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Fertilizer-induced pathogenicity of mycorrhizal fungi to sweetgum seedlings
0038-0717#83:030?57-06?d3.00:0 Copyright (’ IY83 Pergamon Pm\ Ltd
FERTTLIZER-INDUCED PATHOGENICITY OF MYCORRHIZAL FUNGI TO SWEETGUM SEEDLINGS JENNIFEK Departments
M.
KIERNAN,
HENDRIXand
JAMES W.
of Plant Pathology
DALE
and Horticulture, University KY 40546, U.S.A.
M.
MARONEK*
of Kentucky,
Lexington.
Summary-One isolate of Glomus clurus, two of G. efunicafus, and one of G. claroideum, obtained from plants growing on abandoned stripmine sites in Kentucky, and an isolate of G. fasciculurus known to stimulate growth of various woody plants, were evaluated for their influence on growth of sweetgum seedlings in a mixture of sand and stripmine soil. Soils were supplemented with various rates of a complete slow-release fertilizer. Throughout the growth period, G. f~cicu~az~, and most of the stripmine isolates, stimulated growth at low fertilizer rates. At higher fertilizer rates, including the level optimum for non-mycorrhizal plants, the stripmine isolates inhibited plant growth. After 14 weeks, plants inoculated with one of the four stripmine isolates overcame the early growth depression, and those inoculated with a second isolate appeared to bc overcoming the growth depression. G. ,fusciculufu.s was not inhibitory at any fertilizer rate. Root colonization by all three isolates evaluated was inhibited by the highest fertilizer rate, but this effect was not related to growth inhibition of plants. The other two isolates colonized roots at an extremely low rate (< lo/,). Sporulation of all the stripmine isolates, but not G. f~eicM~utu.~, was also inhibited by the highest fertilizer rate. The G. ,fusciculafus isolate used in this study may be atypical of mycorrhizal fungi occurring randomly in nature in its mutualistic or neutral effect on plants under a wide range of growth conditions.
Mycorrhizal fungi are considered beneficial and sometimes essential to growth of most plants. In the absence of an eIIective fungal symbiont, many trees and herbaceous plants growing on stripmine sites are particularly susceptible to adverse conditions such as extremes of pH. low P and N, high temperature and poor drainage (Daft et uf., 1975; Marx, 1976; Schramm, 1966). Failure to establish hardwood species such as sweetgum (Liquidumhar styruc~~ua L.) in reclamation of mined lands may be due to problems with endomycorrhizal fungi. Sweetgum has been found highly dependent on endomycorrhizal fungi (Brown et ul., 1981; Kormanik er al., 1977). We have found that trees on abandoned stripmine sites (orphan lands) in Kentucky, which have become naturally revegetated over several decades, have ectoor endomycorrizal fungal associates. These ecologically-adapted fungi may permit survival and growth of trees planted on mined land superior to that permitted by the mycorrhizai fungi acquired by the seedlings in the nursery. We isolated several pure endomycorrhizal fungal isolates from vigorous plants growing on orphan sites in Kentucky and determined their effects on growth of sweetgum seedlings on stripmine soil under greenhouse conditions. MATERlALS
AND METHODS
The isolates used and their origins appear in Table I. The isolate of Glomus $usciculutus obtained from *Present address: Studebaker Nurseries, lisle Road. New Carlisle. OH 45344,
I 1140 New U.S.A.
Car-
Dr J. W. Gerdemann, University of Illinois, has been shown to improve growth of plants (Hattingh and Gerdemann 1975; Maronek and Hendrix, 1978; Maronek ef al., 1981). Inoculum was produced on a Sudan grass-sorghum hybrid (Sudex), cv. FFR-66 in 15-cm clay pots containing steamed sand-soil mixture (2: 1 by volume), over a ICweek growing period. The inoculum was broken up by hand and the Sudex roots were cut into small pieces f-OScm) and incorporated into the soil. The inoculum soil containing spores and root pieces was homogenized with a commercial food mixer, and samples were removed for determination of spore populations. Inoculum was used immediately. The growing medium was steamed sand:mine soil mixture (2: 1 by volume). The mine soil was obtained from the Press Howard mine of Falcon Coal Co., Rreathitt County, Kentucky. The resultant mix had a pH of 8.6; organic matter 0.54%; NO,-N, less than I mg I-‘; available P, less than 0. I mg I‘.‘; K, Ca and Mg were 2.2, 231, and 9.3 mg I-‘, respectively (Spurway and Lawton, 1949). Because reclamation practices include fertilization, the influence of fertilizer rate was studied. The fertilizer used was a slow release fertilizer, 18-6-12 Osmocote, 8-9 month release rate (ISN-2.6P-10K) manufactured by Sierra Chemical Co., Milpitas, California. The fertilizer, at different rates, was incorporated throughout the sand-mine soil mix. Sweetgum seeds were obtained from the Kentucky Division of Forestry. They were stratified for 6 weeks in moist, sterilized peat at 5°C. They were then surface sterilized in 3% HzO, for IO min. rinsed with deionized water, and germinated in flats containing sterilized peat:perlite (1: I by volume). Two weeks after germination the seedlings were transplanted to
257
JENNIFERM. KIEKNANet (I/.
25x Table lsolatc
Spccics
I. Mycorrhizal
Glomus &ru.r Nicol. & Schenck
7X-I I
G. c~twricwlus Bcckcr & Gerd. G. canicu1u.s
7x-30
(i. c~/~/roirlcWrrr Schcnck & Smith
EN-I?
Black
(Rohitk
<;. /tr.vc~/cYllrrrlr.v (Thax. .WIISII Gcrd.) Gcrd. & Trappc
G-I
used in this study Soil pH
Location Surf~c
Locust
pwuhucuciu
L.)
Brcathitt
mine site.
County,
Kentucky
C’tr.v.ritr Ihsciculalu
Surface mine silt, Muhlcnberg County. Kentucky Surface mine site, Muhlenberg County, Kentucky Surfttcc mint sltc. Kockcastlc County. Kcnluchy
Michx. Cilrus
Cahfornla
Swectgum
(Liquidumhor 78-33
isolates
Host
No.
st~~c~jiuu
Unidentilied Partridge
legume Pen
L.)
5.4 4.3 7.05
* *
*Unknown.
165 ml
Cell” tube containers (Ray Nursery, 1787 North Pine St, containing the sand-mine Oregon), Canby, soil-Osmocote mix. Each tube contained a 20 ml band of inoculum, 2 cm deep covered by a 2 cm layer of sand-soil mix. Control plants were not inoculated. There were seven replicates per treatment. Plants were grown in the greenhouse using a 16-h photoperiod. Greenhouse temperatures ranged from a night low of 21°C to a daytime high of 35°C over the 1Cweek growing period (May-August). The average temperatures were 23°C night and 31°C daytime. The plants were hand-watered daily with deionized water. Heights of each seedling, measured from soil surface to apical meristem, were recorded at 6, 10 and 14 weeks after transplanting. Stem diameters at the first internode were measured at 14 weeks. At 14 weeks from the date of transplanting, the seedlings were carefully removed from the growth tubes, and the soil was carefully shaken from the root systems and retained. The roots were washed with deionized water, cleared, and stained with acid fuschin, using a modified version of Phillips and tiayman’s (1970) technique suggested for heavily-pigmented roots. Each root system was cut into 1.5 cm segments. Percentage mycorrhizal colonization in 25 randomly selected root segments was assessed by determining the presence of vesicles, spores and mycelium at 2-mm intervals. For each treatment, 150 intersects were rated. Soilhorne spore populations were determined on IO-g samples by use of a modification of a common nematological procedure. The soil was weighed directly into a 50-ml centrifuge tube, stirred in 25 ml deionized water, and centrifuged for 5 min at 2900 rev min ‘. The supernatant liquid was decanted, and the pellet was suspended by thorough stirring in 25 ml of 65”,, (w/v) sucrose. The suspension was centrifuged for 2 min at 2900 rev min ‘. and the supernatant liquid was decanted through a 45 pm pore size sieve to collect the spores. The spores were washed with deionized water to remove the sucrose and then washed into 5-cm plastic Petri dishes with parallel lines etched on the bottom to facilitate counting. Counting was done with a binocular microscope. Oven dry weights of the soil were determined for each tube, and the spore populations are given on an oven-dry weight basis. Leach
Leach
“Super
“Conetainer”
RESULTS
At a low level of fertilizer (1.1 g I-‘), nearly all isolates stimulated growth early, and this effect persisted for 14 weeks (Fig. 1). In contrast, at the manufacturer’s recommended rate of 4.5 g I-’ (for light, coarse-textured, well drained soil mixes), and with some at twice that rate, the stripmine isolates inhibited plant growth at 6 weeks when compared to nonmycorrhizal plants. Plants inoculated with two of the stripmine isolates, G. claws and isolate 78-30 of G. etunicatus, and grown with 2.2 g I-’ of fertilizer or more, remained severely stunted at 14 weeks, while plants inoculated with the other two stripmine isolates appeared to catch up with the control plants. G. clarus, isolate 78-30, and G. claroideum also significantly reduced plant stem diameters (Fig. 2). The isolate of G.fasciculatus was never pathogenic at any time in the experiment, and it was usually stimulatory early. In contrast to their effects on height, none of the isolates increased plant stem diameter at any fertilizer rate (Fig. 2). At higher fertilization rates, three of the four stripmine isolates, including the two which inhibited height, reduced stem diameter. Three of the isolates produced abundant vesicles, hyphae, and what appeared to be spores inside the roots. Roots of plants inoculated with these three isolates were rated for colonization (Table 2). Isolate 78-30 of G. etunicafus and G. cluroideum colonized roots heavily at 14 weeks, while G. jhscicula/us colonized roots at much lower levels. With two of the three isolates, roots were more heavily colonized if plants received low fertilization than if they received none. Colonization by all three isolates was much reduced at 9 g fertilizer I ‘. Reduction in colonization occurred for isolate 78-30 at 4.5 g I ‘. G. clurus and G. etunicutus isolate 78-33 colonized roots extremely lightly (less than I”,; at all fertilizer rates). The data on sporulation at 14 weeks (Table 3) are difficult to interpret because we did not initially inoculate with a low, standardized number of spores and because the final spore numbers were extremely variable. Variances for different treatments were unequal, and therefore the use of LSD’s or multiple range tests was invalid. Consequently, t-test comparisons between pairs of means were made. Significant increases in spores per plant from the inoculum were observed as follows: G. clurus at
Fertilizer
b
effects on mycorrhizal
sweetgum seedlings
259
Weeks
80
60
3 Weeks
too _ E ”
80
60
L 0 r”
40
20
0 14 0
4 Weeks
80
60
40
n !”
20
‘L i *
;,o ,”
0
Ferllllzer
(g/l
)
Fig. I. lnlluencc of fcrtilizcr ralc on height of sweetgum seedlings inoculated with various isolates of mycorrhizal fungi at three stages in the growth period. Isolates are from left: control (none), G. c/urus. G. c~tunicutus isolate 7X-30, G. erunicutus isolate 78-33, G. cluroideum and G. fasciculutus, for each fertilizer rate. tsignificant increase in height, compared to the uninoculated control for that fertilizer rate. *Significant decrease, compared to uninoculated control for that fertilizer rate. P = 0.05.
I.1 gl ‘; G. crunicorus isolate 78-30 at 0 and 1. I g I ‘; G. crunicufus isolate 78-33 at 0, I. I, 2.2 and 4.5 g I ‘; G. claroideum at 2.2 g I ‘; and G.,fuscicuhus at 0 and 2.2 g 1~~ ‘. High fertilization tended to decrease sporulation. Significant decreases from the treatment with peak sporulation (treatments from 0, I.1 or 2.2 g I-‘, according to isolate) were found with G. clurus at 4.5
and 9.0 g I- ‘, G. crunicaius isolate 78-30 at 4.5 and 9.0 g I- ‘, G. cfunicutus isolate 78-33 at 9.0 g 1~’ and G. cluroideum at 4.5 and 9.0 g IF’. Sporulation of G. ,fusciculurus was not affected by high fertilization. Roots of all plants, including those inoculated with isolates for which colonization ratings were not obtained, were examined, both in the fresh state and
M. KIERNANef ul.
JENNIFEK
0 Fertlllzer
(g/l
)
Fig. 2. Influence of fertilizer rate on stem diameter of sweetgum seedlings inoculated with various isolates of mycorrhizal fungi at 14 weeks. Isolates are from left: control (none), G. clurus. G. etunicatus isolate 78-30, G. ct~njcut~.~ isolate 78-33, G. c~ur~jdeuz~ and G. ~z.~~iculutu.~,for each fertilizer rate. *Signi~cant decrease, compared to unin~ulatcd control for that fertilizer rate. P = 0.05.
Table 2. Influence of fertilizer rate on percentage colonization of roots of sweetgum seedlings inoculated with various isolates of mycorrhizal fungi Mycorrhi~l Isolate
colonization (“/,f at indicated fertilizer rate (g I- ‘I* ____..LSD 2.2 9.0 I.1 4.5 (P = 0.05)
0
G. erunicurus (78-30) Control G. ciaroideum G. ,~u.~~~cutatus 6. riirrus~ G. rtunicutus (78-33)t LSD$ (P = 0.05)
52.3 0 8.4 18.6 0 0 13.9
77.4 0 57.0 16.0 0 0 15.7
59.1 0 49.6 14.3 0 0 18.5
39.4 0 50.9 15.0 0 0 16.8
i.8 2.9 2.7 0 0 6.1
18.4 13.4 10.8 0 0
*I g-6- I2 Osmocote (8-9-month release rate). tFor these two isolates, colonization was not observed at any intersects. However in qualitative observations of entire root systems (not just intersects), occasionally arbuscules, pelotons, internal hyphae or external hyphae were observed. SStatistical analyses were done only for the three isolates with positive coloni~tion ratings.
Table 3. Influence of fertilizer rate on sporulation of mycorrhizal fungi Spores per plant* with indicated fertilizer rate (g I ‘) 0 ckurus cfzmicutus etunicatus ctaroideum G. .frcscicularus
G. G. G. G.
9.0
4253 3114
2.2 ~_._ 2162 2499 6099
4.5
4269 3840 5239
1503 I.519 4916
96: 710 1698
3251 161 I
2553 1168
3953 1740
519 825
129 1232
1568
(78-30) (78-33)
.~
1.1
*Lnocuium rates (spores per plant) were: G. clurus, 1605; G. etunicufzk (isolate 78-30), 99; G. eru~ie~fu~~(isolate 78-33), 222: G. efar~jdeum, 109; G. ~scicu~utus. 375.
Fertilizer clfccts on mycorrhizal after clearing and staining. No evidence of root necrosis or the presence of pathogenic fungi such as fusaria or chytrids was found with any treatment. DISCUSSION
At low rates of fertilization, we usually observed a growth increase in response to mycorrhizal fungi; however, the magnitude of the increases was less than that reported by others. Kormanik et ul. (1977) obtained at least six-fold increases in height with inoculation with G. ~~zo.sscuc’,and fertilizer rate was inconsequential. We observed growth increases no larger than two-fold as a result of mycorrhizal fungi. In addition, our uninoculated seedlings reached twice the height of those of Kormanik et ol., who also never observed growth inhibition by mycorrhizal fungi at extremely high fertilization as we did. Our experiments dilrered from theirs in numerous experimental conditions, including a wider range of available P, which may account for the differences. However, we cannot conclude that sweetgum seedlings grow better with than without mycorrhizal fungi under all conditions. Plant growth inhibition by mycorrhizal fungi has often been observed (Schenck and Kellam, 1978). Usually this effect is treated as a curiosity, but perhaps mycorrhizal fungi are pathogenic in some modern crop production systems. Frequently, growth inhibition is rclatcd to high soil P (Crush, 1976). Mycorrhizal fungi may be deleterious to crops which are heavily fertilized, a concept consistent with that of Crush (1976). Growth inhibition does not appear to be related to root colonization characteristics. Growth inhibition occurred with G. crunicurus (isolate 78-30) at 2.2 g fertilizer I ‘, with 59% root colonization; and 9.0 g fertilizer I ~‘, with 4’2, colonization (Fig. I, Table 2). A similar situation occurred with G. cluroideum with respect to stem diameter (Fig. 2, Table 2). Percentage root colonization was also extremely low at the high fertilizer rate with the isolate of G. ,/&c.ic.ulu~us. but plants were not stunted. Stunting occurred in plants treated with the isolate of G. clurus at fertility rates of 2.2 gl ’ and above, although mycorrhizal infection was too low to evaluate as a percentage using standard procedures. The isolate of G. etunicurus (78-33) also produced extremely low infection at all rates. However, seedlings, although initially stunted at 4.5 g I-’ and 9.0 g I-‘, showed signs of recovery at I4 weeks. Growth inhibition thus was not related to colonization. Pathogenicity has been observed with sparse colonization (Crush, 1976; Hall er al., 1977). Hall e/ u/. observed that mutualistic associations are characteristically arbuscular, while nonmutualistic ones are vesicular. In our study, one of the isolates pathogenic at I4 weeks was vesicular, the other produced only arbuscules and pelotons. Edaphic factors may influence the effect of isolates on plant growth. The isolates most inhibitory to plant growth in our study were obtained from low pH soils, although the experiments were conducted with an alkaline soil. Craw (1979) observed a pH-dependent inhibition or stimulation ofgrowth of Guimtiu ubywinicu. Lambert c/ al. (1980) found that birdsfoot trefoil yielded better when inoculated with isolates
261
sweetgum seedlings
indigenous to the soil in which the plants were grown than when inoculated with isolates from other soils. In selecting mycorrhizal fungal isolates for improving plant establishment and growth for stripmine reclamation, or any other situation, an empirical approach is necessary at present. Probably no single isolate will be superior for all of the diverse conditions characteristic of mined lands. The deleterious effect of high fertilization combined with stripmine mycorrhizal fungi observed in our study may not occur with current reclamation practices, at least over the long term. The important effect will be the long-term effect, after intensive management of reclaimed land has ceased; and the long-term effects can be determined only by planting specifically-infected mycorrhizal seedlings on reclaimed land and observing them over a number of years. Our studies do indicate that fertilization practices employed in land reclamation should be evaluated in relation to their short-term effects on seedling growth and the survival of the ecologically-adapted fungal symbiont. A~X-,~o,~,/~,d~er?:mls-We appreciate the a&lance of Janet Finley and John Mattingly. We thank N. C. Schenck. University of Florida. for assisting m the identitication of the stripmine isolates of mycorrhizal fungi. Financial supporl was provided by the Office of Surface Mining, U.S. Dcpartmcnt of Interior and Ihe University of Kentucky Inslitulc for Mining and Minerals Rcscarch. REFERENCES
Brown R. W., Schultz R. C. and
Kormamk P. P. (1981) Response of vesicular-arbuscular endomycorrhizal sweetgum seedlings to three nitrogen fertilizers. Forest Science 27, 4 13420. Crush J. R. ( 1976) Endomycorrhizas and legume growth in some soils of the Mackenzie Basin. Canterbury, New Zealand. Nebc Zcalund Journal of A~rmdrural Reseurch 19, 473476. Daft M. J., Hacskaylo E. and Nicolson T. H. (1975) Arbuscular mycorrhizas in plants colonizing coal spoils in Scotland and Pennsylvania. In Endorqcorrhkus (F. E. Sanders, B. Mosse and P. 8. Tinker, eds). pp. 561-580. Academic Press, London. Graw D. (I 979) The influence of soil pH on the efficiency of vesicular-arbuscular mycorrhiza. New Phylologkr 82, 687-695. Hall I. R., Scott R. S. and Johnstone P. D. (1977) Effect of vesicular-arbuscular mycorrhizas on response of ‘Grasslands Huia’ and ‘Tamar’ white clovers to phosphorus. NCVV Zerrlrmd Journul II/ Agricultural Reseurch 20, 349-355. Hattingh M. J. and Gerdemann J. W. (1975) Inoculation of Brazilian sour orange seed with an endomycorrhizal fungus. P/r~topo/ho/og~* 65, IO I 3-l 0 16. Kormanik P. P., Bryan W. C. and Schultz R. C. (1977) Influence seedlings
of endomycorrhizae from eight molher
on growth of sweetgum trees. Foresr Sc~ienw 23,
500-506. Lambert D. H., Cole H. Jr and Baker D. E. (1980) Adaptation of vesicular-arbuscular mycorrhizae to cdaphic factors. NPM. Pllyrologist 85, 5 13-520. Maronek D. M. and Hendrix J. W. (1978) Mycorrhizal fungi in relation to some aspects of plant propagation. Proceedings o/ rhe Inkrnutionul Plmr Propugotors Sociei) 28, 506514. Maronck D. M., Hendrix J. W. and Kiernan J. M. (1981) Mycorrhizal fungi in horticultural crops. Horticuliural Rwiews 3. 172-2 I 3.
26’
JENNIFERM. KIERNANet ul.
Marx D. H. (1976) Use of specific mycorrhizal fungi on tree roots for forestation of disturbed lands. In Proceedings of the Co@rence on Forest&ion of Disturbed Swface Areus, Birmingham, Alabama, April 14-15, 1976 (K. A. Utz, Ed.), pp. 47-65. USDA Forest Service, Atlanta. Phillips J. M. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for the rapid assessment of infection. Transactions of the British Mycologicol Sociery 55, IX- I6 I Schenck N. C. and Kcllam M. K. (1978) The influence
of vesicular-arbuscular mycorrhizae on disease development. Unioersi/y of Florida Technicul Bulk/in No. 798. Schramm J. R. (1966) Plant colonization studies on black wastes from anthracite mining in Pennsylvania. Transaciions of the American Philosophical Society, Philadelphia 56, I-194. Spurway C. H. and Lawton K. (1949) Soil testing-a practical system of soil diagnosis. Michigan Agricultural Experiment Station, East Lansing, Michigan. Technical Bulletin No. 132 (4th revision).
FERTTLIZER-INDUCED PATHOGENICITY OF MYCORRHIZAL FUNGI TO SWEETGUM SEEDLINGS JENNIFEK Departments
M.
KIERNAN,
HENDRIXand
JAMES W.
of Plant Pathology
DALE
and Horticulture, University KY 40546, U.S.A.
M.
MARONEK*
of Kentucky,
Lexington.
Summary-One isolate of Glomus clurus, two of G. efunicafus, and one of G. claroideum, obtained from plants growing on abandoned stripmine sites in Kentucky, and an isolate of G. fasciculurus known to stimulate growth of various woody plants, were evaluated for their influence on growth of sweetgum seedlings in a mixture of sand and stripmine soil. Soils were supplemented with various rates of a complete slow-release fertilizer. Throughout the growth period, G. f~cicu~az~, and most of the stripmine isolates, stimulated growth at low fertilizer rates. At higher fertilizer rates, including the level optimum for non-mycorrhizal plants, the stripmine isolates inhibited plant growth. After 14 weeks, plants inoculated with one of the four stripmine isolates overcame the early growth depression, and those inoculated with a second isolate appeared to bc overcoming the growth depression. G. ,fusciculufu.s was not inhibitory at any fertilizer rate. Root colonization by all three isolates evaluated was inhibited by the highest fertilizer rate, but this effect was not related to growth inhibition of plants. The other two isolates colonized roots at an extremely low rate (< lo/,). Sporulation of all the stripmine isolates, but not G. f~eicM~utu.~, was also inhibited by the highest fertilizer rate. The G. ,fusciculafus isolate used in this study may be atypical of mycorrhizal fungi occurring randomly in nature in its mutualistic or neutral effect on plants under a wide range of growth conditions.
Mycorrhizal fungi are considered beneficial and sometimes essential to growth of most plants. In the absence of an eIIective fungal symbiont, many trees and herbaceous plants growing on stripmine sites are particularly susceptible to adverse conditions such as extremes of pH. low P and N, high temperature and poor drainage (Daft et uf., 1975; Marx, 1976; Schramm, 1966). Failure to establish hardwood species such as sweetgum (Liquidumhar styruc~~ua L.) in reclamation of mined lands may be due to problems with endomycorrhizal fungi. Sweetgum has been found highly dependent on endomycorrhizal fungi (Brown et ul., 1981; Kormanik er al., 1977). We have found that trees on abandoned stripmine sites (orphan lands) in Kentucky, which have become naturally revegetated over several decades, have ectoor endomycorrizal fungal associates. These ecologically-adapted fungi may permit survival and growth of trees planted on mined land superior to that permitted by the mycorrhizai fungi acquired by the seedlings in the nursery. We isolated several pure endomycorrhizal fungal isolates from vigorous plants growing on orphan sites in Kentucky and determined their effects on growth of sweetgum seedlings on stripmine soil under greenhouse conditions. MATERlALS
AND METHODS
The isolates used and their origins appear in Table I. The isolate of Glomus $usciculutus obtained from *Present address: Studebaker Nurseries, lisle Road. New Carlisle. OH 45344,
I 1140 New U.S.A.
Car-
Dr J. W. Gerdemann, University of Illinois, has been shown to improve growth of plants (Hattingh and Gerdemann 1975; Maronek and Hendrix, 1978; Maronek ef al., 1981). Inoculum was produced on a Sudan grass-sorghum hybrid (Sudex), cv. FFR-66 in 15-cm clay pots containing steamed sand-soil mixture (2: 1 by volume), over a ICweek growing period. The inoculum was broken up by hand and the Sudex roots were cut into small pieces f-OScm) and incorporated into the soil. The inoculum soil containing spores and root pieces was homogenized with a commercial food mixer, and samples were removed for determination of spore populations. Inoculum was used immediately. The growing medium was steamed sand:mine soil mixture (2: 1 by volume). The mine soil was obtained from the Press Howard mine of Falcon Coal Co., Rreathitt County, Kentucky. The resultant mix had a pH of 8.6; organic matter 0.54%; NO,-N, less than I mg I-‘; available P, less than 0. I mg I‘.‘; K, Ca and Mg were 2.2, 231, and 9.3 mg I-‘, respectively (Spurway and Lawton, 1949). Because reclamation practices include fertilization, the influence of fertilizer rate was studied. The fertilizer used was a slow release fertilizer, 18-6-12 Osmocote, 8-9 month release rate (ISN-2.6P-10K) manufactured by Sierra Chemical Co., Milpitas, California. The fertilizer, at different rates, was incorporated throughout the sand-mine soil mix. Sweetgum seeds were obtained from the Kentucky Division of Forestry. They were stratified for 6 weeks in moist, sterilized peat at 5°C. They were then surface sterilized in 3% HzO, for IO min. rinsed with deionized water, and germinated in flats containing sterilized peat:perlite (1: I by volume). Two weeks after germination the seedlings were transplanted to
257
JENNIFERM. KIEKNANet (I/.
25x Table lsolatc
Spccics
I. Mycorrhizal
Glomus &ru.r Nicol. & Schenck
7X-I I
G. c~twricwlus Bcckcr & Gerd. G. canicu1u.s
7x-30
(i. c~/~/roirlcWrrr Schcnck & Smith
EN-I?
Black
(Rohitk
<;. /tr.vc~/cYllrrrlr.v (Thax. .WIISII Gcrd.) Gcrd. & Trappc
G-I
used in this study Soil pH
Location Surf~c
Locust
pwuhucuciu
L.)
Brcathitt
mine site.
County,
Kentucky
C’tr.v.ritr Ihsciculalu
Surface mine silt, Muhlcnberg County. Kentucky Surface mine site, Muhlenberg County, Kentucky Surfttcc mint sltc. Kockcastlc County. Kcnluchy
Michx. Cilrus
Cahfornla
Swectgum
(Liquidumhor 78-33
isolates
Host
No.
st~~c~jiuu
Unidentilied Partridge
legume Pen
L.)
5.4 4.3 7.05
* *
*Unknown.
165 ml
Cell” tube containers (Ray Nursery, 1787 North Pine St, containing the sand-mine Oregon), Canby, soil-Osmocote mix. Each tube contained a 20 ml band of inoculum, 2 cm deep covered by a 2 cm layer of sand-soil mix. Control plants were not inoculated. There were seven replicates per treatment. Plants were grown in the greenhouse using a 16-h photoperiod. Greenhouse temperatures ranged from a night low of 21°C to a daytime high of 35°C over the 1Cweek growing period (May-August). The average temperatures were 23°C night and 31°C daytime. The plants were hand-watered daily with deionized water. Heights of each seedling, measured from soil surface to apical meristem, were recorded at 6, 10 and 14 weeks after transplanting. Stem diameters at the first internode were measured at 14 weeks. At 14 weeks from the date of transplanting, the seedlings were carefully removed from the growth tubes, and the soil was carefully shaken from the root systems and retained. The roots were washed with deionized water, cleared, and stained with acid fuschin, using a modified version of Phillips and tiayman’s (1970) technique suggested for heavily-pigmented roots. Each root system was cut into 1.5 cm segments. Percentage mycorrhizal colonization in 25 randomly selected root segments was assessed by determining the presence of vesicles, spores and mycelium at 2-mm intervals. For each treatment, 150 intersects were rated. Soilhorne spore populations were determined on IO-g samples by use of a modification of a common nematological procedure. The soil was weighed directly into a 50-ml centrifuge tube, stirred in 25 ml deionized water, and centrifuged for 5 min at 2900 rev min ‘. The supernatant liquid was decanted, and the pellet was suspended by thorough stirring in 25 ml of 65”,, (w/v) sucrose. The suspension was centrifuged for 2 min at 2900 rev min ‘. and the supernatant liquid was decanted through a 45 pm pore size sieve to collect the spores. The spores were washed with deionized water to remove the sucrose and then washed into 5-cm plastic Petri dishes with parallel lines etched on the bottom to facilitate counting. Counting was done with a binocular microscope. Oven dry weights of the soil were determined for each tube, and the spore populations are given on an oven-dry weight basis. Leach
Leach
“Super
“Conetainer”
RESULTS
At a low level of fertilizer (1.1 g I-‘), nearly all isolates stimulated growth early, and this effect persisted for 14 weeks (Fig. 1). In contrast, at the manufacturer’s recommended rate of 4.5 g I-’ (for light, coarse-textured, well drained soil mixes), and with some at twice that rate, the stripmine isolates inhibited plant growth at 6 weeks when compared to nonmycorrhizal plants. Plants inoculated with two of the stripmine isolates, G. claws and isolate 78-30 of G. etunicatus, and grown with 2.2 g I-’ of fertilizer or more, remained severely stunted at 14 weeks, while plants inoculated with the other two stripmine isolates appeared to catch up with the control plants. G. clarus, isolate 78-30, and G. claroideum also significantly reduced plant stem diameters (Fig. 2). The isolate of G.fasciculatus was never pathogenic at any time in the experiment, and it was usually stimulatory early. In contrast to their effects on height, none of the isolates increased plant stem diameter at any fertilizer rate (Fig. 2). At higher fertilization rates, three of the four stripmine isolates, including the two which inhibited height, reduced stem diameter. Three of the isolates produced abundant vesicles, hyphae, and what appeared to be spores inside the roots. Roots of plants inoculated with these three isolates were rated for colonization (Table 2). Isolate 78-30 of G. etunicafus and G. cluroideum colonized roots heavily at 14 weeks, while G. jhscicula/us colonized roots at much lower levels. With two of the three isolates, roots were more heavily colonized if plants received low fertilization than if they received none. Colonization by all three isolates was much reduced at 9 g fertilizer I ‘. Reduction in colonization occurred for isolate 78-30 at 4.5 g I ‘. G. clurus and G. etunicutus isolate 78-33 colonized roots extremely lightly (less than I”,; at all fertilizer rates). The data on sporulation at 14 weeks (Table 3) are difficult to interpret because we did not initially inoculate with a low, standardized number of spores and because the final spore numbers were extremely variable. Variances for different treatments were unequal, and therefore the use of LSD’s or multiple range tests was invalid. Consequently, t-test comparisons between pairs of means were made. Significant increases in spores per plant from the inoculum were observed as follows: G. clurus at
Fertilizer
b
effects on mycorrhizal
sweetgum seedlings
259
Weeks
80
60
3 Weeks
too _ E ”
80
60
L 0 r”
40
20
0 14 0
4 Weeks
80
60
40
n !”
20
‘L i *
;,o ,”
0
Ferllllzer
(g/l
)
Fig. I. lnlluencc of fcrtilizcr ralc on height of sweetgum seedlings inoculated with various isolates of mycorrhizal fungi at three stages in the growth period. Isolates are from left: control (none), G. c/urus. G. c~tunicutus isolate 7X-30, G. erunicutus isolate 78-33, G. cluroideum and G. fasciculutus, for each fertilizer rate. tsignificant increase in height, compared to the uninoculated control for that fertilizer rate. *Significant decrease, compared to uninoculated control for that fertilizer rate. P = 0.05.
I.1 gl ‘; G. crunicorus isolate 78-30 at 0 and 1. I g I ‘; G. crunicufus isolate 78-33 at 0, I. I, 2.2 and 4.5 g I ‘; G. claroideum at 2.2 g I ‘; and G.,fuscicuhus at 0 and 2.2 g 1~~ ‘. High fertilization tended to decrease sporulation. Significant decreases from the treatment with peak sporulation (treatments from 0, I.1 or 2.2 g I-‘, according to isolate) were found with G. clurus at 4.5
and 9.0 g I- ‘, G. crunicaius isolate 78-30 at 4.5 and 9.0 g I- ‘, G. cfunicutus isolate 78-33 at 9.0 g 1~’ and G. cluroideum at 4.5 and 9.0 g IF’. Sporulation of G. ,fusciculurus was not affected by high fertilization. Roots of all plants, including those inoculated with isolates for which colonization ratings were not obtained, were examined, both in the fresh state and
M. KIERNANef ul.
JENNIFEK
0 Fertlllzer
(g/l
)
Fig. 2. Influence of fertilizer rate on stem diameter of sweetgum seedlings inoculated with various isolates of mycorrhizal fungi at 14 weeks. Isolates are from left: control (none), G. clurus. G. etunicatus isolate 78-30, G. ct~njcut~.~ isolate 78-33, G. c~ur~jdeuz~ and G. ~z.~~iculutu.~,for each fertilizer rate. *Signi~cant decrease, compared to unin~ulatcd control for that fertilizer rate. P = 0.05.
Table 2. Influence of fertilizer rate on percentage colonization of roots of sweetgum seedlings inoculated with various isolates of mycorrhizal fungi Mycorrhi~l Isolate
colonization (“/,f at indicated fertilizer rate (g I- ‘I* ____..LSD 2.2 9.0 I.1 4.5 (P = 0.05)
0
G. erunicurus (78-30) Control G. ciaroideum G. ,~u.~~~cutatus 6. riirrus~ G. rtunicutus (78-33)t LSD$ (P = 0.05)
52.3 0 8.4 18.6 0 0 13.9
77.4 0 57.0 16.0 0 0 15.7
59.1 0 49.6 14.3 0 0 18.5
39.4 0 50.9 15.0 0 0 16.8
i.8 2.9 2.7 0 0 6.1
18.4 13.4 10.8 0 0
*I g-6- I2 Osmocote (8-9-month release rate). tFor these two isolates, colonization was not observed at any intersects. However in qualitative observations of entire root systems (not just intersects), occasionally arbuscules, pelotons, internal hyphae or external hyphae were observed. SStatistical analyses were done only for the three isolates with positive coloni~tion ratings.
Table 3. Influence of fertilizer rate on sporulation of mycorrhizal fungi Spores per plant* with indicated fertilizer rate (g I ‘) 0 ckurus cfzmicutus etunicatus ctaroideum G. .frcscicularus
G. G. G. G.
9.0
4253 3114
2.2 ~_._ 2162 2499 6099
4.5
4269 3840 5239
1503 I.519 4916
96: 710 1698
3251 161 I
2553 1168
3953 1740
519 825
129 1232
1568
(78-30) (78-33)
.~
1.1
*Lnocuium rates (spores per plant) were: G. clurus, 1605; G. etunicufzk (isolate 78-30), 99; G. eru~ie~fu~~(isolate 78-33), 222: G. efar~jdeum, 109; G. ~scicu~utus. 375.
Fertilizer clfccts on mycorrhizal after clearing and staining. No evidence of root necrosis or the presence of pathogenic fungi such as fusaria or chytrids was found with any treatment. DISCUSSION
At low rates of fertilization, we usually observed a growth increase in response to mycorrhizal fungi; however, the magnitude of the increases was less than that reported by others. Kormanik et ul. (1977) obtained at least six-fold increases in height with inoculation with G. ~~zo.sscuc’,and fertilizer rate was inconsequential. We observed growth increases no larger than two-fold as a result of mycorrhizal fungi. In addition, our uninoculated seedlings reached twice the height of those of Kormanik et ol., who also never observed growth inhibition by mycorrhizal fungi at extremely high fertilization as we did. Our experiments dilrered from theirs in numerous experimental conditions, including a wider range of available P, which may account for the differences. However, we cannot conclude that sweetgum seedlings grow better with than without mycorrhizal fungi under all conditions. Plant growth inhibition by mycorrhizal fungi has often been observed (Schenck and Kellam, 1978). Usually this effect is treated as a curiosity, but perhaps mycorrhizal fungi are pathogenic in some modern crop production systems. Frequently, growth inhibition is rclatcd to high soil P (Crush, 1976). Mycorrhizal fungi may be deleterious to crops which are heavily fertilized, a concept consistent with that of Crush (1976). Growth inhibition does not appear to be related to root colonization characteristics. Growth inhibition occurred with G. crunicurus (isolate 78-30) at 2.2 g fertilizer I ‘, with 59% root colonization; and 9.0 g fertilizer I ~‘, with 4’2, colonization (Fig. I, Table 2). A similar situation occurred with G. cluroideum with respect to stem diameter (Fig. 2, Table 2). Percentage root colonization was also extremely low at the high fertilizer rate with the isolate of G. ,/&c.ic.ulu~us. but plants were not stunted. Stunting occurred in plants treated with the isolate of G. clurus at fertility rates of 2.2 gl ’ and above, although mycorrhizal infection was too low to evaluate as a percentage using standard procedures. The isolate of G. etunicurus (78-33) also produced extremely low infection at all rates. However, seedlings, although initially stunted at 4.5 g I-’ and 9.0 g I-‘, showed signs of recovery at I4 weeks. Growth inhibition thus was not related to colonization. Pathogenicity has been observed with sparse colonization (Crush, 1976; Hall er al., 1977). Hall e/ u/. observed that mutualistic associations are characteristically arbuscular, while nonmutualistic ones are vesicular. In our study, one of the isolates pathogenic at I4 weeks was vesicular, the other produced only arbuscules and pelotons. Edaphic factors may influence the effect of isolates on plant growth. The isolates most inhibitory to plant growth in our study were obtained from low pH soils, although the experiments were conducted with an alkaline soil. Craw (1979) observed a pH-dependent inhibition or stimulation ofgrowth of Guimtiu ubywinicu. Lambert c/ al. (1980) found that birdsfoot trefoil yielded better when inoculated with isolates
261
sweetgum seedlings
indigenous to the soil in which the plants were grown than when inoculated with isolates from other soils. In selecting mycorrhizal fungal isolates for improving plant establishment and growth for stripmine reclamation, or any other situation, an empirical approach is necessary at present. Probably no single isolate will be superior for all of the diverse conditions characteristic of mined lands. The deleterious effect of high fertilization combined with stripmine mycorrhizal fungi observed in our study may not occur with current reclamation practices, at least over the long term. The important effect will be the long-term effect, after intensive management of reclaimed land has ceased; and the long-term effects can be determined only by planting specifically-infected mycorrhizal seedlings on reclaimed land and observing them over a number of years. Our studies do indicate that fertilization practices employed in land reclamation should be evaluated in relation to their short-term effects on seedling growth and the survival of the ecologically-adapted fungal symbiont. A~X-,~o,~,/~,d~er?:mls-We appreciate the a&lance of Janet Finley and John Mattingly. We thank N. C. Schenck. University of Florida. for assisting m the identitication of the stripmine isolates of mycorrhizal fungi. Financial supporl was provided by the Office of Surface Mining, U.S. Dcpartmcnt of Interior and Ihe University of Kentucky Inslitulc for Mining and Minerals Rcscarch. REFERENCES
Brown R. W., Schultz R. C. and
Kormamk P. P. (1981) Response of vesicular-arbuscular endomycorrhizal sweetgum seedlings to three nitrogen fertilizers. Forest Science 27, 4 13420. Crush J. R. ( 1976) Endomycorrhizas and legume growth in some soils of the Mackenzie Basin. Canterbury, New Zealand. Nebc Zcalund Journal of A~rmdrural Reseurch 19, 473476. Daft M. J., Hacskaylo E. and Nicolson T. H. (1975) Arbuscular mycorrhizas in plants colonizing coal spoils in Scotland and Pennsylvania. In Endorqcorrhkus (F. E. Sanders, B. Mosse and P. 8. Tinker, eds). pp. 561-580. Academic Press, London. Graw D. (I 979) The influence of soil pH on the efficiency of vesicular-arbuscular mycorrhiza. New Phylologkr 82, 687-695. Hall I. R., Scott R. S. and Johnstone P. D. (1977) Effect of vesicular-arbuscular mycorrhizas on response of ‘Grasslands Huia’ and ‘Tamar’ white clovers to phosphorus. NCVV Zerrlrmd Journul II/ Agricultural Reseurch 20, 349-355. Hattingh M. J. and Gerdemann J. W. (1975) Inoculation of Brazilian sour orange seed with an endomycorrhizal fungus. P/r~topo/ho/og~* 65, IO I 3-l 0 16. Kormanik P. P., Bryan W. C. and Schultz R. C. (1977) Influence seedlings
of endomycorrhizae from eight molher
on growth of sweetgum trees. Foresr Sc~ienw 23,
500-506. Lambert D. H., Cole H. Jr and Baker D. E. (1980) Adaptation of vesicular-arbuscular mycorrhizae to cdaphic factors. NPM. Pllyrologist 85, 5 13-520. Maronek D. M. and Hendrix J. W. (1978) Mycorrhizal fungi in relation to some aspects of plant propagation. Proceedings o/ rhe Inkrnutionul Plmr Propugotors Sociei) 28, 506514. Maronck D. M., Hendrix J. W. and Kiernan J. M. (1981) Mycorrhizal fungi in horticultural crops. Horticuliural Rwiews 3. 172-2 I 3.
26’
JENNIFERM. KIERNANet ul.
Marx D. H. (1976) Use of specific mycorrhizal fungi on tree roots for forestation of disturbed lands. In Proceedings of the Co@rence on Forest&ion of Disturbed Swface Areus, Birmingham, Alabama, April 14-15, 1976 (K. A. Utz, Ed.), pp. 47-65. USDA Forest Service, Atlanta. Phillips J. M. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for the rapid assessment of infection. Transactions of the British Mycologicol Sociery 55, IX- I6 I Schenck N. C. and Kcllam M. K. (1978) The influence
of vesicular-arbuscular mycorrhizae on disease development. Unioersi/y of Florida Technicul Bulk/in No. 798. Schramm J. R. (1966) Plant colonization studies on black wastes from anthracite mining in Pennsylvania. Transaciions of the American Philosophical Society, Philadelphia 56, I-194. Spurway C. H. and Lawton K. (1949) Soil testing-a practical system of soil diagnosis. Michigan Agricultural Experiment Station, East Lansing, Michigan. Technical Bulletin No. 132 (4th revision).