LU502326B1 - Isaria javanica strain for preventing and treating citrus pests and use thereof - Google Patents

Abstract

The present invention belongs to the technical field of microorganisms, and relates to an Isaria javanica strain for preventing and treating citrus pests and use thereof. The strain is classified and named Isaria javanica MSC-f1, has been deposited at the China General Microbiological Culture Collection Center on 19 March 2020, and has the deposit number of CGMCC No. 19601. The strain has strong pathogenicity to Aphis citricola, Icerya purchasi, Diaphorina citri and Panonychus citri, can be developed into a biological antibacterial formulation for biological prevention and treatment of various citrus pests, and has the characteristics of high efficiency, low toxicity, low residue, no pollution, being not easy to produce drug resistance and the like compared with chemical agents; in addition, the Isaria javanica strain has a fast growth speed, a large spore production capacity, is easy to prepare, and has a good market application prospect.

Description

DESCRIPTION LU502326

ISARIA JAVANICA STRAIN FOR PREVENTING AND TREATING CITRUS PESTS AND USE THEREOF

FIELD OF TECHNOLOGY The present invention belongs to the technical field of microorganisms, and relates to an Isaria javanica strain for preventing and treating citrus pests and use thereof.

BACKGROUND Aphis citricola, Icerya purchasi, Diaphorina citri, and Panonychus citri are important pests on citrus. Among them, imagoes and nymphs of Aphis citricola intensively gather on the back of young buds, twigs and young leaves to pierce and suck juice, causing the young leaves to roll back, the new buds to shortage, the young leaves to shrink and deform, the leaves to fall and the buds to bald, and the tree vigor to weaken when they occur seriously; in addition, Aphis citricola can also secrete honeydew, inducing sooty moulds; more importantly, Aphis citricola is also a natural propagation medium for pathogenic organisms such as citrus tristeza virus and citrus yellow pulse virus, and pests are vulnerable to move between diseased and healthy tree, causing spread and dissemination of diseases such as citrus tristeza virus and citrus yellow pulse virus. Nymphs and female imagoes of Icerya purchasi cluster on the branches, both sides of the midvein on the back of leaves and fruits of citrus and other plants, ingest juice, yellow leaves and wither branches, shrink the branches, rough the cortex, or even die, causing fallen leaves and fruits; and Icerya purchasi can also excrete a large amount of honeydew, inducing sooty moulds and affecting photosynthesis. Diaphorina citri is a main pest in the shoot-growing stage of Rutaceae plants such as citrus, and can directly feed on the young shoots and damage the young leaves and produce secretions to induce sooty moulds. In particular, Diaphorina citri is a natural propagation medium for citrus Huanglongbing. Citrus Huanglongbing is the most difficult and threatening devastating disease in production of citrus, and its population density is positively correlated with the severity of citrus Huanglongbing. Imagoes, nymphs and iarvas of Panonychus citri pierce the epidermis of host leaves by means of the mouthparts to suck juice, the damaged leaves show numerous small grey and white spots, and in serious cases the whole leaves lose green and turn gray white, resulting in a large number of fallen leaves, damaging the fruits and green branches, and seriously affecting the tree vigor and yield. LU502326 The effective prevention and treatment of pests such as Aphis citricola, Icerya purchasi, Diaphorina citri, and Panonychus citri is of great significance to eliminate the damage of pests themselves to citrus trees and to inhibit spread and dissemination of diseases such as citrus tristeza virus and citrus yellow pulse virus. Currently, chemical pesticides are mainly used to prevent and treat these pests. However, the frequent use of chemical pesticides has brought about increasingly serious 3R, 1e, resistance, resurgence and residue, problems. However, biopesticides have attracted more and more attention due to their characteristics of no residue, high specificity, no harm to insects and beneficial organisms, being not easy to produce drug resistance, good environmental compatibility, simple production process and the like. Microbial pesticides are the most recently researched biological pesticides. In the application of fungal pesticides, Beauveria bassiana is used to prevent and treat more than 40 kinds of pests such as pine caterpillar, corn borer and rice leathopper, Metarhizium is used to prevent and treat Locusta migratoria moniliasis and other locust pests, and Metarhizium anisopliae is used to prevent and treat sweet potato froghopper. All of these have achieved remarkable effects. It can be determined that fungal pesticides have a good application prospect in the prevention and treatment of plant diseases and insect pests.

Isaria javanica is a fungus of Isaria of Conlycipitaceae. It has been reported that different strains of Isaria javanica can infect various pests such as Bemisia tabaci, Spodoptera litura, Hyphantria cunea, Icerya aegyptiaca, Phenacoccus Solenopsis, Megalurothrips usitatus, Empoasca vitis and Nilaparvata lugens. However, there are few reports on the prevention and treatment of citrus pests by this fungus. It has been proved by the research that the Isaria javanica strain MSC-f1 provided by the present invention has strong pathogenicity to Aphis citricola, Icerya purchasi, Diaphorina citri and Panonychus citri, has the potential to be developed into a biological antibacterial formulation, and is applied to biological prevention and treatment of Aphis citricola and other pests.

SUMMARY An object of the present invention is to provide an Isaria javanica strain for preventing and treating citrus pests and use thereof. The strain has the potential of being developed into biopesticides, and has a good market application prospect.

The object of the present invention is achieved by the following technical solutions:

The present invention first provides an Isaria javanica strain, wherein the strain is classifigdj502326 and named as Isaria javanica MSC-f1, has been deposited at the China General Microbiological Culture Collection Center on 19 March 2020, and has the deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing and the deposit number of CGMCC No.19601.

Further, the described Isaria javanica MSC-f1 is obtained by isolating Diaphorina citri bodies collected from orange orchards in Huangyan District, Taizhou City, Zhejiang Province. The strain has the following results of morphological characteristics, culture characteristics, gene sequence analysis and strain classification.

Morphological characteristics of strain MSC-f1: the strain MSC-f1 grows well in a potato dextrose agar (PDA) medium, with a colony diameter of 4.62 cm after dark culture at 25°C for d. On a PDA medium plate, in the initial stage of culture, the colonies are round with well-developed aerial hyphae and white cotton flocs; in the middle and late stages of culture, the colonies are layered with concentric rings, and the edges of the colonies are mostly thick white flocculent hyphae, the middle layer of the colonies is thin, and is a gray-purple spore layer, and the back of the colonies is light yellow. It is observed under an optical microscope that the hypha of the strain MSC-f1 is transparent and colorless with a width of 1.82 + 0.48 um; conidiophore is thick and short without coremium and with a length of 10.38 + 3.26 um and a width of 3.26 +

0.35 um; conidiogenous cells are flask shaped, the base of a phialide is elliptic and expanded and gradually becomes thinner in an upward direction with a length of 7.52 + 1.31 um and a diameter at the widest position of 2.75 + 0.35 um, and the whorl is directly planted at the top of the hypha or conidiophore; and conidiospore is planted on the phialide, is colorless, smooth, oblong, and arranged in chains with a length of 4.57 + 1.43 um and a width of 2.17 + 0.36 pm.

Biological characteristics of strain MSC-f1: The strain MSC-f1 can grow on a PDA medium in the temperature range of 15°C to 30°C, in which the growth rate of the hyphae at 25°C and 30°C is obviously higher than that at other temperatures, and the colonies hardly grow at low temperatures of 5°C and 10°C. The strain MSC-f1 is capable of growing on a tested PDA medium, a Sabouraud dextrose agar yeast extract (SDAY) medium, a yeast extract malt extract agar (YMA) medium, a corn meal agar (CMA) medium, a malt extract agar (MEA) medium, a Czapek Dox agar (CDA) medium, and a Sabouraud's dextrose agar (SDA) medium, among which the strain MSC-f1 grows fastest on SDAY, PDA and CDA and other media; the back of the colonies is yellow on the seven tested culture media, with the darkest color on the SDAY medium; conidia are produced on all seven culture media with the largest amount of conidta,502326 produced on the SDAY medium, followed by the PDA medium.

Analysis of gene sequence of strain MSC-f1: according to the sequencing results, the strain MSC-f1 has the sequence of ITS1-5.8S rDNA-ITS2 as shown in SEQ ID NO: 1, the sequence of the B-tubulin gene as shown in SEQ ID NO: 2, and the sequence of the TEF1-a gene as shown in SEQ ID NO: 3. Upon comparative analysis by NCBI blast, it is showed that the similarity between the ITS1-5.8S rDNA-ITS2 sequence of the strain MSC-f1 and the corresponding gene sequences (such as KM234215, JN204422 and MH5S32893) of a plurality of strains of Isaria javanica in GenBank reaches 99.8% to 100%; the similarity between the partial sequence of TEF1-a gene of the strain MSC-f1 and the corresponding gene sequences (such as KY587207, KT225600 and KT225598) of a plurality of strains of Isaria javanica in GenBank reaches 100%; and the similarity between the partial sequence of B-tubulin gene and the corresponding gene sequences (such as KT225602, MN215882, and KT225605) of strains different from Isaria javanica in GenBank is at most 99.41%, indicating that the B-tubulin gene sequence of strain MSC-f1 has a base difference from the reported gene sequences of the strains.

According to the above results of the morphological characteristics, biological characteristics and gene sequence analysis regarding the strain MSC-f1, the strain MSC-f1 can be identified as Isaria javanica of Isaria of Cordycipitaceae of Hypocrellales of Sordariomycetes of Pezizomycotina.

The present invention also provides use of the described Isaria javanica for preventing and treating citrus pests.

Further, the described citrus pests comprise Aphis citricola, Icerya purchasi, Diaphorina citri, and Panonychus citri.

The present invention also relates to a biological antibacterial formulation for citrus pests, comprising conidia of the described Isaria javanica MSC1-f1.

Further, Isaria javanica MSC1-f1 in the described biological antibacterial formulation has the contents of 1 x 10® conidia/mL.

The present invention also provides use of the described biological antibacterial formulation for preventing and treating citrus pests.

Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) the Isaria javanica strain MSC-f1 provided in the present invention is obtained by isolating infected Diaphorina citri bodies collected from orange orchards [ny502326 Huangyan District, Taizhou City, Zhejiang Province, has strong pathogenicity to Diaphorina citri, and also has strong pathogenicity to Aphis citricola, Icerya purchasi and Panonychus citri; (2) the Isaria javanica MSC-f1 of the present invention is an insect pathogenic fungus, and a conidial suspension thereof can directly act on pests such as Aphis citricola, Icerya purchasi, Diaphorina citri and Panonychus citri, and the strain has the characteristics of high efficiency, low toxicity, low residue, no pollution, being not easy to produce drug resistance and the like compared with chemical agents; and (3) the strain MSC-f1 has a fast growth speed, a large spore production capacity and a high spore germination rate, and is easy to prepare. In conclusion, the strain MSC-f1 has a good market application prospect when being developed into a biological antibacterial formulation for biological prevention and treatment of pests such as Aphis citricola, Icerya purchasi, Diaphorina citri, and Panonychus citri.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows colony characteristics of Isaria javanica MSC-f1 cultured on PDA medium for 20d.

Fig. 2 shows mycelium, sporulation structure and conidia characteristics of Isaria javanica MSC-f1 observed under an optical microscope.

Fig. 3 shows infestation and pathogenicity of Isaria javanica MSC-f1 on Aphis citricola (A), Icerya purchasi (B), Diaphorina citi (C) and Panonychus citri (D)observed under a stereomicroscope.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention will be further described below in combination with specific embodiments, but the scope of protection of the present invention is not limited thereto.

An Isaria javanica strain disclosed by the present invention, wherein the strain is classified and named as Isaria javanica MSC-f1, has been deposited at the China General Microbiological Culture Collection Center on 19 March 2020, and has the deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing and the deposit number of CGMCC No.19601.

The culture medium used in the present invention is as follows: Potatose glucose agar (PDA) solid medium: peeling 200 g of potato, adding 1000 mL of water, and boiling same for half an hour, filtering same with a gauze, adding 20 g of glucose and g of agar into a filtrate, fully dissolving same and filtering same with the gauze while hot,

packing the mixture into a conical flask or a glass tube, and sterilizing same at 121°C under highjs02326 pressure for 20 min; Sabouraud dextrose agar yeast extract (SDAY) medium: 10 g of yeast extract, 40 g of glucose, 10 g of peptone, 20 g of agar, 1 L of distilled water, pH of 6.0; Yeast extract malt extract agar (YMA) medium: 4 g of yeast extract, 10 g of malt extract, 4 g of glucose, 20 g of agar, 1 L of distilled water, pH of 7.2; Corn meal agar (CMA) medium: 7 g of corn extract, 15 g of agar, 1 L of distilled water, pH of 6.0; Malt extract agar (MEA) medium: 30 g of malt extract, 3 g of soybean peptone, 15 g of agar, 1L of distilled water, pH of 5.6; Czapek Dox agar (CDA) medium: 3 g of sodium nitrate, 1 g of dipotassium phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of potassium chloride, 0.01 g of ferrous sulfate, 30 g of sucrose, 15 g of agar, 1 L of distilled water, pH of 7.3; and Sabouraud's dextrose agar (SDA) medium: 10 g of peptone, 40 g of glucose, 20 g of agar, 1 L of distilled water, pH of 5.6.

Example 1: Isolation and Screening of Entomopathogenic Fungi

1. Isolation and purification of pathogenic bacteria: Diaphorina citri carcasses are collected from citrus orchards in Huangyan District, Taizhou City, Zhejiang Province, and the following operations are performed in a laboratory ultra-clean workbench: first soaking the carcasses in 70 vol% ethanol for 30 s, and then soaking same in 0.1 wt% aqueous mercuric chloride for 3 min, finally rinsing same with sterile water for 3 times, absorbing moisture on the carcasses by means of sterile filter paper, and then placing same in a potato dextrose agar (PDA) solid medium plate; placing a culture dish in a microbial incubator and culturing same at 25°C, after mycelium grows around the insect, picking up the mycelium and transfer same to a new PDA plate for culture (repeating this operation for 2 to 3 times) for about 10 d, continuous culturing same for about 10 d, after conidia are produced on the colony, eluting the conidia with sterile water so as to prepare the conidial suspension (the concentration of 3.0 x 10° conidia/ml), then performing isolation of monospora by referring to a dilution and purification method (Fangzhongda. Research Method for Plant Disease (Third Edition) [m] Beijing: China Agricultural Press, 1998: 122-140), China Agricultural Press, 1998: finally storing the strain obtained by isolation and purification in a PDA slant medium, culturing same for about 5 d, and storing same in a refrigerator at 4°C. LU502326

2. Screening of pathogenic bacteria Inoculating the isolated, purified and preserved strain onto a PDA plate, culturing same at 25°C for about 10 d, eluting the conidia produced on the colonies with sterile water, preparing the conidial suspension with a concentration of 3.0 x 10° conidia/mL, and adding tween-80 with a final concentration of 0.1 vol% into same; taking 30 healthy imagoes of Diaphorina citri and immersing same in the conidial suspension containing 0.1vol% Tween-80 for 10 to 15 s, picking up the Diaphorina citri that can still move freely after treatment, and placing same on the leaves of thyme in the culture dish (placing two layers of filter paper moistened with sterile water at the bottom of the culture dish on which is placed a young branch of thyme with about 10 leaves, and wrapping the bottom of the branches with absorbent cotton moistened with sterile water) for culturing, then culturing same in a light incubator at 25°C with14 h of light/10 h of darkness every day and the humidity of above 95%, setting up Diaphorina citri treated by a sterile water containing 0.1% Tween-80 as a control; and observing the infestation and death of pathogenic bacteria to Diaphorina citri after culturing for 7 d. Then according to the method for isolating and purifying the described pathogenic bacteria, isolating the pathogenic bacteria from the treated Diaphorina citri, and comparing the morphological characteristics and the culture characteristics of the re-isolated pathogenic bacteria with the initially inoculated pathogenic bacteria.

By means of the described operations, screening a strain pathogenic to Diaphorina citri according to the Koch's Rule, and excluding non-pathogenic bacteria such as saprophytic bacteria isolated from Diaphorina citri bodies. By screening, obtaining one strain having pathogenicity to Diaphorina citri, and naming same as MSC-f1.

3. Rejuvenation of strain: Preparing a conidial suspension of the described screened strain MSC-f1 capable of pathogenicity to Diaphorina citri with a concentration of 3.0 x 10° conidia/mL, inoculating same onto healthy conidia bodies, and then isolating and purifying pathogenic bacteria from the diseased Diaphorina citri bodies, the specific operation method being the same as that mentioned above, and finally obtaining the isolated and purified strain MSC-f1.

Example 2: Identification and Analysis of Strain MSC-f1

1. Morphological characteristics of strain MSC-f1 Inoculating the described isolated and purified strain MSC-f1 into the center of a PDA medium plate, placing same and culturing same at 25°C, observing growth conditions pf)j502326 colonies every day, and recording the color and morphology of colonies. Inoculating the strain MSC-f1 onto the center of another PDA culture medium plate, inserting obliquely sterilized cover glass of 1 cm x 1 cm into the culture medium at a distance of about 1 cm from the inoculation point, culturing same at 25°C until hyphae climb onto the cover glass, and then taking out the cover glass and placing same under an optical microscope to observe the hyphae and spore morphology of the strain MSC-f1.

Strain MSC-f1 grows well on a potato dextrose agar (PDA) medium with a dark culture at 25°C for 10 d and a colony diameter of 4.58 cm.

As shown in figure 1, the colony characteristics of the strain MSC-f1 after culturing for 20 d at 25°C on a PDA medium are as follows: it can be determined that the aerial hyphae of the strain are developed and is white cotton flocs; the colonies are round and layered with concentric rings, and the edges of the colonies are mostly thick white flocculent hyphae, the middle layer of the colonies is thin, and is a gray-purple spore layer, and the back of the colonies is light yellow.

As shown in figure 2, the morphological characteristics of the strain MSC-f1 observed under an optical microscope can be seen as follows: the hypha thereof are transparent and colorless with a width of 1.82 + 0.48 um; conidiophore is thick and short without coremium and with a length of 10.38 + 3.26 um and a width of 3.26 + 0.35 um; conidiogenous cells are flask shaped, the base of a phialide is elliptic and expanded and gradually becomes thinner in an upward direction with a length of 7.52 + 1.31 um and a diameter at the widest position of 2.75 +

0.35 um, and the whorl is directly planted at the top of the hypha or conidiophore; and conidiospore is planted on the phialide, is colorless, smooth, oblong and arranged in chains with a length of 4.57 + 1.43 um and a width of 2.17 + 0.36 um.

2. Biological characteristics of strain MSC-f1 (1) Growth conditions of the strain MSC-f1 at different temperatures: eluting the strain MSC-f1 after culturing on a PDA plate for 10 d with 10 mL of sterile water, filtrating same with a sterile gauze and removing hypha, and collecting the filtrate so as to prepare a conidial suspension with a concentration of 3.0 x 10° conidia/mL. transferring 1 ml of conidia suspension with a pipette and adding same into a PDA culture medium cooled to below 50°C and mixing same well, pouring same into a sterile culture dish under a ultra-clean workbench so as to prepare a bacteria-carrying plate, placing same and culturing same at 25°C until the hypha visible to the naked eye grow, punching bacteria cakes with a perforator with a diameter of 0;3)502326 cm, and respectively placing the bacteria cakes on the center of several freshly prepared PDA plate with a diameter of 9 cm, and culturing same in an inverted manner in 5°C, 10°C, 15°C, 20°C, 25°C, 30°C and 35°C constant temperature incubator respectively with three replicates at each temperature. Culturing same for 10 days, observing the growth of colonies every 24 hours and recording the diameter of colonies by means of a cross-over method.

The results are shown in Table 1, and it can be determined that the strain MSC-f1 can grow on a PDA medium in the temperature range of 15°C to 30°C, in which the growth rate of the hyphae at 25°C and 30°C is obviously higher than that at other temperatures, and the colonies hardly grow at low temperatures of 5°C and 10°C. Therefore, it can be determined that 25-30°C is an appropriate growth temperature for strain MSC-f1.

Table 1. Growth of strain MSC-f1 at different temperatures temperature 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 5°C 050 050 050 050 050 050 050 050 050 050 10°C 050 050 050 050 057 059 0.61 064 067 070 15°C 050 050 058 0.71 0.93 1.25 1.53 1.71 195 2.16 20°C 058 082 1.02 1.37 1.72 198 239 275 313 3,1 25°C 0.70 1.13 175 2.13 265 308 339 381 417 4.62 30°C 0.64 095 1.42 1.85 228 275 3.07 360 392 439 35°C 050 058 083 1.12 1.25 1.40 1.72 196 222 2.50 (2) Growth and sporulation of MSCs-f1 in different media: Preparing potatose glucose agar (PDA) solid medium, a Sabouraud dextrose agar yeast extract (SDAY) medium, a yeast extract malt extract agar (YMA) medium, a corn meal agar (CMA) medium, a malt extract agar (MEA) medium, a Czapek Dox agar (CDA) medium, and a Sabouraud's dextrose agar (SDA) medium respectively, and sterilizing same under high pressure so as to prepare a culture medium plate under a ultra-clean workbench. Taking colonies (as mentioned above) with a size of 0.5 cm from the bacteria-carrying PDA plate of strain MSC-f1 with a sterile perforator, then placing the bacterial cake in the center of each medium plate, sealing the culture dish and placing same in the incubator for dark culture for 10 d, and measuring and recording the diameter of colonies by means of a cross-over method. Final culting conidia with 10 mL of sterile water, recording the number of conidia and calculating the concentration of conidia by means of a hemacytometer under an optical microscope.

The results are shown in Table 2, and it can be determined that the strain MSC-f1 can grow on seven kinds of tested media, among which the strain MSC-f1 grows fastest on SDAY, PDAj502326 and CDA and other media; the back of the colonies is yellow on the seven tested culture media with the darkest color on the SDAY medium, followed by the CDA medium, and lighter in color on the remaining media; conidia are produced on all seven culture media with the largest amount of conidia produced on the SDAY medium, followed by the PDA medium. Table 2. Growth and Sporulation of Strain MSC-f1 on Different Media Culture Growth Growth Colonyback Sporulation concentration _ medium diameter (cm) condition color (conidia/mL) SDAY 4.95 Better Dark yellow 3.63 x 10° PDA 4.71 Better Light yellow 3.25 x 10° YMA 2.95 Good Light yellow 9.25 x 107 CMA 3.10 Good Light yellow 3.25 x 108 MEA 3.05 Good Light yellow 2.63 x 10° CDA 4.69 Better Yellow 1.15 x 10° SDA 3.48 Good Light yellow 5.32 x 10°

3. Analysis of gene sequence of strain MSC-f1 inoculating the strain MSC-f1 into a PDA culture medium plate, placing same and culturing same at 25°C until the colonies grow up the whole culture dish, scraping the fungal hyphae grown on the PDA plate in a sterile mortar by means of a sterile medicine spoon, adding liquid nitrogen and milling same to powder, and extracting the total DNA of the strain MSC-f1 by means of a modified CTAB method.

Using purified DNA as a template, the ribosomal DNA internal transcribed spacer (ITS1-5.8S rDNA-ITS2) of strain MSC-f1 is amplified with fungal universal primers ITS5(S'-GGAAGTAAAAGTCGTAACAAGG-3"/ITS4 (S'-TCCTCCGCTTATTGATATGC-3"), the P-tubulin gene of strain MSC-fl is amplified with universal primers Tub-T1(5-AACATGCGTGAGATTGTAAGT-3")/Tub-T22(5'-TCTGGATGTTGTTGGGAATCC -3"), and the translation elongation factor 1-a, TEF1-a, of strain MSC-f1 is amplified by universal primers EF1-983F (5'-GCYCCYGGHCAYCGTGAYTTYAT-3')/EF1-2218R(5'-ATGACACCRACRGCRACRGTY TG-3").

The PCR reaction system is as follows: 10 x Buffer 2.5 uL, dNTPs 2 uL, 1.5 uL each of upstream and downstream primers (10 umol/L), 0.2 uL of Ex-Taq DNA polymerase, 1 pL of DNA template, and sterile water to a total volume of 25 pL. The reaction conditions are as follows: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and 35 cycles; and finally extending at 72°C for 10 min. (For specific operations, see: Lu Lianming et al4502326 Polymorphism and phylogenetic analysis of ribosomal protein gene of citrus Huanglong pathoge. Journal of Zhejiang University, 2011, 37(2):125-132). The PCR product is subjected to agarose gel electrophoresis and then sent to Shanghai Sangong Bioengineering Co., Ltd. for sequencing.

According to the sequencing results, the strain MSC-f1 has the sequence of ITS1-5.8S rDNA-ITS2 as shown in SEQ ID NO: 1, the sequence of the B-tubulin gene as shown in SEQ ID NO: 2, and the sequence of the TEF1-a gene as shown in SEQ ID NO: 3. Upon comparative analysis by NCBI blast, it is showed that the similarity between the ITS1-5.8S rDNA-ITS2 sequence of the strain MSC-fl and the corresponding gene sequences (such as KM234215, JN204422 and MHS32893) of a plurality of strains of Isaria javanica in GenBank reaches 99.8% to 100%; the similarity between the partial sequence of TEF1-a gene of the strain MSC-f1 and the corresponding gene sequences (such as KY587207, KT225600 and KT225598) of a plurality of strains of Isaria javanica in GenBank reaches 100%; and the similarity between the partial sequence of P-tubulin gene and the corresponding gene sequences (such as KT225602, MN215882, and KT225605) of strains different from Isaria javanica in GenBank is at most

99.41%, indicating that the B-tubulin gene sequence of strain MSC-f1 has a base difference from the reported gene sequences of the strains.

According to the results of the morphological characteristics, biological characteristics and gene sequence analysis regarding the strain MSC-f1, in combination with relevant literature (Cabanillas HE, et al. Isaria poprawskii sp. nov. (Hypocreales:Cordycipitaceae), a new entomopathogenic fungus from Texas affecting sweetpotato whitefly. Mycoscience, 2013, 54(2):158-169 et al), it's reported that the strain MSC-f1 was finally identified as Isaria javanica of Isaria of Cordycipitaceae of Hypocrellales of Sordariomycetes of Pezizomycotina.

Example 3: Determination of Pathogenicity of Strain MSC-f1 to Citrus Pests inoculating the strain MSC-f1 onto a PDA solid medium plate, placing same in a 25°C incubator for culturing over 10 d, adding 10 mL of sterile water into the PDA plate, eluting the cultured strain MSC-f1, filtering the eluted product by means of sterile gauze to remove hyphae, and collecting the filtrate so as to prepare a conidial suspension. counting the concentration of conidia by means of a hemacytometer under an optical microscope and determining the concentration of conidia, appropriately diluting the suspension with sterile water so as to finally obtain a conidial suspension with a concentration of 1.0 x 10% conidia/mL, and adding tween-80 with a final concentration of 0.1 vol% thereto for use. LU502326 Determination for pathogenicity of Aphis citricola: cutting a newly extracted branches of Ponkan orange, with a total length of the branches of about 12 cm and a length of the lower old branches of 3 to 5 cm, keeping 5 to 6 leaves, inserting same into a centrifuge tube containing 10 mL of hydroponic nutrient solution, fixing the bottom of the centrifuge tube on a square pearl cotton with a side length of about 8 cm, and laying a piece of white paper directly below the pearl cotton. Picking healthy Aphis citricola with a brush onto prepared branches of Ponkan with 50 Aphis citricola per branch. Placing 30 mL of the prepared conidial suspension of the strain MSC-f1 into a small-size sterile sprayer, and uniformly spraying the tested pests on the branches, and treating Aphis citricola with a sterile water spray containing only 0.1 vol% Tween-80 as a control, all setting for 5 replicates. finally putting a transparent plastic bottle with a volume of 5 L (two ends of the plastic bottle are open, and one end is covered with an 80-mesh pest-proof mesh netting) and placing same in an artificial climate box with a temperature of 25°C and a humidity of 90% or more for cultivation.

Determination for pathogenicity of Icerya purchasi: cutting several citrus branches with Icerya purchasi in the field, wrapping cotton balls soaked with sterile water at the cut of branches, and then placing same in different preservation boxes, with the number of Citrus Icerya purchasi in each preservation box being not less than 50. Placing 30 mL of the prepared conidial suspension of the strain MSC-f1 into a small-size sterile sprayer, and uniformly spraying the tested pests on the branches, and treating Icerya purchasi with a sterile water spray containing only 0.1 vol% Tween-80 as a control, all setting for 5 replicates. Finally putting the opening of each perservation box with 40 mesh insect-proof mesh, placing same in an artificial climate box at a temperature of 25°C and a humidity of 90% or more for cultivation.

Determination for pathogenicity of Panonychus citri: cutting several citrus branches of Panonychus citri on the leaves in the field, with a total length of the branches of about 12 cm and a length of the lower old branches of 3-5 cm, keeping 5 to 6 leaves, and the number of Panonychus citri being not less than 50. Inserting same into a centrifuge tube containing 10 mL of hydroponic nutrient solution, fixing the bottom of the centrifuge tube on a square pearl cotton with a side length of about 8 cm, and laying a piece of white paper directly below the pearl cotton. Placing 30 mL of the prepared conidial suspension of the strain MSC-f1 into a small-size sterile sprayer, and uniformly spraying the tested pests on the branches, and treating Panonychus citri with a sterile water spray containing only 0.1 vol% Tween-80 as a control, all setting for|$)502326 replicates. finally putting a transparent plastic bottle with a volume of 5 L (two ends of the plastic bottle are open, and one end is covered with an 80-mesh pest-proof mesh netting) and placing same in an artificial climate box with a temperature of 25°C and a humidity of 90% or more for cultivation.

Determination for pathogenicity of Diaphorina citri: The treatment method is the same as determination for pathogenicity of Aphis citricola except that the tested pest is replaced with Diaphorina citri.

After culturing, observing the infestation and lethal condition of the conidial suspension of the strain MSC-f1 on the tested pests at different times, and after culturing for 7 d, counting the number of dead pests of the treat and control tested pest respectively, and calculating the mortality rate, and calibrating the mortality.

Mortality =Number of dead pests/Total number of pests* 100% Calibrated mortality = (Mortality in treat group - Mortality in control group) / (1- Mortality in control group) * 100% The prevention and treatment effect of the conidial suspension of the strain MSC-f1 on the various citrus pests is shown in Table 3. It can be determined from the table that the conidial suspension of the strain MSC-f1 has a significant prevention and treatment effect on citrus pests, after treated with the conidial suspension with a concentration of 1.0 x 10® conidia/mL for 7 d, the calibrated mortality of the four tested citrus pests reaches more than 90%, among which the mortality of Aphis citricola and Icerya purchasi could reach 100%. Infestation and pathogenicity of Isaria javanica strain MSC-fl to Aphis citricola, Icerya purchasi, Diaphorina citri and Panonychus citri observed under a stereomicroscope is as shown in Figures 3A, 3B, 3C and 3D, respectively.

Table 3. Prevention and treatment effect of strain MSC-f1 conidial suspension on citrus pests Tested pest Mortality in treat Mortality in control Calibrated mortality group (%) group (%) (%) Aphis citricola 100 0.83 100 Icerya purchasi 100 1.20 100 Diaphorina citri 97.50 16.46 97.01 Panonychus citri 92.63 6.15 92.15

SEQUENCE LISTING LU502326 <110> Zhejiang Citrus Research Institute <120> ISARIA JAVANICA STRAIN FOR PREVENTING AND TREATING CITRUS

PESTS AND USE THEREOF <130>PT2169 <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 613 <212> DNA <213> Isaria javanica <400> 1 ggaagtaaaa gtcgtaacaa ggtctccgtt ggtgaaccag cggagggatc attaacgagt 60 tttttcaact ccctaaccct ttgtgaacat acctatcgtt gecttcggegg actcgececg 120 gcgtecggac ggccetgege cgeecgegac ccggacccag gcggccgecg gagacccaca 180 aattctgttt ctatcagtct ttetgaatec gccgcaaggc aaaacaaatg aatcaaaact 240 ttcaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcgaaat gcgataagta 300 atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattge geccgccage 360 attetggcgg gecatgectgt tecgagcgtca tttcaaccet cgacaccect tcgggggagt 420 cggegttggg gaccggeage ataccgccgg ceccgaaata cagtggegge ccgtecgcgg 480 cgacctetgc gtagtactcc aacgegcacc gggaacccga cgeggecacg ccgtaaaaca 540 cccaacttet gaacgttgac ctcggatcag gtaggactac ccgctgaact taagcatatc 600 aataatcgga gga 613 <210> 2 <211> 1358 <212> DNA <213> Isaria javanica <400> 2 ttcttgttga cecetecteg acgegttect gagagettgt gcagecectg agtggtacce 60 cgeecgegete ccggettgea ccagcacaaa cgetttette tcgagatgga gggecatccaa 120 gagctecgaa taccgtgaat tgttgectaac gacttgegte tttttgegte tgtaggttca 180 cctccaaacc ggtcagtgcg taagtagett cegaggteet gcattgtgge aagtgtecggc 240 LU502326 tctaacagtg tttgtttcga tagggtaacc aaattggtge tgetttetgg caaaccatet 300 ctggcgagca cggectegac tccageggtg tctacaatgg cacttetgag cttcagetecg 360 agcgcatgaa tgtctacttc aacgaggttt gctataccgg cacaacgegt agettggett 420 cattgtggat actaaccgcg aatttcttca ggcctctggt aacaagtatg ttcctegege 480 cgtcctegte gatcttgage ccggtaccat ggacgetgte cgtgecggte cetteggteca 540 gctctteege cecgacaact tegttttegg tcagtcecggt gctggcaaca actgggecaa 600 gggtcactac actgagggtg cggagetcgt tgaccaggte ctcgacgttg ttcgtegega 660 ggccgaagge tgegactgee ttcaggettt ccagatcacce cactctetgg gtggtggtac 720 cggtgccggt atgggtacte tgctcatete caagatccge gaggagttee ccgaccgeat 780 gatggccacc ttetecgttg tecccteecc cggcaactec gacaccgttg tcecgageccta 840 caacgccact ctetecgtec accagetcgt tgagaactec gacgagacct tctgtatcga 900 caaccaggec ctttacgaca tctgcatgeg taccctgaag ctgtccaace cctecgtacgg 960 tgacctgaac caccttgtct ccgtegtcat gtecggcate accacctgee tgegttteee 1020 tggtcagete aactctgacc ttcgecaaget cgccgtcaac atggttecett teccecgtet 1080 tcacttettec atggtecgget ttgctecect gaccageecgt ggtgetcact cettecgege 1140 cgtcteggtt cctgagetca ctcagcagat gttcgaccce aagaacatga tggetgette 1200 tgacttecgt aacggteget acctgacctg ctetgecatt ttgtaagtge accttgetag 1260 caccaattga cacacaacta acaacccatt agccgtggta aggttgccat gaaggaggtt 1320 gaggaccaga tgcgtaatgt gcagaacaag aactccac 1358 <210> 3 <211> 1017 <212> DNA <213> Isaria javanica <400> 3 ggccaccgtg atttcatcaa gaacatgatt actggtactt cccaggecga ttgegetatt 60 ctcatcatcg ctgccggtac tggtgagttc gaggctggta tctccaagga tggccagacc 120 cgtgagcacg ctctgetege cttcaccete ggtgtcaage agctcategt tgccatcaac 180 aagatggaca ctgctcagtg ggccgaggct cgttaccagg aaatcatcaa ggagacttec 240 aacttcatca agaaggtcgg ctacaacccc aagactgttg cettegtece catctctggt 300 ttcaacggcg acaacatget ggececctec accaactgec cctggtacaa gggttgggag 360 aaggagacca aggccggcaa gtccaccgge aagaccctce tcgaggetat tgacgccate 420 LU502326 gagcccccca agcgteecet cgacaagece cteegtette ctettcagga tgtctacaag 480 atcggtggta tcggaacggt gcccgtecggt cgtgtcgaga ctggtatcat caagceccgge 540 atggtcgtca cetttgetec tteccaacgte accactgaag tcaagtecgt cgagatgcac 600 cacgagcagce tgcccgaggg tgttcececggt gacaacgteg gcttcaacgt gaagaacgtt 660 tccgtcaagg aaatecgtecg tggtaacgte getggtgact ccaagaacga cecteccaac 720 ggtgctgect cettcaacge ccaggtcatt gtcatcaace accctggeca gatcggtaac 780 ggttacgeec ctgttettga ctgccacact geccacattg cttgcaagtt ctececgagete 840 ctcgagaaga tcgaccgecg tactggtaag tcggttgaga acaaccccaa gttcatcaag 900 tctggtgact ccgeccatcgt caagatggtt ccctccaage ccatgtgegt tgaggettte 960 accgactacc cccctetggg cegtttegee gtecgtgaca tgegtcagac cgtegee 1017 <210> 4 <211> 22 <212> DNA <213> Primer ITSS <400> 4 ggaagtaaaa gtcgtaacaa gg 22 <210> 5 <211> 20 <212> DNA <213> Primer ITS4 <400> 5 tectecgctt attgatatge 20 <210> 6 <211> 21 <212> DNA <213> Primer Tub-T1 <400> 6 aacatgcgtg agattgtaag t 21 <210> 7 <211> 21

<212> DNA LU502326 <213> Primer Tub-T22 <400> 7 tctggatgtt gttgggaatc c 21 <210> 8 <211> 23 <212> DNA <213> Primer EF1-983F <400> 8 gcyccygghe aycgtgaytt yat 23 <210> 9 <211> 23 <212> DNA <213> Primer EF1-2218R <400> 9 atgacaccra crgcracrgt ytg 23

Claims (6)

CLAIMS LU502326

1. An Isaria javanica strain, wherein the strain is classified and named as Isaria javanica MSC-f1, has been deposited at the China General Microbiological Culture Collection Center on 19 March 2020, and has the deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing and the deposit number of CGMCC No.19601.

2. Use of the Isaria javanica strain of claim 1 for preventing and treating citrus pests.

3. The use of the Isaria javanica strain of claim 2 for preventing and treating citrus pests, wherein the citrus pests comprise Aphis citricola, Icerya purchasi, Diaphorina citri, and Panonychus citri.

4. A biological antibacterial formulation for citrus pests, comprising conidia of the Isaria javanica MSC1-f1 of claim 1.

5. The biological antibacterial formulation of claim 4, wherein Isaria javanica MSC1-f1 in the biological antibacterial formulation has the contents of 1 x 10$ conidia/mL.

6. Use of the biological antibacterial formulation of claim 4 for preventing and treating citrus pests.