CA1316856C - Biological method and compositions for control of fruit tree crown and root rot with enterobacter aerogenes - Google Patents

Abstract

NOVEL BIOLOGICAL METHOD AND COMPOSITIONS
FOR CONTROL OF FRUIT TREE CROWN AND ROOT
ROT WITH ENTEROBACTER AEROGENES

ABSTRACT OF THE INVENTION

This invention relates to the novel use of Enterobacter aerogenes and compositions thereof for the control and prevention of pathogenic crown and root rot of fruit bearing and non-fruit beaning trees. Particu-larly, in one embodiment, the invention relates to the control of crown and root rot disease of fruit trees caused by the soil borne fungus Phytophthora cactorum.
A method of prevention and control root and crown rot in fruit bearing and non-fruit bearing trees due to fungus that utilizes a biocidal bacterial strain of the genus Enterobacter.

Description

NOVEL BIOLOGICAL METHOD AND COMPOSITIONS
FOR CONTROL OF FRUIT TREE CROWN AND ROOT

This invention relates to the novel use of Enterobacter aeroqenes and compositions thereof for the control and prevention of pathogenic crown and root rot disease of ruit and non-fruit bearing trees. Particu-larly, in one embodiment, the invention relates to the control of crown and root rot disease of fruit trees caused by the soil borne fungus Phytophthora cactorum.

Crown and root rot disease of fruit trees is caused primarily by the soil-borne fungus Phytophthora cactorum (Leb. & Cohn) Schroet. This disease is of ; 20 world wide distribution. Tree loss in mature apple trees from crown rot averages 2~ per year in British Columbia, Canada. In other words, about 2% of the current population of mature apple trees in British Columbia (3 million) require premature replacement at a cost of about five and a half dollars per tree per year.
In addition, crown rot not only kills the tree but also reduces productivity. It is estimated that the equiva-lent of 1% of the value of the annual British Columbia apple crop is lost to the disease, that is, about $930,000 annually. ~n the absence of better knowledge about crown rot, it is assumed that the disease would be at least as persistent and damaging in the future as it is currently.
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The fungicide Ridomil (trade mark) is currently registered and recommended for control of crown rot on non-bearing apple trees. The degree of control is good to excellent. Current cost of Ridomil treatment per tree per year is 50 cents. For bearing apple trees, current recommendations are relatively ineffective and costly in terms of labour, so much so that they are not widely used. As a protectant aqainst infection, a suspension of mancozeb fungicide is injected into the soil around the crown of trees using a probe and high pressure pump. For trees exhibiting symptoms of infec-tion, the soil is removed from around the crown area, the infected tissue is removed and the wound is treated with mancozeb.
Such chemical control is not satisfactory.
Pesticide use has a number of problems, some of which are: (1) pesticide resistance; (2) development of new secondary pests; (3) pesticide residues in the environ-ment; (4) increasing cost; and (5) most importantly,human health concerns.

The inventor is aware of the following refer-ences and patents which disclose inventions and prior teachings which are potentially relevant to the subject matter of this invention.

Canadian Patent No. 1,106,201 Jacques Ricard, August 14, 1981, discloses a product with mycofungicidal activity containing propagules or spores of immunizing commensals and cereal flour. The product is charac-terized in that it contains propagules or spores of Scytalidium liqnicola ATCC 16,675, of Trichoderma polYsporum Rifai ATCC 20,475, of Trichoderma viride sensu Bisby ATCC 20,476, or of Trichoderma viride senser '' ' ' ~ :

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Bisby strain CG/BI~AB-INRA I 030 (Institut Pasteur, Paris - France).

Canadian Patent No. 1,119,512, Michael G~
Boosalis, March 9, 1982, refers to the effectiveness of biological agent Corticum sensu lato against Rhizoctonia. There is, however, no data to show the efficacy of this produc~ against Phytophthora, _Ythium, Fusarium or Sclerotia species, although it was claimed to be effective. The method of application of this agent is entirely different from the invention herein. To the applicant's knowledge, Corticum sensu lato has never been tested for effectiveness against crown or root rot of apple trees caused by PhYtophthora cactorum. The crops on whlch the eEfectiveness of Corticum sensu lato has been shown are sugar beet and soybeans.

Canadian Patent No. 1,176,861, Rolf O.
Hultman, October 30, 1984, relates to a method of protecting flowering plants, bushes, trees and like plants against attack from pathogenic fungi, by treating the plants with fungi which are antagonistic toward the pathogenic fungi. The invention also relates~to a fungicide for carrying out the method, and to a method of producing the fungicide. Specifically disclosed is a method of protecting flowering plants, bushes, trees and like plants against attack from pathogenic fungi by treating the plants with funqi antagonistic towards the pathogenic fungus. The method is characterized by treating the soil in which the flowering plant, bush, tree or like plant is intended to grow, or grows, with a product obtained by cultivating the antagonistic fungus on a cultivating substrate comprising compost material obtained by biologically decomposing organic compostible material and treated at a temperature of at least about ;

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70C over a period of about 50 hours (about 2 calendar days) or longer to kill microorganisms which inhibit the cultivation of the antagonistic fungus.

S V.S. Patent No. 3,819,829, Elton W. Mann, June 25, 1974, claims a method for the control of fungal diseases of plants which comprises applying to the seeds of the plants a fungistatically effective amount of a viable culture of Bacillus uniflaqellatus, (ATCC 15134).
The method is specifically intended for controlling black-hull disease in peanuts.

The antagonistic zone of Bacillus uniflaqellatus against Phytophthora (species unknown) is only 8 mm. In comparison, the antagonistic zone between Enterobacter aeroqenes and PhYtophthora cactorum on a 60 mm petri plate is 31 mm. This zone of inhibition by E.
aeroqenes is much larger than Bacillus uniflaqellatus.
It is known that B. uniflaqellatus is effective in soil only against Fusarium wilt and damping off of cotton and peanuts.
i U.S. Patent No. 3,285,807 relates to chemical control of PhYtophthora infestans, which is a causal agent of late blight on tomato. Efficacy data has demonstrated that this product is only preventative against P. infestans.

SUMMARY OF THE INVENTION
A method of prevention and control of root and crown rot in fruit bearing and non-fruit bearing trees due to fungus that utilizes a biocidal bacterial strain.
The root and crown rot in fruit trees may be due to Phytophthora cactorum. The bacterial strain may be ~`:

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Enterobacter aeroqenes. The strain of Enterobacter aeroqenes may be propagated and reselected so that it is resistant to fungicide. The strain of Enterobacter aeroqenes may be B8 as maintained at Agriculture Canada, Summerland, British Columbia. The fruit bearing tree may be a bearing or a non-bearing apple tree.

The method may involve drenching the fruit tree or the soil surrounding the fruit tree with a suspension comprising a root and crown rot control effective amount of Enterobacter aeroqenes and a suit-able carrier. The carrier may be water. The concentra-tion of Enterobacter aeroqenes in the carrier may be between about 2.0 X 10~ and 2.0 X 107 colonY forming units (CFU) per ml. The suspension may be used for the control of apple replant disease. The suspension may be applied sequentially with the fungicide Ridomil.

The invention includes a strain of Enterobacter aeroqenes which is useful in the control of crown and root rot in fruit bearing trees. The strain is produced by propagating an Enterobacter aeroqenes strain and reselecting the strain so that it is resis-tant to fungicide and antibiotics. The strain may be B8.

The invention also includes a composition which is useful in the control of plant pathogens com-prising Enterobacter aeroqenes and a suitable carrier such as water. The strain of Enterobacter aeroqenes in the composition may be propagated and reselected so that it is resistant to fungicide and antibiotics. The strain may be B8.

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A deposit of the B8 strain of Enterobacter aeroqenes has been made at Agriculture Research Culture Collection, Peoria, Illinois, under No. NR~L B-18268.
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DETAILED DESCRIPTION OF SPECIFIC
EMBODIMENTS_OF THE INVENTION

Enterobacter aeroqenes (Kruse) Hornaeche and Edwards, has been found to have the potential for pre-ventative and curative control of the crown and root rot of apple trees. The applicant's Enterobacter aeroqenes is different from any of the products mentioned above and is specifically designed for control of _hYtophthora cactorum on apples. Nonetheless, there is a potential of using this bacterium for control of other plant pathogenic Eungi such as PYthium ultimum, Rhizoctonia solani, Penicillium exansum, BotrYtis cinerea, Monilinia fructicola, Botrytis allii, Venturia inequalis, Alternaria alternata and Collitotrichum lindemuthianum as is indicated by in vitro tests. The applicant's method of culturing and applying the bacterium under field conditions is entirely different from the methods described for the above mentioned products. The product has preventative as well as curative ability against PhYtophthora cactorum on apples.
' Through a survey of naturally occurring soil microflora for antagonistic and hyperparasitic proper-ties against PhYtophthora cactorum, which is a fungus responsible for crown and root rot disease of fruit trees, the applicant has isolated a strain of Enterobacter aeroqenes, which has been designated as B8, that has been found to be highly antagonistic to P.
cactorum. B8 has been propagated in the laboratory and reselected so that the current strain B8 is highly ' :
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resistant to Ridomil and Mancozeb which are fungicides commonly used in orchards. Moreover, this naturally occurring bacterium has been altered by selection pressure to develop resistance to Streptomycin sulfate, Cyclohexamide and senlate (trade mark), 200, 100 and 30 ppm, respectively. Naturally occuring bacterium is jsensitive to these antibiotics. Through a series of greenhouse and orchard experiments, the inventor has demonstrated that B8 applied to the crown and root zone as a soil drench of living bacteria provides a high degree of protection against infection of apple trees by P. cactorum. In the case of apple trees with infection by P. cactorum in the initial stages, this treatment is curative.
The treatment product is produced and applied in the following manner. E. aeroqenes is grown on dextrose broth for 72 hours. Stock bacterial suspen-sions were made by pouring 5 mililitres of sterile water on the plate containing the bacteria, loosening the bacteria with a sterile rubber spatula, and then pouring the contents into 100 ml of sterile water. Bacterial counts in suspension were made by haemacytometer. Each infected tree was then drenched around the base and on the trunk with 5 L of water containing the appropriate concentrations of bacterial population. The treatments were applied with a pesticide sprayer. The bacterial population was 2.0 x 10l per tree. The trees were in the initial stage of infection with 1/4 of the bark at the crown region infected by P. cactorum. The trees were fifteen years old and were spaced 6 x 4 m. The rootstock was MMl06 and the scion cultivar was McIntosh.
A completely randomized block design consisting of ten single tree applications was used. Infected bark on all trees was removed before application of the product.

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The product was applied biannually in late April and in mid October for three years.

A11 naturally infected trees treated with the product containing E. aerogenes (B8) remained alive and produced normal fruit, while the untreated check trees were terminally diseased and were pulled out in three years. E. aeroqenes (B8) has also shown antagonism on agar plates to several serious pathogens like PYthium ultimum, Rhizoctonia solani, Penicillium expansum, Botrytis cinerea, Monilinia fructicola, BotrYtis allii, Venturia inequalis, Alternaria alternata, Collitotricum lindemuthianum. This antagonist has also shown poten-tial for the control of apple replant disease under field conditions. Apple replant disease may be caused by biotic or abiotic factors such as fungi, bacteria, nemotodes, nutrition deficiencies or low or high pH of the soil. Replant disease relates to the poor growth of replanted apple trees on old orchard sites.
In one test, bacterial isolate B8 from local soil, identified as Enterobacter aeroqenes, produced a diffusible substance antagonistic to the growth of Phytophthora cactorum, the causal agent of apple crown and root rot. This substance appeared to be fungicidal in vitro. A11 biological and chemical treatments signi-ficantly reduced the percent infection of McIntosh seedlings under greenhouse conditions but metalxyl, fosetyl-A1, and metalxyl + E. aeroqenes controlled the disease (Table 1). E. aeroqenes applied alone as a soil and trunk drench for field control of crown rot signi-ficantly reduced the percent infection of artifically inoculated apple trees on MMl06 rootstock (Table 2).
Metalaxyl and fosetyl-Al applied alone completely controlled the disease in field tests. When metalaxyl :
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and E. aeroqenes were applied alternately, control of the disease was comparable to that of metalaxyl alone.

In another field test, trees naturally infected with P. cactorum and treated with either E.
aeroqenes or metalaxyl as a soil drench remained alive and produced normal fruit while the untreated trees were terminally diseased and were removed in three years.
The level of protection provided by metalaxyl and fosetyl-Al applied alone, and E. aeroqenes alone or in combination with metalaxyl suggests potential avenues for practical field control of crown and root rot of apple trees.

Crown and root rot disease of apple trees (Malus Pumila~ Mill.) is primarily caused by PhYtophthora cactorum (Leb. & Cohn) Schroet., although P. cambivora (Petri) Buisman, P. sYrinqae Klebahn and PhYthium ultimum Trow also have been implicated (Ref.
8). Phytophthora cactorum is the primary casual agent of crown and root rot in the Okanagan, Similkameen, and Kootenay valleys of British Columbia, Canada (Ref. 8 and 17). Biological control of apple crown rot has been obtained by applying a siderophore-producing Pseudomonas sp. directly on the lesion caused by P. cactorum (Ref.
6). In addition, a strain of Enterobacter aeroqenes (Kruse) Hornaeche and Edwards designated B8, was found to be antagonistic to P. cactorum on corn meal agar (Ref. 13). Under greenhouse conditions using sterile field soil, this strain of E. aeroqenes significantly reduced infections on apple seedlings by three isolates of P. cactorum (Ref. 14). The growth and antagonistic ability of E. aeroqenes were not significantly affected when 50 and 100 mg/L of metalaxyl, fosetyl-Al or mancozeb were added to the corn mean agar (Ref. 13).

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This suggests that control of crown rot may be possible by a combination of biological and chemical treatments.

The following specifically describes in detail the fungicidal and fungistatic activity of substances produced by E. aeroqenes and the evaluation of this antagonist and of fungicides applied singly or in combination for the control of crown rot in sandy loam soils of British Columbia, Canada.
Example 1 A strain of Enterobacter aeroqenes, which has been designated as B8, has been found to be highly antagonistic to P. cactorum. B8 was propagated in the laboratory and reselected so that the current strain B8 was highly resistant to Ridomil and Mancozeb which are fungicides commonly used in orchards. Moreover, this naturally occurring bacterium was altered by selection pressure to develop resistance to Streptomycin sulfate, Cyclohexamide and Benlate (trade mark), 200, 100 and 30 ppm, respectively.

Example 2 A local virulent isolate (PhO3) of P. cactorum was used in all ln virto and greenhouse tests. P.
cactorum and E. aeroqenes (B8), prepared as described above, were placed on corn meal agar (CMA) 40 mm apart on a 60 x 15 mm petri plate, to test fungistatic activity of the latter. The fungal inoculum consisted of a 5 mm disk cut from an actively-growing culture on CMA. To test the fungicidal activity of diffusates produced by E. aeroqenes, a plug P. cactorum was grown on CMA for two weeks in the presence of the bacterium :

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and then was transferred to unamended CMA. The check consisted of a P. cactorum plug grown alone on CMA for two weeks. The plates were incubated at 18C for two weeks after inoculation. All tests were replicated ten times and each experiment was repeated twice.

The fungicides evaluated were metalaxyl ~'Ridomil 2~'TM, methyl N-(2-methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate, Ciba-Geigy Canada Ltd.];
fosetyl-aluminum ['Aliette'TM, aluminum tris (ethyl phosphonate), Rhone-Poulenc Sanitaire, Lyon, France];
mancozeb ['Dithane M-45'TM, a complex of 2.5% zinc and manganese ethylene bis (dithiocarbamate), DuPont Canada Inc.]; THIS [flowable copper 4.4~ and sulphur 50%, a nutrient fertilizer supplement, Stoller Chemical Inc.]; and captafol ['Difolatan 4F'TM, cis-N-(1,1,2,2,-tetra chloroethylthio)-4cyclohexene-1~

2-dicarboximide), Chevron Chemical Company, U.S.A.].

Example 3 Three fungicides, metalaxyl, fosetyl-Al and mancozeb, were evaluated separately. E. aeroqenes was evaluated separately and in combination with metalaxyl in a randomized complete block design. Three replicates consisting of five seedlings in each replicate, growing in autoclaved (121C, 20 min.) field soil (pH 7.2) in 10 cm plastic pots were used. The test was conducted in a greenhouse at 22 ~ 3C with 13 hour photoperiod.
E. aeroqenes (B8) suspensions were prepared as described above. Bacterial suspensions averaged 3.0 x 107 colony-forming units (CFU)/pot. The fungicides were applied as a soil drench in 50 ml of sterile dis-tilled water. Three-week-old McIntosh seedlings were .

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planted in each plastic pot, 10 days after the soil treatments.

Cultures of P. cactorum Eor inoculating seed-lings in greenhouse test were prepared as describedearlier. Counts of sporangia were made and then stock suspensions containing 100 sporangia/ml were prepared.
Each seedling was inoculated with 5 ml of the stock suspension 10 days after planting. The seedlings were flooded for 48 hours by closing the drain holes of the pots with tape and adding water to a depth of 1 cm above the soil surface. After 48 hours the tape was removed and the soil was allowed to drain freely. The seedlings were watered as needed thereafter.

The seedlings were removed from their contain-ers three months after treatment, the roots washed in running tap water and disease severity evaluated.
Symptomatic roots were plated on PlOVP selective medium (Ref. 12) and colonies typical of P. cactorum were examined under a microscope to confirm root infection by the pathogen. Percent infection was calculated from the infected seedlings divided by total number of seedlings in each pot and transformed to arc sine for analysis of variance-Example 4 The test was conducted in a local sandy loam soil (pH 7.6) in the Okanagan Valley of British Columbia. Five fungicides, E. aeroqenes, and a combi-nation of E aeroqenes and metalaxyl were evaluated for control of crown rot. Two-year-old trees on the crown - rot susceptible rootstock MM106, were planted on June 4, 1981 and budded with 'MacSpur' in August 1981. Tree . ' ~

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spacing was 4 x 1.5 m. Treatments were arranged in a 3 x 3 balanced lattice design with 4 replications. Each tree in each treatment was drenched around the base and on the trunk with 4.5 L of water containing the appro-priate material. The treatments were applied with ahome made plot sprayer that included a by-pass valve, mechanical agitator, and a flow restrictor valve (9 litres/min) on the discharge hose. Each plot consisted of eight trees. The first application of chemical and biological material was made at the time of planting and the second one in the fall (mid October) of 1981. The biological and chemical treatments were then applied biannually in subsequent years in late April and in mid October. For the combination treatment, E. aeroqenes and metalaxyl were applied alternately (E. aeroqenes in late April~ metalaxyl in mid October). Stock suspen-sions of E. aeroqenes were prepared as described earlier.

Each tree was artifically inoculated as described by Sewell and Wilson (Ref. 9) with a single 5 mm plug containing actively growing P. cactorum (viru-lent local isolate PhO3) on May 26, 1982 and on June 8, 1983. The soil was removed from the crown region to a depth of about 6 cm, and the inoculum plug was inserted in a hole (5 mm diameter, 1 mm deep) made with a cork borer at the crown region. The soil was replaced immediately after inoculation.

The test plot was irrigated for 8 hours every 10 days with sprinklers located between the tree rows.
Insect, weed, and foliar disease control were carried out uniformly over all treatments as prescribed for apples in the Product Guide for Interior Districts of British Columbia (Ref. 1).

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The presence of P. cactorum in discolored bark was confirmed by plating on a selective medium as described previously (Ref. 14). Ten plates were used per sample and were incubated at 18C. Three days later, typical colonies of P. cactorum could easily be recognized at 40x magnification.

Trunk diameter and shoot length were measured in the last week of August for 1982, 1983 and 1984. For shoot length observations, five randomly selected shoots on each tree were measured. Percent of trees infected by P. cactorum were determined on September 18, 1985.
All percentages were transformed into arc sine values and analysed for statistical significance by analysis of variance (Ref. 3).

Example 5 Metalaxyl and E. aeroqenes treatments applied - 20 separately were tested in a grower's orchard in the Okanagan Valley, British Columbia, Canada, for efficacy on apple trees that were naturally infected with P.
cactorum. The trees were in the initial stage of infection with 1/4 of the bark area at the crown region infected by P. cactorum. Trees were fifteen years old, spaced 6 x 4 m, the rootstock was MM106 and the scion cultivar was McIntosh. A completely randomized block design consisting of ten single tree replicates was used. Infected bark on all trees was removed before application of treatments. Check trees were not treated with biological or chemical agents but infected bark was removed. Biological and chemical treatments were applied biannually as described in Field Trial 1 (Example 4) beginning in late April, 1983. Presence of P. cactorum in the discolored bark of infected trees was confirmed using selective media as described in Field Trial 1 (Example 4). Fertilization, insect, weed and foliar disease control were carried out by the grower as prescribed for apples in the Production Guide for Interior Districts of sritish Columbia (Ref. 2).
Observations on treated and untreated trees were made on May 20, 1986.

Results E. aeroqenes produced an inhibition zone (16.8 mm) when tested against P. cactorum on CMA plates. All plugs of P. cactorum which initially had been incubated in the presence of P. cactorum for two weeks, failed to grow when transferred to CMA. Those incubated in the absence of E. aeroqenes grew and covered the entire CMA
plate two weeks after transferring.

A11 chemical and biological treatments signi-ficantly reduced the percent infection by P. cactorum onMcIntosh seedlings under greenhouse conditions (see Table 1). Metalaxyl and fosetyl-Al alone, and metalaxyl + _. aeroqenes controlled the disease. E. aeroqenes significantly reduced percent infection (33.3~) on McIntosh seedlings compared with the mancozeb treatment (70.0~).

Metalaxyl and fosetyl-Al completely prevented the development of crown rot symptoms on MM106 apple rootstock in Field Trial 1 (Example 4 and Table 2).
Metalaxyl alternated with E. aeroqenes was as effective as metalaxyl applied at each treatment date. Captafol, metalaxyl + mancozeb, and E. aeroqenes (19.8% infection) significantly reduced the disease compared with the ~ . .

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control (34.6~ infection). Mancozeb and THIS were ineffective treatments.

Metalaxyl significantly increased trunk diame-ter in all the three test years and Fosetyl-Al only in 1984 (Table 3). Trees in the metalaxyl treatment showed significant increases in shoot length in 1982 and 1984.
Fosetyl-Al, metalaxyl + E. aeroqenes, and THIS showed significant increases only in 1984 when compared with the check.

In Field Trial 2 (Example 5), all naturally infected trees treated with E. aeroqenes and metalaxyl applied separately remained alive and produced normal fruit, while the untreated check trees were terminally diseased and produced only a few small fruits. All untreated trees were dead and were removed in three years.

Discussion And Conclusions The fact that all plugs of P. cactorum grown for two weeks in the presence of E. aeroqenes failed to grow when subsequently transferred to CMA suggests that the substance produced by the antagonist is fungicidal.
It appears that this antifungal substance kills P.
cactorum and thus controls the disease. Other mecha-nisms of action such as competition or nutrients are less likely but are not ruled out by this study. The antifungal substance was characterized as neutral and of low molecular weight (Ref. 15). Earlier work by the applicant has shown that under sterile soil conditions E. aeroqenes multiplied and produced an antifungal - substance (Ref. 16) inhibitory to the growth of P.
cactorum. The population densities in non-sterile soil .
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11 31~6 are not known because of lack of precise methods for enumerating this bacteriumO

Both field trials (Examples 4 and 5) have established that metalaxyl provided complete control of artificially inoculated young trees and also of natur ally infected fifteen-year-old trees at the initial stage of infection. This indicates curative ability of metalaxyl, if it is applied in the initial stage of infection. Control of Phytophthora species by metalaxyl has also been reported by Ellis and Porpiglia in apple (Ref. 5), Davis in Citrus (Ref. 4), Timmer and Castle in sweet orange (Ref. 11), Taylor and Washington in peach (Ref. 10), and Matheron and Mircetich in walnut (Ref.
7). In the present work metalaxyl and E. aeroqenes applied alternately in the spring and the fall control-led the disease even when metalaxyl was used at a half-rate. It is possible that this practice of combining a chemical fungicide with the biological control agent may delay the development of resistance by P. cactorum to metalaxyl.

E. aeroqenes provided substantial but not complete control of crown rot in artifically inoculated young trees (Table 2). Recovery of naturally infected fifteen-year-old trees after treatment with E.
; aeroqe_es suggests a curative type of control on the part of the organism and the antifungal substance that it produces. Biological control of the P. cactorum crown rot has been reported by Janisiewicz and Covey (Ref. 6) using a siderophore-producing Pseudomonas sp.
They removed decayed bark from the lesion and applied the bacterium directly. This treatment arrested the spread of the lesion and resulted in the formation of wound-healing callus. The applicant's observations are . . .
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similar to theirs; the lesions in treated trees were healed and the cured trees remained alive and produced normal fruit.

The use of metalaxyl + mancozeb resulted in significantly higher infection than with metalaxyl alone (Table 2). Possibly mancozeb, a broad spectrum soil fungicide, may have had a deleterious effect on natural antagonistic inhabitants of the soil.
The recovery of naturally infected fifteen-year-old trees after treatment with E. aeroqenes was comparable to that obtained with metalaxyl. However, in artifically inoculated young trees, the control of crown rot with E. aeroqenes was not as good as that obtained with naturally infected trees (Table 2). Since infected bark in the naturally infected trees was removed before the application of the antagonist, the inoculum level may have been reduced resulting in better control than with trees where infected bark was not removed.

Application of the biocontrol agent is a simple procedure and growers can apply this antagonist with pesticide sprayers. The protection provided by E.
aeroqenes, metalaxyl and fosetyl-Al suggests a practical approach for the control of crown and root rot of apple trees.

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Table l Effects of chemical and biological treatments applied as a soil drench on P. cactorum infected roots of McIntosh seedlings grown in pots.

Treatment Rate/Year Chemical Biological Chemical Biological Infection (ai~treelY (CF-u~tree2y (%) Metalaxyl ~ 0.1 g - O.OZ*

15 Fosetyl-Al - 0.8 g _ 6,7*

Metalaxyl E. aeroqenes 0.1 g 3.0 x 107 13.3*

-E. aeroqenes - 3.0 x 107 33.3*

Mancozeb - 0.2 g - 70.0*

Check - - - 100.0 L.S.D. at 1% 26.9 . . . _ _ Leqend y ai (active ingredients) and CFU (colony-forming units) Z* Significantly less than check using Least Significant Difference (L.S.D.) at the 1~ level.

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Table 2 Effects of chemical and biological treatments applied as soil and trunk drenches from 1981-85 on control of crown rot of artifically inoculated MM106 rootstock under orchard conditions.

Treatment Rate/Year Chemical Biological Chemical Biological Infection (ai/tree)y (CFU/tree)y (%) _ Metalaxyl - 2.0 g - 0.0Z*

Fosetyl-Al - 10.0 g - 0.0*

MetalaxylE. aeroqenes0.1 g1.0 x 101 9.6*

Captafol - 5.0 g - 13.6*

Metalaxyl -~
Mancozeb - 2.0 y + 3.6 g - 15.5*

- E. aeroqenes - 2.0 x 101 19.8*

THIS - 30 ml - 28.6 Mancozeb - 3.6 g - 33.7 Check - - - 34.6 _ Leqend y ai (active ingredient) and CFU (colony forming units) z* Significantly less than check using Least Signi~icant Difference .~`, ' . . ..
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As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

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REFERENCES

1. Anonymous. 1981. Tree Fruit Production Guide for Interior Districts. British Columbia Ministry of Agriculture & Food, Victoria, B.C. 67 pp.

2. Anonymous. 1983. Tree Fruit Production Guide for Interior Districts. British Columbia Ministry of Agriculture and Food, Victoria, B.C. 73 pp.

3. Cochran, W.G., and G.M. Cox. 1964. Experimental Design. John Wiley & Sons, New York. 611 pp.

4. Davis, R. M. 1982. Control of Phytophthora root and foot rot of citrus with systemic fungicides metalaxyl and phosethyl aluminum. Plant Dis. 66:
218-220.

5. Ellis, M.A. and P.J. Porpiglia. 1984. Ef~ects oE
application method on uptake of metalaxyl (Ridomil 2E) by apple trees. Research Circular Ohio Agric.
; Res. Dev. Cent. p. 5-8.

6. Janisiewicz, W.J. and R.P. Covey. 1983. Biological control of collar rot caused by PhYtophthora cactorum. Phytopathology 73: 822 (Abstr.).

7. Matheronl M.E., and S.M. Mircetich. 1985. Control oE Phytophthora root and crown rot and trunk canker in walnut with metalxyl and fosetyl Al. Plant Dis.
69: 1042-1043.

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8. McIntosh, D.L. 1975. Proceeding of the 1974 APDW
Workshop on crown rot of apple trees. Can Plant Dis. Surv. 55: 109-116.

9. Sewell, G.W.F.and J.F. Wilson. 1959. Resistance trials of some apple rootstock varieties to PhYtophthora cactorum (Leb & Cohn) Schrot. J.
Hortic. Sci. 34:51-58.

10. Taylor, P.A., and W.S. Washington. 1984. Curative treatments for Phytophthora cactorum in peach trees using ~etalaxyl and phosethyl Al. Austral. Plant Pathol. 13: 31-33.

11. Timmer, L.W. and W.S. Castle. 1985. Effectiveness of metalaxyl and fosetyl-Al against PhYtophthora parasitica on sweet orange. Plant Dis. 69:
741-743.

12. Tsao, P.H. and G. Ocana. 1969. Selective isolation of species of Phytophthora from natural soils on an improved antibiotic medium. Nature (London) 223:
636-638.

13. Utkhede, R.S. 1983. Inhibition of PhYtophthora cactoru by bacterial isolates and effects of chemical fungicides on their growth and antagonism.
Z. Pflanzenkra. Pflanzensch. 90: 140-145.

14. Utkhede, R.S. 1984. Effect of bacterial antagonist on Phytophthora cactorum and apple crown rot.
Phytopathol. Z. 109: 169-175.

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15. Utkhede, R.S. and ~.P. Gaunce. 1984. Inhibition of Phytophthora cactorum by a bacterial antagonist.
Can. J. Bot. 61: 3343-3348.

16. Utkhede, R.S. 1985. Factors affecting the production of antifungal compound by Enterobacter aeroqenes and Bacillus subtilis, antagonists of Phytophthora cactorum. Phytopathol. Z. 117: 9-16.

17. Welsh, M.F. 1942. Studies of crown rot of apple trees. Can. J. Res. 20: 457-490.

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Claims (15)

WHAT IS CLAIMED IS:

1 . A method of controlling crown and root rot in fruit trees due to Phytophthora cactorum comprising con-tacting the bark and roots of the trees with an effective amount of a strain of Enterobacter aerogenes (No. NRRL B-18268) preselected to be resistant to antibiotics and fungicides antagonistic to Phytophthora cactorum.

2. A method of claim 1 wherein the Enterobacter aerogenes is viable in a culture medium comprising 100 ppm Metalaxyl or 100 ppm Mancozeb and combinations thereof.

3. A method of claim 1 wherein the Enterobacter aerogenes is viable in a culture medium comprising 200 µg/ml streptomycin sulfate or 100 µg/ml cycloheximide or a combination thereof.

4. A composition of claim 2 wherein the Enterobacter aerogenes is viable in a culture medium comprising 200 µg/ml streptomycin sulfate or 100 µg/ml cycloheximide or a combination thereof.

5. A method of claim 1 wherein the contacting comprises drenching the roots with an aqueous suspension comprising Enterobacter aerogenes.

6. A method of claim 5 wherein the drenching con-sists of an effective amount of Enterobacter aerogenes in an aqueous suspension in excess of five litres for each tree.

7. A method of claim 5 wherein the aqueous suspen-sion has a concentration of Enterobacter aerogenes in excess of about 2.0 x 104 colony forming units per unit

8. A method of claim 1 wherein the trees are of the family Malus.

9. A method of claim 1 wherein the trees are apple trees.

10. A composition for the control of phytopathogenic soil bacteria comprising a strain of Enterobacter aerogenes (No. NRRL B-18268) preselected to be resistant to anti-biotics and fungicides antagonistic to Phytophthora cac-torum.

11. A composition of claim 10 wherein the Entero-bacter aerogenes is viable in a culture medium comprising 100 ppm Metalaxyl or 100 ppm Mancozeb or a combination thereof.

12. A composition of claim 10 wherein the Entero-bacter aerogenes is viable in a culture medium comprising 200 µg/ml streptomycin sulfate or 100 µg/ml cycloheximide or a combination thereof.

13. A composition of claim 11 wherein the Entero-bacter aerogenes is viable in a culture medium comprising 200 µg/ml streptomycin sulfate or 100 µg/ml cycloheximide or a combination thereof.

14. A composition of claim 10 wherein the Entero-bacter aerogenes is an aqueous suspension.

15. A composition of claim 14 wherein the aqueous suspension has a concentration of Enterobacter aerogenes in excess of about 2.0 x 104 colony forming units per unit dose.