Streptomyces olivaceus and application thereof

Abstract

The present application relates to the field of plant disease biological control, and specifically discloses a streptomyces olivaceus and application thereof. The prepared streptomyces olivaceus biocontrol agent can prevent and control sclerotinia disease and clubroot disease, and the prevention and control effects reach more than 50%, and the bacteria have strong salt and alkali resistance, indicating that the strain or the biocontrol agent has good application prospect in the fields of sclerotinia disease and clubroot disease prevention and control and in the salt and alkali environment.

Description

Technical Field

[0001] This invention relates to the field of biological control of plant diseases, specifically to a strain of Streptomyces oliveii and its application. Background Technology

[0002] Sclerotinia rot is a widespread plant disease occurring globally, in countries and regions that grow oilseed crops such as rapeseed and soybeans, including China, Canada, Brazil, and Argentina. In recent years, with the continuous increase in the planting area of ​​oilseed crops in my country, the scope of sclerotinia rot has expanded, and the disease trend has become increasingly severe, seriously threatening the development of the oilseed crop industry. Sclerotinia rot is a fungal disease caused by *Sclerotinia sclerotiorum*, which can occur throughout the entire growth and development period of the crop. *Sclerotinia sclerotiorum* mainly infects leaves, stem bases, and fruits. Infected plant tissues develop water-soaked spots, which change from green to light brown to dark brown. As the disease progresses, white mycelium appears in the diseased tissues and forms sclerotia, leading to lodging or plant death. Sclerotia and diseased plant debris in the soil are the primary source of infection for the following year.

[0003] Clubroot disease has been a serious threat to the production of cruciferous crops such as rapeseed and cabbage in recent years, with reports of its occurrence in China, Russia, Australia, and other regions. Clubroot is an important cruciferous disease caused by *Plasmodiophora brassicae*. Because *Plasmodiophora brassicae* can persist in the soil for extended periods and spread through agricultural tools, the incidence of clubroot has been increasing year by year, severely hindering the healthy development of the cruciferous crop industry. Clubroot primarily damages the root system. In the early stages, the above-ground parts of infected plants show signs of water shortage and slow growth; later, the leaves wilt, turn yellow, and lose water. The underground roots initially appear smooth, but later crack and turn yellow, eventually leading to the rotting of the entire root system. After the root tissue of infected plants rots, a large number of dormant *Plasmodiophora brassicae* spores are released into the diseased soil, serving as a major source of infection and disease spread the following year.

[0004] Chemical control methods for sclerotinia rot and clubroot are limited. Commonly used chemical agents mainly include carbendazim, sclerotinia sulfadiazine, and imazalil for sclerotinia rot, and fluazinam and cyazofamid for clubroot. Long-term overuse of chemical agents increases the risk of pathogen resistance, leading to reduced control efficacy. Simultaneously, pesticide residues cause environmental pollution and pose significant risks to humans and animals. The ban on highly toxic pesticides further complicates the selection of chemical control methods. In recent years, with increased environmental awareness in agricultural production, the exploration and utilization of more environmentally compatible biological control resources will benefit sustainable agricultural development, better meet the needs of green control and ecological security, and has attracted widespread attention from researchers. In recent years, biocontrol resources for sclerotinia stem rot control have mainly included *Clonostachys rosea*, *Fusarium oxysporum*, *Bacillus subtilis*, and *Trichoderma harzianum*; while those for clubroot control have mainly included *Phoma glomerata*, *T. viride*, and *B. amyloliquefaciens*. There is a lack of biocontrol bacteria that are effective against both sclerotinia stem rot and clubroot. Summary of the Invention

[0005] To obtain a strain capable of controlling sclerotinia rot and clubroot disease, this invention provides a strain of *Streptomyces oliveri* and its application. This *Streptomyces oliveri* strain can control sclerotinia rot and clubroot disease with a control efficacy exceeding 50%. Furthermore, the strain exhibits strong salt and alkali tolerance, indicating that this strain or biocontrol agent has good application prospects in the control of sclerotinia rot and clubroot disease, as well as in saline-alkali environments.

[0006] This invention provides a strain of Streptomyces olivaceus, MCNB77232, which was deposited at the China General Microbiological Culture Collection Center on April 12, 2023, with accession number CGMCCNO:27095.

[0007] The present invention also provides a biocontrol agent prepared from the aforementioned Streptomyces oliveii.

[0008] This invention also provides a method for preparing the aforementioned biocontrol agent, comprising activating and culturing the *Streptomyces oliveri*, and then preparing OD using PBS buffer. 600nm =0.7-0.8 bacterial solution, which is the biocontrol agent.

[0009] The present invention also provides an application of the aforementioned biocontrol agent in antibacterial activity.

[0010] Furthermore, the antibacterial effect refers to the inhibition of the following bacteria:

[0011] The pathogens include Sclerotinia sclerotiorum, Botrytiscinera, Monilinia laxa, Alternaria solani, Ceratocystis fimbriata, Rhizoctonia cerealis, Fusarium equiseti, Fusarium oxysporum, and Plasmodiophorabrassicae.

[0012] Furthermore, the aforementioned Streptomyces oliveii can be used for the prevention and control of plant diseases.

[0013] Furthermore, the aforementioned Streptomyces oliveii is used to control sclerotinia disease and clubroot disease.

[0014] Furthermore, the pathogen causing sclerotinia disease is *Sclerotinia sclerotiorum*.

[0015] Furthermore, the pathogen causing clubroot disease is *Plasmodiophora brassicae*.

[0016] The present invention also provides an application of the aforementioned biocontrol agent in the prevention and control of saline-alkali environments.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. This invention isolates a strain of Streptomyces olivaceus, and tests show that this biocontrol bacterium has lecithinase and cellulase activities, proving that it has the potential to be used as a biocontrol agent.

[0019] This invention involves culturing a biocontrol agent prepared from *Streptomyces oliveri* against *Sclerotinia sclerotiorum*. The experimental results show that the biocontrol agent has a significant inhibitory effect on the growth of *Sclerotinia sclerotiorum*, with an inhibition rate of 73.2%. The agent can control sclerotinia disease, reducing the disease index from 75.0 to 36.7, with a control effect of 51.1%.

[0020] The biocontrol agent prepared in this invention was co-cultured with *Platycotyle brassicae*. The experimental results showed that the biocontrol agent had a significant inhibitory effect on the germination of dormant spores of *Platycotyle brassicae*, with an inhibition rate of 54.5%. The agent could control clubroot disease, reducing its disease index from 63.3 to 30.0, with a control effect of 52.6%.

[0021] The biocontrol agent prepared by the biocontrol bacterium MCNB77232 disclosed in this invention has good control effects on sclerotinia rot and clubroot, providing a new solution for the control of sclerotinia rot and clubroot, and has good market application prospects.

[0022] 2. The *Streptomyces olivaceus* strain provided by this invention is tolerant of saline-alkali environments and can serve as a potential strain for the prevention and control of plant diseases in saline-alkali soils. It can be used in planting in saline-alkali lands and secondary salinized soils.

[0023] Information on the Preservation of Biological Materials

[0024] MCNB77232, referred to as *Streptomyces olivaceus* in this application, was deposited on April 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 27095. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is *Streptomyces olivaceus*. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is an electron microscope image of the cell morphology of Streptomyces oliveri MCNB77232 in this invention.

[0027] Figure 2 This invention demonstrates the inhibitory effect of Streptomyces oliveri MCNB77232 on the growth of different fungi.

[0028] In the figure, a, b, c, d, e, f, g, and h represent the inhibitory effects of Streptomyces oliveri MCNB77232 on the growth of Sclerotium sclerotiorum, Botrytis cinerea, Streptomyces sclerotiorum, Alternaria solanacea, Rhizoctonia solani, Rhizoctonia graminearum, Fusarium equisetifolium, and Fusarium oxysporum, respectively.

[0029] Figure 3 This invention demonstrates the inhibitory effect of Streptomyces oliveii MCNB77232 on the germination of Plasmodium brassicae spores.

[0030] In the figure, a represents the inhibitory effect of Streptomyces oliveri MCNB77232 on the germination of Plasmodium brassicae spores;

[0031] b is the PBS buffer control group.

[0032] Figure 4 The activity of lecithinase and cellulase in *Streptomyces oliveii* MCNB77232 in this invention was detected.

[0033] In the figure, a represents the detection of lecithinase activity in Streptomyces oliveii MCNB77232;

[0034] b represents the detection of cellulase activity in Streptomyces oliveii MCNB77232.

[0035] Figure 5 This study investigates the salt and alkali resistance of Streptomyces oliveri MCNB77232 in this invention.

[0036] In the figure, a represents the alkali resistance test of Streptomyces oliveri MCNB77232;

[0037] b represents the salt tolerance test of Streptomyces oliveii MCNB77232.

[0038] Figure 6 Safety testing of Streptomyces oliveri MCNB77232 in this invention. Detailed Implementation

[0039] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0040] Example 1: A strain of Streptomyces oliveii and its application.

[0041] I. Isolation and Identification of Strains

[0042] The bacterium was isolated from soil in Shenyang City, Liaoning Province. The specific method is as follows: 1g of soil was added to 9mL of sterile distilled water under sterile conditions, shaken, centrifuged and serially diluted. 100μL of the 100-fold and 1000-fold dilutions were evenly spread on LB solid medium, dried in a clean bench, and incubated upside down in a 25℃ constant temperature incubator. Single colonies were picked with sterile toothpicks and transferred to new medium. Sclerotinia sclerotiorum was used as the target bacterium for preliminary screening using the plate confrontation method. Among them, the bacterium MCNB77232, which has an antagonistic effect on Sclerotinia sclerotiorum, will be identified as a potential biocontrol bacterium.

[0043] LB solid medium formula: 10g tryptone, 5g yeast extract, 10g NaCl, 16g agar, 1L distilled water.

[0044] Based on the morphological characteristics, physiological and biochemical features, 16S rRNA sequence, and specific primer analysis of the *Streptomyces* genus, it was ultimately identified as *Streptomyces olivaceus*. The identification results are as follows:

[0045] 1. Morphological characteristics of bacteria

[0046] Gram-positive, the colonies appear as white colonies on Gao's No. 1 medium, turning reddish-brown after 2 days. The colony surface is rough, with aerial hyphae forming. After 5 days, yellowish-brown and reddish-brown spores appear on the colony surface. Under a microscope, the spores are spherical, with several spores linked together to form spore chains. Electron microscopy reveals the bacterial cells to be filamentous (see...). Figure 1 ).

[0047] 2. Physiological and biochemical characteristics

[0048] The physiological and biochemical characteristics of Streptomyces oliveri MCNB77232 are shown in Table 1.

[0049] Table 1. Physiological and biochemical characteristics of Streptomyces oliveri MCNB77232

[0050] characteristic result characteristic result Gram reaction + VP - Starch hydrolysis + protease - <![CDATA[H2S generation]]> + Urease - lecithinase + Indole production - Cellulase + Methyl red - catalase - Oxidase - Gelatin hydrolysis -

[0051] Note: + indicates a positive reaction; - indicates a negative reaction.

[0052] 3. 16S rRNA sequence analysis

[0053] Methods: DNA was extracted, and the 16S RNA fragment was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR system (50 μL) consisted of: 10 pmol primers, 0.2 mM dNTPs, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase (TaKaRa), 1× PCR buffer (TaKaRa), and 25 ng genomic DNA template. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 90 s, and 30 cycles followed by a final extension at 72℃ for 10 min. The PCR amplification products were separated and purified by 1% agarose gel electrophoresis, and then analyzed by an ABI 3730 DNA sequencer from Sangon Biotech Co., Ltd. The sequencing results identified the bacteria as Streptomyces olivaceus.

[0054] II. Broad-spectrum antibacterial analysis

[0055] 1. Selection of pathogens

[0056] The tested pathogens were *Sclerotinia sclerotiorum*, *Botrytis cinera*, *Monilinia laxa*, *Alternaria solani*, *Ceratocystis fimbriata*, *Rhizoctonia cerealis*, *Fusarium equiseti*, *Fusarium oxysporum*, and *Plasmodiophora brassicae*. *Plasmodiophora brassicae* was used in the spore germination inhibition experiment.

[0057] 2. Antibacterial test

[0058] Streptomyces oliveri MCNB77232 was cultured in LB liquid medium at 28°C with shaking for 48 hours to obtain a bacterial suspension. After activation of the tested pathogen, a 5 mm diameter mycelial disc was placed at the center of a PDA medium, and the bacterial suspension was streaked 30 mm from the center. Simultaneously, a mycelial disc was inoculated at the center of a plate. A control plate with a mycelial disc inoculated only at the center served as a reference. The plates were sealed with sealing film. Each tested pathogen was repeated three times, and the plates were incubated upside down at 25°C until the fungal hyphae in the control plates were just fully colonized. Results showed (e.g.) Figure 2 Streptomyces oliveri MCNB77232 was able to inhibit the growth of the tested fungi (pathogens). The average colony diameter and inhibition rate of the control and treatment groups are shown in Table 1.

[0059] Table 1. Inhibitory effect of Streptomyces oliveri MCNB77232 on the hyphal growth of different fungi.

[0060]

[0061]

[0062] 3. Inhibition of spore germination

[0063] Add 1 mL of dormant spores of *Platycodon brassicae* (concentration 1×10⁻⁶). 6After mixing 10 mL of rapeseed root exudate with a mixture of spores / mL, 1 mL of biocontrol agent was added. The mixture was then incubated in the dark at 25°C for 5 days. PBS buffer was used as a control instead of the bacterial culture. 20 μL of the culture solution was placed on a glass slide and dried. 50 μL of 1% lichen red staining solution was added to cover and stain for 3 min. The stain was washed off with 95% ethanol, and 10 μL of 10% glycerol was added. The slide was then covered and observed under a microscope. Ungerminated spores stained black or red, while germinated spores remained transparent. Results are as follows: Figure 3 As shown in Table 2, Streptomyces oliveii MCNB77232 can inhibit the germination of dormant spores of Plasmodium brassicae (Table 2).

[0064] Table 2. Inhibitory effect of Streptomyces oliveri MCNB77232 on spore germination of Plasmodium brassicae.

[0065] Group Number of spores that germinate. Spore germination rate inhibition rate (%) Processing group 60±5 54.5 control group 132±8 -

[0066] III. Detection of biocontrol-related traits

[0067] Lecithinase activity was assessed using egg yolk agar plates (under sterile conditions, egg yolk was mixed with an equal volume of physiological saline, shaken well, and then added to 50°C meat gravy peptone medium at a 1:20 ratio, mixed thoroughly, and poured into petri dishes) to determine the presence of lecithinase activity. Biocontrol bacteria MCNB77232 (Streptomyces oliveri) was inoculated into the center of the agar plate and incubated at 28°C for 7 days. A clear zone (e.g., a clear zone around the bacterial colony) appeared. Figure 4 a) indicates that the biocontrol bacterium MCNB77232 has lecithinase activity.

[0068] Cellulase activity: Cellulase activity was detected using cellulase detection plates (0.25 g / L magnesium sulfate heptahydrate, 0.25 g / L dipotassium hydrogen phosphate, 1.88 g / L carboxymethyl cellulose, 15 g / L agar). Biocontrol bacteria MCNB77232 was inoculated into the center of the plate and incubated at 28°C for 7 days. 5 mL of 0.2 mg / mL Congo red staining solution was added to the culture medium, and staining was performed for 1 hour. After discarding the Congo red staining solution, 5 mL of 1 M NaCl was added for washing for 1 hour. The washing solution was then discarded. A clear zone appeared around the colony (e.g., a clear zone around the colony). Figure 4 b) indicates that the biocontrol bacterium MCNB77232 has cellulase activity.

[0069] IV. Preparation of Biocontrol Agents

[0070] The biocontrol bacterium MCNB77232 was activated by incubating overnight at 28°C with shaking in LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl). The activated culture was then diluted with 0.01 mol / L PBS buffer to prepare an OD (diethyltoluene) solution. 600nm =0.8 bacterial solution, which is the biocontrol agent.

[0071] V. The efficacy of biocontrol agents against sclerotinia stem rot and clubroot disease

[0072] Rapeseed seeds were planted in sterilized potting soil and watered regularly. Two weeks later, the plants were used to evaluate the biocontrol potential of the biocontrol agent. Treatment groups were established where leaves were inoculated with *Sclerotinia sclerotiorum*, followed by spraying with the biocontrol agent 24 hours later; the control group was inoculated with *Sclerotinia sclerotiorum*, followed by spraying with PBS buffer. For inoculation using live leaves, a 5mm mycelial cake was placed on the same side of the leaf vein, and each plant was sprayed with 5mL of the biocontrol agent. After inoculation, the plants were moved to a 25℃ greenhouse and cultured under a 16-hour light-8-hour dark day-night cycle. Three days after inoculation, 15 leaves were randomly collected, and the severity of sclerotinia sclerotiorum disease was assessed using a 0-4 classification system: 0: no disease; 1: lesions covering less than 10% of the entire leaf; 2: lesions covering 11-30% of the entire leaf; 3: lesions covering 31-50% of the entire leaf; 4: lesions covering more than 51% of the entire leaf.

[0073] In addition, dormant spores of *Plasmodiophora brassicae* were mixed with sterile nutrient soil to prepare inoculation soil (1×10⁻⁶). 6 (Spores / g soil) were evenly spread into 72-well trays, with one rapeseed seed per well and 1 mL of biocontrol agent injected. PBS buffer was used as a control. After inoculation, the plants were moved to a greenhouse at 25℃ and cultured under a 16-hour light-8-hour dark cycle. After 5 weeks, 30 roots were randomly collected, and the severity of clubroot disease was assessed using a 0-3 classification system: Grade 0: no swelling in either the taproot or lateral roots; Grade 1: root swelling only on lateral roots; Grade 2: swelling only on the taproot; Grade 3: root swelling on both the taproot and lateral roots.

[0074] Disease index = Σ(Disease level × Number of corresponding plants) / (Highest disease level × Total number of plants surveyed)

[0075] Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0076] The experimental results (Table 3) showed that the disease index of the sclerotinia disease treatment group sprayed with biocontrol agent 24 hours after inoculation with Sclerotinia sclerotiorum was 36.7, while the disease index of the control group sprayed with PBS buffer 24 hours after inoculation with Sclerotinia sclerotiorum was 75.0. The biocontrol agent achieved a control effect of 51.1% on sclerotinia disease, indicating that the biocontrol agent has a good control effect on sclerotinia disease.

[0077] Table 3 shows the efficacy against sclerotinia stem rot.

[0078] Group Disease index Preventive efficacy (%) Processing group 36.7 51.1 control group 75.0 -

[0079] The experimental results (Table 4) showed that the disease index of the clubroot treatment group was 30.0 five weeks after inoculation, while the disease index of the control group was 63.3 five weeks after inoculation. The biocontrol agent achieved a control effect of 52.6% on clubroot, indicating that the biocontrol agent has a good control effect on clubroot.

[0080] Table 4 shows the efficacy against clubroot disease.

[0081] Group Disease index Preventive efficacy (%) Processing group 30.0 52.6 control group 63.3 -

[0082] VI. Salt and Alkali Resistance Test

[0083] The biocontrol bacterium MCNB77232 was cultured in liquid LB medium with shaking for 48 hours, and the bacterial culture was then diluted to OD. 600 =0.8, and it was inoculated into LB liquid medium (pH values ​​of 7, 8, 9, 10, 11 and NaCl concentrations of 0.1%, 0.2%, 0.5%, 1.0%, 2.0%, 5.0%), and the OD values ​​of the bacterial culture were measured after 24 h and 48 h. The results showed (e.g. Figure 5 The biocontrol bacteria exhibit salt and alkali tolerance, demonstrating their potential application as a biocontrol resource in saline-alkali environments. As the pH value increases, the bacterial concentration initially rises and then decreases. The highest concentration is observed at pH = 9, indicating the fastest reproduction rate and optimal growth for the biocontrol bacteria. Furthermore, as the NaCl concentration increases, the bacterial concentration initially rises and then decreases, reaching its highest point at a NaCl concentration of 0.2%, again indicating the fastest reproduction rate and optimal growth for the biocontrol bacteria.

[0084] VII. Safety Testing of Biocontrol Bacteria

[0085] Biocontrol bacteria MCNB77232 was inoculated onto blood agar medium. After sealing the medium with aluminum foil, it was incubated overnight at 25°C. No clear zone was observed around the colonies of the biocontrol bacteria. Figure 6 The result indicates that the biocontrol bacteria do not have hemolytic effects, demonstrating that the biocontrol bacteria have good biocontrol safety.

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Streptomyces olivaceus (ATCC 27438), characterized in that, Streptomyces olivaceus The Streptomyces olivaceus is MCNB77232, preserved in China General Microbiological Culture Collection Center on April 12, 2023, with a preservation number of CGMCC NO: 27095; the Streptomyces olivaceus can simultaneously inhibit Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ) and Plasmodiophora brassicae ( Plasmodiophora brassicae ). ​ 2. A biocontrol agent prepared from the Streptomyces olivaceus of claim 1.

3. A method for preparing the biocontrol agent of claim 2, characterized by, The activated and cultured S. olivaceus is prepared into OD 600nm =0.7-0.8 bacterial solution, which is a biocontrol agent.

4. Use of the Streptomyces olivaceus of claim 1 or the biocontrol agent of claim 2 in inhibiting bacteria.

5. The use of claim 4, wherein the bacteria are inhibited are: Sclerotium sclerotiorum ( Sclerotinia sclerotiorum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinera ), Sclerotium sclerotiorum ( Monilinia laxa Alternaria alternifolia ( ) Alternaria solani ), Small long-beaked mold ( Ceratocystis fimbriata ), Rhizoctonia granatum ( Rhizoctonia cerealis Fusarium equisetifolium ( Fusarium equiseti Fusarium oxysporum ( Fusarium oxysporum ) and Plasmodium brassicae ( Plasmodiophora brassicae ).

6. Use according to claim 4, characterized in that, The Streptomyces olivaceus can be used for plant disease control.

7. Use according to claim 6, characterized in that, The Streptomyces olivaceus is used for controlling Sclerotinia sclerotiorum and Plasmodiophora brassicae in Brassica napus.

8. Use according to claim 7, characterized in that, The pathogenic bacteria of the sclerotinia disease is sclerotinia sclerotiorum (lib.) de bary Sclerotinia sclerotiorum ).

9. Use according to claim 7, characterized in that, The pathogenic bacteria of the root rot is Plasmodiophora brassicae (Woronin) Tulasne Plasmodiophora brassicae ).

10. Use of the biocontrol agent of claim 2 in controlling plant diseases in saline-alkali environment.

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