Effects of Nitrogen Addition on the Abundance of Bacterial Phosphatase Encoding Genes in the Soil of Pinus sylvestris var. mongolica Plantation

doi:10.13560/j.cnki.biotech.bull.1985.2021-1604

Abstract

Abstract:

In order to reveal the microbial processes regulating the responses of soil organic phosphorus（P）mineralization to nitrogen（N）addition, abundances of 3 soil bacterial phosphatase-encoding genes（phoC, phoD and appA）and corresponding acid phosphomonoesterase, alkaline phosphomonoesterase and phytase activities, as well as related soil physiochemical properties were measured in a Pinus sylvestris var. mongolica plantation that has subjected to 10 years of field N addition（100 kg N ha^-2year^-1）. The results show that N addition reduced acidic and alkaline phosphomonoesterase activities in the soil of Pinus sylvestris var. mongolica plantation by 18.09% and 55.29%, and the phytase activity decreased by 41.88%. Nitrogen addition reduced the copy number of each gene by 40.97%（16S-rRNA）, 78.38%（phoD）, 67.92%（phoC）, and 74.37%（appA）, respectively. The proportion of the copy number of functional gene in the total bacterial gene 16S-rRNA decreased significantly by 61%（phoD）, 44%（phoC）, and 55%（appA）. The contents of soil microbial biomass carbon and microbial biomass phosphorus were significantly positively correlated with the activities of acid phosphomonolipase, alkaline phosphomonoesterase, phytase and gene abundances of 16S rRNA, phoD, phoC and appA. Soil ammonium nitrogen content was significantly negatively correlated with the activities of acid phosphomonoesterase and alkaline phosphomonoesterase, as well as the abundance of 16S rRNA, phoC and appA genes. The activity of acid phosphomonoesterase was significantly positively correlated with its gene abundance, and the other two enzyme activities had no significant correlation with its gene abundance.The above results indicate that the N addition not only reduced abundance of soil phosphate-dissolving bacteria, but also reduced the proportion of phosphate-dissolving bacteria in the total bacteria, thereby greatly reduced phosphatase activities and depressed organic phosphorus mineralization.

Key words: nitrogen addition, phosphomonoesterase, phytase, gene abundance

WU Lin-hui, GENG Bi-miao, WANG Yan-jie, ZHOU Guo-wei, SUN Qing-ye, ZHAO Qiong. Effects of Nitrogen Addition on the Abundance of Bacterial Phosphatase Encoding Genes in the Soil of Pinus sylvestris var. mongolica Plantation[J]. Biotechnology Bulletin, 2022, 38(11): 202-209.

Figures/Tables 8

References 38

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引物Primer	引物名称Primer name	引物长度Primer length/bp	引物序列Primer sequence（5'-3'）	参考文献Reference
phoD	ALPS-F730	370	CAGTGGGACGA CCACGAGGT	[17]
phoD	ALPS-1101	370	GAGGCCGATCG GCATGTCG
phoC	phoC-A-F1	155	CGGCTCCTATCCGTCCGG	[17]
phoC	phoC-A-R1	155	CAACATCGCTTTGCCAGTG
appA	appA-F	247	CAGATACGTCCAGTCCCGAT
appA	appA-R	247	AGTTCCGATGGTAATGCCTG
16S rRNA	338F	468	ACTCCTACGGGAGGCAGCAG	[18]
16S rRNA	806R	468	GGACTACHVGGGTWTCTAAT

引物Primer	引物名称Primer name	引物长度Primer length/bp	引物序列Primer sequence（5'-3'）	参考文献Reference
phoD	ALPS-F730	370	CAGTGGGACGA CCACGAGGT	[17]
phoD	ALPS-1101	370	GAGGCCGATCG GCATGTCG
phoC	phoC-A-F1	155	CGGCTCCTATCCGTCCGG	[17]
phoC	phoC-A-R1	155	CAACATCGCTTTGCCAGTG
appA	appA-F	247	CAGATACGTCCAGTCCCGAT
appA	appA-R	247	AGTTCCGATGGTAATGCCTG
16S rRNA	338F	468	ACTCCTACGGGAGGCAGCAG	[18]
16S rRNA	806R	468	GGACTACHVGGGTWTCTAAT

引物Primer	扩增体系Amplification system	扩增程序Amplification procedure
phoD	10 μL体系：Bestar Sybr Green qPCR Master Mix 5 μL，引物0.4 μL，DNA样品2 μL（10 ng·μL^-1），灭菌水2.6 μL	预变性95℃ 300 s，变性 95℃ 15 s，退火58℃ 30 s，延伸 72℃ 20 s，40 个循环
phoC		预变性 95℃ 300 s，变性 95℃ 15 s，退火57℃ 30 s，延伸 72℃ 20 s，40 个循环
appA		预变性 95℃ 300 s，变性 95℃ 15 s，退火60℃ 30 s，延伸 72℃ 20 s，40 个循环
16S rRNA		预变性 95℃ 300 s，变性 95℃ 15 s，退火55℃ 30 s，延伸 72℃ 20 s，40 个循环

引物Primer	扩增体系Amplification system	扩增程序Amplification procedure
phoD	10 μL体系：Bestar Sybr Green qPCR Master Mix 5 μL，引物0.4 μL，DNA样品2 μL（10 ng·μL^-1），灭菌水2.6 μL	预变性95℃ 300 s，变性 95℃ 15 s，退火58℃ 30 s，延伸 72℃ 20 s，40 个循环
phoC		预变性 95℃ 300 s，变性 95℃ 15 s，退火57℃ 30 s，延伸 72℃ 20 s，40 个循环
appA		预变性 95℃ 300 s，变性 95℃ 15 s，退火60℃ 30 s，延伸 72℃ 20 s，40 个循环
16S rRNA		预变性 95℃ 300 s，变性 95℃ 15 s，退火55℃ 30 s，延伸 72℃ 20 s，40 个循环

处理Treatment	测定指标Measurement indicators
处理Treatment	pH	铵态氮 NH₄⁺-N/（mg·kg^-1）	硝态氮 NO₃^--N/（mg·kg^-1）	有效磷 AP/（mg·kg^-1）	总磷 TP/（g·kg^-1）	总氮 TN/（g·kg^-1）	有机质 SOM/（g·kg^-1）
ZCK	6.58±0.14	2.62±0.18	0.09±0.01	2.06±0.36	0.16±0.02	0.38±0.04	10.66±0.72
ZN	5.78±0.05**	17.89±1.09**	0.60±0.12*	1.12±0.05*	0.10±0.02*	0.45±0.04	12.18±0.55