摘要: 目的望天树是我国特有的濒危一级保护树种，人工林栽培是扩大其种群数量的重要手段，磷素供应是林木生长发育的主要影响因子，而溶磷菌在磷转化中起到重要作用。本研究从不同林龄望天树人工林根际土壤中筛选高效溶磷细菌，探究其在不同培养条件下的溶磷特点，以期为望天树微生物肥料的开发与应用提供菌种资源和理论依据、�/sec> 方法�?）利用无机磷固体培养基从不同林龄望天树林分根际土中分离筛选溶磷细菌，挑�?株高效溶磷菌，结合生理生化试验和16SrDNA基因序列对其进行鉴定。（2）通过检测溶磷动态，研究溶磷细菌溶磷量与菌液pH的相关关系。（3）采用单因素试验探究高效溶磷菌在不同环境因子和营养因子下的溶磷特性、�/sec> 结果�?）共分离筛选出18株溶磷菌，其中溶磷能力较强菌株为P4、P8、P12和P30（溶磷量分别�?52.87�?59.78�?48.53�?98.89 mg/L）。（2）经过形态观察、生理生化鉴定及系统发育树分析，菌株P8鉴定为唐菖蒲伯克霍尔德菌，P4和P12鉴定为洋葱伯克霍尔德菌，P30鉴定为蜡状芽孢杆菌。（3）P4、P8、P12和P30菌株的溶磷量与培养液pH值之间均存在极显著的负相关性（ P< 0.01），相关系数分别为−0.995、−0.990、−0.985和−0.997。（4）单因素试验结果显示：各溶磷菌株在温度为30 ~ 35 ℃，pH�?.5 ~ 8.5，NaCl质量分数�? ~ 2.5%，碳（C）源为蔗糖、乳糖和葡萄糖，氮（N）源为草酸铵和硫酸铵时，表现出较好的溶磷效果；菌株P30、P12和P4最适C∶N�?0�?，P8最适合C∶N�?0�?；菌株P12最佳磷源为FePO ₄，P30、P8和P4最佳磷源均为Ca ₃(PO ₄) ₂、�/sec> 结论不同培养条件会显著影响溶磷细菌的溶磷能力，筛选得到的4株高效溶磷菌具有良好的溶磷能力，且能够溶解多种难溶性无机磷酸盐，可用于望天树专用微生物肥料的研发，具有良好的应用潜力、�/sec>

关键诌�
• 望天栐�/a> /
• 溶磷细菌/
• 溶磷能力/
• 培养条件

Abstract: ObjectiveThe Parashorea chinensisis an endemic and endangered species in China, and it is classified as a class � protected wild plant. Plantation cultivation is an important means to expand its populations. Phosphorus (P) supply is a major influence on tree growth and development, and P dissolving bacteria plays an important role in P transformation. This research aimed to screen high-efficient phosphate-solubilizing bacteria (PSB) from the rhizosphere soil of P. chinensisplantations of different ages, and explore its P-solubilizing characteristics, so as to provide bacterial resources and culture conditions for the development of microbial fertilizer suitable of P. chinensis. Method(1) PSB was isolated and screened from the rhizosphere soil of P. chinensisin different ages by inorganic P solid medium. The physiological and biochemical tests and 16S rDNA gene sequence were used to further identify 4 strains of PSB. (2) The relationship between the amount of P dissolved by PSB and the pH of the bacterial solution was studied by detecting the dynamics of P solubilization. (3) A single factor experiment was conducted to investigate the P-solubilizing characteristics of high efficiency PSB under different environmental and nutritional factors. Result(1) A total of 18 strains of phosphorus solubilizing bacteria were isolated and screened, and the 4 strains with the strongest P-solubilizing capacity were P4, P8, P12 and P30 (P-solubilizing capacity was 552.87, 559.78, 548.53 and 598.89 mg/L, respectively). (2) Strain P8 was identified as Burkholderia gladioli, P4 and P12 as Burkholderia cepacia, and P30 as Bacillus cereusby physiological, biochemical identification and combined with phylogenetic tree analysis. (3) There was a highly significant ( P< 0.01) negative correlation between the pH of the cultures and the amount of phosphorus dissolved by the P4, P8, P12 and P30 strains, with correlation coefficients of �?.995, �?.990, �?.985 and �?.997, respectively. (4) The results of single factor test showed that the PSBs had the preferable P-solubilizing effects at a temperature of 30�?5 �? the pH of 5.5�?.5, the NaCl mass fraction was 0�?.5%, the carbon (C) sources were sucrose, lactose and glucose, and the nitrogen (N) sources were ammonium oxalate and ammonium sulfate. The optimal C∶N of strain P30, P12 and P4 was 20�?, and that of P8 was 40�?. The optimal P source for strain P12 was FePO ₄, and Ca ₃(PO ₄) ₂for P30, P8 and P4. ConclusionThe P-solubilizing ability of PSBs can be significantly affected under different culture conditions. The 4 strains of high efficiency PSB have preferable P-solubilizing ability, which can dissolve a variety of insoluble inorganic phosphates. Therefore, it is expected to provide germplasm resources for the development of high-efficiency microbial phosphate fertilizers with a good application potential.

Key words:
• Parashorea chinensis/
• phosphate-solubilizing bacteria/
• phosphate solubilization capacity/
• culture condition

�?nbsp; 1部分溶磷细菌溶磷圇�/p>

Figure 1.Phosphate solubilizing circle of partial phosphate solubilizing bacteria

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�?nbsp; 2溶磷细菌溶磷能力的测宙�/p>

图中不同小写字母表示不同菌株间溶磷量差异显著＇�i>P< 0.05）。Different lowercase letters in the figure indicate significant differences in phosphorus solubility between different strains (P< 0.05).

Figure 2.Determination of phosphate solubilizing ability of phosphate solubilizing bacteria

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�?nbsp; 3溶磷细菌系统发育栐�/p>

Figure 3.Construction of phylogenetic tree of phosphate solubilizing bacteria

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�?nbsp; 4溶磷菌中溶磷量和pH的动态变匕�/p>

Figure 4.Dynamic changes of phosphorus dissolved amount and pH in phosphorus dissolving bacteria

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�?nbsp; 5pH对溶磷菌溶磷能力的影哌�/p>

Figure 5.Effects of pH on the ability of phosphorus dissolving bacteria to dissolve phosphorus

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�?nbsp; 6温度对溶磷菌溶磷能力的影哌�/p>

Figure 6.Effects of temperature on phosphorus dissolving ability of phosphorus dissolving bacteria

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�?nbsp; 7NaCl质量分数对溶磷菌溶磷能力的影哌�/p>

Figure 7.Effects of NaCl mass fraction on phosphorus solubilization ability of phosphorus solubilizing bacteria

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�?nbsp; 8不同碳源对溶磷菌溶磷能力的影哌�/p>

GL. 葡萄糖；SU. 蔗糖；L. 乳糖；SS. 可溶性淀粉；MAN. 甘露醇。不同小写字母表示同一菌株在不同条件下溶磷量差异显著（P< 0.05）。下同。GL, glucose; SU, sucrose; L, lactose; SS, soluble starch; MAN, mannitol. Different lowercase letters indicate that the phosphate solubilizing ability of the same strain under different treatments is significantly different (P< 0.05). The same below.

Figure 8.Effects of different carbon sources on phosphorus dissolving ability of phosphorus dissolving bacteria

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�?nbsp; 9不同氮源对溶磷菌溶磷能力的影哌�/p>

UR. 尿素；AS. 硫酸铵；AO. 草酸铵；PN. 硝酸钾；SO. 蛋白胨。UR, urea; AS, ammonium sulfate; AO, ammonium oxalate; PN, potassium nitrate; SO, peptone.

Figure 9.Effects of different nitrogen sources on phosphorus dissolving ability of phosphorus dissolving bacteria

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�?nbsp; 10不同C∶N对溶磷菌溶磷能力的影哌�/p>

Figure 10.Effects of different C � N on phosphorus dissolving ability of phosphorus dissolving bacteria

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�?nbsp; 11不同磷源对溶磷菌溶磷能力的影哌�/p>

Figure 11.Effects of different phosphorus sources on phosphorus dissolving ability of phosphorus dissolving bacteria

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�?nbsp; 1试验地林分特征与土壤理化性质

Table 1.Stand characteristics and soil physicochemical properties of the experimental sites

林龄/a Stand age/year	平均树高 Average tree height/m	平均胸径（地径） Average DBH (ground diameter)/cm	pH	含量 Content
				全氮 Total N/(g·kg�?)	全磷 Total P/(g·kg�?)	全钾 Total K/(g·kg�?)	氨态氮 Ammoniacal N/(mg·kg�?)	有效磶�br/>Available P/(mg·kg�?)	速效钽�br/>Available K/(mg·kg�?)
1	1.72	2.28	4.53	1.52	0.32	9.44	6.66	3.58	40.47
2	2.20	3.16	3.49	2.06	0.23	6.60	6.12	5.10	72.74
41	40.60	48.70	3.69	2.04	0.78	11.90	3.13	2.63	61.19

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�?nbsp; 2溶磷细菌的初筛结枛�/p>

Table 2.Preliminary screening results of phosphate solubilizing bacteria

林龄/a Stand age/year	菌株编号 Strain No.	D/d		林龄/a Stand age/year	菌株编号 Strain No.	D/d
1	P1	1.28 ± 0.05f		2	P13	1.02 ± 0.03i
1	P2	1.55 ± 0.09d		2	P14	1.17 ± 0.05h
1	P3	1.62 ± 0.04c		2	P15	2.04 ± 0.03b
1	P4	2.03 ± 0.03b		41	P25	2.05 ± 0.02b
2	P8	2.12 ± 0.02a		41	P26	1.35 ± 0.07ef
2	P9	1.41 ± 0.02e		41	P27	1.20 ± 0.04gh
2	P10	2.01 ± 0.01b		41	P28	1.32 ± 0.02f
2	P11	1.52 ± 0.02d		41	P29	1.27 ± 0.02fg
2	P12	2.18 ± 0.01a		41	P30	2.04 ± 0.02b
注：D表示溶磷圈直径，d表示菌落直径。同列数值后不同字母表示处理间差异显著（P< 0.05）。Notes:Dmeans soluble phosphorus diameter,dmeans colony diameter.Different letters in the same column mean significant differences atP< 0.05 level.

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�?nbsp; 3溶磷细菌的生理生化鉴宙�/p>

Table 3.Physiological and biochemical identification of phosphate solubilizing bacteria

菌株编号 Strain No.	V-P反应 Voges-Proskauer reaction	明胶液化 Gelatin hydrolysis	淀粉水觢�br/>Amylolysis	柠檬酸盐 Citrate	吲哚试验 Indole production	反硝匕�br/>Denitrification
P4	∑�/td>	+	∑�/td>	+	∑�/td>	∑�/td>
P8	∑�/td>	+	∑�/td>	+	∑�/td>	∑�/td>
P12	∑�/td>	+	∑�/td>	+	∑�/td>	∑�/td>
P30	+	+	+	+	+	∑�/td>
注： + 表示阳性反应；−表示阴性反应。Notes: + indicates positive reaction; � indicates negative reaction.

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�?nbsp; 4基于16S rDNA 序列鉴定结果

Table 4.Identification based on 16S rDNA sequence

菌株编号 Strain No.	模式菌株 Type strain	相似�?br/>Identity/%	登录叶�br/>Accession No.
P4	洋葱伯克霍尔德菌Burkholderia cepacia	99.79	AY741348.1
P8	唐菖蒲伯克霍尔德�Burkholderia gladioli	99.65	MW320460.1
P12	洋葱伯克霍尔德菌Burkholderia cepacia	99.79	FJ907187.1
P30	蜡状芽孢杆菌Bacillus cereus	99.72	EU857430.1

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