Genetic manipulation in vesicular-arbuscular mycorrhizal fungi

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages. The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO₂ and plant flavonols.     

Interactions between vesicular-arbuscular mycorrhizal fungi and asparagus

Summary The vesicular-arbuscular mycorrhizal (VAM) fungus,Glomus versiforme increased significantly the growth ofAsparagus officinalis under controlled conditions using Turface as the growth medium. The growth responses, including increases in root fresh weight, numbers of shoots, shoot dry weight, and shoot height follow a pattern similar to other mycorrhizal systems. Indigenous VAM fungi appeared to have negative effects on average shoot fresh and dry weight, number of shoots per pot and average shoot height on one year oldA. officinalis seedlings obtained from the field and grown under controlled conditions. These results may be due either to the high levels of soluble phosphate present in the soil or the ineffectiveness of the particular indigenous fungi as mycorrhizal fungi in asparagus. Indigenous mycorrhizal fungi overwinter in asparagus root crown as vesicles and as external and internal hyphae. Soil obtained from the same fields as the one year old crowns was a good source of mycorrhizal inoculum for sterile seedlings.     

Occurrence of nitrogen-fixing Azospirillum in vesicular-arbuscular mycorrhizal fungi

Nitrogenase activity measured by acetylene reduction, was detected when surface-sterilized spores of vesicular-arbuscular (VA) mycorrhizal fungi (Glomus fasciculatum, G. intraradices, G. scientillans, G. mosseae, Gigaspora gilmorei andEndogone dusii) were inoculated into nitrogen-free liquid medium containing malic acid and incubated under microaerophilic conditions (99% N₂+1% O₂) at 30°C.Azospirillum species were isolated from the nitrogenase-active cultures.     

The Beltsville method for soilless production of vesicular-arbuscular mycorrhizal fungi

A low-cost, low-maintenance system for soilless production of vesicular-arbuscular mycorrhizal (VAM) fungus spores and inoculum was developed and adapted for production of acidophilic and basophilic isolates. Corn (Zea mays) plants were grown with Glomus etunicatum, G. mosseae or Gigaspora margarita in sand automatically irrigated with modified Hoagland's solution. Sand particle size, irrigation frequency, P concentration, and buffer constituents were adjusted to maximize spore production. Modified half-strength Hoagland's solution buffered with 4-morpholine ethane-sulfonic acid (MES) automatically applied 5 times/day resulted in production of 235 G. etunicatum spores/g dry wt. of medium (341000 spores/pot) and 44 G. margarita spores/g dry wt. of medium (64800 spores/pot). For six basophilic isolates of G. mosseae, CaCO₃ was incorporated into the sand and pots were supplied with the same nutrient solution as for acidophilic isolates. The increased pH from 6.1±0.2 to 7.2±0.2 resulted in spore production ranging from 70 to 145 spores/g dry wt. (102000–210000 spores/pot). Spore production by all isolates grown in the soilless sand system at Beltsville has exceeded that of traditional soil mixtures by 32–362% in 8–12 weeks.     

Re-establishment of indigenous vesicular-arbuscular mycorrhizal fungi after topsoil storage

Summary The effect of lead and cadmium on the dry matter yield and nutrient concentration of tomato (Lycopersicum esculentum) and egg-plant (Solanum melongena) was studied in a greenhouse experiment. The results showed some benificial effects of Pb and Cd at their lower doses and toxicity at higher levels. The threshold concentration (C₁₀), toxicity index (T₁₀) and loading rate to produce C₁₀ were also calculated. These values suggested the tomato to be more tolerant than egg-plant for heavy metals whose toxicity was found in the order of Cd>Pb in both the plants.The application of the heavy metals was found to effect the nutrient concentration and their uptake by plants. The results have been explained on the basis of their complexation and translocation through the plant sap.     

Distribution of vesicular-arbuscular mycorrhizal fungi in the natural ecosystem

Natural occurrence of vesicular-arbuscular mycorrhizal (VAM) fungi in Haryana soils showed that VAM sporulation was more intensive in the rhizosphere of nonlegumes than of legumes. Maximum number of spores (342 spores per 50 g of soil) was observed in the rhizosphere of mustard, followed by chickpea, wheat, pearl millet and pigeonpea. Four VAM generaviz. Glomus, Gigaspora, Sclerocystis andAcaulospora, were present there. Soil pH, total soil P, available P, type of soil, soil moisture and cropping season all variables influenced the VA mycorrhizal population in the natural ecosystem. Numbers of VAM spores highly correlated with the presence of total soil P and soil pH indirectly affected the VAM population through the total soil P. The spore population was abundant in sandy soils as compared to loamy sands. Drier soils had higher number of VAM spores. In summer, the VAM population in soil was less as compared to winter season.     

Effect of vesicular-arbuscular mycorrhizal fungi on verticillium wilt of cotton

The development of vesicular-arbuscular mycorrhizal fungi (VAMF): Glomus mosseae (Nicol and Gerd.) Gerdemann and Trappe, Glomus versiforme (Karsten) Berch, Sclerocystis sinuosa Gerdemann and Bakhi and Verticillium dahliae and the effects of the VAMF on the verticillium wilt of cotton (Gossypium hirsutum L. and Gossypium barbadense L.) were studied with paper pots, black plastic tubes and clay pots under natural growth conditions. All of the tested VAMF were able to infect all the cotton varieties used in the present experiment and typical vesicles and arbuscules were formed in the cortical cells of the cotton roots after inoculation. The cap cells, meristem, differentiating and elongating zones of the root tip were found to be colonized by the VAMF. In the case of most V. dahliae infection, the colonization occurred mostly from the root tip up to 2 cm. VAMF and V. dahliae mutually reduced their percentage of infection when inoculated simultaneously. VAMF inoculation reduced the numbers of germinable microsclerotia in the soil of the mycorrhizosphere, while the quantity of VAM fungal spores in the soil was not influenced by infection of with V. dahliae. The % of arbuscule colonization in roots was negatively correlated with the disease grades, while the numbers of vesicles in roots were not. These results suggest that certain vital competition and antagonistic reactions exist between VAMF and V. dahliae. VAMF reduced the incidence and disease indices of verticillium wilt of cotton during the whole growth phase. It is evident that cotton seedling growth was promoted, flowering was advanced, the numbers of flowers and bolls were increased, and this resulted in an increase in the yield of seed cotton. Among the VAMF species, Glomus versiforme was the most effective, and Sclerocystis sinuosa was inferior. So far as the author is aware, such an effect of VAMF on the increase of cotton wilt tolerance/resistance is reported here far the first time.     

The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATP content. A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.     

Interaction of vascular plants and vesicular-arbuscular mycorrhizal fungi across a soil moisture-nutrient gradient

Summary Abundance and distribution of vascular plants and vesicular-arbuscular mycorrhizal (VAM) fungi across a soil moisture-nutrient gradient were studied at a single site. Vegetation on the site varied from a dry mesic paririe dominated by little bluestem (Schizachyrium scoparium) to emergent aquatic vegetation dominated by cattail (Typha latifolia) and water smartweed (Polygonum hydropiperoides). Plant cover, VAM spore abundance, plant species richness, and number of VAM fungi represented as spores, had significant positive correlations with each other and with percent organic matter. The plant and VAM spore variables had significant negative correlations with soil pH and available Ca, Mg, P and gravimetric soil moisture. Using stepwise multiple regression, Ca was found to be the best predictor of spore abundance. Test for association between plant species and VAM fungal spores indicated that the spores of Glomus caledonium are associated with plants from dry, nutrient poor sites and spores of gigaspora gigantea are positively associated with plants occurring on the wet, relatively nutrient rich sites. Glomus fasciculatum was the most abundant and widely distributed VAM fungus and it had more positive associations with endophyte hosts than the other VAM fungi. We found no relationship between beta niche breadth of plant species and the presence or absence of mycorrhizal infection. However, our data suggest that some plant species may vary with respect to their infection status depending upon soil moisture conditions that may fluctuate seasonally or annually to favor or hinder VAM associations.     

Effect of vesicular-arbuscular mycorrhizal fungi and ofagrobacterium radiobacter on maize growth

An influence of dual inoculation with the rhizosphere bacteriumAgrobacterium radiobacter, and the VAM fungi,Glomus mosseae andGlomus sp., on maize growth and mycorrhizal infection was observed. Separate inoculations of bacteria or fungi showed significant positive effects on the shoot biomass production of pot-cultured plants only at the last of three consecutive harvests. Plant biomass production was enhanced substantially after a dual inoculation with bacteria and fungi. Synergistic interaction of fungal and bacterial inoculation and growth stimulation was evident at all three harvests compared to uninoculated plants and also compared to plants inoculated with fungi or bacteria only. The dual inoculation increased the shoot biomass of plants by approximately 30% as compared with control. No significant differences were found in mycorrhizal infection between plants uninoculated and inoculated with bacteria.Agrobacterium radiobacter seems to be compatible with mycorrhizal symbiosis and can act a synergistic partner of some VAM fungi.     

Plant life span and response to inoculation with vesicular-arbuscular mycorrhizal fungi

Summary This study sought to investigate the relationships among residual soil phosphorus (P) pools, plant life span, successional persistence and responsiveness to vesicular-arbuscular mycorrhizal (VAM) infection. Plants of five species which varied in life span, persistence, and VAM responsiveness were grown in nutrientpoor soils in a glasshouse for 8 weeks and given weekly feedings with either high- or low-P solutions. There was little effect of plant life span or VAM status on changes in residual available (1 M KCl extractable) P. In contrast, there were strong correlations between VAM responsiveness and changes in the exchangeable P pool (1 M NH₄C₂H₃O₂ extractable). Plants with greater VAM responsiveness and greater persistence through succession were able to reduce this potentially available P pool by as much as 50% in 8 weeks. In contrast, plants with poor or negative responsiveness to mycorrhizal infection and little successional persistence exhibited little control over potentially available P pools. These data confirm other studies which demonstrate that VA mycorrhizae access insoluble forms of P, thereby controlling potential supply rate over the longer term. Such control over soil P pools may contribute to successional persistence via either inhibition or tolerance mechanisms, and should be considered in any comprehensive theory of the mechanisms underlying succession.     

Seasonal dynamics of the association between sweet potato and vesicular-arbuscular mycorrhizal fungi

To better understand the behavior of selected vesicular-arbuscular mycorrhizal (VAM) isolates in the field, we documented the growth of roots, root hairs, and VAM colonization of inoculated and noninoculated sweet potato plants (Ipomea batatas (L.) Lam. cv White Star) over a growing season. We also determined the seasonal dynamics of P and Zn uptake, and shoot and storage-root growth. Shoot cuttings were inoculated with an isolate of either Glomus etunicatum Becker and Gerdemann or Acaulospora rugosa Mortan, or were not inoculated, and were harvested 2, 4, 8, 13, 20, and 27 weeks after planting (WAP). At each harvest, roots were sampled at 0 to 30, 30 to 60, and 60 to 90 cm depths and at 0, 23, 83, and 116 cm from the base of the shoot. At the end of the study, the roots of three non-inoculated plants were sampled by soil horizon. Inoculation had no affect on shoot growth or total shoot uptake of P and Zn; shoot dry mass and P and Z content increased rapidly up to 20 WAP, while shoot length continued to increase through 27 WAP. Shoot-P concentration of plants inoculated with A. rugosa at 2 and 8 WAP were higher than the noninoculated plants, while shoot-Zn concentration was not affected by inoculation. Storage-root yields of inoculated plants were higher than yields for noninoculated plants. Root length density, and percentage of root length with root hairs and VAM colonization were highest and most dynamic near the base of the plant. Percentage of root length colonization by VAM fungi was highest in the E2 horizon, intermediate in the Bh horizon, and lowest in the Ap horizon. Percentage of root length with root hairs had the opposite pattern. Intensive measurements of root characteristics close to the base of the plant, and shoot P-content and concentration during the period of rapid yield production, provided the most useful data for evaluating the activity of effective isolates.Published as Florida Agricultural Experimental Station Journal Series No. R-02576     

Field application of vesicular-arbuscular mycorrhizal fungi improved garlic yield in disinfected soil

 The production of certified garlic propagation material requires measures to be taken against pathogenic nematodes. Methyl bromide (MB) may be used for this purpose, but is known to cause stunting in Allium spp. Vesicular-arbuscular mycorrhizal (VAM) fungal inoculum was applied to the planting furrow after MB treatment. VAM-inoculated plants were larger, had more green leaves, an increased photosynthesis rate, especially at low light intensities, and higher fresh and dry weights than plants in uninoculated plots. The mean bulb weights from uninoculated and VAM-treated plots were 27 g and 51 g respectively. The native or an improved VAM population should be reintroduced after soil disinfection to ensure satisfactory garlic yields. Accepted: 15 January 1997