Plant Growth Promotion and Biocontrol of Leaf Blight Caused by Nigrospora sphaerica on Passion Fruit by Endophytic Bacillus subtilis Strain GUCC4

Abstract

Passion fruit (Passiflora edulis Sims) is widely cultivated in tropic and sub-tropic regions for the production of fruit, flowers, cosmetics, and for pharmacological applications. Its high economic, nutritional, and medical values elicit the market demand, and the growing areas are rapidly increasing. Leaf blight caused by Nigrospora sphaerica is a new and emerging disease of passion fruit in Guizhou, in southwest China, where the unique karst mountainous landscape and climate conditions are considered potential areas of expansion for passion fruit production. Bacillus species are the most common biocontrol and plant-growth-promotion bacteria (PGPB) resources in agricultural systems. However, little is known about the endophytic existence of Bacillus spp. in the passion fruit phyllosphere as well as their potential as biocontrol agents and PGPB. In this study, 44 endophytic strains were isolated from 15 healthy passion fruit leaves, obtained from Guangxi province, China. Through purification and molecular identification, 42 of the isolates were ascribed to Bacillus species. Their inhibitory activity against N. sphaerica was tested in vitro. Eleven endophytic Bacillus spp. strains inhibited the pathogen by >65%. All of them produced biocontrol- and plant-growth-promotion-related metabolites, including indole-3-acetic acid (IAA), protease, cellulase, phosphatase, and solubilized phosphate. Furthermore, the plant growth promotion traits of the above 11 endophytic Bacillus strains were tested on passion fruit seedlings. One isolate, coded B. subtilis GUCC4, significantly increased passion fruit stem diameter, plant height, leaf length, leaf surface, fresh weight, and dry weight. In addition, B. subtilis GUCC4 reduced the proline content, which indicated its potential to positively regulate passion fruit biochemical properties and resulted in plant growth promotion effects. Finally, the biocontrol efficiencies of B. subtilis GUCC4 against N. sphaerica were determined in vivo under greenhouse conditions. Similarly to the fungicide mancozeb and to a commercial B. subtilis-based biofungicide, B. subtilis GUCC4 significantly reduced disease severity. These results suggest that B. subtilis GUCC4 has great potential as a biological control agent and as PGPB on passion fruit.

Keywords: antagonisms; endophytic Bacillus; enzyme activity; passion fruit; plant growth promotion.

Figure 1

Inhibition of N. sphaerica by 11 Bacillus strains in comparisons to the control, synthetic fungicide mancozeb, and reference strain B. subtilis NCD_2. Treatments, (ck): control, (a): mancozeb, (b): B. subtilis NCD_2, (c): B. subtilis GUCC8, (d): B. subtilis GUCC4, (e): B. cereus GUCC7, (f): B. cereus GUCC3, (g): B. cereus GUCC6, (h): B. cereus GUCC9, (i): B. cereus GUCC11, (j): B. cereus GUCC2, (k): B. cereus GUCC5, (l): B. cereus GUCC1001, (m): B. cereus GUCC10.

Figure 2

Phylogenetic tree based on 16S rRNA (A) gene and gyrB (B) gene sequences. The strains from this study were highlighted in bold.

Figure 3

(A). Inhibition of N. sphaerica by volatile compounds of 11 Bacillus strains treatments, ck: control, a: B. subtilis GUCC8, b: B. subtilis GUCC4, c: B. cereus GUCC7, d: B. cereus GUCC3, e: B. cereus GUCC6, f: B. cereus GUCC9, g: B. cereus GUCC11, h: B. cereus GUCC2, i: B. cereus GUCC5, j: B. cereus GUCC1001, k: B. cereus GUCC10. (B). Inhibition of N. sphaerica by culture filtrate of 11 Bacillus strains treatments, ck: control, a: B. subtilis GUCC8, b: B. subtilis GUCC4, c: B. cereus GUCC7, d: B. cereus GUCC3, e: B. cereus GUCC6, f: B. cereus GUCC9, g: B. cereus GUCC11, h: B. cereus GUCC2, i: B. cereus GUCC5, j: B. cereus GUCC1001, k: B. cereus GUCC10. (C). Inhibition of N. sphaerica by culture filtrate of 11 Bacillus strains. Data in the table are presented as the means ± standard deviation. Different letters in the same column indicate statistical significance (p < 0.05).

Figure 4

The effects of different Bacillus strains on total chlorophyll content (A), SOD (B), POD (C), CAT (D) enzyme activities, total MDA (E), and Pro content (F) in passion fruit leaves. All data represents the means ± standard deviation for three biological replicates. Values with different letters in the same column indicate statistical significance (p < 0.05).