CN105112495A - In-vitro leaf disc inoculation method for identifying zantedeschia aethiopica soft rot resistance - Google Patents
In-vitro leaf disc inoculation method for identifying zantedeschia aethiopica soft rot resistance Download PDFInfo
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- CN105112495A CN105112495A CN201510583677.7A CN201510583677A CN105112495A CN 105112495 A CN105112495 A CN 105112495A CN 201510583677 A CN201510583677 A CN 201510583677A CN 105112495 A CN105112495 A CN 105112495A
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Abstract
The invention discloses an in-vitro leaf disc inoculation method for identifying the zantedeschia aethiopica soft rot resistance. According to the method, leaf discs containing leaf main veins serve as materials, through the processing method that internal and external injection and soaking with carrot soft rot pectin bacillus carrot subspecies bacterial suspension are combined, time for identifying the zantedeschia aethiopica soft rot resistance can be shortened to 60 hours, the identification efficiency is improved, and the resistance statistics and analytical method which is more convenient and easier to grasp is provided. Compared with former methods, it is guaranteed that plants are not damaged, the identification cost is low, operation procedure is simple and easy to control, and furthermore, it is achieved that the evaluation result is reliable and accurate. Requisite technical support and assistance for selection and cultivation of resistant varieties in the zantedeschia aethiopica breeding process in the future are provided.
Description
Technical field
The present invention relates to the Disease Resistance authentication method of a kind of garden crop, be specifically related to a kind ofly identify that the tooth in vitro dish of common calla soft rot resistance connects bacterium method, belong to agricultural plant protection technical field.
Background technology
Common calla is Araeceae (Araceae) per nnial herb, is the general designation of planting in water chestnut Nelumbo (ZantedeschiaSpreng.).It originates in the African middle and south, gains the name because of the funnel-form spathe had like horseshoe type.Due to features such as form are graceful, beautiful in colour, be described as " colored lily ".Its application widely, except doing cut-flower and potted flower, also can be floriation, bouquet and the gaily decorated basket etc. and provides distinctive material.At present, America and Europe such as New Zealand, Holland and the U.S. etc. are the major countries that common calla breed and production is cultivated.
Carrot soft rot Pectinatus Radix Dauci Sativae subspecies (Pectobacteriumcarotovorasubsp.Carotovora) is that one has destructive Micobial Disease to common calla, common calla can be made because of tissues such as blade, petiole and ball (root) stems for it and organ necroses, water logging is softened, rot and lodge death and the underproduction, and can rapid spread until cause common calla to have no harvest completely.This disease can be rated as " cancer " of common calla, has a strong impact on the yield and quality of common calla, significantly limit large-scale production and the application of common calla.Its control just like becomes global problem, there is no effective ways up to now, how to adopt chemical agent such as the measures such as Streptomycin sulphate etc. and enhancement of field management to remedy before and after onset.
In fact, there is susceptibility in various degree to soft rot germ in common calla different varieties or pedigree, this just means that the resistance against diseases between them exists huge difference.Therefore, the selection and popularization strengthening disease-resistant variety controls one of very promising means of common calla soft rot.But in default of effective Resistance Identification method, we do not understand the soft rot resistance feature of common calla kind in current production.
Utilize plants for material, to the injection of the tissues such as its blade, petiole and kind ball or organ or to spray carrot soft rot Pectinatus be the current common soft rot resistant screening methods of common calla.But it exists following problem: one is because carrot soft rot Pectinatus is endophyte, after live body inoculation, finally can cause the death of whole test plant or take bacterium existence carrying out secondary propagation; Two is because the common calla kind ball of China is most from external import, often kind of about 30-50 unit/grain of ball, and expensive, live body inoculation can cause the wasting of resources, and appraisal cost increases; Three is in anti-disease enzyme process, there is scab and to invade and the index such as to rot cannot be weighed, the problems such as quantitative analysis inaccurate and evaluation effect difference.
For overcoming the above problems, to be organized by vitro inoculation common calla or the technology of organ is developed successively.The method also can realize the accurate evaluation resisting susceptible characteristic while protection plant, has and avoids waste and take into account effective advantage.Wherein one just comprises leaf disc infusion method (Snijderetal.2002; EuropeanJournalofPlantPathology), first it obtain the disk of diameter 2.2cm on blade along blade master pulse both sides by punch tool, then be soaked in the porous plate comprising bacterium liquid and cultivated under 20 DEG C of conditions, utilize iteration to repeat the remaining maximum likelihood method (IRREML, IterativeReweightedResidualMaximumLikelihoodAlgorithm) of weighting according to statistics onset area after 4 days and carry out statistical study and evaluation.But the method also has following shortcoming: be first that the observation cycle is long, four days (96h) is at least needed to observe; Next is statistical method more complicated, needs certain mathematical statistics to gain knowledge, and common breeding man or investigator are difficult to effective grasp.
Summary of the invention
The present invention not easily comes to harm the existing methodical plant of reservation, appraisal cost is low, operation steps is simple and while being easy to the advantages such as controls, establishes that a kind of observing time is shorter, statistical and analytical method is simpler but evaluation result equally reliably authentication method accurately.Final object of the present invention wishes to provide effective technical support and help by the screening of resistant variety for realizing domestic common calla breeding for disease resistance from now on.
The present invention is achieved by the following technical solutions:
A kind ofly identify that the tooth in vitro dish of common calla soft rot resistance connects bacterium method, it is characterized in that, each identified kind or the ripe healthy blade of pedigree random choose three parts, first be the leaf disc that axis punch tool obtains that diameter is 1.5cm with master pulse in blade middle and upper part after cleaning and sterilizing, repeat to get 12 times; Utilize syringe to inject 100 μ l carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspensions along the master pulse bottom of leaf disc afterwards, and be immersed in 12 well culture plates comprising 10ml carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension; Place it in 28 DEG C of constant temperature culture in biochemical cultivation case again, period supplements 1 time with sterilized water simultaneously, and taken out by expert evidence after 60 hours, visual observations leaf dish onset area also carries out classification according to grade scale; Calculate disease index, the last disease index according to calculating determines the anti-sense property variation situation of each kind or pedigree.
In the present invention, preferably, comprise the steps:
(1) preparation of carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension
Carrot soft rot Pectinatus Radix Dauci Sativae subspecies are inoculated on gravy peptone (NA) plate culture medium, 28 DEG C of cultivations, single colony inoculation on picking flat board is in LB substratum, activation is carried out and enlarged culturing 24h in 37 DEG C of constant incubators, the centrifugal 10min of 4000rpm, abandon supernatant, add sterilized water in bacterial sediment and be mixed with carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension;
(2) the blade process of tested variety
Choose 15 common calla kinds or pedigree, each kind selects the blade of three parts of healthy mature, and the alcohol disinfecting of rear use 75% is dried in cleaning; Be that to get the diameter comprising rib be the leaf disc of 1.5cm to axis punch tool afterwards with master pulse in blade middle and upper part, each kind repeats to get 12 times;
(3) inoculated identification
Utilize syringe to inject 100 μ l carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspensions respectively along its underpart master pulse respectively to 12 parts of leaf discs, have overflow to be advisable with top; It is placed on respectively afterwards in 12 well culture plates containing 10ml carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension, be positioned over by culture plate in 28 DEG C of biochemical cultivation cases and cultivate, period adds sterilized water 1 time for avoiding evaporating;
(4) resistance O&A
After inoculation 60h, 12 orifice plates are taken out, on LED case, appearance method is observed and is recorded the value of each leaf dish susceptible (water stain shape) area/roundleaf sheet total area, carry out classification according to grade scale and calculate disease index, the last disease index according to calculating determines the anti-sense property variation situation of each kind or pedigree.
In the present invention, preferably, the concentration of described carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension is 1 × 10
8cfu/mL.
In the present invention, preferably, described grade scale is: 0 grade, leaf dish without or less symptom, the 0 < lesion area/total area≤10%; 1 grade, the 10% < lesion area/total area≤30%; 2 grades, the 30% < lesion area/total area≤50%; 3 grades, the 50% < lesion area/total area≤70%; 4 grades, 70% < lesion area/total area.
In the present invention, preferably, determine that the anti-sense property variation situation of each kind or pedigree refers to according to the disease index calculated: disease index≤20 are " high resistance "; 20 < disease index≤40 are " disease-resistant "; 40 < disease index≤60 are " in anti-"; 60 < disease index≤80 are " susceptible "; 80 < disease indexs are " high sense ";
Compared with prior art, beneficial effect of the present invention is embodied in:
The present invention is to comprise the leaf disc of rib for material, injected and soak the treatment process that outstanding bacterium liquid phase combines by inside and outside, can by the time shorten of qualification common calla soft rot resistance to 60h, improve determination rates, and provide convenient and hold wield resistance statistical and analytical method.Compared with method before, not only ensure that plant not easily comes to harm, appraisal cost is low, and operation steps is simple and be easy to control, but also it is reliably accurate to achieve evaluation result equally.This will provide very necessary technical support and help for the selection of resistant variety in common calla breeding process from now on and cultivation.
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments only for the object of illustration, never limit the scope of the invention.
Embodiment 1
Carrot soft rot Pectinatus Radix Dauci Sativae sp. strain; this bacterial strain is at document (Deng Yamin; Du Shuo, Wang Huan, Yang Zhongling; Liu Fengquan; Fan Jiaqin. the functional analysis of carrot soft rot Pectinatus Radix Dauci Sativae subspecies mreB gene. Agricultural University Of Nanjing's journal, 02 phase in 2014) in be disclosed, be the bacterial strain of plant protection institute of Agricultural University Of Nanjing Laboratories Accession; if the public, based on test or research, can be obtained by Agricultural University Of Nanjing.
(1) preparation of carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension
Carrot soft rot Pectinatus Radix Dauci Sativae sp. strain is inoculated on gravy peptone (NA) plate culture medium, 28 DEG C of cultivations, single colony inoculation on picking flat board is in LB substratum, activation is carried out and enlarged culturing 24h in 37 DEG C of constant incubators, finally the bacterium liquid of acquisition is placed in centrifuge tube, the centrifugal 10min of 4000rpm, abandons supernatant, and bacterial sediment adds appropriate sterilized water and is mixed with 1 × 10
8the bacterial suspension of cfu/mL (colony-formingunitsperml) is for subsequent use.
(2) the blade process of tested variety
With table 1 comprise 15 common calla commercial cultivars or pedigree for material, each kind selects the blade of three parts of healthy mature, and the alcohol disinfecting of rear use 75% is dried in cleaning; Be that to get the diameter comprising rib be the leaf disc of 1.5cm to axis punch tool afterwards with master pulse in blade middle and upper part, each kind repeats to get 12 times.
The source of a table 115 common calla Cultivar or pedigree and soft rot evaluation of resistance
(3) inoculated identification
First syringe is utilized to inject 10 of 100ul respectively along its underpart master pulse respectively to 12 parts of leaf discs
8cfu/mL carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension, has overflow to be advisable with top; Afterwards it is placed on respectively the same concentrations bacterial suspension (10 containing 10ml
8cfu/mL) in 12 well culture plates; Finally be positioned over by culture plate in 28 DEG C of biochemical cultivation cases and cultivate, period adds sterilized water 1 time for avoiding evaporating.
(4) resistance O&A
Compare with the responsive soft rot kind FlorexGold of height.After inoculation 60h, 15 orifice plates are taken out, on LED case, appearance method is observed and is recorded the value of each leaf dish susceptible (water stain shape) area/roundleaf sheet total area, carry out classification according to grade scale and calculate disease index, evaluating according to the anti-sense characteristic of disease index to each kind.Mainly determine according to following standard:
1. according to the value of the susceptible area/roundleaf sheet total area of leaf dish, carry out classification according to following grade scale: 0 grade, leaf dish without or less symptom, the 0 < lesion area/total area≤10%; 1 grade, the 10% < lesion area/total area≤30%; 2 grades, the 30% < lesion area/total area≤50%; 3 grades, the 50% < lesion area/total area≤70%; 4 grades, 70% < lesion area/total area.
2. according to following formulae discovery disease index:
3. common calla resistance level is determined according to following grade scale: high resistance (disease index≤20); Disease-resistant (20 < disease index≤40); In anti-(40 < disease index≤60); Susceptible (60 < disease index≤80); High sense (80 < disease index).
4. the anti-soft rot evaluation of common calla 15 kinds or pedigree: as can be seen from Table 1, Z.aethiopica ' HB01 ' in white common calla pedigree, ' TW01 ' and ' K4-1 ' resistance rank to soft rot is higher, is respectively high resistance or disease-resistant; And in calla lily cultivation hybrid, soft rot resistance against diseases is obviously deteriorated, only have CallaforniaRed, Picasso and Vermeer shows middle water resistant and puts down, and remaining is susceptible and high sense, comprise respectively and 15#, Flame, Camaro, HotCherry and Kloon, Rehmannii, Merlot, FlorexGold, BlackMagic.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and can carry out many changes in the spirit and scope that the claims in the present invention limit to it, amendment, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (5)
1. identify that the tooth in vitro dish of common calla soft rot resistance connects bacterium method for one kind, it is characterized in that, each identified kind or the ripe healthy blade of pedigree random choose three parts, first be the leaf disc that axis punch tool obtains that diameter is 1.5cm with master pulse in blade middle and upper part after cleaning and sterilizing, repeat to get 12 times; Utilize syringe to inject 100 μ l carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspensions along the master pulse bottom of leaf disc afterwards, and be immersed in 12 well culture plates comprising 10ml carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension; Place it in 28 DEG C of constant temperature culture in biochemical cultivation case again, period supplements 1 time with sterilized water simultaneously, and taken out by expert evidence after 60 hours, visual observations leaf dish onset area also carries out classification according to grade scale; Calculate disease index, the last disease index according to calculating determines the anti-sense property variation situation of each kind or pedigree.
2. tooth in vitro dish according to claim 1 connects bacterium method, it is characterized in that, comprises the steps:
(1) preparation of carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension
Carrot soft rot Pectinatus Radix Dauci Sativae subspecies are inoculated on gravy peptone (NA) plate culture medium, 28 DEG C of cultivations, single colony inoculation on picking flat board is in LB substratum, activation is carried out and enlarged culturing 24h in 37 DEG C of constant incubators, the centrifugal 10min of 4000rpm, abandon supernatant, add sterilized water in bacterial sediment and be mixed with carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension;
(2) the blade process of tested variety
Choose 15 common calla kinds or pedigree, each kind selects the blade of three parts of healthy mature, and the alcohol disinfecting of rear use 75% is dried in cleaning; Be that to get the diameter comprising rib be the leaf disc of 1.5cm to axis punch tool afterwards with master pulse in blade middle and upper part, each kind repeats to get 12 times;
(3) inoculated identification
Utilize syringe to inject 100 μ l carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspensions respectively along its underpart master pulse respectively to 12 parts of leaf discs, have overflow to be advisable with top; It is placed on respectively afterwards in 12 well culture plates containing 10ml carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension, be positioned over by culture plate in 28 DEG C of biochemical cultivation cases and cultivate, period adds sterilized water 1 time for avoiding evaporating;
(4) resistance O&A
After inoculation 60h, 12 orifice plates are taken out, on LED case, appearance method is observed and is recorded the value of each leaf dish susceptible (water stain shape) area/roundleaf sheet total area, carry out classification according to grade scale and calculate disease index, the last disease index according to calculating determines the anti-sense property variation situation of each kind or pedigree.
3. tooth in vitro dish according to claim 1 and 2 connects bacterium method, it is characterized in that, the concentration of described carrot soft rot Pectinatus Radix Dauci Sativae subspecies bacterial suspension is 1 × 10
8cfu/mL.
4. tooth in vitro dish according to claim 1 and 2 connects bacterium method, it is characterized in that, described grade scale is: 0 grade, leaf dish without or less symptom, the 0 < lesion area/total area≤10%; 1 grade, the 10% < lesion area/total area≤30%; 2 grades, the 30% < lesion area/total area≤50%; 3 grades, the 50% < lesion area/total area≤70%; 4 grades, 70% < lesion area/total area.
5. tooth in vitro dish according to claim 1 and 2 connects bacterium method, it is characterized in that, determines that the anti-sense property variation situation of each kind or pedigree refers to according to the disease index calculated: disease index≤20 are " high resistance "; 20 < disease index≤40 are " disease-resistant "; 40 < disease index≤60 are " in anti-"; 60 < disease index≤80 are " susceptible "; 80 < disease indexs are " high sense ";
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| CN108486033A (en) * | 2018-02-23 | 2018-09-04 | 云南省德宏热带农业科学研究所 | A kind of in-vitro verification method of coffee germplasm rust resistance |
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| CN103333944A (en) * | 2013-07-08 | 2013-10-02 | 江苏徐州甘薯研究中心 | Identification method of sweet potato soft rot resistance |
Non-Patent Citations (2)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108486033A (en) * | 2018-02-23 | 2018-09-04 | 云南省德宏热带农业科学研究所 | A kind of in-vitro verification method of coffee germplasm rust resistance |
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Application publication date: 20151202 |