Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth

Abstract Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N₂ fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.     

Mycorrhizal associations in Pinus massoniana Lamb. and Pinus elliottii Engel. inoculated with Pisolithus tinctorius

Dichotomous mycorrhizas were induced in Pinus massoniana and Pinus elliottii seedlings inoculated with Pisolithus tinctorius growing under non-axenic conditions. Six months after inoculation, Pinus massoniana seedlings exhibited a higher degree of infection, bore more mycorrhizas and had developed more abundant extramatrical mycelium than seedlings of Pinus elliottii. Nevertheless, seedlings of Pinus massoniana were stunted and exhibited chorosis of the needles, indicating a possible nutrient deficiency. Histological examination of these pine mycorrhizas showed an ectomycorrhizal association typical of gymnosperms with an intercellular Harting net penetrating between several layers of cortical cells close to the endodermis. However, strong polyphenolic reactions, intracellular hyphae and wall modifications were occasionally observed, indicating that both host-tissue incompatibility and ectendomycorrhizal association can occur in pine species under stressed conditions.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

The unique root-nodule symbiosis between Rhizobium and the aquatic legume,Neptunia natans (L. f.) Druce

We examined the development of the aquatic N₂-fixing symbiosis between Rhizobium sp. (itNeptunia) and roots of Neptunia natans L. f. (Druce) (previously N. oleracea Lour.) under natural and laboratory conditions. When grown in its native marsh habitat, this unusual aquatic legume does not develop root hairs, the primary sites of rhizobial infection for most temperate legumes. Under natural conditions, the aquatic plant floats and develops nitrogen-fixing nodules at emergence of lateral roots on the primary root and on adventitious roots at stem nodes, but not from the stem itself. Cytological studies using various microscopies revealed that the mode of root infection involved an intercellular route of entry followed by an intracellular route of dissemination within nodule cells. After colonizing the root surface, the bacteria entered the primary root cortex through natural wounds caused by splitting of the epidermis and emergence of young lateral roots, and then stimulated early development of nodules at the base of such roots. The bacteria entered the nodule through pockets between separated host cells, then spread deeper in the nodule through a narrower intercellular route, and eventually evoked the formation of infection threads that penetrated host cells and spread throughout the nodule tissue. Bacteria were released from infection droplets at unwalled ends of infection threads, became enveloped by peribacteroid membranes, and transformed into enlarged bacteroids within symbiosomes. In older nodules, the bacteria within symbiosomes were embedded in an unusual, extensive fibrillar matrix. Cross-inoculation tests of 18 isolates of rhizobia from nodules of N. natans revealed a host specificity enabling effective nodulation of this aquatic legume, with lesser affinity for Medicago sativa and Ornithopus sp., and an inability to nodulate several other crop legume species. Acetylene reduction (N₂ fixation) activity was detected in nodules of N. natans growing in aquatic habitats under natural conditions in Southern India. These studies indicate that a specific group of Rhizobium sp. (Neptunia) occupies a unique ecological niche in aquatic environments by entering into a N₂-fixing root-nodule symbiosis with Neptunia natans.We thank J. Whallon for technical assistance, G. Truchet, J. Vasse, S. Wagener, J. Beaman, F. DeBruijn, F. Ewers, and A. Squartini for helpful comments, and N.N. Prasad and G. Birla for assistance in conducting field observations. This work was supported by the Michigan Agricultural Experiment Station and National Science Foundation grants DIR-8809640 and BIR-9120006 awarded to the MSU Center for Microbial Ecology. This study is dedicated to the memory of Dr. Joseph C. Burton, a friend and colleague who made many contributions to the study of the Rhizobiumlegume symbiosis.     

The root-nodule symbiosis between Sarothamnus scoparius L. and its microsymbionts

When nitrogen fixing root nodules are formed, Sarothamnus scoparius (broom) is inoculated with its microsymbionts. Nodules studied under light and electron microscopy exhibited typical indeterminate nodule histology with apical, persistent meristem, age gradient of nodule tissues, and open vascular bundles, and also with some particular features such as: the presence of mitotic activity in the infected meristematic cells, lack of infection threads, distribution of bacteria by process of host cell division, and occurrence of a large bacteroid zone only with infected cells. The results of cross-inoculation tests have shown a broad host range for S. scoparius microsymbionts including not only the native host but also species such as: Lupinus luteus, Ornithopus sativa, Lotus corniculatus, Genista tinctoria, Chamaecitisus ratisbonensis, Macroptilium atropurpureum, and Phaseolus vulgaris. In addition, our data established a close symbiotic relationship of S. scoparius nodulators to Bradyrhizobium sp. (Lupinus) by comparison of the partial sequence of nodC gene of the strain CYT7, specific for the broom, to those from Bradyrhizobium sp. (Lupinus) strain D1 and others available in the public databases.     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.     

The role of Rhizobium conserved and host specific nodulation genes in the infection of the non-legume Parasponia andersonii

Summary Rhizobium and Bradyrhizobium bacteria gain intercellular entry into roots of the non-legume Parasponia andersonii by stimulating localized sites of cell division which disrupt the epidermis. Infection threads are then initiated from intercellular colonies within the cortex. Infection via the information of infection threads within curled root hairs, which commonly occurs in legumes, was not observed in Parasponia. The conserved nodulation genes nodABC, necded for the curling of legume root hairs, were not essential for the initiation of infection, however, these genes were required for Parasponia prenodule development. In contrast, the nodD gene of Rhizobium strain NGR234 was essential for the initiation of infection. In addition, successful infection required not only nodD but a region of the NGR234 symbiotic plasmid which is not needed for the nodulation of legumes. Agrobacterium tumefaciens carrying this Parasponia specific region, as well as legume nod genes, was able to form nodules on Parasponia which reached an advanced stage of development.     

Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

Summary The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.Abbreviations EH smallest emergent root hair - EM electron microscope - enod2 early nodulin2 gene - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract-mannitol-gluconate     

INTERACTION BETWEEN THE ECTOMYCORRHIZAL FUNGUS PISOLITHUS TINCTORIUS AND ROOT HAIRS OF PICEA MARIANA (PINACEAE)

The ectomycorrhizal fungus Pisolithus tinctorius interacts with roots of Picea mariana to form a typical mantle and Hartig net. Hyphae alter their growth pattern when in contact with susceptible root hairs in the mycorrhizal infection zone and grow acropetally, gradually covering the length of the hair to form a mantlelike structure. Initial contact with the hair may be influenced by a fibrillar material on the root hair surface. Although many root hairs become surrounded by fungal hyphae, they are not penetrated, and therefore are not entry points for this symbiotic fungus.     

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

Influence of aluminum on in vitro formation of Pinus caribaea mycorrhizae

The ability of Pinus caribaea var. hondurensis to form mycorrhizae was determined in vitro with seven species of ectomycorrhizal fungi in the presence of six levels of Al (added as AlCl₃) in a nutrient solution. The time required for mycorrhizal formation, the number of mycorrhizal root tips and the percent mycorrhizae were measured after 15, 30, 60, 90 and 120 days. Cenococcum graniforme was susceptible to Al toxicity at all Al concentrations. Pisolithus tinctorius and Suillus sp. were depressed at lower but stimulated at higher Al concentrations. The inverse was shown for Rhizopogon reaii and Hebeloma cylindrosporum. Tolerance to Al was verified for R. nigrescens and H. crustuliniforme. Pisolithus tinctorius had the largest mycorrhizal capacity, defined as the sum of the values for time, percent and number of mycorrhizae. Ectomycorrhizal fungi appeared to ameliorate Al damage to plant roots even in treatments where no mycorrhizae formed. Inoculation of pine seedlings with Al-tolerant mycorrhizal fungi is likely to improve reforestation efforts in highly-weathered tropical soils.     

Aluminum-mycorrhizal interactions in the physiology of pitch pine seedlings

Aluminum (Al) in the rhizosphere adversely affects plant nutrition and growth. Although many conifer species, and pitch pine (Pinus rigida) in particular, have evolved on acidic soils where soluble Al is often high, controlled environment studies often indicate that Al interferes with seedling growth and nutrient relations. Under normal field conditions, conifer roots grow in a symbiotic relationship with ectomycorrhizal fungi, and this association may modulate the effects of Al on root physiology. To investigate the influence of mycorrhizal infection on Al toxicity, pitch pine seedlings were grown with or without the ectomycorrhizal symbiont Pisolithus tinctorius and were exposed to low levels of Al in sand culture. Aluminum at 50 μM reduced nonmycorrhizal seedling growth and increased foliar Al concentrations, but did not alter photosynthetic gas exchange or other aspects of seedling nutrition. Nonmycorrhizal seedlings exposed to 200 μM Al exhibited decreased growth, increased transpiration rates, decreased water use efficiency, increased foliar Al and Na levels, and reduced foliar P concentrations. Seedlings inoculated with P. tinctorius exhibited unaltered growth, physiological function, and ionic relations when exposed to Al. The fungal symbiont evidently modulated ionic relations in the rhizosphere, reducing Al-P precipitation reactions, Al uptake, and subsequent root and shoot tissue Al exposure.     

Root nodulation of Sesbania rostrata.

The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.     

Mycorrhizal associations in Hong Kong Fagaceae

Castanopsis fissa Rehd. & Wils. is widely distributed from the tropics to the temperate regions of China and Japan and is an important forest component in Hong Kong. Pot-grown C. fissa seedlings inoculated with vegetative mycelial inocula of seven ectomycorrhizal fungi for 20 weeks were analysed for growth performance and mineral nutrient uptake of N, P, K and Ca. Shoot growth stimulation in all fungal treatments generally occurred in the first 4–8 weeks of seedling development. Uptake of P was generally enhanced by all fungi inoculated. Seedlings inoculated with Pisolithus tinctorius (Pers.) Coker and Couch and Cenococcum geophilm (Sow.) Fredinard et Winge, which colonized 22% and 33% of roots respectively, exhibited growth stimulation. The results indicate that P. tinctorius and C. geophilum are suitable for use in large-scale nursey inoculation.     

The infectivity ofRhizobium trifolii into a minute excised root of white clover

The degree of root cell mass necessary for normal infection thread production and nodule formation by rhizobia was studied. Excised white clover root tissues of 5.0, 2.5 and 1.0 mm in length, obtained from two days seedlings, were cultured in the dark withRhizobium trifolii 4S. A culture period of seven days was separated into an initial period of three days and a later period of four days. Culture media of liquid on agar (0.8%) were used including Fåhraeus inorganic medium and an organic medium containing vitamins, sucrose, and an extrinsic substance isolated fromR. trifolii 4S cells (ES-6000). When ES-6000 was added in culture medium for the initial period and root segments had an apical meristem, infection threads and nodules were most numerous.     

Influence of auxin and mycorrhizal fungi on thein vitro formation and growth ofPinus pinaster roots

Summary Experiments, performed withPinus pinaster cloned shoots submitted to an auxin treatment (NAA 10^–6 M, 18 days), demonstrated that rooting abilityin vitro persists over 5 successive induction cycles (through out a 9-month period). Rooting ability needs a permanent synthesis of auxin synergists which activate the metabolism of cell dedifferentiation and root primordium initiation. Agar culture permitted intense meristem initiation, but prevented active root elongation. In the presence of a mycorrhizal fungus,Pisolithus tinctorius orHebeloma cylindrosporum, roots resumed growth and short lateral root formation was stimulated. These two phenomena induced by fungal association improve the quality of the root systems required to facilitate successful transplantation from test-tubes to field conditions.     

Signaling and Gene Expression for Water-Tolerant Legume Nodulation

In the symbiotic interaction with rhizobia, legumes develop nodules in which nitrogen fixation takes place. Upon submersion, most temperate legumes are incapable of nodulation, but tropical legumes that grow in waterlogged soils have acquired water stress tolerance for growth and nodulation. One well-studied model plant, the tropical, semi-aquatic Sesbania rostrata, develops stem-located adventitious root primordia that grow out into adventitious roots upon submergence and develop into stem nodules after inoculation with the microsymbiont, Azorhizobium caulinodans. Sesbania rostrata also has a nodulated underground root system. On well-aerated roots, nodules form via root hair curling infection in the zone, just above the root tip, where root hairs develop; on hydroponic roots, an alternative process is used, recruiting a cortical intercellular invasion program at the lateral root bases that skips the epidermal responses. This intercellular cortical invasion entails infection pocket formation, a process that involves cell death features and reactive oxygen species. The plant hormones ethylene and gibberellin are the major signals that act downstream from the bacterial nodulation factors in the nodulation and invasion program. Both hormones block root hair curling infection, but cooperate to stimulate lateral root base invasion and play a role in infection thread formation, meristem establishment, and differentiation of meristem descendants.