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EFFECT ON BENOMYL 0N SOYBEAN ENDO-MYCORRHIZAE
AND UPTAKE OF BENOMYL BY MYCORRHIZAL ROOTS

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
JACK EUGENE BAILEY
1977

 

I§.L “A

 

' LIBRA R Y
Michis. AC
UlllVL‘I’fflEy

 

ABSTRACT

EFFECT OF BENOMYL ON SOYBEAN ENDO-MYCORRHIZAE
AND UPTAKE OF BENOMYL BY MYCORRHIZAL ROOTS

BY

Jack Eugene Bailey

Soil drenches of benomyl (methyl—l-(butylcarbamoyl)-2-benzimidazoler
carbamate) at 2.5, 25, 125, and ZSO/ug/g soil were added to pots newly

planted to soybeans (Glycine max (L.) Merrill) and containing 120

 

chlamydospores of the vesicular-arbuscular (VA) mycorrhiza-forming

fungus Glomus fasciculatus per 100 9 soil. Mycorrhizal infection,

 

recorded as percent of root length colonized, was decreased from 70 -
80% to approximately 45% by concentrations of 25’Pg/g in one experiment
and 2.5’yg/g in another. Concentrations as high as 250’Pg/g did not
further decrease infection. Benomyl prevented increased growth due to
the VA mycorrhizae even with colonization as high as 48%. Benomyl
uptake was measured in a bioassay using the benomyl-sensitive fungus

Penicillium digitatum and extracts of plant parts. No consistent

 

uptake differences between mycorrhizal and non-mycorrhizal plants were

found.

EFFECT ON BENOMYL ON SOYBEAN ENDO—MYCORRHIZAE

AND UPTAKE OF BENOMYL BY MYCORRHIZAL ROOTS

BY

Jack Eugene Bailey

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirement
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1977

To my wife and parents

ii

ACKNOWLEDGMENTS

I would like to thank Dr. Gene Safir for his advice and encourage-
ment in helping me to prepare this manuscirpt, and in his guidance in
all matters for the last two and one half years.

I would also like to thank Drs. G. W. Bird, J. L. Lockwood, and
M. V. Wiese for their very valuable comments on the technical aspects
of this thesis preparation.

Thanks are also conveyed to all my fellow students that took a
constructive interest in my research.

And to my wife for her appreciation and understanding of the trials

of research, I express my gratitude.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES .................................................. V
LIST OF FIGURES . ............. i... ......... I ....................... Vi
INTRODUCTION AND LITERATURE REVIEW .............................. 1
MATERIALS AND METHODS ............. .. ............................ 9
Preparation of Inocula ........ . ............................ 9
Planting and Inoculation .......... ' ......................... 10
Benomyl Application ........................................ lO
Bioassay Procedures .. ........ i ...... O ........................ 11
Infection Rating ........................................... 12
RESULTS ................ . ......................................... l3
Fungal Tolerance to Benomyl ...... -........ ....... . .......... 13
Uptake of Benomyl by Soybean Plants ........... I ............. 15
DISCUSSION ................... .... ..... ....... ................... 24
LITERATURE CITED ...... ................. .......... ........ ....... 27
APPENDIX A, Mycorrhizal Infection Rating Systems ............... 34
APPENDIX B, Bioassay procedures ............ ° ..... ... ............ 36
APPENDIX C, Preliminary Testing of Benomyl Uptake .............. 44
APPENDIX D, Supplemental Data from Results ..................... 51

iv

LIST OF TABLES

Table Page

1. Effect of leaf plug location and freezing on area of
zone of inhibition .................................... 38

2. Benomyl distribution within plants vs. time after soil
treatment ............................................. 45

3. Growth parameters and uptake of benomyl by M and NM plants
after a soil drench of benomyl ........................ 50

4. Relationship of plant growth parameters, benomyl uptake,
and time .............................................. 57

5. Relationship of plant growth parameters to benomyl uptake .. 58

Figure

1.

10.

11.

LIST OF FIGURES

 

 

 

Page
Effect of various concentrations of benomyl applied to
soil on G, fasciculatus infection of soybeans ........ 14
Effect of various concentrations of benomyl applied to
soil on the leaf area of soybean plants with
(mycorrhizal) or without (non-mycorrhizal) a
g. fasciculatus infection . ..... ... .................... l6
Benomyl uptake with relation to leaf area ...... ............ l7
Benomyl distribution in roots vs. leaves of soybeans with
(M) or without (NM) a g, fasciculatus infection ....... l9
Benomyl concentration in leaves-vs. days exposure to a
25.0/pg/g soil drench ..... ..... . ...................... 20
Benomyl concentration in roots vs. days exposure to a
25.01Pg/g soil drench ..... .... ..... . ........ . ......... 21
Leaf area as a percentage of non-treated controls vs.
exposure time to 25.0/pg/g benomyl ............. . ...... 23
Illustration of middle leaflet of second trifoliate leaf
showing location for-leaf sampling in the leaf plug
bioassay metthd one. 00000 cooc‘co-c'oooo ooooooooooooooooo 37
Zone of inhibition (cmz) about leaf plugs with relation
to exposure time and benomyl concentration ............ 40
Zone of inhibition (cmz) about acetone disks with relation
to exposure times and benomyl concentrations .......... 41
Benomyl concentration in leaves vs. days exposure to a
25 .0 )19/9 soil drench ................................. 46

vi

Figure Page

12.

13.

14.

15.

16.

17.

18.

19.

20.

Benomyl concentration in roots vs. days exposure to a
25.0/pg/g soil drench ................. . ............... 47

Distribution of benomyl in leaves two days after
application ........................................... 49

Effect of various concentrations of benomyl applied to
soil on G, fasciculatus infection of soybean .......... 52

 

Effect of various concentrations of benomyl applied to
soil on the leaf area of soybean plants with
(mycorrhizal) or without (non-mycorrhizal) a
G, fasciculatus infection ............................. 53

 

Benomyl concentration in leaves vs. days exposure on a
25.0/pg/g soil drench ....................... . ......... 54

Benomyl concentration in roots vs. days-exposure to a
25.0’pg/g soil drench ................................. 55

Leaf area as a percentage of non-treated controls vs.
exposure time to 25.0/ug/g benomyl .................... 56

Benomyl distribution in roots vs. leaves of soybeans
with (M) or without (NM) a g, fasciculatus infection .. 59

 

Benomyl distribution in roots vs. leaves of soybeans
with (m) or without (NM) a g, fasciculatus infection .. 60

 

vii

INTRODUCTION AND LITERATURE REVIEW

Vesicular arbuscular (VA) mycorrhizal fungi are common in soil
and in plant roots. They occur on wild herbaceous plants, shrubs,
trees and agricultural crops (30). The first descriptions of these
organisms appeared in the late 1800's, however, their investigation
was limited until the 1950's (30). From 1955 to 1968 most research
dealt with distribution and morphology. More recently emphasis has
been placed on their symbiotic and other effects on plant growth and
development (56).

Fungi which form VA mycorrhizae with plant roots are members of
the family Endogonaceae (29). The large amount of diversity within
this family may eventually lead to the displacement of some species to
other families (29), such as the Mortierelaceae (47,29,l,83). There
are seven genera in the Endogonaceae as defined by Gerdemann (29):

Endogone, Gigaspora, Acaulospora, Glomus, Sclerocystis, Glaziella and

 

 

Modicella. All but Modicella and Glaziella are known to form VA
mycorrhizae (29).

The beneficial effect of VA mycorrhizal infection on plant growth
is well documented (6,7,26,27,14,30,56,3l,4l,82). Increases in

nutrient uptake can occur via the fungal hyphae (37) which increase

the growth of mycorrhizal plants (37,67). Recently, Rhodes and
Gerdemann (67) showed that phosphorus (P) absorption, via hyphae,

can take place up to 7 cm from the root surface. Greater P uptake
is associated with increased plant growth (6,26,49,50,7l), making

P fertilization less beneficial to mycorrhizal than non-mycorrhizal
plants. In some cases P reduces mycorrhizal-related growth
stimulation (7,15,49,50,55,58,70), and mycorrhizal infection (55,57).
Safir gt_al, (72) showed decreased resistance to water transport in
mycorrhizal compared to non-mycorrhizal soybean roots. This
phenomenon may result from increased P concentration of the plant (73).
Other nutrients such as manganese, magnesium, copper, zinc, sulphur,
and strontium also can be taken up more readily by mycorrhizal plants
(30,32,36,45). Their uptake, like accelerated water recovery (72,73),
may not only be due to direct hyphal translocation. For example,
sulphur uptake may not be as greatly dependent on an intact hyphal
network when sulphur is present near the root, indicating a general
increase in root permeability to this compound (68).

Since mycorrhizal plants generally grow and yield better than NM
plants the effect of chemicals on mycorrhizal fungi is an important
consideration when dealing with agricultural systems. Soil fumigants,
for example, have sometimes been detrimental to mycorrhizal fungi (11,
52,71). Nesheim and Linn (59) tested eight different soil fungitoxi-
cants (arasan, botran, captan, lanstan, mylone, terraclor, vapam, and
vorlex). Captan was the least and terraclor the most harmful to the
mycorrhizal formation. Kleinschmidt and Gerdemann found chemical

toxicity detrimental to VA mycorrhizae. Stunting of citrus seedlings

after fumigation in nurseries could be partially overcome by
fertilization. Inoculation of fumigated soils with VA mycorrhizal
fungi restored growth and reduced demand for fertilizer. The
workers suggested that mycorrhizal inoculum could be produced en
masse for agricultural purposes.

From 1966 to 1968, oxathiins, pyrimidines, and benzimidazoles
were introduced as the basis for systemic fungicides (22). In 1968
Delp (16) demonstrated that benomyl showed promise as a soil and
seed treatment in the greenhouse and as a foliar spray and seed
treatment in both the greenhouse and field against a wide variety of
diseases (16,18,19,33,80). Benomyl apparently acts on cell spindle
fibers (personal communication, Charles Delp, DuPont de Nemours and
Co.) as an antimetabolite (2) competing with purine-like compounds
which prevents mitotic division (35,36).

Benomyl is toxic to a wide spectrum of fungi and in many cases its
effectiveness appears to follow taxonomic lines (17). In 1971,
Edgington et_al, (17) did a comprehensive in_zitrg_study to identify
taxonomic/fungitoxicity relationships for the three benzimidazoles:
benomyl, thiabendazole and furidazol. He found a striking similarity
between the activity spectrum of the three compounds and concluded that
the benzimidazole moeity was responsible for these similarities. Five

phycomycetes had a ED 0 greater than 100 ppm, and were considered to be

5
insensitive. This supported the observation made by Delp (16) that
phycomycete ‘caused diseases were not controlled by benomyl.

Jalali and Domsch (46) showed that systemic compounds such as

benomyl and trifoline had ill effects on mycorrhizal development when

used as seed treatments or foliar sprays. Foliar sprays were thought
to induce a physiological incompatibility since systemic fungicides
are generally not translocated downward to any appreciable extent (22,
60,63,54). Inhibition from the seed treatments may be due to passive
translocation of the fungicide during root growth as well as a
physiological incompatibility.

In 1976, Sutton and Shepard (79) showed that benomyl and
thiabendazol were toxic to an unidentified Glomus species. Concen-
trations as low as 15 ppm of benomyl (dry soil) prevented mycorrhizal
development. Forty spores of the fungus served as inoculum in the
planting medium consisting of sand dune soil and silica sand (3:1), a
mixture virtually void of organic matter. The fact that benomyl is
toxic to a fungus in the class Zygomycetes (Phycomycetes) is not
consistent with previous views (17,16). However, in 1970,Bollen and
Fuchs (10) observed a great deal of variation in Mortierella sensitivity
to benomyl in_vit£g. This variability may have been an indication
that strict class adherence to benomyl resistance was on overgenerali-
zation, and that possibly the Morierellaceae-Endogonaceae line of
evolution (1) may represent an exception to the rule. In light of
Sutton and Shepard's report (79), it is of interest to see what effects
benomyl has on other soil microorganisms, including other VA mycorrhizal
fungi. Siegel (75) found that benomyl was metabolized by soil
microorganisms in nutrient-amended soil. In addition, he found a 2- to
3-fold reduction in the number of soil fungi and actinomycetes but not
bacteria. Berg and Bollen (8) also found quantitative decreases in the

mycoflora when 10 ppm benomyl was added to potting soil.

Benomyl stimulates the growth of some fungi both i§_vivo and
iigvitro. Robinson gt_al. (34,69) found that subinhibitive concentra-

trations increased the growth of Ustilago striiformis in vitro.

 

Smith §t_a1, (77) noted that a turf disease incited by an unidentified
Basidiomycete became more prevalent following benomyl treatments and
that the fungus grew on potato dextros agar ammended with 25 ppm

benomyl. Jackson (44) reported that benomyl enhanced Helminthosporium

 

leaf spot on Kentucky bluegrass. Peeple's work (62) showed no
alteration in microbial populations in field soil amended up to
89.6 kg benomyl/hectar. He also found only a small initial rise in
respiration rates of soil in the laboratory when 10 ppm benomyl was

added. Benomyl-sensitive fungi such as Penicillium, Asperigillus, and

 

 

Trichoderma were consistently isolated from benomyl treated soils.

 

‘ Ponchet and Tramier (66) found no change in the number of species
after benomyl application. In addition, Kaastra and Gams showed (48)
no qualitative or quantitative changes in soil microflora amended with

10 ppm benomyl. Among the fungi most commonly isolated from amended

 

soils were Trichoderma pseudokoningii, T. viride, Monilia pruinosa,

 

Penicillium janthinellum, Volutella ciliata, and Chrysosporium

 

 

meridarium which according to Bollen and Fuchs (10L are very sensitive

 

to benomyl. Kaastra and Gams indicated that the low level of metabolic
activity in the soil may have accounted for the lack of sensitivity in
their experiment. Weeds and Hedrick (84) found that an undefined
microorganism population in greenhouse potting soil had an increased
respiration rate when 100 ppm benomgl was added. However, they made

no attempt to qualitatively or quantitatively measure their population.

These works show benomyl to have a low level of toxicity to
soil microorganisms even those known to be highly sensitive in_vit£g.
Two factors probably explain why sensitive fungi can persist after
application of benomyl to soil: benomyl's fungistatic nature (10,48),
and its affinity to organic matter (5,42). Baude (4) found that the
majority of benomyl and its breakdown products, methyl-2-benzimidazole-
carbamate (MBC) and 2-aminobezimidazolecarbamate (AB), did not penetrate
three different soil types more than 10 cm after a soil drench
application. Consequently, there is an inverse relationship between
organic matter and uptake of benomyl by plants (43,73). Peat moss,
for example, added to sand, reduced uptake of benomyl by cotton plants
(21). Leach gt_al, (53) suggested that the inability of benomyl to
control Verticillium wilt of cotton in the field was due to lack of
contact with the compound. They mixed the same amount of benomyl into
100, 50, 25, 12, 6, or 3% of the soil before potting. After 119 days,
the greatest uptake was achieved from the most thorough mixing (100%),
and no uptake occurred in the mixture involving only 3% of the soil.
Benomyl movement in soil was increased by acidification, increasing
soil moisture, or adding a surfactant such as Tween 20 (61).

Benomyl in its MBC form is rapidly translocated apoplasticly (24,
65) from the roots to existing leaves. Thus, new leaves are unprotect-
ed because the fungicidal reservoir is quickly depleted (24). However,
benomyl itself tends to concentrate in the roots and is slowly released
to the leaves as it degrades to MBC (24). As the compound reaches the
leaves it moves with the transpiration stream and becomes concentrated

at leaf margins (65).

Much of benomyl's persistence in soil is due to its retention
and slow release from the soil. Erwin (20) showed that cotton plants
grown in 50% peat and 50% sandy loam were protected against
Verticillium wilt four months after treatment of 50 ppm. Rates of
100 and 500 ppm gave even longer protection.

Soil applications have proven to be much more successful in the
greenhouse than in the field (22), where uneconomically high rates are
needed. Even distribution in the soil is important so that a large
portion of the root system can come into contact with the fungicide.

The possibility that mycorrhizal plants might be more permeable
to benomyl could be valuable in field situations. The possible ill
effects of benomyl on mycorrhizal fungi would not necessarily negate an
increase in absorption rates. Pentachloronitrobenzene (PCNB), a
fungicide that has been used to render external hyphae non-functional
(31), does not decrease water uptake in mycorrhizal plants (72,73)
after short term applications. Since benomyl uptake by soybean plants
is proportional to water uptake (personal communication, Dr. L. V.
Edgington) indirect effects on general root permeability may allow for
increase absorption.

To date the only two works (48,79) which have dealt with the
effect of benomyl on endomycorrhizae have had sand as their planting
medium. In an agricultural situation, organic matter is present, at
least to some degree, and would be expected to reduce benomyl toxicity.
In addition, in neither study was benomyl used as a soil drench, which
is the usual way mycorrhizae come in contact with the compound. Thus

far, seed treatments, foliar sprays (46), and thorough mixing in the

soil (43) have been tested, the latter which would be found only in a
greenhouse situation. Both studies used unidentified species of a
VA mycorrhizal fungus and the numbers of chlamydospores were not
adjusted to levels commonly found in the field (39). Furthermore,
the influence of mycorrhizae on benomyl uptake by plants was not
tested. Finally, there have been no studies of the relationship
between mycorrhizal-stimulated growth and benomyl application (i.e.,
effect on the symbiosis). Sutton gt a1. (79) and Jalali gt E£' (46)
dealt with infection per se, but not with mycorrhizal growth responses.
Hayman and Mosse (40) found no correlation between plant growth
stimulation and rates of infection in some cases, therefore, if growth
data along with infection data are not collected, as was the case with
Sutton gt_§1, and Jalali gt_213, the effects of a given chemical on
mycorrhizal symbioses cannot be estimated.

This study sought to: l) evaluate the effects of benomyl on the
development of a functional VA mycorrhizal relationship involving

soybean and the mycorrhizal fungus Glomus fasciculatus and 2) evaluate

 

the relationship between VA mycorrhizal infection and the uptake of

benomyl by soybean plants.

MATERIALS AND METHODS

Preparation of Inocula

Glomus fasciculatus (29) was maintained on soybean, Glycine max

 

 

(L.) Merril, (cv. Hark) plants in a growth chamber and in the
greenhouse. The plants were grown to senescence then discarded and
the soil retained. The soil was combined, mixed and sampled for

chlamydospores of g, fasciculatus using a centrifugation technique.

 

One hundred cc samples of soil were placed in a 5.5 1 plastic bucket
filled 3/4 full with water and swirled by hand to suspend all spores.
The mixture was allowed to settle approximately 20 seconds to remove
larger soil particles and decanted through a 45’pm sieve. The fraction
retained on the sieve was transferred to centrifuge tubes containing
.35 ml water and centrifuged at 450 g for four minutes. The super-
natant fluid was decanted and the pellets resuspended in a sucrose
solution of 1.14 specific gravity. The resultant suspensions were
centrifuged again at 450 g for one minute, quickly decanted into a
45’Pm.sieve, washed with distilled water, rinsed into a petri dish

and the Chlamydospores counted at 30 X magnification. The pellets
remaining in the test tubes were resuspended in sucrose and reextracted

several times until all spores were recovered. The number of spores

10

per gram of soil was calculated. This "calibrated" soil was

stored at 00C and used as inocula for all experiments.

Planting and Inoculation

Soybean seeds were inoculated with a suspension of Rhizobium
japonicum and planted into 1 Kg autoclaved soil in one liter plastic
(Sweetheart) cups. Each cup had a drainage hole in the bottom covered
by a nylon screen. The soil contained 5% organic patter and had a
pH of 7.7. The soil had the following nutrient status: nitrates,

51 ppm; phosphorus, 8 ppm; potassium, 4 ppm; calcium, 143 ppm; and
magnesium, 29 ppm. Three seeds were planted in each cup.

Controls (NM) were prepared as above but the mycorrhizal inoculum
was autoclaved for 30 minutes at 120°C. To insure that organisms
other than the mycorrhizal fungus present in the chlamydospore'
inoculum were also present in the NM pots, new soil-chlamydospore
inoculum was passed three times through a 451nm sieve and the screened

liquid was added to each pot.

Benomyl Application

A stock solution of benomyl at 500 ppm (in water) was prepared.
Benomyl suspensions were added to each pot in enough additional water
to saturate the soil and give the appropriate soil concentration.
Benomyl concentrations in soil are reported as’pg/g soil.

Benlate is formulated as a 50% wettable powder. The active

ingredient, benomyl (methyl-1(butylcarbamoyl)-2-benzimidazole-carbamate),

11

hydrolyses to MBC in water (12) and in plant tissue when applied as a

root dip or soil drench (63,75).

Bioassay Procedures

In order to test for fungistatic activity in the roots and leaves

of treated plants, a bioassay technique was utilized. Penicillium

 

digitatum (Link) was used as the bioassay organism and maintained in
stock culture on 3% potato-dextrose-agar (PDA) at 2°C until needed.
To obtain spores, the plates were grown at ca. 22°C for approximately
one week. Spores were washed from the plates with distilled water
containing Tween 20. The spores were transferred to Klett
spectrophotometer tubes and adjusted to 50 Klett units using a 420 nm
filter.

One 9 samples of finely chopped leaf or root tissue were placed
into 30 ml test tubes, frozen overnight at 0°C, and extracted in 10 ml
of acetone. Extraction was done by placing the test tubes on a
reciprocal-shaker at 100 cycles per minute for 24 hours. Aliquots
(loo/pl) of the extract were spotted on bioassay disks (12.7 mm; No.
740-E Schleicher & Schuell) with a micro-syringe. The disks were held
by No. 325 insect pins which were placed through the middle of each
disk and stuck into a flat sheet of styrofoam. Once the acetone had
evaporated completely, each disk was placed in the petri dish (100 x
15 mm) containing 6 m1 of 3% water agar. The disks were subsequently
covered with 4 m1 of PDA containing conidia of P, digitatum (PDA-P).

This bilayer agar method is similar to that of Thornberry (81). The

12

PDA-P mixture consisted of one part spore suspension (as described
above) to 100 parts (1.5%) PDA. The spore suspension was added to
molten PDA held between 45-500C and stirred on a magnetic stirrer to
insure a uniform mixture. The petri plates were then stored at room
temperature and zones of inhibition were measured two to three days

later (see also Appendix B).

Infection Rating

This procedure is similar to that of Sutton (78). Root samples
were washed, blotted dry, weighed, and placed in a small glass petri
dish. The roots were cleared by heating in 10% KOH (wt/wt) in an
autoclave at 120°C with 15 pounds pressure for five minutes. The KOH
was decanted and the roots acidified using 0.01 N HCL. This was
decanted and a solution of acid fuchsin stain in lactophenol was
added and autoclaved as above. The roots were removed from the stain
and placed in fresh lactophenol without acid fuchsin stain and allowed
to destain overnight. Before observation, the roots were placed in
petri dishes which had been marked off into 1 mm increments, and
covered with fresh lactophenol. The roots were separated, aligned
perpendicularly to the markings, and 200 mm of the roots were examined
under 112X for any internal signs of the fungus (i.e., hyphae and
vesicles). Infection was reported as a percentage of root length

infected (see also Appendix A).

RESULTS

Fungal Tolerance to Benomyl

Preliminary testing showed that infections of soybean roots by

Glomus fasciculatus was highly tolerant. to benomyl. When benomyl was

 

applied to heavily infested soil (600 g, fasciculatus chlamydospores/

 

100 9 soil) to which soybean seeds had been sown, high levels of
infection resulted even when 250.0’pg/g soil was applied.

In another experiment, the effect of different benomyl concentra-
tions on mycorrhizal development was evaluated. Four concentrations
of benomyl were used (2.5, 25.0, 125.0 and 250.0’Pg/g soil) with five
to six M and NM plants per concentration. Four to six M and NM plants
not treated with benomyl served as controls. Benomyl was added directly
after the seeds and spores, and infection was rated 70 days later. The
percent root infection decreased with increasing concentrations of
benomyl applied as a soil drench up to 25.0/yg/g (Figure 1). At
25.0’yg/g and above no additional decreases below 39% infection were
found. Infection in mycorrhizal plants was significantly less than that
in non-treated mycorrhizal controls when 25.0, 125.0, and 250.0/pg/g
benomyl was applied. Non-mycorrhizal plants showed no infection at any
level of benomyl application. This experiment was repeated once with

13

14

100.0
J

80-0
1

 

 

 

 

(O
.—
D
O
M
cat?
3 e
8
:2: c3 liffl'
l-O ‘5.‘ ‘ZVTL, “4‘,
*_ “'
z
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32
Iu‘9
O. a“
2 ' . r :1
0.0 50-0 100.0 150-0 200.0 250.0

BENOMYL (UG/G SOIL)

Figure 1. Effect of various concentrations of benomyl applied to soil
on g, fasciculatus infection of sonean. Each point
represents the average infection rating of 4-6 plants after
70 days growth.

 

 

 

9v

15

essentially the same results. However the lowest level of infection
in this instance (between 32% and 50%) was reached at 2.5}yg/g benomyl
(Appendix D, Figure 14).

Using leaf area as a growth parameter of M and NM plants, the M
plants were significantly (P < 0.01) larger than the NM plants for
0, 2.5, and 25.0/Pg/g soil (Figure 2). These differences, however,
were smaller with increasing benomyl concentrations. At 125.0 and
250.0’Pg/g soil, total leaf area of M and NM plants were similar.
The growth of NM plants remained unchanged at all elvels of benomyl.
When this experiment was repeated similar results were obtained;
however, like the percent infection data, the reduction in M infection

and plant size occurred at lower concentrations (Appendix D, Figure 15).

Uptake of Benomyl by Soybean Plants

Since M plants have larger leaf areas than do NM plants the effect
of leaf area on benomyl uptake was tested. Six to seven M and NM plants
were grown for 50 days when all but the third, fourth, and fifth
trifoliate leaves were removed from half of the M and NM plants. All
plants were treated with 45’pg/g soil drench of benomyl. Two days
later each plant was bioassayed. There was no apparent relationship
between leaf area and amount of benomyl taken up (Figure 3). This
experiment was repeated with similar results. Hence, leaf area was not
considered as a major source of variability in subsequent experiments.

Preliminary testing indicated that M plants may have a greater

ability than NM plants to take up benomyl. To further investigate this

16

450 .0

400.0

350-0
‘
\.

LERF RREH (50 ON)
300.0

 

280-0
E:
\

 

 

020030

I 1' I’ 71’ ’1
.0 50.0 100.0 150.0 200.0 250.0
BENOMYL (UG/G SOIL)

Figure 2. Effect of various concentrations of benomyl applied to soil
on the leaf area of soybean plants with (mycorrhizal) or
without (non-mycorrhizal) a E. fasciculatus infection. Each
point represents the average leaf area (cm?) of 4-6 plants.

 

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:= 9
o .‘3‘ .=N III
C! EENH
(D
523 3_ at] at
S d
9.
§ 0
H .34 m
E; a:
m t "
2 o
S ‘0‘ . .
El
9
°-0.0 20.0 40.0 00.0 00.0 100.0

TOTRL LERF HREH 80 Cfl!10‘

Figure 3. Benomyl uptake with relation to leaf area. Leaf bioassays
were conducted two days after a soil drench of benomyl.

= NM, = M.

18

possibility benomyl was added as a soil drench (ZSIPg/g soil) to
plants which were 45 days old. Plants were bioassayed five days later.
Four M and NM plants were used per treatment, with one additional
untreated M and NM plant used as controls. The experiment was done
three times (Appendix D, Table 5).

Even though fungal colonization of the roots had taken place, no
mycorrhizal growth stimulation occurred (Appendix D, Table 6). Figure
4 shows the results of one of these experiments. No differences in
uptake could be demonstrated. All experiments had similar results
except in one experiment where the leaves of the NM plants contained
approximately 2X as much benomyl (Appendix D, Figures 22, 23). This
was not a repeatable phenomenon.

In order to determine if varying the period of exposure to benomyl
would allow detection of differing capabilities of M and NM plants to
take up the fungitoxicant, benomyl was applied as a drench (25/pg/g soil)
on four different dates. Benomyl was added on days 19, 26, 33, 40, and
assayed on day 45, resulting in 26, 19, 12, and 5 days exposure,
respectively. Five M and NM plants were drenched on each date. Four
M and NM plants were left untreated as controls. The experiment was
conducted two times. These two experiments showed similar concentra-
tions of benomyl in roots and leaves of M and NM plants at all exposure
times and it was concluded that under the conditions tested there were
no differences in the ability of M and NM plants to take up benomyl.
Figures 5 and 6 show the results of one of these experiments. The

results of the other experiment were similar (Appendix D, Figures 19, 20)

Figure 4.

BENOMYL_EOUIVHLENTS

0-0
I

 

M

NH

19

 

H

N"

 

" NH

 

 

25 00/0

 

ROOTS

 

25 UG/G
LERVES

If ‘r
0.0 UO/O
ROOTS 9ND

LEHVES

Benomyl distribution in roots vs. leaves of soybeans with

(M) or without (NM) a G, fasciculatus infection.

Each bar

 

represents the mean of five plants except the 0.0/pg/g bar
which represents one plant.

V
.0.10(-

 

050.50 PER 0 LEHF TISSUE

10-0
J

 

20

 

 

Ci} . _ . A A“ 4%
6: T I r T 1
030 5.0 10-0 16-0 20-0 25.0
OHYS EXPOSURE
Figure 5. Benomyl concentration in leaves vs. days exposure to a

25.0 9/9 soil drench. Each point represents the average
of five soybean plants either with (mycorrhizal) or with-
out (non-mycorrhizal) a g, fasciculatus infection.

 

.0 CI

‘0..-

.n-llv.|.

.o‘.

 

 

 

I. . . ‘- .4
.
. .. I W;
t . ‘ ..
. . uOu .
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-- .6. cl} 14 . n. o. L- .. '
.-
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-A
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s .
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,-
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21

 

 

9
"‘
u:
:3
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an
.—
.—
O
D
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c5
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m
a.
C!
m u
z
m
a:
O 4
c3 -- I I 1 r l
0.0 5.0 10-0 18 -0 20-0 25.0
DAYS EXPOSURE
Figure 6. Benomyl concentration in roots vs. days exposure to a

25.0 9/9 soil drench. Each point represents the average
of f ve soybean plants either with (mycorrhizal) or with-
out (non-mycorrhizal) a‘gf fasciculatus infection.

 

\ V ‘ - ’ “Q. \ 0. 0 . U... f n. , I r V . 0‘ .0 .6- .. 0..
..........:..i 4:...1 L .H. 2. L...
O Iv as... .l.... . V L. W pal. A. t. 6. ml. ‘ . at! n - t‘ u a ‘. h.-
u 1.. a. 3 a. .4. .3 .n g. V. a...
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.. r L a 0.1. t.
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. 7..-

(-¥-~t'0"d.w'fi“ .I“ ‘5‘ O’— ‘3‘“... U
Q

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.Iu.

22

There was an interesting trend for the plants to form less
foliage with increasing exposure periods to benomyl (Figure 7). This
was much more pronounced for the M plants than the NM plants and
would appear to support the results in the tolerance studies (Figure 2)
which also showed that M plants developed more poorly in the presence

of benomyl then when no benomyl was present.

23

04-0 101-0 100-0 118-0
”6
‘ a
‘9

LERF HREH (PRCT OF CONTROLS)
07-0
1

 

 

0.0 510 10.0 10.0 20.0 20.0
DRYS EXPOSURE

Figure 7. Leaf area as a percentage of non-treated controls vs.
exposure time to 25.0 g/g benomyl. Each point represents
the average of five p ants.

 

 

‘4.

-. _
I d
.-
-
v
.
~ n
o
\-
.

 

DISCUSSION

This paper showed that mycorrhizal spores germinated and infected
soybean plants when up to 250/pg/g soil benomyl was applied. The
infection process was hindered however, as evidenced by the decrease
in mycorrhizal infection after benomyl application. The reduction
in infection could be due to an overall reduced germination of the
chlamydospores and/or hyphal growth in the presence of benomyl or
to an effect on infection per se. Even if 250.0’Pg/g of benomyl was
initially too high for infection to occur dilution via watering, binding
to organic matter, degradation, etc., could have reduced the effective
dose to a level allowing germination and infection. No significant
change in growth occurred when benomyl was applied to NM plants, thus,
phytotoxicity or the reduction of an unnoticed disease cannot account
for these differences. Therefore, the increases in growth were
attributable to the mycorrhizal associations and the reductions in
growth were attributed to the ill effects of benomyl on the host fungus
relationship.

The fact that 50% infection did not increase plant growth may
preclude "percentage-type" measures of infection as valid indices of
symbiotic efficiency. Future studies on soil-plant ecology must

24

25

include both infection rating and growth response measurements.

Total transpiration usually increases with increasing leaf area,
which was expected to result in more benomyl uptake. However, the
larger leaf area also provides additional tissue to be supplied from
a finite pool of benomyl. Under the conditions used in this work
there was no discernible relationship between leaf area or root
wieght with concentration of the compound in the leaves. Therefore,
any true differences in sizes of zones of inhibition in the experiments
would probably have been a result of increased efficiency of the
root systems to take up benomyl. Since there were no consistent
differences in uptake of benomyl by M and NM plants in any of the
experiments, mycorrhizal infection apparently can increase growth
without increasing the uptake of benomyl.

An interesting trend was seen in growth of M plants when exposed
to benomyl for various lengths of time. With increasing exposure,
the M plants showed decreasing rates of leaf area formation. Here,
the mycorrhizal system may no longer function normally resulting in
slower growth rates.

In Sutton's and Sheppard's work (79) sand was used as the
planting medium, which would not have retained benomyl to any great
extent. Unless the chlamydospores took up the fungitoxicant there
should have been an extremely low concentration of benomyl 73 days
after a 15/ug/g soil drench. Nevertheless, after this period of time,
there was no infection of the plants in their experiments. Several

genera of fungi which are known to be sensitive to benomyl (17,10)

26

were found abundantly in their treated soil. These were species of

Cladosporium, Fusarium, Penicillium, Colletotrichum, and Humicola.

 

 

 

Evidently, the benomyl had fungistatic effects on these organisms in
that they were able to grow once removed from the soil and placed on
artificial media. It would have been interesting had they removed the
Glomus chlamydospores and applied them to untreated plants to test for
viability. Sutton's and Sheppard's results, along with those of this
paper, indicate that there may be a wide difference in susceptibility
of Glomus species to benomyl. Possibly a less sensitive fungus was
used in this work than in theirs. Comparisons of these two works are
complicated by differences in soil type, method of benomyl application
and the mycorrhiza-forming fungus used.

The results of this work indicate that mycorrhizal plants do not
have an increased ability to take up benomyl, at least under the condi-
tions tested. However, extensive colonization of the root cortex by

VA fungal hyphae did not hinder benomyl uptake. In addition it was

 

shown that G: fasciculatus can withstand a 250/yg/g soil drench of
benomyl, a concentration much higher than normally used, and still
infect soybean roots. A most important finding is that a seemingly
healthy mycorrhiza loses its ability to increase plant growth when

benomyl is added to the soil.

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thology 60: 1373-1375.

Thornberry, H. A. 1950. A paper-disk plate method for the
quantitative evaluation of fungicides and bacteriocides.
Phytopathology 40: 419-429.

Tinker, P. B. H. 1975. Effects of vesicular-arbuscular
mycorrhizas on higher plants. pp. 325-349. In: Symposia of
the Society for Experimental Biology.

Walker, L. B. 1923. Some observations on the development of
Endogone malleola Hark. Mycologia 15: 245-257.

 

Weeks, R. E. and H. G. Hedrick. 1975. Influence of a systemic
fungicide on oxygen uptake by soil microorganisms. Soil Sci.
119: 280-284.

APPENDICES

APPENDIX A

Mycorrhizal Infection Rating Systems

 

Growth Observation Method

 

The entire ball of dirt containing the roots was carefully
removed from the pots and the free soil was removed by submerging
in water and gently massaging. The M roots remained black with
organic matter in areas where M hyphae were dense. A rating system
of 0 to 3 was used to designate the amount of organic matter clumping.
A 1 represented the amount of organic matter normally adhering to NM
plants with up to 25% of the root system being covered, 2 was from
25 to 75% and a 3 from 75 to 100% of the root system covered by dark

clumps.

ClumpiMethod

 

A clump was defined as an area of root which had organic matter
attached, yet was able to have limited movement in running water.
These areas were found through microscopic observation to be approxi-
mately 98% due to mycorrhizal hyphae.

Such areas were not always distinct. When a continuous length of

root had clumping for more than 0.5 cm, each 0.5 cm of length was

34

35

counted as one clump.

Roots were washed as above and three samples of the root system
were removed. The samples were taken from the top left, lower left,
and right center of the root system. The samples were then placed
in a large petri dish (150 mm diameter) which was divided into 2 cm
square sections. A small amount of water was placed in the dish and
the roots were distributed as evenly as possible. Once distributed,
a piece of paper with ten dots was slid under the dish. Every
square which corresponded with a dot was rated for clumps on a
lighted slide viewing tray at 10 X magnification. Ten squares were
counted and the total clumps for the whole sample were calculated.
The sample was then patted dry, weighed, and the infection was reported

as clumps per gram of root.

APPENDIX B

Bioassay Procedures

 

Leaf Plug Method

 

To sample plant tissue a paper punch (6.0 mm diameter) was used
to remove a circle of tissue from the second trifoliate leaf of each
plant. The plugs were removed from the center leaflet with the
right margin forming a tangent to the punched circle. The plugs of
tissue were frozen on dry ice for 60 seconds, and placed in petri
plates containing approximately 8 ml of (1.5%) PDA. The plates were
sprayed with equal amounts of a spore suspension as used in the
acetone extract method (see methods). Zones of inhibition were
measured after three days.

The effect of varying the location of the sample plug and the
effect of freezing leaf plugs were tested. To accomplish this,
benomyl was added as a soil drench to four soybean plants thirty days
after planting. One plant was left untreated to serve as a control.
Two days later, four plugs were taken from the middle leaflet of the

second trifoliate leaf of each plant as illustrated in Figure 8.

36

37

 

Figure 8. Illustration of middle leaflet of the
second trifoliate leaf. A, A', B, C
mark the locations for the plugs taken
two days after drenching with benomyl.

One of the "A" plugs was frozen by holding it to a block of dry
ice for 60 seconds. The rest of the plugs were left unfrozen. All
plugs were bioassayed as above. This experiment was done twice
(Table 1).

No statistical differences in zone of inhibition were found (t
test P 1<.05) between the frozen and non-frozen leaf plugs taken

from various locations (Table 1). Leaf tissue alone had no fungitoxic

effect on the’P, digitatum spores.

Comparison of the Leaf Plug and Acetone Disk Methods

To determine if the uptake of one leaflet was proportional to
total plant uptake, the leaf plug and acetone extraction methods were
compared for different benomyl concentrations and times of application.
Benomyl at 0.2, 2.0, 5.0, 10.0, and 25.0/pg/g soil was applied 24 hours
before and two to four weeks after seeding. This gave exposure times

of 35, 21, and 7 days respectively, before the assay.

38

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0.0 0.0 ,o.o _ 0.0 00.0 Haeccmm oz

o.m H.0H H.m o.m oo.HH assocmm m a H

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mmwuo>¢ cououmlsoz namuoum uswswnwmxm

 

amoeufinflnsH mo GGON

 

.GOHUAQASCA mo moon mo comm so mcfiwomum cam sowumooH msHm mama mo uowmmm .H magma

39

Both methods showed maximum recoverable benomyl at seven days
after treatment. When exposure periods were longer than seven days I
recovery was greatly reduced with no recoverable benomyl at 35 days
(Figure 9 and 10). The leaf plug method was able to detect benomyl
applied at 2/P9/9 soil whereas the acetone method could detect benomyl
at Slug/g soil. The acetone disk method better differentiated higher
concentrations of benomyl as reflected by the disproportionally larger
increases in zone areas over the leaf plug method. The variance re-
mained small enough with the acetone disk method to show statistical
differences between the zones at 14 days exposure whereas this does
not hold true for the leaf plug method at the same exposure time.

When qualitative indications of fungitoxicity in the leaves was
sought, the leaf plug biomass method was utilized due to its ease and
quick results. However, when quantitative measurements were needed,
the acetone extract method was used. As was seen in the experiment
comparing the two methods (Figures 9 and 10), the leaf plugs gave zones
of inhibition at low concentrations of benomyl. As soil drench
concentrations increased, the leaf plug method rapidly lost resolution.
The acetone method represented a larger amount of tissue and consequently
produced larger zones at the same tissue/fungitoxicant ratios. This
resulted in a more definitive system for measuring total protection of
the leaves.

Other sorts of inaccuracy may be encountered using the leaf plug
method. It has been shown (65) that the movement of benomyl within a

leaf follows the transpiration stream. This results in a clearing of the

zone 0F INHIBIT10N so cn

1-0

-0

40

5-0

I

 

{D
.0 \

 

4

0.2 PP"

0.0 7.0 11.0 721.0 20.0 30.0
DHYS EXPOSURE

 

Figure 9. Zone of inhibition (cm?) about leaf plugs with relation

to exposure time and benomyl concentration. Each point
represents the average of five plants.

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41

 

2.0aEND 0.2 PPM

I I
0.0 7.0 14.0 21 .o 20.0 35.0
DAYS EXPOSURE

Zone of inhibition (cmz) about acetone disks with relation
to exposure times and benomyl concentrations. Each point
represents the average of three plants.

 

42

base and mid-vein and concentrating along the margins of the leaf.
There are differences in the amount of transpiration with different
ages of leaves, thus different rates of benomyl accumulation would be
expected according to the age of the leaf. Finally, the form of the
fungitoxicant (i.e., benomyl or MBC) alters uptake characteristics
(51, 25, 63, 24). Benomyl is taken up into the roots and slowly
released as MBC which travels to the leaf tissues, which are
transpiring (24, 63). This process is gradual and results in
distribution to new leaves which were not present at the time of
application as well as to leaves already present. MBC, however, is
rapidly translocated to existing leaves, depleting the source thusly
rendering leaves formed later unprotected. The ability of the
acetone method to reflect the fungitoxic status of the entire leaf
area rather than just a minute portion of one leaf (i.e., leaf plug)
is desirable in any study equating inhibition with theoretical disease
resistance.

The acetone method has a major advantage for analytical work, its
ease of comparison to a standard curve. During zone of inhibition
formation, benomyl cu: MBC diffuses from a leaf plug through a maze
of cell wall material. To compare the zones of inhibition of this
system to those of a compound diffusing from filter paper disks is at
best an extremely crude comparison. It isluunnithat not all of the
fungitoxicant in plant tissue is removed by acetone. However, the
ratios of extractable to non-extractable compound can be determined

(65), and a correction factor can be applied. An additional advantage of

43

acetone extraction is its versatility in that any part of a plant can
be assayed. This eliminates variation due to differences in thickness
of the parts to be assayed. Moreover, small concentrations may be

analyzed with simple concentration procedures.

 

APPENDIX C

Preliminary Testing of Benomyl Uptake

 

Variable Time

 

Twenty five’Pg/g soil benomyl was added to each pot at various
times to give 21, 14, 7, and 3 days exposure. Two days later, plants
were analyzed using the acetone disk method and infection was measured
by the visual estimation method (Table 2).

No consistent growth stimulation occurred in the M plants. There
did, however, appear to be a difference in uptake patterns (Figures 11 and
12L M plants tended to accumulate more benomyl in leaves per unit time
than did NM plants. The uptake continued to increase until 14 through
21 days after treatment. At 21 days, the M plant leaves had accumulated
2.5 times more of the fungitoxic compound than the NM plants. The root
bioassay results were quite different (Figure 12), in that NM roots
appeared to retain the compound at a higher level for a longer period of
time than did the M plants. The concentrations found in the roots were

quite low when compared to the leaves.

44

45

Table 2. Benomyl distribution within plants vs. time after soil

 

 

 

 

 

 

treatment.
Days Exposurea Leaf Areab Root Weight
to ,
Benomyl Type of Plant (cmz) (g)
M 184.7 3.6
21
NM 248.0 4.8
M 267.0 6 2
14
NM 189.3 5.0
M 171.3 4.5
7
NM 206.0 5.2
M 206.5 4.4
3
NM 244.3 5.6
M 244.8 4.6
0
NM 287.5 7.0
M 219.1 4.7
Average
NM 238.3 5 6

 

aTime after a 25.0/pg/g soil drench.

bAverage of three M and NM plants.

 

46

 

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(C) GD
(I)
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x6
2 o
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C3.
6 T r I I 1
0.0 6.0 10.0 15.0 20.0 28.0
DHYS EXPOSURE
Figure 11. Benomyl concentration in leaves vs. days exposure to a

25.0 g/g soil drench. Each point represents the average
of five soybean plants either with (mycorrhizal) or without
(non-mycorrhizal) a g, fasciculatus infection.

 

 

47

8.0

 

BENOMYL EQUIVHLENTS

 

 

_._—#-A
-

0.0 5:0 15.0 15.0 20.0 25.0
DRYS EXPOSURE

 

Figure 12. Benomyl concentration in roots vs. days exposure to a
25.0 g/g soil drench. Each point represents the average
of five soybean plants either with (mycorrhizal) or without
(non-mycorrhizal) a g. fasciculatus infection.

 

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48

Multiple Concentration

 

Tb compare the ability of mycorrhizal and non-mycorrhizal plants
to take up benomyl over a wide range of concentrations, six M and NM
plants were grown for 44 days before benomyl was added. Benomyl was
applied to individual plants at 8.5, 17.0, 34.3, 68.5, and 137.0’pg/g {1

soil. One concentration was used per plant and the experiment was 9

:14 ~4-

conducted twice. One M and NM plant served as controls and were

drenched with an equal amount of distilled water. Plants were

 

assayed two days later using the leaf plug method.

I! I‘IJ

The M shoots were found to be significantly larger (P .10) than
NM shoots in both tests. Root growth of M and NM plants were not
significantly different. In the bioassays of the first experiment
the M plants showed a greater amount of uptake at all concentrations.
In the second experiment, the NM plants showed either the same or a

greater amount of uptake than did the M plants (Figure 13).

Single Concentration

 

To test the abilities of M and NM plants to take up and trans-
locate benomyl from soil five to ten M and NM plants were grown for
30 to 40 days. Soil was treated with 45/pg/g soil benomyl leaving one
to two plants of each type untreated to serve as controls. Two to three
days after benomyl treatment the plants were bioassayed using the leaf
plug method. This experiment was done twice.

In these preliminary experiments no growth differences occurred
in any of the treatments (Table 3). M plants took up more benomyl than

NM plants in every test, however, the differences were not significant.

49

25-0
J

 

q

ZONE OF INHIBITION (80 CM)
5.0

 

 

0.0

I I 15 ’T' l
0.0 30.0 00.0 90.0 120.0 150.0
BENOMYL (UG/G SOIL)

Figure 13. Distribution of benomyl in leaves two days after
application. Each point represents the average of
two experiments at one plant/experiment/concentration.

 

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APPENDIX D

Supplemental Data From Results

 

51

 

52

100.0

 

00.0

PERCENT INFECTED ROOTS

1

=2 b @N-NYCORRH I 20%
CD

0.0 50.0 100.0 150.0 200.0 250.0
BENOMYL (UG/G SOIL)

 

 

Figure 14. Effect of various concentrations of benomyl applied to
soil on g, fasciculatus infection of soybean. Each point
represents the average infection of four to six plants.

 

0.,

 

n:l.y.

a o~~4<

000.0 050.0

750.0

 

LEHF HREH (30 CH)

050.0

700.0

53

 

 

 

000.0

0.

Figure 15.

0

60.0 100.0 100.0 200.0 200.0
BENOMYL (UG/G SOIL)

Effect of various concentrations of benomyl applied to
soil on the leaf area of soybean plants with (mycorrhizal)
or without (non-mycorrhizal) a g. fasciculatus infection.
Each point represents the average leaf area (cm2) of four
to six plants.

 

54

00.0
14

20.0 30.0

BEN E0 PER G LERF TISSUE
10.0

 

 

 

l r 1 1 I *1
0.0 8.0 12.0 13.0 7.4-0 30-0
DHYS EXPOSURE

Figure 16. Benomyl concentration in leaves vs. days exposure to a
25.0 g/g soil drench. Each point represents the average
of five soybean plants either with (mycorrhizal) or
without (non-mycorrhizal) a E, fasciculatus infection.

 

‘L

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BEN E0 PER G ROOT TISSUE
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Figure 17.

55

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r 1’ If PET
.0 0.0 12-0 10-0 24.0 30-0

DHYS EXPOSURE

Benomyl concentration in roots vs. days exposure to a
25.0 g/g soil drench. Each point represents the average
of five soybean plants either with (mycorrhizal) or
without (non-mycorrhizal) a E, fasciculatus infection.

 

56

 

100-0

 

 

70-0

LERF HREH IPRCT OF CONTROLS)
00-0
Li

 

 

'7 1 I 1’ I
5,00? I500 ‘1’00’ 06h1’ Ell-0' 2&500
DRYS EXPOSURE

Figure 18. Leaf area as a percentage of non-treated controls vs.
exposure time to 25.0 g/g benomyl. Each point
represents the averag of five plants.

 

57

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BENONYL EQUIVHLENTS

Figure 19.

59

25-0
.iJ

J

15-0
l

 

 

NN

 

 

:1 NH :1

 

fl NH

 

25 UG/G 25 00/0

ROOTS LERVES

I
0.0 00/0

ROOTS’RND
LERVES

Benomyl distribution in roots vs. leaves of soybeans with
(M) or without (NM) a g. fasciculatus infection. Each bar

 

represents the mean of five plants except the 0.0/pg/g bar
which represents one plant.

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BENOMYL EQUIVHLENTS

 

6O

 

 

 

 

 

 

c:

g.

CD

3..

‘2

23..

C3

9:.

9

“D-
—-T

9 11 NM 0 m1 ." m1 .

° 26 00/0 25 00/0 0.0 00/0
ROOTS LEHVES ROOTS RNO-

LERVES
Benomyl distribution in roots vs. leaves of soybeans with

(M) or without (NM) a g, fasciculatus infection. Each bar
represents the mean of five plants except the 0.0/pg/g bar
which represents one plant.

 

31.!

 

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