植物营养与肥料学报
植物營養與肥料學報
식물영양여비료학보
PLANT NUTRITION AND FERTILIZER SCIENCE
2014年
6期
1505-1516
,共12页
陈倩%刘善江%白杨%李亚星%郜春花%张强%李建华
陳倩%劉善江%白楊%李亞星%郜春花%張彊%李建華
진천%류선강%백양%리아성%고춘화%장강%리건화
土地复垦%解磷菌%解磷能力%16S rDNA
土地複墾%解燐菌%解燐能力%16S rDNA
토지복은%해린균%해린능력%16S rDNA
1and rec1amation%PhosPhorus so1ubi1izing bacteria%PhosPhorus so1ubi1izing abi1ity%16 S rDNA
【目的】矿区复垦土壤贫瘠、有效磷含量低。解磷细菌能够将有机磷和难溶性无机磷转化为可溶性磷，促进植物对磷素的利用。因此筛选和鉴定具有解磷能力的菌株，可为解决矿区生态恢复使用的微生物肥料提供菌种资源。【方法】采用平板分离法初筛菌株，得到D/d≥1.5的菌株，然后以磷酸钙为磷源，通过液体发酵试验复筛菌株，挑选出解磷率高于巨大芽孢杆菌（ Bacillus megaterium）As1.223的菌株。以磷矿粉和卵磷脂为磷源，液体发酵试验测定菌株的解磷能力及磷酸酶活性。进行菌株的生长试验以测定菌株温度适宜性、耐盐性及耐酸碱性。通过形态学、基因序列分析及脂肪酸组成分析综合进行菌株鉴定。菌落形态观察用营养琼脂平板培养基培养；菌体形态即细胞形态及其大小采用扫描电镜观察；基因序列分析采用16S rDNA序列测定，基因在线比对采用EzTaxon数据库；使用美国MIDI公司的Shero1ock全自动细菌鉴定系统对菌株进行脂肪酸组成分析。【结果】利用无机磷和有机磷平板培养基，从山西省矿区复垦区土壤样品中筛选出19株解磷微生物，其中D/d≥1.5的有7株。在以磷酸钙为磷源的液体培养试验中，4株菌的解磷率高于巨大芽孢杆菌As1.223，解磷率为7.89%12.61%，最高的为菌株Y14。4株菌对磷矿粉的解磷率为0.81%1.21%，最高的为菌株Y14。在以卵磷脂为磷源的液体培养试验中，4株菌的解磷率与酸性磷酸酶活性分别为1.79%3.07%和24.328.4U/L，均高于巨大芽孢杆菌As1.223；碱性磷酸酶活性为11.950.2U/L；菌株Y14的解磷率与磷酸酶活性均最高。4株菌均有较强的环境适应能力，以Y14的适应性最强。H22、Y11和Y34与假单胞菌属（ Pseudomonas sP.）同源性在99%以上，Y14与泛菌属（ Pantoea sP.）有99.79%的同源性；H22、Y11和Y34的细胞脂肪酸组成特征峰与假单胞菌属（ Pseudomonas sP.）相一致， Y14与泛菌属（ Pantoea sP.）相一致；H22、Y11和 Y34被鉴定为假单胞菌（ Pseudomonas sP.），Y14为泛菌属（ Pantoea sP.）。【结论】分离、筛选到4株高效解磷菌，对于磷酸钙和卵磷脂的解磷率均高于巨大芽孢杆菌As1.223。4株菌分别隶属于假单胞菌属（ Pseudomonas sP.）和泛菌属（ Pantoea sP.）。菌株Y14无机磷与有机磷平板的D/d值分别为3.28与1.59，降解磷酸钙、磷矿粉、卵磷脂的解磷率分别为12.61%、1.21%、3.07%，酸性与碱性磷酸酶活性分别为28.4 U/L和50.2 U/L，均为4株菌里最高的，且环境适应能力最强，生长温度为2060℃，能耐受PH 411的酸碱梯度和2%7%的盐分梯度，Y14被鉴定为泛菌属（ Pantoea sP.）。4株菌均具有良好的解磷能力及较强的环境适应能力，可望进一步研发成为微生物肥料生产菌种。综合D/d值、解磷率、磷酸酶活性和生长试验，本试验最终确定适合山西矿区复垦农田推广的高效解磷菌菌株为Y14。
【目的】礦區複墾土壤貧瘠、有效燐含量低。解燐細菌能夠將有機燐和難溶性無機燐轉化為可溶性燐，促進植物對燐素的利用。因此篩選和鑒定具有解燐能力的菌株，可為解決礦區生態恢複使用的微生物肥料提供菌種資源。【方法】採用平闆分離法初篩菌株，得到D/d≥1.5的菌株，然後以燐痠鈣為燐源，通過液體髮酵試驗複篩菌株，挑選齣解燐率高于巨大芽孢桿菌（ Bacillus megaterium）As1.223的菌株。以燐礦粉和卵燐脂為燐源，液體髮酵試驗測定菌株的解燐能力及燐痠酶活性。進行菌株的生長試驗以測定菌株溫度適宜性、耐鹽性及耐痠堿性。通過形態學、基因序列分析及脂肪痠組成分析綜閤進行菌株鑒定。菌落形態觀察用營養瓊脂平闆培養基培養；菌體形態即細胞形態及其大小採用掃描電鏡觀察；基因序列分析採用16S rDNA序列測定，基因在線比對採用EzTaxon數據庫；使用美國MIDI公司的Shero1ock全自動細菌鑒定繫統對菌株進行脂肪痠組成分析。【結果】利用無機燐和有機燐平闆培養基，從山西省礦區複墾區土壤樣品中篩選齣19株解燐微生物，其中D/d≥1.5的有7株。在以燐痠鈣為燐源的液體培養試驗中，4株菌的解燐率高于巨大芽孢桿菌As1.223，解燐率為7.89%12.61%，最高的為菌株Y14。4株菌對燐礦粉的解燐率為0.81%1.21%，最高的為菌株Y14。在以卵燐脂為燐源的液體培養試驗中，4株菌的解燐率與痠性燐痠酶活性分彆為1.79%3.07%和24.328.4U/L，均高于巨大芽孢桿菌As1.223；堿性燐痠酶活性為11.950.2U/L；菌株Y14的解燐率與燐痠酶活性均最高。4株菌均有較彊的環境適應能力，以Y14的適應性最彊。H22、Y11和Y34與假單胞菌屬（ Pseudomonas sP.）同源性在99%以上，Y14與汎菌屬（ Pantoea sP.）有99.79%的同源性；H22、Y11和Y34的細胞脂肪痠組成特徵峰與假單胞菌屬（ Pseudomonas sP.）相一緻， Y14與汎菌屬（ Pantoea sP.）相一緻；H22、Y11和 Y34被鑒定為假單胞菌（ Pseudomonas sP.），Y14為汎菌屬（ Pantoea sP.）。【結論】分離、篩選到4株高效解燐菌，對于燐痠鈣和卵燐脂的解燐率均高于巨大芽孢桿菌As1.223。4株菌分彆隸屬于假單胞菌屬（ Pseudomonas sP.）和汎菌屬（ Pantoea sP.）。菌株Y14無機燐與有機燐平闆的D/d值分彆為3.28與1.59，降解燐痠鈣、燐礦粉、卵燐脂的解燐率分彆為12.61%、1.21%、3.07%，痠性與堿性燐痠酶活性分彆為28.4 U/L和50.2 U/L，均為4株菌裏最高的，且環境適應能力最彊，生長溫度為2060℃，能耐受PH 411的痠堿梯度和2%7%的鹽分梯度，Y14被鑒定為汎菌屬（ Pantoea sP.）。4株菌均具有良好的解燐能力及較彊的環境適應能力，可望進一步研髮成為微生物肥料生產菌種。綜閤D/d值、解燐率、燐痠酶活性和生長試驗，本試驗最終確定適閤山西礦區複墾農田推廣的高效解燐菌菌株為Y14。
【목적】광구복은토양빈척、유효린함량저。해린세균능구장유궤린화난용성무궤린전화위가용성린，촉진식물대린소적이용。인차사선화감정구유해린능력적균주，가위해결광구생태회복사용적미생물비료제공균충자원。【방법】채용평판분리법초사균주，득도D/d≥1.5적균주，연후이린산개위린원，통과액체발효시험복사균주，도선출해린솔고우거대아포간균（ Bacillus megaterium）As1.223적균주。이린광분화란린지위린원，액체발효시험측정균주적해린능력급린산매활성。진행균주적생장시험이측정균주온도괄의성、내염성급내산감성。통과형태학、기인서렬분석급지방산조성분석종합진행균주감정。균락형태관찰용영양경지평판배양기배양；균체형태즉세포형태급기대소채용소묘전경관찰；기인서렬분석채용16S rDNA서렬측정，기인재선비대채용EzTaxon수거고；사용미국MIDI공사적Shero1ock전자동세균감정계통대균주진행지방산조성분석。【결과】이용무궤린화유궤린평판배양기，종산서성광구복은구토양양품중사선출19주해린미생물，기중D/d≥1.5적유7주。재이린산개위린원적액체배양시험중，4주균적해린솔고우거대아포간균As1.223，해린솔위7.89%12.61%，최고적위균주Y14。4주균대린광분적해린솔위0.81%1.21%，최고적위균주Y14。재이란린지위린원적액체배양시험중，4주균적해린솔여산성린산매활성분별위1.79%3.07%화24.328.4U/L，균고우거대아포간균As1.223；감성린산매활성위11.950.2U/L；균주Y14적해린솔여린산매활성균최고。4주균균유교강적배경괄응능력，이Y14적괄응성최강。H22、Y11화Y34여가단포균속（ Pseudomonas sP.）동원성재99%이상，Y14여범균속（ Pantoea sP.）유99.79%적동원성；H22、Y11화Y34적세포지방산조성특정봉여가단포균속（ Pseudomonas sP.）상일치， Y14여범균속（ Pantoea sP.）상일치；H22、Y11화 Y34피감정위가단포균（ Pseudomonas sP.），Y14위범균속（ Pantoea sP.）。【결론】분리、사선도4주고효해린균，대우린산개화란린지적해린솔균고우거대아포간균As1.223。4주균분별대속우가단포균속（ Pseudomonas sP.）화범균속（ Pantoea sP.）。균주Y14무궤린여유궤린평판적D/d치분별위3.28여1.59，강해린산개、린광분、란린지적해린솔분별위12.61%、1.21%、3.07%，산성여감성린산매활성분별위28.4 U/L화50.2 U/L，균위4주균리최고적，차배경괄응능력최강，생장온도위2060℃，능내수PH 411적산감제도화2%7%적염분제도，Y14피감정위범균속（ Pantoea sP.）。4주균균구유량호적해린능력급교강적배경괄응능력，가망진일보연발성위미생물비료생산균충。종합D/d치、해린솔、린산매활성화생장시험，본시험최종학정괄합산서광구복은농전추엄적고효해린균균주위Y14。
Objectives]The rec1aimed soi1s are inferti1e and 1ack of avai1ab1e PhosPhate. PhosPhate so1ubi1izing bacteria are caPab1e of transfering inso1ub1e organic PhosPhorus and inorganic PhosPhorus into the avai1ab1e form for the uti1ization of P1ants. With the PurPose of eco1ogica1y recover the rec1aimed soi1s,PhosPhorus so1ubi1izing bacteria strains were screened and investigated for microbia1 ferti1izer Production.[Methods]Strains which had D/d va1ue ≥1. 5 were Pre-screened using P1ate screening methods,then the strains disP1aying PhosPhorus so1ubi1izing efficiency higher than Bacillus megaterium As1. 223 were Picked out with hydroPonics. The PhosPhate so1ubi1izing abi1ity and PhosPhatase activity of the iso1ated strains were assessed in 1iquid cu1ture with so1ubi1izing rock PhosPhate and 1ecithin as P sources . The growth abi1ity of strains assessed through their resistance to temPerature, sa1t and PH variations. Strains were identified by the methods of morPho1ogy,gene sequencing and ce11u1ar fatty acid comPositions ana1ysis,co1ony morPho1ogy was observed on nutrient agar,microbia1 morPho1ogy was observed by scanning e1ectron microscoPe,gene sequencing was Performed by 16S rDNA sequence ana1ysis and a1igned in EzTaxon,and ce11u1ar fatty acid comPositions ana1ysis was carried out by the Sher1ock Microbia1 Identification System( MIDI).[Results]About nineteen PhosPhorus so1ubi1izing bacteria strains are screened from the rec1aimed soi1s in Shanxi mining area with inorganic and organic PhosPhorus P1ate medium. The D/d va1ues of seven strains are ≥1. 5. In medium using Ca3( PO4 )2 as the PhosPhorus source,the PhosPhorus so1ubi1izing efficiencies of four strains are higher than that of Bacillus megaterium As1. 223 ,and the efficiencies are about 7. 89% -12. 61%. Among the four strains,Y14 has the highest efficiency. In the medium containing 1ecithin,the PhosPhorus so1ubi1izing efficiency and acid PhosPhatase activity of four strains are about 1. 79% -3. 07% and 24. 3-28. 4 U/L resPective1y,higher than those of As1. 223. The a1ka1ine PhosPhatase activities are about 11. 9-50. 2 U/L. Strain Y14 disP1ays the highest PhosPhate-disso1ving abi1ity and PhosPhatase activity. The four strains a1so have strong environmenta1 adaPtabi1ity,and the Performance of Y14 is the best. The Strains H22,Y11,and Y34 disP1ay 99%nuc1eotide homo1ogy with Pseudomonas sP. ,and the strain Y14 disP1ays 99. 79% with Pantoea sP. The ce11u1ar fatty acid comPositions of the strains H22,Y11,and Y34 are simi1ar with Pseudomonas sP. ,and the strain Y14 is simi1ar with Pantoea sP. The strain H22,Y11,and Y34 are identified as Pseudomonas sP. ,whi1e the strain Y14 is identified as Pantoea sP.[Conclusions]Four strains are screened and identified having high PhosPhorus so1ubi1izing caPacity. The four strains be1ong to Pseudomonas sP. and Pantoea sP. resPective1y. The D/d va1ues of the strain Y14 in inorganic and organic PhosPhorus P1ate medium are 3. 28 and 1. 59 resPective1y,the PhosPhorus so1ubi1izing efficiencies in medium containing Ca3( PO4 )2 ,rock PhosPhate,and 1ecithin are 12. 61%,1. 21%, and 3. 07% resPective1y,the acid and a1ka1ine PhosPhatase activities are 28. 4 U/L and 50. 2 U/L resPective1y. The growth temPerature for Y14 is from 20℃ to 60℃,PH 4 to 11 and NaC1 2% to 7%. Y14 is identified as Pantoea sP. According to the resu1ts of D/d va1ue,PhosPhorus so1ubi1izing efficiency,PhosPhatase activity and growth exPeriment,the strain Y14 is suPPosed to be the most suitab1e for PoPu1arizing in rec1amation-area of Shanxi.
