CN114480138A

CN114480138A - Beauveria bassiana LZ2-1a and application thereof

Info

Publication number: CN114480138A
Application number: CN202210025276.XA
Authority: CN
Inventors: 翟立峰; 幸月; 李景明; 张美鑫; 李嘉利; 李昌满; 张元珍; 张嘉渝
Original assignee: Yangtze Normal University
Current assignee: Yangtze Normal University
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-05-13
Anticipated expiration: 2042-01-11
Also published as: CN114480138B

Abstract

The invention discloses beauveria bassiana LZ2-1a and application thereof, and relates to the technical field of biological control. The strain of the invention is preserved, and the preservation number is CCTCC M20211576. The invention also provides application of the strain in controlling fruit tree pest litchi chinensis; and a biocontrol microbial inoculum, the effective component is spore suspension prepared from the beauveria bassiana strain; the viable count of beauveria bassiana strain in spore suspension is not less than 3 × 10⁵cfu/mL. The invention relates to pathogenic fungiThe muscardine globisporus LZ2-1a is obtained by separating lethal muscardine litchi insect, the strain infects healthy litchi insect through conidia to cause death of the litchi insect, the insect forms muscardine insect after death, spores grow from insect bodies, the spores can be spread in the field by wind and rain, and then infect more litchi insect bodies, so that the harm of the litchi insect bodies to litchi and longan is effectively controlled, the strain is harmless to ecological environment, the drug resistance of the insect is not caused, the culture condition is simple, and the storage is easy.

Description

Beauveria bassiana LZ2-1a and application thereof

Technical Field

The invention belongs to the technical field of biological control, and particularly relates to beauveria bassiana LZ2-1a and application thereof.

Background

Fructus litchi chinensis (fructus litchi chinensis)Tessaratoma papillosaDrury), also known as Clerodendrum obtusifolius, belongs to the family of Hemiptera (Hemiptera) stinkbug (Pentatamidae), and is one of the major pests harming the production of litchi and longan fruit trees. The insect is widely distributed in southern Asia, southeast Asia, Fujian, Taiwan, Guangdong, Guangxi, Hainan, Yunnan, Guizhou, Sichuan and Chongqing, and its main hosts include litchi, longan, goldenrain tree, Sapindaceae plants, etc. The insect stabs young shoots, flower ears and fruits of litchi and longan with imago and nymph, resulting in flower and fruit dropping; adults and nymphs of more than 3 years old are disease-transmission vector insects for transmitting pathogens of the longan broomcorn fruit rot, and the nymphs are wounds caused by the pests and are easy to cause the occurrence of the downy mildew. When being disturbed, the odor liquid is sprayed at the tender leaves, young fruits and the like to cause burn, which can cause pistil and tender leaves to wither or shells to be brown, and reduce the quality of fruits and change the shells into brown; the smelly liquid touches eyes and skin of people, causing burning pain, red swelling and inflammation and affecting orchard operation. In the case of severe occurrence, the yield of the litchis and the longans is seriously influenced.

In areas such as Fujian, Guangdong, Guangxi, Hainan and the like, the litchi chinensis bugs occur 1 time in one year, overwinter with immature adult forms, generally overwinter in dense crown-leaf clusters of the litchi trees is selected, the overwinter adult forms begin to gradually recover when the outdoor temperature exceeds 16 ℃ until 3 months of the next year, juice is absorbed from litchi branches or flower spikes to feed on the litchi trees, after the adult forms become sexual maturity, the adult forms copulation and spawn, generally, worm eggs are produced on the leaf backs of the litchi trees, and a few worm eggs are produced on the branch tips. In 4-5 months of each year, the period of the stink bugs in the full spawning period is about 6 middle-late months, and new adults can appear successively. The nymph period is about 80 d, the adult life is 200-300 d, and the total egg laying amount of single female is about 190 grains, and obvious aggregative property, phototaxis, chromotaxis and tenderness are shown on the habit.

Because the seasonal restriction and the occurrence of the insect source of the lygus lucorum are the region restriction of the pest, the prevention and the control of the insect still take chemical prevention and control as the main strategy and take agricultural prevention and control, physical prevention and biological prevention and control as the auxiliary strategy. Among them, the use of organophosphorus and pyrethroid insecticides is still the most common, but in the control, multiple treatments and multiple applications of chemical pesticides in one growth cycle are required to better control the harm. The pest generates stronger drug resistance, and the dosage and the use times are increased year by year, so that the pesticide residue of fruits exceeds the standard, and the pollution-free production of the litchi and the longan is influenced. Therefore, the search for new economic, efficient, green and environment-friendly prevention and treatment technologies and ways has become a problem to be urgently solved by the current litchi and longan planting industry.

Over 1000 pathogens have been isolated from insects and many have the potential to develop nuisance free potent insecticides. To date, 13 species or subspecies of entomopathogenic fungi have been formulated and registered as fungal pesticides (mycoinsectides), mainly Metarhizium anisopliae of ascomycetes (Metarhizium anisopliae)Metarhizium anisopliae) And Beauveria bassiana (balsamo) Vuillemin: (B)Beauveria bassiana). Beauveria bassiana belonging to the genus Beauveria of the class Aphyllophorales, class Hyphomycetes, order Moniliales of the sub-phylum Deuteromycotina (b.), (Beauveria). Beauveria bassiana has been used as a biological material for a long time because of its advantages of low production cost, good prevention and treatment effect, high safety to human and livestock, difficult generation of drug resistance to pests, reproducibility and compatibility and the likeThe important means for preventing and controlling pests is widely used for biological prevention and control of agricultural and forestry pests. Beauveria bassiana has larger specificity to the toxicity of a host, and the toxicity of separate strains from different cadavers of the same host has a certain difference, so that the selection of a strain with high toxicity to a target pest becomes the key for preventing and controlling the pest.

Therefore, the beauveria bassiana is different in controlling plant pests due to different strains, and has no foresight and corresponding revelation.

At present, related researches on the beauveria bassiana for preventing and treating litchi and longan from being harmed by lygus lucorum are still in a blank stage.

In litchi and longan planting, stinkbug symptoms occur seriously all the year round and mainly damage litchi, longan twigs, young stems, flowers and fruits and leaves, so that plant growth is retarded, flowers and fruits fall off and virus of the ghost broom of longan is spread, and the serious yield reduction of the litchi and the longan is caused even the whole plant is not harvested. Because the seasonal restriction and the occurrence of the insect source of the lygus lucorum are the region limitation of the pest, the prevention and the control of the insect are still mainly chemical prevention and control.

However, the chemical pesticide control is time-consuming and expensive, and has poor control effect, thereby influencing the pollution-free production of longan. Therefore, the application of the beauveria bassiana strain LZ2-1a in controlling lygus lucorum is developed and disclosed. The biocontrol microbial inoculum provided by the invention is simple to prepare, low in cost, easy for industrial production, good in control effect, free of environmental pollution, capable of replacing chemical pesticides to control fruit trees harmed by lygus lucorum, and good in development and application prospects. Ensuring pollution-free production of litchi and longan and improving yield and quality.

Disclosure of Invention

The invention aims to provide beauveria bassiana LZ2-1a and application thereof, which aim to solve the existing problems: in the prior art, the control of stinkbug of litchi and longan is mainly chemical control, which is time-consuming, expensive and poor in control effect, and affects pollution-free production.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the invention provides a Beauveria bassiana strain (Beauveria bassiana) LZ2-1a, which is preserved and has the preservation unit: china center for type culture Collection; the address of the depository: wuhan, Wuhan university; the preservation date is as follows: 12 months and 9 days 2021; the preservation number is: CCTCC M20211576.

The beauveria bassiana LZ2-1a is applied to preventing and controlling fruit tree pests.

In the application, the fruit tree pest is the lygus lucorum.

The active ingredient of the biocontrol microbial inoculum is spore suspension prepared from the strain of beauveria bassiana LZ2-1 a.

Further preferred is: the viable count of the beauveria bassiana strain LZ2-1a in the spore suspension is not less than 3 x 10⁵ cfu/mL。

A method of preparing a spore suspension comprising the steps of:

activating the beauveria bassiana strain LZ2-1a on a PDA plate, and then resuspending the strain with a sterile PD liquid culture medium to obtain spore and hypha suspension;

then inoculating the bacterial suspension into a PD liquid culture medium in an inoculation amount of 1%, and performing shake culture at the temperature of 25-30 ℃ for 48-72 h at the rotation speed of 100-250 rpm, so as to obtain the spore suspension from fermentation liquid.

Further preferred is: wherein the shaking culture is carried out for 48-72 h at the temperature of 25-30 ℃ at the speed of 100-250 rpm, and comprises the following steps:

the culture was carried out at 25 ℃ for 60 hours at 200 rpm with shaking.

The invention has the following beneficial effects:

1. according to the invention, the beauveria bassiana LZ2-1a is separated from the muscardine litchi bugs dead by pathogenic fungi, the strain infects healthy litchi bugs through conidia to cause death of the litchi bugs, the muscardine bugs are formed after death of pests, spores grow from polypide, and the spores can be spread in the field by wind and rain to further infect more litchi bugs, so that the damage degree of the litchi bugs to litchi and longan can be inhibited.

2. After the healthy lygus sinensis is treated by using the beauveria bassiana LZ2-1a spore suspension, the feeding activity of the lygus sinensis is obviously reduced, then the polypide dies and generates a large number of conidia, and the beauveria bassiana LZ2-1a is proved to be capable of efficiently inhibiting the harm of the lygus sinensis, has a good biological control effect, has a good effect of preventing and treating the harm of the lygus sinensis and longan, and is harmless to the ecological environment, free of causing the drug resistance of pests, simple in culture condition and easy to store.

3. The biocontrol microbial inoculum provided by the invention is simple to prepare, low in cost, easy for industrial production, good in control effect, free of environmental pollution, capable of replacing chemical pesticides to control fruit trees harmed by lygus lucorum, and good in development and application prospects.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a characteristic diagram of the fleahopper field muscardine (arrows indicate fungal hyphae);

FIG. 2 is a colony morphology of Beauveria bassiana LZ2-1a (A, colony morphology at the time of culture for 7 d; B, colony front enlargement; C colony back enlargement);

FIG. 3 is a diagram of conidiophore morphology of Beauveria bassiana LZ2-1 a;

FIG. 4 is a diagram of an ITS sequence-based evolution analysis of Beauveria bassiana LZ2-1 a;

FIG. 5 is a diagram of the disease symptoms of spore suspensions of different concentrations of Beauveria bassiana LZ2-1a inoculated to lygus lucorum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention aims to provide a Paenibacillus LZ2-1a for cribraria, which can prevent and control the damage of lygus bugs to litchis and longans.

The invention provides a beauveria bassiana strain, which is a beauveria bassiana strain (a)Beauveria bassiana) LZ2-1a, deposited in the China center for type culture Collection, with the address of the Collection Unit: wuhan university in Wuhan, China, the preservation date is 2021, 12 months and 9 days, and the preservation number is CCTCC M20211576.

The invention also provides application of the beauveria bassiana strain LZ2-1a in controlling fruit tree pest lygus sinensis.

The invention also discloses a biocontrol microbial inoculum, the effective component of which is the beauveria bassiana strain LZ2-1 a. The beauveria bassiana strain LZ2-1a is a culture spore suspension of the strain. The viable count of the beauveria bassiana strain LZ2-1a in the spore suspension is not less than 3 x 10⁵ cfu/mL。

In addition, the spore suspension is prepared by the following steps: activating a beauveria bassiana strain LZ2-1a on a PDA (personal digital assistant) flat plate, then re-suspending the strain on a sterile PD (PD) liquid culture medium to obtain spore and hypha suspension, then inoculating the bacterial suspension into the PD liquid culture medium in an inoculation amount of 1%, carrying out shake culture at the temperature of 25 ℃ for 48-72 h at the rpm of 120-200, and obtaining the spore suspension from fermentation liquor.

To further illustrate the invention, the following specific examples are now provided:

EXAMPLE 1 isolation, screening and characterization of Beauveria bassiana LZ2-1a

The separation method comprises the following steps: the method comprises the steps of collecting the stinkbug which is killed by pathogenic fungi from Hejiang county of Luzhou city, Sichuan province (figure 1), after surface disinfection by 75% alcohol, cutting tissue blocks with the square of 5 mm, sequentially treating the tissue blocks with sodium hypochlorite for 1 min, treating the tissue blocks with 75% alcohol for 1 min, washing the tissue blocks with sterile water for 3 times, placing the tissue blocks in sterile filter paper, airing the tissue blocks, and inoculating the tissue blocks to a PDA solid culture medium (200 g of potatoes, 20 g of glucose, 15 g of agar and 1L of distilled water) for culture. The plate was incubated at 25 ℃ for 5 to 7 days. Selecting different fungi according to the forms, hypha colors and the like of fungus colonies screened on the plate, and transferring the fungi to a fresh PDA plate for culture.

The identification method comprises the following steps: morphological characteristics of the strain LZ2-1a which is separated, purified and cultured are identified, and the strain plate is observed that the colony on the front side of the strain plate is milky white at the initial stage of culture and slightly changed into light sand yellow after culture, and the spore layer is thick and uniform and covers the whole colony (figure 2A). The colony had fewer spores at the periphery and a small amount of mycelium was seen (FIG. 2B). When cultured for 7 days at 25 ℃ on PDA medium, the diameter of the colony reaches 30.1 mm, and concentric rings raised from the center to the edge 1/2 of the colony (FIG. 2B). The colony had a yellow back (FIG. 2C). The vegetative hyphae are colorless, conidium peduncles are grown on the vegetative hyphae, the conidia are on hypha or vesicle clusters, and the conidia are transparent, smooth, spherical or nearly spherical and have the diameter of 2.3-3.5 mu m (figure 3).

The DNA of the strain LZ2-1a is extracted by a fungal genome DNA rapid extraction kit of Shanghai biological engineering Limited company, then the eukaryotic ITS-rDNA universal primers ITS1 and ITS4 are adopted for PCR amplification to obtain an amplified transcription spacer ITS sequence fragment, and the amplified fragment is sequenced, wherein the specific sequence is shown as SEQ ID NO. 1. The sequence is aligned with beauveria bassiana (B) through BLAST in GenBankBeauveria bassiana) The most closely related, homology is highest. The results of the phylogenetic tree construction of the ITS sequences for LZ2-1a and other representatives of the genus Beauveria showed that LZ2-1a and that of BeauveriaBeauveria bassianaRepresentative strains are gathered in the same branch (figure 4) and are new strains, and the strain is determined to be beauveria bassiana (Beauveria bassiana) according to morphological identification resultsBeauveria bassiana) Named Beauveria bassiana LZ2-1 a.

Example 2 application of Beauveria bassiana LZ2-1a

1. Spore-producing fermentation of strain beauveria bassiana LZ2-1a

After the beauveria bassiana LZ2-1a is transferred to a fresh PDA plate for activation, the scraped spores are prepared into a spore suspension, the spores are inoculated into an equal amount of PD liquid culture medium (200 g of potato, 20 g of glucose and 1L of distilled water) and are cultured in a shaking way under the temperature gradients of 200 rpm, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃, the initial spore concentration is about 4.61 multiplied by 10⁴Taking 20 mu L of culture solution every 24 hours for detecting sporulation condition per mLIn this case, spore concentration of the strain was measured using a hemocytometer. The results show that: the culture broth cultured at 25 ℃ and 30 ℃ became turbid after 24 hours, but no conidia were produced, and only the mycelia were observed. The culture solution cultured at 10 ℃, 15 ℃ and 35 ℃ has no obvious change; when cultured for 48 h, the strains cultured at 20 deg.C, 25 deg.C and 30 deg.C had produced spores with a spore concentration of about 2.02X 10⁷、5.28×10⁷And 1.23X 10⁸one/mL, only mycelium was seen at 15 ℃ and only a few spores germinated at 10 ℃. When the culture is carried out for 72 hours, the germination rates of the spores under the culture conditions of 10 ℃ and 35 ℃ are 84.66 percent and 17.02 percent respectively, while the culture solution of 15 ℃, 20 ℃, 25 ℃ and 30 ℃ contains a large number of conidia, and the concentration reaches 2.75 multiplied by 10 respectively⁷、5.59×10⁷、8.75×10⁸And 2.09X 10⁸Therefore, the suitable fermentation spore production temperature of the beauveria bassiana LZ2-1a is 25-30 ℃.

Will contain a spore concentration of about 4.61X 10⁴Spore suspension of each mL is subjected to shaking culture at the rotating speed gradients of 50 rpm, 100 rpm, 150 rpm, 200 rpm and 250 rpm at the temperature of 30 ℃, 20 mu L of culture solution is taken for culture for 60 h to detect sporulation, and the spore concentration of the strain is measured by a hemocytometer. The results show that: when the culture is carried out for 48 hours at the rotating speed of 50 rpm, the PA culture medium is clear, flocculent hyphae exist in the culture medium, and no conidium is generated. When the rotation speed was 100 rpm, 150 rpm, 200 rpm and 250 rpm, the culture medium became cloudy and milky white, and the concentration of conidia was measured to be 1.72X 10⁸、1.69×10⁸、2.02×10⁸And 1.68X 10⁸Therefore, the suitable fermentation spore-producing culture rotating speed of the beauveria bassiana LZ2-1a can be 100-250 rpm, but 200 rpm is most suitable.

In conclusion, the spore-producing fermentation conditions of the strain beauveria bassiana LZ2-1a are as follows: culturing at 25-30 ℃ and 250 rpm for 60 h at 100-⁸The above molecular spores.

2. In vivo prevention and treatment effect determination of strain LZ2-1a

Treating Lagotis Linnaeus larva and imago by soaking method, and preparing 1 × 10 with sterilized 0.05% Tween-80 solution⁴、1×10⁵、1×10⁶、1×10⁷And 1×10⁸spores/mL total 5 concentration gradients of spore suspension, one treatment per concentration, treated with sterile 0.05% tween-80 solution as a control. Selecting 3-4 instar larvae and adults with consistent individual sizes, putting the larvae and adults into a suspension with the concentration of test spores, soaking the larvae and adults for 30 s, picking out, putting the larvae and adults on filter paper, absorbing excessive water, transferring the larvae and adults into a culture dish (d =30 cm) with a wet filter paper sheet at the bottom of the dish, and putting sufficient fresh litchi and longan tender leaves into the culture dish for eating. The treated insects are placed in a climatic chamber for feeding (25 +/-1 ℃, RH 80% +/-5%). Each 10 larvae and adults are treated, the treatment is repeated for 3 times, observation is started after inoculation is carried out for 24 hours, observation is continuously carried out for 6 days, the death number of the larvae is recorded every day, and the dead larvae are subjected to moisture preservation observation to determine whether the larvae are killed by the susceptible bacteria. And recording the morbidity of the lygus sinensis.

As can be seen from FIG. 5, after 3 d of culture, both lygus sinensis larvae and adults can be infected by the strain LZ2-1 a. In the initial stage of fungus infection, the eating behavior and the external form of the insect body of the stinkbug are not different from those of the healthy insect body. After 2 days of inoculation, feeding of the larvae is obviously reduced or stopped, and part of the larvae die. After 4 days of inoculation, the larvae and adults of the lygus sinensis all have beauveria bassiana to infect the lethal individual. After 6 days of inoculation, the surface of the susceptible polypide is covered with a large number of hyphae and conidia, and the conidia are white (figure 5). Measurement of 1X 10⁶The viable count of spore suspension is about 3.19X 10⁵cfu/mL. The concentration of inoculated spores is 1X 10⁴、1×10⁵And control worms did not develop disease.

In summary, the present invention relates to Beauveria bassiana (balsamo) VuilleminBeauveria bassiana) The strain LZ2-1a can efficiently kill the lygus lucorum, has a good biological control effect, does not cause environmental pollution, and can replace pesticides to control the lygus lucorum.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

SEQ ID NO.1

GCCCTCGGGCTTACTCCCTACCCTTCTGTGACCTACCTATCGTTGCTTCGGCGGACTCGCCCCAGCCCGGACGCGGACTGGACCAGCGGCCCGCCGGGGACCTCAAACTCTTGTATTCCAGCATCTTCTGAATACGCCGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATCCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGACCTCCCCTTGGGGAGGTCGGCGTTGGGGACCGGCAGCACACCGCCGGCCCTGAAATGGAGTGGCGGCCCGTCCGCGGCGACCTCTGCGTAGTAATACAGCTCGCACCGGAACCCCGACGCGGCCACGCCGTAAAACACCCAACTTCTGAACGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAAAAGGCGGGAGGAACCACCTAATACCTCT

Sequence listing

<110> Changjiang college of teachers and schools

<120> beauveria bassiana LZ2-1a and application thereof

<130> FA1606

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 555

<212> DNA

<213> Beauveria bassiana (Beauveria bassiana)

<400> 1

gccctcgggc ttactcccta cccttctgtg acctacctat cgttgcttcg gcggactcgc 60

cccagcccgg acgcggactg gaccagcggc ccgccgggga cctcaaactc ttgtattcca 120

gcatcttctg aatacgccgc aaggcaaaac aaatgaatca aaactttcaa caacggatct 180

cttggctctg gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa 240

tccagtgaat catcgaatct ttgaacgcac attgcgcccg ccagcattct ggcgggcatg 300

cctgttcgag cgtcatttca accctcgacc tccccttggg gaggtcggcg ttggggaccg 360

gcagcacacc gccggccctg aaatggagtg gcggcccgtc cgcggcgacc tctgcgtagt 420

aatacagctc gcaccggaac cccgacgcgg ccacgccgta aaacacccaa cttctgaacg 480

ttgacctcga atcaggtagg actacccgct gaacttaagc atatcaaaag gcgggaggaa 540

ccacctaata cctct 555

Claims

1. The beauveria bassiana LZ2-1a is characterized in that: the strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M20211576 at 12 months and 9 days in 2021.

2. The use of beauveria bassiana LZ2-1a as claimed in claim 1 for controlling pests of fruit trees.

3. Use according to claim 2, characterized in that: the fruit tree pest is lygus lucorum.

4. A biological control agent is characterized in that: the active ingredient of the biocontrol microbial inoculum is spore suspension prepared from the beauveria bassiana LZ2-1a strain as claimed in claim 1.

5. The biocontrol microbial inoculum of claim 4 wherein: the viable count of the beauveria bassiana strain LZ2-1a in the spore suspension is not less than 3 x 10⁵ cfu/mL。

6. A method of preparing a spore suspension, comprising: the method comprises the following steps:

activating the beauveria bassiana strain LZ2-1a of claim 1 on a PDA plate, and then resuspending with a sterile PD liquid culture medium to obtain spore and hypha suspension;

7. The method of claim 6, wherein: wherein the shaking culture is carried out for 48-72 h at the temperature of 25-30 ℃ at the speed of 100-250 rpm, and comprises the following steps:

the culture was carried out at 25 ℃ for 60 hours at 200 rpm with shaking.