Passion fruit endophytic marine streptomyces BXG368 and application thereof in prevention and control of passion fruit diseases

Abstract

The invention belongs to the field of agricultural microorganisms, and discloses passion fruit endophytic marine streptomyces BXG368 and application of the passion fruit endophytic marine streptomyces BXG368 in prevention and control of passion fruit diseases, and the preservation number of the marine streptomyces BXG368 is CCTCC NO: M 20242382. The bacterial strain is reported for the first time, can be used for preventing and treating passion fruit virus diseases, epidemic diseases, basal stem rot, gray mold and canker at the same time, has the effects of killing aphids and thrips, and has no cross resistance with existing sterilization and bactericide agents. Compared with the existing chemical synthetic pesticide for prevention and treatment, the compound is from the natural environment, has the advantages of high efficiency, low toxicity, no environmental pollution, low possibility of generating drug resistance and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganisms, and specifically relates to an endophytic marine streptomyces BXG368 of passion fruit and application thereof in passion fruit disease prevention and control. Background Art

[0002] Passion fruit (Passiflora edulis Sims), also known as passion fruit, is a herbaceous vine of the genus Passiflora in the family Passifloraceae. Due to its unique flavor, high nutritional value and economic value, however, with the extension of the planting period and the increase of the planting area, coupled with the high temperature and high humidity climate conditions in tropical and subtropical regions, passion fruit diseases and insect pests are more serious, such as virus disease, blight, stem base rot, anthracnose, gray mold and ulcer disease, which have caused serious losses to fruit farmers.

[0003] At present, chemical pesticides are mainly used for the prevention and control of passion fruit diseases and pests. However, the large-scale use of chemical pesticides causes pesticide residues, environmental pollution, and resistance to pests and diseases, and there is an urgent need for safer and more effective prevention and control methods. Biological control has the advantages of safety and efficiency and has been widely used in crop disease prevention and control in recent years. Guo Junqing et al. reported that Bacillus Velez PG1-2 has a good antibacterial effect on the pathogen of passion fruit stem base rot, and Qi Ruibin et al. reported that Bacillus subtilis has a good prevention and control effect on passion fruit stem base rot. There are few reports on biological control of passion fruit diseases, and they are mainly based on Bacillus, lacking excellent biological control strains. In addition, passion fruit aphids and thrips can harm leaves and fruits, and spread viruses at the same time, causing outbreaks of viral diseases. Therefore, the prevention and control of passion fruit aphids and thrips is also the key to the prevention and control of viral diseases.

[0004] Based on the above situation, the inventors classified an endophytic marine Streptomyces from the roots of passion fruit in Wenshan, Yunnan. It has good control effects on the main pathogens of passion fruit diseases, and has control effects on aphids and thrips. It has good colonization ability in the soil and passion fruit roots, and has good application prospects. Summary of the invention

[0005] The present invention aims to provide an isolated marine streptomyces BXG368, the marine streptomyces has a deposit number of CCTCC NO: M20242382. The streptomyces can be used for the prevention and treatment of passion fruit virus diseases, blight, stem base rot, gray mold and canker, and has the function of preventing and controlling aphids and thrips.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An isolated marine streptomyces was isolated from the root of passion fruit and identified as marine streptomyces Streptomyces maritimus. It was sent to the China Center for Type Culture Collection for preservation on October 30, 2024, and its classification name is Streptomyces maritimus BXG368; the preservation number is CCTCC NO: M20242382; location: Wuhan University, Wuhan, China.

[0008] Streptomyces maritimus BXG368 strain grew well on Gao's medium No. 1-2. The aerial hyphae were white to grayish white, and the colonies were flat without protrusions. Figure 1 ).

[0009] The protection content of the present invention includes:

[0010] Fermentation product of Streptomyces maritimus BXG368.

[0011] A composition containing Streptomyces maritimus BXG368 or a fermentation product thereof.

[0012] Use of Streptomyces maritimus BXG368, fermentation products of Streptomyces maritimus BXG368 or a combination thereof in the prevention and control of passion fruit diseases;

[0013] In the above application, the diseases include passion fruit virus disease, passion fruit blight, passion fruit stem rot, passion fruit gray mold, passion fruit canker, aphid pests and / or thrips pests.

[0014] In the above application, preferably, the pathogenic virus of the passion fruit virus disease is Cucumber mosaicvirus, the pathogen of passion fruit blight is Phytophthora nicotianae, the pathogen of passion fruit stem rot is Fusarium solani, the pathogen of passion fruit gray mold is Botrytis cinerea, and the pathogen of passion fruit canker is Xanthomonassp.

[0015] Application of Streptomyces maritimus BXG368, fermentation products of Streptomyces maritimus BXG368 or a composition thereof in the preparation of passion fruit biocontrol drugs.

[0016] Use of Streptomyces maritimus BXG368, a fermentation product of Streptomyces maritimus BXG368 or a composition thereof in the preparation of an insecticide, wherein the insecticide kills aphids and / or thrips.

[0017] Use of Streptomyces maritimus BXG368, a fermentation product of Streptomyces maritimus BXG368 or a composition thereof in the preparation of a fungicide, wherein the fungicide kills Cucumber mosaicvirus, Phytophthora nicotianae, Fusarium solani, Botrytis cinerea, and / or Xanthomonas sp.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The marine streptomyces of the present invention is endogenous to passion fruit. This strain can not only be used to simultaneously prevent and treat passion fruit diseases caused by various viruses and pathogens, but is also reported for the first time to kill aphids and thrips, thus being able to prevent and control viruses carried by pests.

[0020] (2) Compared with the currently used chemical synthetic pesticides, since it comes from the natural environment, it has the advantages of high efficiency, low toxicity, no pollution to the environment, and is not easy to produce drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the colony morphology of marine Streptomyces BXG368.

[0022] Figure 2 Phylogenetic tree of Streptomyces marineus BXG368.

[0023] Figure 3 Antibacterial activity of marine Streptomyces BXG368. DETAILED DESCRIPTION

[0024] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. The technical solutions of the present invention, if not otherwise specified, are conventional techniques in the art, and the reagents or materials, if not otherwise specified, are all derived from commercial channels.

[0025] Embodiment 1:

[0026] Acquisition and identification of Streptomyces maritimus BXG368

[0027] The applicant isolated a strain of Streptomyces from the root system of passion fruit in Wenshan, Yunnan. After 16s rDNA combined with morphological, physiological and biochemical identification, it belonged to Streptomyces maritimus (Table 1, Figure 1 , 2), the bacteria was sent to the China Center for Type Culture Collection for preservation on October 30, 2024, and the classification name is Streptomyces maritimusBXG368; the preservation number is CCTCC NO:M20242382; location: Wuhan University, Wuhan, China.

[0028] In the present invention, Streptomyces maritimus BXG368 is referred to as BXG368.

[0029] Morphological characteristics: Aerial hyphae are grayish white, spore threads are straight or flexible, and spores are oval.

[0030] Table 1 Physiological and biochemical test results

[0031] feature result Gelatin liquefaction test + Starch hydrolysis test + Indole test - VP test - Nitrate reduction test + Citrate utilization test + Mannitol + Catalase test +

[0032] Embodiment 2:

[0033] Fermentation of Streptomyces maritimus BXG368:

[0034] Preparation of Streptomyces maritimus BXG368 seed solution: including activating the slant-stored strain on Gao's No. 1 plate, picking and inoculating into 100 ml of seed culture medium, and culturing on a shaker at 28°C and 160 rpm for 5 days; thus obtaining the seed solution. The seed culture medium formula is: 1L of culture medium includes 20g sucrose, 8g soy peptone, 0.1g magnesium chloride, 0.30g potassium dihydrogen phosphate, 0.5g calcium carbonate, and the rest is water.

[0035] Solid-state fermentation method of Streptomyces maritimus BXG368:

[0036] Preparation of liquid fermentation liquid: 10% of Streptomyces maritimus BXG368 seed liquid was inoculated into solid fermentation medium at 30°C, pH 7, and fermented for 7 days.

[0037] The formula of the fermentation medium includes: 55% bran, 10% soybean meal powder, 10% rice bran, 23% rice husk, 1% magnesium sulfate and 1% calcium carbonate, pH value is 7, and the material-water ratio is 1:1.

[0038] After fermentation, the spore count of Streptomyces maritimus BXG3687 was 6.0×10 10 spores / g, separated and streaked, free of foreign bacteria, fermented evenly and completely, used in the following examples;

[0039] Embodiment 3:

[0040] Antibacterial activity of Streptomyces maritimus BXG368

[0041] The fungal strains on the test slant were transferred to the PDA plate for activation and cultured at 30°C for 6 days. The BXG368 bacterial disc was placed at both ends of the PDA plate. After 24 hours, the pathogenic bacterial disc was connected with a 4mm puncher. The bacterial disc connected to the culture medium was used as a blank control. The distance between the pathogen and the BXG368 bacterial disc was 25mm. After the control plate was fully grown, the colony diameters of each treatment and the control were counted, and the inhibition rate was calculated. The specific pathogens and inhibition rates are shown in Table 2. The in vitro antibacterial test showed that BXG368 had good antibacterial activity against the pathogens of passion fruit blight, stem base rot, and gray mold (Table 2, Figure 3 ).

[0042] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%.

[0043] After streaking a single colony from a glycerol tube, the ulcer pathogen (i.e., Xanthomonas) was transferred to a NB liquid culture medium shaker at 180 r / min and 37°C for 48 hours for use. After the NA culture medium was melted and poured into a plate at about 45°C, the Xanthomonas pathogenic bacteria solution was then added, with the ratio of NA to the bacteria solution being 50:1, and mixed thoroughly. The plate with bacteria was then punched with a sterilized Oxford cup, and 200 μL of the BXG368 fermentation liquid prepared in Example 2 was added to the hole, and it was placed at 37°C for 48 hours to observe whether an inhibition zone was produced. The results showed that Streptomyces maritimus BXG368 had good antibacterial activity against passion fruit ulcer pathogens.

[0044] Table 2 Antibacterial activity of BXG368 against pathogenic bacteria

[0045]

[0046] Embodiment 4:

[0047] Control of passion fruit virus disease with marine Streptomyces maritimus BXG368

[0048] The test was conducted in Wenshan, Yunnan. The variety was the main passion fruit variety Qinguo No. 9. On July 20, 2022, before the disease occurred, the bacterial agent prepared in Example 2 was diluted 200 times and sprayed on the whole plant. The second spray was performed at an interval of 7 days, and the control was sprayed with water. The test was repeated 3 times, with 15 passion fruits in each repeat, and 15 leaves were randomly counted for each plant. After the control was fully diseased (i.e., the disease index was ≥30), the control and treatment disease indexes were counted, and the prevention effect was calculated.

[0049] Disease index grading: Level 0: no obvious symptoms; Level 1: clear veins, mild mosaic; Level 2: mosaic of heart leaves and middle leaves; Level 3: mosaic of heart leaves and middle leaves; Level 5: mosaic of heart leaves and middle leaves, a few leaves are deformed, wrinkled or the plant is slightly dwarfed. Level 7: severe mosaic, most leaves are deformed, wrinkled or the plant is dwarfed; Level 9: severe mosaic, obvious deformed leaves, linear leaves, severe dwarfing of the plant, or even death.

[0050] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

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

[0052] The results of the field test showed that the BXG368 fungicide had a certain control effect on passion fruit virus disease, with a control efficiency of 56.92% (Table 3).

[0053] Table 3 Field control effect of BXG368 fungicide on passion fruit virus disease

[0054] deal with Disease index Prevention effect (%) BXG368 15.70 56.92 Comparison 36.44 -

[0055] Embodiment 5:

[0056] Control of passion fruit blight with marine streptomyces maritimus BXG368

[0057] The test was conducted in Wenshan, Yunnan. The main variety of passion fruit was Qinguo No. 9. On May 10, 2022, the bacterial agent prepared in Example 2 was diluted 200 times before the disease occurred and sprayed on the whole plant. The second spraying was performed after an interval of 7 days, and the control was sprayed with water. The test was repeated 3 times, with 15 passion fruits in each repeat, and 30 fruits were randomly counted for each plant. After the control was fully diseased (disease index ≥ 50), the number of control and treated diseased fruits was counted, and the prevention effect was calculated.

[0058] The results of the field test showed that the BXG368 fungicide had a certain control effect on passion fruit blight, with a control efficiency of 71.63% (Table 4).

[0059] Table 4 Field efficacy of BXG368 fungicide against passion fruit blight

[0060] deal with Diseased fruit rate (%) Prevention effect (%) BXG368 14.79 71.63 Comparison 52.13 -

[0061] Embodiment 6:

[0062] Control of passion fruit stem rot by marine streptomyces maritimus BXG368

[0063] The test was conducted in Wenshan, Yunnan. The main variety of passion fruit was Qinguo No. 9. On May 30, 2022, the bacterial agent prepared in Example 2 was diluted 200 times before the disease occurred. The roots of the plants were irrigated with water for the second time after an interval of 7 days. The control was sprayed with water. The test was repeated 3 times, with 15 passion fruits in each repeat. After the control was fully diseased (disease index ≥ 40), the number of control and treated diseased fruits was counted, and the prevention effect was calculated.

[0064] Disease index grading: Level 0: healthy plants; Level 1: stem lesions are less than 25% of the stem; Level 3: stem lesions are greater than 25% of the stem and less than 50%; Level 5: stem lesions are greater than 50% of the stem and less than 75%; Level 7: stem lesions are greater than 75% of the stem and the plant has not died yet; Level 9: the entire plant is dead.

[0065] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0066] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0067] The results of the field test showed that the BXG368 fungicide had a certain control effect on passion fruit stem rot, with a control efficiency of 71.06% (Table 5).

[0068] Table 5 Field control effect of BXG368 fungicide on passion fruit stem rot

[0069] deal with Diseased fruit rate (%) Prevention effect (%) BXG368 11.66 71.06 Comparison 40.29 -

[0070] Embodiment 7:

[0071] Control of passion fruit gray mold with marine Streptomyces maritimus BXG368

[0072] The test was conducted in Wenshan, Yunnan. The main variety of passion fruit was Qinguo No. 9. In July 2022, the bacterial agent prepared in Example 2 was diluted 200 times before the disease occurred and sprayed on the whole plant. The second spray was performed after an interval of 7 days, and the control was sprayed with water. The test was repeated 3 times, with 15 passion fruits in each repeat, and 30 fruits were randomly counted for each plant. After the control was fully infected (the diseased fruit rate was ≥30%), the number of control and treated diseased fruits was counted, and the prevention effect was calculated.

[0073] The results of the field test showed that the BXG368 fungicide had a certain control effect on passion fruit gray mold, with a control efficiency of 73.22% (Table 6).

[0074] Table 6 Field control effect of BXG368 fungicide on passion fruit gray mold

[0075] deal with Diseased fruit rate (%) Prevention effect (%) BXG368 10.02 73.22 Comparison 37.42 -

[0076] Embodiment 8:

[0077] Control of passion fruit canker by Streptomyces maritimus BXG368

[0078] The test was conducted in Wenshan, Yunnan. The variety was the main passion fruit variety, Qinguo No. 9. On August 25, 2022, before the disease occurred, the bacterial agent prepared in Example 2 was diluted 200 times and sprayed on the whole plant. The second spray was conducted at an interval of 7 days, and the control was sprayed with water. Three plants were investigated each time, and each plant was investigated at 5 points in the east, south, west, north, and center. All leaves and fruits of three new shoots were investigated at each point; the number of diseased leaves (fruits) and the corresponding disease level were recorded, and the prevention effect was calculated. After the control was fully diseased (disease index ≥ 30%), the control and treatment disease indexes were counted, and the prevention effect was calculated.

[0079] The grading standards are as follows:

[0080] Level 0: no lesions;

[0081] Level 1: There are 1 to 5 lesions on each leaf (fruit);

[0082] Level 3: There are 6 to 10 lesions on each leaf (fruit);

[0083] Level 5: There are 11 to 15 lesions on each leaf (fruit);

[0084] Level 7: There are 16 to 20 lesions on each leaf (fruit);

[0085] Level 9: There are more than 21 spots on each leaf (fruit).

[0086] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0087] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0088] The results of the field test showed that the BXG368 bacterial agent had a certain control effect on passion fruit canker, with a control efficiency of 66.86% (Table 7).

[0089] Table 7 Field efficacy of BXG368 fungicide against passion fruit canker

[0090] deal with Disease index Prevention effect (%) BXG368 11.66 66.86 Comparison 35.18 -

[0091] Embodiment 9:

[0092] Marine Streptomyces maritimus BXG368 for controlling passion fruit aphids

[0093] The experiment was conducted in Wenshan, Yunnan. The main passion fruit variety was Qinguo No. 9. There were two treatments: BXG368 and clean water. Each treatment had four replicates, and the area of ​​each replicate was 20m 2 , the liquid volume is 30L / mu. Dilute BXG368 fungicide 200 times and spray it evenly on the tree body with an electric sprayer on July 20, 2022, and spray it with clean water as a control. Investigate the insect population base before application, and investigate the number of residual insects after application. Take samples at 5 points in each plot, select 10 to 30 leaves at each point, and investigate the number of live aphids 1d, 3d, 5d and 7d after application. The survey results are used to calculate the insect population reduction rate and control effect according to the following formula.

[0094] Insect population reduction rate = (number of live aphids before application - number of live aphids after application) / number of live aphids before application × 100%

[0095] Control effect = (pest reduction rate in the treatment area - insect reduction rate in the control area) / (1 - insect reduction rate in the control area) × 100%

[0096] The results of the field test showed that BXG368 had a good control effect on passion fruit aphids, with the control effects of 80.48%, 84.44%, 88.85% and 91.81% at 1d, 3d, 5d and 7d after application, respectively (Table 8). This function of BXG368 helps control the virus transmission by aphids to infect passion fruit.

[0097] Table 8 Field control effect of BXG368 fungicide on passion fruit aphids

[0098]

[0099] Embodiment 10:

[0100] Marine Streptomyces maritimus BXG368 for controlling passion fruit thrips

[0101] The experiment was conducted in Wenshan, Yunnan. The main passion fruit variety was Qinguo No. 9. There were two treatments: BXG368 and clean water. Each treatment had four replicates, and the area of ​​each replicate was 20m 2, the liquid volume is 30L / mu. Dilute BXG368 fungicide 200 times, and use an electric sprayer to spray it evenly on the tree on May 20, 2022, and spray it with clean water as a control. Investigate the insect population base before application, and investigate the number of residual insects after application. Take samples at 5 points in each plot, select 10 to 30 leaves at each point, and investigate the number of live aphids 1d, 3d, 5d and 7d after application. The survey results are used to calculate the insect population reduction rate and control effect according to the following formula.

[0102] Insect population reduction rate = (number of live aphids before application - number of live aphids after application) / number of live aphids before application × 100%

[0103] Control effect = (pest reduction rate in the treatment area - insect reduction rate in the control area) / (1 - insect reduction rate in the control area) × 100%

[0104] The results of the field test showed that BXG368 had a good control effect on passion fruit thrips, with the control effects of 75.63%, 80.38%, 80.50% and 81.71% at 1d, 3d, 5d and 7d after application, respectively (Table 9). This function of BXG368 helps control the virus spread by thrips and infect passion fruit.

[0105] Table 9 Field control effect of BXG368 fungicide on passion fruit thrips

[0106]

[0107]

[0108] Embodiment 11:

[0109] Colonization of passion fruit roots by Streptomyces maritimus BXG368

[0110] The BXG368 inoculant was prepared with clean water into a suspension of 50 million cfu / mL, and 20 ml / plant was used to irrigate the roots of passion fruit. Each treatment was repeated 3 times, with 10 plants in each repeat. The control plant was pitaya. After 15 days, 5 plants were randomly dug out from each repeat pot, the roots were cut off, and the roots were rinsed with tap water, and then the moisture on the root surface was dried with absorbent paper. The fibrous roots were cut off, and 1.00 g of fibrous root samples were weighed on a balance, tied into small bundles and immersed in 75% (V / V) alcohol for 1-2 seconds to remove bubbles on the root surface, and then the roots were placed in a concentration of 1g·L -1Soak in mercuric chloride solution for 2 minutes, take out and rinse with sterile water 5 times. Put it into a sterile mortar with a small spoon of sterile quartz sand and 10mL sterile water and grind it thoroughly to release the marine streptomyces colonized in the roots. After appropriate dilution, spread it on Gao's No. 1 plate and culture it at 28℃. Before pouring Gao's No. 1 medium into the dish, add sterilized K2Cr2O7 to make the final mass concentration of K2Cr2O7 in the medium be 80mg / L). Calculate the number of viable streptomyces colonized per gram of root (colonization density) by comparing the morphology with the corresponding streptomyces strains on the synchronous culture plate and counting them.

[0111] The experimental results showed that the strain BXG368 had a good colonization ability in the root system of passion fruit, and the colonization density in the root system was 4.1×10 5 cfu / g, but it cannot colonize in the root system of pitaya. This good colonization ability helps it play a better role in disease prevention and control.

Claims

1. An isolated strain of Streptomyces marineus Streptomyces maritimus BXG368; the preservation number of the marine Streptomyces BXG368 is CCTCC NO: M20242382.

2. The fermentation product of marine Streptomyces BXG368 according to claim 1.

3. A composition comprising the marine Streptomyces BXG368 according to claim 1 and / or the fermentation product according to claim 2.

4. Use of the marine streptomyces BXG368 described in claim 1, the marine streptomyces BXG368 described in claim 1 or the composition described in claim 3 in passion fruit disease prevention and control.

5. The method according to claim 4, wherein the disease is passion fruit virus disease, passion fruit blight, passion fruit stem base rot, passion fruit gray mold, passion fruit canker, aphid pests and / or thrips pests.

6. The use according to claim 5, wherein the pathogenic virus of the passion fruit virus disease is Cucumber mosaic virus The pathogen of passion fruit blight is Phytophthora nicotianae The pathogen of passion fruit stem rot is Fusarium solani The pathogen of passion fruit gray mold is Botrytis cinerea The pathogen of passion fruit canker is Xanthomonas sp.

7. Use of the marine streptomyces BXG368 according to claim 1, the marine streptomyces BXG368 according to claim 1 or the composition according to claim 3 in preparing a passion fruit biocontrol drug.

8. Use of the marine streptomyces BXG368 according to claim 1, the marine streptomyces BXG368 according to claim 1 or the composition according to claim 3 in the preparation of an insecticide, wherein the insecticide kills aphids and / or thrips.

9. Use of the marine Streptomyces BXG368 of claim 1, the marine Streptomyces BXG368 of claim 1 or the composition of claim 3 in the preparation of a fungicide, wherein the fungicide kills objects including Cucumber mosaic virus, Phytophthora nicotianae, Fusarium solani, Botrytis cinerea, and / or Xanthomonas sp.

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