Dudeja, S.S., Giri, Rupa, Saini, Ranjana, Suneja-Madan, Pooja and Kothe Erika. 2012. Interaction of...

248 Journal of Basic Microbiology 2012, 52, 248–260

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Review

Interaction of endophytic microbes with legumes

S. S. Dudeja1, Rupa Giri1, Ranjana Saini1, Pooja Suneja-Madan2 and Erika Kothe3

1 Department of Microbiology, CCS Haryana Agricultural University, Hisar, India 2 Department of Microbiology, Maharishi Dayanand University, Rohtak, India 3 Microbial Phytopathology, Institute of Microbiology, Faculty for Biology and Pharmacy,

Friedrich Schiller University of Jena, Jena, Germany

Large numbers of bacterial and fungal endophytes have been reported from different plant tissues: roots, nodules, leaves, flowers and sprouts of legumes, with numbers ranging from few to more than 150. Endophytes can accelerate seedling emergence, promote plant establishment under adverse conditions and enhance plant growth. Endophytic microbes promote plant growth by helping plants in acquiring nutrients, e.g. via nitrogen fixation, phosphate solubil-ization or iron chelation, by preventing pathogen infections via antifungal or antibacterial agents, by outcompeting pathogens for nutrients by siderophore production, or by establishing the plant’s systemic resistance. Further growth promotion is affected by producing phytohor-mones such as auxin or cytokinin, or by producing the enzyme 1-aminocyclopropane- 1-carbo-xylate (ACC) deaminase, which lowers plant ethylene levels. For establishment of endophytes in different tissues, endophytic microbes must be compatible with the host plants and able to colonize the tissues of the host plants without being recognized as pathogens. A particular bac-terium or fungus may affect plant growth and development using one or more of these mecha-nisms, and they may use different mechanisms at various times. The population density of endophytes is highly variable, depending mainly on the microbial species and host genotypes, developmental stage and environmental conditions. Genotypic and cultivar specific endophytes have also been reported. The quantum benefit derived by plants from an endophyte and vice versa is still not clear. It seems that the endophytic genus or species best adapted for living in-side a plant is naturally selected. Here, we concentrate on soil or rhizosphere-derived endophy-tes recruited out of a large pool of soil or rhizospheric microbes. Some endophytes are more aggressive colonizers and displace others, but seeming lack of strict specificity has been observed. However, the processes of host-microbe signaling and colonization and the mecha-nisms leading to mutual benefits are less-well characterized. It is still not clear which popu-lation of microorganisms (endophytes or rhizospheric) promotes plant growth and the way the interactions among endophytes influence plant productivity. Though attempts to know the molecular ecology and interactions are underway, a high amount of progress is required to fully understand the mechanism of establishment, the way interactions take place in planta, between different microbes and plants and exlusive benefits by endophytes and plants.

Keywords: Endophyte / Bacterial / Fungal / Legume / Nodules / Roots / Interaction

Received: February 04, 2011; accepted: April 27, 2011

DOI 10.1002/jobm.201100063

Introduction*

Plants sustain the life in soil and are required for natu-ral soil development. Since plants never live alone in Correspondence: Prof. S. S. Dudeja, Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, India E-mail: [email protected]

soil, they release signals to interact with other organ-isms. The plant root system, apart from anchorage and uptake of nutrients and water, mediates numerous underground interactions with beneficial microbes, such as rhizobia, mycorrhizae, endophytes and plant-growth promoting rhizobactertia (PGPR) and parasitic interactions with other plants, pathogenic microbes or

Journal of Basic Microbiology 2012, 52, 248–260 Legume endophytes 249


sustaining soil microbes. Plants constitute vast and diverse niches for endophytic organisms and there is not a single plant species devoid of endophytes. Actu-ally, it is still not clear whether plants benefit from an endophyte or from a rhizospheric bacterium, or if the advantage is higher for the bacteria to become endo-phytic compared with rhizospheric. Both the endo-phytic or rhizospheric populations of microorganisms may promote plant growth, or both work jointly; but benefits conferred by endophytes are well recognized. There is increasing interest in developing biofertilizers for enhancing crop productivity. The term endophyte describes (micro) organisms liv-ing inside plant tissues, and in a strict sense, without causing symptoms to the plant. However, in many in-stances, the mere isolation from surface sterilized plant tissue is taken as the proof for an endophytic nature of an isolated bacterial or fungal strain. In this review, we use the term endophyte for bona fide growth in planta, although for some of the original literature cited the proof of re-infection is still pending. The growth in planta might be associated with phytopathogens, com-mensals, or mutually beneficial interactions. In this review, we will concentrate on interactions specifically promoting plant growth. The need for an update of current work is especially valid for legumes which carry the specific trait to be able to form root nodules. The family Fabaceae con-tains 630 genera and 18,000 species, making it the third largest family of flowering plants [1]. It is convention-ally divided into three distinct sub-families, mainly according to differences in floral structure. The sub-families Mimosoideae and Caesalpinoideae consist pri-marily of trees, shrubs and vines but few herbs, contain many species that are valuable as ornamentals or for their timber, dyes, resins and medicinal properties. They are especially abundant in tropical rainforests, where they may account for half the trees present and are probably fixing vast quantities of nitrogen. The third sub-family, Papilionoideae, contains many species of agricultural importance in temperate and tropical regions. They are a highly nutritional food source for humans and animals, and they are also used as ground cover in plantations and on eroded soils, and as green manures. Taken together the family Fabaceae is second only to the Poaceae in economic importance. Plant tissues and plants are benefited extensively by endo-phytic microbes; they promote plant growth and yield and confer enhanced resistance to various pathogens. Endophytes also produce unusual secondary metabo-lites of plant importance and activate the stress re-sponse system more rapidly and strongly in plants lead-

ing to higher resistance against pathogens. Endophytes may also help to remove contaminants, solubilize phos-phate, or contribute assimilable nitrogen to plants. In addition, endophytic bacteria supply essential vitamins to plants. The production of auxin-like compounds increases seed production and germination along with increased shoot growth and tillering. Other effects of endophyte infection on the host plant include osmotic adjustment, stomatal regulation, modification of root morphology, enhanced uptake of minerals and altera-tion of nitrogen accumulation and metabolism [2]. Both bacteria and fungi are the most common mi-crobes existing as endophytes. Meanwhile, endophytic bacteria of legumes other than rhizobia have been poorly studied. Previous studies on pea and soybean plants have demonstrated that endophytic bacteria can induce the resistance of pea plants to pathogenic fungi or improve the growth of soybean [3–5]. Based on these and other reports, it seems that Enterobacter spp., Pseu-domonas spp., and Bacillus spp. are the most abundant endophytic bacteria in legumes and other plants, such as potato or maize. Using selective media, many other bacteria such as the free-living nitrogen-fixing bacteria and actinomyces also can be isolated from inside the plants. Leguminous plants developed a couple of sys-tems to attain mutual symbiosis with rhizobia, endo-phytes and mycorrhizae. The genetic requirements for rhizobial and mycorrhizal interactions in plants over-lap in a common symbiotic pathway leading to success-ful symbiotic association [6]. The population density of endophytes is highly vari-able, depending mainly on the bacterial species and host genotypes, but also on the host developmental stage, inoculum density, and environmental conditions. Endophytic N2-fixing bacteria seem to constitute only a small proportion of total endophytic bacteria and in-creasing N2-fixing populations in plants has been con-sidered as a possibility to increase nitrogen fixation. It seems that the bacteria best adapted for living inside plants are naturally selected. Since soil represents a rich pool of inocula and roots are especially vulnerable to invasion, the soil- or rhizophere-derived endophytic community is of special importance to the plants. Endophytic bacteria in a single plant host are not restricted to a single species, but comprise several gen-era and species. It is not known whether these multiple endophytic microorganisms inside the plants interact. However, it has been speculated that beneficial effects are the combined effect of activities of these microbes influencing plant productivity. It seems likely that inter-actions among endophytes takes place in planta, specifi-cally those that are depending on quorum sensing. In

250 S. S. Dudeja et al. Journal of Basic Microbiology 2012, 52, 248–260


addition, these communities are prone to be affected by signals exchanged between host and symbionts. In the rhizosphere, which includes plant roots and the sur-rounding area of soil influenced by the roots, plants exude chemicals to effectively communicate with their neighboring soil organisms [7]. All plants in nature harbor a diverse range of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phyto-hormone levels induced by endophytes. Similar mecha-nisms may not be operative in all plant or tissue types. Therefore, natural endophytic bacteria with plant growth promoting traits do not have general and pre-dictable effects on the growth and fitness of all host plants, although the underlying mechanisms may be similar. Legumes are an important crop, specifically with regard to soil fertiliy, as nodule forming rhizobia will lead to a supply with fixed nitrogen thereby en-hancing crop productivity also for subsequent cultiva-tion of non-legumes. Hence, the microbial population of legumes might also be effectively enriched for other endophytic species which then would impact other crops in rotation. It becomes very important to know whether legume endophytes are present in nodules as well as in roots and perform similar functions, or whether depending upon tissue localization they per-form different functions. Further, the presence of endophytes in all tissues is highly dependent upon the type of soil microflora available in the soil. Whether irrespective of host and type of tissue, all endophytes perform similar functions seems questionable. There-fore, proper studies on the interactions among these endophytic bacteria and leguminous plants are needed. In this review, ecology, interaction and mechanisms of microbial establishment and benefits derived by leg-umes will be discussed.

Occurrence of bacterial endophytes in legumes A specific trait of legumes is the ability to form nod-ules. This tissue is a supreme habitat for bateria, not only for nitrogen fixing rhizobia. With respect to nod-ules, the rhizobia leading to nodule formation have to be reviewed, before associated bacterial species should be considered. In legumes, numerous studies to clarify the taxonomy and phylogeny of bacteria that are able to induce or inhibit nodulation are being carried out. This has led to the identification of 12 bacterial genera able to nodulate different legumes [8]. Nine rhizobial genera belong to the α-proteobacteria while three be-long to the β-proteobacteria [9, 10]. Most endophytes appear to originate from the rhizosphere or phyllos-phere; however, some may be transmitted through the

seed. Very rare reports are available regarding the pres-ence of other microorganisms (associated or endo-phytic) in the nodules in spite of the fact that nodules are much richer in nutrients as compared to roots. During the present decade, large number of culturable as well as non cultureable rhizobial and non rhizobial endophytes in legume roots and nodules have been reported [11, 12] and even in our laboratory in roots and nodules of chickpea and pea, large numbers of bacterial endophytes were isolated by proper steriliza-tion of nodules and taking every care to avoid surface contaminants (Fig. 1). The picture becomes confusing if rhizobia failed to nodulate the legume from which they were isolated. Thus, they may not have been involved in the formation of the nodules, but are merely co-occurring as associated endophytes. They may poten-tially supply signals leading to nodulation by the plant but with insufficient invasion skills if not associated by helper bacteria. In the laboratory, we could isolate two types of rhizobia based on growth from chickpea nod-ules (Fig. 2). One type of rhizobia showed growth in 3–4 days, while the other showed growth only after 20 days from nodules. The fast growing rhizobia upon re infec-tion were able to form nodules, whereas slow growing rhizobia did not form any nodules, indicating the pres-ence of endophytic rhizobia in chickpea nodules. In addition to the rhizobia, endophytic bacteria have been isolated from different legume plants such as al-falfa, clover, soybean, Argyrolobium uniflorum, Vicia, Oxy-tropis, Medicago, Melilotus, Onobrychis, peanut and Acacia. Large numbers of bacterial genera have been reported to be associated with different legumes (Table 1). Over 24 non-rhizobial and about 10 rhizobial genera have been isolated from legume tissues including Agrobacte-rium, Bacillus, Curtobacterium, Enterobacter, Erwinia, Myco-bacterium, Paenibacillus, Pseudomonas, Phyllobacterium, Ochro-bactrum, Sphingomonas, Rhizobium, Ensifer, Mesorhizobium, Burkholderia, Phyllobacterium and Devosia [12, 25]. Considering other parts of legume plants, endophytic bacteria were isolated from the foliage, tap roots and nodules of red clover plants (Trifolium pratense L.) [23, 35–37]. Thirty-one bacterial species of 14 different genera were recovered. Genera diversity and species number were greatest in foliage tissues. Pantoea agglom-erans (59.6%) was the most frequent species recovered in foliage tissues, Agrobacterium rhizogenes in the tap root (49.2%) and R. leguminosarum bv phaseoli and Me-sorhizobium loti in the nodules (27.2% each). Clover root nodules were host to 12 bacterial species other than rhizobia, of which 8 were specific to this tissue. In root bacterization experiments, species of nodule bacteria promoted growth of red clover more often when ap-



a) b)

c) d)

Figure 1. Endophytic bacteria isolated from chickpea roots (a); chickpea nodules (b); pea roots (c) and pea nodules (d).

plied in combination with R. leguminosarum bv trifolii than when applied singly. However, Bacillus megaterium, Bordetella avium and Curtobacterium luteum consistently promoted growth either individually or in combination with rhizobia. Similarly, Zakhia et al. [46] described the association of 14 bacterial genera with wild legume nodules in Tunisia. Agrobacterium tumefaciens strains were reported to be endophytic bacteria in the roots, stems, and root nodules of Melilotus dentatus and other legumes [24, 47]. Interactions in the rhizophere may have a pronounced effect on plant growth. Plant growth promoting rhizobacteria (PGPR) were isolated from bean (Phaseolus vulgaris L.) nodules [20]. One of the

strain was found to increase bean yield and growth when plant were co-inoculated with Rhizobium legumino-sarum bv. phaseoli under nitrogen-free conditions. This helper strain was a gram-positive, spore-forming and rod-shaped bacterium. Biolog test and phylogenetic analysis of 16S rRNA gene hypervariant region sequen-ces demonstrated the strain to be Bacillus subtilis. Forty-two bacterial isolates from root nodules of Argyrolobium uniflorum growing in the arid areas of Tu-nisia were characterized by phenotypic features, RFLP and sequencing of PCR-amplified 16S rRNA genes [47]. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew

a) b)

Figure 2. Rhizobia isolated form chickpea nodules appeared after 3–4 days (a) and appeared after 20 d (b) of growth.



Table 1. Bacterial endophytes occurring in different tissues of legumes.

Plant species Plant tissue Bacterial endophytes References

Acacia Root nodules Bradyrhizobium, Ensifer, Mesorhizobium, Burkholderia, Phyllo- bacterium and Devosia and non rhizobial bacteria

[13]

Alfalfa (Medicago sativa L.)

Roots, nodules, seed coat and lateral-root junctions

15 non rhizobial and 5 Gram + ve bacteria; Bravibacillus chosinensis, Bacillus megaterium, Microbacterium trichothecenolyticom, Escherichia coli, Salmonella enterica, Klebsiella, Azospirillum sp. B510, Ensifer (Sinorhizobium) meliloti

[2, 14–18]

Bean

Root and stem nodules

Azorhizobium caulinodans, Bacillus subtilis, Burkholderia cepacia, Ensifer (Sinorhizobium) meliloti, R. leguminosarum bv. phaseoli, R. tropici

[11, 12, 20]

Chickpea Root nodules Bradyrhizobium sp. [21, 22] Clovers Roots, nodules,

stem and foliage R. leguminosarum bv. trifolii, Agrobacterium rhizogenes [23]

Conzattia multiflora Root and nodules Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella [24] Cowpea Nodules Bradyrhizobium sp., Rhizobium sp. NGR 234 [21, 22] Fenugreek Nodules Ensifer (Sinorhizobium) meliloti [21, 22] Hedysarum glomeratum Nodules Agrobacterium, Enterobacteriaceae, Pseudomonas,

Rhizobium, R. sullae, Ralstonia, Xanthomonas [25]

Kennedia nigricans Roots 39 actinomycetes with 95% Streptomyces [26] Leucaena Roots Mesorhizobium loti, Ensifer (Sinorhizobium) meliloti, Rhizobium sp.

NGR 234, R. tropici, Ensifer (Sinorhizobium) meliloti 1021 [27, 28]

Lotus Nodules Mesorhizobium loti [28] Mungbean Nodules Bradyrhizobium sp. [21, 22] Mimosa pudica Nodules Burkholderia [29] Medicago truncatula Roots Microbial communities [30] Ornithopus compressus Nodules Curtobacterium, Enterobacteriaceae, Microbacterium, Pseudomonas [25] Pea Roots Pantoea agglomerans, Pseudomonas fluorescens, R. leguminosarum bv.

viciae., Streptomyces lydicus [3, 31]

Peanut

Root apoplast and nodules

Bradyrhizobium sp., Enterobacter spp., Klebsiella spp., Pseudomonas sp., Burkholderia cepacia

[32, 33]

Psoralea bituminosa Nodules Arthrobacter [34] Red clover

Root and nodules

22 rhizobial and non rhizobial bacteria, Agrobacterium rhizogenes, Pantoea agglomerans

[23, 35–37]

Scorpiurus muricatus Nodules Bacillus, Thiobacillus [25] Sesbania

Root and stem nodules

Azorhizobium caulinodans, Ensifer (Sinorhizobium) saheli, Ensifer (Sinorhizobium) teranga bv sesbania.

[38, 39]

Siratro Roots Rhizobium sp. NGR 234 [40] Soybean Roots and

Nodules 98 non symbiotic bacteria including Pantoea, Serratia, Acineto- bacter, Bacillus, Agrobacterium, and Burkholderia, and other bacteria Bradyrhizobium elkanii, Bradyrhizobium japonicum, Enterobacter agglomeransa, Enterobacter sakazakiia, Erwinia sp.; Klebsiella oxytocab, Klebsiella pneumonia, Pantoea sp., Pseudomonas citronellolis R. fredii, Rhizobium sp. NGR 234, Ensifer (Sinorhizobium) fredii, Bacillus subtilis, Bacillus thuringiensis

[4, 5, 19, 38, 41–45]

Sweet clover Roots Ensifer (Sinorhizobium) meliloti [21, 22] Tetragonolobus purpureus Roots Mesorhizobium, Staphylococcus [21, 22] Vicia – wild species Nodules 154 bacterial genera – R. leguminosarum R. gallicum, Mesorhizobium

huakuii, Ensifer meliloti and Bradyrhizobium spp., and 14 strains related to Agrobacterium, Phyllobacterium, Ensifer, Shinella and R. tropici, 22 strains of R. leguminosarum

[11, 20]

Wild legumes Roots and nodules 14 bacterial genera and 24 non rhizobial genera [25, 46] Mediterranean legumes Nodules 100 unculturable rhizobia and 24 non rhizobial [38, 45]

at temperatures up to 40 °C. Phylogenetically, the new isolates were grouped in the genera Ensifer (Sinorhizo-bium) (27), Rhizobium (13), and Agrobacterium (2). Except for the two Agrobacterium isolates, all strains induced nodulation on Argyrolobium uniflorum, but the number of nodules and nitrogen fixation efficiency varied among

them, with Ensifer (Sinorhizobium) sp. strains being the most effective symbionts. The Qinghai-Tibet plateau is the highest place in the world, and the environment in that plateau is harsh for both animals and plants, with low temperature, low concentration of oxygen and high solar radiations. In a



study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai-Tibet and a loess plateau were comparatively characterized [48]. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA-DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Ensifer (Sinorhizobium) meliloti, Ensifer (Sinorhizobium) fredii, Mesorhizobium sp., Phyllobac-terium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from the Qinghai-Tibet plateau and from the loess plateau were intermixed in these species or groups. Oxytropis spp. and Medicago archiducis-nicolai grown on the Qinghai-Tibet plateau were recorded as new hosts for R. leguminosarum, as well as Oxytropis glabra and Medicago lupulina for Ensifer (Sinorhizobium) fredii. Non-rhizobial endophytes from the surface sterilized root nodules of alfalfa (Medicago sativa L.) were isolated and characterized [14]. Out of 15 endophytic non-rhizobial strains isolated, three isolates were gram-positive strains and identified as Bacillus megaterium, Brevibacillus chosinensis and Microbacterium trichothe-cenolyticum. None of these isolates was able to nodulate alfalfa when re-inoculated under sterilized conditions. However, co-inoculation of all non-rhizobial strains with Ensifer (Sinorhizobium) meliloti positively influenced nodule number, while shoot and root parameters were comparable to those of uninoculated alfalfa plants. Several bacterial isolates were recovered from sur-face-sterilized root nodules of Arachis hypogaea L. (pea-nut) growing in soils from Argentina [33]. The 16S rDNA sequences of seven fast-growing strains were ob-tained and the phylogenetic analysis showed that these isolates belonged to the γ-proteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. These strains were unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were found as endopytes in the nodules and supported the idea that these isolates are opportunistic endophytes. Various rhizobial and non rhizobial strains were isolated from root nodules of two widespread south eastern Australian tree legumes (Acacia salicina, A. steno-phylla). This legume was nodulated primarily by Bradyr-hizobium, while the results indicate significant associa-tions with other root-nodule forming bacterial genera, including Rhizobium, Ensifer, Mesorhizobium, Burkholderia, Phyllobacterium and Devosia. Genetic analyses also re-vealed a diverse suite of non-nodulating bacterial endo-phytes, only a subset of which have been previously

recorded [13]. All in all, these investigations could show that nodules form a prime habitat for a wide variety of bacteria. Some of these may perform a helper function for nodule-inducing rhizobia, while others can be viewed as mainly opportunistic in the nitrogen-rich niche.

Occurrence of fungal endophytes in legumes In addition to bacteria, fungal symbionts are known for legumes. This makes legumes as an important plant functional group since they can form a tripartite sym-biosis with nitrogen-fixing Rhizobium bacteria and phos-phorus-acquiring arbuscular mycorrhizal fungi. How-ever, not much is known about arbuscular mycorrhizal fungal composition in legume root nodules. Legumes, and their root nodules, have relatively high nitrogen concentrations and thus a high phosphorus demand. Accordingly, the presence of legumes as well as nodule-related arbuscular mycorrhizal fungi can be explained by the specific nutritional requirements of legumes or by host specific interactions among legumes, root nod-ules, and arbuscular mycorrhizal fungi. Arbuscular mycorrhizal fungi form a symbiosis with up to 90% of all terrestrial plant species including food crops. In exchange for carbohydrates, they provide plants with phosphorus and nitrogen protect plants against patho-gens and increase drought resistance [49–52]. Interac-tions among endophytic bacteria and fungi are thus very important, as this will determine the extent of inoculation effect and potential in different legumes [53]. Different plant species are colonized by different

arbuscular mycorrhizal fungal populations and some plant-arbuscular mycorrhizal combinations are more likely to occur than others under field conditions.

Whether these plant-arbuscular mycorrhizal combina-tions have a functional relationship is unclear. Hel-gason et al. [54] have shown that Glomus hoi, which was found almost exclusively in the roots of Acer pseudopla-tanus in the field, was the only fungal species (of four species tested) to benefit this plant species in pot ex-periments. Diversity of fungi associated with mangrove legume Sesbania bispinosa was also reported [55]. There have been relatively few studies of mycorrhizal infectivity in legumes, and even less is known about mycorrhizal colonization in the rhizobial root nodules, the root organs in which nitrogen fixation takes place and that is rich in nutrients as compared to roots [56]. Though mycorrhizal fungi can colonize root nodules under laboratory conditions [57], it is not known whether root nodules are colonized under field condi-tions and which species are responsible for the coloni-



zation, though colonization of arbuscular mycorrhizal fungi in roots of angiosperms has been reported to produce mycorrhizal nodules (58). Nodules are different from roots in structure, function, and nutritional sup-ply or requirements. Nodules contain nitrogen-fixing

bacteria and have higher energy and phosphorus de-mands than the roots [59]. Nodule formation also re-sults in physiological changes, including responses related to plant defense reactions [60], however it seems that the defence in nodules is not necessarily more efficient than in roots. Further nutrient exchange in arbuscular mycorrhizal symbiosis seems to be highly specific and it is not known whether mycorrhizal fungi will have better acess to plant carbohydrates, if they form arbuscules in nodules as opposed to root cortical cells. These differences further suggest that the my-corrhizal fungi in roots and nodules might differ as well.

Occurrence of non-cultureable microbial endophytes in legumes Ikeda et al. [38, 45] reported shifts in the microbial com-munity of field-grown soybeans with different nodula-tion genotypes and nitrogen levels, suggesting that soybean accommodates a taxonomically characteristic microbial community by a system of autoregulation of nodulation involving symbiotic signaling networks. A unique procedure of bacterial enrichment from plant tissues assisted this study significantly, although profil-ing techniques currently used to assess the microbial community structure in environmental microbiology require further development [61]. These efforts could provide a platform for dissecting the organization and functions of rhizosphere, root, nodules, phyllosphere and endophytic microbial communities, and for identi-fying the plant loci that contribute to community struc-ture in planta [62]. In addition, a deeper understanding of plant-associated microbial communities offers excit-ing opportunities for controlling crop growth in sus-tainable agricultural settings. Although useful information has been provided by culture-dependent methodologies, the application of culture-independent methodologies using environmen-tal DNA has now revealed that the majority of envi-ronmental microbes, including plant-associated micro-bes, have not yet been cultured in the laboratory [63]. Plant DNA and PCR inhibitors present in a plant tissues are the main causes of interference in culture-indepen-dent analyses of endophytic microbes. Studies of the above-ground parts of plants and underground parts such as seeds, endophytes in roots and nodules have been particularly hampered by this technical difficulty,

Legume roots and nodules

Maceration of tissue in buffer using blender

Differential centrifugation

Density gradient centrifugation

Enriched non-cultureable bacterial cells

DNA extraction

Analysis for identification of microorganisms, genes, markers and complete genome sequencing, protein-protein interactions

Figure 3. Procedure for enrichment of bacterial DNA from plant tissue.

because these tissues contain a lower amount of micro-bial biomass and the plant DNA often out compete the bacterial 16S rRNA gene as a template in PCR amplifica-tion [64–68]. Hence, a technique was developed (Fig. 3) to evaluate the phylogenetic diversities of bacteria as-sociated with legume roots and nodules by a clone li-brary analysis of the bacterial 16S rRNA gene [44]. To adapt this enrichment method to other tissues and plants, fine tuning of the procedures is required in terms of detergent concentration and centrifugation conditions. This information, together with compari-sons of nodulation mutant plants, would enable signifi-cant progress in this field in the next decade. Some of the recent progress made in understanding plant–microbe symbiosis and its contribution to the plant and microbial worlds can be deduced from the articles re-viewed above.

Molecular interactions among microbial endophytes in legumes Our knowledge of the molecular interplay between plant and arbuscular mycorrhizal symbiosis and plant-root nodule symbionts has developed over the past decade [69, 70]. Diverse microbes are able to associate with plants in natural habitats as endophytes and epi-phytes. The cellular signaling networks determine over-lap of both symbionts in legumes, suggesting that the molecular components are shared. Extensive information is available on the molecular mechanisms of plant microbe interactions in the soil [71, 72], however, there is limited data on endophyte-



host molecular interactions. The plant response to endophytes seems to be conditioned, to a large extent, by the plant genotype which makes available different quality and quantity of exudates to the microbes. Plant gene expression may be modulated by the presence of the bacteria, and the genes so expressed provide clues as to the effects of endophytes in plants. Studies of different genes expressed in response to endophytic colonization revealed that genes for nitrogen assimila-tion, for carbon metabolism, for plant growth, as well as genes for a limited plant defense are induced [3]. Mutants of Medicago truncatula and Arabidopsis thaliana showed that plant defense-response pathways regulate the number of endophytic bacteria. Ethylene, a signal molecule for induced systemic resistance in plants, decreases endophytic colonization. An ethylene-insen-sitive Medicago truncatula mutant was hypercolonized, while the addition of the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid to wildtype Medicago truncatula and wheat reduced the amount of endophytic bacteria, a phenotype that was reversed by the use of ethylene inhibitors [73]. Flagella and type III secretion systems of the microbes are also involved in determin-ing the extent of colonization by a microbe. Plants can detect the presence of molecules from bacteria through chemoperception systems [74]. Flagellin, e.g., acts as an elicitor in Arabidopsis thaliana, inducing an oxidative burst, callose deposition, ethylene production and leads to the induction of defense-related genes. The modulation of plant defense molecules largely affects microbial colonization. Antimicrobial peptides have been isolated from maize and rice [75] and could have a role in controlling endophytic populations. Lim-iting carbon sources in the carbon apoplastic fluid may also restrict endophytic growth [76]. Endophytes may differentially express genes that are required to enter and colonize the plant, to grow and survive within plant tissues and to stimulate plant growth, compete and suppress pathogens, or produce different substan-ces. Endophytic bacteria provide useful and rich mod-els to study the genetic regulation of expression pro-files of bacteria in their natural niches or habitats, in this case in planta, which are more structured and vari-able than culture media under controlled laboratory conditions. Nevertheless, very little work has been done on this. To further understand molecular interactions of microbes with plants the development of new in vivo expression technologies is required exploiting different niches [77, 78] like endophytic growth or growth in the rhizosphere [79]. Such new reporter systems may be used to study gene expression while in situ hybridiza-tion studies demonstrate that particular genes are ex-

pressed in a specific tissue, but not in other tissues indicating a suitable environment for specific gene expression [80]. Bacteria can communicate through quorum sensing, a term applied to the production of diffusible signal molecules that control gene expression in a manner dependent upon bacterial population density (quorum) [81, 82]. Until now, phytopathogenic bacteria have been reported to respond to quorum by production of extra-cellular polysaccharides, degradative enzymes, antibiot-ics, siderophore, and pigments, as well as Hrp protein secretion, Ti plasmid transfer, motility, biofilm forma-tion, and epiphytic fitness. Interestingly, plants can perceive quorum sensing signals from the bacteria and control quorum-regulated bacterial responses (e.g., quorum interference) [83, 84]. It would be interesting to determine if endophytes produce quorum sensing molecules in planta and the effects they cause. Inside the plant, exchange of signal molecules among micro-organisms and of bacteria with the host is likely, al-though this has not yet been reported. It would also be interesting to address whether some of the well-studied molecular mechanisms used by phytopathogenic bacte-ria [85] are to some extent shared with endophytes or separate mechanism may be operative. In root bacterization experiments, species of nodule bacteria promoted growth of red clover more often when applied in combination with R. leguminosarum bv trifolii than when applied singly. However, Bacillus mega-terium, Bordetella avium and Curtobacterium luteum consist-ently promoted growth either individually or in combi-nation with rhizobia. Available reports indicate im-proved plant yield, plant health and nodulation when co-inoculated with nodule endophytes, compared to inoculation with rhizobia alone [5, 23, 86].

Benefits of occurrence of microbial endophytes in legumes The processes of host-microbe signaling and coloniza-tion in endophytic microbes, and the mechanisms lead-ing to mutual benefits are less-well characterized. Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubi-lize phosphate, or contribute assimilable nitrogen to plants. In addition, endophytic bacteria supply essential vitamins to plants. The production of auxin-like com-pounds increases seed production and germination along with increased shoot growth and tillering. Other effects of endophytes infection on the host plants in-clude osmotic adjustment, stomatal regulation, modifi-cation of root morphology, enhanced uptake of miner-als and alteration of nitrogen accumulation and meta-



bolism. Bacterial endophytes can accelerate seedling emergence, promote plant establishment under adverse conditions and enhance plant growth. Endophytic bac-teria promote plant growth indirectly by helping plants in acquiring nutrients, e.g. via nitrogen fixation, phos-phate solubilization or iron chelation, by preventing pathogen infections via antifungal or antibacterial agents, by out competing pathogens for nutrients by siderophore production, or by establishing the plant’s systemic resistance. Influence the plant growth directly by producing phytohormones such as auxin or cyto-kinin, or by lowering the plant ethylene levels. In addi-tion to these plant-growth-promoting traits, endophytic bacteria must also be compatible with host plants and able to colonize the tissues of the host plants without being recognized as pathogens [2]. Mechanisms of bioaugmentation with plant growth-promoting rhizobacteria include rhizospheric and endophytic bacteria [87, 88]. Bacterial endophytes are capable of suppressing nematode proliferation and this may benefit other crops in rotation with the host plants [88]. Some of the endophytic isolates are able to pro-duce antibiotics and siderophores in vitro [89]. Ethylene and indole-acetic acid are implicated in virtually all aspects of plant growth and development, ranging from seed germination to shoot growth and leaf abscission. Therefore, production of ACC deaminase and indole-acetic acid is likely an important and efficient way for endophytes to manipulate their plant hosts. Endophytic bacteria containing ACC deaminase are usually located in the plant root apoplast. The cleavage of ACC results in ammonia and α-ketobutyrate which are readily me-tabolized by the bacteria. In this way, these bacteria act as a sink for ACC. By lowering ethylene levels, the bac-teria increase the growth of plant roots and shoots and reduce the inhibitory effects of ethylene synthesis. In addition to being produced by plants, indole-acetic acid is also produced by root associated bacteria such as Enterobacter spp., Pseudomonas spp., Azospirillium spp. or Streptomyces ssp. Further lowering of ethylene levels in plant roots also relieves the suppression of auxin re-sponse factor synthesis, and indirectly increases plant growth [90]. Endophytes also can have allelopathic effects. For example, endophytic bacteria of red clover seem to be responsible for the allelopathic effects observed with these plants over maize, causing reduced plant emer-gence and plant height. Endophytes may contribute to their host plant defenses against phytopathogenic or-ganisms through plant physiology control. An increase in plant growth will prevent a variety of abiotic and biotic stresses, reflecting plant vigor or persistence and

may be considered as a potential protection to patho-gen challenge. Many studies demonstrated that plants infected with endophytes showed growth promotion, resistance to drought stress and tolerance to unsuitable soil conditions. Endophytes enhanced phytoremedia-tion of the organochlorine herbicide [91]. Fungal endophytes have the ability to colonize inter- or intracellular spaces in planta and often are localized in a single cell. The colonization of plant tissues by endophytes involves several steps, including host rec-ognition, spore germination, penetration of the epi-dermis and tissue colonization. After endophytes suc-cessfully colonized host tissue, the endophytic niche becomes established. In this endophytic niche, endo-phytes will obtain a reliable source of nutrition pro-vided from plant metabolites, exudates and leachates and in return the endophyte will protect its host against other microorganisms. Fungal endophytes are generally thought to protect plants by rapid coloniza-tion and thereby exhausting the limited available sub-strates so that none would be available for pathogens to grow. Furthermore, the plants produce lignin and other cell-wall deposits to limit the growth of endophytes, preventing it to become virulent. As a result, the cell wall becomes re-reinforced after endophytic coloniza-tion, which will make it more difficult for pathogens to infect the tissue. Endophytes promote the growth of plants in various ways, for example through secretion of plant growth regulators; e.g. indole-acetic acid [92, 93], via phosphate solubilizing activity, by enhancing hyphal growth and mycorrhizal colonization, produc-tion of siderophores and by supplying biologically fixed nitrogen. Enhancing plant growth may also be considered by phytohormones produced by fungal endophytes. In many cases, this will include fungi known to be phyto-pathogenic on other plants. Colletotrichum sp. produces substances like indole acetic acid to regulate plant processes. Research has indicated that Fusarium sp. E5 extract acts as an auxin. The mechanisms of fungal endophyte stimulation of host growth might be attrib-uted to the phytohormone produced by endophytic fungi. As defense responses are associated with in-creased demands for energy, reducing equivalents and carbon skeletons are provided by primary metabolic pathways. It is reasonable to believe that enhancement of plant growth induced by fungal endophytes will increase indirect protection to pathogens. Many fungal endophytes produce secondary metabo-lites and some of these compounds are antifungal and antibacterial molecules which strongly inhibit the growth of other microorganisms including plant patho-



gens. A group of biocontrol strains can produce single or multiple antibiotics, including terpenoids, alkaloids, aromatic compounds and polypeptides. Endophytic fungi also may produce many volatile organic com-pounds including tetrohydofuran, 2-methyl furan, 2-bu-tanone and aciphyllene which have antibiotic activities. Several antimicrobial metabolites, such as colletotric acid or griseofulvin, are also reported from endophytic fungi. Nematicidal activity has been reported for the culture filtrate of Fusarium oxysporum. Other metabolites of endophytic fungi are known to be responsible for pesticidal activity [94–97]. Colletotrichum truncatum causes disease in most of the leguminous plants. It occurs world-wide, causing dis-ease in several plants including soybean, broad bean, lentil, cowpea, Pisum sativum, and urd bean. Despite the pathogenic nature of the fungus, it has been reported as a biocontrol agent against several noxious weeds. Sesbania exaltata, a noxious weed of soybean, has been successfully managed by the pathogen. Its activity against plant pathogenic fungi, such as S. sclerotiorum and F. oxysporum, has also been reported. As an addi-tional benefit, endophytic fungi are known to produce biomolecules of pharmaceutical and agricultural im-portance. Many of the bioactive metabolites from endo-phytic fungi act as plant defence activator and have been useful in novel drug discovery. A near-future application may consider the use of genetically engineered endophytes with biological con-trol potential in agricultural crops. The endophytes Herbaspirillum seropedicae and Clavibacter xylii have been genetically modified to produce and excrete the δ-endo-toxin of Bacillus thuringensis to control insect pests. Bac-teria degrading recalcitrant compounds are more abun-dant among endophytic populations than in the rhi-zosphere of plants in contaminated sites which could mean that endophytes have a role in metabolizing these substances. Engineered endophytic Burkholderia cepacia strains improved phytoremediation and pro-moted plant tolerance to toluene. However, in most countries, the use of genetically modified organisms is restricted which prevents large-scale application of engineered microbes for biocontrol purposes.

Conclusion

The natural condition of plants seems to be in a close interaction with endophytes. Endophytes seem promis-ing to increase crop yields, remove contaminants, in-hibit pathogens, and produce fixed nitrogen or novel substances. The repertoire of their effects and functions

in plant has not been comprehensively defined. The challenge and goal is to be able to manage microbial communities to favor plant colonization by beneficial bacteria. This would be amenable when a better knowl-edge on endophyte ecology and their molecular interac-tions is attained. The contributions of this research field may have economic and environmental impacts.

References

[1] Allen, O.N., Allen, E.K., 1981. The Leguminosae: A Source Book of Characteristics, Uses and Nodulation. University of Wisconsin Press, Madison, WI/Macmillan Publishing, London.

[2] Rosenblueth, M., Martinez-Romero, E., 2006. Bacterial endophytes and their interactions with hosts. Mol. Plant Microbe Interact., 19(8), 827–837.

[3] Elvira-Recuenco, M., van Vuurde, J.W.L., 2000. Natural incidence of endophytic bacteria in pea cultivars under field conditions. Can. J. Microbiol., 46, 1036–1041.

[4] Oehrle, N.W., Karr, D.B., Kremer, R.J., Emerich, D.W., 2000. Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms. Can. J. Microbiol., 46, 600–606.

[5] Bai, Y., D’Aoust, F., Smith, D.L., Driscoll, B.T., 2002. Isola-tion of plant-growth-promoting Bacillus strains from soy-bean root nodules. Can. J. Microbiol., 48, 230–238.

[6] Kawaguchi, M., Minamisawa, K., 2010. Plant–microbe communications for symbiosis. Plant Cell Physiol., 51(9), 1377–1380.

[7] Long, H.H., Schmid, D.D., Baldwin, I.T., 2008. Native bacterial endophytes promote host growth in a species-specific manner, Phytohormone manipulations do not re-sult in common growth responses PLoS ONE. Available on the web: http://www.plosone.org, 3(7), e2702.

[8] Weir, B.S., 2011. The current taxonomy of rhizobia New Zealand rhizobia website. (http://www.rhizobia.co.nz/taxo-nomy/rhizobia.html).

[9] Dudeja, S.S., Narula, N., 2008. Molecular diversity of root nodule forming bacteria. In: Khachatourians, G.G., Arora, D.K., Rajendran, T.P., Srivastava, A.K. (eds.), Agriculturally Important Microorganisms, Vol. 2. Academic World In-ternational, Bhopal, India. pp. 1–24.

[10] Dudeja, S.S., Singh, N.P., Sharma, P., Gupta, S.C. et al., 2011. Biofertilizer technology and productivity of chick-pea in India. In: Singh, A., Parmar, N., Kuhad, R.C. (eds.), Bioaugmentation, Biostimulation and Biocontrol. Soil Bio-logy 28, DOI 10.1007/978-3-642-19769-7_3, # Springer-Verlag Berlin Heidelberg.

[11] Sun, L., Qiu, F., Zhang, X., Dai, X. et al., 2008. Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microb. Ecol. 55(3), 415–424.

[12] Lei, X., Wang, E.T., Chen, W.F., Sui, X.H., Chen, W.X., 2008. Diverse bacteria isolated from root nodules of wild Vicia species grown in temperate region of China. Arch. Microbiol., 190, 657–671.



[13] Hoque, M.S., Broadhurst, L.M., Thrall, P.H., 2011. Genetic characterisation of root nodule bacteria associated with Acacia salicina and Acacia. stenophylla (Mimosaceae) across south eastern Australia. Int. J. Syst. Evol. Microbiol., 61(2), 299–309.

[14] Stajković, Olivera, De Meyer, Sofie, Miličić, Bogić, Wil-lems, Anne, Delić, Dušica, 2009. Isolation and characteri-zation of endophytic non-rhizobial bacteria from root no-dules of alfalfa (Medicago sativa L.). Botanica SERBICA, 33(1), 107–114.

[15] Gagn’e, S., Richard, C., Rousseau, H., Antoun, H., 1987. Xylem residing bacteria in alfalfa roots. Can. J. Microbiol., 33, 996–1000.

[16] Charkowski, A.O., Barak, J.D., Sarreal, C.Z., Mandrell, R.E., 2002. Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts. Appl. Environ. Microbiol., 68, 3114–3120.

[17] Cooley, M.B., Miller, W.G., Mandrell, R.E., 2003. Coloniza-tion of Arabidopsis thaliana with Salmonella enterica and en-terohemorrhagic Escherichia coli O157:H7 and competition by Enterobacter asburiae. Appl. Environ. Microbiol., 69, 4915–4926.

[18] Dong, Y., Iniguez, A.L., Triplett, E.W., 2003. Quantitative assessments of the host range and strain specificity of en-dophytic colonization by Klebsiella pneumoniae 342. Plant Soil, 257, 49–59.

[19] Kuklinsky-Sobral, J., Araujo, W.L., Mendes, R., Geraldi, I.O., et al., 2004. Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environ. Microbiol., 6, 1244–1251.

[20] Lee, K.D., Bai, Y., Smith, D., Han, H.S., Supanjani, 2005. Isolation of plant-growth-promoting endophytic bacteria from bean nodules. Res. J. Agric. Biol. Sci., 1(3), 232–236.

[21] Kobayashi, D., Palumbo, J.D., 2000. Bacterial endophytes and their effects on plants and uses in agriculture. In: Ba-con, C.W., White, J.F. (eds.), Microbial Endophytes. Mar-cel, Dekker, New York, 199–233.

[22] Provorov, N.A., 2009. Plant-microbe symbioses as an evo-lutionary continuum. Zh. Obshch. Biol., 70(1), 10–34.

[23] Sturz, A.V., Christie, B.R., Matheson, B.G., Nowak, J., 1997. Biodiversity of endophytic bacteria which colonized clover nodules, roots, stems and foliage and their influen-ce on host growth. Biol. Fertil. Soils, 25, 13–19.

[24] Wang, L.L., Wang, E.T., Liu, J., Li, Y., Chen, W.X., 2006. Endophytic occupation of root nodules and roots of Melilo-tus dentatus by Agrobacterium tumefaciens. Microb. Ecol., 52, 436–443.

[25] Muresu, R., Polone, E., Sulas, L., Baldan, B. et al., 2008. Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes. FEMS Microbiol. Ecol., 63, 383–400.

[26] Castillo, U., Myers, S., Browne, L., Strobel, G. et al., 2005. Scanning electron microscopy of some endophytic strep-tomycetes in snakevine – Kennedia nigricans. Scanning, 27(6), 305–311.

[27] Nimnoi, P., Pongsilp, N. 2009. Genetic diversity and plant-growth promoting ability of the indole-3-acetic acid (IAA) synthetic bacteria isolated from agricultural soil as well as rhizosphere, rhizoplane and root tissue of Ficus religiosa L., Leucaena leucocephala and Piper sarmentosum Roxb. Res. J. Agric. Biol. Sci., 5(1), 29–41.

[28] Maruya, J., Saeki, K., 2010. The bacA gene homolog, mlr7400, in Mesorhizobium loti MAFF303099 is dispensable for symbiosis with Lotus japonicus but partially capable of supporting the symbiotic function of bacA in Sinorhizobium meliloti. Plant Cell Physiol., 51, 1443–1452.

[29] Pandey, P., Kang, S.C., Maheshwari, D.K., 2005. Isolation of endophytic plant growth promoting Burkholderia sp. MSSP. from root nodules of Mimosa pudica. Curr. Sci., 89, 177–180.

[30] Enis-James Neil, M.S. 2008. Composition and diversity of Medicago truncatula root bacterial endophyte populations resulting from growth in different Oklahoma soils. Dis-sertation submitted to Oklahoma State University, Okla-homa. USA – publication No 457167 pp. 251.

[31] Tokala, R.K., Strap, J.L., Jung, C.M., Crawford, D.L. et al., 2002. Novel plant-microbe rhizosphere interaction invol-ving Streptomyces lydicus WYEC108 and the pea plant (Pi-sum sativum). Appl. Environ. Microbiol., 68, 2161–2171.

[32] Kishore, G.K., Pande, S., Podile, A.R., 2005, Phylloplane bacteria increase seedling emergence, growth and yield of field-grown groundnut (Arachis hypogaea L.). Lett. Appl. Microbiol., 40(4), 260–268.

[33] Ibanez, F., Angelini, J., Taurian, T., Tonelli, M.L., Fabra, A., 2009. Endophytic occupation of peanut nodules by oppor-tunistic Gammaproteobacteria. Syst. Appl. Microbial., 32(1), 49–55.

[34] Sturz, A.V., Christie, B.R., Nowak, J., 2000. Bacterial en-dophytes: potential role in developing sustainable systems of crop production. Crit. Rev. Plant Sci., 19, 1–30.

[35] Sturz, A.V., Christie, B.R., 1995. Endophytic bacterial systems governing red clover growth and development. Ann. Appl. Biol., 126, 285–290.

[36] Sturz, A.V., Christie, B.R., 1996. Endophytic bacteria of red clover as agents of allelopathic clover-maize syndro-mes. Soil Biol. Biochem., 28, 583–588.

[37] Sturz, A.V., Nowak, J., 2000. Endophytic communities of rhizobacteria and the strategies required to create yield enhancing associations with crops. Appl. Soil Ecol., 15, 183–190.

[38] Ikeda, S., Okubo, T., Anda, M., Nakashita, H. et al., 2010. Community and genome-based views of plant-associated bacteria: plant–bacterial interactions in soybean and rice. Plant Cell Physiol., 51, 1398–1410.

[39] O’Callaghan, K.J., Davey, M.R., Cocking, E.C., 1997. Xylem colonization of the legume Sesbania rostrata by Azorhizobi-um caulinodans. Proc. Royal Soc., London (B), 264, 1821–1826.

[40] Adriana, S.L., Rafeala Simao, A.N., Alexandre, B., Krisle, D.A.S. et al., 2009. Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon Region as indicated by nodulation of siratro (Macroptilium atropurpureum). Plant Soil, 319, 127–145.

[41] Bai, Y., Zhou, X., Smith, D., 2003. Enhanced soybean plant growth due to coinoculation of Bacillus strains with Bra-dyrhizobium japonicum. Crop Sci., 43, 1774–1781.

[42] Li, J.H., Wang, E.T., Chen, W.F., Chen, W.X., 2008. Genetic diversity and potential for promotion of plant growth de-tected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol. Biochem., 40, 238–246.



[43] Kuklinsky-Sobral, J., Araujo, W.L., Mendes, R., Pizzirani-Kleiner, A.A., Azevedo, J.L., 2005. Isolation and characte-rization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide. Plant Soil, 273, 91–99.

[44] Ikeda, S., Kaneko, T., Ohkubo, T., Rallos, L.E. et al., 2009. Development of a bacterial cell enrichment method and its application to the community analysis in soybean stems. Microbiol. Ecol., 58, 703–714.

[45] Ikeda, S., Okubo, T., Kaneko, T., Inaba, S. et al., 2010. Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N le-vels. ISME J., 4, 315–326.

[46] Zakhia, F., Jeder, H., Domergue, O., Willems, A. et al., 2006. Characterisation of wild legume nodulating bacte-ria (LNB) in the infra-arid zone of Tunisia. Syst. Appl. Mi-crobiol., 27, 380–395.

[47] Mahdhi, M., Nzoué, A., Gueye, F., Merabet, C. et al., 2007. Phenotypic and genotypic diversity of Genista saharae mic-rosymbionts from the infra-arid region of Tunisia. Lett. Appl. Microbiol., 45, 604–609.

[48] Kan, F.L., Chen, Z.Y., Wang, E.T., Tian, C.F. et al., 2007. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet plateau and in other zones of China. Arch. Microbiol., 188, 103–115.

[49] Borowicz, V.A., 2001. Do arbuscular mycorrhizal fungi alter plant-pathogen relations? Ecology, 82, 3057–3068.

[50] Davies, F.T., Potter, J.R., Linderman R.G., 1993. Drought resistance of mycorrhizal pepper plants independent of leaf P concentration-response in gas exchange and water relations. Physiol. Plant. 87, 45–53.

[51] Newsham, K.K., Fitter, A.H., Watkinson, A.R., 1995. Multi-functionality and biodiversity in arbuscular mycorrhizas. Tree, 10, 407–411.

[52] Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis, 2nd edition, Academic Press, London, United Kingdom.

[53] Bandara, W.M.M.S., Seneviratne, G., Kulasooriya, S.A., 2006. Interactions among endophytic bacteria and fungi: effects and potentials. J. Biosci., 31(5), 645–650.

[54] Helgason, T., Merryweather, J.W., Denison, J., Wilson, P. et al., 2002. Selectivity and functional diversity in arbuscu-lar mycorrhizas of co-occurring fungi and plants from a temperate deciduous woodland. J. Ecol., 90, 371–384.

[55] Anita, D.D., Sridhar, K.R., Bhat, R., 2009, Diversity of fungi associated with mangrove legume Sesbania bispinosa (Jacq.) W. Wight (Fabaceae). Livestock Research for Rural Development. Volume 21, Article #67. http://www.lrrd. org/lrrd21/5/anit21067.htm.

[56] Kouchi, H., Imaizumi-Anraku, H., Hayashi, M., Hakoya-ma, T. et al., 2010. How many peas in a pod? Legume genes responsible for mutualistic symbioses under-ground. Plant Cell Physiol., 51, 1381–1397.

[57] Vidal-Dominguez, M.T., Azco’n-Aguilar, C., Barea, J.M., 1994. Preferential sporulation of Glomus fasciculatum in the root nodules of herbaceous legumes. Symbiosis, 16, 65–73.

[58] Duhoux, E., Rinaudo, G., Diem, H.G., Auguy, F. et al., 2001. Angiosperm Gymnostoma tree produce root nodules colonized by arbuscular mycorrhizal fungi related to Glo-mus. New Phytol., 149, 115–125.

[59] Almeida, J.P.F., Hartwig, U.A., Frehner, M., Nosberger, J., Lüscher, A., 2000. Evidence that P deficiency induces N feedback regulation of symbiotic N2 fixation in white clo-ver (Trifolium repens L.). J. Exp. Bot., 51, 1289–1297.

[60] Schultze, M., Kondorosi., A., 1998. Regulation of symbio-tic root nodule development. Annu. Rev. Genet., 32, 33–57.

[61] Saito, A., Ikeda, S., Ezura, H., Minamisawa, K., 2007. Mic-robial community analysis of the phytosphere using cul-ture-independent methodologies. Microbes Environ., 22, 93–105.

[62] Bisseling, T., Dangl, J.L., Schulze-Lefert, P., 2009. Next-generation communication. Science, 324, 691.

[63] Araujo, W.L., Marcon, J., Maccheroni, W.J., Van Elsas, J.D. et al., 2002. Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl. Environ. Microbiol., 68, 4906–4914.

[64] Normander, B., Prosser, J.I., 2000. Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions. Appl. Envi-ron. Microbiol., 66, 4372–4377.

[65] Sessitsch, A., Reiter, B., Peifer, U., Wilhelm, E., 2002. Cul-tivation independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. FEMS Microbiol. Ecol., 39, 23–32.

[66] Dent, K.C., Stephen, J.R., Finch-Savage, W.E., 2004. Mole-cular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet). J. Micro-biol. Methods, 56, 17–26.

[67] Seghers, D., Wittebolle, L., Top, E.M., Verstraete, W., Siciliano, S.D., 2004. Impact of agricultural practices on the Zea mays L. endophytic community. Appl. Environ. Microbiol., 70, 1475–1482.

[68] Mano, H., Morisaki, H., 2008. Endophytic bacteria in the rice plant. Microbes Environ., 23, 109–117.

[69] Lugtenberg, B.J.J., Chin-A-Woeng, T.F.C., Bloemberg, G.V., 2002. Microbe-plant interactions: Principles and mecha-nisms. Antonie van Leeuwenhoek, 81, 373–383.

[70] Oldroyd, G.E., Downie, J.A., 2008. Coordinating nodule morphogenesis with rhizobial infection in legumes. An-nu. Rev. Plant Biol., 59, 519–546.

[71] Singh, M, Dudeja, S.S., 2009. Soil-plant-microbe interacti-ons for ecological sustainability. In: Ali, M., Gupta, S., Ba-su, P.S., Naimuddin (eds.), National symposium on legu-mes for Ecological sustainability: Emerging challenges and opportunities. Nov. 3-5, 2007. Indian Society of Pulses Research and Development. IIPR, Kanpur, pp. 154–174.

[72] de Matos, Nogueira, E., Vinagre, F., Masuda, H.P. et al., 2001. Expression of sugarcane genes induced by inocula-tion with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans. Genet. Mol. Biol., 24, 199–206.

[73] Iniguez, A.L., Dong, Y., Carter, H.D., Ahmer, B.M.M. et al., 2005. Regulation of enteric endophytic bacterial coloniza-tion by plant defenses. Mol. Plant Microbe Interact., 18, 169–178.

[74] Boller, T., 1995. Chemoperception of microbial signals in plant cells. Annu. Rev. Plant Physiol. Plant Mol. Biol., 46, 189–214.



[75] Fuentes-Ramírez, L.E., Caballero-Mellado, J., Sepúlveda, J., Martínez-Romero, E., 1999. Colonization of sugarcane by Acetobacter diazotrophicus is inhibited by high N-fertiliza-tion. FEMS Microbiol. Ecol., 29, 117–128.

[76] Rediers, H., Rainey, P.B., Vanderleyden, J., De Mot, R., 2005. Unraveling the secret lives of bacteria: Use of in vivo expression technology and differential fluorescence in-duction promoter traps as tools for exploring niche-specific gene expression. Microbiol. Mol. Biol. Rev., 69, 217–261.

[77] Dudeja, S.S., Narula, N., Anand, R.C., 2009. Plant Microbe Interactions – A practical Manual for Laboratory Studies. Published by CCS Haryana Agricultural University, Hisar. pp. 1–140.

[78] Ramos-Gonzalez, M.I., Campos, M.J., Ramos, J.L., 2005. Analysis of Pseudomonas putida KT2440 gene expression in the maize rhizosphere: In vivo expression technology cap-ture and identification of root-activated promoters. J. Bac-teriol., 187, 4033–4041.

[79] Roncato-Maccari, L.D.B., Ramos, H.J.O., Pedrosa, F.O., Alquini, Y. et al., 2003. Endophytic Herbaspirillum seropedi-cae expresses nif genes in gramineous plants. FEMS Micro-biol. Ecol., 45, 39–47.

[80] Swift, S., Throup, J.P., Williams, P., Salmond, G.P., Ste-wart, G.S., 1996. Quorum sensing: A population-density component in the determination of bacterial phenotype. Trends Biochem. Sci., 21, 214–219.

[81] Badri, D.V., Weir, T.L., van der Lelie, D., Vivanco, J.M., 2009. Rhizosphere chemical dialogues: plant–microbe in-teractions. Curr. Opin. Biotechnol., 20, 642–650.

[82] Von Bodman, S.B., Bauer, W.D., Coplin, D.L., 2003. Quo-rum sensing in plant-pathogenic bacteria. Annu. Rev. Phytopathol., 41, 455–482.

[83] Mathesius, U., Mulders, S., Gao, M., Teplitski, M. et al., 2003. Extensive and specific responses of a eukaryote to bacterial quorum-sensing signals. Proc. Natl. Acad. Sci., USA, 100, 1444–1449.

[84] Van’t Slot, K.A.E., Knogge, W., 2002. A dual role for mic-robial pathogen-derived effector proteins in plant disease and resistance. Crit. Rev. Plant Sci., 21, 229–271.

[85] Parniske, M., 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat. Rev. Microbiol., 6, 763–775.

[86] Rajendran, G., Sing, F., Desai, A.J., Archana, G., 2008. Enhanced growth and nodulation of pigeon pea by co-

inoculation of Bacillus strains with Rhizobium spp. Bio-resour. Technol., 99(11), 4544–4550.

[87] Compant, S., Reiter, B., Sessitsch, A., Nowak, J. et al., 2005. Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl. Environ. Microbiol., 71, 1685–1693.

[88] Compant, S., van der Heijden, M.G.A., Sessitsch, A., 2010. Climate change effects on beneficial plant-microorganism interactions. FEMS Microbiol. Ecol., 73, 197–214.

[89] Sessitsch, A., Reiter, B., Berg, G., 2004. Endophytic bacte-rial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities. Can. J. Microbiol., 50, 239–249.

[90] Gao, F., Chuan-chao Dai, C., Liu, X., 2010. Mechanisms of fungal endophytes in plant protection against pathogens. African J. Microbiol. Res., 4(13), 1346–1351.

[91] Germaine, K.J., Liu, X., Cabellos, G.G., Hogan, J.P. et al., 2006. Bacterial endophyte-enhanced phytoremediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid. FEMS Microbiol. Ecol., 57, 302–310.

[92] Das, A., Varma, A., 2009. Symbiosis: the art of living. In: Varma, A., Kharkwal, A.C., (eds.), Symbiotic Fungi Prin-ciples and Practice. Springer, Berlin., pp. 1–28.

[93] Lee, S., Flores-Encarnación, M., Contreras-Zentella, M., Garcia-Flores, L. et al., 2004. Indole-3-Acetic acid biosyn-thesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in Cytochrome c biogenesis genes. J. Bacte-riol., 186, 5384–5391.

[94] Stancheva, I., Geneva, M., Zehirov, G., Tsvetkova, G. et al., 2006. Effects of combined inoculation of pea plants with arbuscular mycorrhizal fungi and rhizobium on nodule formation and nitrogen fixing activity. Gen. Appl. Plant Physiol., Special Issue, 32, 61–66.

[95] Seena, S., Sridhar, K.R., 2004. Endophytic fungal diversity of two sand dune wild legumes from the southwest coast of India. Can. J. Microbiol., 50(12), 1015–1021.

[96] Anita Dorothy, D., Sridhar, K.R., 2009. Assemlage and diversity of fungi associated with mangrove wild legume Canavalia cathartica Trop.Sub Trop. Agrosys., 10(2), 225–235.

[97] Selvaraj, T., Chellappan, P., 2006. Arbuscular mycorrhi-zae: a diverse personality. J. Central European Agric., 7, 349.

Dudeja, S.S., Giri, Rupa, Saini, Ranjana, Suneja-Madan, Pooja and Kothe Erika. 2012. Interaction of...

Documents

Transcript of Dudeja, S.S., Giri, Rupa, Saini, Ranjana, Suneja-Madan, Pooja and Kothe Erika. 2012. Interaction of...