CN102851219A - Paecilomyces lilacinus and application thereof - Google Patents
Paecilomyces lilacinus and application thereof Download PDFInfo
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Abstract
The invention discloses paecilomyces lilacinus and an application thereof. The provided paecilomyces lilacinus refers to paecilomyces lilacinus E16 with a register number of CGMCC No.5951 in the common microorganism center of the China microorganism culture preservation management committee. According to the paecilomyces lilacinus E16, in potting experiments, the control effect on second-instar banana root-knot nematodes is 86.37%, the growth rate to banana root weight is 37.76%, the growth rate to banana plant height is 56.47%, the growth rate to overground parts of banana is 38.03%; and in area experiments, the control effect on banana root-knot nematodes is 83.03% and the growth rate to banana fruit is 28.05%.
Description
Technical field
The present invention relates to strain Paecilomyces lilacinus and an application thereof, the particularly application of Paecilomyces lilacinus in the tropical fruit tree root knot nematode disease such as prevention and control tropical and subtropical zone area banana and banana blight.
Background technology
Nemas is the Plant diseases that generally occurs in a kind of world wide, endangers 3000 various plants.The annual loss that causes because of nematode in the whole world is up to more than 1,000 hundred million dollars.In China, nearly all crops such as eelworm harm fruit tree, flower, vegetables, grain, oil, woods, tobacco, medicinal material become one of critical limitation factor in the agroforestry production.Banana is important tropical fruit, but the banana nematode generally occurs in China South China, brings out simultaneously the multiple soil-borne diseases such as blight, root rot and bacterial soft rot.The development of serious threat China banana industry.For a long time, mainly be to adopt chemical prevention to banana oxyuriasis in the production, these nematocides strong toxicities, contaminate environment, residual content is high, lack safe and effective preventions, so biological control is in widespread attention.
At present, Wang Jun and Huang Junsheng etc. have been separated to two strains the banana root knot nematode have been had pathogenic Paecilomyces lilacinus E7 bacterial strain (Wang Jun, Liu Lei, Li Chuandong, Liu Changyan, Huang Junsheng. Paecilomyces lilacinus E7 bacterial strain is to the pathogenic and biocontrol effect of banana root knot nematode. Chinese the 10th phase of plant protection guide .2008) and Paecilomyces lilacinus PL04 bacterial strain (Wang Jun, Huang Jun gives birth to *, Deng Guoping, Zhao Peijing, Yang Laying, Chen Xide. Paecilomyces lilacinus PL04 bacterial strain resistance and to the parasitization of banana root knot nematode. northwest agricultural journal .2009,18 (3): 263-267).Although, obtained that the banana root knot nematode is had pathogenic bacterial strain, also need separate the banana root knot nematode is had more highly pathogenic bacterial strain, with the development process of the GR that accelerates efficient stable.
Summary of the invention
A technical problem to be solved by this invention provides a strain Paecilomyces lilacinus.
Paecilomyces lilacinus provided by the present invention is Paecilomyces lilacinus (Paecilomyces lilacinus) E16, and it is numbered CGMCC No.5951 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.This bacterial classification has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 04 06th, 2012.
Paecilomyces lilacinus of the present invention (Paecilomyces lilacinus) E16 CGMCC No.5951 cultivates 7d 28 ℃ of Cha Shi substratum, and colony diameter is 40mm, and bacterium colony is very dry to be connected closely with substratum, is difficult for picking, and central authorities are incarnadine, is white on every side; Mycelia is the fine hair shape, and is long and thin.Conidiophore is the broom shape, and mycelia is longer, and conidium is oval, and bunchiness is the not chain of branch.
Another technical problem to be solved by this invention provides a kind of nematocides.
Nematocides provided by the present invention, its activeconstituents are above-mentioned Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951.
Another technical problem to be solved by this invention provide take above-mentioned Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 as activeconstituents following 1)-5) and in any product:
1) product of increase banana output;
2) increase the heavy product of Banana Root;
3) product of increase banana plant height;
4) product of increase banana over-ground part weight;
5) product of the Plant diseases that caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense) of control.
In one embodiment of the invention, described Plant diseases is specially banana blight.
E16CGMCCNo.5951 specifically can be conidium or/and mycelium for described Paecilomyces lilacinus (Paecilomyces lilacinus).
Described nematocides can be used for control by at least a insect pest of the plant that causes in Meloidogyne incognita (Meloidogyne incognita) and the pawl furrow root knot nematode (Meloidogyne Goeidi).
In one embodiment of the invention, described insect pest of the plant is specially the Banana Root Root-knot.
Paecilomyces lilacinus (Paecilomyces lilacinus) the E16 CGMCCNo.5951 spore suspension that experiment showed, 4 * 106cfu/mL is 39.43% to the Relative Parasitic rate of Meloidogyne incognita ovum; Paecilomyces lilacinus (Paecilomyces lilacinus) E16 CGMCC No.5951 solid fungicide, in potted plant experiment to 2 age the banana root knot nematode prevention effect be 86.37%, be 37.76% to the heavy rate of increase of Banana Root, rate of increase to the banana plant height is 56.47%, is 38.03% to the rate of increase of banana over-ground part; Prevention effect to the banana root knot nematode in the residential quarter experiment is 83.03%, is 28.05% to the stimulation ratio of banana.And Paecilomyces lilacinus (Paecilomyces lilacinus) E16 CGMCC No.5951 reaches 83.2% to the prevention effect of banana blight.
Biomaterial information:
Classification And Nomenclature: Paecilomyces lilacinus (Paecilomyces lilacinus)
Strain number: E16
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 04 06th, 2012
The preservation center numbering of registering on the books: CGMCC No.5951
Describe the present invention in detail below in conjunction with specific embodiment, these embodiment are used for understanding rather than restriction the present invention.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Paecilomyces lilacinus E7 bacterial strain (Wang Jun among the following embodiment; Liu Lei; Li Chuandong; Liu Changyan; Huang Junsheng. Paecilomyces lilacinus E7 bacterial strain is to the pathogenic and biocontrol effect of banana root knot nematode. Chinese the 10th phase of plant protection guide .2008), the public can obtain from Chinese Academy of Tropical Agricultural Sciences's environment and Plant Protection Institute.
Paecilomyces lilacinus PL04 bacterial strain (Wang Jun among the following embodiment; Huang Jun gives birth to *; Deng Guoping; Zhao Peijing; Yang Laying; Chen Xide. Paecilomyces lilacinus PL04 bacterial strain resistance and to the parasitization of banana root knot nematode. northwest agricultural journal .2009,18 (3): 263-267), the public can be from Chinese Academy of Tropical Agricultural Sciences's environment and Plant Protection Institute acquisition.
No. 4 physiological strains of Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense race 4 among the following embodiment; FOC4) B2 bacterial strain (FOC4-B2) (Qi Xingzhu; Yang Laying; Huang Jun gives birth to the cloning and expression analysis of 2 catalase genes of .FOC4. and the banana seedlings oxidative burst Study of China agronomy that causes circular 2012; 28 (15): 163-169), the public can obtain from Chinese Academy of Tropical Agricultural Sciences's environment and Plant Protection Institute.
The Isolation and Identification of embodiment 1, Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951
1, the separation of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951
Retransmit Tanaka's banana plant from Chinese Hainan Province banana planting ground selection wire parasitosis, gather the root soil sample, adopt the transparent circle sieve method in soil, to separate Paecilomyces varioti.Choose a certain amount of soil sample and place the triangular flask that is added with sterilized water and granulated glass sphere, 180r/min, 28 ℃ of concussion 1h allow soil sample fully break up, leave standstill 10min, be diluted to different dilution Soil Slurries with sterilized water again, get a certain amount of Soil Slurry and coat in the chitosan selective medium, cultivated 4～6 days for 28 ℃, picking list bacterium colony carries out the plate streaking purifying, and place on the slant medium and to cultivate 3 days, picking list bacterium colony carries out repeatedly purifying, obtains the E16 bacterial strain.
2, the evaluation of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951
1) identification of morphology
The streak inoculation of E16 bacterial strain on the Cha Shi substratum, is cultivated 7d for 28 ℃, observe colonial morphology.Other gets the bacterium colony of cultivation, suitably observes in microscopically after the dilution.The result shows that E16 bacterial strain colony diameter on the Cha Shi substratum is 40mm, and bacterium colony is very dry to be connected closely with substratum, is difficult for picking, and central authorities are incarnadine, is white on every side; Mycelia is the fine hair shape, and is long and thin.The morphological specificity that microscopically is observed: conidiophore is the broom shape, and mycelia is longer, and conidium is oval, and bunchiness is the not chain of branch.
2) Molecular Identification
The E16 bacterial strain is behind purifying repeatedly, streak culture on the Cha Shi substratum, single bacterium colony is carried out pcr amplification, amplified production is reclaimed test kit with gel reclaim purifying, measure to get the ITS sequence of Paecilomyces varioti E16 by precious biotechnology (Dalian) company limited, sequence in institute's calling sequence and the GenBank database is carried out BLAST to be analyzed, the result shows, that approach the most with this bacterial strain ITS sequence is Paecilomyces lilacinus (Paecilomyces lilacinus), its ITS sequence homology is 97%, in conjunction with the Morphological Characteristics of bacterial strain E16, be Paecilomyces lilacinus (Paecilomyces lilacinus) with this identification of strains.Wherein, the ITS sequence of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCCNo.5951 is shown in the sequence 1 in the sequence table.
Paecilomyces lilacinus (Paecilomyces lilacinus) E16 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 04 06th, 2012, it is numbered CGMCC No.5951 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.
3, the adverse-resistant characteristic of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951
Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 has stronger resistance, the optimal temperature of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 colony growth and product spore is 5～40 ℃, and optimum temperuture is 29 ℃; All can grow in the PDA substratum in pH value 4～13 scopes, its most suitable growth pH value is 7; Be under 0.05～0.25mol/L condition in NaCl concentration, NaCl has certain promoter action that stronger salt resistance ability is arranged to the growth of bacterial strain E16; Paraxin there is stronger tolerance.
Embodiment 2, Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 are to the parasitics of Meloidogyne incognita (tomato) ovum
1, the preparation of Meloidogyne incognita ovum grain suspension
To clean with the tomato old complaint of a large amount of Meloidogyne incognita (Meloidogyne incognita) root knots, picking egg capsule under anatomical lens, clorox table with 1% and the 3min that sterilizes, rear rinsed with sterile water 5 times is drawn broken egg capsule and is regulated with aqua sterilisa and make that the ovum grain is 100/mL in the suspension.
2, the preparation of Paecilomyces lilacinus spore suspension:
With Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 bacterial strain, Paecilomyces lilacinus E7 bacterial strain and Paecilomyces lilacinus PL04 bacterial strain respectively on the PDA substratum, 25 ℃ of lower cultivations 7d(days) after, with sterilized water the spore on the substratum is washed out, calculate spore concentration with the blood counting chamber counting, being mixed with concentration is 4 * 10
6The spore suspension of cfu/mL.
3, the Paecilomyces lilacinus spore suspension is on the impact of Meloidogyne incognita ovum hatching
Four processing are established in experiment, are respectively Paecilomyces lilacinus E16 group, Paecilomyces lilacinus E7 group, Paecilomyces lilacinus PL04 group and control group.
Paecilomyces lilacinus E16 group is processed as follows: adding 1mL ovum grain suspension through autoclaved culture dish, every ware adds 4mL Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 spore suspension (4 * 10
6Cfu/mL), 25 ℃, the microscopy next day of cultivating behind the 3d are added up hatching larva number in each ware.
Paecilomyces lilacinus E7 group is processed as follows: adding 1mL ovum grain suspension through autoclaved culture dish, every ware adds 4mL Paecilomyces lilacinus E7 bacterial strain spore suspension (4 * 10
6Cfu/mL), 25 ℃, the microscopy next day of cultivating behind the 3d are added up hatching larva number in each ware.
Paecilomyces lilacinus PL04 group is processed as follows: adding 1mL ovum grain suspension through autoclaved culture dish, every ware adds 4mL Paecilomyces lilacinus PL04 bacterial strain spore suspension (4 * 10
6Cfu/mL), 25 ℃, the microscopy next day of cultivating behind the 3d are added up hatching larva number in each ware.
Control group is processed as follows: adding 1mL ovum grain suspension through autoclaved culture dish, every ware adds the 4mL sterilized water, and 25 ℃, the microscopy next day of cultivating behind the 3d are added up hatching larva number in each ware.
Wherein, process each 3 repetition for above-mentioned four, each repeats to establish 3 culture dish.The 9th day, be calculated according to the following formula egg hatching rate and ovum Relative Parasitic rate, utilize Excel 2003 to carry out data processing:
Egg hatching rate (%)=[hatching larva number/(hatching larva number+do not hatch ovum grain number)] * 100.
Ovum Relative Parasitic rate (%)=[(contrast egg hatching rate one is processed egg hatching rate)/contrast egg hatching rate] * 100.
Test-results is as shown in table 1, shows that Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCCNo.5951 spore suspension has obvious inhibition to the hatching of Meloidogyne incognita ovum.When cultivating 9 days, the egg hatching rate of contrast (CK) reaches 97.22%, all ovum grains are hatched into larva substantially, and Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951's only have 59.22% hatching, and Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 spore reaches 39.08% to the Relative Parasitic rate of ovum and is better than Paecilomyces lilacinus E7 and Paecilomyces lilacinus PL04 bacterial strain.
Table 1. Paecilomyces lilacinus spore suspension is on the impact of the hatching of Meloidogyne incognita ovum
Annotate: through the Duncan multiple comparisons, same column letter representation significant difference in the table, wherein, different lowercases represent P＜0.05.
Potted plant and the residential quarter experiment of embodiment 3, Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 control banana root knot nematode
Reagent agent: Paecilomyces lilacinus E16 solid fungicide, Paecilomyces lilacinus E7 solid fungicide, Paecilomyces lilacinus PL04 solid fungicide and blank.
Test plant: Brazilian Banana.
One, the preparation of Paecilomyces lilacinus solid fungicide
1. slant strains:
Solid slant culture base: potato 200g, sucrose 20g, peptone 10g, KH
2PO
40.5g, MnSO
40.05g, agar 18g, water be settled to 1L.121 ℃ of steam sterilizings 20 minutes.Paecilomyces lilacinus is inoculated in the test tube slant substratum, in 28 ℃ of cultivations of going down to posterity, activation, covers with the lavender spore to the inclined-plane.
2. seed culture:
Seed culture medium forms: chitosan 20g, sucrose 15g, peptone 10g, soybean cake powder 15g, KH
2PO
40.5g, MnSO
40.05g, acetic acid 0.5ml, water 1L.Chitosan is available from Zhejiang Province gold shell Biochemie Co., Ltd, and catalog number is K071113289, and deacetylation is 95%; Peptone is available from Hainan Qingniao Co. bio tech ltd, and catalog number is 050170; Soybean cake powder is available from the market of farm produce.
Put into the 60ml seed culture medium in each 250ml triangular flask, 121 ℃ of steam sterilizings 20 minutes.
Choose a fritter lawn from the solid inclined-plane, dispose conidial suspension with seed culture medium, conidial suspension is accessed the 250ml triangular flask again, starting point concentration is 10
5Individual/ml, an inclined-plane can connect 6-8 bottle; 27 ℃ of shaking table shaking culture 48 hours, to growing up to lavender thickness mycelium.
3, solid culture:
Culturing rack: have four column shelfs of nylon gauze interlayer in the chamber, long 3.5m, wide 0.6m, high 2.5m are divided into 6 layers of structure with nylon gauze, interlamellar spacing 30cm, and four column materials are carbon steel plastic-blastings.
A. solid medium (ECK): Testa Tritici 20kg, is settled to 20L with tap water at Semen Maydis powder 60kg, bagasse 30kg, coconut palm chaff 10kg, sucrose 15g, ammonium sulfate 15g, potassium primary phosphate 10g, manganous sulfate 1g.
Compound method: first water stirs ammonium sulfate, potassium primary phosphate, manganous sulfate dissolving, the pH nature, and again with other material mixing, 121 ℃ of high pressure steam sterilizations 30 minutes.
B. inoculation: press the 20%(volume percent) inoculum size, pour into respectively the product of Paecilomyces lilacinus (Paecilomyces lilacinus) E16 CGMCCNo.5951, Paecilomyces lilacinus E7 and Paecilomyces lilacinus PL04 seed culture in the solid medium, stir, place on the nylon gauze of culturing rack, place together in the proving room (proving room is sterilized in advance);
C. cultivation is 2 days cultural method: at 24 ℃ of temperature, fermentation indoor humidity 30%(volumn concentration) and under the air tight condition, to covering with the lavender mycelia; Then all substances on the culturing rack are smashed to pieces, under the condition of 24 ℃ of temperature, cultivation indoor humidity 50%, continued to cultivate 3-5 days.
From culturing room, all substances are taken out, be placed on the shady and cool ventilation place and dry, obtain respectively Paecilomyces lilacinus (Paecilomyces lilacinus) E16 CGMCCNo.5951 solid fungicide (Paecilomyces lilacinus E16 solid fungicide), Paecilomyces lilacinus E7 solid fungicide and Paecilomyces lilacinus PL04 solid fungicide.The blood counting chamber count results shows that the content of Paecilomyces lilacinus in the Paecilomyces lilacinus E16 solid fungicide (Paecilomyces lilacinus) E16CGMCC No.5951 is 2.5 * 10
9Cfu/g, the content of Paecilomyces lilacinus E7 is 2.5 * 10 in the Paecilomyces lilacinus E7 solid fungicide
9Cfu/g, the content of Paecilomyces lilacinus PL04 is 2.5 * 10 in the Paecilomyces lilacinus PL04 solid fungicide
9Cfu/g.
Two, Paecilomyces lilacinus is to the biocontrol effect of potted plant Banana Root Root-knot
Sand is mixed the Brazilian Banana seedling that fertilizer cultivates after two weeks cultivate in 25 * 20cm flowerpot every basin one strain.Cultivation matrix is without sick soil, sand and fertilizer 10:5:1 mixed preparing in mass ratio.Above-mentioned sand is mixed fertilizer and cultivation matrix after testing all without nematode.200 basin Brazilian Banana seedlings are divided into four groups, are respectively Paecilomyces lilacinus E16 group, Paecilomyces lilacinus E7 group, Paecilomyces lilacinus PL04 group and blank group, every group of 50 strains.In the cultivation matrix of above-mentioned 200 basin banana seedlings every basin all apply 250 2 age the banana root knot nematode, Paecilomyces lilacinus E16 organizes the Paecilomyces lilacinus E16 solid fungicide that every basin applies the 20g step 1 again, Paecilomyces lilacinus E7 organizes the Paecilomyces lilacinus E7 solid fungicide that every basin applies the 20g step 1 again, Paecilomyces lilacinus PL04 organizes the Paecilomyces lilacinus PL04 solid fungicide that every basin applies the 20g step 1 again, applies the solid medium (ECK) of 20g step 1 in every basin of blank group.Behind the Routine Management 90d, investigate 4 processing potted plant banana of totally 200 strains, will all process plant button basin and pour out, statistics plant height and fresh weight; Carry out the root knot Severity gradation, calculate disease index and prevention effect.
Wherein, every basin apply 2 age the banana root knot nematode by Meloidogyne incognita (Meloidogyne incognita) and pawl furrow root knot nematode (Meloidogyne Goeidi) according to the number of 3:2 than forming.
Wherein, the root knot Seriousness gradation standard is as follows: the root knot severity divides the 0--4 level, and grade scale is with reference to the grade scale of the Meloidogyne incognita 0-4 level root knots such as Ma Chengzhu and egg capsule, and slightly adjusts.Banana Root Root-knot occurring degree is divided into the 0-4 level: 0 grade: do not have root knot or egg capsule; 1 grade: 1-5 root knot or egg capsule/strain; 2 grades: 6-15 root knot or egg capsule/strain; 3 grades: 16-50 root knot or egg capsule/strain; 4 grades: 50 above root knots or egg capsule/strain.
The prevention effect calculation formula is: prevention effect=(contrast disease index-processing disease index)/contrast disease index.
Results from pot experiment test is shown in table 2 and table 3, show that Paecilomyces lilacinus E16 solid fungicide, Paecilomyces lilacinus E7 solid fungicide, Paecilomyces lilacinus PL04 solid fungicide all can suppress breeding and the harm of banana root knot nematode, and Paecilomyces lilacinus E16 solid fungicide obviously is better than Paecilomyces lilacinus E7 solid fungicide, Paecilomyces lilacinus PL04 solid fungicide, preventive effect is respectively 86.67% behind the 90d, 81.48%, 77.04%.The heavy rate of increase of the root of Paecilomyces lilacinus E16 solid fungicide, plant height rate of increase and over-ground part weight rate of increase are all apparently higher than Paecilomyces lilacinus E7 solid fungicide and Paecilomyces lilacinus PL04 solid fungicide (table 2 and table 3).
Each medicament of table 2. is to banana root knot nematode 2 instar larvae preventive effects
| Process | Disease index | Prevention effect (%) |
| Paecilomyces lilacinus E16 group | 9.0d | 86.67a |
| Paecilomyces lilacinus E7 group | 12.5c | 81.48b |
| Paecilomyces lilacinus PL04 group | 15.5b | 77.04c |
| The blank group | 67.5a | —— |
Annotate: through the Duncan multiple comparisons, same column letter representation significant difference in the table, wherein, different lowercases represent P＜0.05.
The impact that each medicament of table 3. is grown Banana Growth
Annotate: through the Duncan multiple comparisons, same column letter representation significant difference in the table, wherein, different lowercases represent P＜0.05.
Three, Paecilomyces lilacinus is to the biocontrol effect of Banana Root Root-knot residential quarter
Reagent agent: the Paecilomyces lilacinus PL04 solid fungicide of the Paecilomyces lilacinus E16 solid fungicide of step 1, the Paecilomyces lilacinus E7 solid fungicide of step 1, step 1 and the solid medium (ECK) (blank) of step 1.
The randomized block experiment design is adopted in this experiment, 12 mu of open-air bases (matrix is sandy soil) that the Banana Root Root-knot is occured front stubble are divided into three district's groups, 4 mu of each district's groups, each district's group is all established 4 residential quarters (1 mu of each residential quarter), use respectively the Paecilomyces lilacinus E16 solid fungicide of step 1, the Paecilomyces lilacinus E7 solid fungicide of step 1, the Paecilomyces lilacinus PL04 solid fungicide of step 1 and the solid medium (ECK) (blank) of step 1 to process 1 mu of each residential quarter.Concrete treatment process is as follows: each residential quarter kind 160 strain Brazilian Banana, and before banana planting that reagent agent and base manure is mixed, to spread manuer in holes, every strain banana 20g reagent agent imposes reagent agent one time by same dose and method behind the field planting 50d again.Each residential quarter 50 strain bananas of all sampling, the output that begins to add up banana collection period, the investigation disease index calculates disease index and prevention effect.
Wherein, after testing, nematode in this base has Meloidogyne incognita (Meloidogyne incognita), pawl furrow root knot nematode, in the residential quarter of processing with the solid medium (ECK) (blank) of step 1, the population density of Meloidogyne incognita when gathering (Meloidogyne incognita) is 73/100 gram soil, and the population density of pawl furrow root knot nematode is 34/100 gram soil.
Wherein, the root knot Seriousness gradation standard is as follows: the root knot severity divides the 0--4 level, and grade scale is with reference to the grade scale of the Meloidogyne incognita 0-4 level root knots such as Ma Chengzhu and egg capsule, and slightly adjusts.Banana Root Root-knot occurring degree is divided into the 0-4 level: 0 grade: do not have root knot or egg capsule; 1 grade: 1-5 root knot or egg capsule/strain; 2 grades: 6-15 root knot or egg capsule/strain; 3 grades: 16-50 root knot or egg capsule/strain; 4 grades: 50 above root knots or egg capsule/strain.
The prevention effect calculation formula is: prevention effect=(contrast disease index-processing disease index)/contrast disease index.Wherein, disease index is as follows with the calculation formula of relative prevention effect:
Plot experiment result shows that strong through banana well developed root system, the stalwartness of control, the blank district banana root knot of control is not serious, and root growth is fragile.The banana in control district only has a small amount of root knot to come across on old root and the top root, and its severity and disease index are starkly lower than does not prevent and treat the residential quarter.The stimulation ratio of Paecilomyces lilacinus E16 solid fungicide and relative prevention effect are all apparently higher than Paecilomyces lilacinus E7 solid fungicide and Paecilomyces lilacinus PL04 solid fungicide (table 4).
Each medicament plot experiment prevention effect of table 4.
Annotate: through the Duncan multiple comparisons, same column letter representation significant difference in the table, wherein, different lowercases represent P＜0.05.
Embodiment 4, Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 ferment filtrate are to Fusarium oxysporum FOC4-B2 affects on the growth
Two processing are established in experiment, are respectively Paecilomyces lilacinus E16 group and control group.
The treatment process of Paecilomyces lilacinus E16 group is as follows: with Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 fermention medium (Semen Maydis powder 4g, peptone 3g, ammonium sulfate 2g, potassium primary phosphate 4g, sal epsom 3g, Calcium dichloride dihydrate 3g, tween 0.2ml, yeast extract 0.5g, water is settled to 1000mL with substratum, 121 ° of C autoclavings 30 minutes, inoculation after 40 ℃ to be cooled) in 28 ℃, 3d is cultivated in the 180r/min concussion, gets fermented liquid through the centrifugal 25min of 10000r/min, and the gained supernatant liquor is crude extract.Get the PDA substratum that 100mL is cooled to 50 ℃ of molten states and add respectively 3ml through the crude extract of biofilter filtration sterilization, shake up, fall 5 flat boards.After flat board solidifies, place the FOC4-B2 bacterium piece of a diameter 3mm in dull and stereotyped central authorities, this culture dish is placed in 28 ℃ of incubators cultivates.Respectively at 7 days after processing, measure the colony diameter of Fusarium oxysporum with the cruciform method of masurement, calculate bacteriostasis rate.Bacteriostasis rate (%)=(control group colony diameter-treatment group colony radius) * 100/ control group colony radius.
The treatment process of control group is except the crude extract that does not add Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCCNo.5951 fermented liquid, and the treatment process that other and Paecilomyces lilacinus E16 organize is identical.
The result is as shown in table 5, and the crude extract of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 fermented liquid reaches 56.58% to the inhibiting rate of FOC4-B2.
The crude extract of table 5. Paecilomyces lilacinus (Paecilomyces lilacinus) E16 CGMCC No.5951 fermented liquid is to the inhibition of FOC4-B2
| Colony diameter (mm) | Inhibiting rate (%) | |
| Paecilomyces lilacinus E16 group | 33 | 56.58 |
| Control group | 76 |
Embodiment 5, the test of potted plant control banana blight
(content of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 is 2.5 * 10 to Paecilomyces lilacinus (Paecilomyces lilacinus) the E16 solid fungicide for preparing for examination microbial inoculum: embodiment 3
9Cfu/ restrains microbial inoculum).
Banana seedlings for examination is Brazilian any of several broadleaf plants seedling, is provided by Chinese Academy of Tropical Agricultural Sciences group training center.
Potted plant design:
Two processing are established in experiment, are respectively Paecilomyces lilacinus E16 group and control group.
The treatment process of Paecilomyces lilacinus E16 group is as follows: (it is 10 that Paecilomyces lilacinus (Paecilomyces lilacinus) the E16 solid fungicide for preparing with aqueous suspension embodiment 3 makes the content of Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 all to apply 100ml Paecilomyces lilacinus (Paecilomyces lilacinus) E16 solid fungicide suspension in the soil with the every basin of 20 basins (5 leaves) Brazilian any of several broadleaf plants seedling (1 strain/basin)
7CFU/ml), after three days, (making the content of FOC4-B2 spore with aqueous suspension FOC4-B2 spore is 10 all to apply 100ml FOC4-B2 spore suspension in the soil of every basin
6CFU/ml), normal temperature is cultivated in warmhouse booth afterwards, after 50 days, and investigation banana incidence and disease index.
The treatment process of control group is as follows: (making the content of FOC4-B2 spore with aqueous suspension FOC4-B2 spore is 10 all to apply 100ml Fusarium oxysporum Cuba specialized form No. 4 physiological strain B2 bacterial strain (FOC4-B2) spore suspension in the soil of the identical every basin of Brazilian any of several broadleaf plants seedling (1 strain/basin) when 20 basin seedling ages are applied FOC4-B2 with above-mentioned Paecilomyces lilacinus E16 group
6CFU/ml), normal temperature is cultivated in warmhouse booth afterwards, after 50 days, and investigation banana incidence and disease index.
The result is as shown in table 6, illustrates that Paecilomyces lilacinus (Paecilomyces lilacinus) E16CGMCC No.5951 has good protection effect to banana blight.
Wherein, banana blight is divided into following 5 standards:
0 grade: plant is without withered and yellow symptom.
1 grade: slight withered and yellow symptom appears in the plant lower blade, and tender leaf is intact, the slight brown stain of small part root system, and water stain shape brown stain appears in stem.
2 grades: obvious withered and yellow symptom appears in the plant lower blade, but tender leaf is intact, and brown stain appears in root system, and water stain shape brown stain appears in stem and false stem.
3 grades: withered and yellow symptom appears in whole plant, and the root system brown stain is rotted, stem and the brown stain of false stem in flakes, the minority petiole occurs reddish brown.
4 grades: the withered death symptom appears in plant, and the serious brown stain of root system is rotted.
Table 6, Paecilomyces lilacinus (Paecilomyces lilacinus) E16 CGMCC No.5951 are to the prevention effect of banana blight
| Process | Disease index | Prevention effect (%) |
| Control group | 68.3 | — |
| Paecilomyces lilacinus E16 group | 11.5 | 83.2 |
Claims (10)
1. Paecilomyces lilacinus (Paecilomyces lilacinus), it is characterized in that: the bacterial strain of described Paecilomyces lilacinus (Paecilomyces lilacinus) number is E16, and it is numbered CGMCC No.5951 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. the application of Paecilomyces lilacinus claimed in claim 1 (Paecilomyces lilacinus) in producing conidium and/or mycelium.
3. product following 1)-6), its activeconstituents is Paecilomyces lilacinus claimed in claim 1 (Paecilomyces lilacinus):
1) nematocides;
2) product of increase banana output;
3) increase the heavy product of Banana Root;
4) product of increase banana plant height;
5) product of increase banana over-ground part weight;
6) product of the Plant diseases that caused by Fusarium oxysporum Cuba specialized form (Fusairium oxysporum f.sp.cubense) of control.
4. product according to claim 3 is characterized in that: described Paecilomyces lilacinus (Paecilomyces lilacinus) for conidium or/and mycelium.
5. it is characterized in that according to claim 3 or 4 described products: described nematocides is used for control by at least a insect pest of the plant that causes of Meloidogyne incognita (Meloidogyne incognita) and pawl furrow root knot nematode (Meloidogyne Goeidi).
6. product according to claim 5, it is characterized in that: described insect pest of the plant is the Banana Root Root-knot.
7. the application of Paecilomyces lilacinus claimed in claim 1 (Paecilomyces lilacinus) in preparation plant nematocides.
8. application according to claim 7 is characterized in that: the target nematode of described plant nematocides is at least a in Meloidogyne incognita (Meloidogyne incognita) and the pawl furrow root knot nematode (Meloidogyne Goeidi).
9. it is characterized in that according to claim 7 or 8 described application: described plant nematocides is used for control Banana Root Root-knot.
10. the application of Paecilomyces lilacinus claimed in claim 1 (Paecilomyces lilacinus) in the following at least a product of preparation:
1) product of increase banana output;
2) increase the heavy product of Banana Root;
3) product of increase banana plant height;
4) product of increase banana over-ground part weight;
5) product of the Plant diseases that caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense) of control.
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| CN104371949A (en) * | 2014-08-21 | 2015-02-25 | 北京世纪阿姆斯生物技术股份有限公司 | Root-knot nematode killing composite microbial inoculum and preparation method thereof |
| CN104818216A (en) * | 2015-01-20 | 2015-08-05 | 鲁东大学 | Paecilomyces lilacinus for prevention and control of meloidogyne diseases of tomato and grape |
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