Formation of Phenolic Compounds in Apogeotropic Roots of Cycad Plants

Formation and location of phenolic compounds in apogeotropic roots (coralloid roots) were studied in six cycad species, which belong to the genera Cycas, Encephalartos, and Ceratozamia. Total contents of soluble phenolic compounds in coralloid roots in all species studied varied insignificantly, with except for Ceratozamia mexicana that accumulated three times higher amounts of phenolic compounds. Phenolic compounds were accumulated in cell walls of cortical parenchyma of coralloid roots, in intercellular spaces, and in specialized storage cells, found in all zones of apogeotropic roots. The greatest number of phenol-storing cells was situated in the cortical parenchyma of the central part of coralloid roots, adjacent to a zone where active symbiotic cyanobacteria were localized, and in the coralloid root basal region lacking viable forms of cyanobionts. It was suggested that phenolic compounds affect the formation of symbiosis between cyanobacteria and apogeotropic roots of cycad plants, as well as their metabolism.     

Resistance Responses of Potato to Vesicular-Arbuscular Mycorrhizal Fungi under Varying Abiotic Phosphorus Levels

In mycorrhizal symbioses, susceptibility of a host plant to infection by fungi is influenced by environmental factors, especially the availability of soil phosphorus. This study describes morphological and biochemical details of interactions between a vesicular-arbuscular mycorrhizal (VAM) fungus and potato (Solanum tuberosum L. cv Russet Burbank) plants, with a particular focus on the physiological basis for P-induced resistance of roots to infection. Root infection by the VAM fungus Glomus fasciculatum ([Thaxt. sensu Gerdemann] Gerdemann and Trappe) was extensive for plants grown with low abiotic P supply, and plant biomass accumulation was enhanced by the symbiosis. The capacity of excised roots from P-deficient plants to produce ethylene in the presence or absence of exogenous 1-amino cyclopropane-1-carboxylic acid (ACC) was markedly reduced by VAM infection. This apparent inhibition of ACC oxidase (ACC_ox) activity was localized to areas containing infected roots, as demonstrated in split-root studies. Furthermore, leachate from VAM roots contained a potent water-soluble inhibitor of ethylene generation from exogenous ACC by nonmycorrhizal (NM) roots. The leachate from VAM-infected roots had a higher concentration of phenolics, relative to that from NM roots. Moreover, the rates of ethylene formation and phenolic concentration in leachates from VAM roots were inversely correlated, suggesting that this inhibitor may be of a phenolic nature. The specific activity of extracellular peroxidase recovered in root leachates was not stimulated by VAM infection, although activity on a fresh weight basis was significantly enhanced, reflecting the fact that VAM roots had higher protein content than NM roots. Polyphenol oxidase activity of roots did not differ between NM and VAM roots. These results characterize the low resistance response of P-deficient plants to VAM infection. When plants were grown with higher abiotic P supply, the relative benefit of the VAM symbiosis to plant growth decreased and root infection was lower. The in vivo ACC_ox activity was also greater in roots of plants grown on high levels of P compared with those grown on low levels, although the influence of VAM infection was partially to counteract the nutritional effect of P on ACC_ox activity. Similar to ACC_ox activity, extracellular peroxidase activity of roots increased linearly with increasing abiotic P supply, thus indicating a greater potential for resistance to VAM infection. These findings suggest that VAM fungi may alter phenolic metabolism of roots so as to hinder ethylene production and the root's ability to invoke a defense response. Raising the abiotic P supply to plants at least partially restores the capacity of roots to produce ethylene and may, in this way, increase the root's resistance to VAM infection.     

Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum

Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space. Received: 26 July 1997 / Accepted: 9 September 1997     

Preinoculation of lettuce and onion with VA mycorrhizal fungi reduces deleterious effects of soil salinity

The hypothesis that inoculation of transplants with vesicular-arbuscular mycorrhizal (VAM) fungi before planting into saline soils alleviates salt effects on growth and yield was tested on lettuce (Lactuca sativa L.) and onion (Allium cepa L.). A second hypothesis was that fungi isolated from saline soil are more effective in counteracting salt effects than those from nonsaline soil. VAM fungi from high- and low-salt soils were trap-cultured, their propagules quantified and adjusted to a like number, and added to a pasteurized soil mix in which seedlings were grown for 3–4 weeks. Once the seedlings were colonized by VAM fungi, they were transplanted into salinized (NaCl) soil. Preinoculated lettuce transplants grown for 11 weeks in the saline soils had greater shoot mass compared with nonVAM plants at all salt levels [2 (control), 4, 8 and 12 dS m^–1] tested. Leaves of VAM lettuce at the highest salt level were significantly greener (more chlorophyll) than those of the nonVAM lettuce. NonVAM onions were stunted due to P deficiency in the soil, but inoculation with VAM fungi alleviated P deficiency and salinity effects; VAM onions were significantly larger at all salt levels than nonVAM onions. In a separate experiment, addition of P to salinized soil reduced the salt stress effect on nonVAM onions but to a lesser extent than by VAM inoculation. VAM fungi from the saline soil were not more effective in reducing growth inhibition by salt than those from the nonsaline site. Colonization of roots and length of soil hyphae produced by the VAM fungi decreased with increasing soil salt concentration. Results indicate that preinoculation of transplants with VAM fungi can help alleviate deleterious effects of saline soils on crop yield.     

Growth of Glomus intraradices and its effect on linseed (Linum usitatissimum L.) in hydroponic culture

This paper presents a hydroponic system for culturing and maintaining the VAM fungus Glomus intraradices in symbiosis with linseed (Linum usitatissimum L.) under greenhouse conditions in pure nutrient solution. It was possible to obtain large quantities of mycorrhizal host plant roots as well as extramatrical mycelium and chlamydospores free of impeding residues of solid substrate components. Starting from linseed donor plants inoculated in sand and transferred to the nutrient solution, new infections arose within the fast growing root system, hyphae spread out into the liquid and infected mycorrhiza-free receptor plants. Data for infection rates and plant growth parameters are presented. In comparsion to other culture systems for VAM fungi, the advantages of this hydroponic system are discussed and potential uses suggested.     

Degradation of aromatic compounds through the β‐ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β‐ketoadipate pathway. In this study, we investigated the role of 3‐carboxy‐cis,cis‐muconate lactonizing enzyme (CMLE), an enzyme of the β‐ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β‐ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β‐ketoadipate pathway in plant‐pathogenic, soil‐borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.     

Topsoil storage effects on primary production and rates of vesicular-arbuscular mycorrhizal development inAgropyron trachycaulum

Summary A greenhouse study was conducted to determine the effects of stockpiling prairie grassland topsoil for 3 years on mycorrhizal development and root and shoot production of slender wheatgrass. The vesicular-arbuscular mycorrhizal (VAM) fungi involved in the symbiosis were also assessed as was the decomposition potential of the soil. During the first week of growth, VAM development in grasses grown in the stockpiled soil lagged behind that observed for grasses in the undisturbed soil. However, by 3 weeks, the mycorrhizal infection in plants in the stockpiled soil had reached levels similar to that in plants in the undisturbed soil. The dominant species of VAM fungi involved in the symbiosis at 8 weeks after planting shifted fromGlomus fasciculatum in the undisturbed soil toG. mosseae in the stockpiled soil. The delay in initial VAM infection and shift in VAM fungal species did not significantly affect plant productivity which was greatest in the stockpiled soil. The greater shoot production exhibited by grasses in the stockpiled soil was attributed to higher levels of NO₃-N in the stockpiled than undisturbed soil. The potential of the soil to decay dead slender wheatgrass roots was not altered by stockpiling.     

Vesicular-arbuscular mycorrhizal fungi,particularly Glomus tenue,in Venezuelan bromeliad epiphytes

The mycorrhizal status of water-impounding tank bromeliad epiphytes from three locales differing in altitude and moisture regime within Venezuelan cloud forest was examined. Species of vesicular-arbuscular mycorrhizal (VAM) fungi found in arboreal soils were compared to VAM fungi found in terrestrial soils. Sixteen of the 19 epiphytes examined for the presence of VAM fungi had roots with infection stages; 14 of these specimens showed growth of the fine endophyte Glomus tenue. Fine endophyte was the only VAM fungus found associated with epiphytes in the driest locale studied, while coarse VAM fungi (Gigaspora and Scutellospora spp.) were found at sampling locales receiving more moisture. Root infection was usually composed of intercellular hyphae and peletons; few arbuscules were observed. However, abundant extracellular hyphae were often observed tangled about roots in arboreal soil. It is concluded that epiphytic bromeliads probably benefit, at least periodically, from VAM fungi scavenging for sporadically available nutrients in arboreal soils. Glomus tenue may be particularly important as a colonizing VAM fungus in drier sites of Venezuelan cloud forest. The species composition of VAM fungi in arboreal soils was different to that of terrestrial soils sampled directly under epiphytic bromeliad perches, suggesting that VAM fungi species associated with bromeliads are dispersed to their hosts by vagile animal vectors.     

Influence of temperature on colonization of spring barleys by vesicular arbuscular mycorrhizal fungi

The effects of three soil temperatures on growth of spring barleys (Hordeum vulgare L.) and on their root colonization by vesicular arbuscular mycorrhizal (VAM) fungi from agricultural soils in Montana (USA) or Syria at different inoculum concentrations were tested in soil incubators in the greenhouse. The number of mycorrhizal plants as well as the proportion and intensity of roots colonized increased with higher soil temperatures. VAM fungi from Montana, primarily Glomus macrocarpum, were cold tolerant at 11°C while those from Syria, primarily G. hoi, were heat tolerant at 26°C. Inoculum potential of Montana VAM fungi was higher than Syrian VAM fungi in cool soils. Harmal, selected from Syrian barley land races, had the highest colonization by mycorrhizal fungi of the cultivars tested.Journal Series Paper: J-2532 Montana Agricultural Experiment Station.     

Role of Calcium on Phenolic Compounds and Enzymes Related to Lignification in Soybean (Glycine max L.) Root Growth

Changes in soluble and cell wall bound peroxidases activities, phenylalanine ammonia-lyase activity and phenolic compounds and lignin contents in roots of calcium-treated soybean (Glycine max (L.) Merr.) seedlings and their relationships with root growth were investigated. Three-day-old soybean seedlings were cultivated in nutrient solution with or without 0.025–5.0 mM calcium for 24 h. In general, length and fresh and dry weights of roots increased, while activities of enzymes (soluble and cell-wall peroxidases and phenylalanine ammonia-lyase) and phenolic compounds and lignin contents decreased against calcium concentrations. In the absence of calcium, phenylalanine ammonia-lyase and peroxidases activities increased by accumulating phenolic compounds and lignin due to restricted growth of roots. Enhanced calcium supply reduced the production of phenolic compounds and lignification due to low phenylalanine ammonia-lyase and peroxidases activities, reinforcing the essential role of calcium to improve the soybean root growth.     

In vitro production of Glomus deserticola in association with Ziziphus nummularia

Glomus deserticola Trappe, Bloss & Menge, one of the most commonly occuring VAM fungi of arid and semiarid regions, was cultured and multiplied in root organ cultures of Ziziphus nummularia under in vitro conditions. The In vitro produced VAM fungi established efficient symbiosis with in vitro raised plantlets of Z. nummularia. This VAM strain improved the biomass production, nutrient uptake, and acclimatization of the in vitro produced plantlets of Z. nummularia in pots under green house conditions.Abbreviations VAM Vesicular Arbuscular Mycorrhiza - N Nitrogen - P Phosphorus - MS Murashige and Skoog(1962) medium     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.     

Growth stimulatory substances for vesicular-arbuscular mycorrhizal fungi in Bahia grass (Paspalum notatum Flügge.) roots

Three kinds of the growth stimulatory substances for vesicular-arbuscular mycorrhizal (VAM) fungi were isolated from the 25% methyl alcohol (MeOH) eluates of Bahia grass roots fractionated by flash chromatography. One of them was identified as eupalitin (3,5-dihydroxy-6,7-dimethoxy-4-hydroxy flavone). At 10 ppm, this compound significantly stimulated the in vitro hyphal growth of a VAM fungus, Gigaspora ramisporophora Spain. The others were not identified. These results suggest that the flavonoids such as eupalitin, and the unidentified compounds may act as signal molecules in VAM symbiosis.     

Glomalean mycorrhizal fungi from tropical Australia

 The isolation of vesicular-arbuscular mycorrhizal (VAM) fungi from natural (savanna, rocky hill, wetland and rainforest) and disturbed (minesite) habitats in a seasonally-dry tropical region in the Northern Territory of Australia into open-pot cultures was undertaken to supplement knowledge about the diversity of these fungi. This experiment considered factors affecting the diversity of fungi obtained in trap cultures started using diluted soil from field sites and two host plants. A range of soil phosphorus and other nutrient levels from severely deficient to sufficient for maximal growth was used to determine the impact of nutrition on mycorrhizal associations of sorghum (Sorghum sp.) and clover (Trifolium subterraneum). Soil cores taken from pots at 6-week intervals provided roots and soil to assess mycorrhiza formation and sporulation without substantial damage to plants. The identification of VAM fungi to genus by observing morphological patterns within clover roots revealed substantial differences in fungus populations between soils and a moderate effect of nutrient levels on fungal diversity. Changes in the proportion of different fungi in roots over the 31 weeks of the experiment were also observed. Glomus spp. were initially the most abundant fungi within roots, but Scutellospora spp. gradually became more dominant at later harvests, while colonisation by Acaulospora spp. was limited at all times. For both clover and sorghum, sporulation was limited and was dominated by single species of Scutellospora and Acaulospora. This contrasted with the much higher diversity of spore types in the original field soils.     

Positive correlation between VAM-induced changes in root exudation and mycorrhizosphere mycoflora

The influence of vesicular-arbuscular mycorrhizal (VAM) fungi on rhizosphere mycoflora was studied together with the possible mechanism involved in this process. Six combinations of VAM fungi and phosphorus fertilizer treatments were applied to Leucaena leucocephala roots and quantitative and qualitative observations were made periodically of the rhizosphere mycoflora and constituents of root exudates. The results obtained indicate that the presence of specific mycoflora in the rhizosphere of mycrorrhizal roots is mediated through root exudates rather than being an outcome of improved P nutrition.     

Residual toxicity of soil-applied chlorothalonil on mycorrhizal symbiosis in Leucaena leucocephala

The residual effect of the fungicide chlorothalonil on the vesicular-arbuscular mycorrhizal (VAM) symbiosis was evaluated in a greenhouse experiment. The soil used was an oxisol (Tropeptic Eutrustox) treated with P to obtain target levels near-optimal for VAM activity or sufficient for nonmycorrhizal host growth. In the uninoculated soil treated with the former P level, the fungicide reduced VAM colonization of roots and completely suppressed symbiotic effectiveness measured in terms of pinnule P content. When this soil was inoculated with Glomus aggregatum, symbiotic effectiveness was significantly reduced but not eliminated by 50 mg of the fungicide kg⁻¹. At higher chlorothalonil levels, VAM effectiveness but not VAM colonization was completely suppressed in the inoculated soil. The pattern with which chlorothalonil influenced tissue P content and dry matter yield at the time of harvest closely paralleled its effect on VAM effectiveness. In the soil treated with P level sufficient for nonmycorrhizal host growth, the adverse effect of the fungicide on the above variables was appreciably milder than when the host relied on VAM fungi for its P supply. The toxic effect of the fungicide, therefore, was partly offset by P fertilization, suggesting that VAM fungi were more sensitive to chlorothalonil than the host. Our results demonstrate that although the toxic effect of chlorothalonil declined as a function of time, a significant level of toxicity persisted 12.5 weeks after the chemical was applied to soil. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3625. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3625.     

Chitinase in roots of mycorrhizal Allium porrum: regulation and localization

Chitinase (EC 3.2.1.14) activity was measured in roots of Allium prorrum L. (leek) during development of a vesicular-arbuscular mycorrhizal symbiosis with Glomus versiforme (Karst.) Berch. During the early stages of infection, between 10 and 20 d after inoculation, the specific activity of chitinase was higher in mycorrhizal roots than in the uninfected controls. However, 60–90 d after inoculation, when the symbiosis was fully established, the mycorrhizal roots contained much less chitinase than control roots. Chitinase was purified from A. porrum roots. An antiserum against beanleaf chitinase was found to cross-react specifically with chitinase in the extracts from non-mycorrhizal and mycorrhizal A. porrum roots. This antiserum was used for the immunocytochemical localization of the enzyme with fluorescent and gold-labelled probes. Chitinase was localized in the vacuoles and in the extracellular spaces of non-mycorrhizal and mycorrhizal roots. There was no immunolabelling on the fungal cell walls in the intercellular or the intracellular phases. It is concluded that the chitin in the fungal walls is inaccessible to plant chitinase. This casts doubts on the possible involvement of this hydrolase in the development of the mycorrhizal fungus. However, fungal penetration does appear to cause a typical defense response in the first stages that is later depressed.     

Detection of high-phosphorus tolerant VAM-fungi colonizing hops and peppermint

Summary In a continuing search for field sources of endomycorrhizal fungi that tolerate high levels of available phosphorus in soil, samples were obtained from long-established liberally fertilized hops (Humulus lupulus L.) and peppermint (Mentha piperita L.) fields. In hops fields, where extractable P (Bray) levels ranged from 21–196 ppm, spores of twoAcaulospora spp. and sixGlomus spp. were isolated. From peppermint fields with P levels of 44 to 244 ppm, spores of oneAcaulospora sp., fourGlomus spp. and oneGigaspora sp. were obtained. Vesicular-arbuscular mycorrhizal (VAM) fungi were found colonizing most roots to some extent, despite the very high fertility of several sites. Although percentages of colonization in hops tended to be low, ranging from 0 to 9.3%, colonizations in peppermint roots were considerably higher averaging 26.5% between the sampled fields, notwithstanding P levels as high as 244 ppm. Curiously, considerable external VAM hyphae were found adherent to or entangling roots in many cases, even where percentage of root length with internal VAM structures was low. The functional benefit, neutrality, or detriment to plants of such high-P tolerant species remains to be determined.Contribution from the USDA-ARS, Horticultural Crops Research Laboratory and the Dept. of Soil Science, Oregon State University, in cooperation with the Oregon State University Agricultural Experiment Station, Corvallis, OR 97331. Technical paper no. 6979 of the latter     

Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g^?1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD-malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non-mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea-Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non-VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.     

The Glycine–Glomus–Rhizobium symbiosis. IV. Interactions between the mycorrhizal and nitrogen-fixing endophytes

Abstract Soybean (Glycine max (L.) Men) plants were grown under controlled conditions in an experiment designed as a 4 × 4 factorial. The factors were N or P nutrition, with different strains of Rhizobium japonicum or N-fertilization as levels of the first factor and different species of vesicular-arbuscular mycorrhizal (VAM) fungi or P fertilization as levels of the other. Organisms used were R. japonicum strains USDA 110, USDA 136, and 61A118, and the VAM fungi Glomus versiforme (Karst.) Berch, Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe, and Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. There were 16 treatments: nine Rhizobium + Glomus combinations, three Rhizobium + V and three Glomus+ N combinations, and one non-symbiotic set of plants supplied with N + P. The tripartite symbioses were evaluated by analysis of variance against the Rhizobium + P and Glomus + N comparison treatments for effects on root and leaf dry mass, root N and P content, nodule mass and activity, and VAM colinization. Significant to highly significant main effects and interactions were found in virtually all evaluations due to both Rhizobium strain and VAM–fungal species. We conclude that different endophyte isolates affect not only the host plant, but also the development and function of their co-endophytes. These findings establish the existence of inter-endophyte compatibility, an important consideration when selecting or engineering for desirable endophyte traits.