CN102732435B - Masson pine rhizosphere solubilizing phosphorus fungus aspergillus awamori and application thereof - Google Patents

Abstract

The invention discloses a rhizosphere phosphorus-solubilizing fungus Aspergillus awamori of Pinus massoniana and its application, which is classified as Aspergillus awamori ( Aspergillus awamori ) JP-NJ1, and has been preserved in the China Center for Typical Culture Collection, with the preservation number CCTCCNO: M 2012166 and the preservation date As of: May 14, 2012. The aspergillus awamori JP-NJ1 of the present invention has a strong dissolving ability to insoluble phosphate calcium phosphate; it is confirmed by a greenhouse pot test that the JP-NJ1 bacterial agent can obviously promote the growth of masson pine seedlings. Therefore, the Aspergillus awamori JP-NJ1 disclosed in the present invention provides an excellent strain resource for the development of the growth-promoting bacterial fertilizer of Pinus massoniana, and has a good prospect for application and development.

Description

A kind of rhizosphere phosphorus-solubilizing fungus Aspergillus awamori of Pinus massoniana and its application

technical field

The invention belongs to the technical field of microbes, and in particular relates to Aspergillus awamori, a phosphorus-dissolving fungus in the rhizosphere of Pinus massoniana and an application thereof.

Background technique

Phosphate-solubilizing microorganisms (PSMs) are a special functional group of microorganisms that can convert insoluble phosphorus into soluble phosphorus that plants can absorb and utilize in soil, including phosphorus-solubilizing fungi, phosphorus-solubilizing bacteria, and phosphorus-solubilizing actinomycetes. wait. Although the number and types of Solubilizing phosphorus fungus are less than those of Phosphorus-dissolving bacteria, studies have shown that the Phosphorus-dissolving ability of Phosphorus-dissolving fungi is generally stronger than that of bacteria, sometimes even dozens of times that of bacteria, and their genetic traits are more Stablize. So far, the phosphorus-solubilizing microorganisms found are mainly concentrated in crops and soil, and a few reports come from ecosystems such as forest land or pasture. And the current research on phosphorus-solubilizing microorganisms is mainly focused on phosphorus-solubilizing bacteria, and the distribution, phosphorus-solubilizing ability and mechanism of phosphorus-solubilizing bacteria have been relatively clear. Phosphorus-solubilizing fungi have been less studied.

Masson pine, as a pioneer tree species in forest succession, is one of the most representative forest types in southern my country, and it is also the largest degraded type in the forest system structure in southern my country. The main cause of this phenomenon is soil fertility decline. In the process of masson pine woodland decline, insufficient phosphorus supply is a prominent and common problem. In forestry, phosphorus has a greater impact on the quality of seedlings, especially coniferous species. Phosphorus is conducive to the smooth transfer of plants from vegetative growth to reproductive growth, promotes the formation of reproductive organs, and can flower and bear fruit early. Phosphorus effectively promotes the development of the root system, enhances the absorption capacity of the roots of seedlings, accelerates the growth of seedlings, and improves the stress resistance and disease resistance of seedlings to a certain extent. Phosphorus-solubilizing microorganisms can not only improve the phosphorus nutrition of plants, but also promote the metabolic activities of beneficial microorganisms in the soil, significantly improve the nutrition of plant roots, and achieve the effect of increasing yield. Using microorganisms to improve the utilization rate of soil solid phosphorus is undoubtedly an effective method. At present, how to develop and utilize the phosphorus fixed by the soil by using phosphorus-solubilizing microorganisms is an urgent problem to be solved in my country's agricultural and forestry production, and it is also one of the hot spots in the field of soil fertilizer and plant nutrition research at home and abroad.

Contents of the invention

Purpose of the invention: Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide the rhizosphere phosphorus-dissolving fungus Aspergillus awamori JP-NJ1 of Pinus massoniana, which has strong phosphorus-dissolving ability. Another object of the present invention is to provide an application of Aspergillus awamori JP-NJ1, a phosphorus-solubilizing fungus in the rhizosphere of Pinus massoniana, in promoting the growth of Pinus massoniana.

Technical solution: In order to realize the above-mentioned purpose of the invention, the technical solution adopted in the present invention is as follows:

Aspergillus awamori, a rhizosphere phosphorus-solubilizing fungus of Pinus massoniana, is classified as Aspergillus awamori (Aspergillus awamori) JP-NJ1, and has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2012166, and the preservation date is: May 14, 2012 date, and the preservation address is: Wuhan University, Wuhan, China.

Aspergillus awamori JP-NJ1 was screened from the rhizosphere soil of 30-40-year-old Pinus massoniana in Nanjing Forestry University.

Biological characteristics of JP-NJ1: As shown in Figure 1, the colony grows rapidly on the PDA medium, and the diameter of the colony is about 65 mm after 7 days of culture at 25 ° C. The colony is black, with radial grooves and velvety texture; the opposite side of the colony Slightly pale yellow in color. The conidiophores are mostly radial when they are young, with a diameter of 70-150um, and loose when they are old, with a diameter of about 300-500um; conidia are produced in large numbers, and they are densest in the middle of the colony and appear dark brown. Conidiophores occur in the stroma, about 600-800um×10um, pale brown when old, and fertile on the entire surface; the spore-producing structure is double-layered, the conidia are spherical or nearly spherical, 3.5-5um in diameter, and the wall is smooth.

As shown in Figure 2, the strain grows slowly on NBRIP medium, cultured at 25°C for 7 days, with a diameter of 35-40mm, a phosphorus-dissolving circle diameter of 46-65mm, a velvety texture, and sparse hyphae. The conidia were less produced, and the colonies were densest in the middle and appeared black. The opposite side of the colony is colorless, and the phosphorus-dissolving circle is large and transparent.

The 18SrDNA ITS sequence of the JP-NJ1 strain is shown in SEQ ID NO.1. Sequences were BLAST aligned with those in the GenBank database. The results showed that the JP-NJ1 strain was 99% similar to Aspergillus awamori. Combined with morphological characteristics and 18SrDNA ITS sequence analysis, it was identified as Aspergillus awamori.

The above-mentioned Aspergillus awamori JP-NJ1 has a strong ability to solubilize tricalcium phosphate, and has a good application in promoting the growth of Pinus massoniana seedlings. The pot plant inoculation test of JP-NJ1 bacterial agent showed that the bacterial agent can obviously promote the growth of Pinus massoniana seedlings.

Beneficial effects: the advantages of Aspergillus awamori JP-NJ1 of the present invention include: it has a strong dissolving ability for the insoluble inorganic phosphate tricalcium phosphate; it has been confirmed by pot experiments that inoculation of pine seedlings with bacteria agent can significantly promote the growth of pine pine . Therefore, the Aspergillus awamori JP-NJ1 of the present invention can provide excellent strain resources for the development of microbial fertilizers, and has good application and development prospects.

Description of drawings

Fig. 1 is the bacterium colony characteristic of Aspergillus awamori JP-NJ1 bacterial strain on PDA medium;

Figure 2 is the phosphorus-dissolving circle of Aspergillus awamori JP-NJ1 strain on NBRIP medium; wherein, the left picture is the back side, and the right picture is the front side;

Fig. 3 is the result figure of the dynamic change of phosphorus solubilizing ability and pH value when Aspergillus awamori JP-NJ1 bacterial strain is cultivated in shake flask;

Fig. 4 is the impact result figure of Aspergillus awamori JP-NJ1 strain on the growth of Pinus massoniana seedlings;

Fig. 5 is a graph showing the effect of the bacterial agent made by Aspergillus awamori JP-NJ1 strain on the growth of Pinus massoniana.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

In the following example, the formula of the PDA medium is: 20 g of potatoes, 20 g of sucrose, 18 g of agar, and 1000 mL of distilled water. The formula of phosphate growth medium (NBRIP) is: glucose 10g, Ca ₃ (PO ₄ ) ₂ 5g, MgCl ₂ .6H ₂ O 5g, KCl 0.2g, MgSO ₄ .7H ₂ O 0.25g, (NH ₄ ) ₂ SO ₄ 0.1g, distilled water 1000mL, pH7.0.

Example 1

The rhizosphere phosphorus-solubilizing fungus Aspergillus awamori (Aspergillus awamori) JP-NJ1 was screened from the rhizosphere soil of 30-40-year-old Pinus massoniana in Nanjing Forestry University. As shown in Figure 1, the colony grows rapidly on the PDA medium, and the diameter of the colony is about 65mm after 7 days of culture at 25°C. The colony is black, with radial grooves and velvety texture; The conidiophores are mostly radial when they are young, with a diameter of 70-150um, and loose when they are old, with a diameter of about 300-500um; conidia are produced in large numbers, and they are densest in the middle of the colony and appear dark brown. Conidiophores occur in the matrix, about 600-800um×10um, light brown when old, and fertile on the entire surface; the spore-producing structure is double-layered, the conidia are spherical or nearly spherical, 3.5-5um in diameter, and the wall is smooth.

As shown in Figure 2, the strain grows slowly on NBRIP medium, cultured at 25°C for 7 days, with a diameter of 35-40 mm, a phosphorus-dissolving circle diameter of 46-65 mm, a velvety texture, and sparse hyphae. Spores were less produced, and the center of the colony was densest and black. The opposite side of the colony is colorless, and the phosphorus-dissolving circle is large and transparent.

Example 2

Inoculate Aspergillus awamori JP-NJ1 on the NBRIP plate, culture at 25°C, observe whether there is still a lysing zone after 7-10 days, measure the diameter of the colony (D) and lysing zone (d), and calculate the diameter of the lysing zone and the diameter of the colony The ratio (d/D) of the phosphate-solubilizing fungi can be preliminarily estimated. Generally, the larger the ratio, the stronger the phosphorus-solubilizing ability of the fungus. According to Table 1, those whose phosphorus dissolving ability is "++" are considered to have stronger phosphorus dissolving ability. The d/D value of Aspergillus awamori JP-NJ1 was measured according to the above method, and the measured ratio was 1.44, indicating that the JP-NJ1 strain has a strong phosphorus-solubilizing ability.

Table 1 Grading standard of phosphorus-solubilizing ability of phosphorus-solubilizing fungi

d/D Phosphorus dissolving ability d/D>1 +++ 0.5<d/D<1 ++ d/D<0.5 +

Example 3

Inoculate the Aspergillus awamori JP-NJ1 strain on the PDA plate, cultivate it for 10 days, and after activation, take the bacterial block (60mm in diameter) from the plate and inoculate it into a 100mL Erlenmeyer flask containing 50mL NBRIP liquid medium. The NBRIP medium is the control (CK), and each bottle is inoculated with 6 bacterial blocks of the same volume, and each strain to be tested is set up in triplicate, 25°C, 200r min ^-1 shaking culture, after 7 days, the fermentation broth is centrifuged 10min (4°C, 10000r·min ^-1 ), using the molybdenum-antimony anti-colorimetric method, that is, take 100uL of the supernatant obtained by the above centrifugation and add it to a 50mL volumetric flask, then add about 25mL of deionized water, and then add 1 ~2 drops of 2,4-dinitrophenol indicator, add 5mL of molybdenum antimony anti-chromogenic agent, add deionized water to volume, mix well, let stand for 0.5h, measure OD value. And calculate the available phosphorus content and phosphorus dissolving rate in the supernatant. The results are shown in Table 2. Among them, phosphorus solubilization rate (%) = (inoculation soluble phosphorus content - control soluble phosphorus content) ÷ the amount of added tricalcium phosphate × 100.

Table 2 Phosphorus-solubilizing ability of the phosphate-solubilizing fungus Aspergillus awamori JP-NJ1

Inoculation strain Available phosphorus content (mg·L ^-1 ) Phosphorus dissolving rate (%) JP-NJ1 1051.69±50.13a 19.3 CK 88.70±22.81d -

Note: P<0.05.

Example 4

Activate the JP-NJ1 strain on NBRIP first, and then activate it twice on PDA. After the JP-NJ1 strain grows on the PDA for 7 days, use a sterilized puncher to punch a 6mm diameter bacterial block on the medium. Inoculate in a 100mL Erlenmeyer flask containing 50mL of NBRIP medium, culture with shaking at 25°C and 200r.min ^-1 , do 7 repetitions for each strain, and use blank medium as a control. Every 24 hours, take the fermentation broth, centrifuge the fermentation broth for 10min (4°C, 10000r.min ^-1 ), take 100uL of the centrifuged supernatant and add it to a 50mL volumetric flask, then add about 25mL of deionized water, and then Add 1 to 2 drops of 2,4-dinitrophenol indicator, add 5ml of molybdenum antimony anti-chromogenic agent, add deionized water to make up volume, mix well, let it stand for half an hour, and then measure the OD value. Then the phosphorus solubilizing ability of the strain was converted according to the phosphorus standard curve. Every 24 hours, the fermentation broth was taken, and the pH value of the fermentation broth was directly measured with a pH meter (Basic ph Meter PB-10).

The results are shown in Figure 3. After 8 days of dynamic measurement of the phosphorus-dissolving ability of the JP-NJ1 strain, it was found that the phosphorus-dissolving capacity of the strain gradually increased from the 2nd to the 6th day, and reached the maximum value on the 6th day, which was 1363.57mg/ L, and then the amount of dissolved phosphorus gradually decreased. During the dynamic fermentation process, it can also be clearly seen that the pH value of the fermentation broth of the JP-NJ1 strain has an obvious downward trend with the increase in the amount of dissolved phosphorus, and reached the lowest value of 2.36 on the 7th day, and then rose slightly. However, it remains at a low pH level.

Example 5

First activate the JP-NJ1 strain on NBRIP, and then activate it twice on PDA, use a puncher to punch out a 6mm diameter bacterial block on the medium, inoculate it in a 100mL Erlenmeyer flask containing 50mL of PD medium, 25 ℃, 200r.min ^-1 shaking culture 7d. The fermentation broth (4°C, 6000r.min ^-1 ) was centrifuged for 5min, the mycelium was crushed with a magnetic stirrer, and the bacterial suspension was adjusted with sterile physiological saline (7～8×10 ⁸ cfu.mL ^-1 ). Bacteria. The supernatant after centrifugation was collected into a sterile Erlenmeyer flask for use.

The inoculation test was divided into four treatments, and 15mL of the corresponding substance was applied to each treatment, A: bacterial suspension, B: supernatant obtained after centrifugation of the fermentation broth, C: blank medium (PD), D: sterile Normal saline (CK). There were 20 replicates for each treatment. After inoculation, the seedlings were placed in the greenhouse (20°C) for unified management and watered at the right time.

After the masson pine seedlings to be inoculated were grown for 150 days, the plant height, ground diameter, plant fresh weight and root weight were measured.

The results are shown in Figure 4 and Table 3, both the bacterial suspension and the fermentation supernatant of the JP-NJ1 strain have obvious growth-promoting effects on Pine massoniana seedlings (p<0.05). The bacterial agent made by JP-NJ1 strain has a particularly significant effect on promoting the growth of Pinus massoniana seedlings. Compared with CK, the seedling height and ground diameter increased by 19.36% and 27.83%, and the fresh weight and root weight increased by 104.13% and 153.12% respectively compared with CK. .

Table 3 Effect of Phosphorus Solubilizing Fungus Aspergillus awamori JP-NJ1 on the Growth of Pinus massoniana

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Claims (3)

1. Pinus massoniana Lamb rhizosphere phosphorus decomposing fungi Aspergillus awamori, its classification called after Aspergillus awamori ( Aspergillus awamori) JP-NJ1, be preserved in Chinese typical culture collection center, deposit number CCTCC NO:M 2012166, preservation date is: on May 14th, 2012, the preservation address is: Chinese Wuhan Wuhan University.

2. the application of the described Pinus massoniana Lamb rhizosphere of claim 1 phosphorus decomposing fungi Aspergillus awamori JP-NJ1 in promoting the growth of Pinus massoniana Lamb seedling.

3. the application of the described Pinus massoniana Lamb rhizosphere of claim 1 phosphorus decomposing fungi Aspergillus awamori JP-NJ1 in dissolving insoluble inorganic phosphate calcium phosphate.

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