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Effect of inoculation level and sieving on the Gigaspora gigantea-soybesm mycorrhizal symbiosis
0038.0717~81,060539-02102.OO;O CopyrIght 0 1981 Pergamon Press Ltd
Soil Bid. Biochem. Vol. 13. pp. 539 to 540. 1981 Printed in Great Britain. All rights reserved
SHORT COMMUNICATION Effect of inoculation level and sieving on the Gigaspora gigantea-soybean mycorrhizal symbiosis GARRIET
Microbiology
Department,
Clemson (Accepted
W. SMITH University, Clemson, 10 April 1981)
Gigaspora gigantea was found by Schenck and Hinson (1971) to produce endomycorrhiza on soybeans (Glycine max, L.Z. Merr) as well as on other plant species (Clark, 1969; Gerdemann, 1955; Nicolson and Gerdemann, 1968). In general, soybeans exhibited a growth response favorably to inoculation with vesicular-arbuscular mycorrhiza (VAM) in pot cultures and field tests (Ross, 1971; Ross and Gilliam, 1973; Ross and Harper, 1970), but not always (Jackson et al., 1972). Schenck et al. (1975) reported that different soybean cultivars responded differently to various VAM fungal species. Similarly, Skipper and Smith (1979) reported different responses of various cultivars to different VAM fungi and the degree of response was related to soil pH. To my knowledge, no study has concentrated on the type and inoculum level required for effective infection of soybeans by VAM fungi. Daft and Nicolson (1969) observed little difference in the response of tomato to various amounts of inoculum (225, 55 and 26 spores per plant) of Glomus macrocarpus. I have determined the effect of IO-fold increases in inoculum level of Gigaspora gigantea on the response of two soybean cultivars. The relative effect of sieving the inoculum (eliminating nurse-crop mycorrhizal roots) was also investigated. The soybean cultivars “Bossier” and “Bragg” were inoculated with G. gigantea infested soil or soil and mycorrhizal corn (Zea mays L., “Pioneer 3369A”) roots. Mycorrhizal inoculum soil averaged four spores and 0.3 g mycorrhizal corn roots g-’ inoculum as determined by the plate method of Smith and Skipper (1979). Soil, growing conditions and replication were as described by Skipper and Smith (1979). Dolomitic limestone (0.5 g kg-’ soil) was added to the soil at planting to yield a final soil of pH 6.1. Inocula were prepared at a concentration of 0, 1, 10 and
Table 1. Elfect of inoculum
Inoculum type With spores and VAM corn roots With spores but no VAM corn roots
SC 29631, U.S.A.
Inocula were prepared at a concentration of 0, 1, 10 and 1OOg mycorrhizal inoculum soil kgg’ autoclaved soil. Inocula were also prepared using soil that was wet sieved to remove mycorrhizal corn roots but not azygospores. A 5OOpm mesh sieve was used to remove mycorrhizal corn roots and a 105 pm mesh sieve to retain the spores. The sieved material was air-dried, then mixed with autoclaved soil and lime before planting. Plants were harvested 53 days after planting, when number of pods, shoot and root fresh weights and root-to-shoot ratios were recorded. At harvest, root segments were selected at random from each treatment, cleared using NaOH, stained with acid fuchsin (Phillips and Hayman, 1970), and the proportion of mycorrhizal infection was estimated (Daft and Nicolson, 1972). soybeans had significantly All inoculated “Bossier” greater shoot fresh weights than controls except those inoculated with I g sieved inoculum (Table 1). The same was true with “Bragg” soybeans except that the 1OOg sieved inoculum was not significantly higher than controls (Table 2). Unlike shoot fresh weight, root fresh weight did not appear to reflect the mycorrhizal status of the soybeans (Tables 1 and 2). In my study, the value most useful to predict yields was the number of pods per plant. Using this character, inoculation of “Bossier” soybeans again appeared effective at all levels except using 1 g sieved material (Table 1). The response of “Bragg” soybeans was again similar to the response of “Bossier” in this respect (Table 2). Likewise, rootto-shoot ratios of inoculated “Bossier” and “Bragg” were significantly lower than controls and plants receiving the I g sieved inoculum. It would appear, from the results, that four azygospores kg-’ soil is not sufficient to induce adequate mycorrhizal
type and level on “Bossier” soybeans. Results are given as means per pot (three replicate pots, four plants per pot)
Inoculum level (g infected soil kg-’ dry wt autoclaved soil)
Shoot fresh weight
Root fresh weight
(g)
(9)
100 10 1 100 10 1 0
i Means within the same vertical column by Duncan’s new multiple range test.
4.59 ab 4.58 a 5.22 a 2.79 b 4.42 ab 3.66 b 4.00 b
2.51 b’ 2.43 b 3.08 a 2.81 a 2.98 a 1.78 c 1.59 c followed
Number of pods per plant 4.6 ab 6.3 a 6.3 a 5.9 a 7.8 a 3.7b 3.6b
Root-to-shoot ratio (fresh wt) 1.85 b 1.89 b 1.70b 0.96 c 1.48 bc 2.06 ab 2.52 a
by the same letter do not differ significrntly 539
(5% level)
540
Short communications Table 2. Effect of inoculum
type and level on “Bragg” soybeans. Results are given as means replicate pots, four plants per pot)
Inoculum level (g infected soil kg-’ dry wt autoclaved soil)
Inoculum type With spores and VAM corn roots With spores but no VAM corn roots
Shoot fresh weight
Root fresh weight
(8)
(g)
100 10
1 loo 10 1 0
’ Means within the same vertical column by Duncan’s new multiple range test.
3.43 a’ 3.38 a 3.37 a 2.42 b 2.99 a 2.21 b 2.I7b followed
infection unless preinfected mycorrhizal roots are included. For this reason. it appears that at low inoculum levels VAM infection occurred primarily via the corn VAM roots. Stained root samples supported this observation. All inoculated roots were from 50 to SO:< infected except those receiving the sieved 1g kg-’ soil inoculum. These roots were only about IO“, infected. Control roots (0 inocutum level) were not infected. My results support the observations made by Daft and Nicolson (1969) that spore concentrations in inocula have little effect on the final degree of VAM infection. It seems that inoculum level is not of major importance as long as enough spores or mycorrhizal roots are present to assure that approximately SO’/<,infection takes place. Sieved G. ~li+~~reu inocula at the level of 1 g kg- ’ soil did not significantly stimulate soybean growth and the proportion of roots infected with VAM was much lower than other inoculation levels including the unsieved 1 g kg- ’ soil level. It would appear. then, that an inoculation level of four G. &t~ttee azygosporcs kg ’ soil is less than adequate to obtain a soybean response unless mycorrhizal roots (30 mg kg-‘) are also included.
REFERESCES CLARK F. B. (1969) Endotrophic
mycorrhizal infection of tree seedlings with ~~~~~~~~~~~ spores. Forrsr Science 15, 134 137. DAFT M. J. and NI~OLSON T. H. (1969) Effect of Endogone mycorrhiza on plant growth. III. Influence of inoculum concentration on growth and infection in tomato. New Phrrologisr 68, 953-963. DA& M. J. and NICOLSON T. H. (1972) Effect of Endoyonr mycorrhiza on plant growth. IV. Quantitative relationships between the growth of the host and the development of the endophyte m tomato and maize. New PhyfoI(XjSf 71. 287-295.
4.68 a 4.00 a 4.23 a 3.08 b 3.57 ab 4.64 a 4.24 a
Number of pods per plant 7.5 a 7.5 a 6.5 a 6.1 b 8.9 a 5.4 c 3.9d
per pot (three
Root-to-shoot ratio (fresh wt) 1.37b 1.18 b 1.25 b 1.28 b 1.19 b 2.10a 1.96 a
by the same letter do not differ significantly
(5% level)
GERDEMANN J. W. (1955) Relation of a large soil-borne spore to phycomycetous mycorrhizal infections. Mycoloyia 47, 619-632. JACKSON N. E., FRANKLIN R. E. and MILLER R. H. (1972) Effects of vesicular-arbuscular mycorrhizae on growth and phosphorus content of three agronomic crops. Procwdings. Soil Scirmw Society C$ America 36. 6467. NICOL~ON 7. H. and GERDEMANN J. W. (1968) Mycorrhi~l Endogone species. ~1pcoloyir~ 60, 313-325. PHILLIPS J. M. and HAYMAN D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycologicul Society 55, 158161. ROSS J. P. (1971) Effect of phosphate fertilization on yield of mycorrhizal and non-mycorrhizal soybeans. Phytoput/~~~~g~! 61, 140&1403. Ross J. P. and GILLIAM J. W. (1973) Effect of ~~~~o~~i~~ mycorrhiza on phosphorus uptake by soybeans from inorganic phosphate. Proceedings. Soil Science Society of Amuica 37, 237~.239. Ross J. P. and HARPER J. A. (1970) Effect of Endogone mycorrhiza on soybean yields. Phytoprcthologp 60, 1552-1556. SCBENCK N. C. and HINSON K. (1971) Endotrophic vesiculararbuscular mycorrhizae on soybean in Florida. h~f~~o~o~iu 63, 672-675. SCXEN~K N. C., Ktstoc~ R. A. and DI~KSO?~ D. W. (1975) Interaction of endomycorrhizal fungi and root-knot nematode on soybean. In ~nd~~z~c~r~~~i~as (F. E. Sanders, B. Mosse and P. B. Tinker. Eds), pp. 607-618. Academic Press, London. SKIPPER H. D. and SMITH G. W. (1979) Influence of soil pH on the soybean-endomycorrhiza symbiosis. PIant & Soil 53, 559563. SMITH G. W. and SKIPPER H. D. (1979) Comparison of methods to extract spores of vesicular-arbuscular mycorrhizal fungi. foumrrl. Soif Sciewe Socier~ of America 43, 722-725.
Soil Bid. Biochem. Vol. 13. pp. 539 to 540. 1981 Printed in Great Britain. All rights reserved
SHORT COMMUNICATION Effect of inoculation level and sieving on the Gigaspora gigantea-soybean mycorrhizal symbiosis GARRIET
Microbiology
Department,
Clemson (Accepted
W. SMITH University, Clemson, 10 April 1981)
Gigaspora gigantea was found by Schenck and Hinson (1971) to produce endomycorrhiza on soybeans (Glycine max, L.Z. Merr) as well as on other plant species (Clark, 1969; Gerdemann, 1955; Nicolson and Gerdemann, 1968). In general, soybeans exhibited a growth response favorably to inoculation with vesicular-arbuscular mycorrhiza (VAM) in pot cultures and field tests (Ross, 1971; Ross and Gilliam, 1973; Ross and Harper, 1970), but not always (Jackson et al., 1972). Schenck et al. (1975) reported that different soybean cultivars responded differently to various VAM fungal species. Similarly, Skipper and Smith (1979) reported different responses of various cultivars to different VAM fungi and the degree of response was related to soil pH. To my knowledge, no study has concentrated on the type and inoculum level required for effective infection of soybeans by VAM fungi. Daft and Nicolson (1969) observed little difference in the response of tomato to various amounts of inoculum (225, 55 and 26 spores per plant) of Glomus macrocarpus. I have determined the effect of IO-fold increases in inoculum level of Gigaspora gigantea on the response of two soybean cultivars. The relative effect of sieving the inoculum (eliminating nurse-crop mycorrhizal roots) was also investigated. The soybean cultivars “Bossier” and “Bragg” were inoculated with G. gigantea infested soil or soil and mycorrhizal corn (Zea mays L., “Pioneer 3369A”) roots. Mycorrhizal inoculum soil averaged four spores and 0.3 g mycorrhizal corn roots g-’ inoculum as determined by the plate method of Smith and Skipper (1979). Soil, growing conditions and replication were as described by Skipper and Smith (1979). Dolomitic limestone (0.5 g kg-’ soil) was added to the soil at planting to yield a final soil of pH 6.1. Inocula were prepared at a concentration of 0, 1, 10 and
Table 1. Elfect of inoculum
Inoculum type With spores and VAM corn roots With spores but no VAM corn roots
SC 29631, U.S.A.
Inocula were prepared at a concentration of 0, 1, 10 and 1OOg mycorrhizal inoculum soil kgg’ autoclaved soil. Inocula were also prepared using soil that was wet sieved to remove mycorrhizal corn roots but not azygospores. A 5OOpm mesh sieve was used to remove mycorrhizal corn roots and a 105 pm mesh sieve to retain the spores. The sieved material was air-dried, then mixed with autoclaved soil and lime before planting. Plants were harvested 53 days after planting, when number of pods, shoot and root fresh weights and root-to-shoot ratios were recorded. At harvest, root segments were selected at random from each treatment, cleared using NaOH, stained with acid fuchsin (Phillips and Hayman, 1970), and the proportion of mycorrhizal infection was estimated (Daft and Nicolson, 1972). soybeans had significantly All inoculated “Bossier” greater shoot fresh weights than controls except those inoculated with I g sieved inoculum (Table 1). The same was true with “Bragg” soybeans except that the 1OOg sieved inoculum was not significantly higher than controls (Table 2). Unlike shoot fresh weight, root fresh weight did not appear to reflect the mycorrhizal status of the soybeans (Tables 1 and 2). In my study, the value most useful to predict yields was the number of pods per plant. Using this character, inoculation of “Bossier” soybeans again appeared effective at all levels except using 1 g sieved material (Table 1). The response of “Bragg” soybeans was again similar to the response of “Bossier” in this respect (Table 2). Likewise, rootto-shoot ratios of inoculated “Bossier” and “Bragg” were significantly lower than controls and plants receiving the I g sieved inoculum. It would appear, from the results, that four azygospores kg-’ soil is not sufficient to induce adequate mycorrhizal
type and level on “Bossier” soybeans. Results are given as means per pot (three replicate pots, four plants per pot)
Inoculum level (g infected soil kg-’ dry wt autoclaved soil)
Shoot fresh weight
Root fresh weight
(g)
(9)
100 10 1 100 10 1 0
i Means within the same vertical column by Duncan’s new multiple range test.
4.59 ab 4.58 a 5.22 a 2.79 b 4.42 ab 3.66 b 4.00 b
2.51 b’ 2.43 b 3.08 a 2.81 a 2.98 a 1.78 c 1.59 c followed
Number of pods per plant 4.6 ab 6.3 a 6.3 a 5.9 a 7.8 a 3.7b 3.6b
Root-to-shoot ratio (fresh wt) 1.85 b 1.89 b 1.70b 0.96 c 1.48 bc 2.06 ab 2.52 a
by the same letter do not differ significrntly 539
(5% level)
540
Short communications Table 2. Effect of inoculum
type and level on “Bragg” soybeans. Results are given as means replicate pots, four plants per pot)
Inoculum level (g infected soil kg-’ dry wt autoclaved soil)
Inoculum type With spores and VAM corn roots With spores but no VAM corn roots
Shoot fresh weight
Root fresh weight
(8)
(g)
100 10
1 loo 10 1 0
’ Means within the same vertical column by Duncan’s new multiple range test.
3.43 a’ 3.38 a 3.37 a 2.42 b 2.99 a 2.21 b 2.I7b followed
infection unless preinfected mycorrhizal roots are included. For this reason. it appears that at low inoculum levels VAM infection occurred primarily via the corn VAM roots. Stained root samples supported this observation. All inoculated roots were from 50 to SO:< infected except those receiving the sieved 1g kg-’ soil inoculum. These roots were only about IO“, infected. Control roots (0 inocutum level) were not infected. My results support the observations made by Daft and Nicolson (1969) that spore concentrations in inocula have little effect on the final degree of VAM infection. It seems that inoculum level is not of major importance as long as enough spores or mycorrhizal roots are present to assure that approximately SO’/<,infection takes place. Sieved G. ~li+~~reu inocula at the level of 1 g kg- ’ soil did not significantly stimulate soybean growth and the proportion of roots infected with VAM was much lower than other inoculation levels including the unsieved 1 g kg- ’ soil level. It would appear. then, that an inoculation level of four G. &t~ttee azygosporcs kg ’ soil is less than adequate to obtain a soybean response unless mycorrhizal roots (30 mg kg-‘) are also included.
REFERESCES CLARK F. B. (1969) Endotrophic
mycorrhizal infection of tree seedlings with ~~~~~~~~~~~ spores. Forrsr Science 15, 134 137. DAFT M. J. and NI~OLSON T. H. (1969) Effect of Endogone mycorrhiza on plant growth. III. Influence of inoculum concentration on growth and infection in tomato. New Phrrologisr 68, 953-963. DA& M. J. and NICOLSON T. H. (1972) Effect of Endoyonr mycorrhiza on plant growth. IV. Quantitative relationships between the growth of the host and the development of the endophyte m tomato and maize. New PhyfoI(XjSf 71. 287-295.
4.68 a 4.00 a 4.23 a 3.08 b 3.57 ab 4.64 a 4.24 a
Number of pods per plant 7.5 a 7.5 a 6.5 a 6.1 b 8.9 a 5.4 c 3.9d
per pot (three
Root-to-shoot ratio (fresh wt) 1.37b 1.18 b 1.25 b 1.28 b 1.19 b 2.10a 1.96 a
by the same letter do not differ significantly
(5% level)
GERDEMANN J. W. (1955) Relation of a large soil-borne spore to phycomycetous mycorrhizal infections. Mycoloyia 47, 619-632. JACKSON N. E., FRANKLIN R. E. and MILLER R. H. (1972) Effects of vesicular-arbuscular mycorrhizae on growth and phosphorus content of three agronomic crops. Procwdings. Soil Scirmw Society C$ America 36. 6467. NICOL~ON 7. H. and GERDEMANN J. W. (1968) Mycorrhi~l Endogone species. ~1pcoloyir~ 60, 313-325. PHILLIPS J. M. and HAYMAN D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycologicul Society 55, 158161. ROSS J. P. (1971) Effect of phosphate fertilization on yield of mycorrhizal and non-mycorrhizal soybeans. Phytoput/~~~~g~! 61, 140&1403. Ross J. P. and GILLIAM J. W. (1973) Effect of ~~~~o~~i~~ mycorrhiza on phosphorus uptake by soybeans from inorganic phosphate. Proceedings. Soil Science Society of Amuica 37, 237~.239. Ross J. P. and HARPER J. A. (1970) Effect of Endogone mycorrhiza on soybean yields. Phytoprcthologp 60, 1552-1556. SCBENCK N. C. and HINSON K. (1971) Endotrophic vesiculararbuscular mycorrhizae on soybean in Florida. h~f~~o~o~iu 63, 672-675. SCXEN~K N. C., Ktstoc~ R. A. and DI~KSO?~ D. W. (1975) Interaction of endomycorrhizal fungi and root-knot nematode on soybean. In ~nd~~z~c~r~~~i~as (F. E. Sanders, B. Mosse and P. B. Tinker. Eds), pp. 607-618. Academic Press, London. SKIPPER H. D. and SMITH G. W. (1979) Influence of soil pH on the soybean-endomycorrhiza symbiosis. PIant & Soil 53, 559563. SMITH G. W. and SKIPPER H. D. (1979) Comparison of methods to extract spores of vesicular-arbuscular mycorrhizal fungi. foumrrl. Soif Sciewe Socier~ of America 43, 722-725.