Screening of Endophytic Antagonistic Bacterium from Phellodendron amurense and Their Biocontrol Effects against Canker Rot

Abstract

Thirty-four strains of bacteria were isolated from Phellodendron amurense. Using Nectria haematococca as an indicator strain, the best strain, B18, was obtained by the growth rate method. The morphological, physiological and biochemical characteristics of strain B18 and its 16S DNA gene sequence were identified, and the biocontrol effect of strain B18 was assessed in pot and field tests, as well as in a field-control test. Drilling methods were used to determine the antibacterial activity of metabolites from strain B18 and their effects on the growth of pathogen mycelia and spores. The best bacteriostatic rate was 85.4%. B18 can hydrolyse starch and oxidize glucose but does not produce gas; a positive result was obtained in a gelatine liquefaction test. According to 16S DNA gene sequencing, strain B18 is Bacillus methylotrophicus (GenBank accession number: MG457759). The results of pot and field-control trials showed 98% disease control when inoculating $10^8cfu/ml$ of the strain. The disease control effect of the B18 culture liquid (concentrations of $10^8$, $2{\times}10^6$, $10^6$, $5{\times}10^5$ and $2.5{\times}10^5cfu/ml$) in the field-control test was higher than 80%, and the cure rate of the original delivery solution was 96%. Therefore, in the practical forestry production, a $2.5{\times}10^5cfu/ml$ culture liquidshould be applied in advance to achieve good control effects.

Fig. 1. Neighbour-joining tree based on analysis of partial 16S rDNA nucleotide sequences of the selected bacterial strain B18. The percentage of replicate trees in which associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980); the results are presented in units of the number of base substitutions per site. In phylogenetic tree, Bacillus subtilis is one type strain.

Fig. 2. Antagnistic effect of culture filtrate of B. methylotrophicus B18. (A) Growth inhibition curve by the B18 solution on mycelium of the pathogenic fungusError bars indicate means ± SD (n = 3); ddifferent lowercase letters indicate significant difference between controls and treatments at different times at P < 0.05 by the LSD test. (B) Antagonistic effect of the B18 bacterial solution (10⁶ cfu/ml) against a pathogenic fungus. (C) Effect of the fermentation sterile culture filtrate on mycelial morphology of the pathogenic fungus. a: Normal mycelium; b: The concentration of biocontrol bacteria B18 is 10⁶ cfu/ml; c: The concentration of biocontrol bacteria B18 is 10⁸ cfu/ml. (D) Effect of the fermentation sterile culture filtrate on spores of the pathogenic fungus. a: Germination of spores at a concentration of 1%; b: Germination of spores at a concentration of 50%.

Table 2. Physiological and biochemical characteristics of strain B18

Table 1. Inhibitory effects of five bacterial strains (culture 5 days)

Table 3. Control effect of the endophytic bacterium strain B18 after 30 days in a pot experiment

Table 4. Effect of the endophytic bacterium strain B18 on canker rot in Phellodendron amurense in a field experiment

Table 5. Inhibitory effect of B18 sterile culture filtrate on the germination of Nectria haematococca spores