CN114606140A

CN114606140A - Isaria fumosorosea and application thereof in controlling diaphorina citri

Info

Publication number: CN114606140A
Application number: CN202210368303.3A
Authority: CN
Inventors: 念晓歌; 王德森; 何余容; 罗亚茹; 武书杰
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-06-10
Anticipated expiration: 2042-04-08
Also published as: CN114606140B

Abstract

The invention discloses isaria fumosorosea and application thereof in controlling diaphorina citri. The Isaria fumosorosea (Cordyceps fumosorosea) SCAU-CFDC01 is obtained by separating from the body of diaphorina citri, and the preservation number is GDMCCNO.62330; the strain has the characteristic of efficiently preventing and controlling diaphorina citri insect pests, and particularly has an obvious effect on preventing and controlling diaphorina citri nymphs; and in the application process, the strain is pollution-free, residue-free, biological and environment-friendly, and has good application prospect in the field of entomogenous fungi.

Description

Isaria fumosorosea and application thereof in controlling diaphorina citri

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to isaria fumosorosea and application thereof in controlling diaphorina citri.

Background

Citrus yellow shoot is a devastating citrus disease caused by bacteria of the genus phlobacterium and is called "cancer" of citrus because of its serious damage and high mortality. The diaphorina citri is an important propagation medium of the citrus greening disease in the field, and is mainly obtained and propagated by piercing and absorbing citrus phloem juice. Since the liberobacter citrii can not be artificially cultured, an effective strategy for preventing and controlling the liberobacter citrii is difficult to be provided from the liberobacter citrii, and the control of the liberobacter citrii is still the most effective means for preventing the liberobacter citrii from spreading all over the world at present. At present, the chemical prevention is mainly used for preventing and controlling the diaphorina citri, and a series of problems such as increase of drug resistance of the diaphorina citri, pesticide residue, environmental pollution, destruction of biological diversity and the like are caused by long-term use of a large amount of chemical insecticides.

The entomopathogenic fungi can be parasitized in an insect host body until the insect is killed, so that the entomopathogenic fungi has good disease epidemic potential and production convenience, and the prevention and treatment of the diaphorina citri by using the entomopathogenic fungi has good development prospect.

Disclosure of Invention

The invention aims to provide isaria fumosorosea with high pathogenicity to diaphorina citri and use the isaria fumosorosea to prevent and control diaphorina citri pests and diseases caused by diaphorina citri, namely citrus yellow dragon disease.

The invention separates a strain SCAU-CFDC01 with high pathogenicity to diaphorina citri from diaphorina citri.

The morphological characteristics of the SCAU-CFDC01 strain are as follows:

the SCAU-CFDC01 strain is milk white and opaque in the early stage on the SDAY culture medium, the colony is smooth and round, and the edge is complete; after 10 days, a clear pale purple spore was observed, and after 15 days, the whole plate appeared pale purple, and the spore yield per plate was about 5.8X 10⁸conidium/mL; the mature conidia are rod-shaped.

Molecular characterization of SCAU-CFDC01 Strain:

the SCAU-CFDC01 strain belongs to the Cordyceps genus by amplifying the 16S rDNA sequence TS1/TS5 (the nucleotide sequence is shown as SEQ ID NO. 1) of the SCAU-CFDC01 strain genome DNA and then performing homology alignment analysis on the sequence in the NCBI gene library. Based on phylogenetic analysis, the SCAU-CFDC01 strain was found to belong to Isaria fumosorosea. Close affinity relationship with three strains Cordyceps fumosorosea IfTS02(KX057373.1), Cordyceps fumosorosea ARSEF 3302(HM209050.1) and Cordyceps fumosorosea CNRCB1(HM 209049.1). The SCAU-CFDC01 strain is determined to belong to the genus Cordyceps (Cordyceps) by combining the morphological characteristics and 16S rDNA of the strain, and is named as Isaria fumosorosea (Cordyceps fumosorosea) SCAU-CFDC 01.

Therefore, the first objective of the invention is to provide an Isaria fumosorosea SCAU-CFDC01 with the collection number GDMCC NO. 62330.

The invention also provides application of isaria fumosorosea SCAU-CFDC01 in preventing and treating diaphorina citri and diseases caused by diaphorina citri.

Preferably, the disease caused by the diaphorina citri is citrus yellow dragon disease.

Preferably, the application comprises the step of contacting the living thallus of the isaria fumosorosea SCAU-CFDC01 or the culture containing the living thallus of the isaria fumosorosea SCAU-CFDC01 with the diaphorina citri.

Preferably, the culture containing the living bacteria of the isaria fumosorosea SCAU-CFDC01 is a bacterial suspension of the isaria fumosorosea SCAU-CFDC01, and the concentration is 1 x 10³-1×10⁸spores/mL.

Preferably, the diaphorina citri is diaphorina citri nymph.

Preferably, the diaphorina citri nymphs are three-to five-year-old nymphs.

The invention also provides a biocontrol agent for controlling diaphorina citri and diseases caused by diaphorina citri, which contains a culture of isaria fumosorosea SCAU-CFDC01 and/or isaria fumosorosea SCAU-CFDC01 as an active ingredient.

Preferably, the biocontrol agent is a bacterial suspension of Isaria fumosorosea SCAU-CFDC01 with the concentration of 1 x 10³-1×10⁸spores/mL.

The Cordyceps fumosorosea SCAU-CFDC01 (Isaria fumosorosea SCAU-CFDC01) has the characteristic of efficiently preventing and treating diaphorina citri insect pests, and particularly has obvious effect of preventing and treating diaphorina citri nymphs; and in the application process, the strain is pollution-free, residue-free, biological and environment-friendly, and has good application prospect in the field of entomogenous fungi.

Preservation description:

the Cordyceps fumosorosea SCAU-CFDC01 (Isaria fumosorosea SCAU-CFDC01) is preserved in Guangdong province microorganism culture collection center (GDMCC) at 28 days 03 and 2022, and the preservation number is as follows: GDMCC NO.62330, accession number: xieli Zhonglu 100 Dazhou No. 59 Lou 5 Lou, Guangzhou province academy of sciences microbial research institute.

Drawings

FIG. 1 is a diagram of the colony morphology and the conidium morphology of the strain SCAU-CFDC01 on SDAY medium; wherein, the picture a is the colony morphology of the strain SCAU-CFDC01 growing for 5 days on the SDAY culture medium, the picture b is the colony morphology of the strain SCAU-CFDC01 growing for 15 days on the SDAY culture medium, and the picture c is the morphology of the conidia of the strain SCAU-CFDC01 under an optical microscope.

FIG. 2 is a morphological diagram of infestation of psyllids by strain SCAU-CFDC 01; wherein the picture a is a morphological picture of the strain SCAU-CFDC01 infecting the diaphorina citri for 3 days, the picture b is a morphological picture of the strain SCAU-CFDC01 infecting the diaphorina citri for 5 days, and the picture c is a morphological picture of the strain SCAU-CFDC01 infecting the diaphorina citri for 9 days.

FIG. 3 is a phylogenetic tree of strain SCAU-CFDC01 based on the 16S rDNA sequence TS1/TS 5; in the figure, the numbering of the corresponding strains in the Genbank database is in parentheses.

FIG. 4 is a graph showing the cumulative mortality of diaphorina citri nymphs and adults as a function of time under the treatment of SCAU-CFDC01 bacteria at different concentrations; FIG. a is a graph of cumulative mortality of third instar nymphs treated with spore suspension; panel b is the cumulative mortality of five instar nymphs under spore suspension treatment; panel c is the cumulative mortality of adults treated with spore suspension.

FIG. 5 is the effect of SCAU-CFDC01 strain on the growth and development of third instar nymphs; in a figure: influence of the SCAU-CFDC01 strain on the daily survival rate of the third-instar nymphs; and (b) figure: influence of the SCAU-CFDC01 strain on the emergence rate of the third-instar nymphs; and (c) figure: influence of SCAU-CFDC01 strain on imago ratio of emergence of the third-instar nymphs; FIG. d: effect of SCAU-CFDC01 strain on adult life of third instar nymphs.

FIG. 6 is the effect of SCAU-CFDC01 strain on the growth and development of five-instar nymphs; in a figure: influence of the SCAU-CFDC01 strain on the daily survival rate of five-year-old nymphs; and (b) figure: influence of SCAU-CFDC01 strain on five-instar nymph emergence rate; and (c) figure: influence of SCAU-CFDC01 strain on imago ratio of five-instar nymph eclosion; FIG. d: effect of SCAU-CFDC01 strain on adult life of five-instar nymphs.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

The inventor isolated a strain with high pathogenicity to diaphorina citri from diaphorina citri (figure 2), which is numbered SCAU-CFDC 01.

1. The preparation method of the culture medium adopted by the strain SCAU-CFDC01 characteristic determination is as follows:

saka (SDAY) plate medium: 40g of glucose, 10g of peptone, 2g of yeast extract and 1.5g of agar were added to sterile water to adjust the total volume to 1000mL and pH to 7.0-7.2. Cooling and pouring the mixture into a flat plate for later use after sterilization.

2. Identification of strain SCAU-CFDC01

According to the fungus identification handbook compiled by Weijing super, the morphological characteristics of SCAU-CFDC01 strain are clear. The strain SCAU-CFDC01 was identified by referring to the method described in "microbiology laboratory Manual" of Zhoudeqing et al (Shanghai: science and technology Press) and 16S rDNA.

The morphological characteristics of SCAU-CFDC01 strain are as follows:

the SCAU-CFDC01 strain appears milky white, opaque, smooth and round in the early stage on SDAY medium, and has complete edge (FIG. 1 a); after 10 days, a distinct pale purple spore was visible, and after 15 days, the entire plate appeared pale purple (FIG. 1b), with a spore yield of about 5.8X 10 per plate⁸conidium/mL; mature conidia are rod-shaped (FIG. 1 c). The thallus is white in early stage on PDA culture medium plate, and becomes rose smoke color after 11-12 daysClear, smooth and round colony.

The culture characteristics of the SCAU-CFDC01 strain are as follows: the growth temperature range is 4-32 ℃, and the optimal growth temperature is 25-27 ℃; the growth pH range is 5.0-8.0, the growth is good under the condition of partial acid, and the optimal growth pH range is 6.0-7.0; the spore yield was 5X 10 after 10 days of culture on SDAY medium⁸conidia/mL.

16S rDNA sequence TS1/TS5 of SCAU-CFDC01 strain

A small number of single colonies were picked and genomic DNA of the strain SCAU-CFDC01 was extracted using a fungal kit. The genome DNA of the strain SCAU-CFDC01 is taken as a template, and the following primers are adopted: ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS 5: 5'-GGTTACCTTGTTACGACTT-3', performing PCR amplification in a system of 50. mu.L containing 2 XPri-113

Max DNA 25. mu.L, forward and reverse primers 2. mu. L, ddH each₂O19. mu. L, DNA template 2. mu.L, and finally deionized ultrapure water was used to adjust the total reaction volume to 50. mu.L. Amplification conditions: 5min at 95 ℃; at 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 1min, and 35 cycles; 5min at 72 ℃. The fragments with the size of about 500bp are amplified by PCR and sent to a company Limited in Biotechnology engineering (Shanghai) for sequencing, and the sequencing result shows that the sequence contains 564 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 1. The sequence is corrected by Chromas sequence splicing software, homology comparison is carried out on NCBI (http:// blast. NCBI. nlm. nih. gov /) gene library, a phylogenetic tree is constructed by adopting MEGA7.0 software and using a Neighbor-Joining method (Neighbor-Joining), phylogenetic analysis is carried out, and the number of each support is the support percentage of 1000 Bootstrap resampling analyses.

The SCAU-CFDC01 strain was found to belong to the genus Cordyceps by homology alignment analysis at the NCBI (http:// blast. NCBI. nlm. nih. gov /) gene bank. Based on phylogenetic analysis (FIG. 3, Table 1), the strain SCAU-CFDC01 was found to belong to Isaria fumosorosea. Close relationship with three strains Cordyceps fumosorosea IfTS02(KX057373.1), Cordyceps fumosorosea ARSEF:3302(HM209050.1) and Cordyceps fumosorosea CNRCB1(HM 209049.1). Determining that the SCAU-CFDC01 strain belongs to the genus Cordyceps (Cordyceps) by combining morphological characteristics and 16S rDNA of the strain, and is named as Isaria fumosorosea (Cordyceps fumosorosea) SCAU-CFDC 01; and the strain was deposited at 2022, 03.28 days in the culture collection of microorganisms of Guangdong province (GDMCC), accession number: GDMCC NO. 62330.

Table 1 is the information on the strains used to construct phylogenetic trees, including the strain names, numbering in the Genbank database, collection, sequence size of the 16S rDNA sequence TS1/TS5 and similarity analysis to SCAU-CFDC01 strain.

TABLE 1 information on the strains used for the construction of phylogenetic trees

5, measuring the indoor pathogenicity of the SCAU-CFDC01 strain to the diaphorina citri

Adding SCAU-CFDC01 strain dry spore powder into 0.05% Tween-80 solution, stirring with magnetic stirrer, determining spore concentration with blood count plate, and diluting to 1 × 10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³6 spores/mL are used for detecting the pathogenicity of the SCAU-CFDC01 strain to three-age and five-age nymphs of the diaphorina citri; setting a blank control for each bioassay, wherein the blank control is 0.05% of tween-80, selecting three-year nymphs/five-year nymphs with consistent individual size and activity, selecting the nymphs to be tested into the spore suspension liquid subjected to different treatments by using a soft brush pen, soaking the nymphs for 20s, picking the nymphs out, placing the nymphs on clean filter paper, absorbing redundant water, transferring the nymphs to citrus leaves, placing the citrus leaves into a culture dish (the diameter is 9cm) with the bottom padded with wet filter paper, sealing the culture dish by using a preservative film, puncturing holes, ventilating, inoculating the nymphs, placing the nymphs in an artificial climate box (25 +/-1 ℃, 60% -70% of Relative Humidity (RH)), and the light cycle is 14L: 10D) and observing and recording the number of dead insects after 24 hours, and continuously observingAnd observing for seven days, and picking the dead insects into a culture dish with moist filter paper at the bottom of the culture dish. Each treatment had 30 nymphs and the experiment was repeated three times.

Diluting dry spore powder in 0.05% Tween-80 solution to 1 × 10⁸、5×10⁷、1×10⁷、5×10⁶、1×10⁶5 spores/mL were used to test the virulence of SCAU-CFDC01 strain on adult diaphorina citri, in a manner similar to treatment of nymphs.

The measurement result shows that: the SCAU-CFDC01 strain has strong pathogenicity to both diaphorina citri nymphs and adults (figure 4), and is at different concentrations (1X 10)³-1×10⁸spores/mL) 7 days after the treatment, the death rate of the third-instar nymphs is 53.19-100%, and the death rate of the fifth-instar nymphs is 41.82-100%; at different concentrations (1X 10)⁶-1×10⁸spores/mL) was 30% -62.5% adult mortality. Wherein, 1 × 10 is used⁸After two days after the treatment of the spore/mL spore suspension, the death rate of the third-instar nymphs reaches 96.2 percent, and the death rate of the fifth-instar nymphs reaches 86.2 percent; by 1X 10⁷Two days after the treatment of each spore/mL spore suspension, the death rate of the third-instar nymphs is 88.89 percent, and the death rate of the fifth-instar nymphs is 70.13 percent; by 1X 10⁶After two days after the treatment of each spore/mL spore suspension, the death rate of the third-instar nymphs reaches 59.32 percent, and the death rate of the fifth-instar nymphs reaches 35.48 percent; by 1X 10⁵Two days after the treatment of each spore/mL spore suspension, the death rate of the third-instar nymphs is 59.26%, and the death rate of the fifth-instar nymphs is 26.69%. By 1X 10⁸spore/mL and 1X 10⁷3 days after the treatment of each spore/mL spore suspension, the death rate of three-year-old nymphs and five-year-old nymphs is up to 100 percent. By 1X 10⁶Three days after the treatment of each spore/mL spore suspension, the mortality rate of the three-instar nymphs is 88.89 percent, and the mortality rate of the five-instar nymphs is 72.58 percent; by 1X 10⁵Three days after the spore/mL spore suspension is treated, the death rate of the third-instar nymphs is up to 81.48 percent, and the death rate of the fifth-instar nymphs is 69.23 percent. By 1X 10⁵The mortality rate of the third instar nymphs was up to 100% and the mortality rate of the fifth instar nymphs was 80.77% 6 days after treatment with one spore/mL spore suspension (table 2).

TABLE 2 cumulative mortality of diaphorina citri nymphs on different treatment days and at different treatment concentrations

LC50s (Table 3) and LT50s (Table 4) both show that the SCAU-CFDC01 strain has strong pathogenicity and high death speed to diaphorina citri nymphs, wherein the pathogenicity to the trichuris is strongest, and the trichuris citri nymphs are five-instar diaphorina citri and are also adult diaphorina citri.

Table 3 shows the lethal middle concentration of Trionyx citriodorus, Trionyx citriodorus and Trionyx citriodorus in adults after the SCAU-CFDC01 strain is treated for different days, and LC (liquid chromatography) is performed along with the increase of the treatment days₅₀The smaller the value.

TABLE 3 lethal middle concentration of Trionyx citriodora for three, five and adults after treatment with SCAU-CFDC01 strain for different days

Table 4 shows LT at various concentrations of SCAU-CFDC01 in the cases of three-year old, five-year old and adult diaphorina citri killing after treatment with increasing treatment concentration₅₀The smaller the value.

TABLE 4SCAU-CFDC01 Strain at different concentrations treatment Trinity, quintuple and lethal time of adults

Determination of greenhouse pathogenicity of SCAU-CFDC01 Strain to Tri-and penta-age nymphs of diaphorina citri

On the basis of indoor toxicity measurement, the field effect of the SCAU-CFDC01 strain on Tri-instar nymphs/five-instar nymphs of diaphorina citri is evaluated.

Experiments are carried out in insect cages (120cm is multiplied by 120cm) in a greenhouse, the experimental conditions are Guangzhou June-September, Murraya paniculata seedlings with consistent growth vigor and consistent young shoots are placed in the insect cages, nine pots are placed in each cage, about 300 diaphorina citri in the mating flourishing stage are placed in the insect cages for free mating for 12 hours, then all adults are completely removed, and when eggs grow to the third-instar nymphs/fifth-instar nymphs, the nymphs are used for the experiments. Experimental treatment group was 1X 10⁵spores/mL SCAU-CFDC01 strain spore suspension, and control group is 0.05% Tween-80. Before treatment, the population base numbers of three-year nymphs and five-year nymphs of diaphorina citri are recorded, and the nymphs are sprayed on the front and back surfaces of murraya paniculata seedlings by using a manual sprayer, and each seedling is sprayed with 20mL of pesticide. Observing and recording the number of larvae, the death number and the death reasons of the larvae on each treatment every day until all the larvae are eclosized, calculating the daily survival rate of the nymphs, the eclosion rate of adults and the sex ratio of adults, and counting the service life of the eclosion adults.

The results show that the SCAU-CFDC01 strain has good control effect on the Trionycis citricida nymphs in the greenhouse (figure 5), the survival rate of the Trionycis is only 9.20%, the eclosion rate of adults is only 7.17%, and the life of females is 16.45 days after the treatment of 5 days, which are all significantly lower than that of a control (P <0.001), but the ratio of the eclosion adults to five-year nymphs and the life of males after the treatment of the five-year nymphs have no significant influence (P > 0.05).

The SCAU-CFDC01 strain also has good control effect on five-instar nymphs of diaphorina citri in a greenhouse (figure 6), the survival rate of the five-instar nymphs is only 33.53 percent, the eclosion rate of adults is only 26.32 percent, and the life of females is 16.47 days after the treatment on the 5 th day, which are all obviously lower than that of a control (P <0.001), but the ratio of the eclosion adults to the five-instar nymphs after the treatment and the life of males have no obvious influence (P > 0.05).

Sequence listing

<110> southern China university of agriculture

<120> Isaria fumosorosea and application thereof in control of diaphorina citri

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 564

<212> DNA

<213> Isaria fumosorosea SCAU-CFDC01(Cordyceps fumosorosea SCAU-CFDC01)

<400> 1

gtgctacgag cttttcactc cctaaccctt tgtgacatac ctatcgttgc ttcggcggac 60

tcgccccggc gtccggacgg ccctgcgccg cccgcgaccc ggacccaggc ggccgccgga 120

gacccacaaa ttctgtttct atcagtcttt ctgaatccgc cgcaaggcaa aacaaatgaa 180

tcaaaacttt caacaacgga tctcttggtt ctggcatcga tgaagaacgc agcgaaatgc 240

gataagtaat gtgaattgca gaattcagtg aatcatcgaa tctttgaacg cacattgcgc 300

ccgccagcat tctggcgggc atgcctgttc gagcgtcatt tcaaccctcg acaccccttc 360

gggggagtcg gcgttgggga ccggcagcat accgccggcc ccgaaataca gtggcggccc 420

gtccgcggcg acctctgcgt agtactccaa cgcgcaccgg gaacccgacg cggccacgcc 480

gtaaaacacc caacttctga acgttgacct cggatcaggt aggactaccc gctgaactta 540

agcatatcaa gggggaaaga ggaa 564

Claims

1. Isaria fumosorosea SCAU-CFDC01 is characterized by being deposited with GDMCC NO. 62330.

2. Application of isaria fumosorosea SCAU-CFDC01 in preventing and treating diaphorina citri and diseases caused by diaphorina citri.

3. The use according to claim 2, wherein the disease caused by diaphorina citri is citrus yellow dragon.

4. The use according to claim 2, comprising the step of contacting live biomass of isaria fumosorosea SCAU-CFDC01 or a culture comprising live biomass of isaria fumosorosea SCAU-CFDC01 with diaphorina citri.

5. The use of claim 4, wherein the culture containing living cells of Isaria fumosorosea SCAU-CFDC01 is a suspension of Isaria fumosorosea SCAU-CFDC01 at a concentration of 1X 10³-1×10⁸spores/mL.

6. The use according to claim 4, wherein the diaphorina citri is diaphorina citri nymph.

7. The use according to claim 6, wherein the diaphorina citri nymph is a three to five-year old nymph.

8. A biocontrol agent for controlling diaphorina citri and diseases caused by diaphorina citri characterized in that it contains a culture of Isaria fumosorosea SCAU-CFDC01 and/or Isaria fumosorosea SCAU-CFDC01 as an active ingredient.

9. The biocontrol agent of claim 8 wherein said biocontrol agent is a bacterial suspension of Isaria fumosorosea SCAU-CFDC01 at a concentration of 1 x 10³-1×10⁸spores/mL.