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Evaluation of vesicular-arbuscular mycorrhizal fungi in diverse plants and soils
Soil Bid. Biochem. Vol. 25, No. 6, pp. 705-713, Printed in Great Britain. All rights reserved
1993
0038-0717/93
$6.00 + 0.00
Copyright 0 1993Pergamon Press Ltd
EVALUATION OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI IN DIVERSE PLANTS AND SOILS D. M. SYLVIA,‘* D. 0. WILSON,’ J. H. GRAHAM,~J. J. MADDOX,~P. MILLNER,’ J. B. MORTON,~ H. D. SKIPPER,‘% F. WRIGHTB~ and A. G. JARSTFER’ ‘Soil and Water Science Department, University of Florida, Gainesville, FL ‘32611-0290, 2Agronomy Department, University of Georgia, Griffin, GA 30223, ‘Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, 4National Fertilizer and Environmental Research Center, Tennessee Valley Authority, Muscle Shoals, AL 35660, jSoi1 Microbial Systems Laboratory, USDA, ARS, Beltsville, MD 20705, 6Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506, ‘Agronomy and Soils Department, Clemson University, Clemson, SC 29634 and 8Appalachian Soil and Water Laboratory, USDA, ARS, Beckley, WV 25801, U.S.A. (Accepted 20 December 1992) Summary-A regional study was made to identify vesicular-arbuscular mycorrhizal (VAM) fungi effective in promoting plant growth in diverse plant and soil systems. Eight cooperators in six states of the eastern United States evaluated six VAM fungal isolates on soybean (Glycine mux L. Merr.) and sorghum (Sorghum bicolor L. Moench) in a shared soil and in at least one regional soil from each location. Plants were grown with high VAM inoculum densities (minimum of 20 VAM propagules ml-‘) for 42-57 days in pasturized soils in greenhouses or growth chambers. Shoot and root dry masses, total and colonized root lengths and shoot-P concentrations were determined at harvest. Under the experimental conditions tested, the VAM fungal isolate was more important than the soil or host plant in determining effectiveness. In the shared soil, inoculation with two isolates of Glomus (GE329 and GENPI) resulted in the greatest shoot masses for soybeans, while the same two isolates and GE312 provided maximum response in sorghum. In the regional soils, GE329 and GENPI had the widest range of growth promotion with both soybean and sorghum; however, for both plant species the mycorrhizal response was greatest in soils with less than 10 mg extractable P kg-‘. For soybeans, colonized root length was not related to VAM growth response. For sorghum, there was a positive correlation between colonized root length and plant growth response. We conclude that VAM isolates exist which are effective in promoting plant growth over a range of edaphic and host conditions.
INTRODUCTION Little is known about the stability of the plant growth with vesicular-arbuscular mycorrhizal response (VAM) fungi over a range of host plants and soil
environments. While it is well-known that VAM fungi lack host specificity (Gianinazzi-Pearson, 1984), their effectiveness under uniform experimental conditions can vary widely (Mosse, 1972; Graham et al., 1982; Lamar and Davey, 1988; Haas and Krikun, 1985; Boerner, 1990; Hung et al., 1990). Considerable data indicate that VAM fungi are adapted to different edaphic conditions (Lambert et al., 1980; Hayman, 1982; Gianinazzi-Pearson et al., 1985; Henkel et al., 1989; Stahl and Christensen, 1991) and may possess unique properties based on their biology (Bethlenfalvay et al., 1989). It is important, therefore, to screen isolates of VAM fungi to insure that each inoculum source is effective on the host and in the soil where it will be utilized. Mass production of VAM inoculum requires a living host or tissue explant due to the obligate nature
of the symbiosis (Jarstfer and Sylvia, 1992). This drives up the cost of inoculum production and, consequently, commercially-feasible inoculum requires that fungal isolates be effective over a range of edaphic and host conditions. In the summer of 1990, eight cooperators participated in a study to screen six isolates of VAM fungi on two hosts in a range of soils found in the eastern United States. Our objective was to identify fungal isolates which are effective in promoting plant growth in diverse host-soil systems.
MATERIAL AND METHODS
At each of the eight locations, six VAM isolates were evaluated on soybean (cv. Centennial) and sorghum (cv. Funk G-522DR) in a shared soil (limed Cecil from Georgia) and in at least one regional soil (Table 1). The treatments within each soil were completely randomized, with five replicates per treatment. Soils and inoculu
*Author for correspondence. TPresent address: Soil Microbial Systems Laboratory, USDA, ARS, Beltsville, MD 20705, U.S.A.
Soils were sieved (2 mm) and pasturized (at least 70°C for 6 h, except for fumigation with methyl 705
D. M. SYLVIA et 01.
706
Table 1. Soil descriotions and selected orooerties
series Clarendon Cecil Gilpin Lily Candler Mountview Galestown Pahokee Dothan Arredondo
Collection site County, State
Description
Tift, Ga Pike, Ga Wyoming, W.Va Monongalia, W.Va Polk, Fla Lawrence, Tenn. Prince Georges, Md Palm Beach, Fla Sumter SC. Alachua, Fla
Fine loamy sand, Plinthaquic Paleudult Clayey, Typic Hapludult Typic Hapludult’ Typic Hapludult’ Fine sand, Typic Quartzipsamment Silt loam, Typic Hapludult Typic Quartzipsamment’ Muck, Lithic Medisaprist Fine loam, Plinthic Paleudult Loamy sand, Grossannic Paleudult
PH’ 5.4 6.4 5.8 5.9 6.4 7.1 6.2 7.0 6.0 5.8
CECb (cm01 kg - ‘) 2.05 1.82 2.95 ND 0.69 2.27 0.71 106.7 ND 3.51
;:)’ 1.1 0.8 1.2 0.9 0.5 0.9 0.3 63.0 1.1 2.2
Nd
F
(w kg- ‘)
(mg kg‘ 9
16.4 41.9 34.6 27.6 4.1 56.9 5.8 1826.0 19.9 94.4
5.0 5.2 5.4 8.1 8.5 9.4 20.0 24.5 36.9 61.6
*l: 1 H,O:soil. bNeutral 1 M NH,OAc. ‘Walkley-Black wet combustion. dNH,-N plus NO,-N, 2 N KCI, 5: 1 extractant to soil. ‘Mehlich I extractable. ‘Mixed 1: 1 with sand. ND = not determined.
bromide of the Pahokee soil). Soil chemical properties ranged as follows: pH from 5.4 to 7.1, organic matter from 0.3 to 63% and Mehlich I-extractable (50 mM HCl and 10 mu H,S03 P from 5 to 62 mg kg-’ (Table 1). Dolomitic limestone (4 : 1 CaCO, : MgC09) was added to the Cecil soil at a rate of 25Omg kg-’ soil, 10-14 days prior to nutrient addition and then the soil was subjected to several wet and dry cycles to achieve a stable pH. All soils received nutrient amendments that included 60 mg (NH&SO, kg-‘, 270 mg KNO, kg-’ for soils planted with sorghum and 222 mg K,S04 kg- ’ for soils planted with soybeans. A complete micronutrient mixture was added to all soils (mg kg - ‘): Mg, 4.6; Fe, 4.7; Mn, 3.7; Zn, 4.2; Cu, 1.2; B, 0.5; MO, 1.0; and S, 12.7. All nutrients were mixed throughout each soil just prior to planting. Three weeks after planting, (NH&SO,, KNO, and KC1 were added to pots with sorghum plants at 180, 90 and 125 mg kg-‘, respectively. Inocula of VAM fungi consisted of root and soil mixtures from established pot cultures. Inoculum density in each culture produced by cooperators was estimated by most-probable number (MPN) assays (Porter, 1979) made just prior to distribution. Inocula were shipped to all locations via overnight express mail. Inoculum (50 ml except for one isolate, GENPI,
where, due to limited supply, 25-37 ml were used) was mixed with 450ml of test soil and the mixture was placed into a 600ml DeePot (J. M. McConkey & Co. Inc., 1615 Puyallup Street, Sumner, WA 98390). Inoculum densities ranged from 20 to 192 propagules ml-i while the total number of propagules added to each pot ranged from 1000 to 9600 (Table 2). All pots received 5ml of a composite microbial filtrate in an attempt to equalize the background microflora. The filtrate was prepared by mixing together 10 ml of each inoculant, adding 1.2 1. of water and filtering the resultant slurry (< 10 pm). The controls contained 500 ml of the pasteurized test soil along with the microbial filtrate. Culture methodr and harvest Seeds were surface disinfested with full-strength household bleach (5.25% NaOCl) for 5 min and rinsed 5 times (20-30min each) with sterile water. Four seeds were placed 1.5 cm below the soil surface. For soybeans, granular bradyrhizobial inoculant (USDA 110) was added with the seed to the planting hole. After emergence, plants were thinned to one per pot and then grown in cooperators’ greenhouses or growth chambers for 42-57 days (Table 3). Soil moisture was maintained near field capacity by daily watering throughout the experiment.
Table 2. Funnal isolates used in screeninn studies
Fungal species Glomus ehmicatum Becker & Gerdemann
Glomus sp.E G. etunicatum G. etunicatum G. claroides
Schcnck & Smith
Isolate code
INVAM’ code
MPNb (propagules ml-‘)
GE312 GE329 GENPI GETVA
FL312 FL906 UT316 TN101
45 192 72 29
2250 9600 1BOO-2664 1450
GC
SC186
20
1000
EC
GA101
63
3150
Propagules added per pot
Entrophospora columbiama
Spain & Schenck
‘International Culture Collection of VA Mycorrhiil Fungi INVAM, 401 Brooks Hall, West Virginia University, Morgantown, WV 26506-6057, U.S.A. bMost probable number. COriginally classified as GIomus etunicotum.
Evaluation
of VA mycorrhizal
107
fungi
Table 3. Summarv of slant-Prowth environments Air temperature Location (soil tested)
Length of experiment (days)
Mean maximum (“C)
Mean minimum (“C)
42
33
20
43
28
12
ND
42
35
26
1800
4s
34
21
ND
42
38
25
1705
48, 57b
32
23
600
42
28
22
450
46
27
18
444
Max. PPFD* @mol m-* s-‘)
Greenhouse
Morgantown, W.Va (Lily) Eeckley, W.Va (Gilpin) Muscle Shoals, Ala (Mountview) Griffin, Ga (Clarendon) Gainesville, Fla (Arredondo, Pahokee) Lake Alfred, Fla (Candler)
728
Growth chamber
Beltsville, Md (Galestown) Clemson, SC. (Dothan) ‘Photosynthetic photon flux density. bFor sorghum and soybeans, respectively.
At harvest, shoots were cut at the soil surface and dried to constant weight at 70°C. The relative mycorrhizal dependency (RMD) of each host with each soil and VAM isolate was calculated using the equation from Plenchette et al., (1983). The relative mycorrhizal dependency of plants will range from 100% (for obligate mycotrophic plants) to 0% (for plants that have no growth response to mycorrhizal inoculation). The dried tissue was ground and P concentration determined after wet or dry ashing or sealed-chamber digestion (Anderson and Henderson, 1986). Roots were removed from soil by soaking and washing with water. The roots were blotted dry and fresh masses determined. A fresh-mass subsample of 250-500 mg was removed from each root system and used to estimate colonization by VAM fungi. The remainder of the root system was dried to constant weight at 70°C. Total and colonized root length were estimated using the gridline-intersect method (Giovannetti and Mosse, 1980). At one location (Gainesville), nodule numbers and masses were also determined. Statistical
analysis
All data was analyzed using the General Linear Model procedure (SAS Institute, 1985a). The inoculum effect in Cecil soil was tested after ranking the inocula means by host and location. The Rank procedure sorted the mean values from the smallest to the largest, assigning the rank of 1 to the smallest, 2 to next largest, and so on up to the value of n (SAS
Institute, 1985b). The ranked values were then used in an analysis of variance and parameters without significant interactions were separated by Duncan’s Multiple Range Test (P < 0.05). The ranked values from the regional soils were also used in an analysis of variance. Pearson product-moment correlation coefficients were determined on ranked data between selected variables. RESULTS
Performance
in shared Cecil soil
Significant inoculum effects (without location interactions) were found for ranked means of shoot and root masses and total and VAM-colonized root lengths (Table 4). For soybean grown in the shared Cecil soil, inoculation with GE329 and GENPI resulted in the greatest shoot masses, followed by GE312, GETVA, GC and finally EC (Fig. 1). The EC inoculum was not different from the control. Root mass varied little, with the exception of EC, which did not differ significantly from the control. Colonized root length was not related to mycorrhizal growth response in that colonized root length of the most effective isolates did not differ from that of the least effective isolates. Colonization by GE329 and GENPI significantly increased nodule numbers (30 plant-‘) compared to the other isolates and the control (13 plant -I). Specific nodule mass was also significantly increased by GE329, GENPI and GE3 12 (2.3 mg nodule-‘) compared to the other isolates and the control (0.8 mg nodule - I).
Table 4. Probabilitv values and coefficients of variation (CV) for ranked data from Cecil soil
sourceof variation Inoculum (I) I x host I x location cv (%)
Shoot mass
Root mass
Root length co.01 0.12 0.10 28
Colonized length
Percent colonization
co.01 0.03 0.68 28
P concn
P uptake
co.01 co.01 co.01 21
to.01 co.01
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D. M.
SYLVIA
et al.
0
SOYBEAN
20 SHOOT
hs
ROOT
m
COL.
MASS MASS ROOT
LENGTH
16
12 C
I
0.5 -
0 0.0
i
2.5
4’W
rQ
1
‘wx
I
a
g 5; Z
D
hl4
K-l
c
E
z
I
O
: 0
50
I
--(
L
SORGHUM
oz 40
iz T
30
-
20
10
GE312
GE329
GENPI
GETVA
GC
EC
0 CONT
ISOLATE Fig. 1. The effect of six isolates of vesicular-arbuscular mycorrhizal fungi (see Table 2) on shoot and root dry masses and root length colonized by the mycorrhizal fungi for soybean and sorghum plants in Cecil soil. Columns represent the mean of 40 replicates. Columns with the same letter within each graph are not significantly different (P Q 0.05).
For sorghum grown in the shared Cecil soil, inoculation with GE312, GE329 and GENPI
resulted in the greatest shoot and root masses, followed by GETVA, GC and EC (Fig. 1). Again, EC did not differ significantly from the control. Among the plants colonized by VAM fungi, those with the greatest lengths of colonized root had the greatest shoot and root masses (r = 0.82, P < 0.01).
Performance in regional soils All variables had a VAM inoculum by soil interaction, and shoot and root biomass and P concentration had an inoculum by host interaction (Table 5). Therefore, results were evaluated separately by each host and soil (See Appendix, Tables Al-A2). The most relevant aspects of this analysis are summarized in Table 6.
Soybean did not respond to inoculation
Table 5. Probability values and coefficients of variation (CV) for ranked data from regional soils source of variation lnocuhm I x soil 1 x host cv (%)
(I)
Shoot mass
Root
40.01 40.01 0.04 30
IIUSS
Root length
Colonized length < 0.01 to.01 0.74 19
Percent colonization
P concn
P uptake
by any
Evaluation of VA mycorrhizal fungi
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Table 6. Best isolate responses in regional soils. Soils are listed in ascending order relative to extractable P content (see Table 11 Soybean Soil Clarendon Gilpin Lily Candler Mountview Galestown Pahokee Dothan Arredondo
Sorghum
Best isolates’
RMD (%)b
GENPI GE312, GE329, GETVA GENPI, GE329, GE312 GE312, GE329, GENPI GE329 No response No response GENPI No response
63 48 62 70 80 0 0 22 0
Best isolates’
RMD (%)”
GENPI GE329 GENPI GENPI, GE329, GE312 GE329 GENPI, GE329, GE312, GC No response GENPI, GE329 No response
94 85 71 94 94 20 0 23 0
‘Those isolates that produced the greatest shoot biomass (P d 0.05) in each soil. bRelative mycorrhizal dependency of each host plant averaged over its response to the best fungal isolates in each soil.
VAM fungal isolate in three regional soils (Galestown, Pahokee, and Arredondo) where P concentrations ranged from 20 to 62 mg kg-’ (Table 1). Isolates GE329 and GENPI best promoted soybean growth (i.e. increased shoot mass) in four of the six regional soils. Isolates GE312 and remaining GETVA were among the best performers in three soils and one soil, respectively. As in the shared Cecil soil, there was little response of soybean roots to VAM inoculation in the regional soils. Phosphorus uptake (r = 0.70, P < 0.01) and shoot-P concentration (r = 0.48, P < 0.01) were related to growth responses. Sorghum had no growth response to VAM inoculation in Pahokee and Arredondo soils. Isolates GE329 and GENPI produced the greatest response in sorghum shoot and root mass in five of the seven remaining soils. Isolates GE312 and GC were among the best performers in only two soils and one soil, respectively. Sorghum plants with the greatest length of VAM colonized root had the greatest shoot and root masses (r = 0.80, P < 0.01). Phosphorus uptake was positively correlated with growth response (r = 0.83, P < 0.01). For both hosts, RMD was highest in soils with less than 10 mg extractable P kg- I. In soils with higher P contents, sorghum responded to mycorrhizal inoculation only in Galestown soil. Sorghum consistently had higher RMD than soybean.
Fungal isolates The results of spore extractions from soil when plants were harvested at West Virginia, Maryland and Georgia locations indicated that isolate integrity was maintained for all isolates except GE312 and GE329. A few spores of GIomus occultum Walker and a fungus tentatively identified as Acaulospora mellea Spain & Schenck appeared in experimental soils inoculated with GE329. These fungi and Scutellospora dipapillosa (Walker & Koske) Walker & Sanders were found in experimental soils inoculated with GE312.
DISCUSSION
Two isolates of Glomus were found to be effective on two host species representing widely separated plant taxa in a range of low- to moderate-P soils from the eastern United States. These results are in contrast to those of others which suggest that isolates have only a limited range of adaptation (Bethlenfalvay et al., 1982; Sainz and A&es, 1988; Stahl et al., 1988). In our study, the VAM fungal isolate was the most important determinant of effectiveness. These results should be encouraging for those who want to produce VAM inoculum on a commercial scale since, at least some, effective isolates appear to have utility in a range of soils on a range of crops. Sen et al. (1989) found that isolates of VAM fungi differ in their competitive ability which further emphasizes the need to select for fungi that can grow in a range of edaphic conditions. Our results only indicate the potential of these VAM isolates to enhance P uptake and growth under the conditions tested. These results need to be validated in field trials where plants are exposed to agronomic rates of P fertilizer, as well as environmental stresses and microbial competition. In the field, environmental factors interact to alter plant growth responses to inoculation. For example, Sylvia et al. (1993) have shown that the mycorrhizal response of field-grown plants can be substantial, even in a high-P soil, if plants are grown in fumigated soil to reduce indigenous VAM fungi and then exposed to water stress. In studies such as ours, differential inoculum densities may be a confounding factor (Daniels et al., 1981). However, it is often not possible to equalize inoculum densities in an experiment because, in the 6-8 wk required to conduct the MPN assays, inoculum densities can change considerably (O’Donnell et al., 1992). We believe the best strategy for conducting screening studies is to determine the densities of the inocula when the experiment is initiated, and then to use those data to help interpret the results. The VAM inocula densities in our study were all high ( > 20 propagules ml - ‘) and should not have been a
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limiting factor. Evidence for this is that all VAM isolates, regardless of effectiveness, colonized soybean roots to nearly the same extent. Furthermore, others have reported a plateau in colonization with between 1 and 2 propagules ml-’ (Haas and Krikun, 1985; Khan, 1988). Species-level taxonomy based on spore morphology provides no information on the ecophysiology of isolates from different soils (Morton, 1990). Stahl et al. (1990) and Bethlenfalvay et al. (1989) observed that populations of Glomus mosseae from different soils were distinct in physiology. We also found that isolates within a species had different effects on plant growth. Physiological properties of different VAM isolates clearly cannot be generalized to all members of a species. This is not surprising, since these fungi reproduce asexually and each population is genetically independent of all others (Morton, 1990). Our results, using soils from different locations in the eastern United States, corroborate many reports of the strong negative influence of P concentration on fungal effectiveness. Host genotype is also an important factor in fungal effectiveness, as indicated by the responsiveness of sorghum and not soybean to four fungal isolates in the Galestown soil. Overall, sorghum was more responsive to VAM inoculation than soybean, despite the high-P requirement of the latter for nodulation and N, fixation. Differences in responsiveness were more likely related to other factors such as nutrient allocation and source-sink relationships. Sorghum and soybean responded differently to colonization. For sorghum there was a relationship between colonized root length and shoot growth response; however, this was not the case for soybean. Nonetheless, root colonization by the effective VAM isolates increased the number and size of nodules. Since legumes support the rhizobial symbiosis in addition to VAM fungi, they may have stricter control of carbon allocation to roots than do grasses (Brown and Bethlenfalvay, 1988; Brown et al., 1988). Mechanisms underlying the differential soybean responses to the various VAM fungi require further study. Factors contributing to fungal effectiveness may include the rate of root colonization (Abbott and Robson, 1981) and the distribution of hyphae in soil (Jakobsen et al., 1992). The mode of root infection (e.g. relative abundance of vesicles vs arbuscules or intramatrical vs extramatrical colonization) should also be quantified in future studies. The other VAM fungi found in soil that had been inoculated with GE312 and GE329 had not been introduced from external sources, because they were found at several locations and were unique to those locations. These other fungi may have been present in very low numbers in the original inoculum, but were non-sporulating under environmental conditions in Florida. The occurrence of different organisms in the same root system is common in nature (Brundrett, 1991) and similar situations are more likely the rule
rather than the exception in many inocula. These contaminant VAM fungi are detectable only when cultural conditions favor sporulation, and this may happen only when inocula are tested in distinct environments. The fungi found in GE312 and GE329 cultures were much more aggressive in northern environments, completely replacing GE3 12 in serial subcultures at the West Virginia location. The A. mellea and S. dipapillosa isolates have subsequently been established in monospecific cultures (INVAM codes FL312B and FL312C, respectively). A comparison of the effectiveness of these isolates with GE312 and GE329 are needed to determine if they contributed to the responses observed in our experiments. Acknowledgements-This research was nartiallv supported by the U.S. Department of Agriculture@oop&ati;d State Research Service. Published as Florida Agricultural Experiment Station Journal Series No. R-02378.
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and Feriiliry Lf Soils 6, 55-60. SAS Institute (1985al SASISTATTM Guide for Persona1 Computers, Version, 6 Ediiion. SAS Institute, Inc., Cary. SAS Institute (1985b) Procedures Guide for Personal Computers, Version 6 Edition. SAS Institute, Inc., Cary.
Sen R., Hepper C. M., Azcon-Aguilar C. and Rosendahl S. (1989) Competition between introduced and indigenous mycorrhizal fungi (Glomus spp.) for root colonization of leek. Agriculture, Ecosystems and Environmenr 29, 355-359.
Stahl P. D., Williams S. E. and Christensen M. (1988) Efficacy of native vesicular-arbuscular mycorrhizal fungi after severe soil disturbance. New Phyrologisr 110, 347-354. Stahl P. D., Christensen M. and Williams S. E. (1990) Population variation in the mycorrhizal fungus Glomus mosseae : uniform garden experiments. Mycological Re search 94, 1070-1076.
Stahl P. D. and Christensen M. (1991) Population variation in the mycorrhizal fungus Glomus mosseae: breadth of environmental tolerance. Mycological Research 95, 3Oa-307.
Sylvia D. M., Hammond L. C., Bennett J. M., Haas J. H. and Linda S. B. (1993) Field response of corn to water management and inoculation with a VAM fungus. Agronomy Journal. In press.
Appendix overleaf
D. M. SYLVIAet al.
712
APPENDIX Table Al. Soybean
Shoot mass Inoculant Arredondo GE312 GE329 GENPI GETVA GC EC Control Candler GE312 GE329 GENPI GETVA GC EC Control Clarendon GE312 GE329 GENPI GETVA GC EC Control Dothan GE312 GE329 GENPI GETVA GC EC Control Galestown GE312 GE329 GENPI GETVA GC EC Control Gilpin GE312 GE329 GENPI GETVA GC EC Control Lily GE312 GE329 GENPI GETVA GC EC Control Mountview GE312 GE329 GENPI GETVA GC EC Control Pahokee GE312 GE329 GENPI GETVA GC EC Control
(9)
response
Root mass (9)
to inoculants
Root length (m)
(see Table 2) in regional
c010nimd length (m)
Colonized length (W
soils Tissue P concn 019 g-‘)
Total P uptake 018)
2525 2360 2339 2061 1230 1865 925
a ab ab bc d c d
3555 a 36Q6a 3588 a 2999 b 1896 c 2815 b 1456 d
895 ab 970 a 664cd 682 cd 767 bc 869 ab 565 d
2061 a 2062 a 1366 b 1179 b 879 c 660d 373 e
1.41 1.54 1.53 1.40 1.54 1.51 1.57
ab* ab ab b ab ab a
0.49 0.55 0.58 0.54 0.50 0.60 0.48
bc abc ab abc bc a c
55 abc 48bc 63 a 60 ab 55 abc 57 abc 44c
41 a 30 b 42 a 41 a 30 b 45 a OC
73 ab 64bc 66bc 68 b 55 c 79 a oc
2.33 2.15 2.05 1.72 1.14 0.78 0.66
a a a b c d d
1.45 1.56 1.55 1.96 1.32 0.72 0.21
b b b a b c d
35 32 30 26 35 25 21
21 a 22 a 20 a 8b 8b 17 a oc
60a 68 a 66a 29 b 24 b 71 a oc
0.77 0.79 1.26 0.96 0.51 0.50 0.46
bc lx a b c c c
0.27 0.28 0.43 0.36 0.17 0.22 0.16
bed bc a ab cd cd d
NA NA NA NA NA NA NA
NA NA NA NA NA NA NA
1696 1494 1013 1270 793 1052 685
2.29 2.52 3.41 2.54 2.52 2.40 2.27
b b a b b b b
0.66 0.73 1.31 0.74 0.73 0.79 1.02
b b a b b b ab
4.9 4.2 4.8 5.5 5.0 6.1 4.4
ab b ab ab ab a ab
0.7 b 1.0 b 2.6 a 0.9 b 0.1 b 0.2 b Ob
20 b 24 b 60a 17 b 2b 3b Ob
NA NA NA NA NA NA NA
2.92 3.41 3.64 3.15 3.51 2.41 3.08
ab a a ab a b ab
0.65 0.77 0.73 0.69 0.75 0.48 0.74
ab a a a a b a
14 32 25 19 23 12 I5
de a b cd bc e de
5.2 b 15.2 a 3.8 bc 4.8 b 4.1 bc 2.4 c Od
36 b 48 a 14 d 25 c 18 cd 18 cd Oe
1723 1567 1360 1637 1587 1786 1898
ab ab b ab ab a a
4967 5299 4860 5093 5559 4250 5566
ab a ab ab a b a
1.28 1.28 0.09 1.02 0.93 0.58 0.62
a a bc ab bc d cd
0.56 0.55 0.43 0.46 0.38 0.32 0.25
a a ab ab bc bc c
25 20 17 15 19 11 11
a ab ab ab ab b b
16 a 12 ab 6bc 5bc 10 ab 4bc oc
63 a 53 a 39 a 37 a 57 a 33 a Oa
1871 2104 1568 1035 1395 867 636
ab a bc de cd e e
1976 871 1442 1069 1242 458 391
a bc ab abc abc c c
1.75 1.76 1.82 1.55 1.19 0.93 0.68
a a a b c d e
0.73 0.68 0.69 0.83 0.54 0.70 0.54
ab ab ab a b ab b
16 22 22 18 19 19 I5
a a a a a a a
2.4 b 1.5 bc 4.4 a 0.9 cd 0.3 cd 0.4 cd Od
14 b 7c 21 a 5cd 1 de 2 de Oe
1058 890 722 1072 988 986 372
a b c a ab ab d
1847 1563 1313 1654 1166 923 256
a b c b c d e
3.30 3.93 2.37 1.98 1.58 0.85 0.80
b a c d e f f
1.18 1.25 0.89 0.82 0.60 0.43 0.36
a a b b c cd d
28 32 25 20 21 I5 12
ab a bc cd 0-J de e
NA NA NA NA NA NA NA
NA NA NA NA NA NA NA
685 682 638 501 975 665 477
b b bed cd a bc d
2333 2627 1560 953 1699 580 436
b a c d c e e
1.67 1.66 1.76 1.83 1.87 1.84 1.83
a a a a a a a
0.52 0.50 0.51 0.57 0.51 0.57 0.51
a a a a a a a
40a 46 a 50 a 52 a 45 a 55 a 46a
28 a 30 a 21 a 21 a <1 b 4b Ob
69 a 63 a 41 b 41 b 2c 9c oc
1720 1859 718 1853 1199 1202 1212
ab a c a bc bc bc
2892 2994 1286 3327 2244 2182 2222
ab ab c a bc bc bc
*Means followed by the same letter are not significantly NA = not available.
a a ab ab a ab b
NA NA NA NA NA NA NA
different at the P 6 0.05 level.
a ab cde bc de cd e
1351 1188 1289 1177 380 526 318
a a a a b b b
NA NA NA NA NA NA NA
Evaluation of VA mycorrhizal fungi Table AZ.
Inoculant
Sorghum
response
to inoculants
Shoot mass
Root mass
Root length
(S)
(S)
(m)
713
(see Table 2) in regional
Colonized length (m)
Colonized length W)
soils Tissue P concn (!u Lx-‘)
Total P uptake @S)
Arredondo GE312 GE329 GENPI GETVA GC EC Control Candler GE312 GE329 GENPI GETVA GC EC Control Clarendon GE312 GE329 GENPI GETVA GC EC Control Dothan GE312 GE329 GENPI GETVA GV EC Control Galestown GE312 GE329 GENPI GETVA GC EC Control Gilpin GE312 GE329 GENPI GETVA GC EC Control Lily GE312 GE239 GENPI GETVA GC EC Control Mountview GE312 GE329 GENPI GETVA GC EC Control Pahokee GE312 GE329 GENPI GETVA GC EC Control
4.11 4.43 4.58 3.53 3.90 4.20 4.50
ab+ a a b ab a a
2.59 2.28 2.87 2.39 2.60 3.37 2.75
ab b ab b ab a ab
183 181 262 194 281 289 286
b b a b a a a
1.48 1.88 2.06 0.46 0.52 1.22 0.11
ab ab a d cd bc d
1.17 2.12 1.18 0.59 0.75 0.89 0.09
b a b bc b b c
175 142 145 70 78 113 11
a ab ab cd bc abc d
1.28 0.99 1.90 1.07 0.47 0.41 0.12
b bc a b cd d d
0.88 0.74 1.43 0.88 0.39 0.91 0.12
ab ab a ab b ab b
143 bc 98 cd 224 a 166 b 13 d 45 de 8e
3.41 3.89 4.32 3.53 3.23 3.39 3.17
bc ab a bc c bc c
3.91 3.83 6.30 3.17 3.00 2.50 2.91
b b a b b b b
1.3 3.1 2.1 1.8 0.9 2.3 1.4
b a ab ab b ab ab
2.27 2.62 2.65 1.85 2.26 1.49 1.95
ab a a bc ab c bc
1.56 1.56 1.61 1.16 1.73 0.91 1.44
ab ab ab bc a c ab
42 54 46 27 49 27 29
b a ab c ab c c
0.76 1.03 0.63 0.22 0.20 0.13 0.15
b a b c c c c
0.89 0.85 0.56 0.24 0.28 0.15 0.15
a a b c c c c
1.67 1.65 1.98 1.60 0.51 0.73 0.57
b b a b c c c
0.85 0.85 1.08 0.85 0.47 0.68 0.52
4.70 5.18 2.60 1.55 2.01 0.22 0.29
b a c e d f f
4.14 4.17 3.50 3.76 3.65 3.81 4.00
a a b ab ab ab ab
13 cd 25 c 51 b 16 cd 3d 68 a Od
8cd 16 ab 20 a 9bc Id 24 a Od
1253 1048 1168 1290 1128 1097 1029
ab ab ab a ab ab b
5169 4684 5412 4557 4341 4614 4619
a a a a a a a
163 a 120 ab 114 ab 28 cd 9d 73 bc Od
92 a 85 ab 79 ab 41 c 11 d 64b Od
790 853 545 647 709 740 832
a a b ab ab ab a
1158 ab 1589 a 1171 ab 292 cd 362 cd 853 bc 1OOd
19 b 6bc 37 a I2 bc lbc 18 bc oc
13 bc 6bc 16 b 7bc 2bc 40a oc
1213 1150 651 654 671 1422 462
ab b c c c a c
1384 1036 1227 629 311 617 53
a a a b bc b c
NA NA NA NA NA NA NA
11 bc 19 b 43 a 6bc 3a 8bc oc
NA NA NA NA NA NA NA
11 b 14 a 9b 4c 3cd 3cd Od
26 ab 27 a 20 bc 14 cd 6 ef 11 de Of
1365 1346 1716 2076 1743 2228 2030
b b ab a ab a a
2934 3468 4502 3833 3935 2903 3847
c bc a ab ab c ab
34 ab 39 a 22 bc 10 cd 19 bed 5d 8 cd
22 a 20 a I1 b 4bc 4bc
65 a 52 ab 51 ab 45 b 20 c 2d 2d
1506 b 1093 bc 1444b 1148 b 1938 a 712 cd 573 d
1309 1138 921 268 402 65 63
a ab abc cd bed d d
ab ab a ab c bc c
25 26 32 27 20 14 17
3.8 ab 1.9 cd 4.9 a 2.3 bc 0.2 de 0.2 de Oe
16 a 7b I5 a 8b 1C 2c oc
1316 1178 1062 1094 1730 1388 735
b c c c a b d
2200 1959 2082 1756 1010 1017 429
a a a a b b c
1.99 2.06 1.31 0.92 1.15 0.23 0.25
a a b c bc d d
68 a 78 a 51 b 31 c 51 b 7d 8d
NA NA NA NA NA NA NA
NA NA NA NA NA NA NA
543 468 470 460 593 599 494
ab ab ab b ab a ab
2682 2422 1171 723 1251 1427 140
a a b c b d d
1.69 1.53 1.63 1.88 1.69 3.00 1.61
b b b b b a b
170 158 118 173 182 218 188
40a 28 b 17 c 7d 2d Od Od
23 a 18 b 15 b 4c IC oc oc
1209 1232 1215 1249 1343 1278 1119
a a a a a a a
5006 5163 4178 4704 4783 4864 4486
a a a a a a a
*Means followed by the same letter are not significantly NA = not available.
abc ab a ab abc c bc
abc bc c ab ab a ab
different
0.1 0.6 0.9 0.1
b ab a b b ab b
at the P < 0.05
level.
1993
0038-0717/93
$6.00 + 0.00
Copyright 0 1993Pergamon Press Ltd
EVALUATION OF VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI IN DIVERSE PLANTS AND SOILS D. M. SYLVIA,‘* D. 0. WILSON,’ J. H. GRAHAM,~J. J. MADDOX,~P. MILLNER,’ J. B. MORTON,~ H. D. SKIPPER,‘% F. WRIGHTB~ and A. G. JARSTFER’ ‘Soil and Water Science Department, University of Florida, Gainesville, FL ‘32611-0290, 2Agronomy Department, University of Georgia, Griffin, GA 30223, ‘Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, 4National Fertilizer and Environmental Research Center, Tennessee Valley Authority, Muscle Shoals, AL 35660, jSoi1 Microbial Systems Laboratory, USDA, ARS, Beltsville, MD 20705, 6Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506, ‘Agronomy and Soils Department, Clemson University, Clemson, SC 29634 and 8Appalachian Soil and Water Laboratory, USDA, ARS, Beckley, WV 25801, U.S.A. (Accepted 20 December 1992) Summary-A regional study was made to identify vesicular-arbuscular mycorrhizal (VAM) fungi effective in promoting plant growth in diverse plant and soil systems. Eight cooperators in six states of the eastern United States evaluated six VAM fungal isolates on soybean (Glycine mux L. Merr.) and sorghum (Sorghum bicolor L. Moench) in a shared soil and in at least one regional soil from each location. Plants were grown with high VAM inoculum densities (minimum of 20 VAM propagules ml-‘) for 42-57 days in pasturized soils in greenhouses or growth chambers. Shoot and root dry masses, total and colonized root lengths and shoot-P concentrations were determined at harvest. Under the experimental conditions tested, the VAM fungal isolate was more important than the soil or host plant in determining effectiveness. In the shared soil, inoculation with two isolates of Glomus (GE329 and GENPI) resulted in the greatest shoot masses for soybeans, while the same two isolates and GE312 provided maximum response in sorghum. In the regional soils, GE329 and GENPI had the widest range of growth promotion with both soybean and sorghum; however, for both plant species the mycorrhizal response was greatest in soils with less than 10 mg extractable P kg-‘. For soybeans, colonized root length was not related to VAM growth response. For sorghum, there was a positive correlation between colonized root length and plant growth response. We conclude that VAM isolates exist which are effective in promoting plant growth over a range of edaphic and host conditions.
INTRODUCTION Little is known about the stability of the plant growth with vesicular-arbuscular mycorrhizal response (VAM) fungi over a range of host plants and soil
environments. While it is well-known that VAM fungi lack host specificity (Gianinazzi-Pearson, 1984), their effectiveness under uniform experimental conditions can vary widely (Mosse, 1972; Graham et al., 1982; Lamar and Davey, 1988; Haas and Krikun, 1985; Boerner, 1990; Hung et al., 1990). Considerable data indicate that VAM fungi are adapted to different edaphic conditions (Lambert et al., 1980; Hayman, 1982; Gianinazzi-Pearson et al., 1985; Henkel et al., 1989; Stahl and Christensen, 1991) and may possess unique properties based on their biology (Bethlenfalvay et al., 1989). It is important, therefore, to screen isolates of VAM fungi to insure that each inoculum source is effective on the host and in the soil where it will be utilized. Mass production of VAM inoculum requires a living host or tissue explant due to the obligate nature
of the symbiosis (Jarstfer and Sylvia, 1992). This drives up the cost of inoculum production and, consequently, commercially-feasible inoculum requires that fungal isolates be effective over a range of edaphic and host conditions. In the summer of 1990, eight cooperators participated in a study to screen six isolates of VAM fungi on two hosts in a range of soils found in the eastern United States. Our objective was to identify fungal isolates which are effective in promoting plant growth in diverse host-soil systems.
MATERIAL AND METHODS
At each of the eight locations, six VAM isolates were evaluated on soybean (cv. Centennial) and sorghum (cv. Funk G-522DR) in a shared soil (limed Cecil from Georgia) and in at least one regional soil (Table 1). The treatments within each soil were completely randomized, with five replicates per treatment. Soils and inoculu
*Author for correspondence. TPresent address: Soil Microbial Systems Laboratory, USDA, ARS, Beltsville, MD 20705, U.S.A.
Soils were sieved (2 mm) and pasturized (at least 70°C for 6 h, except for fumigation with methyl 705
D. M. SYLVIA et 01.
706
Table 1. Soil descriotions and selected orooerties
series Clarendon Cecil Gilpin Lily Candler Mountview Galestown Pahokee Dothan Arredondo
Collection site County, State
Description
Tift, Ga Pike, Ga Wyoming, W.Va Monongalia, W.Va Polk, Fla Lawrence, Tenn. Prince Georges, Md Palm Beach, Fla Sumter SC. Alachua, Fla
Fine loamy sand, Plinthaquic Paleudult Clayey, Typic Hapludult Typic Hapludult’ Typic Hapludult’ Fine sand, Typic Quartzipsamment Silt loam, Typic Hapludult Typic Quartzipsamment’ Muck, Lithic Medisaprist Fine loam, Plinthic Paleudult Loamy sand, Grossannic Paleudult
PH’ 5.4 6.4 5.8 5.9 6.4 7.1 6.2 7.0 6.0 5.8
CECb (cm01 kg - ‘) 2.05 1.82 2.95 ND 0.69 2.27 0.71 106.7 ND 3.51
;:)’ 1.1 0.8 1.2 0.9 0.5 0.9 0.3 63.0 1.1 2.2
Nd
F
(w kg- ‘)
(mg kg‘ 9
16.4 41.9 34.6 27.6 4.1 56.9 5.8 1826.0 19.9 94.4
5.0 5.2 5.4 8.1 8.5 9.4 20.0 24.5 36.9 61.6
*l: 1 H,O:soil. bNeutral 1 M NH,OAc. ‘Walkley-Black wet combustion. dNH,-N plus NO,-N, 2 N KCI, 5: 1 extractant to soil. ‘Mehlich I extractable. ‘Mixed 1: 1 with sand. ND = not determined.
bromide of the Pahokee soil). Soil chemical properties ranged as follows: pH from 5.4 to 7.1, organic matter from 0.3 to 63% and Mehlich I-extractable (50 mM HCl and 10 mu H,S03 P from 5 to 62 mg kg-’ (Table 1). Dolomitic limestone (4 : 1 CaCO, : MgC09) was added to the Cecil soil at a rate of 25Omg kg-’ soil, 10-14 days prior to nutrient addition and then the soil was subjected to several wet and dry cycles to achieve a stable pH. All soils received nutrient amendments that included 60 mg (NH&SO, kg-‘, 270 mg KNO, kg-’ for soils planted with sorghum and 222 mg K,S04 kg- ’ for soils planted with soybeans. A complete micronutrient mixture was added to all soils (mg kg - ‘): Mg, 4.6; Fe, 4.7; Mn, 3.7; Zn, 4.2; Cu, 1.2; B, 0.5; MO, 1.0; and S, 12.7. All nutrients were mixed throughout each soil just prior to planting. Three weeks after planting, (NH&SO,, KNO, and KC1 were added to pots with sorghum plants at 180, 90 and 125 mg kg-‘, respectively. Inocula of VAM fungi consisted of root and soil mixtures from established pot cultures. Inoculum density in each culture produced by cooperators was estimated by most-probable number (MPN) assays (Porter, 1979) made just prior to distribution. Inocula were shipped to all locations via overnight express mail. Inoculum (50 ml except for one isolate, GENPI,
where, due to limited supply, 25-37 ml were used) was mixed with 450ml of test soil and the mixture was placed into a 600ml DeePot (J. M. McConkey & Co. Inc., 1615 Puyallup Street, Sumner, WA 98390). Inoculum densities ranged from 20 to 192 propagules ml-i while the total number of propagules added to each pot ranged from 1000 to 9600 (Table 2). All pots received 5ml of a composite microbial filtrate in an attempt to equalize the background microflora. The filtrate was prepared by mixing together 10 ml of each inoculant, adding 1.2 1. of water and filtering the resultant slurry (< 10 pm). The controls contained 500 ml of the pasteurized test soil along with the microbial filtrate. Culture methodr and harvest Seeds were surface disinfested with full-strength household bleach (5.25% NaOCl) for 5 min and rinsed 5 times (20-30min each) with sterile water. Four seeds were placed 1.5 cm below the soil surface. For soybeans, granular bradyrhizobial inoculant (USDA 110) was added with the seed to the planting hole. After emergence, plants were thinned to one per pot and then grown in cooperators’ greenhouses or growth chambers for 42-57 days (Table 3). Soil moisture was maintained near field capacity by daily watering throughout the experiment.
Table 2. Funnal isolates used in screeninn studies
Fungal species Glomus ehmicatum Becker & Gerdemann
Glomus sp.E G. etunicatum G. etunicatum G. claroides
Schcnck & Smith
Isolate code
INVAM’ code
MPNb (propagules ml-‘)
GE312 GE329 GENPI GETVA
FL312 FL906 UT316 TN101
45 192 72 29
2250 9600 1BOO-2664 1450
GC
SC186
20
1000
EC
GA101
63
3150
Propagules added per pot
Entrophospora columbiama
Spain & Schenck
‘International Culture Collection of VA Mycorrhiil Fungi INVAM, 401 Brooks Hall, West Virginia University, Morgantown, WV 26506-6057, U.S.A. bMost probable number. COriginally classified as GIomus etunicotum.
Evaluation
of VA mycorrhizal
107
fungi
Table 3. Summarv of slant-Prowth environments Air temperature Location (soil tested)
Length of experiment (days)
Mean maximum (“C)
Mean minimum (“C)
42
33
20
43
28
12
ND
42
35
26
1800
4s
34
21
ND
42
38
25
1705
48, 57b
32
23
600
42
28
22
450
46
27
18
444
Max. PPFD* @mol m-* s-‘)
Greenhouse
Morgantown, W.Va (Lily) Eeckley, W.Va (Gilpin) Muscle Shoals, Ala (Mountview) Griffin, Ga (Clarendon) Gainesville, Fla (Arredondo, Pahokee) Lake Alfred, Fla (Candler)
728
Growth chamber
Beltsville, Md (Galestown) Clemson, SC. (Dothan) ‘Photosynthetic photon flux density. bFor sorghum and soybeans, respectively.
At harvest, shoots were cut at the soil surface and dried to constant weight at 70°C. The relative mycorrhizal dependency (RMD) of each host with each soil and VAM isolate was calculated using the equation from Plenchette et al., (1983). The relative mycorrhizal dependency of plants will range from 100% (for obligate mycotrophic plants) to 0% (for plants that have no growth response to mycorrhizal inoculation). The dried tissue was ground and P concentration determined after wet or dry ashing or sealed-chamber digestion (Anderson and Henderson, 1986). Roots were removed from soil by soaking and washing with water. The roots were blotted dry and fresh masses determined. A fresh-mass subsample of 250-500 mg was removed from each root system and used to estimate colonization by VAM fungi. The remainder of the root system was dried to constant weight at 70°C. Total and colonized root length were estimated using the gridline-intersect method (Giovannetti and Mosse, 1980). At one location (Gainesville), nodule numbers and masses were also determined. Statistical
analysis
All data was analyzed using the General Linear Model procedure (SAS Institute, 1985a). The inoculum effect in Cecil soil was tested after ranking the inocula means by host and location. The Rank procedure sorted the mean values from the smallest to the largest, assigning the rank of 1 to the smallest, 2 to next largest, and so on up to the value of n (SAS
Institute, 1985b). The ranked values were then used in an analysis of variance and parameters without significant interactions were separated by Duncan’s Multiple Range Test (P < 0.05). The ranked values from the regional soils were also used in an analysis of variance. Pearson product-moment correlation coefficients were determined on ranked data between selected variables. RESULTS
Performance
in shared Cecil soil
Significant inoculum effects (without location interactions) were found for ranked means of shoot and root masses and total and VAM-colonized root lengths (Table 4). For soybean grown in the shared Cecil soil, inoculation with GE329 and GENPI resulted in the greatest shoot masses, followed by GE312, GETVA, GC and finally EC (Fig. 1). The EC inoculum was not different from the control. Root mass varied little, with the exception of EC, which did not differ significantly from the control. Colonized root length was not related to mycorrhizal growth response in that colonized root length of the most effective isolates did not differ from that of the least effective isolates. Colonization by GE329 and GENPI significantly increased nodule numbers (30 plant-‘) compared to the other isolates and the control (13 plant -I). Specific nodule mass was also significantly increased by GE329, GENPI and GE3 12 (2.3 mg nodule-‘) compared to the other isolates and the control (0.8 mg nodule - I).
Table 4. Probabilitv values and coefficients of variation (CV) for ranked data from Cecil soil
sourceof variation Inoculum (I) I x host I x location cv (%)
Shoot mass
Root mass
Root length co.01 0.12 0.10 28
Colonized length
Percent colonization
co.01 0.03 0.68 28
P concn
P uptake
co.01 co.01 co.01 21
to.01 co.01
708
D. M.
SYLVIA
et al.
0
SOYBEAN
20 SHOOT
hs
ROOT
m
COL.
MASS MASS ROOT
LENGTH
16
12 C
I
0.5 -
0 0.0
i
2.5
4’W
rQ
1
‘wx
I
a
g 5; Z
D
hl4
K-l
c
E
z
I
O
: 0
50
I
--(
L
SORGHUM
oz 40
iz T
30
-
20
10
GE312
GE329
GENPI
GETVA
GC
EC
0 CONT
ISOLATE Fig. 1. The effect of six isolates of vesicular-arbuscular mycorrhizal fungi (see Table 2) on shoot and root dry masses and root length colonized by the mycorrhizal fungi for soybean and sorghum plants in Cecil soil. Columns represent the mean of 40 replicates. Columns with the same letter within each graph are not significantly different (P Q 0.05).
For sorghum grown in the shared Cecil soil, inoculation with GE312, GE329 and GENPI
resulted in the greatest shoot and root masses, followed by GETVA, GC and EC (Fig. 1). Again, EC did not differ significantly from the control. Among the plants colonized by VAM fungi, those with the greatest lengths of colonized root had the greatest shoot and root masses (r = 0.82, P < 0.01).
Performance in regional soils All variables had a VAM inoculum by soil interaction, and shoot and root biomass and P concentration had an inoculum by host interaction (Table 5). Therefore, results were evaluated separately by each host and soil (See Appendix, Tables Al-A2). The most relevant aspects of this analysis are summarized in Table 6.
Soybean did not respond to inoculation
Table 5. Probability values and coefficients of variation (CV) for ranked data from regional soils source of variation lnocuhm I x soil 1 x host cv (%)
(I)
Shoot mass
Root
40.01 40.01 0.04 30
IIUSS
Root length
Colonized length < 0.01 to.01 0.74 19
Percent colonization
P concn
P uptake
by any
Evaluation of VA mycorrhizal fungi
709
Table 6. Best isolate responses in regional soils. Soils are listed in ascending order relative to extractable P content (see Table 11 Soybean Soil Clarendon Gilpin Lily Candler Mountview Galestown Pahokee Dothan Arredondo
Sorghum
Best isolates’
RMD (%)b
GENPI GE312, GE329, GETVA GENPI, GE329, GE312 GE312, GE329, GENPI GE329 No response No response GENPI No response
63 48 62 70 80 0 0 22 0
Best isolates’
RMD (%)”
GENPI GE329 GENPI GENPI, GE329, GE312 GE329 GENPI, GE329, GE312, GC No response GENPI, GE329 No response
94 85 71 94 94 20 0 23 0
‘Those isolates that produced the greatest shoot biomass (P d 0.05) in each soil. bRelative mycorrhizal dependency of each host plant averaged over its response to the best fungal isolates in each soil.
VAM fungal isolate in three regional soils (Galestown, Pahokee, and Arredondo) where P concentrations ranged from 20 to 62 mg kg-’ (Table 1). Isolates GE329 and GENPI best promoted soybean growth (i.e. increased shoot mass) in four of the six regional soils. Isolates GE312 and remaining GETVA were among the best performers in three soils and one soil, respectively. As in the shared Cecil soil, there was little response of soybean roots to VAM inoculation in the regional soils. Phosphorus uptake (r = 0.70, P < 0.01) and shoot-P concentration (r = 0.48, P < 0.01) were related to growth responses. Sorghum had no growth response to VAM inoculation in Pahokee and Arredondo soils. Isolates GE329 and GENPI produced the greatest response in sorghum shoot and root mass in five of the seven remaining soils. Isolates GE312 and GC were among the best performers in only two soils and one soil, respectively. Sorghum plants with the greatest length of VAM colonized root had the greatest shoot and root masses (r = 0.80, P < 0.01). Phosphorus uptake was positively correlated with growth response (r = 0.83, P < 0.01). For both hosts, RMD was highest in soils with less than 10 mg extractable P kg- I. In soils with higher P contents, sorghum responded to mycorrhizal inoculation only in Galestown soil. Sorghum consistently had higher RMD than soybean.
Fungal isolates The results of spore extractions from soil when plants were harvested at West Virginia, Maryland and Georgia locations indicated that isolate integrity was maintained for all isolates except GE312 and GE329. A few spores of GIomus occultum Walker and a fungus tentatively identified as Acaulospora mellea Spain & Schenck appeared in experimental soils inoculated with GE329. These fungi and Scutellospora dipapillosa (Walker & Koske) Walker & Sanders were found in experimental soils inoculated with GE312.
DISCUSSION
Two isolates of Glomus were found to be effective on two host species representing widely separated plant taxa in a range of low- to moderate-P soils from the eastern United States. These results are in contrast to those of others which suggest that isolates have only a limited range of adaptation (Bethlenfalvay et al., 1982; Sainz and A&es, 1988; Stahl et al., 1988). In our study, the VAM fungal isolate was the most important determinant of effectiveness. These results should be encouraging for those who want to produce VAM inoculum on a commercial scale since, at least some, effective isolates appear to have utility in a range of soils on a range of crops. Sen et al. (1989) found that isolates of VAM fungi differ in their competitive ability which further emphasizes the need to select for fungi that can grow in a range of edaphic conditions. Our results only indicate the potential of these VAM isolates to enhance P uptake and growth under the conditions tested. These results need to be validated in field trials where plants are exposed to agronomic rates of P fertilizer, as well as environmental stresses and microbial competition. In the field, environmental factors interact to alter plant growth responses to inoculation. For example, Sylvia et al. (1993) have shown that the mycorrhizal response of field-grown plants can be substantial, even in a high-P soil, if plants are grown in fumigated soil to reduce indigenous VAM fungi and then exposed to water stress. In studies such as ours, differential inoculum densities may be a confounding factor (Daniels et al., 1981). However, it is often not possible to equalize inoculum densities in an experiment because, in the 6-8 wk required to conduct the MPN assays, inoculum densities can change considerably (O’Donnell et al., 1992). We believe the best strategy for conducting screening studies is to determine the densities of the inocula when the experiment is initiated, and then to use those data to help interpret the results. The VAM inocula densities in our study were all high ( > 20 propagules ml - ‘) and should not have been a
710
D. M.
SYLVIA et al.
limiting factor. Evidence for this is that all VAM isolates, regardless of effectiveness, colonized soybean roots to nearly the same extent. Furthermore, others have reported a plateau in colonization with between 1 and 2 propagules ml-’ (Haas and Krikun, 1985; Khan, 1988). Species-level taxonomy based on spore morphology provides no information on the ecophysiology of isolates from different soils (Morton, 1990). Stahl et al. (1990) and Bethlenfalvay et al. (1989) observed that populations of Glomus mosseae from different soils were distinct in physiology. We also found that isolates within a species had different effects on plant growth. Physiological properties of different VAM isolates clearly cannot be generalized to all members of a species. This is not surprising, since these fungi reproduce asexually and each population is genetically independent of all others (Morton, 1990). Our results, using soils from different locations in the eastern United States, corroborate many reports of the strong negative influence of P concentration on fungal effectiveness. Host genotype is also an important factor in fungal effectiveness, as indicated by the responsiveness of sorghum and not soybean to four fungal isolates in the Galestown soil. Overall, sorghum was more responsive to VAM inoculation than soybean, despite the high-P requirement of the latter for nodulation and N, fixation. Differences in responsiveness were more likely related to other factors such as nutrient allocation and source-sink relationships. Sorghum and soybean responded differently to colonization. For sorghum there was a relationship between colonized root length and shoot growth response; however, this was not the case for soybean. Nonetheless, root colonization by the effective VAM isolates increased the number and size of nodules. Since legumes support the rhizobial symbiosis in addition to VAM fungi, they may have stricter control of carbon allocation to roots than do grasses (Brown and Bethlenfalvay, 1988; Brown et al., 1988). Mechanisms underlying the differential soybean responses to the various VAM fungi require further study. Factors contributing to fungal effectiveness may include the rate of root colonization (Abbott and Robson, 1981) and the distribution of hyphae in soil (Jakobsen et al., 1992). The mode of root infection (e.g. relative abundance of vesicles vs arbuscules or intramatrical vs extramatrical colonization) should also be quantified in future studies. The other VAM fungi found in soil that had been inoculated with GE312 and GE329 had not been introduced from external sources, because they were found at several locations and were unique to those locations. These other fungi may have been present in very low numbers in the original inoculum, but were non-sporulating under environmental conditions in Florida. The occurrence of different organisms in the same root system is common in nature (Brundrett, 1991) and similar situations are more likely the rule
rather than the exception in many inocula. These contaminant VAM fungi are detectable only when cultural conditions favor sporulation, and this may happen only when inocula are tested in distinct environments. The fungi found in GE312 and GE329 cultures were much more aggressive in northern environments, completely replacing GE3 12 in serial subcultures at the West Virginia location. The A. mellea and S. dipapillosa isolates have subsequently been established in monospecific cultures (INVAM codes FL312B and FL312C, respectively). A comparison of the effectiveness of these isolates with GE312 and GE329 are needed to determine if they contributed to the responses observed in our experiments. Acknowledgements-This research was nartiallv supported by the U.S. Department of Agriculture@oop&ati;d State Research Service. Published as Florida Agricultural Experiment Station Journal Series No. R-02378.
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Graham J. H., Linderman Development of external mycorrhizal Glomus spp. and growth of Troyer
R. G. and Menge J. A. (1982) hyphae by different isolates of in relation to root colonization citrange. New Phytologisr 91,
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Haas J. H. and Krikun J. (1985) Efficacy of endomycorrhizal fungal isolates and inoculum quantities required for growth response. New Phytologisr 100, 613-622. Hayman D. S. (1982) Influence of soils and fertility on activity and survival of vesicular-arbuscular mycorrhizal fungi. Phytopathology 72, 1119-l 125. Henkel T. W., Smith W. K. and Christensen M. (1989) Infectivity and effectivity of indigenous vesicular-arbuscular mycorrhizal fungi from contiguous soils in southwestern Wyoming. U.S.A. New Phyrologisl 112, 205-214. Hung L. L., Sylvia D. M. and O’Keefe D. M. (1990) Isolate selection and phosphorus interaction of vesicular-arbuscular mycorrhizal fungi in biomass crops. Soil Science Society
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Jakobsen I., Abbott L. K. and Robson A. D. (1992) External hyphae of vesicular-arbuscular mycorihizai fungi associated with Trifolium sublerraneum L. 1. Soread of l$phae and phosphorus inflow into roots. New Piytologist 120, 371-380. Jarstfer A. G. and Sylvia D. M. (1992) Inoculum production and inoculation strategies for vesicular-arbuscular mycorrhizal fungi. In Soil Microbial Ecology: Applications in Agriculture and Environmental Management-(B. Meeting, Ed.). __ DO. 349-377. Marcel Dekker, New York. Khan A. G. (1988) Inoculum density of Glomus mosseae and growth of onion plants in unsterilized bituminous coal spoil. Soil Biology & Biochemistry 20, 749-753. Lamar R. T. and Davey C. B. (1988) Comparative effectivity of three Fraxinus pennsylvanica Marsh. vesicular-arbuscular mycorrhizal fungi in a high-phosphorus nursery soil. New Phytologist 109, 171-181. Lambert D. H., Cole H. and Baker D. E. (1980) Adaptation of vesicular-arbuscular mycorrhizae to edaphic factors. New Phyrologist 85, 513-520. Morton J. B. (1990) Species and clones of arbuscular mycorrhizal fungi (Glomales, Zygomycetes): their role in
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Appendix overleaf
D. M. SYLVIAet al.
712
APPENDIX Table Al. Soybean
Shoot mass Inoculant Arredondo GE312 GE329 GENPI GETVA GC EC Control Candler GE312 GE329 GENPI GETVA GC EC Control Clarendon GE312 GE329 GENPI GETVA GC EC Control Dothan GE312 GE329 GENPI GETVA GC EC Control Galestown GE312 GE329 GENPI GETVA GC EC Control Gilpin GE312 GE329 GENPI GETVA GC EC Control Lily GE312 GE329 GENPI GETVA GC EC Control Mountview GE312 GE329 GENPI GETVA GC EC Control Pahokee GE312 GE329 GENPI GETVA GC EC Control
(9)
response
Root mass (9)
to inoculants
Root length (m)
(see Table 2) in regional
c010nimd length (m)
Colonized length (W
soils Tissue P concn 019 g-‘)
Total P uptake 018)
2525 2360 2339 2061 1230 1865 925
a ab ab bc d c d
3555 a 36Q6a 3588 a 2999 b 1896 c 2815 b 1456 d
895 ab 970 a 664cd 682 cd 767 bc 869 ab 565 d
2061 a 2062 a 1366 b 1179 b 879 c 660d 373 e
1.41 1.54 1.53 1.40 1.54 1.51 1.57
ab* ab ab b ab ab a
0.49 0.55 0.58 0.54 0.50 0.60 0.48
bc abc ab abc bc a c
55 abc 48bc 63 a 60 ab 55 abc 57 abc 44c
41 a 30 b 42 a 41 a 30 b 45 a OC
73 ab 64bc 66bc 68 b 55 c 79 a oc
2.33 2.15 2.05 1.72 1.14 0.78 0.66
a a a b c d d
1.45 1.56 1.55 1.96 1.32 0.72 0.21
b b b a b c d
35 32 30 26 35 25 21
21 a 22 a 20 a 8b 8b 17 a oc
60a 68 a 66a 29 b 24 b 71 a oc
0.77 0.79 1.26 0.96 0.51 0.50 0.46
bc lx a b c c c
0.27 0.28 0.43 0.36 0.17 0.22 0.16
bed bc a ab cd cd d
NA NA NA NA NA NA NA
NA NA NA NA NA NA NA
1696 1494 1013 1270 793 1052 685
2.29 2.52 3.41 2.54 2.52 2.40 2.27
b b a b b b b
0.66 0.73 1.31 0.74 0.73 0.79 1.02
b b a b b b ab
4.9 4.2 4.8 5.5 5.0 6.1 4.4
ab b ab ab ab a ab
0.7 b 1.0 b 2.6 a 0.9 b 0.1 b 0.2 b Ob
20 b 24 b 60a 17 b 2b 3b Ob
NA NA NA NA NA NA NA
2.92 3.41 3.64 3.15 3.51 2.41 3.08
ab a a ab a b ab
0.65 0.77 0.73 0.69 0.75 0.48 0.74
ab a a a a b a
14 32 25 19 23 12 I5
de a b cd bc e de
5.2 b 15.2 a 3.8 bc 4.8 b 4.1 bc 2.4 c Od
36 b 48 a 14 d 25 c 18 cd 18 cd Oe
1723 1567 1360 1637 1587 1786 1898
ab ab b ab ab a a
4967 5299 4860 5093 5559 4250 5566
ab a ab ab a b a
1.28 1.28 0.09 1.02 0.93 0.58 0.62
a a bc ab bc d cd
0.56 0.55 0.43 0.46 0.38 0.32 0.25
a a ab ab bc bc c
25 20 17 15 19 11 11
a ab ab ab ab b b
16 a 12 ab 6bc 5bc 10 ab 4bc oc
63 a 53 a 39 a 37 a 57 a 33 a Oa
1871 2104 1568 1035 1395 867 636
ab a bc de cd e e
1976 871 1442 1069 1242 458 391
a bc ab abc abc c c
1.75 1.76 1.82 1.55 1.19 0.93 0.68
a a a b c d e
0.73 0.68 0.69 0.83 0.54 0.70 0.54
ab ab ab a b ab b
16 22 22 18 19 19 I5
a a a a a a a
2.4 b 1.5 bc 4.4 a 0.9 cd 0.3 cd 0.4 cd Od
14 b 7c 21 a 5cd 1 de 2 de Oe
1058 890 722 1072 988 986 372
a b c a ab ab d
1847 1563 1313 1654 1166 923 256
a b c b c d e
3.30 3.93 2.37 1.98 1.58 0.85 0.80
b a c d e f f
1.18 1.25 0.89 0.82 0.60 0.43 0.36
a a b b c cd d
28 32 25 20 21 I5 12
ab a bc cd 0-J de e
NA NA NA NA NA NA NA
NA NA NA NA NA NA NA
685 682 638 501 975 665 477
b b bed cd a bc d
2333 2627 1560 953 1699 580 436
b a c d c e e
1.67 1.66 1.76 1.83 1.87 1.84 1.83
a a a a a a a
0.52 0.50 0.51 0.57 0.51 0.57 0.51
a a a a a a a
40a 46 a 50 a 52 a 45 a 55 a 46a
28 a 30 a 21 a 21 a <1 b 4b Ob
69 a 63 a 41 b 41 b 2c 9c oc
1720 1859 718 1853 1199 1202 1212
ab a c a bc bc bc
2892 2994 1286 3327 2244 2182 2222
ab ab c a bc bc bc
*Means followed by the same letter are not significantly NA = not available.
a a ab ab a ab b
NA NA NA NA NA NA NA
different at the P 6 0.05 level.
a ab cde bc de cd e
1351 1188 1289 1177 380 526 318
a a a a b b b
NA NA NA NA NA NA NA
Evaluation of VA mycorrhizal fungi Table AZ.
Inoculant
Sorghum
response
to inoculants
Shoot mass
Root mass
Root length
(S)
(S)
(m)
713
(see Table 2) in regional
Colonized length (m)
Colonized length W)
soils Tissue P concn (!u Lx-‘)
Total P uptake @S)
Arredondo GE312 GE329 GENPI GETVA GC EC Control Candler GE312 GE329 GENPI GETVA GC EC Control Clarendon GE312 GE329 GENPI GETVA GC EC Control Dothan GE312 GE329 GENPI GETVA GV EC Control Galestown GE312 GE329 GENPI GETVA GC EC Control Gilpin GE312 GE329 GENPI GETVA GC EC Control Lily GE312 GE239 GENPI GETVA GC EC Control Mountview GE312 GE329 GENPI GETVA GC EC Control Pahokee GE312 GE329 GENPI GETVA GC EC Control
4.11 4.43 4.58 3.53 3.90 4.20 4.50
ab+ a a b ab a a
2.59 2.28 2.87 2.39 2.60 3.37 2.75
ab b ab b ab a ab
183 181 262 194 281 289 286
b b a b a a a
1.48 1.88 2.06 0.46 0.52 1.22 0.11
ab ab a d cd bc d
1.17 2.12 1.18 0.59 0.75 0.89 0.09
b a b bc b b c
175 142 145 70 78 113 11
a ab ab cd bc abc d
1.28 0.99 1.90 1.07 0.47 0.41 0.12
b bc a b cd d d
0.88 0.74 1.43 0.88 0.39 0.91 0.12
ab ab a ab b ab b
143 bc 98 cd 224 a 166 b 13 d 45 de 8e
3.41 3.89 4.32 3.53 3.23 3.39 3.17
bc ab a bc c bc c
3.91 3.83 6.30 3.17 3.00 2.50 2.91
b b a b b b b
1.3 3.1 2.1 1.8 0.9 2.3 1.4
b a ab ab b ab ab
2.27 2.62 2.65 1.85 2.26 1.49 1.95
ab a a bc ab c bc
1.56 1.56 1.61 1.16 1.73 0.91 1.44
ab ab ab bc a c ab
42 54 46 27 49 27 29
b a ab c ab c c
0.76 1.03 0.63 0.22 0.20 0.13 0.15
b a b c c c c
0.89 0.85 0.56 0.24 0.28 0.15 0.15
a a b c c c c
1.67 1.65 1.98 1.60 0.51 0.73 0.57
b b a b c c c
0.85 0.85 1.08 0.85 0.47 0.68 0.52
4.70 5.18 2.60 1.55 2.01 0.22 0.29
b a c e d f f
4.14 4.17 3.50 3.76 3.65 3.81 4.00
a a b ab ab ab ab
13 cd 25 c 51 b 16 cd 3d 68 a Od
8cd 16 ab 20 a 9bc Id 24 a Od
1253 1048 1168 1290 1128 1097 1029
ab ab ab a ab ab b
5169 4684 5412 4557 4341 4614 4619
a a a a a a a
163 a 120 ab 114 ab 28 cd 9d 73 bc Od
92 a 85 ab 79 ab 41 c 11 d 64b Od
790 853 545 647 709 740 832
a a b ab ab ab a
1158 ab 1589 a 1171 ab 292 cd 362 cd 853 bc 1OOd
19 b 6bc 37 a I2 bc lbc 18 bc oc
13 bc 6bc 16 b 7bc 2bc 40a oc
1213 1150 651 654 671 1422 462
ab b c c c a c
1384 1036 1227 629 311 617 53
a a a b bc b c
NA NA NA NA NA NA NA
11 bc 19 b 43 a 6bc 3a 8bc oc
NA NA NA NA NA NA NA
11 b 14 a 9b 4c 3cd 3cd Od
26 ab 27 a 20 bc 14 cd 6 ef 11 de Of
1365 1346 1716 2076 1743 2228 2030
b b ab a ab a a
2934 3468 4502 3833 3935 2903 3847
c bc a ab ab c ab
34 ab 39 a 22 bc 10 cd 19 bed 5d 8 cd
22 a 20 a I1 b 4bc 4bc
65 a 52 ab 51 ab 45 b 20 c 2d 2d
1506 b 1093 bc 1444b 1148 b 1938 a 712 cd 573 d
1309 1138 921 268 402 65 63
a ab abc cd bed d d
ab ab a ab c bc c
25 26 32 27 20 14 17
3.8 ab 1.9 cd 4.9 a 2.3 bc 0.2 de 0.2 de Oe
16 a 7b I5 a 8b 1C 2c oc
1316 1178 1062 1094 1730 1388 735
b c c c a b d
2200 1959 2082 1756 1010 1017 429
a a a a b b c
1.99 2.06 1.31 0.92 1.15 0.23 0.25
a a b c bc d d
68 a 78 a 51 b 31 c 51 b 7d 8d
NA NA NA NA NA NA NA
NA NA NA NA NA NA NA
543 468 470 460 593 599 494
ab ab ab b ab a ab
2682 2422 1171 723 1251 1427 140
a a b c b d d
1.69 1.53 1.63 1.88 1.69 3.00 1.61
b b b b b a b
170 158 118 173 182 218 188
40a 28 b 17 c 7d 2d Od Od
23 a 18 b 15 b 4c IC oc oc
1209 1232 1215 1249 1343 1278 1119
a a a a a a a
5006 5163 4178 4704 4783 4864 4486
a a a a a a a
*Means followed by the same letter are not significantly NA = not available.
abc ab a ab abc c bc
abc bc c ab ab a ab
different
0.1 0.6 0.9 0.1
b ab a b b ab b
at the P < 0.05
level.