短小芽胞杆菌DX01菌株Tn5转座突变株的抑菌活性筛选体系的建立

Establishment of a screening system for Tn5 insertion mutants of Bacillus pumilus DX01 with different anti-phytopathogenic activities

为建立短小芽胞杆菌Bacillus pumilus DX01菌株的Tn5转座突变株抑菌活性的高效筛选体系,采用发酵液平板抑菌法研究了各突变株发酵液对稻瘟病菌Magnaporthe grisea生长的影响,并测定了目标突变株的几丁质酶和蛋白酶活性及发酵液对稻瘟病菌分生孢子萌发的抑制率。从2 633个突变株中筛选出6个抑菌活性较对照菌株DX01显著变化的突变株。对照DX01发酵液对真菌孢子萌发的抑制率为36%,而突变株Tn5-901和Tn5-194则分别为96%和3%,抑菌活性与对照的差异达到极显著水平。其余4个突变株的几丁质酶、蛋白酶活性多重比较结果与发酵液平板抑菌结果并不完全一致,但发酵液平板抑菌法简单、高效,适用于短小芽胞杆菌突变株的抑菌活性初筛。

Inhibition of mycelial growth and conidial germination of the phytopathogenic fungus, Magna- porthe grisea by zymotic fluids of Bacillus pumilus mutants and enzymatic activities of chintinase and pro- teinase of the identified mutants were analyzed in order to establish an efficient screening system for an- tagonistic abilities in Tn5 transposon library of the B. pumilus strain DX01 and further to clone genes asso- ciated with anti-phytopathogenic activity. A total of 2 633 mutants were subjected to anti-phytopathogenic activity screening and six mutants were preliminarily identified. The tests revealed that the inhibition per- centage of M. grisea conidia that respectively treated with zymotic fluids of mutants TnS-901 and Tn5-194 and the wild-type strain DX01 were 96% , 3% , and 36%. A statistically significant difference in anti- bacterial activity between the two mutants and the control strain DX01 were found, but the other four mu- tants did not exhibit a unanimous result in the multiple comparison tests for enzymatic activities and inhi- bition of mycelial growth with bacterial zymotic fluids. In conclusion, the evaluation of inhibition of fun- gal mycelial growth with spreading zymotic fluids of biocontrol bacterium is simple and high efficient, and this approach is optimal for preliminary screening of anti-phytopathogenic activity in B. pumilus.