A Penicillium citrinum strain and its application

Abstract

The present invention relates to a Penicillium citrinum strain, which is Penicillium citrinum PC-1, which is currently deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit date of July 29, 2024, and a deposit number of CGMCC No.41480. The Penicillium citrinum strain of the present invention is separated from the common giant thrips nymph infected with Penicillium citrinum, and can be used to prevent and control the common giant thrips. A large number of biological experiments have shown that the Penicillium citrinum strain of the present invention can not only parasitize the second-instar nymphs of the common giant thrips, but also has a control effect of more than 80% on the common giant thrips.

Description

Technical Field

[0001] The invention relates to a Penicillium citrinum strain and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Megalurrothrips usitatus (Bagnall) is an insect belonging to the genus Megalurrothrips (Bagnall) of the subtribe Thriptridae of the order Thysanoptera. In China, the insect is distributed in Hainan, Taiwan, Shaanxi, Guangxi, Guizhou, Hubei and other provinces (regions), and abroad in Japan, the Philippines, India, Sri Lanka, Australia, Malaysia, Fiji and other countries. The common giant thrips mainly harms legumes. In recent years, the insect has become the main pest of cowpea in Hainan Province. Its nymphs and adults harm tender shoots, flowers and young pods, causing cowpea growth stagnation, leaf atrophy and deformity, flower and pod drop, etc., resulting in a yield reduction of 20%-30% at the mildest, 50%-80% at the severest, or even a total loss of production. When thrips cause serious damage, farmers usually increase the amount of pesticides used, increase the frequency of application, or use highly toxic pesticides for control, which seriously threatens the quality and safety of agricultural products. In addition, the long-term use of chemical control measures based on pesticides will lead to a decrease in the sensitivity of common giant thrips to pesticides, and even produce a certain degree of resistance.

[0003] Penicillium citrinum is a widely distributed fungus belonging to the phylum Ascomycota, class Anomala, order Eurotiophorales, family Trichodermaceae, genus Penicillium. The species was first described and named by Thom in 1910. It is very similar to P. chrysogenum, but the two can be distinguished. At present, the research on P. citrinum at home and abroad mainly includes classification and identification, pathogenicity determination, and application of biological control, but its toxicity to common giant thrips has not been reported. Summary of the invention

[0004] The invention aims to provide a Penicillium citrinum strain, which has a highly effective biological control function against common giant thrips.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] A Penicillium citrinum strain, which is Penicillium citrinum PC-1, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 29, 2024, with the deposit number: CGMCC No.41480.

[0007] The Penicillium citrinum strain of the present invention is separated from the common giant thrips adults infected with Penicillium citrinum. The inventor sterilizes the surface of the common giant thrips nymphs infected with Penicillium citrinum collected from the main cowpea production area of ​​Hainan Province and places them on a culture dish for culture. The cultured colonies grow slowly on the PDA culture medium. After growing hyphae, the colony shape morphological observation shows that the colony diameter can reach 3-5 cm, with a small amount or a large amount of radial wrinkles, or several concentric rings; the texture is usually velvety or flocculent in the center; the conidia structure is usually produced in large quantities (the growth is very sparse if the nutrient-deficient isolate is encountered occasionally), the conidia surface usually presents typical blue-green, and there are also gray-green ones, close to bean green, wormwood green to lily green and light gray olive; the mycelium is white to light yellow; there are usually appropriate or large amounts of light yellow or yellow exudate droplets; the reverse side is orange-yellow, yellow-brown or reddish-brown; the soluble pigment is light yellow-brown or yellow, and occasionally lacks.

[0008] The rDNA-ITS sequence of the Penicillium citrinum strain of the present invention is shown in SEQ ID NO: 1. Through molecular biological identification, ITS sequence alignment and phylogenetic analysis, the strain is determined to be Penicillium citrinum and is named PC-1.

[0009] The application of the above-mentioned Penicillium citrinum strain in preventing and controlling common giant thrips has been proved by a large number of biological experiments that the Penicillium citrinum strain of the present invention can not only parasitize the second-instar nymphs of common giant thrips, but also achieve a control effect of more than 80% on the common giant thrips.

[0010] Beneficial effects of the invention: The invention provides a Penicillium citrinum PC-1, which has a high parasitic ability on the second-instar nymphs of common giant thrips and has a highly effective insecticidal activity. The Penicillium citrinum strain can be used for biological control of common giant thrips, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a colony morphology diagram of the Penicillium citrinum strain of the present invention after being cultured on SDAY medium for 12 days;

[0012] Figure 2 It is an agarose gel electrophoresis diagram of the PCR amplification product of the Penicillium citrinum strain of the present invention;

[0013] Figure 3 The phylogenetic tree of the Penicillium citrinum strain of the present invention and the related strain groups;

[0014] Figure 4 This is an optical microscopic examination of common Macrothrips 96 hours after inoculation with PC-1 strain. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] Example 1 Acquisition of strains

[0017] Adult common thrips infected with citrinum fungus collected from the main cowpea producing areas of Hainan Province were disinfected with 75% alcohol for 30 seconds and 0.1% mercuric chloride solution for 30 seconds, then rinsed with sterile water for three times, placed on sterile filter paper to dry, and the dried adult common thrips were placed on the prepared SDAY culture medium, with 5 adults placed in each dish; then the culture dishes were placed in a constant temperature incubator at 25°C for culture, observed regularly, and the grown hyphae were picked onto new SDAY culture medium, and stored at 4°C after the hyphae were fully grown.

[0018] The SDAY medium (Sabroyo dextrose agar medium) formula: 40g glucose, 10g peptone, 10g yeast extract, 20g agar, 1mL egg yolk, 10mL low-fat milk, add water to 1000mL. When the medium is 45°C, add lactic acid, shake well and pour into a 9cm diameter culture dish for standby use.

[0019] Example 2 Colony Characteristic Cultivation and Morphological Identification of Penicillium citrinum Strain

[0020] (1) Colony characterization culture: The strain stored in a 4°C refrigerator was transferred to a 9-cm diameter PDA plate and activated at 27°C for 5 days. The bacterial block was cut off at the edge of the colony with a 6-mm diameter puncher and transferred to a new SDAY plate. The plate was cultured in a dark box at 27°C for 3 times. The colony morphology, color, and growth were recorded from the second day until the colonies filled the plate.

[0021] After the colonies were cultured on SDAY medium for 12 days, the colony morphology was shown in the figure. Figure 1 ,in, Figure 1 A is the front picture of the colony. Figure 1 B is a picture of the back of the colony. It can be seen that the colony has a diameter of 3.3 to 4.5 cm, with a small or large number of radial wrinkles. The texture is usually velvety or flocculent in the center, white, and turns green-gray after 10 days. The back is white at first and turns light yellow after 10 days.

[0022] (2) Morphological identification of strains: Using the slide culture method, SDAY medium was divided into small slices of 5 mm × 5 mm and placed on a glass slide. The activated hyphae of the fungus were then placed on the culture medium and placed in a constant temperature incubator at 27°C for moisturizing culture. The formation of conidia, phialides, crystals, etc. of the fungus was continuously observed, measured and photographed using a Nikon ECLIPSE E200MV R digital biological microscope system. The hyphae were white to light yellow, and the conidiophores grew upright from the hyphae with branches at the top forming a broom-like structure. The conidia were spherical or elliptical with a smooth or slightly rough surface.

[0023] Example 3 Molecular Biology Study on Penicillium citrinum PC-1:

[0024] The PCR amplification of rDNA-ITS of the Penicillium citrinum strain of the present invention adopts the conventional method in the art. The DNA of the Penicillium citrinum is extracted by the kit method, and the primers used for amplification are universal primers synthesized by Shanghai Shenggong Bioengineering Service Co., Ltd.

[0025] Universal PCR primers:

[0026] ITS1-F: 5'-CTTGGTCATTTAGAGGAAGT-3'

[0027] ITS4-R: 5'-CCTCCGCTTATTGATATGC-3'

[0028] PCR reaction system (25 μL): template DNA 1 μL, 2×Eco Taq PCR SuperMix (Tiangen) 12.5 μL, upstream and downstream primers 1 μL each, add deionized water to 25 μL. Reaction conditions: 94℃ 4min pre-denaturation; 94℃ 1min, 55℃ 30s, 72℃ 90s, 35 cycles; 72℃ extension 10min. PCR products were detected by 1% agarose gel electrophoresis. The results are shown in Figure 2 ,Depend on Figure 2 It can be seen that the strain is a fungus and was successfully amplified with good quality using universal fungal primers ITS1-F and ITS4-R; the PCR amplification product was sent to Shanghai Sangon Biotechnology Technology Service Co., Ltd. for sequencing, and the sequencing result is shown in SEQ ID NO: 1.

[0029] The sequencing results were compared with the existing nucleic acid sequences in GenBank using the Blast program. The results showed that the rDNA-ITS sequence of the strain (numbered PC-1) was 99% homologous to the corresponding sequences of the four existing Penicillium citrinum strains. In order to determine the relationship between the strain and the same genus and other species, representative DNA sequences of the genus Penicillium published in GenBank were selected, aligned using ClustalX, and analyzed using MEGA-11. The phylogenetic tree was constructed using the Neigbor-joining method. Figure 3As shown in the figure, it can be seen that the PC-1 strain is most closely related to Penicillium citrinum strain 146N2, and is also relatively close to Penicillium citrinum strain 242N and Penicillium citrinum isolate 2010F2, but is relatively distant from strains such as Uncultured fungus clone S12T 64 and Penicillium sp. strain SYX14. Therefore, based on the rDNA-ITS sequence comparison results, the strain is consistent with Penicilium citrinum.

[0030] The PC-1 strain was sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation. The preservation date was July 29, 2024, and the preservation number was CGMCC No.41480.

[0031] Example 4 Pathogenicity test of Penicillium citrinum strain PC-1 against second instar nymphs of common Macrothrips

[0032] The modified "spore bath" method was used to inoculate the tested common Thrips. Take a 3.5cm diameter and 2.0cm high PP plastic transparent lidded box, soak and scrub with 75% alcohol, dry naturally to make the alcohol completely evaporate, and place the box in a blast drying oven for dry heat sterilization at 80℃ for 5h. Pour 2mm thick SDAY culture medium into the sterilized small box by aseptic operation in the clean bench. After the culture medium is completely solidified, this box can replace the culture dish and become a culture box. Make a sufficient number of culture boxes, inoculate the PC-1 strain respectively, and place it in an incubator for culture at a temperature of 25±1℃ for 10 days. After 10 days, the colony completely covers the bottom of the culture box and produces a large number of spores. At this time, turn the culture box upside down and inoculate the common Thrips into the culture box, 30 heads per box, and repeat three boxes. Put a piece of untreated cowpea cake in each box as food to prevent the common Thrips from starving to death. Such common thrips can be exposed to the conidia of the spore-forming strain, just like showering with spores, so it is also called "spore bath" inoculation. Take three other culture boxes and pour only the culture medium into them without inoculating the strain as the control group, and each box is also inoculated with 30 common thrips. After the inoculation is completed, the culture box is placed in a constant temperature incubator for cultivation at a temperature of 25±1℃. The mortality rate of common thrips in each culture box is counted at 24h, 36h, 48h, 60h, 72h, and 96h after inoculation. The death standard of the test insect is that there is no reaction when the insect body is lightly touched with a small brush, which is considered dead. The experimental quality standard is that the mortality rate of the control group is less than 10% for an effective experiment.

[0033] The test results are shown in Table 1:

[0034] Table 1 Pathogenicity test results of PC-1 against the second instar nymphs of common Thrips

[0035]

[0036] Example 5 Observation of the infection process of Penicillium citrinum strains on common Macrothrips under an optical microscope

[0037] The PC-1 strain was inoculated into common Thrips by the "spore bath" method and observed under an optical microscope 96 hours after inoculation.

[0038] Figure 4 This is an optical microscopic examination of common Macrothrips 96 hours after inoculation with PC-1 strain. Figure 4 A is the common Thrips nymph, Figure 4 B is an adult of Macrothrips philadelphica. It can be seen that 96 hours after inoculation, a large number of PC-1 conidia attached to the body surface of Macrothrips philadelphica and completed the infection of Macrothrips philadelphica.

Claims

1. A Penicillium citrinum strain, characterized in that The strain is Penicillium citrinum ( Penicilliumcitrinum )PC-1, the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 29, 2024, with the deposit number: CGMCC No.41480.

2. Use of the Penicillium citrinum strain according to claim 1 for controlling common giant thrips.

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