Application of trichoderma harzianum in prevention and treatment of potato black scurf and scab

Abstract

The invention relates to application of trichoderma harzianum to prevention and treatment of potato black scurf and scab. The trichoderma harzianum with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.40017 can well inhibit rhizoctonia solani, so that the effect of preventing and treating potato black scurf is achieved; trichoderma harzianum with the preservation number of CGMCC No.40017 can also inhibit streptomycete, such as streptomyces scabies, streptomyces scabies and streptomyces griseus, so that the effect of preventing and treating potato scab is achieved, the strain or the microbial agent containing the strain is applied to the planting process of potatoes, potato black scurf and scab can be effectively prevented and treated, and the effect of preventing and treating potato scab is achieved. The method has important significance in agricultural production.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, and more specifically, to the application of Trichoderma harzianum in the control of potato black scurf and scab. Background Technology

[0002] potato( Solanum tuberosum L. (Solanum) is an annual herbaceous plant belonging to the genus Solanum in the family Solanaceae. Potato black scurvy is a potato disease caused by Rhizoctonia solani, primarily affecting young shoots, stem bases, and tubers. Infected young shoots may rot before emergence, leading to missing seedlings. After emergence, the lower leaves turn yellow, and brown sunken spots form at the stem base. In severe cases, purplish-red or green aerial tubers develop from axillary buds at stem nodes, or numerous small, economically worthless potatoes appear at the base of the underground stem, with many blackish-brown sclerotia scattered on the surface. Severe disease outbreaks can result in 70-80% morbidity in severely affected fields. In normal years, this can cause potato yield reductions of over 15%, and in some years, it can destroy the entire field, severely impacting potato yield and quality.

[0003] Potato scab is one of the most important potato diseases worldwide, caused by various Streptomyces fungi. It is prevalent in potato production areas in China. Although the lesions are limited to the skin, infected potatoes are not suitable for storage and have an unsightly appearance. Infection significantly impacts potato quality, reducing commercial value and generally causing yield losses of 10-30%, with some areas experiencing losses exceeding 40%. It primarily affects the tubers, initially causing brown spots on the skin, which gradually enlarge into large, nearly circular or irregular brown spots, often scattered. The lesions exhibit a reticulated and cracked pattern, with a rough, woody skin. After cracking, the edges of the lesions are raised, and the center is sunken, resulting in rust-colored, dark brown, or black scab-like hard patches.

[0004] Therefore, it is necessary to develop safe biosecurity products. Summary of the Invention

[0005] The purpose of this disclosure is to provide the application of Trichoderma harzianum in the control of potato black scurf and scab.

[0006] This disclosure provides the application of *Trichoderma harzianum* in the control of potato black scurf and scab, wherein the *Trichoderma harzianum* is classified as *Trichoderma harzianum*. Trichoderma harzianum The preservation number of the *Trichoderma harzianum* is CGMCC No. 40017.

[0007] Optionally, the application includes applying Trichoderma harzianum to the soil of potato plants.

[0008] Optionally, the application includes any one of spraying, root drenching, and mixing with soil.

[0009] Optionally, the dosage of *Trichoderma harzianum* is 10.7 -10 11 CFU / strain.

[0010] Optionally, the application includes: applying a microbial inoculant containing Trichoderma harzianum with preservation number CGMCCNo.40017 to the soil of potato plants.

[0011] Optionally, the application includes any one of spraying, root drenching, and mixing with soil.

[0012] Optionally, the viable count of *Trichoderma harzianum* with accession number CGMCC No. 40017 in each gram of the microbial inoculant is 10. 7 -10 11 CFU.

[0013] Optionally, the amount of the microbial agent used is 1-2 kg / mu.

[0014] Through the above technical solutions, *Trichoderma harzianum* with accession number CGMCC No. 40017 can effectively inhibit *Rhizoctonia solani*, thereby achieving the effect of preventing and controlling potato black scurf. *Trichoderma harzianum* with accession number CGMCC No. 40017 can also inhibit *Streptomyces*, such as *Streptomyces scabii*, *Streptomyces scabii*, and *Streptomyces griseus*, thereby achieving the effect of preventing and controlling potato scab, which is of great significance in agricultural production.

[0015] Other features and advantages of this disclosure will be described in detail in the following detailed description section.

[0016] Preservation of biological materials The inventors of this disclosure isolated and cultured a new fungal species from soil samples taken in Koktokay, Fuyun County, Altay Prefecture, Xinjiang. The fungus was identified as belonging to the genus *Trichoderma*. Trichoderma (spp.), named Trichoderma harzianum. Trichoderma harzianum This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), the designated depository of the State Intellectual Property Office, on December 28, 2021, with accession number CGMCCNo.40017. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a colony morphology diagram of Trichoderma PTDN-1. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0019] This disclosure provides the application of *Trichoderma harzianum* in the control of potato black scurf and scab, wherein the *Trichoderma harzianum* is classified as *Trichoderma harzianum*. Trichoderma harzianum The preservation number of the *Trichoderma harzianum* is CGMCC No. 40017.

[0020] Trichoderma harzianum ( Trichoderma harzianum Trichoderma is a type of fungus widely distributed in moist environments such as soil and decaying wood. It exhibits antagonistic activity against various plant pathogenic fungi. The inventors of this disclosure have discovered that *Trichoderma khatz* with accession number CGMCC No. 40017 can effectively inhibit *Rhizoctonia solani*, thereby achieving the effect of controlling potato black scurf. *Trichoderma khatz* with accession number CGMCC No. 40017 can also inhibit *Streptomyces*, such as *Streptomyces scabica*. Streptomyces scabies ), Streptomyces scabii ( Streptomyces turgidiscabies ) and Streptomyces griseus ( Streptomyces griseus Streptomyces and other fungi can be used to prevent and control potato scab.

[0021] In one embodiment, the application includes applying Trichoderma harzianum to the soil of potato plants.

[0022] In one embodiment, the application includes any one of spraying, root irrigation, and soil mixing.

[0023] In one embodiment, the amount of *Trichoderma harzianum* used is 10... 7 -10 11 CFU / strain.

[0024] In one embodiment, the application includes: applying a microbial inoculant containing Trichoderma harzianum with preservation number CGMCC No. 40017 to the soil of potato plants.

[0025] In the above embodiments, the microbial inoculant can be in liquid or solid form, such as a wettable powder. The microbial inoculant may include: Trichoderma harzianum with preservation number CGMCC No. 40017 and a culture medium. The culture medium can be selected from those conventionally used by those skilled in the art, such as potato dextrose agar solid medium (PDA) and / or potato dextrose liquid medium (PD).

[0026] In one embodiment, the application includes any one of spraying, root irrigation, and soil mixing.

[0027] In one embodiment, the viable count of *Trichoderma harzianum* with accession number CGMCC No. 40017 is 10 per gram of the microbial inoculant. 7 -10 11 CFU.

[0028] In one embodiment, the amount of the microbial agent used is 1-2 kg / mu.

[0029] The present invention will be further described in detail below with reference to the embodiments, but the scope of the present invention is not limited to the following embodiments.

[0030] The method for preparing potato dextrose agar (PDA) solid medium used in this disclosure is as follows: Take 20g of peeled potatoes, boil for 20 minutes to extract the juice, add 2g of glucose and 1.5g of agar to the juice, add water to 100mL, the pH is 7.0, put it into two Erlenmeyer flasks, 50mL each, sterilize and make slant or plate.

[0031] The fermenter formula for the thick-walled spore fermentation medium is as follows: by weight, it contains 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO3, 0.01% ZnSO4, and 0.03% MgSO4.

[0032] Example 1 This example illustrates the isolation and identification of Trichoderma harzianum PTDN-1 with accession number CGMCC No. 40017: I. Soil Collection and Treatment: Soil samples were collected in Koktokay, Fuyun County, Altay Prefecture, Xinjiang. The collection method was as follows: at each sampling point, 10 m of vegetation community... 2 Within the quadrats, the five-point diagonal sampling method was used for soil collection. During sampling, after removing the top 2cm of soil, soil around the plant roots was collected, and soil from the rhizosphere of the host plant at a depth of 20cm was taken. After mixing, 200g of the soil was placed in a sterilized resealable bag, brought back to the laboratory, and recorded (including: collection location, latitude and longitude, altitude, and cover vegetation). The samples were stored at 4°C.

[0033] II. Isolation and purification of strain PTDN-1: 1. Soil sample processing: Using the dilution plate method, 10 μL of each sample was taken. -3 100 μL of a soil sample dilution was spread onto a PDA medium plate, and then spread evenly using a sterile spreader. This process was repeated three times on the PDA medium. The plate was incubated at 28°C in a mold incubator. Fungal colonies were observed after 5-10 days. Suspected Trichoderma colonies appearing on the plates were counted and further purification was performed.

[0034] 2. Culture and purification of bacterial strains: Suspected Trichoderma strains were picked from the plates and placed on PDA medium for multiple rounds of purification culture until the strain was completely purified, with a total culture period of more than three weeks. The purified Trichoderma strains were temporarily stored in a PDA slant at 4°C, while three aliquots were inoculated into 30% glycerol tubes and stored in an ultra-low temperature freezer.

[0035] III. Identification of Trichoderma harzianum PTDN-1: 1. Morphological identification of the isolated suspected Trichoderma colonies: The morphology of hyphae, the morphology and color of spore hyphae, the manner of spore attachment, the arrangement and morphology of spores, etc., are observed under an optical microscope, and the diameter of hyphae, the size of spore hyphae and spores are measured.

[0036] Take a mycelial cake of the same diameter from the edge of a purified colony of strain PTDN-1, place it in the center of a PDA medium, and observe the growth rate of the strain, its growth status during the culture process, colony color, mycelial morphology, and other characteristics.

[0037] The results showed that after culturing at 27℃ for 5-7 days, the isolated and purified strain PTDN-1 produced radially round colonies with a green surface, regular edges, and a light green back. The spores were green, and the sporulation rate was high (see...). Figure 1 ).

[0038] 2. Molecular biological identification: DNA extraction: Scrape an appropriate amount of hyphae of strain PTDN-1 and place them in a sterile centrifuge tube. Incubate at 0°C for at least 30 minutes. Before starting DNA extraction, place one steel ball in each centrifuge tube and oscillate at 50 Hz for 3 minutes using a freeze-thaw cell disruptor. After extraction, follow the steps of the Fungal Genome Extraction Kit (Adley Fungal DNA Rapid Extraction Kit) sequentially. Collect the DNA in a new centrifuge tube and determine the concentration using an ultra-micro spectrophotometer. Once the DNA reaches the acceptable level, PCR amplification can be performed.

[0039] PCR amplification: This was applied to the internal transcribed spacer (ITS) region of ribosomal RNA-encoding genes and translation elongation factor 1α (...). tef The fourth and fifth introns and most of the last exon of 1α), and rpb2 The gene for (the second subunit of RNA polymerase) was amplified, and the primer sequences are shown in Table 1: Table 1

[0040] In the primers mentioned above, Y is T or C; R is G or A; M is A or C; and W is A or T.

[0041] PCR reaction system: 2 μL each of upstream and downstream primers, 25 μL of Taq PCR StarMix (with Loading Dye), 1 μL of bacterial DNA template, 20 μL of ddH2O, and a total system of 50 μL.

[0042] PCR amplification program for RPB and TEF genes: 95℃ for 3 min, 95℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min (35 cycles), 72℃ for 5 min.

[0043] PCR amplification program for the ITS sequence: 95℃ for 3 min, 95℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min (35 cycles), 72℃ for 5 min.

[0044] PCR product detection and sequencing: Prepare a 1% agarose gel, add 6 μL of DNA marker to the first or last well, and add 6 μL of PCR product to the other wells. Electrophoresis is performed at 125V for 30 min, and the bands are observed and images are acquired using a gel imaging system.

[0045] After successful sample amplification, it was sent to the company (Qingke Biotechnology) for sequencing to obtain the ITS region sequence of strain PTDN-1. rpb 2 genes and tef 1α gene sequence. PTDN-1 sequence was determined. rpb The sequences of the two genes are as follows: >PTDN-1- rpb 2SEQ ID NO.1:

[0046] >PTDN-1- tef 1αSEQ ID NO.2:

[0047] >PTDN-1-ITSSEQ ID NO.3: .

[0048] In 2021, Cai and Druzhinina proposed new criteria for classifying Trichoderma species: all of the following must be met simultaneously. rpb 2≥99%, tef A Trichoderma species can only be identified if three conditions are met: α ≥ 97%, ITS ≥ 76% (In honor of John Bissett: authoritative guidelines on molecular identification of Trichoderma, CAI et al., 2021). Using this method for Trichoderma species identification, strain PTDN-1 was... rpb 2 genes and The 1α gene and ITS region sequences were compared using NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cg), and strain PTDN-1 was found to have...tef The similarity of gene 2 to *Trichoderma harzianum* is ≥99% (99.05-99.68%), and strain PTDN-1... The 1α gene shows a high degree of similarity to Trichoderma harzianum. rpb The 1α sequence showed a similarity of ≥97%, and the ITS sequence of PTDN-1 shared ≥76% similarity with *Trichoderma harzianum*. Therefore, we confirmed that the *Trichoderma* species was classified as *Trichoderma harzianum*.

[0049] Based on the above morphological and molecular biological identification, strain PTDN-1 was identified as *Trichoderma*. tef tef Trichoderma harzianum This strain was deposited on December 28, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40017.

[0050] Example 2 This example illustrates the preparation process of microbial inoculants: Trichoderma harzianum with preservation number CGMCC No. 40017 was inoculated from the preservation slant into PDA and incubated at 25°C for 7 days. Three to four mycelial blocks were then added to a seed bottle. The mixture was shaken at 25°C and 180 rpm for 48 hours to prepare a seed culture. The seed culture was added to a seed fermentation tank at a volume ratio of 1:150 and then added to a fermentation tank containing chlamydospore fermentation medium at a volume ratio of 1:20. The mixture was incubated at 28°C for 132 hours until 90% of the mycelia formed chlamydospores, yielding a chlamydospore fermentation broth. The broth was then adsorbed with diatomaceous earth, centrifuged or filtered through a plate and frame filter, and dried to form a wettable powder. This wettable powder is the microbial inoculum of this embodiment, containing 4 × 10⁻⁶ viable Trichoderma harzianum cells. 9 CFU / g.

[0051] Comparative Examples 1-5 Five Trichoderma harzianum strains isolated together with the Trichoderma harzianum strain with accession number CGMCC No. 40017 of this application were used to prepare microbial inoculants according to the method of Example 2 (referred to as Trichoderma harzianum 1, Trichoderma harzianum 2, Trichoderma harzianum 3, Trichoderma harzianum 4 and Trichoderma harzianum 5, respectively).

[0052] Test Example 1 This test example illustrates that the Trichoderma of this disclosure can control black scurf in potatoes during the seedling and tuber stages: The wettable powders of Example 2 and Comparative Examples 1-5 were tested respectively, and a control group was set up without the application of wettable powder.

[0053] The experimental setting was a field. Microbial wettable powders of *Trichoderma harzianum* 1, 2, 3, 4, and 5 (from Example 2) were applied to potato seedlings planted in the soil. Each individual plant was treated with 10... 7 The total number of spores was determined, and an investigation of potato rhizome black spore disease was initiated 20 days after transplanting. Data on black spore disease were then collected three months later at harvest time.

[0054] Grading standards for the incidence of potato black scurf in underground stems: Grade 0: No disease spots on the stem; Grade 1: Stem lesions cover 1% to 5% of the underground stem area; Grade 3: Stem lesions cover 6% to 25% of the underground stem area; Grade 5: Stem lesions cover 26% to 50% of the underground stem area; Grade 7: Stem lesions cover 51% to 75% of the underground stem area; Grade 9: The area of ​​stem lesions covers 76% to 100% of the underground stem area.

[0055] Disease index = 100 × ∑ (number of diseased stems at each level × representative value at each level) / (total number of stems surveyed × highest representative value); Relative prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0056] Potato black scab tuber disease grading standards: Grade 0: No sclerotia are present on the surface of the tuber; Grade 1: The sclerotia area on the tuber surface accounts for less than 1% of the total tuber area; Grade 3: The sclerotia area on the tuber surface accounts for 1% to 10% of the total tuber area; Level 5: The sclerotia area on the tuber surface accounts for 11%~20% of the total tuber area; Level 7: The sclerotia area on the tuber surface accounts for 21%~50% of the total tuber area; Level 9: The sclerotia area on the tuber surface accounts for more than 50% of the total tuber area.

[0057] Disease index = 100 × ∑ (number of diseased tubers at each level × representative value at each level) / (total number of tubers surveyed × highest representative value); Relative prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0058] The results are listed in Table 2.

[0059] Table 2

[0060] As shown in Table 2, compared with the control group and Comparative Examples 1-5, Example 2 demonstrated excellent disease control effects in both the potato rhizome black scurf disease index and the potato tuber black scurf disease index, with relative control efficiencies reaching 88.08% and 73.40%, respectively. These data further indicate that the *Trichoderma harzianum* PTDN-1, with accession number CGMCC No. 40017, can effectively control potato black scurf during the seedling and tuber stages.

[0061] Test Example 2 This test example illustrates that the Trichoderma of this disclosure can prevent and control potato tuber scab disease: The wettable powders of Example 2 and Comparative Examples 1-5 were tested respectively, and a control group was set up without the application of wettable powder.

[0062] The experimental setting was a field. Microbial wettable powders of *Trichoderma harzianum* 1, 2, 3, 4, and 5 (from Example 2) were applied to potato seedlings planted in the soil. Each individual plant was treated with 10... 7 The total number of spores will be counted, and the data on potato scab disease will be collected when potatoes are harvested 3 months later.

[0063] Grading standards for potato scab disease in tubers: Grade 0: No disease spots on the surface of the tuber; Grade 1: 1-2 lesions on the surface of the tuber; Grade 3: 3-4 lesions on the surface of the tuber, with the lesion area not exceeding 1 / 4 of the tuber surface area; Grade 5: 5-7 lesions on the tuber surface, with the lesion area accounting for 1 / 4 to 1 / 3 of the tuber surface area; Grade 7: 8-10 lesions on the tuber surface, with the lesion area accounting for 1 / 3 to 1 / 2 of the tuber surface area; Grade 9: More than 10 lesions on the surface of the tuber, with the lesion area accounting for more than 1 / 2 of the tuber surface area.

[0064] Disease index = 100 × ∑ (number of diseased tubers at each level × representative value at each level) / (total number of tubers surveyed × highest representative value); Relative prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0065] The results are listed in Table 3.

[0066] Table 3

[0067] As shown in Table 3, Example 2, compared with the pathogen control group and Comparative Examples 1-5, exhibited a significantly better disease control effect in the potato scab disease severity index, with a relative control efficacy of 60.73%. These data further demonstrate that the Trichoderma harzianum PTDN-1, with accession number CGMCC No. 40017, can effectively control potato scab disease.

[0068] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0069] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0070] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. The application of *Trichoderma harzianum* in the control of potato black scurf and scab, characterized in that, The fungus *Trichoderma harzianum* is classified as *Trichoderma harzianum*. Trichoderma harzianum The preservation number of the *Trichoderma harzianum* is CGMCC No. 40017.

2. The application according to claim 1, wherein, The application includes applying Trichoderma harzianum to the soil of potato plants.

3. The application according to claim 2, wherein, The application includes any one of spraying, root irrigation, and mixing with soil.

4. The application according to claim 2, wherein, The dosage of the *Trichoderma harzianum* was 10. 7 -10 11 CFU / strain.

5. The application according to claim 1, wherein, The application includes: applying a microbial inoculant containing Trichoderma harzianum with preservation number CGMCC No. 40017 to the soil of potato plants.

6. The application according to claim 5, wherein, The application includes any one of spraying, root irrigation, and mixing with soil.

7. The application according to claim 5, wherein, The viable count of *Trichoderma harzianum* with accession number CGMCCNo.40017 in each gram of the microbial inoculant is 10. 7 -10 11 CFU.

8. The application according to claim 5, wherein, The dosage of the microbial agent is 1-2 kg / mu.

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