EVALUATING MYCORRHIZAL INOCULUM LEVELS IN SOIL

LITERATURE REVIEW

The term ‘mycorrhiza’ (Gr: fungus root) was coined by Frank to describe associations between certain non-pathogenic fungi and roots of higher plants. Peyronel et al. (1969) proposed grouping mycorrhizae into three broad categories: ectomycorrhizae, endomycorrhizae, and ectendomycorrhizae. In ectomycorrhizae, the fungus forms a compact mantle over the root surface from which the hyphae arise and grow into the cortex intercellularly. Endomycorrhizae have external hyphae which are not aggregated to any great extent and there is extensive growth within the root cortex. Ectendomycorrhizae are similar to ectomycorrhizae but have both intercellular and intracellular hyphae. Lewis (1973) suggested grouping ectotrophic and endotrophic mycorrhizae together and classified them as 'sheathing', 'ericaceous', 'orchidaceous', 'vesicular-arbuscular', or 'miscellaneous'. Over the past twenty years it has become evident that the most common and widespread mycorrhizal infections are the vesicular-arbuscular (VA) type caused by the Phycomycete group (Nicolson, 1967). Despite their wide occurrence and ecological importance, only recently have agronomists and soil scientists begun to recognize the importance of VA mycorrhizae to the nutrition of crops; such importance is underscored by the statement of Wilhelm (1966) '... under agricultural field conditions, crops do not, strictly speaking, have roots, they have mycorrhizae.'

Gerdemann and Trappe (1975) reviewed the history of the taxonomy of the genus Endogone. The genus was first described by Link in 1809 and later revised by Thaxter (1922). According to this classification, all species form sporocarps and are distinguished by the structure of the sporocarps and the spores they contain. Peyronel first suggested in 1923 that Endogone spp. produce VA mycorrhizae, but it was not until the work of Mosse in 1962 that this was generally accepted (Gerdemann, 1968). The genus Endogone was revised by Nicolson and Gerdemann (1968) to include species that produce ectocarpic resting spores. Mosse and Bowen (1968a; 1968b) surveyed 250 samples of Australian and New Zealand soils and some Rothamsted field soils. They described nine types of spores and devised a key for the identification of Endogone spores using several diagnostic features: spore attachment, spore contents, spore wall, spore color, and spore size and shape. Gerdemann and Trappe (1974) surveyed the Pacific Northwest USA and proposed a new classification of Endogonaceae that divides the family into five genera: Glomus, Gigaspora, Acaulospora, Sclerocystis, and Endogone. Endogone is a zygosporic genus and the only one that does not contain VA endophytes. The two classification systems just described, Mosse and Bowen (1968a) and Gerdemann and Trappe (1974), are the most widely used. Hall and Fish (1979) have recently proposed a new key to the Endogonaceae which was compiled using a computer program. The program assigns weights to the diagnostic characteristics, assigning high weights to characteristics which vary least and are easily observed.

As other surveys are completed, new species are being described. The taxonomy may undergo further revision to accommodate these additions (Gerdemann, 1976; Redhead, 1977). It should be stressed that the present taxonomy of Endogonaceae is tentative. Walker (1979) suggested that researchers place specimens of fungi in herbaria so that the identification can be checked as taxonomic knowledge increases. Undoubtably new methods will be developed which will facilitate the identification of theendophytes. One promising method is the fluorescent antibody technique which is being tested on fungi (Malajczuk et al., 1978).

During the past several years there has been an interest in surveying soils around the world to determine the presence of VA endophytes. The resulting information from several continents, encompassing a wide variety of natural and agricultural ecosystems, has provided insight regarding the ecological significance of VA mycorrhizae. Recently two papers have reported the occurrence of VA mycorrhizae in aquatic plants (Sondergaard and Laegaard, 1977; Bagyaraj et al., 1979a), an environment where mycorrhizae were previously thought to be absent (Harley, 1969).

When studying the niche mycorrhizal fungi occupy in a given ecosystem, it is important to understand the adaptations of the endophytes to that ecosystem. If more is known about the behavior of mycorrhizal fungi in different environments, then more sensitive methods can be employed to enumerate the fungi. Three methods have been used to characterize the inoculum level: 1. extracting and counting spores; 2. direct observation of infection levels in the plant population; and 3. measuring the rate that test seedlings become infected (Mosse, 1979). As Mosse has pointed out, the method selected depends upon the objectives of the inquiry. Unfortunately no single method satisfactorily assesses the inoculum level of soils. This deficiency is being filled by modifying serial dilution techniques and most probable number (MPN) methods in order to enumerate the viable propagules in the soil (Moorman and Reeves, 1979; Porter, 1979).

Mycocrhizal spores are usually extracted from soil by wet sieving and decanting (Gerdemann and Nicolson, 1963). This method leads to variable results when duplicate samples are handled by two individuals and besides, the method is laborious when large samples are handled. The method preferentially selects for spores that are easily extracted (Harley, 969). Modified procedures involve adding agents to disperse heavy-textured soils (Sanders, 1976). Differences between replicate samples analyzed by the wet sieving method may be so great that statistical comparisons are futile (Nicolson, 1967; Crush, 1973); furthermore, some VA endophytes produce Spores so small that extraction and counting are difficult. A flotation-adhesion method (Sutton and Barron, 1972) resulted in 94-98% efficiency in the recovery of spores. This method has the advantage of recovering spores regardless of size, but results in the recovery of organic debris which interferes with spore counting. Separating spores from organic debris, either by centrifuging in a sucrose solution (Ohms, 1957) or by differential sedimentation on gelatin columns (Mosse and Jones, 1968), permits a more quantitative measure of the spore population. Smith and Skipper (1979) compared several spore extraction methods and described a new plating method. Their study points out sources of error in each of the methods studied and suggests conditions under which one method may be preferred over another. These methods do not distinguish between viable and nonviable spores and it is this distinction that is necessary for a practical assessment of the soil inoculum level.

Mosse (1973a) and Tinker (1975a) have reviewed the work on factors which influence spore populations in soils. Spore populations are dynamic, being influenced by season, soil type, soil moisture, light intensity, nutrient availability and land usage. Whether these factors influence the fungi directly or indirectly through effects on the host plant is largely unknown. The system is complex. Interactions between the strain of fungi, the host plant, and soil-environment conditions make generalizations difficult. As one might expect, the correlation between spore population and infection is strong under certain conditions and weak under others. Daft and Nicolson (1972) evaluated three methods for estimating infection levels in plants and concluded that counting spores produced on external mycelia was the most accurate and convenient. However, Mosse (1979) has pointed out that correlations between number of spores and infection are usually good in experimental situations but much less reliable in situations involving various soils and various strains of fungi. Owusu-Bennoah and Mosse (in press) suggested that spore number was determined by inherent characteristics of the fungi and specific interactions between the fungi and the soil. In a field inoculation trial involving two fungal species and three crop plants, they concluded that spore number was not a good index of infection.

Another difficulty in relying on spore numbers as a measure of soil infectivity is that spores are not the only infecting propagules in the soil. Powell (1976a) showed that hyphae from infected root segments cause infection. Read et al. (1976) surveyed the major vegetation types in east-central England and concluded that the major source of inoculum was infected roots or mycelia. While testing systemic fungicides, Boatman et al. (1978) found that new roots are infected from mycelia in the soil. Observations such as these point to an additional difficulty: fungi may have distinctly different life cycles in cultivated and in fallow or noncultivated soil. Mason (1964) observed increased spore numbers in a cultivated field as new root growth ceased and old roots senescenced. Mosse and Bowen (1968b)

suggested that spores were formed where root growth is intermittent. In a lowland rainforest in Nigeria, seedlings were heavily infected while the soil contained no spores at all (Redhead, 1977). Hayman and Stovold (1979) surveyed 73 sites in New South Wales and found great variability in spore numbers. Spore population varied for the same crop at different sites; they found more spores in agricultural soils than in native grassland-bush soils. Thus agricultural field conditions may select sporulating endophytes, while natural fallow soil conditions may select non-sporulating endophytes.

Soil infectivity can be semi-quantitatively estimated by examining the extent of infection in sample plants. Soil infectivity may be defined as a property of the soil which determines the rate and extent plants form mycorrhizae. A quantitative measure of soil infectivity may be possible provided the same host plant is used and careful, thorough sampling procedures are followed. The semi-quantitative visual evaluation of infection in the plant roots needs to be standardized, and even then duplication of results will be difficult. There are, however, differences in the ease and the degree in which different host species become infected. There is also variation in the amount of vesicles, arbuscules, and hyphae formed by different strains of fungi. Precisely what to look for when evaluating infection is a problem. Hayman (1974) suggested that the total arbuscular formation may be more important than total infection per se; unfavorable light and temperature conditions resulting in slow growth of onion was associated with a deficiency of arbuscules. Nicolson (1960) developed a root slide technique to quantitatively measure infection. He cut the roots into small segments and collected the following data: percentage of infection; the number of infected and noninfected segments; percentage of moribund roots (roots which showed loss of cortical cells); percentage of external mycelia; the proportion of all roots which showed mycelia; and the percentage of roots with brown septate mycelia. Researchers have since used this technique, often with modifications, to inspect roots for both extent and intensity of infection. Read et al. (1976) used the root slide technique to estimate the percent of VA infection by the expression:

% VA infection= No. infected segments x 100.

total No. segments examined

They noted that this procedure describes the distribution of mycelia throughout the root system but does not describe the intensity of infection in the system. Hayman (1970) attempted to measure both parameters of infection by recording length of infected root in each segment, percent root segments with infection, and percent root segments with attached Endogone hyphae, spores, or vesicles. Not only are these techniques time consuming but the relationship of the results to soil inoculum levels is difficult to determine. Strzemska (1974) noted that the occurrence of VA infection in a given species varied considerably from year to year. If we accept the concept of a dynamic population of mycorrhizal fungi, then we must concern ourselves with the implications regarding the infectivity of the soil. The relation of soil inoculum to variation in infection needs to be investigated.

Giovannetti and Mosse (in press) compared four methods for evaluating root infection. They compared: 1. the gridline intersect method; 2. visual estimate of percentage cortex occupied by fungi; 3. estimate of length of cortex infected from a sample mounted on a slide; and 4. recording presence or absence of infection on a sample mounted on a slide. They indicated that the visual estimate of infection, although subjective, can give reliable results. All methods probably overestimate the extent of infection; because after clearing and staining, roots appear as two dimensional rather than three dimensional objects.

Measuring the rate a test seedling becomes infected is rarely reported, although this approach is a promising method for evaluating soil infectivity. Hayman and Stovold (1979) measured the rate of mycorrhizal development in clover seedlings in soils from 23 sites. Infectivity of the VA population was not well correlated with spore population, especially in the native grassland-bush soils. Moorman and Reeves (1979) made 1/4 and 1/40 dilutions of disturbed and nondisturbed soils. After thirty days corn roots were 77% infected on the nondisturbed soil but were only 1% infected on the disturbed soil. The effect of dilution was to reduce the amount of infection accordingly; however in the disturbed soil this effect was not apparent until 90 days because of the low inoculum density in the soil. Porter (1979) adopteda most probable number (MPN) technique to estimate the infective propagules of VA mycorrhizal fungi. Clover and medic seedlings were planted in sterile soil which contained serial dilution of sterile and non-sterile soil. The MPN method was designed to be used with aqueous solutions where the distribution of the organism to be enumerated is assumed to be spatially uniform and random. This assumption may not apply in soil where severe clumping of propagules occurs. The ability of this technique to generate reproducible results remains to be tested. Nevertheless a bioassay is needed that detects only the viable propagules in the soil. Such a method would avoid the obvious difficulties in relying on spore numbers as an estimate of soil inoculum level or soil infectivity.

The evaluation of variations in soil inoculum level interests agronomists. Although VA endophytes are present in most soils, there is evidence that the level is suboptimal under certain conditions. Further research is needed to identify these conditions. Ross (1979) has observed that colonization of soybean roots by naturally-occurring mycorrhizal fungi is lower compared with inoculated soybeans which are grown in sterile soil. He concluded that low sporulation of these fungi in field soil probably results in low inoculum level for subsequent crops. The inoculum level in some Nigerian soils was so low that Stylosanthes guyanensis seedlings did not become infected during the course of the experiment (Mosse, 1977). However S. guyanensis does not appear to be very mycotrophic, thus it is probably a poor indicator of soil inoculum levels.

The standing vegetation or the preceding crop may have an impact on the soil inoculum level. Khan (1972) made use of this fact and transplanted infected and noninfected maize seedlings into unfertilized plots which had previously been occupied by weeds of the Chenopodiaceae family, reported to be non-mycorrhizal (Gerdemann, 1968). P uptake and dry weight of mycorrhizal plants were much greater than the controls; grain weight was almost 12 times greater on mycorrhizal plants. In a study designed to measure the rate of spread of an introduced VA fungi, the effect of growing nonmycorrhizal plants in the soil was to reduce the vigor of the indigenous fungi thereby enhancing the spread of the introduced species (Powell, 1979b). Kruckelmann (1975) found that fertilizers, soil tillage, and crop rotations affected the number of spores in arable soil. His results showed spores were more frequent in loamy soils than in sandy soils. Spore populaticn correlated better with pH than with K, carbon,

or nitrogen content of the soils. Spore numbers increased with higher pH values and decreased with increasing phosphate contents. It certainly would be desirable to know what effect, if any, flooding the soil has on the soil infectivity.

In citrus culture, and some other perennial plantation crops, it is a common practice to fumigate soil or use sterilized growth media to grow seedlings. Heavy P fertilization is necessary to relieve stress resulting from the lack of mycorrhizae (Kleinschmidt and Gerdemann, 1972). Under these conditions inoculation with mycorrhizal fungi can partially substitute for P fertilization (Menge et al., 1978).

The importance of mycorrhizae for eroded lands has not been experimentally determined. However there are several reports on the vertical distribution of mycorrhizal spores in the soil. Sutton and Barron (1972) found that the number of spores changed little with soil depth to 16-24 cm, but declined with further increase in depth. Spores occurred mostly in the top 15 cm of soil in Nigeria (Redhead, 1977). The mean number of spores per 500 cm³at various depths were 2 cm, 748; 7.5 cm, 1946; 15 cm, 1064; 30 cm, 55. Spore numbers in eroded soils were 25% of adjacent non-eroded sites, and a response to inoculation was obtained in 8 out of 10 eroded soils (Hall and Armstrong, 1979). In soils disturbed by strip mining operations the inoculum level was suboptimal (Reeves et al.,1979). Daft et al.(1975) postulated that a mycorrhizal association may be essential for the survival of most herbaceous plants growing in coal spoils. Theyobtained a significant response to inoculation. The implication is that where the surface soil has been removed the inoculum level of exposed soil may be suboptimal for plant growth.

Effect of VA Mycorrhizae on the Host Plant

Increased phosphate absorption by plants infected with VA mycorrhizae when compared with noninfected plants and the increase in P concentration in plant tissue has been well established (Mosse, 1973a; Tinker, 1975a). Although reports of increased uptake of other nutrients and increased water absorption possibly indicate multiple effects of mycorrhizae on plant nutrition, nearly all host growth responses have been attributed to improved phosphorous nutrition.

Experiments using insoluble phosphates have demonstrated that enhanced growth and P uptake was associated with mycorrhizal plants (Murdoch et al., 1967). It has been inferred by same investigators that mycorrhizal fungi may possess P-solubilizing mechanism by which mycorrhizal plants utilize forms of P unavailable to nonmycorrhizal plants. Although no such mechanisms have been demonstrated there is data to suggest that mycorrhizal plants absorb sparingly soluble P more readily than nonmycorrhizal plants. Working with a high P sorbing soil in Hawaii, Yost and Fox (1979) indicated that the threshold concentration for P uptake (the concentration of P in the soil solution below which no P is absorbed) is lower for mycorrhizal plants. Data from Cress et al.(1979) raises the possibility that a major factor contributing to the increased uptake of phosphorus by mycorrhizal plants is a greater ion affinity by the mycorrhizal absorbing sites. The relative importance of this increased ion affinity in soil situations where diffusion of phosphorus is rate limiting can not be determined from their study. Jackson et al.(1972) studied utilization of rock phosphate and did not observe a response to inoculation with VA fungi unless the rock phosphate were mixed in the soil, indicating the importance of the spatial proximity of the association and the nutrient source.

To identify the source of P for mycorrhizal and nonmycorrhizal plants, soil was labeled with ³²Pand the specific activity of absorbed P in infected and noninfected plants was determined. The results indicated that mycorrhizal and nonmycorrhizal plants obtain phosphorus from the same source (Sanders and Tinker, 1971). Such data support the idea that the effect of mycorrhizae results from the hyphae forming a better distributed surface for absorbing phosphorus than roots alone.

Hattingh et al.(1973) provided direct evidence of hyphal uptake and translocation of phosphorus. ³²P-labeled phosphate which had been placed 27 mm from the root surface was absorbed when the roots were mycorrhizal; when the hyphae were severed mycorrhizal roots did not differ significantly in content of ³²P-labeled phosphate content from nonmycorrhizal roots. Growth chamber results such as these should be interpreted with caution. It is probable that hyphal growth is more profuse because of conditions on the soil plane (Hattingh, 1975). Owusu-Bennoah and Wild (1979) used autoradiography to demonstrate phosphate depletion zones around mycorrhizal and nonmycorrhizal roots. They concluded that the main increase of phosphate uptake by mycorrhizae was from soil within 2 mm of the root surface. However the experimental conditions of such work demand a closer look before extrapolations are made to other soil-plant systems. Finely crushed soil with small pore spaces which may be water saturated probably restricts hyphal growth. The increased absorbing; surface of fungal hyphae is important as well as the distribution of absorbing surface in the soil. The specific interaction between the fungal strain and the soil properties will affect the relative importance of these two parameters.

The diffusion of phosphorus in soil and uptake by plants has been studied in detail. Bhat and Nye (1974) indicated that a phosphorus depletion zone surrounds the active absorbing root; hence the value of external mycelia may be that they extend beyond the depletion zone and absorb phosphorus in non-depleted soil. Realization of this prompted Rhodes (1979) to write "...nutrients most likely to be involved in plant growth responses to VA mycorrhizal infection are those for which the rate-limiting step for uptake by plants is movement to roots through soil by diffusion."

Properties of mycorrhizal hyphae are becoming better understood. Pearson and Tinker (1975) demonstrated P transport and measured a mean steady state flux of P in the external hyphae of 0.3-1.0 x 10^-9 moles cm^-2s^-1.They did not determine a value for absorbing power of the hyphae per unit length. Cooper and Tinker (1978) studied the uptake and translocation of P, Zn, and S. In clover external hyphae translocated molar amounts of P, Zn, and S in the ratio of 35:5:1 and the mean fluxes in the ratio of 50:8:1 which suggests high relative efficiency in the uptake and translocation mechanisms for P. Their results also indicated that the phosphorus demand of the host affected the flow of P

in the hyphae, and that the amount of external hyphae in the soil (i.e. the total hyphae length) was secondary in importance.

From an ecological perspective, Baylis (1975) suggested that mycorrhizal fungi have exercised a controlling influence on the evolution of roots. He submitted that magnolioid roots are more dependent on mycorrhizae for P uptake in low P soils than are graminoid roots. Magnolioid roots are coarsely branched and the ultimate roots are rarely less than 0.5 mm in diameter. The roots have a compact stele and normally do not have root hairs. Graminoid roots are finely divided, with ultimate branches often less than 0.1 mm in diameter. They are densely covered with root hairs 1-2 mm in length. Data from Yost and Fox (1979) lend support to this hypothesis. Tinker (1975b) went a step further by pointing out that root hairs may be less effective than hyphae because root hairs have short lives and inter-hair competition is keen; hyphae are more dispersed and hence will have fewer overlapping P depletion volumes.

Evaluating the mycorrhizae-legume symbiosis is particularly challenging. Legumes may play a central role in increasing food production in tropical soils. Because of their ability to obtain nitrogen through symbiotic association with Rhizobia, it may be possible for farmers to obtain good yields with a minimum of expensive chemical fertilizers. Symbiotic nitrogen fixation by legumes may have a high P requirement (Munns, 1977). Phosphorus content of nodules may be 2-3 times more than the P content of the roots on which they are formed (Mosse et al., 1976). Also other micronutrients, notably Cu and Zn, have been shown to enhance or be necessary for nodulation (Hallsworth, 1958; McIlveen et al., 1975). It is not surprising that the, literature reports instances of legumes halving better nodulation, higher nitrogen percentage, and greater nitrogenase activity when they were inoculated with VA mircorrhizal fungi (Abbott and Robson, 1977; Mosse, 1977; Daft and El-Giahmi, 1976). Bagyaraj et al. (1979b) attempted to directly test the effect of VA fungi on N-fixation and plant growth. Four inoculation treatments were used: 1. uninoculated control, 2. inoculated with Rhizobium japonicum, 3. inoculated with a VA fungus Glomus fasciculatus, and 4. inoculated with Rhizobium and Glomus. After 60 days nodule mass and nodule nitrogen content from treatment 4 were double that from treatment 2. Shoot dry weight from treatment 4 was increased 64% over treatment 2; however the increase in grain yield was not significant (at P = 0.05). Waidyanatha et al. (1979) found that inoculation with VA fungi stimulated nodule weights and nitrogenase activity far more than plant growth. They believed that this effect on N fixation may be the most important effect of mycorrhizae on legumes.

The relative progression of Rhizobium nodulation and mycorrhizal infection is not known. The first infection units in soybean seedlings appeared 10-12 days after planting which corresponded with the appearance of root nodules (Carling et_.al., 1979a). Cox and Sanders (1974) defined an infection unit to include the internal mycelia relating to a single entry point. In another study Carling et al. (1979b) worked with nodulating and non-nodulating isolines of soybeans. Total plant and nodule dry weight and nitrate reductaset and nitrogenase activities were increased significantly in mycorrhizal, nodulating plants as compared to nonmycorzhizal nodulating plants. When phosphorus was substituted for mycorrhizae, similar growth and enzyme activities were observed. They concluded that the effects resulted from an improved nutritional environment for the plant rather than a direct interaction between the fungus and the bacterium. These results emphasize that our knowledge about the mycorrhizosphere is limited. The competition and the synergism among mycorrhizae, Rhizobia, and the host plant apparently are complex and are not adequately understood at present.

The significance of mycorrhizae in plant nutrition is probably greatest when fertility is low. Generalizations about the role of mycorrhizae in high fertility situations are more difficult to make. There appears to be a critical value of soil phosphorus for each species in certain growth conditions above which plants will grow well without mycorrhizae. This critical value may be determined in part by the diffusion rate of phosphorus in a particular soil (Cooper, 1975). However it appears that it is the concentration of P in the plant that regulates the mycorrhizal association rather than the concentration of P in the soil. Sanders (1975) induced high P concentrations in the plant by foliar feeding which inhibited infection. Menge et al. (1978a) used a 'split root' technique to demonstrate that number of spores, vesicles, arbuscules, and hyphae were not influenced by high soil P levels but were negatively influenced by high concentrations of P in the root.

The physiology of the root is likely to change as the percentage phosphorus content increases. This may be a self-regulatory mechanism of the plant. Ratnayake et al. (1978) examined root exudates, phospholipid content of root tissue, root P content, and membrane permeability. They concluded that a major consequence of low P nutrition is a decline in membrane phospholipids, an increase in membrane permeability, and increased exudation of metabolites. With increased P nutrition membrane permeability decreases as does the exudation of metabolites.

Growth depressions resulting from inoculation with VA mycorrhizae have been reported. Mosse (1973b) attributed this to phosphorus toxicity and specific interactions between the soil and the endophyte. However there are reports of growth suppresions when P toxicity was clearly not a problem. Cooper (1975) suggests the suppresions were related to the P status of the soil: at high phosphorus levels little infection develops and there is no growth response to mycorrhizae; at low levels of soil P a large response to fungi occurs because of an overriding improvement in the P nutrition of the plant; at intermediate levels, fungal infection is infrequent and transient reductions in growth occur which are followed by increases in growth response to the fungi with time as infection increases and P demand increases. A similar line of reasoning was pursued by Yost and Fox (1979) who suggested that at intermediate soil P levels decreased P uptake can occur if, with increasing levels of soil P, effectiveness of the endophyte decreases faster than P uptake increases by the uninfected root. Sparling and Tinker (1978) reported a decrease in shoot weight of three grasses which had been inoculated with VA mycorrhizae; they thought the decrease was understandable in light of the branched root system with many root hairs and relatively small P requirement.

Although there are many aspects of VA mycorrhizae that are not understood, research is moving ahead rapidly-toward developing inoculation techniques, testing relative efficiency of fungal strains, evaluating field inoculation trials, and attempting to define soil and plant conditions when a response to inoculation may be expected. The objective of this study was to characterize the soil inoculum level of some soils and to relate this to the quantity of phosphorus contributed by mycorrhizae to plants.

the plants would further accentuate the differences.

With the soil columns the bulk densities of the soil materials were calculated to be 0.76, 0.88, and 1.09 for the noncultivated, cultivated, and subsoil respectively. The combination of increased bulk density and poor aeration created conditions for plant growth that were very different in the three soil materials. The source of these differences may not be solely attributed to bulk density. Soils containing volcanic ash materials and high amounts of organic matter may exhibit 'hard to wet' properties. Capillary rise is not as great in soils exhibiting such properties; and is related to the contact angle at the water-solid interface. Where soil surfaces are coated with volcanic ash and organic surfactants, the contact angle can be greater than 90^o, in which case the surfaces will resist wetting. The Wahiawa soil has exhibited these properties (Fox, personal communication), and it is possible that surface soil materials are inherently different from subsoil materials with respect to the ability of water to move by capillary rise.

The differences among the soils are likely to have an effect on the diffusion of solutes in the soil. Nye and Tinker (1977) defined the diffusion coefficient of nonvolatile solutes as

F = D₁ θ f_l dC₁/dx + F_e where

D₁ is the diffusion coefficient of the solute in free solution

θ is the fraction of soil volume occupied by solution

f_l is an impedence factor

C₁ is the concentration of solute in the soil solution

F_E is the excess flux created by surface diffusion

The water content of the soil thus exerts a major effect upon the diffusion coefficient of a solute in soil. With the information from the water retention curves, we may speculate that the diffusion coefficient for phosphorus was greatest in the subsoil, and least in the noncultivated soil.

The effect of diffusion coefficients on the ability of plants to absorb phosphorus will be greatest when soil P levels are low. Nonmycorrhizal plants growing in noncultivated soil were less able to absorb phosphorus than comparable plants in cultivated and subsoils when the soil P levels were low (Fig, 14). The threshold value of soil P for a particular soil, that value below which the plant is unable to extract phosphorus from the soil, may be determined by both the P diffusion coefficient in that soil as well as the plant species. Yost and Fox (1979) reported similar threshold values of .012 mg P/liter for nonmycorrhizal Allium cepa, Stylosanthes, and Leuceaena leucocephala growing in the field. If the roots affinity for phosphorus is similar among plant species, then the predominant factor influencing the threshold value for P absorption may be the P diffusion coefficient.

The principle advantage of mycorrhizae is increased P uptake. This is possible because the hyphae extend into the soil beyond the volume of P depletion by the root. The relative advantage of mycorrhizae should be greatest in soils where the P diffusion coefficients are least and the volume of P depletion is the smallest. For example, in the subsoil where the diffusion coefficient should have been relatively high, mycorrhizae may not be as advantageous to the host as in the noncultivated soil where the diffusion of P should be relatively less. The concept of specific interactions between the soil and the fungi has not received the attention it deserves. Cooper (1975) noted that the effects of soil properties need to be considered when evaluating plant response to mycorrhizae, including P diffusion rates which are not reflected in extractable P values.

The critical P level for mycotrophy may have been influenced by the P diffusion rate in the soil. In the subsoil this critical P level was approximately .008 mg P/liter, and in the cultivated soil it was .062 mg P/liter. Given the more similar physical characteristics of these two soils the relative difference in their critical P levels for mycotrophy may also reflect different inoculum densities. In the noncultivated soil, with a relatively high inoculum density and relatively low P diffusion coefficient, the lower critical level for mycotrophy is unexpected. However soil structure has other effects besides affecting water retention and diffusion rates. Root development can be impeded in soils with high bulk densities. In the noncultivated soil, aeration was undoubtedly more favorable for root development relative to the cultivated and subsoils. The lower P levels for mycotrophy together with the higher growth potential may indicate that mycorrhizae are not a substitute for a well developed root system. The unfavorable soil structure for root proliferatioa may have been the limiting factor in plant growth in the subsoil material. The lack of response to inoculation in an environment not favorable for root growth is understandable.

Another factor which may have contributed to different growth potentials in the soils was the available nitrogen supply. During early growth, leaves of plants growing in sterile soils were a dark green color, while leaves of plants in non-sterile soils were a pale green color. Differences in nitrogen nutrition were suspected, perhaps due to the competition-free environment in sterile soil which allowed for rapid growth of introduced Rhizobia. Other contributing factors may have been the release of NH₄⁺caused by y-irradiation (Singh and Kanehiro, 1970) and the Birch effect. After 22 days the differences in color among the plants disappeared. By the end of the experiment total N uptake was greater in plants in non-sterile soil than sterile soil (Table 4). The warm moist conditions in the pots may have promoted mineralization of organic matter relatively more in non-sterile soils. The differences in organic carbon in the soils (Table 1) should be considered a factor in the different growth potentials of the soil materials. Organic matter may not only have contributed to the 'hard to wet' properties of the noncultivated soil material, but also to the greater uptake of N by plants relative to plants in the other soil materials.

LITERATURE CITED

1. Abbott, K.L., and K.D. Robson. 1977. Growth stimulation of subterranean clover with vesicular-arbuscular mycorrhizas. Aust. J. Agric. Res., 28: 639-649.

2. Bagyaraj, D.J., A. Manjunath, and R.B. Patil. 1979a. Occurrence of vesicular-arbuscular mycorrhizas in some tropical aquatic plants. Trans. Brit. Mycol. Soc. 72: 164-167.

3. Bagyaraj, D.J., A. Manjunath, and R.B. Patil. 1979b. Interaction between a vesicular-arbuscular mycorrhiza and Rhizobium and their effects on soybean in the field. New Phytol. 82: 141-145.

4. Baylis, G.T.S. 1975. The magnolioid mycorrhiza and mycotrophy in root systems derived from it. In F.E. Sanders, B. Mosse and P.B. Tinker (eds.) Endomycorrhizas. Academic Press, New York. pp. 373-389.

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