Application of a protozoan Trichomonas cercopithecidae in improving wheat yield and soil organic matter content

Abstract

The present invention provides a protozoan Trichomonas cercoides for use in improving wheat yield and soil organic matter content. Protozoan Trichomonas cercoides NJAU‑Z1 (Cercomonas direct) NJAU‑Z1), with a preservation number of CCTCC NO: C202443. Field trials have found that the average yield of wheat treated with protozoan Trichomonas cercoides NJAU‑Z1 increased by 11.23%. At the same time, the organic matter content of the soil treated with protozoan Trichomonas cercoides NJAU‑Z1 increased by 9.2%. The present invention shows that protozoan Trichomonas cercoides Cercomonas direct The application of NJAU‑Z1 has great potential in increasing soil organic matter content, soil fertility and grain yield.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganisms and relates to the application of a protozoan Trichomonas cercopithecidae in improving wheat yield and soil organic matter content. Background Art

[0002] Wheat is one of the most important food crops in the world and the main food source for more than one-third of the world's population. Wheat has a long growth cycle and requires a large amount of fertilizers such as nitrogen, phosphorus and potassium. Producers often use chemical fertilizers to supplement the insufficient nutrients in the soil. However, the excessive use of traditional chemical fertilizers has led to many negative agricultural effects, such as soil compaction and reduced soil fertility, and may even have an adverse effect on wheat yield and quality.

[0003] In addition, the long-term and large-scale application of chemical fertilizers inhibits the growth of many key microorganisms in the soil, reduces the diversity of soil microorganisms, and destroys the soil micro-ecosystem. Microorganisms play an important role in soil nutrient transformation and organic matter decomposition, especially straw degradation. The reduction in their number leads to a decrease in the soil's nutrient recycling capacity and weakened soil fertility. This not only restricts the growth and development of wheat, but also makes the soil's fertilizer retention and buffering capacity worse, resulting in a bottleneck in wheat yield increase.

[0004] Soil organic matter content is an important indicator of soil fertility. Soil organic matter is mainly composed of carbon-containing organic compounds and is an important source of nutrients and slow-release nutrient pool in the soil. High levels of soil organic matter can provide a rich source of carbon and energy for soil microorganisms and maintain soil biodiversity. Soil organic matter can not only improve soil structure, soil aeration and water retention capacity, but also increase soil fertilizer retention and buffering capacity, and resist the adverse effects of changes in the external environment. These are extremely beneficial to crop growth, help increase crop yields, and maintain the long-term health of the soil.

[0005] So far, there are no reports on protozoa that can both increase wheat yield and soil organic matter content. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a protozoan Trichomonas cercopithecidae for improving wheat yield and soil organic matter content.

[0007] Protozoa Trichomonas cercopithecidae NJAU-Z1 (Cercomonas directa NJAU-Z1) in improving wheat yield and / or soil organic matter content, the protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas directaNJAU-Z1) was deposited in China Center for Type Culture Collection on January 20, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202443.

[0008] Protozoa Trichomonas cercopithecidae NJAU-Z1 (Cercomonas directa NJAU-Z1) in the preparation of products for improving wheat yield and / or soil organic matter content, the protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas directa NJAU-Z1) was deposited in China Center for Type Culture Collection on January 20, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202443.

[0009] Protozoa Trichomonas cercopithecidae NJAU-Z1 (Cercomonas directa NJAU-Z1) culture for improving wheat yield and / or soil organic matter content, the protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas directa NJAU-Z1) was deposited in the China Center for Type Culture Collection on January 20, 2024. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC NO: C202443. The preparation method of the culture comprises inoculating the Trichomonas NJAU-Z1 into NMAS culture medium, adding inactivated Escherichia coli as food, placing it in an incubator at 20°C, and standing for 48 hours.

[0010] As a preferred embodiment of the present invention, the NMAS culture medium is prepared by: 0.12 g / L sodium chloride, 0.004 g / L magnesium sulfate heptahydrate, 0.006 g / L calcium chloride hexahydrate, 0.142 g / L sodium phosphate, and 0.136 g / L potassium phosphate.

[0011] Beneficial effects:

[0012] The present invention separates and screens the protozoan Trichomonas cercophora that promotes straw degradation from soil Cercomonas directa Field trials have shown that Trichomonas Cercomonas directa NJAU-Z1 can significantly increase wheat yield. Cercomonas directa NJAU-Z1 can also significantly increase the content of soil organic matter. Cercomonas directa It plays an important role in improving crop yield and soil fertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0014] Figure 1 These are photographs of the morphological characteristics of the protozoan Trichomonas cercopithecidae NJAU-Z1 vegetative body (left), dormant cysts (middle) and reproductive cysts (right) of the present invention.

[0015] Figure 2 The phylogenetic tree of the 18S rDNA gene sequence of Trichomonas cerevisiae NJAU-Z1 of the present invention is shown in FIG.

[0016] Figure 3 This is a bar chart comparing wheat yield between the field test of Trichomonas NJAU-Z1 and the blank control in Example 3 of the present invention.

[0017] Figure 4 This is a comparison of wheat seedlings during the field test of Trichomonas NJAU-Z1 (right) and the control (left) in Example 3 of the present invention.

[0018] Figure 5 This is a bar graph comparing the soil organic matter content of the field test Trichomonas NJAU-Z1 and the blank control in Example 4 of the present invention.

[0019] Figure 6 This is a statistical graph of the correlation between wheat yield and soil organic matter content in the field test in Example 4 of the present invention.

[0020] Biomaterial Deposit Information

[0021] Protozoa Trichomonas cercopithecidae NJAU-Z1 Cercomonas directa NJAU-Z1, deposited in China Center for Type Culture Collection, the deposit date is January 20, 2024, the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: C202443. DETAILED DESCRIPTION

[0022] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The experiments in the following examples were repeated four times.

[0023] NMAS culture medium: sodium chloride 0.12 g / L, magnesium sulfate heptahydrate 0.004 g / L, calcium chloride hexahydrate 0.006 g / L, sodium phosphate 0.142 g / L, potassium phosphate 0.136 g / L.

[0024] Example 1 Isolation and purification of protozoan NJAU-Z1:

[0025] The inventor of the present application collected soil from the field of the experimental base of the Sanya Research Institute of Nanjing Agricultural University. The depth of the soil was 0-20 cm, and it was sealed in a sterile ziplock bag and brought back to the laboratory. The soil dilution method was used to isolate protozoa. The specific method is as follows: the crop rhizosphere soil sample was mixed, 1g of soil was weighed and added to a 50 mL centrifuge tube and 30 mL of sterile deionized water was added, and then the centrifuge tube was placed in a shaker at 250 rpm and 20 ° C for 15 minutes to fully mix the rhizosphere soil and release the protozoa in the soil. After the centrifuge tube was removed from the shaker and allowed to stand for 10 minutes, the upper layer of liquid was added to a 96-well plate and inactivated Escherichia coli (OD=0.04) was added as food, and then cultured at 20 ° C for 2 days in dark conditions. The growth of protozoa was examined under an inverted microscope at 100×, 200× and 400×, and gradient dilutions were performed and cultured at 20 ° C for 2 days. Finally, a single protozoan cell was picked up with a capillary tube and placed in a new 96-well plate to obtain a pure culture of the protozoan. In the NMAS culture medium, the protozoan NJAU-Z1 was active, had a single flagellum, and swam rapidly ( Figure 1 Left); its dormant cyst is round, static, and without flagella ( Figure 1 Its reproductive cyst is round, rotating and trembling, without flagella ( Figure 1 right).

[0026] Example 2 Identification of protozoan NJAU-Z1:

[0027] The isolated protozoa were identified by molecular biology methods. PCR reaction system (50 μL system): 2 μL of each primer, 2× Mix 25 μL, add ddH2O to 50 μL. The primers are universal primers P-FLA-F: SEQ ID NO.1 / P-FLA-R: SEQ ID NO.2. Reaction procedure: 94℃ pre-denaturation for 3min, 94℃ denaturation for 55s, 50℃ annealing for 50s, 72℃ extension for 1min, 72℃ extension for 10min, and 16℃ storage after 35 cycles. The PCR product was detected and recovered on 1.5% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The 18S rDNA sequencing result is: SEQ ID NO.3.

[0028] The obtained 18S rDNA gene sequences were compared with the NT (Nucleotide Sequence Database) database on the NCBI website using BLAST software, and the phylogenetic analysis of the isolated protozoa was performed using MEGA software. Cercomonas directa The similarity was the highest, reaching 99.18%, so the isolated protozoan NJAU-Z1 was identified as Cercomonas directa . Figure 2 This is a phylogenetic tree constructed based on 18S rDNA gene sequences.

[0029] The strain was deposited in the China Center for Type Culture Collection on January 20, 2024, with the deposit number: CCTCCNO:C202443.

[0030] Example 3 Field trial of Trichomonas cerci NJAU-Z1 in increasing wheat yield

[0031] The experiment set up two treatments: (1) blank control group CK; (2) treatment group inoculated with Trichomonas cercariae NJAU-Z1. Each treatment had 4 replicates.

[0032] The wheat used for the test was Nongmai 88, and the protozoa used for the test were Trichomonas cerci NJAU-Z1 in Example 2, with the deposit number: CCTCCNO: C202443.

[0033] Preparation of Trichomonas cervicornis NJAU-Z1: Inoculate Trichomonas cervicornis NJAU-Z1 into liquid NMAS medium, add inactivated Escherichia coli (OD=0.04), place in a constant temperature incubator at 20°C, and culture. After 48 hours, take 100 μL of the culture solution and place it under an inverted microscope for counting.

[0034] The field was located in Taizhou Hantu Farm. Each treatment contained four 0.5*0.5m stainless steel frames. The inoculation concentration in each frame was 6×10 5 The vaccine was administered three times, in November, December and January.

[0035] Wheat is harvested from the boxes in June, and the yield in the boxes is calculated per mu. Figure 3 The average per-acre yield of the Trichomonas CK treatment group was 1013.867 catties, and the average per-acre yield of the NJAU-Z1 treatment group was 1127.71 catties, an increase of 11.23% over the control treatment. This shows that the protozoan NJAU-Z1 can significantly increase wheat yield. Figure 4 The results show that the growth of Trichomonas cercariae NJAU-Z1 ( Figure 4 Right) compared with the blank control ( Figure 4 Left) can promote the growth of seedlings.

[0036] Example 4 Field trial of protozoan NJAU-Z1 to improve soil organic matter content

[0037] The experiment set up two treatments: (1) blank control group CK; (2) treatment group inoculated with protozoa NJAU-Z1, each treatment had 4 replicates.

[0038] The protozoa used for the test were Trichomonas cerci NJAU-Z1 of Example 2, with a deposit number of CCTCC NO: C202443.

[0039] Protozoa preparation: Protozoa were inoculated into 25 cm 2 Add 50 ml of liquid NMAS medium and 5 ml of inactivated E. coli (OD = 0.04) to the cell culture bottle. Place it in a constant temperature incubator at 20°C and culture it statically. After 48 hours, take out 100 μL of the culture solution and place it under an inverted microscope for counting.

[0040] The field was located in Taizhou Hantu Farm. Each treatment included four 0.5*0.5 m stainless steel frames to isolate the surrounding soil. The inoculation concentration in each frame was 6×10 5 The protozoan NJAU-Z1 was administered in three doses, in November, December, and January.

[0041] In June, part of the 0~20cm surface soil was dug from the stainless steel frame to measure the soil organic matter content. Figure 4 As shown, the average organic matter content of the CK treatment group was 44.275 g / kg, and the average organic matter content of the group treated with Trichomonas cerci NJAU-Z1 was 48.35 g / kg, which was 9.2% higher than that of the control group. This shows that the application of protozoa NJAU-Z1 can significantly increase the content of soil organic matter. Figure 6 The results show that there is a certain positive correlation between wheat yield per mu and the average organic matter content in the soil.

Claims

1. Protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas direct) NJAU-Z1) in increasing wheat yield and / or soil organic matter content, characterized in that , the protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas) direct NJAU-Z1) was deposited in China Center for Type Culture Collection on January 20, 2024 at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C202443.

2. Protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas direct) NJAU-Z1) in the preparation of products for increasing wheat yield and / or soil organic matter content, characterized in that: The protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas direct) NJAU-Z1) was deposited in China Center for Type Culture Collection on January 20, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202443.

3. Protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas direct) NJAU-Z1) in improving wheat yield and / or soil organic matter content, characterized in that: The protozoan Trichomonas cercopithecidae NJAU-Z1 (Cercomonas direct) NJAU-Z1) was deposited in the China Center for Type Culture Collection on January 20, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202443. The preparation method of the culture comprises inoculating the Trichomonas NJAU-Z1 into NMAS culture medium, adding inactivated Escherichia coli as food, placing it in an incubator at 20°C, and standing for 48 hours; wherein the preparation method of the NMAS culture medium is 0.12 g / L of sodium chloride, 0.004 g / L of magnesium sulfate heptahydrate, 0.006 g / L of calcium chloride hexahydrate, 0.142 g / L of sodium phosphate, and 0.136 g / L of potassium phosphate.

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