Isolation, characterization and copper binding of Gaeumannomyces graminis var. graminis melanin
mutants
by Barbara Anne Frederick
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Microbiology
Montana State University
© Copyright by Barbara Anne Frederick (1997)
Abstract:
Gaeumannomyces graminis var. graminis is a filamentous soil ascomycete that synthesizes
1,8-dihydroxynapthalene (DHN) melanin as a secondary metabolite. We isolated two mutant strains
affected in the melanin biosynthetic pathway, using either chemical or UV mutagenesis. One of these
mutants, JH4300, was unable to synthesize DHN-melanin. Because it accumulated 2-hydroxyjuglone, a
melanin pathway shunt product, this mutant was most likely defective in the reductase that catalyzes
the conversion of 1,3,8-trihydroxynapthalene to vermelone, the penultimate reaction in DHN synthesis.
Genetic crosses with our wild-type strain indicated that this deficiency was the result of a single
mutation. Another slow-growing mutant, JH4301, constitutively synthesized DHN-melanin and
produced more mucilage surrounding the cell wall than our wild-type strain. Genetic crosses with our
wild-type strain suggested that the heavily melanized mutant had a single mutation responsible for its
phenotype.
The melanized wild-type and dark mutant strain JH4301 were more resistant to the lytic enzymes
chitanase and glucanase, and to UV damage than the unmelanized mutant. The heavily melanized
mutant JH4301 secreted fewer lytic enzymes, and tricyclazole inhibition of melanin restored its
secretory ability. Both mutants were unaltered in pathogenicity to rice compared to the wild-type strain,
but the wild-type was a better competitor in mixed rhizosphere communities of rice.
The light mutant JH4300 absorbed less copper than our wild-type as determined by
inductively-coupled plasma atomic emission spectroscopy analysis and silver staining, while the dark
mutant bound significantly more copper when grown with copper or when exposed to copper following
growth. The heavily melanized mutant was more sensitive to the toxic effects of copper than either the
wild-type or unmelanized strains. All three strains bound significant amounts of copper, lead, zinc and
iron from tailings material. Inoculation of range grass rhizospheres with the three strains did not affect
the uptake of copper, iron or zinc from tailings into plant tissue. However, melanized strains increased
the uptake of lead from tailings into shoot tissue. These results suggest that melanized G. graminis var.
graminis may have application in phytoremediation of lead-contaminated sites. ISOLATION, CHARACTERIZATION AND COPPER BINDING OF
G a eu m a n n o m yces gram inis var. gram inis MELANIN MUTANTS
by
B a rb a ra A nne F rederick
A th e s is su b m itte d in p a rtia l fulfillm ent
of th e re q u ire m e n ts for th e degree
of
D octor of P hilosophy
in
Microbiology
MONTANA STATE UNIVERSITY-BOZEMAN
B ozem an, M o n tan a
S e p tem b er 1997
11
fg19,U
APPROVAL
of a th e sis su b m itte d by
B a rb a ra A nne Frederick
T his th e s is h a s been re a d by each m em b er of th e th e sis
com m ittee a n d h a s been found to be satisfac to ry regarding
c o n te n t, E nglish u sa g e, form at, c itatio n s, bibliographic style, a n d
c o n sisten cy , a n d is ready for su b m issio n to th e College of
G ra d u a te S tu d ies.
J o a n M. H enson
D ate
Approv ’
" e D e p a rtm e n t of Microbiology
K eith C ooksey
(Signature)
"""
D ate
A pproved for th e College of G ra d u ate S tu d ie s
J o s e p h Fedock
D ate
Ill
STATEMENT OF PERMISSION TO USE
In
p re se n tin g
th is
th e s is
in
p a rtia l fulfillm ent of th e
re q u ire m e n t for a doctoral degree a t M o n tan a S ta te U niversityB ozem an, I agree th a t th e L ibraiy sh all m ak e it available to
b o rro w ers u n d e r ru le s of th e Library. I fu rth e r agree th a t copying
of th is th e s is is allow able only for scholarly p u rp o se s, c o n siste n t
w ith "fair use" a s p rescrib ed in th e U.S. C opyright Law. R equests
for extensive copying or rep ro d u c tio n of th is th e s is sh o u ld be
referred to U niversity M icrofilms In tern a tio n al, 300 N orth Zeeb
R oad, A nn A rbor, M ichigan 4 8 1 0 6 , to w hom I have g ra n te d "the
exclusive rig h t to rep ro d u c e a n d d istrib u te m y d isse rta tio n in a n d
from m icroform along w ith th e non-exclusive rig h t to rep ro d u ce
a n d d istrib u te m y a b s tra c t in a n y fo rm at in w hole or in part."
w A,
nr
iv
ACKNOWLEDGMENTS
I th a n k m y th e s is advisor, Dr. J o a n H enson, a n d com m ittee
m em b ers D rs. T heC an C aesar, Cliff B ond, Tim M cD erm ott, Dave
Dooley a n d E llen K reig h b au m .
I also th a n k Dr. G reg O lsen of
Little B ear L abs for th e ICP-AES an aly sis, Dr. Mike W heeler for
th e TLC a n a ly sis a n d Dr. C a e sa r for th e b e a u tifu l electron
m icro g rap h s. The s u p p o rt of a P a tricia R o b erts-H arris fellowship
is g ratefully acknow ledged, a n d I th a n k Adele P itten d rig h a n d
D iane C attrell for guiding m e th ro u g h th e com plexities of th e
fellowship.
T h a n k s to T re sa G oins for h e r advice, su p p o rt a n d th e coffee
b re a k s.
W ith o u t th e frien d sh ip of fellow lab r a ts D e a n n a N ash
a n d A n a sta s sia Livintseva, m y lab tim e w ould have b een m u c h
less enjoyable.
T h a n k s also to fellow g ra d u a te s tu d e n ts S cott
K obayashi, W endy C ochran, R obin W illiam s, Tom F o u b ert, M aria
K ohler, a n d
K athy B illings-S andoval for th e ir to leran ce a n d
su p p o rt.
I reserve a special th a n k y o u for m y h u s b a n d Tom, a n d
ch ild ren ,
R achel a n d
u n d e rs ta n d in g .
A ndrew ,
for th e ir p a tien c e,
love a n d
V
TABLE OF CONTENTS
Page
ACKNOWLEGMENTS........................... .......................................................iv
LIST OF TA B L E S........................................................................................vii
LIST OF FIG U R E S.......................................................................................ix
ABSTRACT.................................................................................................... xi
CHAPTER
Oi -K to to
1. IN TR O D U C TIO N ...................................................................................... I
C o m p etitio n .....................................................
E xploitation C o m p e titio n ..............
In terferen ce C o m p e titio n ................
M etal A ccum ulation in F u n g al B iom ass
E n v iro n m en ta l M etal C o n ta m in a tio n . . . . : ..............
10
H istorical Overview of th e A naconda
T ailings P o n d s ...............................
10
P hytorem ediation of M etalliferous S ites. ......................12
The R hizosphere a n d M etal C ycling. ............................. 15
H y p o th e s e s .............................................
17
L itera tu re C ited ...................
19
2. ISOLATION AND CHARACTERIZATION OF
G a eum annom yces gram inis v&r.graminis
MELANIN MUTANTS____ ! ............................................................ 27
I n t r o d u c t io n ....................................................................................27
M aterials a n d M e th o d s.................................................................. 30
S tra in s, M edia a n d G row th C onditions ..................... 30
M u ta n t Is o la tio n ................................................
31
S tra in C ro ss e s....................................................................... 33
E lectro n M icroscopy
...................
33
M elanin Q u a n tita tio n .........................................................34
Novozyme D ig e s tio n ............................... .,........................35
UV P r o te c tio n ....................................................................... 36
E nzym e S e c re tio n ................................................................ 37
TABLE OF CONTENTS - (Continued)
Page
P ath o g en icity ....................................................................... 37
R e s u l t s ................
38
M u ta n t C h a r a c te r iz a tio n ............................................ . 3 8
UV Light S e n s itiv ity .....................
44
Lytic Enzym e P ro te c tio n ................... ' ...........................44
Enzym e S e c re tio n ......................................
46
P a th o g e n ic ity .....................................................................46
D i s c u s s i o n ....................................................
50
L itera tu re C ite d ...............................................................................55
3. COPPER BINDING AND LOCALIZATION
OF C uS BY SILVER STAINING OF G aeum annom yces
gram inis var. g fa m in is MELANIN MUTANTS...............................60
I n t r o d u c t io n ................................................................................... 60
M aterials a n d M e th o d s .....................
63
F u n g al S tra in s a n d G row th C o n d itio n s .....................63
G row th C urves in M inim al M edium
w ith C opper A m en d m en t . ................................. 63
C opper B inding a n d A c c u m u la tio n ............................ 64
Inductively C oupled Plasm a-A tom ic E m issio n
S pectroscopy (IC P-A E S)......................................64
Silver S t a i n i n g ...................................................' .............65
R e s u l t s .............................................................................................65
G. gram inis var. gram inis G row th w ith
C o p p e r....................................................................... 65
C opper Binding, to H y p h a e .............................................67
Silver E n h a n c e m e n t of C opper S u l f i d e ....................6 7
D i s c u s s i o n .......................
69
L itera tu re C ite d .............................................................................. 71
4. CONCLUSIONS
76
V ll
TABLE OF CONTENTS - (Continued)
Page
A P P E N D IC E S ........................................................................................... 78
APPENDIX A -R H IZO SPH ERE
COMPETITIVENESS OF G aeum annom yces
gram inis var. gram inis MELANIN MUTANTS . . . . . 79
M aterials a n d M e th o d s ........................................ 82
C om petition A s s a y ................................... 82
R hizosphere DNA E x tractio n
a n d PCR A m p lific a tio n ................ 84
R e su lts ..................................................................... 85
C om petitiveness .........................................85
P C R ....................................................
85
D isc u ssio n ................................................................ 86
L iteratu re C ite d ...................................................... 91
APPENDIX B—PHYTOREMEDIATION
APPLICATION OF G aeum annom yces
gram inis var. gram inis MELANIN MUTANTS . . . . 93
M aterials a n d M eth o d s....................................... 95
M etal S orption of Tailings
M aterial to B io m a ss........................95
M etal U ptake by P lan t B io m a s s ......... 96
R hizosphere H yphal L e n g th s ................ 98
R e s u l t s .....................................................................99
T ailings S orption to B io m a s s ................99
T ailings M etal U ptake by P la n ts
In o cu lated w ith G. gram inis
var. g ra m in is................................... 99
D isc u ssio n ........................................
L iteratu re C ite d .................................................... 105
102
V lll
LIST OF TABLES
T able
Page
2.1. G enetic c r o s s e s ..........................................................................40
2.2. Enzym e secretio n of G. gram inis var. gram inis
w ild-type a n d m elan in m u ta n t s tra in s grow n
w ith or w ith o u t tric y c la z o le .............................................49
2.3. P athogenicity of w ild-type a n d m u ta n t s tra in s
of G. gram inis var. gram inis to ric e .................................49
3.1. C u a c c u m u la tio n of w ild-type G. gram inis var.
gram inis a n d two m elan in m u ta n t s tra in s
following 14 days grow th in m inim al m ed iu m
w ith C u .....................................................................
68
3.2. C opper b o u n d to 14 day-old c u ltu re s of G. gram inis
var. gram inis a n d two m elan in m u ta n ts
following I h ex p o su re of h y p h ae to 40 mM
CuSCk after 14 days in m inim al m ed iu m w ith
tra c e C u ..............................................................
68
B. I M etal c o n c e n tra tio n s of tailin g s pond m ate ria l
so rb ed to w ild-type (JH 2033) a n d m elan in
m u ta n t s tra in s of G. gram inis var. gram inis............100
B .2. Significant tre a tm e n t effects of p la n t m eta l u p ta k e
e x p erim e n t............................................................................ 101
B.3. S ignificant m a in effect of p la n t species on ro o t a n d
a n d sh o o t b i o m a s s ............................................................102
ix
LIST OF FIGURES
F igure
Page
1.1. A n aco n d a tailings p o n d s y s te m . ........................................... 13
2.1. 1 ,8 -d ih y d ro x y n ap h th alen e (DHN) m elan in
p a th w ay a n d s h u n t p ro d u c ts p ro d u ce d by
m u ta tio n s in th e p a th w a y ..................................................30
2.2. E lectro n m icro g rap h s of w ild-type a n d m elan in
m u ta n t cell w a l l s .................................................................. 42
2.3. H yphal tip s of w ild-type a n d m elan in m u ta n t
s t r a i n s .....................
45
2.4. H yphopodia of w ild-type a n d m elan in
m u ta n t s tra in s . . ■.........................................
46
2.5. G row th cu rv es a n d m elan in c o n c e n tra tio n s
of G. gram inis var. gram inis w ild-type a n d
m e lan in m u ta n t s t r a i n s ...................................................... 47
2.6. B io m ass of G. gram inis var. gram inis wildtyp e (JH 2033) a n d m elan in m u ta n t s tra in s
following tre a tm e n t w ith v ario u s levels of
UV l i g h t ..................................................
48
2.7. N-acetylglucosam ine re le ased from wildtype or m elan in m u ta n t s tra in s of G. gram inis
var. gram inis w ith or w ith o u t Novozyme 234
digestion ..........................................
48
3.1. G gram inis var. gram inis w ild-type a n d m elan in
m u ta n t grow th in m in im al m ed iu m c o n ta in in g
varying c o n c e n tra tio n s of C uS 0 4 ................................... 66
LIST OF FIGURES - C o n tin u ed
F igure
Page
3.2. S ilv er-stain ed h y p h a e show ing localization of
C u S ........................ . . . . ^ ......................................................... 69
A. I In te rn a l tra n s c rib e d sp a c e r (ITS) region of
rib o so m al D N A ................................................................... 81
A.2. Significant interactive effect betw een s tra in
in o cu latio n a n d rh izp sp h ere in o cu lu m on
ro o t a n d sh o o t dry w e i g h t ............................
88
A.3. PCR- am plified ITS regions of DNA e x tra cted
from rh izo sp h ere m a te ria l w ith or w ith o u t
rh izo sp h ere in o cu lu m ."................
89
A. 4. PC R-am plified ITS regions of JH 2 0 3 3 ,
rh izo sp h ere in o cu lu m e x tra c t a n d rh izo sp h ere
in o c u lu m spiked w ith JH 2 0 3 3 ....................................... 90
B. I. In teractiv e tre a tm e n t effect of p la n t sp ecies a n d
tailin g s ad d itio n on iro n u p ta k e into sh o o t
t i s s u e .................................
...103
B .2. Significant in te ra c tio n betw een stra in
in o cu lu m a n d tailings ad d itio n on lead
c o n c e n tra tio n in sh o o t b i o m a s s ....................................105
Xl
ABSTRACT
G aeum annom yces graminis var. graminis is a filam entous soil
ascom ycete th a t synthesizes 1,8-dihydroxynapthalene (DHN) m elanin
a s a secondary m etabolite. We isolated two m u ta n t stra in s affected
in the m elanin biosynthetic pathw ay, using either chem ical or UV
m utagenesis.
One of these m u ta n ts, JH 4300, w as u nable to
synthesize DHN-melanin. B ecause it accum ulated 2-hydroxyjuglone,
a m elanin pathw ay sh u n t product, th is m u ta n t w as m ost likely
defective in the red u ctase th a t catalyzes the conversion of 1,3,8trihydroxynapthalene to verm elone, th e penultim ate reaction in DHN
synthesis. Genetic crosses w ith our wild-type stra in indicated th a t
th is deficiency w as the resu lt of a single m utation. Another slowgrowing m u ta n t, JH 4301, constitutively synthesized DHN-melanin
a n d p roduced m ore m ucilage su rro u n d in g the cell wall th a n our wildtype strain. Genetic crosses with our wild-type stra in suggested th a t
th e heavily m elanized m u ta n t h a d a single m u tatio n responsible for
its phenotype.
The m elanized wild-type an d d a rk m u ta n t stra in JH 4301 were
m ore re sista n t to the lytic enzym es chitanase an d glucanase, an d to
UV dam age th a n th e unm elanized m u tan t. The heavily m elanized
m u ta n t JH 4301 secreted fewer lytic enzymes, an d tricyclazole
inhibition of m elanin restored its secretory ability. B oth m u ta n ts
were u n a ltere d in pathogenicity to rice com pared to th e wild-type
strain , b u t th e wild-type w as a b etter com petitor in m ixed rhizosphere
com m unities of rice.
The light m u ta n t JH 4300 absorbed less copper th a n Our wildtype a s determ ined by inductively-coupled p lasm a atom ic em ission
spectroscopy analysis an d silver staining, while th e d a rk m u ta n t
b o u n d significantly m ore copper w hen grown w ith copper or w hen
exposed to copper following growth. The heavily m elanized m u ta n t
w as m ore sensitive to the toxic effects of copper th a n either the wildtype or unm elanized strains. All three strain s b o u n d significant
am o u n ts of copper, lead, zinc a n d iron from tailings m aterial.
Inoculation of range grass rhizospheres with the th ree stra in s did no t
affect th e u p tak e of copper, iron or zinc from tailings into p lan t
tissue. However, m elanized stra in s increased the u p ta k e of lead from
tailings into shoot tissue. These re su lts suggest th a t m elanized G.
gram inis var. graminis m ay have application in phytorem ediation of
lead-contam inated sites.
I
CHAPTER I
INTRODUCTION
M elanins
a re
p ig m en ts
se c o n d a ry . m etab o lites.
sy n th esized
by m a n y
fungi a s
T hese p ig m en ts are n o t . req u ired for
grow th, b u t have b een im plicated in e n h a n c e d survival in th e
e n v iro n m en t (reviewed in ref. 4).
p olym ers
of
M elanins a re h etero g en eo u s
d ih y droxyphenylalanine
(DOPA)i
y-glutam inyl-4-
h y d roxybenzene (GHB), catechol, or d ih y d ro x y n ap h th ale n e (DHN)
th a t p ro b ab ly c o n trib u te to th e com petitive fitn e ss of fungi by
providing a b a rrie r to e n v iro n m en tal stre sso rs.
However, m elan in
p ro tectio n a g a in st enzym atic d eg rad atio n , UV lig h t dam age a n d
oxidizing a g e n ts have only b een stu d ie d w ith n o n-isogenic stra in s.
M elanins have also b een im plicated in m etal b in d in g in solution,
b u t m o st e m p h a sis h a s focused on sy n th etic or e x tra c te d L-DOPA
m elan in . T he focus of th is review will be on th e role of m elan in in
sa p ro p h y tic com petition a n d m etal bin d in g in th e rh izo sp h ere, w ith
referen ce to p h y to rem ed iatio n of m etalliferous sites.
2
C om petition
In ecological te rm s, com petition is co m p rised of two types:
ex p lo itation a n d interference.
E xploitation com p etitio n is th e
d ep letio n of re so u rc e s by one o rg an ism w ith o u t lim iting th e access
of a n o th e r
org an ism ,
and
in terferen ce
co m p etitio n
in clu d es
m e c h a n ism s to red u c e acc ess to a reso u rce (35).
E x p lo itation C om petition
In general, re so u rc e lim itation is th e p rim a ry com petitive
stra te g y
of
exploitation.
For
exam ple,
w ood-decom posing
b asid io m y cetes w ith h igh foraging ten d e n cies exclude o th e r wooddecaying
fungi
from
s u b s tra te s
(28).
A spergillus
fla v u s
colonization a n d aflatoxin Bi form ation in barley g rain are affected *
by co m petition
by o th e r colonizing fungi (42), a n d
n u trie n t
co m p etitio n w ith Trichoderma harzianum s u p p re s s e s F usarium w ilt
of m elon a n d co tto n (49). A ntagonism of n o n -p ath o g e n ic F usarium
oxysporum a g a in st p athogenic F. oxysporum is a sso c ia te d w ith th e
co m p etition for glucose (34).
In v estig atio n s of th e biological control of ro o t d ise a se s u sin g
a n ta g o n istic m icro o rg an ism s su g g e sts th a t th e in te ra c tio n betw een
th e p a th o g e n a n d th e a n ta g o n is t ta k e s place in th e rh izo sp h ere
3
(35). .W eakly p ath o g en ic fungi w ith th e sam e rh izo sp h ere n iche a s
stro n g e r
p a th o g e n s
can
offset pathogenicity.
For
exam ple,
Phialophora gram inicola p ro te c ts a g a in st G. gram inis var. tritici,
c a u se of tak e -all in w h e a t (13), a n d s u b s tra te com petition w ith
M icrodochium bolleyi re d u c e s tak e -all lesions (29).
In addition,
co m p etition for th ia m in e by a sterile red fu n g u s su p p re s s e s tak eall
(54).
A n o th er
im p o rta n t
d e te rm in a n t
in
s u b s tra te
co m p etitiv en ess in th e rhizo sp h e re is th e ability to utilize cellulose
(I, 22).
Lockwood (1992) su g g ested th a t th e c o rrelatio n betw een
rhizo sp h e re com petence a n d cellulolytic activity is b e c a u se th e
m ucilage overlay of th e root su rface is com posed of cellulosic
p rim a ry cell w alls of ep id erm al cells.
Infection site com petition by p la n t p ath o g en ic fungi c a n also
be c o n sid ered a type of s u b s tra te com petition.
R hizoctonia solani
su ccessfu lly o u tco m p etes P ythium ultim um in th e p e a (Pisum
sativum ) rhizo sp h e re b e c a u se it is m ore aggressive a t com peting for
infection sites (62).
B inucleate Rhizoctonia spp. a n d h y povirulent
R. solani a re effective in biocontrol of R. solani by com peting for
in v asio n sites (26).
S u p p re ssio n of tak e-all a n d com m on root ro t
by p la n t grow th p rom oting fungi (PGPF) is d u e to com petitive root
4
colonization of PGPF, w hich blocks sites available for infection by
p a th o g e n s (55).
F u n g a l p ro tein
secretion
o c cu rs exclusively a t growing
a p ices (60), a n d com petitive ability is e n h a n c e d by efficient enzym e
se cretio n
se cre ted
for s u b s tra te
enzym es,
acq u isitio n .
M elanins
interfere w ith
in clu d in g cellu lases, by in h ib itin g enzym e
activity (10, 56) a n d th u s m ay c o n trib u te to d e c rea se d com petition
for n u trie n ts .
However, b e c a u se h y p h a l tip s a re u n m elan ized ,
a u to c h th o n o u s enzym es m ay n o t be inactivated, w hile m elaninized
fu n g al s tru c tu re s m ay in activ ate enzym es from o th e r sources.
T his w ould allow m elanized fungi a com petitive ad v an tag e in
re so u rc e a cq u isitio n , by lim iting a cc e ss of fu n g al com petitiors to
s u b s tr a te s via enzym e inactivation.
In terferen ce C om petition
A ccording to W icklow (1992), interference com petition is th e
re s u lt of a chem ical or p hysical in te rac tio n betw een individuals
before re so u rc e u se .
T his p h en o m en o n , also called an tib io sis, is
m ed iate d by specific or non-specific m etabolites, by lytic ag en ts,
enzym es, volatile c o m p o u n d s or toxic s u b s ta n c e s (17).
5
Soil
m icro o rg an ism s
secrete
lytic
enzym es,
su c h
as
c h ita n a se a n d g lu c a n a se s, th a t are active a g a in st fungi. M elanized
fungi a re m ore re s is ta n t to lysis by th e se enzym es (6, 31) a n d
su g g e st th a t m elan in a c ts a s a p ro tectio n a g a in st interference
com petition.
V iable re stin g sp o re s of th e obligate p ath o g en
S yn ch ytriu m endobioticum p e rs ist in soil for long p e rio d s (24), a n d
re s ista n c e a n d p ro tectio n provided by m elan izatio n of th e s e sp o res
is im p licated in th e ir longevity in soil (25).
M elanin m ay also c o n trib u te to com petitive interference.
Oxidized m e lan in p re c u rso rs ex hibit a n tib io sis (23, 51) th a t could
p o ten tially in h ib it com peting fungi.
The a c c u m u la tio n of DHN-
m elan in m etab o lite s flaviolin a n d 2-hydroxyjuglone. re s u lts in
su p p re ss io n of M agnaporthe grisea sp o ru la tio n (57).
In su m m ary , it is evident th a t m elan in s do n o t significantly
e n h a n c e exploitation com petition in fungi.
However, m elan in s
c o n trib u te to in terferen ce com petition by v a rio u s m ec h an ism s,
in clu d in g in ac tiv a tio n of enzym es from com peting fungi a s well a s
by blocking lytic enzym es s u c h a s c h itin ase a n d g lu can ases.
M elanins a n d m elan in p re c u rso rs are antibiotic to o th e r org an ism s
a n d p ro b ab ly c o n trib u te to in terferen ce w ith o th e r o rg an ism s in
co m p etition for th e sam e s u b s tr a t e s ., In addition, m e la n in s non-
6
specifically e n h a n c e com petitive abilities by providing p rotection
a g a in st e n v iro n m en tal s tre s s o rs s u c h a s UV light (59).
M elanins
also b in d m e ta ls th a t m ay provide a toxic defense m ec h an ism
a g a in st o th e r soil in h a b ita n ts (45).
M etal A ccum ulation in F u n g al B iom ass
A c cu m u latio n of heavy m e ta ls by m icrobial b io m a ss is a well
c h arac te riz e d p h en o m e n o n , a n d h a s received re c e n t a tte n tio n a s a
m e c h a n ism for en v iro n m en tal p ro tectio n a g a in st m e ta l toxicity a n d
in th e recovery of heavy m etals (reviewed in ref. 58).
W hile som e
m e ta ls (Fe3+, Z n2+, C u2+ a n d Co2+) a re e ssen tial, a n o v era b u n d an c e
of th e s e ele m e n ts lea d s to cell toxicity. M etals c a n ex h ib it toxicity
above
in clu d e
m etal-specific
th re sh o ld
co n ce n tra tio n s.
Toxic
effects
fu n ctio n al group blockage of m etabolically im p o rta n t
m olecules, d e n a tu ra tio n of enzym es, a n d d isru p tio n of m em b ran e
in teg rity (20).
B ecau se m etal io n s frequently flu c tu a te in th e
en v iro n m en t, m a n y m icro o rg an ism s have developed m ec h an ism s
to co n tro l m etal u p ta k e a n d a t th e sam e tim e se q u e s te r elem ents
for
fu tu re
u se .
M icrobial m etal
u p ta k e
and
detoxification
p ro c e sse s include:
u p ta k e of m e ta ls into th e cell by active or
p assiv e
bio so rp tio n
p ro ce sses,
to
cell
a sso c ia te d
m aterials,
7
e n tra p m e n t in e x tra ce llu lar c a p su le s, p recip itatio n , activation of
m etal-specific b in d in g p ro tein s a n d o x id atio n -red u ctio n reactio n s
(19).
H eavy m e ta ls e n te r th e soil, en v iro n m en t a t levels toxic to
m icro o rg an ism s th ro u g h m ining, a n d in d u stria l a n d a g ric u ltu ral
d isc h arg e s.
H igh m etal levels ex ert a selective p re s s u re on
m icro o rg an ism s
th a t
n e c e ssita te s
ex p ressio n
of
a
m etal
detoxification m e c h a n ism to e n su re survival (19). T here is a stro n g
co rrelatio n b etw een C u2+, Cd2+ a n d Z n2+ in soil a n d th e level of
m etal to leran ce in soil m icrobial co m m u n ities (14).
F ungi are
generally c o n sid ered to be less sensitive to m etal pollution th a n
b a c te ria (15), a n d have b een show n to be th e d o m in a n t fraction of
th e m icrobial c o m m u n ity in som e m etal c o n ta m in a te d soils (18).
M etal io n s a re actively tra n s p o rte d into fu n g al cells, w here
th ey b in d specific p ro tein s. The m etallo th io n ein s (MT's) are sm all
c y stein e-rich polypeptides th a t b in d e sse n tia l heavy m e ta ls su c h a s
co p p er a n d zinc, a n d n o n -e sse n tia l m etals s u c h a s m ercu ry a n d
cad m iu m .
M etallothionein e x p ressio n is in d u c e d by th e sam e
m e ta ls th a t b in d to th a t p rotein, th e re b y providing a m ec h a n ism of
p ro tec tio n a g a in st toxicity. O th er m etals are ta k e n u p into fungal
b io m a ss by p assiv e p ro cesses.
Pb2+ u p ta k e into S. cerevisiae is
8
e n e rg y -in d ep e n d en t a n d th e re s u lt of affecting th e perm eab ility of
y e a s t cell m e m b ra n e s to K+ a n d Mg2+ (58).
Fungal
cell
w alls
b io so rp tio n of m etals.
c o n ta in
p o ly sacch arid es
active
in
T hese polym ers in clu d e glycans a n d
cellulose w ith hydroxyl g ro u p s a s p o ten tial m etal b in d in g sites,
c h ito sa n a n d c h itin w ith m etal b in d in g p o ten tial a t am ino a n d
hydroxyl g ro u p s, a n d polyuronide w ith carboxyl a n d hydroxyl
g ro u p s (50). Negative su rface charge on m icrobial cell w alls c a u se d
by th e d isso c iatio n of chem ical g ro u p s, allow for c a tio n exchange
o r co o rd in atio n of m etal catio n s (7). M etals are classified according
to w h e th e r th e y p referentially form com plexes w ith nonpolarizable
lig an d s (hard) or polarizable (soft) lig an d s (41).
C opper a n d zinc
are b o rd erlin e h a r d / soft m etal io n s a n d a d so rp tio n of th ese ions
to b io m a ss is c o rrelated w ith com plexation to soft a n io n s (2).
B io m ass c o n ta in s m an y of th e softer bind in g sites s u c h a s carboxyl
a n d am ino g ro u p s th a t are involved in m etal b in d in g , suggesting
th a t p ro tein a n d c arb o h y d rate fractio n s of cell w all b in d catio n s
(
7) .
F u n g a l p ig m en ts su c h a s m e lan in b in d m e ta ls a n d serve a s
a protective m e c h a n ism a g a in st m etal toxicity. M elanins are high
m o lecu lar w eight polym ers th a t e n h a n c e th e survival of fungi in
9
ad v erse e n v iro n m en tal conditions, in clu d in g h igh c o n c e n tra tio n s of
heavy m etals.
M elanin e x p ressio n is in d u ced in som e fungi by
Pb2+, Fe2+, Al3+, Hg2+ (A ureobasidium pullulans, 21) a n d C u2+ (G.
gram irds var. g r a m in is ,ll; A ureobasidium pu llulans, 21), suggesting
th a t th e s e p ig m en ts provide p ro tectio n a g a in st m etal toxicity.
M elan in s c o n ta in negatively ch arg ed hydroxyl g ro u p s th a t could
p o ten tially b in d c atio n s, a s well a s am ino acid a n d carboxyl gro u p s
p re s e n t in m acrom olecules tra p p e d in th e m e lan in polym er (47).
The s tru c tu re a n d c h a ra c te ristic s of m e lan in s a re sim ilar to w eak
c atio n e x ch an g ers, a n d m etal io n s a n d m elan in s p ro b ab ly in te ra c t
by ion exchange (9).
M etals c a n be d esorbed from m elanized
fu n g al s tru c tu re s by th e a p p licatio n of c h elato rs or pH ch an g es
(36, 45)
A ttractio n of m etal ions to m elan in in c re a se s w ith
in c re a sin g v alance, w hich is c h a ra c te ristic of c a tio n exchange
re a ctio n s. T h u s, of th e divalent m e ta ls Pb2+, C u2+, Ni2+, Co2+, a n d
M n2+, Pb2+ h a s th e h ig h est affinity for m elanin, followed by C u2+
(32). M elanized fu n g al b io m ass a b so rb s 2.5 to 4 -fold m ore Ni, Cu,
Zn C d a n d Pb a n d a t hig h er ra te s th a n u n m elan iz ed c u ltu re s (50).
F u n g a l m e la n in s also show a h igh absorptive c ap a city for Fe3+ (48).
10
E n v iro n m en tal M etal C o n tam in atio n
A nthropogenic deposition of heavy m etals h a s d ram atically
in c re a se d
as
a re s u lt of m ining, in d u stria l a n d
p rac tic es, a n d fossil fuel c o n su m p tio n (2).
im p a cte d
soils
re m a in s
one
of th e
a g ric u ltu ral
R eclam ation of m etal-
m o st
difficult
a re a s
of
bio rem ed iatio n . T ech n iq u es in c u rre n t u se are generally b a sed on
im m obilization by lim ing, or ex tra ctio n of m e ta ls by acid-leaching
or electro -o sm o sis (4).
Som e of th e m ore sp e c ta c u la r m ining-
c o n ta m in a te d site s a re th e tailin g s p o n d s of A naconda, M ontana.
H isto rical Overview of th e A n aco n d a T ailings P onds
Large scale co p p er m ining b e g an in th e B u tte m in in g d istric t
d u rin g th e early 1880's.
B oth m ining a n d sm elting of th e B u tte
co p p er o res req u ire d a d ep en d ab le w a ter so u rce, a n d com panies
typically located th e ir p ro cessin g facilities n e a r or a d ja c e n t to th e
Silver Bow C reek. T hese co m p an ies d eposited th e w aste m aterial
or tailin g s from th e ir sm elters into p o n d s n e a r th e ir p la n ts or
directly into Silver Bow Creek.
Som e of th is m a te ria l eventually
w a sh e d into th e C lark F ork River.
The early sm e lte rs did n o t
efficiently e x tra c t copper or o th e r m in erals from th e ore a n d
c o n seq u e n tly pilings co n ta in e d significant c o n c e n tra tio n s of m etal.
11
In ad d itio n , early sm elting o p e ra tio n s se n t m in e ral lad e n fum es
in to th e air. By th e early 1900's, sm elting o p e ra tio n s w ere m oved
to A n a co n d a (39).
In 1883, c o n stru c tio n beg an on a sm elting facility 26 m iles
w e st of B u tte on W arm S prings C reek in th e D eer Lodge Valley.
W ithin two y e ars, th e A n aco n d a "U pper W orks" w as ex p an d ed a n d
d o u b led th e daily ore capacity.
In 1887, a n a d d itio n al sm elting
facility (the "Lower Works") w as c o n stru c te d s o u th e a s t of th e first
sm elter.
Finally th e c o n stru c tio n of th e A n aco n d a R eduction
W orks s o u th of W arm S prings C reek in 1902 re s u lte d in th e
la rg e st co p p er sm e lter in th e w orld (39).
The U pper a n d Lower W orks (collectively called th e "Old
Works") a t A n aco n d a d eposited w aste into W arm S p rin g s Creek, a
trib u ta ry of th e C lark. Fork River, or into p o n d s a d ja c e n t to th e
creek.
W ith th e c o n stru c tio n of th e A naco n d a R ed u ctio n W orks
sm elter, th e c o m p an y began c o n stru c tio n of a sy ste m of tailings
p o n d s n o rth a n d e a s t of th e sm elter, n e a r th e C lark Fork River.
D u rin g th e early 1900's, p o rtio n s of th e tailin g s p o n d w aste
overflowed into th e C lark Fork River (39).
T he A n aco n d a C opper C om pany b u ilt five tailin g s p o n d s to
c a tc h th e overflow in 1910. T h en in 1917, th e p o n d s B I, B2, C l
12
a n d C2 (O pportunity Ponds), a n d W S l a n d WS2 (W arm S prings
Ponds) w ere c o n stru c te d .
A bout 75% to 80% of th e tailings were
se ttled in th e B a n d C p o n d s, th e re m a in d e r into Silver Bow C reek
a n d th e n in to th e W arm S prings P o n d s (39).
In 1956, c o n stru c tio n b eg an on a new tailin g s p o n d th a t
w ould cover th e en tire a re a of th e A, B a n d C p o n d s, by raisin g
d ik es a ro u n d th e a re a to 90 feet.
T he a re a s p a n n e d 4200 acres
a n d h a d th e c ap a city to hold 4 0 0 m illion to n s.
T he "New River
Ponds" w ere com pleted a ro u n d 1957 (Fig. 1.1).
T he A n aco n d a tailings p o n d sy stem is p a rt of th e larg e st U.S.
E.P.A. S u p e rfu n d in th e U nited S ta te s, a n d c o n ta in significant
levels of copper, a rsen ic , lead a n d zinc. C u rre n t efforts to reclaim
th e tailin g s site in clu d e stab ilizatio n by revegetation following
lim ing a n d organic m a tte r ad d itio n , a n d p h y to rem ed iatio n to
e x tra c t th e m etals (F. M unshow er, p ers. comm.).
P h y to rem ed iatio n of M etaliferous S ites
P h y to rem ed iatio n is th e u s e of green p la n ts to rem ove
p o llu ta n ts from th e en v iro n m en t or re n d e r th e m h a rm le s s (43).
Aside from th e relative in ex p en se of th is a p p ro a c h , from a n
a e sth e tic view point, revegetation of th e se sites is m ore desirable
13
th a n
rem oval
of
P h y to rem ed iatio n
c o n ta m in a te d
of soil c an
soil
for
tre a tm e n t
be divided into
off-site.
two processes:
p h y to ex tractio n , in w hich m etal-a cc u m u latin g p la n ts are u se d to
tra n s p o rt a n d c o n c e n tra te m etals from soil into h a rv e sta b le p la n t
p a rts; a n d p h y to stab ilizatio n , in w hich heavy m etal to le ra n t p la n ts
are u s e d to red u c e th e m obility of heavy m etals, th e re b y reducing
th e risk of leach in g into g ro u n d w ater (46).
ARM SP
R WORKS
WARM S 3RINGS
SETTLING PONDS
PPE R WORKS
W
l fW
XtAldlXiW
ttW
.
Figure 1.1. A n aco n d a tailings p o n d system (39).
14
R ecent review s have d isc u sse d th e u s e of p la n ts a s a
stra te g y for th e reclam atio n of soil e n v iro n m en ts c o n ta m in a te d
w ith toxic m e ta ls (43, 46). O n heavy m e ta l-c o n ta m in a te d sites, th e
s ta tu s of heavy m etal c o n te n t d e te rm in e s th e degree a n d n a tu re of
heavy m etal to leran ce in p la n ts
(8);
p la n ts w ith high lead
to leran ce, for exam ple, a re fo u n d on sites w ith h igh lead co ntent.
M etal to le ra n t p la n ts em ploy th re e
stra te g ie s for grow th on
m etallifero u s soils.
M etal e x clu d ers p rev e n t m etal from en tering
th e ir a eria l p a rts .
N on-excluders a c c u m u la te m e ta ls in above­
g ro u n d t i s s u e s .. M etal n o n -ex c lu d ers c an be divided into two
g ro u p s
te rm e d
in d ic a to rs
and
h y p e ra c c u m u la to rs.
M etal
c o n c e n tra tio n in tis s u e s of in d ic ato r species reflect m e ta l levels in
th e soil.
H y p e rac cu m u lato rs c o n c e n tra te m etals in aboveground
tis s u e s (43).
Species of m etal to le ra n t p la n ts in clu d e A grostis
stolonifera, A. tenuis, F estuca rubra (47), F. ovina (8) a n d Minuaria
h irsu ta (41). R evegetation of m etalliferous soils w ith n o n -to le ra n t
sp ecies re q u ire s th e control of m etal u p ta k e by p la n t roots.
The
m ajo r facto r governing th e availability of m etals to p la n ts in soils is
th e solubility a n d th erm o d y n am ic activity of th e u n co m p lex ed ion,
sin ce in o rd er for root u p ta k e to occur, a soluble sp ecies m u s t be
p re s e n t
a d ja c e n t
to
th e
root
interface
(12),
Therefore,
15
c h a ra c te ristic s of th e ro o t zone, or rh izo sp h ere will d ete rm in e p la n t
m etal u p ta k e .
The R h izosphere a n d M etal Cycling
H iltn er originally in tro d u c e d th e co ncept of th e rhizo sp h ere
a s a zone of e n h a n c e d b a cterial grow th a ro u n d ro o ts of th e
L egum inoseae (27),
b ro a d e n e d
to
Since th e n , th e te rm "rhizosphere" h a s been
in clu d e
th e
en v iro n m en t
in h a b ite d
by
m icro o rg an ism s a s influenced by p la n t roots, th ro u g h e x u d atio n or
leakage of s u b s ta n c e s th a t affect m icrobial activity. T he release of
organic c o m p o u n d s by ro o ts in clu d e ex u d ates, w h ich are low
m o le cu la r w eight su b s ta n c e s th a t lea k o u t of roots;
secretions,
w hose release is m ed iated by biological activity; a n d m ucilages,
m ucigel a n d ly sates. E x u d a te s of ran g e g ra sse s in clu d e carboxylic
acid s, c a rb o h y d ra te s, a n d am ino acid s (30).
T he m o st im p o rta n t fea tu re of th e rh izo sp h ere w ith resp ec t
to m e ta l cycling is th e m odification of its chem ical p ro p erties by
th e root.
R hizosphere p ro ce sses m ediate c h a n g e s in pH, ionic
stre n g th , a n d com position of th e soil solution. The soil solu tio n pH
in th e rh izo sp h ere c h an g e s by ionic u p ta k e followed by charge
b a la n c e co rrectio n in th e root. P ro to n s m u s t be ex creted if excess
c a tio n s a re ab so rb ed , a n d hydroxyl or b ica rb o n ate io n s m u s t be
16
ex creted if excess anionic ch arg e h a s been ta k e n u p .
The
rh izo sp h ere pH of active ro o ts m o st often in c re a se s a s th e re s u lt of
an io n ic u p ta k e (53). T his in cre ase could lead to e n h a n c e d m etal
co m p lex ation by solids s u c h a s organic m a tte r w ith carboxyl or
hydroxyl g ro u p s on th e ir su rfaces.
In ad d itio n , th e release of
o rg an ic c o m p o u n d s in th e rhizo sp h e re c an influence m obility of
m eta ls in th e soil solution. M etal com plexes th a t a b so rb w eakly to
ro o t su rfa c e s will have g rea ter m obility a n d less likely to be ta k e n
u p by roots.
M etal bioavailability will in c re a se if e x u d atio n
in clu d e s c o m p o u n d s th a t form strongly a b so rb ed m eta l com plexes
(53).
However, only soluble m etal com plexes a re ab so rb ed by
ro o ts.
In ad d itio n , p la n t ro o ts are em bedded in a m ucilag in o u s
layer th a t b in d s tra c e m etals Pb2+ a n d C u2+, a n d c a n re s u lt in a
d e crea se of Pb a n d C u tra n s p o rt to ro o ts (38) .
H y d rated , soluble catio n s io n s are co n sid ered to be th e m etal
sp ecies a b so rb e d by p la n ts. To a large extent, m e ta l chelatio n by
o rganic a n d in o rg an ic soil c o m p o n e n ts controls th e m e ta l solubility
a n d availability to p la n ts (33).
Clay m in erals c a n a d so rb heavy
m etal c atio n s, b u t th e influence of clays on to ta l a b so rp tio n
c ap acity in soil is negligible co m p ared to th e c o n trib u tio n of
o rganic m a tte r (37). M etals so rb ed to clay m in e rals s u c h a s oxide
17
su rfa c e s a re inso lu b le, while so rp tio n to organics re s u lts in soluble
com plexes.
C helating a g en ts s u c h a s h u m ic acid e n h a n c e m etal
so lubility b u t in h ib it a b so rp tio n by p la n ts, a n d d isso ciatio n of
th e s e m etal com plexes is a p rere q u isite for u p ta k e (33).
F u n g a l m e la n in s c o n trib u te to soil organic m a tte r a n d sh a re
m a n y c h a ra c te ristic s w ith h u m ic acid s (5, 16).
F u n g i su c h a s G.
gram inis var. gram inis p ro d u ce m elan in in re sp o n se to copper (11),
a n d th e organic lig an d s provided by m elan in p ro b ab ly b in d m etals
a s a detoxification m ec h an ism .
In th e rh izo sp h ere, m elan in
p ro d u c tio n could provide sim ilar o p p o rtu n ities for m e ta l chelation,
th e re b y in c re a sin g com plex form ation a n d d ecrea sin g m etal u p ta k e
by p la n ts.
However, m elan in s have b een im plicated in m obilizing
m etal io n s from soil m in e rals into biological sy ste m s (63) a n d th u s
in c re a s e s in p la n t u p ta k e .
It is n o t know n w h a t role fungal
m elan o g en esis play s in m etal availability for p la n t a b so rp tio n .
H y potheses
My first h y p o th esis is th a t w ild-type m elanized G. gram inis
var. gram inis s tra in s will be m ore saprophytically com petitive th a n
heavily- or n o n -m elan ized stra in s , a n d th u s m ore p ath o g en ic to
rice in m ixed rh izo sp h ere com m unities.
In s tu d ie s u sin g non-
18
isogenic s tra in s , m elan in provides p ro tectio n from e n v iro n m en tal
s tre s s facto rs su c h a s lytic enzym es a n d UV light. U sing isogenic
s tra in s , m elanized fungi sh o u ld be able to su ccessfu lly com pete
w ith
o th e r rh izo sp h ere
in h a b ita n ts.
However,
m elan in
also
in ac tiv a te s enzym es n e c e ssa ry for n u trie n t a cq u isitio n , so a n
o v erp ro d u ctio n of th e se p ig m en ts will d ecrease th e com petitive
ability for s u b s tra te s .
My seco n d h y p o th esis is th a t m elanized G. gram inis var.
gram inis s tra in s
will a b so rb
m ore
copper in
liquid m edium
c o n ta in in g CuSCk th a n u n m elan iz ed stra in s. Previous w ork in o u r
lab o ra to ry h a s show n th a t by tre a tin g G. gram inis var. gram inis
w ith tricyclazole, a n in h ib ito r specific to D H N -m elanin sy n th esis,
C u 2+ a b so rp tio n to b io m a ss d e c re a se s (11). Isolation of G. gram inis
var. gram inis s tra in s w ith m u ta tio n s in th e D H N -m elanin p ath w ay
h av in g e ith e r no m elan o g en esis or over-expression of m elan in , will
a d d to o u r know ledge of th e re la tio n sh ip betw een m eta l binding
a n d m elanin.
My final h y p o th esis is th a t m elanized
G. gram inis var.
gram inis s tra in s will a b so rb m etals from m ine tailin g s in range
g ra s s rhizo sp h e re s, re su ltin g in re d u c e d m etal c o n c e n tra tio n s in
p la n t tis su e .
M elanins are chem ically a n d stru c tu ra lly sim ilar to
19
h u m ic a cid s fo u n d in soils, a n d h u m ic acid s provide lig an d s th a t
b in d heavy m etals. It follows th a t m elanized fungi will b in d heavy
m eta ls in a sim ilar fashion, prev en tin g m etal u p ta k e in to p lan ts.
L iteratu re Cited
!
1.
A hm ad, J . S a n d R. B aker. 1987. C om petitive sap ro p h y tic
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2.
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6.
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Ann. Rev. P h y to p a th o l.
20
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H olm er, L. a n d J . Stenlid. 1996. D iffuse com petition for
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Kirk, J . J . a n d J . W. D eacon. 1987. C ontrol of th e take-all
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1988. R hizosphere m icroorganism effects on soluble am ino
acid s, s u g a rs a n d organic acid s in th e root zone of Agropyron
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Kuo, M .-J. a n d M. A lexander. 1967. In h ib itio n of th e lysis
of fungi by m elan in s. J . B acteriol. 9 4 :6 2 4 -6 2 9 .
32.
L a rrso n , B. a n d Tjalve.
1978. S tu d ies in th e m elaninaffinity of m e ta l ions. Acta. Physiol. S c a n d . 1 0 4 :4 7 9 -4 8 4 .
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role of m etal ion c h elatio n in p la n t u p ta k e p ro cesses. In J .
A. M anthey, D. E. Crowly a n d D. G. L u ster (ed.),
B iochem istry of m etal m ic ro n u trie n ts in th e rhizosphere.
CRC P ress, B oca R aton, FL.
34.
L e m a n c e a u , P., P. A. H. M. B akker, W. J . deKogel, C.
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n o n p a th o g e n ic F usarium oxysporum Fo47 a n d p se u d o b a c tin
3 5 8 u p o n p ath o g en ic F usarium oxysporum f. sp. dianthi.
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35.
Lockwood, J . L. 1992. E xploitation ,com petition. In G. C.
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M errington, G. a n d B. J . Alloway. 1994. T he flux of Cd, Cu,
Pb a n d Z n in m ining po llu ted soils. W ater Air Soil Pollut.
7 3 :3 3 3 -3 4 4 .
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Morel, J ., M. M ench a n d A. G uckert. 1986. M e asu rem en t of
Pb, C u, Cd b in d in g w ith m ucilage e x u d ates from m aize [Zea
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O u z o u n id u o , G. L., L. Sym eonidis, D. B a b a lo n a s a n d S.
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P earso n , R. G. 1963. H ard a n d soft acids a n d b a se s. J Am.
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A sp e r g illu s fla v u s colonization a n d aflatoxin B i form ation in
b arley
g rain
d u rin g
in te ra c tio n w ith
o th e r fungi.
M ycopathologia 136 :5 3 -6 3 .
43.
R ask in , I., N. P. B. A. K um ar, S. D uchenkov a n d D. E. Salt.
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45.
Rizzo, D. M., R. A. B lan c h ette a n d M. A. P alm er. 1992.
B iosorption of m etal ions by Armillaria rh izo m o rp h s. C an. J .
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S enesi, N., G. Sposito a n d J . P. M artin. 1987. C opper (II)
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S iv an , A. a n d I. C het.
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The p o ssib le role of
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26
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Z u n in o , H. a n d J . P. M artin. 1977. M etal-binding organic
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27
CHAPTER 2
ISOLATION AND CHARACTERIZATION OF G aeum annom yces
gram inis var. gram inis MELANIN MUTANTS
In tro d u ctio n
F acu ltativ e fungal p a th o g e n s a re th o se fungi able to c au se
d ise ase a n d live saprophytically in th e a b sen c e of a h o s t organism .
T h ese p a th o g e n s com pete for n u trie n ts w ith free-living decom poser
p o p u latio n s.
G a rre tt (1970) listed a ttrib u te s c o n trib u tin g to th e
su c c e ss of fungi a s com petitive sap ro p h y tes.
T hey ,are: I) rap id
g e rm in atio n of p ro p ag u les a n d ra p id h y p h al grow th to rea ch
s u b s tra te s ;
2) enzym e p ro d u ctio n to utilize s u b s tra te s ;
3)
ex cretio n of e ith e r b a cterio static or fu n g istatic su b s ta n c e s to
red u c e grow th of com petitors;
a n d 4) to leran ce to d eleterious
s u b s ta n c e s p ro d u ce d by o th er o rg an ism s.
G a eum annom yces gram inis var. gram inis is a hom othallic
fila m e n to u s
m any
ascom ycete th a t colonizes root a n d crow n tissu e of
m em b ers
of th e
Poaceae
p la n t
sap ro p h y tically in soil or p la n t litter.
fam ily
and
survives
Som e s tra in s invade h o st
v a sc u la r tis s u e to c a u se root d isease in rice a n d o rn a m e n ta l
28
tu rfg ra ss e s (32, 36).
G. gram inis var. gram inis p ro d u c e s m elanin,
via th e d ih y d ro x y n ap h th ale n e (DHN) p ath w ay (Fig. 2.1) in resp o n se
to s tre s s fac to rs (7, 13). M elanin also a c c u m u la te s in older h yphal
cell w all a n d
infection
in
cells
sex u al s tru c tu re s
(hyphopodia).
W ang
(perithecia)
and
and
adhesive
C asad ev all
(1994)
d e m o n s tra te d th a t m elan in serves a s a protective layer a g a in st UV
irra d ia tio n ,
by
ad d in g
d ih y d ro x y p h en y lalan in e
th e
(L-DOPA)
m elan in
to
p re c u rso r
Cryptococcus
L-
neoform ans
c u ltu re s. C u ltu re s grow n w ith L-DOPA w ere m ore re s is ta n t to UV
lig h t d am age th a n control c u ltu re s. At p re se n t, sim ilar stu d ie s of
D H N -m elanin p ro tectio n a g a in st UV irrad iatio n , e ith e r by u sin g
m e lan in p re c u so r ad d itio n or isogenic stra in s , have n o t been
p u b lish ed .
M elanin c o n te n t of fungal h y p h a e positively co rre lates w ith
e n h a n c e d re s ista n c e to c h itin ase a n d g lu ca n ase d e g ra d atio n (25),
a n d m elanized R hizotonia solani s tra in s are less sensitive to lysis
th a n u n m elan iz ed s tra in s
(23).
In addition, w h e n m elanin-
c o n ta in in g sp icu les of A spergillus p h o en cis
sp o re s a re rem oved,
th e y a re m ore sensitive to lytic activity (4).
W hile th e s e stu d ies
su g g e st a c o rrelatio n betw een m elan izatio n a n d lytic enzym e
p ro tectio n , e x p erim en ts u sin g isogenic stra in s a re lacking.
In
29
su m m ary ,
m elan in
serves
as
a
protective
lay er a g a in st UV
irra d ia tio n a n d lytic enzym es a n d m ay c o n trib u te to com petitive
su c c e ss in a m ixed m icrobial com m unity.
E x tra ce llu lar enzym es se cre ted by G. gram inis a re n e ce ssa ry
for n u tr ie n t acq u isitio n a n d im plicated in th e d ise ase p ro ce ss (9).
Infection re q u ire s p e n e tra tio n of h o s t cells a n d
involves th e
d e g ra d atio n ‘ of cell w all co m p o n e n ts by th e p ath o g e n .
enzym es
se cre ted
at
th e
growing
apex
aid
d eco m p o sition a n d s u b s e q u e n t p ath o g en esis.
in
cell
Lytic
wall
Previous stu d ie s
(Goins a n d H en so n , subm itted) su g g e st th a t s p o n ta n e o u s m elanin
m u ta n ts of G. gram inis var. tritici p ro d u ce less e x tra ce llu lar pro tein
a n d a re less p ath o g en ic th a n th e lightly p ig m en ted w ild-type
stra in . In ad d itio n , m elan in h a s b een show n to provide s tru c tu ra l
rigidity req u ire d
to m a in ta in
infection s tru c tu re s
tu rg o r p re s su re
(20, 21).
of ap p re sso ria l
G. gram inis d o es n o t develop
a p p re sso ria , b u t does p ro d u ce m elanized a d h e sio n stru c tu re s
(hyphopodia) th a t are believed to serve a sim ilar fun ctio n a s
a p p re sso ria .
B e ca u se
c h arac terize
c h arac terize d
m e lan in s
and
are
q u a n tita te ,
h etero g en eo u s
in
th is
stu d y
and
we
difficult
isolated
to
and
G. gram inis var. gram inis m u ta n t s tra in s affected in
30
th e m elan in p ath w ay to d eterm in e th e effect of lack of m elanin or
o v erm elanization in protection from en v iro n m en tal s tre sso rs an d
th e ability to p ro d u ce disease in rice p lan ts.
A C eT A T C
M E L A N IN
i.a .e .a -T M N
ji° ]
F le v lo H n
[H )| j[0 ]
j[° ]
3 . S 1- B m e v i o l l n
[H ijjr o j
T
T
OH
1 .* .4 .5 .7 - P M N
I | m
1 . 2 , 4 . 6 —T M N
ij[ H ]
[H ]| J[0 )
fH jj j[0 ]
-SB
as L~as
OM
OH
OH
OH
1.3 . 4 ^ - T H N
j[ H ]
OH
4 . 8 —O H T
iJ lH l
OH
2 .4 .6 - T H T
Figure 2.1.
1 ,8 -d ih y d ro x y n ap h th alen e (DHN) m elanin
p ath w ay a n d s h u n t p ro d u c ts p ro d u ced by m u ta tio n s in the
p ath w ay (modified from reference 2).
T = site of tricyclazole
in h ib itio n. G enetic n o m en c la tu re is from references 27 a n d 34. 2H J = 2 - hy droxyj u g lo n e ; 1,3,8-THN = 1 ,3 ,8 -trih y d ro x y n ap h th alen e.
M aterials a n d M ethods
S tra in s, m edia a n d grow th cond itio n s
O u r wild type G. gram inis var. gram inis stra in , JH 2 0 3 3 , is a
single a sco sp o re derivative of a n isolate collected by Dr. Craig
R o th ro ck from soybean (Glycine m a x L.).
31
D efined liquid m inim al m ed iu m co n ta in e d in g p e r liter:
su c ro se , 3; K2HPO4, I; MgSCk, 0.05; KC1, 0.05; CaClg^ 0-1; FeCls,
0 .0 0 0 5 ;
and
trac e
e lem en ts
[ZnSO^HgO,
0.0125;
Fe(NH4)(SO4)2-SH2O, 0.0025; C uSO 4- SH2O, 2 x 10-6; M nSO4 H2O,
0 .0 0 0 1 ; H3BO3, 0 .0001; NaMoO4-H2O, 0.0001; biotin, 0.001 a n d
th ia m in e , 0.001].
The sto ck tra c e elem en t so lu tio n w as filter
sterilized a n d d ilu te d 1:4000 in to autoclaved m edium .
Stock
so lu tio n s of MgSO4, KCl a n d CaCl2 w ere filter sterilized a n d d ilu ted
1:1000 into au to clav ed m edium .
F ilter sterilized FeCl3 solution
w as d ilu te d 1:200000 into auto clav ed m edium .
T he pH w as
a d ju s te d to 5.5 w ith HCl for b o th solid a n d liquid m ed ia before
autoclaving.
O th e r m ed ia u s e d w ere p o tato d ex tro se a g a r (PDA;
Difco, D etroit, M ichigan), p o tato
dextrose b ro th
(PDB; Difco,
D etroit, M ichigan) a n d L u ria-B ertan i (LB) w ith or w ith o u t 1.0 %
a g a r (Meer C orp., New B ergen, N J ). C u ltu re s w ere in o cu late d from
LB a g a r into 200 m l liquid m ed iu m in I liter flask s, a n d sh a k e n
(50 cycles m in -1) a t 25 C w ith a 12 h p hotoperiod a n d 3 0 0 0 Lux.
H yphopodia
w ere
grow n
by
in o cu latin g
m ylar
(14C
th ic k n e ss, a gift from D uP ont, W ilm ington, DB) m e m b ra n e s th a t
w ere e th an o l-sterilized a n d placed on 1% w ater agar.
M u ta n t isolation
A five-day c u ltu re of w ild-type h y p h ae grow n in 100 m l LB
b ro th w a s h a rv e ste d by filtration th ro u g h a W h atm a n # I filter a n d
re s u s p e n d e d in 75 m l p ro to p la st buffer (PBI- 0.2 M NaH2PO4 a n d
32
0 .6 M KC1, pH 5.8) w ith 0 .3 7 5 g e a c h of Novozyme 2 3 4 (InterSpex
P ro d u c ts, Inc) a n d cellulase (Sigm a C-8546).
slowly for
1.5 h
H yphae w ere sh a k e n
a t 25 C, filtered th ro u g h g lass wool, a n d
p ro to p la sts in th e filtrate w ere w a sh ed twice w ith PBI a n d
re s u s p e n d e d in 5 m l PBL
P ro to p lasts w ere su b je cted to
chem ical m u ta g e n e sis u sin g
4 -n itro q u in o len e oxide (NQO) or UV light.
A pproxim ately IO8
p ro to p la sts w ere in c u b a te d for I h a t room te m p e ra tu re in 5 m l
PBI c o n ta in in g
I p g /m l NQO (3).
NQO w as n e u tra liz ed by
a d d itio n of a n eq u al volum e of 5% so d iu m th io su lfa te in PBI, th e n
w a sh e d twice w ith PBI a n d re s u sp e n d e d in 5 m l of PBL
For UV
m u ta g e n e sis, IO8 p ro to p la sts in 5 m l PBI w ere exposed to 0.09 J
per
cm 2
in
a
S tratalin k er®
UV C rosslinker,
Model
1800
(S tratag ene, La Jo lla, CA).
T reated p ro to p la sts w ere p lated onto reg e n era tio n m edium
(17) a n d in c u b a te d a t room te m p e ra tu re in th e d a rk for 7 days,
a n d reg e n e ra te d p ro to p la sts w ere tra n sfe rre d to PDA w ith 1%
so rb o se.
S orbose in h ib its h y p h a l sp read in g (16) so th a t m ore
p ro to p la sts could be screen ed on a 90 m m 2 p e tri plate.
M u ta n ts
w ith p ig m e n t p ro d u c tio n different from o u r w ild-type s tra in w ere
tra n s fe rre d to PDA a n d LB agar.
Two m u ta n ts w ere c h o sen for
fu rth e r c h aracterizatio n : JH 4 3 0 0 , a m u ta n t th a t did n o t produce
m e lan in on PDA a n d JH 4301, th a t co n stitutively synth esized
m elan in .
33
S tra in c ro sse s
JH 2 0 2 8 , a b leo m y cin -resistan t tra n s fo rm a n t of JH 2 0 3 3 (28)
w as in o c u la te d w ith JH 4 3 0 0 or JH 4 3 0 1 onto sterile, m o ist filter
p a p e r w ith a layer of to o th p ick s onto w hich au to clav ed rice leaves
w ere placed.
P lates w ere in c u b a te d a t 25 C w ith a
12 h
p h o to p erio d a t 3 0 0 0 Lux. Single, m a tu re p e rith ec ia w ere sq u a sh e d
on slid es u n d e r g lass coverslips a n d sp re a d onto LB ag ar. After 24
h g e rm in ate d a sco sp o res were tra n sfe rre d to PDA p la te s to detect
p ig m e n tatio n p h en o ty p es. A scospore derivatives w ere screen ed for
PhleoR/ Phleos on LB a g a r+2.5 g /m l Bleom ycin (a gift from B ristolM eyer, Evansville, IN).
E lectro n m icroscopy
Nine day-old c u ltu re s grow n in m inim al m ed iu m
collected for m icroscopy.
were
S pecim ens w ere p ro ce ssed for freeze
s u b s titu tio n by freezing in liquid p ro p an e a t -1 9 0 C to -193 C,
s u b s titu tin g in 2% o sm iu m tetroxide a n d 0.05% u ra n y l a cetate in
aceto n e, a n d em b ed d in g betw een two m icroscope slid es in E pon
812-A raldite 6 0 0 5 epoxy re sin (18, 19, 20).
Cells w ere exam ined
for freeze d am age by p h a se c o n tra s t optics a t IOOOx. U ndam aged
cells
w ere
m o u n te d
onto
epoxy
stu b s,
th in -sec tio n e d
longitudinally, a n d sta in e d w ith u ra n y l a ce ta te a n d lead citrate.
S p ecim en s w ere exam ined w ith a Je o L JE M I OOCX tra n sm issio n
m icroscope o p e ra ted a t 80 kV. At le a st 15-30 cell w alls a n d one
h y p h a l tip from e a c h s tra in w ere viewed.
34
M elanin Q u a n tita tio n
M elanin c o n c e n tra tio n of th e th re e s tra in s w ere e stim ate d by
th e m e th o d of B u tle r a n d L achance (1986).
For p re p a ra tio n of
m e lan in for a s ta n d a rd curve, 10 p lu g s of JH 2 0 3 3 from a n LB a g ar
p late w ere in o cu late d into 250 m l of LB b ro th .
T he c u ltu re w as
in c u b a te d for 7 d a y s a t room te m p e ra tu re w ith sh a k in g (150 rpm ).
H yphae w ere rem oved by filtration th ro u g h a W h a tm a n #1 filter
a n d th e s u p e rn a te acidified to 4 N HCl w ith c o n c e n tra te d HCL
After allow ing p recip itatio n a t 4° C for 4 days, m elan in w as
recovered by c en trifu g atio n a t 5000 x g for 15 m in u te s. The pellet
w as w a sh e d 3X w ith 0.1 N HCl followed by 5 w a sh e s w ith distilled
w ater.
The m elan in w as lyophilized overnight to rem ove resid u a l
w a te r a n d sto re d a t -20 °C in th e d ark .
The s ta n d a rd m elanin
curve w a s p re p a re d by w eighing d u p lic ate 250, 500 a n d 1000 pg
sa m p le s into 15 m l Corex® tu b e s.
T hree m l of A zure A (Sigma)
so lu tio n w ere a d d e d to each tu b e a n d allowed to s ta n d for 30
m in u te s.
S o lu tio n s w ere th e n filtered th ro u g h a 0 .4 5 pm syringe
filter. A zure A so lu tio n w as p re p a re d from a sto ck so lu tio n of 0.1 g
A zure A in 500 m l 0.2 N HCl th a t w as d iluted 1:30 in 0.2 N HC1.
M elanin
c o n c e n tra tio n
a b so rb a n c e a t 6 2 8 nm .
w as
d e term in ed
by
a
d ecrease
in
35
M elanization
of
w ild-type,
JH 4 3 0 0
and
JH 4 3 0 1
w as
d e te rm in e d a t 3, 4, 5, 6 a n d 7 d ay s following in o cu latio n of 3 plugs
into 6 0 m l LB b ro th in 300 m l E rlenm eyer flasks.
F la sk s were
s h a k e n a t 150 rp m a t room te m p e ra tu re in th e d a rk . At harv est,
h y p h a e w ere collected by filtration th ro u g h a W h a tm a n #1 filter
a n d lyophilized overnight. T riplicate 2 m g sam p les of dried hyp h ae
w ere a ssa y e d a s d escrib ed for th e s ta n d a rd m elan in curve.
Novozyme D igestion
A
s ta n d a rd
N -acetylglucosam ine
s ta n d a rd
curve
w as
p re p a re d from I mM N -acetylglucosam ine in 0 .0 3 3 M p h o sp h a te
buffer (pH 6.1). N -acetylglucosam ine w as d e tected by th e m ethod
of R eissieg e t al. (1955).
*
To tu b e s co n tain in g 500 pi of solution,
100 pi of p o ta s siu m b o rate (49.64 g H3BO3 p e r liter, pH 9.1) w ere
ad d ed , th e n h e a te d in a boiling w a ter b a th for 3 m in u te s a n d
cooled
in
a
cold
w a ter
b a th .
T hree
m is
of
d im eth y lam in o b en zad eh y d e w ere ad d ed , th e c o n te n ts m ixed, th e n
h e a te d a t 37 °C for 20 m in u te s. After cooling in a cold w ater b a th ,
a b so rb a n c e w as re a d a t 544 a n d 585 nm .
D u plicate w ild-type, JH 4 3 0 0 a n d JH 4 3 0 1 s tra in s w ere grown
for 7 d a y s in 50 m l LB b ro th w ith sh a k in g a t room tem p era tu re .
36
H yphae w ere h a rv e ste d by filtration th ro u g h a W h atm a n #1 filter,
w a sh e d 2X w ith sterile p h o sp h a te buffer a n d tra n s fe rre d to sterile
2 5 0 m l E rlen m ey er flask s co n ta in in g 50 m g Novozyme 234 in 50
m l p h o sp h a te buffer. F la sk s w ere in c u b a te d w ith sh a k in g a t room
te m p e ra tu re a n d aliq u o ts of th e su p e rn a te ta k e n a t I, 2 a n d 3
h o u rs.
S u p e rn a te s w ere analyzed for N -acetylglucosam ine a s
d e scrib e d for th e s ta n d a rd curve.
C ontrols c o n siste d of h yphae
w ith o u t Novozyme 234.
UV p ro tectio n
D u ra p o re type GV 0 .22pm 47 m m d iam eter filters (Millipore
c a t no. GVMP 047 00) w ere au toclaved, w eighed a n d placed on 90
m m d ia m e te r p e tri p la te s c o n tain in g LB agar.
O ne plug of
JH 2 0 3 3 , JH 4 3 0 0 or JH 4 3 0 1 from LB a g ar w as in o c u la te d onto th e
c e n te r of th e filter a n d th e p la te s in c u b a te d in th e d a rk for 3 days
a t room te m p e ra tu re . Lids w ere rem oved from p la te s, placed in a
UV S tra ta lin k e r, th e n exposed to 200, 400 or 6 0 0 p J.
Plate lids
w ere rep laced , a n d in c u b a te d for 3 d ay s a t room te m p e ra tu re in
th e d a rk .
Following in cu b a tio n , filters w ere rem oved, dried
o v ern ig h t on a lyophilizer a n d th e n w eighed.
37
E nzym e S ecretion
P o ly g alactu ro n ase (PGU; EG 3.2.1.15) secretio n w as detected
o n solid m ed iu m co n ta in in g 1% so d iu m polypectate (Sigma) a t pH
6 .5 in 10 mM so d iu m p h o sp h a te buffer w ith 0.1% y e a s t extract,
0.125% e a c h of b iotin a n d th ia m in , a n d 2% B acto agar.
C learing
a ro u n d fu n g al colonies, in d icatin g enzym e secretion, w a s detected
by flooding p late w ith 5 N HCl (40). B ase m ed iu m for th e detection
of a-cellu lo se c o n ta in e d 2.5% C zapek's Dox b ro th (Difco), 0.125%
e a c h of b iotin a n d th ia m in , 1% a-cellulose (Sigma) a n d 2% Bacto
ag ar.
H ydrolysis w as d etected by flooding p la te s w ith LugoPs
iodine a n d m e a su rin g th e cleared a re a (I). H ydrolysis of skim m ilk
w as a s s e s s e d on p la te s c o n tain in g 2% skim m ilk, a n d 2% Bacto
agar.
C learing a ro u n d colonies in d icated hydrolysis.
Filter-
sterilized tricyclazole from a 2.5 m g /m l 100 % EtO H sto ck w as
ad d ed a t a c o n c e n tra tio n of 16 p g /m l to cooled m edia.
P ath o g enicity
H ulled rice
se ed s
[Oryza sa tiv a Gulfport)
w ere
surface
sterilized in 1% AgNOs for 7 m in a n d rin se d 2X in sterile deionized
w ater.
Sterile verm iculite in 10 cm d iam eter p o ts w a s in o cu lated
w ith h y p h a l on p lu g s from LB a g a r a n d seeds w ere p laced n ex t to
38
th e p lu g s.
P la n ts w ere grow n for 28 days a t 25 °C w ith a 12 h
p h o to p erio d a t 3 0 0 0 Lux a n d a w aterin g sch ed u le of 100 m l sterile
w a te r every 5 days. At h a rv e st, ro o ts a n d sh o o ts w ere se p ara ted ,
lyophilized o vernight a n d w eighed.
R esu lts
M u ta n t c h arac te riz a tio n
M u ta n t s tra in JH 4 3 0 0 , iso lated after tre a tm e n t of wild-type
p ro to p la sts w ith 4-nitroqU inolene, did n o t p ro d u ce th e d a rk green
D H N -m elanin pig m en t th a t is c h a ra c te ristic of th e w ild-type stra in
on m in im al, LB or p o tato d ex trose
m edia.
M u ta n t JH 4 3 0 0 w as
oran g e on PDA a s th e re s u lt of 2-hydroxyjuglone
a n d flaviolin
a c c u m u la tio n , w hich are d a rk orange a n d brow n, respectively a n d
confirm ed by th in layer c h ro m a to g ra p h y (TLC, d a ta n o t show n, ref.
2). T his re s u lt su g g ested th a t its m elan in p ath w ay w a s blocked a t
th e re d u c ta s e ste p th a t converts 1 ,3 ,8 -trih y d ro x y n ap h th alen e to
verm elone (Fig. 2.1). M oreover, feeding e x p erim en ts w ith scytalone
did n o t re sto re th e w ild-type m e lan in p henotype (d ata n o t shown),
a n ex p ected finding if th e p ath w ay w as blocked d o w n stre am of th e
scy talo n e b io sy n th etic step (2).
39
W hen we c ro ssed m u ta n t 4 3 0 0 w ith th e w ild-type stra in ,
pro g en y disp lay ed a 1:1 wild type: m u ta n t m e la n in p henotype
(Table 2.1), a n d su g g ested th a t th is m u ta n t p h e n o ty p e resu lte d
from
a
single
m u ta tio n
affecting
th e
gene
encoding
trih y d ro x y n a p h th a le n e re d u c ta se , or th e TH Rl gene {Colletotrichum
lagenarium n o m en c la tu re, ref. 27).
The bleom ycin re sista n c e
m a rk e r did n o t segregate 1:1, a re s u lt c o n siste n t w ith o th e r stu d ie s
of G. gram inis var.
h etero lo gous,
gram inis tra n s fo rm a n t c ro sse s
tra n sfo rm e d
se q u en c es
su c h
as
th e
in w hich
BLE gene
so m etim es did n o t segregate in a 1:1 fash io n (28).
We iso lated
JH 4 3 0 1
following tre a tm e n t
of wild-type
p ro to p la sts w ith UV light a n d it constitutively p ro d u c e d a d a rk
g reen -b lack p ig m e n t on m inim al, LB a n d p o tato d ex tro se m edia.
H yphae of th is s tra in w ere m ore heavily p igm ented th a n w ild-type
h y p h a e a n d a c c u m u la te d a variety of m elan in m etab o lite s in PDA
c o n ta in in g 8 a n d 30 p g /m l tricyclazole, a specific D H N -m elanin
p a th w ay in h ib ito r (Fig. 2.1, ref. 2). In ad dition to flaviolin a n d 2hydroxyjuglone,
and
JH 4 3 0 1
a c c u m u la te d
4 ,8 -d ihydroxytetralone
while
3 ,4 ,8 -trih y d ro x y tetralo n e
tricyclazole-treated
w ild-type
c u ltu re s a c c u m u la te d flaviolin a n d 2-hydroxyjuglone, th e oxidative
p ro d u c ts
of
1 ,3 ,6 ,8 -tetrah y d ro x y n a p h th a len e
and
. 1,3,8-
40
trih y d ro x y n a p h th a le n e , respectively.
All s h u n t p ro d u c ts w ere
identified by TLC w ith Haviolin a n d 2-hydroxyjuglone a s sta n d a rd s
(d ata n o t show n, ref. 2).
T able 2.1. G enetic C rosses.
Cross
Total
Hybrid
Total
Perithecia Perithecia Ascospores
Phenotype
Number
Progeny
JH 2028
X
JH 4300
26
3
262
BleS ThrlBleR ThrlBles T h rl+
BleRThrl+
92
35
86
49
JH 2028
X
JH4301
3
I
233
BleS MoeBleR MoeBleS Moe+
BleR Moe+
27
51
88
67
JH 4302
X
JH 2033
9
I
69
BleS MoeBleR MoeBleS Moe+
BleR Moe+
14
11
25
19
J H 4 3 0 1 did n o t p ro d u ce m a tu re a sco sp o res w h en selfed, b u t
th e c ro ss betw een w ild-type a n d m u ta n t JH 4 3 0 1 displayed 1:2
m u ta n t: w ild-type m elan in p h en o ty p e (Table 2.1).
ratio w a s
P e rh a p s th is
skew ed b e c a u se JH 4 3 0 1 a sco sp o res g erm in ate d m ore
slowly th a n th o se of th e w ild-type a n d som e viable ' JH 4301
a sc o sp o re s w ere possibly overgrow n w ith h y p h ae from m ore rapidly
g e rm in atin g w ild-type asco sp o res. A b ack c ro ss of a PhleoR m u ta n t
JH 4 3 0 1 derived from th is in itial c ro ss w ith th e w ild-type stra in
41
gave ap p ro x im ately 1:1 w ild-type:m u ta n t progeny.
m u ta n t asco sp o re
germ in atio n
Again, slow
possibly in flu en ced
th is ratio.
Progeny of th e c ro sse d p erith ec ia w ith JH 4 3 0 1 m e lan in phenotype
grew a t a sim ilar ra te a n d p ro d u ce d sim ilarly s h a p e d h y phopodia
a s JH 4 3 0 1 .
T hese re s u lts su g g ested th a t JH 4 3 0 1 co n ta in e d a
single m u ta tio n (designated moe) affecting th e m e lan in biosynthetic
p ath w ay .
E lectro n
m icro g rap h s
of
fre e z e -su b stitu te d
hyphae
d e m o n s tra te d cell w all differences betw een th e w ild-type stra in a n d
two m e lan in m u ta n ts .
Nine day-old w ild-type c u ltu re s grown in
th e p re sen c e of tra c e copper h a d only two layers: a n electro n d en se
o u te r m ucilage layer a n d a n electron tra n s lu c e n t in n e r c h itin layer
(Fig. 2.2a.) previously found to b in d w h e a t germ agg lu tin in (7).
S tra in JH 4 3 0 0 cell w alls w ere com posed of two layers: a n in n e r
tr a n s lu c e n t c h itin layer a n d a th in d en se m ucilage o u te r layer (Fig.
2.2c).
By co m p ariso n , d a rk m u ta n t J H 4 3 0 1 h a d a n in term ed iate
m elan ized layer even w h en grow n in trace co p p er a n d a d en se
o u te r layer w ith th ic k e r m ucilage closely a sso c ia te d w ith th e cell
w all (Fig. 2.2b).
42
Figure 2.2. E lectron m icro g rap h s of w ild-type a n d m elan in m u ta n t
cell w alls. A. W ild-type JH 2 0 3 3 , B. D ark m u ta n t JH 4 3 0 1 , C. Light
m u ta n t JH 4 3 0 0 . * is th e in te rm e d iate m elanin layer, i is th e in n er
c h tin layer a n d 0 is th e o u te r m ucilage layer. M agnification =
66,000X .
T hin sectio n s of h y p h al tip s also show ed differences in
m ucilage betw een th e th ree s tra in s (Fig. 2.3). W hile th e w ild-type
d isp lay ed a n electron d e n se m ucilage layer closely a sso c ia te d w ith
43
th e ap ical region (Fig. 2.3a.), th e d a rk m u ta n t show ed a d e n se r
m ucilage th a t ex ten d ed to th e ap ical tip (Fig. 2.3b).
The light
m u ta n t exhibited a loose layer of m ucilage a sso c ia te d w ith th e cell
(Fig. 2.3c).
H yphopodia of th e th re e s tra in s differed in p ig m en tatio n or
sh a p e (Fig. 2.4).
W ild-type h y p h o p o d ia on m y lar w ere m elanized
a n d lobed (Fig. 2.4a.) The light m u ta n t p ro d u ced bro w n ish -o ran g e
lobed h y p h o p o d ia (Fig. 2.4c.) w hile th e d a rk m u ta n t developed
highly p igm ented, ro u n d s tru c tu re s (Fig. 2.4b). P erith e cia of selfc ro sse d m u ta n t JH 4 3 0 0 w ere o range-light brow n a n d u n d e r th e se
co n d itio n s d a rk m u ta n t JH 4 3 0 1
p ro d u ce d im m a tu re p erith ecia
w ith o u t m a tu re a sco sp o res u n le s s cro sse d w ith th e w ild-type (data
n o t show n).
O u r w ild-type s tra in re a c h e d th e sta tio n a ry grow th p h a se by
d ay 6 w h en grow n in LB (Fig. 2.5b), w ith m elan in co n ce n tra tio n
ro u g h ly in c re a sin g in p arallel w ith biom ass.
T he light m u ta n t
JH 4 3 0 0 h a d n o t re a c h e d s ta tio n a iy p h a se by th e e n d of 7 days of
grow th (Fig. 2.5a), a n d m elan in c o n c e n tra tio n s rem ain ed low
th ro u g h o u t th e grow th curve. T he d a rk m u ta n t b io m a ss leveled off
b etw een d a y s 6 a n d 7 (Fig. 2.5c), w hile m elan in c o n c e n tra tio n w as
44
h ig h e s t a t day 5.
E xcept for day 7, th e m elan in c o n c e n tra tio n of
J H 4 3 O lw as significantly h ig h er th a n o u r w ild-type stra in .
UV Light S ensitivity
T he heavily m elanized JH 4 3 0 1 s tra in w as less sensitive to
UV lig h t th a n e ith e r o u r w ild-type s tra in or th e light JH 4 3 0 0 stra in
(Fig. 2.6). UV ex p o su re red u c ed JH 4 3 0 0 b io m ass by 55-62% a n d
w as n o t d o se -d ep e n d en t.
At low ex p o su re (200 a n d 400 p J /c m 2)
th e w ild-type b io m a ss w as re d u c e d by appro x im ately 40% , a n d
54%
a t th e
600
p J /c m 2 exposure.
UV ex p o su re
did
not
significantly red u c e th e d a rk m u ta n t biom ass.
Lytic E nzym e P rotection
S u p e rn a te s from th e d a rk m elan in m u ta n t h a d higher Nacety lg lu cosam ine initially, a n d following I h o u r digestion w ith
Novozyme 234, th a n e ith er o u r w ild-type s tra in or JH 4 3 0 0 (Fig.
2.7).
However,
significantly
m ore
N- acetylgluco sam in e
w as
re le ased from th e light m elan in m u ta n t th a n th e m elanized stra in s
afte r 2 or 3 h o u rs of in c u b a tio n w ith Novozyme 234. After 3 h o u rs
of digestion, th e a m o u n t of N-acetylglucosam ine rele ased from
JH 4 3 0 1 in th e p resen c e of Novozyme w as n o t significantly different
from th e J H 4 3 0 1 control.
45
Figure 2.3. H yphal tip s of w ild-type a n d m elanin m u ta n ts strain s.
A) W ild-type, B) D ark m u ta n t JH 4 3 0 1 , a n d C) L ight m u ta n t
JH 4 3 0 0 . Arrows in d icate m ucilage. M agnification = 33,000X .
46
F igure 2.4. H yphopodia of w ild-type a n d m elan in m u ta n t stra in s.
A) W ild-type JH 2 0 3 3 , B). D ark m u ta n t JH 4 3 0 1 , C) Light m u ta n t
J H 4 3 0 1 1. M agnification = 400X.
Enzym e S ecretion
The light m u ta n t s tra in JH 4 3 0 0 secreted m ore enzym es th a n
th e m elanized s tra in s w hen grow n w ith o u t tricyclazole, w hile the
d a rk m u ta n t show ed very little enzym e secretion (Table 2.2).
Tricyclazole e n h a n c e d enzym e secretion of th e two m elanized
stra in s , b u t did n o t affect th e light m u ta n t.
Pathogenicity
All th re e
s tra in s
decreased
th e
p e rc en t of rice
seeds
g erm in ated (Table 2.3), a n d JH 4 3 0 0 red u c ed sh o o t le n g th s and
b io m a ss eq u iv alen t to th e w ild-type stra in .
significantly red u c ed
sh o o t len g th s
The d a rk m u ta n t
com pared
to
th e
control
tre a tm e n t, b u t less th a n th e w ild-type or light m u ta n t, and
d e crea se d
sh o o t b io m ass to a n
co n tro l a n d w ild-type tre a tm e n ts.
in term ed iate w eight betw een
47
A.
J H 4 3 OO
2 5 0
3 0 0
B i o m
2 0 0
a s s
2 5 0
M e la n in
2 0 0
£ 15 0
I 5 0
I
00
I 00
5 0
0
D a y s
B .
J H2 0 3 3
2 5 0
3 0 0
— Biom ass
2 0 0
2 5 0
M e l a n in
2 0 0
£ 15 0
I 5 0
I
00
IS'
<1
+ 10 0
I
D a y s
C .
J H4 30 I
3 0 0
2 5 0
— Biom ass
2 0 0
2 5 0
M e la n in
2 0 0
I 5 0
I
00
I 00
5 0
0
0
1
2
3
4
5
6
7
D a y s
F igure 2.5.
G row th cu rv es a n d m elan in c o n c e n tra tio n s of G.
gram inis var. gram inis w ild-type a n d m elanin m u ta n t stra in s.
B io m ass is to tal b io m ass in 60 ml c u ltu re , a n d e rro r b a rs are ±
s ta n d a rd error. E ach poin t is th e average of trip licate c u ltu res.
48
JH4300
JH2033
JH4301
Strain
Figure 2.6.
B iom ass of G. gram inis var. gram inis wild-type
(JH 2033) a n d m elan in m u ta n t s tra in s following tre a tm e n t w ith
v a rio u s levels of UV light. Average of triplicate p lates, erro r b a rs
a re ± s ta n d a rd error.
Figure 2.7.
N -A cetylglucosam ine released from w ild-type or
m u ta n t s tra in s of G. gram inis var. gram inis w ith or w ith o u t
Novozyme 234 digestion. E ach p o in t is the average of dup licate
tre a tm e n ts , a n d e rro r b a rs are ± s ta n d a rd error.
49
T able 2.2. E nzym e secretio n of G. gram inis var. gram inis w ild-type
a n d m elan in m u ta n t s tra in s grow n w ith or w ith o u t tricyclazole. - =
no enzym e activity, + = enzym e secretio n only b e n e a th h y p h ae, ++
= enzym e secretio n beyond h y p h a l grow th.______________________ _
JH 4 3 0 0
Tricyclazole
JH 2 0 3 3
JH 4 3 0 1
0
16
0
16
0
16
a - C ellulase
H—h
++
++
++
+'
++
PGU
++
++
-
++
-
+
Milk P ro tease
++
H—h
++
++
(pg/m l)
+
T able 2.3. P athogenicity of w ild-type a n d m u ta n t s tra in s of G.
gram inis var. gram inis to rice. P a ra m e te r m e a n s followed by
differen t le tte rs are significantly different a t P < 0 .0 5 (Tukey's
S tu d e n tiz ed R ange te st, HSD), average of five rep licates.____________
S tra in
JH 4 3 0 0
JH 4301
P a ra m e te r
C ontrol
JH 2 0 3 3
% se ed s
67 a
64 a
65 a
82 b
g erm in ated
12.4 b
Shoot
L en g th s1
16.5 c
7.8 a
8.8 a
Shoot
W eight2
7.3 b
5.3 a
5.3 a
6.1 ab
4.3 a
5.0 a
Root
W eight2
i average p e r p la n t (cm)'
2Average dry w eight p e r p la n t (mg)
4.0 a
4.2 a
50
D iscu ssio n
We iso lated G. gram inis var. gram inis
m u ta n ts affected in
th e ir m e lan in b io sy n th etic p a th w ay to exam ine th e role of m elan in
in p la n t pathogenicity.
The two m u ta n ts d escrib ed h e re differed
from th e w ild-type s tra in in several a sp e c ts of th e ir p ig m entation,
cell w all s tru c tu re a n d h yphopodial m orphology. O u r light m u ta n t
JH 4 3 0 0 a c c u m u la te d 2 -hydroxyjuglone a n d flaviolin, probably a s
th e re s u lt of a single m u ta tio n in th e TH R l gene. T he d a rk m u ta n t
JH 4 3 0 1 co n stitu tiv ely p ro d u ced d a rk p ig m en tatio n on all m edia
w ith o u t tricyclazole, w h e rea s o u r w ild-type s tra in w as heavily
p ig m en ted on PDA a n d lightly m elanized on LB a n d m inim al
m edia.
O u r genetic c ro sse s w ith JH 4 3 0 1
a n d th e w ild-type
su g g e ste d th a t th e JH 4 3 0 1 p h en o ty p e is th e re s u lt of a single
m u ta tio n ; th is m u ta tio n m ay be in a reg u lato ry gene asso c iated
w ith th e m e lan in sy n th e sis p ath w ay ra th e r th a n a s tru c tu ra l gene,
since th is s tra in co nstitutively sy n th esized m elanin.
M icroscopic
ex am in atio n
of th e
th re e
s tra in s
revealed
differences in h y phopodia, p e rith ec ia m orphology a n d m ucilage. G.
gram irds var. gram inis p ro d u ce s brow n, lobed h y p h o p o d ia on
m ylar,
w hile
th e
light
m u ta n t
had
o range-brow n,
lobed
h y p h o p o dia, w hich su g g ested th a t polym erized DHN w as n o t
req u ire d
for in itial
hyphopo dial
developm ent.
O ur
heavily-
m elan ized m u ta n t p ro d u ced d a rk hyp h o p o d ia w ith little or no
51
lobing o n m y lar m em b ran e s. T ricyclazole-treated m u ta n t JH 4301
h y p h o p o d ia w ere
lobed
and
orange
w ith
little
m elanization.
P e rh a p s m a tu re , u n tre a te d J H 4 3 0 1 h y p h o p o d ia a c c u m u la te d m ore
m e lan in th a n th e w ild-type a s th ey aged, a n d th e s tru c tu ra l rigidity
a n d c o n se q u e n t tu rg o r in cre ase a sso c ia te d w ith m e la n in (19, 20)
c a u se d th e lobes to unfold.
The d a rk m u ta n t a p p e a re d to eith er
secrete or re ta in m ore m ucilage a ro u n d its cell w alls th a n th e wildtype stra in , a n d th e light m u ta n t displayed m ore fibrillar m ucilage.
M ucilage is u su a lly p re s e n t on y o u n g h y p h ae, sy n th esized a t or
n e a r th e h y p h a l tip (14, 26, review ed in 31), a n d typically c o n sists
of p ro te in or glycoprotein (26).
P e rh a p s th e m ucilage in th ese
s tra in s w as of sim ilar com position a n d a m o u n t, b u t differences in
cell w all m elan in affected its a p p e a ra n c e a n d /o r re te n tio n to th e
cell wall.
Ellis a n d G riffiths (1975) observed m e la n in g ran u le s
w ith in cell w alls of Phom opsis, b u t also found m e lan in deposition
w ith in m ucilage.
However, w hile th e com position of G. gram inis
var. gram inis m ucilage is u n k n o w n , its a c c u m u la tio n by th e d a rk
m u ta n t m ay have c o n trib u te d to its slow grow th.
In ad d itio n to
differences in h y p h o p o d ia a n d cell w all s tru c tu re betw een stra in s,
o u r lig h t m u ta n t h a d
orange-brow n p erith ec ia b u t p ro d u ced
m a tu re a sc o sp o re s w h en selfed, w hile th e heavily pigm ented
52
m u ta n t developed very sm all, d a rk , ro u n d p e rith e c ia w ith o u t
m a tu re a sco sp o res, a n d req u ire d th e p resen c e of w ild-type to
p ro d u ce viable asco sp o res.
M elanin
serves
as
en v iro n m en ta l stre sso rs .
a
p ro tectio n
a g a in st
a
num ber
of
M elanized sp o res are m ore re s is ta n t to
UV irra d ia tio n (10), a n d m elanized Cryptococcus neofom nans y e a st
cells a re less su sce p tib le to UV light (37).
A rom atics su c h a s
n a p h th a le n e a re know n to a b so rb UV light, a n d D H N -m elanin
p ro b ab ly
provides
p ro tectio n
to
h y p h ae,
survivability in th e soil environm ent.
th e re b y
en h an c in g
O u r heavily m elanized
m u ta n t w as m ore re s is ta n t to UV irra d iatio n th a n th e w ild-type
stra in , a n d th e light m u ta n t w as highly su scep tib le to UV dam age.
UV d am ag e e n h a n c e s m elan o g en esis in m am m a lia n cells (11),
w h ich sy n th esize L-DOPA m elanin. We observed a sim ilar in crease
in D H N -m elanin p ro d u ctio n of o u r m e la n in -c o n ta in in g stra in s
(wild-type a n d d a rk m u ta n t JH 4 3 0 1 , d a ta n o t show n).
M elanins in fungi are im p o rta n t for re s ista n c e to m icrobial
a tta c k by lytic enzym es. M elanized stru c tu re s , in clu d in g sclerotia,
conidia, a n d h y p h a e, are less su scep tib le to lysis th a n hyaline
s tru c tu r e s (4, 23, 25, 29).
A ttem p ts to digest G. gram inis var.
gram inis w ith c h itin a se (Sigm a C7809) w ere u n s u c c e s s fu l (data n o t
53
show n), p e rh a p s b e c a u se c h itin w as a sso c iated w ith glucan.
We
d ig ested o u r w ild-type a n d m elan in m u ta n t s tra in s w ith Novozyme
2 3 4 , w h ich is a m ix tu re of c h itin a se a n d g lu ca n ases, a n d found a
sim ilar c o rrelatio n
betw een
m elan izatio n
of G. gram inis var.
gram inis s tra in s a n d re sista n c e to lytic a ttac k .
m elan ized
m u ta n t
initially
had
W hile o u r heavily
significantly
m ore
N-
acety lg lu cosam ine a t th e o n se t of lysis by Novozyme 234, after
th re e h o u rs of e x p o su re very little c h itin d e g ra d atio n w as detected.
T his re s u lt su g g ested th a t c h itin w as released in th e a b sen c e of
Novozyme, a n d th e enzym es h a d little effect on c h itin d egradation.
By
co m p ariso n ,
Novozyme
tre a tm e n t
in c re a se d
N-
acety lg lu cosam ine release from c h itin from o u r lig h t m u ta n t, a n d
to a le sse r e x te n t from th e w ild-type, in d icatin g th a t m elanin
serves a s a protective layer su rro u n d in g chitin.
Lytic enzym es are secreted by vesicles th a t fu se w ith th e
p la s m a m e m b ra n e a t th e growing ap ex (38), a n d it is believed th a t
p ro tein secretio n is restric te d to h y p h a l tip s (31). B e ca u se h y p h al
tip s a re u n m elan iz ed , it is u n likely th a t m elan in a c ts a s a physical
b a rrie r to enzym e secretion.
E nzym es su c h a s cellulase a n d
p o ly g alac tu ro n ase c a n be in h ib ited by p h en o ls like th o se found in
m e la n in s (5), so p e rh a p s m elan in a c ts a s a n enzym e inactivator.
54
M elanin e x tra cted from G. gram inis in h ib its lytic enzym e activity of
S trep to m yces lavendulae (35).
Tricyclazole
in h ib itio n
of o u r
m elan ized s tra in s re su lte d in in c re a se d enzym e activity, suggesting
th a t
phenolic
polym ers,
r a th e r
th a n
p h e n o ls
such
as
2-
hydroxyjuglone a n d flaviolin, a re resp o n sib le for enzym e inhibition.
A lternatively, tricyclazole m ay directly e n h a n c e enzym e secretion
by som e u n k n o w n m ech an ism .
P athogenicity is a sso c ia te d w ith m elan in sy n th e sis for a
n u m b e r of p la n t p ath o g en ic fungi.
M elanization of M agnaporthe
grisea a p p re ss o ria (sim ilar to hyphopodia) is req u ire d to s u s ta in
th e tu rg o r p re s s u re n e c e ssa iy to p e n e tra te leaves via infection pegs
th a t p ro tru d e from th e adhesive su rface of th e a p p re sso riu m (8,
19, 20). A sim ilar m elan in fu n ctio n is su g g ested for Colletotrichum
lagenerium (24) a n d C. gloesporiod.es (22) a p p re sso ria .
In c o n tra st
to th e s e fu n g al p a th o g e n s, o u r re s u lts do n o t su g g e st th a t m elan in
is req u ire d for G. gram inis var. gram inis p ath o g en icity to rice, a t
le a s t in lab o ra to ry infections. H yphopodia, u n lik e a p p re sso ria , are
n o t re q u ire d for p la n t p e n e tratio n , a s G. gram inis h y p h ae c an
invade ro o t tis su e of h o s t p la n ts via enzym atic digestion (32).
O th e r stu d ie s also d e m o n stra te d th a t co m p o u n d s th a t in h ib it
D H N -m elanin sy n th e sis (tricyclazole, pyroquilon a n d fthalide) do
55
n o t a lte r G. gram inis var. gram inis patho g en icity to lab o rato iy infected w h e a t seedlings (13).
In th e field, m elan in m ay c o n trib u te to p ath o g en icity by
p ro tectin g th e fu n g u s from en v iro n m en tal s tre sso rs a n d p redation.
We have show n th a t m elanized G. gram inis var. gram inis h yphae
a re p ro tec te d from lytic enzym es a n d UV light. However, m elanized
s tra in s also have less lytic enzym e activity, a n d enzym atic lysis is
e sse n tia l
for
ro o t
p e n e tra tio n
w hich
se ts
th e
stage
for
p ath o g e n esis. Therefore, to be a su c ce ssfu l p a th o g en ic com petitor
in a m ixed m icrobial com m unity, G. gram inis var. gram inis m u s t
sy n th esize en o u g h m elan in for p ro tectio n w ith o u t com prom ising
its ability to secrete active lytic enzym es. B ased on th is a rg u m en t,
w hile m e la n in sy n th e sis m ay n o t directly e n h a n c e G. gram inis var.
gram inis pathogenicity, m o derately m elanized s tra in s are m ore
likely to survive saprophytically a n d becom e p ath o g e n ic sh o u ld a
su ita b le h o s t be available.
L iteratu re C ited
I.
A d d lem an , K., T. D u m onceaux, M. G. P aice, R. B o u rb o n n ais
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of T ram etes versicolor m u ta n ts u n a b le to b le a c h hardw ood
k ra ft p u lp . A ppl. E nviron. M icrobiol. 6 1 :3 6 8 7 -3 6 9 4 .
56
2.
Bell, A. A. a n d M. H. W heeler.
fu n c tio n s of fungal m elan in s.
2 4 :4 1 1 -4 5 1 .
1986. B io sy n th esis a n d
A nn. Rev. P h y to p a th o l.
3.
Bowyer, P., R. M usker, A. E. O sborne, B. C lark, C. C aten
a n d M. J . D aniels.
1992.
P roduction of m u ta n ts of
G a eum annom yces gram inis var. tritici a n d var. a ven a e by 4n itroquinolene-oxide tre a tm e n t of p ro to p la sts.
F ungal
G enet. New slett. 3 9 :1 3 -1 4 .
4.
Bloomfield, B. J . a n d M. A lexander. 1967. M elanins a n d
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5.
Bull, A. T.
1970.
K inetics of cellulase in activ atio n by
m elan in . Enzym ologia 3 9 :3 3 3 -3 4 7 .
6.
B u tler, M. J . a n d M.-A. L achance.
1986.
b in d in g of A zure A to m elan in of th e
Phaecoccom yces. Exp. Mycol. 10:166-170.
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C aesar-T onT hat, T. C., F. v an O m m en K loeke, G. G. G eesey
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fila m e n to u s soil fu n g u s in re sp o n se to copper a n d
localization of copper sulfide by silver-staining. AppL
E nviron. Microbiol. 6 1 :1 9 6 8 -1 9 7 5 .
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C h u m ley , F. G. a n d B. V alent. 1990. G enetic an aly sis of
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grisea. Mol. P la n t M icrobe In tera ct. 3 :1 3 5 -1 4 3 .
9.
Dopi, S., Z. Solel a n d I. B a ra sh . 1995. Cell w all-degrading
enzym es p ro d u ce d by G aeum annom yces gram inis var. tritici in
vitro a n d in vivo. Physiol. Mol. P la n t Pathol. 4 6 :1 8 9 -1 9 8 .
10.
D urrell, L. W. 1964. The com position a n d s tru c tu re of d a rk
fu n g u s sp o res. M ycopathol. M ycol. A ppl. 2 3 :3 3 9 -4 5 .
11.
Eller, M. S. K. O strom a n d B. A. G ilchrest. 1996. DNA
d am ag e e n h a n c e s m elanogenesis.
P roc. Natl. Acad. Sci.
USA. 9 3 :1 0 8 7 -1 0 9 2 .
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b lac k y e a st
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12.
Ellis, D. H. A nd D. A. G riffiths. 1975. M elanin deposition in
th e h y p h a e of a species of Phom opsis. C an. J . Microbiol.
2 1 :4 4 2 -4 5 2 .
13.
Elliott,
M. L.
1995.
in h ib itin g
com pounds
Mycologia. 8 7 :3 7 0 -3 7 4 .
14.
E v an s, R. C., S tem pen, H., a n d P. F ra sc a .
1981.
D evelopm ent of h y p h al s h e a th s in Bipolaris m a y d is race T.
C an. J . Bot. 5 9 :4 5 3 -4 5 9 .
15.
G a rre tt, S. D.
1970.
P athogenic root-infecting fungi.
C am bridge U niv. P ress. London.
16.
Gold, M. H. A nd T. M. C heng. 1978. In d u c tio n of colonial
grow th a n d rep lica p latin g of th e w hite ro t B asidiom ycete
P hanaerochaete chrysosporium .
AppL E nviron. Microbiol.
3 5 :1 2 2 3 -1 2 2 5 .
17.
H arling, R., L. Kenyon, B. G. Lewis, R. P. Oliver, J . G. T u rn e r
a n d A. C oddington. 1988. C onditions for efficient isolation
a n d reg e n era tio n of p ro to p la sts from Fulvia fu lva .
J.
P h y to p a th . 1 2 2 :143-146.
18.
H och, H. C. 1986. Freeze s u b s titu tio n of fungi, p. 183-212.
In H. C. A ldrich a n d W. J . Todd (ed.), U ltra s tru c tu re
te c h n iq u e s of m icroorganism s. P lenum P re ss, New York.
19.
H ow ard, R. J . a n d M. A. F errari. 1989. Role of m elan in in
a p p re ss o riu m function. Exp. Mycol. 1 3 :4 0 3 -4 1 8 .
20.
H ow ard, R. J ., M. A. F errari, D. H. Roach, a n d N. P. Money.
1991. P e n e tra tio n of h a rd s u b s tra te s by a fu n g u s em ploying
e n o rm o u s tu rg o r p re ssu re .
P roc. NatL A cad, Sci. USA
8 8 :1 1 2 8 1 -1 1 2 8 4 .
21.
H ow ard, R. J . a n d K. L. O'Donnell.
1987.
Freeze
su b s titu tio n of fungi for cytological an aly sis. Exp. Mycol.
1 1 :2 5 0 -2 6 9 .
Effect of m elan in b io sy n th esis
on
G aeum annom yces
species.
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22.
H w ang, C .-S., M. A. F lia sh m a n a n d P. E. K o la ttu k u d y .
1995. C loning of a gene e x p ressed d u rin g a p p re sso riu m
fo rm atio n by Colletotrichum gloeosporiod.es a n d a m ark e d
d e crea se in virulence by d isru p tio n of th is gene. P lan t Cell
7 :1 8 3 -1 9 3 .
23.
H y ak u m ach i, M. K. Y okoyam a a n d T. Ui. 1987. Role of
m elan in in su scep tib ility a n d re sista n c e of R hizoctonia solani
■ to m icrobial lysis. T ran s. Br. Mycol. Soc. 8 9 :2 7 -3 3 .
22.
K ubo, Y. a n d I. F u ru sa w a . 1991. M elanin b io sy n th esis, p.
2 0 5 -2 1 8 . In G. T. Cole a n d H. C. H och (ed.), The fungal
sp o re a n d d ise ase in itia tio n in p la n ts a n d a n im als. P lenum
P ress, New York.
25.
Kuo, M .-J. a n d M. A lexander. 1967. In h ib itio n of th e lysis
of fungi by m elan in s. J . B acterio l. 9 4 :6 2 4 -6 2 9
26.
N icholson, R. L. arid L. E p stein . 1991. A dhesion of fungi to
th e p la n t su rface, p.3-23 . In G. T. Cole a n d H. C. H och (ed.),
T he fu n g al spore a n d d ise ase in itiatio n in p la n ts a n d
a n im als. P len u m P ress, New York.
27.
P e rp e tu a , N. S., Y. K ubo, N. Y asuda, Y. T ak a n o a n d I.
F u ru sa w a . 1996. Cloning a n d c h arac te riz a tio n of a m elan in
b io sy n th etic TH R l re d u c ta se gene e sse n tia l for a p p re sso ria l
p e n e tra tio n of Colletotrichum lagenarium . Mol. Plant-M icrobe
In tera ct. 9 :3 2 3 -3 2 9 .
28.
Pilgram , A. L., T. G oins a n d J . M. H enson. 1993. The fate of
in te g ra te d DNA in G aeum annom yces gram inis tra n sfo rm a n ts.
FEMS Microbiol. Let. 1 1 3 :3 0 9 -3 1 4 .
29.
Potgeiter, H. J . a n d M. A lexander. 1966. S usceptibility a n d
re s ista n c e of several fungi to m icrobial lysis. J . B acteriol.
9 1 :1 5 2 6 -1 5 3 2 .
30.
R eissig, J . L., J . L. Strom inger, a n d L. F. Leeloir. 1955. A
m odified colorim etric m eth o d for th e e stim a tio n of Nacetylam ino su g a rs. J . Biol. C hem . 2 1 7 :9 2 9 -9 6 6 .
59
31.
S ietsm a, J . H., H. A. B. W osten a n d J . G. H. W essels. 1995.
Cell wall grow th a n d p ro tein secretion in fungi. C an J . Bot.
7 3 :8 3 8 8 -8 3 9 5 .
32.
Skou, J . P. 1981. M orphology a n d cytology of th e infection
p ro cess, p. 175-198. In M. J . C. A sher a n d P. J . S hipton
(ed.), Biology a n d control of take-all.
A cadem ic P ress,
L ondon.
33.
Sm iley, R. W., P. H. D ernoeden, a n d B. B. C larke. 1992.
C o m p en d iu m of tu rfg ra ss d ise ases, 2nd.
Ed. A m erican
P hytopathological Society, St. P au l M inn.
34.
T a k a n o , Y., Y. K ubo, K. Shim izu, K. Mise, T. O kuno a n d I.
F u ru sa w a . 1995. S tru c tu ra l a n aly sis of P K S l, a polyketide
sy n th a se gene involved in m elan in b io sy n th esis in
Colletotrichum lagenarium . Mol. Gen. G enet. 2 4 9 :1 6 2 -1 6 7 .
35.
T sch u d i, S. a n d H. Kern.
1979.
Specific lysis of th e
m ycelium of G aeum annom yces gram inis by enzym es of
Streptococcus lavendulae. P p . 611-615. I n B . S ch ip p ers a n d
W. G am s (ed.), Soil-borne p la n t p a th o g en s. A cadem ic P ress,
L ondon.
36.
W alker, J . 1981. T axonom y of tak e-all fungi a n d related
g e n era a n d species, p. 15-74. JnM. J . C. A sh er a n d P. J .
S h ip to n (ed.), Biology a n d control of tak e-all. Academ ic
P ress, London.
37.
W ang, Y. A nd A. C asadevall. 1994. D ecreased su sceptibility
of m elanized Crytococcus n e o fo m a n s to UV light. A ppl.
E nviron. Microbiol. 6 0 :3 8 6 4 -3 8 6 6 .
38.
W essels, J . G. H. 1993. W all grow th, p ro te in secretion a n d
m o rp h o g en esis in fungi.
T ansley Review No. 45.
New
Phytol. 1 2 3 :3 9 7 -4 1 3 .
39.
W este, G. 1970. E x tra ce llu lar enzym e p ro d u c tio n by v arious
iso lates of Ophiobolus gram inis a n d O phiobolus a venae I.
E n zym es p ro d u ce d in c u ltu re . P hytopath. Zeit. 6 7 :1 8 9 -2 0 4 .
60
CHAPTER 3
COPPER BINDING AND LOCALIZATION OF C uS BY SILVER
STAINING OF G auem annom yces gram inis var. gram inis
MELANIN MUTANTS
In tro d u ctio n
H eavy m etal c o n c e n tra tio n s in soil are in c re a sin g a s a re s u lt
of fossil fuel c o m b u stio n , a n d m ining, in d u stria l a n d a g ric u ltu ral
p ra c tic e s (I).
A lthough som e m etallic elem en ts a re e sse n tia l for
m icrobial grow th, th e ir o v e ra b u n d an c e lead s to excessive cellular
a c c u m u la tio n , w ith s u b s e q u e n t toxic effects (reviewed in reference
16). E n v iro n m en ta l m etal c o n c e n tra tio n s frequently flu c tu a te a n d
m a n y fungi have developed m e c h a n ism s to cope w ith higher, m ore
toxic levels of m eta ls th ey e n co u n ter. Wall a n d m e m b ra n e layers of
e u k ary o tic m icro b es provide th e m ajo r b a rrie r to th e influx a n d
efflux of m etal io n s (25). M etals excluded by v a rio u s m etal to le ran t
m icro b es in clu d e lead, m ercury, copper, cobalt, zinc, c ad m iu m a n d
m a n g a n e se (12, 18, 19).
F u ngi also p ro d u ce m etallo th io n ein s,
sm all c y stein e-rich p ro tein s th a t b in d a n d se q u e s te r heavy m etals
in tra ce llu larly (15, 32).
Saccharom yces cerevisiae g e n erate s HsS
61
th a t tra p s m etal io n s a s inso lu b le m etal sulfides e ith e r extra- or
in tra c e llu la rly (8, 24).
Citric acid is also p ro d u ce d by m an y fungi
a n d c h e la te s co p p er a n d o th e r m etal ions (21).
F u n g i also p ro d u ce cell w all m a teria ls a n d ex tracellu lar
s u b s ta n c e s ,
in clu d in g
phenolic
polym ers,
m ela n in s, c h itin s a n d c h ito sa n s th a t m ay
m e ta ls from so lu tio n (23).
g lu c a n s,
m a n n a n s,
rem ove or b in d heavy
In m etal bin d in g s tu d ie s w ith S.
cerevisae cell w all polym ers, m a n n a n s w ere th e m o st significant
a c c u m u la to rs of m etals, p a rtic u la rly copper (5).
O th e r stu d ie s
d e m o n s tra te th a t m etal b in d s to carboxyl a n d am ino g ro u p s, w hich
su g g e sts th a t p ro tein a n d c arb o h y d rate fractio n s a re involved in
m etal b in d in g (3).
F u n g a l p ig m en ts su c h a s m elan in are also im plicated in
m etal b in d in g a n d m ay serve a s a protective m e c h a n ism a g a in st
m etal
toxicity
(reviewed
in
reference
16).
M elanins
are
h ete ro g en o u s, h igh m o lecu lar w eight p igm ents p ro d u c e d by m an y
fungi a n d often th e y a re m ad e in re sp o n se to s tre s s (reviewed in
referen ce 2).
T hese dark ly pigm ented polym ers m ay serve a s a
protective b a rrie r a g a in st adverse en v iro n m en tal co n d itio n s su c h
a s UV light, lytic enzym es, p la n t defense m e c h a n ism s, p red atio n ,
d esiccatio n , a n d m etals. F ungi p ro d u ce different ty p es of m elan in
62
in clu d in g
catech o l,
polym ers
of
L -dihydroxyphenylalanine
dihydroxybenezene,
(L-DOPA),
g lu tam in y l-3 ,4 - dihydroxybenzene
a n d 1,8 -d ih y d ro x y n ap h th alen e (DHN).
B ecau se of its negatively
c h arg ed hydroxyl gro u p s, D H N -m elanin could po ten tially bind
c a tio n s a n d exclude th e m from cells. In addition, am in o acid s a n d
carboxyl g ro u p s th a t a c t a s b in d in g sites for positively ch arg ed ions
m ay be p re s e n t in o th e r m acrom olecules th a t a re tra p p e d in th e
m elan in polym er (30).
ch em ical
s tru c tu re s
B oth th e reactio n c h a ra c te ristic s a n d
of m elan in
and
of w eak
m etal
cation
e x c h a n g ers a re sim ilar a n d su g g e st th a t m etal io n s a n d m elan in
in te ra c t by ion
exchange
(6)
In
vivo a n d
in
vitro stu d ie s
d e m o n stra te th a t fu n g al m elan in s a re efficient m e ta l b io so rb a n ts
(17, 18, 20, 26, 28), a n d su g g est th a t th is m etal a c c u m u la tio n by
fu n g al b io m a ss m ay be exploited for heavy m e ta l rem oval from
c o n ta m in a te d soils or w a ter (3, 4, 13, 14).
G. gram inis var.
gram inis is a hom othallic
filam entous
ascom ycete th a t p ro d u ce s m elan in via th e d ih y d ro x y n ap h th ale n e
(DHN) p athw ay. We previously show ed th a t c u ltu re s of G. gram inis
var. gram inis grow n in th e p re sen c e of tricyclazole, a specific
in h ib ito r of D H N -m elanin sy n th e sis (2), b o u n d significantly less
co p p er
th a n
c u ltu re s
grow n w ith o u t tricyclazole,
and
silver
63
sta in in g d e m o n s tra te d localization of C uS w ithin th e m e lan in layer
(7). In th e p re s e n t stu d y , we exam ined th e role of m elanized fungi
in co p p er b in d in g u sin g isogenic G. gram inis var. gram inis m elan in
m u ta n ts .
M aterials a n d M ethods
F u n g a l S tra in s a n d G row th
C o n d itio ns
G en eratio n , grow th a n d c h a ra c teriza tio n of G. gram inis var.
gram inis. m elan in w as previously d escrib ed in C h a p te r 2. The th ree
s tra in s c h o se n for s tu d y w ere JH 2 0 3 3 (wild-type), JH 4 3 0 0 (a
m u ta n t derived from JH 2 0 3 3 th a t does n o t sy n th esize D H N -,
m elanin), a n d JH 4 3 0 1
(a m u ta n t derived from JH 2 0 3 3 th a t
o v erp ro d u ces m elanin).
G row th C urves in M inim al M edium
w ith C opper A m en d m en t
Two h u n d re d m is of m inim al m edium w ere su p p le m e n te d
w ith tra c e (80 nM Cu), 20 p,M C u, 40 jaM C u or 80 pM C u from a
1% (w/v) C uSG 4 sto c k solution. Five LB ag ar p lu g s w ere ad d ed to
trip licate flask s, w hich w ere s h a k e n a t 50 rp m a n d h a rv e ste d a t 9,
64
14, 18 a n d 24 days. H yphae w ere recovered by filtratio n th ro u g h
a W h a tm a n #1 filter, lyophilized overnight a n d w eighed.
C opper B inding a n d A ccum ulation
F o u rte e n day-old c u ltu re s grow n in m in im al m ed iu m w ere
h a rv e ste d by filtration th ro u g h a W h atm a n #1 filter. H yphae were
re s u s p e n d e d in 100 m l 40 mM CuSCU, s h a k e n a t 50 rp m for I h a t
25 C, w a sh e d th re e tim es w ith 200 m l distilled w a te r a n d frozen a t
-20 C u n til ICP-AES a n aly sis of copper binding.
C opper
a c c u m u la tio n
by
fungal
b io m a ss
w as
also
d e te rm in e d by ICP-AES following 14 days of grow th in m inim al
m ed iu m co n ta in in g tra c e (8 nM), 20, 40, 60 or 80
pM CuSCU.
C u ltu re s w ere h a rv e ste d by filtration th ro u g h a W h a tm a n # I filter,
w a sh e d 3X w ith distilled HzO a n d th e n lyophilized overnight a n d
frozen a t -20 C u n til analysis.
Inductively C oupled P lasm a
Atom ic E m ission
S p ectro scopy (ICP-AES)
S am p les w ere dried a t 105 C for 2 h, w eighed (16-104 mg)
in to g lass vials a n d digested in I m l of 1:1 n itric acid a t 70 C. After
2 h , I m l of 1:1 HCl w as ad d ed a n d h e atin g c o n tin u e d for 10 m in.
S am p les w ere d ilu te d to 10 m l w ith distilled w a ter a n d analyzed on
a
L eem an
L abs
P S 950
ICP
sp e ctro m eter
w ith
b a ck g ro u n d
correction. S am p les w ere d ilu ted a s n eeded in 2% HNOs p rior to
65
ICP m e a s u re m e n ts .
ICP a n aly sis w ere perform ed a t Little B ear
L ab o rato ries by Dr. Greg O lson.
Silver S tain in g
14 day-old c u ltu re s w ere h a rv e ste d from m in im al m edium
c o n ta in in g 40 pM CuSCk.
H yphae w ere fixed a n d sta in e d for 10
m in a s d escrib e d elsew here (7) following m a n u fa c tu re r's protocol
(HQ SILVER, N anoprobes).
R esu lts
G. qram inis var. gram inis G row th
w ith C opper
T he w ild-type s tra in a n d th e light m u ta n t J H 4 3 0 0 w ere n o t
in h ib ite d by co p p er c o n c e n tra tio n s u p to 80 pM; afte r 24 days of
grow th, th e s e two s tra in s h a d approxim ately e q u iv alen t b io m ass
w ith tra c e or h ig h er copper in th e m ed iu m (Fig, 3 .1 a a n d 3.1b).
C u ltu re s of th e d a rk m u ta n t JH 4 3 0 1
w ere in h ib ite d by all
c o n c e n tra tio n s of copper (Fig. 3.1c).
At th e
h ig h er copper
c o n c e n tra tio n s (60 a n d 80 pM CUSO 4), grow th of th is m u ta n t w as
a lm o st com pletely inhibited. T his s tra in also grew a t a slow er ra te
in m in im al m ed iu m w ith trac e co p p er th a n th e o th e r two stra in s.
66
A. JH 4 3 0 0
B. JH 2 0 3 3
#
- IO ug/m I C u
C. JH 4 3 0 1
H -----0 u g m l C u
5 ug/ m I C u
-----I O u g m l C u
— 20 u g m l Cu
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
D ays
F igure 3.1.
G. gram inis var. gram inis w ild-type a n d m elanin
m u ta n t
grow th
in
m inim al m edium
c o n ta in in g
varying
c o n c e n tra tio n s of CuSCX B iom ass is th e total b io m a ss in 200 ml
c u ltu re . E ac h p o in t is th e average of 3 replicates.
67
C opper B inding to Hypfaae
C opper a c c u m u la tio n w ith th e d a rk m u ta n t w as g rea ter th a n
w ith th e light m u ta n t or w ild-type for all c o p p e r. c o n ce n tra tio n s,
a n d J H 4 3 0 0 a c c u m u la te d less co p p er th a n th e two pigm ented
s tra in s (Table 3.1) following 14 d a y s grow th in c o p p er-co n tain in g
m in im al m edium .
The d a rk m u ta n t JH 4 3 0 1 a c c u m u la te d m ore
co p p er th a n th e o th e r s tra in s a t all copper c o n ce n tra tio n s.
In
ad d itio n , fo u rtee n day-old c u ltu re s of JH 4 3 0 1 grow n in m inim al
m ed iu m w ith o u t co p p er a b so rb ed significantly m ore copper th a n
lig h t m u ta n t JH 4 3 0 0 or th e w ild-type after I h ex p o su re to 40 mM
C uSO 4 (Table 3.2).
Silver E n h a n c e m e n t of C opper Sulfide
Silver sta in in g d e m o n stra te d th e p resen ce of co p p er sulfide
in h y p h a e of th e w ild-type a n d s tra in JH 4 3 0 1 following grow th for
14 d a y s w ith 40 p,M C uSO 4 (Fig. 3.2).
Silver d eposition w as
ob serv ed w ith in se p ta , w hich sta in e d a d a rk brow n, a n d w ithin cell
w alls w h ich im p a rte d a yellow ish-brow n color to h y p h a e. No silver
d e p o sits w ere observed in th e light s tra in JH 4 3 0 0 afte r grow th in
40 pM C uSO 4, a n d th is su g g ested th a t C uS did n o t a c c u m u la te in
th is stra in .
68
T able 3 .Ii C u a c c u m u la tio n of w ild-type G. gram inis var. gram inis
a n d two m e lan in m u ta n t s tra in s following 14 d a y s grow th in
m in im al m ed iu m w ith Cu. C o n c en tratio n s are m g C u /g fungal
b io m a ss a n d th e average of 4 rep licates.
M eans followed by
d ifferent le tte rs a c ro ss a row are significantly different by T ukey1s
S tu d e n tiz ed R ange (HSD) a t P < 0.05.______________________________
S tra in
M edium C u
C o n c en tratio n
(pM)
T race (8 nM)
Light M u ta n t
JH 4 3 0 0
0 .0 0 6 2 5 a
20
0 .6 6 5 a
2.17 b
4.36 c
40
2.57 a
4.73 b
7.97 c
60
3.09 a
9.82 b
16.2 c
80
3.72 a
9.42 b
19.3 c
W ild-type
D ark M u ta n t
JH 2 0 3 3
JH 4301
0.027 b
0 .0 0 3 7 5 a .
Table 3.2. C opper b o u n d to 14 day-old c u ltu re s of w ild-type G.
gram inis var. gram inis a n d two m elan in m u ta n ts following I h
e x p o su re of h y p h a e to 40 mM CuSCk after 14 d a y s grow th in
m in im al m ed iu m w ith tra c e Cu. C o n c en tratio n s a re m g C u /g
fu n g al b io m a ss a n d th e average of 2 replicates. M eans followed
a c ro ss tre a tm e n t row s w ith different le tters a re significantly
differen t a t P < 0 .0 5 (Tukey's S tu d e n tiz ed Range te s t, HSD).________
S tra in
T re a tm e n t
. Light
M u ta n t
JH 4 3 0 0
W ild-type
JH 2 0 3 3
D ark
M u ta n t
JH 4301
No C u
0* a**
0 a
0 a
3 2 .1 5 b
6 7 .3 0 c
Cu
. 2 2 .8 5 a
69
Figure 3.2. S ilver-stained h y p h ae show ing localization of CuS.
S tra in s w ere grow n for 14 days in m inim al m ed iu m co n tain in g 40
gM C uS 04. A. Light m u ta n t JH 4 3 0 0 (500 X), B. W ild-type
JH 2 0 3 3 (500 X), C. W ild-type JH 2 0 3 3 (1250 X), D. D ark m u ta n t
JH 4 3 0 1 (1250 X), E. W ild-type u n s ta in e d (500 X).
D iscu ssio n
Previous stu d ie s show th a t L-DOPA m elan in a b so rb s copper
(22, 30, 31).
In addition, tricyclazole-treated G. gram inis var.
70
gram inis c u ltu re s b in d less co p p er th a n u n tre a te d c u ltu re s (7), a
finding th a t su g g e sts th a t D H N -m elanin is involved in copper
binding. O u r heavily-m elanized m u ta n t s tra in b o u n d significantly
m ore co p p er th a n th e w ild-type s tra in or th e light m u ta n t w hen
grow n
in
Cu.
T hese
findings
su g g ested
o v erp ro d u ctio n w as a sso c iated w ith copper binding.
th a t
pigm ent
A lternatively,
in c re a se d m ucilage secretio n by th e d a rk m u ta n t m ay have
c o n trib u te d to co p p er binding.
O u r light m u ta n t did n o t b in d a s
m u c h co p p er a s th e w ild-type s tra in w hen grow n in C u or w hen
grow n w ith tra c e co p p er a n d th e n exposed. The m u cilage layer or
o th e r c ellu lar co m p o n e n t of th e light m u ta n t m ay have b o u n d
som e copper.
Silver sta in in g is c a u se d by silver p recip itatio n a ro u n d C uS
n u c le a tio n sites in w hich 10 or m ore m olecules of C uS exist (9-11)
a n d confirm ed th e p resen c e of C uS in se p ta a n d cell w alls of th e
w ild-type a n d over-m elanized m u ta n t grow n in CUSO 4 . CuS w as
n o t a p p a re n t in h y p h a l cell w alls or se p ta of th e u n m elan ized
m u ta n t, a finding th a t su g g ested th a t cell wall m elan in is required
for co p p er sulfide acc u m u latio n .
C uS sta in in g m ay be m ore
indicative of co p p er a cc u m u la tio n asso c iated w ith m elan in th a n
ICP-AES a n a ly sis of w hole cells, w hich m ay have b o u n d copper in
form s o th e r th a n C u S .
M elanin serves a s general p rotection a n d m ay provide a
p h y sical b a rrie r a g a in st copper.
W hile o u r heavily m elanized
m u ta n t a c c u m u la te d 1.6-2X a s m u c h copper a s th e w ild-type a n d
71
3 -6 .6X a s m u c h co p p er a s o u r light m u ta n t (Table 3.1), grow th w as
a lm o st com pletely in h ib ited w ith th is s tra in a t h ig h er copper
c o n c e n tra tio n s.
We observed a sim ilar delayed grow th of th e
m elan ized w ild-type in
th e
p resen c e
of 80
pM copper, b u t
su rp risin g ly , th e u n m elan iz ed m u ta n t w as n o t in h ib ite d by higher
c o n c e n tra tio n s of Cu.
C opper b in d in g to m elan in m ay actu ally
in h ib it grow th if it re a c h e s levels toxic to th e fu n g u s a n d , while th e
w ild-type s tra in w as able to overcom e th is toxicity, th e slow
grow ing d a rk
m u ta n t could
not.
We sp e cu la te
th a t since
m elan ized s tra in s a c c u m u la te d m ore C u th a n th e u n m elan ized
m u ta n t, m e lan in m ay be involved in seq u esterin g m eta ls, su c h a s
C u o r Fe th a t a re n e c e ssa ry for grow th.
Rizzo e t al. (1992)
su g g e ste d th a t a coating of m etal io n s on fu n g al su rfaces of
A m arillia also a c t a s p ro tectio n from a n ta g o n istic m icroorganism s.
P e rh a p s co p p er b in d in g to G. gram inis var. gram inis provides a
sim ilar fu n ctio n in th e environm ent.
L iteratu re C ited
1.
Ayers, R. U. 1992. Toxic heavy m etals: M aterials cycle
optim ization. P roc. Natl. Acad. Sci. USA 8 9 :8 1 5 -8 2 0 .
2.
Bell, A. A. a n d M. H. W heeler.
fu n c tio n s of fungal m elan in s.
2 4 :4 1 1 -4 5 1 .
3.
B rady, D. a n d J ; R. D u n c an . 1994. B inding of heavy m etals
by th e cell w alls of Saccharom yces cerevisiae. Enyz. M icrob.
T echnol. 16:6 3 3 -6 3 8 .
1986. B io sy n th esis a n d
Ann. Rev. P h y to p a th o l.
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4.
B rady, D. a n d J . R. D u n c a n . 1994. B io accu m u latio n of
m etal c a tio n s by Saccharom yces cerevisiae. A ppl. Microbiol.
B iotechnol. 4 1 :1 4 9 -1 5 4 .
5.
B rady, D., A. D Stoll, L. S ta rk e a n d J . R. D u n c a n .
1994.
C hem ical a n d enzym atic ex tra ctio n of heavy m etal binding
polym ers from isolated cell w alls of Saccharom yces
cerevisiae. B iotechnol. Bioeng. 4 4 :2 9 7 -3 0 2 .
6.
B ru en g er, F. W., B. J . Stover a n d D. R. A therton. 1967. The
in co rp o ra tio n of v ario u s m etal ions in in vivo- a n d in vitrop ro d u c e d m elan in . Rad. Res. 3 2 :1 -1 2 .
7.
C aesar-T onT hat, T. C., F. v a n O m m en Kloeke, G. G. G eesey
a n d J . M. H enson.
1995. M elanin p ro d u c tio n by a
fila m e n to u s soil fu n g u s in re sp o n se to copper a n d
localization of copper sulfide by silver-staining.
AppL
E nviron. Microbiol. 6 1 :1 9 6 8 -1 9 7 5 .
8.
C erv an tes, C. a n d F. G utierrez-C orona.
re s ista n c e m e c h a n ism s in b a c te ria a n d
M icrobiol. Rev. 1 4 :121-138.
9.
D a n sc h e r, G. 1981. H istochem ical d e m o n stra tio n of heavy
m etals. H isto ch em istry 7 1 :1 -1 6 .
10.
D a n sc h e r, G. 1984. A utom etallography. A new tech n iq u e
for light a n d electron m icroscopic v isu alizatio n of m etals in
biological tis s u e s (gold, silver, m etal sulfides a n d m etal
selenides). H isto ch em istry 8 1 :3 3 1 -3 3 5 .
11.
D a n sc h e r, G.
1991.
D etection of m e ta ls in tissu e s.
H istochem ical trac in g of zinc, m ercury, silver a n d gold.
Prog. H isto c h em . C ytochem . 2 3 :2 7 3 -2 8 5 .
12.
F o ster, P. L. 1977. C opper exclusion a s a m e c h a n ism of
heavy m etal to leran ce in a green algae. N a tu re 2 6 9 :3 2 2 323.
1994.
fungi.
C opper
FEMS
73
13.
F o u re st, E. a n d J.-C . Roux. 1992. Heavy m etal bio sorption
by fu n g al b y -p ro d u cts: m e c h a n ism s a n d influence of pH.
A ppl. Micobiol. B iotechnol. 3 7 :3 9 9 -4 0 3 .
14.
F o u re st, E., C. C anal a n d J.-M . Roux. 1994. Im provem ent
of heavy m etal b iosorption by m ycelial d e a d b io m asses
(R hizopus
arrhizus,
M ucor
m iehei
and
Penicillium
chyrsogenum ): pH control a n d cationic activation. FEMS
M icrobiol. Rev. 14:3 2 5 -3 3 2 .
15.
F u rs t, P., S. H u, R. H ack ett a n d D. H am er. 1988.
C opper
activ a tes m etallo th io n ein gene tra n s c rip tio n by a lterin g th e
co nform ation of a specific DNA binding pro tein . Cell 5 5 :7 0 5 717.
16.
G a d d , G. M. 1993. In te ra c tio n s of fungi w ith toxic m etals.
New P hytol. 1 2 4 :2 5 -6 0 .
17.
G a d d , G. M. a n d L. deRom e. 1988. B iosorption of copper by
fu n g al m elanin. A ppl. Microbiol. B iotechnol. 2 9 : 610-617.
18.
G a d d , G. M. a n d A. J . Griffiths. 1978. M icroorganism s a n d
heavy m etal toxicity. Microb. Ecol. 4 :3 0 3 -3 1 7 .
19.
G a d d , G. M. A nd A. J . G riffiths. 1980. Influence of pH on
toxicity a n d u p ta k e of co p p er by A ureobasidium pullulans.
T ran s. Br. M ycol. Soc. 7 5 :9 1 -6 .
20.
G a d d , G. M. a n d J . L. Mowll. 1985. C opper u p ta k e by
y east-lik e
cells,
hyphae
and
chlam ydo sp o res
of
A u reobasidium pullulans. Exp. Mycol. 9 :2 3 0 -2 4 0 .
21.
H u g h es, M. N. a n d R. K. Poole. 1989. M etals a n d m icro­
o rg an ism s. C h a p m a n a n d Hall, London, New York.
22.
L arsso n , B. a n d H. Tjalve. 1978. S tu d ies o n th e m elaninaffinity of m etal ions. A cta Physiol. S cand. 1 0 4 :4 7 9 -4 8 4 .
23.
Lepp, N. W. 1992. U ptake a n d reg u latio n of m etals in
b a c te ria a n d fungi, p. 2 7 7 -2 9 8
In D. C. A driano (ed.),
B iogeochem istry of tra c e m etals. CRC P ress, B oca R aton, FL.
74
24.
Lin, C.-M ., B. F. C raw ford a n d D. J . K osm an.
1993.
D istrib u tio n of 64C u in Saccharom yces cerevisiae: cellular
locale a n d m etabolism . J . Gen. Microbiol. 1 3 9 :1 6 0 5 -1 6 1 5 .
25.
R ogers, H. J ., H. R. P erk in s a n d J . B. W ard.
1980.
M icrobial cell w alls a n d m em b ran e s.
C hapm an-H all,
L ondon, New York.
26.
Rizzo, D. M., R. A. B lan ch ette, a n d M. A. P alm er. 1992.
B iosorption of m etal ions by Armillaria rh izo m o rp h s. C an. J .
Bot. 7 0 :1 5 1 5 -1 5 2 0 .
27.
R ogers, H. J ., H. R. P erk in s a n d J . B. W ard.
1980.
M icrobial cell w alls a n d m em b ran es.
C hapm an-H all,
L ondon, New York.
28.
Saiz-Jim en e z, C. a n d F. S hafizadeh. 1984. Iro n a n d copper
b in d in g by fungal phenolic polym ers: a n electro n sp in
re so n a n c e stu d y . C u rr. Microbiol. 10:2 8 1 -2 8 6 .
29.
S a rn a , T., W. Fronicz a n d J . S. Hyde. 1980. C u 2+ probe of
m etal-b in d in g sites in m elan in u sin g electro n p ara m ag n e tic
re so n a n c e spectroscopy. Arch. B iochem . B io p h y s. 2 0 2 :3 0 4 313.
30.
S a rn a , T., J . S. Hyde a n d H. M. Sw artz. 1976. Ion exchange
in m elan in . An electron sp in stu d y w ith la n ta n id e probes.
Science 1 9 2 :1 1 3 2 -1 1 3 4 .
31.
S enesi, N., G. Sposito a n d J . P. M artin. 1987. C opper (II)
a n d iro n (III) com plexation by h u m ic acid-like polym ers
(m elanins) from soil fungi- Sci. Total E nviron. 6 2 :2 4 1 -2 5 2 .
32.
Thiele, D. J .
1988.
A C E l reg u lates e x p re ssio n of th e
Saccharom yces cerevisiae m etallo th io n ien gene. Mol. Cell.
Biol. 8 :2 7 4 5 -2 7 5 2 .
75
33.
W hiteside, S. G. A nd D. J . Plocke. 1992. Selection a n d
c h a ra te riz a tio n of a c o p p e r-re sista n t su b p o p u la tio n of
S chizosaccharom yces pom be. J . Gen. M icrobiol. 1 3 8 :2 4 1 7 242.
76
CHAPTER 4
CONCLUSIONS
1.
M elanin p ro te c ts fungi from UV light d am ag e a n d lytic
enzym e activity.
O verm elanization r e s u l ts ’ in d ecreased
enzym e secretion.
M elanin c o n trib u te s to th e com petitive
ability
of fungi
p ro tectio n
in
a g a in st
m ixed
co m m u n ities
e n v iro n m en tal
by
en h an c in g
stre sso rs ,
but
o v erexpression of m elan in com prom ises enzym e secretion
n eed e d for n u trie n t acq u isitio n a n d pathogenicity.
2.
i
M elanized G. gram inis var. grafninis s tra in s b in d m ore C u2+
th a n u n m elan iz ed stra in s , a n d th e a m o u n t of co p p er binding
is p ro p o rtio n a l to th e m elan in co n cen tratio n .
3.
M elanized G. gram inis var. gram inis stra in s b in d m ore m etals
in m ine tailin g s th a n th e u n m elan iz ed stra in , b u t w ith th e
exception of lead, th e p resen c e of th e m elanized stra in s do
n o t affect m etal u p ta k e into p la n t tissu e .
M elanized G.
76
gram inis var. gram inis
u p ta k e into p la n t tissu e .
in th e rh izo sp h ere e n h a n c e s ■lead
78
APPENDICES
79
APPENDIX A
RHIZOSPHERE COMPETITIVENESS OF G a e u m a n n o m y c e s
g r a m in is var. g r a m in is MELANIN MUTANTS
80
T he rh izo sp h ere is th e zone su rro u n d in g p la n t ro o ts a n d th e
site of in te n se m icrobial activity. P la n ts release n u trie n ts into th e
rhizo sp h e re
th a t
a re
utilized
by
soil
m ic ro o rg an ism s,
and
rhizo sp h e re in h a b ita n ts in tu r n m ineralize so il-b o u n d n u trie n ts for
p la n t u p ta k e (3).
M any soil-borne fungal p a th o g e n s c an exist
sap ro p h y tically in th e rhizo sp h ere, w here th ey a re in com petition
w ith o th e r rhizo sp h e re m icro o rg an ism s for s u b s tra te s . In addition,
co m p etin g m icro o rg an ism s m ay p o ss e s s inhibitory or a n tag o n istic
m e c h a n ism s
th a t
can
affect
o th e r
o rg an ism s
in
th e
sam e
en v iro n m ent.
F u n g a l m e la n in s are h e te ro g en eo u s p ig m en ts t h a t serve a s a
p ro tectio n
from
en v iro n m en tal
s tre sso rs
(I).
Som e
p la n t
p a th o g e n s s u c h a s Colletotrichum lagenarium a n d M agnaporihe
grisea req u ire m elan izatio n for effective patho g en icity (4, 8). T hese
fungi develop a m elanized a d h e sio n s tru c tu re t h a t allows th e
tu rg o r p re s s u re n eed ed for leaf p e n e tra tio n (6, 7). T he filam entous
soil fu n g u s
G aeum annom yces gram inis var.
gram inis is
also
m elanized, b u t th e re is no evidence to su g g est a d ire c t relatio n sh ip
betw een m elan o g en esis in th is fu n g u s a n d p ath o g en icity (5).
However, m elan in m ay e n h a n c e survivability in a com petitive
e n v iro n m en t a n d th u s c o n trib u te to patho g en icity indirectly.
In
81
th is ex p erim en t, we a tte m p te d
gram inis
var.
gram inis
to determ in e th e effect of G.
m elanization
on
rhizo sp h ere
co m p etitiv en ess, u sin g m u ta n ts affected in th e m elan in pathw ay.
We also tried to ch aracterize th e rhizo sp h ere fungal com m unity
u sin g PCR am plification of th e ITS region (Figure A. I). E ukaryotic
rib o so m al gen es are a rra n g ed in a ta n d e m re p e a t w ith th e 5.8S
coding
region
flanked
by in te rn a l
tra n sc rib e d
sp a c e rs
(ITS).
In se rtio n s a n d /o r s u b s titu tio n s o ccu r w ithin th e seq u en ce of th e
m u ltip le ITS copies (2).
We expected to tentatively d etect the
survivability of G. gram inis var.
gram inis in th e
p resence of
co m p etin g fungi by am plification of th e ITS region c o n siste n t in
size w ith G. gram inis var. gram inis.
IT SIF
ITS5
ITSI
ITS3
5.8 S
ITS2
ITS 2
ITS4
ITS48
50 100 150
bp
F igure A. I. In te rn a l tra n sc rib e d sp a c e r (ITS) region of ribosom al
DNA (2).
S hadow ed boxes re p re se n t ribosom al s u b u n its , a n s
arro w s in d ic ate position a n d direction of PCR p rim ers.
82
M aterials a n d M ethods
C om petition A ssay
R hizosphere in o cu lu m w as p re p a re d by w eighing 50 g of
verm iculite in to eac h of 4 p o ts, ad d in g 20 g of g a rd e n soil,
and
m ixing well. T w enty n o n -ste rile rice seed s (O. sa tiv a Lemont) w ere
a d d ed to e a c h pot, th e p o ts tra n s fe rre d to a g ree n h o u se a n d
w a tered w ith 100 m l deionized w a ter a n d 50 m l 1A stre n g th
H o ag lan d ’s solution. Pots w ere w atered on a lte rn a te d a y s w ith 50
m l 1A stre n g th H oag lan d ’s or w a ter a n d p la n ts allow ed to grow for
28 days. At h a rv e st, p la n ts w ere rem oved a n d c o n te n ts of th e four
p o ts w ere tu rn e d
into a sterile g lass p a n .
T he in o cu lated
v erm iculite w as th o ro u g h ly m ixed w ith a flam e-sterilized sp a tu la
a n d u s e d for th e rh izo sp h ere inoculum .
Sterile rice seedlings w ere p re p a re d by rem oving h u lls from
seed s, so ak in g th e seed s overnight in 100 m l sterile deionized
w ater, th e n a d d in g 25 m l Clorox® b leach a n d 250 pi Tween 80
(Sigma).
After sw irling to mix, se ed s w ere left in th e Tween 8 0 /
b lea ch so lu tio n for 20 m in u te s, w ith occasional sw irling. C o n ten ts
of flask s w ere p o u re d into sterile p e tri d ish e s a n d aseptically
tra n s fe rre d to p e tri d ish e s co n ta in in g sterile deionized w ater.
83
S eeds w ere
tra n s fe rre d
rin se d
again
w ith
sterile
to p e tri p la te s co n ta in in g
deionized
w ater,
1A stre n g th
th e n
H oagland’s
so lu tio n w ith 0.75% B acto agar. Five day-old seedlings w ere u se d
for th e com petition assay .
T he u n in o c u la te d (no rh izo sp h ere inoculum ) tre a tm e n t w as
p re p a re d by w eighing 50 g of verm iculite into 20 cm 2 p lastic pots
w ith W h a tm a n #1 filter p a p e r in th e botto m of th e p o ts to cover
d ra in holes.
P ots w ere covered w ith a lu m in u m foil a n d sterilized
by au toclaving.
T w enty seedlings w ere tra n s fe rre d to each pot,
along w ith 1 2 X 3 m m LB a g ar p lu g s c o n tain in g w ild-type, JH 4 3 0 0
o r JH 4 3 0 1
or no in o cu lu m sp a ce d betw een seedlings.
The
in o c u la te d (with rhizo sp h e re inoculum ) tre a tm e n t c o n sisted of
p lastic p o ts w ith 40 g sterile verm iculite w ith 20 g rhizo sp h ere
in o c u lu m a d d ed to th e top of th e p o t after autoclaving.
Seedling
a n d fu n g al in o cu latio n w ere th e sam e a s for th e no rhizosphere
tre a tm e n t. All tre a tm e n ts were p re p a re d in triplicate.
P la n ts w ere grow n for 28 d a y s in a g ree n h o u se w ith w atering
every o th e r day, a lte rn a tin g w ith 50 m l sterile
H o ag lan d 's so lu tio n or sterile deionized w ater.
w ere rem oved from
pots, verm iculite
1 /4
stre n g th
At h a rv e st, p la n ts
ad h erin g
to
ro o ts w as
collected a sep tically into sterile tu b e s , a n d frozen a t -20 0C u n til
84
fu rth e r an aly sis. P la n t roots w ere w a sh e d u n d e r ru n n in g w ater to
rem ove a n y a d d itio n al verm iculite, th e n s e p a ra te d from shoots,
a n d b o th ro o ts a n d sh o o ts freeze-dried overnight to o b tain dry
w eights.
R h izo sp here DNA E xtractio n
a n d PCR A m plification
R h izosphere DNA w as e x tra cted a s d escrib ed by T sai a n d
O lson (1991) w ith th e following m odifications:
w ere ex tracted ; following th e IysiS step,
2 g of verm iculite
I m l of a solution
c o n ta in in g 1% CTAB a n d .0.1 M NaCl w as ad d ed , m ixed a n d
in c u b a te d a t 60 °C for I h o u r w ith gentle shaking; a n d finally, th e
e x tra c te d DNA w as b ro u g h t u p to a I m l volum e a n d purified by
p a ssin g th ro u g h a S ep h ad ex G 200 colum n.
Purified
DNA
w as
PCR-am plified
(TCCTCCGCTTATTGATATGC)
and
ITS5
u sin g
p rim e rs
ITS4
(GGAAGTAAAGTCGTAA
CAAGG). T he te m p e ra tu re p ro g ram w as 94 °C, 2 .5 m in u te s for I
cycle, th e n 94 °C (15 s), 53 °C (30 s), 72 0C (1.5 m in u tes) for 40
cycles, followed by 72 0C for 10 m in u te s.
electro p h oretically
se p a ra te d
on
a
3.5%
PCR p ro d u c ts w ere
a g aro se
tre a tm e n t trip licate w as am plified individually.
gel.
E ach
85
R esu lts
C o m p etitiveness
In th e com petition assay , th e in te rac tio n betw een G, gram inis
v a r . "gram inis s tra in in o cu latio n a n d rh izo sp h ere in o cu lu m w as
sig n ificant for b o th sh o o t (P < 0.018) a n d root (P < 0.036) biom ass.
R h izo sp h ere in o cu latio n significantly d e creased ro o t a n d sh o o t
b io m a ss in co n tro l a n d J H 4 3 0 1-tre a te d p la n ts (Fig. A.2), a s well a s
ro o t b io m a ss of JH 4 3 0 0 -in o c u la te d p la n ts.
O u r w ild-type
stra in
JH 2 0 3 3 significantly d ecrea se d b o th root a n d sh o o t b io m ass in
th e
a b se n c e
of rhizo sp h ere
com petition.
However,
in
th e
com petition, p la n t b io m a ss w as n o t significantly re d u c e d com pared
to p la n ts in o cu late d w ith JH 2 0 3 3 alone.
PCR
PCR su ccessfu lly am plified a PCR p ro d u c t c o n siste n t in size
w ith th e ITS region of JH 2 0 3 3 for som e tre a tm e n ts , b u t n o t o th ers
(Fig. A.3). More PCR p ro d u c ts w ere g en erated from DNA ex tracted
from sa m p le s w ith o u t rhizo sp h e re inoculum .
In addition, G.
gram inis var. gram inis w as am plified in m o st n o n -rh izo sp h ere
tre a tm e n ts in o cu late d w ith G. gram inis var. gram inis:
w ith o u t seq u en ce
an aly sis of th e
PCR p ro d u c ts,
However,
we c a n n o t
86
definitively co n clu d e th a t th e 600 bp b a n d re p re s e n ts G. gram inis
var.
gram inis.
To d eterm in e
th e
ability of ITS p rim ers
to
su ccessfu lly am plify G. gram inis var. gram inis DNA in soil ex tracts,
JH 2 0 3 3 DNA w as purified th ro u g h a S ephadex G 200 colum n in
th e sa m e m a n n e r a s soil DNA, th e n PCR-am plified w ith or w ith o u t
soil DNA from th e rh izo sp h ere in o cu lu m . The PCR p ro d u c ts from
JH 2 0 3 3 DNA w ere less c o n c e n tra te d th a n th o se fo u n d in th e soil
in o cu lu m , a lth o u g h it did n o t a p p e a r th a t am plification of JH 2 0 3 3
w as in h ib ited in th e spiked rhizo sp h e re in o cu lu m (Fig.A.4).
D iscu ssio n
M elanized G. gram inis var. gram inis s tra in s a re n o t m ore
p ath o g en ic th a n u n m elan iz ed s tra in s in p ath o g en icity te s ts w ith G
gram inis var. gram inis a s th e sole in o cu lu m (see C h a p te r 2).
However, b e c a u se m elan in provides p rotection from enviro n m en tal
facto rs s u c h a s UV light a n d lytic enzym es, p ig m en ted stra in s m ay
be b e tte r c o m p etito rs in m ixed com m unities; t h u s m elan in m ay
in d irectly e n h a n c e pathogenicity.
We a d d re sse d th is issu e by
devising a com petition ex p erim en t com paring p a th o g en ic effects of
th e th re e s tra in s on rice in th e p re sen c e a n d a b se n c e of additio n al
rh izo sp h ere m icroorganism s. At h a rv e st, we e x tra c te d rhizosphere
87
DNA in
an
a tte m p t
to
ch arac terize ' th e
rh izo sp h ere
fungal
c o m m u n ities of th e different tre a tm e n ts , u sin g PCR am plification
of th e ITS regions (Figure A. I). B ecau se th e ITS reg io n s are sites of
in se rtio n s a n d deletions, we h o p ed to determ in e differences in
fu n g al c o m m u n ities b a se d on th e size differences of th e PCR
p re d ic ts.
from
W hile we su ccessfu lly am plified a 600 b p PCR p ro d u ct
rhizo sp h e re
DNA
(Figure
A:3,
JH 2033),
it
sh o u ld
be
e m p h a size d th a t seq u en ce a n aly sis of th e PCR p ro d u c t w ould be
req u ire d for u n eq u iv o cal identification.
am plification
of DNA e x tra c ts
It is ev ident from PCR
th a t even
tre a tm e n ts
w ith o u t
rhizo sp h e re in o c u lu m h a d a cq u ired fungal rhizo sp h e re in h a b ita n ts
in a d d itio n to G. gram inis var. gram inis;
c o n ta m in a tio n probably
o rig in ated from th e g reen h o u se. However, o u r two m e lan in m u ta n t
s tra in s w ere u n a b le to sufficiently com pete w ith th e s e rh izo sp h ere
in h a b ita n ts to c a u se significant p la n t b io m ass red u ctio n .
The
rh izo sp h ere in o cu lu m a p p a re n tly co n ta in e d p a th o g e n s th a t were
m ore v iru le n t th a n G. gram inis var. gram inis.
PCR re s u lts also
su g g e st a sh ift in rh izo sp h ere co m m u n ities w h e n th e initial
rh izo sp h ere in o cu lu m ITS "profile" is co m p ared to profiles a t
h a rv e st (Figure A.3b).
88
a. S h o o t dry w eights
I
£
I
CQ
I
Control
JH2033
JH4300
JH4301
Strain
b. Root dry w eights
9
□ No Rhizosphere
Inoculum
8
I
£
I
I
I
o
Control
JH2033
JH4300
JH430I
Strain
Figure A.2. S ignificant interactive effect betw een s tra in inoculation
a n d rh izo sp h ere in o cu lu m on root a n d shoot dry w eight. D ifferent
le tte rs above b a rs in dicate significant differences a t P < 0.05
(Tukey's S tu d e n tiz ed Range test).
89
a. W ith out R hizosphere Inoculum
S Ggg RI
Control
JH 2033
■
Q
JH 4300
JH 4301 Ggg S
Q _____ _______ r>_____ Q_____ C'___________ Q _____ ______________ Q____ V l
600 bp
RH
■
200 bp
b. W ith R hizosphere Inoculum
S Ggg RI
600 bp
Control
a b c
a
P
JH 2033 JH 4300
b c a b c
JH 4301 Ggg S
a b c
r':"$
»
#
# mm»
200 bp
Figure A.3. PCR-am plified ITS regions of DNA e x tra cted from
rh izo sp h ere m ateria l w ith or w ith o u t rh izo sp h ere inoculum .
T riplicate p o ts = a, b a n d c. S = 100 bp s ta n d a rd ; Ggg = PCRam plified JH 2 0 3 3 ; RI = rh izo sp h ere inoculum . Negative controls
(no tem plate) show ed no am plification (not show n).
90
Figure A.4. PCR-am plified ITS regions of JH 2 0 3 3 , rhizosphere
in o cu lu m e x tra c t a n d rh izo sp h ere in o cu lu m spiked w ith JH 2033.
S = 100 bp s ta n d a rd ladder; RI = rh izo sp h ere in o cu lu m .
W hile th is ex p erim en t provides som e evidence to su p p o rt th e
in d ire ct effect of m elan in
on
pathogenicity,
m ore
controlled
e x p erim en ts are need ed to a d d re ss th e u n c e rta in tie s asso ciated
w ith th e re s u lts .
rep licate
From th e PCR am plification, it is evident th a t
tre a tm e n ts
probably
had
different
rhizosphere
m icro o rg an ism s, a n d p e rh a p s a defined rhizo sp h ere in o cu lu m th a t
91
is n o t itself p a th o g en ic a s th e com petition facto r w ould am eliorate
rep lication.
In addition, ste p s to elim inate c o n ta m in a tio n in th e
g re e n h o u se w ould e n h a n c e th e q u ality of d a ta collected.
From
F igure A. 4, it is also a p p a re n t th a t th e PCR rea ctio n is n o t
optim ized for am plification of th e G. gram inis v a r ." gram inis ITS
region.
L iteratu re C ited
1.
Bell, A. A. a n d M. H. W heeler.
fu n c tio n s of fungal m elan in s.
2 4 :4 1 1 -4 5 1 .
1986. B io sy n th esis a n d
Ann. Rev. Phytopathol.
2.
B oysen, M., M. Borja, C. del M oral, O. S a laz ar a n d V. Rubio.
1996. Identification a t s tra in level of R hizoctonia solani AG4
iso lates by d irec t seq u en ce of asym m etric PCR p ro d u c ts of
th e ITS region. C urr. G enet. 2 9 :1 7 4 -1 8 1 .
3.
C olem an, D. C., C. P. P. Reid a n d C. V. Cole.
1983.
Biological stra te g ie s of n u trie n t cycling in soil sy stem s. Adv.
EcoL Res. 13:1-55.
4.
C h u m ley , F. G. a n d B. V alent. 1990. G enetic an aly sis of
m elanin-deficient, n o n -p ath o g en ic m u ta n ts of M agnaporthe
grisea. Mol. P la n t M icrobe in te rac t. 3 :1 8 9 -1 9 8 .
5.
Elliott, M. L.
1995.
in h ib itin g
com pounds
Mycologia 8 7 :4 5 3 -4 5 9 .
6.
H ow ard, R. J . a n d M. A. F errari. 1989. Role of m elan in in
a p p re ss o riu m function. Exp. Mycol. 1 3 :4 0 3 -4 1 8 .
Effect of m elan io n b io sy n th esis
on
G aeum annom yces
species.
92
7.
H ow ard, R. J ., M. A. F errari, D. H. R oachi a n d N. P. Money.
1991. P e n e tra tio n of h a rd s u b s tra te s by a fu n g u s em ploying
e n o rm o u s tu rg o r p re ssu re .
P roc. Natl. Acad. Sci. USA
8 8 :1 1 2 8 1 -1 1 2 8 4 .
8.
T a k a n o , Y., Y. K ubo, K. Shim izu, K. Mise, T. O kuno a n d I
F u ru sa w a . 1995. S tru c tu ra l an aly sis of P K S l, a polyketide
sy n th a s e gene involved in m elan in b io sy n th esis in
Colletotrichum lagenarium . Mol. Gen. G enet. 2 4 9 :1 6 2 -1 6 7 .
9.
T sai, T.-L. a n d B. H. O lson. 1991. R apid m eth o d for d irect
e x tra ctio n of DNA from soil a n d sed im en ts. AppL Environ.
M icrobiol. 5 8 :1 0 7 0 -1 0 7 4 .
)
93
APPENDIX B
PHYTOREMEDIATION APPLICATION OF G a e u m a n n o m y c e s
g r a m in is var. g r a m in is MELANIN MUTANTS
94
T he A n aco n d a C opper C om pany extensively m in e d th e B utte,
M o n ta n a a re a for approxim ately 80 y e ars, a n d sm elted th e ore a t
th e A n a co n d a sm elter, 30 m iles w e st of B u tte.
T he re su ltin g
tailin g s from th e sm elting p ro ce ss w ere d ep o sited into tailings
p o n d s covering several sq u a re m iles. The tailings p o n d s are p a rt of
th e la rg e st U.S. E.P.A. S u p e rfu n d site in th e U n ited S ta te s, a n d
stra te g ie s a re c u rre n tly being developed to rem e d ia te a n d /o r
reclaim th e tailin g s po n d s.
O ne s u c h stra te g y is p h y to rem ed iatio n of th e c o n ta m in a te d
site.
P h y to rem ed iatio n is defined a s th e u s e of green p la n ts to
rem ove p o llu ta n ts s u c h a s m e ta ls from th e en v iro n m en t or. to
re n d e r th e m h a rm le s s (8).
T he m ajo r factor in availability of
m e ta ls to p la n ts in soil is th e solubility a n d th erm o d y n am ic activity
of th e u n co m p lex ed ion.
Only a soluble species a d ja c e n t to th e
ro o t is likely to be tra n slo c a te d in to th e root (I).
S table m etal-
o rganocom plexes s u c h a s th o se u s e d in fertilization m ay be highly
so lu b le (6) a n d readily ta k e n u p by roots.
P la n ts m ay secrete
m etal-c h elatin g p ro te in s (10) or p h y to ch e la tin s (9) th a t a c t a s
p h y to sid ero p h o re s
and
solubilize
"soil-bound"
m etals.
P lan t
u p ta k e of n o n -e s se n tia l heavy m e ta ls m ay also be e n h a n c e d by
rh izo sp h ere m icro o rg an ism s (8, 12). R hizosphere fungi have been
95
sh o w n to acidify soil su rro u n d in g ro o ts (2), w hich w ould in crease
m e ta l availability for p la n t u p ta k e .
O rganic fu n g al co m p o n en ts
s u c h a s m e lan in m ay also b in d m etals a n d influence m etal
solubility. In th is stu d y , we investig ated th e in te ra c tio n s betw een
m elan ized a n d u n m elan iz ed G. gram inis var. gram inis s tra in s a n d
tailin g s m etal u p ta k e by ran g e g rasses.
M aterials a n d M ethods
M etal S orp tio n of T ailings M aterial
to B io m ass
T ailings m ate ria l w as collected from th ree site s in th e "New
River Pond" n e a r A naconda, M o n ta n a in J u n e
1996, by th e
M o n ta n a S ta te U niversity R eclam ation R esearch D ep artm en t. The
to p 5cm of m a te ria l w as rem oved a n d tailings w ere collected to a
d e p th of 20 cm. M aterial w as sieved th ro u g h a 2 m m screen, a n d
sto re d a t 4 °C in covered c o n ta in e rs u n til u se.
Shake
flask s
su p p le m e n te d w ith
c o n tain in g
200
ml
m in im al
m edium
1% A n aco n d a tailings p o n d m ateria l were
in o c u la te d a n d in c u b a te d a s d e scrib e d for copper b in d in g stu d ies.
At h a rv e st, fu n g al b io m a ss a n d a sso c ia te d tailin g s m ate ria l w ere
collected by filtratio n th ro u g h a W h atm a n #1 filter, rin se d 2X to
96
rem ove re s id u a l tailings, th e n dried overnight.
S am p les w ere
d ig ested a s previously d escrib ed in C h a p te r 3, a n d analyzed by
ICP-AES for iron, zinc, copper a n d lead.
M etal U p tak e by P la n t B iom ass
T he e x p erim en t w as d esigned a s a 2 X 4 X 5 factorial, w ith
th e following tre a tm e n t factors: I) tailin g s p o n d ad d itio n , 2) wildtype o r m elan in m u ta n t s tra in s of G. gram inis var. gram inis, a n d 3)
p la n t species,
P ots w ere p re p a re d by rin sin g 500 g (diy weight) sa n d
(Pakm ix, Inc., Toledo, OH) 2X w ith ta p w ater, th e n tra n sfe rrin g into
10 cm p o ts w ith W h atm a n #1 filter p a p e r covering th e d ra in holes.
Air b u b b le s w ere rem oved by gently sh a k in g th e p o ts a n d excess
su rface w a te r w as p o u re d off.
O u te r seed h u lls of Agropyron d a sy sta c h y u m , A. elongatum,
A. trachycaulum , E ly m u s cinereus, or E. ju n e u s w ere rem oved a n d
se ed s w ere p laced in 250 m l E rlynm eyer flasks c o n ta in in g 25 m l
Clorox® b lea ch a n d 1% Tween 80. After sh a k in g for 20 m in u tes,
c o n te n ts of flask s w ere tra n s fe rre d to sterile p e tri d ish e s.
Seeds
w ere w a sh e d in sterile w ater, th e n aseptically tra n s fe rre d to 25 X
150 m m slip -cap tu b e s c o n tain in g 20 m l of % stre n g th n u trie n t
97
a g a r (Difco). Five to six day-old seedlings w ere tra n s fe rre d to p o ts
along w ith 4 - Ic m d iam ete r p lu g s of LB a g a r co n ta in in g no
in o c u lu m (control), JH 2 0 3 3 , JH 4 3 0 0 or J H 4 3 0 1.
Plugs w ere
p laced 0 .5 cm below th e s a n d su rface a t a d ista n c e of 1.5 cm from
th e p la n t. P ots w ere tra n sfe rre d to a g reen h o u se, a n d w atered on
a lte rn a te d ay s w ith e ith e r 50m l deionized H 2O or 1A stre n g th
H o ag lan d ’s solution. After 14 days, 50 g of s a n d su rro u n d in g th e
p la n t w ere rem oved a n d m ixed w ith 50 g of A n aco n d a tailings pond
m aterial.
The m ix tu re w as rep laced in th e p o t a n d th e p la n ts
grow n for a n a d d itio n al 14 days. At h a rv e st, p la n ts w ere rem oved
a n d m ate ria l a d h e rin g to ro o ts w as collected into sterile tu b e s a n d
sto re d a t -20 °C. P la n ts w ere th e n rin se d w ith ta p w a ter, roots a n d
sh o o ts se p a ra te d , lyophilized overnight, th e n w eighed.
S hoots
w ere c u t into Ic m pieces a n d d igested for m etal a n a ly sis by EPA
m e th o d 3050A , w ith a final sam p le volum e of 50 ml.
G row th
m ate ria l tightly a d h e re d to ro o ts even following extensive w ashing,
m ak in g a n y a s s e s s m e n t of m etal c o n te n t w ith in ro o t tissu e
su sp e c t.
98
R h izo sp here H yphal L engths
F u n g a l h y p h a l len g th s w ere d eterm in ed by th in a g ar film
p re p a ra tio n (5) followed by m icroscopic e x am in atio n of h y p h al
se g m en t in te rse c ts on a n o c u la r grid (7).
A 0.5 g s u b sam ple of
rh izo sp h ere m ate ria l w as w eighed in to a 50 m l screw -cap p ed tu b e
c o n ta in in g 4.5 m l sterile deionized w ater.
The s u s p e n s io n w as
vortexed vigorously for 30 s, th e n allow ed to settle for 30 s.
One
m l of s u p e rn a te w as p ip etted into a screw -capped tu b e co ntaining
4 m l of m o lten 2% agar. The tu b e c o n te n ts w ere m ixed well, a n d
th e n p o u re d onto hem ato cy to m eters. A slide w as p laced over th e
c e n te r of th e h e m ato cy to m eter a n d th e a g a r w as allow ed to solidify.
E x cess a g a r w a s c u t aw ay from a ro u n d th e c e n te r section of th e
h e m a to c y to m e te r a n d th e a g ar film w as floated o nto a m icroscope
slide in a shallow d ish of w ater.
The film s w ere air-d ried a n d
ob serv ed w ith p h a se c o n tra s t m icroscopy a t 100X m agnification.
F o u r film s w ere p re p a re d for e a c h sam ple, a n d 10 ra n d o m fields
p e r film w ere co u n ted .
99
R e su lts
T ailings S orption to B iom ass
More iron a n d copper in th e tailings p o n d .m ateria l were
b o u n d to th e d a rk m u ta n t JH 4 3 0 1 th a n th e light m u ta n t, while th e
w ild-type so rb ed a n in te rm e d iate c o n c e n tra tio n n o t significantly
d ifferent from e ith e r m u ta n t (Table B .l). T his re s u lt is in c o n tra st
to ex p erim e n ts w ith copper a c c u m u la tio n of c u ltu re s grow n w ith
CuSCk (Table 4.1) a n d su g g est th a t w hile th e ionic co p p er species
b in d s to m elanized fungal b io m ass, o th e r copper sp ecies m ay b in d
to G. gram inis var. gram inis b io m a ss reg a rd less of m elan in sta tu s .
Lead a n d zinc c o n c e n tra tio n s w ere sim ilar for th e th re e stra in s.
T ailings M etal U ptake by P la n ts
In o cu la te d w ith G. gram inis var.
pram inis
A su m m a ry of significant tre a tm e n t effects is given in Table
B .2.
G. gram inis var. gram inis in o cu latio n significantly in creased
h y p h a l le n g th s in th e rh izo sp h ere.
C ontrol tre a tm e n ts h a d less
h y p h a e (2.39 m /g ) th a n JH 4 3 0 0 , JH 2 0 3 3 or JH 4 3 0 1 (3.84, 3.49
a n d 3.21 m /g , respectively).
significantly different.
In o cu lated tre a tm e n ts w ere n o t
100
T able B .l. M etal c o n c e n tra tio n s of tailings p o n d m ateria l a n d
m e ta ls so rb ed to w ild-type (JH 2033) a n d m elan in m u ta n t stra in s of
G. qram inis var. gram inis.______________
B iom ass S orp tio n
M etal
T ailings P ond
JH 4 3 0 0
JH 2 0 3 3
JH 4301
Species
19.34 a b
Fe (m g/g) 2 0 .0 0 (0.54)*
17.66 a**
26.10 b
Z n (pg/g) 9 6 .6 8 (15.28)
121.40 a
1 16.56 a
95.39 a
157.78
(4.52)
139.07
a
141.20
a
b
C u (pg/g)
185.21 b
3 8 3 .5 0 a
3 9 4 .0 4 a
4 7 2 .2 7 a
Pb (pg/g) 3 9 7 .8 0 (9.99)
* s ta n d a rd e rro r
** m e a n s followed by different le tte rs a c ro ss a row a re significantly
differen t a t P < 0.05 (Tukey's S tu d e n tiz ed Range te s t, BSD).
P la n t b io m a ss w as n o t affected by e ith e r tailings pond
a d d itio n or by in o cu latio n , su g g estin g th a t th e ap p lied tailings
c o n c e n tra tio n w as n o t toxic, a n d th a t G. gram inis var. gram inis w as
n o t p a th o g en ic to a n y of th e p la n t species tested . Not surprisingly,
b o th ro o t a n d sh o o t b io m a ss differed according to p la n t species
(Table B.3).
A. elongatum p la n t b io m a ss w as g re a te s t of th e five
sp ecies teste d .
A. d a sy sta c h y u m p ro d u ced th e le a s t aboveground
b io m ass, w hile E. ju n e u s p ro d u ce d th e le a st below ground.
In o cu latio n h a d no effect on iron u p ta k e a n d tra n s p o rt to
sh o o t tis s u e
(Table B.2).
However, a sig n ifican t in te rac tio n
b etw een p la n t species a n d tailin g s ad dition su g g e ste d th a t th ese
five p la n t sp ecies re sp o n d ed differently in iron u p ta k e from tailings
m ateria l.
All species grow n w ith o u t tailings h a d sim ilar iron
101
c o n c e n tra tio n s in sh o o t tis su e (Figure B .l).
O nly one species (A.
elongatum ) h a d a n in c re a se d iron c o n ce n tra tio n w h en grow n w ith
th e tailin g s ad d itio n , su g g estin g th a t th is sp ecies w a s p e rh a p s
e ith e r u n a b le to reg u late iron u p ta k e , or h a d a n e n h a n c e d iron
u p ta k e m ec h an ism .
In c o n tra st, th e only significant tre a tm e n t
effect o n zinc c o n c e n tra tio n w as tailings ad d itio n (Table B.2). Zinc
sh o o t c o n c e n tra tio n of u n a m e n d e d tre a tm e n ts w as 4 2 .4 2 p g /g
b io m a ss a n d a m e n d e d tre a tm e n ts h a d a zinc c o n c e n tra tio n of
9 4 .2 9 p g /g b io m ass.
T able B .2. Significant tre a tm e n t effects of p la n t m etal u p ta k e
ex p erim ent. P = p la n t species, I = F u n g al s tra in in o cu lu m , T =
tailin g s addition. * P < 0.05, ** P < 0.01, *** P < 0.001.
T re a tm e n t Effect
P a ra m e te r
H yphal L engths
S h o o t B iom ass
Root B iom ass
S h o o t Iron
S h o o t Zinc
S h o o t C opper
S h o o t Lead
P
*** .
**
**
-
I
***
T
PxI
—
PxT
—
—
-
***
-
**
***
-
Ix T P x I x T
-
*
-
T he only significant effect of G. gram inis in o cu latio n w as a n
in te ra c tio n
b etw een
in o cu lu m
and
tailings
a d d itio n
on lead
c o n c e n tra tio n in sh o o t biom as (Figure B.2). C ontrol a n d JH 4 3 0 0 in o c u la te d tre a tm e n ts gave sim ilar resp o n ses; lea d co n ce n tra tio n
102
w as significantly h ig h er in sh o o ts of u n a m e n d e d co m p ared to
ta ilin g s-am en d e d tre a tm e n ts .
B oth th e w ild-type JH 2 0 3 3 a n d
JH 4 3 0 1 s tra in s h a d less sh o o t lead in th e no tailin g s tre a tm e n t
th a n th e u n in o c u la te d (control) a n d JH 4 3 0 0 -in o c u la te d p lan ts.
In terestingly, th e heavily m elanized m u ta n t J H 4 3 0 1 significantly
in c re a se d lead c o n te n t in sh o o ts w ith ad d ed tailings.
T able B.3. Significant m ain effect of p la n t sp ecies on root a n d
sh o o t b io m ass. M eans in co lu m n followed by different le tters are
significantly different a t P < 0.05 (Tukey's S tu d e n tiz ed Range test,
HSD).____________________________ ________ i__________________ '
P la n t B iom ass (mg d ry weight)
S hoot
P la n t Species
Root
136.11
ab
A. d a s y s ta c h y u m
6 4 .9 9 a
2 6 0 .0 0 c
A. elongatum
134.39 b
107.61 a
170.75 ab
A. trachycaulum
189.34 b
118.13 ab
E. cinereus
112.20 a
7 4 .4 4 a
E. ju n eu s
D iscu ssio n
We fo u n d a positive co rrelatio n betw een m e la n in a n d m etal
b in d in g of tailin g s m aterial, in d icatin g th a t m elan ized fungal
b io m a ss a re cap ab le of sorbing m ixed m etal sp ecies, p a rticu la rly
iro n a n d copper.
B ecau se o u r ICP-AES an aly sis did n o t provide
in fo rm atio n c o n cern in g th e type of m etal com plexes in th e tailings
m ateria l, c o m p a riso n s betw een CuSCk bin d in g e x p erim en ts a n d
tailin g s so rp tio n sh o u ld be view ed w ith cau tio n . M ost likely m an y
103
d ifferent m etal com plexes exist w ithin th e tailings m ateria l th a t
m ay re a c t w ith fungal co m p o n e n ts o th e r th a n m elanin.
No Tailing;
I With Tailings
Adasystad^uti
A dongahtn
A trachyccnJuii
E dmreus
Ejmeus
PiantSpecies
Figure B .l.
Interactive tre a tm e n t effect of p la n t species a n d
tailin g s ad d itio n on iron u p ta k e into sh o o t tissu e . M eans followed
by d ifferent le tte rs a re significantly different a t P < 0 .0 5 (Tukey's
S tu d e n tiz ed Range te st, HSD).
F u n g al m elan in s c o n stitu te a large portion of th e soil h u m u s
a n d th erefore affect th e tra n slo c a tio n a n d availability of m etal ions
to p la n ts (11).
H um ic acid s form w ater-so lu b le a n d w ater-
in so lu b le com plexes w ith m etal ions. Stability c o n s ta n ts of m etalh u m ic acid com plexes su g g est th a t am ino a cid s a n d hydroxy
carboxylic acid s secreted by p la n t ro o ts will com pete favorably w ith
h u m ic a cid s for m etal ions a n d prom ote th e ir u p ta k e by p la n t roots
(13). However, C u a n d Pb a c c u m u la tio n in p la n ts grow n in sludge-
104
a m e n d e d soils show only sm all in c re a se s co m p ared to th o se grown
on u n c o n ta m in a te d soils (3).
We fo u n d no c o rrelatio n betw een
m elan ized fu n g al in o cu lu m a n d co p p er a c c u m u la tio n in sh o o t
tis su e w ith tailin g s a m e n d m e n t.
O u r re s u lts su g g e st th a t fungal
m elan in does n o t significantly e n h a n c e co p p er availability by
in c re a sin g
solubility,
or
d e crea se
p la n t
co p p er
u p ta k e
by
se q u e stra tio n . We w ere u n a b le to a c c u ra te ly d ete rm in e root tissu e
c o n c e n tra tio n s b e c a u se of m a te ria l tightly a d h e re d to th e foot
su rface, a n d th u s c a n n o t re a c h a n y c o n clu sio n s a b o u t m etal
c o n c e n tra tio n s
of w hole
p la n ts.
P resu m ab ly
h ig h er
m etal
c o n c e n tra tio n s in ro o ts w ould lead to g reater sh o o t tran slo c atio n
a s well, p a rtic u la rly w h en m etal c o n c e n tra tio n of ro o ts is in excess.
W hile co p p er c o n c e n tra tio n s did n o t vary w ith tre a tm e n t, we did
find in c re a se d lead a cc u m u la tio n in p la n ts from a m e n d ed soils
in o c u la te d w ith th e heavily-m elanized m u ta n t, in d ic atin g th a t
m e la n in m ay have in c re a se d solubility of lead in tailin g s m aterial.
S oil-applied c h elatin g a g en ts s u c h a s EDTA have b e en show n to
greatly in c re a se th e tra n slo c a tio n of heavy m etals, in clu d in g lead
from soil in to sh o o ts (4). P e rh a p s fu n g al m elan in a c ts in a sim ilar
m a n n e r a s a chelator.
T he re s u lts of th e se m etal b in d in g ex p erim e n ts w ith o u r
m e lan in m u ta n ts provide evidence to su p p o rt th e relatio n sh ip
b etw een fu n g al m elan in a n d m etal binding. S orp tio n of m etals to
D H N -m elanin v aries w ith
com plexes, a n d
m etal
species
and
different m etal
m elan in m ay a c t a s a c h e la to r in th e
soil
105
en v iro n m en t to in cre ase m etal solubility (particularly lead) an d
s u b s e q u e n t p la n t u p ta k e on m etal-c o n ta m in a te d sites.
e x p erim en ts
w ith
defined
m etal
com plexes
are
F u rth e r
req u ired
to
d ete rm in e th e exact re la tio n sh ip betw een fungal m elan o g en esis in
th e rh izo sp h ere a n d m etal u p ta k e into p la n t tissu e.
Control
JH4300
JH2033
JH4301
Strain
F igure B.2. S ignificant in te rac tio n betw een stra in in o cu lu m a n d
tailin g s a d d itio n on lead c o n ce n tra tio n in sh o o t b io m ass. M eans
followed by different le tters are significantly different a t P < 0.05
(Tukey's S tu d e n tiz ed Range te st, HSD).
L iterature Cited
I.
C ataldo, D. A. a n d R. E W ildung. 1978. Soil a n d p la n t
facto rs influencing th e a c c u m u la tio n of heavy m etals by
p la n ts. E nviron. H ealth Perspec. 2 7 :1 4 9 -1 5 9 .
106
2.
Devevre, J . G arb ay e, a n d B. B ottom .
1996. R elease of
com plexing organic acid s by rh izo sp h ere fungi a s a factor in
N orw ay sp ru c e yellowing in acidic soils.
Mycol. Res.
1 0 0 :1 3 6 7 -1 3 7 4 .
3.
H ooda, P. S., D. McNulty, B. J . Alloway, a n d M. N. A itken.
1997. P la n t availability of heavy m etals in soils previously
a m e n d ed w ith heavy ap p lica tio n s of sew age sludge. J . Sci.
Food A gric. 7 3 :4 4 6 -4 5 4 .
4.
H uang, J . W., S. D. C u n n in g h am .
1996.
p h y to ex tractio n : species v ariatio n in lead u p ta k e
tra n slo c a tio n . New P hytbl. 134 :7 5 -8 4 .
5.
J o n e s , P. C. T. A nd J . E. M ollison. 1948. A T echnique for
th e q u a n tita tiv e estim atio n of Soil m icro o rg an ism s. J . Gen.
M icrobiol. 2 :5 4 -6 9 .
6.
Norvell, W. A. a n d W. L. Lindsay. 1969. R eactio n s of EDTA
com plexes o f Fe, Zn, Mn, a n d C u w ith soils. Soil Sci. Soc.
Am. P ro c. 3 3 :8 6 .
7.
O lson, F. C. W. 1950. Q u an titativ e e stim a te s of filam entous
algae. T ran s. Am. M icro..Soc. 6 9 :2 7 2 -2 7 9 .
8.
R askin, I., R. D. S m ith a n d D. E. Salt.
1997.
P h y to rem ed iatio n of m etals:
u sin g p la n ts to rem ove
p o llu ta n ts from th e en vironm ent. C urr. O p in . B iotechnol.
8 :2 2 1 -2 2 6 .
9.
R a u se r, W. E. 1995. P h y to ch elatin s a n d re la te d peptides.
P la n t Physiol. 1 0 9 :1 4 1 1 -1 4 1 9 .
10.
R obinson, H. J ., A. M. T om m ey, C. K uske a n d P. J . Ja c k so n .
1993. P la n t m etallo th io n ein s. B iochem istry 2 9 5 :1 -1 0 .
11.
S aiz-Jim enez, C. a n d F. S hafizadeh. 1984. Iron a n d copper
b in d in g by phenolic polym ers: a n electrbn sp in reso n an c e
stu d y . C urr. Microbiol. 10:2 8 1 -2 8 6 .
Lead
and
107
12. . S alt, D. E., M. Blaylock, N. P, B. A. K um ar, V. D ushenkov, B.
D. E n sley , I. C het, a n d I. R askin. 1995. P hytorem ediation:
a novel stra te g y for th e rem oval of toxic m e ta ls from th e
e n v iro n m en t u sin g p la n ts. B iotechnology 1 3 :4 6 8 -4 7 4 .
13.
S tev en sen , F. J . 1976. S tability c o n s ta n ts of C u 2+, Pb2+ a n d
C d2+ com plexes w ith h u m ic acids. Soil Sci. Soc. Am. J .
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