CN102640671A - Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm - Google Patents
Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm Download PDFInfo
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Abstract
The invention discloses a method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm, which comprises the following steps: (1) preparing Alternaria altemata (Fr.) Keissler pathogen, and mainly performing culture medium preparation, Alternaria altemata (Fr.) Keissler separation identification, Alternaria altemata (Fr.) Keissler pathogen transfer and Alternaria altemata (Fr.) Keissler pathogen activation and enlarging culture; (2) preparing a Alternaria altemata (Fr.) Keissler resistance material, and mainly performing pear variety grafting and pear grafting variety transplanting; (3) performing artificial inoculation identification, and mainly utilizing Alternaria altemata (Fr.) Keissler pathogen spore suspension to perform spraying inoculation to grafted seedling blades of an annual pear variety; and (4) performing operation of disease investigation and grading standard. The method is practical and simple and convenient to operate, and the Alternaria altemata (Fr.) Keissler resistance of the pear variety can be identified rapidly and accurately. By means of the method, the Alternaria altemata (Fr.) Keissler resistance of the pear variety in scientific research, breeding and production of pears is identified rapidly.
Description
Technical field
The invention belongs to the fruit tree technical field, more specifically relate to the method that the anti-black spot of a kind of pears germplasm is identified, handle the problem that can effectively solve the anti-black spot Rapid identification of pears kind in pears scientific research, breeding and the production through this method.This method is applicable to R&D institution, company and the individual who is engaged in pears scientific research, breeding and production.
Background technology
Pear for tall this deciduous fruit tree, is that China three one of cultivates apple trees greatly in the rose family (Rosaceae) apple subfamily (Maloideae) pear (Pyrus) plant, also occupies critical role in the world.According to Chinese yearbook statistics, China's pears cultivated area was 1063.1 thousand hectares in 2010, and 1505.71 ten thousand tons of output occupy first place in the world.It is the mainstay industry that China's increasing peasant income are got rich that the operatic circle produces always, also is one of main farm produce of China's foreign exchange earning.But along with the expansion of Chinese pear area under cultivation, pears black spot (Alternaria alternata (Fr.) Keissler) develops into disease main in China theatre gradually, and particularly morbidity is even more serious in southern Chinese pear producing region.Pears black spot main harm leaf, fruit and young sprout, it is weak to cause setting body, shortens the time limit as a result, causes serious economy loss, but also causes sick fruit of storage period, and in the foreign trade of pears, receives watching with the deepest concern of importer.
The pears black spot is prevented and treated improper meeting and is caused the pear tree blade to come off in advance, makes pear tree form the secondary flower easily, has a strong impact on next year the operatic circle output, and general pears black spot causes orchard worker's loss can reach 20%-30%, reaches 50%-60% when serious.The pears producing region in China south, preventing and treating the successful key of disease mainly is the effect of seeing pears black spot control in the theatre, pear tree pesticide control cost over half is used to prevent and treat the pears black spot in agricultural 1 year of the pears.The method of control pears black spot is to use chemical agents such as thiophanate methyl, mancozeb and Difenoconazole at present; Long-term use these chemical agents, if improper use will inevitably cause pathogen to chemical agent develop immunity to drugs, contaminated environment and health produced problem such as harm.In order to overcome chemical agent corrosion-resistant toxicity shortcoming; Formulate the effectively comprehensive preventive health measures of pears black spot rationally; Seed selection disease resistance production kind is imperative; But how identifying fast and accurately that the pears kind has the characteristic of anti-black spot in large tracts of land popularization process, has been the key subjects that pendulum must solve in face of the scientific research personnel.
For many years, we rely on national fruit tree germ plasm resource garden, have carried out a large amount of scientific researches and test round the anti-black spot characterization and evaluation of pears germplasm, sum up the method that the anti-black spot of a cover pears germplasm is identified of inventing out.This method is to utilize pears black spot pathogen spore suspension that annual pears kind grafting blade is carried out spray inoculation; Inoculate and keep air humidity in back 24 hours more than 95%; Inoculating back 20 days investigates; According to state of an illness SERIES CALCULATION disease index, the pears germplasm is carried out anti-black spot characterization and evaluation, adopt this method to carry out anti-black spot to the pears kind fast and accurately and identify.
Summary of the invention
The objective of the invention is to be to provide a kind of pears germplasm method that anti-black spot is identified, easy to implement the method, easy and simple to handle, use this method this method can carry out anti-black spot evaluation to the pears kind fast and accurately.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
The present invention utilizes pears black spot pathogen spore suspension that annual pears kind grafting blade is carried out spray inoculation; Inoculate and keep air humidity in back 24 hours more than 95%; Inoculating back 20 days investigates; According to state of an illness SERIES CALCULATION disease index, the pears germplasm is carried out anti-black spot characterization and evaluation.
The method that the anti-black spot of a kind of pears germplasm is identified the steps include:
1. the preparation of blackspot pathogen:
(1) preparation of medium:
Potato culture (PSA): potato (peeling) 200g, agar powder 20g, sucrose 20g, water 1000mL.Compound method is following: with peeling potatoes, be cut into the fritter of about 2cm2, boil 0.5h, note stirring in case stick with paste with glass bar.Filter with double gauze then, get it and consider liquid adding agar powder, the dissolving back adds sucrose, supplies water again to 1000mL, and PH 7.0.
(2) pears black spot isolation identification:
The blade or the fruit that have pears black spot illness gathered, auspicious with reference to Du Lian in 2005, Lu Fuping chief editor " microbiology experimental technique " carried out separate tissue, monospore purifying, cultivation, preservation.To the tender pears blade of children, can observation produce typical black spot scab, and then scab is carried out separation and purification with the inoculation that separate to obtain.The pathogenic strains of separation and purification rule on the PSA medium cultivate 6-8d, observe colonial morphology; Continue to cultivate mycelia and the spore shape characteristic of examining under a microscope pathogen after 19-21 days,, identify the kind of separating obtained pathogen with reference to Wei Jingchao chief editor's in 1979 " fungi morphology identification handbook ".In order further to confirm the kind name of pears black spot pathogen bacterial strain, adopt rDNA-ITS Molecular Identification method that strain separated is analyzed.With the DNA of disease fungus as template; With universal primer ITS1:TCCGTAGGTGAACCTGCGG (sequence 5-3 ') and ITS4:TCCTCCGCTTATTGATATGC (sequence 5-3 ') document " the ITS sequence analysis is increased in the classification of fungi application in identification " (Chen Jian mountain in 2007, primer sequence source; Zheng obeys clump), with measured rDNA-ITS sequence in GenBank with Clustalx 2.0.10 to separation pears alternaria and logined the pears black spot pathogen rDNA-ITS sequence of delivering and carried out the multiple sequence compare of analysis.Comprehensive pathogen morphological feature and rDNA-ITS sequencing comparison result are identified the pears alternaria.
(3) pears blackspot pathogen switching:
With separate, purifying and the blackspot pathogen of identifying rule on fresh inclined-plane PSA medium, illumination box was cultivated 19-21 days for 28 ℃, was put in 4 ℃ of preservations of refrigerator after growing spore.
(4) activation of pears blackspot pathogen and enlarged culture:
The blackspot pathogen that preserves is taken out from refrigerator, in culture dish, rule with oese, and in 28 ℃ of cultivations of illumination box.Behind mycelia confluent cultures ware, under aseptic condition with method enlarged culture blackspot pathogen spore in culture dish of inoculated by hypha block.Cultivate 2-3 time so repeatedly, turn out 2 * 10
5The spore suspension inoculation pears blade of individual spore/mL.
2, pears disease resistance material is prepared:
(1) pears kind grafting:
10-11 when pears are in hibernation, gathers the branch that has selected the pears kind between the month; Adopt budding method with reference to Hu Hongju in 2010; Cultivating seedlings in Wang Youping chief editor " Chinese pear improved seeds and standardized planting technology ", grafting on beans pears stock, each kind grafting 50 strain.
(2) pears grafting variety transplanting:
1-2 before sleety weather arrives, prevents kind bud frostbite with pears grafting kind nursery stock transplanting between the month in easy green house, temperature is controlled at the 10-15 degree.
3. artificial infection is identified:
(1) cause of disease spore liquid preparation:
With the culture dish enlarged culture after 19-21 days; Add the 20mL sterile water in each culture dish, with 2cm type plate brush down, focus in the beaker of 1L pears black spot spore brush; Use blood counting chamber to count, and to use distilled water that pears black spot spore is diluted to concentration be 2 * 10 at microscopically
5The spore suspension of individual spore/mL is put into and is cultivated 6h under 28 ℃ of cultivation constant temperature, and germination rate is more than 80%, and is for use.
(2) artificial infection:
Do not spray bactericide in the pears kind whole growing to be inoculated, between June-July, will wait to inoculate pears kind terminal bud and remove, use 2 * 10
5The pears black spot spore suspension spray inoculation of individual spore/mL is sprayed to leave dual sides and all drenches on grafting pear kind to be identified.Inoculate in back 24 hours and to keep air humidity, inoculate back 20 days and investigate,, the pears germplasm is carried out anti-black spot characterization and evaluation based on state of an illness SERIES CALCULATION disease index more than 95%.
4. the state of an illness is investigated and grade scale:
Inoculate 20 days, the whole blades of the investigation inoculation pears kind rank of being injured.Investigate the disease index of the natural occurrence of identification of species simultaneously, by every strain East, West, South, North four direction investigation, each direction is investigated 50 leaves immediately.
State of an illness investigation and grade scale
0 grade of no scab;
1 grade of scab accounts for below 10% of leaf area;
3 grades of scabs account for 10%～25% of leaf area;
5 grades of scabs account for 26%～40% of leaf area;
7 grades of scabs account for 41%～65% of leaf area;
9 grades of scabs account for more than 65% of leaf area.
According to the rank of being injured, calculate the black spot disease index.
In the formula: BI---the black spot disease index
x
i---sick leaves at different levels are represented progression
n
i---the sick numbers of sheets at different levels
N---total number of sheets
According to disease index and standards, confirm the black spot resistance of germplasm.
Pear tree is divided into 5 grades to the resistance of black spot according to disease index.
1 high anti-(HR) (disease index＜10.0)
3 disease-resistant (R) (10.0≤disease index＜25.0)
Anti-(M) R (25.0≤disease index＜40.0) in 5
7 susceptible (S) (40.0≤disease index＜65.0)
9 high sense (HS) (disease index >=65.0).
Generally can carry out anti-black spot evaluation to introducing a fine variety the pears kind fast and accurately with said method.Easy to implement the method, easy and simple to handle, handle the problem that can effectively solve the anti-black spot Rapid identification of pears kind in pears scientific research, breeding and the production through the present invention, and qualification result is accurate, is suitable for the early stage evaluation of anti-black spot in pears kind seedling stage.
Description of drawings
Fig. 1 is a kind of pears black spot pathogen strains A, H, the spore shape sketch map of C bacterial strain under * 400 power microscopes.
Fig. 2 is that the colonial morphology of a kind of pears black spot pathogen on the PSA medium identified sketch map.
Fig. 3 is a kind of pears black spot pathogen ITS sequence Molecular Identification electrophoresis sketch map.
Fig. 4 is that a kind of pears black spot bacterial strain A, C, H, GQ249171.1, GU797144.1 and GU566303.1 carry out ITS sequence homology sequence alignment sketch map.
Fig. 5 is that the anti-black spot of a kind of Chinese pear germplasm is identified sketch map.
Fig. 6 be a kind of natural occurrence with artificial inoculation conditions under different Chinese pear kind disease index sketch mapes.
Experiment illustration 1:2008,2009,2010 and 2011 adopt this method that 331 parts of pears germ plasm resources in the Chinese pear garden, national fruit tree germplasm Wuchang are carried out anti-pears black spot characterization and evaluation.
Fig. 7 is a different pears black spot resistance Chinese pear variety identification sketch map as a result under a kind of artificial inoculation conditions.
Can find out from Fig. 6, Fig. 7; Through using continuous 4 years of this method that 331 parts of pears germ plasm resources in the Chinese pear garden, national fruit tree germplasm Wuchang are carried out anti-pears black spot characterization and evaluation, filter out 9 parts of high anti-black spot kinds, 71 parts in disease-resistant variety, in 165 parts of anti-kinds, 68 parts of susceptible variety, 18 parts of high sense kinds.Qualification result will be established experimental basis and theoretical foundation for the seed selection of the anti-black spot kind of pears.
Embodiment
The method that the anti-black spot of a kind of pears germplasm is identified the steps include:
1. the preparation of blackspot pathogen:
(1) preparation of medium:
Potato culture (PSA): potato (peeling) 200g, agar powder 20g, sucrose 20g, water 1000mL.Compound method is following: with peeling potatoes, be cut into about 2cm
2Fritter, boil 0.5h, note stirring in case stick with paste with glass bar.Filter with double gauze then, get it and consider liquid adding agar powder, the dissolving back adds sucrose, supplies water again to 1000mL, and PH 7.0.
(2) pears black spot isolation identification:
The blade or the fruit that have pears black spot illness gathered, auspicious with reference to Du Lian in 2005, Lu Fuping chief editor " microbiology experimental technique " carried out separate tissue, monospore purifying, cultivation, preservation.To the tender pears blade of children, can observation produce typical black spot scab, and then scab is carried out separation and purification with the inoculation that separate to obtain.The pathogenic strains of separation and purification rule on the PSA medium cultivate 6 or 7 or 8d, observe colonial morphology; Continue to cultivate mycelia and the spore shape characteristic of examining under a microscope pathogen after 19 or 20 or 21 days,, identify the kind of separating obtained pathogen with reference to Wei Jingchao chief editor's in 1979 " fungi morphology identification handbook ".In order further to confirm the kind name of pears black spot pathogen bacterial strain, adopt rDNA-ITS Molecular Identification method that strain separated is analyzed.With the DNA of disease fungus as template; With universal primer ITS1:TCCGTAGGTGAACCTGCGG (sequence 5-3 ') and ITS4:TCCTCCGCTTATTGATATGC (sequence 5-3 ') document " the ITS sequence analysis is increased in the classification of fungi application in identification " (Chen Jian mountain in 2007, primer sequence source; Zheng obeys clump), with measured rDNA-ITS sequence in GenBank with Clustalx 2.0.10 to separation pears alternaria and logined the pears black spot pathogen rDNA-ITS sequence of delivering and carried out the multiple sequence compare of analysis.Comprehensive pathogen morphological feature and rDNA-ITS sequencing comparison result are identified the pears alternaria.
(3) pears blackspot pathogen switching:
With separate, purifying and the blackspot pathogen of identifying rule on fresh inclined-plane PSA medium, illumination box was cultivated 19 or 20 or 21 days for 28 ℃, was put in 4 ℃ of preservations of refrigerator after growing spore.
(4) activation of pears blackspot pathogen and enlarged culture:
The blackspot pathogen that preserves is taken out from refrigerator, in culture dish, rule with oese, and in 28 ℃ of cultivations of illumination box.Behind mycelia confluent cultures ware, under aseptic condition with method enlarged culture blackspot pathogen spore in culture dish of inoculated by hypha block.Cultivate so repeatedly 2 or 3 times, turn out the spore suspension inoculation pears blade of 2 * 105 spore/mL.
2, pears disease resistance material is prepared:
(1) pears kind grafting:
10-11 when pears are in hibernation, gathers the branch that has selected the pears kind between the month; Adopt budding method with reference to Hu Hongju in 2010; Cultivating seedlings in Wang Youping chief editor " Chinese pear improved seeds and standardized planting technology ", grafting on beans pears stock, each kind grafting 50 strain.
(2) pears grafting variety transplanting:
1-2 before sleety weather arrives, prevents kind bud frostbite with pears grafting kind nursery stock transplanting between the month in easy green house, temperature is controlled at 10 or 12 or 13 or 15 degree.
3. artificial infection is identified:
(1) cause of disease spore liquid preparation:
With culture dish enlarged culture 19 or after 20 or 21 days; Add the 20mL sterile water in each culture dish, with 2cm type plate brush down, focus in the beaker of 1L pears black spot spore brush; Use blood counting chamber to count, and to use distilled water that pears black spot spore is diluted to concentration be 2 * 10 at microscopically
5The spore suspension of individual spore/mL is put into and is cultivated 6h under 28 ℃ of cultivation constant temperature, and germination rate is more than 80%, and is for use.
(2) artificial infection:
Do not spray bactericide in the pears kind whole growing to be inoculated, between June-July, will wait to inoculate pears kind terminal bud and remove, use 2 * 10
5The pears black spot spore suspension spray inoculation of individual spore/mL is sprayed to leave dual sides and all drenches on grafting pear kind to be identified.Inoculate in back 24 hours and to keep air humidity, inoculate back 20 days and investigate,, the pears germplasm is carried out anti-black spot characterization and evaluation based on state of an illness SERIES CALCULATION disease index more than 95%.
5. the state of an illness is investigated and grade scale:
Inoculate back 20 days, the whole blades of the investigation inoculation pears kind rank of being injured.Investigate the disease index of the natural occurrence of identification of species simultaneously, by every strain East, West, South, North four direction investigation, each direction is investigated 50 leaves immediately.
State of an illness investigation and grade scale
0 grade of no scab;
1 grade of scab accounts for below 10% of leaf area;
3 grades of scabs account for 10%～25% of leaf area;
5 grades of scabs account for 26%～40% of leaf area;
7 grades of scabs account for 41%～65% of leaf area;
9 grades of scabs account for more than 65% of leaf area.
According to the rank of being injured, calculate the black spot disease index.
In the formula: BI---the black spot disease index
x
i---sick leaves at different levels are represented progression
n
i---the sick numbers of sheets at different levels
N---total number of sheets
According to disease index and standards, confirm the black spot resistance of germplasm.
Pear tree is divided into 5 grades to the resistance of black spot according to disease index.
1 high anti-(HR) (disease index＜10.0)
3 disease-resistant (R) (10.0≤disease index＜25.0)
Anti-(M) R (25.0≤disease index＜40.0) in 5
7 susceptible (S) (40.0≤disease index＜65.0)
9 high sense (HS) (disease index >=65.0).
Claims (1)
1. the method that the anti-black spot of pears germplasm is identified the steps include:
The preparation of A, blackspot pathogen:
At first be the selection of medium, select potato culture, the preparation method is potato 200g, agar powder 20g, and sucrose 20g, water 1000Ml, compound method is following: with peeling potatoes, be cut into 2cm
2Fritter, boil 0.5h, stir with glass bar, filter with double gauze then, get it and consider liquid and add agar powder, the dissolving back adds sucrose, supplies water again to 1000mL, PH 7.0; Next is a pears black spot isolation identification step; The blade or the fruit that have pears black spot illness of authentication method for gathering carries out separate tissue, monospore purifying, cultivation, preservation by conventional method, with separating the inoculation that obtains to the tender pears blade of children; Observe the black spot scab; Scab is carried out separation and purification, the pathogenic strains of separation and purification is rule on the PSA medium cultivate 6-8d, observe colonial morphology; Continue to cultivate mycelia and the spore shape characteristic of examining under a microscope pathogen after 19-21 days; With reference to fungi morphology identification handbook; Identify the kind of separating obtained pathogen; In order to confirm the kind name of pears black spot pathogen bacterial strain, adopt rDNA-ITS Molecular Identification method that isolated strains is analyzed, with the DNA of disease fungus as template; Increase with universal primer ITS1 and ITS4; With measured rDNA-ITS sequence in GenBank with Clustalx 2.0.10 to separating the pears alternaria and logined the pears black spot pathogen rDNA-ITS sequence of delivering and carried out the multiple sequence compare of analysis, comprehensive pathogen morphological feature and rDNA-ITS sequencing comparison result, evaluation pears alternaria; Be pears blackspot pathogen switching once more, the switching step for will separate, purifying and the blackspot pathogen of identifying rule on fresh inclined-plane PSA medium, 28 ℃ of cultivations of illumination box 19-21 days are put in 4 ℃ of preservations of refrigerator after growing spore; Be pathogen activation of pears blackspot and enlarged culture at last; Cultural method is rule in culture dish with oese for the blackspot pathogen that preserves is taken out from refrigerator, and in 28 ℃ of cultivations of illumination box; Behind the mycelia confluent cultures ware; Under aseptic condition,, cultivate 2-3 time repeatedly, turn out 2 * 10 with method enlarged culture blackspot pathogen spore in culture dish of inoculated by hypha block
5The spore suspension inoculation pears blade of individual spore/mL;
B, pears disease resistance material are prepared:
At first be the grafting of pears kind, 10-11 when pears are in hibernation, gathers the branch that has selected the pears kind between the month, adopts budding method grafting on beans pears stock, each kind grafting; Secondly be pears grafting variety transplanting, 1-2 is between the month, before the sleety weather arriving, with pears grafting kind nursery stock transplanting to green house;
C, artificial infection are identified:
At first be the preparation of cause of disease spore liquid; With the culture dish enlarged culture after 19-21 days; Add the 20mL sterile water in each culture dish, with pears black spot spore brush down, focus in the beaker of 1L; Use blood counting chamber to count, and to use distilled water that pears black spot spore is diluted to concentration be 2 * 10 at microscopically
5The spore suspension of individual spore/mL is put into and is cultivated 6h under 28 ℃ of cultivation constant temperature, and germination rate is 80%; Next is artificial infection, and inoculation method is not spray bactericide in the pears kind whole growing to be inoculated, and between June-July, will wait to inoculate pears kind terminal bud and remove, with 2 * 10
5The pears black spot spore suspension spray inoculation of individual spore/mL is on grafting pear kind to be identified; Being sprayed to leave dual sides all drenches; Inoculate and keep air humidity in back 24 hours 95%; Inoculate back 20 days and investigate,, the pears germplasm is carried out anti-black spot characterization and evaluation according to state of an illness SERIES CALCULATION disease index;
Investigation of D, the state of an illness and grade scale are inoculated back 20 days, and the whole blades of the investigation inoculation pears kind rank of being injured is investigated the disease index of the natural occurrence of identification of species simultaneously, and by every strain East, West, South, North four direction investigation, each direction is investigated 50 leaves immediately;
State of an illness investigation and grade scale are with reference to as follows:
0 grade of no scab;
1 grade of scab accounts for below 10% of leaf area;
3 grades of scabs account for 10%～25% of leaf area;
5 grades of scabs account for 26%～40% of leaf area;
7 grades of scabs account for 41%～65% of leaf area;
9 grades of scabs account for 65% of leaf area;
According to the rank of being injured, calculate the black spot disease index:
In the formula: BI---the black spot disease index;
x
i---sick leaves at different levels are represented progression;
n
i---the sick numbers of sheets at different levels;
N---total number of sheets;
According to disease index and standards, confirm the black spot resistance of germplasm:
Pear tree is divided into 5 grades to the resistance of black spot according to disease index:
1 is high anti-: disease index＜10.0;
3 is anti-: 10.0≤disease index＜25.0;
Anti-in 5: R25.0≤disease index＜40.0;
7 is susceptible: 40.0≤disease index＜65.0;
9 high senses: disease index >=65.0.
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