AU2020103320A4

AU2020103320A4 - Microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata and preparation method and use thereof

Info

Publication number: AU2020103320A4
Application number: AU2020103320A
Authority: AU
Inventors: Qu DU; Ting He; Songzhu Hu; Shan Liu; Wei Liu; Shuping Tu; Qiong Wang; Jiahai WU; Jinlian Zhang; Weiwei Zhang; Xiaomin Zhao
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2019-12-09
Filing date: 2020-11-09
Publication date: 2021-01-28
Anticipated expiration: 2028-11-09
Also published as: CN110938549B; CN110938549A

Abstract

The disclosure belongs to the technical field of microbial preparations, and provides a microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata and preparation method and use thereof, where the microbial agent includes Claroideoglomus claroideum (Cc) and Septoglomus viscosum (Sv). The microbial agent of the disclosure can form a symbiotic system of arbuscular mycorrhiza (AM), so as to significantly promote growth of the container-grown seedlings of Euscaphis konishii Hayata, improve phosphorus nutrients, and enhance external stress resistance of the container-grown seedlings of Euscaphis konishii Hayata. The microbial agent of the disclosure in use can significantly increase biomass of the container-grown seedlings of Euscaphis konishii Hayata, where leaf number, plant height and ground diameter of the container-grown seedlings are all obviously increased.

Description

MICROBIAL AGENT FOR PROMOTING GROWTH OF CONTAINER-GROWN SEEDLINGS OF EUSCAPHIS KONISHII HAYATA AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

[001] The disclosure relates to the technical field of microbial preparations, in particular to a microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata and preparation method and use thereof.

BACKGROUND

[002] Euscaphis konishii Hayata is an evergreen small tree of the genus Euscophis in the Staphyleaceae family. It is an excellent native gardening tree species with ornamental fruits. Seedling resources of Euscaphis konishii Hayata have a promising broad market and are important for adjusting proportions of individual greening tree species and enriching precious broadleaftree resources.

[003] Yearly increment of seedlings of Euscaphis konishii Hayata is not large, and growth characteristics of a shallow root system make its seedlings suitable for breeding by container-grown seedling technology. However, there are few researches on efficient planting and seedling cultivation of container-grown seedlings of Euscaphis konishii Hayata. There is a lack of a microbial agent which can promote growth of the container-grown seedlings of Euscaphis konishii Hayata and an efficient cultivation method.

SUMMARY

[004] An objective of the disclosure is to provide a microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata and preparation method and use thereof. The microbial agent of the disclosure can significantly promote growth of the container-grown seedlings of Euscaphis konishii Hayata, improve phosphorus nutrients, and enhance external stress resistance of the container-grown seedlings of Euscaphis konishii Hayata.

[005] To achieve the above objective, the disclosure provides the following technical solutions:

[006] The disclosure provides a microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata, including Claroideoglomus claroideum (abbreviated as Cc) and Septoglomus viscosum (abbreviated as Sv), where a ratio of a number of Cc spores to that of Sv spores is (0.5-1.5):(0.5-1.5).

[007] Preferably, the microbial agent has 300-480 effective spores /60 g.

[008] Preferably, the Cc includes a Cc strain Cc-01 deposited with an accession number of

CGMCC No. 18593; and the Sv includes an Sv strain Sv-01 deposited with an accession number of CGMCC No. 18594.

[009] The disclosure also provides a method for preparing the microbial agent according to the above solution, including the following step: mixing the Cc and the Sv to obtain the microbial agent.

[010] The disclosure also provides use of the microbial agent according to the above solution in promoting growth of container-grown seedlings of Euscaphis konishii Hayata, including the following steps:

[011] filling a container with a substrate and the microbial agent in sequence from bottom to top, planting seedlings of Euscaphis konishii Hayata, overlaying with an additional amount of the substrate, watering, conducting singling of the seedlings when growth of the seedlings of Euscaphis konishii Hayata is stable, and culturing.

[012] Preferably, the substrate includes the following raw materials in parts by volume: 2.5-3.5 parts of garden soil, 0.5-1.5 parts of peat, and 0.5-1.5 parts of river sand.

[013] Preferably, the container includes a plastic flowerpot with an upper diameter of 8-10 cm, a lower diameter of 5-7 cm, and a height of 7-9 cm.

[014] Preferably, 110-130 g of the substrate, 50-70 g of the microbial agent, and 50-70 g of the substrate for overlaying are filled in each container.

[015] Preferably, there are 1-2 seedlings left in each container after the singling of the seedlings.

[016] Preferably, the culturing is carried out at 24-28 °C with optical density of 700-900 mmol-m-2-s- 1, lighting duration of 15-17 h/d, and humidity of 60%-70%.

[017] Beneficial effects of the disclosure: The disclosure provides a microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata, including Cc and Sv. The microbial agent of the disclosure can form a symbiotic system of arbuscular mycorrhiza (AM), so as to significantly promote growth of the container-grown seedlings of Euscaphis konishii Hayata, improve phosphorus nutrients, and enhance external stress resistance of the container-grown seedlings of Euscaphis konishii Hayata. The microbial agent of the disclosure in use can significantly increase biomass of the container-grown seedlings of Euscaphis konishii Hayata, where leaf number, plant height and ground diameter of the container-grown seedlings are all obviously increased.

[018] Description of Biological Deposit

[019] The Cc-01 was deposited with an accession number of CGMCC No. 18593 on October 14, 2019 in the China General Microbiological Culture Collection Center located at No. 3, NO.1 West Beichen Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences.

[020] The Sv-01 was deposited with an accession number of CGMCC No. 18594 on October 14, 2019 in the China General Microbiological Culture Collection Center located at No. 3, NO.1 West Beichen Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences.

DETAILED DESCRIPTION

[021] The disclosure provides a microbial agent for promoting growth of container-grown seedlings of Euscaphis konishii Hayata, including Cc and Sv, where a ratio of a number of Cc spores to that of Sv spores is (0.5-1.5):(0.5-1.5), preferably 1:1. The microbial agent has preferably 300-480 effective spores /60 g, more preferably 350-400 effective spores /60 g. The Cc preferably includes a Cc strain Cc-01 deposited with an accession number of CGMCC No. 18593; and the Sv includes an Sv strain Sv-01 deposited with an accession number of CGMCC No. 18594.

[022] The microbial agent of the disclosure can form a symbiotic system of AM, so as to significantly promote growth of the container-grown seedlings of Euscaphis konishii Hayata, improve phosphorus nutrients, and enhance external stress resistance of the container-grown seedlings of Euscaphiskonishii Hayata.

[023] The disclosure also provides a method for preparing the microbial agent according to the above solution as follows: inoculating the Cc strain and the Sv strain on a sterilized medium for multiplication, planting corn and/or shamrock to ensure full multiplication of the Cc strain and the Sv strain between the medium for multiplication and plant roots, and finally harvesting the microbial agent of the Cc strain and the Sv strain including dry soil of the medium for multiplication, extraradical mycelium, intraradical mycelium, and infected root segments, where the microbial agent includes preferably 300-480 effective spores/60 g.

[024] The disclosure has no special requirements for the medium for multiplication, and a medium for multiplication conventional in the field can be used. During specific implementation of the disclosure, the medium for multiplication includes river sand and garden soil in a mass ratio of 1:1. The river sand is common local river sand for construction, and a source of the garden soil is local red soil without application of fertilizers. The medium for multiplication is preferably sterilized. The inoculating the Cc strain and the Sv strain on a sterilized medium for multiplication is carried out with an inoculation amount conventional in the art. During specific implementation of the disclosure, based on a weight ratio, the inoculation amount is preferably 3%-5% of the medium for multiplication.

[025] The disclosure also provides use of the microbial agent according to the above solution in promoting growth of the container-grown seedlings of Euscaphis konishii Hayata, including the following steps:

[026] filling a container with a substrate and the microbial agent in sequence from bottom to top, planting the seedlings of Euscaphis konishii Hayata, overlaying with an additional amount of the substrate, watering, and conducting singling of the seedlings when growth of the seedlings of Euscaphis konishii Hayata is stable, and culturing.

[027] In the disclosure, the substrate preferably includes the following raw materials in parts by volume: 2.5-3.5 parts of garden soil, 0.5-1.5 parts of peat, and 0.5-1.5 parts of river sand (a source of the garden soil is local red soil without application of fertilizers; a source of the peat is imported Denmark Pinstrup peat 0-10 mm; and the river sand is common local river sand for construction). More preferably, the substrate includes the following raw materials in parts by volume: 3 parts of garden soil, 1 part of peat, and 1 part of river sand. The substrate is preferably screened through a 2 mm sieve, and has a pH value of preferably 5.5-6.5 and more preferably 5.99, an organic matter content of preferably 35-45 g-kg- 1 and more preferably 40.46 g-kg-1 , an organic carbon content of preferably 65-75 g-kg- 1 and more preferably 69.75 g-kg-1 , a total phosphorus content of preferably 0.5-0.9 g-kg- 1 and more preferably 0.75 g-kg-1 , a total nitrogen content of preferably 1.7-2.1 g-kg-1 and more preferably 1.90 g-kg-1 , an ammonium nitrogen content of preferably 13-18 mg-kg-1 and more preferably 15.07 mg-kg-1 , and a nitrate nitrogen content of preferably 4-4.5 mg-kg-1 and more preferably 4.21 mg-kg-1 .

[028] In the disclosure, the substrate is preferably prepared by the following method: mixing the garden soil, the peat and the river sand, passing through a 2 mm sieve, and sterilizing to obtain the substrate. The disclosure has no special limitations on a mixing method as long as uniform mixing can be achieved. The sterilizing is carried out by preferably high-temperature high-pressure steam sterilization at preferably 115-130°C and more preferably 121°C at preferably 0.1 MPa for preferably 0.3-0.8 h and more preferably 0.5 h.

[029] In the disclosure, the container preferably includes a plastic flowerpot with an upper diameter of preferably 8-10 cm and more preferably 9 cm, a lower diameter of preferably 5-7 cm and more preferably 6 cm, and a height of preferably 7-9 cm and more preferably 8 cm.

[030] In the disclosure, based on the container (plastic flowerpot) having an upper diameter, a lower diameter and a height of 9 cm, 6 cm and 8 cm respectively, each container is filled with preferably 110-130 g and more preferably 120 g of the substrate, preferably 50-70 g and more preferably 60 g of the microbial agent, and then 50-70 g and more preferably 60 g of the substrate for overlaying. The planting the seedlings of Euscaphis konishii Hayata is carried out with preferably 2-4 and more preferably 3 seedlings. The watering is carried out with preferably 150-250 mL and more preferably 200 mL water per container. The singling of the seedlings is carried out to maintain preferably 1-2 seedlings in each container.

[031] In the disclosure, the seedlings of Euscaphis konishii Hayata are preferably prepared by the following method: disinfecting seeds of Euscaphis konishii Hayata, washing, soaking in water, accelerating germination until the seeds crack to show white contents, placing in river sand, and cultivating to obtain the seedlings of Euscaphis konishii Hayata. The seeds of Euscaphis konishii Hayata are preferably full and collected in a current year. The disinfecting is carried out by preferably soaking the seeds of Euscaphis konishii Hayata in a disinfectant for preferably 10 min. The disinfectant is preferably a H 2 0 2 aqueous solution having preferably 10% H 2 0 2 by volume percentage. The washing is carried out with preferably sterile water to remove residual H2 0 2 solution. The soaking in water is carried out at an initial temperature of preferably 65-68°C and more preferably 66-67°C which drops by preferably natural cooling to a termination temperature of preferably 20-30°C and more preferably 25°C. The soaking in water is carried out for preferably 5 d with water changed once a day. The accelerating germination is carried out at preferably 25°C. The river sand is preferably sterilized at preferably 121C for preferably 30 min. The culturing is carried out at preferably 24-28°C and more preferably 26°C with humidity of preferably 60%-70% and more preferably 65% for a time period based on emergence of the seedlings.

[032] In the disclosure, the culturing is carried out with water holding capacity (mass ratio) of soil of preferably 18%-25% and more preferably 20%, optical density of preferably 700-900 mmol-m-2-s-I and more preferably 800 mmol-m-2 s-1, and lighting duration of preferably 15-17 h/d and more preferably 16 h/d. The culturing is carried out at preferably 24-28°C and more preferably 26°C with humidity of preferably 60%-70% and more preferably 65%.

[033] The technical solution provided by the disclosure will be described in detail in connection with the following examples, but they should not be construed as limiting the claimed scope of the disclosure.

[034] Example 1

1. Garden soil, peat, and river sand in a volume ratio of 3:1:1 were selected, passed through a 2 mm sieve, mixed uniformly, steam sterilized at a high temperature (121C) and a high pressure

(0.1 MPa) for 0.5 h, and cooled naturally to obtain a substrate. The substrate had basic physical and chemical properties as follows: pH 5.99, organic matter content: 40.458 g-kg- , organic carbon content: 69.750 g-kg- 1, total phosphorus content: 0.754 g-kg- 1, total nitrogen content: 1.903 g-kg-1 , ammonium nitrogen content: 15.068 mg-kg-1 , and nitrate nitrogen content: 4.209 mg-kg-1 .

2. Full seeds of Euscaphis konishii Hayata collected that year were selected, soaked with a 10% H2 0 2 solution for 10 min to achieve disinfection, washed with sterile water to remove residual H2 0 2 solution and soaked in warm water cooled to 65°C. After naturally cooling to room temperature (25°C), soaking was carried out at room temperature with sterile water for continuous 5 d (the water was changed once a day). Germination acceleration was carried out in a °C incubator. When the seeds cracked to show white contents, they were cultivated in sterile river sand that had been sterilized at 121C for 30 min and cooled. A temperature was controlled at 26°C with humidity controlled at 65%. The seeds were cultivated to emergence for later use.

3. A total of 20 plastic flower pots were used in the experiment where the pots had an upper diameter, a lower diameter, and a height of 9 cm, 6 cm and 8 cm respectively. Each pot was filled with 120 g of the substrate, and then inoculated with 60 g of microbial agent. The microbial agent contained 300 spores of Cc-01 (with an accession number of CGMCC No. 18593) and 300 spores of Sv-01 (with an accession number of CGMCC No. 18594). 3 seedlings of Euscaphis konishii Hayata were planted. 60 g of substrate was used for overlaying. Watering was carried out with 200 mL water per pot, and singling of seedlings was carried out with 1 seedling left when seedling growth was stable.

4. Watering was carried out in determined quantity every week. Water holding capacity of soil was maintained at 20% (mass ratio), and optical density was maintained at 800 mmol-m-2-s-. Light supplement was implemented with an agricultural sodium lamp. A light duration was 16 h a day. A temperature was 26°C with humidity of 65%. Weighing was carried out every 3 d. Growth of aboveground part of the Euscaphis konishii Hayata was recorded (compound leaf number, plant height, and ground diameter), and biomass of the Euscaphis konishii Hayata was calculated (root dry weight, stem dry weight, leaf dry weight, total dry weight and root/shoot ratio). Results were shown in Table 1 and Table 2.

[035] Comparative Example 1

[036] Settings were the same as those in Example 1, except that no microbial agent was inoculated. Results were shown in Table 1 and Table 2.

[037] Comparative Example 2

[038] Settings were the same as those in Example 1, except that the microbial agent was replaced by a microbial agent including 600 spores of Claroideoglomus etunicatum (abbreviated as Ce, provided by "Bank of Arbuscular Mycorrhizal Fungi in China" in Beijing Academy of Agriculture and Forestry Sciences, with no.1511C0001BGCAM0053 inNational Infrastructure of Microbial Resources). Results were shown in Table 1 and Table 2.

[039] Comparative Example 3

[040] Settings were the same as those in Example 1, except that the microbial agent was replaced by a microbial agent including 600 spores of Cc-01. Results were shown in Table 1 and Table 2.

[041] Comparative Example 4

[042] Settings were the same as those in Example 1, except that the microbial agent was replaced by a microbial agent including 600 spores of Sv-01. Results were shown in Table 1 and Table 2.

[043] Comparative Example 5

[044] Settings were the same as those in Example 1, except that the microbial agent was replaced by a microbial agent including 300 spores of Ce (provided by "Bank of Arbuscular Mycorrhizal Fungi in China" in Beijing Academy of Agriculture and Forestry Sciences, with no.1511C0001BGCAM0053 in National Infrastructure of Microbial Resources) and 300 spores of Cc-01. Results were shown in Table 1 and Table 2.

[045] Comparative Example 6

[046] Settings were the same as those in Example 1, except that the microbial agent was replaced by a microbial agent including 200 spores of Ce (provided by "Bank of Arbuscular Mycorrhizal Fungi in China" in Beijing Academy of Agriculture and Forestry Sciences, with no.1511C0001BGCAM0053 in National Infrastructure of Microbial Resources), 200 spores of Sv-01 and 200 spores of Cc-01. Results were shown in Table 1 and Table 2.

Table 1 Effect of different treatments on growth of aboveground part of Euscaphis konishii Hayata

Compound leaf Ground diameter Treatment Inoculation Plant height (cm) number (mm)

Example 1 Cc and Sv 18.71+ 0.79 a 24.70 + 1.39 a 4.74 + 0.09 ab

Control (CK), Comparative inoculated with no AM 4.70 + 0.30 c 6.30 + 0.24 d 2.03 + 0.04 c Example 1 fungi Comparative Ce only 17.50 + 0.61 a 22.11 + 0.90 abc 4.95 + 0.09 a Example 2

Comparative Cc only 15.00 + 0.64 b 19.88 + 0.83 c 4.74 + 0.12 ab Example 3 Comparative Sv only 17.61 + 0.65 a 20.46 + 1.05 bc 4.91 + 0.11 ab Example 4 Comparative Ce and Cc 17.81+ 0.61 a 20.28 + 0.94 bc 4.74 + 0.09 ab Example 5

Comparative Ce, Cc and Sv 18.67+ 0.73 a 23.39 + 0.8 ab 4.83 + 0.06 ab Example 6

Note: Different letters in a same column indicated significant differences (at a level of 0.05).

[047] It can be seen from Table 1 that, compared with Comparative Example 1, inoculation with AM fungi can significantly promote growth of biomass of container-grown seedlings of Euscaphis konishii Hayata, where leaf number, plant height and ground diameter of the container-grown seedlings were all obviously increased. The treatment of Example 1 (inoculation with Cc and Sv) resulted in the largest leaf number, reaching 18.71 and an optimum height of the seedlings of Euscaphis konishii Hayata, with an average seedling height of 24.7 cm. The treatment of Comparative Example 2 (inoculation with Ce only) had the best ground diameter of the container-grown seedling, reaching 4.91 mm. Based on comprehensive consideration, the treatments of Example 1 (inoculation with Cc and Sv) and Comparative Example 2 (inoculation with Ce) had the best promotion effects on the biomass of the container-grown seedlings of Euscaphis konishii Hayata. However, the biomass growth of Comparative Example 2 mainly came from the underground part. Compared with Comparative Example 2, the Example was 6.9% higher in leaf number, 11.7% higher in plant height, and 4.4% smaller in ground diameter.

Table 2 Effect of different treatments on biomass of Euscaphis konishii Hayata Root dry Stem dry Leafdry Total dry Root/shoot Group Inoculation weight (g) weight (g) weight (g) weight (g) ratio

0.95+0.14 2.93+0.28 0.66+0.04 Example 1 Cc and Sv 1.69 + 0.14 ab 0.52+ 0.03 a abc ab b

Control (CK), Comparative 0.18+0.01 1.14+0.16 inoculated with no 0.04 + 0.00 c 0.06 + 0.00 d 0.01 + 0.00 b Example 1 c a AM fungi

Comparative 3.32+ 0.79+0.05 Ce only 1.88 + 0.10 a 1.13 + 0.07 a 0.53 + 0.06 a Example 2 0.24a b

Comparative 2.79 +0.24 0.78 +0.05 Cc only 1.6 + 0.11 ab 0.85 + 0.09 bc 0.45 + 0.04 a Example 3 ab b

Comparative 2.99+0.17 0.74+0.04 Emplie Sv only 1.52+ 0.09 b 1.04 + 0.08 ab 0.41 + 0.04 a ab Example 4 ab b

Comparative CCe and Cc 1.55 + 0.11 ab 0.73 + 0.06 c 0.42+ 0.04 a 2.57+0.14 0.85+0.05

Example 5 b

Comparative 2.93 +0.14 0.82+ 0.05 Ce,CCcandSv 1.56 + 0.08 ab 0.87+ 0.05 bc 0.44 + 0.03 a Example 6 ab b

[048] It can be seen from Table 2 that, inoculation of AM fungi can significantly increase the biomass of different organs of the container-grown seedlings of Euscaphis konishii Hayata. The treatment of Comparative Example 2 (inoculation with Ce) resulted in the largest root biomass 1.88 g, the largest leaf biomass 0.53 g, and the largest total biomass 3.32 g of the container-grown seedlings. The container-grown seedlings treated in Comparative Example 1 had the highest root/shoot ratio (1.14), while the ones treated in Example 1 had the lowest root/shoot ratio (0.66) (a lower root/shoot ratio indicated that a plant can support higher aboveground growth with fewer roots, and more energy can be used for the aboveground vegetative growth while reduced nutrients can be used to maintain underground growth), which proved that inoculation of AM fungi did improve nutrient absorption of the Euscaphis konishii Hayata.

[049] The above descriptions are merely preferred implementations of the disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the disclosure.

Claims

1UUdIlU34U CLAIMS

1. A method of promoting growth of container grown seedlings of Euscaphis konishii, comprising contacting the seedlings of Euscaphis konishii with a microbial agent comprising Claroideoglomus claroideum (Cc) and Septoglomus viscosum (Sv), wherein the microbial agent comprises Cc and Sv spores, and wherein a ratio of a number of the Cc spores to that of the Sv spores is (0.5-1.5):(0.5-1.5).

2. The method of claim 1, wherein the microbial agent comprises 300-480 effective spores /60 g.

3. The method of claim 1 or 2, wherein the Cc comprises a Cc strain Cc-01 deposited with an accession number of CGMCC No. 18593; and the Sv comprises a Sv strain Sv-01 deposited with an accession number of CGMCC No. 18594.