Fungicidal compositions comprising chitinase and enterobacter cloacae, and a method for stimulation proliferation of E. Cloacase

Combination of fungal cell wall degrading enzymes (e.g., chitinolytic enzymes) and antifungal bacteria (e.g., E. cloacae) which bind to fungal cell walls in the presence of said enzymes even when sugars are present, increases potency and range of use of the bacteria and fosters proliferation thereof.

TECHNICAL FIELD 
This invention is directed at antifungal synergistic combinations of fungal 
cell wall degrading enzyme and fungal cell wall binding antifungal 
bacteria and use thereof for topical application in agriculture to inhibit 
replication, germination or growth of fungi and to a method of 
proliferating the growth of antifungal bacteria. 
1. Background of the Invention 
Various antifungal bacteria are known, e.g., Enterobacter cloacae, 
Pseudomonas fluorescens and Pseudomonas putida. The biocontrol ability of 
Enterobacter cloacae has been associated with its ability to bind to 
hyphal walls. It has been reported that some sugars such as D-glucose and 
sucrose inhibit this binding. Consequently, E. cloacae has been able only 
to protect seeds and plants with low sugar exudation. 
2. Summary of the Invention 
It is an object of this invention to increase the potency of bacteria where 
antifungal function is associated with ability to bind to fungal cell 
walls and to expand the range of use of such bacteria so that antifungal 
function occurs even when normally interfering sugars are present. 
The antifungal composition of the invention herein comprises 
(a) purified fungal cell wall degrading enzyme, and 
(b) antifungal bacteria which bind to fungal cell walls in a 2% dextrose 
aqueous solution in the presence of enzyme (a) but which do not bind to 
fungal cell walls in a 2% dextrose aqueous solution in the absence of 
enzyme (a), 
these being present in a ratio of (b) to (a) ranging from 1 cell of (b) to 
1 .mu.g of (a) to 200,000 cells of (b) to 1 .mu.g of (a), 
the combination of (a) and (b) being present in an antifungal effective 
amount. 
A method of the invention herein is directed to inhibiting the replication, 
germination or growth of a fungus and comprises contacting such fungus or 
a locus to be protected from such fungus, with an antifungal effective 
amount of composition of the invention herein. 
A method of another embodiment of the invention herein is directed to 
causing proliferation in the growth of Enterobacter cloacae and comprises 
the steps of reacting fungal cell wall degrading enzyme with a substrate 
therefor to obtain a nutrient for Enterobacter cloacae and growing 
Enterobacter cloacae in the presence of said nutrient so as to cause said 
proliferation. 
The term "fungal cell wall degrading enzyme" is used herein to mean enzyme 
that effects lysis of fungal cell walls. 
The term "purified fungal cell wall degrading enzyme" is used herein to 
mean cell wall degrading enzyme which is purified to a specific activity 
greater than its specific activity in a culture filtrate of the 
microorganism from which it is obtained and thus distinguishes the case 
where bacteria exude cell wall degrading enzymes in nature. 
The term "antifungal bacteria" is used herein to mean bacteria which 
inhibit the replication, germination or growth of a fungus. 
The term "bind to fungal cell walls" is used herein to mean physical 
attachment of bacteria to fungi. 
The term "inhibit" is used herein to mean reduce the growth and/or 
development of fungi compared to where inhibiting agent is not present. 
The term "locus to be protected from such fungus" includes seeds, roots, 
stems, leaves, flowers and fruits to be protected and to the soil 
surrounding seeds and roots to be protected. 
The term "causing proliferation in the growth of" is used herein to mean 
increasing the number of bacterial cells at least five-fold compared to 
where fungal cell wall degrading enzyme or substrate therefor is not 
present. 
The term "growing Enterobacter cloacae" is used herein to include not only 
culturing but replication in nature.

DETAILED DESCRIPTION 
The fungal cell wall degrading enzymes for the compositions herein include, 
for example, chitinolytic enzymes and .beta.-1,3-glucanolytic enzymes for 
degrading cell walls of fungi where the cell walls contain, as major 
structural components, chitin and .beta.-1,3-glucans, and cellulases for 
degrading cell walls of lower fungi (Oomycetes) where the cell walls 
contain, as a major structural component, cellulosic polysaccharides. 
These enzymes are found in fungi, bacteria and higher plants. The 
compositions herein are limited to containing purified enzymes to 
distinguish the case where bacteria exude cell wall degrading enzymes in 
nature. The purified enzymes can be partially purified, i.e., purified 
compared to what occurs in nature or in a culture filtrate of the 
microorganism from which it is obtained, but with other protein present. 
However, the enzyme component of the composition herein is preferably used 
in biologically pure form, that is, purified to be free of contaminating 
protein (purified to homogeneity). Fungal cell wall degrading enzymes are 
readily obtained in biologically pure form from source microorganisms by 
culturing the source microorganism, concentrating the culture filtrate, 
fractionating by gel filtration chromatography, concentrating, and further 
purifying by chromatofocusing, followed, if necessary, by 
isoelectrofocusing in a Rotofor cell (BioRad, Richmond, Calif.). 
The chitinolytic enzymes cleave chitin, and include, for example, 
endochitinases, chitin 1,4-.beta.-chitobiosidases and 
N-acetyl-.beta.-D-glucosaminidases. These can be obtained from fungi, for 
example, from the genera Trichoderma, Gliocladium, Lycoperdon and 
Calvatia; from bacteria, e.g., from the genera Streptomyces, Vibrio, 
Serratia and Bacillus; and from higher plants, e.g., Nicotiana, Cucumis 
and Phaesolus. 
The endochitinases are enzymes that randomly cleave chitin. Endochitinase 
activity is readily measured by determining optical density at 510 nm as 
reduction of turbidity of a 1% suspension of moist purified colloidal 
chitin in 100 mM sodium acetate buffer, pH 5.5, or in 50 mM KHPO.sub.4 
buffer, pH 6.7, after 24 hours of incubation at 30.degree. C. For 
calculation of specific activity, one unit is defined as the amount of 
enzyme required to obtain a 5% turbidity reduction. 
A very preferred endochitinase is isolated from Trichoderma harzianum 
strain P1 having accession No. ATCC 74058 and has a molecular weight of 36 
kDa (as determined by sodium dodecyl sulfate polyacrylamide gel 
electrophoresis after the protein was prepared under reducing conditions, 
on direct comparison to migration of a 36 kDa protein) and an isoelectric 
point of 5.3.+-.0.2 as determined based on its elution profile from a 
chromatofocusing column, and a molecular weight of 40 kDa (as determined 
by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the 
protein was prepared under reducing conditions, from a regression based on 
the log of molecular weight of standard proteins) and an isoelectric point 
of 3.9 as determined by isoelectric focusing electrophoresis from a 
regression of distance versus the isoelectric point of standard proteins. 
The specific activity of the purified endochitinase was determined to be 
0.86 units/.mu.g protein with the turbidity reducing assay and 2.2 
nkatal/.mu.g protein when 
nitrophenyl-.beta.-D-N,N',N"-triacetylchitotriose was used as a substrate. 
The production and purification to homogeneity of this endochitinase are 
described in Harman et al U.S. Pat. No. 5,173,419, and also in Ser. No. 
07/919,784, filed Jul. 27, 1992. 
Another endochitinase is isolated from Gliocladium virens strain 41 having 
accession No. ATCC 20906 and has a molecular weight of 41 kDa (as 
determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis 
after the protein was prepared under reducing conditions, from a 
regression based on the log of molecular weight of standard proteins) and 
an isoelectric point of 7.8 as determined by isoelectric focusing from a 
regression of distance versus the isoelectric point of standard proteins. 
The procedures used for molecular weight determination and isoelectric 
point determination are the same as those described in detail in Ser. No. 
07/919,784. The enzyme is active in citric acid/K.sub.3 PO.sub.4 buffer 
over a pH range of 3.5 to 7.0 and shows a 90-100% activity between pH 4.0 
and 6.0 and shows maximum activity at pH 4.5. The optimum temperature for 
endochitinase activity at pH 5.5 is between 30.degree. and 37.degree. C., 
and activity drops off sharply at temperatures above 40.degree. C. The 
production and purification to homogeneity of this enzyme are described in 
detail in the patent application of Harman et al, Ser. No. 07/990,609, 
filed on Dec. 15, 1992. The enzyme was purified to an activity 105-fold 
that of its activity in the culture filtrate. 
The chitin 1,4-.beta.-chitobiosidases cleave dimeric units from chitin from 
one end, i.e. cleave chitobiose units from chitin. Chitin 
1,4-.beta.-chitobiosidases are sometimes referred to for convenience 
hereinafter as chitobiosidases. Chitobiosidase activity is readily 
determined by measuring the release of p-nitrophenol from 
p-nitrophenyl-.beta.-D-N,N'-diacetylchitobiose, e.g., by the following 
procedure. A substrate solution is formed by dissolving 3 mg of substrate 
in 10 ml 50 mM KHPO.sub.4 buffer, pH 6.7. Fifty .mu.l of substrate 
solution is added to a well in a microtiter plate (Corning). Thirty .mu.l 
of test solution is added, and incubation is carried out at 50.degree. C. 
for 15 minutes. Then the reaction is stopped by the addition of 50 .mu.l 
of 0.4M Na.sub.2 CO.sub.3, and the optical density is read at 410 nm. An 
activity of one nanokatal (nkatal) corresponds to the release of 1 nmol 
nitrophenol per second. 
A chitobiosidase is isolated from Trichoderma harzianum strain P1 having 
accession No. ATCC 74058 and in its most prevalent form has a molecular 
weight of 36 kDa (as determined by sodium dodecyl sulfate polyacrylamide 
gel electrophoresis after the protein was prepared under reducing 
conditions, on direct comparison to migration of a 36 kDa protein), and an 
isoelectric point of 4.4.+-.0.2 as determined based on its elution profile 
from a chromatofocusing column and a molecular weight of 40 kDa (as 
determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis 
after the protein was prepared under reducing conditions, from a 
regression based on the log of the molecular weight of standard proteins), 
and an isoelectric point of 3.9 as determined by isoelectric focusing 
electrophoresis from a regression of distance versus isoelectric point of 
standard proteins. Conditions for molecular weight determination and 
isoelectric point determination are described in detail in patent 
application Ser. No. 07/919,784. It has an optimum pH for activity of 
about 3 to 7. The production and purification of this chitobiosidase are 
described in Harman et al U.S. Pat. No. 5,173,419 where it is referred to 
as a chitobiase and also in patent application Ser. No. 07/919,784, filed 
Jul. 27, 1992 where it is referred to as a chitobiase and also as a 
chitobiosidase. The enzyme obtained in Ser. No. 07/919,784 has a specific 
activity of 127 nkatal/mg protein and is purified to greater than a 
200-fold increase in specific activity compared to its activity in the 
culture filtrate. Ser. No. 07/919,784 refers to the presence also of a 
minor band at 36 kDa. It has since been discovered that the chitobiosidase 
from Trichoderma harzianum strain P1 (ATCC 74058) gives three closely 
spaced protein bands with molecular weights of 40 kDa (staining most 
intensely), 38 kDa (faintest stain) and 35 kDa (intermediate intensity 
stain), as determined by sodium dodecyl sulfate polyacrylamide gel 
electrophoresis after the protein was prepared under reducing conditions, 
from a regression based on the log of the molecular weight of standard 
proteins, and that the three bands represent different levels of 
N-glycosylation of the same protein. The term "biologically pure" as used 
herein includes the 40 kDa enzyme isolated as described above with or 
without the same protein with different level of glycosylation also being 
present. 
The N-acetyl-.beta.-D-glucosaminidases cleave monomeric units from chitin 
from one end, i.e., release N-acetylglucosamine from chitin. 
N-Acetyl-.beta.-D-glucosaminidases may be referred to for convenience 
hereinafter as glucosaminidases. Glucosaminidase activity is readily 
determined by measuring the release of p-nitrophenol from 
P-nitrophenyl-.beta.-D-N-acetylglucosaminide, e.g., by the same procedure 
as described above for assaying for chitobiosidase activity except for the 
substitution of substrate. An activity of one nanokatal (nkatal) 
corresponds to the release of 1 nmol nitrophenol per second. 
Glucosaminidase activity is present in culture filtrates from Trichoderma 
harzianum strain P1 having accession No. ATCC 74058 and from Gliocladium 
virens strain 41 having accession No. ATCC 20906. 
The .beta.-1,3-glucanolytic enzymes include, for example, glucan 
1,3-.beta.-glucosidases. The glucan 1,3-.beta.-glucosidases cleave 
1,3-.beta.-glucans. The sources for these enzymes are typically the same 
as the sources for chitinolytic enzymes and are preferably microorganisms 
from the genera Trichoderma and Gliocladium. Glucan 1,3-.beta.-glucosidase 
activity is readily determined by measuring the amount of reducing sugar 
release from laminarin in a standard assay containing 250 .mu.l of enzyme 
solution and 250 .mu.l of a 0.1% solution of laminarin in 50 mM potassium 
phosphate buffer, pH 6.7, wherein incubation is carried out at 30.degree. 
C. for 1 hour whereupon 250 .mu.l of a copper reagent (prepared by 
dissolving 28 g Na.sub.2 PO.sub.4 and 40 g potassium sodium tatrate in 700 
ml deionized water, adding 100 ml of 1N NaOH, then adding 80 ml of a 10% 
(w/v) solution of CuSO.sub.4.5H.sub.2 O with stirring, then adding 180 g 
Na.sub.2 SO.sub.4, when all the ingredients have dissolved, bringing to 1 
L with deionized water, then allowing to stand for 2 days, then decanting 
and filtering) is added, and the admixture is covered with foil and heated 
for 20 minutes in a steam bath, whereupon, after cooling, 250 .mu.l of 
arsenomolybdate reagent (prepared by dissolving 25 g of (NH.sub.4).sub.6 
Mo.sub.7 O.sub.24.4H.sub.2 O in 450 ml deionized water, adding 21 ml 
concentrated H2SO.sub.4 with mixing, then adding a solution containing 3 g 
Na.sub.2 HAsO.sub.4.7H.sub.2 O in 25 ml distilled water and mixing, 
incubating at 37.degree. C. for 2 days and storing in a brown bottle until 
used) is added with mixing, followed by adding 5 ml deionized water, and 
reading color in a spectrophotometer at 510 nm, and wherein appropriate 
controls without either enzyme or substrate may be run simultaneously; the 
quantity of reducing sugar is calculated from glucose standards included 
in the assay. An activity of one nkatal corresponds to the release of 1 
nmol glucose equivalent per second. Glucan 1,3-.beta.-glucosidase activity 
is present in culture filtrates from Trichoderma harzianum strain P1 
having accession No. ATCC 74058 and from Gliocladium virens strain 41 
having accession No. ATCC 20906. 
A glucan 1,3-.beta.-glucosidase is isolated from Trichoderma harzianum 
strain P1 having accession No. ATCC 74058 and has a molecular weight of 78 
kDa (as determined by sodium dodecyl sulfate polyacrylamide gel 
electrophoresis after the protein was prepared under reducing conditions, 
from a regression based on the log of molecular weight of standard 
proteins) and an isoelectric point of 6.2 as determined by isoelectric 
focusing electrophoresis from a regression of distance versus the 
isoelectric point of standard proteins. The procedures for molecular 
weight determination and isoelectric point determination are the same as 
those described in Ser. No. 07/919,784. The enzyme has activity against 
.beta.-1,3 glucan laminarin between pH 4 and 7, with the strongest 
activity between 4.5 and 5.5. The enzyme is obtained and purified as 
generally described above with the medium for culturing of the 
microorganism being SMCS medium (described in comparative Example 2 
hereinafter). After the chromatofocusing step, several peaks with glucan 
1,3-.beta.-glucosidase activity are detected and fractions from major 
activity peaks are pooled, dialyzed, concentrated and applied to the 
Rotofor cell to obtain an electrophoretically pure exoglucosidase. The 
production and purification of the enzyme are described in detail in the 
patent application of Harman et al, Ser. No. 07/990,609, filed on Dec. 15, 
1992. The enzyme was purified to a specific activity 35-fold that of its 
activity in the culture filtrate. 
The cellulases are enzymes which cleave cellulosic polysaccharides. 
Cellulase activity is readily measured by the reducing group assay 
described previously except that cellulose or a cellulose derivative is 
substituted for laminarin. Other assays are known to those skilled in the 
art. One nkatal of activity corresponds to the release of 1 nmol glucose 
equivalent per second. 
Cellulases are produced, for example, by fungi of the genera Aspergillus, 
(e.g., Aspergillus niger), Trichoderma (e.g., Trichoderma viride) and 
Thielatia (e.g., Thielatia terrestris). 
Combinations of the above-described enzymes are useful herein. A preferred 
combination is provided by endochitinase and chitobiosidase in a weight 
ratio ranging from 3:1 to 1:1.2, very preferably ranging from 2:1 to 1:1. 
We turn now to the antifungal bacteria for use as component (b) of the 
composition herein. As indicated above, these bind to fungal cell walls in 
a 2% dextrose aqueous solution in the presence of enzyme (a), e.g., when 
fungal cells are cultured in potato dextrose broth in the presence of 
enzyme (a), but which do not bind to fungal cell walls in a 2% dextrose 
aqueous solution in the absence of enzyme (a), e.g., when fungal cell 
walls are cultured in potato dextrose broth without enzyme (a) present. 
Potato dextrose broth, when not further qualified, is used herein to mean 
the aqueous admixture formed by dissolving 24 g of a powder (obtained from 
Difco Laboratories, Detroit, Mich.) consisting of 20 g dextrose and 4 g 
solids, obtained from filtering and drying 200 g of infusion from 
potatoes, in 1 liter of water and sterilizing by autoclaving, final pH of 
5.1.+-.0.2. Preferably, the antifungal bacteria herein are selected from 
those of the genus Enterobacter and very preferably are not species which 
are pathogenic to humans or animals. Very preferred species of 
Enterobacter for use as antifungal bacteria (b) herein, include, for 
example, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter 
agglomerans, Enterobacter dissolvens, Enterobacter intermedius and 
Enterobacter sakazakii. Enterobacter cloacae is most preferred. 
Enterobacter cloacae is a frequent organism in the spermosphere and 
rhizosphere of plants and is rhizosphere competent. Enterobacter cloacae 
strain E6 having accession No. ATCC 39978 is especially preferred. The 
antifungal bacteria (b) of the composition herein do not include 
Pseudomonas fluorescens and Pseudomonas putida which unlike the bacteria 
of (b) do not bind to fungal cell walls when enzyme (a) is absent even in 
the absence of the sugars D-glucose and sucrose or when cultured in potato 
dextrose broth in the presence of enzyme (a). 
As indicated above, the enzyme (a) and bacteria (b) are present in a ratio 
of (b) to (a) ranging from 1 cell of (b) to 1 .mu.g of (a) to 200,000 
cells of (b) to 1 .mu.g of (a). Preferably the enzyme (a) and bacteria (b) 
are in a ratio of (b) to (a) which is at least 3 cells of (b) to 1 .mu.g 
of (a). 
The compositions herein are readily formulated by admixing the fungal cell 
wall degrading enzymes and antifungal bacteria with non-toxic carriers 
appropriate for the particular use for the composition, e.g., 
agriculturally acceptable carriers for agricultural uses. They may be 
formulated as liquids (solutions or suspensions) or as solids. Suitable 
carriers include, for example, water, adhesives such as 
carboxymethylcellulose, methyl cellulose, gum arabic, and polyethylene 
glycol, talc, peat moss, simple carbohydrates and particulate cellulose. 
The carrier can also be a medium which supports the growth of bacteria 
(b), such as potato dextrose broth, Richard's medium, Czapek's broth or 
trypticase soy broth, or medium which is dissolvable or suspendible or 
reconstitutable to be such bacteria growth supporting medium. The enzyme 
(a) should be present in an amount effective for binding of bacteria (b) 
to fungal cell walls when D-glucose and/or sucrose is present. For liquid 
compositions, the enzyme component (a) is preferably present at a 
concentration ranging from 5 to 100 .mu.g/ml, very preferably from 20 to 
50 .mu.g/ml for endochitinase, 50 to 75 .mu.g/ml for chitobiosidase and 5 
to 25 .mu.g/ml for at 1:1 combination of chitobiosidase and endochitinase, 
depending on the target fungus, for in vitro uses. Concentrations for 
practical agricultural uses can differ according to application and 
delivery system and may range up to 10 times those listed above as 
preferred for in vitro uses. 
We turn now to the method herein which is directed to inhibiting the 
replication, germination or growth of a fungus and comprises contacting 
such fungus, or a locus to be protected from such fungus, with an 
antifungal effective amount of appropriate composition herein. 
In its broad aspect, the method utilizing composition herein comprises 
contacting the target fungus, or a locus to be protected from such fungus, 
with a composition comprising (a) purified fungal cell wall degrading 
enzyme, and (b) antifungal bacteria as broadly described above, in a ratio 
of (b) to (a) ranging from 1 cell of (b) to 1 .mu.g of (a) to 200,000 
cells of (a) to 1 .mu.g of (a), with the combination of (a) and (b) being 
present in an antifungal effective amount. 
Preferably, the method utilizing the composition herein comprises 
contacting the target fungus, or a locus to be protected from such fungus, 
with a composition comprising purified fungal cell wall degrading enzyme 
(a) selected from the group consisting of endochitinase isolated from 
Trichoderma harzianum strain P1 having accession No. ATCC 74058, chitin 
1,4-.beta.-chitobiosidase isolated from Trichoderma harzianum strain P1 
having accession No. ATCC 74058 and combinations thereof, and antifungal 
bacteria (b) which are Enterobacter cloacae strain E6 having accession No. 
ATCC 39978, in a ratio of (b) to (a) ranging from 3 cells of (b) to 1 
.mu.g of (a) to 200,000 cells of (b) to 1 .mu.g of (a), with the 
combination of (a) and (b) being present in an antifungal effective 
amount. 
For agricultural purposes, application of antifungal composition can be to 
seeds, foliage, roots or fruit to be protected or to the soil surrounding 
a plant or seed to be protected, or to fungus thereon which is to be 
inhibited. Normally application is topical. 
The method of use herein utilizes compositions containing fungal cell wall 
degrading enzymes (a), which are chitinolytic enzymes or 
.beta.-1,3-glucanolytic enzymes, for application to fungi containing, as 
major structural components, chitin and .beta.-1,3-glucan, e.g., species 
from genera including Fusarium, Gliocladium, Rhizoctonia, Trichoderma, 
Uncinula, Ustilago, Erysiphe, Botrytis, Saccharomyces, Sclerotium and 
Alternaria. Example I hereinafter is directed to application to species 
from the genera Fusarium, Uncinula and Botrytis, which were selected in 
the work supporting this invention as model test fungi. 
The method of use herein utilizes compositions containing fungal cell wall 
degrading enzymes (a) which are cellulases for application to lower fungi 
(i.e., Oomycetes) where the cell walls contain, as major structural 
component, cellulosic polysaccharides, e.g., species from the genera 
Pythium and Phytophthora. 
In the composition herein and in the method of its use, interaction is 
provided between the cell wall degrading enzyme component and the 
antifungal bacteria component to increase the potency of the antifungal 
bacteria against pathogenic fungi and so that the antifungal bacteria bind 
to fungal cell walls even in the presence of sucrose and D-glucose to 
expand the range of use to seeds and plants which excrete these sugars. 
We turn now to the method herein for causing proliferation in the growth of 
Enterobacter cloacae which comprises the steps of reacting fungal cell 
wall degrading enzyme with a substrate therefor to obtain a nutrient for 
Enterobacter cloacae and growing Enterobacter cloacae in the presence of 
said nutrient so as to cause said proliferation. In the case of all 
substrates, the nutrient is made up of monosaccharide and/or 
oligosaccharide hydrolysis product. When the substrate is a fungus, the 
nutrient additionally contains cytoplasm (including lipids, proteins, 
carboxhydrates and nucleic acids) released from the substrate by action 
thereon of the cell wall degrading enzyme. 
In this method, the cell wall degrading enzymes are the same as those 
described for component (a) above except that the enzymes can be used in 
purified or natural form, i.e., not separated from the source (e.g., by 
utilizing source microorganisms in the composition herein). Even with this 
breadth, the method is not found in nature as indicated by the results of 
Example III wherein the control medium (E. cloacae and no substrate for 
enzyme) produced about the same or greater growth of E. cloacae as where 
E. cloacae and substrate for enzyme, but no added enzyme, were present. 
The substrates for the chitinolytic enzymes include moist purified 
colloidal chitin and for the .beta.-1,3-glucanolytic enzymes include 
laminarin and for chitinolytic and .beta.-1,3-glucanolytic enzymes include 
living hyphae of Fusarium, Gliocladium, Rhizoctonia, Trichoderma, 
Uncinula, Ustilago, Erysiphe, Botrytis, Saccharomyces, Sclerotium and 
Alternaria. The substrates for cellulases include species from the genera 
Pythium and Phytophthora. 
The reaction of cell wall degrading enzyme and substrate therefor is 
preferably carried out in a growth supporting medium for Enterobacter 
cloacae, e.g., potato dextrose broth, Richard's medium, Czapek's broth or 
trypticase soy broth. The reaction is carried out by utilizing substrate 
in a nutrient producing amount, e.g., 10 to 30 mg chitin, preferably 20 mg 
chitin, per ml of medium or 1.times.10.sup.4 to 5.times.10.sup.6 conidia 
of fungal substrate per .mu.l of medium. The reaction is preferably 
carried out by utilizing cell wall degrading enzyme at a concentration of 
1 to 500 .mu.g per ml of medium and incubating for 24 to 30 hours at 
25.degree. to 30.degree. C. Monosaccharide and oligosaccharide hydrolysis 
product, and when hyphae constitute substrate, also released cytoplasm, 
are preferably recovered, e.g., by centrifuging and filtering, and 
retaining the filtrate, and E. cloacae is preferably grown in the presence 
of the said filtrate by incubating for 24 to 48 hours at 25.degree. to 
30.degree. C. at greater than 80% relative humidity. 
The invention is illustrated in the following examples. 
In the Examples, the following applies. The results are the average between 
at least two experiments with three replicates for each experiment. The 
standard deviations were calculated from at least two experiments. For the 
analysis of spore germination, the values obtained for the control were 
taken as 0% inhibition, and all other values were divided by these values 
and multiplied by 100 to obtain percent of inhibition. Limpel's formula as 
described by Richer, D. L., Pestic. Sci. 19:309-315 (1987) was used to 
determine antifungal synergistic interactions. This formula is E.sub.e 
=X+Y-XY/100 where E.sub.e is the expected effect from the additive 
responses of two inhibitory agents, and in the present case, X and Y are 
the percentages of inhibition relative to each agent used alone, and a 
value greater than E.sub.e indicates synergism exists. 
EXAMPLE I 
This experiment involved determining any synergistic effect obtained in 
inhibiting conidia germination and germ tube elongation of Botrytis 
cinerea, Fusarium solani and Uncinula necator by combination of 
endochitinase described hereinbefore which is isolated from Trichoderma 
harzianum strain P1 having accession No. ATCC 74058, chitin 
1,4-.beta.-chitobiosidase described hereinbefore having a molecular weight 
of 40 kDa and an isoelectric point of 3.9 which is isolated from 
Trichoderma harzianum strain P1 having accession No. ATCC 74058, or a 1:1 
weight mixture of these enzymes, and Enterobacter cloacae strain E6 having 
accession No. ATCC 39978, Pseudomonas fluorescens strain TL-3 (Burr, T. 
J., et al, Phytopathology 68, pages 1377-1383, 1978) or Pseudomonas putida 
strain BK-1 (Burr, T. J., et al, Phytopathology 68, pages 1377-1383, 
1978). 
Conidia of B. cinerea and F. solani were grown at 20.degree.-25.degree. C. 
on potato dextrose agar (Difco Laboratories, Detroit, Mich.), suspended in 
water, filtered through sterile Kimwipes (Kimberly-Clark, Roswell, Ga.). 
Conidia of U. necator were produced on grapes grown aseptically in tissue 
culture, suspended in 0.4 osmol mannitol, filtered through sterile 
Kimwipes. 
Cells of E. cloacae strain E6 (ATCC 39978) were grown in potato dextrose 
broth at 28.degree.-30.degree. C. on a rotary shaker at 200 rpm to mid log 
phase (optical density of 600 nm=0.4 to 0.6), harvested by centrifugation, 
washed in 0.85% NaCl solution and resuspended in sterile water for the 
bioassays. 
Cells of the Pseudomonas spp were grown in King's Medium B at 
28.degree.-30.degree. C. on a rotary shaker at 200 rpm to mid log phase 
(optical density of 600 nm=0.4 to 0.6), harvested by centrifugation, 
washed in sterile water and resuspended in sterile water for the 
bioassays. 
Enzyme solutions were kept at 4.degree. C. and utilized for the bioassays 
within two weeks. Otherwise they were concentrated to dryness in a 
SpeedVac apparatus (Savant Instruments, Farmingdale, N.Y.) and stored at 
-20.degree. C. until dissolved to provide solutions for the bioassays. 
Test solutions or suspensions were prepared that contained in sterile water 
either a single enzyme, a 1:1 mixture of the two enzymes, bacterial cells 
from a single strain or enzyme(s) and bacterial cells from a single 
strain, and sterile water was used as a control. 
The enzyme solutions were made up to provide in an assay a concentration of 
enzyme(s) with an ED.sub.20-30 for spore germination for the test fungus 
participating in the assays (i.e., a dose effective to inhibit 20% to 30% 
of spore germination). The enzyme concentrations for the assays for U. 
necator, F. solani and B. cinerea were respectively 50, 75 and 50 .mu.g 
ml.sup.-1 for the chitobiosidase, 25, 50 and 25 .mu.g ml.sup.-1 for the 
endochitinase and 9, 25 and 8 .mu.g ml.sup.-1 for the total protein 
constituting the 1:1 mixture of the chitobiosidase and the endochitinase. 
Since the enzyme test solution constituted one-third of an assay mixture, 
the enzyme test solutions were made up at 3 times the strength desired in 
the assays. 
The test suspensions of bacterial cells were made up to initially contain 
1.5 to 2.5.times.10.sup.4 bacterial cells ml.sup.-1 so as to provide 
300-500 bacterial cells in an assay mixture. For E. cloacae, this amount 
of cells produced about 30% inhibition of the spore germination for all of 
the test fungi and 45 to 50% inhibition of germ tube elongation for all of 
the test fungi. Increasing or decreasing the initial inoculation for E. 
cloacae proportionally affected the level of inhibition, except that the 
highest level of inhibition of spore germination that could be obtained by 
increasing the initial inoculum of E. cloacae was 65%. For the Pseudomonas 
spp., the highest level of inhibition of spore germination that could be 
obtained by increasing the initial inoculum was not more than 10%. 
Suspensions of test fungus containing 10.sup.6 -10.sup.7 conidia ml.sup.-1 
were made up for the assays. 
Additionally a potato dextrose broth solution was made up of strength three 
times that to be present in the assay mixtures as medium for the assays. 
For the assays, 20 .mu.l of test solution or control, 20 .mu.l of conidia 
suspension and 20 .mu.l of potato dextrose broth solution medium were 
admixed in a sterile Eppendorf tube. The tubes were incubated at 
25.degree. C., and after 24 to 30 hours, the percentage of germinating 
conidia for each tube (percentage of the first 100 spores seen on a 
microscope slide), and the lengths of 20 germ tubes were measured and 
averaged. Percent inhibition of spore germination was calculated according 
to the following equation: %I=(1-%S.sub.t /%S.sub.c).times.100, where %I 
represents the percentage inhibition, %S.sub.t represents percentage 
germination of spores in the treatment of interest, and %S.sub.c 
represents the percentage germination of spores in the control (i.e., with 
neither antifungal bacteria nor enzyme). A similar equation was used to 
calculate inhibition of germ tube elongation, except that germ tube length 
in .mu.m for treatment of interest and for the control were respectively 
substituted for %S.sub.t and %S.sub.c. 
Results are set forth in FIGS. 1A and 1B. 
FIGS. 1A and 1B are sets of bar graphs depicting % inhibition for enzymes, 
E. cloacae and the combination of enzymes and E. cloacae, in respect to 
the three target fungi, respectively of spore germination and germ tube 
elongation. In FIGS. 1A and 1B, the solid black bars represent assays 
involving B. cinerea, the diagonally hatched bars represent assays 
involving F. solani and the horizontally hatched bars represent assays 
involving U. necator and "E6" means E. cloacae strain E6, "Chitobios" 
means the chitobiosidase described in this example, "Endochit" means the 
endochitinase described in this example and "Chitobios/Endochit" means the 
1:1 mixture of chitobiosidase and endochitinase described in this example. 
In FIGS. 1A and 1B, the error bars indicate standard deviations. 
The data of FIGS. 1A and 1B demonstrates synergy according to Limpel's 
formula. 
Turning firstly to FIG. 1A, even assuming 30% inhibition of spore 
germination for enzyme alone and for E. cloacae alone in the case of each 
fungus, Limpel's formula gives 30+30-30(30)/100 which equals 51. Even if 
the effect of E. cloacae is maximized by increasing the initial inoculum 
to provide 65% inhibition, Limpel's formula gives 65+30-65(30)/100 which 
equals 75.5. However, as shown in FIG. 1A, the combination even without 
increased inoculum of E. cloacae provides levels of inhibition close to 
100%, thereby demonstrating synergy. 
Turning now to FIG. 1B and considering it as showing 30% inhibition of germ 
tube elongation for enzyme alone and 50% inhibition of germ tube 
elongation for E. cloacae alone, Limpel's formula gives 65%. On the other 
hand, the result shown for the combination is about 90% inhibition or 
more, thereby demonstrating synergy. 
In summary, when E. cloacae or enzyme was added to the target fungi alone, 
there was some effect. However, when enzyme and E. cloacae were combined, 
the target fungi were largely destroyed; spore germination was reduced to 
very low levels, and the surviving germ tubes grew poorly. Furthermore, 
synergy was evident with the combination of either or both enzymes and E. 
cloacae. 
The addition of cells of Pseudomonas spp to samples containing either of 
the enzymes from T. harzianum strain P1 or combination thereof, did not 
increase the level of inhibition for any fungus tested. This demonstrates 
that the synergistic result is not obtained when antifungal bacterium 
which does not bind to fungal cell walls is substituted for the E. cloacae 
(which as shown in Example III binds to fungal cell walls in the presence 
of cell wall degrading enzyme even when interfering sugars are present). 
COMATIVE EXAMPLE 1 
Cells of E. cloacae strain E6 (ATCC 39978) were grown on potato dextrose 
broth at 28.degree.-30.degree. C. on a rotary shaker at 200 rpm until mid 
to late log phase was reached (optical density at 600 nm of 0.7 to 1.0). 
The culture filtrate was harvested by centrifugation, filtered through a 
0.45 .mu.m pore size filter, dialyzing the filtrate against distilled 
water overnight at 4.degree. C. and concentrating by dialysis (6-8 kDa 
cutoff) about 20- to 25-fold using polyethylene glycol (35,000 MW, Fluka 
Chemika Biochemika, Buchs, Switzerland). 
Test solutions were made up of the dialyzed, concentrated culture filtrate, 
with and without enzymes (chitobiosidase, endochitinase and a 1:1 mixture 
of chitobiosidase and chitobiase as in Example I). 
Suspensions of conidia of B. cinerea, F. solani and U. necator were made up 
as in Example I. 
Potato dextrose broth solution of three times strength was made up as in 
Example I. 
Assays were carried out as in Example I except that culture filtrate from 
E. cloacae was utilized in place of living bacteria. 
No synergistic antifungal effect was noted for the combinations of E. 
cloacae culture filtrate and enzymes from T. harzianum strain P1 against 
any of the test fungi. 
For example, with culture filtrate alone, 21% inhibition of spore 
germination of B. cinerea was obtained. For endochitinase alone at 25 
.mu.g ml.sup.-1 in the assay, 28% inhibition of spore germination of B. 
cinerea was obtained. For the combination, 38% inhibition was obtained. 
Substituting 21 and 28 in Limpel's formula gives 43%. Thus, no synergy was 
present. The above indicates that the presence of intact bacterial cells 
is required for a synergistic effect rather than extracellular metabolite 
thereof. 
EXAMPLE II 
Assays were carried out as in Example I except that E. cloacae cell density 
was determined rather than % inhibition. The cell density was determined 
for bacterial cells not in clusters and was carried out microscopically 
using a Petroff-Hauser counting chamber. Results are shown in FIG. 2 which 
is a set of bar graphs depicting amount of E. cloacae cells determined for 
the various test solutions. In FIG. 2, "Chitobios" means chitobiosidase, 
"Endochit" means endochitinase, "Chito/Endo" means a 1:1 mixture of 
endochitinase and chitobiase, fungus means B. cinerea, F. solani or U. 
necator (results were similar for the test fungi) and "Control" means 
sterile water. As shown in FIG. 2, the density of cells of E. cloacae not 
associated in clusters was about 10-fold higher in samples containing test 
fungus and chitinolytic enzymes, compared to controls without enzyme 
and/or fungus. This shows that chitinolytic enzyme in the presence of 
substrate (in this case the fungus) stimulated the replication of E. 
cloacae. 
EXAMPLE III 
1.times.10.sup.6 conidia of test fungi or 2% moist purified chitin 
(prepared as described in Vessey, J. C., et al, Trans. Br. Mycol. Soc. 
60:133-143, 1973 by grinding and washing crab shell chitin with distilled 
water, and then with a mixture containing ethanol:diethyl ether:HCl, 
50:50:1, precipitating by diluting with ice water, and then repeatedly 
washing with water adjusted to pH 8.5 until the pH of the chitin equals at 
least 3) were placed in 200 .mu.l potato dextrose broth in sterile 
Eppendorf tubes at 25.degree. C., and after 24 hours endochitinase from T. 
harzianum strain P1 and/or the 40 kDa chitobiosidase from T. harzianum 
strain P1 were added to the tubes at a final concentration of 50 .mu.g 
ml.sup.-1 for endochitinase and 100 .mu.g ml.sup.-1 for chitobiosidase. 
Control samples contained medium, substrate and sterile water instead of 
enzyme or medium, enzyme and sterile water instead of substrate or only 
medium. After this addition the tubes were reincubated at 25.degree. C. 
After 24 hours, all samples were centrifuged and the supernatant was 
recovered and filtered through a 0.45 .mu.m pore size polysulfone filter. 
For each sample, one hundred .mu. l of the resulting filtrate was placed 
in a well of a standard ELISA plate containing about 500 cells of E. 
cloacae strain E6 suspended in 10 .mu.l of potato dextrose broth. For each 
sample, the absorbance at 560 nm was determined initially. Then each plate 
was incubated at 30.degree. C. over moistened towels to maintain high 
relative humidity. Bacterial growth was monitored at 24, 29 and 48 hours 
by measuring the absorbance at 560 nm and subtracting the initial value 
from the readings obtained. 
Results for 29 hours are set forth in FIG. 3 which is a set of bar graphs 
depicting change in absorbance for various substrates and enzymes. 
As shown in FIG. 3, after 29 hours, the absorbance at 560 nm was much 
higher in samples containing a chitinous substrate (chitin or fungi) in 
the presence of chitinolytic enzymes compared with a control medium or 
with controls containing a substrate incubated without enzyme or enzyme 
incubated without a substrate, indicating a much higher bacterial cell 
density where enzyme and substrate were present. Differences in bacterial 
cell density between samples were also confirmed microscopically in a 
Petroff-Hauser counting chamber. 
The results suggest that the enzymes released sufficient nutrients from the 
substrates (chitin and fungi) to proliferate the E. cloacae to high 
levels. 
EXAMPLE IV 
Young hyphae of B. cinerea in potato dextrose broth supplemented with 100 
mM sucrose were inoculated with cells of E. cloacae strain E6 in the 
absence of enzyme. The E. cloacae cells did not bind to the B. cinerea 
hyphae. 
In one case endochitinase from T. harzianum strain P1 at 50 .mu.g ml.sup.-1 
and in a second case the 40 kDa chitobiosidase from T. harzianum strain P1 
at 75 .mu.g ml.sup.-1 were added. Binding of the E. cloacae cells to the 
B. cinerea starts to occur within 2 to 5 hours and occurs in almost all 
cases within 4 to 5 hours. The binding is coincident with a great 
increment in antifungal activity. 
COMATIVE EXAMPLE 2 
E. cloacae strain E6 bacteria were grown for 3 days at 28.degree. to 
30.degree. C. to mid to late log phase (optical density at 600 nm=0.7 to 
1.0) in 250 ml of SMCS medium (680 mg KH.sub.2 PO.sub.4, 870 mg K.sub.2 
HPO.sub.4, 200 mg KCl, 1 g NH.sub.4 NO.sub.3, 200 mg CaCl.sub.2, 200 mg 
MgSO.sub.4.7H.sub.2 O, 2 mg FeSO.sub.4, 2 mg ZnSO.sub.4, 2 mg MnSO.sub.4, 
42 g moist colloidal chitin prepared as described in Example III, in 1 L 
distilled water, final pH 6.0) and in another case in SMS medium (same as 
SMCS medium but with 5 g sucrose substituted for colloidol chitin). The 
culture filtrate was harvested by centrifugation and filtration through a 
0.45 .mu.m pore size filter and the filtrate was dialyzed against 
distilled water overnight at 4.degree. C., and then concentrated by 
dialysis (6-8 kDa cutoff) about 20- to 25-fold using polyethylene glycol 
(35,000MW; Fluka Chemika-Biochemika, Buchs, Switzerland) and assayed for 
enzyme activity. No chitobiosidase, glucosaminidase or glucanase activity 
was detected. A low level of endochitinase activity was detected; 500 
.mu.l of the dialyzed concentrated culture filtrate reduced the turbidity 
of a suspension of colloidal chitin 28% when the bacteria were grown on 
SMCS medium and 14% when the bacteria were grown on SMS medium. 
As indicated in FIG. 3, the endochitinase activity produced by E. cloacae 
is not such as to provide synergy as indicated by comparison of results 
for control medium with those for substrate (chitin or fungi). 
As indicated in Example IV, the endochitinase activity produced by E. 
cloacae is not such as to effect binding to fungal hyphae when interfering 
sugars are present. 
Variations of the invention will be obvious to those skilled in the art. 
For example, genes coding for chitinolytic enzymes can be added to bacteria 
that adhere to fungal cell walls (in the absence of interfering sugars but 
not in the presence of D-glucose and sucrose) so that resulting transgenic 
bacteria produce chitinolytic enzymes thereby to provide potent control of 
pathogenic fungi and protect plant seeds regardless of level of 
carbohydrates exuded during seed germination. 
Therefore, the invention is defined by the claims.