CN112592833B

CN112592833B - Trichoderma polyspora and application thereof

Info

Publication number: CN112592833B
Application number: CN202010249214.8A
Authority: CN
Inventors: 吴令上; 斯金平; 陈秋燕; 周莹
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2022-05-24
Anticipated expiration: 2040-04-01
Also published as: CN112592833A

Abstract

The invention discloses a trichoderma polyspora strainTrichoderma polysporum) DCR2 strain, depository: china center for type culture Collection, preservation date: 24 days 3 and 24 months in 2020, and the preservation number is CCTCC M2020058. The strain is cultured on a Potato Dextrose Agar (PDA) plate at 25 ℃, the colony is white, felty and sparse, and the back of the colony is yellow. Trichoderma polyspora (Trichoderma polysporum) DCR2 can effectively inhibit the growth of Sclerotium delphinii, obviously reduce the incidence of Dendrobium officinale seedlings and improve the disease resistance of Dendrobium officinale seedlings. Meanwhile, the pesticide composition has a certain effect on preventing and treating various crop diseases caused by the fungus Sclerotium delphinii.

Description

Trichoderma polyspora and application thereof

Technical Field

The invention relates to trichoderma polyspora and application thereof, belonging to the technical field of microorganisms.

Background

Dendrobium officinale Dendrobium catenatum (D.officinale) is a traditional famous and precious medicinal material and has the effects of tonifying stomach, promoting the production of body fluid, nourishing yin, clearing heat, inhibiting tumors and the like. With the continuous increase of the cultivation scale and the operation intensity of the dendrobium officinale, the harm of pests is increasingly aggravated, wherein sclerotium delphinii is fast in disease occurrence and difficult to control through infecting southern blight caused by leaves, stem bases and roots of the dendrobium officinale, and serious economic loss is caused. Southern blight is a common and destructive disease, and the pathogenic bacteria of southern blight have wide host range, and can cause serious diseases of more than 500 crops such as peanut, chickpea, tobacco, oil tea, sesame, strawberry, bighead atractylodes rhizome and the like. In recent years, research on the utilization of endophytic fungi for preventing and treating plant diseases is focused, the dendrobium officinale as an orchid plant has the growth specificity and is closely related to fungi in vivo, however, at present, the research on the influence of the endophytic fungi of the dendrobium officinale on the disease resistance of plants is lacked, and the application of the endophytic fungi in the disease prevention and treatment of the dendrobium officinale is not seen.

Trichoderma (Trichoderma sp) is one of important microorganisms in biological control research of plant diseases, has various and wide-range types, and is a dominant population of soil and plant microecology. The microorganism plays a plurality of beneficial roles in preventing and treating plant diseases by growth competition, heavy parasitism and secretion of antibiotics. The antagonistic Trichoderma species studied at present are many, including Trichoderma hamatum (Trichoderma hamatum), Trichoderma viride (Trichoderma viride), Trichoderma longibrachiatum (Trichoderma longibrachiatum), Trichoderma harzianum (Trichoderma harzianum), and Gliocladium virens (Gliocladium virens) classified as Gliocladium. The invention patent of China invention with the patent number of '201410797356.2' discloses a trichoderma polyspora and application thereof, and discloses a trichoderma polyspora (Hypomeapachybasioides) FSR-97 and application thereof in prevention and treatment of main pathogenic bacteria of ginseng root rot, epidemic disease, sclerotinia rot, rust rot, black spot, damping off and gray mold. However, the use of trichoderma polyspora for preventing and treating diseases of the dendrobium officinale plant fungus micronucleus delphinii is not reported.

Disclosure of Invention

The invention aims to provide a novel trichoderma polyspora, provides application of the trichoderma polyspora in resisting host southern blight, and provides a material basis for preventing and treating host diseases by utilizing endophytic fungi.

The trichoderma polyspora is obtained by separating and purifying endophytic fungi from a living plant of Dendrobium officinale Dendrobium catenatum (D.officinale) of the Dendrobium of the orchid family by adopting an endophytic fungi separation and purification technology, and is identified as trichoderma polyspora DCR2 by microbiological classification. The strain is preserved, the preservation date is 2020 and 03 months and 24 days, the preservation number is CCTCC No. M2020058, the preservation unit is China center for type culture Collection, address: wuhan city Wuchang Lojia mountain, post code: 430072.

the solid culture of the trichoderma polyspora is characterized in that: culturing on potato glucose agar (PDA) medium at 25 deg.C, wherein the colony is white felt and sparse, and the back of the colony is yellow.

The ITS and 5.8S rDNA base sequences of the trichoderma polyspora are shown as SEQ ID NO. 1: ggatcattac cgagtttaca actcccaaac ccctatgtga acgttaccaa aatgttgcct cggcggggaa tttattcatg ccccgggcgc gtcgcagccc cggaccaagg cgcccgccgg aggaccaacc aaaactcttt tgtatgtccc ctcgcggact tttataattc tgaaccatct ggcgcccct agcgggcgtt tcgaaaatga atcaaaactt tcaacaacgg atctcttggt ctggcatcg atgaagaacg cagcgaaatg cgataagtaa tgtgaattgc agaattcagt gaatcatcga atctttgaac gcacattgcg cccgccagta ttctggcggg catgcctgtc cgagcgtcat ttcaaccctc gaacccctcc gggggtacgg cgttggggat cggcccttt cggggccggc cccgaaatac agtggcggtc tcgccgcagc ctctcctgcg cagtagtttg cacactcgca tcgggagcgc ggcgcgtcca cgtccgtaaa acacccaact ttctgaaatg ttgacctcgg atcaggtagg aatacccgct gaacttaagc atatca are provided.

The invention also provides application of the Trichoderma polyspora DCR2 in resistance to sclerotium rolfsii disease.

Because the trichoderma polyspora DCR2 provided by the invention can effectively inhibit the growth of the fungus Sclerotium delphinii, the morbidity of dendrobium officinale seedlings is obviously reduced, and the disease resistance of the dendrobium officinale seedlings is improved. Meanwhile, the pesticide composition has a certain effect on prevention and treatment of more than 500 crop diseases such as peanut, chickpea, tobacco, oil tea, sesame, strawberry, bighead atractylodes rhizome and the like caused by the fungus Sclerotium delphinii, and has a great application prospect.

Drawings

FIG. 1 is a morphological diagram of Trichoderma polyspora of the present invention on a PDA plate medium;

FIG. 2 is a plate-confronted view of pathogenic bacteria of Trichoderma polyspora and sclerotium candidum southern blight of the invention;

FIG. 3 is a diagram showing the growth of the dendrobium officinale inoculated with the endophytic fungi and pathogenic bacteria according to the invention for one week, two weeks and three weeks.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

Example 1 Trichoderma polyspora of the present invention is a strain obtainable from leaf, stem or root of Dendrobium officinale Kimura et Migo cultivated in Zhejiang province in three-dimensional manner. The trichoderma polyspora is obtained by separation according to the following steps: washing collected fresh and healthy dendrobium officinale stems, leaves and roots in tap water, sucking surface water by using sterile filter paper, cutting the roots and stems into small sections with the length of 3cm, cutting the leaves into small blocks with the length of 3cm multiplied by 3cm, and carrying out three-step surface disinfection treatment of 1min → 2.5% sodium hypochlorite 3min → 75% ethanol for 30 s. After sterilization, the surface of the material is washed with sterile water for 3 times and then is dried by sterile filter paper, roots and stems are cut into small segments (1.0cm) by using sterilized scissors, leaves are cut into small blocks (0.5cm multiplied by 0.5cm), and the small blocks are placed on PDA (potato 200g/L, glucose 20g/L and agar 15g/L) culture medium containing 50mg/L penicillin to be cultured at the temperature of 26 +/-2 ℃. The samples were observed daily for fungal growth. Hyphae grow out after 5 to 7 days, the hyphae tips are picked and transferred to a new PDA culture medium for purification and culture, and the hyphae tips are combined according to the shape to finally obtain the trichoderma polyspora strain. The morphology of trichoderma polyspora on PDA plate medium is shown in fig. 1.

Activating pathogenic bacteria and endophytic fungi, beating 5mm fungus cakes, respectively inoculating the endophytic fungi and the pathogenic fungi cakes into two points on the same straight line at a position 20mm away from the center on a PDA (personal digital assistant) plate, setting the inoculated pathogenic bacteria plate as a control, repeating the treatment for 3 times, observing the growth condition of colonies day by day, and measuring the corresponding radiuses r1 and r2 of the growth of the pathogenic bacteria and antagonistic fungi colonies and the control radius r0 by using a vernier caliper. After the pathogenic bacteria and antagonistic fungus colonies are intersected, the inhibition of the antagonistic fungus on the pathogenic bacteria is observed and recorded, the process of occupying the nutrition space of the pathogenic bacteria is occupied, the test period is 4 days, and the growth inhibition rate of the pathogenic bacteria is calculated [ inhibition rate (%) - (r 0-r 1)/r0 & 100% ]. The confronting of Trichoderma polyspora and pathogenic bacteria is shown in FIG. 2. The inhibition ratio is shown in table 1.

Table 1: incidence rate of dendrobium officinale seedlings inoculated by different modes of trichoderma polyspora and southern blight pathogenic bacteria

。

Healthy and consistent-growth dendrobium officinale aseptic seedlings are selected and inoculated into tissue culture bottles, one for each bottle. The endophytic fungi and the pathogenic bacteria are inoculated into the tissue culture bottle by adopting 3 inoculation modes.

The inoculation of the dendrobium candidum aseptic tissue culture seedling with the trichoderma polyspora and southern blight pathogenic bacteria comprises the following steps:

(1) taking a trichoderma polyspora strain, picking a small amount of hypha by using an inoculating needle under the aseptic condition, inoculating the hypha into a sterilized solid PDA culture medium flat plate, and performing activated culture at the temperature of 25 +/-1 ℃ for 7 days;

(2) preparing the dendrobium officinale aseptic tissue culture seedling, inoculating endophytic fungi and pathogenic bacteria into a tissue culture bottle by adopting 3 inoculation modes when hypha grows over a flat plate:

1) the inoculation cake method comprises the following steps: the two sides of the tissue culture seedling are respectively connected with the pathogenic bacteria and the endophytic fungi cakes with the diameters of 5mm, and the pathogenic bacteria, the endophytic fungi and the non-inoculated tissue culture seedling are treated by 5 bottles each by taking the single-inoculated pathogenic bacteria, the endophytic fungi and the non-inoculated tissue culture seedling as a control.

2) Spraying mixed bacteria liquid: directly spraying 1mL of mixed bacteria liquid (preparation of mixed bacteria liquid, that is, respectively beating 5mm fungus cakes on plates of pathogenic bacteria and endophytic fungi, inoculating the fungus cakes into a conical flask containing 100mLPDB culture medium, placing the conical flask in a shaking table, culturing at the temperature of 28 ℃ for 5 days at 180 r.min < -1 >, and homogenizing to prepare mixed bacteria liquid). 5 bottles of each treatment were treated with a single spray of pathogenic bacteria, endophytic fungi and blank PDB as controls.

3) The acupuncture bacteria liquid coating method comprises the following steps: puncturing tissue culture seedling leaves by using an injector, respectively puncturing 2 wounds of pathogenic bacteria and endophytic fungi of each leaf, puncturing 3 leaves of each plant, then coating double-bacterium mixed bacterium liquid on the wounds, and treating 5 bottles each by taking single-puncturing pathogenic bacteria, endophytic fungi and only-puncturing non-inoculating bacteria as controls and coating PDB. And culturing the inoculated tissue culture seedlings at the temperature of 25 ℃ under the photoperiod L/D of 12 h/12 h, regularly observing and recording the growth conditions of the seedlings and the bacteria, and counting the morbidity. The growth conditions of the endophytic fungi and the pathogenic bacteria inoculated dendrobium officinale for one week, two weeks and three weeks are respectively shown in figure 3.

Test results show that in the culture process of the dendrobium officinale tissue culture seedling, after pathogenic bacteria are inoculated into a sterile tissue culture seedling bottle, the dendrobium officinale tissue culture seedling can generate diseases, and meanwhile, obvious inhibition effect on the pathogenic bacteria is achieved when the dendrobium officinale tissue culture seedling is inoculated into a tissue culture seedling bottle of a trichoderma polyspora strain. The fungus cake inoculation method and the needle bacterium liquid application method by the mixed bacterium liquid spraying method have good effects, the inhibition effect is generated in the second week after inoculation, the morbidity is only 20% in the fourth week after inoculation, and the possible reason is that endophytic fungi can degrade cell walls of host pathogenic bacteria or compete with pathogenic bacteria resources by producing enzymes such as cellulase, protease, amylase and the like. In addition, the synergistic effect of the metabolite of the endophytic fungi and the hydrolase can also influence the antagonistic effect of the strain on pathogenic bacteria, and plays an important role in the biological control of plant diseases.

In addition, different processing modes produce different effects. Tests show that the treatment effect of the inoculation cake method and the acupuncture liquid-applying method is relatively poor, and the incidence rate is 60-80% after the fourth inoculation.

Therefore, the trichoderma polyspora obtained by the method is sprayed on a protected plant body by adopting a proper method, and can be effectively used for preventing and treating the sclerotium rolfsii of the dendrobium officinale.

Sequence listing

<110> Zhejiang agriculture and forestry university

<120> Trichoderma polyspora and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 583

<212> DNA

<213> Trichoderma polyspora (2 Ambystoma laterale x Ambystoma jeffersonoanum)

<400> 1

ggatcattac cgagtttaca actcccaaac ccctatgtga acgttaccaa aatgttgcct 60

cggcggggaa tttattcatg ccccgggcgc gtcgcagccc cggaccaagg cgcccgccgg 120

aggaccaacc aaaactcttt tgtatgtccc ctcgcggact tttataattc tgaaccatct 180

ggcgccccta gcgggcgttt cgaaaatgaa tcaaaacttt caacaacgga tctcttggtc 240

tggcatcgat gaagaacgca gcgaaatgcg ataagtaatg tgaattgcag aattcagtga 300

atcatcgaat ctttgaacgc acattgcgcc cgccagtatt ctggcgggca tgcctgtccg 360

agcgtcattt caaccctcga acccctccgg gggtacggcg ttggggatcg gccctttcgg 420

ggccggcccc gaaatacagt ggcggtctcg ccgcagcctc tcctgcgcag tagtttgcac 480

actcgcatcg ggagcgcggc gcgtccacgt ccgtaaaaca cccaactttc tgaaatgttg 540

acctcggatc aggtaggaat acccgctgaa cttaagcata tca 583

Claims

1. Trichoderma polyspora (Trichoderma polysporum) DCR2, characterized in that it has a collection number of CCTCC number M2020058.

2. The trichoderma polyspora DCR2 of claim 1, wherein said trichoderma polyspora DCR2 is cultured on PDA media at 25 ℃ with the colonies being matted and sparse and the back of the colonies being yellow.

3. The application of the trichoderma polyspora DCR2 in preventing and treating the dendrobium candidum disease according to claim 1 or 2, wherein the dendrobium candidum disease is caused by sclerotium delphiniiSclerotium delphiniiAnd the resulting southern blight.