1 Introduction

The microbes most frequently found in plants belong to eubacteria, ­cyanobacteria, and fungi. Some of these microbes induce the formation of visible nodules on roots, but many others do not reveal their presence by external symptoms on the host. Most of them provide the host with nitrogen and obtain protection against predators and competing organisms in the soil. In other cases, the symbiont promotes the production of plant growth substances and receives protection against soil-competing organisms. However, it is difficult to define all the types of plant–microbe associations as the relationships between the associated partners are ­multiple and various.

A focus concerning the plant–microbe association is the response of the host to the various steps of the association, from the adhesion to the localization of the microbe in the host cytoplasm or tissues or in the apoplast. Other questions concern the microbe behavior during the steps from outside until localization and cooperation with the host. Among these problems, those less known are the final stages of the microbes in the host and their survival in the soil.

In order to answer the last question, we compared the behavior of bacteria and cyanobacteria in different association systems such as Cycad-Nostoc; Azolla-Anabaena; Legume-rhizobia; and tomato-Azospirillum. There are associations in which the symbionts are intercellular in coralloid roots or in leaf cavities, or intracellular in special structures as root nodules or only in cortex or other organs and tissues, without visible modifications in the plant. Due to this, we mainly pinpoint the ways in which microbes undergo and overcome the stress conditions when inside the host. More specifically, we look at a group of proteins and other compounds, such as Superoxide dismutase (SOD), Poly-β-hydroxybutyrate (PHB), trehalose, and melanin, found in the associated microbes. We also review the formation of spore-like cysts and akinetes to interpret the stress symptoms ­that predict the final fate of the microbes in the plant.

In all the above-cited systems, bacteria or cyanobacteria adhere to the plant roots after being attracted by plant-excreted molecules and penetrate the host via passive ways, as through broken hairs in Azospirillum, or modified hair wall and the resulting formation of thread infection in rhizobia. During these steps, ­cyanobacteria and bacteria often undergo changes in their shape, and dimensions, as well as in their metabolism. Similarly, other changes characterizing situations of microbial stress occur in the microbes once they are inside the host (Grilli Caiola, 1992; Grilli Caiola et al., 2004).

Stress symptoms in microbes have been reported in cultures grown in ­different nutrient concentrations, mainly in nitrogen-fixing microorganism; moreover, carbon compound availability, pH or salt and water variation induce stress (Gerson et al., 1978; Tung and Watanabe, 1983; Rai and Rai, 2000).

Trehalose (alpha-D-glucopyranosyl-(1-1)alpha-glucopyranoside) has been discovered in many bacteria and cyanobacteria, in which it often builds high tolerance levels to different abiotic stresses (Benaroudj et al., 2001; Garg et al., 2002; Wingler, 2002). Trehalose and its hydrolyzing enzyme trehalase appear to be less frequent in plants but common in plant–symbiotic microbes (Mellor, 1992). In symbiotic organs, trehalose seems to prevent phagolysosome fusion in host cells, could act as a reserve or storage form of reduced carbon, or may help in thermotolerance, in resistance to water stress, e.g., desiccation, or in the stabilization of biological structures. It occurs in Nostoc associated with cycads, in the Nostoc-Gunnera symbiosis, in Rhizobiaceae that live in legume root nodules (Mellor, 1992; Muller et al., 1995; Aeschbacher et al., 1999).

PHB is a polymer related metabolically to lipids. It is a highly reduced ­polymer made up exclusively of D-β-hydroxybutyric acid units in ester linkage. Its tertiary structure is a compact right-handed coil with a twofold screw axis and a pitch of 0.60 nm. PHB appears as small or large roundish-shaped electron transparent granules surrounded by a single membrane, which appears as a dense line approximately 3 nm in thickness under a transmission electron microscope. It occurs only rarely in free-living cyanobacteria, whereas it is common in other free-living and symbiotic bacteria. It is considered a reserve material in bacteria involved in the maintenance of nitrogen fixation when the photosynthesis is restricted (Gerson et al., 1978; Trainer and Charles, 2006). In Azospirillum brasilense, the synthesis and utilization of PHB as a carbon and energy source under stress conditions apparently favors the establishment of this bacterium and its survival in competitive environments (Kadouri et al., 2003).

Melanin is a dark pigment produced by some free-living and/or plant-­associated bacteria. It is a polymer occurring inside the cytoplasm as small electron-dense granules. It is formed often in nitrogen-fixing bacteria as well as in aging vegetative cells in culture and symbiosis. Melanin production is the result of the oxidative polymerization of phenolic compounds by the polyphenol oxidases:tyrosinase that has monophenol monooxygenase (EC 1.18.14.1) and o-diphenol:oxygenoxidoreductase (EC 1.10.3.1) activities, and by laccase (EC 1.10.3.2) that has p-diphenol:oxygen-oxidoreductase ­activity. Tyrosinase has been reported in Rhizobium and Synorhizobium. Laccase has been found in Azospirillum and Rhizobium, and the beneficial effects of coinoculation of both bacteria have been reported in legumes.

Superoxide dismutases (SODs: EC 1.15.1.1) are metalloproteins that rapidly convert superoxide O2 to hydrogen peroxide (H2O2) and molecular oxygen in all aerobic organisms(Fridovich, 1995). They prevent damage caused to ­cellular membranes by oxygen species (ROS), and act as a primary defense ­during oxidative stresses to which organisms are exposed. Due to their ­superoxide detoxifying capacities, SODs are considered a hallmark of plant defense responses to pathogens. Moreover, convincing evidence has demonstrated a positive correlation between levels of FeSOD and nitrogenase activity of cyanobionts, supporting the hypothesis that FeSOD protects nitrogenases against ROS damage (Canini et al., 1992).

Spores are formed by free-living bacteria and less frequently in associations. No spores or cysts have been reported in rhizobia in root nodules legume. Cysts (spore-like) have been described in Azospirillum free-living in the soil as well as when inside the roots of tomato (Sadavisan and Neyra, 1987; Grilli Caiola et al., 2004). A cyst derives from a vegetative cell, which after losing motility, develops a thickened outer coat, assumes an enlarged spherical form, and accumulates abundant PHB granules. The encystation is accompanied by a production in the cytoplasm of a dark brown pigmentation due to melanin.

Akinetes of Anabaena occur in Azolla sporocarps, but rarely in the leaves (Grilli et al., 1992), and Nostoc, too, rarely forms akinetes inside the cycad coralloid roots (Grilli Caiola and De Vecchi, 1980; Grilli Caiola, 1992, 2002). Akinetes derive from vegetative cells whose differentiation is controlled by a critical level of nitrogen and carbohydrate. They contain high amounts of cyanophycin and ­glycogen, both facilitating long-term survival under difficult environmental conditions.

Of course, the behavior of stressed microorganisms depends on their own ability to react to the strong influence of the host. In the following, we present a brief summary of associations in which the microbes are localized inside a newly formed structure of their symbiotic partners.

2 Cycad-Nostoc

Cycad coralloid roots house cyanobacteria belonging to Nostoc or Anabaena. These are localized in the cyanobacterial zone, the intercellular spaces of the zone formed by radially elongated cells (Grilli Caiola, 2002). Mainly vegetative cyanobacterial cells are developed in the apical part of coralloid roots, whereas the number of heterocysts increases from the median parts of the coralloid toward the basal ones (Fig. 1a), where most of the heterocysts degenerate.

Figure 1.
figure 1

Cyanobacteria in Cycas revoluta coralloid root at TEM. (a) Healthy vegetative cell (vc) and heterocyst (h) in the median part of coralloid. Bar = 1 µm. (b) Basal part of coralloid with healthy cyanobacterial vegetative cells (vc) and degenerated heterocysts (h). Bar =1.2 µm.

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The association allows the host to access nitrogen even when it is poorly available in the soil. On the other hand, cyanobionts benefit from the nutrient availability and the more stable conditions inside the coralloids. Coralloid roots form independently from the presence of the symbiont. They represent apogeotropic roots with a short life. Because they are usually annually developed, the advantage for the symbiont is limited to one or a few years after the coralloid degenerates, and the cycad utilizes the product of the reduced nitrogen, e.g., in the form of glutamine or citrulline.

In this system, the cyanobiont accumulates trehalose (Lindblad and Bergman, 1989) and cyanophycin at first. The latter is detectable as large granules inside the nucleoplasm or in the symbiont’s thylakoidal apparatus. Both compounds disappear in the vegetative cells present in the oldest parts of the coralloid, suggesting sharp changes of the metabolic conditions inside the host. Together with these metabolic changes in the oldest part of the coralloid, many vegetative cells and, above all, heterocysts undergo degenerative processes, and probably autolysis. Only a few of the small vegetative cells survive and probably return alive to the soil (Fig. 1b). Akinetes are very rare in cyanobacteria–cycad association, and no bacteria or other microbes usually accompany cyanobionts.

Cyanobacterial relation to the host is regulated in a manner that suggests a dominance of the cycad over the partner. In fact, high oxygen pressure in the heterocysts inhibits the nitrogenase activity and the utilization of derived nitrogen compounds, inducing the degenerative process in these specialized cells (Canini and Grilli, 1993).

The synthesis and regulation of SOD appears to be a possibility to defend heterocysts against the superoxide anion originating from the high respiration necessary to provide the energy for N2 reduction. A pattern of FeSOD labeling has been revealed in the cycad cyanobiont: FeSOD particle densities in heterocysts are in line with nitrogenase activity. FeSOD was never localized in ­degenerate, non-nitrogen-fixing heterocysts. No melanin or PHB has been observed in cyanobacteria associated with cycads.

In cycad–cyanobacteria association, the cyanobacteria cell morphology and metabolism is different from the free-living stages. The reduction of size and the relative composition of cell types from the apical parts to the basal ones of the coralloid, lead to the conclusion that the conditions of the cyanobacteria inside the coralloid are rather stressful, and for a majority of the cells, survival is difficult (Grilli Caiola, 2002).

3 Azolla-Anabaena-Bacteria

In previous papers (Grilli Caiola, 1992; Canini and Grilli Caiola, 1995; Grilli Caiola and Forni, 1999), the life of the prokaryotes, Anabaena and bacteria, in the leaf cavities of Azolla has been examined. Azolla is a genus of small aquatic fern. The species are distributed in almost every continent as spontaneous or cultured plants much appreciated for their water and soil enrichment in nitrogen due to nitrogen fixation occurring in their leaf cavities. The leaves are overlapping, each with a dorsal floating chlorophyllous lobe and a ventral submerged lobe. Their floating lobe has a cavity (Fig. 2a, b) containing many simple hairs, one primary branched hair, one secondary branched hair, the cyanobacterium Anabaena azollae, and bacteria. Recent studies have pointed out the existence of different cyanobionts strains among Azolla species, and diversity within a single Azolla species (Papaefthimiou et al., 2008). Based on the latter, cyanobiont seems to have more genotypic affinity to the genus Anabaena than to other Nostocaceae.

Figure 2.
figure 2

Azolla leaf cavity. (a) Leaf cavity of Azolla Mexicana with a simple hair (ha), Anabaena (a) Azolla mexicana and bacteria (arrow). SEM. Bar = 6 µm. (b) Vegetative cells (vc) of Anabaena azollae, bacteria (b), and apex of an hair (ha) in a leaf cavity of Azolla caroliniana. TEM. Bar = 2 µm.

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The Cyanobiont is hosted inside Azolla during all its life cycle (Canini and Grilli Caiola, 1995). In fact, the Azolla sporocarps present beneath the indusium Anabaena akinetes and bacteria. When the megaspore germinates it give rise a gametophyte which after sexual reproduction originates a new sporophyte. On this latter, small colonies of Anabaena vegetative cells derived by akinete germination occur. Such colonies are capable of maintaining the symbiont in the host so that Azolla and Anabaena are transmitted together from Azolla sporophyte via gametophyte to a new sporophyte.

The Anabaena heterocysts fix nitrogen. A coordinated work inside the leaf cavities results in the reduction of nitrogen molecules and the uptake of nitrogen compound by Azolla. Bacteria accompanying Anabaena cooperate in the reduction of oxygen concentration inside the leaf cavities to enhance nitrogenase ­activity (Grilli Caiola and Forni, 1999).

However, by comparing young and old leaves of Azolla, we can conclude that cyanobionts and bacteriobionts could not have an easy life in the host. In fact, Azolla acts on its “own behalf.” In addition, whenever the host’s life becomes too stressful, the symbiont can be eliminated.

Symptoms from suffering of the cyanobacterial symbiont can be recognized in the old leaves where reduced number of vegetative cells, degeneration of ­heterocysts, the appearance of akinetes under the indusium of micro- and megasporocarps are observed (Grilli Caiola et al., 1992). No trehalose has been found in Anabaena vegetative cells (Newton and Herman, 1979). Isolated A. azollae can grow on glucose and fructose, and synthesize glycogen; its mixotrophic growth results in an increased growth rate, higher heterocysts frequency, and nitrogen fixation.

The SOD activity increases in the symbiont from the median leaves to the basal ones. The accumulation of cyanophycin and the reduction of dimension in the vegetative cells of the old leaves are the aspects that can be related to a ­stressful condition of the Anabaena and its endeavor to survive the hostile conditions when the vegetative cycle of the host is approaching its end. A typical pattern of FeSOD distribution was evidenced in Anabaena living in Azolla cavities: higher particle densities of FeSOD in heterocysts than in vegetative cells; the FeSOD labeling trend overlaps that of nitrogenase activity of leaf cavity heterocysts. Moreover, low or degenerated, non-nitrogen fixing heterocysts exhibited low or zero FeSOD labeling (Canini and Grilli Caiola, 1995).

4 Legume–Rhizobia Association

In 1988, the first centenary of the discovery of symbiotic nitrogen fixation in leguminous plants by Hellriegel and Wilfarrh and of the isolation of the rhizobia by the Dutch bacteriologist M.W. Beijerinck, was held in Cologne (Quispel, 1988). Updated reviews by Hirsch (1992), van Rhijn and Vanderleyden (1995), Michiels and Vanderleyden (1994), and Sprent (2007) on the rhizobia associated with the legumes have well documented the morphology and the molecular basis of the establishment and functioning of the nitrogen-fixing root nodule.

Nodule origins on legume roots by association with soil bacteria of the ­genera Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, and Azorhizobium. Rhizobia are rod flagellate Alphaproteobacteria free-living in the soil (see also Hirsch, this volume). Most of the natural and cultivated leguminous plants attract rhizobia by means of root exudates containing molecules specific for each species. However, a legume plant can host one or more rhizobia strains. Bacteria penetrate the host through root hairs, which undergo a curling process thereafter. Hairs cooperate in forming a thread infection in which bacteria are present. After infection, the cells of root cortex tissues begin divisions, thus leading to a nodule emerging on the root. The root nodule shape varies from roundish to elongated and on the root, it can be isolated or in groups, this in relation to the presence or not of a nodule meristematic zone. In the spherical shape, the growth of nodule is determinate and the nodule has no meristematic zone. In indeterminate nodules, a meristem allows growth and lengthening for a long period of time and the shape of nodule becomes elongated. In a transversal section, a nodule shows a medullar zone in which the host cells contain numerous bacteria whose morphology depends on the steps and age of nodule. In fact, once the thread infection has reached a cell of the nodule, the bacteria are released into the cytoplasm ­embedded in a mucilage envelope. Here they divide (Fig. 3a) and contain electron transparent inclusions of poly-β-hydroxybutyrate, often resembling vacuoles due to their electron transparency.

Figure 3.
figure 3

Rhizobia in legume root nodules at TEM. (a) Initial stage of infected cell of pea (Pisum sativum) nodule with dividing rhizobia (b). Host cell shows abundant endoplasmic reticulum (er) and mitochondria (m). Bar = 0.15 µm. (b) Symbiosomes of indeterminate nodule of broad bean (Vicia faba). Bacteroids (ba) are elongated, singly enclosed in the peribacteroid membrane (pm and arrow), and in a reduced peribacteroid space (ss). Bar = 0.15 µm.

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Inside the host, the rhizobia undergo deep changes in shape and metabolism. Frequently, bacteria in this stage also have electron-dense granulations of dark material comparable to melanin. At a subsequent stage however, when they are released from the infection thread into the host cell cytoplasm, rhizobia enlarge and form variously branched and unbranched bodies known generally as bacteroids. Similar forms are produced when young rhizobia are subjected to certain environmental stresses (Jordan and Coultier, 1965). Bacteroid is a modified form of rhizobium inside the nodule cell. It contains nitrogenase and occurs in the symbiosome, the structural unit for the nitrogen fixation in association with the host plant. A symbiosome consists of the bacteroid, the peribacteroid space and the peribacteroid membrane (Fig. 3b), where the leghemoglobin is synthesized to regulate the entry of oxygen inside the symbiosome. The organization of the symbiosome is different in the various legumes as it results from the ­comparison of nodules (Fig. 4a–c) of bean (Phaseolus vulgaris) to pea (Pisum sativum), cowpea (Vigna sinensis), broad bean (Vicia faba), and lupine (Lupinus albus), or Robinia pseudoacacia (Grilli, 1963, 1964). However, not all the nodules are active in nitrogen fixation (ineffective nodules). The life of rhizobia in nodule is related and limited to the vegetative period of the host, from flowering to fruit maturation, if the legume has annual cycle. In the oldest nodule or in the oldest medullar zone, bacteroids undergo degeneration processes concluding with the lysis of nodule and the contained bacteria (Grilli, 1963, 1964).

Figure 4.
figure 4

(a) Symbiosomes in a cow pea (Vigna sinensis) determinate nodule. Several bacteroids (ba), are enclosed in a peribacteroid membrane (pm). The large peribacteroid space (ss) and the vesiculated host cell cytoplasm indicate the senescent condition of the nodule. Among the symbiosomes some free vacuolated (v) bacteria occur. Bar = 0.5µm. (b) Symbiosomes in indeterminate nodule of lupine (Lupinus albus). The elongated bacteroids (ba) contain dark granules of melanin (me) and are individually enclosed in a peribacteroid membrane (arrow) with a reduced peribacteroid space (ss). Bar = 1µm. (c) Symbiosomes in a determinate nodule of bean (Phaseolus vulgaris). Several bacteroids (ba) are enclosed in a peribacteroid membrane (pm and arrow) and in a rather large peribacteroid space (ss). Bacteroids contain numerous PHB granules (PHB). Bar = 1 µm.

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During the invasion phase, inside the thread infection and in the host cell release, many dividing rhizobia contain numerous granulations of PHB, whereas melanin granules have been detected in the bacteroid, mainly in the final stage of nodules. These granules are sometimes accompanied by the presence of dark brown pigmentation of the old part of the nodule. PHB seems to indicate that inside the nodule, there is abundant carbohydrate compound available for the symbionts, compared to the amount present in the soil. PHB are numerous in the bacteria of pea, bean, and particularly in bacteroids of bean and in those of lupine and pea in the final stage.

Melanin, on the other hand, represents a product of phenol metabolisms related to age and stress conditions for bacteria inside the nodule. They are very abundant in the bacteroids of bean and lupine. It is unclear whether melanin production by Rhizobium plays any role in the symbiotic process (Cubo et al., 1988). Probably, it is involved in the detoxification of the phenolic compound in nodules and roots of senescent bean plants.

During the elongation of the thread, bacteria are exposed to an oxidative burst due to the host plant with releasing of hydrogen peroxide and superoxide (ROS). For proper symbiotic development, bacteria encode a set of enzymes to defend against ROS, including SODs and catalases. Several SOD isoenzymes have been detected. Sinorhizobium meliloti, for example, encodes an SOD, SodB that can use either Fe2+ or Mn2+ with a strong role for manganese to carry its protective physiological role (Davies and Walker, 2007). Several pieces of evidence suggest an active role of SOD for the development of effective and efficient symbioses (Tavares et al., 2007). However, a model by which ROS and antioxidants ­interacting with hormones should orchestrate the nodule senescence has been proposed by Puppo et al. (2005).

5 Endophytes in Higher Plants: Tomato-Azospririllum

The tissues of healthy plants were originally considered to be sterile by Pasteur (1876) and subsequent authors such as Fernbach (1888). However, since then a number of investigators have reported instances in which bacteria were found in various parts of healthy plants such as the storage organs (Hollis, 1951; Tonzig and Bracci Orsenigo, 1955), fruits (Samish and Dimant, 1959; Samish et al., 1961), ovules and seeds (Mundt and Hinkle, 1976), root xylem, or many different organs (Schanderl, 1939). After Dobereiner (1961) isolated bacteria from sweet cane roots, a new viewpoint arose about the significance of microbes in the plants. Endophytes was the term generally used to indicate the microbes present in different organs and tissues of plants without disease symptoms. Although it is commonly used, this term has been criticized by some authors and the significance of the presence of many microbes inside the host plant is still unknown. An increasing ­number of bacteria belonging to different taxa such as Acetobacter, Herbaspirillum, Pseudomonas, Azoarcus, have been found associated to spontaneous and cultivated monocotyledonous and dicotyledonous plants (e.g., Reinhold-Hurek and Hurek, 1998). In some cases, they prove to be associated to the external root surface; in others, they are found inside the plant as small colonies spread in different tissues or organs from which they have been isolated in higher amounts (Chi et al., 2005).

Rhizobacteria are bacteria living in the soil in a zone termed rizoplane where roots also grow and, due to their capacity to invade the roots, many of them are considered endophytes. Bacteria and fungi inhabit this space in large number and in different relation to plant roots. Many fungi are able to envelop roots and penetrate the plant’s cortical tissues, thus giving rise to the well-known symbiosis mycorrizae. Rhizobia and Actynomycetes can also penetrate the roots and invade the inner tissues of the plant, causing the formation of structures as tubercules as they occur in legumes and Alnus. Rhizobacteria adhere to the plant roots without inducing apparent modification in the host. Their relationship with the host can be limited to adhering to the external surface or penetrating the cortex root. Root penetration has been reported in many plants, whereas the ­diffusion inside the tissues of the host is until now limited to cortex and, in a few instances, to xylem tissue, thus reaching the foliar ­system. Ascending migration of endophytic rhizobia from roots to leaves inside rice plants has been recently reported (Chi et al., 2005). Colonization of ­internal plant tissues is thought to be largely intercellular and, more rarely, intracellular in living plant cytoplasm.

Among the rhizobacteria, Azospirillum is the most studied because of its effect on producing indolacetic acid (IAA) and stimulating optimal growth of the host. In some conditions, it is also capable of nitrogen fixation. Apparently, Azospirillum seems to help the penetration in obtaining a more convenient ­environment ­regarding the nutrient availability and protection from the competitors in the soil. However, the symbiotic condition is not free from some complications as it results from a study carried out on a system set up between tomato (Lycopersicum esculentum Mill.) and Azospirillum brasilense Cd (Grilli Caiola et al., 2004).

Azospirillum brasilense is capable of a vegetative phase with motile flagellate cells, but in aged culture, it produces brown colonies forming cysts. Thus, it is a good model for following the bacteria variation when outside in the soil compared to when it lives inside the plant.

Information was obtained through experiments: (a) on tomato seeds inoculated with Azospirillum brasilense Cd grown on agarized medium with and ­without combined nitrogen; (b) on 30-day old tomato plantlets inoculated with Azospirillum brasilense grown on modified Okon agarized medium supplied or not with combined nitrogen and (c) on comparing young Azospirillum cultures in exponential growth phase and old brown culture in the late stationary phase. Useful results were obtained through an analysis of the previous material at OM, SEM, and TEM.

Comparison of Azospirillum in young and old brown cultures and also inside tomato roots has provided important information about the relation between rhizobacteria and the host plant. Young Azospirillum cells show a single polar flagellum and several lateral ones (Fig. 5a) that are only synthesized in solid growth medium. Old cultures show aflagellate cells containing large electron-dense forms enclosed within a thick capsule, housing two or more smaller cells that are then released in the medium.

Figure 5.
figure 5

(a) Periflagellate Azospirillum brasilense Cd in young culture. SEM. Bar = 1.1 µm. (b) Azospirillum brasilense inside tomato root. Bacteria have a thick coat (c), numerous melanin (me) and PHB granules. TEM. Bar = 0.6 µm.

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In the brown mature cultures, Azospirillum has very electron-dense colonies, with smaller cells and a thicker envelope than observed in younger cultures. Such structures have been identified as the cysts previously reported in A. brasilense. The brown color of the cultures was attributed to the melanin also present in other bacteria (Cubo et al., 1988; Castro-Sowinski et al., 2002). The bacteria penetrate the host plant via the root hairs and epidermis cells and localize between the host cells, aligned in rows or aggregated into large colonies in the intercellular spaces. Some bacteria occur within cells that appear to be lysed and dead. The morphology and activity of the bacteria inside the tomato roots change with respect to those observed in culture. They do not divide, have a very thick capsule, numerous large PHB (Fig. 5b) and glycogen granules, similarly to cysts observed in aged brown cultures. Cysts and pigmentation have been observed in A. brasilense in aging cultures under carbon and nitrogen limitation (Sadavisan and Neyra, 1987). These cysts differ from those observed in cultures rich in melanin, which are frequently divided into smaller cells and lack glycogen.

However, some other aspects of these cysts suggest that they may instead be active forms, with high levels of oxidative metabolisms, as revealed by the large amounts of SOD present and the accumulation of reserves (PHB, glycogen) (Grilli Caiola et al., 2004).

6 Concluding Remarks

The analyzed associations suggest reconsidering the concept of symbiosis. De Bary defined symbiosis as “unlike organisms living together,” among other examples, by observing the presence of “algae” in the leaves of Azolla. The systems reported above are formed of a cormophyte with one dominant microorganism of a microbial community. The fates of these associations are different, but some common aspects can be recognized. With the exception of the Azolla-Anabaena-bacteria association, where the partners live and are transmitted together, in the other examples reported here, there is a defined duration of association. Usually, the host survives, whereas most microbionts die and only a few bacteroids (McDermott et al., 1987) and perhaps vegetative cells of cyanobionts (Grilli, 1992, 2002) survive. Thus, the host prevails over the partner and the association results in a temporary combination for the host’s benefit. In this situation, microbes show, more or less pronounced symptoms of suffering until cells lyse and its content is utilized by the host after a first phase, during which it seems to take advantage of the host. The symptoms of such a suffering state are the absence of the microbial cell divisions, the changes in morphology and the production of compounds for a defense against the host metabolism. In addition, the influence of the host on the microbiont can also affect the microbial genotype, some of which are modified, as in the rhizobia so that the surviving forms sometimes show different genetic composition compared to the infecting ones (Simms et al., 2006; Sprent, 2007). This stressed condition becomes evident in the production of storage compound such as PHB, glycogen, or specialized resting cells such as akinetes in cyanobacteria and cysts in bacteria. All these aspects, although not exclusive to microbes in association with plants, occur during all or most of the symbiont life cycle in the host.

Concerning the causes of the stress in microbiont associated to plants, they can be identified in biotic and abiotic origin. Nitrogen deprivation induces heterocyst differentiation and nitrogen fixation both in cyanobacteria as well in bacteroids. In addition, host affects symbiont metabolism, inducing the synthesis of new metabolites. Moreover, high temperature, high salinity, phosphorus deprivation can affect both host as well as symbiont (Tung and Watanabe, 1983; Rai et al., 2006).

In the light of new research results, it is tempting to revise the concept of symbiosis. Recently, Sapp (2004) introduced the concept “symbiome,” which defines the organism as a “functional field that includes microbial communities.” Carrapico (2002) interpreted the Azolla-Anabaena-bacteria symbiosis as a ­“natural microcosm.”

When considering the fate of microbes in plants, a question arises: “Why are most microbes beneficial to their plant hosts, rather than parasitic?” Concerning rhizobia, Denison and Kiers (2004) suggest that multiple strains per plant and root-to-root transmission favor rhizobia, which invest in their own reproduction, rather than symbiotic N2-fixation. Legumes seems to select for mutualistic strains by controlling nodule O2 supply and reducing reproduction of rhizobia, which fixes less N2. A mechanism to suppress non-mutualistic strains could be by sanctions against undifferentiated or less active rhizobia in the nodule (Denison, 2000; West et al., 2001). In this context, an approach based on bargaining theory has been presented as a model for negotiation of benefits (Akçay and Roughgarden, 2007) and as mechanism to prevent exploitation between partners with conflicting interest (Simms et al., 2006).

Despite the numerous studies devoted to researching the deep relations between host and partners and the resulting beneficial effects for the plant, the association of plants with microbes remains an intriguing world to explore.

7 Summary

Many associations of plants with microbes are considered to be mutually beneficial for both partners. The authors of this paper suggest that associations of plants with oxyphototrophic or heterotrophic bacteria are beneficial mainly for plants, the microbes being eliminated at the end of symbiosis. To prove this, different plant–microbe associations such as cycad-cyanobacteria, Azolla-Anabaena, legume-rhizobia, and tomato-Azospirillum have been examined. All the systems considered show elimination of most microbes in course of time. In fact, only a few individual symbionts may survive in the soil or inside the host as resting form.

The life of the symbiont appears to be more comfortable inside the host than in the free-living stage outside, especially during the first steps of the association. However, afterward, symptoms of stress such as a decrease in growth and division, appearance of trehalose, PHB, melanin, SOD, resting cells like akinetes, and spore-like cysts occur in the microbe. All these mechanisms are involved in response to stress conditions.