Strain SGSF339 and application of biocontrol reagent prepared from strain SGSF339 in insect resistance and bacterium resistance

Abstract

The invention provides a strain SGSF 339 and application of a biocontrol reagent prepared from the strain SGSF 339 in insect resistance and bacterium resistance, and belongs to the technical field of microorganisms. The invention provides a biocontrol reagent prepared from the strain SGSF 339, and the strain SGSF 339 and the biocontrol reagent prepared from the strain SGSF 339 can be applied to resistance to storage pests and resistance to phytopathogens. The strain SGSF 339 and the biocontrol reagent prepared from the strain SGSF 339 not only enrich the variety of hypocrea fungi, but also have the accumulative corrected death rate of 67.32 + / -0.03% and the bombyx batryticatus rate of 69.68 + / -0.04% in a callosobruchus chinensis resistance activity test. The biocontrol reagent prepared from the strain SGSF 339 has a remarkable effect in resisting phytopathogens, and the phytopathogens comprise ralstonia solanacearum, xanthomonas oryzae, rhizoctonia solanacearum or alternaria alternata.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to the application of strain SGSF339 and the biocontrol reagent prepared therefrom Background Art

[0002] Fungal resources are rich, with diverse species and wide distribution, and are applied in various fields such as medicine, agriculture, and food (Hyde KD 2020, Pozo MJ 2021). Forest litter is a good habitat for fungi. There are many unexploited fungi in the litter, and they may have various activities such as antibacterial activity, insecticidal activity, phosphate-solubilizing activity, and cellulose-degrading activity. The spore suspension of the newly established genus Parametarhizium isolated from litter by Gao et al. (Gao S 2021) has the activity of resisting insects such as Rhopalosiphum maidis. Among the 40 strains of fungi isolated from forest litter by Wu Tong (Wu Tong 2022), 3 strains of fungi have strong activity against Meloidogyne incognita, and the control effect on the eggs of Meloidogyne incognita in tomato pot experiments reached 61.75%. Forest litter fungi have diverse activities and great development and utilization value, and need to be continuously explored

[0003] Fungi in the order Hypocreales are one of the most species-rich orders in the fungal kingdom. They have diverse lifestyles and can live as saprophytes, parasites, epiphytes, endophytes, and symbionts, and can inhabit various environments such as air, soil, water, animals, and plants (Gao S 2021, Wu T 2022, Mu Y T 2022, Hou L W 2023). Hypocreales fungi have a rich species diversity, and many of them can be developed and utilized. They have been widely applied in the fields of industry, pharmaceuticals, and biological control (Hyde K D 2019, Sullivan C F 2022). Fungi in the family Cordycipitaceae have antibacterial and insecticidal activities. The methanol extract of Cordyceps nidus has antibacterial activity against Gram-positive bacteria, including Kocuria, Staphylococcus, and Enterococcus (Chiriví J 2017). Beauveria bassiana has insecticidal activity against the larvae or adults of various insects and has been registered as a fungal insecticide. For example, it is used as a biocontrol agent against the banana pseudostem weevil (Odoiporus longicollis) that damages bananas (Shikano I 2017, Alagesan A 2019). Among the fungi in the family Ophiocordycipitaceae, there are well-known medicinal fungi such as Ophiocordyceps sinensis (Xu J 2016). Fungi in this family also have insecticidal activity. In field trials, when the spore suspension of Hirsutella citriformis was used to treat the Asian citrus psyllid (Diaphorina citri), the mortality rate of the Asian citrus psyllid reached 80% (Pérez-González O 2023). The fungi in this order have rich species diversity and diverse activities, which are worthy of in-depth study.

[0004] Among the Hypocreales fungi, the Clavicipitaceae fungi have been reported the most and are relatively well-studied. They have been widely applied in practice, especially playing a huge role in agricultural applications. Under suitable temperature and humidity, the spores of Metarhizium spp. can germinate on the surface of insects, forming infection peg structures. As these structures grow, they become appressoria to further infect the insects. In this process, the mad1 gene of Metarhizium spp. plays a role. This gene is related to the formation of appressoria, and the MAD1 protein can help the spores of Metarhizium spp. attach to the insect cuticle (Shiina M 2016, Jiang An 2018, Ortiz-Urquiza A 2013). As an entomopathogenic fungus, Metarhizium spp. can infect insects of multiple orders such as Orthoptera, Lepidoptera, Hemiptera, and Coleoptera, including Brontispa longissima, Nilaparvata lugens, and Plutella xylostella (Samuels K DZ 1989, Qiu Hui 2004, Godonou I 2009, Lian T 2021). However, among the currently reported Hypocreales fungi, there are relatively few that have both insect resistance and antibacterial properties. Therefore, it is very necessary to further explore Hypocreales fungi with both insect resistance and antibacterial properties. Summary of the Invention

[0005] The strain SGSF339 provided by the present invention has both insect resistance and antibacterial properties, enriching the species of Hypocreales fungi.

[0006] Application of the strain SGSF339 in controlling stored-product pests.

[0007] Furthermore, the stored-product pests include Callosobruchus chinensis.

[0008] A biocontrol reagent prepared from the strain SGSF339, and the preparation method of the biocontrol reagent is as follows:

[0009] S1: Activate and culture the strain SGSF339 in a potato dextrose agar medium, and take a mycelial plug from the edge of the colony and inoculate it into a potato dextrose broth medium to obtain a seed solution;

[0010] S2: Inoculate the seed solution into four media, namely a potato dextrose agar medium, a vermiculite medium containing yeast extract and sucrose, a rice medium, or a potato dextrose agar medium containing 10% nicotinamide, and ferment and culture in these four media;

[0011] S3: Add ethyl acetate equal to the fermentation volume to the medium after fermentation is completed and extract for 24 h. Use a rotary evaporator to evaporate and concentrate the extract of the ethyl acetate layer under reduced pressure to obtain the crude extract.

[0012] Further, the seed liquid is inoculated into a potato dextrose medium or a vermiculite medium or a rice medium containing yeast extract and sucrose, and then statically fermented at 25 °C for 21 d.

[0013] Further, the seed liquid is inoculated into a potato dextrose medium added with 10% nicotinamide and cultured in a shaker at 180 r / min at 25 °C for 14 d.

[0014] Application of the biocontrol reagent prepared from strain SGSF339 in resisting plant pathogens.

[0015] Further, the plant pathogen is Ralstonia solanacearum.

[0016] Further, the plant pathogen is Xanthomonas oryzae.

[0017] Further, the plant pathogen is Rhizoctonia solani.

[0018] Further, the plant pathogen is Alternaria alternata.

[0019] Beneficial effects

[0020] The present invention provides the application of strain SGSF339 and the biocontrol reagent prepared therefrom in resisting storage pests and plant pathogens, enriching the species of Hypocreales fungi in biocontrol agents.

[0021] The strain SGSF339 provided by the present invention has a significant effect in resisting storage pests. In the anti-Callosobruchus chinensis activity test, the cumulative corrected mortality rate reaches 67.32 ± 0.03%, and the rate of mummified insects reaches 69.68 ± 0.04%.

[0022] The present invention provides a biocontrol reagent prepared from strain SGSF339, which has a significant effect in resisting plant pathogens, and the plant pathogens include Ralstonia solanacearum, Xanthomonas oryzae, Rhizoctonia solani or Alternaria alternata. Description of the drawings

[0023] Figure 1 It is a graph showing the insecticidal activity results of strain SGSF339. Detailed implementation manners

[0024] I. Materials used in the experiment.

[0025] 1. Test media.

[0026] Fungal isolation medium: 1 / 4 Potato Dextrose Agar (1 / 4PDA).

[0027] Potato Dextrose Agar (PDA) was used for morphological observation of fungi. Six media used for small-scale fermentation of fungi: Potato Dextrose broth (PD), Potato Dextrose broth and Nicotinamide (PD+Nic), Super Malt extract (SM), Potato Dextrose Agar (PDA), Rice, Yeast Extract Sucrose and Vermiculite (YES+vermiculite);

[0028] 2. Tested biocontrol strains.

[0029] Strain SGSF339 was isolated from forest litter in Daxing'an Mountains. It was identified as belonging to the genus Albacillium, A. hingganense, and was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20232069 (Ding et al., 2024: DING, M., LI, Y., BAI, Y., GAI, P., WANG, J., JIANG, Z., HU, Y. and XU, L., 2024. Albacillium hingganense gen. et sp. nov. (Clavicipitaceae) from forest litters in Northeast China. Phytotaxa, 650(2), pp. 157-168.).

[0030] 3. Tested pests and plant pathogens.

[0031] Tested stored-product pest: Callosobruchus chinensis.

[0032] Tested plant pathogenic bacteria: Ralstonia solanacearum and Xanthomonas oryzae.

[0033] Tested plant pathogenic fungi: Rhizoctonia solani and Alternaria alternata.

[0034] Example 1. Morphological observation and molecular biological identification of strain SGSF339.

[0035] Strain SGSF339 was inoculated onto media such as PDA for culture. The morphological characteristics such as the size of the fungal colony, the color characteristics of the front and back sides, the texture of the colony, and whether there were wrinkles were observed. The size of the hyphae in the colony, whether there were septa, the morphology of the spore-producing structure, and the size and shape of the conidia were observed using a microscope. The genomic DNA of strain SGSF339 was extracted, and its ITS, LSU, RPB1, RPB2, SSU, and TEF sequences were amplified. The most closely related strain of strain SGSF339 was queried using BLAST on the NCBI website, and its morphological characteristics were compared with it.

[0036] The colony of strain SGSF339 on media such as PDA was nearly circular or circular, white in color, villous, with relatively sparse hyphae, smooth edges, no pigment production, and yellow on the back. Its hyphae were transparent, smooth, with branches and septa, and the hyphal diameter was 0.8 - 2.5 μm. Its phialide structure was directly produced from the aerial hyphae, transparent, solitary, the spore-producing structure was mostly upright, occasionally curved, tapering gradually from the base to the top, with a length of 8.6 - 60.1 μm × 0.9 - 1.5 μm. The top of the phialide structure was a spherical structure, which was a sticky ball composed of multiple conidia and was not easily dispersed. The conidia were oval to crescent-shaped, straight or slightly curved, transparent, with smooth walls, and some had 1 - 2 droplet-like substances, with a size of 2.0 - 4.3 μm × 0.8 - 1.3 μm.

[0037] Six fragments of ITS, LSU, RPB1, RPB2, SSU, and TEF in strain SGSF339 were amplified and sequenced, and each sequence fragment was aligned. The results are shown in Table 1. Through NCBI alignment, it was found that the similarity of the above six fragments in strain SGSF339 to the sequences in known species was low, except for SSU. In particular, the similarities of ITS, RPB1, and RPB2 to the sequences in known species were all below 85%. The most similar genera to the ITS fragment and RPB1 fragment of strain SGSF339 were Metarhizium and Regiocrella, respectively, which belong to the Clavicipitaceae family. The most similar genus to the LSU fragment and SSU fragment was Chlorocillium. The most similar genus to the RPB2 fragment was Purpureocillium. The closest genus to the TEF fragment was Rotiferophthora. The morphological characteristics of SGSF339 were different from those of the above six genera, and it had a unique sporulation structure (the top of the phialide structure was a spherical structure, which was a sticky ball composed of multiple conidia and was not easy to disperse; the conidia were oval to crescent-shaped). Therefore, a new genus: Albacillium and a new species: A. hingganens were established. For the detailed information on this new genus and new species, please refer to the literature DING, M., LI, Y., BAI, Y., GAI, P., WANG, J., JIANG, Z., HU, Y. and XU, L., 2024. Albacillium hingganense gen. et sp. nov. (Clavicipitaceae) from forest litters in Northeast China. Phytotaxa, 650(2), pp. 157 - 168.

[0038] Table 1

[0039]

[0040]

[0041] Note: The accession number is the accession number of the most similar genus of strain SGSF339.

[0042] Example 2. Insecticidal activity test.

[0043] The anti-insect activity test was carried out by the method of dipping insects in spore suspension. Five strains of SGSF339 were inoculated on PDA plates by streaking with a sterilized cotton swab dipped in conidia. After culturing for 15 days, 5 mL of 0.05% Tween 80 solution was added to the center of the colonies on the plates respectively. After standing for 1 min to make the spores easier to collect, the spores were suspended in 0.05% Tween 80 solution with a cotton swab, poured into a 50 mL centrifuge tube, shaken well and filtered through sterile cotton to remove excess mycelia. The concentration was adjusted to 1×10 7 cells / mL with a hemocytometer. The adult Callosobruchus chinensis were immersed in the spore suspension of 5 strains of SGSF339 for 5 s respectively, and then transferred into a conical flask filled with sufficient mung beans. 20 adult Callosobruchus chinensis were treated in each group, and 3 repeated experiments were carried out. The number of dead Callosobruchus chinensis was observed and counted every day. After the Callosobruchus chinensis died, they were transferred to a petri dish containing moist sterilized filter paper, and whether there were mummified insects was observed every day.

[0044] The anti-Callosobruchus chinensis activity of strain SGSF339 was tested by the method of dipping insects in spore suspension. Strain SGSF339 showed certain insecticidal activity, and the results are shown in Table 2, and it can make Callosobruchus chinensis form mummified insects( Figure 1 ).

[0045] Table 2

[0046] Strain number Accumulative corrected mortality rate (%) Median lethal time (d) Rate of dead pupae (%) SGSF339 67.32±0.03 8.18±0.31 69.68±0.04

[0047] Note: The mummified insect rate is the number of mummified insects / the number of dead insects. Lowercase letters represent significant differences in the same column (P<0.05), the same below.

[0048] Example 3. Antifungal activity test of fungi.

[0049] The strain SGSF339 was activated and cultured in PDA medium for 14 days. Two 6-mm mycelial discs were taken from the edge of the colony and inoculated into 20 mL of PD medium, and then cultured in a shaker at 25 °C and 180 r / min for 4 days to prepare a seed solution. Six kinds of media (PDA, YES + vermiculite, rice, PD, PD + Nic, and SM) with a volume of 20 mL were prepared and inoculated with 200 μL of the seed solution respectively. Among them, three liquid media (PD, PD + Nic, and SM) were cultured in a shaker at 25 °C and 180 r / min for 14 days after inoculating the seed solution; the other three solid media (PDA, vermiculite + YES, and rice) were statically fermented at 25 °C for 21 days after inoculating the seed solution. After the fermentation of the strain was completed, an equal volume of ethyl acetate to the fermentation volume was added to the six media. After 24 hours, the extract of the ethyl acetate layer was concentrated by rotary evaporation under reduced pressure to obtain a crude extract. 1 mL of dimethyl sulfoxide (DMSO) was added to each to prepare a crude extract test solution. The antibacterial and antifungal activities of the crude extracts obtained from the strain SGSF339 in different fermentation media were tested by the punching agent diffusion method.

[0050] Six crude extracts were prepared by fermenting the strain SGSF339 with six different media, and the antibacterial activities of these six crude extracts against two plant pathogenic bacteria (Ralstonia solanacearum, Xanthomonas oryzae) and two plant pathogenic fungi (Rhizoctonia solani, Alternaria alternata) were tested. The results are shown in Table 2. The crude extract from the rice fermentation of the strain SGSF339 showed antibacterial activities to varying degrees against the four tested plant pathogenic bacteria, while the fermentation products of PD and SM media did not show antibacterial activities against the four tested plant pathogenic bacteria.

[0051] Table 3

[0052] A P N R S Y Ralstonia solanacearum - - - + - + Xanthomonas oryzae - - - + - + Rhizoctonia solani + - - + - - Alternaria alternata ++ - + ++ - +

[0053] Note: “—” indicates no activity; “+” indicates that the diameter of the inhibition zone is 1 - 6 mm; “++” indicates that the diameter of the inhibition zone is more than 6 mm; A, P, N, R, S, and Y represent the crude extracts of the cultures of PDA, PD, PD + Nic, rice, SM, and YES + vermiculite respectively.

[0054] In this study, a fungus SGSF339 was isolated and cultured from the forest litter in the Greater Khingan Mountains. It was identified as a new species belonging to a new genus (Leucotrichum), and was named Leucotrichum xinganense. Leucotrichum xinganense SGSF339 has insecticidal activity against the stored-grain pest Callosobruchus chinensis and antibacterial activity against four plant pathogenic bacteria (Ralstonia solanacearum, Xanthomonas oryzae, Rhizoctonia solani, and Alternaria alternata).

Claims

1. Application of strain SGSF339 in resistance to storage pests.

2. The use according to claim 1, characterized in that: The storage pests include green bean weevils.

3. A biocontrol agent prepared from strain SGSF339, characterized in that: The preparation method of the biocontrol agent is: S1: The strain SGSF339 was activated and cultured in potato glucose medium, and the bacterial cake at the edge of the colony was inoculated into potato glucose liquid medium to obtain seed liquid; S2: The seed solution was inoculated into potato glucose medium, vermiculite medium containing yeast extract sucrose, rice medium, or potato glucose medium containing 10% nicotinamide, and fermented in the four culture media; S3: Add ethyl acetate in an amount equal to the fermentation volume to the culture medium at the end of fermentation and extract for 24 hours. Use a rotary evaporator to evaporate and concentrate the extract of the ethyl acetate layer under reduced pressure to obtain a crude extract.

4. The biocontrol agent according to claim 3, characterized in that The seed liquid is inoculated into a potato glucose medium or a vermiculite medium or a rice medium containing yeast extract sucrose and then left to ferment at 25° C. for 21 days.

5. The biocontrol agent according to claim 3, characterized in that: The seed solution was inoculated into a potato glucose medium containing 10% nicotinamide and then cultured in a shaking incubator at 25° C. and 180 r / min for 14 days.

6. Use of the biocontrol agent prepared by strain SGSF339 as claimed in claim 3 in combating plant pathogens.

7. The use according to claim 6, characterized in that: The plant pathogen is Ralstonia solanacearum.

8. The use according to claim 6, characterized in that: The plant pathogen is Xanthomonas oryzae.

9. The use according to claim 6, characterized in that: The plant pathogen is Rhizoctonia solani.

10. The use according to claim 6, characterized in that: The plant pathogen is Alternaria alternata.

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