TRICHODERMA COMPOSITIONS AND METHODS OF USING THE SAME

Abstract

Provided herein is a Trichoderma strain designated as TM17 and deposited under accession number RREC-TM17 (ATCC #). The Trichoderma strain TM17 and a carrier may be used of make agricultural compositions. Plants comprising the Trichoderma strain TM17 or the agricultural compositions provided herein are also contemplated. Methods for inhibiting the growth of a microorganism on a plant are also provided and may include contacting the plant with an effective amount of the Trichoderma strain TM17 or agricultural compositions including the Trichoderma strain TM17 to inhibit the growth of the microorganism on the plant.

Claims

1. A Trichoderma strain designated as TM17 and deposited under accession number RREC-TM17 (ATCC #).

2. An agricultural composition comprising the Trichoderma strain of claim 1 and a carrier.

3. The agricultural composition of claim 2, wherein the carrier comprises a buffer.

4. The agricultural composition of claim 2, wherein the concentration of the Trichoderma strain is between 10.sup.7 to 10.sup.10 conidia per milliliter.

5. The agricultural composition of claim 2, wherein the agricultural composition is a liquid suspension, a granular form, or a powder.

6. A plant comprising the composition of claim 2.

7. The plant of claim 6, wherein the Trichoderma strain is present on or within the leaves of the plant.

8. The plant of claim 6, wherein the plant is selected from the group consisting of a rice plant, a cotton plant, a soybean plant, a wheat plant, a sorghum plant, and a corn plant.

9. A method for inhibiting the growth of a microorganism on a plant comprising contacting the plant with an effective amount of the composition of claim 2 to inhibit the growth of the microorganism on the plant.

10. The method of claim 9, wherein the plant is a rice plant.

11. The method of claim 9, wherein the microorganism is a fungal plant pathogen.

12. The method of claim 9, wherein the microorganism is selected from the group consisting of a Magnaporthe species and a Rhizoctonia species.

13. The method of claim 9, wherein the microorganism is Magnaporthe oryzae or Rhizoctonia solani.

14. The method of claim 9, wherein the contacting is carried out before the microorganism grows on the plant.

15. The method of claim 9, wherein the contacting is carried out after the microorganism grows on the plant.

16. The method of claim 9, wherein the contacting is carried out by spraying the plant with the composition.

17. The method of claim 9, wherein the contacting is carried out by dusting the plant with composition.

18. The method of claim 9, wherein the leaves of the plant are contacted with the composition.

19. The method of claim 9, wherein the seeds of the plant are contacted with the composition.

20. The method of claim 9, wherein the plant is contacted at least 2 times with the composition.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1 shows the methodology used to isolate, characterize and the final identification protocol for the endophyte Trichoderma sp. TM17.

[0009] FIGS. 2A-2D show the dual confrontation result of TM17 against Magnaporthe oryzae, FIGS. 2A, 2B, 2C, and 2D are day 3, 4, 6, and 10, respectively. BL-Blast pathogen (Magnaporthe oryzae), TR (Trichoderma sp.-TM17) and TR+BL=dual plate for TR and BL in a single plate.

[0010] FIGS. 3A-3C show the dual confrontation result of TM17 against Rhizoctonia solani AG1-1A. FIGS. 3A, 3B, 3C are day 3, 4, 6, respectively. SB-Sheath blight (Rhizoctonia solani AG1-1A), TR-Trichoderma sp.-(TM17) and TR+SB=dual plate for TR and SB in a single plate.

[0011] FIG. 4 shows the growth performance of BL, SB, TR and dual inoculated plates after 10 days of inoculation.

[0012] FIGS. 5A-5D show the performance of rice plants treated in accordance with the present invention. FIG. 5A shows control treatment that was inoculated with the blast pathogen but with no TM17 applied. FIG. 5B shows plants pre-treated with TM17 and then inoculated with the blast pathogen. FIGS. 5C and 5D show inoculated plants but the healthy looking plants on the left are treated with TM17 prior to inoculation with the blast pathogen.

[0013] FIG. 6 shows the visible lesion size on the rice leaves. The top picture shows the results when TM17 is sprayed 24 h before inoculation with the blast pathogen. The middle picture shows the results of post-treatment with TM17. The TM17 was applied 48 h after the blast pathogen inoculation. The picture at the bottom shows the control plants inoculated with the blast pathogen and not treated with TM17.

[0014] FIGS. 7A and 7B show two photographs to demonstrate the contrast between TM17 treated and untreated rice plants from two different views. The plants on the left of each picture were inoculated with Rhizoctonia solani AG1-1A agar plugs 24 h after TM17 treatment and the plants on the right were inoculated with Rhizoctonia solani AG1-1A agar plugs, but not treated with TM17.

DETAILED DESCRIPTION

[0015] Here, in the non-limiting Examples, the present inventors have identified a Trichoderma strain (referred to as TM17) that may be applied to plants to suppress the growth of microbial plant pathogens such as Magnaporthe oryzae and Rhizoctonia solani. Without being bound by theory, the present inventors show in the non-limiting Examples that spraying plants with TM17 allows TM17 to colonize the plant surface thereby creating a plant-Trichoderma interaction system. The plant-Trichoderma interaction system protects the plants from being colonized by, and damaged by, pathogenic microbes such as fungi. Given this data, the present inventors show that TM17 may provide a broad-spectrum control of multiple diseases in plants and provide a safe alternative to chemical pesticides.

[0016] In one aspect of the present invention, a new Trichoderma strain designated as TM17 and deposited under accession number RREC-TM17 (ATCC #) is provided. A viable culture of the new strain of the fungus Trichoderma sp. designated as TM17 has been deposited in the Culture Collection at Rice Research and Extension Center (RREC), Stuttgart, A R under the accession number is RREC-TM17. Alternatively, the strain will be deposited at the American Type Culture Collection. Trichoderma strain TM17 produces abundant spores and has been shown to suppress and control the rice blast and sheath blight diseases caused by Magnaporthe oryzae and Rhizoctonia solani AG1-1A, respectively. The present invention relates to a method of protecting plants from infection, which comprises contacting the plant with Trichoderma strain TM17 under conditions effective for the Trichoderma strain to colonize the surface of the plant, thereby creating a plant-Trichoderma interaction system. A biologically pure culture of fungus Trichoderma sp. isolate TM17 for inducing suppression of rice plant diseases caused by Magnaporthe oryzae and Rhizoctonia solani AG1-1A is provided. The culture produces abundant mycelium, conidia and chlamydospores.

[0017] In another aspect, the present invention relates to agricultural compositions. An agricultural composition is a composition formulated for application to a plant or plant part. An agricultural composition is commonly in liquid form for application by spraying or soaking, but may be in a solid, granular, or powder form for rehydration or application by dusting or dry coating. The agricultural composition may be concentrated for dilution in water or other solvent.

[0018] The agricultural compositions may include Trichoderma strain TM17 and a carrier. As used herein, a carrier may be solid or liquid and may include substances ordinarily employed in formulations applied to plants. Carriers may include a buffer, water, oil, nonionic surfactants, ionic surfactants such as cationic or anionic surfactants, or available agricultural byproducts from, for example and without limitation, rice.

[0019] In some embodiments, the agricultural compositions may also include an additional active ingredient such as, without limitation, a fungicide, an herbicide, a biosanitizer product or fertilizer.

[0020] The agricultural compositions may include Trichoderma strain TM17 at a concentration between 10.sup.5 to 10.sup.12 or more conidia per milliliter or any range therein. Suitably, the concentration of the Trichoderma strain TM17 in the agricultural composition may be 10.sup.7 to 10.sup.10 conidia per milliliter. As detailed in the Examples, the concentration of Trichoderma strain TM17 conidia in the agricultural composition may be quantified using a hemocytometer.

[0021] In a further aspect of the present invention, plants are provided. The plants may include Trichoderma strain TM17 or any one of the agricultural compositions described herein. The Trichoderma strain TM17 or any one of the agricultural compositions described herein may be present on or within at least a part of the plant.

[0022] As used herein, a plant includes any portion of the plant including, without limitation, a whole plant or a portion of a plant such as a part of a root, leaf, stem, seed, pod, flower, cell, tissue plant germplasm, asexual propagate, or any progeny thereof. For example, a rice plant refers to the whole rice plant or portions thereof including, without limitation, the leaves, roots, or otherwise. Suitable plants may include, without limitation, a rice plant, a cotton plant, a soybean plant, a wheat plant, a sorghum plant, or a corn plant. In some embodiments, the plant is a rice plant.

[0023] In a further aspect of the present invention, methods for inhibiting the growth of a microorganism on a plant are provided. The methods may include contacting the plant with an effective amount of Trichoderma strain TM17 or any one of the agricultural compositions described herein to inhibit the growth of the microorganism on the plant.

[0024] The microorganism may be any bacterial or fungal plant pathogen. Suitably, the microorganism may be a fungal plant pathogen including, without limitation, a Magnaporthe species or a Rhizoctonia species. In some embodiments, the microorganism is Magnaporthe oryzae or Rhizoctonia solani. The Rhizoctonia solani may be Rhizoctonia solani AG1-1A. Magnaporthe oryzae is the causative agent of blast disease in rice and Rhizoctonia solani AG1-1A is the causative agent of sheath blight.

[0025] As used herein, contacting may be carried out through any of the variety of procedures used to apply compositions to plants that will be apparent to the skilled artisan. Suitable application methods may include, without limitation spraying or dusting. Other suitable application procedures can be envisioned by those skilled in the art. Contacting may also be carried out indirectly via application, for example, to the soil surrounding a plant, trunk injection, or other plant media or substrates. The contacting of the present methods may be carried out before or after the microorganism grows on the plant.

[0026] In the present methods, various parts of the plant may be contacted with or by Trichoderma strain TM17 or any one of the agricultural compositions described herein. Suitably and without limitation, the leaves or seeds of the plant may be contacted with Trichoderma strain TM17 or any one of the agricultural compositions described herein.

[0027] In some embodiments, the plant may be contacted at least 2, 3, 4, 5, or more times with with Trichoderma strain TM17 or any one of the agricultural compositions described herein during a single growing season. Different parts of the plant may be contacted at different points within the growing season. The plant may be contacted with the Trichoderma strain TM17 or the agricultural compositions using different formulations or means of contacting at different points within a growing season.

[0028] Effective amount is intended to mean an amount of a composition described herein sufficient to inhibit the growth of a microorganism on a plant by, for example, 10%, 20%, 50%, 75%, 80%, 90%, 95%, or 1-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more compared to a negative control plant not treated with the Trichoderma strain TM17 or one of the agricultural compositions provided herein. In some embodiments, the effective amount of Trichoderma strain TM17 either alone or in an agricultural composition may be 10.sup.5 to 10.sup.12 or more conidia per milliliter or any range therein. Suitably, the concentration of Trichoderma strain TM17 either alone or in an agricultural composition is 10.sup.7 to 10.sup.10 conidia per milliliter. A negative control refers to a sample that serves as a reference for comparison to a test sample. For example, a test sample can be taken from a test condition including the presence of Trichoderma strain TM17 and compared to negative control samples lacking Trichoderma strain TM17 or including a composition not expected to inhibit microbial growth. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters.

[0029] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms including, comprising, or having, and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as including, comprising, or having certain elements are also contemplated as consisting essentially of and consisting of those certain elements.

[0030] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word about to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0031] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference in their entirety, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

[0032] Unless otherwise specified or indicated by context, the terms a, an, and the mean one or more. For example, a protein or an RNA should be interpreted to mean one or more proteins or one or more RNAs, respectively.

[0033] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

EXAMPLES

Example 1Isolation of Trichoderma sp. TM17

[0034] Although the concept of biological control of plant diseases has been known for a number of years, the idea of selecting endophytic biological control agents is relatively new. In fact, Trichoderma sp.-TM17 is the first microorganism isolated from rice seed as an endophyte that showed a potential to control both Blast and Sheath blight disease of rice in Arkansas.

[0035] The Trichoderma strain disclosed herein, Trichoderma sp.-TM17, was isolated from rice seed as an endophyte. The isolation of the endophytic strain was accomplished by the following procedure. See, e.g., FIG. 1. Rice seeds were collected from different sites of major rice growing fields of Arkansas at rice crop maturity and were kept in paper bags at 4 C. until use. Seed samples randomly selected from the mixed samples were set for processing for the test. Isolation of endophyte fungi was according to the protocol of Mysore et al. 2005. Briefly, seeds were cleaned under running tap water to remove debris and then air-dried on a sterile filter paper. From each sample, seeds were surface-sterilized by immersing them in 95% ethanol (2 min), followed by sodium hypochlorite (4% available chlorine; 5 min), and were then washed three times with sterile water. Samples were then allowed to dry on a sterile filter paper for 10 min in a sterile laminar flow chamber. Seeds were plated horizontally on potato dextrose agar (PDA) supplemented with streptomycin sulphate (0.4 mg/mL). In a course of 5 days incubation at 25 C., individual hyphal tips of the developing colonies were re-plated onto PDA plates and were further incubated for 8-10 days. Eventually, single spore cultures for each strain were prepared to assure sample purity. Isolates are currently stored in 30% glycerol at 80 C. in the Collection of Plant Pathology at Rice Research and Extension Center (RREC).

Example 2Preparation of TM17 Samples for Experimentation

[0036] Conidia and mycelia of TM17 were produced by growing the isolate on PDA agar for 7 days under continuous fluorescent light at room temperature. Conidia were removed from the agar surface by pipetting 5 ml of sterile distilled water on the surface and gently rubbing the surface with a sterile cotton-tipped applicator. Conidia were counted in a hemocytometer, and the suspensions adjusted with water to provide the desired concentration of conidia in each test. There were five replications in all experiments, and each experiment was done twice.

Example 3Biocontrol Activity of TM17 In Vitro

[0037] Biocontrol activities of Trichoderma sp. strain TM17 against the two fungal rice pathogens were determined in vitro on potato dextrose agar (PDA) (20 ml/plate). See FIGS. 2A-2D, 3A-3C, and 4. The two pathogens were Magnaporthe oryzae (FIGS. 2A-2D and 4) and Rhizoctonia solani AG1-1A (FIGS. 3A-3C and 4). A plug of the biocontrol agent (8 mm diameter) from the edge of 5-day old colonies of the Trichoderma cultures and of 10-day old colonies of the pathogens were paired on the medium on opposite sides of 91.5 cm petri plates and then were incubated at room temperature. Also fresh culture agar plugs of prey and predator fungi were placed on a PDA plate on opposite poles, each, 1 cm away from the edge of the plate. The disks from TM17 were placed 5 cm from those of the pathogens. Isolates of Magnaporthe oryzae and Rhizoctonia solani were placed on the agar 48 hours before TM17. All other combinations were done simultaneously. Cultures were incubated at room temprature under continuous fluorescent light of the same intensity and monitored for inhibition every day from 3 to 10 days after placement of the TM17. Antagonistic potential was estimated as a reduction of the prey growth rate corrected for the growth rate of a prey when confronted with itself. Antagonistic potential presented as averaged growth inhibition or overgrowth of each plant pathogen (prey) by Trichoderma strain TM17 and was expressed in percentages. In both cases 100% represented unrestricted growth of the prey corrected for the confrontation with itself. Trichoderma sp.-TM17 showed an aggressive biocontrol potential growing faster and eventually overwhelming the tested pathogen isolates compared to the control plates inoculated with the respective pathogen alone under the same conditions of incubation. The Trichoderma sp.-TM17 did not produce zones of inhibition against any of the pathogens on plates. However, its competitive growth overwhelmed the pathogen growth. See FIGS. 2A-2D, 3A-3C, and 4.

Example 4Biocontrol Activity of TM17 In Vivo

[0038] The ability of Trichoderma sp.-TM17 to suppress diseases was also determined in vivo. This endophyte Trichoderma isolate revealed high inhibition potential on the growth of the two prey isolates. Greenhouse experiments were carried out for both blast (FIGS. 5A-5D, FIG. 6) and sheath blight (FIGS. 7A-7B) control by TM17. To test for blast, seeds of M206 planted in hill plots in a 0.02 m.sup.2 flat containing greenhouse soil:sand:loam mixture in 3:1:1 proportion, respectively. There were 81 hill plots in a flat. The flats were kept in the greenhouse at an average temperature of 78 F. and 85% relative humidity. Flats were watered for 3 weeks and fertilized as needed. Conidia of TM17 harvested from 8 to 10-day-old cultures growing on PDA were washed off using cotton-tipped applicator. Suspensions of conidia were counted using a hemocytometer and adjusted to contain 10.sup.9 conidia per milliliter for spray application.

[0039] To test for sheath blight, 10 to 15 seeds were planted in a half-gallon pot, watered, and fertilized as needed. After a month, sheath blight disease causing pathogen Rhizoctonia solani AG1-1A was grown on PDA plates. For pre-inoculation treatment, TM17 was sprayed on rice seedlings at full tillering developmental stage and then after 24 hours the plants were inoculated following standard greenhouse procedure by placing 10 days old mycelial agar plugs of Rhizoctonia solani AG1-1A at the base of each culm. For post-inoculation treatment, rice of similar age were inoculated using mycelial agar plugs in a similar way except TM17 was sprayed 24 hours after inoculation at a concentration of 10.sup.9 conidia per ml. On the other hand, a conidial suspension of M. oryzae was prepared by growing it on rice bran agar at 27 C. for 10 days, washed with sterilized distilled water and strained through sterile filter papers to remove mycelia. Conidial concentrations of M oryzae were adjusted to 10.sup.5 conidia per ml. Then the suspensions were kept at 4 C. until foliage spray. Similar to Rhizoctonia solani AG1-1A, for pre-inoculation treatment, TM17 was sprayed on rice seedlings at 24 h before spray-inoculation by M. oryzae suspension. For post-inoculation treatment, rice seedlings of similar age were spray-inoculated with the spore suspension of M. oryzae at 24 h before spraying with TM17 at 10.sup.9 conidia per ml.

[0040] Application of Trichoderma sp.-TM17:

[0041] Conidial suspension of TM17 was prepared for protective and therapeutic treatments. In the former, TM17 was sprayed 48 hours before inoculating the respective pathogens (FIGS. 5B-5C). In the latter rice plants were inoculated with isolates of Magnaporthe oryzae and Rhizoctonia solani AGJ-JA, then 48 h after the rice plants were sprayed with TM17 suspension (FIGS. 5A-5B). Experimental controls were treated with water in place of TM17.

[0042] Data Collected:

[0043] The number of rice plants with blast lesions and without lesions were counted. For the sheath blight, the lengths of lesions were measured in proportion to height of the plant. This invention relates to Trichoderma sp.-TM17, which is seen potentially useful in controlling the two major rice diseases caused by Magnaporthe oryzae and Rhizoctonia solani AG1-1A. To suppress the blast and sheath blight rice diseases, TM17, a strain of Trichoderma sp. was applied to greenhouse grown rice plants in an amount sufficient to colonize and populate the plant surface, thereby reducing/suppressing the growth of the pathogens. See FIGS. 5A-5D, FIG. 6, and FIG. 7A-7B.

REFERENCES

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