Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1990 Interactions of Rhizoctonia solani Kühn and Trichoderma spp populations in soil Maria Esther de la Fuente Prieto Iowa State University
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Interactions of Rhizoetonia soiani Ktthn and Trichodtrma spp. populations in soil
de la Fuente Prieto, Maria Esther, Ph.D. low» State Univenity, 1990
UMI SOON.ZeebRd. Ann Aibor, MI 48106
Interactions of Shizoctonia solani Kflhn and Trichoderma spp.
populations in soil
by
Maria Esther de la Fuente Prieto
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Major: Plant Pathology
Approved:
Signature was redacted for privacy. In Charge of Major Work
Signature was redacted for privacy. For the Majw Department
Signature was redacted for privacy. For the
Iowa State University Ames, Iowa
1990 11
TABLE OF CONTENTS
Page
INTRODUCTION 1
LITERATURE REVIEW 5 Biological Control 5 Parasitism 6 Antibiosis 9 Competition 11 Biological control with Trichoderma .... 12 Suppressiveness 15 RhLzoctonia solanl 18 Description and taxonony 18 Ecology of Rhizoctonla solanl and epidemiology . 21 Enumeration of Rhizoctonla solanl 24 Trichoderma spp 26 Taxonomy and description 26 Ecology 33 Antagonistic attributes 34 Enumeration 36
MATERIALS AND METHODS 37 Fungal Cultures 37 Media 38 Inoculum and Inoculation 40 PDB 40 PDA 40 GBM 41 Red beet seed 41 Soil 41 Procedures 43 Studies of fungi in dual cultures 43 Rhlzoctonla-Trlchoderma dual system with different types of soils and containers ... 45 Effect of levels of infestation of mycoparasite and type of baiting on R. solanl and Trichoderma spp. populations in soil systems 49 Effect of different Rhizoctonla isolates on the population levels of Trichoderma spp. in soil 50 Development of a Rhizoctonla-avcppteaaive soil with and without consecutive cropping 51 Measurement of Conduciveness Indices 53 Statistical analyses 56 ill
RESULTS AND DISCUSSION 57 Fungal Cultures and Media 57 Studies of Fungi In Dual Cultures 60 Single medium system 60 Two media system 65 Rhizoctonla'Trichoderma Dual System In Different Types of Soils and Containers .... 69 Plastic pots (direct contact system) .... 69 Glass petrl dishes (contact and non-contact systems) 71 Effect of Levels of Infestation of Mycoparaslte and Type of Baiting used on R. solani and Trichoderma spp. Populations In Soil Systems 74 Effect of Different Rhizoceonia Isolates on the Population Levels of Trichoderma spp in Soil 86 Development of Rhizoctonia Suppressive Soils with and without Consecutive Cropping .... 90 Measurement of Conduclveness Indices 100
CONCLUSIONS 104
LITERATURE CITED 106
ACKNOWLEDGEMENTS 118
APPENDIX 119 1
INTRODUCTION
In 1932, Velndllng (1932) reported for the first time a species of
Trichoderma parasitizing other soli fungi, among them was Rhizoctonia
solanl Kûhn. The mycoparasltlc activity of Trichoderma spp. has been
studied extensively In many laboratories and many countries. In this genus
are probably the most widely documented mycoparasltes.
Cook (1985) In his presidential address at the 76^^ Annual Meeting of
the Canadian Phytopathologlcal Society, noted the first experiments on
biological control of plant pathogens with antagonists were conducted In
Canada nearly 65 years ago by G. B. Sanford. Sanford's first paper which
concerned the factors affecting the pathogenicity of the potato scab
organism, was published in Phytopathology in 1926. Later, Sanford and W.
C. Broadfoot published on the "biological control" of the wheat take-all
fungus; this was the first usage of the term "biological control" in plant
pathology.
K. F. Baker and R. J. Cook in both of their books (Baker and Cook,
1982; Cook and Baker, 1983) on biological control of plant pathogens,
define biological control in the broadest sense to Include the use of any
organism to control a pathogen. This definition Includes the use of higher
plants as well as microorganisms, and they Include host plant resistance
as one of the best and most effective biological controls. Biological control of plant pathogens must no longer be recognized as a science based 2
mainly on the disciplines of ecology, taxonomy, and soil microbiology. It
currently involves the disciplines of plant and microbial genetics,
molecular biology, cytology, biochemistry, plant physiology, and agronomy
among others. Moreover, biological control Is no longer applicable to a
few plant parasitic nematodes and plant pathogenic fungi; bacterial and
virus diseases are among the most extensively studied. ' Biological control
can be accomplished by manipulation (ecological or genetic) of the host,
antagonist, or even the pathogen Itself and may be directed at the ecosystem, population, or individual level. Biological control, broadly defined, may occur remote from the plant, it may occur on the plant, or it may take place inside the plant. Althou^ it often depends on antagonistic microorganism in the classic sense, it also depends on the plant and may even use the pathogen against itself. It is the broad concept of biological control that makes it a fascinating field of study and potentially useful plant-disease-management strategy.
The fungal pathogen, R. solani as presently understood, probably
Induces a greater number of diseases in hosts from more plant families over a larger part of the world than any other plant pathogenic species
(Tompkins, 1975).
During the last 40 years considerable knowledge has become available on the ecology of R. solani and the epidemiology of diseases caused by this pathogen. The growth, saprophytic behavior, and survival of R. solani in soil have been studied extensively (Baker et ai., 1967; Benson, 1974;
Benson and Baker, 1974; Blair, 1943; Coley-Smith and Cooke, 1971; Flower, 3
1976; Henis and Ben-Yephet, 1970; Henls et al., 1978b; Lewis, 1979; Naiki,
1985; Naiki and Ui, 1975, 1978; Papavizas, 1968; Papavizas and Davey, 1961,
1962; Papavizas et al., 1975; Ui et al., 1976; Weinhold, 1977).
The vast literature on biological control of R. solanl covers seed
treatments, modification of cultural practices, amending soil with plant
residues and specific substances to induce changes in soil microflora, and
direct Introduction of biological antagonists into soil. However, no
single treatment provides a satisfactory control of X. solani. With the
exception of resistance, crop sequence, tillage and fertilization practices
that capitalize upon biological control phenomena, utilization of
biological measures for root disease control have been doubted both
theoretically and practically.
The development of soils suppressive to R. solani has been
demonstrated in controlled environments. Repeated culture of a susceptible
crop (radish) has resulted in soils suppressive to development of and
pathogenic activity by R. solani (Liu and Baker, 1980; Kadir, 1985; Henis
et al., 1979). This repeated culture of a susceptible crop causes a
decline in the inoculum potential of R. solani. Suppressiveness to R.
solani was correlated with an increase in the population of Trichoderma
spp. in the soil (Liu and Baker, 1980; Kadir, 1985). The pathogen, R. solani, was introduced into the soils in the above experiments, but it was never demonstrated whether or not the fungus or cropping was needed for development of suppressive soils because insufficient controls were employed. There are reports that R. solani may attract the hyphae of 4
Trichoderaa spp. (Chet et al,, 1981), but stimulatory effects on the population dynamics of R. aolani on Trichodenaa spp. are unknown.
The objectives of this research were:
1) To determine the involvement of Rhizoctonla solani and host plants in the soil ecosystem during the development of soils suppressive to R. solani with repeated cropping.
2) To assess the stimulatory activity of R. solani in the development of Trichoderaa spp. populations in soil. 5
LITERATURE REVIEW
Biological Control
Biological control is defined by Baker and Cook (1982) and Cook and
Baker (1983) in the broadest sense to include the use of any organism to
control a pathogen. This definition includes the use of higher plants as
well as microorganisms, and host plant resistance is included as one of the
best and most effective biological controls.
The soil microflora constitutes an extremely diverse community of
beneficial and deleterious microorganisms. The microorganisms are
constantly interacting among themselves and are affecting roots of higher
plants. According to Baker and Snyder (1965), rhizosphere microflora can be manipulated to enhance certain beneficial microorganisms and to reduce the activity of plant pathogens. The mechanisms of biological control of soilbome plant pathogens, as discussed by Cook and Baker (1983), are based on the activity of antagonistic microorganisms which were isolated and studied out of their natural balanced habitat; special care must be taken when data collected in vitro are interpreted to describe the situation in situ. Soil microorganisms are mainly heterotrophic, therefore their growth and activity are dependent on nutrients derived from living plant roots, plant debris, and other flora and fauna under favorable environmental conditions. Therefore microbial interactions are influenced by many factors. Antagonism of soil borne pathogens, according to Park (1960), 6
plays a major role In biological control, and can be based on three types
of interactions: parasitism, antibiosis, and competition. These three
Interactions are probably never exclusive. An antagonist in the broad
sense, may reduce the activity, efficiency and/or Inoculum density
(viability) of a plant pathogen and thereby reduce its inoculum potential
(Cook, 1985).
ParasItiam A mycoparasite is an organism which parasitizes fungi, and often it
is another fungus (Welndling, 1932). A mycoparasite that parasitizes a
parasitic fungus, is considered a hyperparaslte (fioosalis, 1964). The
mycoparasitlc and predatory traits of bacteria, fungi, nematodes,
protozoans, arachnids. Insects, and other microflora are well documented In
the literature (Ayers and Adams, 1981; Boosalis, 1964; Boosalis and Mankau,
1965; Cook and Baker, 1983; Elad ec al., 1983b, 1983f; Kuhlman, 1980;
Sundhelm and Tronsmo, 1988). All fungal structures may be parasitized, and
this includes the highly melanized survival structures like rhizomorphs,
sclerotla, and chlamydospores (Snyder eC al., 1976).
Among mycoparasites, the species of TrLchoderma that parasitize sollbome plant pathogens such as SclerotLim rolfsii and Ehizoctonia solani have been studied extensively (Allen and Haenseler, 1935; Chet et al.,
1981; Dennis and Webster 1971a, 1971b, 1971c; Durrell, 1968; Elad çt al.,
1980; Jager et al., 1979; Lewis and Papavizas, 1985). Mycoparasitlsm begins after mycoparasite hyphae make physical contact with the host 7
hyphae. The hyphae of TrLchoderaa spp. coll around the hyphae of its host
and then penetrate them by enzymatlcally digesting the cell wall (Chet et
al., 1981; Elad et al,, 1982a, 1983e). Contact in some cases may involve
recognition.
Recognition between fungal host and mycoparasites only recently has
been demonstrated. Elad et al. (1983a, 1983b) and Barak et al. (1985)
isolated an agglutinin from the hyphae of RhizoctonLa solanl and the
cultural filtrate of the fungus, which was capable of agglutinating "0"
erythrocytes with a high degree of specificity. Mycelial extracts showed activity at a high titer in comparison to the culture filtrate. However, culture filtrate was the preferred agglutinin source throughout the studies because it had comparatively low protein content and low viscosity, and these properties facilitated the agglutination tests and the purification.
The agglutination was Ca'*"*' and Mn"*^ dependent and was inhibited by added galactose; galactose is present in the cell walls of the mycoparasite,
Trichoderma. The agglutinin was specifically inhibited by D-glucose and D- mannose. The cations Mn^ and Ca"*^ also reversed the inhibitory effect on agglutination by the chelating agent Na^-EDTA. Barak et al. (1985) later showed that Sclerotlua rolfsLL agglutinin agglutinated washed conidia of
Trichoderma. Barak et al. (1985) also found that the hyphae of two host fungi, S. rolfsil and R. solani, possess lectins which bind to carbohydrates on Trichoderma cell walls. The ability of different isolates of Trichoderma spp. to attack S. rolfsil or R. solani was correlated with the ability of the lectin from the host fungus to agglutinate Trichoderma 8
conldla. The agglutinin of the host fungus Is strongly bound to the
extracellular polysaccharide In culture filtrates of Trichodenaa spp.
In the Trlchoderma-RhlzocConla systems described above it appears
that the agglutinin-carbohydrate binding is an initial recognition step
which may be involved in specificity and leads to further events in
mycoparasitlc processes, such as enzymatic degradation of host cell walls
and penetration of the hyphae. Toyama and Ogawa (1968) have purified and
defined the properties of Trlchoderaa mycolytlc enzymes. Elad et al.
(1982a) showed that Trichodenaa spp. excreted lytic extracellular beta-
(l,3)glucanase and chltinase into the growth medium and even into the soil.
Production of fungal inhibitory compounds may assist the biocontrol agents during the interaction (Carter and Lockwood, 1957; Jackson, 1965).
Lectins are sugar-binding proteins or glycoproteins of non-immune origin, which agglutinate cells and/or precipitate glycoconjugates.
Discovered first in plants and later in other categories of living things, lectins are Involved in the interactions between the cell's surface components and its extracellular environment (Elad et al., 1983b). Plant lectins specifically bind to hyphal tips and septa of Trichodenaa viride and other fungi and inhibit fungal growth as well as spore germination
(Barak et al., 1985).
The interacting sites of dual cultures of either Rhizoctonia solani or Sclerotixm rolfsii and Trichodenaa species were observed by scanning and transmission electron microscopy by Elad et a.1. (1983b, 1983c, 1983d), and a significant accumulation and deposition of intercellular fibrillar 9
materials was observed outside the Interacting cells shortly after
Triehoderma hyphae made contact with the host hyphae. This Is considered
as a secondary effect of the recognition process.
The mycoparasltlc Triehoderma spp. are natural antagonists of plant
pathogenic fungi like R. solani and have been successfully applied as a
blocontrol agent against this fungus In field crops.
Antibiosis
Although many microorganisms produce antimicrobial substances in
vitro, antibiosis as a mechanism of blocontrol of plant pathogens In the
soil has not been demonstrated by direct evidence. However, It Is
reasonable to expect that antimicrobial substances enhance the capacity of
their producers to colonize root surfaces or other organic matters and to
compete for nutrients and space In the rhlzosphere. Baker et al (1967) and
Park (1960) suggest that the effective range of these compounds Is limited
to an Immediate area or microsite.
Inhibitory substances are produced by certain mycoparasites and,
thus, may aid mycoparasltism. Culture filtrates of Triehoderma harzianum
and volatiles produced In cultures of the fungus, had an inhibitory effect
on the linear growth of Shizoctonia soLani and other fungi (Fedorlnk et ai., 1975). Other studies revealed that production by Triehoderma of a fungal inhibitory factor is isolate rather than species-dependent (Dennis and Webster, 1971a, 1971b). 10
Antagonists were grown from an Inoculum disk over the surface of a
cellophane membrane laid on an agar medium, and the metabolites produced
were allowed to diffuse through the cellophane into the agar. Antibiotic
activity was then assessed by growing a test fungus on the medium after
removal of the antagonist. Mughogho, cited by Dennis and Webster (1971a),
and then Dennis and Webster (1971a), reported that isolates from the '
particular species-group of T. harzXanum did not affect the growth of R.
solanl and that r. virlde affected the growth of R. solani more than any
other species-group. The inhibited colonies were often compact and dense,
the hyphae were generally more branched and sometimes thicker than normal
hyphae and vacuolation of the hyphae occurred. Severe inhibition of growth
of surface nycelium was often accompanied by abundant aerial growth from
the inoculum disk.
Antibiotics that have been isolated and characterized from
Trichoderma spp or their culture filtrates include a group of antagonistic chemical products that are expected to be produced and released into the environment by living Trichoderaa spp. These antibiotic compounds are:
"Trichodermin" (sesquiterpene), "Suzukacillin" (peptide), "Dermatine"
(unsaturated monobasic acid), "Alamethicine" (peptide), "Gliotoxin",
"Viridin", "Glioviridin", and acetaldehyde (Iwasaki et al., 1964; Ricard,
1981; Roth, 1969; Weindling, 1932). 11
Competition.
Competition as a mechanism of biological control of soil-borne
pathogens was described by Baker (1968) as an active demand in excess of
immediate supply of material or condition on the part of two or more
organisms. Clark (1965) clarified principles relating to competition in
microbial ecology by systematically eliminating water and space as factors
in competition, circumscribing oxygen, and calling for greater definition
of effects resulting from limiting substrate. Dormancy itself may be
wholly or partially due to the phenomenon of competition (Baker, 1968).
Two ideas currently are being used to explain induced dormancy of
propagules (the phenomenon of soil fungistasls) in soil: (a) the nutrient
deficiency hypothesis, which contends that the observed effects can be
wholly explained by lack of nutrients in soil essential for germination;
(b) the presence of a low level of diffusible Itihibltory substances in soil, the effects of which may be annulled by application of suitable nutrients (Baker, 1968). Other biological-control systems also appear to reduce nitrogen to a limiting level. Blair (1943) attributed depressed growth of RhizocConia to nitrogen starvation of the mycelium through rapid utilization of available soil nitrogen by a cellulose-decomposing microorganism, such as Trlchoderma spp. Simple additions of inorganic nitrogen reversed the effect. Saprophytic activity and survival of
Rhizoctonla were increased with substrate enrichment with nitrogen
(Fapavizas and Davey, 1961). Weinhold at ai. (1972) have demonstrated the influence of such exogenous nutrients, like nitrogen in the soil, on the 12
virulence of J!, solani. Competition for space has been suggested as being
responsible for suppression of microorganisms. Insufficient space is
available on suitable substrates to accommodate competing organisms
(Clark, 1965; Park, 1960). Microscopic examination of soil and decomposing
organic material in It indicates that non-colonized areas are present, and
that space in this sense is not limiting. However, spatial relationships
may have a profound Influence on colonization of either dead or living
substrates. Suitable substrates become available at distances significant
in relation to the three-dimensional habitat of their thalll. Earlier
occupation of organic matter or host tissue usually means ultimate
possession (Baker, 1968). Therefore, the ability to extend and rapidly
reach discontinuous habitats in three-dimensional space may impart
competitive advantage. In some cases, when Trichoderma has been added as
biological control agent and no effect of it can be directly observed or
detected even after isolation through dilution series, the possibility of
competition (Alexander, 1982) between the biocontrol agent and the pathogen should be considered.
Biological control with Trichoderma
The species of Trichoderma capable of hyperparasitlzlng plant pathogenic fungi are highly efficient antagonists (Bamett and Binder,
1973). Effective biological control depends, among other things, on the presence of living cells of Trichoderma spp. and the presence of a food base (Backman and Rodriguez-Kabana, 1975; Hadar et al., 1979a). Successful 13
biological control of ScleroClum rolfsll Sacc. and JZ. solani Kûhn by
infesting fields with cultures of Trichoderma spp. has been described by
Backman and Rodriguez-Kabana (1975), Elad et al. (1980a, 1981a, 1981c,
1982c), and Wells et al. (1972).
The effective introduction of antagonists into soils continues to be
a major problem for biological control of soilbome plant pathogens. In
most studies, conidla of Trichoderma spp. or a nondescript biomass produced
on various organic substrates, have been applied to soil (Elad et al.,
1980a; Hadar et al., 1979a; Papavlzas et al., 1982). Some success has been
obtained with these preparations. Lewis and Papavlzas (1984b) demonstrated
that population densities of isolates of Trichoderma spp. and other
potential blocontrol fungi increased up to 10^-fold in natural soils amended with mycelial preparations of young cultures on a food base (bran) but Increased only 10^with conldial preparations. Populations of isolates of Trichoderma spp. also Increased in soils amended with fermentor biomass preparations that consisted mostly of chlamydospores (Davet et al., 1981).
Mycelial preparations of Trichoderma spp. containing young actively-growing hyphae either embedded in (or in possession of) a food base (bran) were more effective than conldial preparations, conidla, or free mycelium, in reducing both activity and survival of R. solani in soil and preventing damping-off (Lewis and Papavlzas, 1985). They suggested that hyphae already occupying the food base do not appear to be subject to funglstasls and that intimate contact between mycelium and food base enabled the antagonists to grow relatively unimpeded through the soil. The activity 14
of mycelial preparations In enhancement of antagonist density coupled with
suppression of pathogen and prevention of damplng-off may be explained by
the principle of substrate possession as described by Bruehl (1975). The
specific activities of the antagonist that occur during Its growth are
probably more Important In pathogen suppression than the population
proliferation of the antagonist. These activities may be enzymatic (Hadar
et al., 1979a) or antibiotic production (Dennis and Webster, 1971a, 1971b),
or mycoparasltlsm (Artlgues et al., 1984; Boosalls and Mankau, 1965).
The form of the host fungus may also be Important In studying disease
control with antagonists. It has been suggested (Papavlzas, 1968) that the
response of a soll-borne host fungus to TrLchodema spp. depended on the
developmental stage, physiological state, and age of the host fungus. For
example, JZ. solani can exist In soil In several forms (mycelium, barrel cells, and sclerotla). However, Lewis and Papavlzas (1985) effectively reduced the survival of R. solani when the host fungus and sclerotla were added to soil either embedded In organic matter (bran, seeds) or as free mycelium (In sand/commeal). Growth of R. solani from a food base Into soil was Inhibited greatly. These observations would suggest that mycelial preparations of antagonists may be able to attack and destroy a pathogen that exists In soil as various propagules.
Although most biological control studies are based upoon dual culture assays In the laboratory that show antibiosis or mycoparasltlsm on a specific medium (Dennis and Webster, 1971a, 1971b, 1971c; Papavlzas and
Lumsden, 1980), yet generally there Is little or no correlation between 15
laboratory phenomena and the ability to reduce or prevent disease in the
greenhouse or field. Microorganisms apparently responsible for biocontrol
have been Isolated from suppressive soils (Alabouvette et al., 1979; Chet
and Baker, 1980, 1981; Elad et al., 1982b; Homma et al., 1981; Liu and
Baker, 1980). When these are added to conducive soils, they induce
suppresslveness. These microorganisms have the potential of being superior
biocontrol agents. Some have been added as a slurry treatment on seeds and
they were as affective as fungicide seed treatments (Elad et al., 1982d;
Harman, 1982; Harman et al., 1980, 1981). The population of T. hamatixm
added with the seed is retained in the rhizosphere for a time after
planting and it may control root rots and other soilbome diseases. The
ability of r. hamatum to be effective in small populations as a seed
treatment is probably associated with its ability to grow and sporulate on
seeds, and thereafter to become established in large numbers in soil
(Wells, 1988).
Suppresslveness
The incidence of plant disease is generally Increased by monoculture and by high density planting. Although monoculture of a crop plant species generally is thought to promote Increased damage from soilborne plant pathogens, disease control may occur with monoculture. Development of antifungal suppresslveness in soils during monoculture has been reported for several plant pathogenic fungi. Including R. solanL (Alabouvette et 16
al., 1979; Baker, 1982; Bruehl, 1987; Castanho and Butler, 1978; Chet and
Baker, 1980; Cook, 1982; Henls et al., 1979; Homma et al, 1981, 1986;
Homby, 1983; Jager and Velvls, 1983; Kadlr, 1985; Llu and Baker, 1980;
Lumsden et al., 1981; Nelson and Holtlnk, 1982, 1983; Nelson et al., 1983;
Schneider, 1982; Schroth and Hancock, 1981). For most plant pathogens the
mechanism of suppresslveness was related to the biological characteristics
of certain soils.
Methods for precise measurement of suppresslveness In soils are
needed In epidemiology to determine the amount of biological control
generated In contrast to other methods. The simplest assay may consist of
growing the crop host (radish) In soil Infested with the pathogen and
determining subsequent disease Incidence. For R. solani. Inoculum density
profoundly Influences disease incidence, as established by Benson and Baker
(1974); at least an initial Inoculum level of five propagules per gram of
soil needs to be used because that is the minimum level capable of inducing
almost 100% damping-off. Any increase in suppresslveness (reflected by
decreased disease Incidence) in the soil should be detectable at this
inoculum density. The magnitude of suppression becomes more impressive
when inoculum density-disease incidence curves are generated to compare
responses in conducive and suppressive soils (Benson and Baker, 1970).
Soils amended weekly with several organic amendments became slightly suppressive to R. solani (Henis ec al., 1979). Thus far, suppressive soils have been generated only from soils containing a high population of viable fungal thalll; specifically, in the radish monoculture system in 17
which development and activity of R. solani was stimulated by the presence
of the plant host and by weekly supplements of viable yeast. This could be
interpreted to indicate that an antagonistic microflora develops in
suppressive soils specifically in response to activity by living entitles
and that these entities may provide substrates for mycoparasltes (Chet,
1987a).
Henis at ai. (1978a) proposed that both pathogen (R. solani) and host
(radish) had to be present to Induce suppresslveness. This was supported
by Liu and Baker (1980). They clearly indicated that the pathogen had to
be active to induce the development of postulated biological antagonists
which benefited from the association of host and pathogen.
In the several studies, there has been no correlation between
suppresslveness of the soil to R. solani and antibiosis, antagonism, or
lytic ability to the pathogen by Isolates of the soil microflora. However,
all Trichoderma isolates from suppressive soil were parasitic to R. solani
in two-membered cultures. Then, Henis et al. (1979) detected no
correlations between suppresslveness of soil and the antagonism of various
soil microflora with one exception, viz,, Increase in soil lytic properties
was associated with Increase in propagule population density of Trichoderma
spp.
Soil suppressive to Rhlzoctonla solani has been generated by
monoculture planting of successive crops of radishes at weekly Intervals
in soil Infested with the pathogen (Liu and Baker, 1980). Numbers of
Trichoderma spp. propagules in the soil Increased as suppresslveness
increased, whereas inoculum density of jZ. solani was Inversely 18
proportional to the density of these Trichoderma spp. following radish
monoculture (Kadir, 1985; Liu and Baker, 1980). Successive plantings of
cucumber also generated suppressiveness which was associated with
population of TrLchoderma spp. propagules. Suppressiveness did not
develop and Trichoderma was undetectable with sugar beet, alfalfa, and
wheat monoculture.
In the radish monoculture system, disease incidence Increased more
rapidly in the first three successive replantlngs when host tissue was
reincorporated in soil than when this tissue was removed (Liu and Baker,
1980). This suggested that the suppression of disease that occurred In
later successive replantlngs was not due to a buildup of a compound In
radish that is active against R. solani (Liu and Baker, 1980).
Suppressiveness with radish monoculture could be induced more rapidly in acidified than in alkaline soils.
BhLzoctonLa solani
Description taxonomy
A brief search through Biological Abstracts and the Review of
Applied Mycology reveals that papers mentioning Bhizoctonia solani are currently appearing at the rate of about 100 per year. The reference- card files of the Mycology Division of the United States Department of
Agriculture report over 5,000 entries for Ehizoctonia solani. 19
The fungus causes a variety of diseases to many plants and occurs
In most climatic zones (Tompkins, 1975). The species Is an amalgam of
many strains with like vegetative characteristics used In taxonomy, a
common tellomorph, Thanatephorus cucumerLa (Frank) Donk, and Is variable
In pathogenicity, sclerotlal morphology, cultural appearance on media
and physiology (Ogoshl, 1987).
According to Ogoshl (1987), the genus Rhizoctonla, which was
vaguely described by De Candole in 1815, is a grouping of
basidlomycetous imperfect fungi characterized as follows: (a) branching
near the distal septum of cells in young vegetative hyphae; (b)
formation of a septum in the branch near the point of origin; (c)
constriction of the branch at the point of origin; (d) dolipore septa;
(e) no clamp connections; (f) no conldla, except monilioid cells; (g)
sclerotla not differentiated into rind and medulla; and (h) no
rhizomorph. Many of the described species are not Rhizoctonla and some
species described in other genera are actually Rhizoctonla. The
taxonomlc concept of Ogoshl (1987), which appears to be well accepted,
could allow division into three sub-generic groups: (1) species with
three or more nuclei per cell in young vegetative hyphae, hyphae of 6-10 ttm diameter, and teleomorph genus of Thanatephorus Donk; (2) species
with usually two nuclei per cell, hyphae of 4-7 pm diameter, and
teleomorph genus of Ceratobasidium Rogers; and (3) R. oryzae and R. zeae with three or more nuclei per cell and teleomorph genus of Waitea Uarcup
& Talbot. R. solanl is typified by the first sub-generic group (Ogoshi, 20
1987; Parmeter and Whitney, 1970). Field Isolates possess all of the
characters described and have some shade of brown pigmentation (Parmeter
and Whitney, 1970).
Criteria for positive identification of R. solanL, especially for
field isolates, are: hyphal size, branching, and septation (Ogoshi,
1987), and septal pore characters and numbers of nuclei per cell, which
can be observed with a modified Giemsa stain, according to Whitney
(cited by Papavizas, 1968), or with a rapid staining technique developed
by Tu and Kimbrough (1973).
Intraspecific grouping of R. solanL has been attempted by several
persons. These can be separated into two categories: (1) grouping
based on pathogenicity, cultural appearance, morphology, physiology, and
ecology: and (2) grouping based on compatibility for hyphal anastomosis on culture media (Ogoshi, 1987).
R. solani is known to be composed of an indefinite number of races distinguished by asexual morphological and physiological characteristics
(Anderson, 1982). The original studies of Anderson (1982) and Bolkan and Butler (1974) showed that in Thanatephorus cucumerls, the perfect state of R. solani, at least four anastomosis groups (AGs) can be found.
This was extended to eight AGs by Ogoshi (1987), with AG 2 divided into
2 sub groups. The population of R. solani, even in a small field, is composed of a number of distinct Isolates with various saprophytic and parasitic potentialities. Isolates within an anastomosis group vary in pathogenicity. Ogoshi (1987) recognized 13 intraspecific groups based 21
upon anastomosis behavior, cultural appearance, and pathogenicity.
It has been shown that the capacity to anastomose provides an
indication of relationship within groups of isolates. Among those
isolates that conform to the present concept of R. solani, there remains
a wide diversity in appearance and behavior (Parmeter et al, 1969).
Isolates belonging to different AGs are unable to anastomose with each
other, and thus cannot form heterokaryons (Bolkan and Butler, 1974).
Heterokaryotlc field isolates Interact with each other to produce new
heterokaryons that are remarkably different morphologically and
pathogenlcally from their parent field isolates. Field Isolates form
heterokaryons with homokaryons (Bolkan and Butler, 1974).
Geology s£ Rhizoctonia solani AQS| enldemlologv
During the past 40 years, considerable knowledge has become
available on the ecology of JZ. solani and the epidemiology of diseases
caused by this pathogen. The growth, saprophytic behavior, and survival of R. solani in soil have been studied extensively (Baker et al., 1967;
Benson, 1974; Benson and Baker, 1974; Blair, 1943; Coley-Smlth and
Cooke, 1971; Flower, 1976; Henis and Ben-Yephet, 1970; Henls et al.,
1978b; Lewis, 1979; Nalkl, 1985; Naikl and Ui, 1975, 1978; Papavlzas,
1968; Papavlzas and Davey, 1961, 1962; Papavlzas et al., 1975; Ui et al., 1976; Weinhold, 1977). Although studies with models may sometimes be extrapolated to the field, such studies often do not reflect the natural conditions and stresses to which pathogens are subjected in the 22
field (Papavlzas et al., 1975).
Rhizoctonia solani apparently survives as sclerotla or thick-
walled hyphae associated with plant debris (Boosalis and Scharen, 1959).
Variation in survival ability very often correlated with soil
temperature and moisture content (Parkinson and Waid, 1980); cool dry
soils favor survival.
Blair (1943) was one of the first suggesting that R. solani is
highly dependent on plant tissue and almost disappears when the latter
is exhausted. The pathogen grows rapidly from a suitable energy base,
but survives poorly in competition once a selective substrate is gone.
The population may range from 0-15 propagules per 100 g of soil, based
upon debris particle infestation of R. solanL (Weinhold, 1977).
The competitive saprophytic ability of Rhizoctonia solani has been
estimated by its ability (i) to grow in natural soil and colonize suitable culture media in screened immersion plates or in soil immersion tubes, as shown by Martinson (1963), (ii) to colonize competitively organic substrates segments buried in soil, as shown by
Oavey and Papavlzas (1962), and (ill) to invade seeds buried in Infested soils, as shown by Kendrick and Jackson (1958). Martinson (1963) showed a significant linear relationship between frequency of radish emergence and saprophytic activity measured by the frequency of holes invaded per microbiological sampling tube by R. solani. There should be a high degree of correlation between soil infestation level and saprophytic activity of R. solani as assessed with the plant segment colonization 23
method, the plant debris particle isolation method, or the immersion
tube method. The degree of parasitism could be predicted by the degree
of saprophytlsm, although saprophytic colonization methods are not
reliable for studying survival of R. solanl in soil (Papavizas, 1968).
Papavizas (1968) established that the immersion tube method was less
sensitive than the seed colonization method for R. solanl detection,
that saprophytic colonization techniques, pathogenicity tests, and
microscopic observations established that activity of R. solani Is
substantially higher in the coarse soil fractions and in the organic
debris particles than in the very fine fractions. Saprophytic
colonization techniques and pathogenicity tests have established that
saprophytic activity (and therefore Inoculum density) of R. solani is at
its peak in the upper 10 cm of soil (Papavizas et al., 1975). Welch and
Weinhold (1976) isolated debris particles from soil and found that the
endogenous nutrient content of the propagules in the particles is
usually quite low. Supplemental nutrients, like asparagine, were needed
to increase the inoculum potential of the pathogen to allow for successful Invasion of plant tissue.
Papavizas et al. (1975) established that competitive saprophytic activity of R. solani had a positive correlation with the magnitude of host root rot in the greenhouse, and to a lesser degree, in the field.
Such correlation enhances Papavizas' view (1968) that competitive saprophytic ability (sensu Garrett, 1960), measured by colonization of a suitable substrate, can be considered a highly reliable criterion of 24
survivability of R. solani in soil. The correlation is also an
indirect, but statistically accurate, measure of its Inoculum density in
soil. Similar conclusions were drawn by Sneh ec «1. (1966), who used a
different statistical approach, and by Martinson (1963), who used a
different colonization method.
Enumeration Rhizoctonia ssdaoL
Population studies of specific soil microorganisms usually involve dilution plates using appropriate media. When low populations are to be determined, however, enrichment techniques, followed by cultivation on selective media, commonly are used. For some soilborne plant pathogenic fungi, baits such as perforated test tubes containing a specific medium, seeds, or plant tissue segments may be buried in soil for a given period of time. These can be examined for the presence of the particular organism either directly or Indirectly after further incubation on a specific medium. Because of the relatively low levels of R. solani found in naturally infested soil, most of the methods used for quantitative estimates of this pathogen are Indirect. Such methods
Include the segment colonization method proposed by Davey and Papavizas
(1962), the Immersion tube method by Martinson (1963), and the seed and/or seedling infection method by Menzies (1963), modified by Roberts and Herr (1979). Other more sophisticated methods such as the disk- plate method for selective isolation of R. solani from soil, described by Herr (1973), or the pellet soil-sampler, designed by Henls et al. 25
(1978b), were used specifically for the study of population dynamics of
R. solanl in soil.
Direct isolation of R. solan! from soil, debris particles, and
plant tissues has been aided with selective media like chloramphenicol-
supplemented water agar (Sneh et al., 1966) or complex selective media
developed by Ko and Mora (1971) and Flower (1976). Ferris and Mitchell
(1977) and Vincelli and Beaupre (1989) made a comparison of these and
some other media for the isolation of R. solanl from soil and found that
they were highly effective for inhibiting other soilbome aerobic fungi
and bacteria. No differences were observed among media in estimates of
populations of R. solanl from different soils. Although efficiency of
recovery of R. solanl from soil was equal among media, it was much
easier to locate and identify colonies of R. solanl on the selective
media than on water-agar. The ethanol-potassium nitrate medium,
developed by Trujillo et al. (1987), although expensive, was shown by
Vincelli and Beaupre (1989) to be useful for quantifying soilborne
populations of R. solanl when restricted growth of R. solanl is
desirable, such as when making standard plate counts of serially diluted
organic matter extracted from soil. They also show that the Ko and Hora
(1971) medium was a preferable medium when using a most-probable-number
technique (Alexander, 1982) to quantify propagules in serially diluted
organic matter, because of the low cost of materials and rapid growth of
R. solanl on the medium. 26
A system Involving pre-emergence damplng-off of radish Incited by
R. solanl can be used for the study of survival and activity of the
pathogen. The short time (3-7 days) required for disease expression
makes this host-pathogen relationship Ideal for accumulation of the data
needed for analysis. R. solanl is considered a primitive parasite
(Garrett, 1960, 1979) surviving in soil as "resistant hyphae" and sclerotia (Boosalls and Scharen, 1959), but capable of growth through soil when provided with a food base (Herr, 1973). Susceptibility to
damplng-off is limited in time, because seedlings become resistant with
age (Benson and Baker, 1974).
Trichoderma spp.
In the last 50 years, more than 2,300 papers on Trichoderma spp.
have been published; currently, the activity of Trichoderma has reached
an intensity sufficient for the HENRY DOUBLEDAY RESEARCH ASSOCIATION to
issue a Trichoderma Newsletter.
Taxonomy gng description
According to Blsby (1939), Persoon described the genus Trichoderma in 1794. r. vlrlde was described briefly as the first species, with Pyrenlum llgnonm var. vulgare Tode, 1790, listed as a synonym. Later
Fries reduced to synonymy the names considered to belong to Trichoderma vlrlde Pers. ex Fr. This is the type species of the genus. Persoon's 27
herbarium contains six specimens marked Trichoderma viride. These
represent his concept of the species, and one of them must be the
lectotype of the genus and species.
Odor of Trichoderma cultures was recorded as "pénétrante,
legérèment camphrée" by Vuillemin as camphorated and by others as that
of coconut oil, coconut-cakes (Blsby, 1939). This odor is often very
marked on certain media. Including PDA (originated from organic volatile
products) but most isolations from soil lack the odor.
Bisby (1939) remarked,
"Young Trichoderma still white, but with a suggestion of green color at the center of many tufts. Tufts in the mature condition, i. e. each tuft (0.5-1.5 mm in diam.) is dark olive-green, and under binocular microscope is seen to consist of a mass of dark spores and a few projecting hyphae. A permanent mount shows the spores to be pale greenish brown, globoid, 2.5-4 /i, or ovoid, 3.5-5 x 2.5-3.5 n, surface (as seen with oil immersion) slightly roughened; clusters of spores still present, and a few typical phialides were seen" (Bisby 1939).
Brefeld in 1891 (cited by Bisby, 1939) proved by pure cultures that the ascospores of Hypocrea rufa developed Trichoderma viride as the conidial stage. Trichoderma viride was very common on piles of branches, and developing young stromata of Hypocrea rufa. Since the studies of Tulasne and Brefeld, it has been accepted that the perfect stage of Trichoderma viride is Hypocrea rufa (Rifai, 1969). Variability may be found in a culture from one ascospore (Bisby 1939; Rifai, 1969).
The genus Trichoderma Pers. ex Fr. is distinguished by considering in conjunction these macroscopic and microscopic characters: tufts of cushions of hyphae normally appear on natural substrata or in artificial 28
culture; the tufts are composed of conldiophores, many spores and some
sterile hyphae; conldiophores Indefinite, consisting of an unbranched or
branched hyphae, bearing phlalldes laterally and terminally; phlalldes
surmounted by heads (rarely by short chains) of slime spores; spores
hyaline or brightly colored under the microscope, one celled (Blsset,
1984). Although there are some similarities between TrLchoderma and the
genus Gliocladium, Blsby (1939) considered TrLchoderma to be a monotyplc
genus. This was later contradicted by Rlfal (1969) In his review:
"Since different species of Hypocrea produce quite similar TrLchoderma conldlal states, It follows that TrLchoderma Is not a monotyplc genus but rather it is composed of a number of morphologically closely related species".
The genus is composed by several species-aggregates (Blssett,
1984). The accepted general parameters for proper identification
include colonies, conldiophores, phlalldes, conidia and chlamydospores.
Trtchoderma colonies TrLchoderma produces either loosely
floccose or compactly tufted colonies; both occasionally occurring on
the same colony. In the majority of species the conldlal areas are
formed in distinct ring-like zones which have been shown to be mediated
by light. The color of the colony is largely due to the pigmentation of
the phialospores. In culture, colonies usually grow rapidly, at first smooth-surfaced and almost translucent or watery white, later becoming floccose or compactly tufted, with tufts of various shades of green or pure white coloration (Rlfal, 1969). According to Blssett (1984), growth occurs between pH 2 and 8.2, with a cardinal temperature from 35 to about 38 C. At temperatures of 20-30 C, growth is rapid, but optimum 29
temperature varies with the Individual cultures and with the medium.
Trichoderma conldiophorea The complicated and highly ramified
conidiophores of Trichoderma are conical or pyramidal in outline,
loosely or very compactly tufted, formed in distinct concentric ring
like conldium-producing zones, or borne singly and Irregularly on the
aerial hyphae. The main branches of the conidiophores produce numerous
shorter side branches which arise either singly or more commonly in
groups of up to three, and these in turn may form further smaller side
branches (Rifal, 1969).
Trichoderma ohlalldes Phialides are flask- or nine-pin-shaped,
slightly narrower at the base than above the middle, and attenuated
towards the apex into a narrow conical or subcylindrical neck. They stand at a wide angle to the conldiophore but may bend slightly. There seems to be a correlation between the type of branching system of conldiophore and phialide position. In older cultures, it is often very difficult, to elucidate the characters of the phialides and the conidiophores because the phialides collapse at maturity (Rifal, 1969).
Trichoderma conidla Conidia are slimy phialospores produced singly and successively, accumulating at the apex of the phlallde to form a globose or subglobose conidial head mostly less than 15 /im diam.
Very rarely they may also form a short chain. The phialospores are smooth or minutely rough-walled, hyaline or yellowish-green to dark green, subglobose, short obovold or obovold, ellipsoidal or elliptic- cylindrical to almost oblong, sometimes appearing angular, with bases 30
occasionally distinctly truncate. When young they may contain one to
several oil globules which disappear at maturity (Rlfal, 1969).
Blsby (1939) originally described them as spherical or broadly ovate,
smooth, "hardly exceeding 3.5 fm In the greater diameter, and growing
solitary, or shortly monlllform-concatenate, or even fasciculate".
Trichoderma chlamvdosoores Chlamydospores are Intercalary or rarely terminal which are mostly globose or less often ellipsoidal, colorless and smooth walled. They are present In most species, but
Isolates differ In frequency, size and location of chlamydospores
(namely whether they are formed In hyphae In the medium or both In submerged and aerial hyphae) (Rlfal, 1969). Lewis and Papavlzas (1984a) have shown formation of chlamydospores of the species of Trichoderma In natural substrates.
The species aggregates commonly associated with biological control phenomena can be characterized as follow.
Ik hamatum (Bon.) Bain, aggr. Colonies are always compactly tufted and the branching system of the conldlophores of this species aggregate Is always very complicated. The main branches of the conldlophores are relatively long but they have short and thick side branches, and the apex of each main branch Is typically terminated by a long, whip-like, sterile hyphal elongation. The presence of these sterile hyphal elongations over the tufts of conldlophores of T. hamatum makes colonies appear characteristically whitish or grayish-green.
Typical phlalldes of this species aggregate (those formed by 31
conldlophores in the compactly tufted conidlal areas) are short and
plump and often pear-shaped to almost ovoid. Normally they are very
close to one another, crowding the short but thick side branches.
Fhialospores are of medium to large size, mostly more than 4 pm long and
may be up to 9 /un long, without wall marking. The growth rate of
colonies of T. hamattm aggregates is generally slightly slower than the
other species aggregates (Rifai, 1969).
XJU harzlanum Rifai aggr. Fhialospores are usually small and
only about 2.7 pm diam., globose or subglobose or short obovoid, with
smooth walls. Conidiophores have a complicated dendroid branching
system. Phialides are quite regularly disposed and almost Verticillium-
like (Rifai, 1969).
L. vlride Pers. ex S. F. Gray apgr. Colonies have a dark green coloration, varying from yellow to yellowish or light green. Most isolates exhibit a different and characteristic type of phialide disposition. The majority of T. viride isolates have subglobose phialospores, but other isolates of this species may constantly produce obovoid or ellipsoidal phialospores. All of them have distinctive rough walls, whereas in the other species aggregates no spore markings can be detected by light microscopy. Almost all isolates with rough-walled phialospores emit a characteristic "coconut" odor at maturity; this character is quite useful in recognizing r. vlride, especially if its colonies fail to sporulate. Typical cultures of T. vlride are capable of secreting pigments into the medium and changing the color of the 32
medium (Rlfai, 1969). Trichoderaa viride, described by Bisby (1939),
was green on acid media, but yellow on alkaline. On PDA the cultures
varied more in appearance, made more luxuriant growth and more
overgrowth of hyphae at the line of contact between any two cultures.
Hyphal fusions have been considered a possible criterion for
distinguishing species of fungi, which occur commonly in a culture of
TrLchoderma, Hyphal fusions readily occur with hyphae from T. viride.
The floccose or tufted appearance, the amount of mucus, and even to some
extent the shape and length of the phialide, vary with the individual
and with the conditions of culture. This association of slime with spores is an important character; it precludes the spread of spores by air currents. The tuft spores are often recorded as "powdery", but it is a damp powder very different from the dust of a dry-spored fungus such as Pénicillium. The mycelium of T. viride may become gelatinous.
The length of the indefinite conidiophore varies from 10-1000 um. The most noteworthy variable character is the shape of the conidia, which are subglobose in some isolates, ovoid in others; the same variation Is found in the shape of the ascospores. Some spores occur in chains, the longer chains occurring with more globoid spores. The physical conditions requisite for the production of chains seem to be damp conditions (Bisby, 1939). 33
ESfilfigX Many Isolates found In the soil do not conform well with any of
the nine species-aggregates described by Rifai (1969), but are on the
"border line" between two species. Most Trichoderma isolates are
tested for their biological activity before being characterized
taxonomically. Some isolates possess enzymes such as beta-
(l,3)glucanase and chitinase (Elad et al., 1982a, 1982c), two types of
cellulases (Iwasaki et ai., 1964), xylanases (Nomura et al., 1968) and
other oncolytic enzymes (Toyama and Ogawa, 1968). These enzymes may be
used for identification of isolates, using the isoenzymes as markers.
The two systems can be used for the identification and characterization of isolates, viz., taxonomic differentiation together with the application of physiological and biochemical markers (Chet, 1987b).
According to soil samples taken from agricultural regions, the natural population of Trichoderma is rather low, usually not exceeding
10^ CFU/g soil. It is possible to generate soil suppressive to R. solani by a successive monoculture of radishes (Henis et al, 1978a) and such suppressiveness is accompanied by a significant increase in
Trichoderma propagule density (Liu and Baker, 1980).
Trichoderma species are strongly favored by acidic conditions
(Papavizas, 1981; Ruppel et ai., 1983). Liu and Baker (1980) and Chet and Baker (1980) found that low pH is favorable to the activity of
Trichoderma spp. against R. solani with both in vitro and soil studies.
At pH levels lower than 6.5, the linear growth rate of Trichoderma spp. 34
was significantly greater than that of R. solan!. Maximal conldlophore
formation and conldlum germination of Trlchoderma spp. occurred at pH
values lower than 6. Acidity also accelerated the generation of
suppresslveness In a radish monoculture system. Survival of Trichoderma
spp. generally Is enhanced and suppressive effects are more persistent
In moist soil than In drier soils, since Trichoderma spp. commonly
Inhabit soils having high moisture content (Ahmad and Baker, 1987; Chet
and Baker, 1981; Chet and Henls, 1985; Davet, 1979). In contrast, R.
solan! persists for the longest periods In soils with low matrlc
potential (Baker and Martinson, 1970; Benson and Baker, 1974).
Antagonistic attributes, Many authors have tested the antagonistic ability of species of
Trichoderma against several sollbome plant pathogens (Allen and
Haenseler, 1935; Chet, 1987b; Chet et al., 1981; Chet and Henls, 1985;
Curl et al., 1977; Elad et al., 1980, 1981a, 1981c, 1982b, 1982d, 1983d;
Hadar et al, 1979a, Harman et al., 1980; Henls et al, 1978a; Lewis and
Papavlzas, 1985; Ruppel et al., 1983; Strashnov et al., 1985a, 1985b;
Tronsmo, 1986). The in vitro activity of Trichoderma spp. against plant pathogens has been Investigated extensively (Bell et al., 1982).
Testing for antagonism in a host-pathogen-antagonist system was efficient and rapid when employed with diseases caused by jZ. solan! in natural habitats. Good examples are damping-off of cotton seedlings
(Curl et al, 1977; Elad et al., 1982d; Lewis and Papavlzas, 1985; Ruppel 35
et al, 1983), rot of strawberry (Elad et al., 1981a), rot of carnation
(Elad et al., 1981c), potato disease (Elad et al., 1982a), damping-off
of tomato seedlings (Elad et al., 1982b; Hadar et al., 1979a; Lewis and
Fapavizas, 1985; Ruppel et al., 1983), fruit rot of tomato (Strashnov et
al., 1985a), damping-off of radish seedlings (Harman et al, 1980 ; Henis
et al., 1978a; Lewis and Fapavizas, 1985; Ruppel et al., 1983), damping-
off of pea seedlings (Harman et al, 1980; Ruppel et al, 1983), peanut
disease (Elad et al, 1982b), and root rot of sugar beet (Ruppel et al,
1983). Chang et al. (1985) have demonstrated the increased growth of
some plants induced by the biological control exerted by some species of
Trichoderaa upon plant pathogens. Chet et al. (1979) have published a
compilation of the biological control ability of Trichoderma spp. upon
many soil borne plant pathogens. However, Allen and Haenseler (1935),
Chet et al. (1981), Chet and Henis (1985), Elad et al. (1980, 1982b,
1983d), and Tronsmo (1986) found no clear correlation between the
parasitism exerted on other parasitic or necrotrophic fungi by
Trichoderaa spp. and the antagonistic ability of the latter. So, for
evaluating antagonistic capacity of Trichoderma spp. a rapid, reliable
test is one that considers the variability in pathogens, soils,
temperatures, and other environmental factors and uses host plants.
Baker and his co-workers (Henis et al., 1978a; Liu and Baker,
1980; Chet and Baker, 1981) used a system of radish, R. solani and antagonist, and found it easy and rapid, because the Rhizoctonia damping-off can be detected within 7 days. 36
Enumeration
Quantitative estimation of TrLchoderma spp. in soil is often
difficult because of the relatively rapid growth of some other soil
fungi on conventional agar media. A selective medium is a very useful
tool for isolating Trichoderma from soils as well as for estimating its survival in soil. Elad et al. (1981b) developed a Trichoderma-selective agar medium (TSM) for the quantitative isolation of Trichoderma from soil. Selectivity was obtained by using chloramphenicol as a bacterial inhibitor and pentachloronitrobenzene, fenaminosulf, and rose bengal as selective fungal inhibitors. The low concentration of glucose was enough to allow relatively rapid growth and sporulation for convenient and rapid identification of Trichoderma colonies. Modifications were made to improve the TSM for specific selective isolation of Trichoderma spp. even in the presence of Fusarium spp. in the soil (Elad and Chet,
1983). Other effective selective media were developed by Davet (1979) and Fapavizas and Lumsden (1982). Davet's (1979) medium was useful for the recovery of both Trichoderma and Gliocladium species. Fapavizas and
Lumsden's (1982) medium is effective for isolations of Trichoderma spp. from soils containing Mucorales. All available media are effective for the detection and estimation of Trichoderma spp. population, but different conditions or soils may prove one relatively superior to the others (Chet, 1987b). 37
MATERIALS AND METHODS
Fungal Cultures
The primary fungal Isolates used In this study were RHA-3 of
Rhizoctonia solanl, a pathogen of many dicotyledonous crop species and TR-2
of Trichoderma viride, a hyperparaslte (Table 1). Other fungal Isolates
that were used In some experiments are listed In Table 1. All cultures
were maintained on Potato Dextrose Agar (PDA) slants at about 5 C after
several weeks growth at about 22 C.
Table 1. Identity and source of fungal Isolates
LAB ID ORGANISM ISOLATE AND AG^, ORIGIN CODE^ and SUPPLIER
RHA-BN-1 Blnucleate Shizoctonia BN-1GM460, H.J. Herr.OH RHA-1 R. solani unknown RHA-2 R. solani AG-4, Phaseolus vulgaris RB, D.F. Bateman, NY RHA-3 R. solani AG-4, Pistm sativum C.R. Grau, MN RHA-4 R. solani AG-1, Pinus sylvestris unknown RHA-6 R. solani H.J. Herr, OH RHA-7 R. solani AG-4 L.H. Tiffany, lA RHA-42 R. solani AG-3 type culture #42, N.A. Anderson, MN RHA-43 R. solani AG-1 type culture #43, N.A. Anderson, MN RHA-48 R. solani AG 2-1 type culture #48, N.A. Anderson, MN RHA-77 R. solani AG 2-2 type culture #H3-77, N.A. Anderson RHA-140 R. solani AG-4 type culture #140, N.A. Anderson,MN RHA-462 R. solani AG-5 type culture #462, N.A. Anderson,MN
TR-2 Trichoderma viride, Zea mays L. N.G. Vaklll, lA TR-3 T. hanacum R. Baker, CO TR-5 T. harzianum, ATCC 60850 T-95, R. Baker, CO
^AG Anastomosis group Is a sub-specific grouping for R. solanL based upon mastomosls compatibility among Isolates (Ogoshl, 1987). "Suppliers code for the Isolate, person providing the culture (not necessarily the person who originally Isolated It), suppliers state. 38
Media
The culture media used in this study were:
(Commercial agars were prepared according to manufacturers directions).
1. Difco Potato Dextrose Agar (PDA), amended with streptomycin
sulfate (135 pg/ml)
2. Potato Dextrose Broth (PDB). The liquid remaining after
autoclaving 200 g diced potatoes in 1000 ml tap water for 1/2 hour;
(volume readjusted to 1000 ml), 20 g dextrose, 1.0 g yeast extract,
and about 50 mg antifoam A (30% aqueous emulsion).
3. Difco Noble Agar (NoA). 2% in deionized water.
4. Bioxon Nutrient Agar (NA).
5. Ko Mora agar (selective medium for R. aoltmi developed by Ko and
Hora, 1971), composed of 1 g K2HFO4, 0.5 g MgS04.7H20, 0.5 g KCl,
10 mg FeS04.7H20, 0.2 g NaN02, 0.4 gallic acid, 90 mg fenaminosulf
[sodium p-(dimethylamino)benzenediazosulfonate, 70% (WP) wettable
powder], 50 mg chloramphenicol, 50 mg streptomycin sulfate, 20 g
agar, in 1 liter distilled water. All the mineral salts are added
before autoclaving, and gallic acid, fenaminosulf,
chloramphenicol, and streptomycin sulfate are added to melted agar
media (50 C) before pouring plates.
6. Trichoderma selective medium (TSM) modified from Elad et al.
(1981b), composed of 0.2 g MgS04.7H20, 0.9 g K2HPO4, 0.15 g KCl,
1.0 g NH4NO3, 3.0 g glucose, 0.25 g chloramphenicol, 0.3 g 39
fenaninosulf (60% WP), 0.2 g FCNB (75% WF), 0.15 g rose bengal, 20.0 g
agar in 1 liter distilled water. Mineral salts and glucose were
dissolved separately in small amounts of waterbefore combining and
adjusting volume to 1,000 ml before autoclaving. Rose bengal, FCNB,
fenaminosulf, and chloramphenicol added to melted-cooled medium.
7. Green Bean Medium (GBM) for the production of R. solani sclerotial inoculum. Method was modified by Voland (1989) from van
Bruggen and Ameson (1985). Frozen, standard-cut green bean pods were emptied from the package into metallic basket, defrosted in an autoclave at 100 kFa (15 psi) for 60 seconds, and immediately returned to ambient pressure. The bean pods were placed in glass petri dishes in a single layer (about 35 g of bean pods per dish).
These dishes were then autoclaved, with no wrapping for 30 minutes at 100 kFa, and dried for 10 minutes in the autoclave. The autoclaving was repeated 24 hr later. They were then placed in a single layer in a laminar flow hood to cool, and to evaporate all external water. The sterile pods were seeded with R. solani and incubated at 27 C for 3 to 4 weeks. Sclerotia of R. solani matured within 2 to 3 weeks in mats of hyphae growing on the bean pods. The cultures were incubated for one additional week in order to reduce required drying time and contamination. Mature mats of R. solani were inverted on paper towels and dried in laminar flow hood for 24 hr. Dried mats were ground in a Waring blender, and stored at room temperature and humidity. 40
8. Red beet seed. Red beet seed [Beta vulgaris L. (garden red beet)
cultiver - Detroit Dark Red] was autoclaved three times with or
without water, 24 hr apart.
Inoculum and Inoculation.
ZDS
Sterile PDB in 250 ml erlenmeyer flasks (50 ml/flask) was seeded with a
5 mm diameter plug of JK. solan! and incubated in still culture at 27 C in
the dark for 21 - 28 days. Mycelia and sclerotia were washed in distilled water, and pressed between paper towels until excess moisture was removed.
Weighed portions of the mycelial mats and sclerotia were ground with
distilled water in a Waring Blender. The ground inoculum was added and kneaded into the soil as a slurry and finally blended by shaking in a plastic bag.
m
PDA plates were used for the conidial production of Trichoderma spp.
Plates were seeded with plugs from young cultures, incubated at 28 C under light, for 7-10 days, when the conidia were harvested. Ten ml of sterile distilled water were poured on top of the fungal growth, conidia were dislodged with a brush, concentration determined with aid of a hemacytometer, and concentration adjusted to the desired inoculum level. 41
The thrice autoclaved GBM In glass petrl dishes was seeded with plugs
from young Ji. solani cultures on PDA. Cultures were Incubated at 28 C In
the dark for about 30-40 days, according with the method used by Voland
(1989) modified from van Bruggen and Ameson (1985). The Inoculum added to soil consisted of 0.3 g of compressed sclerotla, Introduced as a dry pellet with a 5 mm diameter cork borer.
Red beet seed
The thrice sterilized beet seeds were moistened with sterile water
(0.5 ml/g) and seeded with mycelial plugs from fresh cultures of the required fungi. Container flasks were kept in still culture at 27 C for
21-28 days.
Soil
Two soils were employed in this study. Both originated from the Iowa
State University Hinds Research Center near Ames, lA. These were a Harlan fine sandy loam and a Splllvllle loam. The former had been in continuous maize culture for eight years prior to a year of weedy fallow. The latter had been in continuous maize culture for 19 years. The soils were collected in the autumn or spring (at least six months after any herbicide or fertilizer applications). The soil was shredded and sieved through a 6 mesh sieve and stored at 28 C and maintained in a visibly moist condition. 42
In some experiments the soil was diluted with washed river sand at a ratio
of 2 parts soil : 1 part sand.
The soil moisture for experiments was adjusted to 51.12 kPa (-0.5
atm. -0.511 bar -7.42 psi). Assessments of soil moisture content at this
level were performed by pressure plate at the Laboratory of Soil Physics,
l.S.U. Two aluminum rings 4.5 cm in diameter, 4.0 cm in height, with
cheesecloth held with a rubber band around the bottom were filled with
soil. This was pressed by tapping slightly on the table. Examples of
measurements and calculations follow for two soil samples (A & B):
RING Ring Ring+Soil Ring+Soil After 48 hr at After 48 hr weight weight water saturated 0.5 atm pressure oven drying (g) (g) (g)* (g)b (O.D.)(g)C
A 23.35 89.57 117.28 94.72 81.26 A-rlng 0.0 66.22 93.93 71.37 57.91
B 23.11 88.21 116.25 93.13 80.10 B-ring 0.0 65.10 93.14 70.02 56.99
^ Filled from bottom up, displacing all air. Pressure plate previously soaked (for 24 hr) with water. Diffusion membrane, 15 bar ceramic plate extractor, sample left until all water was displaced at this pressure (water stops coming out). ^ Oven set at 105 C, until constant dry weight.
Considering that
g of water % Soil Moisture (by weight)- (100) g of 0.0. the following calculations were done: 43
SAMPLE Water at % Soil Moisture Water In % Soli Moisture 0.5 atm. at 0.5 atm. field sample In field sample (g) (g)
A 13.46 23.24 8.31 14.34 B 13.03 22.86 8.11 14.23
a 23705 14.28
To calculate the ml of water needed to adjust 100 g of the field soil
to 51.12 kPa (0.5 atm) moisture tension:
% Moisture at 0.5 atm - % Moisture in sample
ml Water - (100)
100 + % Moisture in sample
The equivalences used for working with the pressure plate were:
1 bar - 0.978 atm 1 bar - 14.5058 PSI
0.5 atm - 0.5101 bar 0.5101 bar - 7.42 PSI
Procedures
Stvdifs ai. £smgi in dual cultures
Rhizoctonia solanl AG-4 (RHA-3) and Trichodemia viride (TR-2) were studied in dual culture with two systems in petri dishes: 44
1) Single Medium System. A 5 mm plug was taken from the leading edge
of a young PDA culture of R. solanl AG-4 (RHA-3) and T. vLride (TR-2) and
placed near the perimeter (one opposite the other) of FDA, NoA, and NA
media in 15 mm x 100 mm polystyrene petri dishes. Ten plates of each
medium were Incubated at 28 C for 15 days and periodic observations of the
activities recorded.
2) Two Media System. Falcon* (Reg T.M., Becton-Dickinson Co.) compartmented petri dishes (15 mm x 100 mm), which were manufactured with four radial dividers (7.5 mm height), were filled with a different medium in each quarter of the dish (5 ml/quarter), as shown in Table 2. The divider Imposed a physical polystyrene barrier about 3 mm high that a fungus growing in one quarter had to transverse before entering the adjacent quarter. The combination of fungal seedings among the quarters in different dishes is outlined in Table 2. The cultures were Incubated at 28
C and observed periodically.
In this system, due to the smaller area of culture medium in each quarter, the inoculum disks used were only 3 mm.
One of the measured parameters was parasitism. For this a careful scanning throughout each quarter using both dissecting (30X) and the compound microscope (40X) was done. Every suspected parasitic relationship, was isolated and corroborated into a slide and observed at higher magnifications (450X, 600X) in the compound microscope. 45
Rhizoctonia-Trichoderma Aui 5Xa£Sffl !ZUl^ difffCSnt tYPfg fi£ @9&l9 AM
containers
A) Plastic Pots (Direct Contact System). Three types of soil were
used, 1) Unlnfested Splllvllle loam soil, 2) Splllvllle loam Infested with
1 g of 12. solanl sclerotla/Kg soil, and 3) Splllvllle loam Infested with
10^ conldla of T. vlride/g soil. The soils were placed In tared separate
stainless steel containers, 45 x 30 x IS cm, containing approximately
20,000 cc of soil. The soil moisture was monitored and adjusted to 51.12
kPa (0.5 atm) and during Incubation the containers were covered with
aluminum foil to avoid moisture losses. Sterile distilled water was added
when needed to readjust for moisture losses.
In the unlnfested soil. It was previously shown that populations of
both fungi were very low or undetectable by baiting and standard dilution
series assays.
Two weeks after the Infestation (equilibrium time), and when the
soils were still In the metallic containers, the Initial population
level of the two fungi In the soils was assessed. For R. solanL
Isolation, about 4 cm layer of soil was removed from the container and 50
sterile beet seeds were dispersed over a nylon net on the soil surface,
another net was placed over the seeds and the soil replaced. After 12 hr
the seeds were recovered, and placed on PDA (5 seeds/dish). Fungal growth from the seed baits was observed microscopically to detect R. solanL. For
Trichoderma spp. Isolation by standard soil dilution assays was performed.
For the unlnfested soil, monitoring with baits and dilution plates was done 46
Table 2. Distribution of media and fungal seedings in four section petrl dishes in two sets of experiments
SET I MEDIUM® PDA NoA PDA NoA SECTION Quarter I Quarter II Quarter III Quarter IV
COMBINATION 1 RHA-3 TR-2 2 RHA-3 3 - TR-2 4 - - TR-2 RHA-3 5 - - TR-2 6 - - - RHA-3
SET II MEDIUM PDA TSM PDA TSM SECTION Quarter I Quarter II Quarter III Quarter IV
COMBINATION 7 RHA-3 TR-2 8 RHA-3 - - 9 - TR-2 10 - - TR-2 RHA-3 11 - - TR-2 12 - - - RHA-3
® PDA- Potato dextrose agar; NoA- Noble agar; TSM- Trichoderma Selective Medium.
to be sure that the populations of the working fungi were still undetectable.
The soils were then re-blended individually and distributed into 8.2 cm diameter by 6 cm tall, slightly tapered, cylindrlc plastic pots. These 47
experimental units contained about 300 cc of soil. Three different baits
were produced for their Introduction Into the soil systems:
a) Sterile baits, consisting of beet seeds, sterilized by
three dry-autoclavlngs, 24 hr apart.
b) Rhlzoctonia baits, consisting of moist thrice autoclaved beet
seeds, seeded with R. solani, which was allowed to grow for two weeks
c) TrLchoderma baits, consisting of moist thrice autoclaved beet
seeds, seeded with T. viride, which was allowed to grow for two weeks
Before placing the baits in the soil, they were sampled to confirm
100% colonization by the seeded fungi without contamination, and no
contamination in the sterile ones. The baits were blended into the soils
at a rate of 50 per pot.
The treatments were as follow, with eight repetitions per treatment:
1. Uninfested soil with sterile baits,
2. Uninfested soil with Rhizoctonia baits,
3. Uninfested soil with TrLchoderma baits,
4. Rhizoctonia Infested soil with sterile baits,
5. Rhizoctonia infested soil with Trlchoderma baits,
6. Trlchoderma infested soil with sterile baits,
7. Trlchoderma infested soil with Rhizoctonia baits.
Weight of each experimental unit was recorded for maintaining moisture levels. Pots were covered with aluminum foil and kept at 21 to 25
C. 48
After 3 days, four experimental units per treatment were harvested to
recover the baits and assess the populations of the fungi. Soil was washed
through a #8 mesh screen, which retained the baits. Baits were recovered
and transferred to 3.8 cm dlam. x 2.2 cm tall PVC cylinders enclosed on one
end with a fiberglass screen. The cylinders with seeds were inserted into
the bottom of a PVC pipe and washed with running tap water for 5 min.
Then, the seeds and cylinders were submerged for 3 min. in a 150 pg/ml
solution of dichloran (Botran^), to reduce Rhlzopus spp. contamination.
Seed baits were blotted and placed on petri dishes. Half of the recovered
baits were plated on PDA and half on TSN, 5 seeds per plate, and incubated
during 7 to 15 days at 28 C. On PDA plates it was possible to determine
the growth of either fungus, but on TSM plates it was possible to count
only Trlchodezwa spp., but only after being incubated 7 days in the
incubator and then exposed to constant cool-light for the other 7 days.
Percentage colonization of the baits measured was an index of fungal
populations and activity.
Five days after incubation the remaining experimental units were
harvested and processed using procedures noted for the 3 day samples.
B) Glass Petri Dishes (Contact and Non-contact Systems).
Ten g of natural uninfested soil (Harlan fine sandy loam) (51.12 kPa)
was added to petri dishes with a 48 hr old colony of R. solani (covering
the entire surface of the plate) on PDA, with the soil placed either on top of the mycelial growth (contact) or in the lid of the inverted plate (non- contact). The soil was amended or not amended with 10 sterile beet seeds 49
(not used as baits but as an organic source). A set of petrl dishes with
PDA, but without R. solanL, were used as controls. The petrl plates were
sealed with paraffin film and Incubated about 80 cm below four cool white
fluorescent light bulbs that were Illuminated for 3 and 5 days. Five of
the repetitions for each treatment were recovered at the third day and five
more on the fifth day. The soil was suspended in water and serially
diluted In sterile water with sample allquots placed on TSM. After 7 to 10
days of incubation under light, Trlchoderma spp. population assessment was
done and expressed as an average number of propagules per gram of soil.
E££S££ a£ levels Sl Infestation mvcooaraslte and tvoe af baiting gg
SPimi and Trlchoderaa son, populations Iq soil systems
The population of Trlchoderma spp. in naturally infested Spillville
loam soil (51.12 kPa) was determined by dilution plate on TSM. Thereafter
the soil was blended and divided into four lots; conldia from a young r.
viride culture were infested to the separate lots to create soils with 2X,
4X, and 8X the original (IX) populations of Trichodenaa.
Three baits were developed: sterilized beet seeds, sterilized beet
seeds cultured with R. solanL for 2 weeks (living Rhizoctonia baits), and
sterilized beet seeds cultured with Rhizoctonla for two weeks then treated
with propylene oxide (2 ml/1) for 48 hr to kill the fungus (killed
Rhizoctonla baits). These baits were blended into the soils Infested with
Trichodenaa at four levels, at a rate of 50 baits per pot (50 seeds/300 g soil) (see prior section). The experimental units (single pots) were 50
weighed to monitor moisture and covered with aluminum foil, then Incubated
for 3 and 5 days at 28 C.
On the third day, a soil sample (10 g) was taken from each pot,
serially diluted In sterile water, and allquots were spread over TSM on
petrl dishes. Baits were washed and recovered from the soil as described
In the previous section, placed either on PDA or on TSM for the monitoring
of RhLzoctonia and Trlchoderam populations respectively, and Incubated for
7 days at 28 C. TSM dilution plates and those TSM plates with baits had a
7-day subsequent Incubation under constant cool light, for promoting
Trichoderma spp. sporulation.
Effect different Rhizoetonia isolates an population levels si
Trichoderma SPP. IN soil
A set of 13 Isolates of Rhizoctonla spp., mostly Rhizoctonia solani, were used to assess the stimulatory nature of the Isolates to Trichoderma spp. In a baiting system. The Isolates and their Identification code, description and origin are shown In Table 1.
The Isolates were seeded separately Into flasks with sterile beet seeds. Incubated and grown for 2 weeks before being introduced into a soil system.
The experimental units consisted of plastic pots containing 300 cc of soil (Harlan fine sandy loam), with 50 Shizoctonia Infested baits each, covered with aluminum foil. Moisture content was monitored as described before. Eight repetitions were made per Isolate; four were harvested at 72 51
hr of Incubation and the remaining four at 120 hr of Incubation with the
recovering method described earlier. Baits were transferred to TSH plates,
Incubated another 7 days at 28 C and 7 additional days under constant light
for sporulation. The percentage colonization of the baits by Trichoderma
spp. was recorded. Also, a 10 g sample of soil from each pot was taken for
quantifying Trichoderma populations by a 10 fold standard serial dilution
assay on TSM plates.
Development GF & Rhlzoctonla-suppressIve soil with AQJI without consecutive
cropping
Soil for this experiment was the Splllvllle loam which was ground,
screened to pass a #8 mesh screen and mixed 2:1 with washed river sand.
Soil at 51.12 kPa moisture level was Infested (or not Infested) with R.
solani AG-4 (RHA-3) (10 g mycelial mats in a slurry/Kg of soil) and planted
(or not planted, left fallow) with hosts and a non-host (Table 3), and then
replanted or left fallow for six cycles. One of the unplanted treatments
was relnfested with the pathogen at the same rate each cycle. The R.
solani Inoculum for this experiment was produced on PDB. Soil moisture was
monitored and adjusted by weight of the experimental units, which consisted
of rectangular polystyrene boxes (27 x 19 x 9 cm deep) with lids and each contained 2,000 g of soil. The boxes and soil were Incubated in a plant growth chamber with 12 hr fluorescent light daily and constant temperature
28 ± 2 C. Each planting cycle was for 8 days. 52
The planting was done by removing about 500 g of soil from the
crisper, leveling the surface and using a vacuum template for the
deposition of 100 seeds per crisper. Seeds were then covered by the
removed soil, and water needed for moisture adjustment was added. The box
was covered with a lid, which did not fit ti^tly, and then the box was
placed in the growth chamber. Four replications were employed.
The seeds of all the crops (Table 3) were tested for germination In
moist-sterile sand and in moist paper towels. The crops included and
treatments are listed in Table 3.
At the end of each cycle, percentage of seedling emergence (and the
respective index of disease incidence) was measured and every second cycle
Triehodenaa spp. populations were determined. Once the percentage of emergence was determined, seedlings were removed, soil was mixed and
Table 3. Crop and fungus treatments used to develop soils suppressive to R. solani
TREATMENT CROP R. solani ADDED
1 No crop No R. solani 2 No crop Once during cycle 1 3 No crop Every cycle 4 Radish (host) No R. solani 5 Radish (host) Once during cycle 1 6 Wheat (non-host) No R. solani 7 Wheat (non-host) Once during cycle 1 8 Cucumber (host) No R. solani 9 Cucumber (host) Once during cycle 1 53
moisture reestablished before planting again. Every other cycle,
populations of the species of Trlchoderaa were measured by the removal of a
10 g soil sample from every crisper, performing serial dilutions and
plating on TSM.
After 6 cycles, a mycelial slurry of R. solanL was introduced into
the soil of all treatments at the same rate as originally, and radish seed
were planted in all of the units. Percentage emergence was measured. The
planting of radish was repeated for 4 cycles.
Measurement Conduclveneaa Indices
After having developed different suppressive soils, the conduciveness
index for Xhizoctonia-damplng off disease in radishes was measured in each
experimental unit according to two different methods: 1) conduciveness
index sensu Henls et al. (1978a), and 2) by soil pellet weight.
Conduciveness Index (CI) sensu Henls et al. (1978a) was defined as
the incidence of disease in a given plant population [(32 radish seeds
arranged in eight 45° radial lines from a central inoculum source and
planted with aid of a template), see Fig. 1]. The first seed in each line
was 1 cm from the center of the inoculum source and the seeds in each
radial line were 1 cm apart. The Inoculum source for CI sensu Henls et al,
(1978a) consisted of 0.3 g of dry R. solanl sclerotia that were compressed
into a 5 mm diam. cork borer and extruded into the soil as a dry pellet.
CI was expressed by the proportion of healthy seedlings developing in the 54
Infested and in the uninfested treatments. Thus, conduclveness can be
described by the linear function:
A - X X CI - — 1 A A
where CI is the conduciveness index, A is the number of symptomless
seedlings in the non-inoculated control, and X is the number of symptomless
seedlings in the inoculated treatment. The number of plants affected by R.
solanl, therefore, is A - X. By definition, the limits of X are 0
(minimum) and A (maximum), whereas those of CI are 0 and 1. Conduciveness
is an inversely linear function. Since many isolates of R. solani are
capable of growth in soil, the conduciveness index from the equation was
developed with this growth factor in mind as well as the ability of the
pathogen to induce disease upon its arrival at the infection court. Thus, a standard percentage disease rating based only on disease incidence in soil uniformly Infested with the pathogen would not provide as complete a picture of potential suppressiveness of that soil (Henis et al., 1978a).
Conduciveness index based on the soil pellet weight (FW), was determined by weighing the soil (g) aggregated by the fungal mycelium around the inoculum pellet (same pellet as in CI sensu Henis at al.) 8 days after infestation. After CI sensu Henis et al, (1978a) was recorded by counting symptomless and symptomatic seedlings, the seedlings were pulled from the soil. The soil pellet formed by the hyphae radiating from the 53
CONDUCIVENESS INDEX
# Rhizootonia solan/ sclerotial pellet. O Radish seed (seedling).
Figure 1. Design of template used to space radish seeds during planting and locate a pellet of R. solani Inoculum in conduclveness assays (1.5 X) 56 inoculum pellet after 8 days of soil incubation was carefully removed and weighed.
StatiBtisal analyggg Statistics to determine differences among the treatments were applied according to analysis of variance and regression analysis, as suggested by
Johnson and Berger (1982). Statistical significance was established at P -
0.05; means were compared with Fisher's least significant difference analysis. Correlations were performed with Statvlew 516 (Abacus Concepts). 57
RESULTS AND DISCUSSION
Fungal Cultures and Media
Rhizoctonia solanL KOhn, AG-4 (RHA-3) when plated on PDA, NoA, NA
and Ko-Hora medium, showed differences in growth on the different media,
particularly in culture density. In most cases, the daily growth rate was about the same, about 1 cm in colony diameter/day. On NoA, vigor and density were less than in the other media. On Ko-Hora, the vigor was very reduced compared to the vigor on PDA and NA; this was particularly noticeable on the aerial mycelia. Ko-Hora medium stimulated more hyphal growth within the agar medium than on the agar surface.
In all of the media, the initial mycelium of R. solanL was whitish, turning to light brown and dark brown at the edges; once the perimeter of the petri dish was reached with the mycelial growth, sclerotial production was stimulated, mostly at the edges of the plate.
Sclerotial production was related to vigor; with thicker colony growth there was more sclerotla production. Vigorous colonies, those on suitable media, produced sclerotla sooner and in greater numbers than those colonies with sparce growth. Sclerotla were always light brown turning to dark brown or even black, measuring no more than 3 mm in diameter. solanL has the ability to change the color of PDA and NA 58
media, apparently through a pigment secreted by the fungus Into the
medium.
R. solani, when growing on FD Broth, required about 2 1/2 weeks
to start sclerotlal production. At this time, the mycelial mats were
harvested, yielding about 10 g of dry mycelial mat for every 20 g of
dextrose.
Trichoderma behaved differently In every medium and according to
the several species studied:
On PDA, growth of Trichoderma virlde, the type species of the
genus according to Blsby (1939), was smooth-surfaced Initially and
almost translucent or watery white and grew very rapidly, 1.4 cm/day; It
showed some erect whitish nycellum after 24 hr. The cultures were white
Initially and then turned to a pale grayish yellowish green, an obvious sign of sporulation. After about 72 hr, floccose mycelial tufts appeared that were dark green with some yellow, and spores were copiously present. The cultures emitted a characteristic "coconut" odor, which was very marked on PDA, even In Immature colonies before heavy sporulation.
On PDA, T. hamatum had compactly tufted colonies, but the conldlatlon occurred In distinct and characteristic ring-like zones, which apparently were mediated by Intermittent light. The presence of sterile hyphal elongations over the tufts of conldlophores of T. hamatum made colonies appear characteristically whitish or grayish-green. The growth rate (colony diameter) of T. hamatum was slower than the rate for 59
r. vLride.
T. harzLanvm colonies grew rapidly and formed initially a
smooth-surfaced, watery white and sparse oycelial mat, but soon aerial
hyphae developed on the culture surface. The conidial areas were
whitish-green, turning to bright green but ultimately they appeared as
dull green. The reverse of the colony, however, remained uncolored.
When Trlchodezma spp. were growing on NoA, T. viride had a slightly faster growth rate than 12. solani. While sporulation patterns on PDA were uniformly distributed throu^out the plate surface, on NoA they occurred as random tufts that were more concentrated at the very edge of the colony; mycelial growth on NoA was whitish while the tufts of sporulation were whitish turning to greenish.
The growth pattern of Trichoderma spp. on NA was very similar to growth on PDA, but sporulation was much more yellowish on NA than on
PDA. NA is a more alkaline medium than PDA, therefore this observation confirms observations of Hilbum (cited by fiisby, 1939), that the fungus culture was green on acidic media, but yellow on alkaline.
On TSM, all species of Trichoderma required light to get uniform and faster sporulation. Growth rate was much slower on TSM than on PDA, about 2.0 mm colony diameter per day, and sporulation was distributed in circular patches or tufts on the plate surface. Mycelial growth was whitish and spores greenish, but later the white mycelium became pinkish, probably absorbing the rose bengal dye from the medium. 60
All of the Trichoderma species studied secreted a yellow-brownish
pigment into the medium, therefore changing the color of the medium.
This was especially evident on PDA and NA.
Studies of Fungi in Dual Cultures
For the dual culture studies, Trichoderma viride (TR-2) was
selected among the other species aggregates of Trichoderma because of
its vigor, speed of growth, and sporulation. This species has been
reported fewer times parasitizing Rhizoctonia solani, when compared with
T. hamaCum and T. harzianum, but this isolate was very aggressive
against the RHA-3 isolate of R. solani.
Single medium system
When seeded on opposite edges of the media in glass petri dishes,
TR-2 and RHA-3 had essentially the same rate of colony expansion on PDA, with the hyphae of the two fungi making contact in about 48 hr. In some instances, T. viride caused inhibition of the R. solani colony long before or immediately before hyphal contact between the two fungi ; often this occurred when the two colonies were 3-5 mm apart. Some isolates of
T. viride have been shown to produce non-volatile antibiotics (Dennis and Webster, 1971a, 1971b). The leading hyphae of Rhizoctonia (those nearest to the T. viride colony) showed morphological changes similar to those reported by Dennis and Webster (1971a). These changes were 61
vacuolaClon and coagulation of cytoplasm which were especially
noticeable: bursting of Shizoctonia hyphae was occasionally observed to
occur before contact with the T. viride hyphae. Such observations
provide supporting evidence that T. viride produced diffusible
compounds, possibly antibiotics, which were active in advance of the
hyphae.
Immediately after contact, T. viride began coiling around the
hyphae of R. solani and eventually penetrated the cell wall. Coiling
was more frequently observed around aerial hyphae of R. solani. Some
coils were very tight, others very loose. Sometimes the hyperparasite
grew parallel to the host and at Intervals attached Itself to the host
hypha by forming a hook-like, short, branch hypha that encircled much of
the host hypha. These resembled appressoria at the tips of the short
branches. Following these interactions, the mycoparasite sometimes
penetrated the host mycelium at points of close contact, apparently by
partially degrading its cell wall. Detachment of a coiled hypha of T.
viride from around the pathogen by gentle shaking, revealed "footprints"
an area that appeared digested or with partial lysis on the host hyphae.
These penetration sites on the host mycelium have been observed by
scanning electron microscopy by Elad et al. (1983d). The cell walls of
R. solani are composed of beta-l,3-glucan (laminarin) and chitin (Ayers
and Adams, 1981; Bamett and Binder, 1973; Elad, 1986), and Trichoderma releases active lytic enzymes, that can digest these components, as shown by Elad et al. (1982a) and Hadar et al. (1979a). Microorganisms 62
capable of lyslng other organisms are widespread In natural ecosystems.
Henis and Chet (1975) have suggested that the extracellular enzymes may
play a role In microbiological control.
Shizoctonia hyphae generally ceased growth soon after contact
with 7. viride, whether or not Trichoderma penetrated into it. This was
as expected from earlier reports (Dennis and Webster, 1971c). In some
of the observations, JZ. solani hyphae had burst, resulting in the
release of cytoplasm from the hyphae and a possible explanation of the
color change in the medium in the contact zone. The hyphal tips of R.
solani began releasing a brown pigment into the medium soon after
association with T. viride, and this began at the edge of the colony and
progressed back through the R. solani colony with further enlargement of
the T. viride colony, over the R. solani colony. Orynbaev, cited by
Dennis and Webster (1971c), studied cytochemical changes in R. solani
induced by Trichoderma in dual culture experiments. He observed
premature vacuolatlon of the cytoplasm, which then became granular and
finally disintegrated. Because of the leakage of cytoplasm, T. viride may have obtained nutrients from R. solani. Thus, by the action of antibiotics, and enzyme systems, or by the complementary action of both, it was feasible that T. viride gained nutrients from the other fungus without parasitism.
As r. viride began to overgrow the colony of R. solani, a line of. sclerotial production began at the leading edge of the R. solani culture. This did not progress backwards into the R. solani culture. 63
About 48 hr later, a dark line of sclerotla was clearly visible in the
zone of first contact between the two fungi. This is similar to
observations by Dennis and Webster (1971c), where sclerotla were formed
by young hyphae of R. solani in dual culture with TrLchoderma, but not
in pure cultures of the same age. At 120 hr, the greenish-sporulating
growth of r. viride had completely overgrown the entire R. solani
colony, and everywhere in the JZ. solani colony it was possible to find
microscopic evidence of parasitism. Mycelial samples from the
interaction region of dual cultures were observed with a compound
microscope. The diameter of hyphae of T. viride was 1.3 to 3 /um and the
diameter of R. solani was 6 to 12 pm, so they could be distinguished
easily from each other.
When r. viride grew toward R. solani, contact was made and
mycoparasitism occurred. Chet et al. (1981) indicated that hyphae of
Trichoderma spp. were directed towards R. solani, which suggested that
this was a tropism response, not a random phenomenon. None of the
observations in this study would support a hypothesis of hyphal tropism
or a chemotactile response.
When plated on NoA, R. solani grew sparsely, but hyphal
elongation was nearly the same as in PDA. Proportionally less aerial
mycelium or branching occurred on NoA than on PDA. The elongation rate
of r. viride hyphae was about the same as R. solani but the hyphae were
very thin. Sporulation was absent until about 8 days after seeding T, viride. Hyphal contact by the two fungi resulted in hyperparasitism of 64
R. solanl. The parasitic phenomena were the same as in FDA. Hyphae
were sparse, JZ. solanL continued to grow through the T. viride colony
where the R. solani escaped 7. viride, and no sclerotia were formed by
R. solani. There was no evidence of antibiosis. There were few aerial
hyphae present on NoA. Sporulation by 7. viride began around the
Inoculum disk, and spores were whitish; later tufts of sporulation
occurred in the area of contact with R. solani.
The poor growth of R. solani on TSM agar and T. viride on Ko-Hora
medium precluded any antagonism studies between these two fungi on these
media. It was desired to observe the Interaction of the organisms under conditions where one or the other was strongly inhibited by the milieu.
Direct inspection on the petrl plates by light microscopy was carried out before and after contact of the hyphae of the two fungi in
PDA and NoA. It was difficult to select a standard time to examine the cultures for interactions. In some dishes, T. viride hyphae coiled around the hyphae of Rhizoctonia as soon as the two colonies made contact. In others, colling did not occur until the Trichoderma hyphae had penetrated far into the host fungus colony. Also, some variation in coiling behavior was observed at different times. No colling or parasitism by T. viride was observed with subsurface hyphae. In some rare instances, 7. viride hyphae penetrated and grew internally in the
R. solani hyphae. 65
madia system
With the second system using the compartmented petri dishes
divided into four quarters, observations on behavior and growth were
more complicated. Two experimental designs were used. PDA and NoA were
used in the first one, with combinations 1 to 6, and PDA and TSM in the
second one, with combinations 7 to 12 (Table 2).
Combination % After 3 days of incubation, RHA-3 had
completely colonized the quarter were it was seeded (quarter I, PDA),
and had grown over the divider and about 1 cm into the neighboring
quarters (quarters II and IV with NoA, and the opposite quarter III with
PDA), in a radial colony of 3.3 cm radius from the plug, but with less
dense growth on the NoA.
TR-2 had germinated and grown to occupy the quarter where it was
seeded (quarter II with NoA) less branched and less vigorous than the R.
solanl which was spreading into quarter II. There was microscopic
evidence of parasitism by coiling around and penetration of the R. solanL hyphae at the points of contact of the two colonies. T. vLride had sporulated (Immature whitish conidla) in small whitish tufts.
Because of the mixture of colonies, it was difficult to estimate the radius of the growth of Trichoderma. After 4 days of incubation, the radius of the RHA-3 colony had reached 4.5 cm, was still uniform, and radially distributed on the other quarters. The spores of TR-2 were green and present in dispersed tufts only in quarter II (NoA). At this time, the microscopic evidence of parasitism was demonstrated on RHA-3 66
in the other quarters, especially those areas of the R. solani colony
near to the compartment dividers. Although parasitism of R. solani was
evident, sporulation of 7. vlrlde occurred only in quarter II.
After 5 days of incubation, RHA-3 had reached a radial colony of
6.2 cm; evidence of parasitism was found in most samples regardless of
the quarter, but no sporulation of T. vlrlde was observed in any
quarter except the one initially seeded with T. vlrlde, quarter II.
Nine days after seeding, the results were essentially the same as for 5
days. However, after 13 days T. vlrlde was finally beginning to
sporulate on the older aerial hyphae of R. solani, starting initially with those hyphae near the dividers separating quarter II from quarters
I and III.
Combination % Rhlzoctonla seeded alone had grown radially to the other quarters as a colony with a radius of 3.3 cm by the third day and 4.3 and 6.6 cm by the fourth day and fifth day respectively. Radial growth in NoA was equal to growth on PDA, but the density of growth on
NoA was sparse and less branched, with smaller diameter hyphae and less aerial hyphae than on the PDA quarters.
Combination ^ TR-2 was seeded into quarter II (NoA) and by 3 days it had grown throughout the seeded quarter. It continued to develop within the seeded quarter with NoA, showing whitish and later greenish sporulation in tufts, and few aerial hyphae. At seven days, T. vlrlde traversed the quarter II barrier dividers and started growing on both neighboring PDA quarters; on the eighth day after seeding It spread 67
into the opposite NoA quarter. In the petrl dishes with combination 1,
where R. solani was also present, 7. viride was able to traverse the
dividers by 4 days, aided by R. solani hyphae that had traversed the
divider and grown Into the T. viride culture on NoA.
Combination ^ Trlchoderaa was seeded on PDA (quarter III),
and after 3 days of incubation had grown radially into the neighboring
quarters, with a colony radius of 4.1 cm. All of quarter III had dense
sporulation (green conidia) after 3 days. There was microscopic
evidence of parasitism in quarters I and IV where the R. solani colony
(radius of 2.8 cm) had spread, but there was no sporulation of T. viride
in any quarter but quarter III. R. solani did not develop into the
quarter III. Parasitism of R. solani in quarter IV was more extensive
than in quarter II of combination I above. It appeared that r. viride was a better parasite of R. solani growing in NoA when the origin of the r. viride culture was from PDA rather than NoA.
By the fourth day, the T. viride colony radius was 5.0 cm and was more dense on NoA than at the third day. The R. solani colony radius was 4.1 cm, and where the two colonies met in quarter I (PDA) parasitism of the young R. solani hyphae was extensive and T. viride was initiating sporulation. R. solani hyphal elongation was stopped, the medium and culture were beginning to brown. This was typical of the reaction observed in the prior dual culture experiment on PDA. R. solani did not grow into quarter III, the section initially seeded with r. viride. At 5 days 7. viride was aggressively overrunning R. solani 68
in quarter III and sporulation of T. viride on the R. solani in quarter
III was heavy. The culture of R. solani in quarter III formed the dark
zone of pigmentation in the agar near the margin of the colony. T.
viride was sporulating in all quarters but quarter IV, the NoA quarter
initially seeded with JZ. solani, although parasitism of R. solani was
good in quarter IV. By nine days R. solani in quarter III was
completely overrun by T. viride and sporulation was heavy except for R.
solani culture areas that were darkly pigmented (nearly black).
Sporulation of 7. viride in quarter IV (NoA) was only sparse, whereas it
had many tufts of sporulation in quarter II (NoA).
Combination £ The radius of the T. viride colony was equal
in all compartments, measuring 4.2 and 6.2 cm on the third, and fifth
days after seeding on PDA. The green sporulation on the third day was
restricted exclusively to the seeded quarter, but there was immature
white sporulation in all the other quarters. On the fifth day all
sporulation was green, totally distributed over the PDA surface, and
being restricted to small dispersed tufts on NoA. In this combination,
r. viride traversed the dividers surrounding quarter III (PDA) by the
third day. In combination 3, T, viride was seeded into NoA and it was
unable to traverse the dividers until seven days.
• Combination 6 R- solani was seeded on NoA quarter and made sparse growth but grew into a dense colony on the PDA quarters and a sparse colony on the opposite NoA quarter. The radius of the colony was
2.5, 3.0 and 5.2 cm on the third, fourth and fifth days, respectively. 69
Combinations 7-12 In the second system with compartmented
petrl dishes, which contained PDA and TSM and combinations 7 to 12, R.
solani was strongly Inhibited by TSM and did not grow and appeared to
die when seeded on TSM (combinations 10 and 12). When seeded on PDA, R. solani would not establish In the adjoining quarters with TSM. The advantage of T. viride was marked because of the use of TSM.
When R. solani was growing on PDA and T. viride on TSM
(combination 7), the growth rate of the later was faster on TSM than when R. solani was not present In the petrl dish (combination 9). A volatile stimulus may be suggested.
Rhizoctonia-Trichodenaa Dual Systen
In Different Types of Soils and Containers
Plastic (direct contact system)
The activity of JZ. solani and Trichoderaa spp. In soil was assessed by their Isolation by beet seed baits from soil that was
Infested with T. viride, R. solani, or unlnfested. The beet seed baits were previously sterile, or infested with T. viride or R. solani before placing then In the soil.
The sterile baits did not detect Rhizoctonia or Trichoderma spp. from natural populations in the unlnfested soil (Table 4). Therefore, in this naturally buffered soil, the organic matter provided by the sterile baits was Inadequate for stimulating the growth of either fungus in 70
Table 4. Percentage colonization of beet seed baits (sterile or previously Infested with JZ. solanl or T. vlride) in soils (unlnfested or infested with T. vlride or JZ. solanl) after 3 and 5 days incubation in the soil
% Colonization of baits BOZ-S EfiO-S Soil infestation Bait tvne TrishO • * KhiZOS. Tfish?. KhlZPC.
None Sterile 0 0 0 0 None TR-2 infested - 0 - 0 _( None RHA-3 infested 25 - 60 TR-2 Sterile 96 0 100 0 TR-2 RHA-3 infested 91 - 99 - RHA-3 Sterile 5 74 29 77 RHA-3 TR-2 infested - 18 - 8
^ Trichoderaa spp. growing out of the baits plated on TSM after 3 and 5 days incubation of the baits in the soil. " Rhizoctonia spp. growing out of the baits plated on PDA after 3 and S days incubation of the baits in the soil. ^ Never assayed.
competition with the natural microflora. When Rhizoctonia was
Introduced into the soil, either as a prior resident of the baits or through a uniform infestation of the soil with sclerotia, the frequency of Trlchoderma isolations Increased to 60% and to 29% respectively.
Trlchoderma spp. were stimulated to colonize the baits previously colonized with R. solanl, especially after 5 days. Sterile baits placed into a Rhizoctonia infested soil were able to recover Rhizoctonia from
77% of the baits, at 5 days and Trlchoderma spp. were recovered from 29% of the baits. R. solanl Isolation from the previously sterile baits was nearly as frequent at 3 days (74%) as at 5 days (77%), but the frequency 71
of Trichoderma spp. isolation Increased from 5% at 3 days to 29% at 5
days. R. solanl Invasion of the baits evidently stimulated invasion of
the baits by Trichoderma spp. In the soil infested with T. vlride, both
the sterile baits and the baits containing R. solani became infested
with Trichoderma at about the 100% level after 5 days In the soil.
There is an interesting discussion by Baker et al. (1984)
regarding the proper use of controls and the scientific method with
special emphasis on biological control. The biological control agents
are commonly cultured on a sterile medium, and the medium and agent are
then infested into the soil. They suggested that sterile media (sterile
beet seeds) not infested with the biological control agent or the pathogen have been considered as good controls. Yet, the sterile medium is not the same substrate as that acted upon by the agent (fungi) during incubation. Further, the addition of previously undigested organic matter to soil can induce increased disease severity and/or toxin production resulting from the activity of soil microflora leading to plant damage or disguise of results, or, alternatively, can lead to biological control. So, sterile beet seeds have their own
"interference" and may not be an appropriate control.
Glass Petri dishes (Contact and non-contact systems)
It may be suggested, based upon the prior experiments, that R. solanl may stimulate Trichoderma spp. to germinate and reproduce in the natural soil environment. R. solanl was cultured on FDA and soil was 72
placed either on the young culture or in the lid of an inverted petri
dish culture. In the former arrangement, the soil was contiguous with
the R. solanl culture and in the latter there was an air gap of about
1.0 cm between the R. solanl culture and the soil. PDA without R.
solani was used for the no JZ. solanl controls. The lowest population of
TrLchoderma CFU (colony forming units) that could be detected would be
100 CFU/g soil. The population of Trichodermm in the soil was <100 CFU
and never became detectable in the control where soil was placed in the
petri dish lid and it never contacted the PDA (Table 5, Fig. Al). When
sterile beet seeds were added to the soil the population Increased to
400 CFU/g after 3 days and 4000 CFU/g after S days. If R. solanl was
present on the culture, although the soil was placed in the lid, the
population of r. virlde increased to about 15,000 CFU/g in 3 days, and 39,000 CFU/g in 5 days. If R. solanl was not growing on the medium,
even the introduction of a food source, the beet seeds, did not Increase
Trlchoderma populations as much as when R. solanl was present, because
Trlchoderma populations increased to about 2,500 CFU/g in the first 3 days, and about 22,000 CFU/g during the next 2 days. When the soil was placed on top of the growing PDA culture of R. solanl, regardless of the presence or absence of beet seeds, the population of Trlchoderma increased as much as when the soil was placed in the lid (to about
40,000 CFU/g). These data would support the suggestion that a volatile compound produced by R. solanl may be involved in the Increase of
Trlchodermt populations (Table 5). 73
The beet seeds were used to assure that the stimulus was
acting Independently from an organic source. When Rhlzoctonia was
Introduced Into the system, either placing the soil on top, in direct
contact with the grown colony or in the inverted cover of the petri
plate, the Trichoderma spp. population increased regardless of the
amendment with.the beet seeds. The treatment that produced the highest
increase was when the soil was put on top of the Rhlzoctonia colony plus
the beet seed amendment. These data are consistent with a hypothesis
that Rhlzoctonia is producing some substance that promotes the
Table 5. CFU of Trichoderma spp./g soil after 3 and 5 days contact or non contact^with culture of Rhlzoctonia solani RHA-3 on PDA or PDA only, with or without 10 beet seeds/10 g soil amendment TREATMEgTS QTV/g soil Soil location RHA-3° Beet seeds* Pay 3 Pay ? . On culture present 15.800 A 37,500 A< On culture present + 20,000 A 40,500 A On PDA absent 2,500 B 22,800 B On PDA absent + 2,800 B 21,000 B In lid present 14,800 A 38,000 A In lid present + 16,500 A 39,900 A In lid absent 0®C 0 D In lid absent + 400 C 4,000 C
^ Contact is 10 g soil spread over PDA culture in 10 cm petri dish. For non contact the soil was placed in lid of inverted petri dish with PDA culture. ^Whether or not RHA-3 was cultured on PDA in petri dish. ^Whether or not beet seeds were amended to soil. ^ Numbers in each column followed by different letters are significantly different, P-0.05 ® Not detectable; < 100 CFU/g soil. 74
Trichoderma population Increase, and the Trichoderaa spp. populations
are increased additionally with the beet seeds as an added organic
substrate. When the soil was on top of the R. solanL culture, the
Trichoderma spp. could get food not only from Shlzoctonia, but also from
the substratum (PDA) underneath the colony, therefore its ability to
reproduce is greater. The best evidence that there might be a volatile
stimulator of Trichoderma spp. was stimulation of Trichoderma
reproduction when the soil was not in contact with the R. solani
culture. The only food source that the Trichoderma spp. had in this
arrangement was the soil nutrients, except when the beet seeds were
present.
Effect of Levels of Infestation of Mycoparaslte
and Type of Baiting used on R. solani and Trichoderaa spp.
Populations in Soil Systems
The base population of Trichoderma spp. in the soil was assessed
as 2.0 X 10*, based upon dilution plate assays on TSM. T. viride spores
were added to the soil to obtain infestation levels of 2X, 4X, and 8X
the original population (X).
The Introduction of no baits, sterile baits or killed baits
(Table Al, Figs. 2, 3, and 4), resulted in no significant change in the populations of Trichoderma spp. in the soil mass. This is depicted by the horizontal orientation of the graph lines; regardless of the level of r. vlride Infestation, the fungus population did not change
appreciably. Incorporation of baits with living R. solanl Into the soil
Induced a rapid Increase In the population of TrLchoderma propagules in
the soil (Table Al, Fig. 5). The Increase was apparent after 1 day and
continued to Increase with each sampling. The largest population
Increase with R. solanl Infested baits occurred with the natural
population (IX) of Trlchoderaa (Fig. 5). The 7. vlride spores Infested
Into the soil evidently tempered the build up of the natural populations
(Figs. 2, 3, 4). The highest population of 1.12 X 10 ^ CFU/g soil was
obtained with the unlnfested (IX) soil. The Infested 7. vlride may have
Invaded the baits before R. solanl could develop very much from the
baits Into the soil.
When the data are depicted to compare the Influence of the
different baits at an Infestation rate of T. vlride (Figs. 6, 7, 8 and
9), the Increase of Trlchoderma spp. propagules In the presence of the
living Shlzoctonla bait was exponential; the other baits had little
effect comparatively on the populations of Trlchoderma spp. In the soil.
When R. solanl was killed In the baits before adding them to the soil,
the presence of a fungal substrate did not Induce Trlchoderma activity.
The baits recovered 72 and 120 hrs after Incubation were placed
on PDA and on TSN to determine Rhlzoctonla and Trlchoderma populations, respectively. The base population of Trlchoderma spp. in the soil was much higher than in prior experiments and more than 50% of the sterile baits were invaded by Trlchoderma spp. (Table 6). Addition of spores of 76
Table 6. Percentage of baits yielding R. solani and Trlchoderma spp. upon culturing after 3 and 5 days incubation in soils of different initial infestation with Trlchoderma spp.
Bait* Infestation Percentage Recovery from baits Type Level 3 dava 5 davs RhlzoetontaP TrlehodemaC RhiZPCtOn&a Trishodptmp
IX 0 70 0 72 Sterile 2X 0 48 0 SO baits 4X 0 63 0 79 8X 0 62 0 70
IX 0 68 0 70 Killed 2X 0 40 0 60 R. solani 4X 0 60 0 71 ax 0 54 0 72
IX 46 61 21 77 Living 2X 30 73 9 83 R. solani 4X 12 83 0 95 8X 3 94 0 99
* Sterile beet seeds, sterile beet seeds as substrate for R. solani, and beet seeds where R. solani in seed was killed by propylene oxide. °Cultured on PDA. ®Cultured on TSM.
T. viride to the system did not affect the rate of Trlchoderma recovery from the sterile beet seed bait and baits containing killed R. solani.
By contrast, the added inoculum of T. vlrlde was reflected in the increased rate of Trlchoderma spp. recovery from baits with living R. solani. The added T. vlrlde spores evidently were not stimulated to i— MMtaUM - -ùr - M##*#*#» —Q— MMtoUM + - MmIaUm Laval IX U*«l IX LwW 4X LwW tX
O 1000 o o
o« N, —o o — M ©
10
0 1 2 3 4 5 6 7 8
Days of Incubation
Figure 2. Populations of Trichoderma spp. sampled for 8 days by soil dilution assays on TSM from soils infested at four levels with Trichoderaa spp. 4— MMtoUM - "û" - MMUUM —-0-- MMUUM + MMteUM LwW IX Uv«l XX LMM 4X Lmal #X
1000
*• o en
10 o
0 1 2 3
Days of Incubation
Figure 3. Populations of Trichodenaa spp. sampled for 6 days by soil dilution assays on TSH from soils infested at four levels with Trichoderma spp. and amended with sterile beet seed baits i— MMUVM - -ùr - MMUHM —0— IwW IX Uval tx Lnti 4X LwW SX
1000
s - N o
—A
10
0 3
Days of Incubation
Figure 4. Populations of Trlcboderma spp. sampled for 6 days by soil dilution assays on TSH from soils infested at four levels with Trichoderaa spp. and amended with beet seed baits preinfested with R. solanl and then sterilized with propylene oxide •I MmUUM - -ùr - —0-- kfAttadM + InfMtotlM lw«l IX U*«l 2X L«««l 4X l##W *X
1000 o o o T- X
I looif # O o
s 10 o
0 1 2 3 4 6
Days of Incubation
Figure 5. Populations of Trichoderma spp. sampled for 6 days by soil dilution assays on TSH from soils Infested at four levels with Trichoderma spp. and amended with beet seed baits that were infested with R. solani 81
Invade the baits as a heterotroph but invaded them as a parasite of R.
solanL. The ability of Trichoderma to displace a primary colonizer,
seems to depend directly on the inoculum potential or population level
of the antagonist in the soil. When sterile baits and baits with the
killed R. solan! were placed in the soil, the native population of
Trichoderma spp. had sufficient inoculum potential and a good
competitive saprophytic ability for colonizing these baits. The
presence of living Rhizoctonla in the soil helped to trigger the
Trichoderma spp. population explosion and subsequent parasitic activity.
Shizoctonia populations in soil must have been very low and/or
their activity was greatly inhibited (possibly by the high populations
of Trichoderma spp.). R. solani was recovered by the baits only when It
was previously Introduced into the soil with the same bait. The baits
with living R. solani were 100% Infested before introduction into the
soil, yet less than 50% of the baits yielded R. solani when sampled 3
days later. The T. viride inoculum added to the soils caused a further
reduction in the number of baits yielding R. solani; the reduction was
directly related to amount of T. viride Inoculum added to the soil.
This apparent decolonization of the beet seed baits in soil was greater
after 5 days incubation than 3 days incubation in the soil.
The need for using baiting for quantitative estimates of R. solani was previously shown by several authors (Davey and Fapavizas,
1962; Henis et al., 1978b; Martinson, 1963; Menzies, 1963). Single hyphae and propagules of R. solani are rarely detected from soil sample •— No —àt" sterile —Killed • Living Balls baits Rhizoctonia Rlilzoctonla
100 o« e 01
2 10 o
0 1 2 3 4 6 6 7 8
Days of incubation
Figure 6. Populations of Trichoderma spp. sampled periodically by soil dilution assays on TSH from a soil amended or not amended with various beet seed baits •— No —A Sterile —Killed Living BalU balte Rtilzoctenla Rhizoctenla
3 1000 o o X
o 100 m o a
3 10 o
0 1 2 3 6 8
Daye of Incubation
Figure 7. Populations of Trichoderma spp. sampled periodically by soil dilution assays on TSM from a soil infested with T. virlde at 2X the normal population of Trichoderma spp. and amended or not amended with various beet seed baits •— No —A - Sterile —#— Killed •••+.. Living Baits baits Rhizoctonia Rhizoctonia
3 1000 o o X +
o 100 m o a
10
0 1 2 3 4 5 6
Day# If Incubation
Figure 8. Populations of Trichoderma spp. sampled periodically by soil dilution assays on TSH from a soil Infested with T. vlride at 4X the normal population of Trichoderma spp. and amended or not amended with various beet seed baits "•— No —A sterilft —#— Killed + Living Balls balU Rlilzoctonia Rhlzoctonla
o 100 w o a
10
0 2 3 4 6 6
Day# of InciAatlon
Figure 9. Populations of Trlchoderma spp. sampled periodically by soil dilution assays on TSH from a soil Infested with 7. viride at 8X the normal population of Trlchodenm spp. and amended or not amended with various beet seed baits 86
when placed on media after soil dilutions. Their propagule populations
are commonly so low that 10 g of soil or more are needed to detect one
propagule. Larger propagules of R. solanl (large sclerotia), are
capable of producing hyphal growth at a rate of 1 mm/hr (Benson and
Baker, 1974) and are less sensitive to fungistasis and suppression than
small propagules. Selective media have been developed for JZ. solanL (Ko
and Hora, 1971), yet the growth of microorganisms other than J2. solan!
on the media cannot be completely eliminated. Quantitative assays for
jZ. solanL require timely enumeration of the colonies (usually with less
than 24 hrs variance) before other fungi mask the R. solanL colonies.
Effect of Different RhLzoctonLa Isolates
on the Population Levels of TrLchoderma spp. In Soil
The prior research that demonstrated the stimulatory nature of R.
solanL towards TrLchoderma spp. in soil was done with one isolate, RHÀ-3
belonging to the anastomosis group AG-4. The universality of this
stimulatory effect among RhLzoctonLa spp. was tested with 13 isolates,
which were cultured on beet seeds and introduced into a soil. When
baits were recovered after 3 days and plated on TSM bait colonization by
TrLchoderma spp. ranged from 1.0 to 45% with a mean of 22.7% (Table A2,
Fig. 10). The mean number of isolates of TrLchoderma spp. isolated by the baiting procedure after 5days incubation in the soil was still 87
22.8%. The highest percentage of isolation of Trichodemta spp. by
baiting was with RHA*3 after 5 days. Three isolates of Rhizoctonia,
viz., BN-1, RHA-43, AND RHA-48, when infested into the soils with the
baits, were associated with the lowest frequencies of Trichoderma
isolations. These isolates were the bi-nucleate hyperparasitic
ShizoctonLa, an AG-1, and an AG2-1 types respectively (Table 1). When
the isolation frequencies of Trichoderma spp. for 3 days and 5 days were
averaged, the four isolates of the Rhizoctonia that supported the
highest frequencies of Trichoderma spp. isolation by baiting were the
four AG-4 types (Table 1 and Fig. 10). Some of the other isolates were
also good hosts for Trichoderma spp.
The introduction of some Rhizoctonia infested baits into the soils, resulted in a significant increase in the population of
Trichoderma spp. propagules in the soil mass (Fig. 11). After 3 days incubation of the infested baits in the soil, only four Rhizoctonia infested baits induced a two fold increase in the population of
Trichoderma spp. propagules in the soil mass. These were the baits infested with the four AG-4 isolates of R. solani, viz. RHA-2, RHA-3,
RHA-7, and RHA-140 (Table A2, Fig. 11). This phenomenon with the AG-4 isolates was magnified greater after S days incubation of the
Rhizoctonia infested baits in the soil. The five day sampling of the soil showed that slight, but significant increases, in population of
Trichoderma spp. in the soil mass were associated with baits infested with RHA-1, RHA-42, and RHA-77 in the soil (Table A3). Although the AFTER I 1 AFTER 3 day* 5 day*
60 -
d 50 - a w
BN-1 1 2 3 4 6 7 42 43 48 77 140 462 Rhizoctonia isolates (RHA #) in baits
Figure 10. Percentage of beet seed baits precolonized with Shizoctonia sp. that yielded Trlchoderma spp. after 3 and 5 days incubation in soil Inoculation lZ3 After After Day 72 hr 120 hr
o 400 o o
300 -
00
200 -
e# I 100 -
BN-1 1 3 4 6 7 42 43 48 77 140 462 Rhizoctonia iaoiat## (RHA #) in baits
Figure 11. Populations of Trichoderma spp. in the soil mass before and 3 and 5 days j after introduction of beet seed baits precolonized with different isolates of j Rhizoctonia i i 90
baits were removed from the soil before sampling the soil mass for
TrLchoderma spp., some of the population Increase In the soil mass may
have been spores produced during parasitism of the baits.
Most of the Isolates used In this limited study were parasitized
by the TrLchoderma spp. native In the soli, but only those Isolates
belonging to the AG-4 type promoted the reproduction and proliferation
of TrLchoderma spp. In the soil mass.
The correlation coefficients between percentage colonization of
the baits by TrLchoderma spp. and the population of TrLchoderma spp. in
the soil mass were R - 0.62 (a - 0.02) and R - 0.66 (a - 0.01), after 3 and 5 days of incubation respectively.
An AG-4 isolate of R. solanL was used by Baker and his co-workers
(Chet and Baker, 1981; Henis et al., 1978a; Henis 1979; Liu and Baker,
1980) to develop soils suppressive to R. solanL by rapid monocropping.
This suppresslveness was related to a rapid increase in TrLchoderma spp. in soil in these studies.
Development of RhLzoctonLa Suppressive Soils
with and without Consecutive Cropping
The radish and wheat seeds used in this experiment had good germination (Table 7), but the cucumber seed germinated poorly (about
70%), although it had a germination percentage of 90% on the current 91
year label. This was the seed lot with the highest reported
germinability for cucumbers and experiments were initiated before the
germination was checked.
Table 7. Percentage germination of crop seeds by two germination testing procedures
Crop Sterile sand Paper towel Average method method
Radish 100% 100% 100% Wheat 97% 99% 98% Cucumber 65% 75% 70%
The emergence of radish and wheat seedlings in the unlnfested soil during the six cycles of planting averaged about 93 and 87% respectively (Table 8, Fig. A2). Cucumber emergence was about 64% in the absence of added R. solani inoculum during the six cycles of cropping. Although some variability was experienced with each crop, the emergence of radish and wheat remained fairly constant during the experiment. The emergence percentage of the cucumbers was about the same during the first four cycles of planting and then declined during cycles 5 and 6.
Infestation of the soil with R. solani was associated with a reduction in emergence of all three crops, compared to the emergence of 92
the unlnfested controls (Table 8). The decrease in radish seedling
emergence was greater than with the other two crops. Freemergence
damping off decreased with each cycle of cropping and the emergence
differences between infested and non-infested soils disappeared by the
fourth cycle.
Table 8. Percentage emergence of crop seedlings during six consecutive cycles of plantings in soil either infested or not Infested with R. solani
Crop RhizocConia 1 2 3 4 5 6
Radish . » 96.75 90.00 86.00 92.00 95.75 96.25 Radish + 29.50 42.00 54.50 91.00 96.50 98.50 Wheat - 92.75 92.25 92.25 71.50 90.75 83.75 Wheat + 74.75 79.00 84.00 93.50 92.25 92.25 Cucumber - 68.00 68.50 70.25 68.00 56.50 51.00 Cucumber + 52.50 52.00 49.00 54.50 49.50 57.00
^ R. solani inoculum added (+) or not added (-) to the soil.
Only cucumber and radish are considered hosts for the AG-4 type isolate that was used. This Isolate of R. solani had some pathogenic activity towards wheat, althou^ it was never recovered or relsolated from seedlings. Most of the reduced emergence of cucumber (Table 8) was attributed to a very low percentage of germination (Table 7) rather than parasitism by R. solani. Isolations from cucumber seedlings killed by 93
preemergence damping off consistently yielded an unidentified Fusarium
sp. that was also found to be seed borne. The effects of Jt. solani on
emergence were especially noticeable when the loss of these effects are
graphically displayed (Fig. A2). The treatment Including radish and
Shizoctonia showed a marked Increase In emergence during the first four
cycles (from 29.5 to 98.5%). Except for wheat exposed to R. solani, the
other treatments had no effect on seedling emergence.
The dynamics of Trichoderma spp. populations were studied by sampling the soil In every treatment before the first planting and after cropping cycles 2, 4, and 6. All treatments where R. solani inoculum was added to the soil had significant Increases In Trichoderma populations (Table 9, Fig. A3). With all three crops It was possible to
Increase the population of the antagonist, but only when R. solani was added to the system. Amending non cropped soil with R. solani caused the Trichoderma spp. populations to Increase nearly as much as when crops were Included In the system with R. solani (Fig. A3). Adding the
R. solani Inoculum each cycle, resulted In the largest Increase In
Trichoderma spp. populations, which was 1.9 X 10^ CFU/g after six cycles
(Table 9); this was > 1700 X the original population.
When R. solani was added only once, either with or without cropping, the highest populations of Trichoderma spp. In soil occurred with the sampling after two cycles; thereafter the populations decreased somewhat (Fig. A3). The greatest decrease was with the non-cropped soil. This may Indicate that R. solani, developing as a parasite on the 94
crops, was reproducing and providing a continued stimulus for
Trichoderaa spp. to multiply. The amount of R. solani Inoculum added,
each cycle was evidently more than the amount added through parasitic
activities each cycle. The rate of decrease in damping off in soil
cropped to radish (Fig. A2) did not relate directly to the populations
of Trichoderma spp. in the soil (Table 9). The highest population of
Table 9. Populations of Trichoderma spp. sampled periodically from soil cropped for 6 cycles or not cropped and Infested or not Infested with R. solani.
Trichoderma spp. (CFU/g of soil)
Crop Rhizoctonia 0 2 4 6
Radish . b 3,672 8,664 875 5,811 D* Radish + 3,738 985,000 400,000 249,125 B Wheat - 1,038 1,650 956 3,281 D Wheat + 1,288 670,000 330,000 186,000 B Cucumber - 3,967 1,475 981 1,869 D Cucumber + 906 782,500 555,000 223,125 B
None * • 3,315 2,351 2,397 2,375 D None + 3,927 299,375 317,375 41,892 C None 1 M 1 M ® 1,138 772,813 1,155,000 1,937,500 A
^ No seeds planted but soil was maintained in same environment and at same moisture level as the cropped treatments. R. solani added (+) or not added (-) to soil at beginning of experiment. ^ R. solani added to soil every cycle. Population values for cycles 2, 4, and 6 (P - 0.05) followed by different letters are significantly different. 95
TrLchoderma spp. was after 2 cycles although It may have actually peaked
after one or three cycles) in the cropped treatments. Henls et al.
(1979) detected no correlations between suppressIveness of soil and the
antagonism of various soil microflora with one exception: increase in
soil lytic properties was associated with an increase in the propagule
population density of Trichoderaa spp. t After six cycles of cropping (and non-cropping) it was planned to
evaluate the suppressiveness of the soils towards R. solani. This would
involve testing with one crop after reinfesting the soil with R. solani.
Radish was selected as the test crop and was planted in all of the soils
for one cycle before infesting the soil with R. solani.
A checking cycle with radish was needed to know if any other
external factor of the systems, such as Fusarium spp. that was already
demonstrated to be present and affecting cucumber, was going to be
affecting the tester radish. Emergence was good, over 92% with all
soils (Table 10). Four cycles of cropping with radish were done after
infesting or reinfesting all the soils. The amount of R. solani
inoculum added was equal to the amount infested at the beginning of the experiment; however, the inoculum potential may not have been equal for both infestations. Radish emergence was good in most of the soils which may have indicated that the inoculum potential was lower the second time
(Table 10) than originally (Table 8).
The suppressive nature of the soil originally planted to radish with R. solani infestation was very striking as 99.75% of the seeds emerged with the first challenge planting. Those treatments that did 96
not have a previous crop, but were Infested with R. solani either
originally or every cycle, were also truly suppressive soils (Table 10,
Fig. 12). The soil that was never cropped nor Infested with R. solani
during the original treatments, had the most amount of disease during
the first cycle of planting radish as a tester. The original treatments
of radish + RhlzocConla, and no plants + Rhlzoctonla every cycle were highly suppressive even In the first cycle of the challenge, therefore
they were the most suppressive soils of the treatments. The original treatments of no plants + Rhlzoctonla and wheat + Rhlzoctonla appeared to become very suppressive at the second cycle. The suppressive nature of the soil originally cropped to radish but not Infested with R. solani
Is puzzling because this soil did not have high levels of Trlchoderma spp. after the first six cycles of planting (Table 9). There may have been another antagonist developing In this soil. The rapidity of the development of suppresslveness In the tester soils can be compared with the results during the first six cycles In Fig. 13.
There was no change In the suppressive nature of the soils where R. solani had been added Initially, Similarly, no change was observed when successive crops were planted without the pathogen. Thus,
Increase In suppresslveness following repeated crops of Inoculated plants cannot be due to a disease potential factor, as suggested by
Baker (1968) although one can not eliminate the possibility of fungitoxic chemicals produced by the host plant Itself. The suppresslveness may be an example of an Increase in antagonistic 97
Table 10. Percentage emergence of the tester crop, radish, following the original treatment* and then infestation with R. solani followed by cycles of the tester crop
Percentage of emergence of tester Original Treatment ^ Gx&laa Crop Rhlzoctonia Check" 1° 2 3 4
Radish - 99.75 91.00 92.50 94.25 95.75 Radish + 99.75 99.75 100.00 100.00 100.00 Wheat - 95.00 81.25 87.50 92.75 96.25 Wheat + 95.75 80.25 93.50 96.50 99.25 Cucumber - 98.00 67.25 73.50 92.50 95.25 Cucumber + 97.25 82.75 88.25 96.50 97.00
None . 99.25 60.00 72.50 77.50 94.75 None + 97.25 89.50 94.00 95.75 96.25 None 11(11 92.50 94.75 98.75 98.75 100.00
^ See footnotes to Table 9. ° Emergence of radish without reinfestation with R. solani. ° Emergence of radish for first cycle after all soils were infested with R, solani.
populations postulated by Baker and Cook (1982) in areas where both the
pathogen and disease occur; that is, the pathogen must be active to
induce the development of antagonists gaining some benefit from the
association of host and active pathogen. Supposedly wheat is not a host
of R. solani AG-4, and it still is associated with the ability to
produce some suppressiveness. Also, the fallow soil when amended once or weekly with the pathogen developed suppressiveness. This counters statements by Henis et al. (1978a) and Liu and Baker (1980) where they establish that both pathogen and host need to be present to induce Prior Treatment of soils in the absence of crops o 0 atolMi in t flrat oyei» «m No Raolani mdd#d so s aadwii ki 00 1 «v«ry oyol* § I
Cbk IR 2R 3R 4R Cycle* aftar Chtlleiiged wHh R. telanl Rtd-Rh WlM*t-nh Cuo-Rh - -O- - No Pl-Rh Rmd*Rh VVhMt«nh Cuo+Rh No pt«nh 100 r
« I
I vO vO
Testing with radish
6 CHECK 1*Rh 2+Rh 3+Rh 4+Rh Cycles
Figure 13. Emergence of radish, wheat, and cucumber through six cycles of repeated cropping In the presence or absence of R. solaxiL during the regular cycles and emergence of radish in the same soils during the five cycles of repeated planting and all soils infested with R. solani after the first cycle 100
suppresslveness, and the pathogen must be active to Induce the
development of the postulated biological antagonist. Therefore, one may
suggest that other profound factors must Influence the Inoculum
potential, especially those related to the saprophytic ability rather
than with the parasitic ability of a pathogen.
Capacity factors, sensu Olmond (cited by Baker and Martinson,
1970) especially the microbial environment, presumably Influence the
suppresslveness of the soil in this case. Influences on inoculum
changes Involving genetic factors, propagule size, pathogen nutrition,
and age also provide candidate hypotheses for mechanisms; however, the
prime factor may be the Influence of the suppressive factor on inoculum
density.
Measurement of Conduclveness Indices
Immediately following cycle four of the challenge phase in the prior experiment, the soils were analyzed for conduclveness Indices.
Radish was the test crop and compacted sclerotla were employed as the inoculum pellet (Fig. 1).
The Conduclveness Index (CI) sensu Henls eC ai. (1978a) is calculated by the linear function CI - (A-X)/A - 1 - X/A, where A - 32
(symptomless seedlings in the non-Inoculated control), and X is the 101
variable number of symptomless seedlings in the treatments. A second
Conduclveness Index (PU) was based on the wei^t of the soil aggregate associated with the inoculum pellet after 8 days.
The nine soils had widely divergent conduciveness indices (Table
11) indicating that these soils were not equal in actual suppressiveness although after four cycles of radish planting, emergence was nearly equal in all soils (Table 10). The two procedures for determining conduciveness indices produced values that were very similar and in
Table 11. Conduciveness indices of soils employed in the measurement of suppressiveness (Section 6) evaluated following the final challenge cycle with radish.
Original treatment * Conduciveness Indices Crop Rhizoctonia CI " FW
Radish - 0.3071 C 15.86 C' Radish + 0.0625 A 4.64 fi Wheat - 0.2993 C 18.77 C Wheat + 0.3306 C 22.09 C Cucumber - 0.2890 C 18.07 C Cucumber + 0.3071 C 15.36 C None - 0.4975 C 51.38 D None + 0.1537 B 6.05 B None II111 0.0650 A 1.54 A
^ For history of soil treatments see footnotes to Tables 9 & 10. ° Conduciveness index sensu Henis et al. (1978a). ^ Conduciveness index based upon the weight of the soil aggregate surrounding the inoculum pellet after eight days. ^ Numbers in each column followed by different letters are significantly different, P-0.05 102
agreement. The correlation between the values was R - 0.92. The FW
method was a little more sensitive because it separated the treatments
statistically into four different groups, while the CI method would
distinguish only three groups (Table 11). By both methods, the original
treatments of radish + Rhizoctonia, no plants + Rhlzoctonia once, and no
plants + Ehizoctonla every cycle had the least conducive soils. The
balance of the treatments had higher conduclveness Indices, meaning that
the pathogen was able to spread farther in these types of soils during
the test. The soil never cropped nor infested with R. solanl
originally, had the highest conduclveness index. Comparisons were made
between the two conduclveness Indices (Table 11) and the emergence of
radish during cycle 1 and cycle 4 of the challenge test for
suppresslveness (Table 10). The correlation coefficients and alpha
values were:
Comparison Correlation Coefficient a value CI vs. Cycle 1 - 0.85 0.004 CI vs. Cycle 4 - 0.70 0.035 PW vs. Cycle 1 - 0.86 0.003 PW vs. Cycle 4 - 0.59 0.092
There was a strong relationship between conduclveness index and the emergence of radish following R. solani infestation of the soils.
The correlations of CI vs. Cycle 4 was significant even when the radish emergence values were all above 90% for cycle 4 (Table 10). Greater than 70% of the suppresslveness in cycle 1 was inversely related to the eventual conduclveness index. The defeat of conduclveness appears to be 103
an accumulated trait of the soil. The soils with the lowest
conduciveness indices were the soils with the longest history of
potential exposure to R. solani, although little disease was apparent
for many of the cycles (Table 11). The apparent development of
suppressiveness in a non-suppressive soil during the challenge phase of
the experiment (original no plant and no Rhlzoctonla treatment, Table
10) did not render that soil as suppressive (it was the most conducive,
Table 11) as the soil continually infested with R. solani. Therefore the development of suppressiveness must be accumulative.
Suppressiveness in soil to R. solani comes with the addition of
R. solani to the soil, as all the treatments with J2. solani were more suppressive than those without it. 104
CONCLUSIONS
The presence of Fhizoctonia solani Is necessary for the development
of soil suppresslveness against pre-emergence damping off by R. solani,
regardless of the presence or absence of a suitable host. R. solani can be
present as a parasite or a saprophyte and induce suppresslveness. Highly
suppressive soils can be generated by adding R. solani to fallow soil. The
degree of suppresslveness is based upon the frequency of R. solani
additions to the soil because suppresslveness is an additive factor. The degree of suppresslveness in the soils is highly correlated with the population levels of the hyperparasitic antagonists, Trichoderma spp.
The AG-4 anastomosis group of Rhizoctonia solani was unique for the ability to promote a rapid increase in the populations of Trichodexrma spp. in soil. They could quickly generate suppresslveness in soils towards isolates of this particular anastomosis group.
The mechanisms of antagonism have been proven to include a high degree of parasitism and at least in vitro with nutrient media, some antibiosis. This antagonism involves a previously described mechanism of recognition and attraction. Recognition may be related to a volatile substance produced by R. solani.
When a soil has been demonstrated to be uniformly highly suppressive to R. solani, the amount of conduciveness may still vary. Suppresslveness of damping-off and amount of conduciveness were inversely correlated phenomena. 105
Biological control ot R. solan! was related to Trlchoderma spp. in the Iowa soils used in this study. This does not mean that Trichoderma spp. were the only factors involved in disease control. Other undescribed antagonistic phenomena could be important and possibly more important than antagonism by Trichoderma spp. Successful biological control will be dependent upon a thorough understanding of all interactions of the host, pathogen, and environment which includes the antagonist and antagonistic phenomena. 106
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ACKNOWLEDGEMENTS
I wish to thank my major professor, Dr. C. A. Martinson for his
support and suggestions throughout this project, for all the patience and
expertise to Initiate and to plan the research.
I am grateful to the other members of my committee, Drs. L. H.
Tiffany, T. E. Loynachan, N. A. Vaklll, and D. A. McGee, for their time,
advice and suggestions.
I wish to thank Institute Tecnoldglco y de Estudlos Superlores de
Monterrey, In Monterrey, N. L., Méxlco for co-sponsoring my Ph.D. studies
with Iowa State University. Financial support from the Biotechnology
Council (Iowa State University), USDA CSRS RRF (Project 2405, Iowa State
University), Graduate College, Iowa State University, and ITESM is acknowledged.
I am Indebted to my husband, Zak Mousli, and to my family and friends, for their support along all these past years.
Finally I dedicate this thesis to God and to the memory of my father, who have been my models and guiding lights all along my life. 119
APPENDIX 50
40
30
e 20
10
i • 0& 0 1 2 3 4 5 Days of Incubation
—o—Soil on culture, RHA-3 present, no beet seeds. — •—Soil on culture, RHA-3 present, beet seeds added. —-O—-Soil on PDA, RHA-3 absent, no beet seeds. --•'-Soil on PDA, RHA-3 absent, beet seeds added. —Soilin lid, RHA-3 present, no beet seeds. —• Soil in lid, RHA-3 present, beet seeds added. — O—Soil in lid, RHA-3 absent, no beet seeds. — • Soil in lid, RHA-3 absent, beet seeds added.
Figure Al. Populations of Trichoderma spp. in response to stimuli from Shlzoctonia solani in soil systems 20 N»
1 2 3 4 5 6 CYCLES
—A—• —A— Ratfitli —•— WhMt —•— WhMl —O— CiMunk*—#— nwmrti «MIIM «MIIsm «RMIM
Figure A2. Percentage emergence of three crop species during six cycles of planting in soils Infested or not infested with R. solani -•O"- NP-Rh
10000 F - NP+Rh
1000 - or— Rad-Rh
*— Rad+Rh W 100 : Whe-Rh
$ Cuc-Rh
Cuc+Rh
- No Pl+Rh/ cycle
Figure A3. Populations of Trichoderma spp. sampled from soil cropped to radish, wheat, and cucumber or uncropped and infested or not infested with R. solani 123
Table Al. Populations of Trichoderma spp. assayed from soil with or without Infestation with Trichoderma viride at three levels and amended with beet seed baits that were unlnfested or Infested with R. solani
££U X aaii BAITING TYPE Infestation Day 1 Day 2 Day 3 Day 4 Day 6 Day 8 AND Day INFESTATION LEVEL
NO BAITS IX 2.0 0.5 0.7 0.3 0.3 0.5 0.6 2X 8.5 2.7 5.3 4.5 10.5 3.2 4.0 4X 10.5 3.7 3.0 2.0 1.1 4.3 5.2 8X 11.8 4.6 20.0 10.0 9.0 10.0 12.0
STERILE BAITS IX 0.8 3.0 1.3 0.7 0.7 2X 3.6 3.0 2.8 2.1 5.8 4X 8.0 5.0 10.0 13.0 15.0 8X 7.0 9.5 8.1 6.0 4.3
KILLED Shizoctonia IX 0.7 0.9 1.6 1.0 1.2 2X 2.6 2.8 2.0 2.0 2.2 4X 14.0 6.9 4.3 2.0 4.6 8X 13.0 21.0 15.0 13.0 13.0
LIVING Rhizoctonia IX 3.2 80.0 90.0 102.0 112.0 2X 4.4 17.0 28.0 36.0 44.0 4X 36.0 14.0 50.0 86.0 26.0 8X 42.0 46.0 50.0 56.0 68.0 124
Table A2. Percentage colonization of beet seed baits, which were preinfested with different isolates of Hhizoctonia sp., with TrLchoderma. spp. after 3 and 5 days incubation in soil, and populations of TrLchoderma spp. in the soils before and after 3 and 5 days incubation of the infested beet seed baits in the soils
Isolate Day & DflX 1 12fiX 1 Soil OOP.* Bait 1 Soil POP. £fil£ 1 Soil POP.
BN-1 22.5 A 9.67 D 25.5 D® 1.17 G 33.0 C RHA-1 30.5 A 30.17 B 38.0 D 24.00 CD 55.0 B RHA-2 31.5 A 20.50 C 130,0 B 43.34 B 134.0 A RHA-3 19.5 A 39.17 AB 286.7 A 50.00 A 360.0 A RHA-4 19.5 A 18.00 CD 15.5 D 29.17 CD 24.0 C RHÂ-6 20.5 A 22.50 BC 25.5 D 20.00 D 39.5 C RHA-7 30.0 A 29.00 B 80.0 C 33.50 C 170.0 A RHA-42 26.5 A 12.84 CO 22.5 D 17.67 DE 64.0 B RHA-43 36.0 A 8.34 D 30.0 D 6.84 F 27.5 C RHA-48 27.0 A 1.00 E 15.0 D 5.50 F 14.0 C RHA-77 27.5 A 28.34 B 40.0 D 11.17 E 58.0 B RHA-140 32.0 A 45.00 A 153.0 B 24.67 CD 289.7 A RHA-462 34.5 A 31.17 B 11.0 D 29.17 CD 17.5 C
MEAN 22.74 22.78
^ CFU of TrLchoderma spp. in soil (X 10^) ° Percentage of baits yielding TrLchoderma spp. ° Values in each column followed by the same letter are not significantly different (P-0.05).