WO2024148286A1

WO2024148286A1 - Use of streptomyces nigrescens for nematode biological control in plants

Info

Publication number: WO2024148286A1
Application number: PCT/US2024/010518
Authority: WO
Inventors: Mark Roberts; Matthew Kowalski
Original assignee: Blacksmith Bioscience Llc
Priority date: 2023-01-06
Filing date: 2024-01-05
Publication date: 2024-07-11

Abstract

Methods of controlling plant-parasitic nematodes are provided. The methods include contacting a plant, plant part, or solid plant growth medium with a composition including Streptomyces nigrescens 541. In some examples, a seed, plant reproductive tissue or root tissue is contacted with the composition. In other examples, a solid growth medium, including, but not limited to soil is contacted with the composition.

Description

USE OF STREPTOMYCES NIGRESCENS FOR NEMATODE BIOLOGICAL CONTROL

IN PLANTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/478,734, filed January 6, 2023, which is incorporated by reference in its entirety.

FIELD

The present disclosure relates to use of Streptomyces nigrescens for control of plant pathogenic nematodes.

BACKGROUND

Disease caused by microbial pathogens or nematodes reduce plant robustness and productivity, resulting in substantial economic losses in plant-based industries such as agriculture, horticulture, and forestry. Application of pesticides can help control disease, but pesticides are generally expensive, toxic to humans and the environment, and are only effective for a limited amount of time. Furthermore, persistent use of traditional pesticides encourages emergence of pesticide-resistant pathogens. Thus, there is a need for alternative solutions for effective pest control.

SUMMARY

Methods of controlling plant-parasitic nematodes are provided. The methods include contacting a plant, plant part, or solid plant growth medium with a composition including Streptomyces nigrescens 541 (such as the microbes in Agricultural Research Service Culture Collection (NRRL) deposit number NRRL B-67882). In some aspects, the plant, plant part, or solid growth medium is infected with one or more plant-parasitic nematodes. In some examples, a seed, plant reproductive tissue, or root tissue is contacted with the composition. In other examples, a solid growth medium, including, but not limited to soil is contacted with the composition.

In some aspects, the nematode is a root-knot nematode (e.g., Meloidogyne sp.), a cyst nematode (e.g., Heterodera sp. or Globodera sp.), a root lesion nematode (e.g., Pratylenchus sp.), Radopholus sp., Ditylenchus sp., Bursaphelenchus sp., Rotylenchulus sp., Xiphinema sp., Nacobbus sp., or Aphelenchoides sp.

In additional aspects, the methods further include contacting the plant, plant part, or solid plant growth medium with chitin. The plant, plant part, or solid plant growth medium may be contacted with the chitin and the composition comprising Streptomyces nigrescens 541 simultaneously or sequentially (in either order).

The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figure.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the effect of 5. nigrescens 541 and chitin on Meloidogyne incognita J 2 numbers at different egg inoculation rates. UTC = the untreated control, AMV1033 = .S'. nigrescens-only treatment, chitin low + AMV1033 = chitin co-inoculated with .S'. nigrescens, and the chemical control treatment, abamectin (ABA), a, b, c: values are significantly different (p<0.05).

DETAILED DESCRIPTION

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewi ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a microbe” includes singular or plural microbes and can be considered equivalent to the phrase “at least one microbe.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

Contacting: Placement in direct physical association, including in either solid and/or liquid form. For example, contacting can occur with one or more microbes (such as S. nigrescens 541) and a plant, plant part (e.g., seed), or solid growth medium.

Effective amount: An amount of an agent (such as Streptomyces nigrescens 541) that is sufficient to produce beneficial or desired results. For instance, this can be an amount of Streptomyces nigrescens 541 necessary to control plant-parasitic nematodes. Isolated: An “isolated” biological component (such as a nucleic acid, protein or organism) has been substantially separated or purified away from other components (such as other cells, cell debris, other proteins, nucleic acids, or organisms). Biological components that have been “isolated” include those components purified by standard purification methods. The term “isolated” (or “enriched” or “purified”) does not require absolute purity, and can include microbes or molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.

Microbe: A microorganism, including but not limited to bacteria, archaebacteria, fungi, and algae (such as microalgae). In some examples, microbes are single-cellular organisms (for example, bacteria, cyanobacteria, some fungi, or some algae).

Microbial composition: A composition (which can be solid, liquid, or at least partially both) that includes cells of at least one type (or species) of microbe (or a population of cells of at least one type of microbe). In some examples, a microbial composition comprises cells of one or more types (species) of microbes (or one or more populations of microbes) in a liquid (such as a storage, culture, or fermentation medium), for example, as a suspension in the liquid. In other examples, a microbial composition includes cells of one or more types (species) of microbes (or one or more populations of microbes) on the surface of or embedded in a solid or gelatinous medium (including but not limited to a culture plate), or a slurry or paste. In other examples, a microbial composition includes cells of one or more types (or species) of microbes (or one or more populations of microbes) in association with a seed, such as on the surface of or impregnated in a seed.

Plant-parasitic nematode: Roundworms that infect plants and drain nutrients or otherwise stress plants, decreasing yields and in some cases causing plant death. In some examples, a plant- parasitic nematode is a root-knot nematode from the genus Meloidogyne (e.g., M. javanica, M. arenaria, M. incognita, M. hapla). In other examples, a plant-parasitic nematode is a cyst nematode, such as soybean cyst nematodes (e.g. , Heterodera glycines)' , potato cyst nematodes (e.g., Globodera pallida or Globodera rostochiensis), and cereal cyst nematodes (e.g., Heterodera avenae or Heterodera filipjevi). Additional plant-parasitic nematodes include root lesion nematodes (e.g., Pratylenchus sp. ), Radopholus sp., Ditylenchus sp., Bursaphelenchus sp., Rotylenchulus sp., Xiphinema sp., Nacobbus sp., or Aphelenchoides sp.

Streptomyces nigrescens 541: Also referred to as Streptomyces nigrescens MR541. In some aspects, Streptomyces nigrescens 541 is the microbe deposited with the Agricultural Research Service Culture Collection (NRRL; Peoria, Illinois) on November 21, 2019 and assigned deposit number NRRL B -67882. II. Methods of Controlling Plant- Parasitic Nematodes

Methods of controlling plant-parasitic nematodes are provided. In some aspects, the methods include contacting a plant, plant part, or a solid plant growth medium with a composition (such as a microbial composition) comprising Streptomyces nigrescens 541. In some examples, the plant, plant part, or solid growth medium being contacted with the composition comprising .S'. nigrescens 541 is infected with one or more plant-parasitic nematodes. In some examples, the level of nematode infection and the corresponding disease index includes an inoculum of 5000 eggs/50 g soil, 15,000 eggs/50 g soil, or 25,000 eggs/50 g soil representing a low, medium, or high level of infection, respectively. The disease level may be based on a gall index scale from 0 to 5, with 0 representing a low gall infection and 5 indicating a high gall infection. In some examples, using such a scale, a value of 0-1 represents a low disease level of disease, 2-3 represents a medium level of disease, and 4-5 represents a high level of disease. In some aspects, the plant, plant part, or solid growth medium is infected with a high level of infection with plant-parasitic nematodes (e.g., about 25,000 eggs/50 g soil or a gall index of at least 4). Exemplary methods of detecting and assessing nematode infection are provided in the Examples below; however, nematode infection and severity of disease can be determined using any methods known to one of ordinary skill in the art.

In some aspects, Streptomyces nigrescens 541 is the microbe deposited with the Agricultural Research Service Culture Collection (NRRL; Peoria, Illinois) on November 21, 2019, and assigned deposit number NRRL B-67882.

In some aspects, the methods include contacting a plant or plant part with an effective amount of a composition including Streptomyces nigrescens 541. In particular examples, the methods include contacting a seed, plant reproductive tissue (e.g., a tuber, rhizome, corm, bulbil, or bulb), root tissue, or leaf with the composition. In other examples, the methods include contacting a solid plant growth medium with an effective amount of a composition including Streptomyces nigrescens 541. In particular examples, the solid plant growth medium includes soil, agar, peat moss, potting mix, wood residue, bagasse, rice hulls, sand, perlite, vermiculite, or calcined clay. Thus, in some examples, the methods include contacting soil, agar, peat moss, potting mix, wood residue, bagasse, rice hulls, sand, perlite, vermiculite, or calcined clay with the composition. In some examples, the composition is applied to the solid medium (e.g., soil or potting mix) prior to and/or at the time of planting. In other examples, the composition is applied to the solid medium (e.g., soil or potting mix) after planting.

The composition may be formulated as an effective amount of Streptomyces nigrescens 541 and a liquid carrier. In some examples, the liquid carrier is water, for example, irrigation water. In one aspect, an “effective amount” is an amount sufficient to decrease nematode infestation, for example, number of nematodes or nematode eggs (such as encysted eggs), such as decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, or at least 90% for example relative to a control (such as no treatment with Streptomyces nigrescens 541). In another aspect, an “effective amount” is an amount sufficient to decrease root damage (such as size and/or number of galls) to a plant, such as decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, or at least 90% for example relative to a control (such as no treatment with Streptomyces nigrescens 541). In other aspects, an “effective amount” is an amount sufficient to increase plant growth (such as overall plant size, amount of foliage, root number, root diameter, root length) by at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500% as compared to a control (such as no treatment with Streptomyces nigrescens 541). In other aspects, an “effective amount” is an amount sufficient to increase crop yield by at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500% as compared to a control (such as no treatment with Streptomyces nigrescens 541).

In some examples, the composition is applied to soil in furrow or by chemigation. In other examples, the composition is applied to a plant or plant part by chemigation. In other examples, the composition is applied to a plant part (such as a seed or tuber) by application with a dry formulation of spores in a carrier (such as talc). Seeds can also be coated by rotation or shaking in a “pillcoating apparatus” with the inoculum. Another method is spraying the seeds or tuber with the composition in a liquid carrier, which includes in some examples passing the seeds under a spray nozzle on a conveyor system.

In some examples, the composition includes about 10³-10⁷ CFU/g (for example, about 10³- 10⁵, about 10⁴-10⁶, about 10⁵-10⁷, or about 10³, about 10⁴, about 10⁵, about 10⁶, or about 10⁷ CFU/g) Streptomyces nigrescens 541. In some examples, the composition is applied at a rate of about 3 ounces per acre (w/v) to about 12 ounces per acre (w/v) (for example, about 3 ounces per acre (w/v) to about 6 ounces per acre (w/v), about 4 ounces per acre (w/v) to about 8 ounces per acre (w/v), about 6 ounces per acre (w/v) to about 10 ounces per acre (w/v), or about 9 ounces per acre (w/v) to about 12 ounces per acre (w/v)). In a particular example, the composition is applied at about 6 ounces per acre (w/v). However, one of ordinary skill in the art can select other rates of application, for example, based on plant type, location, severity of infection, and other factors.

The methods may include contacting the plant, plant part, or solid medium with the composition one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times). In some examples, the method includes contacting the plant, plant part, or solid medium (such as soil) with the composition about 1-4 times. The composition can be applied weekly, biweekly, monthly, every other month, or less frequently. If the composition is applied to seed or plant reproductive parts, the application may be one or more times prior to planting.

In some aspects, the methods further include contacting the plant, plant part, or solid growth medium with chitin (such as a composition including chitin). In some examples, the plant, plant part, or solid growth medium is contacted with chitin one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times). In some examples, the plant, plant part, or solid growth medium is contacted with about 25 mg chitin/g soil to about 750 mg chitin/g soil (such as about 25 to about 75 mg chitin/g soil, about 50 to about 100 mg chitin/g soil, about 75 to about 125 mg chitin/g soil, about 100 to about 200 mg chitin/g soil, about 200 to about 300 mg chitin/g soil, about 300 to about 400 mg chitin/g soil, about 400 to about 500 mg chitin/g soil, about 500 to about 600 mg chitin/g soil, or about 600 to about 750 mg chitin/g soil). In some examples, 50 mg chitin/g soil is a representative low level and 500 mg chitin/g soil is considered a high level.

In some examples, the plant, plant part, or solid growth medium is contacted with the composition including .S'. nigrescens 541 and the chitin simultaneously, for example, the S. nigrescens 541 are in the same composition or mixture. In other examples, the plant, plant part, or solid growth medium is contacted with the composition including S. nigrescens 541 and the chitin sequentially. In some examples, the plant, plant part or solid growth medium is contacted with the composition including .S'. nigrescens 541, followed by contacting with chitin. In other examples, the plant, plant part, or solid growth medium is contacted with chitin, followed by the composition including S. nigrescens 541. In the case of sequential application, both the composition including S. nigrescens 541 and chitin may be applied each time if multiple applications are utilized.

Exemplary nematodes that may be controlled by the disclosed methods include root-knot nematodes from the genus Meloidogyne (e.g., M. javanica, M. arenaria, M. incognita, M. hapla), cyst nematodes, such as soybean cyst nematodes (e.g., Heterodera glycines.), potato cyst nematodes (e.g., Globodera pallida or Globodera rostochiensis), and cereal cyst nematodes (e.g., Heterodera avenae or Heterodera filipjevi), and root lesion nematodes (e.g., Pratylenchus spp.). Additional nematodes that may be controlled by the disclosed methods include Radopholus sp. (e.g., Radopholus similis), Ditylenchus sp. (e.g., Ditylenchus dipsaci), Bursaphelenchus sp. (e.g., Bursaphelenchus xylophilus), Rotylenchulus sp. (e.g., Rotylenchulus reniformis), Xiphinema sp. (e.g., Xiphinema index), Nacobbits sp. (e.g., Nacobbus aberrans), or Aphelenchoides sp. (e.g., Aphelenchoides besseyi).

The disclosed methods can be used in connection with any crop (for example, for direct crop treatment or for soil treatment prior to or after planting). Exemplary crops include, but are not limited to alfalfa, almond, banana, barley, broccoli, cabbage, canola, carrots, citrus and orchard tree crops, com, cotton, cucumber, flowers and ornamentals, garlic, grapes, hops, horticultural plants, leek, lettuce, melon, oil palm, onion, peanuts and legumes, pineapple, poplar, pine and woodbearing trees, potato, raspberry, rice, sesame, sorghum, soybean, squash, strawberry, sugarcane, sunflower, tomato, turf and forage grasses, watermelon, wheat, and eucalyptus.

III. Additional Aspects

Aspect 1. A method of controlling plant-parasitic nematodes, comprising contacting a plant, plant part, or a solid plant growth medium with a composition comprising Streptomyces nigrescens 541, thereby controlling the plant-parasitic nematodes.

Aspect 2. The method of aspect 1, wherein the plant, plant part, or solid plant growth medium is infected with plant-parasitic nematodes.

Aspect 3. The method of aspect 1 or aspect 2, wherein the plant-parasitic nematode is Meloidogyne sp., Heterodera sp., Globodera sp., Pratylenchus sp., Radopholus sp., Ditylenchus sp., Bursaphelenchus sp., Rorylenchulus sp., Xiphinema sp., Nacobbus sp., or Aphelenchoides sp.

Aspect 4. The method of any one of aspects 1 to 3, wherein contacting the plant part comprises contacting seed with the composition.

Aspect 5. The method of any one of aspects 1 to 4, wherein contacting the plant part comprises contacting plant reproductive tissue with the composition.

Aspect 6. The method of aspect 5, wherein the plant reproductive tissue comprises a tuber, rhizome, corm, bulbil, or bulb.

Aspect 7. The method of any one of aspects 1 to 3, wherein contacting the plant or plant part comprises contacting root tissue with the composition.

Aspect 8. The method of any one of aspects 1 to 7, wherein contacting the solid plant growth medium comprises contacting one or more of soil, agar, peat moss, potting mix, soilless potting medium, wood residue, bagasse, rice hulls, sand, perlite, vermiculite, and calcined clay with the composition. Aspect 9. The method of any one of aspects f to 8, wherein the plant, plant part, or solid plant growth medium is contacted with about 3 ounces of the composition per acre (w/v) to about 12 ounces of the composition per acre (w/v).

Aspect 10. The method of any one of aspects 1 to 9, further comprising contacting the plant, plant part, or solid plant growth medium with chitin.

Aspect 11. The method of aspect 10, wherein the plant, plant part, or solid plant growth medium is contacted with the chitin and the composition comprising Streptomyces nigrescens 541 simultaneously or sequentially.

Aspect 12. The method of aspect 10 or aspect 11, wherein the plant, plant part, or solid plant growth medium is contacted with 50 mg/g or 500 mg/g chitin.

Aspect 13. The method of any one of aspects 1 to 12, wherein the composition comprising Streptomyces nigrescens 541 comprises the microbes in Agricultural Research Service Culture Collection (NRRL) deposit number NRRL B-67882.

EXAMPLES

The following examples are provided to illustrate certain particular features and/or aspects of the disclosure. These examples should not be construed to limit the disclosure to the particular features or aspects described.

Example 1

Control of Nematodes on Potatoes

The objective of this plot field trial was to determine if Streptomyces nigrescens 541 could control the northern root knot nematode (Meloidogyae hapla) on potato (Umatilla). The trial was conducted in the 2016 season near Eltopia, Washington, USA.

Streptomyces nigrescens 541 (Forge™, Blacksmith BIO) was applied (6 oz/acre) in-furrow at the time of planting followed by three more monthly applications applied via chemigation at the same rate. The trial design was a randomized complete block consisting of 4 replications/treatment. Table 1. Forge™ vs. untreated check ratings on nematode incidence, severity, and potato yield

Values with the same letter are not statistically significant

There was heavy nematode pressure, 34.7% of tubers had some incidence of nematode damage (untreated check) and had a rating of 50.5 (untreated check). In comparison, the Forge™ treated potatoes only had an average incidence of 7.9% and a severity rating of 33.5 (Table 1). These results indicate Forge™ can reduce both the incidence and severity of nematode damage on potatoes.

The average weight of potatoes in each untreated plot was 24.7 lbs. as compared to 41.3 lbs. on the Forge™ treated potatoes. This is exemplified in the tons/acre yield data, 6.40 in the untreated check compared to 10.70 with the Forge™ treatment. The yield in tons/acre calculates to a 67% increase in yield with the Forge™ treatment. The F values associated with the yield data indicate the results are statistically significant.

Example 2

Control of Nematodes on Watermelon

The purpose of this study was to determine the potential of co-inoculation of chitin and 5. nigrescens 541 to inhibit or eradicate Meloidogyne incognita (root knot nematode) on watermelon. Chitin induces S. nigrescens 541 to produce chitinase, therefore destroying the egg shell of the nematodes and limiting the emergence of juveniles (J2). An in vitro environmental incubation test and a greenhouse bioassay were conducted to evaluate the efficacy of .S'. nigrescens with or without chitin against root knot nematodes. In the in vitro test, 50 g of pasteurized sandy soil were mixed with M. incognita eggs at a rate of 5000, 10,000, 15,000, 20,000, or 25,000 eggs. Each of these soils (containing differing amounts of eggs) was inoculated with one of four treatments. One treatment consisted of water only, functioning as the untreated control (UTC). A second treatment consisted of 5. nigrescens 541 only (listed as AMV1033, FIG. 1). The third treatment consisted of a co-application of chitin applied at a low level (50 mg/g of soil) and S. nigrescens 541. The fourth treatment functioned as the industry standard chemical for nematodes and consisted of Abamectin (ABA), 20 ppm. Each treatment was replicated eight times (containers arranged in a RCBD). One set of experiments was incubated in an environmental chamber, and the other in a greenhouse. The greenhouse treatment included two additional treatments, chitin at a low level (chitin low) and chitin at a higher concentration (500 mg/g soil). After 2 weeks incubation, one seed of Citrullus lanatus ‘Royal Sweet’ watermelon was sown in each container. After 5 weeks the plants were harvested, the plant tops were severed at ground level, washed and dried. The roots were washed, weighed, and evaluated for nematode- induced galling on a 0-5 scale, and then the galls were counted.

Comparisons of the main treatment effects showed the treatment chitin low + .S', nigrescens had the lowest number of nematode J2’s extracted, followed by similar numbers from the treatments S', nigrescens 541 only, and the industry standard, ABA. All three treatments had significantly fewer juveniles emerging than the UTC. A similar pattern was shown for all egg inoculation levels (FIG. 1).

The second experiment was conducted in the greenhouse to evaluate plant growth effects in addition to galling and nematode egg mass numbers (Table 2). Root galling was significantly reduced compared to the untreated control on all treatments except chitin low (Table 2). The chitin + S. nigrescens treatments and abamectin treatment significantly reduced the number of galls per g root as compared to the untreated control. Differences in egg mass numbers were more distinct, chitin high + 5. nigrescens and abamectin had similar numbers close to the detection limit, lower than all the other treatments. Treatments did not negatively affect plant growth (data not shown). Results of this study showed that amending chitin with inoculation of S. nigrescens 541 leads to nematode suppression without a negative effect on plant growth. Table 2. Effect of S. nigrescens 541 and chitin treatments on gall index, number of galls, and number of nematode egg masses extracted.

Mean ± SEM of original data of two replications were combined. Means followed by a different letter were significantly different with Ismeans P < 0.05.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

1. A method of controlling plant-parasitic nematodes, comprising contacting a plant, plant part, or a solid plant growth medium with a composition comprising Streptomyces nigrescens 541, thereby controlling the plant-parasitic nematodes.

2. The method of claim 1 , wherein the plant, plant part, or solid plant growth medium is infected with plant-parasitic nematodes.

3. The method of claim 1, wherein the plant-parasitic nematode is Meloidogyne spp., Heterodera spp., Globodera spp., Pratylenchus spp., Radopholus spp., Ditylenchus spp., Bursaphelenchus spp., Rotylenchulus spp., Xiphinema spp., Nacobbus spp., or Aphelenchoides spp.

4. The method of claim 1, wherein contacting the plant part comprises contacting seed with the composition.

5. The method of claim 1, wherein contacting the plant part comprises contacting plant reproductive tissue with the composition.

6. The method of claim 5, wherein the plant reproductive tissue comprises a tuber, rhizome, corm, bulbil, or bulb.

7. The method of claim 1, wherein contacting the plant or plant part comprises contacting root tissue with the composition.

8. The method of claim 1, wherein contacting the solid plant growth medium comprises contacting one or more of soil, agar, peat moss, potting mix, soilless potting medium, wood residue, bagasse, rice hulls, sand, perlite, vermiculite, and calcined clay with the composition.

9. The method of claim 1, wherein the plant, plant part, or solid plant growth medium is contacted with about 3 ounces of the composition per acre (w/v) to about 12 ounces of the composition per acre (w/v).

10. The method of claim 1, further comprising contacting the plant, plant part, or solid plant growth medium with chitin.

11. The method of claim 10, wherein the plant, plant part, or solid plant growth medium is contacted with the chitin and the composition comprising Streptomyces nigrescens 541 simultaneously or sequentially.

12. The method of claim 10, wherein the plant, plant part, or solid plant growth medium is contacted with 50 mg/g or 500 mg/g chitin.

13. The method of claim 1, wherein the composition comprising Streptomyces nigrescens 541 comprises the microbes in Agricultural Research Service Culture Collection (NRRL) deposit number NRRL B -67882.