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The incidence of vesicular-arbuscular mycorrhizae in wastewater irrigated and non-irrigated oldfield soils in Michigan was studied. The most common VAM fungal species were Glomus mosseae and G. Fasciculatum. The effects of wastewater irrigation on VAM are due to the effects of both water and nutrients.
The incidence of vesicular-arbuscular mycorrhizae in wastewater irrigated and non-irrigated oldfield soils in Michigan was studied. The most common VAM fungal species were Glomus mosseae and G. Fasciculatum. The effects of wastewater irrigation on VAM are due to the effects of both water and nutrients.
The incidence of vesicular-arbuscular mycorrhizae in wastewater irrigated and non-irrigated oldfield soils in Michigan was studied. The most common VAM fungal species were Glomus mosseae and G. Fasciculatum. The effects of wastewater irrigation on VAM are due to the effects of both water and nutrients.
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Vesicular-arbuscular mycorrhizae in a wastewater-irrigated oldfield ecosystem in Michigan* G.R. SAFIR, J.O. SIQUEIRA ~ and T.M. BURTON Department of Botany and Plant Pathology and the Department of Zoology, Michigan State University, East Lansing, MI 48824, USA. IOn sabbatical leave.from ESAL, Lavras-MG, Brazil Received 16 May 1989. Revised August 1989 Key words: Glomus, irrigation, mycorrhizal fungi, soil microorganisms, wastewater disposal Abstract The incidence of vesicular-arbuscular mycorrhizae (VAM) in wastewater irrigated and non-irrigated oldfield soils in Michigan was studied. Soil and root samples were taken monthly from field plots on the second and third years of consecutive irrigation with municipal wastewater at rates of 0, 5 and 10 cm wk J. The oldfield ecosystem contained a high VAM fungal spore population density, but low species diversity. The most common VAM fungal species were Glomus mosseae and G. fasciculatum. Both spore density and root colonization were higher in irrigated than in non-irrigated plots. Irrigation effects were largest early in the growing season. In addition to increasing VAM incidence, wastewater irrigation shifted VAM fungal species composition. Irrigation favored G. mosseae over G. fasciculatum. Bioassays using either Sorghum vulgate or Daucus carota, an oldfield native species, indicated that the VAM systems were still functioning after the third year of consecutive wastewater irrigation. The data from experiments using nutrient solutions at wastewater concentrations suggest that the effects of wastewater irrigation on VAM are due to the effects of both water and nutrients. Since VAM are a very important component of the plant's water and nutrient uptake system and equally important in structuring plant communities under limiting growth conditions, it is suggested that the stimulatory effect of wastewater irrigation on VAM in an oldfield ecosystem enhances the ecosystem's ability to function as a living filter for wastewater clean up. Introduction Soil and its vegetation act as a wastewater filter and can provide a desirable alternative for disposal of secondary treated wastewater (Bower and Chaney, 1974). Application ofwastewater to either natural or agricultural ecosystems often increases plant biomass production (see overview by Brock- way et al. and other individual papers in D'Itri, 1982). However, this increased biomass production has to be balanced against possible contamination of aquatic and groundwater systems, especially with nitrate and chloride (Burton, 1978; Burton and King, 1981), possible problems with increased * Michigan Agricultural Experimental Station Journal Article No 13137. 187 spread of human pathogens (Kowal et al., 1981; Kristensen and Bonde, 1977; Shuval, 1977), and possible deleterious effects on the plant com- munities within natural ecosystems including changes in species composition (Burton and Hook, 1982) and injury and vegetation decline if wastewater is applied in excess (Burton, 1982). Other adverse effects of wastewater application on vegetation may result from over fertilization (Baier and Fryer, 1973; Neary et al., 1975), increased disease incidence (Epstein and Safir, 1982) and depression of biological nitrogen fixation in legumes (Tesar et al., 1982). Despite these potential problems, reuse of wastewater is a widespread phenomenon worldwide (Heaton, 1981) and offers substantial advantages over discharge into aquatic ecosystems, especially in low precipitation areas of 188 Safir et al. the world. An area of concern to us that has been largely overlooked is the potential impact of wastewater nutrients, toxins, pathogens and anti- microbial agents, such as chlorine, on the desirable microbiological processes in the soil ecosystems. Of particular interest is the impact on vesicular- arbuscular mycorrhizae (VAM). VAM are widely recognized as components of all terrestrial ecosystems (Safir, 1987), and known to be essential to above- (Allen and Allen, 1984) and below-ground processes in oldfield ecosystems (Crowell and Boerner, 1988). Since VAM for- mation and function are sensitive to soil moisture (Nelson, 1987) and fertility (Hayman, 1982), the effect of wastewater irrigation on naturally occur- ing VAM is of interest. In this paper, we report the effects of three consecutive years of wastewater irrigation on the incidence of VAM fungi and VAM infectivity in an oldfield ecosystem in Michigan. Material and methods Field studies This study was conducted at the water quality management facility at Michigan State University, on an abandoned farm field site that had not been cultivated for approximately 10 years. Site veg- etation was domi nat ed by a mixture of quackgrass (Agropyron repens) and goldenrod (Solidago graminifolia and Solidago canadensis) with a diverse mixture of other oldfield species. This field was divided into 24 x 27 m plots that received 0, 5, and 10 cm wk- 1 of chlorinated municipal Table 1. Mean annual concentration (mg L -~) of wastewater applied to the oldfield site (from Hook and Burton, 1978) Constituent Second year Third year Nitrate-nitrogen 10.5 9.1 Ammonium-nitrogen 1.4 1.2 Total nitrogen 13.7 13.6 Total phosphorus 2.7 2.7 Potassium 10.0 10.5 Calcium 69.9 57.9 Magnesium 24.4 25.6 Sodium 86.5 97.1 Chloride I 12.0 120.0 pH = 7.5; alkalinity - equivalent CaCO 3 = 150. Table 2. Average soil analyses/~g g dry soil ~, for the top 30 cm of soil of the oldfield site after two years of wastewater irrigation (after Hook and Burton, 1978) Constituent 0 cm wk -t 5 cm wk -~ 10 cm wk -~ Phosphorus (Bray) 18.1 11.0 17.5 Potassium (NH4OAc) 135.8 75.1 73.6 Calcium (NH4OAc) 806.8 932.4 944.8 Magnesium (NH4OAc) 89.3 130.6 160.4 Sodium (NH4OAc) 27.9 87.0 99.7 Chloride (K2 SO4) 9.6 32.2 32.7 NO 3-nitrogen 1.3 1.9 2.3 (Kjeldahl) wastewater irrigation by spray. The wastewater originated from the East Lansing, Michigan sewage treatment facility from the first of four lakes used to store this wastewater prior to irrigation (see Table' 1 for chemical analysis of this wastewater). Spray irrigation continued from April to Oct ober each year. Soil chemistry for these plots determined after the first and second years of irrigation, indicated that wastewater irrigation increased calcium, mag- nesium, sodium, and chloride in these soils (Table 2). Mass balance budgets for the site also indicated that substantial amount s of N and P were being retained by the vegetation and soils (Burton and Hook, 1982; Hook and Burton, 1978). Precipitation averaged 77cm year -~ with an average snowfall of 124cm year -~. Temperat ure averaged 8.2C with a mean monthly low of - 5. 5 C in January and a mean monthly high of 27.6C in July. The average growing season in this part of Michigan extends from May 7 to October 8 (154 days). To evaluate VAM incidence, 20 soil and root samples were removed mont hl y from May to October, for a two year period from each of six randomly assigned 24 x 27 m plots that received 0, 5 or 10 cm wk- a of wastewater. Wastewater ir- rigation had been applied in the year before sam- pling and was applied at the same rate during the two years of sampling. They are referred to as the second and third years of consecutive wastewater irrigation. Plots were not harvested during the sam- pling period. Samples were taken to a depth of 25cm and 5cm sections were separated and analyzed for VAM spore numbers. Fifteen gram samples from each 5 cm section were thoroughly mixed in 100 mL of distilled water and wet-sieved through 40 and 325-mesh screens (Gerdemann and VA mycorrhizae in a waste-water irrigated oldfield 189 Nicolson, 1963) and the spores separated by sucrose (density = 1.18gcm 3) centrifugation. Spores were grouped by appearance and counted under a dissecting microscope. These groups were later classified to the species level as described in Gerdemann and Trappe (1974) and Trappe (1977). In the third year of consecutive application, root samples were taken from 20 plants, separated by species, in three 2 2m random sections from each plot. Quackgrass (Agropyron repens) the predominant species, was sampled on a monthly basis, while other species were sampled only in September. Roots were separated from soil, washed, cleared, and stained according to Phillips and Hayman (1970). For colonization assessment stained segments were mounted on microscope slides, scanned for VAM fungal structures, and rated as 3, 2 and 1 according to the intensity of fungal structures in the root as follows. Rating 3 when fungal structures were present in at least 130 mm/200 mm of root; 2 if they were present in 60-129mm/200mm and 1 if present in 1-59mm/ 200 mm of root. The data were subjected to a one way ANOVA and the means for each sampling time separated by the Student-Newman-Keuls' multiple range test at the 0.05 level. Greenhouse experiments To evaluate the effects of wastewater application on VAM infectivity and effectiveness of oldfield ecosystems, VAM formation potential and effects on plant growth were assessed using sorghum (Sorghum vulgare) (routinely used for infection studies) and Queen Anne's lace (Daucus carota), a mycorrhizal oldfield species, as test plants. Both of these species are easily produced from seeds. Quackgrass was not used for these studies because of the difficulty in obtaining uniform plant material for propagation. The first experiment was conduc- ted for 10 weeks in waxed cups containing 800 g of soil collected from each irrigation treatment (0, 5 and 10cmwk-I), by the end of the third consecu- tive year of irrigation. In addition soil from the highest irrigation level (10cmwk -t) and a sandy loam soil (with pH = 7.7 and 8 ppm of P) were autoclaved and included as treatments. Each treat- ment had 8 replications. In the second experiment, sorghum remained untreated or was inoculated with VAM fungi and planted in either 10 cm wk -I irrigated or greenhouse autoclaved soil. Highly in- fective VAM fungus inoculum (a mixture of Glomus mosseae and Glomus fasciculatum) originat- ing from sudangrass greenhouse pot cultures, was applied at a rate of 200 g of soil inoculum per 800 g of either field or greenhouse soil. Each treatment had 6 replications and the experiment was conduc- ted for 10 weeks. A third set of experiments was conducted using the same treatments as the second experiment, except that the oldfield native plant Queen Anne's lace (D. carota) was used as a test plant. At the end of each experiment plants were harvested and shoot dry weights were determined. Roots were separated from the soil for assessment of VAM colonization as previously described. In additional greenhouse experiments, nutrient solutions were adjusted to simulate wastewater (ac- cording to Table 1) using the following salts: (NH4)2NO3, KNO3, NaH2PO4, NaC1, CaC12 2H20, Ca(NO3)' 4H20, MgSO4" 7H20, CaSO4, CaCO3, MnSO4.4H2 and FeSOa' 7H20. Pre- germinated seeds of D. carota were planted in waxed cups containing 800 g of oldfield soil, to which the following treatments were applied: 1) water-stressed control (100mL of distilled water); 2) water irrigated (190mL of distilled water); 3) simulated wastewater (100 mL of nutrient solution) and; 4) water and nutrient (190mL of nutrient solution). These treatments were applied in the pres- ence and absence of VAM inoculum (G. mosseae and G. fasciculatum) as previously described. Simulation treatments had 8 replications and were applied 5 times per week to approximate the 5 cm irrigation rates. After 12 weeks growth, plants were harvested and dry weight and root colonization assessed as previously described. Every experiment was repeated once. All the data were subjected to statistical analysis and significant effects separated by the Student Newman-Keuls multiple range test at the .05 probability level. Results Incidence of VAM The oldfield site from Michigan had spore population densities as high as 25 spores/g soil (Table 3, 4). The predominant fungal species 190 Safir et al. Table 3. Effect of wastewater irrigation on soil spore density for the three predominant VAM fungal species in a oldfield ecosystem in Michigan. Data (number of spores/15 g soil) for the second consecutive year of irrigation in the 0- - 10c m top soil layer Sampling G. fasciculatum G. mosseae G. constrictum time 0 5 cm wk -~ 10 cm wk ~ 0 5 cm wk -~ 10 cm wk -t 0 5 cm wk -~ 10 cm wk -~ May 109 be 331 a 392 a 41 cd 154 b 111 b 29 d 84 c 57 cd June 134 b 203 ab 282 a 49 cd 188 ab 170 ab 47 c 22 c 38 c July 167 b 161 b 417 a 53 cd 127 b 411 a 43 b 21b 54 b August 132 c 169 b 163 b 60 cd 170 b 282 a 40 cd 47 cd 50 cd September 98 cde 160 bcd 207 b 105 ode 270 b 353 a 56 de 32 de 58 de October 107 c 151 b 164 b 66 cd 223 a 281 a 57 cd 57 cd 55 cd Means in the same line (month) followed by the same letters are not statistically different by the Student-Newman-Keuls' multiple range test at .05 level. Table 4. Effect of wastewater irrigation on soil spore density for the three predominant VAM fungal species in an oldfield ecosystem in Michigan. Data (number of spores/15 g soil) for the third year of consecutive irrigation in the 0- - 10 cm top soil layer Sampling G. fasciculatum G. mosseae G. constrictum time 0 5 cm wk t 10 cm wk -t 0 5 cm wk -~ 10 cm wk -~ 0 5 cm wk -~ i0 cm wk t May 31 b 33 b 18 c 22 c 81 a 28 b 2 d 5 d 10 d June 13c 44a 23b 7c 75a 29b 4c 4c 7c Jyly 8 c 19 b 15 b 8 c 35 a 23 a 6 c 2 c 4 c August 17 c 34 a 20 bc 14 c 38 a 17 c 6 c 18 c 7 d September 19 c 30 b 20 d 22 cd 60 a 28 bc 6 f 16 de 11 e October 11 c 28 a 20 a 10 c 87 a 23 a 2 d 12 b 6 c Means in the same line (month) followed by the same letters are not statistically different by the Student-Newman-Keuls' multiple range test at .05 level. recovered were identified as Glomus mosseae, Glomus fasciculatum and Glomus constrictum. A fourth species Sclerocystis rubiformis was found in low numbers and for this reason was not con- sidered further. Spore numbers were higher in the second (Table 3) than in the third year of consecu- tive wastewater irrigation (Table 4), even in non- irrigated plots. In the second year of consecutive irrigation (Table 3), the numbers of G. mosseae and G. fasciculatum spores were higher in irrigated than in non-irrigated plots. G. constrictum was only sig- nificantly affected by irrigation in May of the second year. In non-irrigated plots, G. fasciculatum had higher spore numbers in June and July, while in irrigated plots, at both 5 and 10 cm wk-t , spore numbers were higher earlier in the growing season and decreased toward the end of the season. G. mosseae did not change throughout the season in non-irrigated plots, but increased late in the season in irrigated plots. G. constrictum had a lower spore density than the other VAM fungus species and was less affected by either sampling time or irri- gation treatment. In the third year of consecutive irrigation (Table 4), 5 cm wk- ~ plots had more of G. mosseae and G. fasciculatum spores than the 10cmwk -~ plots at most sampling times. G. constrictum spore numbers were significantly different in August and Septem- ber when 5 cmwk -~ plots had more spores than either non-irrigated or 10 cm wk-~ treatments. Despite the monthly variation, irrigated plots had higher numbers of G. mosseae and G. fasciculatum population densities than non- irrigated ones for both G. mosseae and G. fasciculatum during both sampling years but to a lesser extent in the third year (Fig. 1). G. constric- turn, however responded very little to irrigation. Spore numbers were also affected by soil depth. Spores were concentrated in the upper 15 cm of soil and their distribution in the soil profile was dif- ferentially affected by irrigation and sampling time (Figure 2). In May, irrigated plots had an average of 4.6 times more spores than non-irrigated ones in VA mycorrhizae in a waste-water irrigated oldfield 191 400 0 G, fasci cul atum "6 aoo 0' ) t O } iI~ 2OO .1o g 0 5 10 G. mos s eae 0 5 10 I--] Second year [ ] Third year G. consrri ctum o 5 l O I r r i gat i on Tr eat ment s, cm/ wk Fig. 1. Overall effects of wastewater irrigation levels on the spore densities of G. fasciculatum, G. mosseae and G. constrictum in an oldfield soil duri ng the second and third years of consecutive irrigation. 5.0 o t~ 4. 0 I f : "E ~ 3.0 Z ca ~ 2.0 September ,~/ , h , L ' 115 ' 0 i , 0 5 10 2 25 Soi l Dept h, cm Fig. 2. Effect of wastewater irrigation on VAM spore density at increasing soil dept h at the begi nni ng and at the end of the growing season duri ng the second year of consecutive wastewater application. Each data point represents the ratio of mean spore density for irrigated (5 and 10cmwk -t plots) over non-irrigated plots. the 0-5 cm soil layer. This ratio dropped to less than 2.0 at higher depth (20-25 cm). By September this trend reversed, i.e. irrigation favored fungal spore production at deeper layers in the profile. In addition to effects on total spore number, irrigation altered VAM fungal community struc- ture in the oldfield soil (Fig. 3). The calculated ratio between numbers of G. mosseae/G, fasciculatum spores indicated very little effect of sampling time O =. t 2 81 c May a, irrigated - 0 - Non-irrigated qr i ..Q- -- I-- O- -- O-- --8 Second Year Third Year I i I I J L I I I I I Jul Sept May Jul Sept Sampl i ng Ti me Fig. 3. Rat i o of mean spore numbers for G. mosseae (MOS) versus G.fasciculatum (FAS) in irrigated and non-irrigated plots t hroughout the growing season in the second and third year of consecutive wastewater irrigation. in non-irrigated plots, but a considerable effect in irrigated plots. In the second year of wastewater application, the G. mosseae/G, fasciculatum spore number ratio increased from 0.36 early in the season (May) to 2.0-2.5, late in the season (Septem- ber and October). In the third year, however, this ratio was not as high as was found in the second year, but was much less affected by the sampling time. Since plant species composition differs in ir- rigated and non-irrigated plots, individual species can not be compared, except for quackgrass, which 3.0 ca t- e,- i- ._o C 2 O tJ 2.5 2. 0 1. 5 1. 0 o. of A I r r i gat ed ,~ - - - 0- - - Non- i r r i gat ed / / / / / / / I I May June July Sampl i ng Ti me Au;ust I September Fig. 4. VAM root colonization rating of quackgrass (Agropyron repens) after the third year of consecutive wastewater irrigation and from non-irrigated plots in an oldfield ecosystem duri ng the growing season. 192 Safir et al. was present in both situations. The overall species root colonization means for non-irrigated and ir- rigated plots were 1.6 + 0.4 and 2.5 +__6, respec- tively. The monthly colonization rating, for quack- grass from non-irrigated and irrigated plots are given in Figure 4. Irrigated plots were more heavily colonized from May to July, but no differences were evident late in the growing season. Although non-irrigated plots reached the same colonization levels as irrigated ones late in the season, higher VAM formation earlier in the season may be of great advantage for plant nutrient uptake and growth. Effects on VAM formation and function The greenhouse assays using sorghum demonstrated that three years of consecutive wastewater irrigation had no significant effect on either VAM formation or plant growth (Table 5). However, when 10cmwk -~ irrigated soil was autoclaved to eliminate VAM propagules, plant growth was significantly reduced. Re-infestation of the same soil with mixed VAM inoculum, restored its infectivity and greatly increased sorghum and D. carota growth. This suggests that wastewater ir- rigation for three consecutive years at rates as high as 10cmwk -~ had no adverse effects on VAM formation and function. Simulation experiments using D. carota showed significant plant growth responses (Figure 5). Root colonization was at the same level as those of D. carota inoculated plants in Experiment 3 (Table 5) and was not affected by any of the treatments. Plant dry weight was lower when nutrients alone (NUT) were applied in the absence of VAM, but was im- proved by joint application of nutrients and water (DW + NUT). VAM fungus inoculation im- proved plant growth in comparison to non-mycorr- hizal plants in every treatment. Its effects were more pronounced when either distilled water (DW), nutrients (NUT) or both (DW + NUT) were applied. Plant growth in these treatments was equally high and significantly greater than every non-inoculated treatment. Simulated wastewater (DW + NUT) improved plant growth in com- parison to the control (CON) plants whether they Tabl e 5. Infectivity and pl ant growt h pr omot i on of wastewater-irrigated and non-irrigated oldfield soils from Mi chi gan under greenhouse conditions Soil origin Treat ment Pl ant dry wt Root colonization Experi ment t - sorghum - Non-irrigated field soil 5 cm wk -~ irrigated field soil 10 cm wk -~ irrigated field soil 10 cm wk- t irrigated field soil Greenhouse VAM conductive soil Experi ment 2- sorghum - 10 cm wk -t irrigated field soil 10 cm wk -1 irrigated field soil Greenhouse soil Greenhouse soil Experi ment 3 - Daucus carot a - 10 cm wk -I irrigated field soil 10 cm wk- i irrigated field soil 10 cm wk -1 irrigated field soil Greenhouse None 7.0 a b 1.6 a b None 6.6 a 1.2 a None 6.5 a 1.7 a Aut ocl aved 4.0 b 0.0 b Aut ocl aved 3.2 b 0.0 b Aut ocl aved 5.1 b 0.0 b VAM inoculated a 10.5 a 1.8 a Aut ocl aved 5.7 b 0.0 b VAM inoculated a 11.9 a 2.0 a None 0.87 b 1.5 a Aut ocl aved 0.45 c 0.0 c Aut ocl aved and 1.33 a 1.0 b VAM inoculated a Aut ocl aved and 1.05 b 1.0 b VAM inoculated" a Inoculated with highly infective soil i nocul um f r om pot culture cont ai ni ng a mi xt ure of Gl omus mosseae and Gl omusf asci cul at um. b Means followed by t he same letters are not statistically different within experi ment s by the St udent -Keul s' multiple range test at .05 level. 5 I j Non-mycorrhizal ~ Mycorrhizal 4 a iiii~iii~iiii~il b ~ ~ ! 1 c d } i ............ i 0 ............... CON DW O) J i E' l "E 2 a i , ! . . . . i~;~ ~i~i;: !,iiiiiiiiiii~i~i~'i~i ,~' NUT i : : / ~i~ i: ~ I DW + NUT Fig. 5. Growth of mycorrhizal and non-mycorrhizal Daucus carota in a simulated wastewater experiment. CON = control water stressed, DW = distilled water only, NUT = nutrients only and DW + NUT = distilled water + nutrients. Means with similar letters do not differ by the Student-Newman-Keuls' test at the .05 level. were mycorrhizal or not. However, if they were mycorrhizal, biomass product i on was much greater. Discussion As reported for other non-agricultural ecosys- tems (Read et al., 1976; Sparling and Tinker, 1978) VAM fungi occur quite abundantly in oldfields in Michigan. The two predomi nant GIomus species, G. mosseae and G. f asci cul at um, found in our systems are also commonl y found in Michigan cul- tivated soils (Wacker, 1988) and in the grassland/ shrubland of Nort h America and other parts of the world (Miller, 1987). Also, in asparagus fields in Michigan, the relative density of G. mosseae decreases with field age, while G. f asci eul at um is more frequent in older fields (Wacker, 1988). These community shifts can result from the action of selective factors such as moisture (Anderson et al., 1983; Dickman et al., 1983) soil chemical charac- teristics (Schenk and Siqueira, 1987) and al- VA mycorrhi zae in a wast e-wat er irrigated oldfield 193 lelopathic compounds (Wacker, 1988) that accumulate over time. Since this study was concluded, G. f asci eul at um as previously described (Gerdemann and Trappe, 1974), has been shown to include several additional distinct taxa (Walker and Koske, 1987). This suggests that additional taxonomic studies may increase the number of species reported herein. The beneficial effects of VAM on plant-water relationships is well known (Safir et al., 1971) and has been the subject of many recent publications as reviewed by Nelson (1987). Nevertheless, the influ- ence of soil moisture on VAM development and function has not attracted the same attention. The results presented here show that root colonization and spore numbers in the soil increased in response to wastewater irrigation. Wastewater application also resulted in increased plant biomass in these sites (Burton and Hook, 1982). Water shortage in the soil can reduce and delay VAM fungal spore germination (Tommerup, 1984) root growth (Reid and Bowen, 1979), and subsequent VAM for- mation (Paula and Siqueira, 1987; Reid and Bowen, 1979). Because colonization progresses to a maximum t hroughout the season even under limit- ing conditions (Hayman, 1970; Sparling and Tinker, 1978), no effect of irrigation was found in our study late in the growing season as was found early in the season. Soil moisture is known to affect VAM fungal sporulation under controlled environments (Paula and Siqueira, 1987) and to be a domi nant factor in spore abundance in natural ecosystems (Miller, 1987). Soil moisture, however, may have a greater effect on root growth than on the sporulation process (Paula and Siqueira, 1987). Therefore, it is possible that the higher spore numbers in our wastewater irrigated plots are simply a result of an increased amount of root growth (Burton and Hook, 1982) and VAM fungal root colonization. Wastewater application also affected spore distribution in the soil profile. Similar effects have been reported for plain water irrigation of Citrus rootstock (Levi et al., 1983). However, in our study, this effect varied during the growing season in that it increased spore density at lower depths late in the season. Increasing soil infectivity of lower soil layers may be of ecological significance. In addition to increasing VAM spore numbers, wastewater irrigation resulted in a change in spore 194 Safir et al. numbers for the predominate species. Irrigation favored G. mosseae over G. f asci cul at um. After September of the second year of consecutive irri- gation the relative density of G. mosseae was twice as high as that of G. f asci cul at um. This trend con- tinued during the third year, however, the numbers of spores were reduced. Population shifts between these two fungi have been reported to occur in other systems (Visser et al., 1984; Wacker, 1988), and is probably due to changes in the edaphic environment. Hayman and Mosse (1979) reported that lime and phosphate amendments induced a greater reduction in the population of indigenous fungi than of introduced G. mosseae and G. f asci cul at um in grassland soils in Wales. VAM fungi do not exhibit habitat specificity, but may have a narrow range of tolerance to environmental conditions. G. mosseae is well known for its prefer- ence for neutral to alkaline soils (Siqueira et al., 1984), while G. f asci cul at um seems to tolerate a broader pH range. In fact, either lime or Ca 2 application has been shown to increase root colonization by G. mosseae (Hepper and O'shea, 1984; Siqueira et al., 1984). Wastewater has a high pH and base content and its application to the oldfield ecosystem increases Ca 2 and Mg 2 levels in the soil (Table 2), thus favoring G. mosseae over G. f asci cul at um. Greenhouse experiments indicated that both ir- rigated and non-irrigated field soils were very infec- tive and had an effective VAM native population, because plant growth dropped significantly when VAM was eliminated by autoclaving soil. Growth promotion effects were restored by the reinfestation of autoclaved soil with a mixed inoculum of G. mosseae and G. f asci cul at um. This suggests that after three years of consecutive wastewater irri- gation no anti-VAM fungal factor had built up in the soil and that the native VAM fungi were func- tioning. It also suggests that application of chlorinated wastewater by spray irrigation does not lead to reduction in VAM populations, due to chlorination effects. Although D. carota is not the predominant species in our oldfield irrigated plots, the fact that it showed a high degree of mycorrhizal dependency indicates the importance of VAM in this system. The experiment with simulated wastewater suggests that the wastewater irrigation effects on VAM formation as reported here, and biomass production (Burton and Hook, 1982) are due to both nutrients and the water supply. The 3.5-fold increase in D. carota growth due to inoculation with G. rnosseae and G. f asci cul at um when water and nutrients were applied (Fig. 5), is evidence of the importance of VAM in oldfield irrigated ecosystems. Urban and industrial disposal on land has been of major concern among environmentalists because of their potential to harm the ecosystem. For in- stance, the percent of VAM colonization in barley was reduced by 6-fold due to land sludge appli- cation (Boyle and Paul, 1988). Because formation and function of VAM are greatly affected by soil nutrient status, especially P and N availability (Hayman, 1982), and because considerable amounts of these nutrients were applied in the wastewater, between 150-270 kg ha- ~ yr- 1 of N and 30-70kgha -l yr -l of P (Burton and Hook, 1982), it was expected that wastewater irrigation would reduce VAM in the oldfield ecosystem. Instead, it favored root colonization and increased VAM spore population densities in the top soil layer and also deeper in the soil profile. Such effects were also found in a 50 year old beech-maple stand in north- western Michigan with effluent irrigation up to 7.6 cm wk-~ (Otto, 1980). Below-ground activity, is of importance to any system acting potentially as a living filter for wastewater disposal. Since VAM are crucial components of such a system in terms of its nutrient and water uptake capacity, and equally important in structuring plant communities under limiting conditions for adequate growth (Allen and Allen, 1984), the stimulatory action of wastewater on VAM in oldfield ecosystems may enhance the efficiency of these sites as living filters for wastewater clean up. This suggests a need for ad- ditional long term studies. Acknowledgments Here we would like to thank Ms Barbara Car- penter for technical assistance on this project, and CNPq-Brazil for the scholarship to J O S. References Allen E B and Allen M F 1984 Competition between plants of different successional stages: Mycorrhizae as regulators. Can. VA mycorrhi zae in a wast e- wat er i rri gat ed oldfield 195 J. Bot. 62, 2625-2629. Anderson R C, Libert A E, Dickman L A and Katz A J 1983 Spatial variation in vesicular-arbuscular mycorrhiza spore density. Bull. Torrey Bot. Club 110, 519-524. Baier D C and Fryer W B 1973 Undesirable plant response with sewage irrigation. Irr. Drain. Div. Am. Soc. Civil Eng. 99, 133-141. Bower H and Chaney R L 1974 Land treatment of wastewater. Adv. Agron. 26, 133-176. Boyle M and Paul E A 1988 Vesicular-arbuscular mycorrhizal associations with barley on sewage-amended plots. Soil Biol. Biochem. 20, 945-948. Burton T M (Ed.) 1978 The Felton-Herron Creek: Mill Creek Pilot Watershed Studies. EPA-905/9-78--002. U.S. Environ- mental Protection Agency, Great Lakes National Program Office, Chicago, IL, 214 p. Burton T M 1982 Studies of land application in old growth forests in southern Michigan. In Land Treatment of Municipal Wastewater: Vegetation Selection and Manage- ment. Ed. F M D'Itri. pp 181-193. Ann Arbor Sci. Publishers Inc., Ann Arbor, MI. Burton T M and Hook J E 1982 Oldfield and grass management studies on the water quality management facility at Michigan State University. In Land Treatment of Municipal Wastewater: Vegetation Selection and Management. Ed. F M D'ltri. pp 107-133. Ann Arbor Sci. Publishers, Inc., Ann Arbor, MI. Burton T M and King D L 1981 The Michigan State University water quality management facility - A lake-land system to recycle wastewater. In Municipal Wastewater in Agriculture. Eds. F M D'Itri, J A Martinez and M A Lambarri. pp 249-269. Academic Press, New York. Crowell H F and Boerner R E F 1988 Influences of mycorrhizae and phosphorus on belowground competition between two oldfield annuals. Environ. Exp. Bot. 28, 381-392. Dickman L A, Liberta A E and Anderson R C 1983 Ecological interaction of little bluestem and vesicular-arbuscular mycorrhizal fungi. Can. J. Bot. 62, 2272-2277. D'Itri F M (Ed.) 1982 Land Treatment of Municipal Wastewater: Vegetation Selection and Management. Ann Arbor Sci. Publishers Inc., Ann Arbor, MI, 218 p. Epstein L and Safir G R 1982 Plant diseases associated with municipal wastewater irrigation. In Land Treatment of Municipal Wastewater: Vegetation Selection and Manage- ment. Ed. F M D'Itri. pp 195-203. Ann Arbor Sci. Publishers Inc., Ann Arbor, MI. Gerdemann J W and Nicolson T H 1963 Spores of mycorrhizal Endogone species extracted from soil by wet-sieving and decanting. Trans. Brit. Mycol. Soc. 46, 235-244. Gerdemann J W and Trappe J M 1974 Endogonaceae in the pacific northwest. Mycologia Mem. 5, 1-76. Hayman D S 1982 Influence of soils and fertility on activity and survival of vesicular-arbuscular mycorrhizal fungi. Phytopathol. 72, 1119-1125. Hayman D S 1970 Endogone spore numbers in soil and VAM in wheat as influenced by season and soil treatment. Trans. Brit. Mycol. Soc. 54, 53-63. Hayman D S and Mosse B 1979 Improved growth of white clover in hill grasslands by mycorrhizal inoculation. Ann. Appl. Biol. 93, 141-148. Heaton R D 1981 Worldwide aspects of municipal reclamation and reuse. In Municipal Wastewater in Agriculture. Eds. F M D'Itri, J A Martinez and M A Lambarri. pp 43-74. Academic Press, New York. Hepper C M and O'Sheia J 1984 Vesicular-arbuscular mycor- rhizal infection in lettuce (Lactuta sativa) in relation to calcium supply. Plant and Soil 82, 61-68. Hook J E and Burton T M 1978 Land application of municipal effluent on oldfields and grass lands. In The Felton-Herron Creek: Mill Creek Pilot Watershed Studies. Ed. T M Burton. pp 25-65. EPA-905/9-78-002. U.S. Environmental Protection Agency, Great Lakes National Program Office, Chicago, IL. Kowal N E, Pahren H R and Arkin E W 1981 Microbiological health effects associated with the use of municipal wastewater for irrigation. In Municipal Wastewater in Agriculture. Eds. F M D'Itri, J A Martinez and M A Lambarri. pp 271-342. Academic Press, New York. Kristensen K K and Bonde G J 1977 The current status of bacterial and other pathogenic organisms in municipal wastewater and their potential health hazards with regard to agricultural irrigation. In Wastewater Renovation and Reuse. Ed. F M D'Itri. pp 387-419. Marcel Dekker, Inc., New York. Levi Y, Dodd J and Krikun J 1983 Effect of irrigation, water salinity and root stock on the vertical distribution of vesicular-arbuscular mycorrhiza in citrus roots. New Phytol. 95, 397-403. Miller M R 1987 The ecology of vesicular-arbuscular mycor- rhizae in the grass- and shrublands. In Ecophysiology of VA Mycorrhizal Plants. Ed. G R Safir. pp 135-170. CRC Press Inc., Boca Raton, FL. Neary D G, Scheineider G and White D P 1975 Boron toxicity in red pine following municipal wastewater irrigation. Soil Sci. Soc. Am. Proc. 39, 981-982. Nelson C E 1987 The water relations of vesicular-arbuscular mycorrhizal systems. In Ecophysiology of VA Mycorrhizal Plants. Ed. G R Safir. pp 71-91. CRC Press Inc., Boca Raton, FL. Otto P C 1980 The Effects of Sewage Effluent on Acer sp. Mycorrhizal and Related Soil Properties. MS Thesis, Ann Arbor, University of Michigan. 65, 164 p. Paula M A and Siqueira J O 1987 Efeitos da umidade de solo sobre a simbiose endomicorrizica em soja: I. Colonizacao radicular, esporulacao, nodulacao e acumulo de nitrogenio. R. bras. Ci. Solo 1 I, 283-287. Phillips J M and Hayman D S 1970 Improved techniques for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Brit. Mycol. Soc. 55, 158-161. Read D J, Koucheki H K and Hodgson J 1976 Vesicular- arbuscular mycorrhiza in natural vegetation systems. I. The occurrence of infection. New Phytol. 77, 641-653. Reid C P P and Bowen G D 1979 Effects of soil moisture on V/A mycorrhiza formation and root development in Medicago. In The Soil-Root Interface. Eds. J L Harley and R S Russell. pp 211-219. Academic Press, London. Safir G R (Ed.) 1987 Ecophysiology of VA mycorrhizal plants. CRC Press Inc., Boca Raton, FL, 224 p. Safir G R, Boyer J S and Gerdemann J W 1971 Mycorrhizal enhancement of water transport in soybean. Science 172, 581-583. 196 VA mycorrhi zae in a wast e-wat er irrigated oldfield Schenck N C and Siqueira J O 1987 Ecology ofVA mycorrhizal fungi in temperate agroecosystems. In Mycorrhizae in the Next Decade: Practical Application and Research Priorities. Eds. D M Sylvia, L L Hung and J M Graham. pp 2-4. University of Florida. Gainesville. Shuval H I 1977 Public health implications of wastewater reuse for municipal purposes. In Wastewater Renovation and Reuse. Ed. F M D'Itri. pp 349-386. Marcel Dekker, Inc., New York. Sparling G P and Tinker P B 1978 Mycorrhizal infections in pennine grassland. I. Level of infections in the field. J. Appl. Ecol. 15, 943-950. Siqueira J O, Hubbell D H and Mahmud A W 1984 Effect of liming on spore germination, germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi. Plant and Soil 76, 115-124. Tommerup I C 1984 Effect of soil water potential on spore germination of vesicular-arbuscular mycorrhizal fungi. Trans. Brit. Mycol. Soc. 83, 193-202. Tesar M B, Knezek B D and Hook J E 1982 Management studies of annual grasses and perennial legumes and grasses at the Michigan State University Water Quality Management Facility. In Land Management of Municipal Wastewater: Vegetation Selection and Management. Ed. F M D'Itri. pp 79-105. Ann Arbor Sci. Publishers Inc., Ann Arbor, MI. Trappe J M 1977 Three new Endogonaceae: Glomus constrictus, Sclerocystis clavispora, and Acaulospora scrobiculata. Mycotaxon 6, 359-366. Visser S, Griffiths C L and Parkinson D 1984 Topsoil storage effects on primary production and rates of vesicular- arbuscular mycorrhizal development in .4gropyron trachycan- turn. Plant and Soil 82, 51-60. Wacker T L 1988 The Role of Vesicular-Arbuscular Mycor- rhizal Fungi in the Asparagus (Asparagus officonalis L.) Agroecosystem. MS Thesis, East Lansing, Michigan State University, 101 p. Walker C and Koske R E 1987 Taxonomic concepts in the Endogonaceae. IV. Glomus fasciculatum redescribed. Mycotaxon 30, 253-262.