Korean pseudomonas, fermentation liquor, microbial agent and application

Abstract

The invention relates to the technical field of microbial control, in particular to Korean pseudomonas, fermentation liquor, a microbial agent and application. The Korean pseudomonas is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31567. The Korean pseudomonas Sneb2094 can be used for effectively preventing and treating the root knot nematode disease and promoting the plant growth.

Description

Technical Field

[0001] The invention relates to the technical field of microbial control, and in particular to a strain of Korean Pseudomonas, fermentation liquid, a microbial agent and application thereof. Background Art

[0002] Since it was first reported in 1855, root-knot nematode disease has become one of the most difficult soil-borne diseases to control and prevent worldwide. Root-knot nematode disease generally causes a 22% to 55% reduction in crop yields, and in severe cases even leads to crop failure. The global crop losses caused by this disease exceed $100 billion each year. Both the main root and lateral roots of tomatoes are susceptible to root-knot nematodes. In the initial infection, the aboveground part of the plant does not change significantly. In the later stage, the plant grows slowly, becomes short and thin, and the leaves tend to turn yellow, fall off, or wilt, similar to the symptoms of lack of fertilizer and water. When the disease worsens, the plant will gradually die. Root-knot nematodes cause mechanical damage to the host during the invasion process. Infected plants produce giant cells on the phloem or nearby parenchyma tissue at the feeding point of the larvae. The root tissue around the nematodes proliferates and thickens, gradually showing obvious root knots. The wounds left by nematode infection are conducive to the invasion of other soil-borne pathogenic microorganisms, which often occur together with a variety of soil-borne diseases such as vegetable wilt, verticillium wilt, and damping-off, forming a complex disease that increases losses. The main species of root-knot nematodes that harm vegetables in my country are the southern root-knot nematode (Meloidogyne. incognita), followed by the Java root-knot nematode (M. javanica), the peanut root-knot nematode (M. arenaria), and the northern root-knot nematode (M. hapla).

[0003] Root-knot nematodes have always been a major problem due to their strong pathogenicity, fast spread, and wide host range. At present, the use of chemical agents is still the main method for the prevention and control of root-knot nematodes. With the continuous progress of human society, restrictions on the use of chemical agents have become stricter, and some chemical agents have been listed as restricted pesticides due to their high toxicity. The traditional single and large-scale use of highly toxic chemical nematicides has become history. Biological control is favored because of its advantages such as being environmentally friendly and basically safe for humans and animals. At present, more and more biological control agents have been used for the prevention and control of crop root-knot nematodes, and provide an effective choice for the reduction of chemical agents. Although biological control agents have problems such as slow effect, unstable effect, and susceptibility to environmental influences in the early stage of the prevention and control of root-knot nematodes, they are still popular because of their advantages such as being beneficial to crop growth and being green and safe. At present, the microbial resources that have been developed into biological agents are still limited. Therefore, separating, screening and identifying efficient and stable biological control bacteria that have toxic effects on southern root-knot nematodes from the rhizosphere soil of plants will provide more possibilities for the control of root-knot nematode disease. Summary of the invention

[0004] Based on the above technical problems, the present invention takes southern root-knot nematodes as the target and screens out a strain of Pseudomonas koreensisi with high anti-nematode activity from the soil, intending to provide new resources for the research and development of new effective biocontrol agents against root-knot nematodes.

[0005] The specific technical solutions provided by the present invention are as follows:

[0006] In a first aspect, the present invention provides a Korean Pseudomonas (Pseudomonas koreensisi), which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.31567.

[0007] The second aspect of the present invention provides a fermentation method of the Korean Pseudomonas, comprising the following steps:

[0008] The activated Korean Pseudomonas was inoculated into LB broth medium at 28-37°C and 200-250 r·min -1 Culture for 1 to 2 days. The components of LB culture are (g / L): 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, pH: 6.9-7.1.

[0009] The third aspect of the present invention provides a fermentation broth of Pseudomonas koreana obtained according to the above method.

[0010] In a fourth aspect, the present invention provides a microbial agent, which is prepared by compounding the Korean Pseudomonas or the fermentation broth with a carrier.

[0011] In a fifth aspect, the present invention provides a use of the Korean Pseudomonas or the fermentation liquid or the microbial agent in the preparation of a product for controlling root-knot nematodes. The product is prepared by compounding the Korean Pseudomonas or the fermentation liquid or the microbial agent with an agronomically acceptable adjuvant or carrier. The specific type of the adjuvant or carrier is selected according to the preparation form of the pesticide, and the preparation of the pesticide preparation is carried out according to conventional methods in the art, such as microemulsion, liquid preparation or powder preparation.

[0012] As a preferred embodiment of the present invention, when controlling plant root-knot nematodes, the fermentation liquid is used for root irrigation, and the effective viable bacteria count of the fermentation liquid is 1×10 8 cfu / mL, 10-200mL was irrigated into the roots of each plant.

[0013] In a sixth aspect, the present invention provides a use of the Korean Pseudomonas or the fermentation liquid or the microbial agent in promoting plant growth.

[0014] As a preferred embodiment of the present invention, the promoting plant growth is promoting the increase of plant height, root length and root fresh weight.

[0015] More preferably, the plant is tomato.

[0016] In a seventh aspect, the present invention provides a use of the Korean Pseudomonas or the fermentation liquid or the microbial agent in promoting plant seed germination.

[0017] In an eighth aspect, the present invention provides a plant growth promoter, which comprises the Korean Pseudomonas or the fermentation broth or the microbial agent, and a botanically acceptable auxiliary material or carrier.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The present invention targets southern root-knot nematodes and screens out a strain of Korean Pseudomonas Sneb2094 (Pseudomonas koreensisi) with high anti-nematode activity from soil. Potted plant control efficacy tests and field control efficacy tests have shown that the Korean Pseudomonas Sneb2094 can effectively prevent and control root-knot nematode disease and promote plant growth. Seed germination experiments have also shown that the germination rate and bud length of seeds soaked in fermentation liquid of Korean Pseudomonas Sneb2094 are significantly improved.

[0020] Biomaterial deposit information:

[0021] Korean Pseudomonas Sneb2094, with the Latin name Pseudomonas koreensisi, is taxonomically named Korean Pseudomonas koreensisi. The strain was deposited in the General Microbiology Center of China Culture Collection on August 12, 2024, with the deposit number CGMCC No.31567. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0023] Figure 1 These are the morphological characteristics of Korean Pseudomonas; A, morphology of Korean Pseudomonas on NA medium; B, Gram staining (100×);

[0024] Figure 2 The growth of the aboveground and underground parts of the plants after root irrigation with the fermentation liquid of Korean Pseudomonas Sneb2094; A and A1, CK+J2; B and B1, LB+J2; C and C1, Sneb2094+J2; 3 parallel tests were conducted in each group;

[0025] Figure 3 The root conditions of tomato plants in the field test in Wenxiang Town; A, ddH2O; B, LB; C, avermectin; D, Sneb2094;

[0026] Figure 4 This is the root condition of tomato plants in the field experiment in Daheigangzi Town; A, ddH2O; B, LB; C, avermectin; D, Sneb2094. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0028] Root-knot nematode disease has become one of the most difficult soil-borne diseases to control and prevent worldwide. In the early stages of infection, the aboveground parts of the plants do not change significantly. In the later stages, the plants grow slowly, become short and weak, and the leaves tend to turn yellow, fall off, or wilt, similar to the symptoms of lack of fertilizer and water. When the disease worsens, the plants will gradually die.

[0029] At present, the use of chemical agents is still the main method for controlling root-knot nematodes. With the continuous progress of human society, restrictions on the use of chemical agents have become stricter, and some chemical agents have been listed as restricted pesticides due to their high toxicity. Biocontrol agents are increasingly used in the control of crop root-knot nematodes because of their advantages such as being beneficial to crop growth and being green and safe. Currently, the microbial resources that have been developed into biological agents are still limited. Therefore, isolating, screening and identifying efficient and stable biocontrol bacteria that are toxic to southern root-knot nematodes from the rhizosphere soil of plants will provide more possibilities for the control of root-knot nematode disease.

[0030] Based on this, the present invention provides a Korean Pseudomonas (Pseudomonas koreensisi), which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31567.

[0031] Pot plant efficacy tests and field efficacy tests have shown that the Korean Pseudomonas Sneb2094 can effectively prevent and treat root-knot nematode disease and promote plant growth. Seed germination experiments have also shown that the germination rate and bud length of seeds soaked in the fermentation liquid of Korean Pseudomonas Sneb2094 are significantly improved.

[0032] Example 1

[0033] Isolation, Identification and Screening of Pseudomonas koreensisi Sneb2094 Strain

[0034] The Korean Pseudomonas strain was isolated and purified from soybean field soil by serial dilution method. The specific process is as follows:

[0035] 10.0 g of soil sample from Xinglong Primary School, Boli County, Qitaihe City, Heilongjiang Province (130°′30.38″E, 45°94′42.97″N) was weighed and added into a conical flask containing 100 mL of ddH2O. The sample was shaken at 37°C and 180 rpm for 10 min and then allowed to stand for 5 min. 10 μL of the soil suspension was taken and diluted to 10% by adding ddH2O. -4 , 10 -5 Concentration, 100 μL of each was evenly spread on NA solid medium, and cultured at 28° C. for 24 h. According to their different colony morphological characteristics, single colonies were picked and purified on NA solid medium to obtain bacterial strains.

[0036] The identification of biocontrol bacteria was carried out according to the method described in the Manual of Identification of Common Bacterial Systems compiled by Dong Xiuzhu and Cai Miaoying (2001). The purified bacterial strains were inoculated on NA solid culture medium plates by the plate streak method and cultured at 28°C for 1 to 2 days to observe the morphological characteristics of the bacterial colonies, such as size, shape, and color. Gram staining reagents were purchased from Beijing Regen Biotechnology Co., Ltd., and the results were observed with an oil immersion lens after staining. The genomic DNA of the bacteria to be tested was extracted according to the instructions of the bacterial DNA extraction kit (Tiangen). Using the genomic DNA as a template, the bacteria were amplified by PCR using 16S rDNA primers and gyrB primers, and the PCR products were sent to Shenggong Biotechnology Co., Ltd. for sequencing.

[0037] The results show that Figure 1 ), the colonies of this strain on NA medium are small, translucent milky white, round, moist and smooth, rod-shaped, and Gram-negative.

[0038] BLAST system sequence comparison analysis revealed that the strain had the highest homology with Korean Pseudomonas koreensisi accession numbers CP015852.1 and LT629687.1.

[0039] Pseudomonas koreensisi Sneb2094 (Depository: General Microbiology Center, China Culture Collection Administration; Address: Zhongguancun, Beijing, China; Deposit date: August 12, 2024; Deposit registration number: CGMCC No.31567.

[0040] The culture and fermentation method of Pseudomonas koreensisi is as follows:

[0041] The Korean Pseudomonas bacterial liquid stored at -80°C was activated using an inoculation loop on NA solid culture medium. The culture medium formula was beef extract peptone agar medium (NA), and the ingredients were (g / L): 3-5g beef extract; 10g peptone; 5g sodium chloride; 15-20g agar; 1000mL distilled water; pH: 7.4-7.6, and cultured at 28°C for 2d.

[0042] Pick a single colony with good growth and inoculate it into 150mL liquid culture medium for shaking. The culture medium formula is LB broth (LB), and the ingredients are (g / L): tryptone 10.0g; yeast extract powder 5.0g; sodium chloride 10.0g; pH: 6.9-7.1. The fermentation temperature is 28℃, and the shaking speed is 200r·min -1 , fermentation time 2d.

[0043] The fermented liquid or the finished product of the fermented liquid can be directly used to coat tomato seeds or directly used to irrigate the roots of seedlings. After the treatment, the infection degree of root-knot nematode disease on grown tomato plants is reduced.

[0044] Example 2

[0045] Contact Killing Test of Pseudomonas koreensisi Sneb2094 against Root-knot Nematodes

[0046] The fermentation liquid of Pseudomonas koreensisi prepared by the aforementioned liquid fermentation culture method was set aside. The fermentation liquid of the strain was centrifuged at 8000r / min for 5min to prepare the fermentation filtrate of the strain, which was used for the toxicity test of the second-instar larvae (J2) of southern root-knot nematodes.

[0047] The oocysts of the southern root-knot nematode (Meloidogyne incognita) were obtained by propagating tomato roots. J2 were hatched by the shallow tray method and the adult suspension was prepared.

[0048] Add 400 μL of fermentation filtrate to a 24-well plate, then add 100 μL of worm suspension (about 50 J2), and add equal amounts of ddH2O (CK) and LB broth to the worm suspension as a control treatment. Observe the death of nematodes after 12 h and 24 h, record the number of dead nematodes and the total number of nematodes surveyed, and calculate the mortality rate.

[0049] Mortality rate (%) = number of nematodes killed / total number of nematodes × 100%

[0050] The results show that the mortality rates of the second-instar larvae of southern root-knot nematodes treated with the fermentation filtrate of the Sneb2094 strain were 51.11% and 75.91% after 12 hours and 24 hours, respectively. The strain Sneb2094 can effectively kill the second-instar larvae of southern root-knot nematodes.

[0051] Table 1 Contact killing effect of Pseudomonas koreensisi Sneb2094 fermentation liquid on second-instar larvae of southern root-knot nematode

[0052]

[0053] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's method.

[0054] Example 3

[0055] Seed coating germination test of Pseudomonas koreensisi Sneb2094 strain

[0056] Prepare the fermentation broth of Sneb2094 strain according to the aforementioned liquid fermentation culture method and set aside.

[0057] Add 3% sodium hypochlorite solution to the test tube to cover the seeds, shake for 5 minutes, rinse with ddH2O for 3 times, place tomato seeds of variety L-402 on sterile filter paper and wait for the seeds to air-dry, soak the seeds in the strain fermentation liquid for 3 minutes, and set aside after the seeds are dried.

[0058] Instruments and appliances: steam sterilizer, electric constant temperature air drying oven, electric constant temperature incubator, clean workbench, disposable culture dish, shaking incubator, seedling tray.

[0059] The soaked seeds were placed in a sterile culture dish (d = 9 cm) covered with filter paper. Each treatment was repeated 3 times, with 10 seeds in each repeat. ddH2O (CK) and LB broth (LB) were used as controls. The temperature was controlled in a constant temperature and humidity incubator at 25°C for dark culture. After 24 hours, the number of germinated seeds was counted once a day. After 7 days of culture, the germination rate was calculated, and the root length and shoot length were measured.

[0060] Germination rate (%) = number of germinated seeds on a given day / total number of test seeds × 100

[0061] Table 2 Effect of Korean Pseudomonas Sneb2094 fermentation liquid on tomato seed germination after coating

[0062]

[0063] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's method.

[0064] The results of the experiment showed that compared with the control, the germination rate of tomato seeds treated with the fermentation liquid of Pseudomonas koreensisi Sneb2094 was significantly higher than that of the control group, and the root length and shoot length had no significant difference with the LB control. This showed that the fermentation liquid of Pseudomonas koreensisi Sneb2094 was used to treat tomato seeds for seed soaking and coating, which promoted seed germination and had no inhibitory effect on the growth of tomato plants.

[0065] Example 4

[0066] Potted plant control effect experiment of Pseudomonas koreensisi Sneb2094

[0067] The fermentation liquid of Pseudomonas koreensisi prepared by the aforementioned liquid fermentation culture method is set aside. The nematode suspension is prepared by the aforementioned nematode culture method and set aside.

[0068] The tomato variety was L-402. A 3% sodium hypochlorite solution was added to the test tube to cover the seeds. The solution was shaken for 5 minutes and rinsed with ddH2O for 3 times. The seeds were placed on sterile filter paper and air-dried before being buried in a 50-hole seedling tray filled with nutrient soil (the volume ratio of sand to substrate was 1:1). The nutrient soil was sterilized at 121°C for 180 minutes before sowing. The seeds were placed in a greenhouse at a temperature of 25°C and a light cycle of L / / D=16h / / 8h (L represents the light duration and D represents the dark duration) for about two weeks. After the seeds were cultivated to the four-leaf stage, the tomato seedlings were transplanted into pots (9.5cm×11.5cm), with one seedling in each pot, for later use.

[0069] The following treatments were designed in the experiment: ddH2O root irrigation and inoculation with southern root-knot nematode J2 (CK+J2), LB broth root irrigation and inoculation with J2 (LB+J2), and strain fermentation liquid root irrigation and inoculation with J2 (Sneb2094+J2). The concentration of the strain fermentation liquid was about 1×10 8 cfu / mL, 10mL was irrigated to the roots of each seedling, while the control was irrigated to the roots of each seedling with an equal amount of ddH2O or culture medium (LB broth), and 2mL of second-instar larvae suspension (about 1000 J2) was added to each seedling. Five seedlings were placed randomly for each treatment. After 30 days of cultivation, the physiological indicators of tomato plants such as plant height, plant fresh weight, root length, root fresh weight, and the number of root knots were investigated, and the root knot reduction rate was calculated.

[0070] Root knot reduction rate (%) = (number of control root knots - number of treated root knots) / number of control root knots × 100 Table 3 Effect of root irrigation with Korean Pseudomonas Sneb2094 fermentation liquid on the control of tomato root knot nematode disease and plant growth under potted conditions

[0071]

[0072] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's method.

[0073] From Table 3 and Figure 2 It was found that under potted conditions, compared with the control, after the fermentation liquid of Pseudomonas koreensisi Sneb2094 was used to irrigate the roots of tomato seedlings, the number of root knots of tomato plants decreased significantly, the root knot reduction rate was 60.32%, and the plant height and plant fresh weight increased significantly, while the root length and root fresh weight had no significant difference. Therefore, under potted conditions, Pseudomonas koreensisi Sneb2094 effectively prevented tomato root knot nematode disease and promoted plant growth.

[0074] Example 5

[0075] Field test of the efficacy of Pseudomonas koreensisi Sneb2094

[0076] For tomato variety L-402, add 3% sodium hypochlorite solution to cover the seeds in a test tube, shake for 5 minutes, rinse with ddH2O for 3 times, place the seeds on sterile filter paper, and after the seeds are air-dried, bury them in a 50-hole seedling tray filled with nutrient soil (the volume ratio of sand and substrate is 1:1). Before sowing, the nutrient soil is sterilized at 121℃ for 180min, and cultured in a greenhouse at a temperature of 25℃ and a light cycle of L / / D=16h / / 8h (L represents the light duration, D represents the dark duration) for about two weeks, and culture them to the four-leaf stage for use.

[0077] Two field control experiments were conducted in greenhouses with serious root-knot nematode disease in Wenxiang Town, Haicheng City, Liaoning Province (122°31′5″E, 41°2′20″N) and Daheigangzi Town, Liaozhong District, Shenyang City (122°35′30″E, 41°44′45″N). On the day of transplanting, the tomato seedlings were irrigated with the fermentation liquid of the strain at a concentration of 1×10 8 CFU·mL -1The roots of tomato plants were irrigated with ddH2O, LB broth, and 10% avermectin as the control (Yantai Obeisi Biochemical Company). Each seedling was irrigated with 200 mL of water for each treatment, with 3 replicates per treatment and 3 seedlings per replicate, randomly distributed. After 30 days of transplanting, the whole plant was dug out and the complete root system was retained. The physiological indicators of tomato plants such as plant height, plant fresh weight, root length, and root fresh weight were measured, the root knot index was investigated, and the control effect was calculated.

[0078] The disease was graded using the Bridge & Page (1980) criteria:

[0079] Level 0, no root knot;

[0080] Level 1, there are some small knots on the fibrous roots;

[0081] Level 2, there are some obvious small knots on the fibrous roots, and no knots on the taproot;

[0082] Grade 3, there are some obvious large knots on the fibrous roots, but no knots on the taproot;

[0083] Level 4: Most of the fibrous roots are large knots, and there are no knots on the taproot;

[0084] Level 5: Half of the root system has root knots, and there are also root knots on the main root;

[0085] Level 6, a large number of root knots appear on the taproot;

[0086] Level 7: Most of the taproots have root knots;

[0087] Level 8, all taproots have root knots;

[0088] Level 9, all roots have root knots;

[0089] Level 10, severe root knots and no root;

[0090]

[0091] Table 4 Effect of Korean Pseudomonas Sneb2094 fermentation solution on the control of tomato root-knot nematode disease and plant growth under field conditions in Wenxiang Town

[0092]

[0093] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's method.

[0094] Table 5 Effect of Korean Pseudomonas Sneb2094 fermentation solution on the control of tomato root-knot nematode disease and plant growth under field conditions in Daheigangzi Town

[0095]

[0096] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's method.

[0097] Through two field trials (Tables 4-5 and Figures 3-4 ) found that under field conditions, Korean Pseudomonas (Pseudomonas koreensisi) Sneb2094 can effectively prevent and control southern root-knot nematode disease after root irrigation of tomato seedlings. The control efficacy of the two field trials was 75.00% and 71.01% respectively; compared with ddH2O, LB and avermectin treatment, the plant height, root length and root fresh weight of tomato plants were significantly increased. Therefore, in the field trial, Korean Pseudomonas (Pseudomonaskoreensisi) Sneb2094 effectively prevented tomato root-knot nematode disease and promoted plant growth.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Korean Pseudomonas (Pseudomonas koreensisi), characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.31567.

2. A fermentation method of Pseudomonas koreaensis according to claim 1, characterized in that: The following steps are involved: The activated Korean Pseudomonas was inoculated into LB medium and incubated at 28-37°C and 200-250 r·min -1 Culture for 1 to 2 days.

3. A fermentation broth of Pseudomonas koreana obtained according to the method of claim 2.

4. A microbial agent, characterized in that: The method is prepared by compounding the Korean Pseudomonas described in claim 1 or the fermentation liquid described in claim 3 with auxiliary materials.

5. Use of the Korean Pseudomonas according to claim 1, the fermentation liquid according to claim 3, or the microbial agent according to claim 4 in preparing a product for controlling root-knot nematodes.

6. The use according to claim 5, characterized in that: When controlling plant root-knot nematodes, the fermentation liquid is used for root irrigation. The effective viable bacteria count of the fermentation liquid is 1×10 8 cfu / mL, 10-200mL was irrigated into the roots of each plant.

7. Use of the Korean Pseudomonas according to claim 1, the fermentation liquid according to claim 3, or the microbial agent according to claim 4 in promoting plant growth.

8. The use according to claim 7, characterized in that: The promoting plant growth is any of the following uses: (1) Promote the increase of plant height; (2) Promote the increase of plant root length; (3) Promote the increase of plant root fresh weight; (4) Promote plant seed germination.

9. The use according to claim 7, characterized in that: The plant is tomato.

10. A plant growth promoter, characterized in that: The method comprises the Korean Pseudomonas according to claim 1, the fermentation broth according to claim 3, or the microbial agent according to claim 4, and an acceptable auxiliary material or carrier.

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