CN115960724B - Novel trichoderma reesei strain and application thereof in identification of strawberry diseases - Google Patents
Novel trichoderma reesei strain and application thereof in identification of strawberry diseases Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism strains and strawberry disease identification, provides a novel trichoderma reesei fungus strain and application thereof in identification of strawberry diseases, and lays a foundation for diagnosis and control of the novel strawberry hollow disease. The name of the strain is: novel moxaburner with clavulansNeopestalotiopsis clavispora CMSF-1 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.40205. Preservation address: beijing, chaoyang, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, postal code: 100101. the identification of the bacteria has better effect by a kit extraction method. The pathogenic bacteria can be identified by ITS gene sequences.
Description
Technical Field
The invention belongs to the technical field of microorganism strains and strawberry disease identification, and particularly relates to a novel trichoderma reesei fungus strain and application thereof in strawberry disease identification.
Background
In recent two years, a new disease with symptoms appears in strawberry planting areas, most of strawberry planting areas such as Beijing, liaoning, hebei, shanxi, shandong, zhejiang, gansu, yunnan and Henan are found in China, and the strawberry planting areas gradually spread in China along with the transportation of seedlings and other factors, so that the healthy development of strawberry industry in China is seriously affected. The newly discovered disease most appear after the strawberry bud appears, the best seedling supplementing stage is missed at the moment, and once the disease is developed, the disease area is large, so that the loss caused by dead seedlings cannot be compensated.
At present, the new symptom disease has no formal name, no institution at home makes an exact answer strictly according to the Koch law which is recognized by plant diseases, and the strawberry farmer mostly calls the disease of hollow disease or broken head.
According to the field observation result, after the root of the strawberry is infected by pathogenic bacteria, the short stem is diseased, the inside of the strawberry is in a scalding shape, and the strawberry is gradually liquefied, so that a hollow state can be finally generated; the back of the blade can be provided with irregular black plaques, the spots are gradually increased and converged into a piece. When the disease is serious, the whole plant wilts, and the short stem can be broken off by gently breaking off the plant with hands. The invention refers to the disease as empty heart disease.
As the symptoms of empty heart disease on the leaves, it has been inferred that the disease is caused by Xanthomonas [1-2] The infection should occur in practice as bacterial angular leaf spot. However, the existence of angular leaf spot has been found some decades ago abroad, but few reports of the disease causing the symptoms of shortstalk hollow are available. In addition, according to human analysis, the number of times of threshing is frequent in the domestic seedling raising and planting processes of strawberry farmers, once the timely spraying of the pesticide is not carried out to prevent pathogenic bacteria infection, wound generated by splitting leaves gives a multiplicative machine for the infection of xanthomonas to the shortened stems, and hollow symptoms appear on the base parts of strawberries.
The inquiry literature and the data show that the related report of a new disease, namely the hollow disease, is not available abroad, and the related research in China is almost in a blank stage, and even the pathogenic bacteria causing the disease are not exactly reported. Only complaints of tranquility [3] A related study was performed in which the pathogenic bacteria of Anhui strawberry hollow disease were considered to beFusarium solani，But this result is still to be agreed upon. It can be said that the scientific research on the empty heart disease and pathogenic bacteria thereof is quite deficient at present, and no report that the new trichoderma reesei of the corynespora causes the empty heart disease of the strawberry is found, so the invention is the first report that the trichoderma reesei of the corynespora reesei causes the empty heart disease of the strawberry, and lays a foundation for diagnosing and preventing the new disease of the empty heart disease of the strawberry.
Disclosure of Invention
The invention provides a new trichoderma reesei fungus strain and application thereof in identification of strawberry diseases, which lays a foundation for diagnosis and control of the new disease of strawberry hollow diseases.
The invention is realized by the following technical scheme: a new fungus of Pediophora disharmoni, the name of which isThe method comprises the following steps: novel moxaburner with clavulansNeopestalotiopsis clavispora CMSF-1 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 40205; the preservation date 2022, 5 month and 26 days, the preservation address: beijing, chaoyang, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, postal code: 100101.
the morphological characteristics of the strain are as follows: the diameter of the molecular spore disk is about 150-250 mu m, black is scattered on the surface of a bacterial colony, and the epidermis is irregularly cracked to emit conidium. Conidia are spindle-shaped or long spindle-shaped, 18-26 multiplied by 6.5-8.5 mu m, the whole spores are in a straight or slightly bent state, 5 cells are shared, 4 membranes are shared, and the constriction at the separation part is not obvious.
The basal cell length is 4-5 mu m, the color is transparent, and the triangle is conical; the middle 3 cells have darker colors and brown multiple sites, and the lengths of the 3 cells are respectively 4.5-5.4 mu m, 5.4-6.5 mu m and 4.3-5.7 mu m; the cell length at the top is 3.2-4.5 μm, the shape is conical, and the color is transparent. The basal cell has 1-2 colorless auxiliary filaments, and has a tubular shape with a length of 21-30 μm; the top end is provided with 1 accessory filament which is not branched, and the accessory filament is colorless and transparent. The new moxaburner of the corynespora canicolaNeopestalotiopsis clavispora CMSF-1 is separated and purified from a strawberry hollow disease plant, and is subjected to gene detection analysis and belongs toNeopestalotiopsis clavispora。
The invention also aims to provide an application of the novel moxef sterculia pseudodisc as a reference substance in the aspect of identifying the strawberry hollow disease.
The experimental results show that: novel moxaburner with clavulansNeopestalotiopsis clavispora CMSF-1 is pathogenic bacteria of the empty heart disease of the strawberry disease, and can be identified by a morphological method, and the identification of the bacteria has a good effect by a kit extraction method. The pathogenic bacteria can be identified by ITS gene sequences.
The strain of the invention is new moxaburner trichoderma clavatumNeopestalotiopsis clavispora CMSF-1, which has been preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.40205 and a preservation date of2022, 5, 26. Preservation address: beijing, chaoyang, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, postal code: 100101.
drawings
FIG. 1 is a cross-sectional view of a strawberry root part with disease;
FIG. 2 shows the growth state of strain CMSF-1 on PDA medium, wherein: a is the front surface; b is the reverse side;
FIG. 3 is a conidium of strain CMSF-1;
FIG. 4 is a polygenic phylogenetic tree of strain CMSF-1;
FIG. 5 shows the growth of strain CMSF-1 on different media, in which: a: PDA medium; b: OA medium; c: corn culture medium; d: a culture medium; e: starch-based; f: mannitol-formula; g: fructose-observing; h: glucose-monitoring; i: lactose-formula; j: ammonium chloride-r-type; k: urea-urea; l: tryptone-test; m: glutamic acid-scouting; n: arginine-form.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize that equivalents of the specific embodiments described, as well as those known by routine experimentation, are intended to be encompassed within the present application.
The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
1. Collecting, separating, purifying and identifying the disease plants:
1. description of the situation: in recent two years, a new disease with symptoms appears in strawberry planting areas, most of strawberry planting areas such as Beijing, liaoning, hebei, shanxi, shandong, zhejiang, gansu, yunnan and Henan are found in China, and the strawberry planting areas gradually spread in China along with the transportation of seedlings and other factors, so that the healthy development of strawberry industry in China is seriously affected. The investigation and research on strawberry diseases by the penholder in 2020 in eastern yang county of elm in Shanxi province shows that the root and leaf of strawberry are damaged by a fungal disease. When the root of the strawberry is infected by pathogenic bacteria, the short stem is diseased, the inside of the strawberry is in a scalding shape (figure 1), the strawberry is gradually liquefied, and finally a hollow state is generated; irregular black plaques appear on the back of the leaf blades, and the irregular black plaques appear in punctiform, gradually increase and are converged into a piece. When the disease is serious, the whole plant wilts, and the short stem can be broken off by gently breaking off the plant with hands. The invention refers to the disease as empty heart disease.
Collecting local strawberry plants, performing germ separation and purification, morphology and symptom description, performing strawberry potted plant root irrigation tieback experiments, comparing tieback roots with specimen roots, and determining that pathogenic germs are new trichoderma reesei.
2. And (3) germ separation and purification: test medium: potato dextrose agar medium (beijing solebao technologies limited); test instrument: the main instrument is a Nikon optical microscope.
The disease is investigated in the strawberry planting base in eastern yang county of elm province in Shanxi province in 2020, the symptom is described, typical disease plants are collected, and the disease plant forms are described as follows: the disease plants wilt, irregular spots are arranged on the back of the leaves, and black conidium discs can be generated; the stem base of the disease plant is cut, so that the internal tissue can be seen to have a scalding-like lesion, and the serious disease plant has a hollow state. Collecting the disease plants for germ separation and purification.
And (3) separating and purifying pathogenic bacteria: tissue isolation was used. The root of the collected strawberry plant is washed clean, the diseased part is disinfected by 70% alcohol, then is washed three times by sterilized distilled water, a tissue block with the length of 5mm multiplied by 5mm is cut at the junction of the disease and the key, the diseased part is disinfected by 70% alcohol, then is washed three times by sterilized distilled water, is placed in a PDA culture medium, and each culture dish is inoculated with 2-4 blocks for culture at 25 ℃.
And selecting a bacterial colony with good growth state and less mixed bacteria pollution, when the length of the mycelium reaches 2cm, picking the mycelium at the edge of the bacterial colony by using an inoculating hook, inoculating the mycelium to the center of a PDA culture medium again, and separating and purifying for a plurality of times until the bacterial colony grows neatly and consistently without mixed bacteria. Colony morphology was observed and conidial morphology was observed under a microscope. A pathogen (numbered CMSF-1) with good growth and typical status was selected for subsequent experiments.
Bacterial strains separated from the PDA culture medium grow white hyphae, are fluffy, grow rapidly and grow over the whole culture dish, and black conidium discs can grow on the surfaces of the bacterial colonies in the later period. Some of the colonies in the dishes appeared green, black and pale yellow colonies.
Purifying: the colony obtained by purification is white velvet-like, approximately circular, irregular in edge, wavy in surface, no obvious pigment is generated on the front side of the colony at the later stage, and black conidium can be generated about 15 days after the back side of the colony is light orange. Finally obtaining pure strain 5 strains. A strain typical of growth was selected for subsequent experiments and numbered CMSF-1. The growth state of strain CMSF-1 on PDA medium is shown in FIG. 2.
3. The koch's law verifies: and (3) verifying by using the Koch rule, and selecting healthy potted strawberry plants for a tieback experiment. The root-filling inoculation method is used for the potted strawberry plant stabbing. And selecting healthy and disease-free 'red-colored' strawberry plants for experiments.
Root-filling inoculation of potted strawberry plants by stabbing: eluting spores of pathogenic bacteria CMSF-1 to obtain suspension, adding 0.01% Tween 20, slightly shaking until spores are uniformly dispersed, and concentrating spore solution at 1×10 8 Individual mL -1 . Transplanting strawberry plants to a high-temperature sterilized substrateIn the quality, after 7d of seedling, a sterilized scalpel is inserted into the soil near the plants to cause micro-wounds at the roots of the plants, and each strawberry is poured into a plant with the concentration of 100 mL of 1 multiplied by 10 8 Individual mL -1 3 replicates of each treatment and control strawberry plants were filled with 100 mL sterile water. The culture conditions were 25℃until the vaccinated group died. And separating pathogenic bacteria again from the disease-key junction of the disease plant.
Root irrigation inoculation experiments show that after about 40d of root irrigation, strawberries wilt and root of the strawberries are dissected, lesions are found in the short stems, and the lesions are separated and purified from root symptoms of diseased plants all the time, so that the obtained strain (3 strains) is consistent with inoculated strains, bacterial colonies are white villus, the bacterial colonies are wavy, and black conidium discs can be produced in the later period.
4. Morphological identification: and (5) picking isolated and purified single colony hyphae, inoculating the single colony hyphae to a PDA culture medium, culturing for 5 days, and observing colony characters.
The morphological identification result is: the diameter of the molecular spore disk is about 150-250 mu m, black is scattered on the surface of a bacterial colony, and the epidermis is irregularly cracked to emit conidium. As shown in FIG. 3, the conidium is spindle-shaped or long spindle-shaped, 18-26×6.5-8.5 μm, and the whole spore is in straight or slightly curved state, and has 5 cells, 4 membranes, and no constriction at separation part.
The basal cell length is 4-5 mu m, the color is transparent, and the triangle is conical; the middle 3 cells have darker colors and brown multiple sites, and the lengths of the 3 cells are respectively 4.5-5.4 mu m, 5.4-6.5 mu m and 4.3-5.7 mu m; the cell length at the top is 3.2-4.5 μm, the shape is conical, and the color is transparent.
The basal cell has 1-2 colorless auxiliary filaments, and has a tubular shape with a length of 21-30 μm; the top end is provided with 1 accessory filament which is not branched, and the accessory filament is colorless and transparent.
5. Molecular identification (DNA extraction, PCR amplification, phylogenetic tree construction):
DNA extraction: the genome DNA of pathogenic bacteria is extracted by adopting a CTAB method.
And (3) PCR amplification: PCR amplification was performed using primer ITS 1/4. Primer sequence: ITS1 TCCGTAGGTGAACCTGCGG; ITS4 TCCTCCGCTTATTGATATGC.
Amplification system (50.0 ul): genomic DNA (20 ng/ul), 1.0ul 10 XBuffer (containing 2.5mM Mg2+) 5.0ul, taq polymerase (5 u/. Mu.L) 1.0ul, dNTP (10 mM) 1.0ul, ITS1 primer (10 uM) 1.5ul, ITS4 primer (10 uM) 1.5ul, ddH 2 O 39.0 ul。
Reaction conditions: pre-denaturation at 95℃for 3 min, denaturation at 95℃for 30 s, extension at 58℃for 30 s and 72℃for 1 min, total extension at 72℃for 7 min for 35 cycles, and preservation at 4 ℃.
ITS sequence sequencing: the PCR product was sent to Peseno for sequencing, sequencing results: the ITS sequence of the strain CMSF-1 is shown as SEQ ID NO. 1.
(7) Building a development tree: the sequences obtained by sequencing were BLAST aligned in NCBI (https:// www.ncbi.nlm.nih.gov /), and phylogenetic trees were constructed using MEGA 7.0. Phylogenetic tree is shown in fig. 4.
According to the comparison result, a phylogenetic tree is constructed, and the result shows that the experimental strain CMSF-1 and CMSF-1 areNeopestalotiopsis clavisporaGather into one branch and form separate branches, which we define as new species, namedNeopestalotiopsis clavispora CMSF-1. The strain is initially defined as the new Mucor pulmonale.
N.clavisporaBelongs to the genus PediophoraNeopestalotiopsisIs a plant pathogenic bacteria. In the year 2014,Maharachchikumburaet al [4] Based on morphological features and phylogenetic development, pelargonium species are selectedPestalotiopsisDividing into PediophoraNeopestalotiopsisPediophora pseudostellaPseudopestalotiopsisAnd Pediophora sppPestalotiopsisOf these, the new Pediopsis has 36 species. New Pediophora spp at home and abroadNeopestalotiopsisThe scientific research of the related literature is not so much that the related literature has a plurality of index. Ayoubi et al [5] Discovery ofN.iranensisAndN.mesopotamicaassociated with strawberry fruit rot, gilardi et al [6] Report the nameN.clavisporaCan cause the crown rot of the strawberries, shi Jianjun et al report for the first timeN.clavisporaCan causeStrawberry calyx and flower receptacle [7] Wilt disease, sun Qian et al [8] Discovery is madeN.rosaeIs a pathogenic bacterium of strawberry root rot. Notably Li Qingjie et al [9] Is called in the study of (a)N.clavisporaStrawberry root rot can be caused in the baoding area, but the relation between the germ and the hollow disease is not mentioned in the article.
At present, research on prevention and treatment of the strawberry empty heart disease is still in a development stage, and especially, research on pathogenic bacteria of the empty heart disease is still very few in China. The test is to separate and purify pathogenic bacteria causing novel disease and empty heart disease of strawberries, detect pathogenicity, identify the pathogenic bacteria morphologically and molecular system, and finally determine that the pathogenic bacteria are the new trichoderma reesei of the corynespora.
The identification result of pathogenic bacteria shows that the strawberry hollow disease pathogenic bacteria are new moxaburner trichoderma of corynesporaNeopestalotiopsis clavisporaCMSF-1 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.40205.
2. Analysis of biological characteristics of pathogenic bacteria:
1. the experimental method comprises the following steps:
influence of different nutritional conditions on the growth and spore yield of pathogenic bacteria hyphae: test medium selection PDA, OA, CMA and study medium; taking a culture medium as a basic culture medium, and replacing sucrose in the basic culture medium with glucose, fructose, mannitol, soluble starch and lactose with equal carbon content to prepare a culture medium plate with different carbon sources; the sodium nitrate is replaced by urea, ammonium chloride, tryptone, glutamic acid and arginine with equal nitrogen content, and a culture medium plate with different nitrogen sources is prepared. Culturing at 25deg.C in dark condition for 7d.
Influence of environmental conditions on the growth and sporulation of pathogenic hyphae: the strain was cultured using PDA medium. The temperature test is carried out by setting 5 temperature gradients of 10, 15, 20, 25 and 30 ℃ and culturing in dark; the illumination test sets 3 conditions of 24 h continuous illumination, 12h darkness and 24 h continuous darkness, and the constant temperature culture is carried out at 25 ℃. The incubation time was 7d.
2. Results and analysis: the experimental results are shown in fig. 5 and tables 1 and 2.
From FIG. 5 and Table 1, it can be seen that strain CMSF-1 grew well on PDA medium and oat medium, and after 15d of growth, black spores were produced in both media. For different carbon sources, the bacterial strain CMSF-1 has the longest colony diameter on a culture medium with starch as the carbon source, and has better hypha growth vigor on the culture medium with mannitol as the carbon source; in experiments of different nitrogen sources, the strain CMSF-1 has better growth state on a observing culture medium with ammonium chloride and peptone as nitrogen sources. The most suitable culture medium for the strain CMSF-1 is PDA culture medium by comprehensively considering objective factors such as cost and the like. The results in Table 2 show that the most suitable growth temperature for hyphae of strain CMSF-1 is 25℃and the most suitable light culture conditions are dark cultures. In summary, the optimal culture medium of the strain CMSF-1 is a PDA culture medium, the optimal carbon source is soluble starch, the optimal nitrogen source is tryptone, the optimal growth temperature is 25 ℃, and the optimal culture condition is dark culture.
TABLE 1 growth status of strain CMSF-1 on different media
Table 2: growth of strain CMSF-1 under different culture conditions
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Reference is made to:
[1] tian Qian screening of DNA barcodes of Xanthomonas and research on detection technology of important pathogenic varieties [ D ]. National academy of agricultural sciences, 2018.
[2] Noon ali, mai tijiang, xi Xin, ma Zhanhong. Pathogen identification of a new bacterial disease on strawberry [ a ]. Chinese plant pathology, academy of years 2018, chinese plant pathology: chinese plant pathology, 2018:1.
[3] Ningzhi, yixing Kai, wei Fasheng, et al isolation and identification of strawberry hollow disease pathogens in Anhui province [ J ]. Anhui agricultural science, 2020,48 (24): 136-139+160.
[4]S.S.N. Maharachchikumbura et al. Pestalotiopsis revisited[J]. Studies in Mycology, 2014, 79 : 121-186.
[5]Ayoubi, Najmeh, Zare, et al. First report of Curvularia inaequalisand C. spicifera causing leaf blight and fruit rot of strawberry in Iran[J]. Nova Hedwigia: Zeitschrift fur Kryptogamenkunde, 2017, 105(1/2):75-85.
[6]Gilardi G , Bergeretti F , Gullino M L , et al. First Report of Neopestalotiopsis clavispora Causing oot and crown rot on strawberry in Italy[J]. Plant Disease, 2019, 103(11):PDIS-03-19-0673.
[7]Shi J J，Zhang X M ，Liu Ye , et al. First Report of Neopestalotiopsis clavispora Causing calyx and receptacle blight on strawberry in China[J].Plant Disease,2022,106(04):1307.
[8]Sun Qian ,Harishchandra D ,Jia J , et al. Role of Neopestalotiopsis rosae in causing root rot of strawberry in Beijing, China[J]. Crop Protection, 2021, 147:105710.
[9] Li Qingjie, zhu Jiagong, wu Jiajia, et al, identification of strawberry root rot pathogen in Hebei Baoding area and determination of indoor virulence [ J ]. Hebei university of agriculture, 2021,44 (02): 15-20.
Claims (2)
1. A new strain of Cladosporium roseumNeopestalotiopsis clavispora) Fungi, characterized in that: the new Pediophora pseudodiscriminant fungus is named: the new moxaku-like septoria clavulans CMSF-1 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.40205 and the preservation date of 2022, 5 months and 26 days.
2. The use of a novel colletotrichum gloeosporioides fungus as defined in claim 1 as a control for identifying hollow disease of strawberry.
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| CN113980836A (en) * | 2021-09-23 | 2022-01-28 | 中化化肥有限公司临沂农业研发中心 | Bacillus belgii and application thereof in prevention and treatment of strawberry root rot |
| CN115305221A (en) * | 2022-08-02 | 2022-11-08 | 山西农业大学 | Compound microbial agent, preparation method thereof and application of compound microbial agent in prevention and treatment of strawberry hollow core disease |
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