Vesicular-arbuscular Endophytes Research Articles

Re‐establishing life in restored topsoils

AbstractSurface mining activities and other large civil engineering works often involve the removal, storage and reinstatement of topsoil. Disturbance disrupts normal biological functions within the soil and can markedly change the environment within which soil organisms must exist. This has implications for soil fertility and for the overall ecology of replaced land. A number of studies have investigated the impact of opencast coal mining practices on soil organisms and their capacity for recovery following site reinstatement. The extent and duration of any adverse effect varies from one group of organisms to another according to their vulnerability to damage, their capacity for recovery and their ability to recolonize areas from which they have been eliminated. Three groups of organisms—earthworms, aerobic bactería and vesicular arbuscular endophytes—are considered, to illustrate their differing responses to soil disturbance and the various factors limiting their early recovery throughout a restored area. Given favourable management, consistent with good general rehabilitative practice, the re‐establishment of normal earthworm populations depends entirely on the distribution of an inoculum. For aerobic bacteria adverse physical conditions in reinstated soils are the overriding influence determining levels of activity. For mycorrhizal fungi both soil conditions and the absence of an inoculum may delay recovery, although the evidence in support of this view is inconclusive.

The Possible Relationship of Spore-Like Structures on the Mycelium of Glomus Monosporum to Formation of Appressoria

Hyphal penetration of a root epidermal cell by a vesicular-arbuscular endophyte was considered by Nicolson (1959) and Mosse (1959b) to be preceded by hyphal swelling and formation of an appressorium. Observation of early mycorrhizal infections of adventitious roots of leek seedlings obtained using one sporeling of Glomus monosporum Gerdemann & Trappe as inoculum (San Antonio, unpubl. data) indicated that the shape of some appressoria were reminescent of the spherical spore-like structures characteristically present on external mycelium of G. monosporum (Fig. 1) described by Nicolson (1959) and Mosse (1959a). Warner and Mosse (1980) reported that such spore-like structures never became detached naturally from the mycelium, and when deliberately detached, neither germinated nor grew in culture. Warner and Mosse's comment that it is not known whether root infection can

Host and non-host relationships between Arbutus unedo L. And mycorrhizal fungi

The mycorrhizal status of Arbutus unedo L. was tested in its natural ecosystem, and the development of mycorrhizal colonization was followed during the early stages of plant growth. Two different types of ectoendomycorrhizae occurred in nature, only one of which was synthesized in experimental conditions. The behaviour of the ericoid fungus Hymenoscypus ericae and six vesicular-arbuscular endophytes were tested on the non-host plant A. unedo.

Impact of simulated erosion on the abundance and activity of indigenous vesicular-arbuscular mycorrhizal endophytes in an Oxisol

The influence of simulated erosion on the abundance and activity of indigenous vesiculararbuscular mycorrhizal (VAM) populations was evaluated in an Oxisol. Surface-soil losses in excess of 7.5 cm were generally associated with significant decreases in the numbers of total and active VAM propagules and in the symbiotic effectiveness of the active propagules. Surface-soil removal not exceeding 7.5 cm was associated with decreased propagule abundance without adverse effects on VAM colonization of roots and symbiotic effectiveness of the fungi. The extent of VAM colonization of roots and the degree of symbiotic effectiveness observed at this level of simulated erosion were significantly higher than those observed in the soil not subjected to simulated erosion. This stimulation is attributed to the removal of antagonistic biotic factors as the top 7.5 cm of soil was removed. It is concluded that propagules lost during erosional soil losses must be replaced before legumes grown on relatively highly weathered and severely eroded tropical soils could appreciably benefit from the VAM symbiosis.

Some studies relating to "independent" growth of vesicular–arbuscular endophytes

In dual culture with Ri T-DNA transformed carrot roots nutrient conditions were sought that would totally suppress mycorrhizal infection while stimulating "independent" growth of vesicular–arbuscular endophytes. In this system fungal development in the agar was comparable in extent with that normally occurring only after mycorrhizal infection has become well established; it differed qualitatively from that characteristic of germinating spores and extramatrical mycelium in such cultures. There was profuse development of arbuscule-like branches, many anastomoses, and increased branching of extension hyphae. Biomass increased up to fivefold when the mycelium was detached in situ from the inoculum (root pieces or spores). Growth from explants was little more than residual and was hampered by extensive bacterial contamination at the desired pH, near neutrality. The rationale of this approach to the culture of vesicular–arbuscular endophytes is discussed.

Orchidaceous rhizoctonias from roots of nonorchids: mycelial and cultural characteristics of field and pot culture isolates

This paper reports the field isolation of orchidaceous rhizoctonias similar to those found recently in pot cultures of vesicular–arbuscular endophytes. The paper describes the morphology and growth of field and pot-culture isolates on different natural and synthetic media and reports light microscope observations on the nuclei and septal pores of hyphal cells. All isolates were the same in regard to the utilisation of simple and complex carbohydrates and different forms of nitrogen. They were all multinucleate and had an inconspicuous septal pore. Consistent differences were found among isolates in the morphology and frequency of chlamydospores and monilioid hyphae and in the rate of growth, shape, type of margin, and colour of colonies. By using these criteria, the fungi were segregated into 10 entities. The observations are discussed in relation to the natural distribution of the fungi and their resemblance to other fungi in the form-genus Rhizoctonia.

Assessment of field situations for the feasibility of vesicular-arbuscular mycorrhizal inoculation, using a forage legume as test plant

This paper reports experiments aimed at predicting ecological situations where vesicular-arbuscular mycorrhizal (VAM) inoculation would be beneficial. Twelve unsterile soils were assayed and Hedysarum coronarium, a forage legume, was the test plant. Three vesicular-arbuscular endophytes, namely Glomus mosseae, G. fasciculatus and a Glomus sp. were tested, and in all cases G. mosseae was the more efficient, with indications of some plant-fungus specificity. A considerable degree of dependency on mycorrhizas, for suitable growth and N 2-fixation, was also demonstrated for the tested legume- Rhizobium sp. system. The studied soils varied in their concentration of plant-available phosphorus and in the amount, infectivity and effectiveness of indigenous VAM propagules; however, no correlation between these parameters could be found. The level of success of the introduction of G. mosseae in the presence of naturally existing endophytes was evaluated by the extent of the plant response in terms of growth, nodulation and N and P uptake in each soil. The inoculation of G. mosseae was effective in 7 out of the 12 soils; however, it was not possible to find a correlation between any of the soil parameters studied (i.e. soluble P concentration, amount of mycorrhizal root pieces and VAM mycelium and spores) and the plant response to G. mosseae inoculation. Thus, it is necessary to emphasize the need for simple techniques to screen situations where it would be worth attempting VAM inoculation, since each particular soil must be checked for each given plant-fungus combination.

Effect of soil micro-organisms on spore germination and growth of the vesicular-arbuscular mycorrhizal fungus Glomus mosseae

Germination rate of surface-sterilized chlamydospores of the vesicular-arbuscular (VA) mycorrhizal endophyte Glomus mosseae was significantly hastened by the presence of freeliving fungi in a time-course experiment on water-agar. The fungi also stimulated hyphal growth, while vegetative spores formed only on the mycelium arising from resting spores grown in the presence of such free-living micro-organisms. The time of appearance of vegetative spores was related to the time when the free-living fungi were added. The interest in soil biology and ecology of these microbe-microbe relationships involving VA fungi is discussed.

MYCORRHIZAL INFECTION OF BETULA PENDULA AND ACER PSEUDOPLATANUS: RELATIONSHIPS WITH SEEDLING GROWTH AND SOIL FACTORS.

Mycorrhizal infection of two-year-old seedlings of Betula pendula Roth, and Acer pseudoplatanus L. was examined in a pot experiment, in relation to several soil variables (pH, organic matter, N, P, K, Ca, Fe, and phosphatase activity), host uptake of 32 P, and plant performance (height and weight), in 25 Cumbrian soils from natural and semi-natural habitats (such as woodland, carr and upland heath). Soils were used in a fresh state to maintain indigenous mycofloras and seedlings were raised from several seed-lots to include possible ecotypic variation. Infection (incidence and intensity) of both 'immature' and 'mature' mycorrhizas was estimated. Most plants were mycorrhizal, ectomycorrhizas occuring on Betula and vesicular-arbuscular (VA) endophytes in Acer. Differences in the degree of infection between soils, but not between seed-lots, were highly significant. Infection of one or both species was highly correlated with several soil factors (pH, organic matter, phosphorus and iron), and with all the plant variables. In Betula, evidence for most of these relationships was obtained only from 'immature' mycorrhizas, suggesting early physiological activity. Mycorrhizas accounted for a small but significant proportion of the variation in growth of both hosts, and soil properties accounted for a high proportion of the variation in both the plant growth and mycorrhizal infection, as shown by multiple regression analyses. Growth of Acer appeared to have a greater influence than soil factors on the variation in VA infection. 'Beading' of Acer roots was negatively correlated with plant weight and soil phosphorus, suggesting a morphological response to P- deficiency. Possible causal relationships between hosts, fungal symbionts and soils are discussed.

Spore germination and hyphal growth of a vesicular–arbuscular mycorrhizal fungus, Glomus mosseae (Gerdemann and Trappe), under decreased oxygen and increased carbon dioxide concentrations

Spore germination and hyphal growth from spores of the vesicular–arbuscular endophyte Glomus mosseae have been studied under different oxygen and carbon dioxide tensions. Spores failed to germinate at low oxygen tension. This effect was reversible. However, mycelial growth from spores not preincubated in air was irreversibly damaged. The mycelial growth from preincubated spores was affected by oxygen tension. Between 21 and 3% oxygen, growth decreased slightly. Below 3% oxygen it decreased sharply. Again this effect was reversible. A tension of 5% carbon dioxide had no effect on spore germination, but reduced hyphal growth consistently at all oxygen tensions. This effect persisted when spores were subsequently returned to air.

Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Nodulation and Growth of Subclover

Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Nodulation and Growth of Subclover

Response of maize to different inoculum densities of vesicular arbuscular mycorrhizal endophytes

The effect of the inoculum density of vesicular arbuscular mycorrhizal endophytes on growth and development in maize was investigated in sterilized soil under glass-house conditions. Mycorrhizal plants grew robust and produced three times more dry weight than non-mycorrhizal plants. 40 or more endophytes per plant produced the highest mycorrhizal association and the maximum growth in maize. The uninoculated plants exhibited the symptoms of chlorosis.

GROWTH RESPONSES OF ENDOMYCORRHIZAL ONIONS IN UNSTERILIZED COAL WASTE

SummaryThe effects of the widespread vesicular‐arbuscular (VA) endophytes, namely Glomus macrocarpus var macrocarpus, Glomus mosseae, and Sclerocystis rubiformis and those of E3 (a strain of Endogone introduced from Rothamsted Experimental Station) on growth of onions were compared in unsterilized coal washery waste from the Coal‐Cliff Collieries in the Illawarra region of New South Wales. Mycorrhizal onions were significantly larger (P < 0.05), with respect to all the growth measurements than NM controls. Different VA endophytes had quantitatively different effects on the growth; the S. rubiformis was always poorest and E3 most consistently good. This is because S. rubiformis produced more localized infection, while the growth of E3 was more general producing, under similar conditions, more extramatrical hyphae than any other endophyte used. Among widespread species G. mosseae was the most efficient at stimulating plant growth. The possible role of VA mycorrhizas in plant colonization of coal spoil areas is discussed.

Biochemistry of ungerminated and germinated spores of the vesicular-arbuscular mycorrhizal fungus, Glomus caledonius: changes in neutral and polar lipids.

Neutral and polar spore lipids of the vesicular-arbuscular (VA) endophyte Glomus caledonius, were identified and quantitatively determined during spore germination, germ tube growth, and germ tube senescence. There are no previous reports detailing the spore lipid components of any member of the Endogenaceae, which is in the Zygomycotina. The fungus contained 45 to 72% total lipid depending upon its stage of growth. The concentration of neutral lipids decreased during germination while the polar lipids increased. Triacylglycerides were the most abundant neutral lipid, and lesser amounts of diacylglycerides, monoacylglycerides, free fatty acids, bound fatty acids, hydrocarbons, and sterols. The major fatty acids identified by gas--liquid chromatography and mass spectrometry were 16:1, 16:0, and 18:1. The minor fatty acids identified were n-3 and n-6 polyunsaturates. The n-3 polyunsaturated fatty acids have not been reported before in Zygomycetes. The fatty acid composition of the individual lipid classes was examined. The major phospholipids were phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, with smaller amounts of diphosphatidylglycerol and phosphatidic acid. The free sterol fraction was in greater quantity than sterol esters during germination and germ tube elongation. The capacity to synthesize sterols was demonstrated. Approximate net rates of change in the different lipid components were calculated. During spore germination and early germ tube growth, there was a net synthesis of lipids, with a large production of free fatty acids, in the germinating spore. Later in the growth period there was a net degradation of lipid, characterized by a large conversion of free fatty acids to unidentified compounds. During this period net free sterol synthesis ceased and sterol ester synthesis continued using the existing free sterol.

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30 degrees C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30 degrees C for Glomus caledonius or at 35 degrees C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 mug of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 mug/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40 degrees C for 80 min, 55 degrees C for 10 min, and 60 degrees C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 x 10 ergs cm from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.

Preservation of Spores of Vesicular-Arbuscular Endophytes by L-Drying

The spores of four species of vesicular-arbuscular endophytes were L-dried at 22 degrees C, and their viability was tested after heating at 80 degrees C for up to 40 min. L-drying of spores in the soil in which they developed was a very effective method of preservation of all spore types examined. Slow L-drying of spores separated from soil and supported on glass fiber filters also gave high viability for spores of some species. A scheme for the long-term preservation of vesicular-arbuscular endophyte spores is proposed.

The 'most probable number' method for enumerating infective propagules of vesicular arbuscular mycorrhizal fungi in soil

Estimates of the number of infective propagules of vesicular-arbuscular (VA) endophytes in two soils were obtained using a most probable number (MPN) method. These estimates were compared with counts of the number of spores in the same soils obtained using a conventional wet sieving method. In one soil, there was good agreement between the number of coarse endophyte propagules, estimated by the MPN technique, and the number of germinable spores extracted using the wet sieving technique. However, a large population of fine endophyte propagules (more than 230 per 50 g soil) could only be enumerated using the MPN technique. In the second soil, fewer coarse endophyte propagules were found using the wet sieving technique than when using the MPN technique. The MPN technique appears to give a more realistic estimate of the number of infective propagules of VA endophytes in field soils than the conventional method.

GROWTH OF SUBTERRANEAN CLOVER IN RELATION TO THE FORMATION OF ENDOMYCORRHIZAS BY INTRODUCED AND INDIGENOUS FUNGI IN A FIELD SOIL

SUMMARYThree vesicular arbuscular (VA) endophytes differed in their ability to increase the growth of subterranean clover when introduced into a freshly collected field soil. Four indigenous endophytes infected the roots in both the control and inoculated treatments. Two of the introduced endophytes, isolates of Glomus monosporus, differed in their ability to increase growth and phosphorus content of subterranean clover. These endophytes were both superior to the third introduced endophyte, G. fasciculatus.Differences in the growth of subterranean clover when limited by phosphorus supply were related to the rate of mycorrhiza development. Inoculation of soil with the three introduced endophytes did not reduce initial infection by the four indigenous endophytes. Mycorrhizas formed by G. monosporus and G. fasciculatus were distinguished from those formed by indigenous endophytes on the basis of morphology.Plants inoculated with isolates of G. monosporus had a greater proportion of their dry weight and phosphorus content in the tops than plants inoculated with G. fasciculatus or the control plants.

Effect of rate of application of superphosphate on populations of vesicular arbuscular endophytes

The effect of altering the rate of superphosphate application on the population of vesicular arbuscular endophytes in a pasture soil was studied during 1976. Between 1950 and 1965 the pasture near Bakers Hill, Western Australia, had received an average of 150 kg superphosphate ha-1 year-1. For the ten years prior to 1976, five rates of superphosphate were applied to plots within the pasture. Varying the rate of superphosphate affected the number of VA endophyte spores. Adding 56 kg superphosphate ha-1 year-1 increased spore numbers by 40 per cent compared with the unfertilized plots. Spore numbers in plots receiving higher rates of superphosphate than 56 kg ha-1 were similar to those in unfertilized plots. The relative abundance of spores of the four endophyte species found (Glomus monosporus, Acaulospora laevis, Gigaspora sp. and 'White reticulate') was unaffected by superphosphate applications. In a glasshouse experiment vesicular arbuscular endophytes from plots that had received the highest rate of superphosphate (224 kg ha-1 year-1) did not differ from those which had received no superphosphate in their ability to infect and increase the phosphorus uptake and growth of clover. It was concluded that altering the rate of superphosphate application on an old, adequately fertilized pasture does not affect the infectivity or effectiveness of the vesicular arbuscular endophyte population.

A note on the effect of soil tillage on the frequency and vertical distribution of spores of vesicular-arbuscular endophytes

Spores of vesicular-arbuscular endophytes resembling Acaulospora laezis and Glomus monospovus were common in the surface layers of a soil under wheat and pasture, declining in numbers down the profile until rare at 500 mm. Tillage had no effect on total spore numbers, but directly drilled wheat had more spores in the surface 80 mm and fewer from 80 mm to 150 mm than conventionally tilled plots. This is thought to be a consequence of mechanical inversion of the soil during cultivation.