Comparing inoculum potential of vesicular-arbuscular mycorrhiza from three plant species
by Stuart Michael Levit
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Land
Rehabilitation
Montana State University
© Copyright by Stuart Michael Levit (1989)
Abstract:
Many studies completed in recent years point to the potential for increasing land reclamation success
by increasing the role of vesicular-arbuscular mycorrhiza (VAM) in revegetation plans. Understanding
mycorrhizal infection in areas of chemical toxicity may be particularly important to land rehabilitation
because of increased plant stress. To demonstrate the effects that tailings materials have on VAM
infection rates the infectivity of mycorrhiza from three grass species growing in, near, and away from
tailings enriched soil were compared.
Vesicular-arbuscular mycorrhizal infected roots from 1) tufted hairgrass growing in tailings enriched
alluvium, 2) redtop bentgrass growing near tailings enriched soil, and 3) smooth brome growing (away
from river deposited tailings) in agricultural soil were mixed in 1:1, 1:2, 1:4, and 1:8 dilutions (root:soil
by volume) with sterilized greenhouse soil. Control treatments consisted of sterilized greenhouse soil
material with no root inoculum. In the greenhouse, sudangrass, acting as a plant host, was seeded into
these mixtures and its roots were examined for quantitative mycorrhizal infection to assess the impact
that tailings material plays on mycorrhizal infectivity in the field.
In the 1:1 and 1:8 treatments there was significantly lower infectivity between both tufted hairgrass
VAM and redtop bentgrass VAM and VAM from.smooth brome. In the 1:2 there were significant
differences between all three grass species' VAM. In the 1:4 treatment experimental error was probably
the cause of unexpectedly high infection rates of tufted hairgrass. Similar to the 1:1 and 1:8 treatments,
in the 1:4 treatment there was a significant difference between the infectivity of VAM from redtop
bentgrass and smooth brome. Redtop bentgrass VAM was significantly less infective than smooth
brome VAM at all inoculum levels. In all four treatments there were significant differences between
smooth brome VAM and the VAM from one or both of the other two species. It was concluded that
there are significant differences in VAM infectivity based on the source of VAM inoculum material.
Increased examination frequency for infection and a longer growing period may yield more consistent
results in similar infectivity studies. COMPARING INOCULUM POTENTIAL OF VESI CULAR-ARBUSCULAR
MYCdRRHIZA FROM THREE PLANT SPECIES
by
S t u a r t M ic h a e l
L e v it
A t h e s i s s u b m itt e d i n p a r t i a l f u l f i l l m e n t
o f t h e r e q u ir e m e n t s f o r t h e d e g re e
of
M a s te r o f S c ie n c e
in
Land R e h a b i l i t a t i o n
MONTANA STATE U N IV E R S ITY
B ozem an, M o n tan a
May 1989
ii
APPROVAL
o f a th e s is submitted by
S tu a rt L e v it
This th e sis has been read by each member o f the th e s is committee
and has been found to be s a tis fa c to ry regarding content, English usage,
form at, c ita tio n s , b ib lio g ra p h ic s ty le , and consistency, and is ready
fo r submission to the College o f Graduate Studies.
Date
Chairperson, Graduate Committee
Approved fo r the Major Department
Date
Head, Major Department
Approved fo r the College o f Graduate Studies
Z
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting th is th e sis in p a rtia l f u lf illm e n t o f the
requirements fo r a m aster's degree at Montana State U n iv e rs ity , I agree
th a t the L ib ra ry s h a ll make i t a v a ila b le to borrowers under ru le s o f the
L ib ra ry .
B rie f quotations from th is th e s is are allow able w itho ut
special perm ission, provided th a t accurate acknowledgement o f source is
made.
Permission fo r extensive quotation from or reproduction o f th is
th e s is may be granted by my major professo r, or in h is absence, by the
Dean o f L ib ra rie s when, in the opinion o f e ith e r, the proposed use o f
the m a teria l is fo r s c h o la rly purposes.
Any copying or use o f the
m ateria l in th is th e s is fo r fin a n c ia l gain sh a ll not be allowed w itho ut
my w ritte n perm ission.
S ig n a tu re _
Date
/%<✓
7
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iv
ACKNOWLEDGEMENTS
I wish to thank The Spokane Mushroom Club fo r p ro vid in g fin a n c ia l
support o f th is p ro je c t.
I would also lik e to thank Dr. Frank
Munshower, Dr. Douglas D ollho pf, Dr. C liffo r d Mo.ntagne, and Mr. Dennis
Neuman, a ll o f my th e s is committee, fo r t h e ir assistance in many aspects
o f planning and execution.
A d d itio n a l thanks go to Dr. Frank Munshower
fo r his patience and The Reclamation Research U nit fo r m a te ria ls and
work space.
Special thanks go to B ill Grey fo r guidance and m a teria ls
throughout the p ro je c t, Dr. Sharon Eversman fo r advice, enthusiasm, and
tr u s tin g me w ith her microscopes, Dave Baumbauer and Perry H offerber fo r
greenhouse space and assistance, Mike Cormier and Chuck Hardy fo r
a n a ly tic a l assistance, and my fa m ily fo r t h e ir support and tole ran ce.
F in a lly , I would lik e to thank Erich Beagle fo r p roviding in s p ira tio n
and d ire c tio n th a t w ill la s t a life tim e .
V
TABLE OF CONTENTS
Page
LIST OF T A B LE S ...................................................
vi
LIST OF FIGURES....................................................................................................... v i i
ABSTRACT....................................... ■................................................................ . V i i i
INTRODUCTION
......................................................................................................
I
LITERATURE REVIEW...........................................................................,...................
V esicula r-A rbu scular Mycorrhiza .......................................................
VAM Plant R e la tio n s .......................................................................... .
Inoculum and In fe c tio n .......................................................................
Water R e la tio n s ...........................
VAM and. R e cla m a tio n .......................................
VAM and S oil C o n d i t i o n .......................................................................
3
3
5
8
12
14
16
METHODS AND MATERIALS .......................................................................................
Study D e s i g n ..........................................................................................
S ite D e s c r i p t i o n ...................
S o i l s ..........................................................................................................
Primary Vegetation ...............................................................................
C o lle c tio n o f Plant M a teria ls ...........................................................
Root P ro c e s s in g ..........................................................
Greenhouse Care .......................................................................................
P lant H a r v e s t................................................
Root Preparation and A n a ly s is .......................
21
2.1
22
24
27
28
31
33
33
34
RESULTS AND DISCUSSION ...................................................................................
S ta tis tic a l Analysis . . ...................................................................
35
36
SUMMARY AND CONCLUSION.......................................
43
RECOMMENDATIONS ..................................................................................................
46
LITERATURE CITED
..............................................................................................
48
APPENDICES..................................................................
A. In fe c tio n Counts fo r Plant Species By Treatment ................
B. A nalysis o f Variance T a b le s ...............................
54
55
57
vi
LIST OF TABLES
Table
1.
Page
Selected to ta l copper, arsenic, and cadmium
concentrations o f selected s o ils and vegetation
....................
27
2.
Greenhouse conetainer design ............................................................
32
3.
A nalysis o f variance and treatm ent means fo r p la n t and
treatm ent comparison ...........................................................................
37
4.
In fe c tio n counts (%) fo r p la n t species by treatm ent
56
5.
A nalysis o f variance and treatm ent means fo r p la n t
species at the 1:1 treatm ent le v e l ........................
58
A nalysis o f variance and treatm ent means fo r p la n t
species at the 1:2 treatm ent l e v e l ....................................... . . .
58
A nalysis o f variance and treatment-means fo r p la n t
species at the 1:4 treatm ent l e v e l ...............................................
59
A nalysis o f variance and treatm ent means fo r p la n t
species at the 1:8 treatm ent l e v e l ................................................
59
6.
7.
8.
. . . .
vii
LIST OF FIGURES
Figure
1.
Page
Location o f the Grant-Kohrs Ranch National H is to ric
s ite (study s i t e ) ...............................................................................
23
2.
Sample c o lle c tio n lo c a tio n s at the Grant-Kohrs Ranch
. . .
24
3.
Photograph o f tu fte d hairgrass ro o t c o lle c tio n area
. . . .
28
4.
Photograph o f redtop bentgrass ro o t c o lle c tio n area
. . . .
29
5.
Photograph o f smooth brome ro o t c o lle c tio n area ....................
29
6.
In te ra c tio n between Plant Species and Treatment ...................
38
7.
P lant species comparison at the 1:1 treatm ent le v e l
. . . .
39
8.
P lant species comparison at the 1:2 treatm ent le v e l
. . . .
40
9.
Plant species comparison at the 1:8 treatm ent le v e l
. . . .
40
. . .
41
10.
Plant species comparison at the 1:4 treatm ent le v e l
viii
ABSTRACT
Many studies completed in recent years p o in t to the p o te n tia l fo r
increasing land reclam ation success by increasing the ro le o f v e s ic u lararbuscular mycorrhiza (VAM) in revegetation plans. Understanding
m ycorrhizal in fe c tio n in areas o f chemical t o x ic it y may be p a r tic u la r ly
im portant to land r e h a b ilita tio n because o f increased p la n t s tre s s . To
demonstrate the e ffe c ts th a t ta ilin g s m a te ria ls have on VAM in fe c tio n
ra tes the in f e c t iv it y o f mycorrhiza from three grass species growing in ,
near, and away from ta ilin g s enriched s o il were compared.
V e sicu la r-a rb u scu la r m ycorrhizal in fe cte d roots from I) tu fte d
hairgrass growing in ta ilin g s enriched alluvium , 2) redtop bentgrass
growing near ta ilin g s enriched s o il, and 3) smooth brome growing (away
from r iv e r deposited ta ilin g s ) in a g ric u ltu ra l s o il were mixed in 1:1,
1:2, 1:4, and 1:8 d ilu tio n s ( r o o t:s o il by volume) w ith s te r iliz e d
greenhouse s o i l . Control treatments consisted o f s te r iliz e d greenhouse
s o il m ateria l w ith no ro o t inoculum. In the greenhouse, sudangrass,
a ctin g as a p la n t host, was seeded in to these m ixtures and i t s roots
were examined fo r q u a n tita tiv e m ycorrhizal in fe c tio n to assess the
impact th a t ta ilin g s m aterial plays on m ycorrhizal in f e c t iv it y in the
f ie ld .
In the 1:1 and 1:8 treatm ents there was s ig n if ic a n tly lower
i n f e c t iv it y between both tu fte d hairgrass VAM and redtop bentgrass VAM
and VAM from.smooth brome. In the 1:2 there were s ig n ific a n t
d iffe re n c e s between a ll three grass species' VAM. In the 1:4 treatm ent
experimental e rro r was probably the cause o f unexpectedly high in fe c tio n
ra te s o f tu fte d h a irg ra ss. S im ila r to the 1:1 and 1:8 treatm ents, in
the 1:4 treatm ent there was a s ig n ific a n t d iffe re n c e between the
i n f e c t iv it y o f VAM from redtop bentgrass and smooth brome. Redtop
bentgrass VAM was s ig n if ic a n tly less in fe c tiv e than smooth brome VAM at
a ll inoculum le v e ls . In a ll fo u r treatm ents there were s ig n ific a n t
d iffe re n c e s between smooth brome VAM and the VAM from one or both o f the
other two species. I t was concluded th a t there are s ig n ific a n t
d iffe re n c e s in VAM in f e c t iv it y based on the source o f VAM inoculum
m a te ria l. Increased examination frequency fo r in fe c tio n and a longer
growing period may y ie ld more co n siste n t re s u lts in s im ila r in f e c t iv it y
stu d ie s.
I
INTRODUCTION
Vesic u la r-a rb u scu lar mycorrhiza (VAM) are a mutuali s t i c sym biotic
fungi th a t commonly in fe c t species from most p la n t fa m ilie s .
When
n u trie n ts or water are in lim ite d supply VAM provide the p la n t w ith
improved a v a ila b ilit y and in re tu rn receive photosynthate.
Mycorrhiza
are found in a ll geographic regions and under most s o il, topographic,
e co lo g ic, (human) use, and c lim a tic c o n d itio n s .
The ro le and e ffe ctive n e ss o f mycorrhiza va rie s w ith environmental
c o n d itio n s .
T h eir sym biotic re la tio n w ith a p la n t host is most
successful when the p la n t is under some n u tr itiv e or com petitive s tre s s .
Under some con dition s mycorrhiza! in fe c tio n may be detrim ental to the
p la n t host.
D eleterious c o n d itio n s , such as chemical t o x ic it y , th a t
d ir e c t ly e ffe c t p la n t health may also d ir e c tly e ffe c t m ycorrhizal
h e a lth .
Through in o c u la tio n o f seed, p la n ts , and s o il, m ycorrhizal
symbiosis may be u tiliz e d to improve p la n t health and com petitiveness in
a g ric u ltu re , range sciences, and land reclam ation.
Mycorrhiza are
c u rre n tly being used to enhance crop production in some areas.
Current
m ycorrhizal "technology" fo r land r e h a b ilita tio n is m inim al, though much
is known about the e ffe c ts o f land disturbances on m ycorrhizal in fe c tio n
(eg. A lle n and A lle n 1980, Killham and Firestone 1983).
2
The in c lu s io n o f v e s ic u la r-a rb u s c u lar mycorrhiza in land
reclam ation p ro je c t designs has the p o te n tia l to increase revegetation
success because many d ra s tic land disturbances, such as surface m ining,
damage the b io tic p o te n tia l o f the s o il, in c lu d in g m ycorrhiza.
B io tic
p o te n tia l may be reduced by many means in clu d in g the la c k o f to p s o il,
to p s o il storage, s o il m anipulation b rin g in g to x ic m a te ria ls up to the
ro o t zone, and the a d d itio n o f to x ic m in in g /m illin g wastes to the s o il
p r o f ile .
As VAM form mutuali s t i c sym biotic re la tio n s h ip s w ith th e ir
p la n t hosts, th e ir reduction often has measurable negative impacts on
the host p la n t's v ig o r and com petitiveness.
Reclamation p ro je c t
success, th e re fo re , may be lim ite d or hindered where VAM re la tio n s h ip s
are reduced.
The purpose o f th is study was to demonstrate the e ffe c ts th a t mine
t a ilin g s derived alluvium have on VAM in fe c tio n .
The general design was
to compare the in f e c t iv it y o f mycorrhiza from three grass species th a t
had been growing in , near, and away from ta ilin g s d e p o sitio n s.
V e sicu la r-a rb u scu lar m ycorrhizal roots from tu fte d hairgra ss,
redtop bentgrass, and smooth brome growing in or near areas o f a llu v ia l
t a ilin g s deposits were mixed in 1:1, 1:2, 1:4, and 1:8 d ilu tio n s
( r o o t : s o il, v /v ) w ith s te r iliz e d s o il.
Sudangrass, a ctin g as a p la n t
host, was seeded in to these m ixtures and it s roots were examined fo r
q u a n tita tiv e m ycorrhizal in fe c tio n to assess the impact th a t ta ilin g s
m a teria l plays on m ycorrhizal in f e c t iv it y in the f i e ld .
3
LITERATURE REVIEW
Vesicular-Arbuscular Mycorrhiza
Vesic u la r-a rb u scu lar m ycorrhiza, lik e a ll m ycorrhiza, form
mutuali s t i c sym biotic re la tio n s w ith t h e ir p la n t hosts.
They commonly
form endomycorrhizal re la tio n s in most p la n t fa m ilie s o f the
angiosperms, gymnosperms and pterido phytes.
The angiosperms represent
the p la n t group most commonly in fe c te d by VAM, w ith only the
Chenopodiaceae, C rucife rae , Cyperaceae, and Rosedaceae ra re ly in fe c te d .
V e sicu la r-a rb u scu lar mycorrhiza form aseptate fungal s tru c tu re s w ith in
the ro o t cortex o f the host p la n t.
There is no s p e c ific re la tio n known
between VAM species and p la n t species.
Taxonomically d is s im ila r fungi
may have s im ila r e ffe c ts on seedling growth (Johnson 1977).
Mycorrhiza
are common to a ll geographic regions o f the globe and are found in most
eco logica l systems.
Taxonomic a lly , VAM are placed in the Endogonaceae in fo u r genera:
Acaulospora, Gigaspora, Glomus, and S cl er o cys tis.
form ing.
A ll are spore
Acaulospora and Gigaspora reproduce by chlamydospores and
Glomus and S cle ro cys tis species reproduce by azygospores.
Although they
sometimes form ectomycorrhiza and are thought to be re la te d to the VAM,
the m ycorrhiza! forming status o f the Complexipes, Endogone,
Entrophospora, G l a z i e l l a, and Modicella genera is unknown (H a ll, 1984).
V e s ic u la r-a rb u scu lar mycorrhiza! in fe c tio n is not thought to
4
produce any c y to lo g ic a l or anatomical changes to the host p la n ts ' ro o t.
O ccasionally, such as in onions, the in fe c te d ro o t may be colored
y e llo w .
M ycorrhizal in fe c tio n may also re s u lt in s lig h t ly reduced ro o t
size and a reduction in the number o f ro o t h a irs .
This reduction is
presumably because the in fe c tin g fungi is takin g over some o f the
physical gathering/absorbing ca p a c itie s o f the p la n t ro o t.
In fe c tio n
does not occur in the ce n tra l vascular c y lin d e r or m eristem atic regions
o f the ro o t and continuous in fe c tio n throughout the ro o t system ra re ly
occurs.
In fe c tio n is u su a lly sporadic and lo c a liz e d (D aft and N ic o lson
1969).
V esic u la r-a rb u scu lar mycorrhiza fungal s tru c tu re s are
phycomycetous and include in t e r c e llu la r and in tr a c e llu la r hypha,
v e s ic le s , arbuscules, and reproductive c h iamydospores o r zygospores.
These fungi form e xtra m a trica l (outside o f the host ro o t) hyphal
s tru c tu re s but they are d iffe re n tia te d from those o f ectomycorrhiza by
the la ck o f defined hyphal mantle.
c o r tic a l parenchyma.
In te r c e llu la r hypha are found in the
They spread b y .d ila tin g in te r c e llu la r spaces and
form bundles and connections w ith in and between in d iv id u a l hyphal
strands.
Points o f hyphal passage in to the ro ot are often marked by an
appressorium (e xtra m a trica l hyphal spreading) or in tr a c e llu la r fungal
c o n s tric tio n .
When passing between c e lls ; VAM m aintain c e ll wall
c o n tin u ity by fu sin g w ith , and in v a g in a tin g in to , the host c e ll
plasmalemma.
In tr a c e llu la r in fe c tio n is characterized by hyphae passing
s tra ig h t through or looping w ith in a c e ll.
In tr a c e llu la r hyphae do not
branch as a fe a tu re o f hyphal extension.
V esicles are storage organs forming te rm in a lly on hyphae both
5
in te rc e l I u la r ly and in tr a c e lIu la r ly .
T h eir c e ll w a lls are th in at f i r s t
but w ith age the w a lls thicken and t h e ir cytoplasm becomes f i l l e d w ith
lip id s ( f a t t y a c id s ).
Arbuscules form by dichotomous hyphal branching
w ith in a host c e ll and are s ite s o f n u trie n t exchange between mycorrhiza
and p la n t.
Arbuscules survive f o r only a few days and then are
dissolved and absorbed by the p la n t ( ly s is ) .
The repro ductive spores o f
in d iv id u a l mycorrhiza are the prim ary id e n tify in g fe a tu re used fo r
species d e s c rip tio n and c la s s ific a tio n .
VAM Plant Relations
A great deal o f curren t mycorrhiza! in v e s tig a tio n centers on the
basic physiology o f the m ycorrhiza-host p la n t re la tio n s h ip .
While the
mycorrhiza provides the host w ith improved water or mineral n u tr itio n ,
the host provides photosynthate (carbohydrate) to the m ycorrhiza.
The
mycorrhiza q u ic k ly converts these carbon compounds in to fungal carbon
compounds.
This conversion is rapid so th a t there w i l l be a constant
concentration g radie nt from host to heterotroph (Gianinazzi-Pearson and
G ianinazzi 1983).
Concentration g radie nt and photosynthate supply going
to the mycorrhiza may play a ro le in McGonigle and F it t e r 's (1988)
conclusion th a t mycorrhiza do not always supply t h e ir host p la n t w ith
increased n u trie n ts ( p a r tic u la r ly phosphorous) and th e re fo re are not
always m utuali s t i c a l l y sym biotic.
T h e ir experiment involved tra n s p la n t
o f in fe c te d pla nts from greenhouse pots in to the f i e l d .
Mycorrhiza o f
the colonized p la n ts may have th e re fo re been placed in to a s itu a tio n
where they were not b io lo g ic a lly needed.
I t should be noted th a t the
6
ro le o f mycorrhiza is not always b e n e fic ia l.
Phosphate absorption is believed to be the most im portant
m ycorrhizal fu n c tio n .
C arling and. Brown (1982) in d ica te d th a t increased
phosphorous-absorbing surface from hyphae extending in to the rhizosphere
plays a ro le in increased phosphorous uptake.
Mycorrhiza are not
a ttrib u te d w ith the a b i li t y to m o bilize phosphate sources unavailable to
non-m ycorrhizal ro o ts but ra th e r they are thought to possess a pathway
w ith a much higher a f f i n i t y fo r phosphate than non-m ycorrhizal roots
(Gianinazzi-Pearson and Gianinazzi 1983).
The increase in ro o t
absorbing surface provided by e x tra m a tric a l mycorrhiza would also play a
ro le in phosphorous g a the ring.
This would be e s p e c ia lly im portant when
s o il is dry and phosphorous is less m obilg.
Top-dressing w ith
phosphorous f e r t i l iz e r s has been demonstrated to decrease m ycorrhizal
in fe c tio n but the e ffe c t declined two years a fte r a p p lic a tio n on a
Pennine (England) grassland (S parling and Tinker 1978).
Graw (1979)
in d ica te d th a t the phosphorous compound applied, in conjunction w ith pH,
played a ro le in m ycorrhizal in fe c tio n .
S parling and T in ker (1978)
fu r th e r noted th a t lim e, n itro g e n , and potassium f e r t i l iz a t i o n did not
appreciably a ffe c t amounts o f m ycorrhizal in fe c tio n .
D aft and Nicolson (1969) found m ycorrhizal in fe c tio n under
experimental co n d itio n s as high as 90% or as low as 25% (on a g ric u ltu ra l
tomatoes) w ith d iffe r e n t phosphate sources and con centratio ns.
Hayman
and Mosse (1971) s im ila r ly indica ted the e ffe c ts o f mycorrhiza and the
a d d itio n o f a v a ila b le phosphate to be c lo s e ly re la te d and somewhat
s im ila r .
They found th a t in s o ils d e fic ie n t in a v a ila b le phosphate,
responses to added phosphate were s im ila r , equal, or less than those to
7
m ycorrhiza.
The change g e n e ra lly depended on the s o il type and te x tu re
(whether the s o il was a "ph o sp h a te -fixin g " s o il, thus rendering the
phosphorous u n a v a ila b le ).
S oil pH influences the s o lu b ilit y o f diverse
phosphorous compounds d if f e r e n t ly and would also play a ro le in p la n t
a v a ila b ilit y (as w ell as give c o n tra d ic to ry re s u lts on the e ffe c ts o f
s o il pH on VAN) (Graw 1979).
There are no known exclusive VAM-host p la n t re la tio n s h ip s but i t
has been suggested th a t mycorrhiza g e n e ra lly do not grow in the absence
o f a host p la n t (H e tric k 1984).
Powell (1976), however, demonstrated
th a t spores do produce fa n - lik e septate hyphae w itho ut association w ith
a host.
These fa n - lik e s tru c tu re s , however must undergo c y to lo g ic a l
changes before they in fe c t a p la n t.
These changes must occur before
ro o t penetratio n is possible even i f the fungi is a c tu a lly touching the
ro o t (Powell 1976).
V e sicu la r-a rb u scu lar mycorrhiza can form in fe c tio n s
from ro o t fragments (mycelium), spores, and sporocarps (Read et a l .
1976; Sanders and Sheikh 1983) but in fe c tio n rates are fa s te r when ro o t
m a teria l is used as inoculum (H all 1976).
Johnson (1977) fu rth e r
demonstrated th a t detached hyphae were even more in fe c tiv e than ro o t
segments.
Spores, however, are often b e tte r able to m aintain th e ir
v i a b i l i t y during long periods o f storage.
H all (1976) found th a t cessation o f growth by a VAM dependant
shrub (Coprosma robusta) seedling can be re lie v e d by the a p p lic a tio n o f
phosphorous containin g f e r t i l iz e r s or by VAM in o c u la tio n .
When
phosphorous containin g f e r t i l iz e r s were added pla nts had longer growth
.
periods compared to VAM a p p lic a tio n but p la n t weight was lower than when
growth depended only on mycorrhiza.
In terms o f land reclam ation
8
p ro je c t cost and p la n t s u rv iv a l, under many c o n d itio n s , m ycorrhizal
in fe c tio n may be o f g re a te r value than f e r t i l iz a t i o n in shrub and grass
establishm ent.
Because inoculum can be introduced during seeding or
p la n tin g and in tro d u c tio n is a one time treatm ent, costs could be
reduced and reclam ation bond (release) time sta rte d e a r lie r .
Inoculum and Infection
Increasing the amount o f inoculum increases the amount o f ro o t
in fe c tio n (Johnson, 1977; C arling et a l . 1979).
C arlin g and his co­
workers reported th a t in fe c tio n d e n sity in soybean seedlings increased
as inoculum d e n sity increased u n til a plateau was reached: fo r ty grams
o f inoculum produced the same amount o f in fe c tio n as twenty grams.
A fte r th is plateau was reached, no a d d itio n a l increase was reported.
They fu r th e r concluded th a t even extremely low inoculum le v e ls (3
spores) would, by seasons end, be able to produce ro o t c o lo n iz a tio n and
growth response equal to th a t produced by high inoculum (250 spores).
D aft and Nicolson (1969) demonstrated th a t increased inoculum rates
re su lte d in increased in fe c tio n in tomato plants fo r a period o f tim e -w ith the same peak c o lo n iz a tio n rates regardless o f the i n i t i a l inoculum
ra te .
The p a tte rn o f p la n t growth they discovered was unique.
When the
tomato p la n t was inoculated w ith a high number o f spores more apical
leaves developed and more lower leaves were re ta in e d .
Low spore
inoculum pla nts had apical le a f development s im ila r to uninoculated
p la n ts but more basal leaves were re ta in e d .
Daft and Nicolson
postulated th a t the mycorrhiza somehow stim ulated the ontogeny and
9
delayed senescence o f leaves.
T heir a lte rn a tiv e explanation was th a t 4
the e ffe c ts o f inoculum ra te were re la te d to n u trie n t uptake.
As
expected, phosphorous le v e ls in the in fe c te d pla nts were hig her than in
the co n tro l p la n ts .
An im portant d iffe re n c e between the research o f
C arling-and his co-workers and D aft and N ic o lson's study is th a t the
former measured in fe c tio n in seedlings, before growth response would be
d e te cta b le , and the l a t t e r examined mature p la n ts .
C a rlin g and his
associates concluded th a t an increase in the maximum growth y ie ld is
more probable i f the p la n t was in fe c te d from the seedling stage.
Early
in fe c tio n , i t may be expected, would increase p la n t com petitiveness but
since in fe c tio n rates plateaued w ith in one growing season (D aft and
Nicolson 1969; Johnson 1977; C arling et a l . 1979) the com petitive
advantage was sho rt liv e d .
In fe c tio n is u s u a lly associated w ith non-primary ro o t development
(Brown 1956; Rich and B ird 1974; C arlin g et a l . 1979) suggesting th a t a
fix e d period o f time is required fo r m ycorrhizal p e n e tra tio n ,
e lo n g a tio n , and organ development.
There could also be a required delay
between ro o t penetratio n and arbuscule development, which would in d ic a te
n u trie n t exchange (Sutton 1973).
Sutton reported th a t g reate r than 90%
o f m ycorrhizal in fe c tio n o f crop p la n ts occurred in secondary or la te r
ro o ts .
The p o te n tia l advantages o f high inoculum a p p lic a tio n and
subsequent higher e a rly in fe c tio n ra tes include increased p la n t v ig o r
and com petitiveness, e a r lie r m ycorrhizal spore production
(re p ro d u c tio n ), p la n t growth s tim u la tio n , and in the case o f annual or
sh o rt liv e d p la n ts (whether because o f ecology or p la n t p h y s io lo g y ),
maximum m ycorrhizal b e n e fit w ith in the p la n t's growing season (H e tric k
10
1984).
The weight o f VAM-in fe cte d ro ots was determined to be greate st
during summer but the highest percent in fe c tio n was found during w in te r,
suggesting there is a considerable amount o f ro o t turnove r and some
s e lf-re g u la tio n mechanism in inoculum ra te (S parling and Tinker 1978).
The amount ( l i m i t ) o f in fe c tio n o f most p la n t species is also thought to
be constant (Read e t a l . 1976; S parling and Tinker 1978).
Powell (1976)
demonstrated th a t spores g e n e ra lly germinated w ith in 16 days given
adequate co n d itio n s but sporal hyphae did not in fe c t onion roots fo r
long periods o f time or u n til they were in physical co n ta ct.
In
c o n tra s t, hyphae from in fe cte d ro o t segments immediately in fe c te d the
onion ro o ts .
He concluded th is d iffe re n c e between re s tin g spores and
m ycorrhizal ro o t segments is probably because o f d iffe r in g n u trie n t
supplies as w ell as necessary c y to lo g ic a l changes in e a rly sporeproduced hyphae (which are septate and c h a r a c te r is tic a lly d iffe r e n t from
ro o t-a sso cia te d hyphae).
These fin d in g s are supported by A lle n and
A lle n (1980) who ind ica ted th a t in fe c tio n percentages were not h ig h ly
c o rre la te d w ith spore counts.
S ites disturbed by mining often had high
spore counts, even a fte r s o il m ixing, but had low in fe c tio n rates
r e la tiv e to undisturbed range.
This la ck o f c o rre la tio n suggests th a t
many fa c to rs determine inoculum p o te n tia l.
These include p la n t species
genotype, s o il features (n u trie n ts , te x tu re , m o isture ), and s o il
m icro bial a c t iv it y (D aft and N ic o lson 1969; A lle n and A lle n 1980).
Of p a r tic u la r relevance to the cu rre n t study is work by Johnson
(1977) which evaluated the e ffe c ts o f a v a rie ty o f inoculum and s o il
m ixtures on m ycorrhizal in f e c t iv it y .
Johnson found th a t the p ro p e rtie s
11
o f endophytes d i f f e r when growing in d iffe r e n t s o ils (in o ld e r
com m unities).
Host p la n t growth was enhanced when p la n ts were in fe c te d
w ith fungi n a tive to the tested s o il.
Glomus microcarpus (a m ycorrhizal
species) an e ffe c tiv e c o lo n iz e r in a pH 5.7 s o il was compared w ith
Gigaspora gigantea (another m ycorrhizal species), which was an e ffe c tiv e
c o lo n iz e r in pH 3.7 s o il.
Comparing in fe c tio n and p la n t response in
various s o ils both species o f mycorrhiza produced s ig n if ic a n tly la rg e r
p la n ts in the s o il from which they were obtained (adapted).
Two other
experiments by Johnson, however, using d iffe r e n t mycorrhiza and s o ils
showed no s ig n ific a n t d iffe re n c e between in fe c tio n ra te s .
These mixed
re s u lts suggest th a t some mycorrhiza may develop g re a te r s p e c ific it ie s
than others or s o il c h a ra c te ris tic s to which mycorrhiza are s p e c ific may
be a lte re d by experimental con dition s (Johnson 1977).
S im ila rly , Lambert and Cole (1980) concluded th a t there were
d iffe re n c e s between m ycorrhizal species in fe c tin g w hite c lo v e r.
Plant
growth under low pH con dition s was s ig n if ic a n tly g re a te r when plants
were inoculated w ith fungal species from acid s ite s (pH < 4 .0 ).
Growth
o f p la n ts in fe cte d w ith non-acid-adapted mycorrhiza was s ig n ific a n tly
g re a te r at higher pH.
While examining the e ffe c t o f pH on two p la n t
species in fe cte d w ith the same m ycorrhizal s tra in [Glomus macrocarpus)
Graw (1979) found th a t p la n t species has a strong in flu e n c e on the
e ffic ie n c y o f VAM at d iffe r e n t pH values and w ith d iffe r e n t P-compounds.
In most.cases the fungus was more e f f ic ie n t at higher pH values.
Higher
pH values are g e n e ra lly more d e sirab le fo r land reclam ation but these
re s u lts suggest th a t where lower pH is a fa c to r, s p e c ific p la n t,
f e r t i l i z e r , and fungal combinations may increase revegetation success.
12
Lambert and Cole concluded th a t e s ta b lis h in g mycorrhiza in mining wastes
is useful i f indigenous inoculum is absent or non-adapted, a v a ila b le
phosphorous is low, and the reclam ation p la n t species are m yco rrh iza !.
Water Relations
The removal o f e x tra ra d ic a l hyphae from clo ve r and leek was shown
to decrease p la n t water pressure (Hardie 1985).
The author concluded
th a t the reduction in e x tra ra d ic a l hyphae reduced p la n t absorptive
cap acity and hence p la n ts had d i f f i c u l t y m aintaining tra n s p ira tio n
le v e ls a fte r tra n s p la n tin g .
The experimental design was d iffe r e n t from
most oth er VAM response comparison studies because VAM in fe c te d plants
were used fo r both the experimental and co n tro l treatm ents.
A fte r
desired i n i t i a l growth was achieved the e x tra ra d ic a l hyphae o f the
experimental p la n ts were removed.
There is a p o s s ib ility th a t the
physical m anipulation and exposure o f the -roots played a ro le in the
study re s u lts but the method provides a more natural standard fo r VAM
comparison.
The advantage o f using id e n tic a lly in fe c te d pla nts is th a t
the n u tr itio n a l s ta tu s , p a r tic u la r ly the phosphorous status o f both
p la n t groups is ensured (Hardie 1985).
Bradley et a l . (1982) and Gildon
and T inker (1983a) pointed out the need to consider the amount o f
a v a ila b le phosphorous in the s o il when judging m y c o rrh iz a 's e ffe c ts on
pi ants.
A lle n and Boqsalis (1983) reported the presence o f mycorrhiza did
not a ffe c t p la n t dry weight but increased stomata! conductances.
Stomatal closure occurred at lower le a f water p o te n tia ls and a fte r
13
g re a te r de sicca tio n in m ycorrhizal pla nts than in non-mycorrhizal
p la n ts .
Glomus f a s c i c u l a tu s increased w in te r wheat drought tolerance
but G. mosseae did n o t, suggesting th a t both p la n t species and
mycorrhiza species must be taken in to con sideration .
H e tric k et aI (1987) reported th a t big bluestem be nefited by
m ycorrhizal asso ciation under c y c lic and severe drought stre ss but corn
and sudangrass did no t.
They pointed out a re la tio n s h ip between non-
amended and phosphorous amended p la n ts ; drought stressed m ycorrhizal
p la n ts w ith o u t phosphorous amendments were not la rg e r than nonm ycorrhizal p la n ts th a t were f e r t i l iz e d .
The plants apparently were
more dependant on the mycorrhiza fo r phosphorous than fo r water
r e la tio n s .
Nelson and S a fir (1982) demonstrated th a t drought tolerance
was improved by m ycorrhizal association in f e r t iliz e d onions but th is
m ycorrhizal growth response was elim inated under drought stress in a
s im ila r experiment using corn (H e tric k e t . a l. 1987).
Factors c ite d as
leading to these c o n tra d ic to ry re s u lts include the s e v e rity o f drought
s tre s s , d i f f i c u l t y in m aintaining constant la b o ra to ry c o n d itio n s , s o il
type, and in d iv id u a l p la n t responses (H e tric k e t . a l. 1987).
With these fa c to rs in mind, the conditions (m ycorrhizal in fe c tio n ,
s o il type, m oisture regime, e tc .) responsible fo r p la n t v ig o r or growth
may be re la te d to , but not dependant on, m ycorrhizal in fe c tio n .
The
most co n siste n t p la n t responses to VAM in fe c tio n occur as a re s u lt o f an
improved phosphorous n u trie n t regime (Cooper, 1984) but p la n t s p e c ific
response to VAM in fe c tio n would play an equal or g re a te r ro le than s o il
fe r t ilit y
(Azcon and Ocampo, 1981).
14
VAM and Reclamation
The degradation o f the b io tic environment as a re s u lt o f land
disturbances, in c lu d in g mining, may include reduced to p s o il volume,
m ixing to p s o il w ith buried and/or to x ic m a te ria ls , and the a d d itio n o f
to x ic m a te ria ls to the ro o t zone.
Plant succession fo llo w in g land
disturbance also plays a ro le in m ycorrhizal re la tio n s h ip s .
On
d istu rbe d rangeland in northern Colorado, .Reeves and co-workers (1979)
demonstrated th a t prim ary succession is dominated by non-mycorrhizal
p la n ts o f the Chenopodiceae and Brassicaceae fa m ilie s .
M ille r (1979)
also showed th a t species o f the Chenopodiceae fa m ily were the dominant
prim ary co lo n ize rs o f disturbed rangeland in Wyoming.
Because only one
percent o f the prim ary colo nizers on disturbed lands were m ycorrhizal
compared to 99% on adjacent undisturbed rangeland Reeves and his
associates concluded th a t in the disturbed (mycorrhiza d e fic ie n t)
range!and, non-mycorrhizal species were more com petitive than the
m ycorrhizal species.
Furthermore, the non-mycorrhizal species may
hinder successional stages because they do not provide an inoculum
source fo r la te r successional species which re q u ire m ycorrhizal
associations fo r s u rv iv a l (Moorman and Reeves 1979; Reeves et a l . 1979).
M ille r (1979) examined VAM in fe c tio n o f plants on disturbed and
u n d is tu rb e d /s ite s in the red desert (Wyoming) and concluded th a t
re p ro ductive stra te g y and m ycorrhizal in fe c tio n play major ro le s in
species establishm ent and com petitiveness fo llo w in g disturbance.
Of
note was halogeton (Halogeton glomeratus) which comprised over 90% o f
to ta l p la n t cover at the s ite .
VA m ycorrhizal spores {Glomus spp.) were
15
present but through an unknown mechanism, p o ssibly a lle lo p a th y by
halogeton, the spores were not in o c u la tin g plants at the s ite .
A
p la n t's a b i l i t y to become colonized w ith mycorrhiza would be o f no
b e n e fit in an environment such as th is .
Therefore, a p la n t must assume
a non-m ycorrhizal ruderal reproductive stra te g y to e s ta b lis h ( M ille r
1979).
In t h e ir study o f Colorado rangeland, Reeves et a l . (1979) found
th a t the Poaceae fa m ily is one o f the dominant fa m ilie s in primary
succession.
While most o f the pla nts o f Poaceae are m yco rrh iza !, only a
lim ite d number are commonly found growing in to x ic environments.
One explanation fo r non-mycorrhizal p la n t species and p la n t
communities was presented by Tester and co-workers (1987) who proposed
th a t fu n g ito x ic compounds in c o r tic a l ro o t tis s u e or ro o t exudates may
reduce p la n t s u s c e p tib ility to fungal in fe c tio n .
They a d d itio n a lly
proposed th a t in fe c tio n may be lim ite d or halted by a la c k o f ro ot
exudation (the VAM not re ce ivin g adequate -n u trie n ts to develop or
sustain in fe c tio n ) , poor exudate q u a lity , or the la c k o f compounds
required in minute (p u ta tiv e ) q u a n titie s to " s ig n a l" in fe c tio n .
Tester
and his asso ciates' fin a l theory suggests th a t c e ll w all or la m e lla r
in te ra c tio n prevents mycorrhiza! p e n e tra tio n .
This hypothesis is
supported by th e ir observation o f ro ots h e a vily e n c irc le d by hyphae but
w ith no in fe c tio n in to the ro o ts .
Reducing to p s o il through removal or mixing reduces the number o f
spores and the amount o f a v a ila b le inoculum m a te ria l.
Topsoil storage
p r im a rily reduces the v i a b i l i t y o f ro o t inoculum m a teria l but a lso , to a
le sse r degree, spore v ig o r (Rives e t a l . 1980; L ib e rta 1981; Hardie
1985).
A lle n
and A lle n (1980) studied the natural reestablishm ent o f
16
VAM fo llo w in g strip m in e reclam ation.
They found th a t mixing to p s o ils
w ith subsoils reduced i n i t i a l spore counts by 5% to 25% (compared to
undisturbed s ite s ) but th a t m ycorrhizal in fe c tio n was up to 50% o f
undisturbed a fte r three years.
Waal and and A lle n (1987), however,
suggested th a t the kind o f s o il substrate was more im portant than time
in determ ining co n d itio n s fo r spore inoculum.
Comparing I to 6 year and
10 to 31 year old reclaimed s o il, reclaimed s p o il, and undisturbed range
they found th a t there was no re la tio n s h ip between time from
distu rba nce/reclam ation and spore d e n s ity .
The most im portant o f the
s p e c ific experiment re s u lts was th a t to p s o ile d s ite s almost always had
s ig n if ic a n tly higher spore counts than reclaimed or orphaned s ite s
in d ic a tin g the importance o f to p s o il salvage and c re a tin g a favorable
m ic ro b io tic environment through reclam ation.
VAM and Soil Condition
V e s ic u la r-a rb u scu lar m ycorrhizal response to to x ic m a teria ls are
the most d i f f i c u l t m ycorrhiza-reclam ation re la tio n s h ip to study because
many fa c to rs play a ro le in p la n t response.
These fa c to rs include p la n t
species, mineral n u tr itio n , s o il type and chem istry, water supply, and
the type and degree o f to x in .
Tufted hairgrass {Deschampsia
cae spitosa), the p la n t species being considered in th is in v e s tig a tio n ,
is known fo r i t s metal tolerance (Cox and Hutchinson 1980; Cahoon 1983).
In th e ir experiment examining weedy and c o lo n iz e r species'
m ycorrhizal in fe c tio n , Pendleton and Smith (1983) discovered th a t over
o n e -h a lf o f the 75 weedy species they evaluated were m yco rrhiza l.
The
17
presence or absence o f in fe c tio n was s t r i c t l y w ith in fa m ily taxonomic
d iv is io n s and they concluded th a t fa m ily connection was more im portant
than weedy h a b it in determ ining m ycorrhizal in f e c t iv it y .
F la t semi a rid
s ite s were dominated by non-mycorrhizal species whereas rocky sloped
s ite s were dominated by m ycorrhizal species having deeply pe netrating
ro o t systems.
These re s u lts , p a r tic u la r ly w ith respect to the non-
m ycorrhizal sem i-arid s ite s , in d ic a te the importance o f the ro le th a t
water a v a ila b ilit y plays in determ ining the percent c o lo n iz a tio n and
to ta l number o f m ycorrhizal co lo nizers (Reeves et a l . 1979; Pendleton
and Smith 1983; H e tric k 1984).
Among the past th e o rie s to why tu fte d hairgrass and co lo n ia l
bentgrass (Agrostis t e n u i s ), another metal to le ra n t species, are often
found growing in metal to x ic and low pH areas is th a t mycorrhiza act as
f i l t e r s , p ro te c tin g the p la n t from the metals (Gildon and Tinker 1981;
Bradley et a l . 1982; Gildon and Tinker 1983a; Killham and Firestone
1983; Ferns 1984).
The general conclusion o f these studies o f the
m y c o rrh iz a -a s -filte rs the ory, however, is th a t VAM do not p ro te c t the
p la n t from heavy metal, contamination, and may in fa c t act as a pathway
fo r increased p la n t uptake.
Studies examining the ro le o f mycorrhiza. in
to x ic environments can be broken in to two groups: I) m ycorrhiza-plant
symbionts th a t have evolved in a to x ic environment, and 2) mycorrhizapl ant symbionts th a t have survived tog eth er in an environment ra p id ly
made to x ic by human a c t iv it ie s such as m in in g /m illin g .
In serpentine s o ils , which are c h a r a c te r is tic a lly high in iro n ,
n ic k e l, and magnesium but low in n itro g e n , potassium and phosphorous,
Hopkins (1987) determined th a t 26 o f 27 established herbaceous species
18
were m ycorrhizal (23 o f these m ycorrhizal species had almost s o lid
in fe c tio n o f the c o rte x ).
cover.
Annuals made up 84.4% o f the herbaceous
She proposed th a t shallow s o il (20cm) and poor parent m aterial
(u ltra -m a fic rock) may have co n trib u te d to n u trie n t and water stress
thus cre a tin g an obvious niche fo r m ycorrhiza.
The uniqueness o f heavy
c o lo n iz a tio n o f annuals in poor s o il also suggests th a t the ro le o f
p la n t d e n s ity , e a rly c o lo n iz a tio n , and ra pid c o lo n iz a tio n were fa c to rs
a llow in g the symbiosis to succeed (Hopkins 1987).
Gilden and Tinker (1981,1983a,b) and Bradley et a l . (1982) also
performed experiments on n a tu ra lly occurring to x ic s o ils .
Bradley and
co-workers concluded th a t the hyphal complexes o f the mycorrhiza
provided adsorptive surfaces fo r m etals, thereby p ro te c tin g the p la n t by
e ffe c tiv e ly binding to x ic metals in the ro o t c o rte x.
In two o f th e ir
experiments, Gildon and Tinker (1981, 1983a) concluded th a t a p a rtic u la r
s tra in o f c lo v e r-in fe c tin g mycorrhiza th a t had grown n a tu ra lly on
h e a v ily zin c and cadmium contaminated s ite s was p ro te c tin g it s host from
absorbing some o f the metals.
The second o f the two papers (Gildon and
T inker 1983a), however, suggested th a t in the act o f f i l t e r i n g metals
the mycorrhiza may not be providing adequate phosphorous to the p la n t.
Less promising were the conclusions o f Gildon and T in k e r's th ir d paper
(1983b) which in d ica te d th a t copper uptake in leeks could be reduced by
decreasing m ycorrhizal in fe c tio n rates by adding phosphorous.
In c o n tra d ic tio n to the above lit e r a t u r e , studies o f mycorrhizap la n t re la tio n s in environments made to x ic by man suggest th a t
mycorrhiza may increase p la n t absorbance o f heavy m etals.
Plants
in fe c te d w ith VAM and growing in s o il contaminated w ith a c id ic and heavy
19
metal de positions were shown to have higher tis s u e metal le v e ls and
s ig n if ic a n tly reduced growth compared to non-mycorrhizal co n tro ls
(K illham and Firestone 1983).
These authors found th a t under the
co n d itio n s o f a pH o f 5.6 and high m etals, m ycorrhizal. and nonm ycorrhizal p la n ts had s im ila r metal loads (e s p e c ia lly copper and
n ic k e l, and to a le sse r degree, lead and z in c ) .
The same metal load in
p la n ts grown in m a te ria ls e x h ib itin g pH values less than 3.0 re su lte d in
257% and 309% increases in copper and n icke l concentration in
m ycorrhizal p la n ts .
This is supported by Ferns (1984) who found th a t
VAM from p la n ts growing in a ta ilin g s pond increased tra n s p o rta tio n o f
to x ic metals to a non-metal or low pH to le ra n t host.
K illham and Firestone (1983) noted th a t metals e n te rin g the s o il
from the atmosphere could be complexed in a high c la y or organic s o il
but would be more a v a ila b le in sandy s o ils .
Where atmospheric or
uncomplexed metals are common, such as around smokestacks and m illin g
operations, th e re fo re , mycorrhiza would not be a d e s ira b le land
reclam ation fe a tu re .
In fu r th e r support o f these explanations o f metal tolerances,
Cahoon (1983) examined the in tr a s p e c ific d iffe re n c e s in heavy metal
accumulation and d is tr ib u tio n in tu fte d hairgrass.
He concluded th a t a
race o f tu fte d hairgrass th a t was c o lle c te d on a m e ta llife ro u s waste
s ite had g re a te r metal tolerance (accumulated less cadmium or copper)
than a race grown from commercial seed.
P hysiological d iffe re n c e s in
the uptake and tra n s p o rt o f metals w ith in the pla nts were also noted.
The general trend in the lit e r a t u r e is th a t VAM in fe c tio n and
spore production increase, to a p o in t, as s o il pH increases (Graw 1979).
20
Change in pH is lik e ly to a ffe c t p la n t a v a ila b le phosphorous, thereby
in flu e n c in g m ycorrhizal c o lo n iz a tio n .
Mosse and P h illip s (1971)
demonstrated th a t high le v e ls o f phosphorous or nitroge n may in h ib it VAM
in fe c tio n ra te s .
A p la n t's c o n d itio n , w ith regard to n u trie n ts and
w ater, would determine i t s degree o f m ycorrhizal in fe c tio n .
I t has
th e re fo re been concluded by a number o f authors th a t the n u tr itio n a l
health o f the p la n t d ir e c tly c o n tro ls in fe c tio n ra te ra th e r than the
f e r t i l i t y o f the s o il (Menge et a l . 1978; H e tric k 1984).
21
METHODS AND MATERIALS
Study Design
A greenhouse study was implemented to determine whether the
inoculum p o te n tia l o f VAM is impacted when the fungus is found on pla nts
growing in an in h ib ito r y ta ilin g s environment.
The general stra te g y o f
the design was to mix VAM-colonized roots from p la n ts growing in and
near areas o f ta ilin g s de position (as inoculum m a te ria l) w ith s te r iliz e d
s o il and grow host p la n ts whose roots would be examined fo r in fe c tio n .
Roots were chosen as the inoculum m a terial because much o f the s o il
associated w ith s o il/s p o re inoculum fo r tu fte d hairgrass could contain
t a ilin g s m a te ria l.
A d d itio n a lly , because o f time c o n s tra in ts and to
minimize s ite disturbance spore inoculum was not used.
. P lant roots were c o lle c te d from I) tu fte d hairgrass [Deschampsia .
caespitosa (L .) Beauv.) growing in a m ixture o f s o il and ta ilin g s
m a te ria ls deposited by the C lark Fork R iver, 2) Redtop bentgrass
{Agrostis alba L .) growing near the r iv e r but not d ir e c t ly in ta ilin g s
contaminated s o il, and 3) Smooth Brome (Bromus inermis Leyss.) growing
in a g ric u ltu ra l s o il th a t has been unaffected by t a ilin g s c a rrie d in the
r iv e r .
Root c o lle c tio n depth ranged from 15 to 60 centim eters.
S oil
was determined to be ta ilin g s enriched based on S oil Conservation
Service s o il d e s c rip tio n s and metal contamination data from Rice and Ray
22
(1984), and soil pH data taken from across each collec tio n area.
Because A gro sti s alba tends to take on the aspect o f A. s t o l o n i f e r a and
the two species appear to intergrade (Hitchcock and Chase 1971),
A gro sti s alba w ill re fe r to both species fo r the purposes o f th is paper.
Root m ateria l from each grass species was mixed w ith s te r iliz e d s o il in
d ilu tio n s o f ( r o o ts r s o il, by volume) 1:1, .1:2, 1:4, and 1:8 and seeded
w ith sudangrass {Sorghum sudanense (P iper) S ta p f.).
Roots from each o f
the p la n ts and treatm ents were harvested and examined fo r q u a n tita tiv e
VAM in fe c tio n at two d iffe r e n t growth stages (two le a f and fiv e le a f) .
Site Description
When I f i r s t reached Montana, the Deer Lodge Valley
was one o f the most b e a u t i f u l stretches o f bunch
grass country imaginable. The grass waved l i k e a
huge f i e l d o f grain.
Conrad KohrS (NPS 1987)
John Grant brought a herd o f c a ttle to the Deer Lodge V alley in
the e a rly 1800's.
These c a ttle , along w ith others acquired on immigrant
t r a i l s such as the Mormon and Oregon T r a ils , formed the foundation fo r
Montana's C a ttle in d u s try .
Around the tu rn o f the century, the Ranch
had holdings o f 30,000 acres and grazed one to fiv e m illio n acres
throughout the Northern Rockies s ta te s .
The Grant-Kohrs Ranch National
H is to ric s ite was established to provide an understanding o f the
f r o n t ie r c a ttle era and preserve the Grant-Kohrs ranch because o f it s
h is to r ic s ig n ific a n c e (NPS 1987).
The Ranch is located in Deer Lodge,
Montana (Powell County T8N, R9W, Sec28) (Figure I ) .
23
Figure I .
Location o f the Grant-Kohrs Ranch National H is to ric s ite
(study s i t e ) .
Samples were co lle c te d along the C lark Fork River in the west
ce n tra l p a rt o f the Grant-Kohrs Ranch (Figure 2).
The headwaters o f the
C lark Fork is formed by the convergence o f Warm Springs Creek (SW from
Anaconda), S ilv e r Bow Creek (SE from B u tte ), and sm aller creeks and
streams.
The Clark Fork forms the drainage o f the west slope o f the
c o n tin e n ta l d iv id e and the east slope o f the F lin t Creek Range (P in tle r
Mountains).
The S ilv e r Bow and Warm Spring Creeks d ra in the mining and
sm elting areas o f Butte and Anaconda and together w ith the C lark Fork,
have a h is to ry o f ca rryin g and spreading mining waste and ta ilin g s
m a te ria ls along th e ir flo o d p la in s .
24
Powell County, Montana
Section 29
Section 28
T8N, R9W
DeCa C o lle ction Area >>>|,>'>,!>)
AgAl C o lle ctio n Area
B rIn C olle ction Area ' W W
Slickens
025 50
150 200
Meiers
T yp ic Fluvaquent
Figure 2.
Rice and Ray Sam ple Site
1-6
Numbers correspond to date
on Table I
/
T ypic H a p la q u o ll
Sample c o lle c tio n lo c a tio n s at the Grant-Kohrs Ranch.
Soils
The s o ils o f the west cen tral p a rt o f the ranch, where tu fte d
hairgrass and redtop samples were taken, were c la s s ifie d as "Typic
F luvaquent, mixed; f r i g id ; u n d iffe re n tia te d " (SCS 1988).
The c u ltiv a te d
s o il where smooth brome roots were taken was c la s s ifie d as Typic
H ap la qu oll, fine-loam y, mixed.
A number o f areas were c la s s ifie d as
"s lic k e n s " by the S oil Conservation S ervice.
These were defined as "an
25
u n d iffe re n tia te d s o il type c on sisting o f accumulation o f fin e -te x tu re d
m a te ria ls , such as are separated by placer mine and ore m ill operations.
Slickens from ore m i lls consist la rg e ly o f fre s h ly ground rock th a t
commonly has undergone chemical treatment during m i l l i n g or smelting
processes" (SCS 1988).
Slickens s ite s determined by Rice and Ray (1984)
are i d e n t i f i e d in Figure 2.
Rice and Ray discovered th a t arsenic le v e ls
in sediments in the slickens and grasses (tu fte d hairgrass) growing near
t h e i r perimeter were as high as o r ig in a tin g adjacent to actual smelter
s ite s .
Preparation f o r s o il pH determination consisted o f mixing 50 grams
o f s o il (dry) w ith 100 m i l l i l i t e r s o f d i s t i l l e d water and l e t t i n g the
mixture stand f o r 45 minutes (Ruddell and Montagne).
Five samples were
c o lle c te d from across each o f the three p la nt c o lle c tio n areas.
Hydrogen ion concentrations at the c o lle c tio n s ite s ranged from 4.5 to
6.0 f o r tu fte d hairgrass (average 5 .0 ), 5.0 to 6.5 f o r redtop bentgrass
(average 5 .5 ), and 5.0 to 6.8 f o r smooth brome (average 6 .4 ).
numbers in d ic a te l i t t l e
These
change from those values determined by Rice and
Ray (1984) and Ray (1985).
Ray reported pH values to range from
approximately 4.2 to 6.8.
There was no change in f l o r a l composition, s o il d e s c rip tio n , or pH
between the 1984 survey and those established in the course o f th is
study (Figure 2).
For the d e s c rip tiv e purposes o f t h i s study the heavy
metal concentrations they reported were accepted as accurate.
Total
s o il and p la n t accumulated copper, arsenic, and cadmium concentrations
f o r Rice and Ray (1984) sample s ite s located close to the root
c o lle c t io n zones f o r t h is study are presented in Table I .
Table I also
26
presents metal concentrations they found at the Tin Cup Joe Creek Check
P lot (Figure I ) .
Rice and Ray established th is check p lo t on the
f l u v i a l contour o f the Tin Cup Joe Creek because the area had no h is to ry
o f water borne metal contamination and was formed from si mi l i a r parent
m aterial (Quartenary g la c ia l outwash p la in ) as s o ils o f the Grant-Kohrs
Ranch.
Metal contamination le v e ls were s i m i l i a r (highest metals closest
to the r i v e r , decreasing moving away from the r i v e r ) along the length o f
the Clark Fork th a t they examined.
There was a metal gradient in s o ils decreasing away from the Clark
Fork River.
1984).
This was consistent with f l o r a l composition (Rice and Ray
Tufted hairgrass was found most commonly along the Clark Fork in
i n h ib i t o r y zones o f floo d event t a i l i n g s deposition.
Redtop bentgrass
was found in tr a n s itio n s between the rip a r ia n and meadow zones.
I t was
also found in is o la te d c lu s te rs along the r i v e r .
Rice and Ray (1984) reported th a t the concentrations o f copper,
cadmium, and arsenic in s o ils along the Clark Fork River at the GrantKohrs Ranch were g re a tly elevated r e la t iv e to t h e i r control p lo ts .
They
demonstrated th a t metal laden sediments were c arried by the Clark Fork
and deposited on the flo o d p la in o f the Grant-Kohrs Ranch.
The highest
metal concentrations were found in the r ip a ria n flo o d p la in , p a r t i c u l a r l y
along the r i v e r and in old channels and sloughs where sedimentation had
taken place.
The highest metal concentrations in the meadows were found
adjacent to the i r r i g a t i o n d itc h and were concluded by Rice and Ray
(1984) to be a r e s u lt o f flood overflow and sediment deposition or
sediment maintenance removal.
r e s u lt o f i r r i g a t i o n .
These concentrations could also be a
27
Table I .
Selected t o t a l copper, a r s e n ic ,. and cadmium concentrations o f
selected s o ils and vegetation (Dry w t . ) (Rice and Ray 1984).
I
uq/q in
s o ils (0-25cm)
Cu
2851
As
684
Cd
4.6
2
Samole S ite i
4
5
_^
1721
332
3.8
2701
50
4.8
uq/q in
qrass (A qrostis alba)
Cu
12
15
As
1.9
1.0
Cd
0.2
0.1
8.1
0.6
0.06
10
1.3
0.03
6
92
40
2.0
6.2
1.2
0.07
Tin Cup Joe
Creek3
94
22
1.8
5.9
3.5
0.03
51
20
1.7
7.2
1.6
0.05
1Sample s it e numbers correspond to those labeled in Figure 2.
2Soil metal contamination data fo r t h is s ite unavailable.
3Tin Cup Joe Creek Check P lo t. See Figure I .
Primary Vegetation
Slicken areas in the western parts o f the Ranch were e ith e r barren
or monocultures o f tu fte d hairgrass.
Fallen and dead stands o f slender
w illo w [ S a l i x exigua N u t t . ) were scattered through these areas.
Other
rip a r ia n areas were c la s s ifie d as redtop bentgrass-tufted hairgrass and
redtop bentgrass-slender w illo w communities (Rice and Ray 1984).
The
only associated species in the c o lle c tio n area was thread rush (Juncus
f i l i f o r m i s L .) , although smooth brome, Kentucky bluegrass [Poa pratensis
L . ) , white clove r { T r i f o l i u m repens L . ) , and black cottonwood {Populus
trichocarpa T&G.) were noted near rip a ria n areas f a r th e r away from
slickens (Rice and Ray 1984).
Some redtop bentgrass roots were taken
from areas close r to the hay meadows than the rip a ria n and slicken zone.
These redtop bentgrass c o lle c tio n s ite s were populated by Kentucky
28
bluegrass, meadow f o x t a il [Hordeum brachyantherum Nevski), and smooth
brome.
Rice and Ray characterized the hay meadow c o lle c tio n area as a
meadow foxtail-sm ooth brome community.
The observed associated species
were Kentucky b l uegrass, redtop bentgrass, crested wheatgrass [Agropyron
cristatum (L .) Gaertn.), and white clove r.
Collection of Plant Materials
Tufted hairgrass roots were randomalIy collected from areas along
approximately 100 meters o f the Clark Fork River (Figure 3).
Redtop
bentgrass roots were collected along a roughly p a ra lle l lin e but fu r th e r
away from the r i v e r than the tu fte d hairgrass (Figure 4).
Smooth brome
roots were collected along a visual transect 75 meters long (Figure 5).
Figure 3.
Photograph of tu fte d hairgrass root c o lle c tio n area.
29
Figure 5.
Photograph of smooth brome root c o lle c tio n area.
30
The tu fte d hairgrass c o lle c tio n area was sparsely to densely
populated w ith tu f te d hairgrass and surrounded by slender w illo w .
Tufted hairgrass stands not selected f o r ro ot c o lle c tio n were generally
monocultures and were surrounded by dead w illow s.
resembled slickens areas.
Fork River.
These areas often
Many plants were w ith in 5 meters o f the Clark
The redtop bentgrass c o lle c tio n area was densely populated
w ith redtop bentgrass, thread rush, and c lu s te rs o f slender w illo w .
Tufted hairgrass was sometimes present, most notably on the frin g e of
the less dense redtop bentgrass stands.
This observation is in keeping
w ith tu fte d ha irg ra ss' c h a ra c te ris tic poor competitiveness except under
adverse s o il con dition s.
The smooth brome c o lle c tio n area was densely
populated w ith smooth brome, meadow f o x t a i l , and kentucky bluegrass.
On fo u r occasions p r io r to ro o t inoculum c o lle c t io n , sample roots
from a l l three species were colle c te d from across a wide area and
analyzed f o r mycorrhizal in fe c tio n in order to guarantee th a t infected
roots were used in the experiment.
Only monoculture stands o f tu fte d
hairgrass and plants growing along the r i v e r ' s edge were found to be
non-m ycorrhizal.
As a metal to le ra n t but not otherwise competitive
species (Gaboon 1983), i t was no surprise th a t tu fte d hairgrass plants
growing w ith no species competition (suggesting extreme in h ib it o r y
c on dition s) were non-m ycorrhizal.
Tufted hairgrass apparently loses i t s
com petitive edge as t o x i c i t y decreases.
The roots from at le a s t 35 plants throughout each p la n t's area
were c o lle c te d to assure th a t a representative sample was obtained.
most cases the e n tire ro ot mass was gathered.
shaken to remove as much s o il as possible.
In
The ro o t mass was g e ntly
Al I ro ot c o lle c tio n and
31
preparation was done separately f o r each p la nt species.
Soil samples
were taken approximately every 10 meters along the c o lle c tio n area f o r
pH determ ination.
In the course o f searching f o r appropriate s ite s f o r root
c o lle c t io n , plants from many areas along the S ilv e r Bow Creek (flow ing
west through Butte, Montana) were examined fo r VAM in fe c tio n .
In fe c tio n
was g e nerally found in those plants growing above the creek's flood
leve l or in mixed p la nt groups.
Areas adjacent to the creek or those
he avily flooded were most often found to be tu fte d hairgrass
monocultures and they were ra re ly in fe c te d .
Although the water ta b le in.
the areas examined was often only 20 to 40 centimeters below the
surface, these areas were presumably too to x ic fo r mycorrhizal s u rv iv a l.
Root Processing
Roots (and attached s o i l ) were cut in to one to two centimeter
pieces and l i g h t l y agitated over 4 mm and 2 mm screens to remove s o i l .
The mixture was homogenized, placed over 2 mm and .991 mm screens and
l i g h t l y washed w ith d i s t i l l e d water to remove as much remaining s o il as
possible w ithout damaging fin e roots or extram atrical hyphae.
The water
rin s e was intended to remove s o il and spores from the ro o t inoculum.
The ro o t material was mixed with an equal volume o f steam s t e r i li z e d
greenhouse s o il (30% Bozeman S i l t Loam, 25% Humus, 35% Sand),
homogenized, allowed to stand fo r 30 minutes, and homogenized again.
The 30 minute standing period was provided to minimize ro o t material
32
"clumping" by allowing the s o il to absorb some o f the moisture from the
wet ro ots.
The r o o t - s o il mixture was added to s t e r i li z e d greenhouse s o il and
homogenized to create d ilu t io n s (v /v ) o f 1:1, 1:2, 1:4, and 1:8.
P la s tic conetainers (15 cm X 2.5 cm) were f i l l e d w ith these root s o il
m ix tu re s .
Controls were prepared from s t e r i li z e d s o il alone.
c o n tro ls were prepared fo r each treatment and species.
Three
Ninety
conetainers were f i l l e d with the s o il mixtures (Table 2), comprising
three r e p e titio n s o f the fiv e treatments (fo u r r o o t : s o il d ilu t io n s and
the c o n t r o l) fo r each o f the three p la nt species and two complete
harvests.
The conetainers were randomly placed in holders.
Four
sudangrass seeds were placed approximately 3 mm in to the s o il o f each
conetainer.
Table 2.
Greenhouse conetainer design.
Plant Species
Repetitions
Di I u t io n s 1
Harvests
Tufted hairgrass
3
3
3
3
1:1
1:2
1:4
1:8
2
2
2
2
Redtop bentgrass
3
3
3
3
1:1
1:2
1:4
1:8
2
2
2
2
Smooth brome
3
3
3
3
1:1
1:2
1:4
1:8
2
2
2
2
12
1:0
2
Control
1RootiSoil by volume.
33
Greenhouse Care
Plants began emerging in the greenhouse a ft e r fo u r days.
A dditio n a l seeds were placed in to conetainers where less than three
plants emerged.
Six days a ft e r emergence, the smallest p la nt was cu lle d
from conetainers where a l l fou r o r ig in a l seeds had germinated.
Plants
were watered every one to three days, depending on drying conditions,
w ith 5 to 20 ml o f d i s t i l l e d water.
o f water at any one given time.
A ll plants received the same amount
T h ir ty one days a f t e r emergence yellow
spots began forming on the leaves o f a number o f p la n ts , suggesting
c h lo ro s is ( F o l l e t t et a l . 1981).
To a lle v ia te the p o te n tia l fo r
nitrogen d e fic ie n c y damage, 7 ml o f a 70 mg/kg s o lu tio n o f reagent grade
potassium n i t r a t e was applied to each conetainer.
This treatment
reduced the y ellow in g.
Plant Harvest
Three r e p e titio n s o f the fou r treatments and control were
harvested f o r each o f the three p la nt species 15 days a f t e r germination
when a l l plants had reached the two l e a f stage o f development.
The
remaining plants were harvested a f t e r 49 days when a l l plants had
reached at le a s t the f iv e leave stage.
At each harvest, the roots from
each r e p e titio n were placed in separate p la s tic containers and stored at
4° C.
34
Root Preparation and Analysis
Root preparation and analysis were based on Kormanik and McGraw
(1982) and Grey (pers. comm. 1988).
Roots were cleared in a 2.5%
s o lu tio n o f heated potassium hydroxide.
They were then a c id if ie d in a
1% h yd roch loric acid s o lu tio n and stained in .05% trypan blue in a
mixture o f l a c t i c a c id :g ly c e rin :w a te r (1:2:1 v / v / v ) .
V esicular-
arbuscular mycorrhizal q u a n tific a tio n was completed by placing the ro ot
on a s lid e marked w ith Chartpak cross-pattern (87 lin e s /d e c im e te r)
contact f i l m .
Chartpak f i l m was used to f a c i l i t a t e permanent mounting
o f roots on s lid e s w hile reducing costs.
As a m o d ific a tio n o f the
g r id l i n e in te rs e c t method o f Kormanik and McGraw (1982), the top f iv e
c e lls at each g r id lin e were examined along the f u l l length o f the ro o t.
Approximately 125 observations (o f 5 c e lls each) were made f o r each o f
fo u r s lid e s prepared f o r each r e p e t it io n . ‘ Each s lid e consisted o f a
complete p la nt ro o t length-.
Kormanik and McGraw c ite d 100 observations
as a minimum when the g r id lin e in te rs e c t method is used.
was made on a light-m icroscope at IOOX m a gnifica tion.
Examination
When there was
any doubt about the in fe c tio n o f a c e l l , because o f o b s c u rity from
nearness to the g r id l i n e , depth o f f i e l d , s tru c tu re c l a r i t y , etc.
examination was made at 400X m a gnifica tion.
The primary d i f f i c u l t y
encountered was d is tin g u is h in g VAM from non-mycorrhizal septate f u n g i.
S p e c ific id e n tify in g features such as vesic le s , arbuscules, and bending
w ith in c e lls were located and followed along hyphae when possible.
Data
were c o lle c te d from two plants f o r each o f the three r e p e titio n s f o r a l l
f i v e treatments f o r a ll species.
35
RESULTS AND DISCUSSION
The f i r s t set o f roots was harvested 15 days a f t e r germination.
Each p la n t had entered the two le a f stage o f development.
Three
re p e titio n s o f the fo u r treatments f o r each plant and three con trols per
treatment were included in the harvest.
roots revealed no mycorrhizal in fe c tio n .
Microscope examination o f the
There were, however, non-
mycorrhizal in fe c tio n points on many o f the ro ots.
fungus, possibly an Opidium species.
These were septate
No attempt was made to
s t a t i s t i c a l l y q u a n tify them but they were present on about one quarter
o f the ro o ts .
The septate hyph.ae observed were c la s s ifie d as non-mycorrhizal
based on two fa c to rs .
F i r s t , they had no features associated w ith VAM,
such as vesicles and arbuscules.
Second, Powell's (1976) study o f spore
and ro o t inoculum material demonstrated th a t most septate hyphae were
associated w ith growth from spore inoculum and were not a ttra c te d to
p la n t (onion) roots u n t il they were almost touching them.
mycorrhiza also did not successfully in fe c t the p la n t.
The septate
Powell found
th a t septate mycorrhizal fungi were branched and fan shaped.
those observed were fan shaped.
None o f
Hyphae from infected ro o t segment
inoculum were always aseptate (Powell 1976).
The second set o f roots was harvested 49 days a f t e r germination.
A ll plants had f iv e leaves th a t had s u b s ta n tia lly elongated.
Only a few
36
plants began growing a s ix th le a f.
As w ith the f i r s t harvest, three
re p e titio n s and three con trols f o r each treatment f o r each plant species
were harvested.
Percent in fe c tio n f o r p la n t species and treatments fo r
the second harvest are presented in Appendix A.
Because only one
harvest yie ld e d actual in fe c tio n data, the two harvests were not
s t a t i s t i c a l l y compared.
Control plants from both harvests had no
observable mycorrhizal or non-mycorrhizal in fe c tio n .
This lack of
c o lo n iz a tio n demonstrated the effectiveness o f the s o il s t e r i l i z a t i o n
procedure and ind ica tes th a t the non-m ycorrhizal/septate fungi colonized
from the ro o t inoculum and was present on plants in the f i e l d .
The non-mycorrhizal fungi found during the f i r s t harvest was also
found during examination o f roots from the second p la n t harvest.
They
were not q u a n tifie d a ft e r the second harvest but were notably more
common in those roots w ith fewer mycorrhizal in fe c tio n s .
Tufted
hairgrass, w ith fewer VAM in fe c tio n s had the greatest q u a n tity o f these
f u n g i.
Smooth brome appeared to have the fewest septate in fe c tio n s .
Statistical Analysis
S t a t i s t i c a l analysis was completed w ith the computer program
MSUSTAT (Lund 1988).
Analysis o f variance was performed on in fe c tio n
rates (Appendix A) by p la n t species and treatment (Table 3 ).
This was
to compare the VAM i n f e c t i v i t y based on both species and amount o f
inoculum together.
"Performance" or " i n f e c t i v i t y " by the VAM o f a given
p la n t species re fe rs to the a b i l i t y o f the VAM to act as inoculum and
colonize VAM in to the host p la nt (Sudangrass).
A p l o t t i n g o f these
37
Table 3.
Analysis o f variance and treatment means f o r p la nt and
treatment comparison.
Analysis o f Variance
D.F.
Plant Species
Treatment
Plant X T r t .
Residual
M.S
S.S.
2
3
6
24
52.78
88.54
26.88
21.71
26.39
29.51
4.48
0.91
F-Value
P-Value
29.18
32.63
4.95
.0000
.0000
.0020
Plant and Treatment Means
Plant Species
Mean
Treatment I
Mean
DeCa
AgAl
BrIn
1.93
2.05
4.56
1:1
1:2
1:4
1:8
4.86
3.27
2.78
0.48
1R ootrsoil by volume.
r e s u lts however, showed th a t there was in te ra c tio n between the plant
species (Figure 6)'.
At the 1:4 treatment level tu fte d hairgrass broke
the downward trend o f a ll three p la nt species' VAM.
Therefore, t h is
method o f p la n t and treatment analysis was unacceptable.
A p la n t by treatment analysis o f variance was then performed.
This compared the d iffe re n c e in i n f e c t i v i t y between the three plant
species at each inoculum/treatment le v e l.
Data (Appendix B) examination
indicated th a t at c e rta in treatment le v e ls VAM i n f e c t i v i t y was
s i g n i f i c a n t l y d i f f e r e n t between p la n t species.
The Least S ig n ific a n t
D ifference was calculated at the f iv e percent confidence, l e v e l .
38
DeCa
H - AgAI
H^- Brln
Treatment
Figure 6.
In te ra c tio n between Plant Species and Treatment.
Treatments at the 1:1, 1:2, and 1:8 levels yielded somewhat
p re d ic ta b le i n f e c t i v i t y responses.
At the 1:1 treatment le v e l, tu fte d
hairgrass VAM's i n f e c t i v i t y did not d i f f e r s ig n if ic a n t ly from redtop
bentgrass VAM (Figure 7).
Smooth brome VAM colonized s ig n if ic a n t ly more
c e lls than both tu fte d hairgrass VAM and redtop bentgrass VAM.
At a ll
treatment le v e ls , smooth brome VAM had greater i n f e c t i v i t y than redtop
bentgrass VAM.
39
I DeCa
c
6-
O
V/A
AgAI
EEHiBrIn
I
Different letters denote significance
P ■ .05
U
CD
A
C4—
C
C
CD
U
SCD
O-
2
-
Plant Species
Figure 7.
Plant species comparison at the 1:1 treatment l e v e l .
The 1:2 treatment's i n f e c t i v i t y was s i m i l i a r to the i n f e c t i v i t y
response hypothesized at the s ta r t o f the experiment and the 1:8
tre atm e nt's response was s i m i l i a r to the 1:1 treatment (Figures 8 and
9).
In the 1:2 treatment there were s ig n if ic a n t diffe re n c e s between the
i n f e c t i v i t y of a ll three grass species' VAM.
Though d i f f e r i n g
c o lo n iz a tio n percents between a ll three grass species' VAM was i n i t i a l l y
hypothesized fo r a ll four treatments, only th is one treatment responded
in th is manner. In the 1:8 treatment tu fte d hairgrass and redtop
bentgrass VAM performed in a s im ila r manner.
Both o f t h e i r VAM
independently colonized s i g n if ic a n t l y fewer sudangrass root c e lls than
smooth brome VAM.
40
I DeOa
C
O
1^22 AqAI
H+HlBrln j
Different letters denote significance
U
CU
C
CU
U
SCU
CL
Plant Species
Figure 8.
Plant species comparison at the 1:2 treatment level
DeCa
Y/A
A gA l
B E B r ln
I
Different letters denote significance
P - .05
DeCa ■ 0.0
Plant Species
Figure 9.
Plant species comparison at the 1:8 treatment le v e l.
41
Tufted ha irg ra ss' response in the th ir d treatment (1:4, r o o t : s o il )
was l i k e l y the product o f experimental e rro r.
There is nothing in the
p e rtin e n t l i t e r a t u r e nor is there any b io lo g ic a l explanation fo r the VAM
from tu fte d hairgrass to s ig n if ic a n t ly outperform redtop bentgrass and
nearly equal smooth brome (LSD = 1.520) (Figure 10).
This discrepancy
could have resulted from incomplete homogenization o f ro o t material
prepared f o r the 1:4 treatment.
Natural explanations f o r the improved
1:4 tu fte d hairgrass in fe c tio n include the p o s s i b i l i t y th a t these plants
made greate r contact w ith the root inoculum and the inoculum colonized
at a greate r ra te .
That Carling and co-workers (1979) discovered that
f i n a l in fe c tio n rates would be s im ila r between plants infe cted with low
Inoculum le v e ls and plants infected with high inoculum le v e ls suggests
Figure 10.
Plant species comparison at the 1:4 treatment le v e l.
42
a l l o f the tu fte d hairgrass plants would reach an equal in fe c tio n level
"plateau" were the experiment to have continued f o r a f u l l growing
season.
43
SUMMARY AND CONCLUSION
V e s ic u lar-arbuscular mycorrhizal roots from tu fte d hairgrass,
redtop bentgrass, and smooth brome growing in or near areas o f t a i l i n g s
derived alluvium were mixed in 1:1, 1:2, 1:4, and 1:8 d ilu t io n s
( r o o t : s o i l , v /v ) w ith s t e r i li z e d s o i l .
Sudangrass, acting as a plant
host, was seeded in to these mixtures and i t s roots were examined fo r
q u a n tita tiv e mycorrhizal in fe c tio n to assess the impact th a t t a i l i n g s
m aterial plays on mycorrhizal i n f e c t i v i t y .
In the 1:1 treatment there was a s ig n if ic a n t d iffe re n c e in
i n f e c t i v i t y between the VAM from tu fte d hairgrass and redtop bentgrass
and smooth brome VAM.
In the 1:4 treatment experimental e r ro r was
believed to be the cause o f unexpectedly high in fe c tio n rates o f tu fte d
ha irgrass.
The re s u lts o f the 1:4 experiment were otherwise s im ila r to
those o f the 1:1 treatment:
There was a s ig n if ic a n t d iffe re n c e between
the i n f e c t i v i t y o f VAM from redtop bentgrass and smooth brome.
In the 1:2 treatment, tu fte d hairgrass VAM in fe c te d s i g n if ic a n t l y
fewer sudangrass ro o t c e lls than redtop bentgrass VAM, which infected
s i g n i f i c a n t l y fewer sudangrass c e lls than smooth brome VAM.
In the 1:8
treatment there was a s ig n if ic a n t d iffe re n c e between both the tu fte d
hairgrass and redtop bentgrass VAM and the VAM from smooth brome.
The
conclusion was made th a t there are s ig n if ic a n t diffe re n c e s in VAM
i n f e c t i v i t y based on the source o f VAM inoculum m a te ria l.
In a l l fou r
44
treatments there were s ig n if ic a n t d iffe ren ces between smooth brome VAM
and the VAM from one or both o f the other two species.
That inoculum
from smooth brome was found in n o n -ta ilin g s enriched a g r ic u ltu r a l s o i l ,
w hile the inoculum from tu fte d hairgrass was selected from a p o t e n t ia lly
i n h ib i t o r y s o il and th a t o f redtop bentgrass roots was found in less
severely p o llu te d s o ils , suggests th a t t a i l i n g s contamination plays some
ro le in inoculum p o te n tia l.
Other fa c to rs th a t may be a ffe c tin g th is study's i n f e c t i v i t y
r e s u lts include the depth to water ta b le associated w ith the three p la nt
species, the amount o f in fe c tio n o f the roots used as inoculum, and
m ic ro c lim a tic and edaphic v a ria tio n th a t could a ffe c t the plants in the
fie ld .
Based on the re s u lts , VAM may o f f e r lim ite d or no improvement to
land reclam ation/revegetation success when low pH, mine t a i l i n g s , and/or
metal contamination' are present.
One l i m i t i n g fa c to r to VAM success
appears to be reduced VAM i n f e c t i v i t y .
Inoculum from plants growing in
t a i l i n g s derived alluvium were demonstrated to have lower i n f e c t i v i t y
than VAM from plants growing in non-contaminated s o i l .
This conclusion
is con sistent w ith Johnson's (1977) fin d in g s th a t pro p e rtie s of
endophytes d i f f e r when growing in d i f f e r e n t s o ils .
Mycorrhizal species
may be more e f f i c i e n t and reproductive in an i n h ib it o r y environment,
such as at the Grant-Kohrs Ranch when they have evolved w ith the
i n h ib i t o r y c o n d itio n .
V esicular-arb uscu lar mycorrhizal c o lo n iz a tio n ,
th e re fo re , may have been in h ib ite d by the steam s t e r i li z e d greenhouse
s o il used in the experiment because i t was d if f e r e n t from the s o ils in
the f i e l d .
A mycorrhizal species chosen f o r revegetation a p p lic a tio n
45
could th e re fo re be expected to reproduce most successfully i f i t was
c o lle c te d from plants growing in s o il conditions s im ila r to the s it e
being reclaimed.
Where plants are growing in extremely contaminated
s o i l , such as some places along the S ilv e r Bow Creek, mycorrhiza are.
apparently unable to survive and/or reproduce and th e re fo re can o f f e r no
improvement to land reclamation.
46
■ RECOMMENDATIONS
Some o f the inconsistency w ith in the VAM response between the
treatments could be reduced i f the ro o t inoculum was placed commonly
w ith in each conetainer.
D ir e c tly below the seed, as is often done w ith
spore inoculum placed on an agar block, is the lo g ic a l lo c a tio n because
i t would be d i r e c t l y and immediately av a ila b le to the emerging seedling
(Grey pers. comm. 1988).
This would insure th a t each p la n t has equal
contact w ith the inoculum.
I t would also remove the p o s s i b i l i t y o f ro o t
clumping or incomplete homogenization.
L a s tly , the immediate contact
w ith the seedling from d ir e c t placement could cause e a r l i e r mycorrhizal
in fe c tio n .
Increasing the harvest frequency would help determine the time
period required f o r in fe c tio n to begin (Sutton .1973) and demonstrate
whether c o lo n iz a tio n speed is impacted by t a i l i n g s m a te ria ls .
Using
smaller conetainers would reduce ro ot material (in d iv id u a l sample size)
re q u irin g examination.
Were th is done, a smaller g r id could be used f o r
microscope examination in order to q u a n tify a minimum o f 100 c e lls
(Kormanik and McGraw 1982).
Examining the i n f e c t i v i t y o f f i e l d inoculum in a s t e r i l e non-toxic
s o il te s ts the impact o f t a i l i n g s on the mycorrhiza already in the
f i e l d , and th e re fo re t h e i r a b i l i t y to in fe c t present and l a t e r
successional p la n t species.
As the ro le o f the m ic ro b io tic environment
47
becomes in c re a s in g ly c le a r, the ro le o f in d iv id u a l i n h ib it o r y and to x ic
conditions must be characterized f o r land reclamation to proceed.
T e s tin g .th e i n f e c t i v i t y o f f i e l d and p o t-c u ltu re d mycorrhizal s tra in s
under s p e c ific to x ic and i n h ib it o r y conditions would advance the
re la tio n s h ip between s o il t a i l i n g s contamination and i n f e c t i v i t y t h is
study began to examine.
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54
APPENDICES
Appendix A
Infection Counts for Plant Species By Treatment
56
Table 4.
Infection counts (%) for plant species by treatment1.
Treatment (Root:Soil v/v )
Plant Species
DeCa
Rep2
I
2
3
AgAl
I
2
3
BrIn
I
2
3
1:1
1:2
1:4
1:8
4.1
1.5
3.9
1.0
7.3
2.2
3.9
1.2
0.0
3.7
1.0
6.3
2.4
1.7
0.0
0.0
1.2
2.5
3.0
3.6
2.5
0.0
1.9
0.0
6.4
5.1
0.0
4.3
4.5
3.1
1.0
2.2
4.4
5.7
5.4
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.8
5.1
3.3
3.0
2.2
5.7
3.9
3.2
8.7
5.2
1.5
5.1
1.5
1.0
3.9
4.9
5.6
2.2
0.0
3.8
1.0
2.7
3.8
4.7
1.4
0.0
0.0
0.0
1.6
5.0
3.8
0.0
0.0
0.0
0.0
0.0
0.0
1.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
14.1
7.4
2.9
6.3
6.8
6.4
3.9
2.8
16.0
10.6
18.7
15.3
1.6
8.1
4.0
5.6
10.8
7.9
6.0
5.4
9.2
2.5
5.7
4.0
6.6
1.0
2.8
5.4
2.0
2.7
2.2
6.2
8.6
0.0
3.2
6.3
1.2
1.0
0.0
4.1
0.0
2.4
1.5
0.0
2.5
2.5
0.0
0.0
1Four counts o f 125 lin e in te rs e c ts each were made f o r each r e p e titio n
^Repetition
57
Appendix B
Analysis of Variance Tables
58
Table 5.
Analysis of variance and treatment means for plant species at
the 1:1 treatment level.
Analysis o f Variance
Source
D.F.
Treatments
Residual
2
6
S.S.
M.S
30.95
14.03
15.47
2.34
F-Value
P-Value
6.62
.0304
Treatment Means and M u ltip le Comparison Based on LSD
Plant Species
SE For Mean
SE For D i f f .
LSD (Cal by t )
3.03
4.13
7.40
DeCa
AgAl
BrIn
Table 6.
Mean
0.883
1.249
3.055
Analysis o f variance and treatment means f o r p la nt species at
the 1:2 treatment le v e l.
Analysis o f Variance
Source
D.F.
Treatments
Residual
2
6
S.S.
32.04
1.83
M.S
16.02
0.31
F-Value
P-Value
52.43
.0002
Treatment Means and M u ltip le Comparison Based on LSD
Plant Species
DeCa
AgAl
BrIn
Mean
1.20
2.87
5.77
SE For Mean
SE For D i f f .
LSD (Cal by t )
0.319
0.452
1.100
59
Table 7.
Analysis of variance and treatment means for plant species at
the 1:4 treatment level.
Analysis o f Variance
Source
D.F.
Treatments
Residual
2
6
S.S.
9.10
3.47
M.S
4.55
0.58
F-Value
P-Value
7.87
.0210
Treatment Means and M u ltip le Comparison Based on LSD
Plant Species
SE For Mean
SE For D i f f .
LSD (Cal by t )
3.50
1.53
3.80
DeCa
AgAl
BrIn
Table 8.
Mean
0.439
0.620
1.520
Analysis o f variance and treatment means f o r p la n t species at
the 1:8 treatment le v e l.
Analysis o f Variance
Source
D.F.
Treatments
Residual
2
6
S.S.
2.59
0.77
M.S
1.29
0.12
F-Value
P-Value
10.03
.0122
Treatment Means and M u ltip le Comparison Based on LSD
Plant Species
DeCa
AgAl
BrIn
Mean
0.00
0.33
1.27
SE For Mean
SE For D i f f .
LSD (Cal by t )
0.207
0.293
0.717
1