Phosphate solubilizing effect by two paraburkholderia bacteria Isolated from button mushroom medium

양송이배지로부터 분리한 두 Paraburkholderia 속 세균에 의한 인산가용화 효과

Abstract

The present study was conducted to investigate the synergistic effects caused by single and co-inoculation of the phosphate solubilizing bacteria (PSB), Paraburkholderia phenazinium YH3 and Paraburkholderia metrosideri YH4. Phosphate solubilization was assessed by measuring the phosphorus contents for 7 days in a single and co-inoculation medium. Co-inoculation of the two strains was found to release the highest content of soluble phosphorus ($1,250{\mu}g\;mL^{-1}$) into the medium, followed by the single inoculation of P. metrosideri YH4 ($1196.59{\mu}g\;mL^{-1}$) and P. phenazinium YH3 ($994.34{\mu}g\;mL^{-1}$). The highest pH reduction, organic acid production and glucose consumption was also observed in the co-inoculation medium of the two strains. A plant growth promotion bioassay revealed that co-inoculation with the two strains enhanced the growth of romaine lettuce more than single inoculation with either of the two strains (28.5% for leaf and 16.6% for root). Although there was no significant difference between single and co-inoculation of bacterial strains in terms of phosphorous release and plant growth, the synergistic effects of co-inoculation with PSB could be beneficial for crop growth.

양송이배지로부터 분리한 인산가용화균 Paraburkholderia phenazinium YH3와 Paraburkholderia metrosideri YH4의 단일접종 및 동시접종에 따른 인산가용화 능력의 시너지효과 및 상추생육효과를 조사하였다. 인산캄슘이 함유된 NBRIP 액체배지에 분리균을 접종하고 해리된 가용인산(soluble phosphorus)함량을 HPLC에 의해 분석하여 인산가용화능을 측정한 결과, 배양 3일차에 동시접종구 $1,250{\mu}g\;mL^{-1}$, P. metrosideri $1196.59{\mu}g\;mL^{-1}$, P. phenazinium $994.34{\mu}g\;mL^{-1}$의 순으로 나타나 두 종의 인산가용화 박테리아간의 동시 접종에 의한 시너지효과가 관찰되었다. 배지 내 pH 와 잔류 glucose 함량 변화도 모든 접종구에서 배양 1일 후 대부분의 변화가 이루어져 pH의 경우 초기 pH 7.0에서 pH 4.0 수준으로 감소하였으며, 잔류 glucose 함량은 초기 $10mg\;mL^{-1}$에서 $4.8mg\;mL^{-1}$ 수준으로 검출되어 인산가용화능과 거의 유사한 경향을 보였다. 배양여액의 유기산 분석결과 gluconic acid 약 $11mg\;mL^{-1}$와 malic acid 약 $4mg\;mL^{-1}$ 수준으로 가장 높게 검출되었으며, Park et al(2016)에 의해 보고된 oxalic acid는 검출되지 않았다. 대부분의 유기산이 배양 1일과 3일 후 생성되는 결과를 보임으로서 유기산이 배지의 pH를 감소시키고 인산 가용화를 유도하는 주요 원인임을 확인할 수 있었다. 접종 4주 후 상추 생육차이를 조사한 결과, TCP를 첨가하지 않은 P. phenazinium 접종구, P. metrosideri 접종구 와 동시 접종구에 비해 TCA 첨가한 모든 처리구에서 10 -20% 수준의 생육증진효과가 확인되었으나 단일접종과 동시접종구간에 유의수준의 차이를 확인할 수 없었다. 결과적으로 인(P) 결핍 토양 및 염류토양에 인산가용화균을 사용하면 식물과 작물 수확량에 의한 P 섭취가 동시에 증가하고 생육이 촉진된다는 다른 연구결과(Rodriguez et al, 1999; Suh et al, 2008; Walpola and Yoon. 2013)와 유사한 경향을 보였다.

Fig. 1. Phylogenetic tree based on 16S rDNA gene sequences, showing the position of isolate phosphate solubilizing bacterial strains YH3 (PSB-C) with respect to related species. The scale bar dindicates 0.005 substitutions per nucleotide position and accession numbers are given in parenthesis.

Fig. 2. Phylogenetic tree based on 16S rDNA gene sequences, showing the position of isolate phosphate solubilizing bacterial strains YH4 (PSB-D) with respect to related species. The scale bar dindicates 0.005 substitutions per nucleotide position and accession numbers are given in parenthesis.

Fig. 3. Comparison of phosphate solubilization between single and co-inoculation of P. phenazinium and P. metrosideri. Values given here are the means of three replicates (n=3).

Fig. 4. Comparison of pH change during single and coinoculation of P. phenazinium and P. metrosideri. Values given here are the means of three replicates (n=3).

Fig. 5. Comparison of residual glucose in the medium between single and co-inoculation of P. phenazinium and P. metrosideri. Values given here are the means of three replicates (n=3)

Table 1. HPLC analysis of organic acid by single and co-inoculation of (Paraburkholderia phenazinium and Paraburkholderia metrosideri) Values given here are the means of three replicates (n=3). ND = Not dectected.

Table 2. Comparison on growth of romaine lettuce after 4 weeks inoculation with Paraburkholderia phenazinium and Paraburkholderia metrosideri. Values given here are the means of three replicates (n=3).