Abstract

Seed endophytic bacteria (SEB) are primary symbionts that play crucial roles in plant growth and development. The present study reports the isolation of seven culturable SEB including Kosakonia cowanii (KAS1), Bacillus subtilis (KAS2), Bacillus tequilensis (KAS3), Pantoea stewartii (KAS4), Paenibacillus dendritiformis (KAS5), Pseudomonas aeruginosa (KAS6), and Bacillus velezensis (KAS7) in pearl millet seeds. All the isolates were characterized for their plant growth promoting activities. Most of the SEB also inhibited the growth of tested fungal phytopathogens in dual plate culture. Removal of these SEB from seeds compromised the growth and development of seedlings, however, re-inoculation with the SEB (Kosakonia cowanii, Pantoea stewartii, and Pseudomonas aeruginosa) restored the growth and development of seedlings significantly. Fluorescence microscopy showed inter and intracellular colonization of SEB in root parenchyma and root hair cells. Lipopeptides were extracted from all three Bacillus spp. which showed strong antifungal activity against tested fungal pathogens. Antifungal lipopeptide genes were also screened in Bacillus spp. After lipopeptide treatment, live-dead staining with fluorescence microscopy along with bright-field and scanning electron microscopy (SEM) revealed structural deformation and cell death in Fusarium mycelia and spores. Furthermore, the development of pores in the membrane and leakages of protoplasmic substances from cells and ultimately death of hyphae and spores were also confirmed. In microcosm assays, treatment of seeds with Bacillus subtilis or application of its lipopeptide alone significantly protected seedlings from Fusarium sp. infection.

Highlights

  • Plants have evolved with continuous interaction with diverse microorganisms

  • A total of seven types of endophytic bacteria were isolated from pearl millet seeds on nutrient agar media

  • Past studies have shown that seedassociated microbes, including bacteria and fungi, improve seed germination and seedling establishment (Puente et al, 2009; FIGURE 5 | Measurement of minimum inhibitory concentration (MIC) of lipopeptide against Fusarium sp. (A)

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Summary

Introduction

Plants have evolved with continuous interaction with diverse microorganisms. Many of these microorganisms actively colonize into the endospheric compartment of plants as endophytes and provide benefits to plants (Glassner et al, 2018; White et al, 2019). Seed endophytic bacteria (SEB) are believed to have more influence on the development of plant because they can be transmitted to the generation and become the first colonizers of roots and shoots of the seedlings after germination (Johnston-Monje and Raizada, 2011; Verma et al, 2019). In comparison to other plant-associated microbes, SEB are more competent in benefiting hosts, but very little has been explored regarding their mechanisms of colonization during a seedling’s formation, or their functional roles in seedling development and protection. We hypothesized that seeds of pearl millet might be inhabited by endophytic bacteria that play a crucial role during the early development of seedlings, and protect them from fungal pathogens. Fluorescent and, scanning electron microscopy (SEM), we examined the lipopeptide effects on fungal hyphae and spores

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