An endophytic Bacillus Velez subtilis N1 and its application

Abstract

The present invention provides an endophytic Bacillus Velez N1 and its application, which belong to the field of microbial technology. The endophytic Bacillus Velez N1 provided by the present invention was deposited in the Guangdong Provincial Microbial Culture Collection Center on November 7, 2023, with a deposit number of GDMCC 63983. The endophytic Bacillus Velez N1 provided by the present invention has a good inhibitory effect on sugarcane tip rot pathogens (Sugarcane Fusarium, Layered Fusarium), sugarcane root rot pathogens (Shared Fusarium) and a variety of crop pathogenic fungi (Rice Sheath Blight, Citrus Expans Penicillium, Corn Seedling Blight Fusarium Verticillium). The extract of the sterile fermentation liquid of the N1 strain also has a significant inhibitory effect on sugarcane tip rot pathogens. Therefore, the endophytic Bacillus Velez N1 can be used for biological control of sugarcane fungal diseases and provide new strain resources for the sugarcane biocontrol bacteria resource library.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an endophytic Bacillus Velez subtilis N1 and an application thereof. Background Art

[0002] In recent years, sugarcane yields per mu have stabilized, while the area under sugarcane cultivation has continued to decline, leaving sugarcane production unable to meet demand. Sugarcane production has been severely limited by pests and diseases such as smut, shoot rot, and borers. As China's largest sugarcane-growing region, Guangxi attaches great importance to promoting disease control technologies in the sugarcane industry.

[0003] Sugarcane disease prevention and control primarily involves chemical pesticides, agronomic practices, breeding of disease-resistant varieties, and biological control. While chemical fertilizers and pesticides offer quick results and significant yield increases, they can also lead to environmental pollution and other issues. Agronomic practices, such as sun-drying and tilling, and crop rotation, consume significant manpower and resources. While effective, breeding disease-resistant varieties is time-consuming and labor-intensive, and disease-resistant varieties are often targeted for resistance, making rapid breeding difficult in actual production. Biological control utilizes the growth-promoting and antibacterial properties of beneficial microorganisms to prevent and control diseases while promoting sugarcane growth. In recent years, biological control, owing to its environmental and sustainable nature, has become a hot topic in disease prevention and control. Therefore, identifying biological resources with antagonistic effects against diseases is a crucial task. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an endophytic Bacillus Velez subtilis N1 and its application. The endophytic Bacillus Velez subtilis N1 provided by the present invention has an inhibitory effect on sugarcane pathogenic fungi and various crop pathogens, and can especially prevent and control sugarcane tip rot.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] An endophytic Bacillus Velez subtilis N1, the endophytic Bacillus Velez subtilis N1 is classified as Bacillus sp., deposited in Guangdong Provincial Microbial Culture Collection on November 7, 2023, with the deposit number GDMCC63983, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In certain embodiments, the endophytic Bacillus Velez-O'Kara N1 is isolated from sugarcane leaves.

[0008] In certain embodiments, the 16S rRNA sequence of the endophytic Bacillus Velez subtilis N1 is shown as SEQ ID NO.1.

[0009] In certain embodiments, the TUF sequence of the endophytic Bacillus Velez subtilis N1 is shown as SEQ ID NO.2.

[0010] In certain embodiments, the endophytic Bacillus Velez subtilis N1 is a Gram-positive bacterium; it can grow in the range of 20°C to 42°C, but cannot grow at 4°C; it can utilize glucose, fructose, sucrose, and starch; it can produce cellulase, amylase, and protease, but cannot utilize inorganic phosphorus and does not produce siderophores.

[0011] In certain embodiments, the initial colony morphology of the endophytic Bacillus Velez subtilis N1 is relatively regular and translucent and circular; after culturing for 24 hours, the colony morphology is milky white and wrinkled, and a transparent viscous liquid flows out after the colony is punctured, and no pigment is produced during the growth process.

[0012] In certain embodiments, the endophytic Bacillus Velez N1 has an antagonistic effect on sugarcane tip rot pathogens; the sugarcane tip rot pathogens include Fusarium saccharum Fusarium sacchari , Fusarium Fusarium proliferatum .

[0013] In certain embodiments, the endophytic Bacillus Velez-O'Neorhizium sp. N1 has an antagonistic effect against sugarcane root rot pathogens.

[0014] In certain embodiments, the sugarcane root rot pathogen includes a shared Fusarium spp. Fusarium commune .

[0015] In certain embodiments, the endophytic Bacillus Velezii N1 has an antagonistic effect on crop pathogenic fungi; the crop pathogenic fungi include Rhizoctonia solani Rhizoctonia solani, Penicillium expansum Penicillium expansum , Fusarium verticillium Fusarium verticillioides .

[0016] The present invention also provides an application of the endophytic Bacillus Velezii N1 described in the above technical solution, including the following applications:

[0017] As an antagonistic bacterium against sugarcane shoot rot pathogen;

[0018] As an antagonistic bacterium against sugarcane root rot pathogen;

[0019] It is used as an antagonist against crop pathogenic fungi such as rice sheath blight, citrus expansoides, and corn seedling blight.

[0020] The present invention also provides a use of the endophytic Bacillus Velez subtilis N1 or its fermentation products and metabolites in the above technical solution in preparing a product for preventing and treating sugarcane tip rot.

[0021] In certain embodiments, the metabolite of endophytic Bacillus Velez subtilis N1 is an extract of sterile fermentation broth.

[0022] In certain embodiments, the extract of the sterile fermentation broth includes but is not limited to an acid precipitation extraction product, an n-butanol extraction product, and an ethyl acetate extraction product.

[0023] Beneficial technical effect: The present invention provides an endophytic Bacillus Velez subtilis N1, the endophytic Bacillus Velez subtilis N1 is classified as Bacillus sp., deposited with the Guangdong Provincial Microbial Culture Collection on November 7, 2023, with accession number GDMCC 63983. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, 5th Floor, 100 Xianlie Middle Road, Guangzhou. The endophytic Bacillus Velez N1 provided by the present invention has good inhibitory effects against sugarcane tip rot pathogens (Fusarium spp. and Fusarium spp.), sugarcane root rot pathogens (Fusarium spp.), and various crop pathogens (Rhythmia solani, Penicillium expansum, and Fusarium verticillioides). The extract of the sterile fermentation broth of strain N1 also has significant inhibitory effects against sugarcane tip rot pathogens. Therefore, endophytic Bacillus Velez N1 can be used for the biological control of sugarcane fungal diseases, providing a new strain resource for the sugarcane biocontrol fungus library. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony phenotype of Bacillus velez N1 cultured in LB agar medium for 24 hours;

[0025] Figure 2 These are the results of enzyme activity tests for amylase, protease, and cellulase of Bacillus velez N1;

[0026] Figure 3 Phylogenetic tree constructed for the 16S rRNA sequence of Bacillus velez N1;

[0027] Figure 4 Phylogenetic tree constructed for TUF sequences of Bacillus velez N1;

[0028] Figure 5 The antagonistic effect of Bacillus Velez N1 on six pathogens in plate confrontation. The upper figure (referring to 12 culture dishes) shows the growth phenotype of pathogens cultured normally on PDA plates and in dual culture with Bacillus Velez N1. The lower figure is a bar graph of the growth diameter of pathogens cultured normally and in dual culture with Bacillus Velez N1. "***" indicates P <0.001;

[0029] Figure 6The left figure (referring to 5 culture dishes) shows the inhibitory effect of the acid precipitation product, n-butanol extract, ethyl acetate extract of the sterile fermentation broth of Bacillus Velez N1 and carbendazim on the sugarcane tip rot pathogen CNO1 on the plate; the right figure is a bar graph of the growth diameter of the sugarcane tip rot pathogen CNO1 on the PDA plate containing the acid precipitation product, n-butanol extract, ethyl acetate extract of the sterile fermentation broth of Bacillus Velez N1 and carbendazim; "***" indicates P <0.001;

[0030] Figure 7 The figures show the effect of Bacillus Velez-Nepal N1 in preventing and controlling tip rot in pot experiments; among them, a is the sugarcane injected with 100μL CNO1 spore solution and 100μL sterile water for 7 days; b is the sugarcane injected with 100μL CNO1 spore solution and 100μL N1 bacterial solution for 7 days; c is the sugarcane injected with 200μL sterile water for 7 days. DETAILED DESCRIPTION

[0031] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial sources; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0032] Example 1 Isolation and identification of strains

[0033] 1.1 Sample collection

[0034] In June 2022, we traveled to the Agricultural Science City of Guangxi University to collect healthy leaves from the field of Zhongzhe No. 9, a sugarcane variety independently bred by our laboratory. Specifically, we located healthy, disease-free sugarcane from Zhongzhe No. 9, cleaned the scissors with 75% alcohol, cut off a single leaf from the variety, placed it in a clean sampling bag, sealed it, placed it in a sampling box filled with ice, and brought it back to the laboratory.

[0035] 1.2 Isolation and purification of strains

[0036] (1) Rinse the dust off the surface of the sugarcane leaves with purified water, wipe dry the water, and cut the leaves into 1 cm long and wide using sterilized scissors in a clean bench;

[0037] (2) Soak the cut leaves in 75% ethanol for 1 minute, then rinse with sterile water three times, each time for 1 minute;

[0038] (3) After absorbing the moisture on the leaves with sterilized lens paper, place the leaves on LB agar plates and incubate in the dark at 37°C for 48 h;

[0039] (4) The colonies grown on the culture medium are separated and purified according to their size and morphology, and the strains are preserved.

[0040] 1.3 Strain identification

[0041] (1) Morphological observation: The strain was streaked and cultured in a 37°C incubator for 24 h. Single-cell colonies were observed and their characteristics were recorded.

[0042] See the results Figure 1 The initial colony morphology of the N1 strain was relatively regular and translucent and circular. After 24 hours of culture, the colony morphology became milky white and wrinkled. After puncturing the colony, a transparent viscous liquid flowed out. No pigment was produced during the growth process.

[0043] (2) Physiological and biochemical tests: Refer to the Manual of Identification of Common Bacterial Systems and the Bergey Manual of Systematic Bacteriology to conduct tests on the physiological and biochemical characteristics of the strains.

[0044] The results of the physiological and biochemical characteristics test of the Bacillus Velez N1 strain of the present invention are shown in Table 1. As shown in Table 1, the Bacillus Velez N1 strain cannot grow at 4°C, but can grow in the range of 20°C to 42°C. It can utilize a variety of carbon sources such as glucose, fructose, sucrose and starch, and can produce indole-3-acetic acid (IAA) and cellulase, amylase, protease ( Figure 2 ), cannot utilize inorganic phosphorus and does not produce iron carriers.

[0045] Table 1 Physiological and biochemical test results of N1 strain

[0046]

[0047] (3) Molecular biological identification: 16S rRNA and TUF sequences were amplified by PCR and then subjected to next-generation sequencing and NCBI alignment. Primer synthesis and gene sequencing were performed by Shanghai Sangon Biotechnology Co., Ltd.

[0048] Among them, the amplification primers for 16S rRNA gene fragments are:

[0049] 27F (SEQ ID NO.3): 5'-AGAGTTTGATCMTGGCTCAG-3';

[0050] 1492R (SEQ ID NO.4): 5'-GGTTACCTTGTTACGACTT-3';

[0051] The PCR amplification program for the 16S rRNA gene sequence was as follows: 95°C for 3 min; 30 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 30 s; and 72°C for 5 min.

[0052] The primers for amplification of the TUF gene fragment are:

[0053] tufGPF (SEQ ID NO.5): 5'-ACGTTGACTGCCCAGGACAC-3';

[0054] tufGPR (SEQ ID NO.6): 5'-GATACCAGTTACGTCAGTTGTACGGA-3';

[0055] The PCR amplification program for the TUF gene sequence was as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 1 min, and 72°C for 15 s, 30 cycles; and 72°C for 5 min.

[0056] After comparison, the N1 strain was found to be similar to Bacillus velezinis ( Bacillus sp. ) has a homology of 99.79%.

[0057] MEGA7.0 software was used to construct the 16S rRNA sequence of N1 strain using the neighbor-joining (NJ) method. Figure 3 ) and TUF sequence ( Figure 4 ) phylogenetic tree, it was found that the N1 strain was most closely related to Bacillus velez.

[0058] A comprehensive analysis of morphology, physiological and biochemical tests, sequencing results, and phylogenetic tree comparisons revealed that the identified strain N1 belongs to Bacillus velezinii, a member of the genus Bacillus. Bacillus velezinii N1 was deposited with the Guangdong Provincial Microbial Culture Collection on November 7, 2023, under the accession number GDMCC 63983. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, 5th Floor, 100 Xianlie Middle Road, Guangzhou.

[0059] Example 2 Plate confrontation experiment of Bacillus velezensis N1 strain and various pathogenic fungi

[0060] The present invention uses the plate confrontation method to determine the activity of Bacillus Velez N1 strain against sugarcane tip rot pathogen: Fusarium saccharum Fusarium sacchari (CNO1), Fusarium spp. Fusarium proliferatum (YN41); Sugarcane root rot pathogen: Shared Fusarium Fusarium commune (BS46); and various crop pathogenic fungi: Rhizoctonia solani Rhizoctonia solani (WK) 、 Penicillium expansum Penicillium expansum (QM), corn seedling blight pathogen Fusarium verticillium Fusarium verticillioides (SF1) (the above pathogenic fungi were isolated and identified in this experiment and are currently stored in this laboratory) plate inhibition ability.

[0061] The culture medium used was PDA culture medium (pH = 5.4 ~ 5.8), whose ingredients included: potato extract powder 6g / L, glucose 20g / L, agar 20g / L, and sterilized with high-temperature and high-pressure steam at 115 ° C for 20 minutes.

[0062] Preparation of Bacillus Velez N1 bacterial suspension: Inoculate 0.1% (v / v) of Bacillus Velez N1 strain, which has been mixed with 50% (v / v) glycerol and stored at -80°C, into LB broth for fermentation. Incubate the culture in a shaker at 37°C at 220 rpm for 24 h to obtain fresh N1 bacterial suspension.

[0063] The N1 strain was subjected to a plate confrontation experiment with pathogenic fungi using the plate confrontation culture method. The specific method is: inoculate activated pathogenic fungal cakes (d=0.6cm) at the center of the PDA culture medium, draw two perpendicular straight lines with the center point of the culture medium as the intersection, mark the position 2cm away from the center intersection on the straight line, and place a sterile filter paper on it; add 10μL of fresh biocontrol liquid of the N1 strain on the sterile filter paper, let it stand for 30 minutes until the biocontrol liquid on the sterile filter paper no longer flows, then put it in a 28℃ incubator for inverted culture, and repeat each treatment three times. After 6 days of culture, measure the pathogen colony diameters of the treatment group and the control group to determine the antagonistic effect of the Bacillus Velez N1 strain. Figure 5 It can be seen that the strain N1 of Bacillus velezis is effective against Fusarium spp., Fusarium spp., Fusarium spp. and Rhizoctonia solani. 、 The growth inhibition effect on citrus expansins and Fusarium solani was significant.

[0064] At the same time, the present invention measured the antibacterial rate of the N1 strain against the above pathogenic fungi, as shown in Table 2. The Bacillus velezensis N1 strain has a wide antibacterial spectrum.

[0065] Table 2. Inhibitory effect of N1 strain on 6 pathogenic fungi

[0066]

[0067] Note: The formula is: Inhibition rate (%) = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100. The diameters mentioned above are the values ​​obtained by subtracting 0.6 cm of the bacterial cake. The values ​​in the table are mean ± standard deviation. *** indicates that there is a significant difference between the treatment group and the control group at the 0.001% level after t-test.

[0068] Example 3 Antibacterial Detection of Metabolites of Bacillus velezinis N1

[0069] First, 300 mL of fresh bacterial culture of the N1 strain was prepared using the method mentioned in Example 2. Then, the metabolites of the N1 metabolite strain were extracted using three methods. Specifically:

[0070] (1) Acid precipitation method

[0071] The bacterial broth was centrifuged at 10,000 rpm for 20 minutes to remove the bacterial cells, and the upper sterile fermentation broth was collected. The pH of the sterile fermentation broth was adjusted to 2.0 with concentrated hydrochloric acid, refrigerated at 4°C for 12 hours, and centrifuged again at 10,000 rpm for 20 minutes. The waste liquid was discarded, and the precipitate was collected. 25 mL of HPLC-grade methanol was added to the precipitate, and extraction was carried out at room temperature for 8 hours. The supernatant was collected, and 10 mL of HPLC-grade methanol was added to the precipitate again for 4 hours. The supernatant was centrifuged again at 10,000 rpm for 20 minutes. The methanol supernatants from the two extractions were combined and then evaporated to dryness using a rotary evaporator at 45°C. 2 mL of HPLC-grade methanol was added to the dried material for dissolution, and impurities were filtered through a 0.22 μm sterile filter.

[0072] (2) n-Butanol extraction method

[0073] The bacterial liquid was centrifuged at 10,000 rpm for 20 minutes to remove the bacteria and collect the upper sterile fermentation liquid. An equal volume of n-butanol was added to the sterile fermentation liquid, mixed for 30 minutes, and then centrifuged at 5,000 rpm for 20 minutes. The supernatant was collected and the n-butanol and the remaining liquid were evaporated using a rotary evaporator at 60°C. 2 mL of HPLC-grade methanol was added to the evaporated material to dissolve it, and finally impurities were filtered out using a 0.22 μm sterile filter membrane.

[0074] (3) Ethyl acetate extraction method

[0075] The bacterial liquid was centrifuged at 10,000 rpm for 20 minutes to remove the bacteria and collect the upper sterile fermentation liquid. An equal volume of ethyl acetate was added to the sterile fermentation liquid, mixed for 30 minutes, and then centrifuged at 5,000 rpm for 20 minutes. The supernatant was collected and the ethyl acetate and the remaining liquid were evaporated using a rotary evaporator at 45°C. 2 mL of HPLC-grade methanol was added to the evaporated material to dissolve it, and finally impurities were filtered out using a 0.22 μm sterile filter membrane.

[0076] The metabolites obtained by the three methods were added to the PDA culture medium at a rate of 1% (v / v). Carbendazim was used as a control treatment group (0.2g of carbendazim was weighed, dissolved in 2mL of methanol, and impurities were filtered through a 0.22μm sterile filter membrane). It was also added to the PDA culture medium at a rate of 1% (v / v), mixed well, and poured into plates. After the plates solidified, fresh sugarcane tip rot bacteria ( Fusarium sacchari) CNO1 bacterial cake (d = 0.6 cm), and at the same time inoculated CNO1 bacterial cake into PDA medium without additives as a blank control group. After static culture at 28℃ for 6 days, the phenotype was observed and photographed.

[0077] The results are shown in Table 3. Figure 6 As shown in the data, compared with the growth of CNO1 strain in the blank control group (ordinary PDA medium without additives), the acid precipitation extract of the sterile fermentation broth of the N1 strain had the greatest inhibition on the pathogen, with an inhibition rate of 83.07%, followed by 22.64% of the n-butanol extract, and finally 3.74% of the ethyl acetate extract. Among them, the inhibitory effect of the acid precipitation extract and the n-butanol extract on CNO1 reached a significant level, and carbendazim also had a significant inhibitory effect on CNO1. At the same time, compared with carbendazim, the antibacterial effect of the acid precipitation extract of N1 was more significant.

[0078] Table 3 Antibacterial effect of the sterile fermentation broth extract of N1 strain

[0079]

[0080] Note: The formula is: Inhibition rate (%) = (colony diameter of blank control group - colony diameter of treated group) / colony diameter of blank control group × 100. The diameters mentioned above are the values ​​obtained by subtracting 0.6 cm of the bacterial cake; the values ​​in the table are mean ± standard deviation; *** indicates that there is a significant difference between the treated group and the control group at the 0.001% level after t-test.

[0081] Example 4 Disease resistance experiment of Bacillus velez N1 in potted plants

[0082] (1) The preparation of N1 bacterial solution was consistent with the method mentioned in Example 2.

[0083] (2) Centrifuge the prepared N1 bacterial solution at 6000 rpm for 20 min, discard the waste liquid, and resuspend the bacteria with sterile water. Centrifuge again at 6000 rpm for 20 min, discard the waste liquid, and adjust the OD value of N1 with sterile water. 600 The value is adjusted to 1.

[0084] (3) Preparation of sugarcane tip rot fungus spores. The dominant strain CNO1 of sugarcane tip rot fungus was selected as the infective strain. The sugarcane root rot fungus CNO1 stored in a -80℃ refrigerator was inoculated into PDW potato glucose water culture medium at 0.1% (v / v). The culture was shaken at 220 rpm at 28℃ for 48 h to obtain fresh bacterial liquid spore liquid. The fresh CNO1 bacterial liquid was filtered through 4 layers of sterilized filter paper to obtain spores. The filtrate was centrifuged at 6000 rpm for 20 min, the waste liquid was discarded, and the precipitated spores were resuspended with sterile water. The spores were centrifuged again at 6000 rpm for 20 min, the waste liquid was discarded, and the spore count was adjusted to 1×10 with sterile water. 6CFU / mL is reserved.

[0085] (4) Potted experiment: The sugarcane variety Zhongzhe No. 9 (independently bred by our laboratory) susceptible to tip rot was brought back from the Agricultural Science City of Guangxi University. The sugarcane stems were washed with clean water and cut into single bud segments. The single bud sugarcane stems were then completely buried in the substrate. After growing in the single pot substrate for 30 days, the sugarcane was treated as follows: ① Inject 100 μL of 1×10 6 CFU / mL of CNO1 spore solution, and then inject 100μL of sterile water. ② Inject 100μL of 1×10 6 CFU / mL of CNO1 spore solution, and then inject 100μL OD 600 =1 N1 bacterial solution. ③ Inject 200μL of sterile water into the growing point. Repeat 15 plants for each treatment. After one week, observe and count the number of diseased plants and the disease grade. The results are shown in Table 4 and Figure 7 shown.

[0086] The formula for calculating the incidence rate is: incidence rate (%) = number of diseased plants / total number of plants surveyed × 100;

[0087] The disease severity index calculation formula is DSI (Disease severity index, DSI) = [∑(ni×vi) / 4N]×100; where n represents the number of samples at the level, v represents the level, and N represents the total number of samples participating in the test.

[0088] The results showed that N1 could effectively reduce the occurrence of shoot rot and lower the disease index.

[0089]

[0090] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An endophytic Bacillus Velezii N1, characterized in that It was deposited in the Guangdong Provincial Microbiological Culture Collection on November 7, 2023, with the deposit number GDMCC No. 63983, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A use of the endophytic Bacillus Velez subtilis N1 according to claim 1, characterized in that: Select from the following applications: As an antagonistic bacterium against sugarcane tip rot pathogen; the sugarcane tip rot pathogen is selected from Fusarium sacchari , Fusarium proliferatum ; As an antagonistic bacterium against sugarcane root rot pathogen; the sugarcane root rot pathogen is Fusarium commune ; As crop pathogens Rhizoctonia solani Antagonistic bacteria, Penicillium expansum Antagonistic bacteria, Fusarium verticillioides antagonistic bacteria.

3. A use of the endophytic Bacillus Velez N1 according to claim 1 in preparing a product for preventing and treating sugarcane tip rot, characterized in that: The pathogen of sugarcane tip rot is selected from Fusarium sacchari , Fusarium proliferatum .

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