Method for making biocontrol agents rhizosphere-competent

Soilborne rhizosphere-incompetent biocontrol agents can be converted into rhizosphere-competent agents by exposing them to a mutagenic agent and then screening the exposed rhizosphere-incompetent agent for a strain showing increased cellulase production. The increased cellulase production characteristic serves to convert the originally rhizosphere-incompetent agent into one which is rhizosphere-competent. Seeds of plants to be protected against various diseases can then be treated with the rhizosphere-competent strain. The roots of the plant, as well as its original seed, are protected by biocontrol agents produced by the disclosed process.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention generally relates to processes for identifying, developing 
and using new biotypes of soilborne biocontrol agents. More specifically 
it relates to mutagenic processes for converting soilborne 
rhizosphere-incompetent biocontrol agents into rhizosphere-competent 
biocontrol agents. The term "rhizosphere competence" has been employed to 
describe an attribute of rhizobia characterized by their consistent 
association with legume root nodules. Here, we use the term to describe 
the ability of a microorganism to grow and function in the developing 
rhizosphere. 
2. Description of the Art 
The protection of plants from infection by soilborne fungal and bacterial 
pathogens by use of antagonistic microorganisms is well known to the art. 
For example, it is known that various Trichoderma spp. such as Trichoderma 
harzianum Rifai act as biological control agents against certain plant 
diseases. Nonetheless, use of Trichoderma spp., as biocontrol agents has 
been rather limited. This is mainly because seed treatment with 
Trichoderma spp. generally does not provide continued protection for the 
emerging root system of the maturing plant. Such seed treatment does serve 
to reduce preemergence damping-off but the root system is left 
unprotected. It is generally believed that this failure to protect the 
root system is because Trichoderma spp. are not rhizosphere-competent, see 
for example, Papavizas, G. C., Phytopathology 72: 121-125 (1982) and Chao, 
W. L., et al., Phytopathology 76: 60-65 (1986). Therefore, a need clearly 
exists for more effective methods for inducing biocontrol agents to 
colonize a plant's developing root system as well as its spermosphere. 
This need has been partially met in the realm of certain bacterial 
biocontrol agents; see for example, Mendez-Castro, F. A. and Alexander, 
M., Method for establishing a bacterial inoculum on corn roots, Appl. 
Eviron., Microbiol. 45: 254-258 (1983). A similar plant protection 
strategy has been applied to fungal biocontrol agents (Ahmad, J. S., and 
Baker, R., Induction of rhizosphere competence in Trichoderma harzianum 
(Abstr.) Phytopathology 75: 1302 (1985)). This work indicated that when 
benomyl-tolerant mutants of Trichoderma harzianum Rifai were applied to 
seeds, the roots became colonized. However, the reason or reasons as to 
why such mutants are rhizosphere-competent, were not apparent. It seemed 
that many of the results obtained from following this research strategy 
were inconsistent and/or in conflict with what was then known about 
rhizosphere-competence. For example, it should be noted that the Papavizas 
article previously cited discloses the use of benomyl tolerant isolates of 
T. harzianum, obtained by ultraviolet light irradiation to test for 
rhizosphere-competence of bean and pea seedlings. However, Applicants 
found that ultraviolet mutants, tolerant to benomyl were not rhizosphere 
competent. Moreover, reports by other workers (see for example, Garrett, 
S. D., Pathogenic root-infected fungi, Cambridge University Press (1970)) 
postulated the theory that the share of a substrate obtained by any 
particular fungal species is determined partly by its intrinsic 
competitive saprophytic ability and partly by the balance between its 
inoculum potential and that of competing species. This report also 
theorized that production of, and tolerance to, antibiotics is another 
important attribute of successful rhizosphere fungi. 
All of these theories were however, to some degree, inconsistent with the 
results of Applicants' rhizosphere competence tests. For example, 
Applicants found that both the mutants and the wild types had the same 
population density when applied to seeds. Moreover, none of Applicants' 
mutant strains have antibiotic activity in vitro except for a routing 
factor seen in the hyphal cytoplasm, at microscopic levels affecting 
Pythium spp. In trying to reconcile these theoretical and/or evidentiary 
conflicts, as well as those relating to the nature of the plant root 
surfaces themselves (see Foster, R. C., Rovira, A. D., and Cock, T. W., 
Ultrastructure of the Root-Soil Interface. Am. Phytopath. Soc., St. Paul, 
Minn. (1983)), Applicants postulated that rhizosphere-competence was 
somehow related to a possible possession by the mutant strains of enzymes 
for cellulase degradation. The subsequent finding by the Applicants that a 
wide variety of rhizosphere-competent Trichoderma mutant strains do in 
fact possess increased cellulase degradation capabilities, is a key aspect 
of the overall teachings of this disclosure. 
SUMMARY OF THE INVENTION 
This invention provides a process whereby rhizosphere-incompetent 
biocontrol agents such as fungi and bacteria can be mutated into strains 
which are rhizosphere-competent. The disclosed process is particularly 
useful in producing rhizosphere-competent fungi capable of controlling 
diseases caused by Phythium spp., Sclerotuim spp., and Rhizoctonia solani 
in such varied plants as beans, maize, tomato, radish, cucumber, wheat, 
barley, lettuce, and carnations. 
The mutation process of this invention can be induced by known means such 
as mutagenic chemicals. Applicants have found that 
N-methyl-N-nitro-N-nitrosoguanidine is a particularly useful mutagenic 
chemical in producing Trichoderma strains which possess the desired 
increased cellulase production characteristic. Other useful mutagenic 
agents would include, but not be limited to ultraviolet light and 
radiations. 
The most straightforward means for producing and identifying biocontrol 
agents having the characteristic of increased cellulase production is to 
grow them in a nutrient medium where cellulase represents a major part of 
the agent's source of carbon, or more preferably its only source of 
carbon. Representative sources of carbon would include, but not be limited 
to carboxy methyl cellulase, cotton linters and solka floc. 
The most effective cellulase producing strains of Trichoderma spp. thus far 
produced by Applicants have been deposited with the American Type Culture 
Collection, 12301 Parklawn Drive, Rockville, Md. 20852, under the 
following designations. 
______________________________________ 
Trichoderma spp. 
Applicants' Designation 
______________________________________ 
harzianum T-12B 
harzianum T-95 
koningii T-8-7 
ATTC Designation 
20835 
60850 
20836 
______________________________________ 
Other biocontrol agents which could be mutated by Applicants' methods to 
become rhizosphere-competent would include Fusarium spp., Penicillium 
spp., Mvrothecium spp., Chaetomium spp., and Glicladium spp. 
In keeping with the provisions of the directive found on page 638, volume 
886 of the Official Gazette of the United States Patent Office, progeny of 
each and every such strain will be made available during the pendency of 
this patent application to anyone determined by the Commissioner of 
Patents and Trademarks to be entitled thereto under 37 CFR .sctn.1.14 and 
35 USC 12.2. All restrictions on the availability to the public of progeny 
of each and every such strain will be irrevocably removed upon the 
granting of a patent of which these strains are subject.

DESCRIPTION OF PREFERRED EMBODIMENTS 
The competitive saprophytic ability (CSA) of strains of Trichoderma spp. 
was determined by the modified Cambridge method (sensu Garrett, S. D. 
Pathogenic root infection fungi. Cambridge University Press, London 
(1970)). Two rhizosphere-competent mutants of Tharzianum (T-95 and T-12B) 
had higher CSA indices than four rhizosphere-incompetent Trichoderma spp. 
and strains. CSA was directly correlated with rhizosphere competence. When 
the strains were grown for 6 days on Czapek Dox broth with cellobiose, 
carboxy methyl cellulose, or cotton linters as sole sources of carbon, 
mutants produced more cellulase than the wild types. The amount of 
cellulase produced by these strains was directly correlated with CSA and 
rhizosphere competence. Rhizosphere competence of the mutants, therefore, 
can be at least partially explained by their capacity to utilize cellulose 
substrates associated with the root. It should also be noted that the 
above noted Trichoderma are benomyl-tolerant. 
METHODS 
Tests for Rhizosphere Competence. Various methods have been employed to 
test rhizosphere competence. These were primarily based on a comparison of 
the numbers of cfu of microorganisms in the soil associated with roots to 
population densities in non-rhizosphere soil. The rhizosphere competence 
assay used in this research effort was developed to improve measurement in 
time and space of the activity of potential rhizosphere inhabitants. 
Certain criteria were demanded by the experimental questions to be 
examined. Quantitative analysis of population densities at each depth of 
root was necessary. No water was added during incubation obviating the 
possibility of propagules being washed into the rhizosphere. To test 
whether the agent introduced from a seed into the rhizosphere could 
compete under typical ecological condition, raw soil was used. Therefore, 
the system allowed rhizosphere competence to be measured on the basis of 
cfu/mg or g of rhizosphere soil as a function of root depth. 
The nature and quantity of root exudates have been analyzed in the past in 
axenic systems by use of perfusion and filter paper absorption techniques. 
Since such analyses often are obtained under gnotobiotic conditions, it is 
difficult to extrapolate such findings into the ecological conditions 
present in the rhizospheres of plants growing in raw soil. To overcome 
this objection, bioassays relating relative magnitudes of microbial 
population densities in the rhizosphere compared with non-rhizosphere soil 
were developed, the R/S ratio. Such analyses are subjected to many 
variables and, at best, provide only a relative gross assay of the 
activity of the total biomass about the root. The rhizosphere competence 
assay provided a quantitative measurement of a specific 
rhizosphere-competent microorganism at the root tip where exudates are in 
relatively high concentration. In more mature portions of the root, 
however, interpretations based on population densities are confounded by 
maturation of the agent resulting in propagule production, various 
interaction leading to auto- or heterolysis, or changes in characteristics 
of substrates provided by senile tissues of the root. Nevertheless, the 
rhizosphere competence assay provides the best bioassay yet developed for 
the rhizosphere nutrient at root tips. It has potential for use in a wide 
variety of experimental problems related to ecological and nutritional 
interactions in the rhizosphere. 
Several species of Trichoderma were tested for rhizosphere competence by 
coating the seed with each isolate and following population densities of 
the fungus to a root depth of 8 cm. No species grew to greater depth than 
2 cm. This confirms the conclusions of other workers that Trichoderma spp. 
are not rhizosphere-competent. 
Additional evidence for rhizosphere competence of strain T-95 was obtained 
by microscopic observations comparing the length of hyphae on root 
originating from seeds coated with or without conidia of the fungus. Of 
course, it was not possible to identify with certainty the hyphae of T-95; 
however, the total length of hyphae observed was relatively similar to the 
cfu obtained by use of the rhizosphere competence assay. 
Roots were essential for colonization below the site (seed) where the 
strains of Trichoderma were applied. The wild type was recovered at low 
densities to a 4-cm depth; mutants were recovered at all depths of 
rhizosphere sand when applied to seed. Neither the wild type nor a mutant 
was recovered below the glass beads. 
Our particular rhizosphere-competence assays were conducted as follows. 
Polypropylene centrifuge tubes (28.6 by 103.6 mm) were sliced 
longitudinally into two halves. Each half was filled with moistened soil 
(-0.03 bars) and pre-incubated for 48 hours in plastic bags. One treated 
seed was placed on the half-tube 1 cm below the rim. The unseeded 
half-tube was placed on the first half and secured with rubber bands. 
Tubes were completely randomized and lots in portions of six each were 
placed vertically in 10 cm diameter plastic pots. Soil, previously 
moistened to -0.03 bars and of the same pH as in the tubes, was added to 
the pots so that the length of the tube was surrounded by the soil, with 
the top 1 cm of each tube uncovered. No water was added to the tubes or 
the pots after seed were sown. Pots were covered with plastic bags to 
maintain constant matric potential leaving enough space above the tubes 
for the plants to grow. Pot were placed under constant illumination 
supplied by 10 white, 40-watt, 120 cm long fluorescent lamps 
(approximately 5000 lux), at desired temperatures. 
After 8 days, or as desired by the experiment, tubes were removed from the 
pots. After the unseeded half of a tube was carefully lifted, the roots in 
he seeded half, starting from the crown, were excised in 1 cm segments 
with a sterile scalpel. The scalpel was flamed between cuts. After loosely 
adhering soil was shaken off root segments with their adhering rhizosphere 
soil were air dried under a 100-watt lamp for 30 minutes. Each unit was 
weighed and transferred to a 20 ml glass vial containing 1 ml sterile 
distilled water. The contents of the vial were stirred vigorously with a 
sterile spatula. The colony forming units (cfu) of Trichoderma contained 
in the rhizosphere soil at each cm of root were determined by plating a 
series of 10-fold dilutions from the vial of Trichoderma-selective medium. 
Root segments were removed from the dilution flask, blotted on paper towel 
and weighed to determine the dry weight of rhizosphere soil removed 
through washing. In experiments where sand was substituted for soil and 
glass beads were coated with conidia, what would have been rhizosphere 
sand was sampled after 8 days and treated as explained above. Plates were 
incubated at 25.degree. C. for 5 days. Counts of Trichoderma cfu per mg 
rhizosphere soil for each root segment were made with six replicates per 
treatment. All experiments were repeated twice. 
Microscopic observation of roots. Root segments with rhizosphere soil were 
placed in multi-well tissue culture plates. One-half ml of an aqueous 0.3% 
Calcofluor solution (Calcofluor white M2R, Polysciences, Inc., Warrington, 
Pa.) was added to each well. The plate was covered with aluminum foil and 
incubated at 25.degree. C. for 20 hours. Root segments were transferred to 
a microscope slide. After addition of a drop of water and a cover slip, 
the slide was viewed with an Olympus BH microscope (Olympus Optical Co., 
Tokyo, Japan), with a blue exciter filter (8G-12) providing 400 nm light 
supplied by an epifluorescent illuminator. A barrier filter (530 nm) also 
was used when viewing the slides. Each root segment was viewed and the 
total length of hyphae per root cm was measured with the aid of an ocular 
micrometer. The experiment was repeated twice. 
Statistical analysis. The date for weight of mycelium and cellulase units 
produced was subjected to one way analysis of variance and the means were 
separated with an FLSD (P-32 0.05). The date of CSA were subjected to 
multiple regression analysis and the slopes values were separated with an 
FLSD (P=0.05). 
MATERIALS 
Soil. Nunn sandy loam was used in these investigations. Water content of 
43.2 kg portions was adjusted to -0.03 bars and the soil was stored for 48 
hours before use. The soils had the following characteristics: pH 7.0, 
conductivity 0.4 mmhos, lime low, organic matter 1.4%, No.sub.3-N 1 hg/g. 
P 9 hg/g, K198 hg/g, Zn 0.5 hg/g, Fe 3.2 hg/g. Its pH was measured by the 
CaCl.sub.2 method. To adjust soil pH, 10% (v/w) 0.1N H.sub.2SO4 was added 
to a 1 kg portion of soil. The soil was mixed thoroughly, allowed to dry, 
and ground with a mortar and pestel. By this method, soil pH was reduced 
from 7.0 to 2.5. Portions of this soil were added to field soil to adjust 
pH values from 7.0 to 5.0 and 6.0. No change in pH was observed during the 
course of experiments. 
Trichoderma spp. and strains. Various strains of Trichoderma harzianum 
(e.g., T-95 [ATCC 60850] T-12B, WT, and T-12) and one strain each of 
Trichoderma koningii (T-8) and Trichoderma viride (T-S-1) were used in 
these investigations. They were obtained from various sources. For 
example, the T. harzianum designated as WT originally was isolated from a 
soil in Columbia, SA. T. harzianum (T-95) a benomyl tolerant mutant, was 
derived from T. harzianum WT. T. harzianum (T-12) T. koningii (T-8) were 
isolated from a soil in New York, were provided by G. E. Harman (New York 
State Agricultural Experiment Station, Geneva, N.Y.). T. viride (T-S-1) 
was provided by M. T. Dunn (Mycogen Corporation, San Diego, Calif.). T. 
harzianum (T-12B) was a benomyl tolerant mutant derived from T. harzianum 
(T-12). Conidia of the strain being tested were exposed to 100 .mu.g/ml of 
N-methyl-N-nitro-N-nitrosoguanidine (Tredom Chemical, Inc., 255 Oser Ave., 
Hauppauge, N.Y.) for 30 minutes. The conidia were centrifuged at 2500 g 
for 15 minutes and resuspended in sterile water three times. Seeds of 
cucumber (Cucumis sativus L. "Straight Eight"), radish (Raphanus sativus 
L. "Early Scarlet Globe"), tomato (Lycopersicum esculentum L. Burpee's Big 
Boy), beans, (Phaseolus vulgaris L. "Olathe"), and maize (Zea mays L. T. 
E. 6998) were surface disinfested for 10 minutes in 1.1% sodium 
hypochlorite solution and 5% ethanol, washed in distilled water, and air 
dried. Seeds were treated with conidial suspensions of Trichoderma spp. in 
water containing 2% (v/w) Pelgel (The Nitragen Co., Milwaukee, Wis.) as a 
spreader or sticker. Conidial density was adjusted to 106 per seed. 
Controls were treated with Pelgel alone. 
Competitive saprophytic ability assay. To test CSA of Trichoderma spp., the 
Cambridge method was modified. Strains of Trichoderma spp. were grown on 
potato-dextrose agar (PDA). Mutants tolerant to benomyl were grown on PDA 
containing 10 ug a.i. benomyl per ml. Plates were incubated for 8 days at 
25.degree. C., flooded with sterile distilled water and conidia were 
gently freed from the culture with a brush. The suspension was sieved 
through four layers of cheese cloth, centrifuged at 2500 grams for 15 
minutes and resuspended in sterile distilled water three times. Conidia 
were counted with a hemacytometer and then adjusted to the desired 
concentrations. 
Freshly harvested conidia were added to 7.2 kg of previously moistened and 
incubated field soil at the rate of 101, 102, 103 and 104 conidia per 
grams soil. No conidia were added in controls. The soil was mixed 
thoroughly by hand and distributed in nine 11-cm-diameter plastic pots. 
Clean, mature, polished winter wheat straw was cut in 1-cm segments; each 
segment included a node. Twenty pieces were buried randomly in each pot. 
The pots were arranged in a completely randomized design, covered with 
plastic to conserve moisture at -0.03 bars and incubated in the dark. No 
water was added to the pots. All twenty pieces, from each treatment 
including a non-infested control were removed from the pots after 2, 4, or 
6 days; washed in tap water to remove all adhering soil and debris and 
surface-disinfested in a mixture of 1.1% sodium hypochlorite solution and 
5% ethanol for 5 minutes. Segments were plated on medium selective for 
Trichoderma and incubated at 25.degree. C. for 5 days. Percent 
colonization of wheat pieces by Trichoderma for each treatment at a given 
time was determined. There were three replicates per treatment and all 
experiments were repeated twice. 
In experiments where cellophane disks were substituted for straw pieces, 
the disks were obtained by punching holes (6-mm diameter) in an untreated 
cellophane sheet. Disks were removed from the pots after incubation for 2, 
4, or 6 days, washed in sterile distilled water and plated on Trichoderma 
selective medium. 
Growth of Trichoderma spp. in liquid culture. Strains of Trichoderma spp. 
were grown in 250 ml Erlenmeyer flasks containing 50 ml Czapek Dox broth 
on a rotary shaker at 100 rpm at 26.degree. C. for 6 days. Finely ground 
cotton linters, carboxy methyl cellulose or cellobiose (Sigma Chemical 
Co., St. Louis, Mo.) were used as sole sources of carbon. Each flask was 
seeded with a 4-mm diameter disk of PDA on which the strains had been 
grown for 2 days. After 6 days the hyphal mat was removed aseptically and 
dried for 2 days at 60.degree. C. to obtain the weight of mycelium. There 
were six replicates per strain. 
Enzyme assay. Cellulase (E.C. 3.2.1.4) was assayed spectrophotometrically 
(A 340) by following the release of free glucose from the substrates 
listed above according to the manufacturer's directions (Sigma Chemical 
Co., St. Louis, Mo.). Cellulase activity was expressed as units of 
cellulase produced per ml culture filtrate of each strain when grown in 
the substrate for 6 days. There were six replicates per strain. 
Competitive saprophytic ability (CSA) index. A CSA index for each strain 
was developed as follows: 
##EQU1## 
where c is the frequency of isolation of a specific strain of Trichoderma 
from the segments, t is time of incubation, p is the population density of 
conidia added to the soil and n is the number of treatments. 
Rhizosphere competence (RC) index. Rhizosphere competence index (RC index) 
for each strain was developed from the data reported in reference 2 by use 
of the equation: 
##EQU2## 
where p is the population density per mg rhizosphere soil, d is the root 
depth and n is the total root length. 
EXPERIMENTAL RESULTS 
Colonization of straw by Trichoderma spp. When polished wheat straw pieces 
were buried in soil infested with conidia of Trichoderma spp. and removed 
after 2, 4, and 6 days, T. koningii (T-8) and T. viride (T-S-1) were not 
isolated at any population density. T. harzianum T-12 and WT were 
recovered from straw less frequently than the other strains and were slow 
to colonize the straw segments at higher population densities (FIG. 1). 
However, the mutants of these wild types, T-12B and T-95, respectively, 
were isolated from the straw segments at any population density (FIG. 1). 
Strains T-95 and T-12B showed significantly higher percent colonization 
than WT and T-12 at any population density on all days. Strain T-95 showed 
significantly higher percent colonization than T-12B at 101, 102, and 103 
cfu/grams soil on all days but there were no significant differences 
between the two strains at 104 cfu/g. Strain WT showed significantly 
higher percent colonization than T-12 and WT were added at 101 cfu/grams 
soil, and 10.sup.4 cfu/g. When strains T-12 and WT were added at 10.sup.1 
cfu/grams soil, neither were isolated from what straw pieces after 2, 4, 
and 6 days incubation. Trichoderma spp. were not isolated from controls. 
When washed cellophane disks were buried in soil infested with conidia of 
T-95 or WT and removed after 2, 4, and 6 days, both strains could be 
isolated from the disks at any population density (FIG. 2). Strain T-95 
showed significantly higher percent colonization than WT at 101, 102, and 
103 cfu/grams soil on all days but there were no significant differences 
between the two strains at 104 cfu/g. 
Growth of Trichoderma spp. in liquid culture. When strains of Trichoderma 
spp. were grown in Czapek Dox broth with cellobiose as the sole source of 
carbon, the mutant (T-95) mycelium attained significantly higher dry 
weight than all other wild type strains (FIG. 3A). Strains T-12B, T-12, 
and WT had significantly higher dry weight than T-8 and T-S-1. When 
carboxyl methyl cellulose or cotton linters were the sole source of 
carbon, the mutants T-95 and T-12B had significantly higher dry weights 
than the wild types (FIG. 3B and C). In both cases strain T-95 had 
significantly higher dry weight than T-12B. With cotton linters strains WT 
and T-12 had significantly higher dry weights than T-8 and T-S-1. 
Production of Cellulase. All strains produced cellulase when grown in 
Czapek Dox broth with cellobiose as the sole source of carbon (FIG. 4A). 
Mutants T-95 and T-12B produced significantly higher amounts of cellulase 
than the wild type or other strains. When carboxy methyl cellulose was the 
sole source of carbon, strain T-12 failed to produce any cellulase, strain 
T-95 produced significantly higher amounts of cellulase than all other 
strains and mutant T-12B produced significantly more than T-12 (FIG. 4B). 
When cotton linters were the sole source of carbon, the mutants of T. 
harzianum produced significantly higher amounts of cellulase than the wild 
types or other strains and strain T-95 produced significantly higher 
amount than all other strains (FIG. 4C). 
DISCUSSION 
Isolation of fungi from baits of dead plant material buried in field soil 
provides direct evidence that recovered fungi can colonize these 
substrates as competitive saprophytes. Therefore, many investigators have 
used wheat straw pieces rich in cellulose in the Cambridge method to 
determine the competitive saprophytic ability of root infecting fungi. The 
CSA index measured the capacity of different strains and species of 
Trichoderma to compete effectively in the colonization of wheat straw. 
Benomyl-tolerant mutants had higher CSA indices than the wild types. 
Garret has included the ability to produce enzymes for utilization of 
specific substrates among the attributes of fungal species that contribute 
to their CSA. 
Strains of Trichoderma spp. produce cellulase and other cell wall degrading 
enzymes. In our study the CSA indices were correlated directly with 
production of cellulase (FIG. 6A). Also, mutants of T. harzianum produced 
significantly greater amounts of cellulase when (cotton linters) was the 
sole source of carbon. By use of the RC index, rhizosphere competence was 
directly correlated with amount of cellulase units produced by the mutants 
and the CSA of the mutants. These correlations indicate that mutants with 
higher cellulase activity than wild types can utilize cellulose substrates 
on or near the root more efficiently and thus, are rhizosphere-competent. 
Utilization of cellulose substrates is not associated with parasitism 
since microscopic examination revealed no evidence of such a relationship. 
A more likely source of cellulose substrates is the remains of the primary 
cell walls in the mucigel. 
The pattern of hydrolytic enzymes used by strains of Trichoderma spp. for 
the hydrolysis of cellulose has been well studied. Exo and endo 
B-1,4-glucanases act on cellulose that is broken down to cellobiose and 
glucose. Cellobiose is further hydrolized by, B-1,4-glucosidases to 
glucose. In an attempt to distinguish the amount of these enzymes 
produced, different carbon sources were used as substrates. When 
cellobiose was used as the sole carbon source, the mutants produced 
significantly greater amount of B-1,4-glucosidases than the wild types. 
The mutants not only produced greater amounts of B-1,4-glucanases but, 
evidently, also produced significantly greater amounts of 
B-1,4-glucosidases and utilized the substrates more efficiently. This is 
also evident from the dry weight of mycelium produced. 
These results indicate that certain strains of T. Harzianum are 
rhizosphere-competent because of increased enzyme activity which results 
in higher CSA for possession of cellulose substrates on or near the root 
surface. This attribute of rhizosphere-competence has not been previously 
recognized. If the extension rate of fungal thalli are sufficient to keep 
pace with root growth, the attribute of higher efficiency of cellulose 
degradation could be a key factor for inducing these microorganisms to 
become rhizosphere-competent. 
These results also indicate that mutation and selection of other strains of 
other fungi and bacteria based upon an ability to produce rhizosphere 
competence in a wide variety of biocontrol agents.