Strain of spirillum harteri FtiPs-6 and application thereof

Abstract

The invention discloses a strain of spirillum harteri FtiPS-6 and an application of the strain of spirillum harteri FtiPS-6. The bacterial strain is classified and named as Herbaspiridium huttiense FtiPs-6, and the preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) No.37084. The invention further discloses a preparation method of the bacterial strain. The spirillum harteri FtiPS-6 disclosed by the invention can effectively promote the growth of buckwheat and improve the production capacity, so that the plant height, the fresh weight and the yield of a single plant are remarkably increased. The strain can also improve the tolerance of buckwheat to drought stress, so that the survival rate, the relative water content and the proline content of buckwheat are remarkably increased, the catalase activity level is improved, and the malondialdehyde content is reduced. In an in-vitro simulated drought culture environment, the proline production capability of the strain is induced to be improved. And the method has a relatively high application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a strain of Herbaspirillum huttiense FtiPs-6 and application thereof. BACKGROUND

[0002] Buckwheat (Fagopyrum spp.) is an important short-season, nutrient-rich, and versatile crop. Its seeds are rich in high-quality protein, flavonoids (such as rutin), dietary fiber, and minerals, and have significant nutritional and health values. Buckwheat is often planted in marginal lands or as a post-disaster crop due to its short growth period and certain environmental adaptability. However, buckwheat is highly sensitive to drought stress. Drought stress can cause buckwheat plants to grow slowly, leaf wilting and shedding, photosynthetic efficiency to decrease significantly, flower organ development to be abnormal, seed setting rate to decrease, and ultimately yield to decrease sharply and quality to decrease.

[0003] Plant endophytes are microorganisms that colonize inside healthy plant tissues for all or most of their life cycle without causing obvious disease symptoms. Specific beneficial endophytes can establish mutualistic symbiotic relationships with host plants, enhancing the host's tolerance to biotic and abiotic stresses, including drought. Although plant-microbe interactions show great potential in improving crop stress resistance. Patent CN110591924B reports that Morchella crassepsis strain OL-Y-B can promote the growth of buckwheat. Patent CN110358697B discloses that Bacillus megaterium YC4-R4 can promote the growth of buckwheat and enhance its drought resistance. Patent CN109554313B reports that Herbaspirillum sp. H.5-28 can be used as a biological fertilizer and has good effects on resisting maize common blight, wheat sharp eyespot, and aconite root rot. Patent CN110106101B reports that Herbaspirillum huttiense ZL-3 can be used for nitrogen fixation composting under low temperature conditions. IN201717043114A reports that Dothideomycetes and Sordariomycetes can promote wheat growth and improve its drought resistance to some extent, but the improvement of wheat drought resistance is not obvious. It also reports two species of Herbaspirillum, but does not verify their effects. Moreover, due to physiological structure, metabolic network, and immune system, endophytes that can be used for wheat drought resistance cannot be directly used for buckwheat. There is no report on using Herbaspirillum (β-proteobacteria) to enhance the drought resistance of buckwheat. SUMMARY

[0004] The purpose of this invention is to provide a strain of Hericium hartii FtiPs-6 and its applications. This strain can significantly promote the growth and development of buckwheat and increase the yield per plant. Its high proline production characteristic has a significant effect on improving the survival ability of buckwheat under drought stress.

[0005] The technical solution of the present invention is as follows:

[0006] A strain of *Herbaspirillum huttiense*, FtiPs-6, belonging to plant endophytes, was isolated and screened from the best-growing buckwheat stem tissue in barren soil in Xiangtan, Hunan Province. Based on colony morphology and a phylogenetic tree based on 16S rRNA, it was identified as *Herbaspirillum huttiense*. This strain was deposited on December 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) (depository address: Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 37084.

[0007] The present invention also provides the application of the aforementioned buckwheat endophytic Hericium hartii FtiPs-6 in promoting buckwheat growth, development and production.

[0008] The present invention also provides a microbial preparation comprising the aforementioned buckwheat endophytic Hericium hartii FtiPs-6.

[0009] The buckwheat endophytic *Hattella hartiensis* FtiPs-6 and the microbial preparation described in this invention have nitrogen-fixing and phosphorus-solubilizing growth-promoting properties, and can secrete plant growth hormone IAA and siderophores and other active substances that can promote plant growth.

[0010] Furthermore, the concentration of the buckwheat endophytic *Hattella hartiensis* FtiPs-6 described in this invention is 5 × 10⁻⁶. 5 -5×10 6 CFU / mL, preferably a final concentration of 5×10⁻⁶. 6 CFU / mL.

[0011] Furthermore, the application method of the buckwheat endophytic *Hattella hartiensis* FtiPs-6 described in this invention is a spore suspension (preferably at a concentration of 5 × 10⁻⁶). 6 Apply CFU / mL to the roots of buckwheat plants as a root drench, 10 mL per plant, in the evening.

[0012] The present invention further provides the application of the aforementioned buckwheat endophytic Hericium hartii FtiPs-6 in improving buckwheat drought stress resistance.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The strain obtained by the application can stably colonize in buckwheat, can not only promote the growth and development of buckwheat and increase the yield, but also can improve the drought stress resistance of buckwheat, and has a good development and application prospect.

[0015] The buckwheat endophytic Hart grass spirillum FtiPs-6 can significantly improve the growth and yield of buckwheat in the field, increase the plant height by 26.2%, increase the fresh weight of the aboveground part by 78.9%, increase the yield per plant by 91.9%, and increase the thousand-grain weight by 6.0%.

[0016] The buckwheat endophytic Hart grass spirillum FtiPs-6 can significantly improve the drought stress resistance of buckwheat, and the drought stress treatment at the germination stage can increase the survival rate of the seedling by about 5 times, and the treatment at the seedling stage can increase the survival rate of the plant by about 60%.

[0017] The buckwheat endophytic Hart grass spirillum FtiPs-6 increases the content of the osmotic regulation substance proline by 44.6% under the culture of the in-vitro simulated drought stress; in-vivo, the proline content of the buckwheat-FtiPs-6 symbiotic body increases by 157.1%. At the same time, it can also improve the hydrogen peroxide enzyme activity level and reduce the content of malondialdehyde.

[0018] Biological material preservation information

[0019] The Hart grass spirillum Herbaspirillum huttiense FtiPs-6 is preserved in the China General Microbiological Culture Collection Center (No. 3, Beichen West Road, Chaoyang District, Beijing, China) on December 15, 2025, and the preservation number is CGMCC No. 37084. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the colony morphology (a) and the cell morphology (b) of the buckwheat endophytic Hart grass spirillum H. huttiense FtiPs-6 in Example 1 of the application.

[0021] Figure 2 It is a development tree diagram of the buckwheat endophytic Hart grass spirillum H. huttiense FtiPs-6 in Example 1 of the application.

[0022] Figure 3 It is a growth comparison chart of buckwheat after different concentrations of buckwheat endophytic Hart grass spirillum H. huttiense FtiPs-6 in Example 2 of the application.

[0023] Figure 4 It is a comparison chart of the growth and fruiting of buckwheat treated with the optimal concentration of buckwheat endophytic Hart grass spirillum H. huttiense FtiPs-6 in Example 3 of the application and the untreated group.

[0024] Wherein, a is field growth buckwheat; b is single buckwheat and its seeds after collection.

[0025] Figure 5 It is a level diagram of nitrogen fixation, phosphorus dissolution, iron carrier production and IAA of buckwheat endophytic Hart grass spiral bacteria H. huttiense FtiPs-6 in Example 4 of the present application.

[0026] Wherein, a is Ashby nitrogen-free medium; b is Brown nitrogen-free medium; c is inorganic phosphorus dissolution ability detection medium; d is CAS iron carrier detection medium; e is IAA detection reaction solution.

[0027] Figure 6 It is a comparison chart of the influence of buckwheat drought stress tolerance by buckwheat endophytic Hart grass spiral bacteria H. huttiense FtiPs-6 in Example 5 of the present application.

[0028] Wherein, a is buckwheat at the germination stage; b is buckwheat at the seedling stage.

[0029] Figure 7 It is a growth curve of buckwheat endophytic Hart grass spiral bacteria H. huttiense FtiPs-6 in normal and 15% PEG added culture environment in Example 6 of the present application.

[0030] Figure 8 It is a comparison chart of the growth of buckwheat endophytic Hart grass spiral bacteria H. huttiense FtiPs-6 in normal (a) and 15% PEG added culture environment (b) in Example 6 of the present application.

[0031] Figure 9 It is a comparison chart of the influence of other 6 buckwheat endophytic bacteria on buckwheat growth in the field;

[0032] Wherein, a is buckwheat endophytic bacteria AR-2; b is buckwheat endophytic bacteria MS-5; c is buckwheat endophytic bacteria ALb-1; d is buckwheat endophytic bacteria AR-6; e is buckwheat endophytic bacteria Ala-1; f is buckwheat endophytic bacteria AR-1; CK is blank control. DETAILED DESCRIPTION

[0033] The present application is further described below in conjunction with the drawings and specific preferred embodiments of the present application, but the protection scope of the present application is not limited thereby.

[0034] In the following examples, the materials and instruments used are commercially available.

[0035] Example 1: Isolation, purification and identification of the strain

[0036] The rhizome and leaf tissue of buckwheat growing in relatively poor nutrient soil in the Hunan Science and Technology University biological park in Yuhu district of Xiangtan city was collected, washed and surface sterilized, and then cut into 1 cm small pieces and placed in LB medium and cultured at 37 °C. When the bacteria were separated from the tissue, they were picked and transferred to a new LB medium for streaking and purification. After the pure culture of the strain was obtained, it was stored in 25% glycerol at -80 °C. The strain was named FtiPs-6.

[0037] The composition of the LB medium used (g / L) was: yeast extract 5 g, peptone 10 g, sodium chloride 10 g, agar powder 20 g, natural pH, 121 °C high temperature and high pressure sterilization for 25 min.

[0038] The strain was cultured in LB medium, and the colony was milky white, round and convex, with smooth and moist surface and regular edge without halo. Scanning electron microscope observation showed that the bacterial body was micro-curved rod-shaped. The bacterial body and colony morphology of FtiPs-6 are shown in Figure 1 .

[0039] The purified single colony was inoculated into liquid LB medium and shaken overnight. After the bacterial solution was collected, the genomic DNA of FtiPs-6 was extracted using the bacterial total DNA extraction kit of Shanghai Bioengineering Co., Ltd., and the universal primer of 16s rRNA encoding gene (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 16S-1492R: 5'-GGTTACCTTGTTACGACTT-3') was used as a template. The PCR reaction system (20 μL) was: DNA template 2 μL, reaction enzyme system mixed solution 10 μL, forward and reverse primers 1 μL each, and ddH20 6 μL. The PCR reaction conditions were: 95 °C pre-denaturation for 5 min, then heat cycle: 94 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 1 min 30 s, a total of 30 cycles. Finally, 72 °C extension for 10 min. The PCR amplification product was purified by Shanghai Bioengineering Technology Co., Ltd. and sequenced, and its 16rSRNA is shown as SEQ ID No. 1. According to the sequencing results, BLAST sequence alignment analysis was carried out in the NCBI website, and it was identified that the similarity of the strain to Herbaspirillum huttiense was 99.9%. The phylogenetic tree of FtiPs-6 identification is shown in Figure 2 .

[0040] Example 2: Promoting effect of the strain and optimization of application concentration

[0041] Test material: buckwheat (bitter buckwheat variety).

[0042] Planting method: buckwheat was cultured in water in a greenhouse, with a light cycle of 16 hours light / 8 hours darkness, a culture temperature of 26 degrees, and air humidity of 60%.

[0043] Test treatment: The bacterial suspension of FtiPs-6 was obtained according to the method in Example 1, and the bacterial suspension was added to the buckwheat water culture (1 / 2 Hoagland nutrient solution). Three different concentration gradients of 5x10 5 , 5x10 6 , and 5x10 7 CFU / mL were set, and the treatment groups were named E+ (10 5 ), E+ (10 6 ), and E+ (10 7 ) buckwheat, respectively. The non-inoculated endophyte treatment group was named E- buckwheat. After the buckwheat grew to a stable and uniform phenotype (about 2 weeks), the growth indicators such as plant height and fresh and dry weight were counted.

[0044] The test results are shown in Figure 3 When the concentration of FtiPs-6 bacterial solution was 5x10 6 , the promotion effect on buckwheat growth was optimal, and when the concentration was 5x10 5 , the growth-promoting effect disappeared, and when the concentration was 5x10 7 , the growth of buckwheat was significantly inhibited. The growth index statistics are shown in Table 1, and the growth indicators of E+ (10 6 ) were significantly higher than those of the other three treatment groups. For example, the plant height of E+ (10 6 ) increased by 11.56% compared with E- buckwheat, the fresh weight of the aboveground part increased by 57.93%, and the fresh weight of the underground part increased by 51.14%. Therefore, the optimal concentration of FtiPs-6 for promoting buckwheat growth is 5x10 6 CFU / mL.

[0045] Table 1

[0046]

[0047] Note: E- is the water treatment blank control, and E+ refers to the addition of bacterial suspension to the water culture, and the same below.

[0048] Example 3 Field Test of Strain Promoting Effect on Buckwheat Growth

[0049] Test material: buckwheat (bitter buckwheat variety).

[0050] Planting method: buckwheat was cultured in the field.

[0051] Test treatment: The bacterial suspension of FtiPs-6 was obtained according to the method in Example 1 and diluted to a final concentration of 5x10 6CFU / mL. After the buckwheat grew two leaves, the seedlings were treated with 10 mL / strain of bacterial suspension, and the treatment was performed in the evening. After the buckwheat grew to the mature stage, agronomic trait indexes such as plant height, fresh and dry weight of the aboveground part, yield per plant, and thousand-grain weight were determined.

[0052] In the field test, FtiPs-6 had a significant promoting effect on the growth and development of buckwheat. After growing to the mature harvest stage, the plant height of E+ buckwheat was increased by 26.15% compared with E- buckwheat, and the fresh weight of the aboveground part was increased by 78.89% (P<0.05). Figure 4 The related indexes of agronomic traits are shown in Table 2. FtiPs-6 can also effectively increase the yield of buckwheat, and the thousand-grain weight of E+ buckwheat was increased by 6.0%, and the yield per plant was increased by 91.88%.

[0053] Table 2

[0054]

[0055] Example 4 Determination of nitrogen fixation, phosphorus solubilization, siderophore production, and indole acetic acid production abilities of the strain

[0056] Determination of nitrogen fixation ability:

[0057] The glycerol strain FtiPs-6 was activated on an LB plate, and then a single colony was picked and shaken. The OD of the bacterial liquid was adjusted to 0.8-1.0. 100 μL of the bacterial liquid was spread on Ashby and Brown nitrogen-free medium plates, and inverted culture was performed at 29 ℃. After 7 days, it was observed that the strain could grow on both types of nitrogen-free medium (P<0.05), indicating that it had nitrogen fixation ability. Figure 5 a and Figure 5 b.

[0058] Determination of inorganic phosphorus solubilization ability:

[0059] 5 μL of the bacterial liquid was dropped on a PKO medium plate, repeated 3 times, and inverted culture was performed at 29 ℃ for 3-7 days. During the culture, the growth status of the colonies and whether a phosphorus solubilization ring was produced were observed. The results are shown in Figure 5 c. A transparent ring appeared around the colonies of the strain, indicating that it had the ability to dissolve inorganic phosphorus.

[0060] Determination of siderophore production ability:

[0061] 5 μL of the bacterial liquid was dropped on a CAS medium plate, repeated 3 times, and inverted culture was performed at 29 ℃ for 7 days. The results are shown in Figure 5 d. An orange transparent ring appeared around the colonies of the strain, indicating that it had the ability to produce siderophores.

[0062] Determination of indole acetic acid production ability:

[0063] (1) IAA qualitative experiment

[0064] The above bacteria liquid was inoculated into Kings medium with 0.2 g / L tryptophan at an inoculation amount of 1%, three replicates were set, and the culture was carried out at 29°C in a 180 rpm shaker for 3 days, then centrifuged at 12000 r / min for 10 min, 200 μL of the supernatant after centrifugation was taken to a 12-well plate, an equal volume of colorimetric solution was added, and the plate was wrapped with tin foil paper and reacted in the dark for 30 min. The positive result was pink, and the darker the color, the stronger the strain's ability to produce IAA. The results are shown in Figure 5 e, after the reaction, the culture supernatant containing the strain was pink, indicating that it had the ability to produce IAA.

[0065] (2) IAA quantitative experiment

[0066] 200 μL of the supernatant after centrifugation was added to 4 times the volume of colorimetric solution and reacted in the dark for 30 min, and the absorbance at 530 nm was measured. The standard curve was compared, and the yield of IAA of the strain was calculated to be about 49.73 μg / mL.

[0067] Example 5 Influence of the strain on drought stress resistance of buckwheat

[0068] Test material: buckwheat (bitter buckwheat variety).

[0069] Planting method: buckwheat was cultured in water in a greenhouse, the light cycle was 16 hours of light / 8 hours of darkness, the culture temperature was 26 degrees, and the air was moderate at 60%.

[0070] Test treatment: The bacterial suspension of FtiPs-6 was obtained according to the method in Example 1, and the bacterial suspension was added to the buckwheat water culture solution, and the final concentration was set to 5×10 6 CFU / mL. Add 15% PEG to the buckwheat water culture solution to simulate drought stress treatment. After the stress treatment, the buckwheat leaves were almost completely dehydrated and wilted, and then recovered for 7 days, and the relevant indicators were detected.

[0071] (1) Drought stress treatment during germination

[0072] Select the newly emerged but not yet germinated buckwheat seeds and transfer them to the water culture solution for culture, and at the same time add the bacterial suspension and 15% PEG to simulate drought stress treatment. The results are shown in Figure 6 a, After drought stress treatment, the germination rate of E+ buckwheat seeds with bacterial suspension was 31.25%, and the germination rate of E- buckwheat without bacterial suspension was 6.25%, which was increased by 5 times, indicating that FtiPs-6 can effectively improve the survival ability of buckwheat seedlings under drought stress.

[0073] (2) Drought stress treatment during seedling stage

[0074] After drought stress treatment for about 2 weeks, the E-buckwheat was completely dehydrated and wilted, while the E+ buckwheat still had some leaf expansion. After 7 days of recovery with normal water culture solution, nearly 60% of the E+ buckwheat plants recovered normal growth, while the E-buckwheat was almost completely dead. Figure 6 b). After recovery, the relative water content of the E+ buckwheat leaves was 2.93 times higher than that of the E-buckwheat, the MDA content decreased from 63.97 nmol / g to 27.74 nmol / g, the catalase activity increased from 61.02 U / g to 103.96 U / g, an increase of 70.37%, and the proline content increased from 29.19 μg / g to 75.04 μg / g, an increase of 157.07% (Table 3).

[0075] Table 3

[0076]

[0077] Example 6 Determination of the drought resistance of the strain

[0078] Test material: buckwheat endophytic Hart grass Spirochaete FtiPS-6.

[0079] Culture method: the purified FtiPS-6 single colony was inoculated into liquid LB medium containing 15% PEG and liquid LB medium without PEG, respectively, and shaken. The bacterial dry weight was measured every 10 h.

[0080] FtiPS-6 has strong tolerance to PEG in the culture environment, and as the culture time increases, the growth level of the bacterial mass has no significant difference with that of the normal culture (Fig. 2). Figure 7 There is almost no significant change in the morphology of the bacterial mass observed by scanning electron microscopy (Fig. 3). Figure 8 The proline content in the fermentation broth increased significantly from 42.19 μg / mL in the control group to 61 μg / mL, an increase of 44.6%. It is proved that FtiPS-6 has strong drought tolerance.

[0081] Example 7 Field growth-promoting ability test of six other buckwheat endophytic strains

[0082] Test material: buckwheat (bitter buckwheat variety).

[0083] Planting method: buckwheat was cultivated in the field.

[0084] Test treatment: According to the method in Example 1, the bacterial suspensions of six buckwheat endophytic strains FtiAR-2, FtiMS-5, FtiALb-1, FtiAR-6, FtiALa-1, FtiTR-3, FtiAR-1, and FtiAR-1, which were isolated at the same time as FtiPS-6, were diluted to a final concentration of 5 x 106 CFU / mL. After the buckwheat seedlings have grown two leaves, treat them by drenching the roots with 10 mL of bacterial suspension per plant in the evening. After the buckwheat reaches maturity, measure agronomic traits such as plant height, above-ground fresh and dry weight, yield per plant, and thousand-grain weight.

[0085] In field trials, the six tested endophytic bacteria did not significantly promote the growth and development of buckwheat; some strains even significantly reduced the fresh weight and yield per plant of buckwheat. Figure 9 For example, the fresh weight of buckwheat in the FtiAR-1 treatment group decreased significantly by 57.64%, and the yield per plant decreased by 45.02%. This further demonstrates that FtiPS-6 is the functionally dominant strain among the isolated buckwheat endophytic bacteria.

Claims

1. A strain of *Hattella hartenbergii* Herbaspirillum huttiense FtiPs-6 was deposited on December 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37084.

2. A microbial preparation, characterized in that, Contains *Hattella hartenbergii* as described in claim 1 Herbaspirillum huttiense FtiPs-6.

3. The Hartella hartella as described in claim 1 Herbaspirillum huttiense The use of FtiPs-6 or the microbial preparation of claim 2 in promoting buckwheat growth and / or increasing buckwheat yield.

4. The Hartella hartella as described in claim 1 Herbaspirillum huttiense Application of FtiPs-6 or the microbial preparation of claim 2 in improving the drought stress resistance of buckwheat.

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