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This is to certify that the

thesis entitled

The Effect of Some Soil Factors on the Infection and
Severity of Fomes Annosus (Kr.) Karst. Root Rot on
the Seedlings of Loblolly Pine (Pinus Taeda).

 

presented by

Ezekiel Anwuchaepe Uche Umeokafor

has been accepted towards fulfillment
of the requirements for

M.S. Forestry

degree in

 

 

QM; 9‘ @144

Ma' r professor

Date October 16, 1978

 

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@ I979

EZEKIEL ANWUCHAEPE UCHE UMEOKAFOR

ALL RIGHTS RESERVED

 

THE EFFECT OF SOME SOIL FACTORS ON EHE INFECTION
AND SEVERITY OF FOMES ANNOSUS (xr.) Karat.
ROOT ROT ON THE SEEDLINGS OF
LOBLOLLY PINE, PINUS TAEDA

By

Ezekiel Anwuchaepe Uche Umeokafor

A THESIS

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

[ASTER OF SCIENCE

Department of Forestry

1978

 

ABSTRACT
THE EFFECT OF SOME SOIL FACTORS ON THE INFECTION
AND SEVERITY OF FOMES ANNOSUS (Kr.) Karat.

ROOT ROT ON THE SEEDLINGS OF
LOBLOLLY PINE, PINUS TAEDA

By
Ezekiel Anwuchaepe Uche Umeokafor

Egggg annosus is a nonhost-specific, soil-borne
pathogen. The pathogen is more severe in thinned forest
plantations, on some soil types and in forest plantations
established on old agricultural land than in natural un-
thinned stands. This study investigated the effect of
soil texture, soil reaction and soil micro-organisms on
the infection of loblolly pine seedlings by E. annosus.
The tests were made on sandy and clayey soils adjusted to
pH 4.8 and 6.5. Three isolates of Trichoderma viride and
four species of Trichoderma were screened for their antag-
onistic effect on E. annosus. Seeds of loblolly pine were
aseptically planted in autoclaved soil, unautoclaved soil
and soil autoclaved but later contaminated with g. viride.
g. viride isolate myco 10088 was the most antagonistic of
the Trichoderma species and isolates screened. There was
a significantly higher (P . 0.05) infection rate in the
sterilized soil than in the other types of soil. 2. virtue

Ezekiel Anwuchaepe Uche Umeokafor

isolate myco 10088 provided protection to loblolly pine
seedlings against E. annosus throughout the duration of
the study. In an additional study, soil samples collected
in the Kellogg Forest from ten infected and uninfected
spots 15 feet away were analyzed but showed no difference

in characteristics.

To my mother, Mrs. Mgbugo Umeokafor,
who did not go to college but knew
the value of education and instilled in me

the need for and value of an education.

ii

ACKNOWLEDGMENTS

I am deeply indebted to Dr. Victor Rudolph for his
counsel and guidance and for the interest he has taken in
my work throughout my master's program.

I would also like to express my thanks to the fol-
lowing who served on my committee at different times: Dr.
John Hart and Dr. John Lockwood, who substituted for him
on many occasions and offered useful advice; Dr. Michael
Walterscheidt, who initiated the study but left for Texas
A and M University before the completion of the study; and
Dr..James B. Hart, who replaced him on the committee.

Further thanks are due to Dr. Charles Cress for
suggestions on statistical matters and to Dr. Raymond J.
Kunze and Dr. Boyd G. Ellis of the CrOp and Soil Science
Department for their consultancy.

Finally, I wish to acknowledge my wife Ngozi, for
her help in the preparation of this manuscript and for her
continual encouragement throughout this study, and my two
sons Obi and Ogo, who were denied paternal care during the

preparation of this thesis.

iii

TABLE OF CONTENTS

 

Page
CHAPTER I . . . . . . . . . . . . . . . . . . . . 1
INTRODUCTION . . . . . . . . . . . . . . . . 1
CHAPTER 2 . . . . . . . . . . . . . . . . . . . . 4
LITERATURE REVIEW . . . . . . . . . . . . . . 4
Soil Texture . . . . . . . . . . . . . . 4
Soil pH . . . . . . . . . . . . . . . . 5
Soil Microorganisms . . . . . . . . . . 7
Other Soil Factors . . . . . . . . . . . 9
Mode of Entry . . . . . . . . . . . . . 9
CHAPTER 3 . . . . . . . . . . . . . . . . . . . . 11
MATERIALS AND METHODS . . . . . . . . . . . . ll
Collection of Soil Samples . . . . . . . 11
Mechanical Analysis . . . . . . . . . . 11
Soil pH . . . . . . . . . . . . . . . . l2

Isolation of Egmgg annosus from Infected
Roots . . . . . . . . . . . . . . . . 14
Inoculation . . . . . . . . . . . . . . I 14
Identification of the Fungus in Culture. 18

Screening Trichoderma Species for Antag-
onistic Effécts on F. annosus . . . . 19
Preliminary Studies . . . . . . . . . . l9
EXperimental Procedure . . . . . . . . . 22

iv

Field Study . . . . . . . . . . . .
Statistical Methods . . . . . . . .

CHAPTER.4 . . . . . . . . . . . . . . .1. . .
RESULTS AND DISCUSSION . . . . . . . . .

Screening Trichoderma Species for Antag-

onistic Effgcts on F. annosus . .

Soil Sterility and Microorganisms .

Soil pH . . . . . . . . . . . . . .

Soil Texture . . . . . . . . . . . .

Field Study . . . . . . . . . . . .

CHAPTER 5 . . . . . . . . . . . . . . . . . .

SUMMARY AND CONCLUSIONS . . . . . . . . .
LITERATURE CITED .

Page
23
24
25
25

25
26
29
3O
51
53

33
36

LIST OF TABLES

Table ‘ Page

1. Growth Rate of Fomes annosus and Tricho—
derma Species and Isolates in mm of
Radius for 4 Days in 2.5% MEA of pH 6.5 . . 2O

2. Number of Seedlings Infected and Not In-
fected by Fomes annosus . . . . . . . . . . 26

5. The Severity of the Infection Based on
the Sum of All Rankings for Each Treat-
ment 0 O O O O O O O O O O O O O O O O 0 O 27

4. Mean Severity Rating of Infection for
Media, Soil Texture and Soil Reaction . . . 28

5. Soil Data from Diseased and Adjacent
Healthy Spots in the Kellogg Forest . . . . 52

vi

LIST OF FIGURES

Figure Page

1. Amount of HCl acid required to adjust
50 gms. of soil to varying pH levels. . . l5

2. Fomes annosus infected spot, from where
the Inocqum was obtained . . . . . . . . 15

5. Isolation of F. annosus from infected
WOOd O O O O O O O O O O O O O O O O O O 16

4. Oedocephalum lineatum a conidial stage
of E. annosus on 2.0% MEA . . . . . . . . l7

5. Growth of F. annosus and Trichoderma
species after 21 days . . . . . . . . . . 21

vii

CHAPTER I
INTRODUCTION

Egmgg annosus (kr.) is a soil-borne pathogen that
causes root and butt rot diseases in many trees. Sinclair
(1964) reported that 80 species of conifers and 55 species
of hardwoods are susceptible to Fgmgs annosus. Above
ground symptoms of the disease seldom appear until the
tree is virtually dead. Boyce (1961) listed some of the
symptoms when apparent, as root rot, red rot, spongy rot,
brown rot and butt rot. The pathogen attacks trees of all
ages (Miller 1945), although contrary Opinion (Boyce 1962)
holds that it attacks only older trees and pole-sized
trees. This disease causes considerable loss in Europe
and North America.

2. annosus was reported first in the United States
in 1925 by Hedgcock in North Carolina. Killing of slash
pine (Pinus elliottii var elliottii) by Egmgg annosus was
first recorded in the Southeast in 1954 by Campbell and
Hepting. Power, 23 Q; (1961), on their survey of planted
and natural stands in three southeastern states for signs
of F. annosus, have noted that the fungus was killing
slash pines in 73% of the plantations examined. Berry and

Dooling (1962) later surveyed the southern parts of

l

Missouri and found that this pathogen was present in
shortleaf pins in many state and federal thinned pine plan-
tations but not in natural unthinned stands.

Soil microorganisms are very complex. The funda-
mental knowledge of the factors contributing to this com-
plexity is often the key to eventual development of soil
borne disease control measures. Froelich, Dell and Walkin-
shaw (1966) considered soil pH, organic matter, and soil
texture as variables most likely to influence either host
resistance, root infecting fungi or soil inhabiting micro-
organisms that may compete with the pathogen. Other soil
factors believed to play a role in.§. annosus root rot de-
velopment include soil moisture stress (Tower and Stam-
baugh 1968), and organic matter content (Froelich gt 3;
1966; Rishbeth 1961). Similarly, Hook and Eno (1961),
Peace (1956) and Powers and Boyce (1961) reported that the
disease was more pronounced on former agricultural crop-
land than on forest land.

Knowledge of the ecology and biology of z. annosus
as well as the characteristics of its host plants is neces-
sary to deve10p effective control measures. It may well be
that wide-spread fungistatic factors exist in the soil
which inhibit the activities of F. annosus. It is likely
that these fungistatic factors plus other numerous soil

factors may obliterate the effect of F. annosus rather than

destroy it in the soil. The removal of these inhibiting
factors may allow the host species to be susceptible.

Similarly, microorganisms in the rhizosphere may exert a
profound influence on the host plant by associative and
antagonistic relationship, or actual parasitism.

The objectives of this study were to determine:

1. The effect of soil texture, soil micro-
organisms and soil pH on the incidence
of F. annosus root rot on loblolly pine
(Pinus taeda) seedlings.

2. The extent to which an isolated soil
microorganism, Trichoderma viride, could
provide protection to loblolly pine
seedlings from Fgmgs annosus root rot.

It is h0ped that the results of this investigation
will be useful in deveIOping risk rating and selecting
sites for growing loblolly pine seedlings without mortality

due to F. annosus.

CHAPTER 2
LITERATURE REVIEW

Soil Texture

Soil-plant root relationships that lead to root and
butt rot disease development and its severity are influ-
enced among other things by the soil texture, the hydrogen—
ion concentration (pH), the organic matter content, temp-
erature, aeration, moisture and microorganisms. Froelich,
gt g; (1966) considered the soil pH, organic matter con-
tent and texture as soil variables most likely to influ-
ence host resistance. They characterized severely damaged
plots as having extremely deep sand and well drained soils
(high hazard sites), while they generalized that heavy
clay soils with a high water table are low hazard sites.

Alexander and Skelly (1975) studied disease inci-
dence and severity in loblolly pine planted on two soil
hazard types. They found that the sand content and large
pores were directly related to infection. Small pores,
clay and organic matter content were inversely related to
infection. Morris and Frazier (1966) defined a high hazard
soil as being well-drained and having a sandy top soil 10
inches deep or more. Kuhlman gt g; (1976) associated

4

disease severity with deep well-drained soils. These
findings are in agreement with earlier work by Anderson
(1921), who noted that the texture of the soil appears to
influence greatly the mode of Fgmgg attack.

However, Jorgensen, Lund, and Treschow (1959) did
not establish any definite correlation between the inci-
dence of heart rot and soil type although they stated that
the disease tends to assume a more virulent character on

sandy than on clayey soil.

Soil pH

The influence of pH on the incidence and growth of
Egmgs annosus depends on the nature of the substrate
(Treshow 1941). There are therefore varying pH ranges
under which Egggg annosus thrives well. Treshow also re-
ported no variation in the growth of the fungus within a
range of pH 5 to 7 on filter paper in 5% malt extract. He
obtained profuse growth on a sterilized acid spruce litter
(pH 4.5) and on beech mold pH 6.5, but the fungus was
unable to develOp on pure mineral soil. When the pH of
sterile forest humus was varied by the admixture of lime in
amounts ranging from 2 to 20 gm. per 50 gm. of soil, no
difference in the growth of the fungus was detected.
Rennerfelt (1955) in his physiological and ecological ex-
periment with Polyporus annosus (E. annosus) found that the
growth of the fungus takes place between pH 2.9 and 7.0,
and between 2° C and 52° C.

6

Weis and Nielsen (1928) investigated the growth of
the root destroying fungus Pglypgggg radiciperda (g,
annosus) on malt extract culture of pH value from 4.0 to
4.5, and found growth rate to be modified by temperature.
Tilford (1956) found that the Optimum pH value for E.
annosus on malt agar at 20° to 40° C were 4.91 and 5.52,
respectively.

The pH of a site in Britain on which E. annosus
had caused considerable damage was found to be different
from that of adjacent sites by Anderson (1921). He con-
cluded that soil acidity is the dominant predisposing
cause of attack. Hopffgarten (1955) found that the di-
sease occurs on sites in Denmark with pH ranging from 4.6
to 6.0. This compares with pH ranges of 5.7 to 6.7 in
California (Wagner and Cave 1946). Etheridge (1957) stu-
died the relationship between site and decay in subalpine
spruce in Alberta and noted that a pH less than 5.95 had a
positive influence on the incidence of root and butt rot
disease. Grainger (1946) reported that F. annosus prefers
to grow on wood that remains fairly acidic at a pH of 5.0.

On the other hand, Rishbeth (1950) and Wallis (1961)
demonstrated that higher infection rates occur on alkaline
soils than on acid soils. According to them, alkaline soil
has limited fungi colonies and this gives E. annosus an

advantage over other competing microorganisms.

Soil Microorganisms

Soil microorganisms play an important role in the
infection and severity of root-borne diseases. They may
promote the activity of soil-borne pathOgens (Rishbeth,
1951a and 1951b)° Inoculation of plants with Egmgg
annosus in non-sterile soil has yielded poor results
(Rishbeth, 1951b, Treschow, 1941 and Wallis 1961).

Sago and Cabb (1975) studied the influence of soil
biological factors on the mycelial growth of F. annosus
and found that it was dramatically inhibited by antagonis-
tic soil microorganisms. Treschow (1941) reported the
failure of Trametes radiciperda (E. annosus) to grow on
acid (pH 4.2) forest humus from a 20-year-old spruce na-
tural stand, but it developed satisfactorily on the same
medium subjected to sterilization. Egmgg annosus made no
appreciable growth on autoclaved soil inoculated with
Trichoderma species, but when he introduced Bacillus
subtilis or Actinomyces species into the sterilized medium,
the vigorous deveIOpment of E. annosus was not impeded.
Rishbeth (1951a) gave an account of the interaction in cul-
ture of E. annosus with strains of Trichoderma viride, many
of which were antagonistic to the former. The growth of E.
annosus has been found to be antagonized by soil micro—
organisms other than soil fungi. Missen (1956), working on

Actinomycetes antagonistic to Polyporus annosus (F. annosus;
demonstrated that a number of Actinomycetes isolated from

8

forest soil in Denmark proved that a number of Actinomyce-
Egg proved to be antagonistic in vitro. Some Streptomyce-
Egg species also suppressed the mycelial growth of the
pathogen in sterile garden soil. Bjorkman (1949) investi-
gated some soil antibiotics acting against the rot fungus
Polyporus annosus, and reported that a great number of the
soil mycelia tested on malt agar plates were completely
grown over by the E. annosus mycelium. Most of the over
grown mycelia belonged to Phgmg, Clados orium, and several
Actinomycetes species. However, he observed that some
parallel seems to exist between the occurrence of the
root-rot fungus and certain soil microorganisms without
antibiotic effect.

Huppel (1968) worked on the protection of coniferous
seedlings from attack by E. annosus and.showed that 40%»of
the isolates of soil fungi were characterized as mycorrhi-
zal fungi which provided protection to the roots of pine
seedlings. Dobbs and Hinson (1955) reported the presence
of widespread fungistatic factors in the soil. It is
likely that these fungistatic factors may be responsible
for the distribution of Fgmgg annosus on a particular site.

Adam, Witcher and Lane (1964) studied microorganisms
in soils from Fgmgg annosus infected pine stand and reported
that there were significantly fewer colonies of fungi from
the soil around infected trees than from soil around non-
infected trees. Manka (1968) attempted to classify myco-

flora of the soil environment (root, rhizosphere and litter)

into communities arranged into so-called "biotic series"

to show the function of these communities in relation to F.
annosus. Of the 15 species of fungus he isolated from the
soil environment, Trichoderma glaucum showed the highest
degree of biotic influence. Manka (1968) also found that
there were more biotic influences in a healthy stand than
in an infected stand and that communities of fungi were
more abundant in the root and rhizosphere of the healthy

stand.
Other soil factors

Other soil factors such as aeration, temperature,
humus and moisture are also interrelated with the factors
already discussed. In a similar way these factors are
linked to the incidence of Fgmgg annosus. Tower and
Stambaugh (1968) found that soil temperature was a signifi-
cant factor in the longevity of F. annosus inoculum in the
soil. They also found that more seedlings of loblolly pine
die from £9223 infection when subjected to temporary wilt-
ing than in soil maintained at field moisture capacity.
HOpffgarten (1955) observed that permanently moist sites

are not invaded by the fungus in the absence of aeration,

but infection is prevalent on temporarily damp areas.
Mode of Entry

The mode of infection and spread of F. annosus has

been reviewed by Hodges (1969). who stressed that the

lO

spread of the disease is accomplished by spores and by
growth of mycelium. Evidence abounds that roots of fresh-
ly cut stumps and even living trees around them become in-
fected by spores. On the other hand little experimental
evidence is available on infection through above ground
wounds. Besides, tree species differ in their ability to
resist the attack of F. annosus through trunk wounds.
Rishbeth (1951a) reported one case where E. annosus may
have infected a suppressed tree through pruning wounds and
eventually reached the root system. However, he did not
succeed in inoculating cut branches of Scotch pine (Piggg

sylvestris) with F. annosus. Witcher and Beach (1962) on

 

the other hand reported infection initiated from pruning
wounds.

E. annosus spreads from diseased to adjacent trees
by root contact. Rishbeth (1951a) observed that the
mycelium of the fungus passes directly from the bark of
the infected roots to that of living roots. The work of
Dimitri (1962) and Bassette, et a1 (1967) tends to impli-
cate the presence of a toxin in the infection process.
This toxin was named fomanosin, although it has not been

possible to isolate it from infected wood.

CHAPTER 3
mmsams AND METHODS

Collection of Soil Samples

Two soil samples for green house studies were col-
lected from two different sites to reflect differences in
soil texture. The first site was located along College
Road on the University Farms which has clayey soil. The
other site was located next to the University Married Hous-
ing apartments along Mount Hope Avenue and has a sandy
soil. The soil samples were collected with an auger to a

depth of 10 inches.
Mechanical Analysis

The sand, silt and clay content Of the soil samples
were determined by the hydrometer method. Fifty grams of
each sample were mixed with a dispersing agent consisting
of 55.5 grams of sodium oxalate and 2.0 grams of sodium
hydroxide in one liter of water. The soil sample was
placed in a one-liter cylinder and the dispersing agent
added until the whole volume was brought to 1000 ml. The
cylinder was shaken vigorously, a hydrometer inserted, and
readings taken after 40 seconds and one-hour intervals;
the former gave the sand content in percent while the

11

12

latter gave the clay content in percent. The percent silt
was obtained by adding the two readings and subtracting the
total from 100. The clay loam soil has 52.4% sand, 55.2%
clay and 52.4% silt, whereas the sandy loam soil has 72.5%
sand, 15.0% clay and 12.5% silt.

Soil pH

The pH of the soil samples was Obtained by mixing
20 grams of each soil sample with 25 m1. of 0.01 normal
calcium chloride solution and after 10 minutes the pH was
determined with a 9600 Zerometic pH meter.

The pH of the sandy loam soil was 7.2 and that Of
the clay loam 6.7. Two pH levels, 4.8 and 6.5, were se-
lected for this study. To bring the pH Of the soil samples
to those levels, varying concentrations of hydrochloric
acid (O,1,2,4,10,l6,22,52,MeH+) were mixed with 50 gms of
the sample in seven 100 m1. beakers for each sample. These
were left for one week, after which 20 mls. of distilled
water were added to each beaker, stirred and the pH deter-
mined with a 9600 Zeromatic pH meter. The pH values
against the concentrations of hydrochloric acid were plot-
ted for the two samples (Figure 1). These curves were

then used to adjust the two soils to pH 4.8 and 6.5.

l3

 

 

 

l J l

l l

 

 

no IS '50 25 so 35

(Jp-

MeH7|OOgm Soil

Figure I. Amount of HCI acid required to adjust 509ms of
soil to varying ph levels.

14

 

 

Isolation of Fomes annosus from Infected Roots

, Cultural medium for the isolation of Egggg annosus
from infected roots was prepared using the modification of
Russell (1956), with O-phenylphenol medium as applied by
Miller and Arthur (1966). Components of the cultural
medium include 17 gm. agar, 20 gm. malt extract, 60 mg.
O-phenylphenol, 100 mg. streptomycin sulphate, and 1.5 m1.
of 50% lactic acid in 1000 ml. of distilled water. These
components were added asephically after the malt agar was
autoclaved. Fgmgg annosus inoculum was Obtained from the
roots Of Scotch pine within an infected pocket in the Carl
G. Fenner Arboretum along Mount Hope Avenue in Lansing
(Figure 2). The Fgmgg annosus isolate was obtained after
two weeks (Figure 5) and was used in the inoculation of
2.0% of malt extract agar (MEA 2% malt extract, 2% agar,
2% dextrose, and 0.1% peptone in distilled water). This
was used subsequently in the inoculation of test seedlings

(Figure 4).
Inoculation

Egggg annosus isolate was grown in MEA at 25° C in
the dark for 21 days. Then, one m1. of conidial mycelia
suspension was harvested by flooding the inoculated 2%»MEA
plate with distilled water, stroking the surface, separat-
ing the suspension and diluting it with 100 m1. Of distill-

ed water. Ten weeks after germination, the seedlings were

15

I, ,
.w. '13..wa .
. a .

t . is
urine
in

c i.
. cc .
O,

 

Fenner Arboretum, Lansing, Michigan, from where

Fomes annosus infected spot in the Carl G.
the inoculum was obtained.

re 2.

Figu

16

 

Figure 5. Isolation of _F_‘. annosus from infected wood.

1?

 

Figure 4. pedocephalum lineatum a conidial stage of F.
annosus on 2.0% M.E.A.

18

inoculated with 10 m1. of the above suspension.
Identification of the Fungus in Culture

The conidial state of F. annosus (Figure 4) has
been called Oedocephalum lineatum (Bakshi, 1952). The
fungus rarely forms basidiocaps in culture. For this
reason, identification is based on the features of the
conidial stage. The identification of the fungus was
based on Nobels' (1965) method, and all tests performed on
malt agar prepared according to the following formula:
malt extract 12.5, and agar 20.0 gm. in 1000 m1. Of dis-
tilled water. The fungus for identification was grown on
the above medium in a petri dish and incubated for 4 weeks
at 25° C in the dark, being brought out to the light for
Observations only. The test of the presence or absence of
extra cellular oxidase was determined by dropping an al-
coholic solution of guaiacum (0.5 gm. gum guaiac in 50 m1.
of 95%iethyl alcohol) on the fungal mycelia mat. Blue
color which appeared indicated the presence of extra cellu-
lar oxidase. The fungus has an oval conidia attached by a
short sterigmata to a club-shaped conidiosphore. The
mycelial mat has a white color initially which changes to
pale color with time. The fungus has thin-walled hyphae
with simple septa. The rate of growth was slow and the

petri dish was covered within 14 days.

19

Screening Trichoderma Species for Antagonistic Effects on
F. annosus ‘

 

 

Four species of Trichoderma and three isolates of
T. viride from Mycological Service Amersta, Massachusetts
(T. viride myco. 10045, T. viride myco. 10087, T. viride
myco. 10088, T. harzianum myco. 10090, T. koningii myco.
Ga) were each inoculated at one edge of four petri dishes.
The Opposite edge was inoculated with pieces of T. annosus.
The medium was 2.5%»MEA with a pH of 6.5. The control in—
cluded only inoculates Of one species or isolates arranged
as above. The radius Of each fungal growth was determined
for 4 days (Table l) and further observed for 21 days
(Figure 5). Another medium was prepared but acidified
with cactic acid and buffered with potassium sulphate
(KH2P04) to lower the pH to 4.8. The inoculation of this
second medium was as above. On this basis isolate myco.
10088 was the most antagonistic and therefore was selected

for the study.
Preliminary Studies

A pilot experiment was set up to determine soil
moisture levels for the study. A given volume of water
was used to irrigate equal amounts of soil samples. The
water retained after a period of time was highly varied
and it was Observed that it is difficult to maintain a

given moisture level and to have the water uniformly

2O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

tn mm d
on mH n
mH NH N
CH w H
OHmoon .H. mmmmmmm .M hen
Os mm id mm mm s
cm mH m on mH m
mH MH N mH #H N
CH m H m m H
HHwanOM .H unwound .M hen mkbelMoha unwound .M _hmn
mean? .m
m: NN # H# #N #
Nd mH m mm 5H m
MN OH N ON NH N
MH 0 H HH m H
mamosam .M ham mmOOH Ooha Osmoawm .M hen
«.33» .m...
we mm a mw mm d
N: mH m m: 0H m
MN mH N NN HH m
MH 5 H NH m H
azqunHmn .H unwound xm hen mmbelmoha unwound .M ham
32? .H
.me mg no «as and 3 when a new msaeem
HO as OH mopmHomH dam OOHOOQm mauOOOnOHna One annosus mmaom HO ovum upsonu .H OHOOB

Figure 5.

21

 

Growth of T. annosus and Trichoderma species
after 21 days. Trichoderma on the left Of
petridish and Fomes on the right.

Top row: T. harzianum and E. annosus; T. longi—
bracheatum and E. annosus; T. Koningii
and T. annosus; and T. reesei and

E. annosus.

Bottom row: T. viride myco 10078 and T. annosus,
T. viride myco 10045 and T. annosus,
and T. viride myco 10088 and

F. annosus.

22

distributed. For this reason differing soil moisture
levels were not used in the study.

In December 1977, a study was initiated to deter-
mine the length Of period the soil sample would remain
sterile under experimental conditions. For this purpose,
six 125 m1. Erlnmeyer flasks and six 58 mm. culture tubes,
each containing 60 gms. and 50 gms. of soil samples, re-
spectively, were autoclaved for two periods of 60 minutes
each 24 hours apart. The irrigation of the soil was with
sterile water. Small samples Of soil from each container
were tested for sterility every 14 days. After three
months, the samples in the culture tubes still maintained

their sterility.
Experimental Procedure

Loblolly pine seeds from an improved seed source in
Georgia were surface sterilized by immersion and occasional
shaking in 50%ihydrogen peroxide for one hour. They were
rinsed with sterile distilled water and stratified by
soaking in wet, filter paper in petri dishes and kept at
50° F. in a cold room for 55 days.

The soil samples were thoroughly mixed and irri-
gated with water to slightly below field capacity. Sam-
ples were weighed into 58 mm. culture tubes. These were
autoclaved for two periods each lasting 60 minutes 24

hours apart. The tubes were plugged with loose cotton

23

and covered with steel caps in the last autoclaving. Then
5 m1. Of sterile malt extract in distilled water were
added to each tube. The pH of the malt extract corresponds
with that of the soil into which it was poured. The sur-
face sterilized seeds were planted aseptically in the
tubes which were covered as described above and left at
50° C for the duration of the study.

The medium to be treated with T. viride myco.
10088 was first sterilized and after two days was inocu-
lated with T. viride. Two weeks later sterilized loblolly
pine seeds were shown in the medium previously inoculated
with T. viride and other media used in this experiment.
Ten weeks after germination the seedlings were inoculated

with Fomes annosus.
Field Study

Ten Egggg annosus infected spots were located in
the Kellogg Forest near Battle Creek, Michigan, in red
pine, white pine and Scotch pine plantations. A soil
sample was taken within each infected spot and also in an
uninfected spot adjacent to it. A soil core of approxi-
mately four inches in diameter was taken to a depth of ten
inches on each spot. These samples were taken into the
laboratory and analyzed mechanically and the pH deter-
mined with a 9600 Zeromatic pH meter.

24
Statistical Methods

The greenhouse experiment was a 5x2x2 factorial
arranged in a completely randomized design with five seed-
lings per treatment and three replications. In all, 180
seedlings were used. Symptoms of root rot disease began
to show ten days after inoculation. The number of days
it took an infected seedling to die or show wilting symp-
toms was recorded and ranked for virulence as follows:

Number of days after

inoculation that death or Ranking of the
wilting occurred virulence of T. annosus.

 

 

12
14
16
18

C) 0H A) \N .¢

20

The ranked mortality data were subjected to an analysis
of variance and multiple comparisons among means were made
with the least significant difference test set at the 5%

level.

CHAPTER 4
RESULTS AND DISCUSSION

 

Screening Trichoderma Species for Antagonistic Effects on
F. annosus. 4' A

Trichoderma viride myco. 10088 was the most antag-
onistic of the three isOlates of T. viride and four Of
Trichoderma species screened. Antagonism is based on the
width of the bare zone between T. annosus and each Of the
other species or isolates of Trichoderma grown on 2.5%iMEM
in a petri dish for three weeks (Figure 2).

At pH 6.5, Trichoderma species and isolates showed
slightly faster rate of growth on malt extract agar than
they did on the same medium acidified to pH 4.8. On the
other hand, T. annosus' rate of growth was higher on MEA
of pH 4.8 than in one with pH 6.5. These differences may
be partially attributed to the high temperature under
which the fungi grew. This is Similar to the observations
of Rennerfelt and Paris (1955) on the physiology and
ecology Of Polyporus annosus (E. annosus), when they noted
that the mycelium of T. viride overgrew that of T. annosus
at 22° C but at 12° C T. annosus overgrew T. viride.

Bjorkman (1949) worked on soil antibiotics acting against

25

26

the root rot fungus Polyporus annosus and he interpreted
the presence of the bare zone between T. annosus and some
soil‘microorganisms as an indication of antibiotic activ-

ity, even if only temporary.
Soil Sterility and Microorganisms

The number of seedlings infected and not infected
by T. annosus are shown in Table 2, while Table 5 lists
the severity of the disease based on the mortality ranking

of each treatment.

Table 2. Number of Seedlings Infected and Not Infected by
Fomes annosus

 

 

Sterilized soil
Sterilized Unsterilized but treated
with T. viride

 

 

Soil Sandy Clay Sandy Clay Sandy Clay
pH loam loam loam loam loam loam
Infected l4 l2 1 2 2 O
4.8
Non Infected 1 5 14 15 l5 l5
Infected l5 15 l l l l
6.5

Non Infected 2 2 14 14 14 14

 

27

Table 5. The Severity of the Infection Based on the Sum
of A11 Rankings for Each Treatment.

 

 

 

 

Sterilized ‘ Unsterilized Sterilized but treated

 

 

 

 

S 11 Soil , , Soil with T. viride
O ' . . “
pH Sandy Clay Sandy Clay Sandy Clay
loam loam loam loam "loam loam
4.8 39 28 5 2 5 0
6.5 53 26 5 2 1 1

 

The number of seedlings in the sterilized medium
killed by T. annosus was significantly greater (P==0.05)
than those killed in the unsterilized medium and sterilized
but later contaminated with T. viride (Table 4). There
was no significant difference between the infection rate
of seedlings in unsterilized soil and in sterilized soil
later contaminated with T. viride. The high infection
rate of T. annosus on loblolly pine seedlings under ster-
ile conditions is probably due to the absence of antagon-
istic soil microorganisms. This absence of microorganisms
gave T. annosus the ability to express its potential as a
pathogen. The sterile condition may have eliminated
fungistatic factors which are widespread in the natural
soil.

The severity rating was also highest in the steri-
lized soil than in the unsterilized soil later contaminatei
with T. viride (Table 2). Seedlings on the unsterilized

soil did not show any significant difference in either

28

Table 4. Mean Severity Rating of Infection for Media,
, Soil Texture and Soil Reaction.°

 

 

Main Effect Mean Severity Rating
Media _
Unsterilized soil 9.67a
Sterilized soil 0.85b

Sterilized inoculated
soil 0.42b

Soil Texture
Sandy loam 4.56
Clay loam 2.72
Soil Reaction
Sandy loam 4.11
Clay loam 3.17
‘"a" significantly different from "b"

(P - 0.05). Statistical analyses performed on
the CDC 6500 computer.

 

degree Of severity or number Of seedlings infected from
those in Trichodermg contaminated soil (Tables 2 and 5).
This is indicative of T. viride's ability to provide pro-
tection to loblolly pine seedlings from the attack of T.
annosus. The ability to provide protection may be due

to its toxic prOperties which according to Weindling
(1952) are composed of two distinctly different substances,
gliotoxin, which is active both as a bactericide and a
fungicide, and viridin, exclusively a fungicide.

29

The low infection rate and low degree of severity
of T. annosus root rot on unsterilized soil is due to a com-
bination of factors. Perhaps the most important of these
is the presence of soil microorganisms which are antagon-
istic to E. annosus. This is in conformity with Bjorkman
(1949) who noted that some parallel seem to exist between
the occurrence of the root-rot fungus and certain soil
microorganisms.

Although temperature was not monitored in this
study, there is every likelihood that temperature favored
the competitive ability of T. viride particularly if one
takes into consideration the high average daily tempera-

ture during the months of June to August.

Soil pH

The effects Of soil pH together with other factors
are shown in Tables 2 and 5. There is a slight difference
between the severity of infection on pH 4.8 and pH 6.5.

The severity of the disease tends to be greater on soils
with pH 4.8 than on soils of pH 6.5. However, when the
means were compared using the least significant difference,
there was no significant difference between the means at
both (P = 0.05) and (P = 0.01). It is likely that the
range of pH chosen was So small that it did not show any

effect on disease severity and infection rate. In nature,

soil reaction may range from pH 5.4 in acid soil to 8.5

50

or more in alkaline soil. Some workers have shown that
Egggg annosus grows favorably under a wide range of pH.
Rennerfelt, et a1 (1955) stated that the growth rate of

T. annosus takes place between pH 2.9 and 7.0. On 5%imalt
extract there was no variation in the growth of T. annosus
within a range of pH 5.0 to 7.0 (Treschow 1946). He also
did not detect any difference in the growth of the fungus
when he varied the pH of sterile forest humus by adding
lime ranging from 2 to 20 gm per 50 gm of humus soil. In
all cases the growth rate of the fungus is indicative Of
its potential to cause root rot. Etheridge (1955) ob-
served that all the isolates of T. annosus he investigated
had a similar pH optimum for growth, 4.6 to 5.5. This Op-
timum range falls within the range of pH used in this
study. It is not surprising, therefore, that no signifi-

cant difference in the effect of pH was detected.

Soil Texture

The degree of virulence was higher on sandy soil
than on clay soil particularly within the sterilized soil
lot. There was a significant difference (5% level) be-
tween the rate at which the seedlings planted on sandy
soil died and the rate at which those on clay soil died.
More seedlings died on the sandy soil than on clay soil.
The sandy soil not only favored an easy penetration of the

roots into the soil, but it also offered better growth

51

conditions to the fungus. Treschow (49) noted that the
growth of T. annosus depends not only on the pH of the sub-
strate but also on its nature. The puddling tendency of
clay soil after long periods of autoclaving may have re-
duced the porosity of the clay soil to the point that
growth of the fungus may have been slightly affected.

Field Study

The results of soil mechanical analyses and soil
reaction of the infected and apparently healthy spots in
the field are summarized in Table 5. There was no signifi-
cant difference (5%) between % sand, silt and clay from
diseased spots and healthy spots adjacent to them using the
"t" test. Similarly there was no significant difference
between their pH although there are few spots where the
pH of the infected site was less acidic than those of the
healthy spots. The infected spots may have initiated from
inoculum left over on stumps of thinned trees or the stumps
of cutover vegetation prior to plantation establishment.

It is noteworthy to mention that 80% of the infected spots
throughout the plantation occurred at a lower elevation or
at the base of the hills where the soil was deeper than
farther up the hills.

52

Table 5. Soil Data from Diseased and Adjacent Healthy
Spots_in the Kellogg Forest°_

 

 

 

 

 

DiSeased Healthy

SP°t Sand Clay Silt Reaction Sand Clay Silt Reaction
%. %, %. PH %i %. %i pH
a. 65 10 25 5.1 59 14 27 5.0
b. 60 15 25 4.9 60 15 25 5.1
c. 64 12 24 4.8 64 15 25 4.8
d. 65 14 25 4.9 62 15 25 4.7
e. 64 15 25 4.7 64 14 22 4.7
r. 68 12 20 4.7 69 l2 19 4.5
g. 65 15 20 5.2 65 15 20 4.9
n. 61 12 27 5.5 61 12 27 5.1
i. 68 l5 19 4.7 69 l5 18 4.4
3. 59 2o 21 4.6 59 2o 21 4.6

 

‘Approximate distance between the points of sampling
at each site was 15 feet. NO Significant difference be-
tween % sand, silt and clay (5%).

. ._ .§._x.Il..s.’.’ .m‘ cam

 

CHAPTER 5
SUMMARY AND. CONCLUSIONS

The Objective of this study was to determine the
effect of some soil factors, namely texture, pH, and iso-
lated microorganism Trichoderma viride on the incidence
and severity of T. annosus root rot on loblolly pine
seedlings, and to determine the extent to which the iso-
lated case Of soil microorganism could provide protection
to loblolly pine seedlings from T. annosus infection.

Three isolates of T. viride and four species of
Trichoderma were tested with T. annosus conidial mycelium
on a petri dish containing 2.5%»MEA. The fungi were
placed Opposite each other on the plate. The bare zone
between them was the basis for the selection of the most
antagonistic of the Trichoderma isolates and species.
Trichoderma viride myco 10088 was selected as the most
antagonistic because it showed the broadest bare zone.
Two soil samples typical of clay loam soil and sandy loam
soil were collected and their mechanical prOperties con-
firmed by mechanical analysis. The pH of these soils was
determined with a 9600 Zeromatic pH meter and thereafter
adjusted to pH levels 4.8 and 6.5.

33

54

The soil samples were divided into three parts, one
autoclaved, the second unautoclaved and the third auto-
claved but later contaminated with T. viride. Surface
sterilized and stratified seeds of loblolly pine were
aseptically sown in these soils. Ten weeks after germina-
tion the seedlings were inoculated with conidial mycelia
suspension of T. annosus. The numbers of seedlings that
died or wilted were recorded. ‘

Soil samples were collected from ten infected spots
and apparently uninfected spots fifteen feet away from
them. The soil samples were analyzed in the laboratory
for mechanical prOperties and soil reaction.

The soil texture and soil pH levels tested here do
not tend to have much influence on the incidence of the
disease, probably because a very narrow range of pH was
used. More important to the direct effect of soil texture
and pH on the fungus is the effect of these factors on the
seedlings. Any site which stresses the seedlings or trees
eventually leads to susceptibility to T. annosus infection.

It is noteworthy to observe that T. viride was able
to provide protection tO'lOblolly pine seedlings under the
experimental conditions. The high mortality rate of seed-
lings grown on sterilized soil may partially be due to the
absence of mycorrhiza forming fungi as a result of prO-

longed autoclaving. Huppel (1968) has demonstrated that

certain mycorrhiza forming fungi provided protection to

55

pine seedlings from T. annosus attack.

The interaction of T. viride with other soil micro-
organisms and their antagonistic effects are not well
known. Therefore the possibility of using the aggressive
and antagonistic features of T. viride as a method of con-
trol or selecting suitable soil media for raising seed-
lings of loblolly pine is subject to further investigation.
A very important point is that changes in soil temperature
may render T. viride inactive and thus put T. annosus to
an advantage. From the ecological standpoint, the tempera-
ture of the soil may be a selective factor determining the
microorganisms present on a particular site at any given

time.

LITERATURE CITED

37

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Alexander, S. A. and J. M. Skelly. 1975. Disease inci-
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Bakshi, B. K. 1952. Oedocephalum lineatum is a conidial
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58

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41

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JUVHHI'I

 

 

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