CN103160442A - Paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri - Google Patents
Paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri Download PDFInfo
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Abstract
The invention relates to a paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri and belongs to the technical field of the microorganism. The strain is paecilomyceslilacinus ZJPL08 which is registered and conserved in China General Microbiological Culture Collection Center on March 15, 2013, and the conservation number of the strain is CGMCC No. 7318. The paecilomyceslilacinus ZJPL08 provided by the invention has stronger pathogenicity for diaphorina citri and can be developed into a biocontrol agent for biological prevention and treatment of diaphorina citri; and compared with the present chemical agent for preventing and treating diaphorina citri, the paecilomyceslilacinus ZJPL08 has the characteristics of high efficiency, low toxicity, low residue, no pollution and not easy creation of resistance to drugs. The strain ZJPL08 has stronger acting effect on diaphorina citri than other paecilomyceslilacinus strains; the strain ZJPL08 is quick to cultivation, high in sporulation quantity, high in germination rate of spores and easy for preparation, and has excellent market application prospect.
Description
Technical field
The present invention relates to a strain to the Paecilomyces lilacinus of the strong virulence of oranges and tangerines wood louse tool (
paecilomyces lilacinus) bacterial strain, belong to microbial technology field.
Background technology
The oranges and tangerines wood louse (
diaphorina citri) belong to Homoptera (Homoptera), Psyllidae (Chermidae), be the primary pest of the rutaceae young sprout phases such as oranges and tangerines, lemon, Calusena lansium, Leaf and twig of Common Jasminorange, matrimony vine, be also the unique natural entomophila of propagating Citrus Huanglongbing pathogen.Citrus Huanglongbing pathogen is the most difficult as control on current Orange Producing, threaten maximum a kind of destructive disease, and the stability and development of Citrus Industry has been brought to serious threat.But at present this disease is not yet obtained to the effectively preventing medicament, the integrated control that the control oranges and tangerines wood louse of take is the master is the main way of current these disease prevention and control.Therefore, strengthen the control to the oranges and tangerines wood louse, all have great importance for controlling the harm to oranges and tangerines of the popular of Citrus Huanglongbing pathogen or reduction polypide itself.
Anti-Zhiduo County for the oranges and tangerines wood louse adopts Chemical control methods at present, yet the frequent use of chemical pesticide has caused pesticide residue, environmental pollution, species diversity to destroy and the oranges and tangerines wood louse problems such as develop immunity to drugs, and biological pesticide is efficient with it, low toxicity, low residue, pollution-free, be difficult for developing immunity to drugs and the raw material characteristic such as be easy to get be it is believed that it is that the ideal of following chemical pesticide substitutes medicament.Therefore, utilize biological pesticide control oranges and tangerines wood louse also to cause gradually people's attention.At present, both at home and abroad the investigator has carried out many good tries utilizing aspect the control oranges and tangerines wood louses such as plant milk extract, Natural Enemies of Insects, also carrying out certain research aspect oranges and tangerines wood louse entomogenous fungi, as the fungi that the oranges and tangerines wood louse is had to a pathogenic effect of having reported at present have lemon shape by the hair spore (
hirsutella citriformis), paecilomyces varioti (
paecilomyces varioti), paecilomyces fumosoroseus (
paecilomyces fumosoroseus), beauveria bassiana (
beauveria bassiana), aphid bamboo shoot top spore mould (
acrostalagmus aphidium), fusarium culmorum (
fusarium culmorum) and Verticillium lecanii (
lecanicillium lecanii) etc.The current research many places for oranges and tangerines wood louse biocontrol microorganisms are in laboratory stage, and the example that utilizes Biological agents successfully to prevent and treat the oranges and tangerines wood louse in agriculture production not yet has been reported.Therefore, the current further investigation for the further separation screening of oranges and tangerines wood louse entomogenous fungi and biotic potential, for promoting carrying out and having great importance smoothly of oranges and tangerines wood louse biological control in the future.
Paecilomyces lilacinus (
paecilomyces lilacinus) be a kind of important biocontrol fungi, research find this bacterium except the various plants nematode is had stronger pathogenic, also can parasitic lychee stink-bug, the various insects such as leafhopper, brown paddy plant hopper, termite, weevil and cluster caterpillar, in addition, this bacterium has certain antagonistic action to various plants pathogenic bacterias such as fusarium graminearum, cucumber anthracnose and cotton-wilt fusariums, and its meta-bolites also can promote seed germination, promotion root system of plant and plant strain growth etc., and the several functions enzyme of its generation also has certain degradation of pesticide effect.At present, the Paecilomyces lilacinus Biological agents for control root knot nematode etc. of the existing marketization.Yet, for Paecilomyces lilacinus, can parasitize the phenomenon of oranges and tangerines wood louse and utilize the research of Paecilomyces lilacinus control oranges and tangerines wood louse not yet to have been reported at home and abroad.The Paecilomyces lilacinus bacterial strain that the oranges and tangerines wood louse is had to strong virulence provided by the present invention, be that the useful of current oranges and tangerines wood louse Resources of Entomogenous Fungi supplemented, and has the potential that is developed to oranges and tangerines wood louse Biological agents.
Summary of the invention
The object of the present invention is to provide the Paecilomyces lilacinus bacterial strain of a strain to the strong virulence of oranges and tangerines wood louse tool, this bacterial strain has the potential that is developed to biological pesticide, has good market application foreground.
The objective of the invention is to be achieved through the following technical solutions:
Paecilomyces lilacinus bacterial strain provided by the present invention be Paecilomyces lilacinus (
paecilomyces lilacinus) ZJPL08, on March 15th, 2013, at China Committee for Culture Collection of Microorganisms's common micro-organisms center, registered preservation, deposit number is CGMCC No. 7318.
The present invention also provides the application of described Paecilomyces lilacinus bacterial strain in the prevention and control Citrus Huanglongbing pathogen, and the application in the biological pesticide for the preparation of control oranges and tangerines wood louse, and the conidial suspension of bacterial strain ZJPL08 has strong virulence to the oranges and tangerines wood louse.
Described Paecilomyces lilacinus (
paecilomyces lilacinus) bacterial strain ZJPL08 is that the oranges and tangerines wood louse polypide that gathers from the orangery of City of Taizhou Huangyan District separates and obtains, the morphological specificity of this bacterial strain, cultural characters, ITS1-5.8S rDNA-ITS2 sequence and bacterium classification result are as follows:
1, the morphological specificity of bacterial strain ZJPL08: bacterial strain ZJPL08 is at potato dextrose agar (PDA) substratum well-grown, rounded, the protuberance of bacterium colony on the PDA flat board, and mycelia is finer and close, and surface is without secretory product; The Initial stage of culture colony colour is white, and after the generation spore, colony colour is shown as lavender, and conidium is the opaque shape.Along with increasing of sporulation quantity, colony colour is deepened gradually, and to late stage of culture, colony colour becomes intense violet color.At the optical microphotograph Microscopic observation, bacterial strain ZJPL08 conidiophore list is given birth to or is verticillate, and bottle metulae section column or ampuliform, upwards become elongated tubular product, 7.5 ~ 9 * 1.8 ~ 3 μ m.Conidium is unicellular, avette or fusiform, and colourless to yellow, 1.5 ~ 2.8 * 1.3 ~ 2.5 μ m are catenation on sporophore.
2, the biological characteristics of bacterial strain ZJPL08: bacterial strain ZJPL08 all can grow on the PDA substratum under the temperature condition of 15 ℃ ~ 35 ℃, but differing temps has remarkably influenced to the growth of bacterial strain.In the time of 25 ℃ ~ 30 ℃, mycelial growth rate, apparently higher than other temperature, is the suitable growth temperature of bacterial strain ZJPL08.And under 15 ℃ and following low temperature or 35 ℃ and above high temperature, the mycelial growth of bacterial strain ZJPL08 is slow, sporulation quantity reduces.Under full exposure, full dark and 12 h illumination/12 h dark conditions, bacterial strain ZJPL08 is equal well-grown on the PDA substratum, and the length of light application time is not obvious to the growth effect of bacterial strain ZJPL08.After inoculation, the conidium output of 1 d bacterial strain ZJPL08 in potato sucrose (PS) liquid nutrient medium is apparently higher than potato glucose (PD) liquid nutrient medium, Sa Shi glucose yeast medicinal extract (SDY) liquid nutrient medium, Cha Shi (CD) liquid nutrient medium and malt extract (ME) liquid nutrient medium.7 d after inoculation, the mycelial yield of bacterial strain ZJPL08 in the PD liquid nutrient medium is the highest, and the mycelial yield in the PS substratum is lower.The conidium of bacterial strain ZJPL08 all can sprout under the temperature condition of 15 ℃ ~ 35 ℃, and its suitable Germination Temperature is 25 ℃ ~ 30 ℃, and the low temperature below 15 ℃ and the high temperature spore more than 35 ℃ all are difficult to sprout.Prolongation in time of spore germination rate and raising, after cultivating 9 h, the conidia germination rate of 30 ℃ of lower bacterial strain ZJPL08 reaches 99.5%.
3, the ITS1-5.8S rDNA-ITS2 sequence of bacterial strain ZJPL08: shown in the ITS1-5.8S rDNA-ITS2 sequence SEQ ID NO.1 of order-checking obtained strains ZJPL08, utilize NCBI Blast compare of analysis to find, Paecilomyces lilacinus in this sequence and GenBank (
paecilomyces lilacinus) the corresponding sequence similarity of a plurality of isolates (as JN650588, FR751342, HM439952, HM032028, EU553316 etc.) up to 100%, with the similarity of 3 control strains (ACCC30640, ACCC32480 and ACCC31532) of Paecilomyces lilacinus purchased from Chinese agriculture microbial resources storehouse also up to 99.8%.
According to the above morphological specificity about bacterial strain ZJPL08, biological characteristics and ITS1-5.8S rDNA-ITS2 sequence signature, by bacterial strain ZJPL08 be accredited as hyphomycetales (Hyphomycetales) paecilomyces (
paecilomyces) Paecilomyces lilacinus (
paecilomyces lilacinus).
Compared with prior art, advantage of the present invention and beneficial effect are: (1) there is no the report that infects the oranges and tangerines wood louse about Paecilomyces lilacinus at present both at home and abroad, and bacterial strain ZJPL08 of the present invention to be the oranges and tangerines wood louse polypide of being infected from City of Taizhou Huangyan District citrus orchard separate obtains, with the Paecilomyces lilacinus bacterial strain separated from other base thing, compare, Paecilomyces lilacinus ZJPL08 of the present invention has stronger virulence to the oranges and tangerines wood louse; (2) Paecilomyces lilacinus ZJPL08 of the present invention is a kind of insect pathogenic fungus, can be developed to Biological agents for the biological control to the oranges and tangerines wood louse, prevent and treat more at present the chemical agent of oranges and tangerines wood louse and compare, there is efficient, low toxicity, low residue, pollution-free, the feature that is difficult for developing immunity to drugs; (3) conidial suspension of Paecilomyces lilacinus ZJPL08 of the present invention has strong virulence to the oranges and tangerines wood louse, the LC of the 3rd d after inoculation oranges and tangerines wood louse
50be 5.77 * 10
9spores/mL, concentration is 1.0 * 10
8the LT of the spore suspension of spores/mL
50be only 3.1 d.Bacterial strain ZJPL08 has stronger action effect than other Paecilomyces lilacinus bacterial strains to the oranges and tangerines wood louse, has good market application foreground.(4) bacterial strain ZJPL08 cultivates soon, and sporulation quantity is large, and spore germination rate is high, is easy to preparation.
The accompanying drawing explanation
Fig. 1 be Paecilomyces lilacinus (
paecilomyces lilacinus) cultural characteristic of bacterial strain ZJPL08 on the PDA substratum.
Fig. 2 be Paecilomyces lilacinus (
paecilomyces lilacinus) bacterial strain ZJPL08 is to the situation that infects of oranges and tangerines wood louse.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this.
A kind of Paecilomyces lilacinus to the strong virulence of oranges and tangerines wood louse tool, bacterial classification called after ZJPL08, Classification And Nomenclature: Paecilomyces lilacinus (
paecilomyces lilacinus), its deposit number: CGMCC No. 7318, preservation date: on March 15th, 2013, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment 1: the Isolation and screening of insect pathogenic bacteria
1, the separation and purification of germ: in City of Taizhou Huangyan District citrus orchard, gather oranges and tangerines wood louse worm corpse, taking back laboratory is put in it in 70% ethanol and soaks 30 s, soak 3 min through 0.1% mercuric chloride again, finally use aseptic water washing 3 times, blot the moisture on polypide with aseptic filter paper, then (compound method is: take 200 g potatos it to be put in to potato dextrose agar (PDA) substratum, clean the peeling chopping, add water 1000 mL and boil half hour, filtered through gauze, add again 20 g glucose and 20 g agar, filtered through gauze while hot after fully dissolving, divide and be filled in Erlenmeyer flask or glass test tube, 121 ℃, autoclaving 20 min) in, be placed in 28 ℃ of constant temperature culture of microbiological incubator, after growing mycelia on every side to polypide, the picking mycelia goes on new PDA flat board cultivates (repeating this operation 2 ~ 3 times), continue to cultivate 5 d left and right, treat on bacterium colony to produce conidium, be prepared into mitogenetic spore suspension with sterilized water wash-out conidium, then with reference to the dilution method of purification described in " planting the disease research method " (Fang Zhongda. plant disease research method (third edition) [M]. Beijing: Chinese agriculture press, 1998:122-140) germ is carried out to the monospore separation.The bacterial strain finally separation and purification obtained is stored in the PDA slant medium, after cultivating 3 ~ 4 d, is placed in 4 ℃ of Refrigerator stores.
2, the screening of pathogenic bacterium: the inoculation that above-mentioned separation and purification is preserved is to the PDA flat board, behind 28 ℃ of constant temperature culture, 10 d left and right, conidium to produce on sterilized water wash-out bacterium colony, prepare conidial suspension, and adding wherein appropriate tween-80 to final concentration is 0.1%.Being taken at 30 healthy oranges and tangerines wood louse adults raising in greenhouse (this specification sheets embodiment all take oranges and tangerines wood louse adult be tested object) is immersed in containing 10 ~ 15 s in the conidial suspension of 0.1% tween-80, picking still can be freely movable after processing the oranges and tangerines wood louse be placed on the Leaf and twig of Common Jasminorange blade of culture dish and (be covered with 2 layers of filter paper by the moistening mistake of sterilized water bottom culture dish, place a spray of the Leaf and twig of Common Jasminorange with 10 left and right blades on it, the branch bottom is with aseptic water-moistened absorbent cotton parcel), then be put in (h illumination every days 14/10 h dark in 28 ℃ of illumination boxs, humidity is more than 95%) cultivate, set up containing the sterilized water of 0.1% tween-80 simultaneously and process the oranges and tangerines wood louse for contrast, observe germ infecting and lethal situation the oranges and tangerines wood louse after cultivating 7 d.Separate germ by the method in step 1 in oranges and tangerines wood louse polypide from processing again afterwards, then morphological specificity and the cultural characteristic of the germ of the germ separated and initial inoculation.
By above operation, filter out meet the He Shi of section rule to the pathogenic bacterial strain of oranges and tangerines wood louse tool, get rid of the saprophytic microorganism be separated to from oranges and tangerines wood louse polypide etc. non-pathogenic bacteria.By screening, obtain 1 strain and the oranges and tangerines wood louse is there is to the bacterial strain of virulence, by its name ZJPL08.
3, the rejuvenation of bacterial strain: the conidial suspension for preparing the above-mentioned bacterial strain ZJPL08 to oranges and tangerines wood louse tool virulence screened, be inoculated on healthy oranges and tangerines wood louse polypide, and then from the morbidity oranges and tangerines wood louse polypide the separation and purification germ, concrete operation method is the same.Finally obtain the bacterial strain ZJPL08 of separation and purification.
Embodiment 2: the evaluation of bacterial strain ZJPL08
1, the morphological specificity of bacterial strain ZJPL08: the bacterial strain ZJPL08 of above-mentioned separation and purification is inoculated into to PDA culture medium flat plate central authorities, is placed in 28 ℃ of constant temperature culture, observe the colony growth situation every day and record colony colour and form.Bacterial strain ZJPL08 is inoculated into to another PDA culture medium flat plate central authorities, cover glass after sterilizing (1 cm * 1 cm) oblique cutting is entered in the vaccination substratum that approximately 1 cm left and right is located simultaneously, be placed in 28 ℃ of constant temperature culture, 5 d left and right after mycelia climbs on cover glass, take out mycelia and spore shape that cover glass is placed in optical microphotograph Microscopic observation bacterial strain ZJPL08.
Fig. 1 shows the morphological specificity after bacterial strain ZJPL08 cultivates 7 d on the PDA substratum, and visible bacterial strain ZJPL08 bacterium colony is rounded, protuberance, and mycelia is finer and close, and surface is without secretory product; The Initial stage of culture colony colour is white, and after the generation spore, colony colour is shown as lavender, and conidium is the opaque shape.Along with increasing of sporulation quantity, colony colour is deepened gradually, and to late stage of culture, colony colour becomes intense violet color.Can be observed bacterial strain ZJPL08 conidiophore list at opticmicroscope living or verticillate, bottle metulae section column or ampuliform, upwards become elongated tubular product, 7.5 ~ 9 * 1.8 ~ 3 μ m.Conidium is unicellular, avette or fusiform, and colourless to yellow, 1.5 ~ 2.8 * 1.3 ~ 2.5 μ m are catenation on sporophore.
2, the sequential analysis of bacterial strain ZJPL08: bacterial strain ZJPL08 is seeded to the PDA culture medium flat plate, be placed in 28 ℃ of constant temperature culture to bacterium colonies and cover with whole culture dish, with the hypha,hyphae of growing on aseptic spoon scraping PDA flat board in aseptic mortar, add liquid nitrogen grinding to Powdered, adopt improved method of CTAB to extract total DNA of bacterial strain ZJPL08, the DNA of purifying of take is template, with fungi universal primer ITS5(5 '-GGAAGTAAAAGTCGTAACAAGG-3 ') and ITS4(5 '-TCCTCCGCTTATTGATATGC-3 ') carry out pcr amplification (concrete operation method reference: deer Lian Ming etc. polymorphism and the Phylogenetic Analysis of Citrus Huanglongbing pathogen bacterium ribosomal protein gene. journal of Zhejiang university, 2011, 37 (2): 125-132).The PCR product, through agarose gel electrophoresis, utilizes gel to reclaim after test kit reclaims and delivers company's order-checking.
The ITS1-5.8S rDNA-ITS2 sequence of order-checking obtained strains ZJPL08, as shown in SEQ ID NO.1, utilizes NCBI Blast compare of analysis to find, Paecilomyces lilacinus in this sequence and GenBank (
paecilomyces lilacinus) the sequence similarity of a plurality of isolates (as JN650588, FR751342, HM439952, HM032028, EU553316 etc.) up to 100%, with the similarity of 3 control strains (ACCC30640, ACCC32480 and ACCC31532) of Paecilomyces lilacinus purchased from Chinese agriculture microbial resources storehouse also up to 99.8%.
According to the above morphological specificity about bacterial strain ZJPL08 and ITS1-5.8S rDNA-ITS2 sequence signature, bacterial strain ZJPL08 can be accredited as hyphomycetales (Hyphomycetales) paecilomyces (
paecilomyces) Paecilomyces lilacinus (
paecilomyces lilacinus).
Embodiment 3: the biological characteristics of bacterial strain ZJPL08
1, the impact of differing temps on bacterial strain ZJPL08 growth: with aseptic punch tool, in the periphery of bacterial colonies of the bacterial strain ZJPL08 that cultivates 7 d left and right on the PDA substratum, buy the bacterium cake of getting 5 mm sizes, then the bacterium cake is placed in respectively to dull and stereotyped (diameter is 9 cm) central authorities of PDA of several fresh preparations, respectively at being inverted in 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃ of illumination boxs, cultivate, each temperature is divided into 3 repetitions.Cultivate 14 d, every 24 h observe the colony growth situation and adopt the right-angled intersection method to record colony diameter.
The results are shown in Table 1, can find out, bacterial strain ZJPL08 all can grow on the PDA substratum under the temperature condition of 15 ℃ ~ 35 ℃, wherein when 25 ℃ and 30 ℃ mycelial growth rate apparently higher than other temperature, cultivate 14 d bacterium colonies and almost can cover with whole culture dish, and colony growth rate obviously slows down 35 ℃ the time, under 10 ℃ and 5 ℃ of low temperature, bacterium colony is grown hardly.Therefore, 25 ℃ ~ 30 ℃ suitable growth temperatures that are bacterial strain ZJPL08.
The growing state of bacterial strain ZJPL08 under the different number of days differing temps after table 1 inoculation
2, the impact of different illumination conditions on bacterial strain ZJPL08 growth: with aseptic punch tool, in the periphery of bacterial colonies of the bacterial strain ZJPL08 that cultivates 7 d left and right on the PDA substratum, buy the bacterium cake of getting 5 mm sizes, then the bacterium cake is placed in respectively to dull and stereotyped (diameter is 9 cm) central authorities of PDA of several fresh preparations, in 28 ℃ of incubators, in every day 0, under h, 12 h and 24 h illumination conditions, cultivate respectively, cultured continuously 14 d, observe and record colony diameter every 24 h.
Result, as table 2, can find out, bacterial strain ZJPL08 equal well-grown under different illumination conditions, and light application time length is not remarkable to the growth effect of bacterial strain ZJPL08.
The growing state of bacterial strain ZJPL08 under the different number of days different illumination conditions after table 2 inoculation
3, the impact of different culture media on bacterial strain ZJPL08 sporulation quantity and mycelial yield: prepare following substratum: (compound method is potato sucrose (PS) liquid nutrient medium: take 200 g potatos, clean the peeling chopping, add water 1000 mL and boil half hour, filtered through gauze, add again 20 g sucrose and 20 g agar, filtered through gauze while hot after fully dissolving, divide and be filled in Erlenmeyer flask or glass test tube, 121 ℃, autoclaving 20 min), (compound method is potato glucose (PD) liquid nutrient medium: take 200 g potatos, clean the peeling chopping, add water 1000 mL and boil half hour, filtered through gauze, add again 20 g glucose and 20 g agar, filtered through gauze while hot after fully dissolving, divide and be filled in Erlenmeyer flask or glass test tube, 121 ℃, autoclaving 20 min), Sa Shi glucose yeast medicinal extract (SDY) liquid nutrient medium (yeast extract 10 g, glucose 40 g, peptone 10 g, agar 20 g, pH6.0), Cha Shi (CD) liquid nutrient medium (SODIUMNITRATE 3 g, dipotassium hydrogen phosphate 1 g, sal epsom (MgSO
4 .7H
2o) 0.5 g, Repone K 0.5 g, ferrous sulfate 0.01 g, sucrose 30 g, distilled water 1000 mL) and malt extract (ME) liquid nutrient medium (Fructus Hordei Germinatus soaks powder 15 g, distilled water 1000 mL, pH4.7).The bacterium piece of the bacterial strain ZJPL08 of picking fresh culture is seeded in the Erlenmeyer flask that SDY nutrient solution (120 mL/ bottle) is housed, and puts shaking culture on 28 ℃ of shaking tables (150 rpm/min) 2 d, as seed culture fluid.Then get respectively 1 mL seed culture fluid and join (120 mL/ bottle) in the Erlenmeyer flask that PD, PS, SDY, CD and ME nutrient solution are housed, 3 repetitions are established in each processing, put 28 ℃ of shaking table shaking culture (150 rpm/min), 6 d, every 24 h nutrient solution that takes a morsel drops on blood counting chamber at the optical microphotograph Microscopic observation and records spore quantity, then calculates the concentration of each substratum miospore.
Measurement result, as table 3, can be found out 1 d after inoculation, and the spore output of bacterial strain ZJPL08 in the ME substratum is the highest, and, after 1 d, the spore output in the PS substratum is all higher than other substratum.In PD, PS and CD substratum, bacterial strain ZJPL08 the 3rd d sporulation quantity after inoculation peaks, on a declining curve gradually afterwards.Therefore, the best that the PS substratum is bacterial strain ZJPL08 is produced the spore substratum.The 7th d after inoculation, to after each medium centrifugal, collect bacterial sediment, measure the mycelia dry weight after dry in thermostatic drying chamber, result shows that the mycelial yield of bacterial strain ZJPL08 in the PD substratum is the highest, be 32.15 mg/mL, output in the SDY substratum is minimum is 3.8 mg/mL, and at sporulation quantity, the mycelial yield in the highest PS substratum is also relatively low, is only 4.7 mg/L.
The spore concentration of bacterial strain ZJPL08 in the different time different culture media after table 3 inoculation
4, differing temps and the time impact on bacterial strain ZJPL08 spore germination rate: with PD liquid nutrient medium wash-out, in the conidium of the upper bacterial strain ZJPL08 cultivated of PDA, be mixed with conidial suspension, get respectively 30 μ L spore suspensions and drop to the central authorities of each aseptic slide glass, then be placed in 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, in 35 ℃ and 40 ℃ of incubators, cultivate, each temperature condition is established 3 repetitions, count under the microscope 400 ~ 600 spores after 8 h, the germ tube length of take surpasses spore length 1/2 and is considered as spore germination as standard, statistics spore germination number is also calculated spore germination rate.As mentioned above the spore suspension of bacterial strain ZJPL08 is dropped to slide glass central authorities, then be placed in 28 ℃ of constant incubators and cultivate, every 1 h observes and records the spore count of sprouting, and calculates spore germination rate.
Result, as table 4 and table 5, can find out, the conidium of bacterial strain ZJPL08 all can sprout under the temperature condition of 15 ℃ ~ 35 ℃, and wherein the highest at 30 ℃ of lower spore germination rates, 25 ℃ are taken second place, and the low temperature below 5 ℃ and the high temperature spore more than 35 ℃ all are difficult to sprout.Under 28 ℃ of conditions, front 3 h of the conidium of bacterial strain ZJPL08 sprout hardly, and after 5 h, germination rate significantly rises, to 9 h germination rates nearly 100%.Therefore, 25 ℃ ~ 30 ℃ suitable product spore temperature that are bacterial strain ZJPL08.
The conidia germination rate of bacterial strain ZJPL08 under table 4 condition of different temperatures
The conidia germination rate of bacterial strain ZJPL08 under the different incubation times of table 5
Embodiment 4: the Pathogenicity of bacterial strain ZJPL08 to the oranges and tangerines wood louse
1, median lethal concentration(LC&-{50}) and the median lethal time of bacterial strain ZJPL08 to the oranges and tangerines wood louse: bacterial strain ZJPL08 is seeded in the PS liquid nutrient medium, after 28 ℃ of constant-temperature table shaking culture (150 rpm/min), 3 d, the bacterium liquid of cultivating removes by filter mycelium via sterile gauze, collect conidial suspension, carry out serial dilution with sterilized water, obtain concentration and be respectively 1.0 * 10
8spores/mL, 1.0 * 10
7spores/mL, 1.0 * 10
6spores/mL, 1.0 * 10
5spores/mL, 1.0 * 10
4the conidial suspension of the bacterial strain ZJPL08 of spores/mL, and to add wherein appropriate tween-80 to final concentration be 0.1%.By embodiment 1 is described, healthy oranges and tangerines wood louse is put in to processing containing infiltration in the conidial suspension of 0.1% tween-80 of above-mentioned each concentration, be put in afterwards (h illumination every days 14/10 h dark in 28 ℃ of illumination boxs, humidity is more than 95%) cultivate, set up the sterilized water processing oranges and tangerines wood louse of take containing 0.1% tween-80 is contrast simultaneously.3 repetitions are established in above each processing, 30 oranges and tangerines wood louses of each re-treatment.Start to observe every day germ infecting and lethal situation the oranges and tangerines wood louse from cultivating the 2nd d, record oranges and tangerines wood louse mortality, be calculated as follows mortality ratio and corrected mortality, the data obtained carries out regression analysis by SAS software, obtain regression equation and correlation coefficient r, calculate median lethal concentration(LC&-{50}) (LC
50) and median lethal time (LT
50).
The results are shown in Table 6 and table 7.Can find out, the conidial suspension concentration of bacterial strain ZJPL08 is higher, better to the pathogenic effect of oranges and tangerines wood louse.Under the same concentration condition, after inoculation, incubation time is longer, and the mortality ratio of oranges and tangerines wood louse is higher.The LC of each time period after inoculation
50show notable difference, as the 3rd d after inoculating, LC
50be 5.77 * 10
9spores/mL, the 7th d is only 2.20 * 10
4spores/mL.Conidial suspension concentration is higher, the LT of bacterial strain ZJPL08 to the oranges and tangerines wood louse
50also shorter, as concentration is 1.0 * 10
4the LT of the conidial suspension of spores/mL to the oranges and tangerines wood louse
50be 7.15 d, and concentration is 1.0 * 10
8the LT of spore suspension
50be only 3.1 d, oranges and tangerines wood louse the 6th d mortality ratio nearly 100% after cultivation of processing with this concentration spore suspension.
The median lethal concentration(LC&-{50}) of table 6 bacterial strain ZJPL08 to the oranges and tangerines wood louse
The median lethal time of table 7 bacterial strain ZJPL08 to the oranges and tangerines wood louse
2, the control application of bacterial strain ZJPL08 to the oranges and tangerines wood louse: bacterial strain ZJPL08 is seeded in the PS liquid nutrient medium, after 28 ℃ of constant-temperature table shaking culture (150 rpm/min), 3 d, the bacterium liquid of cultivating removes by filter mycelium via sterile gauze, collect conidial suspension, the sterilized water of take is diluted to its concentration as 1.0 * 10
8spores/mL, adding appropriate tween-80 to concentration is 0.1%.Get healthy oranges and tangerines wood louse and be put in (placement 1 strain Leaf and twig of Common Jasminorange seedling in 1 dependent insect cage on the potted plant Leaf and twig of Common Jasminorange seedling in dependent insect cage, put 30 of oranges and tangerines wood louses on 1 strain Leaf and twig of Common Jasminorange seedling, if 5 repetitions), the spore suspension of the above-mentioned bacterial strain ZJPL08 prepared is packed into after miniaturised nebuliser, to the oranges and tangerines wood louse even spraying on Leaf and twig of Common Jasminorange, fully moistening to the insect body surface.With the Paecilomyces lilacinus bacterial strain ACCC30640(purchased from Chinese agriculture microbial resources storehouse, separate from root-knot nematode egg) bacterial strain as a comparison, as stated above preparation with bacterial strain ZJPL08 with the spore suspension of concentration and process the oranges and tangerines wood louse.In addition, process the oranges and tangerines wood louse in contrast with the sterilized water containing 0.1% tween-80.Above-mentioned respectively finish dealing with after, dependent insect cage is put into to glasshouse, controlling temperature in greenhouse is 25 ℃ ~ 30 ℃, humidity is more than 90%, h illumination every days 14/10 h dark.Fig. 2 shows and processes the infect situation of Paecilomyces lilacinus bacterial strain ZJPL08 to oranges and tangerines wood louse on Leaf and twig of Common Jasminorange after 9 d, and visible oranges and tangerines wood louse has been infected lethal by bacterial strain ZJPL08, lilac hypha,hyphae and the whole oranges and tangerines wood louse of conidium nearly cover polypide.Observe and record total borer population and the dead borer population of the above-mentioned oranges and tangerines wood louse that each is processed in the 9th d, calculate as follows mortality ratio and corrected mortality.
By calculating, learn, the 9th d after processing, take concentration as 1.0 * 10
8the corrected mortality of the oranges and tangerines wood louse that the conidial suspension of the Paecilomyces lilacinus bacterial strain ZJPL08 of spores/mL is processed is 91.58%, and take the corrected mortality of the oranges and tangerines wood louse that the conidial suspension of Paecilomyces lilacinus bacterial strain ACCC30640 of same concentration processes, is only 73.68%.Can find out, with the Paecilomyces lilacinus bacterial strain separated from root knot nematode, compare, separation provided by the invention has stronger action effect from the Paecilomyces lilacinus bacterial strain ZJPL08 of oranges and tangerines wood louse for the oranges and tangerines wood louse.
<110 > Zhejiang Citrus Research Institute
<120 > the Paecilomyces lilacinus bacterial strain of a strain to the strong virulence of oranges and tangerines wood louse tool
<160> 1
<170> PatentIn Version 3.3
<210> 1
<211> 509
<212> DNA
<213>Paecilomyces lilacinus (
paecilomyces lilacinus)
<400> 1
ccgagttata caactcccaa acccactgtg aaccttacct cagttgcctc ggcgggaacg 60
ccccggccgc ctgcccccgc gccggcgccg gacccaggcg cccgccgcag ggaccccaaa 120
ctctcttgca ttacgcccag cgggcggaat ttcttctctg agttgcacaa gcaaaaacaa 180
atgaatcaaa actttcaaca acggatctct tggttctggc atcgatgaag aacgcagcga 240
aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgaatcttt gaacgcacat 300
tgcgcccgcc agcattctgg cgggcatgcc tgttcgagcg tcatttcaac cctcgagccc 360
cccccggggg cctcggtgtt gggggacggc acaccagccg cccccgaaat gcagtggcga 420
ccccgccgca gcctcccctg cgtagtagca cacacctcgc accggagcgc ggaggcggtc 480
acgccgtaaa acgcccaact ttcttagag 509
Claims (3)
1. a Paecilomyces lilacinus strain, it is characterized in that this bacterial strain be Paecilomyces lilacinus (
paecilomyces lilacinus) ZJPL08, on March 15th, 2013, at China Committee for Culture Collection of Microorganisms's common micro-organisms center, registered preservation, deposit number is CGMCC No. 7318.
2. the application of Paecilomyces lilacinus bacterial strain as claimed in claim 1 in the prevention and control Citrus Huanglongbing pathogen.
3. the application of Paecilomyces lilacinus bacterial strain as claimed in claim 1 in the biological pesticide for the preparation of control oranges and tangerines wood louse.
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| CN103468579A (en) * | 2013-08-07 | 2013-12-25 | 浙江省柑桔研究所 | New lecanicillium bacteria genus fungi specie providing pathogenicity for diaphorina citri |
| CN103798012A (en) * | 2013-12-12 | 2014-05-21 | 广西大学 | Method for screening Candidatus Liberibacter resisting chemical through catharanthus roseus |
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