A tripartite culture system for endomycorrhizal inoculation of micropropagated strawberry plantlets in vitro

The objective of the current investigation was to develop a reliable method to obtain vesicular arbuscular mycorrhizae (VAM) in micropropagated plantlets and to determine their influence on growth. An in vitro system for culturing the VA mycorrhizal fungus Glomus intraradices with Ri T-DNA-transformed carrot roots or nontransformed tomato roots was used in this study as a potential active source of inoculum for the colonization of micropropagated plantlets. After root induction, micropropagated plantlets grown on cellulose plugs (sorbarod) were placed in contact with the primary mycorrhizae in growth chambers enriched with 5000 ppm CO₂ and fed with a minimal medium. After 20 days of tripartite culture, all plantlets placed in contact with the primary symbiosis were colonized by the VAM fungus. As inoculum source, 30-day-old VA mycorrhizal transformed carrot roots had a substantially higher infection potential than 5-, 10-or 20-day-old VAM. Colonized plantlets had more extensive root systems and better shoot growth than control plants. The VAM symbiosis reduced the plantlet osmotic potential. This response may be a useful pre-adaptation for plantlets during transfer to the acclimatization stage.     

Response ofLeucaena leucocephala to vesicular-arbuscular mycorrhizal colonization and rock phosphate fertilization in an Oxisol

Response ofLeucaena leucocephala (Lam) de Wit to rock phosphate application and inoculation with the vesicular-arbuscular mycorrhizal (VAM) fungusGlomus aggregatum (Schenck and Smith emend Koske) was evaluated in a pot experiment. VAM colonization increased as rock phosphate application increased. Using phosphorus concentration in pinnules as an indicator of VAM activity, significant VAM activity occurred at 25 days after planting at the lower levels of rock phosphate application (0, 0.34 and 0.68 g P kg^–1). The time required for significant VAM activity was shortened by 5 days at the higher P levels (1.36, 2.72 and 5.44 g P kg^–1). The highest VAM activity was associated with the highest rate of rock phosphate application.Inoculation withG. aggregatum significantly increased the uptake of Cu, P and Zn and dry-matter yield at all levels of rock phosphate applied. Copper concentrations in roots of mycorrhizal Leucaena were significantly higher than that of shoots. The results indicated that Leucaena in symbiotic association with VAM fungi effectively utilized P from rock phosphate. However, high rates of rock phosphate are required to attain growth comparable to that obtained with the application of water-soluble phosphate.Contribution from Hawaii Institute of Tropical Agriculture and Human Resources, Journal Series No. 3243.     

Glomus fasciculatum alleviates transplantation shock of micropropagated Sesbania sesban

Investigations were carried out using the vesicular arbuscular mycorrhizal fungus, Glomus fasciculatum, to improve the success in transplanting micropropagated plantlets of Sesbania sesban. Plantlets were developed from somatic embryos and/or adventitious buds (induced from various explants on Gamborg's medium supplemented with 6-benzylaminopurine), in the presence of 10^–7 m α-naphthaleneacetic acid and 5×10^–6 m gibberellic acid. Subsequent to nodulating the roots with Rhizobium, plantlets were transplanted into sterile garden soil and inoculated with or without G. fasciculatum. Only 30% of plantlets transferred to soil without G. fasciculatum survived. In contrast, all the plantlets inoculated with G. fasciculatum survived. Histochemical study revealed the presence of intracellular hyphae with well-developed arbuscules and intercellular hyphae with vesicles, suggesting that G. fasciculatum formed a good mycorrhizal association with S. sesban roots. These observations showed that mycorrhizal association helped to increase the potential of micropropagated plantlets to successfully withstand transplantation shock. Received: 6 January 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Initial and residual toxicity of soil-applied thiram on the vesicular-arbuscular mycorrhizal symbiosis

The effect of the non-systemic fungicide thiram on the vesicular-arbuscular mycorrhizal (VAM) symbiosis and on Leucaena leucocephala was evaluated in a greenhouse experiment. In the uninoculated soil treated with P at a level optimal for mycorrhizal activity, mycorrhizal colonization of roots was low, and did not change as the concentration of thiram in the soil increased with the from 0 to 1000 mg/kg. When this soil was inoculated VAM fungus Glomus aggregatum, with VAM colonization was enhanced significantly, but decreased increase in thiram concentration until it coincided with the level observed in the uninoculated soil. Similarly, symbiotic effectiveness was reduced, its expression delayed or completely eliminated with increase in the concentration of thiram. Amending soil to a P level sufficient for non-mycorrhizal host growth fully compensated for thiram-induced loss of VAM activity if the thiram levels did not exceed 125 mg/kg. In soil treated with 50 mg thiram/kg, the toxicity of the fungicide dissipated within 66 days of application. At higher concentrations, the toxicity of the chemical on the mycorrhizal symbiosis appeared to be enhanced.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3716     

Effects of vesicular-arbuscular mycorrhizal infection and phosphate on Plantago major ssp. pleiosperma in relation to internal cytokinin concentrations

Relations between cytokinin concentrations and effects of P and vesicular-arbuscular mycorrhizal (VAM) infection were investigated in Plantago major L. ssp. pleiosperma Pilger. Both mycorrhizal infection by Glomus fasciculatum (Thaxt. sensu Gerdemann) Gerdemann and Trappe and P addition increased the shoot to root ratio, specific leaf area (SLA), and P concentrations of shoot and roots, and decreased the percentage of dry matter in the shoot during the experiment. In general, P concentration in the shoot and roots of each treatment correlated positively with the shoot to root ratio and specific leaf area, and negatively with the percentage of dry matter in the shoot. Cytokinin concentrations in the tissue of shoots and roots were determined using an enzyme-linked immunosorbent assay. Concentrations of zeatin and zeatin-ribosides in the free base and nucleotide fractions had increased more after P addition than in the case of mycorrhizal infection in both shoot and roots, whereas the P concentrations had increased less. It is suggested that zeatin and zeatin-ribosides are not the primary growth-substances involved in mediating VAM effects.     

Influence of edaphic and climatic factors on dynamics of root colonization and spore density of vesicular-arbuscular mycorrhizal fungi in Acacia farnesiana Willd. and A. planifrons W.et.A.

 A study was conducted to assess the dynamics of vesicular-arbuscular mycorrhizal (VAM) fungi associated with Acacia farnesiana and A. planifrons in moderately fertile alkaline soils. The intensity of root colonization by VAM fungi and the distribution of VAM fungal structures varied with host species over a period of time. The occurrence of vesicles with varied morphology in the mycorrhizal roots indicates infection by different VAM fungal species. This was further confirmed from the presence of spores belonging to different VAM fungal species in the rhizosphere soils. Root colonization and spore number ranged from 56% – 72% and 5 – 14 g^{–  1}soil in A. farnesiana and from 60% – 73% and 5 – 15 g^{–  1} soil in A. planifrons. Per cent root colonization and VAM spore number in the rhizosphere soil were inversely related to each other in both the Acacia species. However, patterns of the occurrence of VAM fungal structures were erratic. Spores of Acaulospora foveata, Gigaspora albida, Glomus fasciculatum, G. geosporum and Sclerocystis sinuosa were isolated from the rhizosphere of A. farnesiana whereas A. scrobiculata, G. pustulatum, G. fasciculatum, G. geosporum and G. microcarpum were isolated from that of A. planifrons. The response of VAM status to fluctuating edaphic factors varied with host species. In A. farnesiana though soil nitrogen (N) was positively correlated with root colonization, soil moisture, potassium and air temperature were negatively correlated to both root colonization and spore number. Per cent root colonization and spore number in A. planifrons were negatively related to each other. Further, in A. planifrons as the soil phosphorus and N were negatively correlated with the density of VAM fungal spores, the same edaphic factors along with soil moisture negatively influenced root colonization. Received: 16 May 1995 / Accepted: 7 February 1996     

Soil Solution Phosphorus Status and Mycorrhizal Dependency in Leucaena leucocephala

A phosphorus sorption isotherm was used to establish concentrations of P in a soil solution ranging from 0.002 to 0.807 μg/ml. The influence of P concentration on the symbiotic interaction between the tropical tree legume Leucaena leucocephala and the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum was evaluated in pot experiments. The level of mycorrhizal infection in Leucaena roots increased as the concentration of P was raised from 0.002 to 0.153 μg/ml. Higher levels of P depressed mycorrhizal infection, but the level of infection never declined below 50%. Periodic monitoring of P contents of Leucaena subleaflets indicated that significant mycorrhizal activity was detected as early as 17 days after planting, with the activity peaking 12 to 16 days thereafter. The highest level of mycorrhizal activity was associated with a soil solution P level of 0.021 μg/ml. Even though the mycorrhizal inoculation effect diminished as the concentration of P in the soil solution was increased, mycorrhizal inoculation significantly increased P uptake and dry-matter yield of Leucaena at all levels of soil solution P examined. The concentration of P required by nonmycorrhizal L. leucocephala for maximum yield was 27 to 38 times higher than that required by mycorrhizal L. leucocephala. The results illustrate the very high dependence of L. leucocephala on VAM fungi and the significance of optimizing soil solution phosphorus for enhancing the benefits of the VAM symbiosis.     

Development of vesicular-arbuscular mycorrhizal infection and the uptake of immobile nutrients inLeucaena leucocephala

The level of vesicular-arbuscular mycorrhizal (VAM) infection in the roots of Leucaena grown in a sand-soil mixture in the greenhouse increased rapidly with time and reached a peak value of 84% at 30 days from planting. The pattern of immobile nutrient uptake and accumulation closely paralleled that of the development of infection, particularly during the first 10–30 days after planting. Significant changes in dry matter yield were also observed only after a significant portion of the root length was colonized byGlomus aggregatum. The development of VAM infection was not accompanied by growth depression at any of the sampling periods. However, VAM roots had very high levels of Cu which was not translocated to shoots. It is hypothesized that such a diversion of Cu by the endophyte from the host could cause growth depression under conditions where the soil volume is supplied with sub-optimal levels of Cu. Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Series No. 3186.     

Enhanced N-Transfer from a Soybean to Maize by Vesicular Arbuscular Mycorrhizal (VAM) Fungi

Using a split-root technique, roots of soybean plants were divided between two pots. In one of the two pots, two maize plants were grown and half of those pots were inoculated with the vesicular arbuscular mycorrhizal (VAM) fungus, Glomus fasciculatus. Fifty-two days after planting, ¹⁵N-labeled ammonium sulfate was applied to the pots which contained only soybean roots. Forty-eight hours after application, significantly higher values for atom per cent ¹⁵N excess were found in roots and leaves of VAM-infected maize plants as compared with the non-VAM-infected maize plants. Results indicated that VAM fungi did enhance N transfer from one plant to another.     

Inhibition of mycorrhizal symbiosis in Leucaena leucocephala by chlorothalonil

The effect of the fungicide, chlorothalonil, on vesicular-arbuscular mycorrhizal (VAM) symbiosis was studied in a greenhouse using Leucaena leucocephala as test plant. Chlorothalonil was applied to soil at 0, 50, 100 and 200 μg g⁻¹. The initial soil solution P levels were 0.003 μg mL⁻¹ (sub-optimal) and 0.026 μg mL⁻¹ (optimal). After 4 weeks, the sub-optimal P level was raised to 0.6 μg mL⁻¹ (high). The soil was either uninoculated or inoculated with the VAM fungus, Glomus aggregatum. The fungicide reduced mycorrhizal colonization of roots, development of mycorrhizal effectiveness, shoot P concentration and uptake and dry matter yields at all concentrations tested, although the highest inhibitory effect was noted as the concentration of the fungicide was increased from 50 to 100 μg g⁻¹. Phosphorus applied after four weeks tended to partially offset the deleterious effects of chlorothalonil in plants grown in the inoculated and uninoculated soil which suggests that the fungicide was interfering with plant P uptake. The results suggest that the use of chlorothalonil should be restricted to levels below 50 μg g⁻¹ if the benefits of mycorrhizal symbiosis are to be expected. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3464. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3464.     

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.     

Effectiveness of mycorrhizal fungi on globe artichoke (Cynara cardunculus L. var. scolymus) micropropagation

The effectiveness of two arbuscular mycorrhizal (AM) fungal isolates (Glomus intraradices and Glomus viscosum) in sustaining plant growth and the physiological activities of the micropropagated globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori) were investigated during acclimatization and 90 days after plant establishment. All the mycorrhizal microplants survived transplant shock thus confirming the positive role of AM fungi colonization on ex vitro establishment. The growth increased in mycorrhizal plants, especially in plants inoculated with Glomus viscosum. Mycorrhizal plantlets showed higher stomatal conductance, which is probably necessary to supply the carbon needs of fungal symbionts. The SPAD (soil plant analysis development) data could be useful for plant management as a predictor for tissue nitrogen levels. The higher SPAD values in mycorrhizal plants are strictly related to a higher photosynthetic potential, and consequently to their better nitrogen nutrient status due to the symbiotic relationship. Regardless of the mycorrhizal performance in the host–fungus combination, the most efficient fungus for the artichoke microplants was Glomus viscosum.     

Dual axenic culture of sheared-root inocula of vesicular-arbuscular mycorrhizal fungi associated with tomato roots

Surface-sterilized sheared-root inocula of two vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus intraradices and G. versiforme) from pot cultures associated with excised tomato roots showed significant sporulation and the production of an extensive hyphal biomass. As many as 10²–10³ axenic mature spores were recovered in Petri dishes during 3 months incubation in the dark. Propagules of both species were able to complete their vegetative life cycle in vitro and efficiently colonize Acacia albida roots after 1 month under greenhouse conditions. The effectiveness of 0.5 cm pieces of VAM roots as starter inocula indicates the high inoculum potential of intravesicle propagules.     

Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum

To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.     

Biochemical changes in Glomus fasciculatum colonized roots of Lycopersicon esculentum in presence of Meloidogyne incognita

Glasshouse experiments were conducted to elicit biochemical substantiation for the observed difference in resistance to nematode infection in roots colonized by mycorrhiza, and susceptibility of the fresh flush of roots of the same plant that escaped mycorrhizal colonization. Tomato roots were assayed for their biochemical profiles with respect to total proteins, total phenols, indole acetic acid, activities of polyphenol oxidase, phenylalanine ammonia lyase and indole acetic acid oxidase. The roots of the same plant (one set) received Glomus fasciculatum and G. fasciculatum plus juveniles of Meloidogyne incognita separately; and half the roots of second set of plants received G. fasciculatum while the other half of roots did not receive any treatment. Roots colonized by G. fasciculatum recorded maximum contents of proteins and phenols followed by that of the roots that received G. fasciculatum plus M. incognita. However, IAA content was lowest in the roots that received mycorrhiza or mycorrhiza plus juveniles of root-knot nematode and correspondingly. Roots that received juveniles of root-knot nematode recorded maximum IAA content and per cent increase over healthy check and mycorrhiza-inoculated roots. The comparative assay on the activities of PPO, PAL and IAA oxidase enzymes in treated and healthy roots of tomato, indicated that PAL and IAA oxidase activities were maximum in G. fasciculatum colonized roots followed by the roots that received mycorrhiza plus juveniles of root-knot nematode, while the activity of PPO was minimum in these roots. The roots that received juveniles of root-knot nematode recorded minimum PAL and IAA oxidase activities and maximum PPO activity. Since the roots of same plant that received mycorrhiza and that did not receive mycorrhiza; and the plant that received nematode alone and mycorrhiza plus nematode recorded differential biochemical contents of proteins, total phenols and IAA, and differential activities of enzymes under study, it was evident that the biochemical defense response to mycorrhizal colonization against root-knot nematodes was localized and not systemic. This explained for the response of plant that differed in root galling due to nematode infection in presence of mycorrhizal colonization. The new or fresh roots which missed mycorrhizal colonization, got infected by nematodes and developed root galls.     

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.     

Host genotype dependency and growth enhancing ability of VA-mycorrhizal fungi for Eleusine coracana (finger millet)

Inoculation of finger millet (Eleusine coracana Gaertn.) plants with one of six different vesicular, arbuscular, mycorrhizal (VAM) fungi increased plant biomass, height, leaf area and absolute growth rate; however, effectiveness of the various VAM fungi varied significantly. Maximum root colonization and mycorrhizal efficacy was observed with plants inoculated with Glomus caledonicum. Among five host genotypes tested for mycorrhizal dependency against G. caledonicum, genotype HR-374 gave the highest plant biomass, mycorrhizal efficacy and root colonization, the inoculation resulting in increased mineral (phosphate, nitrogen, Zn²⁺ and Cu²⁺) content and uptake in shoots.     

Phosphorus effect on phosphatase activity in endomycorrhizal maize

Success of a mycorrhizal symbiosis is influenced by the availability of phosphorus (P) in the soil. Maize ( Zea mays L. cv. Great Lakes 586) plants were grown under five different levels of soil P, either in the presence or absence of formononetin or the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith. We detected physiological differences in mycorrhizal roots very early in the development of symbiosis, before the onset of nutrient‐dependent responses. Under low P levels, VAM roots accumulated a greater shoot dry weight (13%), root P concentration (15%) and protein concentration (30%) than non-VAM roots, although root growth was not statistically significantly different. At higher P levels, mycorrhizal roots weighed less than non-VAM roots (10%) without a concomitant host alteration of growth or root P concentration. Mycorrhizal colonization decreased as soil P increased. Formononetin-treatment enhanced colonization of the root by G. intraradices and partially overcame inhibition of VAM colonization by high soil P concentrations. This is the first report that formononetin improves root colonization under high levels of soil P. Acid phosphatase (ACP) and alkaline phosphatase (ALP) activities were closely related to the level of fungal colonization in corn roots. ACP activity in corn roots responded more to soil P availability than did ALP activity (38% more). These results suggest that ACP was involved in the increased uptake of P from the soil, while ALP may be linked to active phosphate assimilation or transport in mycorrhizal roots. Thus, soil P directly affected a number of enzymes essential in host-endophyte interplay, while formononetin enhanced fungal colonization.     

Mycorrhizal and nonmycorrhizal host growth in response to changes in pH and P concentration in a manganiferous oxisol

Glomus aggregatum and Leucaena leucocephala were allowed to interact in a manganese-rich oxisol at pH 4.3–6.0 and at soil P concentrations considered optimal for mycorrhizal host growth and sufficient for nonmycorrhizal host growth. At 0.02 mg P l^-1, vesicular-arbuscular mycorrhizal fungal (VAMF) colonization of roots increased as soil pH increased from 4.3 to 5.0. However, VAMF colonization of roots did not respond to further increases in pH. At pH 6.0, growth of mycorrhizal Leucaena observed at 0.02 mg P was comparable with that observed at 0.8 mg P l^-1. Increasing P concentration from 0.02 to 0.8 mg P 1^-1 increased target soil pH from 4.3 to 4.7 and reduced the concentration of available soil Mn from 15.1 to 1.9 mg 1^-1. Thus, the normal plant growth observed at the higher P concentration at pH<5 was mainly due to the alleviation of Mn toxicity as a result of its precipitation by excess P. VAMF colonization levels observed at pH 5.0–6.0 were similar, but maximal plant growth occurred at pH 6.0, suggesting that the optimal pH for mycorrhizal formation was substantially lower than for VAMF effectiveness. The poor growth of Leucaena at the lower P concentration in the unlimed soil was largely due to high concentrations of Mn²⁺ and H⁺ ions.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3910     

Effectiveness of VAM fungi in nonsterile soils before and after optimization of P in soil solution

Five tropical soils were either not inoculated or inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus aggregatum. The degree to which VAM effectiveness was expressed in the soils was evaluated prior and after solution P status was adjusted for optimal VAM activity. VAM effectiveness determined by monitoring P concentrations of pinnules of Leucaena leucocephala leaves as a function of time and as dry matter yield determined at the time of harvest, indicated that in three of the soils VAM effectiveness was either very restricted or altogether unexpressed irrespective of vesicular-arbuscular mycorrhizal fungal (VAMF) inoculation if soil solution P was not optimized for VAM effectiveness. After P optimization, effectiveness was significantly increased by VAMF inoculation although in four of the soils, densities of indigenous VAMF propagules greatly exceeded that attained by the inoculum after it was mixed with soil. Mycorrhizal fungal inoculation effects varied from soil to soil, depending on the extent to which the effectiveness of indigenous and introduced endophytes was enhanced by P optimization and the similarity of inherent soil solution P concentrations to the range known to be optimum for VAM effectiveness. Of the indicator variables monitored, VAMF colonization was least sensitive to treatment effects followed by shoot P concentration measured at the time of harvest.Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal series No. 3781.Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal series No. 3781.