Application of rhodopseudomonas palustris in promoting photosynthesis of tomato leaves under weak light

Abstract

The invention relates to the technical field of plant growth regulation, in particular to application of rhodopseudomonas palustris in promoting photosynthesis of tomato leaves under weak light. When the rhodopseudomonas palustris liquid provided by the invention is sprayed on the tomato leaf surfaces, photosynthesis can be promoted, the total chlorophyll content of the tomato leaves can be obviously improved, the aging of the leaves can be delayed, and the rhodopseudomonas palustris liquid has an application value in improving the resistance of tomatoes to weak light stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant growth regulation, and more particularly to an application of Rhodopseudomonas palustris in promoting photosynthesis of tomato leaves under weak light. BACKGROUND

[0002] In natural and agricultural production environments, short day seasons and weather changes cause a decrease in light intensity, which seriously affects crop growth and yield. The main harm is to the leaves, which is manifested as leaf yellowing, reduced chlorophyll content, and decreased photosynthesis. In greenhouse tomato cultivation, 50% shading can reduce yield by 26%. At the present stage, improving tomato photosynthesis under weak light and increasing yield rely on LED light supplementation and plant hormone-based growth regulators. LED light supplementation increases the initial investment cost, and improper use of plant hormones can cause fruit malformation, post-fruiting flower drop, and fruit rot, which seriously affects commodity quality. Therefore, using beneficial microorganisms to improve tomato leaf photosynthesis under weak light is a worthwhile approach to explore.

[0003] Photosynthetic bacteria are the oldest on earth, prokaryotes that can use bacterial chlorophyll to absorb near-infrared light and perform photosynthesis under anaerobic conditions. Photosynthetic bacteria have the advantages of being non-toxic, easy to obtain, and economical. Photosynthetic bacteria have good effects in promoting rice growth and improving the resistance of corn to salt stress. However, there is no report on the use of photosynthetic bacteria to improve tomato leaf photosynthesis under weak light. SUMMARY

[0004] In view of the limitations of the prior art, the present application aims to provide a Rhodopseudomonas palustris strain to improve the resistance of greenhouse tomatoes to weak light stress and ensure tomato growth and yield.

[0005] To achieve the above-mentioned purpose, the present application realizes the following technical solutions: The present application provides an application of Rhodopseudomonas palustris in promoting photosynthesis of tomato leaves under weak light.

[0006] In the preferred scheme, in the above application, the light condition of the weak light condition is: 300 µmol·m -2 ·s -1 .

[0007] In the preferred scheme, in the above application, the weak light condition is simulated by a sunshade net.

[0008] In the preferred scheme, in the above application, it is used to improve the chlorophyll content, net photosynthetic rate, and / or maximum photochemical efficiency of photosystem II of tomato leaves.

[0009] In the preferred scheme, in the above application, the Rhodopseudomonas palustris is Rhodopseudomonas palustris CGA009.

[0010] In the above application, the Rhodopseudomonas palustris bacterial solution is sprayed on the tomato leaves under weak light conditions, and the concentration of the Rhodopseudomonas palustris is 10 7 ~10 9 cfu / mL.

[0011] In the above application, the preparation method of the Rhodopseudomonas palustris bacterial solution comprises the following steps: (1) Take 20 µL of Rhodopseudomonas palustris CGA009 strain and streak on the lower solid culture medium, and stand for 2-3 min; after sterilization, cool the upper solid culture medium to 40°C, pour it into the lower solid culture medium, and seal after cooling and solidification. Culture for 5-7 days to obtain red single colonies; the culture conditions are: temperature 30-35°C, light intensity 3000-4000 lx; (2) Inoculate the red single colonies into the photosynthetic bacterial liquid medium and culture to the logarithmic growth phase to obtain the first photosynthetic bacterial solution; the culture conditions are: temperature 30-35°C, light intensity 3000-4000 lx, anaerobic conditions; (3) The first photosynthetic bacterial solution is inoculated into fresh photosynthetic bacterial liquid medium at a ratio of 1:100 and cultured to the logarithmic growth phase to obtain the second photosynthetic bacterial solution; the culture conditions of the photosynthetic bacterial liquid medium are: temperature 30-35°C, light intensity 3000-4000 lx, anaerobic conditions; (4) Centrifuge the second photosynthetic bacterial solution, collect the Rhodopseudomonas palustris bacterial pellet, wash the bacterial pellet with 0.9% NaCl solution, and resuspend to OD 600nm =1 to obtain the Rhodopseudomonas palustris bacterial solution; The formula of the upper solid culture medium is: peptone 5 g / L, beef extract 1.5 g / L, yeast extract 1.5 g / L, K2HPO4 4.8 g / L, KH2PO4 1.3 g / L, trace metal element stock solution 1.0 ml / L, and agar 18 g / L, with pH value of 7.0-7.2; The formula of the lower solid culture medium is: peptone 5 g / L, beef extract 1.5 g / L, yeast extract 1.5 g / L, K2HPO4 4.8 g / L, KH2PO4 1.3 g / L, trace metal element stock solution 1.0 ml / L, and agar 13 g / L.

[0012] The formula of the photosynthetic bacterial liquid medium is: peptone 5 g / L, beef extract 1.5 g / L, yeast extract 1.5 g / L, K2HPO4 4.8 g / L, KH2PO4 1.3 g / L, trace metal element stock solution 1.0 ml / L, and pH value of 7.0-7.2; Trace metal stock solution: EDTA 2.5g, ZnSO4 6.13g, MgSO4 0.78g, CuSO4 0.25g, CoCl2 0.08g, FeSO4·7H2O 7.0g were dissolved in 1L distilled water.

[0013] In the above application, preferably, in step (4), the obtained suspension after resuspension is diluted 10 times to obtain the Rhodopseudomonas palustris bacterial solution.

[0014] In the above application, preferably, the Rhodopseudomonas palustris bacterial solution is used once every 7 days.

[0015] In the above application, preferably, the centrifugation condition is 5000 rpm for 8 min.

[0016] The Rhodopseudomonas palustris strain is activated, fermented, centrifuged, and resuspended to prepare the Rhodopseudomonas palustris bacterial solution, and the concentration of the Rhodopseudomonas palustris is 10 8 The Rhodopseudomonas palustris solution is used for exogenous leaf spraying treatment of the tomato plants under weak light conditions. After the treatment, the growth of the tomato plants is detected, and the photosynthetic physiological indexes of the leaves are determined. The Rhodopseudomonas palustris treatment is implemented once every 7 days, and the physiological indexes are determined 3 days after the third treatment.

[0017] The results show that under weak light conditions, the Rhodopseudomonas palustris can significantly promote the photosynthesis of the tomato leaves. Compared with the tomato leaves not sprayed with the Rhodopseudomonas palustris solution under weak light conditions, the chlorophyll, net photosynthetic rate, and maximum photochemical efficiency of photosystem II of the tomato leaves treated with the Rhodopseudomonas palustris solution are significantly improved.

[0018] The beneficial effects of the application are as follows: The Rhodopseudomonas palustris CGA009 provided by the application is simple to culture, low in cost, and convenient to handle, can effectively improve the photosynthesis of the tomato leaves under weak light, and is environmentally friendly.

[0019] The Rhodopseudomonas palustris is used for the first time in the application to improve the photosynthesis of the tomato leaves under weak light and delay the leaf senescence. The results show that the Rhodopseudomonas palustris can improve the chlorophyll content of the tomato leaves and significantly improve the photosynthetic capacity of the leaves, indicating that the Rhodopseudomonas palustris has application value in improving the response of the tomato to weak light stress. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1NL group, WL group, and WL+R.p 10 9 NL group, WL group, and WL+R.p 10 8 NL group, WL group, and WL+R.p 10 7 NL group, WL group, and WL+R.p 10 Figure 2 NL group, WL group, and WL+R.p 10 8 NL group, WL group, and WL+R.p 10 Figure 3 NL group, WL group, and WL+R.p 10 8 NL group, WL group, and WL+R.p 10 Figure 4 NL group, WL group, and WL+R.p 10 8 NL group, WL group, and WL+R.p 10 DETAILED DESCRIPTION

[0021] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application will be further described below in combination with specific embodiments.

[0022] Experimental material used in the present application 1. Test strain R. palustris CGA009, Latin name: Rhodopseudomonas palustris CGA009 is a model strain, which is purchased from Shanghai Jiachu Biological Engineering Co., Ltd., and the strain number is SHMCC (SHBCC) D25179.

[0023] 2. Culture medium and its formula The photosynthetic bacterial double-layer solid culture medium is composed of an upper solid culture medium and a lower solid culture medium; The upper solid culture medium: 5 g / L of proteose peptone, 1.5 g / L of beef extract, 1.5 g / L of yeast extract, 4.8 g / L of K2HPO4, 1.3 g / L of KH2PO4, 1.0 ml / L of trace metal element stock solution, and 18 g / L of agar, with a pH value of 7.0-7.2; The lower solid culture medium: 5 g / L of proteose peptone, 1.5 g / L of beef extract, 1.5 g / L of yeast extract, 4.8 g / L of K2HPO4, 1.3 g / L of KH2PO4, 1.0 ml / L of trace metal element stock solution, and 13 g / L of agar.

[0024] The photosynthetic bacterial liquid culture medium: 5 g / L of proteose peptone, 1.5 g / L of beef extract, 1.5 g / L of yeast extract, 4.8 g / L of K2HPO4, 1.3 g / L of KH2PO4, 1.0 ml / L of trace metal element stock solution, and a pH value of 7.0-7.2; Trace element stock: EDTA 2.5 g, ZnSO4 6.13 g, MgSO4 0.78 g, CuSO4 0.25 g, CoCl2 0.08 g, FeSO4·7H2O 7.0 g in 1 L distilled water.

[0025] 3. Test seeds Tomato variety Ailsa Craig (AC) was purchased from the Chinese Academy of Agricultural Sciences, Institute of Vegetables and Flowers.

[0026] Example 1

[0027] The Rhodopseudomonas palustris bacterial solution was obtained by culturing Rhodopseudomonas palustris CGA009, and the culture method included the steps of activation, production culture, and suspension preparation.

[0028] Specifically, the preparation method of the Rhodopseudomonas palustris bacterial solution includes the following steps: (1) Take 20 μL of the preserved Rhodopseudomonas palustris CGA009 strain and streak it on the lower solid culture medium, and let it stand for 2-3 min; after sterilization, cool the upper solid culture medium to 40°C, pour it onto the lower solid culture medium, and seal it after cooling and solidification. After 5 days of culture, a red single colony is obtained; the culture conditions are: temperature 30-35°C, light intensity 3000-4000 lx.

[0029] (2) The red single colony (1 mm in diameter) obtained in step (1) is inoculated into a serum bottle with 5 mL of photosynthetic bacterial liquid medium, and cultured to the logarithmic growth phase, with the logarithmic growth phase being OD 600nm =0.6-0.8; the culture conditions of the photosynthetic bacterial liquid medium are: temperature 30-35°C, light intensity 3000-4000 lx, and anaerobic conditions.

[0030] (3) The bacterial solution obtained in step (2) is inoculated into fresh photosynthetic bacterial liquid medium at a ratio of 1:100 and cultured to the logarithmic growth phase, with the logarithmic growth phase being OD 600nm =0.6-0.8; the culture conditions of the photosynthetic bacterial liquid medium are: temperature 30-35°C, light intensity 3000-4000 lx, and anaerobic conditions.

[0031] (4) The bacterial solution obtained in step (3) is centrifuged at a speed of 5000 rpm for 8 min, and the Rhodopseudomonas palustris bacterial pellet is collected and washed with 0.9% NaCl solution, then resuspended to OD 600nm =1 (10 9 cfu / mL) to obtain the Rhodopseudomonas palustris bacterial solution.

[0032] The prepared Rhodopseudomonas palustris bacterial suspension was divided into three portions. One portion was left untreated, while the other two portions were diluted 10 times and 100 times with 0.9% NaCl solution, respectively, for later use.

[0033] Experimental Example 1 Select plump tomato seeds, soak them in warm water at 55℃ to promote germination, and sow them in seedling trays after they sprout. Transplant the tomato seedlings that have grown to the four-leaf-one-heart stage into nutrient pots and place them in a light incubator for 7 days to allow them to recover. The cultivation conditions of the light incubator are: temperature 26℃ / 18℃ (day / night) and photoperiod 16h / 8h (day / night).

[0034] Tomato plants with good growth and uniform growth were selected and randomly divided into 5 groups of 20 plants each. Each group had 3 replicates. The experimental grouping and treatment are shown in Table 1.

[0035] Table 1 Group Treatment NL Under normal light, the tomato leaves were sprayed with clean water, and the liquid droplets were evenly distributed on the front and back of the leaves but did not drip WL Under weak light, the tomato leaves were sprayed with clean water, and the liquid droplets were evenly distributed on the front and back of the leaves but did not drip WL+R.p 10 9 ]]> Under weak light, the tomato leaves were sprayed with the undiluted Rhodopseudomonas palustris bacterial solution of Example 1, and the liquid droplets were evenly distributed on the front and back of the leaves but did not drip WL+R.p 10 8 ]]> Under weak light, the tomato leaves were sprayed with the 10-fold diluted Rhodopseudomonas palustris bacterial solution of Example 1, and the liquid droplets were evenly distributed on the front and back of the leaves but did not drip WL+R.p 10 7 ]]> Under weak light, the tomato leaves were sprayed with the 100-fold diluted Rhodopseudomonas palustris bacterial solution of Example 1, and the liquid droplets were evenly distributed on the front and back of the leaves but did not drip As shown in Table 1, after the seedling establishment period, except for the NL group which was subjected to normal light (1000 µmol·m⁻¹), -2 ·s -1 In addition to the above, the remaining four groups were covered with shade nets with a 70% shading rate to simulate a low-light environment, WL+Rp 10. 9 Group, WL+Rp 10 8 Group and WL+Rp 10 7 Different concentrations of Rhodopseudomonas palustris bacterial solution were sprayed on the leaves of the tomato groups. The treatment standard was that the liquid droplets were evenly distributed on both sides of the tomato but did not drip. The leaves of the NL and WL groups were treated with water according to the same treatment standard. Spraying was carried out once every 7 days, for a total of 3 treatments. Samples were taken 3 days after the last treatment. The leaf sampling standard was the 7th fully unfolded functional leaf above the first true leaf of the tomato.

[0036] Figure 1 For NL group, WL group, WL+Rp 10 9 Group, WL+Rp 10 8 Group and WL+Rp 10 7 A schematic diagram showing the yellowing of the 7th leaf of a tomato plant. From... Figure 1 It can be seen that, compared with the WL group of tomato leaves, foliar spraying with *Rhodopseudomonas palustris* significantly inhibited leaf yellowing caused by low light. (WL+Rp 10) 8 The group can significantly inhibit leaf yellowing.

[0037] Take NL group, WL group and WL+Rp 10 8 The 7th leaf of the tomato plant was used to test the chlorophyll content, gas exchange parameters, and chlorophyll fluorescence parameters. The test results are as follows: Figures 2-3The test method is as follows: Determination of chlorophyll content: Take 0.5 g of the 7th leaf, grind in liquid nitrogen, and add 10 mL of 80% (v / v) acetone solution, shake and mix for 15 s. Rotate extraction at 4°C for 12 hours. Centrifuge at 4°C, 3000 rpm for 10 min. Take the supernatant and measure the absorbance value at 645 nm and 663 nm wavelengths. Calculate the total chlorophyll content according to the absorbance value, as follows: C (mg·g -1 )=20.21×OD 645nm +8.02×OD 663nm Total Chl content (mg·g -1 )=(C×V×n) / W Wherein, C represents the total chlorophyll concentration (mg·g -1 ), V represents the total volume of extraction (L), n represents the dilution multiple, and W represents the fresh weight of the sample (g).

[0038] Determination of gas exchange parameter Pn: On the third day after the third treatment, three tomato plants with similar growth characteristics were selected from each treatment group. The net photosynthetic rate (Pn) of the 7th functional leaf above the 1st true leaf of the tomato was measured using the CIRAS-2 portable photosynthesis system of the British PP System Company. The instrument settings are as follows: temperature is 25°C, light intensity is 1000 μmol·m -2 ·s -1 , ambient carbon dioxide concentration, and relative humidity is 75%.

[0039] Determination of chlorophyll fluorescence parameter Fv / Fm: The chlorophyll fluorescence parameter of the leaf was determined by using the modulation chlorophyll fluorescence imager IMAGIN-PAM (Germany Heinz WaItz). Three tomatoes were randomly selected from each treatment, and after 30 minutes of dark adaptation, the third fully expanded functional leaf was cut, unfolded and fixed on the measurement table of the fluorometer. The detection parameters of the fluorescence imager are set as follows: measurement light intensity 0.1 μmol·m -2 ·s -1 , photochemical light intensity 81 μmol·m -2 ·s -1 , saturation pulse light intensity 2700 μmol·m -2 ·s -1 , pulse light time 0.8 s. The saturation pulse light is played every 20 s, a total of 15 times. Read and analyze the light adaptation fluorescence parameter value measured under the last saturation pulse light.

[0040] Figure 2 NL group, WL group and WL+R.p 108 The test results of the total chlorophyll content of the 7th leaf of the tomato. Figure 2 It can be seen that, compared with normal light, the chlorophyll content of the leaf under weak light decreased significantly; compared with the WL group, the chlorophyll content of the leaf sprayed with the marsh red pseudomonas under weak light increased significantly.

[0041] Figure 3 The NL group, the WL group and the WL+R.p 10 8 The test results of the net photosynthetic rate of the 7th leaf of the tomato. Figure 3 As shown in the table, under weak light, the Pn of the tomato plant decreased significantly compared with normal light; and after spraying the marsh red pseudomonas on the leaf, the Pn increased significantly. It is shown that the photosynthetic bacteria marsh red pseudomonas has a very good effect on improving the photosynthesis of the tomato leaf under weak light.

[0042] Figure 4 The NL group, the WL group and the WL+R.p 10 8 The test results of the maximum photochemical efficiency (Fv / Fm) of the photosystem II of the 7th leaf of the tomato. Figure 4 As shown in the table, under weak light, the Fv / Fm of the tomato plant decreased significantly compared with normal light; and after spraying the marsh red pseudomonas on the leaf, the Fv / Fm increased significantly. It is shown that the photosynthetic bacteria marsh red pseudomonas has a very good effect on improving the photosynthesis of the tomato leaf under weak light.

[0043] The present application simulates the weak light environment of the sunlight greenhouse by covering the shading net, and sprays the marsh red pseudomonas on the leaf of the tomato plant. After 24 days of treatment, it is found that the marsh red pseudomonas can improve the chlorophyll content and photosynthetic efficiency of the leaf of the tomato plant under weak light. Spraying the marsh red pseudomonas on the leaf has a significant promoting effect on the photosynthesis of the tomato leaf under weak light.

[0044] The main features and advantages of the present application are shown and described above, and for those skilled in the art, the details are not limited to the above-mentioned embodiments, and the present application can be realized in other specific forms without departing from the essential characteristics of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Rhodopseudomonas palustris for use in promoting photosynthesis of tomato leaves under weak light.

2. Use according to claim 1, characterized in that, Light conditions for low light conditions: 300 pmol m -2 ·s -1 .

3. Use according to claim 1, characterized in that, The weak light condition is simulated by using a sunshade net.

4. Use according to claim 1, characterized in that, for increasing the chlorophyll content, net photosynthetic rate and / or maximum photochemical efficiency of photosystem II of tomato leaves.

5. The use according to claim 1, characterized in that, The Rhodopseudomonas palustris is Rhodopseudomonas palustris CGA009.

6. Use according to claim 1, characterized in that, In weak light conditions, the Rhodopseudomonas palustris bacterial solution was sprayed on the tomato leaves, and the concentration of Rhodopseudomonas palustris was 10 7 ~10 9 cfu / mL.

7. Use according to claim 6, characterized in that, A preparation method of the Rhodopseudomonas palustris bacterial liquid comprises the following steps: (1) 20 μL of Rhodopseudomonas palustris CGA009 strain is streaked on a lower solid culture medium and is left to stand for 2-3 min; the upper solid culture medium is sterilized and then cooled to 40℃, and is poured into the lower solid culture medium, and is sealed after being cooled and solidified, and is cultured for 5-7 days to obtain a red single colony; the culture conditions are as follows: a temperature of 30-35℃ and a light intensity of 3000-4000 lx; (2) the red single colony is inoculated into a photosynthetic bacterial liquid medium and is cultured to the logarithmic growth phase to obtain a first photosynthetic bacterial liquid; the culture conditions are as follows: a temperature of 30-35℃, a light intensity of 3000-4000 lx and anaerobic conditions; (3) the first photosynthetic bacterial liquid is inoculated into fresh photosynthetic bacterial liquid medium at a ratio of 1:100 and is cultured to the logarithmic growth phase to obtain a second photosynthetic bacterial liquid; the culture conditions of the photosynthetic bacterial liquid medium are as follows: a temperature of 30-35℃, a light intensity of 3000-4000 lx and anaerobic conditions; (4) centrifuging the second photosynthetic bacterial liquid, collecting the Rhodopseudomonas palustris bacterial block, washing the bacterial block with 0.9% NaCl solution, and resuspending to OD 600nm =1 to obtain a Rhodopseudomonas palustris bacterial liquid; the upper solid culture medium has a formula of 5 g / L of proteose peptone, 1.5 g / L of beef extract, 1.5 g / L of yeast extract, 4.8 g / L of K2HPO4, 1.3 g / L of KH2PO4, 1.0 ml / L of trace metal element stock solution and 18 g / L of agar, and has a pH value of 7.0-7.2; the lower solid culture medium has a formula of 5 g / L of proteose peptone, 1.5 g / L of beef extract, 1.5 g / L of yeast extract, 4.8 g / L of K2HPO4, 1.3 g / L of KH2PO4 and 13 g / L of agar; the photosynthetic bacterial liquid medium has a formula of 5 g / L of proteose peptone, 1.5 g / L of beef extract, 1.5 g / L of yeast extract, 4.8 g / L of K2HPO4, 1.3 g / L of KH2PO4 and 1.0 ml / L of trace metal element stock solution, and has a pH value of 7.0-7.2; the trace metal element stock solution is prepared by dissolving 2.5 g of EDTA, 6.13 g of ZnSO4, 0.78 g of MgSO4, 0.25 g of CuSO4, 0.08 g of CoCl2 and 7.0 g of FeSO4·7H2O in 1 L of distilled water.

8. Use according to claim 7, characterized in that, In step (4), the obtained suspension is diluted 10 times after resuspension to obtain the Rhodopseudomonas palustris bacterial liquid.

9. Use according to claim 8, characterized in that, The Rhodopseudomonas palustris bacterial liquid is used once every 7 days.

10. The use of Rhodopseudomonas palustris according to claim 7 for promoting photosynthesis in tomato leaves, characterized in that, The centrifugation is performed at a speed of 5000 rpm for 8 min.

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