CN105754901B

CN105754901B - Klebsiella with plant drought resistance promoting effect and application thereof

Info

Publication number: CN105754901B
Application number: CN201610228039.8A
Authority: CN
Inventors: 张磊; 辛凯芸; 沈锡辉
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2016-04-13
Filing date: 2016-04-13
Publication date: 2020-07-07
Anticipated expiration: 2036-04-13
Also published as: CN105754901A

Abstract

The invention discloses a Klebsiella with the effect of promoting plant drought resistance and application thereof, wherein the code is LTGPAF-6F, the strain is preserved in China center for type culture Collection at 1 month and 18 days in 2016, the preservation number is CCTCC M2016053, and the strain is classified and named as Klebsiella sp. The strain LGPAF-6F provided by the invention has the growth-promoting and stress-resistant properties of producing extracellular polysaccharide and indoleacetic acid, and being capable of self-generating nitrogen fixation and the like. The strain is prepared into 10⁸～10⁹The CFU/mL viable bacteria preparation is inoculated to the root of the wheat seedling, so that the biomass of the wheat seedling can be obviously increased, and the resistance of the wheat seedling to drought stress is improved. The Klebsiella L-LTGPAF-6F can be used as a biological agent to be applied to the agricultural production practice in the arid region, so that the drought resistance of plants is improved.

Description

Klebsiella with plant drought resistance promoting effect and application thereof

Technical Field

The invention belongs to the technical field of microbial resource utilization and agricultural and crop stress resistance research, and relates to a Klebsiella with a plant drought-resistant promoting effect and application thereof.

Background

With the continuous increase of population and the continuous deterioration of environment, the shortage of water resources, the salinization of soil and the small arable area become main problems in the agricultural production all over the world. In addition, with the frequent occurrence of extreme weather such as high temperature, drought, flood and the like, the method causes great damage to the global grain production. In recent years, research on the adaptability of plants to stress has become a focus of global attention, and how to improve the stress resistance of plants has become an important part of research. Some methods have been developed for improving the stress resistance of crops, such as cultivating plant varieties with strong stress resistance, improving cultivation measures and the like. However, these measures are still far from adequate for solving the problem, and new methods and techniques still need to be explored. Just as the relationship between intestinal microorganisms and human health is receiving increasing attention from the medical field, the role played by endophytes in promoting plant growth and development and improving plant stress resistance is also receiving increasing attention from agricultural researchers and microbiologists.

Most of Klebsiella (Klebsiella) belong to iatrogenic pathogenic bacteria or conditional pathogenic bacteria, and Klebsiella pneumoniae (Klebsiella pnenmonia) has the strongest pathogenicity to people, but researches in the fields of agricultural microorganisms, industrial pollution treatment and the like in recent years show that the Klebsiella does not show pathogenicity to plants, and is a green bacterial fertilizer and a high-efficiency purifying agent. The Klebsiella is widely distributed in roots and soil of gramineous plants, is a rich root system combined nitrogen fixation microorganism resource, establishes a close cooperative relationship with plants in the long-term evolution and phylogenetic processes, and indirectly promotes the growth and metabolism of the plants. Therefore, the excellent Klebsiella germplasm resource is discovered from plant tissues which live in extreme environments for a long time, is used for improving the survival capability of plants in adverse habitat, and has important significance for vegetation recovery and agricultural production under adverse natural conditions.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides the klebsiella with the effect of promoting the drought resistance of plants and the application thereof, which can help crops to grow in a drought environment and improve the drought resistance of the crops.

The technical scheme is as follows:

the Klebsiella with the effect of promoting plant drought resistance has a code of LTGPAF-6F, and the strain is preserved in China center for type culture Collection at 18 days 1 month in 2016, the preservation address is Wuhan university in China, the preservation number is CCTCC M2016053, and the Klebsiella sp.

Preferably, the 16S rRNA gene sequence of the Klebsiella LTGPAF-6F is shown as SEQ ID NO. 1.

Preferably, the nifH gene sequence of the Klebsiella LTGPAF-6F is shown as SEQ ID NO. 2.

Preferably, said Klebsiella LTGPAF-6F is capable of producing indole acetic acid.

Preferably, said Klebsiella LTGPAF-6F is capable of producing exopolysaccharides.

Preferably, the Klebsiella LTGPAF-6F can fix nitrogen spontaneously.

A liquid preparation containing the Klebsiella LTGPAF-6F, wherein the effective number of the bacteria of the strain is 10⁸～10⁹CFU/mL。

The invention discloses application of Klebsiella LTGPAF-6F with the effect of promoting plant drought resistance in the process of promoting crop drought resistance by symbiosis with plants.

Preferably, the drought condition is 12 days without watering.

Preferably, the crop is wheat.

The Klebsiella LTGPAF-6F is applied to the process of promoting the growth of plants.

The Klebsiella in the invention is applied in the process of producing indoleacetic acid.

The application of the Klebsiella in the exopolysaccharide production process is provided.

The Klebsiella in the invention is applied in the process of fixing nitrogen element in the air.

The invention has the beneficial effects that:

the Klebsiella L LTGPAF-6F can improve the drought resistance of plants, the fresh weight of wheat seedlings inoculated with the strain LTGPAF-6F is improved by 54.7 percent compared with that of the wheat seedlings without inoculation after the watering is stopped for 12 days and the watering is resumed for 3 days, and the lengths of the overground part and the underground part are respectively improved by 10.3 percent and 18.9 percent.

The Klebsiella L LTGPAF-6F can produce indoleacetic acid and is beneficial to promoting plant growth. Can produce extracellular polysaccharide and is beneficial to improving the drought resistance of plants.

The Klebsiella LTGPAF-6F can fix nitrogen elements in the air, and is beneficial to improving the utilization of the nitrogen elements by plants.

Drawings

FIG. 1 is a photograph of a colony of Klebsiella sp.LTGPAF-6F cultured in LB medium for 24 hours;

FIG. 2 is a comparison of growth status of non-inoculated wheat seedlings versus Klebsiella sp.LTGPAF-6F-inoculated wheat seedlings under drought stress conditions (second and third pots) and two no-drought controls (first and fourth pots);

FIG. 3 is a comparison of fresh weights of wheat seedlings inoculated with Klebsiella sp.LTGPAF-6F and wheat seedlings not inoculated with bacteria under drought stress conditions;

FIG. 4 is a comparison of root and stem lengths of wheat seedlings inoculated with Klebsiella sp.LTGPAF-6F under drought stress conditions with wheat seedlings not inoculated with bacteria.

Detailed Description

The method of the present invention is described in further detail below with reference to the figures and specific examples.

The Klebsiella sp.LTGPAF-6F is classified and named as Klebsiella sp.LTGPAF-6F, and is preserved in the China center for type culture Collection in 2016, 1 month and 18 days, with the preservation number as follows: CCTCC M2016053, preservation time 30 years.

The method for separating and identifying the Klebsiella comprises the following steps:

the strain LTGPAF-6F is a endophyte, is obtained by separating the roots of Alhagi sparsifolia Shap collected from Xinjiang desert areas in China, and is identified to belong to Klebsiella (Klebsiella).

The separation process is as follows: selecting healthy alhagi sparsifolia root tissues, shearing the alhagifolia root tissues by using scissors, soaking the alhagifolia root tissues in clear water for several minutes, washing off impurities, soaking the alhagifolia root tissues in 75% ethanol for 1min, soaking the alhagifolia root tissues in 2% sodium hypochlorite for 3min, soaking the alhagifolia root tissues in 75% ethanol for 30s, and washing the tissues for 5 times by using sterile water. After surface sterilization, the tissue is ground to a slurry under aseptic conditions. Transfer 1mL of the slurry to 50mL of LPAF medium (peptone 10.0g, acid casein 10.0g, anhydrous MgSO₄1.5g，KH₂PO₄1.5g, glycerol 10mL, distilled water 1000mL) was incubated at 28 ℃ on a rotary shaker at 200rpm for 24 h. Transfer 1mL of the suspension to another 50mL of the AF culture solution, and culture for 24h under the same conditions. At 3d, transfer 1mL of suspension to 50mL DF Medium [4.0g KH₂PO₄，6.0g Na₂HPO₄，0.2g MgSO₄·7H₂O, 2.0g gluconic acid, 2.0g citric acid, 2.0g (NH)₄)₂SO₄0.1mL trace elements (10mg H)₃BO₃，11.19mg MnSO₄.H2O，124.6mg ZnSO₄.7H₂O，78.22mg CuSO₄.5H₂O，10mg MoO₃100mL of water), 0.1mL of FeSO4 & 7H₂O(100mg FeSO₄·7H₂O, 10mL water)]And culturing for 24h under the same condition. 4d, transfer 1mL of suspension to 50mL ADF Medium [ DF Medium basis to remove (NH)₄)₂SO₄Addition of 0.2% ACC]Culturing for 24h under the same condition, then coating the bacterial suspension after gradient dilution on an ADF flat plate, culturing for 72h at 28 ℃, picking out a single colony, purifying and storing.

The physiological and biochemical characteristics of the Klebsiella LTGPAF-6F are as follows: the growth is good on various culture media such as TSB, R2A, Marine Agar2216, Nutrient Agar, MacConkey Agar, LB and the like; as shown in figure 1, after 24h of culture on an LB plate, a round, 2-3 mm-diameter, opaque, semi-moist and convex milky colony is formed; the maximum tolerated concentration of NaCl was 7%; can resist the high temperature of 44 ℃; the growth range of pH is 5.0-8.5; is facultative anaerobe, and can grow well under aerobic and anaerobic conditions; producing catalase and cytochrome oxidase; can reduce nitrate into nitrite.

Inoculating the strain LTGPAF-6F into 5mL of PAF liquid culture medium, carrying out shake culture at 28 ℃ and 150rpm for 48h, centrifuging at 8000rpm, collecting thalli, and extracting the total genomic DNA of the strain according to a CTAB method. The 16S rRNA gene fragment is PCR amplified by using genome DNA as a template and using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-CGGTTACCTTGTTACGACTT-3'), and the 16S rRNA gene sequence of the strain LTGPAF-6F obtained after sequencing is shown as SEQ ID NO. 1. The 16S rRNA gene sequence is submitted to a GenBank database for multiple sequence alignment, and the sequence similarity of the 16S rRNA gene sequence and a related model strain of Klebsiella (Klebsiella) is the highest and is found to be the highest with Klebsiella microorganissis W14^TThe sequence similarity of the sequence reaches 99.4 percent. Thus, the strain LTGPAF-6F was assigned to the genus Klebsiella and namedKlebsiellasp.LTGPAF-6F。

The results of some performance studies of the Klebsiella bacteria of the invention are given below:

determination of Indolylacetic acid (IAA) production ability of Klebsiella LTGPAF-6F

Klebsiella LTGPAF-6F was inoculated into SMS liquid medium [ sucrose10g, (NH) containing 0.5mg/mL L-tryptophan₄)₂SO₄1g、K₂HPO₄2g、MgSO₄0.5g、Yeast extract 0.5g、CaCO₃0.5g, NaCl 30g, 1000mL of distilled water, pH 7.2]Shaking at 30 deg.C and 180rpm for 3 d. Spectrophotometric determination of OD of bacterial suspension₆₀₀Then the bacterial suspension is centrifuged at 10000rpm for 10min, and the supernatant is added with an equal volume of Salkowski colorimetric solution (50mL of 35% HClO)₄+1mL 0.5M FeCl₃) Standing in dark for 30min, and determining OD₅₃₀Values, IAA content was calculated by a standard curve of IAA gradient dilution. Calculating the bacterial concentration OD₆₀₀At a value of 1, the IAA content per volume of fermentation broth. Through determination, the IAA yield of the Klebsiella LTGPAF-6F can reach 30.7 mu g/(mL. OD)₆₀₀)。

Determination of extracellular polysaccharide producing capability of Klebsiella LTGPAF-6F

Inoculating Klebsiella LTGPAF-6F into 0.5 × TSB liquid culture medium, shake culturing at 30 deg.C and 180rpm to stationary phase, centrifuging at 4 deg.C and 6000g for 15min to remove thallus, collecting supernatant, adding 2.5 times volume of ethanol, and obviously showing flocculent precipitate, which indicates that the strain can produce extracellular polysaccharide.

Klebsiella LTGPAF-6F nitrogen fixation capacity determination

Inoculating activated Klebsiella into liquid nitrogen-free culture medium [4.0g KH ]₂PO₄，6.0gNa₂HPO₄，0.2g MgSO₄·7H₂O, 2.0g gluconic acid, 2.0g citric acid, 0.1mL trace elements (10mg H)₃BO₃，11.19mg MnSO₄·H₂O，124.6mg ZnSO₄.7H₂O，78.22mg CuSO₄.5H₂O，10mg MoO₃100mL sterile water), 0.1mL FeSO₄·7H₂O(100mg FeSO₄·7H₂O, 10mL sterile water)]The strain was cultured by shaking at 180rpm at 30 ℃ for 3 days, and the strain grew well in a nitrogen-free medium. Using the genome DNA of the strain as a template, a target fragment of about 300bp was amplified using universal primers F (5'-GGCTGCGATCCAAGGCCGA-3') and R (5 ' -CTGGCCTTGTTTCGCGGATGGCATGGC) of the nitrogen-fixing gene (nifH). Cutting the amplified fragment into gel, recovering and connecting the gel into a pMD19-T vector, transforming escherichia coli competent cells, selecting positive clones for sequencing, and obtaining the nifH gene sequence of the strain LTGPAF-6F as shown in SEQ ID NO. 2. The sequence alignment in GenBank database shows that the similarity of the nifH gene sequence and the nifH gene sequence of Klebsiella sp.AL060224_03 is as high as 97.9%. The growth experiment of the Klebsiella LTGPAF-6F in a nitrogen-free culture medium and the sequencing result of the nifH gene sequence show that the strain has the self-generating nitrogen fixation capacity.

The following experiment of the application of the Klebsiella LtgpaF-6F of the present invention is given

A single colony of Klebsiella LTGPAF-6F cultured in TSA solid medium for 24h was picked, inoculated in TSB liquid medium, cultured at 28 ℃ for 12h at 200rpm, and then transferred into a triangular flask containing 200mL of TSB liquid medium. Placing the mixture on a shaking table at 200rpm and 28 ℃ for shake culture for 24h, and centrifugally collecting the thalli at 6000rpm when the thalli reach the logarithmic phase. The cells were washed twice with sterilized tap water and diluted to 10 deg.C⁸～10⁹CFU/mL is used as the biological agent.

Mixing vermiculite and soil according to a volume ratio of 1: 3 to obtain a planting matrix, filling the planting matrix into strain bags, wherein each strain bag is 450g, sterilizing the strain bags at 121 ℃ for 25min after sealing, and then filling the sterilized mixed soil into a plastic flowerpot.

Selecting seeds of JINMAI 47 with full and consistent size, soaking in 75% ethanol for 30s, removing liquid, and adding 0.1% HgCl₂Soaking for 7min, washing with sterile water for 5-6 times, and placing the wheat seeds on sterilized wet double-layer filter paper, and culturing at 25 deg.C in dark for 2 d. Wheat seeds with consistent bud length and root length are selected, planted into soil, 3 seeds are planted in each pot, and 40mL of water is watered.

Placing the prepared pot at 25 deg.CIn a greenhouse, the illumination is 14h every day, 30mL every other day is watered, after one week, 40mL of the prepared Klebsiella LTGPAF-6F viable bacteria preparation is added (according to 10 per gram of soil)⁷～10⁸Number of live bacteria inoculated) was poured onto the root of wheat while setting 40mL of tap water after the control group was poured and sterilized, and this was repeated for 3 pots per treatment. Culturing for about one week, waiting until the wheat grows to two leaves and one heart stage, performing drought treatment on the butt-inoculated and non-inoculated control groups at the same time, stopping watering for 12 days, and observing and recording the growth condition of the wheat. And then the watering is resumed for 3 days, the health state of the wheat seedlings is recorded and photographed, then the wheat is taken out from the soil, the soil on the surface of the wheat is cleaned, and the growth indexes of average root length, plant height, fresh weight, dry weight and the like of each treatment are measured and counted. And finally, respectively carrying out surface disinfection on the wheat seedlings of the treatment group and the control group under the aseptic condition (1 min of treatment by 70% ethanol, 3min of treatment by 2% sodium hypochlorite, 30S of 70% ethanol, 5 times of washing tissues by sterile water), naturally airing and weighing, grinding the wheat seedlings into homogenate, diluting and coating the homogenate on a TSA solid culture medium, culturing for 48h at 28 ℃, observing and recording the number of colonies, and selecting 2 representative colonies for carrying out 16S rRNA gene sequence PCR amplification and sequencing detection.

As shown in FIG. 2, the first pot from left to right shows the control without drought treatment of Klebsiella L-LTGPAF-6F, the second pot shows the drought treatment of wheat seedlings without Klebsiella L-LTGPAF-6F, the third pot shows the drought treatment of wheat seedlings with Klebsiella L-LTGPAF-6F, and the fourth pot shows the control without drought treatment of Klebsiella L-LTGPAF-6F. As can be seen from FIG. 2, under the same drought stress condition, the wheat seedlings inoculated with the Klebsiella LTGPAF-6F (the third pot) keep fresh green and grow normally, while the wheat seedlings not inoculated with the Klebsiella LTGPAF-6F (the second pot) grow slowly and have obvious withering phenomenon, which shows that the growth development and the health condition of the wheat seedlings inoculated with the Klebsiella LTGPAF-6F (the third pot) are obviously better than those of the wheat seedlings not inoculated with the Klebsiella LTGPAF-6F (the second pot). In FIGS. 3 and 4, CK and LTGPAF-6F represent the drought treatment of wheat seedlings not inoculated with bacteria and wheat seedlings inoculated with Klebsiella LTGPAF-6F, respectively. As shown in FIG. 3, the fresh weight of wheat seedlings subjected to drought treatment and applied with the viable bacteria preparation of Klebsiella LTGPAF-6F increased by 54.7% compared with the control without inoculated bacteria. As shown in FIG. 4, the wheat seedlings inoculated with Klebsiella LTGPAF-6F during drought treatment had a 10.3% increase in the above-ground part and a 18.9% increase in the root length of the below-ground part, compared to the control with different strains.

The separation result of wheat seedling endophyte inoculated with Klebsiella LTGPAF-6F shows that after 48 hours of culture at 28 ℃, a large number of colonies with the same form as the Klebsiella LTGPAF-6F are grown on a TSA separation plate, and the number reaches 2 × 10⁵CFU/g fresh weight. The results of 16S rRNA gene sequencing by picking representative colonies also showed that the 16S rRNA gene sequences of these colonies were consistent with Klebsiella LTGPAF-6F, indicating that Klebsiella LTGPAF-6F could colonize wheat seedlings in large quantities.

The results of the above wheat pot experiments show that the Klebsiella LTGPAF-6F can be colonized in wheat seedlings, improve the resistance of host plants to drought stress and promote the growth and development of the host plants.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims

1. A Klebsiella with the effect of promoting plant drought resistance is characterized in that the code is LTGPAF-6F, the strain is preserved in China center for type culture Collection in 2016, 1, 18 days, the preservation number is CCTCC M2016053, and the strain is classified and named as Klebsiella sp.LTGPAF-6F;

the Klebsiella can produce indoleacetic acid;

the Klebsiella can produce extracellular polysaccharide;

the Klebsiella can fix nitrogen spontaneously.

2. Use of a klebsiella bacterium according to claim 1 for promoting drought tolerance of crops in symbiosis with plants, wherein the drought condition is 12 days without continuous watering;

the crop is wheat.

3. Use of the bacterium klebsiella of claim 1 for promoting plant growth.

4. A liquid preparation comprising the bacterium Klebsiella as claimed in claim 1, wherein the effective number of the bacterium Klebsiella LTGPAF-6F is 10⁸～10⁹CFU/mL。