Agriculturally Beneficial Endophytic Bacteria of Wild Plants.

Abstract

Endophytic bacteria colonize internal plant tissues and can improve plant growth under normal and stressed conditions. Most past work has focused on the endophytic bacteria of agronomic plants. Wild perennial plants remain little investigated for their endophytic diversity. Unlike crop plants, wild plants are constantly challenged by adverse environmental conditions. Choosing the right endophytic partner can abet wild plants to survive better under adversity. Since endophytic bacteria can have broad host range, identifying agriculturally beneficial endophytic bacteria of wild plants can have great agricultural significance. Therefore, the present work focused on the isolation and characterization of plant beneficial endophytic bacteria from wild perennial plants to assess their growth promoting potential on Canola (Brassica napus). Moreover, in-planta gene expression profiling of a model endophytic bacterium was also undertaken to identify the bacterial traits important during endophytic growth. Endophytic bacteria were isolated from two wild perennial plants, Cannabis sativa L. and Dodonaea viscosa L. For C. sativa, two different approaches were used to isolates endophytic bacteria. The first approach involved direct isolation from C. sativa roots while the second novel approach utilized canola plants to selectively isolate endophytes from C. sativa rhizosphere. Selective method isolated 18 distinct bacteria while direct method isolated 16 bacteria. The bacteria were identified using 16S rRNA gene sequence. The selective method yielded 13 unique bacterial genera while the direct method isolated 11 genera. Overall, the most abundant genera were Acinetobacter, Chryseobacterium, Enterobacter, Microbacterium, Nocardioides, Paenibacillus Pseudomonas, Stenotrophomonas. Moreover, six (33.3%) isolates from selective method significantly promoted canola root growth under gnotobiotic conditions, as compared to two (12.5%) isolates from direct method. The bacteria that significantly promoted canola growth were found to be phylogenetically related. Furthermore, C. sativa roots contained 8×107 cfu/gfw bacterial cells. From the second wild plant used for isolating endophytic bacteria, D. viscosa , the bacteria were isolated from roots of the healthy plants, which contained 1×107 cfu/gfw bacterial cells. Bacterial identification based on 16S rRNA gene sequence revealed 11 distinct bacterial genera, where Bacillus, Xanthomonas and Streptomyces were the most predominant. Many isolates (67%) significantly promoted canola root growth; Bacillus, Pseudomonas and Xanthomonas were prominent genera in this regard. These bacteria were also found to be phylogenetically related, strengthening the argument that plant growth promoting ability of the endophytic bacteria is evolutionary conserved. Many traits required for plant growth promotion and colonization were detected in the endophytic isolates from C. sativa and D. viscosa. Most of the bacteria produced plant cell-wall degrading enzymes (cellulase and pectinase) needed for systemic plant colonization. Moreover, all isolates produced phytohormone IAA, where majority of the growth promoting isolates produced IAA in the range 2-10 μg/ml. The isolates also possessed phosphate solubilization and siderophore production. Moreover, seven isolates of C. sativa and D. viscosa inhibited the growth of two phytopathogenic fungi Aspergillus niger and Fusarium oxysporum. Most pronounced antifungal activity was observed for the genera Bacillus, Paenibacillus, Pseudomonas and Streptomyces. All the fungal antagonists produced fungal cell-wall targeting enzymes, chitinase and protease. Burkholderia phytofirmans PsJN is a model endophytic bacterium that can promote growth of a variety of non-host plants. Expression of 15 selected strain PsJN genes was analyzed and compared between bacterial growth in-planta and growth on M9 minimal media. For this, strain PsJN was vacuum infiltrated into tomato plants, and inoculum dose was first optimized for maximum bacterial extraction from leaf tissues. Out of four inoculum doses tested, the dose 109 cfu/ml yielded the highest cells counts (108 cfu/gfw on second day) and was used to inoculate plants for gene expression study. Compared to M9 grown bacteria, total bacterial RNA extracted from in-planta bacteria was lower in quantity and quality, but RNA was analyzable using qPCR. Bacterial gene expression was analyzed and compared between in-planta and M9 bacteria using relative quantification qPCR. Eight genes were upregulated during in-planta growth: Cellulase and Pectinase (plant colonization), IAA degradation and ACC deaminase (plant growth promotion), β-xylosidase and β-galactosidase (sugar metabolism), peroxidase (oxidative stress reduction) and one quorum sensing gene (cell-cell communication); three genes were downregulated: IAA synthesis (phytohormone), flagella (movement), and second quorum sensing gene. Expression of Pili (cell attachment), aerobactin (siderophore), hemagglutinin and Type III secretion system gene (virulence) was similar between the two growth conditions. Gene expression study revealed that the bacterium was active in the tomato leaves and expressed traits that are compatible with plant growth promotion and invasion. The present work concludes that wild perennial plants harbor unique and agriculturally beneficial endophytic bacteria with multiple plant growth promoting traits that are expressed during endophytic growth.