Membrane-mediated decrease in root exudation responsible for phorphorus inhibition of vesicular-arbuscular mycorrhiza formation

The mechanism responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation in sudangrass (Sorghum vulgare Pers.) was investigated in a phosphorus-deficient sandy soil (0.5 micrograms phosphorus per gram soil) amended with increasing levels of phosphorus as superphosphate (0, 28, 56, 228 micrograms per gram soil). The root phosphorus content of 4-week-old plants was correlated with the amount of phosphorus added to the soil. Root exudation of amino acids and reducing sugars was greater for plants grown in phosphorus-deficient soil than for those grown in the phosphorus-treated soils. The increase in exudation corresponded with changes in membrane permeability of phosphorus-deficient roots, as measured by K⁺ (⁸⁶Rb) efflux, rather than with changes in root content of reducing sugars and amino acids. The roots of phosphorus-deficient plants inoculated at 4 weeks with Glomus fasciculatus were 88% infected after 9 weeks as compared to less than 25% infection in phosphorus-sufficient roots; these differences were correlated with root exudation at the time of inoculation. For plants grown in phosphorus-deficient soil, infection by vesicular-arbuscular mycorrhizae increased root phosphorus which resulted in a decrease in root membrane permeability and exudation compared to nonmycorrhizal plants. It is proposed that, under low phosphorus nutrition, increased root membrane permeability leads to net loss of metabolites at sufficient levels to sustain the germination and growth of the mycorrhizal fungus during pre- and postinfection. Subsequently, mycorrhizal infection leads to improvement of root phosphorus nutrition and a reduction in membrane-mediated loss of root metabolites.     

Effects of a vesicular-arbuscular mycorrhizal fungus and other soil microorganisms on growth,mineral nutrient acquisition and root exudation of soil-grown maize plants

Maize (Zea mays L. cv. Alize) plants were grown in a calcareous soil in pots divided by 30-m nylon nets into three compartments, the central one for root growth and the outer ones for hyphal growth. Sterle soil was inoculated with either (1) rhizosphere microorganisms other than vesicular-arbuscular mycorrhizal (VAM) fungi, (2) rhizosphere microorganisms together with a VAM fungus [Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappel], or (3) with a gamma-irradiated inoculum as control. Plants were grown under controlled-climate conditions and harvested after 3 or 6 weeks. VAM plants had higher shootroot ratios than non-VAM plants. After 6 weeks, the concentrations of P, Zn and Cu in roots and shoots had significantly increased with VAM colonization, whereas Mn concentrations had significantly decreased. Root exudates were collected on agar sheets placed on the interface between root and hyphal compartments. Six-week-old VAM and non-VAM plants had similar root exudate compositions of 72–73% reducing sugars, 17–18% phenolics, 7% organic acids and 3% amino acids. In another experiment in which root exudates were collected on agar sheets with or without antibiotics, the amounts of amino acids and carbohydrates recovered were similar in VAM and non-VAM plants. However, threeto sixfold higher amounts of carbohydrates, amino acids and phenolics were recovered when antibiotics were added to the agar sheets. Thus, the high microbial activity in the rhizosphere and on the rhizoplane limits the exudates recovered from roots.     

Stimulation of Vesicular-Arbuscular Mycorrhizal Fungi by Mycotrophic and Nonmycotrophic Plant Root Systems

Transformed root cultures of three nonmycotrophic and one mycotrophic plant species stimulated germination and hyphal growth of the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum (Becker & Gerd.) in a gel medium. However, only roots of the mycotrophic species (carrot) supported continued hyphal exploration after 3 to 4 weeks and promoted appressoria formation by G. etunicatum.     

Effect of onion (Allium cepa) root exudates on the hyphal growth of Gigaspora margarita

 The effect of root exudates from onions differing in P status on spore germination and hyphal growth of arbuscular mycorrhizal fungi was investigated. Onion (Allium cepa) was grown in solution culture at different phosphorus concentrations (0, 0.1, 1.0, 8.0 and 24.0 mg P l^–1) and root exudates were collected. When spores of the arbuscular mycorrhizal fungus, Gigaspora margarita were incubated with these root exudates, spore germination was only slightly affected but hyphal growth was greatly affected, particularly with exudates from P-deficient plants. This suggests that the P nutrition of host plants influences the composition of root exudates and thereby the hyphal growth of arbuscular mycorrhizal fungi. Accepted: 25 June 1995     

Light effects in mycorrhizal soybeans

Soybean (Glycine max. L. Merr.) plants were grown in an experiment with a 3 × 3 factorial design using different levels of light (170, 350, and 700 μE·m⁻²·s⁻¹) and P as factors. Plants were grown in a greenhouse in pot cultures using a soil low in plant-available P under three P regimes: no additional P, P added as KH₂PO₄, or P uptake enhanced by colonization of the host plant with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe. Development of the VAM fungal endophyte and of plants under all three P regimes was depressed by limiting light. However, the growth response of VAM plants to increasing light relative to non-VAM plants in the absence of additional P increased while the response relative to non-VAM plants with additional P decreased slightly. The highly significant interaction between the factors (P < 0.001) of the experiment was due to differences in the magnitude and direction of simple effects of the factors. The implications of these differences in terms of source-sink relationships of the symbionts and the value of different non-VAM controls in interpreting VAM effects are discussed.     

Colonization by Arbuscular Mycorrhizal Fungi of Sorghum Leads to Reduced Germination and Subsequent Attachment and Emergence of Striga hermonthica

Two sorghum cultivars: the Striga-tolerant S-35 and the Striga-sensitive CK60-B were grown with or without arbuscular mycorrhizal (AM) fungi, and with or without phosphorus addition. At 24 and 45 days after sowing (DAS) of sorghum, root exudates were collected and tested for effects on germination of preconditioned Striga hermonthica seeds. Root exudates from AM sorghum plants induced lower germination of S. hermonthica seeds than exudates from non-mycorrhizal sorghum. The magnitude of this effect depended on the cultivar and harvest time. A significantly (88–97%) lower germination of S. hermonthica seeds upon exposure to root exudates from AM S-35 plants was observed at both harvest times whereas for AM inoculated CK60-B plants a significantly (41%) lower germination was observed only at 45 DAS. The number of S. hermonthica seedlings attached to and emerged on both sorghum cultivars were also lower in mycorrhizal than in non-mycorrhizal plants. Again, this reduction was more pronounced with S-35 than with CK60-B plants. There was no effect of phosphorus addition on Striga seed germination, attachment or emergence. We hypothesize that the negative effect of mycorrhizal colonization on Striga germination and on subsequent attachment and emergence is mediated through the production of signaling molecules (strigolactones) for AM fungi and parasitic plants.Key Words: arbuscular mycorrhiza, root exudate, sorghum, striga, strigolactones, germination     

Differential Activity of Striga hermonthica Seed Germination Stimulants and Gigaspora rosea Hyphal Branching Factors in Rice and Their Contribution to Underground Communication

Strigolactones (SLs) trigger germination of parasitic plant seeds and hyphal branching of symbiotic arbuscular mycorrhizal (AM) fungi. There is extensive structural variation in SLs and plants usually produce blends of different SLs. The structural variation among natural SLs has been shown to impact their biological activity as hyphal branching and parasitic plant seed germination stimulants. In this study, rice root exudates were fractioned by HPLC. The resulting fractions were analyzed by MRM-LC-MS to investigate the presence of SLs and tested using bioassays to assess their Striga hermonthica seed germination and Gigaspora rosea hyphal branching stimulatory activities. A substantial number of active fractions were revealed often with very different effect on seed germination and hyphal branching. Fractions containing (−)−orobanchol and ent-2''-epi-5-deoxystrigol contributed little to the induction of S. hermonthica seed germination but strongly stimulated AM fungal hyphal branching. Three SLs in one fraction, putative methoxy-5-deoxystrigol isomers, had moderate seed germination and hyphal branching inducing activity. Two fractions contained strong germination stimulants but displayed only modest hyphal branching activity. We provide evidence that these stimulants are likely SLs although no SL-representative masses could be detected using MRM-LC-MS. Our results show that seed germination and hyphal branching are induced to very different extents by the various SLs (or other stimulants) present in rice root exudates. We propose that the development of rice varieties with different SL composition is a promising strategy to reduce parasitic plant infestation while maintaining symbiosis with AM fungi.     

Acquisition of phosphorus and copper by VA-mycorrhizal hyphae and root-to-shoot transport in white clover

White clover (Trifolium repens L.) plants were grown in a calcareous soil in pots with three compartments, a central one for root growth and two outer ones for growth of vesicular-arbuscular (VA) mycorrhizal (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe) hyphae (hyphal compartments). Phosphorus (P) was applied at three levels (0, 20 and 50 mg kg⁻¹ soil) in the outer compartments in mycorrhizal treatments. Root and shoot dry weight were increased in mycorrhizal plants with hyphal access to outer compartments. Growth of the mycorrhizal hyphae in the outer compartments was not significantly affected by variation in P level in these compartments. However, both concentration and amount of P in roots and shoots sharply increased with increasing P supply in the outer (hyphal) compartments. With increasing P levels the calculated delivery of P by the hyphae from the outer compartments increased from 34% to 90% of total P uptake. Hyphal access to the outer compartments also significantly increased both concentration and quantity of Cu in the plants. The calculated delivery of Cu by the hyphae from the outer compartments ranged from 53% to 62% of total Cu uptake, irrespective of the P levels and the amounts of P taken up and transported by the hyphae. However, the distribution of Cu over roots and shoots was largely dependent on P levels. With increase in P level in the outer compartments the calculated hyphal contribution to the total amount of Cu in the shoots increased from 12% to 58%, but decreased in the roots from 75% to 46%. In conclusion, uptake and transport by VA-mycorrhizal hyphae may contribute substantially not only to P nutrition, but also to Cu nutrition of the host.     

Systemic inhibition of arbuscular mycorrhiza development by root exudates of cucumber plants colonized by<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

The arbuscular mycorrhizal (AM) non-host plants mustard, sugar beet, lupin and the AM host plant cucumber were used as test plants. Cucumber plants were grown either in the absence of the AM fungus (AMF) Glomus mosseae or in a split-root system, with one side mycorrhizal and one side non-mycorrhizal. Root exudates of the AM non-host plants, the non-mycorrhizal cucumber plants and the mycorrhizal and the non-mycorrhizal side of the split-root system of mycorrhizal cucumber plants were collected and applied to cucumber plants inoculated with the AMF. Root exudates of non-mycorrhizal cucumber plants showed a significant stimulatory effect on root colonization, whereas root exudates from the mycorrhizal and the non-mycorrhizal sides of a split-root system of a mycorrhizal cucumber plant did not show this stimulatory effect and were even slightly inhibitory. Root exudates of the two AM non-host plants mustard and sugar beet significantly reduced root colonization in cucumber plants, whereas no such effect was observed when root exudates of the AM non-host plant lupin were applied.     

Root exudates of mycorrhizal tomato plants exhibit a different effect on microconidia germination of Fusarium oxysporum f. sp. lycopersici than root exudates from non-mycorrhizal tomato plants

The effect of root exudates from mycorrhizal and non-mycorrhizal tomato plants on microconidia germination of the tomato pathogen Fusarium oxysporum f. sp. lycopersici was tested. Microconidia germination was enhanced in the presence of root exudates from mycorrhizal tomato plants. The more tomato plants were colonized by the arbuscular mycorrhizal fungus Glomus mosseae, the more microconidia germination was increased, indicating that alterations of the exudation pattern depended on the degree of root AM colonization. Moreover, alterations of the exudation pattern of mycorrhizal plants are not only local, but also systemic. Testing the exudates from plants with a high and a low P level revealed that the alterations of the root exudates from mycorrhizal plants, resulting in a changed effect on microconidia germination, are not due to an improved P status of mycorrhizal plants.     

Effect of root exudates of mycorrhizal tomato plants on microconidia germination of Fusarium oxysporum f. sp. lycopersici

The effect of root exudates from mycorrhizal and non-mycorrhizal tomato plants on microconidia germination of the tomato pathogen Fusarium oxysporum f. sp. lycopersici was tested. Microconidia germination was enhanced in the presence of root exudates from mycorrhizal tomato plants. Tomato plants were colonised by the arbuscular mycorrhizal fungus Glomus fasciculatum, indicating that alterations of the exudation pattern depended on the degree of root AM colonisation. Testing the exudates from plants with a high and a low P level revealed that the alterations of the root exudates from mycorrhizal plants, resulting in a changed effect on microconidia germination, are not due to an improved P status of mycorrhizal plants.     

Interaction Between Meloidogyne arenaria and Glomus fascicuqlatus in Grape

Root zones of grape (Fitis vinifera cv Thompson Seedless) cuttings were infested with chlamydospores of Glomus fasciculatus or eggs of Meloidogyne arenaria or both. Growth of grapevines was greatest in mycorrhizal (G. fasciculatus) plants. Mycorrhizal development and growth of mycorrhizal and nonmycorrhizal plants were reduced in the presence of M. arenaria. At low initial nematode inoculum (PI) levels (approx. 200 eggs/plant), the presence of mycorrhizae enhanced plant growth during 1 yr, but no significant benefit was achieved by mycorrhizae where PI was high (approx. 2,000 eggs/plant). Final nematode populations were highest in mycorrhizal plants.     

Extensive In Vitro Hyphal Growth of Vesicular-Arbuscular Mycorrhizal Fungi in the Presence of CO2 and Flavonols

Various flavonoids were tested for their ability to stimulate in vitro growth of germinated spores of vesicular-arbuscular mycorrhizal fungi. Experiments were performed in the presence of 2% CO₂, previously demonstrated to be required for growth of Gigaspora margarita (G. Bécard and Y. Piché, Appl. Environ. Microbiol. 55:2320-2325, 1989). Only the flavonols stimulated fungal growth. The flavones, flavanones, and isoflavones tested were generally inhibitory. Quercetin (10 μM) prolonged hyphal growth from germinated spores of G. margarita from 10 to 42 days. An average of more than 500 mm of hyphal growth and 13 auxiliary cells per spore were obtained. Quercetin also stimulated the growth of Glomus etunicatum. The glycosides of quercetin, rutin, and quercitrin were not stimulatory. The axenic growth of G. margarita achieved here under rigorously defined conditions is the most ever reported for a vesicular-arbuscular mycorrhizal fungus.     

Glycine-Glomus-Rhizobium Symbiosis: V. Effects of Mycorrhiza on Nodule Activity and Transpiration in Soybeans under Drought Stress

Soybean (Glycine max [L.] Merr.) plants were nodulated (Bradyrhizobium japonicum) and either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or left uncolonized. All plants were grown unstressed for 21 days initially. After this period, some VAM and non-VAM plants were exposed to four 8-day drought cycles while others were kept well watered. Drought cycles were terminated by rewatering when soil moisture potentials reached −1.2 megapascal. Nodule development and activity, transpiration, leaf conductance, leaf and root parameters including fresh and dry weight, and N and P nutrition of VAM plants and of non-VAM, P-fed plants grown under the same controlled conditions were compared. All parameters, except N content, were greater in VAM plants than in P-fed, non-VAM plants when under stress. The opposite was generally true in the unstressed comparisons. Transpiration and leaf conductance were significantly greater in stressed VAM than in non-VAM plants during the first half of the final stress cycle. Values for both VAM and non-VAM plants decreased linearly with time during the cycle and converged at a high level of stress (−1.2 megapascal). Effects of VAM fungi on the consequences of drought stress relative to P nutrition and leaf gas exchange are discussed in the light of these findings and those reported in the literature.     

Effect of vesicular-arbuscular mycorrhizae on the growth and yield of rice-stubble cultured soybeans

Summary Inoculation with vesicular-arbuscular (VA) mycorrhizal fungiGlomus fasciculatus, G. mosseae, G. etunicatus orAcaulospora scrobiculatus, increased plant dry weight and seed yields of pot-grown soybean plants in sterilized soil. Inoculation with a mixture ofG. fasciculatus, G. mosseae andG. etunicatus, orG. fasciculatus alone, increased seed yields and other agronomic traits of soybean plants grown in a no-tillage, rice-stubble field.     

Phosphorus amendment inhibits hyphal branching of the VAM fungus Gigaspora margarita directly and indirectly through its effect on root exudation

 The effect of solution phosphorus (P) concentration upon growth of pregerminated spores of the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita was examined in vitro. P at 1 mM significantly inhibited branching of the primary germ tube. The number of branches and the total hyphal length were both significantly inhibited at 10 mM P. In addition, germinated spores exposed to exudates produced by Ri T-DNA-transformed roots of Daucus carota L. grown in the presence of P showed significantly less hyphal branching than those exposed to exudates produced by P-stressed roots. These phenomena could contribute to the observed inhibition of mycorrhiza formation by high P. Accepted: 31 July 1996     

Effect of root exudate fractions from P-deficient and P-sufficient onion plants on root colonisation by the arbuscular mycorrhizal fungus Gigaspora margarita

 The effect of root exudates from P-deficient onion on root colonisation by an arbuscular mycorrhizal fungus was examined. Onions (Allium cepa L.) were grown in solution culture at phosphorus concentrations of 0 (P0) and 2 (P2) mg P l^–1. Root exudates were collected and fractionated with Amberlite XAD-4 resin to give EtOH and water soluble fractions. Onions inoculated with the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were grown with or without (control) root exudates and exudate fractions in a growth chamber. After 24 days, arbuscular mycorrhiza levels and appressoria formation had increased in plants treated with P0-root exudate or the P0-EtOH fraction when compared to corresponding P2 treatments or control plants. P0 and P2 water-soluble fractions did not significantly affect either aspect of fungal development. These results suggest that hydrophobic compounds found in root exudates from P-deficient onion increase appressorium formation and, therefore, enhance mycorrhiza development. Accepted: 2 June 1998     

Glycine-Glomus-Rhizobium Symbiosis: II. Antagonistic Effects between Mycorrhizal Colonization and Nodulation

Soybean (Glycine max [L.] Merr.) plants grown in pot cultures were inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe and Rhizobium japonicum strain 61A118 at planting (G₁R₁) or at 20 days (G₂₀R₂₀), or with one of the endophytes after the other has colonized the host root (G₁R₂₀, G₂₀R₁). Nodulated (PR₁) and VAM (G₁N) dipartite associations, or nonsymbiotic plants (PN) using nutrient solutions with N, P, or N + P concentrations providing endophyte-equivalent nutrient inputs were used as controls. The delayed tripartite associations received the appropriate N, P, or N + P amendment while one or both endophytes were absent during the first 20 days of growth. Prior inoculation with one endophyte significantly inhibited development of the other. Root hexose sugar concentrations were negatively correlated with VAM colonization (r = −0.89), nodule activity (r = −0.91), and root P content (r = −0.93). Nodule (r = 0.97) and root (r = 0.96) P content correlated positively with VAM colonization. Nodule weight or VAM-fungal biomass were significantly greater in associations grown with only one endophyte. Dry weights of the PN, G₁N, PR₁, and G₂₀R₂₀ plants were significantly greater than those of tripartite plants inoculated at planting with either or both endophytes. Interendophyte inhibition is attributed to competition for root carbohydrates, and this effect apparently also affects overall plant productivity. The objective of the study was to determine if the timing of endophyte introduction and establishment affected the development of the other symbiotic partners.     

Fungal Growth Stimulation by CO2 and Root Exudates in Vesicular-Arbuscular Mycorrhizal Symbiosis

Transformed roots of carrot were used to determine the effects of root metabolites on hyphal development from spores of the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal growth of this obligately biotrophic symbiont was greatly stimulated by a synergistic interaction between volatile and exudated factors produced by roots. Root volatiles alone provided little stimulation, and root exudates alone had no effect. For the first time, carbon dioxide was demonstrated to be a critical root volatile involved in the enhancement of hyphal growth. ¹⁴C-labeled root volatiles were fixed by the fungus and thus strongly suggested that CO₂ served as an essential carbon source.     

Glycine-Glomus-Rhizobium Symbiosis : VI. Photosynthesis in Nodulated, Mycorrhizal, or N- and P-Fertilized Soybean Plants

Soybean (Glycine max [L.] Merr. cv Hobbit) plants were grown in a growth chamber for 56 days in a phosphorus- and nitrogen-deficient soil and were colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd) Gerd. and Trappe and Rhizobium japonicum strain USDA 136, or by either organism alone, or by neither. Non-VAM plants received supplemental phosphorus and nonnodulated plants supplemental nitrogen to achieve the same rate of growth in all treatments. Plants of all four treatments had the same (P > 0.05) dry weights at harvest, but VAM plants had higher rates of CO₂ exchange (CER, P < 0.05) and lower leaf P concentrations (P < 0.01). Leaf nitrogen concentrations were lower in nodulated than in nitrogen-supplemented plants (P < 0.01) while starch concentrations were higher (P < 0.01). There was a significant negative relationship between nitrogen and starch (r = −0.989). Statistical evaluation of the data showed that some parameters (CER, leaf area and phosphorus content) were associated with phosphorus nutrition (or the presence of the VAM fungus), others (leaf fresh weight and root dry weight) with nitrogen nutrition (or the presence of Rhizobium), and some (leaf nitrogen and starch content) by both factors. The development of microsymbiont structures and nodule activity were significantly lower in the tripartite association than in plants colonized by one endophyte only. The findings suggest that endophyte effects go beyond those of simple nutrition and associated source-sink relationships.