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1 土壤 (Soils), 2018, 50(5): DOI: /j.cnki.tr 一株高效解钾脱硅真菌的筛选鉴定及培养条件优化 * ( ) (Stenoloma chusanum) WJ007 rdna ITS Penicillium spinulosum WJ007 WJ ph mg/l WJ ph mg/l WJ007 S A [1-2] [3] [4-7] [8-10] (Stenoloma chusanum) ( ~ E ~ 25 1 N) K 2 O 11.09% Al 2 O % SiO % 100 ( ) (20171BAB204007) * (chenyejjtc@126.com) (1971 ) @qq.com

2 PDA [11-12] ) PDA (Φ=0.5 cm) 28 4 ~ 5 d 2) (Φ=0.5 cm) 50 ml/150 ml r/min 15 d 3 [13] [14] Optima 8000 (Perkin-Elmer ) [15-16] ( ) (mg/l) A =(b 1 b 0 ) A ( ) (mg/l) b 1 ( ) (mg/l) b 0 ( ) (mg/l) 3) d (mg/l) 3 4) SEM (TESCAN VEGIILSU) (EDS) X XRD( Rigaku D/Max 2500 ) [16] ) [17-18] 2) DNA Barnett Hunter [19] Guo [20] SDS-CTAB DNA 2 DNA PCR [21] Primer 1 (ITS1 5 -TCCGTAGGTGAACCTGCGG-3 ) Primer 4(ITS4 5 -TCCTCCGCTTATRGATATGC-3 ) DNA ITS1 ITS2 5.8S rrna (50 μl) Primer 1 Primer μl DNA Buffer 2.5 μl dntps 2 μl Taq E 0.4 μl DNA 1 μl ( ) ddh 2 O 17.6 μl PCR 94 2 min 94 1 min s s min PCR (QiaQuick PCR purification Kit Qiagen) ( ) 3 NCBI MEGA5.0 N-J BootsVap(Bootstrap) WJ ph [21] r/min 15 d 3 L 9 (3 4 ) Origin7.5 MEGA ( WJ005 WJ006 WJ007 WJ010 WJ012 WJ064 WJ067 WJ074) ~ mg/l ~1.86 mg/l WJ mg/l( 1A B) WJ d 8 WJ007

3 Fig. 1 图 1 不同菌株对钾长石作用能力的筛选 (A: 解钾能力 ;B: 脱硅能力 ) Potassium-solubilization capacity (A) and desilication capacity (B) of different strains on potassium feldspar K + Si 4+ [17,22] 图 2 不同菌株在含钾长石培养基中的菌株生物量变化曲线 Fig. 2 Biomass curves of different strains in potassium-bearing feldspar medium 2.3 WJ d ( 3) WJ007 ( 4) WJ007 WJ007 WJ007 EDS ( 5) 5 K + Si 4+ WJ007 K + Si 4+ (A WJ007 B WJ007 ) 图 3 WJ007 菌株对钾长石作用的体视显微镜照片 Fig. 3 Stereomicroscope images of interaction of WJ007 strain on potassium feldspar 2.4 WJ007 [23] Penicillium Subgenus Aspergilloides Penicillium spinulosum Thom( 6) ITS1/ITS4 WJ007 rdna ITS PCR PCR 590 bp GenBank(NCBI) Megablast (blastn) WJ007 ITS P. spinulosum ITS 100% WJ007 P. spinulosum WJ007 GenBank

第5期张红芳等一株高效解钾脱硅真菌的筛选鉴定及培养条件优化 937 (A WJ007 菌株作用前的钾长石 B C WJ007 菌株孢子吸附在钾长石上 D E WJ007 菌株菌丝吸附在钾长石上 F G 发酵培养第 9 天 WJ007 菌株对钾长石的作用 H 发酵培养第 12 天 WJ007 菌株对钾长石的作用 I J

4 第5期张红芳等一株高效解钾脱硅真菌的筛选鉴定及培养条件优化 937 (A WJ007 菌株作用前的钾长石 B C WJ007 菌株孢子吸附在钾长石上 D E WJ007 菌株菌丝吸附在钾长石上 F G 发酵培养第 9 天 WJ007 菌株对钾长石的作用 H 发酵培养第 12 天 WJ007 菌株对钾长石的作用 I J 发酵培养第 15 天后 WJ007 菌株对钾长石的作用 K WJ007 菌株作用后的矿样 L 发酵培养第 15 天接种灭活菌株矿样) 图4 Fig. 4 WJ007 菌株对钾长石作用的扫描电镜照片 SEM images of interaction of WJ007 strain on potassium feldspar

938 50 (A WJ007 B WJ007 ) 图 5 WJ007 菌株作用后钾长石表面 EDS 能谱 Fig. 5 Energy dispersive spectrum of potassium feldspar under WJ007 strain (A B C ) 图 6 WJ007 菌株形态特征 Fig.

5 (A WJ007 B WJ007 ) 图 5 WJ007 菌株作用后钾长石表面 EDS 能谱 Fig. 5 Energy dispersive spectrum of potassium feldspar under WJ007 strain (A B C ) 图 6 WJ007 菌株形态特征 Fig. 6 Morphological characters of WJ007 strain ( 7) WJ007 GenBank P. spinulosum( KM KM KM AF034461) 99% WJ007 P. spinulosum ( 50% ) 图 7 基于 ITS 序列借助最大似然法构建的青霉属系统发育树 Fig. 7 Molecular phylogenetic tree of Penicillium inferred from ITS sequences using maximum likelihood method 2.5 WJ ph 4 WJ WJ ph ph 6

6 5 939 Fig. 8 图 8 不同培养条件对 WJ007 菌株解钾脱硅作用的影响 Effects of different culture conditions on potassium-solubilization and desilication of WJ007 strain ( 1) L 9 (3 4 ) WJ007 3 WJ007 > ph> > T K1 T K2 T K3 A 2 B 3 C 2 D 3 ( 60 1 ph 7) WJ mg/l WJ007 > > ph> T Si1 T Si2 T Si3 A 2 B 1 C 2 D ph 6 WJ007 ( 2) 9.74 mg/l ( 3) 表 1 正交试验水平与因素表 (L3 4 ) Table 1 Level and factor of orthogonal test(l3 4 ) A B C D ph 表 2 WJ007 菌株解钾脱硅正交试验结果 Table 2 Orthogonal experimental results of potassium-solubilization and desilication of WJ007 strains A B C D (mg/l) (mg/l)

7 A B C D (mg/l) (mg/l) T Si1 (T K1 ) 19.67(197.53) 22.91(204.43) 22.72(208.35) 19.98(194.25) T Si2 (T K2 ) 23.00(265.05) 20.64(217.03) 22.99(228.13) 22.94(203.36) T Si3 (T K3 ) 22.48(180.58) 21.60(221.70) 19.44(206.68) 22.23(245.55) t Si1 (t K1 ) 6.56(65.84) 7.64(68.14) 7.57(69.45) 6.66(64.75) t Si2 (t K2 ) 7.67(88.35) 6.88(72.34) 7.66(76.04) 7.65(67.79) t Si3 (t K3 ) 7.49(60.19) 7.2(73.90) 6.48(68.89) 7.41(81.85) R 3.33(84.47) 2.27(17.27) 3.55(21.45) 2.96(51.30) Si C>A>D>B K A>D>C>B Si A 2 B 1 C 2 D 2 K A 2 B 3 C 2 D 3 T=65.18 T= A B C D ph T 1 T 2 T 3 T t 1 t 2 t 3 R 表 3 方差分析 Table 3 Variance analysis SS df MS F P ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ph ( ) ( ) ( ) ( ) ( ) ( ) ( ) WJ007 WJ WJ007 A 2 B 1 C 2 D mg/l 8.74 mg/l WJ007 A 2 B 3 C 2 D mg/l mg/l WJ WJ007 rdna-its Penicillium Penicillium spinulosum WJ ph mg/l 60 1 ph mg/l WJ007 [1],. [J]., 2001, 32(4): [2],,. [J]., 2005, 36(2): [3],,,. [J]., 2012, 24(9): , 785 [4],,. [J]., 2000, 23(1): [5],,,.

8 5 941 [J]., 2016, 48(1): [6] Badr M A, Shafei A M, Sharaf El-Deen S H. The dissolution of K and P-bearing minerals by silicate dissolving bacteria and their effect on sorghum growth[j]. Research Journal of Agriculture and Biological Sciences, 2006, 2(1): 5 11 [7],,,. [J]., 2013, 52(21): [8]. [J]., 2015, 31 (5): [9],,,. [J]., 2016, 36(3): [10] Lugo M A, Reinhart K O, Menoyo E, et al. Plant functional traits and phylogenetic relatedness explain variation in associations with root fungal endophytes in an extreme arid environment [J]. Mycorrhiza, 2015, 25(2): [11],,,. 1 [J]., 2013, 41(10): [12],,,. [J]., 2013, 22(6): [13],,,. [J]., 2017, 49(2): [14],,,. [J]., 2014, 51(3): [15],,,. [J]., 2014, 51(2): [16]. [J]., 2018, 50(1): [17],,,. [J]., 2014, 47(3): [18] Keith S, Gareth M J, Walter G, et al. The genera of hyphomycetes[m]. Utrecht, The Netherlands: CBS-KNAW Fungal Biodiversity Centre, 2011: [19] Barnett H L, Hunter B B. Illustrated genera of imperfect fungi (4 Ed.)[M]. St Paul, Minnesota: APS Press, 1998: [20] GUO Ling-dong, Hyde K D, Liew E C Y Deteetion and identification of endophyte fungi with in frond tissues of Livlstona chinensis based on rdna sequence[j]. Molecular Phylogenetics and Evolution, 2001, 20: 1 13 [21],. (Aspergillus oryzae) [J]., 2011, 36(4): [22],,,. 2 IAA [J]., 2017(3): [23]. ( 35 ): [M]. :, 2007 Screening, Identification and Cultivated Condition Optimization of High Efficiency Potassium Solubilization and Desilication Fungus Strain ZHANG Hongfang, HE Gang, WU Lvying, SUN Desi, CHEN Ye * (School of Pharmacy and Life Sciences, Jiujiang University, Jiujiang, Jiangxi , China) Abstract: In order to excavate the functional endophyte fungi resources, a fungus strain, named WJ007 with high-efficiency potassium solubilization and desilication was screened from Stenoloma chusanum, it was identified as Penicillium spinulosum based on morphological characteristics, rdna ITS sequence and phylogenetic relationships analyses. Its optimal culture conditions obtained by single factor and orthogonal experimens for desiliconization were glucose as carbon source, yeast extract as nitrogen source, carbon and nitrogen ratio of 60 1, initial ph 6, and culture soluble silicon content in liquid is 8.74 mg/l under these conditions; while its optimal culture conditions for potassium solubilization were glucose as carbon source, protein peptone as nitrogen source, carbon and nitrogen ratio of 60 1, initial ph 7, and soluble potassium contents in culture solution is mg/l under these conditions. It is significant for the development and utilization of plant endophytic fungi to optimize fermentation conditions of potassium solubilization and desiliconization for WJ007. Key words: Endophytic fungi; Potassium-solubilization; Desilication; Isolation; Identification; Fermentation condition

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%