CN111621424B - Saline-alkali-resistant neomyces and application thereof - Google Patents
Saline-alkali-resistant neomyces and application thereof Download PDFInfo
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Abstract
The invention discloses a saline-alkali tolerant bakanae and application thereof. The Trichoderma noviride TUCIM 10301 has a preservation number of CGMCC 19611. The strain is a new species of Trichoderma (Trichoderma sp.) and is finally named as Trichoderma intertidal zone (Trichoderma areeneea sp. nov.). Trichoderma koningii TUCIM 10301 can tolerate salt stress caused by 1.5M (namely 8%) salt concentration and alkali stress with the pH value reaching 9.0, and has good saline-alkali tolerance. Under the stress of different salt concentrations, the Twinia tsumadai 10301 removes the influence of salt on the plant growth inhibition, and compared with a control without inoculation, the Twinia tsumadai 10301 inoculated improves the crop biomass by 37% and 81% respectively under the conditions of 0.5% and 0.75% of NaCl content, and promotes the crop root system to elongate and the lateral root to differentiate.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a saline-alkali-resistant neomyces neoformans and application thereof.
Background
The saline soil in China is rich in resources and can be mainly divided into four major types, namely a coastal saline area, a Huang-Huai-Hai plain saline area, a desert grassland saline area and a grassland saline area. Wherein, the total area of the saline soil of Jiangsu coastal beaches is 68.73 hectares, which accounts for 1/4 of the total area of the national beaches, and the net increase area is about 1100 hectares every year, which belongs to a typical silt long coast and is an important reserve land resource. Although the reclamation development history of the coastal beaches in the northern Suzhou province is long, at present, salinization still serves as one of main barrier factors for restricting the development and utilization of the coastal beach soil in the northern Suzhou province, and the coastal beach saline soil has the advantages of heavy salinity, poor water and fertilizer retention, unstable structure and low microbial activity, and is difficult to adapt to the growth of crops without improvement. Therefore, how to promote the growth of the plant root system in saline soil is a key technology.
Trichoderma sp belongs to Hypocrea fungi, and has hypha growth suitable for wide temperature, humidity and pH range, fast growth and propagation, capacity of utilizing all available resource and occupying survival space. Among them, about 20 kinds of trichoderma (about 10-12% of the whole genus) are known to be distributed worldwide, they have high nutritional competitive power and heavy parasitic power, and most of the trichoderma strains used for biological control of plant soil-borne diseases and biofertilizers belong to these few species. At present, the researches on the biocontrol and growth promotion mechanism of trichoderma at home and abroad are still in a continuous perfection stage, and the researches on the application of the trichoderma and the preparation thereof to saline soil agriculture are less. Therefore, the trichoderma strain which can resist saline-alkali stress, promote plant growth and biologically prevent and control soil-borne fungal diseases is obtained by screening, and has important significance for sustainable agricultural development of saline-alkali soil and efficient utilization of saline-alkali soil resources.
Disclosure of Invention
The invention aims to provide a saline-alkali tolerant trichoderma novaeanum TUCIM 10301.
Another objective of the invention is to provide a microbial inoculum of Trichoderma neotimatum TUCIM 10301.
The invention also aims to provide the Trichoderma noviride TUCIM 10301 and application of the microbial inoculum thereof.
The purpose of the invention can be realized by the following technical scheme:
a saline-alkali-resistant Trichoderma neotimatum TuCIM 10301 is separated from coastal beach wetland of Dafenggang Jiangsu, 4 months and 3 days in 2020, and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 19611. The strain is a new species of Trichoderma (Trichoderma sp.) and is finally named as Trichoderma intertidal zone (Trichoderma areeneea sp. nov.).
Trichoderma koningii TUCIM 10301 can tolerate salt stress caused by 1.5M (namely 8%) salt concentration and alkali stress with the pH value reaching 9.0, and has good saline-alkali tolerance.
Under the stress of different salt concentrations, the Trichoderma koningii TUCIM 10301 eliminates the influence of salt on the plant growth inhibition effect, and compared with a control without inoculation, the inoculated Trichoderma koningii TUCIM 10301 improves crop biomass by 37% and 81% respectively under the conditions that the NaCl content is 0.5% and the NaCl content is 0.75%, and promotes the crop root system to elongate and the lateral root to differentiate.
The application of the Trichoderma atroviride (Trichoderma areenea sp. nov.) TUCIM 10301 in resisting pathogenic fungi; the pathogenic fungi is selected from one or more of Pestalotiopsis bacteria, Rhizoctonia solani, Fusarium oxysporum and Alternaria alternata.
The Trichoderma reesei (Trichoderma areenea sp. nov.) TUCIM 10301 is applied to the removal of the inhibition of salt on the growth of crops.
The application of Trichoderma atroviride (Trichoderma areenea sp. nov.) TUCIM 10301 in promoting growth of saline-alkali soil crops and preventing and treating pathogenic fungal diseases of the saline-alkali soil crops; the pathogenic fungi is selected from one or more of Pestalotiopsis bacteria, Rhizoctonia solani, Fusarium oxysporum and Alternaria alternata.
A microbial inoculum prepared from said Trichoderma intertidal (Trichoderma areenea sp. nov.) TUCIM 10301.
The application of the microbial inoculum in antagonizing pathogenic fungi; the pathogenic fungi is selected from one or more of Pestalotiopsis bacteria, Rhizoctonia solani, Fusarium oxysporum and Alternaria alternata.
The microbial inoculum is applied to removing the growth inhibition of salt to crops.
The microbial inoculum is applied to promoting the growth of saline-alkali soil crops and preventing and treating pathogenic fungal diseases of the saline-alkali soil crops; the pathogenic fungi is selected from one or more of Pestalotiopsis bacteria, Rhizoctonia solani, Fusarium oxysporum and Alternaria alternata.
The invention has the beneficial effects that:
the trichoderma strain TUCIM 10301 provided by the invention has the effects of antagonizing various pathogenic fungi, resisting salt and alkali and relieving the inhibition of salt on the growth of crops.
Drawings
FIG. 1 is a phylogenetic tree of Trichoderma tsugae TUCIM 10301 of the present invention.
The Trichoderma atroviride TUCIM 10301 is known as a new species from the evolutionary tree, and is finally named as Trichoderma atroviride TUCIM 10301 (Trichoderma arenera sp. nov.).
FIG. 2 is a morphological diagram of Trichoderma koningii TUCIM 10301 of the present invention.
Wherein a-c in figure 1 are colony morphology diagrams of strains growing in PDA, CMA and SNA culture media respectively, and d-g in figure 1 are morphology diagrams of conidium and phialide cells.
FIG. 3 shows the saline-alkali tolerance of Trichoderma koningii TUCIM 10301 of the invention.
FIG. 4 shows the inhibitory effect of Trichoderma TUCIM 10301 against various plant pathogenic bacteria.
FIG. 5 shows the effect of Trichoderma TUCIM 10301 on promoting the growth of tomato seedling root system under the stress of different salt concentrations.
Biological material preservation information
TUCIM 10301, classified and named as Trichoderma areenerea, and deposited in China general microbiological culture Collection center (CGMCC) at 3.4.2020, with the deposition address of No.3 Hozeh No.1, Kyowa, Yangyang, Beijing, and the deposition number of CGMCC No. 19611.
Detailed Description
Example 1: isolation and identification of trichoderma tsugae TUCIM 10301
Trichoderma koningii TUCIM 10301 was isolated from rhizosphere soil of Phragmites communis in coastal beach of Jiangsu Dafenggang, with a collection number of CGMCC NO. 19611.
1.1 morphological characteristics of Trichoderma TUCIM 10301 intertidal zone
1.1.1 test Medium
PDA: potato dextrose agar medium;
CMA: corn flour agar medium;
SNA: synthetic low-nutrient agar culture medium (formula: KH)2PO4 1.0g l-1；KNO3 1.0g l-1；MgSO4·7H2O 0.5 g l-1；KCl 0.5g l-1(ii) a Glucose 0.2g l-1(ii) a Sucrose 0.2g l-1(ii) a Agar powder 20g l-1(ii) a Adjusting pH to 7.0)
1.1.2 colony morphology
Inoculating Trichoderma tsunami 10301 strain to the culture medium, culturing at 25 ℃ in the dark for 3d, and observing the morphology, wherein the specific description is as follows (the morphological characteristics of Trichoderma tsunami 10301 are shown in FIG. 2):
bacterial colony: the mudflat trichoderma TUCIM 10301 grows fastest on a PDA plate, hyphae grows fastest and produces spores fastest, the plate with the diameter of 9cm can grow full after 3d of culture, aerial hyphae are white and villous, and the front of a colony generates green conidia; the growth speed on the CMA plate is the next time, the plate with the diameter of 9cm can grow after 4 days of culture, the aerial hyphae are in a white villiform shape, but no spore is produced until the 10 th day is observed; the growth is slowest on the SNA plate, the plate with the diameter of 9cm can grow after 7d of culture, aerial hyphae are in a white coiled villus shape, yellow green conidia are generated on the front surface of a colony, and the growth is carried out while sporulation is carried out.
The strain is as follows: the hypha is transparent, the wall is smooth, and small bulges are arranged on the surface; conidiophores are single, intergrown or opposite, the walls are smooth, the bases are expanded, and the conidiophores become thinner towards the tops; conidia are many on PDA culture medium, most of them are nearly spherical or elliptical, the wall is smooth, and the conidia are few rough.
1.2 molecular characterization of Trichoderma TuCIM 10301 intertidal zone
Through morphological identification, alignment based on ITS sequences and phylogenetic tree analysis and identification aiming at rpb2 and tef1 gene fragments, the similarity of the strain and the known nearest Trichoderma is determined to be lower than 97 percent, the strain is a new species of Trichoderma (Trichoderma sp.), and is finally named as Tmochi 10301 (Trichoderma areenea sp. nov.) and the evolutionary tree is shown in figure 1.
The trichoderma is screened from coastal saline soil and is friendly to local environment; the yield of spores is high, the agricultural production and utilization are convenient, and the native trichoderma has strong adaptability in native application and is easy to play a role.
The ITS sequence of TUCIM 10301 is shown as SEQ ID NO.1, the rpb2 sequence is shown as SEQ ID NO.2, and the tef1 sequence is shown as SEQ ID NO. 3.
Example 2: saline-alkali tolerance of trichoderma tsugae TUCIM 10301
The reeds can normally grow in coastal saline soil with the salt content of 1.5% and the pH value of 8.5, so that trichoderma separated from rhizosphere soil samples of the reeds has high possibility of tolerating high-salt and high-alkali environments. The strains which grow fast and produce much spores are selected from the screened trichoderma to carry out NaCl and pH tolerance tests as follows:
2.1 salt resistance test:
the method is characterized in that a Trichoderma tsugae TUCIM 10301 strain is taken as a main research object, and 2 Trichoderma reesei strains which grow fast and produce spores are selected to carry out a salt tolerance test together. Inoculating the three bacteria on PDA plate, culturing at 28 deg.C for 7d to generate large amount of conidia, scraping the conidia under aseptic condition, and diluting with sterile water to OD6000.1. 30% Murashige Skoog medium (not containing agar) containing different NaCl concentrations (0M, 0.5M, 1M, 1.5M) was dispensed into each well of a 96-well plate containing 198. mu.l of medium, and then 2. mu.l of medium diluted to OD was added to each well600Set 8 replicates for conidia suspension 0.1. Culturing at 25 deg.C in dark at constant temperature, and measuring OD (optical density) at 750nm (hyphal growth) with microplate reader at 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, and 168 hr.
2.2 alkali resistance test
The same as that of the Trichoderma strain selected in the salt tolerance testInoculating to PDA plate, culturing at 28 deg.C for 7 days to generate large amount of conidia, scraping the conidia under aseptic condition, and diluting with sterile water to OD6000.1 PDB medium (potato dextrose medium, agar free) at different pH's (5.0, 7.0, 8.0, 9.0) was dispensed into each well of a 96-well plate containing 198. mu.l of medium, and then 2. mu.l of medium diluted to OD was added to each well6000.1 conidia suspension, 8 replicates were incubated at 25 ℃ in the dark at constant temperature, and OD (optical density) at 750nm (hyphal growth) was measured using a microplate reader at 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, 168h, respectively.
2.3 test results
The trichoderma tsugae 10301 has good salt tolerance and alkali resistance (fig. 3), can tolerate salt stress caused by NaCl concentration up to 1.5M (namely 8%), can tolerate alkali stress with pH value up to 9.0, and provides a good biological material for improvement of biological strains in saline-alkali soil.
Example 3: inhibition effect of trichoderma tsugae TUCIM 10301 on various plant pathogenic fungi
Selecting 4 plant pathogenic fungi including Pestalotiopsis fici TUCIM 5788, Rhizoctonia solani TUCIM 3753, Fusarium oxysporum TUCIM 4848, and Alternaria alternata 10233, inoculating the above plant pathogenic fungi into 9cm PDA plate side with sterilizing perforator, inoculating Trichoderma TUCIM 10301 into the other side of the plate after 24h, culturing in dark at 25 deg.C for 14d, and taking pictures to record the confronting condition of fungi.
The results show (figure 4), Trichoderma TUCIM 10301 can inhibit the growth of Alternaria alternata and Rhizoctonia solani colonies, limit the colony radius to 1.5-2.0cm, and completely cover the colonies of the two pathogenic fungi, and produce a small amount of Trichoderma spores on the colonies; the trichoderma TUCIM 10301 can generate antagonism with fusarium oxysporum and polyminergic bacteria to limit the bacterial colonies to grow further, and the trichoderma generates a large amount of green spores around the bacterial colonies of the fusarium oxysporum and the polyminergic bacteria for one week. This facilitates the biological control of soil-borne fungal diseases by trichoderma.
Example 4: growth promoting effect of TuCIM 10301 on tomato seedlings under stress of different salt concentrations
4.1 test materials: trichoderma intertidal TUCIM 10301; tomato seed (Shanghai cooperative 903)
4.2 test methods:
4.2.1 spore suspension preparation:
inoculating Trichoderma tsurui 10301 strain on PDA culture medium, culturing for 7 days until spore is completely produced, scraping spore, adjusting the concentration of spore suspension to 10 with deionized water8cfu ml-1。
4.2.2 seedling culture:
soaking tomato seed at 30 deg.C for 2 hr, and adding 2% H2O2Soaking for 2min, placing the seeds on a culture dish paved with two layers of wet filter paper, and accelerating germination at 28 ℃. The seeds with uniform germination are planted in the matrix and grow for about 3 weeks until two leaves and one heart are used.
4.2.3 transplanting seedlings:
1-3mm vermiculite and 1-3mm perlite 1: 1, mixing and placing the tomato seedlings in 600ml plastic pots, transplanting 250g of tomato seedlings with consistent growth vigor into pots containing a matrix, carrying out trichoderma spore suspension root irrigation treatment after 7d field planting, and inoculating 2.5ml of tomato seedlings with the concentration of 10 at the root parts of each tomato seedling6cfu ml-1The spore suspension of (2) was simultaneously poured with Murashige Skoog nutrient solution containing NaCl, and the NaCl concentrations of the culture mediums treated differently were controlled to 0.00%, 0.5%, and 0.75%, respectively.
4.2.4 test seedlings:
after 6 weeks of cultivation, the growth condition of tomato plants is measured, indexes such as SPAD value (representing photosynthesis intensity), plant height, fresh plant weight, dry plant weight and the like are measured, and the total root length, root circumference and root tip number of tomato seedling root systems are scanned and analyzed. 4.3 test results:
under different degrees of salt stress, the tomato seedlings inoculated with the Trichoderma atroviride TUCIM 10301 are obviously superior to those subjected to control treatment without inoculation in growth vigor (Table 1 and figure 5). Under the salt stress, the tomato seedlings treated by the CK in a contrast manner have slender stalks, less biomass and weak root systems; and the biomass response, the thick stem and the developed root system of the treated biomass response inoculated with the Trichoderma TUCIM 10301 of the mudflat (figure 5).
TABLE 1 growth promoting action of TuCIM 10301 against tomato seedlings under stress of different salt concentrations
Using the CK of the treatment without Trichoderma strain as control, inoculating 2.5ml of 10-concentration CK to the root of each tomato seedling in the treatment group6cfu ml-1The spore suspension of trichoderma intertidal TUCIM 10301. Mean ± standard deviation (n ═ 12). There was no significant difference between treatments with the same letter (p ═ 0.05).
This shows that the TuCIM 10301 has significant growth promoting ability to plants under the stress of different salt concentrations, and compared with the control without inoculation, the inoculation of TUCIM 10301 can increase crop biomass by more than 35%. And the TUCIM 10301 is screened from coastal mudflats, can adapt to soil environments such as high salt, high pH and the like in coastal mudflat areas, and has practical significance for the development of saline soil agriculture.
Sequence listing
<110> Nanjing university of agriculture
<120> saline-alkali-resistant neomyces neoformans and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 601
<212> DNA
<213> Trichoderma atroviride (Trichoderma areenerea)
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ggtctccgtt ggtgaaccag cggagggatc attaccgagt ttacaactcc caaacccaat 60
gtgaaccata ccaaactgtt gcctcggcgg ggtcacgccc cgggtgcgtc gcagccccgg 120
aaccaggcgc ccgccggagg gaccaaccaa actctttctg tagtcccctc gcggacgtta 180
tttcttacag ctctgagcaa aaattcaaaa tgaatcaaaa ctttcaacaa cggatctctt 240
ggttctggca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat tgcagaattc 300
agtgaatcat cgaatctttg aacgcacatt gcgcccgcca gtattctggc gggcatgcct 360
gtccgagcgt catttcaacc ctcgaacccc tccgggggtc cggcgttggg gatcgggaac 420
ccctaagacg ggatcccggc cccgaaatac agtggcggtc tcgccgcagc ctctcctgcg 480
cagtagtttg cacaactcgc accgggagcg cggcgcgtcc acgtccgtaa aacacccaac 540
ttctgaaatg ttgacctcgg atcaggtagg aatacccgct gaacttaagc atatcaataa 600
g 601
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atgcgcagaa tgaatactga gctggcaaac tacctgagac gatgtgttga gggcaaccgc 60
cacttcaatc ttgctgttgg catcaagccc ggcacgcttt ctaacggatt gaagtactca 120
ctcgctactg gaaactgggg tgaccagaag aaggcaatga gctcgaccgc aggtgtatca 180
caggtgctga accgttacac ttttgcttct acactatctc atttgcgtcg taccaataca 240
cccatcggaa gagatggtaa gctggcgaag cctcgacagc tccacaacac acactggggc 300
ttggtgtgcc cggctgagac ccctgaagga caggcttgtg gtctggtcaa gaacttgtct 360
ctgatgtgct acgtcagtgt tggatctcct tctgagcctt tgatcgagtt tatgatcaac 420
agaggcatgg aagtcgttga ggagtacgag ccgctgagat atccccatgc tacaaagatc 480
tttgtgaacg gtgtctgggt tggagtccac caggatccta agcatctggt aaaccaagtt 540
ttggatactc gtcgcaaatc ctatctgcag tacgaagtct ctctgatcag agaaattcga 600
gatcaagaat ttaaaatttt ctctgatgcc ggtcgtgtta tgcgtcccgt cttcactgta 660
cagcaggaag atgacccgga aacgggtatc aacaagggcc acctggtttt gaccaaggaa 720
ctggtcaata ggttggctaa ggagcaggtt gagcctcctg aagacccaag ccagaagctt 780
ggatgggaag ggttgatcag ggctggtgcg gtggaatatc tcgacgccga ggaagaagaa 840
acctctatga tttgcatgac accggaagat cttgagcttt atcgtctgca gaaggctggt 900
attgccacgg acgaagacat aggagataat ccgaaccagc gtctgaagac gaagacaaat 960
ccaacaactc acatgtatac gcattgcgag attcacccga gtatgatctt aggtatctgt 1020
gccagtatca ttcctttccc cgatcacaac caggtatgtc cacccgagag gctgtccttt 1080
cccgttttgc ccaacttttt ctg 1103
<210> 3
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<212> DNA
<213> Trichoderma atroviride (Trichoderma areenerea)
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gtaagctcat ttcactactt tttccaccac gcttggcaca atcgtgtccg acaattctgt 60
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ttggcagcaa atttttctgc tgcctcgttt gactttagtg gggtgtcaaa tttttttttt 180
ggcaaccccg ctattgccac tgtccctcat ccatcgtccc aacaaaatgc actcattcaa 240
tcgcatcgtc ttttgactcg atttctctat ggttcgttgt gctaatcatg cttcaatcaa 300
taggaagccg ccgaactcgg caagggttct ttcaagtatg cgtgggttct tgacaagctc 360
aaggccgagc gtgagcgtgg tatcaccatc gacattgccc tctggaagtt cgagactccc 420
aagtactatg tcaccgtcat tggtatgtta ttcctggctc ttgacatgtc gaaatcatca 480
t 481
Claims (8)
1. Saline-alkali tolerant trichoderma reesei (or trichoderma viride)Trichoderma arenerea) TUCIM 10301, which was deposited in China general microbiological culture Collection center (CGMCC) at 3.4.2020, with the deposit number of CGMCC NO. 19611.
2. The Trichoderma intertidal zone of claim 1 (T. reesei), (T. reesei) and (T. reesei)Trichoderma arenerea) The application of TUCIM 10301 in antagonizing pathogenic fungi; the pathogenic fungi is selected from pestalotiopsis ficuum (A) and P.ficuum (A)Pestalotiopsis fici) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) Fusarium oxysporum (F.), (Fusarium oxysporum) Alternaria alternata (Alternaria alternata) One or more of (a).
3. The Trichoderma intertidal zone of claim 1 (T. reesei), (T. reesei) and (T. reesei)Trichoderma arenerea) The application of TUCIM 10301 in removing salt inhibition on crop growth is provided.
4. The Trichoderma intertidal zone of claim 1 (T. reesei), (T. reesei) and (T. reesei)Trichoderma arenerea) The application of TUCIM 10301 in promoting the growth of saline-alkali soil crops and/or preventing and treating pathogenic fungal diseases of saline-alkali soil crops; the pathogenic fungi is selected from pestalotiopsis ficuum (A) and P.ficuum (A)Pestalotiopsis fici) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) Fusarium oxysporum (F.), (Fusarium oxysporum) Alternaria alternata (Alternaria alternata) One or more of (a).
5. Trichoderma reesei (Trichoderma reesei) (T. reesei) as claimed in claim 1Trichoderma arenerea) TUCIM 10301.
6. Use of the microbial inoculum according to claim 5 for antagonising pathogenic fungi; the pathogenic fungi is selected from pestalotiopsis ficuum (A) and P.ficuum (A)Pestalotiopsis fici) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) Fusarium oxysporum (F.), (Fusarium oxysporum) Alternaria alternata (Alternaria alternata) One or more of (a).
7. Use of the inoculant according to claim 5 for the disinhibition of salt to crop growth.
8. The use of the microbial inoculum of claim 5 in promoting the growth of saline-alkali soil crops and/or preventing and treating pathogenic fungal diseases of saline-alkali soil crops; the pathogenic fungi is selected from pestalotiopsis ficuum (A) and P.ficuum (A)Pestalotiopsis fici) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) Fusarium oxysporum (F.), (Fusarium oxysporum) Alternaria alternata (Alternaria alternata) One or more of (a).
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| WO2015013558A8 (en) * | 2013-07-26 | 2015-02-26 | Adaptive Symbiotic Technologies LLC | Compositions and methods related to isolated endophytes |
| CN111394261A (en) * | 2020-05-07 | 2020-07-10 | 南京农业大学 | Saline-alkali-tolerant trichoderma asperellum and application thereof |
| CN112746024A (en) * | 2021-01-19 | 2021-05-04 | 南京农业大学 | Trichoderma composite microbial agent and bio-organic fertilizer prepared from same |
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