Application of phosphate salt in preparation of fungicide for promoting growth of streptomyces scabies

Abstract

The invention belongs to the technical field of potato soil-borne disease control. The invention discloses application of phosphate salt in preparation of a microbial inoculum for promoting growth of streptomyces scabies. The method provided by the invention can screen antagonistic bacteria with an efficient phosphate solubilizing function and effectively prevent and control potato scab.

Description

Technical Field

[0001] The invention belongs to the technical field of prevention and treatment of potato soil-borne diseases. Background Art

[0002] International research on the prevention and control of potato scab has a history of more than 100 years, but no major breakthroughs have been made so far. On the one hand, the pathogen is a special Streptomyces that causes a few plant diseases. There are many species, soil-borne and seed-borne. More than 20 pathogenic Streptomyces have been reported, and Streptomyces scabies, acidic scab Streptomyces S. acidiscabies and S. turgidiscabies, which causes convex scab, are three common pathogens. Among them, S. scabies is the most representative and widely distributed strain, which is commonly found in the world's major potato producing areas and causes serious damage in soils with a pH range of 5.5-7.5. On the other hand, the pathogens in each ecological zone have obvious compositional diversity and differential distribution. S.scabies has a pathogenicity island (PAI) containing multiple highly conserved sequences of thaxtomins biosynthetic genes (txtA, txtB, txtC, txtD, txtE, txtH and txtR), which can be horizontally transferred between strains of different genotypes, causing non-pathogenic bacteria to cause disease, resulting in the continuous identification and reporting of new pathogens; thirdly, the degree of damage of the disease is not only affected by the resistance of potato varieties, but also closely related to a variety of complex environmental factors such as soil moisture, pH, and previous crops. But in any case, the number of pathogenic Streptomyces in the soil determines the degree of damage of scab. Up to now, the most critical factor that induces the rapid growth and reproduction of pathogens in the soil has never been discovered, resulting in prevention and control being in a blind and passive exploration stage, and no effective measures for modeling and industrial application have been formed.

[0003] Phosphorus is an essential nutrient for the growth and development of all living things. It is involved in the synthesis of proteins and carbohydrates, as well as the production and transportation of photosynthetic products. Applying phosphorus fertilizer is the main means to supplement the lack of soil phosphorus and promote crop yields. Soil colloids have a strong adsorption and fixation effect on phosphorus. Most phosphorus fertilizers (mainly monoammonium phosphate and diammonium phosphate) react with Ca in the soil. 2+ , Fe 2+ , Fe 3+ 、Al 3+ The combination forms insoluble phosphates that cannot be absorbed and utilized by plants, resulting in a utilization rate of less than 25% for phosphate fertilizers in the season, and less than 15% in technologically underdeveloped areas. Potatoes are typical shallow-rooted crops with weaker phosphorus absorption capacity, with a utilization rate of only 11.2%. This phenomenon causes unusable phosphorus to accumulate in the soil year by year, with the content increasing. Summary of the invention

[0004] In view of this, the present invention discloses the use of phosphate in the preparation of a bacterial agent for promoting the growth of Streptomyces scabies.

[0005] In a specific embodiment of the present invention, the phosphate salt includes phosphate, hydrogen phosphate and / or dihydrogen phosphate.

[0006] Furthermore, the dihydrogen phosphate includes Ca(H2PO4)2 and / or KH2PO4; the concentration of Ca(H2PO4)2 is not higher than 3.5 g / L; and the concentration of KH2PO4 is not higher than 2.5 g / L.

[0007] Furthermore, the phosphate includes Ca3(PO4)2 and / or K3PO4; the concentration of Ca3(PO4)2 is not higher than 5 g / L; and the concentration of K3PO4 is not higher than 5 g / L.

[0008] Furthermore, the hydrogen phosphate includes CaHPO4 and / or K2HPO4; the concentration of CaHPO4 is not higher than 5g / L; the concentration of K2HPO4 is not higher than 5g / L.

[0009] The present invention also provides a bacterial agent for preventing and controlling potato scab, which contains phosphate-solubilizing bacteria and antagonistic bacteria; the phosphate-solubilizing bacteria are bacteria with phosphate-solubilizing function; and the antagonistic bacteria are bacteria with antagonistic and inhibitory effects on Streptomyces scab.

[0010] In a specific embodiment of the present invention, the antagonistic bacteria and phosphate-solubilizing bacteria include Bacillus atrophaeus and / or Burkholderia cepacia.

[0011] In a specific embodiment of the present invention, the Bacillus atrophicus includes Bacillus atrophicus DX-9.

[0012] Furthermore, the Burkholderia cepacia includes Burkholderia cepacia BN-4.

[0013] The method of the invention can screen antagonistic bacteria with efficient phosphate-solubilizing function and effectively prevent and control potato scab disease. Description of the drawings:

[0014] Figure 1 This is a diagram of the growth status of Streptomyces scabies at 8 different salt concentrations.

[0015] Figure 2 These are electron micrographs of Streptomyces scabies at different Ca3(PO4)2 contents and at different times.

[0016] Figure 3 The effect of different phosphorus fertilizer dosages on the incidence of scab in potted potatoes.

[0017] Figure 4 The accumulation of Ca3(PO4)2 reduced the abundance and antagonistic activity of antagonistic bacteria of Streptomyces scabies in the soil.

[0018] Figure 5 This is a diagram of the growth status of phosphate-solubilizing bacteria and non-phosphate-solubilizing bacteria on Ca3(PO4)2 medium.

[0019] Figure 6 This is a diagram of the disease states of phosphate-solubilizing bacteria and non-phosphate-solubilizing bacteria in potted potatoes. Specific implementation method:

[0020] Example 1

[0021] Growth of Streptomyces scabiei on different culture media

[0022] 100 μL of S. scabiei bacterial suspension was respectively taken and evenly spread on the adjusted culture media T1, T2, T3, T4 and T5 with different formulations. The growth status of the colony was observed after 7 days of cultivation, and the culture was scraped and suspended in 30 ml of sterile water. The number of live bacteria was counted by the plate count method.

[0023] LB medium: peptone 10 g, yeast extract 5 g, NaCl 10 g, dilute to 1000 mL with distilled water, adjust pH to 7.0.

[0024] Phosphate-solubilizing medium: glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4 0.3g, MnSO4 0.03g, K2SO4 0.3g, FeSO4 0.03g, agar 15g, Ca3(PO4)2 5g, make up to 1000mL with distilled water and adjust the pH to 7.0.

[0025] PDA culture medium: Boil 200 g of potatoes in distilled water for 20 min. After filtering and removing the residue, add 20 g of glucose and 15 g of agar, make up to 1000 mL with distilled water, and adjust the pH to 7.

[0026] Other culture media: Based on the phosphate-dissolving medium, gradually increase the content of phosphate or calcium salt by 25%, with the minimum content being 0g / L and the maximum content being 5g / L.

[0027] Adjust the culture medium: Based on the phosphate-solubilizing medium, gradually increase the content of Ca3(PO4)2 or the salt to be tested by 25%.

[0028] The experiment set up 5 different component treatments, numbered T1, T2, T3, T4 and T5. The phosphate components and contents of each treatment are shown in the following table:

[0029] Table 1 Adjustment of phosphate components and content of culture medium (g / L)

[0030]

[0031] Test strain: Streptomyces scabiei, purchased from the Culture Collection of Institute of Microbiology, Chinese Academy of Sciences (CGMCC), Chinese translation of Streptomyces scabies, numbered CGMCC NO.4.1765, this strain is abbreviated as S. scabiei in this patent.

[0032] Potato varieties tested: Chapoti, Favorita, and Qingshu No. 9.

[0033] Observation of bacterial growth status: A sterile coverslip was inserted obliquely on the surface of the culture medium inoculated with S. scabiei. Samples were taken at 24h and 72h, respectively. The samples were fixed with 2.5% glutaraldehyde for 2h, and then the fixative was gradually replaced with 50%, 75%, 85%, 95% and 100% ethanol, respectively. After drying and gold spraying, the bacterial status was observed using a scanning electron microscope.

[0034] Table 1 Average colony counts of different adjusted culture media

[0035] 0(g / L) 1.25(g / L) 2.5(g / L) 3.75(g / L) 5(g / L) <![CDATA[KH2PO4]]> 0 469.3333 234.6667 17.33333 0 <![CDATA[Ca(H2PO4)2]]> 0 421.3333 464 561 107.3333 <![CDATA[K2HPO4]]> 0 560 592 930.6667 865.3333 <![CDATA[CaHPO4]]> 0 621.3333 658.6667 890.6667 1082.667 <![CDATA[K3PO4]]> 0 680.6667 789.3333 828 625.3333 <![CDATA[Ca3(PO4)2]]> 0 1109.333 1120 1161.333 1234.667 KCl 568 579 552 574 629 <![CDATA[CaCl2]]> 792 812 844 954 888

[0036] It can be seen that S. scabiei has a clear preference for insoluble Ca3(PO4)2, HPO4 2- It is the most suitable phosphorus absorption state for the growth and reproduction of S. scabiei. On the culture medium with CaHPO4 and K2HPO4 as the only phosphorus source, the growth rate of S. scabiei increased and the spore formation period shortened with the increase of phosphate concentration, indicating that HPO4 2- It is the appropriate phosphorus absorption state for the growth and development of S. scabiei. On the culture medium with KH2PO4 and Ca(H2PO4)2 as the only phosphorus source, when the concentration of KH2PO4 is less than or equal to 2.5g / L and the concentration of Ca(H2PO4)2 is less than or equal to 3.75g / L, the growth rate of S. scabiei increases with the increase of their concentrations. When the concentrations of KH2PO4 and Ca(H2PO4)2 reach 5g / L, the growth rate of S. scabiei slows down, indicating that excessive H2PO4 -It is not conducive to the growth of S.scabiei. In the culture medium with Ca3(PO4)2 as the only phosphorus source, the growth and reproduction of S.scabiei is faster than other phosphate culture media; it is worth mentioning that S.scabiei can also grow rapidly and well in the culture medium with a Ca3(PO4)2 content of only 1.25g / L. In the culture medium with K3PO4 as the only phosphorus source, the growth and reproduction rate of S.scabiei did not change significantly with the change of its concentration. When the phosphorus content remained unchanged, the growth rate of S.scabiei did not change significantly with the increase of CaCl2 concentration. The change of KCl concentration had a slight effect on the growth of S.scabiei, but the degree of influence was far less obvious than that of Ca3(PO4)2, indicating that the growth of S.scabiei is not affected by Ca 2+ The impact of changes in content.

[0037] Example 2

[0038] Effects of different phosphate fertilizer dosages on scab of potted potato (potted plants)

[0039] Experiment 1: Liquid adjustment culture medium with different formulations (T1, T2, T3, T4, T5) together with 100 ml of S. scabiei bacterial suspension were evenly mixed into vermiculite in advance, and placed in flower pots with a height of 10 cm and a diameter of 17 cm. Three virus-free tissue culture seedlings of the potato variety Solanum scabiei were transplanted into each pot and cultured under the conditions of a light intensity of 50,000 Lux and a photoperiod of 8h / 16h. After culturing for 30 days and 60 days, the liquid adjustment culture medium was applied to supplement nutrients.

[0040] The experiment was repeated three times, and tubers were harvested at 100 days of growth, and the incidence rate and disease index were calculated.

[0041] Potato scab investigation and grading standards:

[0042] Grade 0, the tuber has no scab;

[0043] Level 1, scab coverage rate 1-2%;

[0044] Grade 2, scab coverage 2.1-5.0%;

[0045] Grade 3, scab coverage 5.1-10.0%;

[0046] Grade 4, scab coverage rate 10.1-25%;

[0047] Level 5: scab coverage rate > 25%.

[0048] The calculation method is as follows:

[0049] Incidence rate (%) = number of diseased tubers / total number of investigated tubers × 100

[0050] Disease index = ∑[(number of tubers at each disease level × representative value of the disease level) / (total number of investigated individuals × highest disease level)] × 100

[0051] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100

[0052] Experiment 2: A flower pot with a height of 10 cm and a diameter of 17 cm was filled with vermiculite pre-mixed with 100 ml of S. scabiei bacterial suspension, and 3 potato variety Shapoti virus-free tissue culture seedlings were transplanted into each pot. The pot was cultured for 20 days under the conditions of a light intensity of 50,000 Lux and a photoperiod of 8 h / 16 h. Four antagonistic bacteria with similar inhibitory effects on S. scabiei in the plate confrontation test were inoculated respectively, among which Bacillus atrophicus DX-9 and Burkholderia cepacia BN-4 had obvious phosphate-solubilizing function; Bacillus atrophicus XJ-3 and Paenibacillus polymyxa HZ-9 had no phosphate-solubilizing function.

[0053] After 100 days of cultivation, the tubers were harvested and the incidence rate and disease index were calculated. The experiment was repeated three times.

[0054] Potato scab investigation and grading standards:

[0055] Grade 0, the tuber has no scab;

[0056] Level 1, scab coverage rate 1-2%;

[0057] Grade 2, scab coverage 2.1-5.0%;

[0058] Grade 3, scab coverage 5.1-10.0%;

[0059] Grade 4, scab coverage rate 10.1-25%;

[0060] Level 5: scab coverage rate > 25%.

[0061] The calculation method is as follows:

[0062] Incidence rate (%) = number of diseased tubers / total number of investigated tubers × 100

[0063] Disease index = ∑[(number of tubers at each disease level × representative value of the disease level) / (total number of investigated individuals × highest disease level)] × 100

[0064] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100 Table 2 Control effect of antagonistic bacteria on scab

[0065]

[0066] As the concentration of Ca3(PO4)2 increases, the incidence of potato scab increases, and the disease index also increases ( Figure 2 ).

[0067] The incidence rate and disease index of T1 treatment, which did not contain Ca3(PO4)2 in the culture medium, were the lowest, at 50% and 12.5, respectively. The incidence rate and disease index of T5 treatment, which contained 5g / L Ca3(PO4)2 in the culture medium, were the highest, at 90.91% and 61.36, respectively. Compared with T1 treatment, the incidence rate of T3 increased significantly, and the incidence rates of T4 and T5 increased extremely significantly; the disease index of T2 and T5 increased significantly, which was consistent with the trend of the plate confrontation test.

[0068] Example 3

[0069] Effects of different phosphate fertilizer application rates on potato scab in the field (field)

[0070] Plot experiment: A single-factor randomized block experiment was set up in potato fields in Huidong County, Guangdong Province (longitude 114.72, latitude 22.98) and Yakeshi City, Inner Mongolia Autonomous Region (longitude 120.71, latitude 49.29), with a plot area of ​​18m 2 The planting density was 75,000 plants / ha. Three different phosphorus fertilizer application treatments were set up in the experiment, namely high phosphorus fertilizer application HP (240 kg / ha), medium phosphorus fertilizer application MP (120 kg / ha) and no phosphorus fertilizer LP. The fertilizer application rates of each treatment are shown in Table 3, and each treatment was repeated three times.

[0071] Table 3 Fertilizer application amount for each treatment (unit: kg / ha)

[0072]

[0073] After harvest, 60 tubers were randomly selected from each treatment to investigate the incidence of scab.

[0074] The potatoes tested in Huidong County in 2020 were first-grade Qingshu No. 9 virus-free seed potatoes, which were sown on October 30 and harvested on February 25, 2021.

[0075] The potatoes participating in the trial in 2021 were first-grade Feureta virus-free seed potatoes. The trial was sown on November 9 and harvested on March 17, 2022.

[0076] The potatoes participating in the trial in Yakeshi City in 2021 were first-grade Feureta virus-free seed potatoes. The trial was sown on May 18 and harvested on September 2 of the same year.

[0077] The soil on the surface of the tubers of each treatment was obtained by multi-point sampling method, frozen with dry ice and transported back to Beijing for 16S amplicon sequencing and analysis.

[0078] The results of soil physical and chemical properties before sowing are shown in Table 4.

[0079] Table 4 Basic physical and chemical properties of soil in field test

[0080]

[0081] Field demonstration: A field demonstration of DX-9 was conducted on 1 hectare of soil in Yakeshi City. The test material was the commercial potato variety Faureta. The dosage of the microbial agent was 30 kg / ha and the spore count was 1.0×10 9 CFU / g, and evaluate its actual field control effect on potato scab.

[0082] The incidence and disease index of two potato varieties (Feureta and Qingshu 9) were statistically analyzed under the condition of increased application of phosphorus fertilizer.

[0083] Potato scab investigation and grading standards:

[0084] Grade 0, the tuber has no scab;

[0085] Level 1, scab coverage rate 1-2%;

[0086] Grade 2, scab coverage 2.1-5.0%;

[0087] Grade 3, scab coverage 5.1-10.0%;

[0088] Grade 4, scab coverage rate 10.1-25%;

[0089] Level 5: scab coverage rate > 25%.

[0090] The calculation method is as follows:

[0091] Incidence rate (%) = number of diseased tubers / total number of investigated tubers × 100

[0092] Disease index = ∑[(number of tubers at each disease level × representative value of the disease level) / (total number of investigated individuals × highest disease level)] × 100

[0093] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100

[0094] In general, both the incidence and the morbidity index were positively correlated with phosphate concentrations, as high phosphate fertilizer (HP) increased the incidence and index of CS compared with low phosphate (LP) or medium phosphate conditions (MP).

[0095] In line with this, primers for the pathogenic gene Txt A of Fusarium scab were designed using soil environmental DNA from potato fields as templates. TxtA in soil was amplified by qRT-PCR using 5'-CACGTACGCGCAGTTCAATG-3' as the forward primer and 5'-AGATGATGTAGGCGGGAC-3' as the reverse primer. It was found that the biomass of Streptomyces scab indicated by the DNA level of the gene txtA was also higher in HP than in LP.

[0096] In conclusion, these results suggest that high amounts of P fertilizer promoted the growth of S. scabies in soil, leading to an increase in the abundance of S. scabies in field soils.

[0097] Example 4

[0098] Accumulation of Ca3(PO4)2 reduces the abundance and antagonistic activity of antagonists of Streptomyces scabiei in soil

[0099] With the increase of Ca3(PO4)2 concentration, the diameter of the inhibition zone of antagonistic bacteria showed a significant downward trend, indicating that high concentration of Ca3(PO4)2 can reduce the antibacterial activity of antagonistic bacteria against S.scabies.

[0100] ( Figure 4 )

[0101] Table 5 Inhibition zone and Ca3(PO4)2 concentration

[0102] HZ-9 XB 8-3 SC-2 T1 25.26667 51.56 58.16667 55.73333 T2 21.61667 47.34333 47.42667 49.96333 T3 20.56 43.44667 41.05333 38.68667 T4 19.7 40.68 36.28333 35.3 T5 20.76667 37.02333 34.45333 33.74333

[0103] Example 5

[0104] Metabolomics analysis of Streptomyces scabiei

[0105] The optimum initial pH for scabies bacteria is 7. Secondly, after adding Ca3(PO4)2 (2.5 or 5 g / L), the culture medium acidified as the pH value decreased. The metabolites in this process were analyzed using HPLC-MS, and the level of organic acids in the culture medium containing Ca3(PO4)2 increased compared to the culture medium without Ca3(PO4)2. More specifically, the organic acids produced with the addition of Ca3(PO4)2 included pyruvic acid, pyrrole-2-carboxylic acid, L-leucine, and p-hydroxybenzoic acid. XPS (X-ray photoelectron spectroscopy) was used to analyze the phosphate content in the medium. In the culture medium containing Ca3(PO4)2 but without pathogenic Streptomyces as a control, Ca3(PO4)2 and PO4 3- Both. Inoculation with Mycobacterium scabies changed HPO4 2-In summary, the use of medium containing Ca3(PO4)2 to culture S. scabies acidifies the medium, which decomposes Ca3(PO4)2 into HPO4 2- As a suitable phosphorus source to support the growth of Streptococcus scabies.

[0106] Example 6

[0107] Effects of phosphate-solubilizing and non-phosphate-solubilizing bacteria on the control of potato scab

[0108] Four antagonistic bacteria with similar inhibition zone diameters and different phosphate solubilization effects in the plate confrontation test were selected for potted disease prevention tests. The results are shown in Table 4. The incidence and disease index of mini-tuber treated with DX-9 and BN-4 with phosphate solubilization functions were significantly lower than those treated with non-phosphate solubilizing bacteria XJ-3 and HZ-9, among which DX-9 had the best prevention effect, reaching 72.48%. It can be seen that screening antagonistic bacteria with phosphate solubilization functions is an important goal for achieving effective prevention and control of scab.

[0109] Potato scab investigation and grading standards:

[0110] Grade 0, the tuber has no scab;

[0111] Level 1, scab coverage rate 1-2%;

[0112] Grade 2, scab coverage 2.1-5.0%;

[0113] Grade 3, scab coverage 5.1-10.0%;

[0114] Grade 4, scab coverage rate 10.1-25%;

[0115] Level 5: scab coverage rate > 25%.

[0116] The calculation method is as follows:

[0117] Incidence rate (%) = number of diseased tubers / total number of investigated tubers × 100

[0118] Disease index = ∑[(number of tubers at each disease level × representative value of the disease level) / (total number of investigated individuals × highest disease level)] × 100

[0119] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100 Table 6 Inhibition zone and Ca3(PO4)2 concentration

[0120]

[0121] The DX-9 strain with the best control effect was demonstrated in a 1-hectare potato field in Yakeshi City. The incidence rate and disease index of potato scab in the field were 25.41 and 14.64, respectively, which were significantly or extremely significantly different from the control without using bacterial agents. The actual control effect in the field reached 71.8%.

[0122] The present invention discloses that pathogenic Streptomyces has an obvious preference for Ca3(PO4)2, and the amount of phosphate fertilizer is a key factor affecting the number, incidence and disease index of pathogenic Streptomyces. The pathogenic Streptomyces is inoculated on a solid phosphate-dissolving culture medium and cultured at 28°C for 7 days. Then the surface bacteria are removed and freeze-dried, and then the type and relative content of phosphate in the culture medium are determined by X-ray photoelectron spectroscopy (XPS). In this case, the uninoculated culture medium is used as a blank control. S.scabies can produce organic acids such as pyruvic acid and pyrrole-2-carboxylic acid through metabolism, and release protons to combine with Ca3(PO4)2 to form HPO4 2- It is the most suitable phosphorus absorption state for the growth and reproduction of pathogenic Streptomyces. Excessive H2PO4 - The method of the invention can screen antagonistic bacteria with efficient phosphate solubilization function and effectively prevent and control potato scab.

Claims

1. Application of phosphate in the preparation of bacterial agents that promote the growth of Streptomyces scabies.

2. The use according to claim 1, characterized in that: The phosphate salt includes phosphate, hydrogen phosphate and / or dihydrogen phosphate.

3. The use according to claim 2, characterized in that: The dihydrogen phosphate comprises Ca(H2PO4)2 and / or KH2PO4; The Ca(H2PO4)2 concentration is not higher than 3.5 g / L; The KH2PO4 concentration is not higher than 2.5 g / L.

4. The use according to claim 2, characterized in that: The phosphate includes Ca3(PO4)2 and / or K3PO4; The Ca3(PO4)2 concentration is no higher than 5g / L. The K3PO4 concentration is not higher than 5g / L.

5. The use according to claim 2, characterized in that: The hydrogen phosphate includes CaHPO4 and / or K2HPO4; The CaHPO4 concentration is not higher than 5g / L; The K2HPO4 concentration is not higher than 5g / L.

6. A bacterial agent for preventing and controlling potato scab, characterized in that: Contains phosphate-solubilizing bacteria and antagonistic bacteria; The phosphate-solubilizing bacteria are bacteria with phosphate-solubilizing function; The antagonistic bacteria are bacteria that have antagonistic and inhibitory effects on Streptomyces scabies.

7. The bacterial agent according to claim 6, characterized in that: The antagonistic bacteria and phosphate-solubilizing bacteria include Bacillus atrophaeus and / or Burkholderia cepacia.

8. The bacterial agent according to claim 6, characterized in that: The Bacillus atrophicus includes Bacillus atrophicus DX-9.

9. The bacterial agent according to claim 6, characterized in that: The Burkholderia cepacia includes Burkholderia cepacia BN-4.

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