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磷是植物生长必需的大量元素,在植物光合作用、根系延伸及能量传递等方面起着重要作用[1-2]。大多数磷以磷酸钙、磷酸铝及磷酸铁等难溶性磷酸盐形式存在于自然界土壤中,只有少数H2PO4-和HPO42-等可溶性磷能被植物吸收利用[3]。磷缺乏是限制植物生产力提高的主要因素之一,大量的磷肥施入土壤中满足了植物的生长需求,却引起了如水体污染等诸多环境问题[4]。另外,磷肥主要是从磷矿石中提取,而磷矿石的储量有限[5],开采矿石和磷肥生产是一种高成本和不可持续的过程。使用一种替代或可再生能源提高磷的循环利用,减少对磷矿石和磷肥的依赖是目前急需解决的问题[4]。
土壤微生物能通过促进植物根系生长和磷的矿化等方式来提高其对养分的利用[6],其中溶磷微生物能通过有机磷矿化和无机磷的增溶从土壤难溶性磷酸盐中水解出可溶性磷[7-8]。如Zhang发现2株溶磷真菌对Ca3(PO4)2、FePO4、AlPO4和植酸钙均具有较好的溶解作用[9];在田间或温室盆栽试验条件下,石灰性土壤中施用巴西青霉菌(Penicillium bilaii)能显著促进小麦、大豆及豌豆等农作物的生长及磷的获取[4, 10]。Wang发现溶磷真菌CS-1能分泌草酸、酒石酸和柠檬酸,对磷酸钙具有较好的溶解能力,且能显著促进小麦生物量的累积[11]。此外,部分溶磷微生物还具有分泌嗜铁素、吲哚乙酸(IAA)和解钾等多种功能进而促进植物生长[3]。李云玲筛选出一株溶磷细菌CHW10B具有产嗜铁素和IAA的能力,且能促进南方红豆杉生长及生物量提高[12]。张扬从毛竹根际土壤中筛选出2株溶磷细菌兼具解钾,分泌IAA及抑制病原菌等功能,对毛竹有较好的促生作用[13]。因此,通过筛选和应用溶磷微生物改善土壤磷素供应或许是解决我国缺磷区养分供应不足的有效途径[14]。
毛竹(Phyllostachys edulis (Carrière) J. Houz.)是我国分布最广,面积最大的竹种[15],主要分布于亚热带地区,占全国竹林面积70%左右,面积已达443万hm2[16]。毛竹是典型的克隆植物,具有生长迅速及更新周期短等特点,是南方山区林农收入的重要来源,也是我国重要的经济林树种[17]。由于长期的不合理经营方式导致毛竹林地养分流失,地力衰退,生产力不稳定,土壤磷素缺乏导致竹材、竹笋产量逐年呈下降趋势,严重威胁着竹林的持续经营利用[18]。研究表明,磷素已成为限制毛竹林资源质量及生产力提升的主要因子之一[9, 19]。目前关于毛竹林地溶磷真菌的研究少有报道。利用溶磷真菌提高竹林磷素利用率,对于改善毛竹林地土壤养分,提高竹林生产力,促进竹农增收具有重要的现实意义,也是一种低成本、环保和可持续的生物技术策略。本课题组前期从毛竹根际土壤中分离到1株解磷真菌新黑曲霉(Aspergillus neoniger)[9],但关于该菌株解磷等相关功能尚未系统研究,对毛竹是否具有促生作用也尚不明确,限制了开发和利用。本研究探讨该新黑曲霉(JXBR16)的解磷特性和解磷条件,并通过盆栽试验研究其对毛竹实生苗的促生作用,揭示该菌株的溶磷效果及其促生机理,以期为该菌株作为生物肥料应用时发挥最佳溶磷功效提供参考,为改善土壤磷素营养和维持毛竹林可持续经营发展提供理论依据和实践指导。
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菌株JXBR16对Ca3(PO4)2、CaHPO4、FePO4、AlPO4和植酸钙等5种难溶性矿质盐均具有较好的溶解能力且差异显著(图 1)。该菌株对FePO4、CaHPO4的溶解能力最强,解磷量分别为3 208.31和3 027.09 mg·L-1;对植酸钙和AlPO4的解磷能力其次,解磷量分别为1 862.73和1 342.43mg·L-1;对Ca3(PO4)2的溶解作用最低,解磷量也达至850.84 mg·L-1。
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不同碳源对菌株JXBR16溶解磷酸钙能力的影响差异显著(图 2A)。以蔗糖和葡萄糖分别为唯一碳源时,菌株JXBR16对磷酸钙的溶解能力达至最强,解磷量分别为1 054.4 mg·L-1和1 021.47 mg·L-1;以麦芽糖、果糖和可溶性淀粉为碳源时,该菌株的解磷量显著降低,尤其是以可溶性淀粉为碳源时,解磷量最小,为567.75 mg·L-1。在不同氮源条件下菌株JXBR16对磷酸钙的解磷能力差异显著(图 2B),以硫酸铵为唯一氮源时,菌株JXBR16的解磷能力最强,解磷量为943.58 mg·L-1;其次是以牛肉膏和蛋白胨为氮源时,该菌株的解磷的能力分别为847.64 mg·L-1和844.27 mg·L-1;而以酵母粉为氮源时解磷能力降至最低,为109.74 mg·L-1。
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菌株JXBR16在不同环境条件下其解磷能力差异显著(图 3)。该菌株在装液量为2/5时对磷酸钙具有较好的溶解能力,解磷量1 053.18 mg·L-1,显著高于其他装液量解磷量(图 3A)。菌株的解磷能力受初始pH值的显著影响;当初始pH值为3.5时,该菌株具有最大的解磷能力,解磷量为749.98 mg·L-1,其次为初始pH值4.5、1.5、和6.5时,而初始pH值5.5和2.5时最低(图 3B)。温度在25 ℃和30 ℃时该菌株对磷酸钙的溶解量显著高于其它温度处理的溶解量,解磷能力分别为758.33和716.67 mg·L-1,其次为温度35 ℃和20 ℃时,而温度为40 ℃时解磷能力最低(图 3C)。随着NaCl浓度的增加,该菌株解磷能力总体呈现下降的趋势,在NaCl浓度为0.0 g·L-1、1.0 g·L-1和2.0 g·L-1时,解磷能力分别为1 035.2、984.13和920.72 mg·L-1,显著高于其余处理(图 3D)。
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与对照相比,施用菌剂JXBR16能显著促进毛竹实生苗的生长(图 4)。苗高、地径及总生物量分别比对照提高了47.27%、44.44%和50.40%,地上和地下生物量分别比对照提高了56.05%和39.2%,总生物量比对照提高了50.4%(表 1)。
图 4 施用解磷真菌JXBR16对毛竹促生效果的盆栽试验(180 d)
Figure 4. Inoculated with phosphate-solubilizing fungi JXBR16 for moso bamboo growth promotion in pot experiment (180 d)
表 1 施用菌剂JXBR16对毛竹实生苗生长的影响
Table 1. The effect of adding strain JXBR16 on the growth of moso bamboo seedling
处理
Treatment苗高
Seedling height/cm地径
Ground diameter/cm地上生物量
Aboveground biomass/g地下生物量
Underground biomass/g总生物量
Total biomass/g施用菌剂Adding strain 35.27±1.21a 2.21±0.26a 3.87±0.16a 1.74±0.09a 5.61±0.14a 对照CK 23.95±0.73b 1.53±0.17b 2.48±0.14b 1.25±0.06b 3.73±0.22b 同时,发现施用菌剂JXBR16后,毛竹根际土壤有效磷、铵态氮及矿质氮都显著提高,有效磷、铵态氮和矿质氮含量分别比对照提高了36.68%、84.26%、41.07%;此外,植株体磷含量也显著提高,毛竹叶片、茎干和根系磷含量分别比对照提高了43.28%、102.17%和95.35%(表 2)。
表 2 施用菌剂JXBR16对毛竹实生苗养分吸收和根际土壤养分的影响
Table 2. Effect of inoculation with strain JXBR16 on nutrient uptake of moso bamboo seedlings and rhizosphere soil nutrient
观测指标Variables 对照CK 施用菌剂Adding strain T检验T-test 叶片磷含量Leaf P/(g·kg-1) 2.01±0.12 2.88±0.21 P < 0.05 茎磷含量Stem P/(g·kg-1) 0.92±0.05 1.86±0.27 P < 0.05 根系磷含量Root P/(g·kg-1) 0.86±0.06 1.68±0.07 P < 0.05 有效磷Available P/(mg·kg-1) 2.29±0.25 3.13±0.11 P < 0.05 铵态氮NH4+-N/ (mg·kg-1) 1.08±0.09 1.99±0.15 P < 0.05 硝态氮NO3--N/ (mg·kg-1) 2.11±0.02 2.51±0.04 P>0.05 矿质氮Mineral N/ (mg·kg-1) 3.19±0.05 4.50±0.18 P < 0.05
毛竹根际新黑曲霉的解磷特性及促生作用
Phosphate Solubilizing Characteristics and Growth Promoting Effect of Aspergillus neoniger on Phyllostachys edulis Seedling
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摘要:
目的 探究毛竹根际微生物新黑曲霉(Aspergillus neoniger)JXBR16对土壤难溶性磷酸盐的溶磷作用及对毛竹的促生效果。 方法 采用液体发酵培养法研究该菌株对Ca3(PO4)2、CaHPO4、FePO4、AlPO4和植酸钙等5种难溶性磷酸盐的溶解能力及碳源、氮源、pH、装液量和盐离子等因素对其解磷能力的影响;并采用温室盆栽法评价该菌株对毛竹实生幼苗的促生作用。 结果 表明,新黑曲霉对5种难溶性磷酸盐均具有较好的溶解能力,其中对FePO4、CaHPO4的溶解能力最佳,解磷量分别达3 208.31和3 027.09 mg·L-1;在碳源为蔗糖或葡萄糖、氮源为硫酸铵、初始pH值3.5、装液量2/5、温度25℃和盐离子浓度为0~1.0 g·L-1条件下新黑曲霉的解磷能力最佳。施用该菌株180 d后毛竹根际土壤有效磷和矿质氮分别提高37%和41%,植株根、茎和叶磷含量分别提高了95%、102%和43%;毛竹地径、苗高和生物量分别比对照显著提高了44%、47%和50%。 结论 新黑曲霉能够有效增强南方红壤区毛竹林土壤磷的供给,促进植株养分吸收利用,并提高毛竹的生长,具有应用于竹林生物菌肥研制和开发的巨大潜力。 Abstract:Objective The aim of this study is to investigate the phosphate-solubilizing characteristics and growth promoting effect of Aspergillus neoniger (JXBR16) from rhizosphere soil of Phyllostachys edulis. Method The phosphate-solubilizing ability of strain JXBR16 on five different types of mineral phosphate, including Ca3(PO4)2, CaHPO4, FePO4, AlPO4 and calcium phytate, were compared and the effects of carbon sources, nitrogen sources, initial pH, liquid filling volume, temperature and salt ions on phosphate solubilizing ability of strain JXBR16 were studied using the liquid fermentation experiment. Also, the effects of strain JXBR16 on nutrient acquirement and growth of Ph. edulis were evaluated using pot experiment. Result The strain JXBR16 showed the greatest ability to solubilize FePO4 and CaHPO4 with 3 208.31 and 3 027.09 mg·L-1, respectively. The strain JXBR16 displayed the highest phosphate-dissolving capacity when the initial pH reached 3.5, the volume of liquid was 2/5, the temperature was 25℃, the NaCl concentration was 0.01.0 g·L-1, sugar and yeast powder were used as carbon and nitrogen sources, respectively. Meanwhile, compared with control, the rhizosphere soil available phosphorus, mineral nitrogen, root, stem, and leaf phosphorus contents increased by 37%, 41%, 95%, 102% and 43%, respectively in Ph. edulis seedling inoculated with strain JXBR16 for 180-days. In addition, the ground diameter, seedling height and biomass accumulation were higher by 44.44%, 47.27% and 50.40%, respectively in strain JXBR16 treatment than control. Conclusion A. neoniger can effectively enhance soil phosphorus supply, promote nutrient uptake and utilization, and improve the growth of Ph. edulis in red soil region. It has great potential to be used in development of biological fertilizer for bamboo forests in southern China. -
表 1 施用菌剂JXBR16对毛竹实生苗生长的影响
Table 1. The effect of adding strain JXBR16 on the growth of moso bamboo seedling
处理
Treatment苗高
Seedling height/cm地径
Ground diameter/cm地上生物量
Aboveground biomass/g地下生物量
Underground biomass/g总生物量
Total biomass/g施用菌剂Adding strain 35.27±1.21a 2.21±0.26a 3.87±0.16a 1.74±0.09a 5.61±0.14a 对照CK 23.95±0.73b 1.53±0.17b 2.48±0.14b 1.25±0.06b 3.73±0.22b 表 2 施用菌剂JXBR16对毛竹实生苗养分吸收和根际土壤养分的影响
Table 2. Effect of inoculation with strain JXBR16 on nutrient uptake of moso bamboo seedlings and rhizosphere soil nutrient
观测指标Variables 对照CK 施用菌剂Adding strain T检验T-test 叶片磷含量Leaf P/(g·kg-1) 2.01±0.12 2.88±0.21 P < 0.05 茎磷含量Stem P/(g·kg-1) 0.92±0.05 1.86±0.27 P < 0.05 根系磷含量Root P/(g·kg-1) 0.86±0.06 1.68±0.07 P < 0.05 有效磷Available P/(mg·kg-1) 2.29±0.25 3.13±0.11 P < 0.05 铵态氮NH4+-N/ (mg·kg-1) 1.08±0.09 1.99±0.15 P < 0.05 硝态氮NO3--N/ (mg·kg-1) 2.11±0.02 2.51±0.04 P>0.05 矿质氮Mineral N/ (mg·kg-1) 3.19±0.05 4.50±0.18 P < 0.05 -
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