A method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi

Abstract

This invention relates to the field of sweet potato cutting cultivation technology, specifically a method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi, comprising the following steps: obtaining a Diaporthe ueckerae spore suspension and diluting it to 1×10⁻⁶. 6 The inoculum was prepared by inoculating sweet potato cuttings with a diameter of 0.5-0.7 mm at the cut end, leaving a node 1 cm above the cut. The cuttings were then immersed in 150 ml of Hoagland's nutrient solution for 6 hours to allow them to recover. After recovery, the cuttings were further immersed in 150 ml of Diaporthe ueckerae spore inoculum for 6 hours for inoculation. The inoculated cuttings were then planted in soil with a NaCl content of 3 g / kg. Inoculating sweet potato cuttings with Diaporthe ueckerae effectively improves their salt tolerance, preventing growth inhibition by saline-alkali soil. Furthermore, the endophytic fungus Diaporthe ueckerae is easy to obtain at low cost, and the inoculation process is simple, thus not significantly increasing the planting cost of sweet potato cuttings and meeting the demand for salt-tolerant sweet potato cuttings in saline-alkali areas.

Description

Technical Field

[0001] This invention relates to the field of sweet potato cutting cultivation technology, specifically a method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi. Background Technology

[0002] Sweet potato, also known as yam or sweet potato, is a perennial vine-like herbaceous plant belonging to the genus Ipomoea in the Convolvulaceae family. With its large tubers and high yield, sweet potato is one of the world's major food crops. Some improved tuber-derived sweet potato varieties, due to their well-developed stems and leaves, tender and crisp texture, and rich content of various proteins, vitamins, and minerals, have also made sweet potato leaves a popular leafy vegetable. Sweet potato vines readily produce fibrous roots, and based on this characteristic, sweet potatoes are mainly propagated through vine cuttings. Sweet potato vines have a strong regenerative ability, and their tender stems and leaves can be harvested and consumed repeatedly during the growing season. Therefore, the annual yield of sweet potato leaves is relatively high, bringing significant economic benefits to farmers.

[0003] However, sweet potato cuttings are not very tolerant to salt stress. When the soil salinity exceeds 2 g / kg, the rooting ability and survival rate of sweet potato vine cuttings decrease significantly, which greatly limits the cultivation and planting of sweet potatoes in vast saline-alkali areas. In order to adapt sweet potato cuttings to saline-alkali soils and expand the planting scale of sweet potatoes, it is necessary to improve the salt tolerance of sweet potato cuttings. Breeding salt-tolerant sweet potato varieties is one of the methods to improve the salt tolerance of sweet potato cuttings. However, this method is technically demanding, requires a large investment, and is time-consuming, which makes the breeding technology of salt-tolerant sweet potato varieties relatively slow. Therefore, a new technology is urgently needed to improve the salt tolerance of sweet potato cuttings. Although a large number of studies have shown that endophytic fungi can improve the salt tolerance of plants, so far there is no literature to report any endophytic fungi that can improve the salt tolerance of sweet potato cuttings. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi, comprising the following steps:

[0005] Obtain a Diaporthe ueckerae spore suspension and dilute it to 1×10⁻⁶. 6 1 cell / ml, as the inoculum;

[0006] Obtain sweet potato cuttings with a cut diameter of 0.5-0.7mm, retaining a node 1cm above the cut end, and insert them into 150ml of Hoagland nutrient solution for 6 hours to allow them to recover.

[0007] After the seedlings have recovered from transplant shock, sweet potato cuttings are inoculated by soaking them in 150ml of Diaporthe ueckerae spore inoculation solution for 6 hours.

[0008] After inoculation, sweet potato cuttings were inserted into soil with a NaCl content of 3 g / kg.

[0009] Preferably, the Diaporthe ueckerae strain was obtained from the *Diaporthe ueckerae* plant, and the method of obtaining it was as follows:

[0010] Healthy petals were collected from the *Gynostemma pentaphyllum* plant, washed with tap water, and then placed in a laminar flow hood and immersed in 75% alcohol for 0.5 minutes, followed by immersion in 5% NaCl for 8 minutes. After removing the petals, they were immersed in 75% alcohol for 0.5 minutes. They were then removed from the laminar flow hood, rinsed three times with sterile water, and dried on filter paper. The petals were cut into small pieces and inoculated onto sterile PDA medium with a NaCl content of 3 g / L. The culture dishes were placed in a 26°C incubator for 7 days of dark incubation. The mycelia growing from the edges of the petal pieces were then transferred to sterile PDA medium without NaCl and placed in a 26°C incubator for 7 days of dark incubation to obtain the strain with colony morphology.

[0011] Preferably, after obtaining the strain, the strain is propagated and cultured. The culture steps are as follows:

[0012] Take a 0.5 cm diameter mycelial cake from the bacterial colony and transfer it to a sterile PDA medium without NaCl for culture. Transfer one small piece of mycelial colony to each PDA medium. Seal the culture dish with sealing film and incubate in the dark at 26°C for 7 days.

[0013] The preferred method for obtaining Diaporthe ueckerae spore suspension is as follows:

[0014] The colony-forming strains were transferred from PDA medium to sterile PDB medium without NaCl, and then incubated at 26°C in a dark incubator for 48 hours. The mycelia were then filtered through three layers of sterile gauze to obtain a Diaporthe ueckerae spore suspension.

[0015] Preferably, a hemocytometer is used to count the spore concentration in the spore suspension.

[0016] Preferably, the sweet potato cuttings are inserted into soil with a NaCl content of 3 g / kg at a depth of 6 cm.

[0017] Preferably, after the sweet potato cuttings are planted, 100ml of deionized water is poured in.

[0018] The present invention has the following beneficial effects:

[0019] By inoculating sweet potato cuttings with Diaporthe ueckerae, the salt tolerance of the cuttings can be effectively improved, and the growth environment of the cuttings can be freed from the inhibition of saline-alkali soil. Moreover, the endophytic fungus Diaporthe ueckerae is easy to obtain and has low cost, and the inoculation process is also simple. Therefore, it will not significantly increase the planting cost of sweet potatoes, and can meet the demand for salt-tolerant sweet potato cuttings in saline-alkali areas, thus expanding the scale of sweet potato planting. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Diaporthe ueckerae colony provided by the present invention.

[0021] Figure 2 This is a statistical chart showing whether Diaporthe ueckerae has halophilicity, as provided in this invention.

[0022] Figure 3 This is a diagram showing the growth results of the sweet potato cuttings provided by this invention under salt stress.

[0023] Figure 4 This is a survival data graph of sweet potato cuttings provided by the present invention under salt stress.

[0024] Figure 5 This is a diagram showing the experimental results of whether Diaporthe ueckerae, provided by this invention, causes disease in sweet potato cuttings. Specific Implementation

[0025] The principles and steps of the present invention are described below. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In one specific embodiment, such as Figure 1-5 As shown, a method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi includes the following steps:

[0028] Obtain a Diaporthe ueckerae spore suspension and dilute it to 1×10⁻⁶. 6 1 cell / mL, as the inoculum;

[0029] Obtain sweet potato cuttings with a cut diameter of 0.5-0.7mm, retaining a node 1cm above the cut end, and insert them into 150ml of Hoagland nutrient solution for 6 hours to allow them to recover.

[0030] After the seedlings have recovered from transplant shock, sweet potato cuttings are inoculated by soaking them in 150ml of Diaporthe ueckerae spore inoculation solution for 6 hours.

[0031] After inoculation, sweet potato cuttings were inserted into soil with a NaCl content of 3 g / kg.

[0032] In this embodiment, the Diaporthe ueckerae strain was obtained from the plant *Gynostemma pentaphyllum*. *Gynostemma pentaphyllum* is an invasive plant, and its acquisition method is simple and low-cost. Furthermore, it is a natural host plant for endophytic fungi, eliminating the need for additional artificial cultivation, making it relatively cost-effective. The method for obtaining the Diaporthe ueckerae strain is as follows:

[0033] Healthy petals were collected from the *Pterocarya stenoptera* plant, washed thoroughly with tap water, and then immersed in 75% alcohol for 0.5 minutes in a laminar flow hood. Next, they were immersed in 5% NaClO for 8 minutes. After this initial immersion, the petals were again immersed in 75% alcohol for 0.5 minutes. The petals were then removed from the laminar flow hood and rinsed three times with sterile water before being air-dried on filter paper. The petals were then cut into 0.5cm x 0.5cm pieces and inoculated onto sterile PDA medium (potato dextrose agar) with a NaCl concentration of 3 g / L. The A culture medium was prepared with the following ratio: 200g potato, 20g glucose, 15g agar, 0.1g streptomycin sulfate, and 1000mL deionized water. Three petal pieces were inoculated onto each PDA medium, for a total of three PDA mediums. The petri dishes were then covered and sealed with sealing film. The petri dishes containing sterile PDA medium were placed in a 26°C incubator for 7 days of dark incubation. The development of mycelium at the edges of the petal pieces on the PDA medium was observed. In this example, mycelium grew on the edge of one petal piece on one of the three PDA mediums. The petal piece with mycelium growth was transferred to sterile PDA medium without NaCl and incubated in a 26°C incubator for 7 days. The resulting bacterial strain exhibited the following colony morphology: Figure 1 As shown.

[0034] To verify the accuracy of the bacterial species, ITS sequencing was performed on the strain. The primers used were ITS1f / ITS4r (ITS1f 5′-CTTGGTCATTTAGAGGAAGTAA-3′, ITS4r 5′-TCCTCCGCTTATTGATATGC-3′), and the obtained ITS sequence was 5′-GGGGTAGTCTCGTTGGTGACCAGCGGAGGGATCATTGCTGGAACGCG-3′. Similarity comparison of this sequence with known bacterial species sequences showed a 100% similarity, thus confirming that the endophytic strain is *Diaporthe ueckerae*.

[0035] In this embodiment, after obtaining the strain, the strain is propagated and cultured to form more treatment groups and control groups. The culture steps are as follows:

[0036] A 0.5 cm diameter mycelial disc was taken from the bacterial colony and transferred to a sterile PDA medium without NaCl for culture. One colony disc was transferred to each PDA medium, for a total of 20 PDA mediums. The culture dishes were sealed with sealing film and placed in a 26°C incubator for dark culture for 7 days to propagate 20 Diaporthe ueckerae colonies.

[0037] In this embodiment, a test was also conducted to determine whether the Diaporthe ueckerae strain exhibits halophilicity. The specific experimental procedure and results are as follows:

[0038] A salt gradient was set up using NaCl, and PDB culture media with NaCl concentrations of 0 g / L, 3 g / L, and 5 g / L were prepared respectively. The preparation ratio of the PDB culture media was 200 g potato, 20 g glucose, and 1000 mL deionized water. The PDB culture media were sterilized in an autoclave at a temperature of 121 °C for 20 min, cooled to room temperature, and then poured into 250 mL Erlenmeyer flasks, with each Erlenmeyer flask containing 100 mL. There were three Erlenmeyer flasks for each salt concentration of PDB culture media, for a total of nine Erlenmeyer flasks. Inside a clean bench, 5mm diameter mycelial pellets were aseptically punched from the edge of Diaporthe ueckerae colonies and inoculated into PDB medium in nine conical flasks. The flasks were sealed with sealing film and placed on a constant-temperature shaker at 250 rpm and 26°C to ensure sufficient contact between the medium and oxygen, thus increasing the dissolved oxygen supply. Three biological replicates were performed for each NaCl concentration condition to ensure a more rigorous experiment. After 60 hours, the fungal culture was filtered through four layers of gauze, and mycelial pellets were collected. The pellets were rinsed with purified water to remove surface fermentation broth and placed on pre-weighed petri dishes. These dishes were then dried in a constant-temperature oven at 80°C until constant weight. After cooling, the pellets were weighed using an analytical balance. The biomass of the strain was calculated by subtracting the pre-weighed petri dish from the constant weight, and the average value of the three biological replicates was calculated. Analysis of variance was performed, and the LSD method was used to detect the significance of differences in bacterial biomass under salt concentrations of 0 g / L, 3 g / L, and 5 g / L. Figure 2 As shown, Figure 2 The median is the mean ± standard error, and different letters indicate significant differences (P < 0.005). Figure 2It can be seen that the biomass of Diaporthe ueckerae in PDB medium with NaCl concentrations of 3 g / L and 5 g / L is significantly higher than that in PDB medium with NaCl concentration of 0 g / L, and the biomass is highest in PDB medium with NaCl concentration of 5 g / L. This result indicates that Diaporthe ueckerae is halophilic.

[0039] In this embodiment, the Diaporthe ueckerae spore suspension was prepared by transferring Diaporthe ueckerae colonies cultured for 7 days from PDA medium to sterile PDB medium without NaCl in a laminar flow hood, and then incubating them at 26°C in a dark incubator for 48 hours. The fungal culture was filtered through three layers of sterile gauze to remove hyphae, and the resulting filtrate was the Diaporthe ueckerae spore suspension. The spore concentration of the suspension was counted using a hemocytometer, and the spore concentration was diluted to 1×10⁻⁶ with sterile PDB. 6 Diaporthe ueckerae spore inoculum was obtained by dividing the spores by 150 ml, with each inoculum containing 150 ml, for a total of ten inoculum.

[0040] In this embodiment, 20 sweet potato cuttings were collected from the field. The upper part of the stem of a healthy sweet potato plant was cut with scissors, retaining the central leaf and one leaf. The cuttings were approximately 13-15 cm long, with a cut diameter of approximately 0.5-0.7 mm at the lower end. A node was retained 1 cm above the cut to maintain similar viability. The prepared cuttings were then inserted into 150 ml of Hoaglandia nutrient solution for 6 hours to allow them to acclimate.

[0041] In this embodiment, to compare the growth of sweet potato cuttings with those not inoculated with *Diaporthe ueckerae* spores, 10 sweet potato cuttings were immersed in 150 ml of *Diaporthe ueckerae* spore inoculation solution as the treatment group, and the remaining 10 sweet potato cuttings were immersed in 150 ml of Hogland's nutrient solution as the control group. In both the treatment and control groups, the cuttings were immersed in either the Hogland's nutrient solution or the *Diaporthe ueckerae* inoculation solution to a depth of 6 cm, and the inoculation time was 6 hours, resulting in 10 inoculated cuttings and 10 uninoculated cuttings.

[0042] In this embodiment, when preparing the soil, in order to test the growth status of sweet potato cuttings under various salt concentrations, soil was first collected from non-saline-alkali fields and air-dried. The air-dried soil was divided into two parts, and NaCl was added to each part. The NaCl content of the two parts of soil was adjusted to 3g / kg and 5g / kg, respectively, to form saline-alkali soil as the culture medium for sweet potato cuttings.

[0043] In this embodiment, before cutting, 3g / kg and 5g / kg of saline-alkali soil were filled into 12×12cm plastic flowerpots, with a soil filling thickness of 10cm. Then, the 10 inoculated sweet potato cuttings were inserted into two different culture media with NaCl contents of 3g / kg and 5g / kg, with 5 cuttings in each media and 1 cutting in each flowerpot. Similarly, for comparison, 10 uninoculated sweet potato cuttings were inserted into two different culture media with NaCl contents of 3g / kg and 5g / kg, with 5 cuttings in each media and 1 cutting in each flowerpot. All cuttings were inserted to a depth of approximately 6cm. Finally, 100ml of deionized water was added to each flowerpot, and all cuttings were placed at room temperature and under normal light conditions for cultivation. Therefore, under NaCl stress conditions of 3 g / kg and 5 g / kg, both the inoculated and uninoculated treatments of sweet potato cuttings contained 5 biological replicates.

[0044] In this embodiment, after 5 days of cultivation, cuttings from all inoculated treatments and non-inoculated treatments under the above-mentioned salt stress conditions were harvested and washed clean with tap water. The growth results are as follows. Figure 3 As shown, Figure 3 In the figures, (a) shows the growth of sweet potato cuttings inoculated with 3 g / kg NaCl, (b) shows the growth of sweet potato cuttings inoculated with 5 g / kg NaCl, (c) shows the growth of sweet potato cuttings without inoculation under 3 g / kg NaCl, and (d) shows the growth of sweet potato cuttings without inoculation under 5 g / kg NaCl. Under 3 g / kg NaCl stress, the stems of the uninoculated sweet potato cuttings rotted and the leaves turned yellow, while the stems of the inoculated sweet potato cuttings did not rot and the leaves remained green. Under 5 g / kg NaCl stress, the stems of the uninoculated sweet potato cuttings rotted severely and the leaves withered severely, while the leaves of the inoculated sweet potato cuttings withered, but the stems did not rot and fibrous roots still developed. This shows that the salt damage suffered by the inoculated sweet potato cuttings was significantly lower than that of the uninoculated sweet potato cuttings.

[0045] In this embodiment, detailed data on growth results were recorded to facilitate more rigorous comparisons. The number of fibrous roots, number of new leaves, and ratio of stem decay length between inoculated and uninoculated sweet potato cuttings were measured, and the average of five biological replicates was calculated. Analysis of variance was performed, and independent samples tests were used to detect the significant differences in the number of fibrous roots, number of green leaves, and ratio of stem decay length between inoculated and uninoculated sweet potato cuttings under NaCl concentrations of 3 g / L and 5 g / L (P < 0.05). The results are as follows: Figure 4 As shown, Figure 4 In the diagram, E+ represents sweet potato cuttings treated with inoculation, and E- represents sweet potato cuttings treated without inoculation. It can be seen that under soil stress of 3 g / kg NaCl concentration, the number of green leaves and fibrous roots of E+ cuttings were significantly higher than those of E- cuttings, while the stem decay length ratio was significantly lower. Under soil stress of 5 g / kg NaCl concentration, there was no significant difference in the number of green leaves between E+ and E- cuttings, but the number of fibrous roots of E+ cuttings was significantly higher than that of E- cuttings, and the stem decay length ratio was significantly lower than that of E- cuttings. Therefore, it can be concluded that inoculation with the endophytic fungus *Diaporthe ueckerae* can improve the salt tolerance of sweet potato cuttings.

[0046] Compared to E+ cuttings grown under soil stress of 5 g / kg NaCl concentration, E+ cuttings grown under 3 g / kg NaCl stress did not exhibit any salt damage symptoms. Therefore, sweet potato cuttings inoculated with the endophytic fungus Diaporthe ueckerae are suitable for planting and cultivation in saline-alkali areas with soil salt content of approximately 3 g / kg.

[0047] To investigate whether the endophytic fungus Diaporthe ueckerae causes disease in sweet potato cuttings and affects their subsequent growth, an experiment was conducted. The specific verification method was as follows:

[0048] In the field, use scissors to cut the upper part of the stem of healthy sweet potato plants as cuttings. Each cutting should retain one central leaf and one leaf blade, and be approximately 13-15 cm long. The diameter of the cut at the lower end of the cutting should be approximately 0.5-0.7 mm, and a node should be retained 1 cm above the cut. Insert the cuttings into 150 ml of Hoagland's nutrient solution for 6 hours to allow them to acclimate. Spray the upper and lower surfaces of the leaves of the sweet potato cuttings with 1 ml of Diaporthe ueckerae spore inoculum as the inoculation treatment group. Additionally, spray the upper and lower surfaces of the remaining sweet potato cuttings with 1 ml of sterile PDB as the control group. The treatment and control groups were cultured separately in their respective light incubators. The temperature in both incubators was 25±1℃, the relative humidity was 85±2%, the light / dark cycle was 14h / 10h, and the light intensity was 200 μmol·m⁻²·s⁻¹. After 5 days, using the disease-free control group as a reference, check the leaves of the treatment groups for disease occurrence. Figure 5As shown, Figure 5 In the diagram, (a) represents the treatment group and (b) represents the control group. It can be seen that, like the control group, the leaves of both groups remained healthy and showed no signs of disease. This confirms that the Diaporthe ueckerae strain is not pathogenic to sweet potato cuttings and will not affect their subsequent growth.

[0049] In conclusion, inoculating sweet potato cuttings with Diaporthe ueckerae can effectively improve their salt tolerance, allowing them to grow without being inhibited by saline-alkali soil. Furthermore, the endophytic fungus Diaporthe ueckerae is easy to obtain at low cost, and the inoculation process is simple, thus not significantly increasing sweet potato cultivation costs. This approach can meet the demand for salt-tolerant sweet potato cuttings in saline-alkali areas and expand the scale of sweet potato cultivation.

[0050] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the description above. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, using the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi, characterized in that: Includes the following steps: Obtain a Diaporthe ueckerae spore suspension and dilute it to 1×10⁻⁶. 6 1 cell / ml, as the inoculum; Obtain sweet potato cuttings with a cut diameter of 0.5-0.7 mm, retaining a node 1 cm above the cut end, and insert them into 150 ml of Hoagland nutrient solution for 6 hours to allow them to recover. After the seedlings have recovered from transplant shock, sweet potato cuttings are inoculated by soaking them in 150 ml of Diaporthe ueckerae spore inoculation solution for 6 hours. After inoculation, sweet potato cuttings were inserted into soil with a NaCl content of 3 g / kg. The Diaporthe ueckerae strain was obtained from the plant *Gynostemma pentaphyllum*, using the following method: Healthy petals were collected from the *Pterocarya stenoptera* plant, washed thoroughly with tap water, and then immersed in 75% ethanol for 0.5 minutes in a laminar flow hood. Next, the petals were immersed in 5% NaCl for 8 minutes. After this initial immersion, the petals were again immersed in 75% ethanol for 0.5 minutes. They were then removed from the laminar flow hood and rinsed three times with sterile water before being air-dried on filter paper. The petals were cut into small pieces and inoculated onto sterile PDA medium containing 3 g / L NaCl. The culture dishes were then incubated in the dark at 26°C for 7 days. The mycelia growing from the edges of the petal pieces were then transferred to sterile PDA medium without NaCl and incubated in the dark at 26°C for 7 days. After d, the strain with colony morphology was obtained; after obtaining the strain, the strain was propagated and cultured. The culture steps were as follows: take a 0.5 cm diameter mycelium from the colony of the strain and transfer it to a sterile PDA medium without NaCl for culture. One colony piece was transferred to each PDA medium. The culture dish was sealed with sealing film and placed in a 26℃ incubator for dark culture for 7 days. The method for obtaining Diaporthe ueckerae spore suspension is as follows: the strain with colony morphology is transferred from PDA medium to sterile PDB medium without NaCl, and then placed in an incubator at 26°C in the dark for 48 h. Then, the PDB medium is filtered through three layers of sterile gauze to obtain Diaporthe ueckerae spore suspension.

2. The method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi according to claim 1, characterized in that: The spore concentration of the spore suspension was counted using a hemocytometer.

3. The method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi according to claim 1, characterized in that: Sweet potato cuttings were inserted 6 cm deep into soil with a NaCl content of 3 g / kg.

4. A method for improving the salt tolerance of sweet potato cuttings using halophilic endophytic fungi according to claim 1 or 3, characterized in that: After the sweet potato cuttings are planted, pour 100 ml of deionized water over them.

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