食细菌线虫促进了N的矿化和硝化作用。矿化作用增强使得硝化作用的底物NH4+-N显著增加可能是AOB数量显著增多的重要原因之一。

Interactions between feeding and feedback of soil microfauna and microbes are one of the core issues in soil ecology.Ammonia-oxidizing bacteria （AOB） are a key group in soils in the transformation of ammonia （NH＋4） to nitrite （NO-2） which is often considered as the rate limiting step of nitrification.It is well known that the activities of bacterial-feeding nematodes are very efficient in promoting nitrification,and one of the reasons is likely by stimulating the enhancement of AOB amount.The objective of the study was to investigate the effects of bacterial-feeding nematodes on ammonia oxidizing bacteria （AOB） colonies and the feedback of AOB by simulating the natural soil in the lab condition.Treatments were inoculated the soil sample with soil bacteria solely （soil suspension） or with AOB as CK.and three different densities,e.g.10ind（individual）g-1 dry soil（SBN10）,20 ind g-1 dry soils （SBN20） and 40 ind g-1 dry soil （SBN40）,of bacterial-feeding nematodes （name of species） were inoculated to compare the dynamic changes of AOB colonies under the grazing of nematodes.The common FISH （oligonucletide probes labeled with Cy3 fluorochrome） and CARD-FISH （fluorescent in-situ hybridization with horseradish peroxidase （HRP）-labeled oligonucleotide probes and tyramide signal amplification） were compared at first to look at the suitability of the methods.Finally,the method chosen in this experiment was CARD-FISH.Results showed that the AOB colonies changed with the density of bacterial-feeding nematodes inoculated significantly.We found that the order was：20 ind （individual） g-1 dry soils （SBN20） 10 ind g-1 dry soil （SBN10） 40 ind g-1 dry soil （SBN40）.However,AOB colony in the treatment with 40 ind g-1 dry soil （SBN40） was lower than that in the treatment with bacteria alone （SB） after 14 days in the experiment and it was significantly lower after 28 days.This was due to the effect of overgrazing of AOB from bacterial-feeding nematodes.This result suggested that the effect of bacterial-feeding nematodes on AOB exhibits a density-dependent regulation.The AOB was stimulated strongly in the presence of bacterial-feeding nematodes with optimum density of SEN.Results also showed that soil nematode numbers in the treatment with 20 ind g-1 dry soil （SBN20） was higher than that in the treatment with 40 ind g-1 dry soil （SBN40） at the time of 14 days.However,overgrazing was not observed at this moment due to the high abundance of AOB.It indicated the presence of a mutual-benefit phenomenon showing the bacterial-feeding nematodes promoted the multiplication of AOB,and the multiplication of the AOB provided more food for the nematodes.Nevertheless,with the reduction of nutrient in the closed system,both nematodes and AOB colonies were decreased at the end of incubation period.Inorganic N data showed that NH＋4-N contents decreased in the treatment with bacteria alone while it was significantly increased in the presence of nematodes in the first two weeks,indicating that bacterial-feeding nematodes could promote the rapid release of NH＋4-N thus increased N mineralization.Meanwhile,NO-3-N concentration was consistently and significantly greater in the treatment with nematodes than in the treatment without them after the beginning of the incubation,indicating that nitrification also was significantly increased in the presence of nematodes.The increase of NH＋4-N resulting from N mineralization was one of the most important facts to indicate the significant effects caused from the complex interactions between AOB colonies and the bacterial-feeding nematodes in soils in the experiment.The enhancement of AOB colonies in the presence of SEN may be an important reason which increased the rate of nitrification relating to the N mineralization.