Penicillium oxalicum strain and application thereof

Abstract

The invention discloses a penicillium oxalicum strain and application thereof. The strain is named as Penicillium oxalicum HYC2101, the Penicillium oxalicum HYC2101 is registered and preserved in the China General Microbiological Culture Collection Center on August 3, 2022, the preservation number is CGMCC No.40263, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The invention also discloses application of the strain in prevention and treatment of plant pests and preparation of a citrus pathogenic fungus inhibitor.

Description

Technical Field

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

[0002] Panonychus citri is the main pest mite on citrus, which harms the leaves and fruits of citrus. When the population is large, it causes citrus to fall leaves and fruits, resulting in serious citrus yield reduction. The two-spotted spider mite (Tetranychus surticae Koch) is an agricultural pest mite with a wide host range, high reproduction rate, and difficulty in prevention and control. It can harm more than 1,100 plant species such as apples, strawberries, and melons. The brown planthopper (Nilaparvatalugens) is a major pest on rice. It has the characteristics of strong virulence, strong reproduction, and long-distance migration. In severe cases, it can cause rice to die and a large reduction in yield. Diaporthe citri, Alternaria alternata, and Colletotrichum gloeosporioides are the pathogens of black spot disease, brown spot disease, and anthracnose on citrus. They are widely present in orange orchards around the world, affecting not only the quality and appearance of citrus, but also the growth and yield of citrus trees. At present, chemical control is still the main means of control for the above-mentioned diseases and pests, but the large-scale and frequent use of chemical pesticides has caused the target pests to develop resistance to most commonly used pesticides, and has also caused varying degrees of harm to the environment, natural enemies of pests and human health. Therefore, in order to solve the above difficulties, screening high-efficiency, environmentally friendly and broad-spectrum insecticidal and fungicidal active strains to develop biocontrol agents has become a major development direction of green agricultural control.

[0003] Penicilium oxalicum is widely found in nature. Studies have shown that Penicilium oxalicum has certain insecticidal activity against citrus psyllids and soybean cyst nematodes, and has antagonistic effects on apple oxysporum, apple moniliforme, apple laminarin, and pepper root rot pathogen Fusarium solani. At present, there are few research reports on this fungus at home and abroad, and there are no reports on the fungus infecting mites such as Panonychus citrus and antagonizing pathogenic fungi such as Aspergillus and Alternaria. Summary of the invention

[0004] The purpose of the present invention is to provide a Penicillium oxalicum strain and application thereof.

[0005] The technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a Penicillium oxalicum strain, which is named Penicillium oxalicum HYC2101, and has been registered and preserved in the General Microbiological Center of the China Microorganism Culture Collection on August 3, 2022, with a preservation number of CGMCC No. 40263, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The second aspect of the present invention provides the use of the above-mentioned Penicillium oxalicum strain in controlling plant pests; Furthermore, the plant pests include Panonychus citri, Bemisia tabaci, and Nilaparvata lugens.

[0007] The third aspect of the present invention provides the use of the above-mentioned Penicillium oxalicum strain in the preparation of citrus pathogenic fungi inhibitors; Furthermore, the citrus pathogenic fungi include Diaporthe citri, Alternaria alternata, and Colletotrichum gloeosporioides.

[0008] The significant advantages of the present invention are: HYC2101 is a strain of Penicillium oxalicum isolated in our laboratory. According to the biological characteristics analysis, the strain has a wide temperature range, strong resistance to low and high temperatures, high spore production, and is easy to cultivate. Pathogenicity analysis found that the strain not only has strong pathogenicity to plant pests such as Panonychus citrus, Bemisia tabaci, and brown planthopper, but also has a strong inhibitory effect on citrus pathogenic fungi such as Aspergillus niger and Alternaria alternata. Therefore, strain HYC2101 has good development and application prospects as a biocontrol bacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Morphological identification of Penicillium oxalicum HYC2101.

[0010] Figure 2 :Effects of different temperatures on the mycelial growth of Penicillium oxalicum HYC2101.

[0011] Figure 3 :Effects of different culture media on mycelial growth of Penicillium oxalicum HYC2101.

[0012] Figure 4 :Morphological characteristics of Penicillium oxalicum HYC2101 in different culture media. A: CDA; B: LCA; C: MEA; D: SDAY; E: PDA; F: PSA.

[0013] Figure 5:Effects of temperature and culture time on spore germination of Penicillium oxalicum HYC2101.

[0014] Figure 6 :Phylogenetic tree of Penicillium oxalicum HCY2101 strain constructed based on rDNAITS gene.

[0015] Figure 7 :Infection status of Penicillium oxalicum HCY2101 on plant pests. A: Panonychus citri; B: Bemisia tabaci; C: Brown planthopper.

[0016] Figure 8 :A, B, C are the inhibitory effects of Penicillium oxalicum HYC2101 on Diaporthe citri, Alternaria alternata, and Colletotrichum gloeosporioides, respectively; D, E, and F are Diaporthe citri, Alternaria alternata, and Colletotrichum gloeosporioides, respectively. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0018] Embodiment 1: 1 Materials and Methods 1.1 Isolation and purification of entomogenous fungi Naturally occurring citrus mites were collected from the field in Taizhou citrus orchard in Zhejiang Province. The samples were placed in sterile sample bags and returned to the laboratory for isolation and screening on the same day.

[0019] 1.2 Culture medium The separation and purification medium is potato agar PDA medium. The preparation method of PDA medium is as follows: weigh 200 g of potatoes, peel and cut into small pieces, add ultrapure water and boil for 20 minutes, filter through double-layer gauze to obtain the supernatant, and add glucose (20 g) and agar powder (20 g) to every 1000 mL of filtrate. The culture media for morphological observation were: PDA medium, potato sucrose agar (PSA) medium (preparation method was the same as PDA medium, except that glucose was replaced by sucrose), malt extract agar (MEA) medium (malt extract powder 30 g, soy peptone 3 g, agar 15 g, distilled water 1 L, pH 5.6), LCA medium (glucose 1 g, magnesium sulfate heptahydrate 0.2 g, potassium chloride 0.2 g, potassium dihydrogen phosphate 1 g, sodium nitrate 2 g, yeast powder 0.2 g, agar 15 g, distilled water 1 L, pH 6.6-7.0), Sabouraud dextrose agar (SDAY) medium (glucose 40 g, peptone 10 g, agar 15 g, distilled water 1 L, pH 5.6) and Czapek (CDA) medium (sodium nitrate 3g, dipotassium hydrogen phosphate 1g, ferrous sulfate 0.01g, sucrose 30g, magnesium sulfate heptahydrate 0.5g, potassium chloride 0.5g, agar 15g, distilled water 1L, pH 7.3). They were separately packed into conical flasks (150mL) and sterilized by high pressure at 121℃ for 25min for use in the experiment.

[0020] 1.3 Isolation and purification of strains Pick up naturally infected Panonychus citri mites, place them in a centrifuge tube containing 200 μL of sterile water, shake and mix, take the supernatant, and dilute 10 -1 , 10 -2 , 10 -3 After doubling, plate culture was performed. When a single colony grew out, bacterial blocks at the edge of the colony were picked and placed in a new culture dish for growth. The strains after single spore separation and purification were transferred to the slant surface of a PDA test tube and stored in a 4°C refrigerator.

[0021] The isolated and purified target strain was inoculated into healthy Panonychus citrifolia mites. After the disease occurred, the hyphae were picked and inoculated onto the PDA medium plate, and compared with the previously isolated strains. After the pathogenic bacteria were verified by Koch's method, the pathogenic strains were cultured again and placed in a 28°C constant temperature incubator for 5 to 14 days. During this period, the colony morphology, color, hyphae texture, etc. were continuously observed and recorded. The microscopic structures of the hyphae and spores of the strains were observed by the insert method. Refer to the "Handbook of Fungal Identification" for preliminary identification.

[0022] 1.4 Molecular identification The molecular identification of the strain was carried out by comparing the nrDNA ITS sequence. The universal primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS (5'-GGAAGTAAAAGTCGTAACAAGG-3') were used for PCR amplification. The PCR reaction system was as follows: DNA template 1.0μL, 10×Buffer 2.5μL, dNTPs 2μL, upstream and downstream primers ITS4 / ITS5 (10μmol / L) 1.5μL each, r-Taq DNA polymerase 0.2μL, and sterile water was added to a total volume of 25μL. The reaction conditions were 94℃5min; 94℃1min, 55℃1min, 72℃1min, 35 cycles; 72℃10min. The amplified product was purified and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results were submitted to NCBI for sequence homology comparison. The phylogenetic tree was constructed using MEGA5 software to determine the taxonomic status of the strain.

[0023] 1.5 Biological characteristics of strains 1.5.1 Effects of different temperatures on mycelial growth and spore germination Sterile water was added to the plate where the strain was cultured for 7 days to wash off the spores of the strain cultured on the plate. The spores were obtained by filtering the mycelium through sterile gauze and made into 1×10 6 1 mL of spore / mL suspension was added to PDA medium to prepare bacterial plates. A 0.5 cm bacterial cake was punched with a cork puncher and placed in the center of the PDA plate. The plates were inverted and cultured in incubators at 10, 15, 20, 25, 30, and 35°C. Three replicates were set for each temperature, and the colony diameters were observed and recorded every day.

[0024] 1.5.2 Effects of different culture media on mycelial growth of strains The preparation of bacterial plates was the same as (1.5.1). Use a hole puncher to punch out 0.5 cm bacterial cakes and place them on PDA, PSA, SDAY, MEA, CDA and LCA culture media. Culture them in a constant temperature incubator at 25°C. Set up 3 replicates for each treatment. Measure and record the colony diameters using the cross method every day.

[0025] 1.5.3 Effect of culture time on spore germination The spores were washed out with PDA liquid medium on the plate after 7 days of culture and 1×10 7Spores / mL spore suspension, aspirate the suspension and place it on a sterile glass slide, then place it on a U-shaped glass tube in a sterile culture dish, cover the bottom with sterile filter paper and add water to keep it moist, place it in a 30℃ constant temperature incubator for dark culture, take samples regularly at 3, 4, 5, 6, and 7 hours, place it under a microscope, and calculate the spore germination rate. According to the above method, place the bacterial suspension in a constant temperature incubator at 20, 25, 30, 35, 40, and 45℃ for dark culture, take samples at different times, and calculate the spore germination rate.

[0026] 1.6 Pathogenicity of strain HYC2101 to various plant pests and pathogens To determine the pathogenicity of citrus mites, select citrus leaves of the same size, clean them, disinfect the surface with 75% alcohol, and place them with the back facing up on sterile filter paper in a sterile culture dish. Sterile filter paper is moisturized with sterile water to prevent citrus mites from crawling out. Then, adult mites of the same age in the population are randomly selected and placed on the leaves, with about 50 mites on each leaf. Sterile water is added to the plate where the strain HYC2101 was cultured for 7 days, and the spores of the strain cultured on the plate are washed off. The spores are obtained by filtering the mycelium through sterile gauze and made into 1×10 8 spores / mL suspension (containing 0.1% Tween-80), clamp the leaf with Panonychus citri with tweezers and put it into the spore suspension, immerse it for 5 seconds, take it out and put it in a culture dish, and treat it with sterile water containing 0.1% Tween-80 as a negative control, and repeat each treatment 3 times. Finally, move the culture dish to a light cycle (16L:8D), temperature (26±2)℃, humidity (70±5%) incubator for culture, observe the death of adult mites at 3, 6, and 9 days after inoculation, and record the effective lethality of mites that grow the biocontrol strain.

[0027] Adult rice brown planthoppers of the same age were randomly placed on potted rice seedlings in insect cages, and white waterproof cloth was laid on the bottom to identify the fallen brown planthoppers (two rice seedlings in one insect cage, 50 rice brown planthoppers, and three replicates). Sterile water was added to the plate where the strain HYC2101 was cultured for 7 days, and the spores of the strain cultured on the plate were washed off. The spores were obtained by filtering the mycelium through sterile gauze and made into 1×10 8 spores / mL suspension (containing 0.1% Tween-80) and put it into a handheld sprayer, and spray the brown planthoppers on the rice seedlings evenly until the insect surface is moistened. The brown planthoppers treated with sterile water containing 0.1% Tween-80 were used as controls. The above treatments were placed in the insect breeding room (indoor temperature 26±228℃, humidity 70±5%, photoperiod 16L:8D). The number of brown planthopper deaths was observed and recorded at 3, 6, and 9 days. Bemisia tabaci was inoculated on pumpkin seedlings, and black cloth was spread on the bottom. The pathogenicity test method was the same as above.

[0028] The antibacterial activity of the antagonistic strain HYC2101 was tested by the plate confrontation method. The bacterial cakes (5 mm) of pathogens such as Diaporthe citri were punched out with a hole puncher and placed on the right side of the center of the culture medium. The bacterial cakes (5 mm) of the strain HYC2101 were inoculated at a distance of 30 mm from the bacterial cakes. The bacterial cakes inoculated with only pathogens were used as controls. Each treatment was repeated 3 times. After culturing in a constant temperature incubator at 28°C for 7 days, the colony diameter was measured and the antibacterial rate was calculated.

[0029] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100 2 Experimental results 2.2.1 Morphological identification Results Figure 1 The colony of strain HYC2101 grew slowly on the PDA culture medium, with a diameter of about 3.44 cm on the 7th day. The colony was round and spread outward in concentric rings. The edges of the colony were irregular. The hyphae were white at first and then dark green. The hyphae were short and villous, rich in spores. The reverse side of the colony was slightly yellow with a transparent circle around it. Under an optical microscope, the conidiophores of strain HYC2101 were relatively thick, the spore-producing cells were slender and bottle-shaped, with clusters of conidia at the end, and the spores were oval and nearly round.

[0030] 2.2.2 Effects of different temperatures on the growth of strain HYC2101 Results Figure 2 The strain HYC2101 can grow at temperatures of 10-35℃ on PDA medium, and the mycelial growth rate at 25-35℃ is significantly higher than that at other temperatures. From the above, it can be seen that the suitable temperature for strain HYC2101 is 25-35℃, and it has good high temperature resistance in growth.

[0031] 2.2.3 Effects of different culture media on the growth of strain HYC2101 Results Figure 3 and Figure 4 The strain HYC2101 grew well on PSA, SDAY, LCA, MEA, CDA and LCA media, and its growth rate on LCA, CDA and SDAY media was higher than that on the other media; the colonies of strain HYC2101 were orange-red on SDAY, white on CDA, and grass green or olive on the other four media.

[0032] 2.2.3 Effects of culture time and temperature on spore germination of strain HYC2101 The spore germination rate of strain HYC2101 was measured after 3, 4, 5, 6, and 7 h of culture at 30°C. The results are shown in Figure 5A. The spore germination rate showed an increasing trend with the increase of time, and the spore germination rate reached more than 91% at 7h. The temperature had a significant effect on the spore germination rate of strain HYC2101. After 6h of culture, the spores basically did not germinate at temperatures below 20℃ and above 45℃. The spores of strain HYC2101 were suitable for spore germination at 25-40℃, and 30-40℃ was the most suitable germination temperature, indicating that strain HYC2101 had a wider temperature range than other strains and was more resistant to high temperatures ( Figure 5 B).

[0033] 2.2.3 Molecular Biological Identification The ITS1-5.8S rDNA-ITS2 sequence (SEQ ID NO.1) of strain HYC2101 was measured and compared with multiple isolates of Penicillium oxalicum in GenBank (such as ON413721.1, OQ692095.1, KY853420.1, MT588795.1, etc.) by NCBI Blast analysis. The sequence similarity was as high as 99.49%. Figure 1 ), biological characteristics and molecular ITS1-5.8S rDNA-ITS2 sequence characteristics, strain HYC2101 was identified as Penicillium oxalicum, and was registered and deposited in the General Microbiology Center of China Microorganism Culture Collection on August 3, 2022, with the deposit number CGMCC No.40263, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] 2.2.4 Pathogenicity of strain HYC2101 to various crop pests was measured at a concentration of 1.0×10 8 The conidia suspension of Penicillium oxalicum HYC2101 with spores / mL was used to treat citrus mites, two-spotted spider mites and rice brown planthoppers. The mortality rate increased with time. On the 9th day, the mortality rate for the three tested pests reached more than 70%, and the mortality rate for citrus mites was the highest, at 94.68%.

[0035] Table 1 Pathogenicity of spore suspension of strain HYC2101 to various crop pests 2.2.5 Inhibitory effect of Penicillium oxalicum on citrus pathogenic fungi The results of antagonistic bacteria plate confrontation showed that Penicillium oxalicum HYC2101 had obvious antibacterial effect on Diaporthe citri, Alternaria alternata and Colletotrichum gloeosporioides, with inhibition rates of 65.21%, 59.42% and 52.86%, respectively, indicating that the antagonistic bacteria had good antagonistic effect on citrus pathogenic fungi and had good biocontrol potential.

[0036] Table 2 Inhibitory activity of Penicillium oxalicum HYC2101 against various citrus pathogenic fungi

Claims

1. A Penicillium oxalicum strain, characterized in that: The strain was named Penicillium oxalicum ( Penicillium Oxalicum )HYC2101 was registered and deposited in the General Microbiology Center of China Microorganism Culture Collection on August 3, 2022, with the deposit number CGMCC No.40263, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Penicillium oxalicum strain as claimed in claim 1 in controlling plant pests.

3. The use according to claim 2, characterized in that: The plant pests include Panonychus citri, Bemisia tabaci, and Nilaparvata lugens.

4. Use of the Penicillium oxalicum strain according to claim 1 in the preparation of citrus pathogenic fungi inhibitors.

5. The use according to claim 4, characterized in that: The citrus pathogenic fungi include Aspergillus niger ( Diaporthe citri )、Alternaria alternata( Alternaria alternata ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ).

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