NL2033035B1

NL2033035B1 - Stachybotrys sp. hn17496 strain, microbial biocontrol agent, and preparation method and use thereof

Info

Publication number: NL2033035B1
Application number: NL2033035A
Authority: NL
Inventors: Kang Guozhang; Geng Yuehua; Guo Yashuang; Gao Wei; Zhang Kunpu; Xu Chao; Zhang Meng; Zhai Niping
Original assignee: Univ Henan Agricultural
Priority date: 2022-05-27
Filing date: 2022-09-15
Publication date: 2023-12-12
Also published as: CN114774293B; CN114774293A

Abstract

The present disclosure relates to the technical ﬁeld of biocontrol strains, in particular to a Slachybolrys sp. HN17496 strain, a microbial biocontrol agent, and a preparation 5 method and use thereof. The HN17496 strain provided by the present disclosure is deposited at China Center for Type Culture Collection (CCTCC) under accession number M2022489. The Slachybolrys sp. HN17496 strain provided by the present disclosure has an excellent biocontrol effect on Bipolarl's sorokiniana, and has no potential safety hazard on human and livestock; from the examples, the Slachybolrys 10 sp. HN17496 strain provided by the present disclosure and metabolites thereof inhibit 65%-72% of B. sorokmicma, with a signiﬁcant biocontrol effect. FIG. 4 is speciﬁed as the abstract drawing.

Description

STACHYBOTRYS SP. HN17496 STRAIN, MICROBIAL BIOCONTROL AGENT,

AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of biocontrol strains, in particular to a Stachybotrys sp. HN17496 strain, a microbial biocontrol agent, and a preparation method and use thereof.

BACKGROUND ART

[0002] Bipolaris sorokiniana is a pathogenic fungus widely distributed in the world and a major pathogen of wheat root rot. The pathogenic fungus can infect roots, basal stalk, leaves and ears of wheat, and has a serious effect on the yield and quality of wheat. The major control method of B. sorokiniana at the present stage is seed soaking with medicaments or spraying of chemical agents, both of chemical methods are harmful to the environment. There are few studies of biocontrol strains of B. sorokiniana. In 2010, it was found that Stenotrophomonas maltophilia strain C3 could produce a chitin-degrading enzyme and had an excellent control effect on B. sorokiniana. However, the strain is a conditioned pathogen that is unsuitable as a biocontrol strain of B. sorokiniana. There are few reports of biocontrol fungi of B. sorokiniana.

SUMMARY

[0003] In order to solve the above problems, the present disclosure provides a

Stachybotrys sp. HN17496 strain, a microbial biocontrol agent, and a preparation method and use thereof. The Stachybotrys sp. HN17496 strain provided by the present disclosure has an excellent biocontrol effect on B. sorokiniana.

[0004] To achieve the above objective, the present disclosure provides the following technical solutions:

[0005] The present disclosure provides a Stachybotrys sp. HN17496 strain, where the

HNI17496 strain is deposited at China Center for Type Culture Collection (CCTCC) under accession number M2022489.

[0006] The present disclosure further provides a microbial biocontrol agent, where the microbial biocontrol agent includes the foregoing HN17496 strain and/or metabolites of the HN17496 strain.

[0007] Preferably, on condition that the microbial biocontrol agent includes the

HN17496 strain, based on per g or mL of the microbial biocontrol agent, a number of spores of the HN 17496 strain in the microbial biocontrol agent may be >105 CFU.

[0008] The present disclosure further provides a preparation method of the foregoing microbial biocontrol agent, including the following steps:

[0009] inoculating the foregoing HN17496 strain on Potato Dextrose Agar (PDA) to culture for 5-7 days to obtain the microbial biocontrol agent.

[0010] Preferably, the culture may be conducted at 25-28°C.

[0011] Preferably, on condition that the microbial biocontrol agent does not include the HN17496 strain, sterilization may be further conducted 5-7 days after the culture.

[0012] Preferably, the sterilization may be conducted at 121°C for 30 min.

[0013] The present disclosure further provides use of the foregoing HN17496 strain, the foregoing microbial biocontrol agent, or a biocontrol agent prepared by the foregoing preparation method in controlling a plant disease, where a pathogen of the plant disease includes B. sorokiniana.

[0014] Preferably, the plant disease may include wheat root rot.

[0015] Preferably, the plant may include wheat.

[0016] Beneficial effects:

[0017] The present disclosure provides a Stachybotrys sp. HN17496 strain, where the

HN17496 strain is deposited at CCTCC under accession number M2022489. The

Stachybotrys sp. HN17496 strain provided by the present disclosure has an excellent biocontrol effect on B. sorokiniana, and has no potential safety hazard on human and livestock; from the examples, the Stachybotrys sp. HN17496 strain provided by the present disclosure and metabolites thereof inhibit 65%-72% of B. sorokiniana, with a significant biocontrol effect.

[0018] Deposit of biological material

[0019] Stachybotrys sp. HN17496 strain has been deposited at China Center for Type

Culture Collection (CCTCC), Wuhan University, No. 299, Bayi Road, Wuchang

District, Wuhan, Hubei Province on Monday, April 25, 2022 under accession number

M2022489. The Stachybotrys sp. HN17496 strain provided by the present disclosure has an excellent biocontrol effect on B. sorokiniana.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To describe the technical solutions in the examples of the present disclosure or in the prior art more clearly, the accompanying drawings required for the examples are briefly described below.

[0021] FIG. 1 partly illustrates primary screening results of biocontrol strains of B. sorokiniand,

[0022] FIG. 2 illustrates morphological characteristics of a re-screened HN17496 strain, where panel A illustrates the morphology of a HN17496 colony on the front surface of PDA, panel B illustrates the morphology of a HN17496 colony on the back of a Petri dish, panels C to G illustrate conidiophores, panels H to I illustrate conidia, and the scale in panels C to I is 10 un;

[0023] FIG. 3 illustrates a phylogenetic tree of an HN 17496 strain;

[0024] FIG. 4 illustrates a biocontrol effect of an HN17496 strain, wherein panels A and B illustrate normally grown B. sorokiniana (without addition of metabolic liquid), panel C illustrates a biocontrol effect of a metabolic liquid of an unautoclaved biocontrol fungus, panel D illustrates an antibacterial effect of a metabolic liquid of an autoclaved biocontrol fungus.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present disclosure provides a Stachybotrys sp. HN17496 strain, where the

HN17496 strain is deposited at CCTCC under accession number M2022489.

[0026] The HN17496 strain provided by the present disclosure may preferably have the following characteristics:

[0027] 1) a colony is black; conidiogenous cells grow on a culture medium, are viscous, and are covered with grey or black sori,

[0028] 2) hyphae produce dark brown secretions that resemble impurities, usually appeared as a bundle, and form a hyphal bridge;

[0029] 3) a conidiophore appears as a huge line, which is solitary or grown in a clustered manner, thin-walled, branched or unbranched, and erect or slightly curviform; 1 or 2 septa are present, smooth, and 18.2-64.0 x 2.8-4.9 um in size; 1-6 conidiogenous cells are apically inserted;

[0030] 4) conidiogenous cells are phialidic, claviform to quasi-claviform, transparent, semitransparent or dark black, smooth-surfaced, 6.6-11.5 x 3.4-5.5 um in size, with an obvious neck ring; and

[0031] 5) acroconidia are present, 0-septum, suborbicular, elliptical to subterete, and transparent, semitransparent to dark olivaceous; the wall is thick, dark olivaceous to pitchy, smooth-surfaced or tuberculate, and 7.0-11.6 x 3.3-7.5 un in size.

[0032] The Stachybotrys sp. HN17496 strain provided by the present disclosure has an excellent biocontrol effect on B. sorokiniana, and has no potential safety hazard on human and livestock; from the examples, the Swchvbotrvs sp. HN17496 strain provided by the present disclosure and metabolites thereof inhibit 65%-72% of B. sorokiniana, with a significant biocontrol effect.

[0033] The present disclosure further provides a microbial biocontrol agent, where the microbial biocontrol agent includes the foregoing HN17496 strain and/or metabolites of the HN17496 strain. The microbial biocontrol agent provided by the present disclosure may preferably achieve the biocontrol effect by inhibiting and/or killing pathogenic microorganisms.

[0034] In the present disclosure, on condition that the microbial biocontrol agent includes the HN 17496 strain, based on per g or mL of the microbial biocontrol agent, a number of spores of the HN17496 strain in the microbial biocontrol agent may preferably be >10° CFU.

[0035] The present disclosure further provides a preparation method of the foregoing microbial biocontrol agent, including the following steps:

[0036] inoculating the foregoing HN17496 strain on Potato Dextrose Agar (PDA) to culture for 5-7 days to obtain the microbial biocontrol agent.

[0037] In the present disclosure, the culture may preferably be conducted at 25-28°C.

[0038] In the present disclosure, on condition that the microbial biocontrol agent does not include the HN17496 strain, sterilization may preferably be further conducted 5-7 days after the culture; the sterilization may preferably be conducted at 121°C for 30 min.

[0039] The present disclosure further provides use of the foregoing HN 17496 strain, the foregoing microbial biocontrol agent, or a biocontrol agent prepared by the foregoing preparation method in controlling a plant disease, where a pathogen of the plant disease includes B. sorokiniana. In the present disclosure, the plant disease may preferably include root rot, and the plant may preferably include wheat.

[0040] To further describe the present disclosure, the Stachybotrys sp. HN17496 strain, the microbial biocontrol agent, and the preparation method and the use thereof will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present disclosure.

[0041] Example 1 5 [0042] A Stachybotrys sp. HN17496 strain was obtained from the soil under weeds in the Huamei Valley, Lushan County, Pingdingshan by dilution-plate method, specifically as follows:

[0043] 10 g of soil was weighed and placed in an Erlenmeyer flask with 90 mL of sterile water; the Erlenmeyer flask was placed on a shaker and oscillated at 120 rpm for 25 min to disperse soil particles in distilled water evenly, and a 10-fold diluted soil suspension was obtained; 1 mL of the soil suspension was pipetted into a test tube with 9 mL of sterile water to obtain a 100-fold diluted suspension.

[0044] Water-agar (WA) medium was autoclaved, cooled down to 45°C, supplemented with 30 pg/mL streptomycin and 4 drops of chloramphenicol, and poured into a Petri dish for curing and cooling; the resulting 100-fold diluted soil suspension was shaken well, 1 mL of the soil suspension was pipetted, and 5 drops of the soil suspension were added to each Petri dish and evenly applied with a sterile curved glass rod; the Petri dish was inverted and cultured in a biochemical incubator at 25°C. The ecological environment of the soil sample collection site was imitated, the temperature was set at 25°C, and observations were made on the WA medium under stereoscope after 30 days. A single spore or colony was picked onto PDA, and the strain was preserved after purification.

[0045] Primary screening of biocontrol strains of B. sorokiniana

[0046] The purified strain and the pathogenic fungus B. sorokiniana were cultured by the plate confrontation method, specifically as follows:

[0047] The purified strain and the pathogenic fungus were inoculated on a 9 cm Petri dish for confrontation culture, respectively. The pathogenic fungus was inoculated in the middle of the plate, and four test strains were inoculated surrounding it. The inoculation diameter of each strain was 0.5 cm, each strain was 1 cm away from the edge of the Petri dish, and the distance between strains was 2 cm. A pathogenic strain without inoculation with the purified strain was used as a control. Three replicates were set for each treatment and control and cultured at 25°C for 4 days. According to the colony growth rate and the antibacterial situation, antibiosis between colonies was observed. Biocontrol strains with significant antibacterial effect (significant limitation of the normal growth of B. sorokiniana was defined as effectiveness, and confluent growth was defined as no antibacterial effect) were picked out for re-screening. Partial primary screening results are shown in FIG. 1.

[0048] From FIG. 1, some purified strains have an inhibitory effect on the growth of

B. sorokiniana, and the inhibitory effect is different.

[0049] Re-screening of biocontrol strains of B. sorokiniana

[0050] The biocontrol strains obtained by primary screening were prepared into mycelial plugs by using a perforator with a diameter of 0.5 cm; the mycelial plugs were cultured on a 9 cm plate by the plate confrontation method, and both mycelial plugs were | cm away from the edge of the Petri dish, respectively; three replicates were set for each treatment; a pathogenic strain without inoculation with the biocontrol strain was used as a control. All groups were cultured at a constant temperature of around 25°C for 5-7 days. The colony radius was regularly measured by the cross method, and the inhibition rate of the strain against the pathogenic fungus was calculated according to the following formula. [OSL] hbo rate = Caan tom phon re colorant teed bons re 100%

[0052] A strain with the optimal inhibition rate was screened and labeled as an

HN17496 strain.

[0053] Example 2

[0054] Identification of the re-screened strain

[0055] The HN17496 strain re-screened in Example | was inoculated on PDA and cultured at 25°C for 7 days to obtain a colony with a diameter of 2.2 cm. The colony had the following morphological characteristics:

[0056] The colony was black; conidiogenous cells grew on a culture medium, were viscous, and were covered with grey or black sori; the back of the Petri dish appeared dark brown. There was no perithecium. Hyphae produced dark brown secretions that resembled impurities, usually appeared a bundle, and formed a hyphal bridge. The conidiophore appeared as a huge line, which was solitary or grown in a clustered manner, thin-walled, branched or unbranched, and erect or slightly curviform; 1 or 2 septa were present and looked smooth; the conidiophore was 18.2-64.0 um long and 2.8-4.9 um wide; 1-6 conidiogenous cells were apically inserted. Conidiogenous cells are phialidic, claviform to quasi-claviform, transparent, semitransparent or dark black, smooth-surfaced, 6.6-11.5 x 34-55 pum in size, with an obvious neck ring.

Acroconidia are present, free of septum, suborbicular, elliptical or subterete, and transparent, semitransparent to dark olivaceous; the wall was thick, dark olivaceous or pitchy, smooth-surfaced or tuberculate; the conidium was 7.0-11.6 um long and 3.3-7.5 um wide. The HN 17496 strain was preliminarily identified as Stachybotrys sp.

[0057] Using Bayesian inference, a phylogenetic tree was jointly constructed on

HN17496 and its similar species based on six genes, cmd A, ITS, LSU, rpb2, tub2, and tef-1a, with PP value >0.50 at a node, specifically as follows:

[0058] The phylogenetic tree contained 100 Stachvbonrys sp. strains. In the phylogenetic tree jointly constructed by cmd A, ITS, LSU, rpb2, tub2, and tef-1a, there were a total of 4,276 bases and 3,772 parsimony-informative characters (cmd A contained 692 bases and 376 parsimony-informative characters; /75 contained 549 bases and 189 parsimony-informative characters; LSU contained 830 bases and 70 parsimony-informative characters, rpb2 contained 777 bases and 2,826 parsimony-informative characters; f#b2 contained 287 bases and 113 parsimony-informative characters; tef~/a contained 1,141 bases and 450 parsimony-informative characters); the outgroup was Peethambara sundara (CBS 646.77), which had a considerable genetic distance away from all species of the genus

Stachybotrys and allied genera, was significantly distinguished in the genus, between species, and in the species, and had a higher node support rate.

[0059] The HN17496 strain became an individual branch in the phylogenetic tree (FIG. 3) and was significantly different; its hyphae were usually bundle-like and formed a hyphal bridge, and the conidium was smooth-surfaced or tuberculate, which was distinguished from other species; combined with the morphology and the molecular systematics, the HN17496 strain was determined as a new species:

Stachybotrys sp. 1.

[0060] The screened strain was named Stachybotrys sp. HN17496 and sent to deposit at CCTCC under accession number M2022489.

[0061] Example 3

[0062] Biocontrol effect of HN17496 strain

[0063] The HN17496 strain screened in Example 2 was inoculated on PDA and cultured at 25°C for 5 days; a mycelial plug (diameter: 5 mm) was cut at the edge of the colony of the HN17496 strain by using a perforator in a clean bench; the mycelial plug was transferred into an Erlenmeyer flask with 150 mL of Potato Dextrose Broth (PDB) and cultured in a 25°C constant temperature incubator shaker for 7 days (at a rotational speed of 100 r/min). At 7 days after culture, the mycelial plug was filtered with sterile gauze (eight-layered) and sterile filter paper successively. 30 mL of filtrate was filtered through a biofilter (0.22 pum) in the clean bench, and a biocontrol strain culture medium (CL) was obtained and stored at 4°C for use. In addition, 30 mL of filtrate was autoclaved in a 121°C autoclave for 30 min, and a crude toxin extract (CT) of the biocontrol strain was obtained and stored at 4°C for use.

[0064] The bioactivity of the metabolic liquid was detected by the mixed plate method. The metabolic liquid and the PDA were mixed evenly in a ratio of 1:4 (V/V); a mycelial plug (d = 5 mm) of the pathogenic fungus (B. sorokiniana) was inoculated in the middle of the plate and cultured at 25°C for 5 days; the change of colonial morphology was observed; the colony diameter was measured by the cross method, and the inhibition rate of the mycelial growth was calculated (according to the following formula). With a plate without metabolic liquid as a negative control, the test was repeated three times.

[0065] inhibition rate = en ESO ADORE 100%

[0066] The inhibition test results are shown in FIG. 4, where Petri dishes A and B show that no metabolic liquid is added and the diameter on Day 5 reaches 5 cm, while

Petri dishes C and D with metabolic liquid show that B. sorokiniana grows slowly, and the diameter on Day 5 of growth is less than 1 cm.

[0067] The biocontrol fungus HN17496 strain had an inhibitory effect on B. sorokiniana: the average inhibition rate of the unautoclaved biocontrol strain culture medium was 65%, and that of the autoclaved biocontrol strain culture medium reached 72%. The autoclaved culture medium had a more significant inhibitory effect on B. sorokiniana, indicating that chemicals that inhibited B. sorokiniana were resistant to high temperature and pressure. However, in the present disclosure, the relative content of antimicrobial substances in the crude toxin extract of the biocontrol strain obtained by sterilization without filtration through the biofilter (0.22 um) was increased, thereby showing a significant increase in inhibition rate.

[0068] In conclusion, the Stachybotrys sp. HN17496 strain provided by the present disclosure has an excellent biocontrol effect on B. sorokiniana, and has no potential safety hazard on human and livestock; from the examples, the Stachybotrys sp.

HN17496 strain provided by the present disclosure and metabolites thereof inhibit 65%-72% of B. sorokiniana, with a significant biocontrol effect.

[0069] Although the present disclosure has been disclosed as above in preferred examples, they are not intended to limit the present disclosure. Those skilled in the art can make various variations and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to that defined by the claims.

Claims

Conclusions l. Microbial biocontrol agent, wherein the microbial biocontrol agent comprises the HN17496 strain and/or metabolites of the HN17496 strain, wherein the HN17496 strain has been deposited at China Center for Type Culture Collection (CCTCC) under accession number M2022489.

The microbial biocontrol agent of claim 1, wherein provided that the microbial biocontrol agent comprises the HN17496 strain, based on g or mL of the microbial biocontrol agent, a trace number of the HN17496 strain in the microbial biocontrol agent is > 10 5 CFU.

A method of preparing the microbial biocontrol agent according to claim 1 or 2, wherein the method comprises the following step: inoculating the HN17496 strain according to claim 1 to culture on potato dextrose agar (PDA) for 5-7 days to obtain the microbial biocontrol agent .

Preparation method according to claim 3, wherein the culture is carried out at 25-28°C.

The preparation method according to claim 3 or 4, wherein provided that the microbial biocontrol agent does not include the HN17496 strain, sterilization is further carried out 5-7 days after culture.

Preparation method according to claim 5, wherein the sterilization is carried out at 121°C for 30 minutes.

Use of the HN17496 strain according to claim 1 or 2, the microbial biocontrol agent according to claim 2 or 3 or a microbial biocontrol agent prepared by the preparation method according to any one of claims 3 to 6 in the control of a plant disease, wherein a pathogen of the plant disease Bipolaris sorokiniana.

S11 -

Use according to claim 7, wherein the plant disease comprises wheat root rot.

Use according to claim 7 or 8, wherein the plant comprises wheat.