Plant-bacteria interaction at the microscope M. del Gallo*, C. Ercole, and F. Matteucci Department of Life, Health and Environmental Sciences, University of L Aquila, Coppito 1, 67100, Italy *Corresponding author: maddalena.delgallo@univaq.it Plants, as do animals, harbor inside, on surfaces, and on root influence areas - the so-called rhizosphere - a very high number of microorganisms that form a unique ecosystem with the plant, the microbiome. Interactions are very tight, even with DNA exchanges. Approximate calculations of symbiotic microorganisms of the human species provide data of 10 microorganisms per human cell. Same it is for plants: all surfaces are covered with microorganisms, particularly the underground ones, and leaf areas around the stomata. Also inside, particularly xylem vessels, there are billions of microorganisms. However, it is difficult quantify them. Inside the stem of the herbaceous plants, we have counted from 10 7 to 10 9 microorganisms per gram of dry matter. Regarding the role that microorganisms can cover in the association, we can mention protection from pathogens, production of plant hormone, nitrogen fixation, production of Fe-chelating molecules, and solubilization of phosphates. A relevant role is played by endophytes, the microorganisms that live within the plant, especially inside the xylematic vessels. In our research conducted over several years, we have dealt with plant-bacterium interactions, endophytes in particular, with the role played by them, the colonization of the host plant and their allocation. Different microscopy techniques were utilized: isolated and inoculated bacteria were checked for plant lectin interactions (FITC-lectin binding) and for biofilm production. Sorghum and chickpea seeds inoculated with arbuscular mycorrhizas Glomus occultum and Scutellospora persica, and four plant-growth promoting bacteria Azospirillum brasilense, Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus and Burkholderia ambifaria. Chickpea plants were regularly nodulated by Mesorhizobium ciceri. Plant colonization was analyzed both by optical and by electron microscopy (SEM) revealing very tight interactions between microorganisms and the host plant since the first stages of the colonization. Keywords: Plant bacteria interactions, Glomus occultum, Scutellospora persica, Azospirillum brasilense, Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, Burkholderia ambifaria, Cicer arietinum, Sorghum bicolor 1. Introduction Plants, as do animals, harbor inside, on surfaces and root influence areas - the so-called rhizosphere - a very high number of microorganisms that form a unique ecosystem with the plant, the microbiome. Interactions are very tight, even with DNA exchanges [1, 2]. Approximate calculations of symbiotic microorganisms of the human species provide data of 10 microorganisms per human cell. These data, however, are very controversial and date back to a study done by Luckey in 1972 [3, 4]. Recently, other studies estimated human body s cells varying from 15 trillion to 724 trillion [5, 6] and that microorganisms associated with the human body are very variable. For instance, the ones present on the skin can vary depending on detergents used in washing. Those associated with the intestine, the most numerous, vary greatly depending on the diet and drop dramatically in case of consumption of antibiotics. Certainly - as Moeller at al. point out in the July 2016 Science the exact number of microorganisms it is not relevant as it is the fact that the gut flora has a fundamental role in human health and has coevolved for millions of years with hominids [2]. The same is true for plants: all surfaces are covered with microorganisms, and in particular roots and leaf areas around the stomata. Also inside, especially in xylem vessels, there are many microorganisms. However, quantify them is difficult. Inside the stem of the herbaceous plants, for example, 10 7 to 10 9 microorganisms per gram of dry matter were counted [7]. Therefore, the numbers of microorganisms associated with plants is very high and their function is fundamental [8]. Concerning the different roles microorganisms can have in association with plants we can mention the following: protection from pathogens, production of plant-growth substances, nitrogen fixation in assimilable form, production of Fe-chelating molecules, solubilization of phosphates and other poorly understood functions. They are commonly referred to by the acronym PGPR (Plant Growth Promoting Rhizobacteria). As endophytes, instead, we define the microorganisms that live inside the plant, especially in the xylem vessels. These microorganisms, in fact, colonize areas of the plant that have transport function of nutrients absorbed by the roots. But if we liken the xylem vessels to the human intestinal system definitely a daring comparison in some respects - we realize that they stabilize in a nutrientrich environment like our intestines and can, therefore, have similar functions: surface protection from attack of pathogens; "digestion" of the long chains of humic molecules easily assimilated by the plant and release the minerals associated with them; nitrogen fixation and production of useful molecules such as plant hormones, phytostimulants, antibiotics and others. On nitrogen fixation and production of hormonal substances, such as auxins, gibberellins, cytokinins, and ethylene, there is plenty of literature [9]. 312
Microbial species associated with plants are bacteria, archaea and fungi. In our research, we focused particularly on bacteria. We only marginally employed fungi. Fungi are excellent carriers of bacteria inside the plants, and play a vital role in almost all plant species through mycorrhizae. We also conducted research on some pathogenic fungi and the role that bacteria play in plant protection. In this article, we describe some of the microscopy techniques used for the study of these interactions and the results obtained. 2. Materials and Methods 2.1 Observations at the optical microscope The bacteria utilized in the experiments were Azospirillum brasilense, Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus and Burkholderia ambifaria. Bacteria were grown in T4 medium, previously described [10]. The fungi utilized were Glomus occultum and Scutellospora persica reproduced in symbiosis with Medicago sativa in pot. The plants used in the experiments were Sorghum bicolor and Cicer arietinum. For the observations at the first stages of the interaction, the seeds, sterilized with 3% of Ca hypochlorite, were germinated in the dark in petri dishes with the bottom covered with discs of absorbent paper soaked with sterile water. Seedling tips were inoculated with A. brasilense under the microscope and were observed at variable times. 2.2 Observations at the Scanning Electron Microscope The bacteria were observed with a scanning electron microscope (SEM) after having made them grow for 48 h, not under stirring, on slides immersed in T4 medium. Bacteria were grown as either pure culture or all four together. In another experiment both bacteria and roots of sterile Sorghum bicolor, 15 days old, inoculated with the four bacteria and Glomus occultum or Scutellospora persica, were used for SEM observations. Samples were fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer, ph 7.2-7.4 at 4 C for 24-48 h; they were then washed in the same buffer for 20 min and dehydrated in alcoholic successions 30, 50, 70, 96%, then in absolute acetone. After dehydration, plant samples were cut into thin sections and then, all plant and bacteria samples were covered with gold. 3. Results and discussion The four bacteria used in the experiments have shown to be excellent plant colonizers. In Figure 1 inoculated and control plants of chickpea, regularly nodulated with their specific rhizobium Mesorhizobium ciceri are shown: inoculated plants show a growth of more than 50% of the controls and, interesting for the farmer, greater precocity in the maturation. Fig.1 Chickpea plants inoculated with the four bacteria, inoculated (left), not inoculated (right). Both inoculated and controls plants were nodulated by the symbiont Mesorhizobium ciceri. As reported in the literature, the bacteria adhere to the plant and colonize plant surfaces and xylematic vessels by slime and biofilm formation [11, 12]. We analyzed these formations by pure and mixed culture of A. brasilense, H. 313
seropedicae and G. diazotrophicus grown separately and together, by SEM. All three bacteria form biofilms, particularly abundant when they grow together (Fig. 2). Among the four bacteria utilized in the experiments, A. brasilense, in particular, has a very striking effect on the plant, by increasing germination of 30%, and with a stimulation of rooting and an increase of root hairs, either as a number than as surface covered by (Fig. 3). a) b) c) d) Fig. 2 Biofilm formed by Azospirillum a), Herbaspirillum b), Gluconacetobacter (c) and a mixed biofilms (d) in which stand out a few cells inside an abundant biofilm (SEM). a) b) Fig. 3 7 days old seedlings of Cicer arietinum grown in Petri dishes and inoculated with the four bacteria. To the left a control root, to the right an inoculated root. The rootlets of inoculated seedlings of are much more developed and branched and shows a larger number of root hairs (10x). We looked at the first stages of colonization by inoculating directly sorghum root tips with azospirillum, under the microscope. Bacteria quickly approach the radical apex, reach it and penetrate into the mucilage that surround it (Fig. 4a and 4b). 314
a) b) Fig. 4 Dark field microscopy of a root apex of Sorghum bicolor inoculated with Azospirillum brasilense under the microscope. Arrows indicate bacteria approaching the root apex (a); bacteria adhere to a root hair in forming a clump (b). (200x) After a few minutes, then, they are wedged in the crumbling area of the apical cells, the calyptra (Fig. 5a), inside the root hairs (Fig. 5b) and begin the penetration within the root reaching the xylem vessels, that they colonize and multiply (fig. 6). We studied this stage of colonization by SEM in 15 days old plant of S. bicolor inoculated both with bacteria and with AM. The cannels of the xylematic area were completely covered by bacterial biofilm and the AM spores were clearly visible above the bacterial biofilm. a) b) Fig. 5 a: phase contrast microscopy of an area of a of Sorghum bicolor rootlet close to the apex, inoculated under the microscope with Azospirillum brasilense: bacteria (little black spots) have settled inside the radical apex mucilage (short dark arrow) that covers the calyptra and form clusters close to flaked cells (longer arrow), from where penetrate within the root. Root cells that have flaked are clearly visible even incorporated into the mucilage (white arrow). b: it is possible to observe bacteria that are forming on a root hairs a structure similar to the infection thread of rhizobia with legumes. Some bacterium is visible also inside a root hair. (400x) The colonization of the plant, therefore, takes place in several phases: - Chemotactic attraction of the bacteria towards the root - Adhesion and penetration of the bacteria on a seedling rootlet through the growth area of the calyptra and of the flaking cells, and colonization, at the same time, of the radical apex mucilage, - Penetration inside the root in the xylem vessels and stabilization with formation of a biofilm. The penetration is favored, in particular, by arbuscular mycorrhizae. The next steps in our research by microscopy methods, will involve the use of genetic probes labeled with fluorescent dyes specific for each bacterium (Fluorescent in situ hybridization, FISH). This technique will allow us to observe the bacteria directly inside the plant, locating them both individually or in association of different species, opening the possibility of more in-depth investigations of the plant microbiota. 315
a) b) c) d) e) f) Fig. 6 Sterile sorghum root (a and b) and inoculated with the four bacteria (c) and a Scutellospora persica spore (d), and the four bacteria and Glomus occultum (d and e). AM spores, dipped in the biofilm (dashed arrow) formed by the bacteria, are visible (indicated by continuous arrow). (Scale bar vary between 10 and 20 μm). Acknowledgement We wish to thank Maria Di Giammatteo, Lorenzo Arrizza and Anna Ragnelli for their suggestions and support with the Scanning Electron Microscopy. References [1] Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature 2000; 405: 299-304. [2] Moeller AH, Caro-Quintero A, Mjungu D, Georgiev AV, Lonsdorf EV, Muller MN, Pusey AE, Peeters M, Hahn BH and Ochman H. Cospeciation of gut microbiota with hominids. Science 2016; 22: 380-382. [3] Abbott A. Scientists bust myth that our bodies have more bacteria than human cells. Nature 2016; 8. [4] Luckey TD. Introduction to intestinal microecology. Am. J. Clin. Nutr. 1972; 25: 1292 1294. [5] Leser TD and Lars Mølbak L. Better living through microbial action: the benefits of the mammalian gastrointestinal microbiota on the host. Environment. Microbiol. 2009; 11: 2194 2206. [6] Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. Plos Biology 2016; 1-14. [7] Isopi R, Fabbri P, Del Gallo M, Puppi G. Dual inoculation of Sorghum bicolor (L.) Moench. ssp. bicolor with Vesicular Arbuscular Mycorrhizas and Acetobacter diazotrophicus. Symbiosis, 1995; 18: 43-55. [8] Schlaeppi K, Bulgarelli D. The plant microbiome at work. Mol. Plant-Microbe Interact. 2015; 28:212-217. [9] Spaepen S. Vanderleyden J. Remans R. Indole-3-acetic acid in microbial and microorganisms-plant signalin. FEMS Microbiol. Rev. 2007; 31:425-448. 316
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