溶磷菌的筛选、溶磷条件优化及对玉米的促生作用研究
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摘要
施用溶磷菌是提高土壤中难溶性磷生物有效性的重要措施之一,为了使施入的溶磷菌在土壤中更好地发挥其活化难溶性磷、改善作物磷素营养状况的作用,所筛选的溶磷菌就必须兼具利用多种形态难溶性磷的能力。本文采用改良的Pikovskaya(?)培养基筛选兼具溶解Ca3(P04)2、磷矿粉和卵磷脂等难溶性磷源的菌株,并通过液体摇瓶试验、生理生化特性和16S rDNA序列分析、盆栽试验、培养及溶磷条件优化等,研究了其在NBRIP培养基中的溶磷效果、分类学地位、对玉米的促生作用及溶磷相关因素的影响。获得的主要结果如下：
(1)从江苏和安徽的5种石灰性土壤中共分离获得16株溶磷菌,其中菌株P1对Ca3(P04)2、磷矿粉和卵磷脂中磷的溶解效果最佳,溶磷量分别为674.5μg·mL-1、132.3μg·mL-1和33.0μg·mL-1。
(2)经过菌落及菌体形态特征、生理生化特性和16S rDNA序列分析,初步鉴定菌株P1和P3分别为唐菖蒲伯克霍尔德氏菌(Burkholderia gladioli)和乙酸钙不动杆菌(Acinetobacter calcoaceticus)。根据pqqE和gabY基因序列设计引物,通过PCR扩增菌株P1、P2、P3、P4和P5中pqqE和gabY基因片段,仅从菌株P3中扩增到与Pseudomonas cepaciaE37(U10242.1)(?)勺溶磷相关基因gabY同源性为95%的基因片段,而其它菌株中未扩增出该基因片段。
(3)菌株P1和P3的最佳培养碳、氮源分别是葡萄糖、谷氨酸和淀粉、蛋白胨,而菌株P1和P3的最佳溶磷碳氮源条件分别是葡萄糖、KN03和葡萄糖、(NH4)2SO4。在NBRIP液体培养的1～7d内,170rpm,30℃,菌株P1和P3培养液的pH值第1d就分别下降至5.03和4.98,并且在后续培养中仍趋于保持该值。随着培养时间的增加,水溶性磷浓度呈增长趋势,而菌体数量变化总体趋势都是先下降,后上升,再下降,最后趋于稳定。此外,加透析袋处理WSP浓度有所减低。
(4)盆栽试验中,5种溶磷菌微生物有机肥均可显著促进玉米生长,株高、叶长、叶宽和干重分别增加5.1%-33.8%、14.0%-38.0%、17.7%-37.6%和31.7%-177.1%,植株全磷含量比对照增加41.6%～366.6%。经抗生素标记的5株溶磷菌在盆栽试验中的定殖效果以菌株P4和P5优于P1、P2和P3,而对玉米的促生作用却以P1的效果最佳,P3次之,P4和P5效果较差。菌株P1和P3的发酵上清液可在一定程度上促进玉米种子发芽。室内平板对峙试验结果显示菌株P1对辣椒疫病、棉花黄萎病、烟草黑胫病和黄瓜立枯病的病原菌都有明显的拮抗作用,而菌株P3只对棉花黄萎病病原菌有拮抗作用。
筛选的菌株P1和P3兼具溶解Ca3(PO4)2、磷矿粉和卵磷脂和拮抗土传病原菌的功能,在盆栽试验和玉米种子发芽试验中也均表现出一定的促生作用,因此,在微生物肥料生产中具有潜在的应用前景。
Applying phosphate-solubilizing bacteria strains (PSBs) into soil was one of the major methods to improve bioavailability of insoluble phosphates. In order to make effectively solubilize insoluble phosphates and improve phosphorus uptake of plants, PSBs should have the abilities to dissolve multifarious forms of insoluble phosphates. PSBs which could dissolve tricalcium phosphate, rock phosphate and lecithin were isolated and screened with modified Pikovskaya media in this paper. Culture conditions of growth and solubilizing insoluble phosphates, physiological characteristics and 16S rDNA gene sequence analysis of the isolated PSBs were carried out. Pot experiment was conducted to evaluate the isolated PSBs roles in dissolving phosphates and growth promotion to maize. The results obtained were as follows:
(1) Sixteen strains, which could solubilize tricalcium phosphate, rock phosphate and lecithin were isolated from five calcareous soils collected from Jiangsu and Anhui province. Among all of the isolated strains, strain P1 was the most effective for solubilizing tricalcium phosphate, rock phosphate and lecithin. After 7 days incubation, strain P1 could produce water-soluble phosphorus (WPS) up to 674.5μg·mL-1,33.0μg·mL-1 and 33.0μg·mL-1, respectively, with tricalcium phosphate, rock phosphate and lecithin as insoluble phosphate source.
(2) Strain P1 and P3 were identified as Burkholderia gladioli and Acinetobacter calcoaceticus, respectively, based on their physiological characteristics and 16S rDNA gene sequence analysis. Amplification of gabY and pqqE in P1 P2, P3, P4 and P5 strains by PCR method was carried, however, the DNA fragment of gabY genes was obtained only in strains P3, which showed 95% similarities with that in strain Pseudomonas cepaciaE37(U10242.1).
(3) The optimal carbon and nitrogen sources for the growth of P1 and P3 were glucose, starch and glutamic acid peptone, respectively. While the optimal carbon and nitrogen sources for strain P1 and P3 dissolving phosphate were glucose, glucose and potassium nitrate and ammonium sulfate, respectively. During seven-day incubation by shaking at 170 rpm and 30℃in the liquid NBRIP media, the culture pH value of strain P1 and P3 fell to 5.03 and 4.98 on 1st day, respectively, and pH value of culture kept steady in the following days. WSP concentration in culture increased along with the incubation time. Simultaneously, WSP contents in tne medium decreased to a certain extent using dialysis tubing. The variation tendency of cell counts was that it declined at first, then raised, declined again, finally maintained at a stable level.
(4) Five PSB bio-fertilizers significantly promoted the growth of maize as compared with organic fertilizer. Shoot height of maize seedling increased by 5.1%-33.8%, leaf length by 14.0%-38.0%, leaf width by 17.7%-37.6% and dry weight by 31.7%-177.1%, and phosphorus accumulation in maize increased by 41.6%～366.6%. The colonization numbers of P4 and P5 in maize rhizosphere soil were more than that of P1, P2 and P3 in pot experiment. Among the five treatments, P1 was the most effective one in promotion of maize growth, then P3, and P4 and P5 were the last. Besides, fermentation supernatants of P1 and P3 showed promotion to maize seed germination. P1 also had strong antagonistic activities against Phytophthoia capsici, Verticillium dahliae, Phytophthora parasitica var. nicotianae and Rhizoctonia solani.
In summary, strains P1 and P3 with the abilities of solubilizing multifarious insoluble phosphates and antagonizing some soil-borne pathogens, showed promotion to maize growth both in pot and seed germination experiment, suggesting that their potential application in bio-organic fertilizer-producing is promising in future.
(1)从江苏和安徽的5种石灰性土壤中共分离获得16株溶磷菌,其中菌株P1对Ca3(P04)2、磷矿粉和卵磷脂中磷的溶解效果最佳,溶磷量分别为674.5μg·mL-1、132.3μg·mL-1和33.0μg·mL-1。
(2)经过菌落及菌体形态特征、生理生化特性和16S rDNA序列分析,初步鉴定菌株P1和P3分别为唐菖蒲伯克霍尔德氏菌(Burkholderia gladioli)和乙酸钙不动杆菌(Acinetobacter calcoaceticus)。根据pqqE和gabY基因序列设计引物,通过PCR扩增菌株P1、P2、P3、P4和P5中pqqE和gabY基因片段,仅从菌株P3中扩增到与Pseudomonas cepaciaE37(U10242.1)(?)勺溶磷相关基因gabY同源性为95%的基因片段,而其它菌株中未扩增出该基因片段。
(3)菌株P1和P3的最佳培养碳、氮源分别是葡萄糖、谷氨酸和淀粉、蛋白胨,而菌株P1和P3的最佳溶磷碳氮源条件分别是葡萄糖、KN03和葡萄糖、(NH4)2SO4。在NBRIP液体培养的1～7d内,170rpm,30℃,菌株P1和P3培养液的pH值第1d就分别下降至5.03和4.98,并且在后续培养中仍趋于保持该值。随着培养时间的增加,水溶性磷浓度呈增长趋势,而菌体数量变化总体趋势都是先下降,后上升,再下降,最后趋于稳定。此外,加透析袋处理WSP浓度有所减低。
(4)盆栽试验中,5种溶磷菌微生物有机肥均可显著促进玉米生长,株高、叶长、叶宽和干重分别增加5.1%-33.8%、14.0%-38.0%、17.7%-37.6%和31.7%-177.1%,植株全磷含量比对照增加41.6%～366.6%。经抗生素标记的5株溶磷菌在盆栽试验中的定殖效果以菌株P4和P5优于P1、P2和P3,而对玉米的促生作用却以P1的效果最佳,P3次之,P4和P5效果较差。菌株P1和P3的发酵上清液可在一定程度上促进玉米种子发芽。室内平板对峙试验结果显示菌株P1对辣椒疫病、棉花黄萎病、烟草黑胫病和黄瓜立枯病的病原菌都有明显的拮抗作用,而菌株P3只对棉花黄萎病病原菌有拮抗作用。
筛选的菌株P1和P3兼具溶解Ca3(PO4)2、磷矿粉和卵磷脂和拮抗土传病原菌的功能,在盆栽试验和玉米种子发芽试验中也均表现出一定的促生作用,因此,在微生物肥料生产中具有潜在的应用前景。
Applying phosphate-solubilizing bacteria strains (PSBs) into soil was one of the major methods to improve bioavailability of insoluble phosphates. In order to make effectively solubilize insoluble phosphates and improve phosphorus uptake of plants, PSBs should have the abilities to dissolve multifarious forms of insoluble phosphates. PSBs which could dissolve tricalcium phosphate, rock phosphate and lecithin were isolated and screened with modified Pikovskaya media in this paper. Culture conditions of growth and solubilizing insoluble phosphates, physiological characteristics and 16S rDNA gene sequence analysis of the isolated PSBs were carried out. Pot experiment was conducted to evaluate the isolated PSBs roles in dissolving phosphates and growth promotion to maize. The results obtained were as follows:
(1) Sixteen strains, which could solubilize tricalcium phosphate, rock phosphate and lecithin were isolated from five calcareous soils collected from Jiangsu and Anhui province. Among all of the isolated strains, strain P1 was the most effective for solubilizing tricalcium phosphate, rock phosphate and lecithin. After 7 days incubation, strain P1 could produce water-soluble phosphorus (WPS) up to 674.5μg·mL-1,33.0μg·mL-1 and 33.0μg·mL-1, respectively, with tricalcium phosphate, rock phosphate and lecithin as insoluble phosphate source.
(2) Strain P1 and P3 were identified as Burkholderia gladioli and Acinetobacter calcoaceticus, respectively, based on their physiological characteristics and 16S rDNA gene sequence analysis. Amplification of gabY and pqqE in P1 P2, P3, P4 and P5 strains by PCR method was carried, however, the DNA fragment of gabY genes was obtained only in strains P3, which showed 95% similarities with that in strain Pseudomonas cepaciaE37(U10242.1).
(3) The optimal carbon and nitrogen sources for the growth of P1 and P3 were glucose, starch and glutamic acid peptone, respectively. While the optimal carbon and nitrogen sources for strain P1 and P3 dissolving phosphate were glucose, glucose and potassium nitrate and ammonium sulfate, respectively. During seven-day incubation by shaking at 170 rpm and 30℃in the liquid NBRIP media, the culture pH value of strain P1 and P3 fell to 5.03 and 4.98 on 1st day, respectively, and pH value of culture kept steady in the following days. WSP concentration in culture increased along with the incubation time. Simultaneously, WSP contents in tne medium decreased to a certain extent using dialysis tubing. The variation tendency of cell counts was that it declined at first, then raised, declined again, finally maintained at a stable level.
(4) Five PSB bio-fertilizers significantly promoted the growth of maize as compared with organic fertilizer. Shoot height of maize seedling increased by 5.1%-33.8%, leaf length by 14.0%-38.0%, leaf width by 17.7%-37.6% and dry weight by 31.7%-177.1%, and phosphorus accumulation in maize increased by 41.6%～366.6%. The colonization numbers of P4 and P5 in maize rhizosphere soil were more than that of P1, P2 and P3 in pot experiment. Among the five treatments, P1 was the most effective one in promotion of maize growth, then P3, and P4 and P5 were the last. Besides, fermentation supernatants of P1 and P3 showed promotion to maize seed germination. P1 also had strong antagonistic activities against Phytophthoia capsici, Verticillium dahliae, Phytophthora parasitica var. nicotianae and Rhizoctonia solani.
In summary, strains P1 and P3 with the abilities of solubilizing multifarious insoluble phosphates and antagonizing some soil-borne pathogens, showed promotion to maize growth both in pot and seed germination experiment, suggesting that their potential application in bio-organic fertilizer-producing is promising in future.
引文
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