Fungal Disease Prevention in Seedlings of Rice (Oryza sativa) and Other Grasses by Growth-Promoting Seed-Associated Endophytic Bacteria from Invasive Phragmites australis

Abstract

Non-cultivated plants carry microbial endophytes that may be used to enhance development and disease resistance of crop species where growth-promoting and protective microbes may have been lost. During seedling establishment, seedlings may be infected by several fungal pathogens that are seed or soil borne. Several species of Fusarium, Pythium and other water moulds cause seed rots during germination. Fusarium blights of seedlings are also very common and significantly affect seedling development. In the present study we screened nine endophytic bacteria isolated from the seeds of invasive Phragmites australis by inoculating onto rice, Bermuda grass (Cynodon dactylon), or annual bluegrass (Poa annua) seeds to evaluate plant growth promotion and protection from disease caused by Fusarium oxysporum. We found that three bacteria belonging to genus Pseudomonas spp. (SLB4-P. fluorescens, SLB6-Pseudomonas sp. and SY1-Pseudomonas sp.) promoted seedling development, including enhancement of root and shoot growth, and stimulation of root hair formation. These bacteria were also found to increase phosphate solubilization in in vitro experiments. Pseudomonas sp. (SY1) significantly protected grass seedlings from Fusarium infection. In co-culture experiments, strain SY1 strongly inhibited fungal pathogens with 85.71% growth inhibition of F. oxysporum, 86.33% growth inhibition of Curvularia sp. and 82.14% growth inhibition of Alternaria sp. Seedlings previously treated with bacteria were found much less infected by F. oxysporum in comparison to non-treated controls. On microscopic observation we found that bacteria appeared to degrade fungal mycelia actively. Metabolite products of strain SY1 in agar were also found to inhibit fungal growth on nutrient media. Pseudomonas sp. (SY1) was found to produce antifungal volatiles. Polymerase chain reaction (PCR) amplification using specific primers for pyrrolnitirin synthesis and HCN (hydrogen cyanide) production suggested presence of genes for both compounds in the genome of SY1. HCN was detected in cultures of SY1. We conclude that microbes from non-cultivated plants may provide disease protection and promote growth of crop plants.

Keywords: antifungal activity; biocontrol; disease suppression; seedling development.

Figure 1

(a) Rice seedlings inoculated with bacteria (H₂O only or strains SLB4, SLB6 and SY1) in magenta boxes containing potting mix. (b) Seedlings showing differences in root and shoot lengths between control and SY1-treated rice seedlings after 15 days grown in potting mix.

Figure 2

Antifungal activity of SY1 (Pseudomonas sp.) against F. oxysporum, Curvularia sp. and Alternaria sp.: Where first set (a–c) are antagonism in dual culture method; second set (d–f) are antifungal by agar diffusion (using bacterial-free plugs of agar from plates where bacteria were grown for five days); third set (g–i) antifungal activity by volatiles produced by SY1.

Figure 2

Antifungal activity of SY1 (Pseudomonas sp.) against F. oxysporum, Curvularia sp. and Alternaria sp.: Where first set (a–c) are antagonism in dual culture method; second set (d–f) are antifungal by agar diffusion (using bacterial-free plugs of agar from plates where bacteria were grown for five days); third set (g–i) antifungal activity by volatiles produced by SY1.

Figure 3

Inoculation of SY1 (Pseudomonas sp.) onto rice seeds, protecting rice seedlings from Fusarium oxysporum infection. First set is in Petri plates with water (three-days-old); second set in 0.7% agarose plates (eight-days-old) and third set in magenta boxes containing potting mix (15-days-old).

Figure 3

Inoculation of SY1 (Pseudomonas sp.) onto rice seeds, protecting rice seedlings from Fusarium oxysporum infection. First set is in Petri plates with water (three-days-old); second set in 0.7% agarose plates (eight-days-old) and third set in magenta boxes containing potting mix (15-days-old).

Figure 4

Protection of Bermuda grass (a,b) and annual bluegrass (c,d) seedlings on 0.7% agarose from F. oxysporum infection by SY1 isolate. More surviving seedlings are evident in (b,d) where bacterium SY1 and the fungus were present, than in (a,c) where only the fungus was present.

Figure 5

Microscopic view of roots (stained with diaminobenzidine tetrachloride (DAB)); In Bermuda grass (a–c): Where control (a) is infected with F. oxysporum (arrow indicates hypha within root hair); SY1-treated (b,c) found free of hyphae, but bacterial L-forms are visible inside root hairs (arrows). In annual bluegrass (d–g): (d,f) are control annual bluegrass root (d) and root parenchyma (f) colonized by fungus (arrows), and (e,g) are treated with SY1 and found free of infection by fungus F. oxysporum.

Figure 6

F. oxysporum from rice seedlings (magnification = 1000×). Where (a,b) are controls (without SY1 bacterial treatment) showing mycelium and conidia without bacteria, and (c–g) are treated with the bacterium SY1 showing degrading mycelium (arrows). ((a–d) are stained with cotton blue and (e–g) are stained with SYTO@13).

Figure 7

Polymerase chain reaction (PCR) products of different antibiotic genes amplified. Where M = marker, C = control, 1 = PHZ, 2 = phzCD, 3 = phze, 4 = phzF (1-4 related with phenezine synthesis), 5 = phlD-2,4-Diacetylphloroglucinol, 6 = prnD-pyrrolnitrin, 7 = hcnBC-hydrogen cyanide, 8 = PLTC-pyoleutirin. Lanes 6 and 7 suggest genes for pyrrolnitrin and HCN synthesis, respectively.