Production of vesicular-arbuscular mycorrhizal sporocarps,Glomus aggregatum,on fiberglass screens

Glomus aggregatum was culture onZea mays in sand. Fiberglass screen layers wrapped around the sand medium effectively provided a framework for sporocarp formation, and facilitated the subsequent harvest of the sporocarps.     

Sucrose mobilization in relation to essential oil biogenesis during palmarosa (<Emphasis Type="Italic">Cymbopogon martinii</Emphasis> Roxb. Wats. var.<Emphasis Type="Italic">motia</Emphasis>) inflorescence development

Palmarosa inflorescence with partially opened spikelets is biogenetically active to incorporate [U-¹⁴C]sucrose into essential oil. The percent distribution of¹⁴C-radioactivity incorporated into geranyl acetate was relatively higher as compared to that in geraniol, the major essential oil constituent of palmarosa. At the partially opened spikelet stage, more of the geraniol synthesized was acetylated to form geranyl acetate, suggesting that majority of the newly synthesized geraniol undergoes acetylation, thus producing more geranyl acetate.In vitro development of palmarosa inflorescence, fed with [U-¹⁴C]sucrose, resulted in a substantial reduction in percent label from geranyl acetate with a corresponding increase in free geraniol, thereby suggesting the role of an esterase in the production of geraniol from geranyl acetate. At time course measurement of¹⁴CO₂ incorporation into geraniol and geranyl acetate substantiated this observation. Soluble acid invertase was the major enzyme involved in the sucrose breakdown throughout the inflorescence development. The activities of cell wall bound acid invertase, alkaline invertase and sucrose synthase were relatively lower as compared to the soluble acid invertase. Sucrose to reducing sugars ratio decreased till fully opened spikelets stage, concomitant with increased acid invertase activity and higher metabolic activity. The phenomenon of essential oil biosynthesis has been discussed in relation to changes in these physiological parameters.     

Response of lettuce to pre- and post-transplant phosphorus and pre-transplant inoculation with a VA-mycorrhizal fungus

Pre-transplant inoculation of lettuce (Lactuca sativa L.) seedlings with the vesicular-arbuscular mycorrhizal fungusGlomus aggregatum (Smith and Schenck emend. Koske) increased P uptake and dry matter yields after transplanting into soil when the concentration of P in the soil solution was 0.02 mg L^–1 but had little affect in soil with 0.30 mg L^–1 solution P. Tissue P concentrations and dry matter yields after transplanting were increased by supplying adequate P prior to transplanting. Adequate levels of pre-transplant P appeared to be more important than maximum mycorrhizal infection of transplants for promoting post-transplant growth of the fast maturing lettuce crop.Journal Series No. 0000 of the Hawaii Institute of Tropical Agriculture and Human Resources.     

Root colonization of different hosts by the vesicular-arbuscular mycorrhizal fungus Glomus dimorphicum

Colonization of the roots of beans, alfalfa, onions, red clover, corn, and four barley cultivars (Bonanza, Klondike, Gateway 63, and Olli) by Glomus dimorphicum Boyetchko and Tewari, a vesicular-arbuscular mycorrhizal fungus isolated from a barley field in Alberta, Canada, was studied under greenhouse conditions. Infection levels were low in all four barley cultivars but were higher in beans, alfalfa, and onions and were highest in red clover and corn roots. The infection patterns of G. dimorphicum varied among all the hosts. Coiling of intracellular hyphae occurred in corn, alfalfa, and red clover roots. Appreciable numbers of intraradical vesicles were found only in red clover and bean roots, while arbuscules formed in all hosts except barley. It was concluded that the pattern of root colonization by G. dimorphicum is influenced by the host genome and that the fungal morphology in the roots is variable and, thus, not diagnostic for the mycorrhizal species.     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of palmarosa grass (Cymbopogon martinii)

A cell suspension culture was established from nodal callus ofCymbopogon martinii (Roxb.) Wats in a liquid medium containing Murashige and Skoog (1962) basal salts, vitamins, 100 mg 1^–1 myo-inositol and 20 g l^–1 of sucrose (MS) that was supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid and 1.15 M kinetin. An initial inoculum density of 2 x 10⁴ cells ml^–1exhibited optimum cell growth. Calli were obtained 12–15 days after the suspension was plated onto semisolid medium of a similar composition. When calli were transferred to semisolid regeneration medium containing MS + 6.7 M N ⁶-benzyl-adenine + 1.15 M kinetin, somatic embryogenesis and plantlet regeneration occurred after 10–25 days. There was no significant decrease in the regeneration potential of the calli even when the cultures were initiated from 47-week-old cell suspensions. Chromosome counts of cells in suspensions, calli and somatic embryos derived from cultures of different ages revealed the presence of diploids, tetraploids and octaploids. However, the 33 regenerated plants tested were all diploid, indicating that only diploid cells were capable of regeneration in vitro.Abbreviations MS Murashige and Skoog (1962) basal salts with vitamins (100 mg1^–1 myo-inositol, 20 g1^–1 sucrose) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N ⁶-benzyl-adenine - Kn kinetin - MSC MS + 13.6 M 2,4-D + 1.15 M Kn - MSR MS +6.7 M BA + 1.15 M Kn     

Variations in water status, gas exchange, and growth in Rosmarinus officinalis plants infected with Glomus deserticola under drought conditions

The influence of the arbuscular mycorrhizal fungus Glomus deserticola on the water relations, gas exchange parameters, and vegetative growth of Rosmarinus officinalis plants under water stress was studied. Plants were grown with and without the mycorrhizal fungus under glasshouse conditions and subjected to water stress by withholding irrigation water for 14 days. Along the experimental period, a significant effect of the fungus on the plant growth was observed, and under water stress, mycorrhizal plants showed an increase in aerial and root biomass compared to non-mycorrhizal plants. The decrease in the soil water potential generated a decrease in leaf water potential (psi(l)) and stem water potential (psi(x)) of mycorrhizal and non-mycorrhizal plants, with this decrease being lower in mycorrhizal water-stressed plants. Mycorrhization also had positive effects on the root hydraulic conductivity (Lp) of water stressed plants. Furthermore, mycorrhizal-stressed plants showed a more important decrease in osmotic potential at full turgor (psi(os)) than did non-mycorrhizal-stressed plants, indicating the capacity of osmotic adjustment. Mycorrhizal infection also improved photosynthetic activity (Pn) and stomatal conductance (g(s)) in plants under water stress compared to the non-mycorrhizal-stressed plants. A similar behaviour was observed in the photochemical efficiency of PSII (Fv/Fm) with this parameter being lower in non-mycorrhizal plants than in mycorrhizal plants under water stress conditions. In the same way, under water restriction, mycorrhizal plants showed higher values of chlorophyll content than did non-mycorrhizal plants. Thus, the results obtained indicated that the mycorrhizal symbiosis had a beneficial effect on the water status and growth of Rosmarinus officinalis plants under water-stress conditions.     

Dual inoculation with Aspergillus fumigatus and Glomus mosseae enhances biomass production and nutrient uptake in wheat (Triticum aestivum L.) supplied with organic phosphorus as Na-phytate

In a pot experiment, wheat was grown for 50 days in two heat-sterilized low-phosphorus (P) soils supplied with organic P as Na-phytate. Seed inoculation with the phosphatase-producing fungus (PPF) Aspergillus fumigatus or soil inoculation with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae increased shoot and root dry weight and root length, phosphatase activity in the rhizosphere and shoot concentrations of P and to a lesser extent of K and Mg. As a rule, the greatest effects on those parameters were most in the combined inoculation treatment (PPF + VAM). Shoot concentrations of Cu and Zn were only enhanced by VAM, not by PPF. At harvest, depletion of organic P in the rhizosphere soil increased in the order of: sterilized soil < PPF < VAM < PPF + VAM which corresponded with the enhanced P concentrations in the plants. The results demonstrate that organic P in form of Na-Phytate is efficiently used by VAM and that use of organic P can be increased by simultaneous inoculation with phosphatase-producing fungi.     

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     

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.     

Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae

The essential oil extracted from palmarosa (Cymbopogon martinii) has proven anti-microbial properties against cells of Saccharomyces cerevisiae. Low concentrations of the oil (0.1%) inhibited the growth of S. cerevisiae cells completely. The composition of the sample of palmarosa oil was determined as 65% geraniol and 20% geranyl acetate as confirmed by GC-FTIR. The effect of palmarosa oil in causing K(+) leakage from yeast cells was attributed mainly to geraniol. Some leakage of magnesium ions was also observed. Blocking potassium membrane channels with caesium ions before addition of palmarosa oil did not change the extent of K(+) ion leakage, which was equal to the total sequestered K(+) in the cells. Palmarosa oil led to changes in the composition of the yeast cell membrane, with more saturated and less unsaturated fatty acids in the membrane after exposure of S. cerevisiae cells to the oil. Some of the palmarosa oil was lost by volatilization during incubation of the oil with the yeast cells. The actual concentration of the oil components affecting the yeast cells could not therefore be accurately determined.     

Influences of Arbuscular Mycorrhizal Fungus Glomus mosseae on Growth and Nutrition of Lentil Irrigated with Arsenic Contaminated Water

Arsenic (As) contamination of irrigation water represents a major constraint to Bangladesh agriculture. While arbuscular mycorrhizal (AM) fungi have their most significant effect on P uptake, they have also been shown to alleviate metal toxicity to the host plant. This study examined the effects of As and inoculation with an AM fungus, Glomus mosseae, on lentil (Lens culinaris L. cv. Titore). Plants were grown with and without AM inoculum for 9 weeks in a sand and terra green mixture 50:50 v/v and watered with five levels of As (0, 1, 2, 5, 10 mg As L⁻¹ arsenate). Inoculum of Rhizobium leguminosarum b.v. Viceae strain 3841 was applied to all plants. Plants were fed with modified Hoagland solution (1/10 N of a full-strength solution and without P). Plant height, leaf number, pod number, plant biomass and shoot and root P concentration/offtake increased significantly due to mycorrhizal infection. Plant height, leaf/ pod number, plant biomass, root length, shoot P concentration/offtake, root P offtake and mycorrhizal infection decreased significantly with increasing As concentration. However, mycorrhizal inoculation reduced As concentration in roots and shoots. This study shows that growing lentil with compatible AM inoculum can minimise As toxicity and increase growth and P uptake.     

Interactions Between Plant Growth Promoting Fungi and Arbuscular Mycorrhizal Fungus Glomus Mosseae and Induction of Systemic Resistance to Anthracnose Disease in Cucumber

Cucumber plants were treated with plant growth promoting fungi (PGPF), Phoma sp. (isolates GS8-2 and GS8-3) and Penicillium simplicissimum (isolate GP17-2) with or without the arbuscular mycorrhizal fungus (AMF) Glomus mosseae. Induction of systemic resistance in cucumber against the anthracnose disease caused by Colletotrichum orbiculare was tested to evaluate the nature of the interaction between the PGPF and AMF. Root colonizing ability of each fungal species as influenced by their interaction was also evaluated. Plant roots were pre-inoculated with each PGPF isolate and/or G. mosseae for four weeks and leaves were then challenge inoculated with the pathogen C. orbiculare. Plants treated with each PGPF isolate showed considerable protection against the disease, but the treatment of G. mosseae had no significant effect on disease development. However, combined inoculation of Phoma GS8-2 or GS8-3 with G. mosseae reduced the level of disease protection induced by single inoculation of each Phoma isolate. In contrast, the high levels of protection induced by the P. simplicissimum GP17-2 were not altered by combining it with G. mosseae. Root colonization of both Phoma sp. isolates was also suppressed by the presence of the G. mosseae, but such an effect was not found on the population development of P. simplicissimum. The percent cucumber root length colonized by G. mosseae was not affected by any of the PGPF isolates tested.     

Cold-storage of mixed inoculum of Glomus intraradices enhances root colonization,phosphorus status and growth of hot pepper

Growth response of hot pepper (Capsicum annuum L.) inoculated with the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenck and Smith was evaluated in a greenhouse study. Three treatments in a soil-based medium amended with rock phosphate were: (1) control (CON), (2) inoculation of G. intraradices as a freshly prepared soil mixture of spores, hyphae and colonized roots of Sorghum vulgare (FM), and (3) inoculation of the fungus as cold-stored mixed inoculum (CM). Colonization at 14 weeks after inoculation with CM was 42.5%, but was significantly lower with FM (14.5%). Inoculation with G. intraradices as FM and CM increased growth of pepper, and total phosphorus and nitrogen uptake in shoots and roots compared with the CON treatment. Inoculation with CM resulted in significant increases in plant dry weight and chlorophyll concentration compared to the FM and CON treatments. Acid phosphatase activity in the rhizosphere was generally increased by AM fungal treatments. Highest acid phosphatase activity occurred at 14 weeks after inoculation with CM. Alkaline phosphatase activity in the CM treatment was significantly higher compared to that in CON and FM treatments throughout the growth period. Thus, cold storage of mixed inoculum enhanced colonization and growth-promoting activity of G. intraradices compared to freshly prepared inoculum.     

Studies on the diversity of the distinct phylogenetic lineage encompassing Glomus claroideum and Glomus etunicatum

Morphological and molecular characters were analysed to investigate diversity within isolates of the Glomus claroideum/Glomus etunicatum species group in the genus Glomus. The inter- and intra-isolate sequence diversity of the large subunit (LSU) rRNA gene D2 region of eight isolates of G. claroideum and G. etunicatum was studied using PCR-single strand conformational polymorphism (SSCP)-sequencing. In addition, two isolates recently obtained from Southern China were included in the analysis to allow for a wider geographic screening. Single spore DNA isolation confirmed the magnitude of gene diversity found in multispore DNA extractions. An apparent overlap of spore morphological characters was found between G. claroideum and G. etunicatum in some isolates. Analysis of the sequence frequencies in all G. etunicatum and G. claroideum isolates (ten) showed that four LSU D2 sequences, representing 32.1% of the clones analysed for multispore extraction (564) were found to be common to both species, and those sequences were the most abundant in four of the ten isolates analysed. The frequency of these sequences ranged between 23.2% and 87.5% of the clones analysed in each isolate. The implications for the use of phenotypic characters to define species in arbuscular mycorrhizal fungi are discussed. The current position of G. claroideum/G.etunicatum in the taxonomy of the Glomeromycota is also discussed.     

Mycorrhizal infection of an Agrostis capillaris population on a copper contaminated soil

To investigate the possible natural development of heavy metal tolerance in VA-mycorrhizal fungi, plants of Agrostis capillaris from an uncontaminated, a copper-contaminated and a zinc/cadmium-contaminated area were examined for VA-mycorrhizal infection. During a period of 5 years (1987 to 1991) the plants of the copper-tolerant population were hardly infected, whereas the population on the uncontaminated soil showed a mean infection of nearly 60% and the zinc/cadmium-tolerant population of 40%. A detailed analysis of the surroundings of the copper-enriched site revealed the presence of VA-mycorrhizal fungi and a negative correlation between the infection rate of A. capillaris and the copper content of the soil. In contrast to the copper-contaminated soil, the abundant presence of VA-mycorrhizal fungi in the area contaminated by zinc and cadmium indicates that these fungi have evolved a zinc and cadmium tolerance and that they may play a role in the zinc and cadmium tolerance of A. capillaris.     

Genetic differentiation in Plantago major L. in growth and P uptake under conditions of P limitation

To study possible adaptive mechanisms inbred lines from three populations of Plantago major from sites that were found to differ in P availability were compared. In a pot experiment the growth and P uptake either in the presence or absence of Glomus fasciculatum was determined. Under these P-limited conditions it was shown by partitioning the relative growth rate (RGR, in mg g^-1 day^-1) in the components root weight ratio (RWR, in g_roots g_plant ^-1), specific P uptake rate (SPUR, in mol P g_roots ^-1 day^-1), and P-efficiency (PEFF, in mg mol P^-1), that the increase in RGR of mycorrhizal infected plants was related to an increase in SPUR, and a decrease in RWR and PEFF. P. major ssp. major had a lower RGR (related to a lower PEFF and SPUR) and a higher RWR than P. major ssp. pleiosperma. In a second experiment three inbred lines were compared upon P depletion in a nutrient solution. The P. major ssp. major line had a lower RGR and higher RWR, and a higher accumulation of P in the roots than the P. major ssp. pleiosperma lines under optimal growing conditions. There were no differences among the inbred lines in the relative contribution of inorganic P to the total P concentration in the shoot. The results are discussed in relation to the characteristics of the habitats of the investigated P. major populations.     

Mycorrhizal inoculation effect on nodulation and N accumulation in Cajanus cajan at soil P concentrations sufficient or inadequate for mycorrhiza-free growth

The interaction of Cajanus cajan with Rhizobium and vesicular-arbuscular mycorrhizal fungi (VAMF) was investigated in a greenhouse experiment. C. cajan was planted in soil that had been inoculated with Glomus aggregatum or treated with benlate to suppress VAMF activity. Initial soil solution P concentrations of 0.06, 0.2, 0.4, and 0.8 mg l^-1 were established to test the interaction at external P levels that ranged from inadequate to nonlimiting for the host plant. At 0.06 and 0.2 mg P l^-1, mycorrhizal inoculation significantly increased plant P concentrations as well as nodule numbers and shoot dry weight. Mycorrhizal inoculation also significantly increased nodule dry weight at a soil P concentration of 0.4 mg l^-1 but did not significantly influence any of the other variables. The mycorrhizal inoculation effect observed at this soil solution P concentration could not be explained by any of the measures of plant P status. At 0.8 mg P l^-1, none of the measured variables were affected significantly by mycorrhizal inoculation. The results indicate that the enhanced nodulation associated with mycorrhizal inoculation at soil P concentrations lower than 0.4 mg l^-1 was explainable by mycorrhizal-mediated P uptake. The small but significant increase in nodule mass due to VAMF inoculation at 0.4 mg P l^-1 suggests that factors not related to plant P nutrition may be involved. On the other hand, the lack of a VAMF inoculation effect at 0.8 mg P l^-1 despite VAMF colonization at a level comparable to that observed at the former P concentration appear to discount this hypothesis. This observation is also supported by the lack of response of plant N status and nodule number to VAMF inoculation at this soil P concentration.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No.4066     

Mycorrhizal responsiveness of aerobic rice genotypes is negatively correlated with their zinc uptake when nonmycorrhizal

Plant Zn uptake from low Zn soils can be increased by Zn-mobilizing chemical rhizosphere processes. We studied whether inoculation with arbuscular mycorrhizal fungi (AMF) can be an additional or an alternative strategy. We determined the effect of AMF inoculation on growth performance and Zn uptake by rice genotypes varying in Zn uptake when nonmycorrhizal. A pot experiment was conducted with six aerobic rice genotypes inoculated with Glomus mosseae or G. etunicatum or without AMF on a low Zn soil. Plant growth, Zn uptake and mycorrhizal responsiveness were determined. AMF-inoculated plants produced more biomass and took up more Zn than nonmycorrhizal controls. Mycorrhizal inoculation, however, significantly increased Zn uptake only in genotypes that had a low Zn uptake in the nonmycorrhizal condition. We conclude that genotypes that are less efficient in Zn uptake when nonmycorrhizal are more responsive to AMF inoculation. We provide examples from literature allowing generalization of this conclusion on a trade off between mycorrhizal responsiveness and nutrient uptake efficiency.     

The impact of carbofuran soil application on growth and mycorrhizal colonization by Glomus clarum of groundnut

The influence of soil application of carbofuran on the growth response of groundnut, and both mycorrhizal colonization and sporulation of Glomus clarum was studied in a pot culture experiment. Carbofuran application with or without mycorrhizal inoculation increased the height of the potted plants measured 8 weeks after sowing. Mycorrhizal plants were significantly taller than nonmycorrhizal plants at the final harvest time (14 weeks). Carbofuran, at the recommmended field dose of up to 2 kg/ha, greatly increased shoot dry matter and pod yield in mycorrhizal groundnut. Colonization and sporulation by this VAM fungus were also enhanced significantly at these dose levels. The application of carbofuran at 5 kg/ha inhibited both growth and mycorrhizal status of groundnut.     

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.