CN110100685A - A method of using conidium inoculated identification Peanut Web Blotch Disease disease resistance - Google Patents
A method of using conidium inoculated identification Peanut Web Blotch Disease disease resistance Download PDFInfo
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Abstract
The invention discloses a kind of methods using conidium inoculated identification Peanut Web Blotch Disease disease resistance, belong to crop resistance identification technology field.The present invention cultivates the conidium of net blotch pathogen under 16 small duration illumination conditions generates a large amount of pycnidias, conidial suspension is made in broken pycnidia, spray inoculation peanut seedling, using leaf spot lesion areal analysis network analysis leaf spot lesion area and disease index is calculated, evaluates the net blotch disease resistance of different peanut materials.This method develops the training method that net blotch pathogen quickly, largely generates pycnidia, solves the problems such as net blotch pathogen production spore is difficult, production spore is slow, sporulation quantity is small, takes spore difficult, manufactured conidial suspension uniformity height, Yi Dingliang, overcome the difficult quantitative problem of hyphal suspension inoculation, the Disease Resistance Identification suitable for indoor pot peanut.
Description
Technical field
The present invention relates to a kind of methods using conidium inoculated identification Peanut Web Blotch Disease disease resistance, belong to crop disease-resistant
Property identification technology field.
Background technique
Peanut (Arachis hypogaea L.) is the economy planted extensively in a kind of world wide and oil crops, I
State is the plantation, consumption and big export country of peanut, and peanut annual output occupies first place in the world, thus the stable yields of peanut and high yield always by
To multi-party concern.Peanut Web Blotch Disease is a kind of Major Diseases of peanut leaf portion, can be occurred in entire breeding time, in peanut growth
Later period can cause blade largely to fall off, and so as to cause yield decline 10%~40%, therefore the disease has become and urgently solves in production
Disease certainly.Peanut Web Blotch Disease is as caused by fungal infection, and this fungi is found in 1934 earliest, and Khokhr is entitled by its
Mycosphaerella arachidicola, is under the jurisdiction of mycosphaerella；And Marasas in 1969 etc. is separated from peanut leaf
To different fungies, Phoma is divided into according to its biomorph, is named as Phoma arachidicola.2010
Phoma is divided into 9 subclass from the angle of evolution using the sequence information of rDNA by Aveskamp etc., and the two are sick
Opportunistic pathogen, which has all been gathered, sends the mould class of human relations (Peyronellaea), therefore is renamed as Peyronellaea arachidicola (flower
Raw Phoma sp).Peanut Phoma sp has very strong cellulase activity, and infection court is mostly extended to the outside along vein with radial, shape
At the reticulate pattern stain of no limbus, so Peanut Web Blotch Disease is also known as blotch.Peanut stem point mould silk is fine and close, white or
Canescence, mitogenetic steamed stuffed bun device are partly to bury raw or bury life, and the conidium of field acquisition is mostly double spores, the conidium manually cultivated
Mostly monospore；Chlamydospore, it is raw among mycelia or top, Dan Sheng or concatenate.Peanut Phoma sp is generally with mycelia and mitogenetic
Spore device is passed the winter in residual leaf texture, and properly largely to break out to condition, conidium can propagate with the wind, drug control effect compared with
Difference, plantation disease-resistant variety become the defence most economical effective mode of Peanut Web Blotch Disease.
The breeding of disease-resistant variety depends on sick nursery and artificial infection idenfication, and Infected Field identification climate and strain influence
Larger, even if artificial creation's onset condition, but sick nursery area is still limiting factor, so artificial infection idenfication operability
It is stronger.Wang Zhenyu etc. develops Peanut Web Blotch Disease hyphal suspension inoculated identification method, but due to mycelial growth densification, is not easy
It scatters, so the concentration of hyphal suspension cannot quantify, the mycelia for spraying application to blade is different in size and be unevenly distributed, pathogenic
There are larger differences, and experimental error is larger, qualification result inaccuracy.It is superfine using conidial suspension inoculated identification to wear biography
Method, the advantages of this method are: the conidium manually cultivated is mostly monospore, and manufactured suspension concentration is easily quantitative, is sprayed application to
The spore of blade is evenly distributed, and spore germination growing way consistency is good, and qualification result is reproducible.It is mitogenetic compared with chlamydospore
Spore is more easily separated, because chlamydospore is transformed by mycelia, makes to isolate these chlamydospores with the physical connection of mycelia
It is technically a challenge；In addition, the quantity that conidium generates is significantly larger than chlamydospore, it can in a pycnidia
There can be a conidiums up to a hundred.But the limitation of this method is, peanut Phoma sp is relatively also easy to produce thick wall under culture conditions
Spore, generation pycnidia is more difficult, and not only the time grows (more than 20 days) and amount is few, but also pycnidia and mycelia, thickness
Together, conidium is not easy to obtain the mixed life of wall spore.Therefore it develops a kind of quick and facilitates a large amount of conidial sides of acquisition
Method becomes the key for promoting the identification method.
Summary of the invention
In view of the deficiencies of the prior art, conidium inoculated identification peanut filigree is used the object of the present invention is to provide a kind of
The method of sick disease resistance, this method develop quick net blotch pathogen, big volume production spore and obtain conidial training method,
Manufactured conidial suspension uniformity height, Yi Dingliang overcome the difficult quantitative problem of hyphal suspension inoculation.
To achieve the goals above, the technical scheme adopted by the invention is that:
A method of using conidium inoculated identification Peanut Web Blotch Disease disease resistance, comprising the following steps:
(1) it is inoculated with using the conidium of net blotch pathogen, cultivates pycnidia under the conditions of alternation of light and darkness；
(2) it is crushed pycnidia, conidial suspension B is made；
(3) by conidial suspension B spray inoculation peanut seedling, leaf spot lesion areal analysis network analysis blade is utilized
Lesion area simultaneously calculates disease index, evaluates the net blotch disease resistance of different peanut materials.
In step (1) for net blotch pathogen conidial of inoculation the preparation method comprises the following steps:
1. picking net blotch pathogen mycelium inoculation is cultivated under alternation of light and darkness on OA culture medium, fragmentary distribution is generated
Pycnidia；
2. picking pycnidia is crushed on new OA culture medium, is cultivated under alternation of light and darkness, obtain dense distribution
Pycnidia；
3. collecting, pycnidia is broken to be made conidial suspension A, is inoculated with for conidium.
The conidium of net blotch pathogen is to be inoculated in OA culture medium in step (1).
Alternation of light and darkness is first to carry out illumination cultivation 16h, then dark culturing 8h, and such cycle alternation carries out.
Cultivation temperature is 25 DEG C, incubation time 7-10d.
Step (2) method particularly includes: pycnidia is collected, the ball milling instrument added with sterile water is put into and draws a design in device, with
30hz shake frequency carries out 40s and draws a design broken, after being filtered with 3 layers of sterile gauze, Tween-20 is added, mixes to obtain the final product.
The concentration of conidial suspension is 1 × 10 in step (3)6-1×107A/ml.
The invention has the advantages that:
The conidium of net blotch pathogen is cultivated a large amount of mitogenetic spores of generation by the present invention under 16 small duration illumination conditions
Conidial suspension is made in sub- device, broken pycnidia, and spray inoculation peanut seedling utilizes leaf spot lesion areal analysis system
System analysis leaf spot lesion area simultaneously calculates disease index, evaluates the net blotch disease resistance of different peanut materials.This method develops
Net blotch pathogen quickly, largely generates the training method of pycnidia, solves net blotch pathogen and produces spore hardly possible, produces spore
Slowly, sporulation quantity is small, the problems such as taking spore difficult, and the manufactured conidial suspension uniformity is high, Yi Dingliang, overcomes hyphal suspension
The difficult quantitative problem of inoculation, the Disease Resistance Identification suitable for indoor pot peanut.
Detailed description of the invention
Fig. 1 is indoor inoculation standard of perfection (sick grade) figure of Peanut Web Blotch Disease.
Fig. 2 is the colonial morphology of mycelium inoculation generation under the conditions of different illumination cultivations.A: alternation of light and darkness L//D=16h/8h；
B: alternation of light and darkness L//D=12h/12h；C: dark culture L//D=0h/24h.
Fig. 3 is the colonial morphology that the inoculation of different illumination conditions conidium generates.A: alternation of light and darkness L//D=16h/8h；B:
Alternation of light and darkness L//D=12h/12h；C: dark culture L//D=0h/24h.
Fig. 4 is the percentage of the total leaf area of each kind lesion area Zhan under different conidium inoculum densities.
Fig. 5 is the disease index of each kind under different conidium inoculum densities.
Fig. 6 is the frequency distribution situation for recombinating self-fertilization family (sff) disease index and opposite disease index.
Fig. 7 is the disease index difference of each kind after mycelia and conidial suspension inoculation.
Fig. 8 is the Difference Between Coefficients of Variation between mycelia and the repetition of conidial suspension inocalation method.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Embodiment 1
A method of using conidium inoculated identification Peanut Web Blotch Disease disease resistance, comprising the following steps:
(1) picking net blotch pathogen mycelium inoculation is on OA (oat agar) culture medium, in alternation of light and darkness (illumination 16h,
Dark 8h, i.e. L//D=16h/8h) 7-10d is cultivated at 25 DEG C of constant temperature, mycelia is paved with entire culture dish, wherein being sporadicly dispersed with one
A little pycnidias；
(2) crushing of picking pycnidia is on new OA culture medium, in alternation of light and darkness (L//D=16h/8h) constant temperature 25
7-10d is cultivated at DEG C, generates a large amount of pycnidias, dense distribution is on OA culture medium；
(3) on superclean bench, the pycnidia on OA culture medium is gently scraped off, is collected into added with sterile water
Ball milling instrument is drawn a design in device, is drawn a design broken with 30hz shake frequency progress 40s, is filtered with 3 layers of sterile gauze, remove agar medium and bacterium
Silk is added Tween-20 and mixes, obtains conidial suspension A；
(4) conidial suspension A is coated on OA culture medium, in 25 DEG C of constant temperature of alternation of light and darkness (L//D=16h/8h)
Lower culture 7-10d, generates a large amount of pycnidias, it is radial it is intensive, be uniformly distributed on OA culture medium；
(5) on superclean bench, the pycnidia on OA culture medium is gently scraped off, is collected into added with sterile water
Ball milling instrument is drawn a design in device, is drawn a design broken with 30hz shake frequency progress 40s, is filtered with 3 layers of sterile gauze, remove agar medium and bacterium
Silk obtains pure conidial suspension B, and Tween-20 is added and mixes, until the concentration of conidial suspension B is 1 × 106-
1×107A/ml；
(6) by gained conidial suspension B spray inoculation peanut seedling (6-8 leaf age), leaf spot lesion face is utilized after 12d
Integral analysis system analysis leaf spot lesion area simultaneously calculates disease index, evaluates the net blotch disease resistance of different peanut materials.
Wherein, the condition of culture of peanut seedling are as follows: after 48h vernalization by chitting piece plantation in 10cm alms bowl basin, by alms bowl basin
It is placed in phjytotron, climate box temperature setting is 25 DEG C, relative humidity 90-95%, illumination 16h, dark 8h.
Disease index calculation formula:
Disease index={ [Σ (sick grade strain number × disease grade)]/(investigation total strain number × 9) } × 100
The indoor inoculation standard of perfection (sick grade) of Peanut Web Blotch Disease is (as shown in Figure 1):
0/I grades: disease-free spot；1/HR grades: lesion area is no more than the 6% of leaf area；3/R grades: lesion area is greater than blade face
Long-pending 6% but it is no more than the 25% of leaf area；5/MR grades: lesion area is more than the 25% of leaf area but is no more than leaf area
50%；7/S grades: lesion area is more than the 50% of leaf area but is no more than the 75% of leaf area；9/HS grades: lesion area is more than leaf
The 75% of area.
The optimization of embodiment 2, condition of culture
2.1 mycelium inoculation
Net blotch pathogen mycelia is taken on OA culture medium, in different illumination conditions culture 10d (25 DEG C), alternation of light and darkness
(L//D=16h/8h) bacterium colony front generates obvious mycelia under, and forms the hypha body (Fig. 2A) of protrusion, and the bacterium colony back side can
Visually to see black dot in culture medium, unarmed tabletting can see a large amount of conidiums and shed from spore device.Brightness is handed over
Obvious mycelia (Fig. 2 B) is generated for bacterium colony front under (L//D=12h/12h), and bacterium under dark culture (L//D=0h/24h)
Falling front is thin (Fig. 2 C).It is found by data statistics, pathogen mycelia cultivates 10d generation under 16h/8h alternation of light and darkness
Pycnidia number is more, and every 10 ware averagely about generates 57 pycnidias；10d is cultivated under 12h/12h alternation of light and darkness
Every 10 ware of the pycnidia number of generation is about 15 pycnidias；The mitogenetic spore that 10d is generated is cultivated under dark condition
Sub- device number is minimum, and every 10 ware averagely about generates 2.3 pycnidias, the raw chlamydospore of fecund.
The inoculation of 2.2 conidiums
Drawing 2 μ l concentration respectively is about 3 × 106(about 6000 points of the net blotch pathogen conidial suspension of a/ml
Raw spore) it is inoculated on OA culture medium, in alternation of light and darkness (L//D=16h/8h and L//D=12h/12h) and dark culture (L//D
=0h/24h) under the conditions of after 25 DEG C of culture 7d of constant temperature, observe the pycnidia formational situation (Fig. 3) on culture medium.Brightness is handed over
Pycnidia is uniformly and intensive on culture medium under the conditions of replacing, and with radial growth, shape is more regular, is distributed in for annular shape
On culture medium (Fig. 3 A/3B)；Pycnidia grows the shape that do not fix under dark culture, and substantially not with annular shape distribution
(Fig. 3 C).By every ware using 10ml sterile water as solvent, statistical result is found, the sporulation quantity highest under 16h/8h alternation of light and darkness is put down
Equal sporulation quantity is 34.23 × 105A/ml；Average sporulation quantity under 12h/12h alternation of light and darkness is 3.14 × 105A/ml；Dark culture
Under average sporulation quantity be 5.66 × 105A/ml.
It is all that mycelia is raw regardless of alternation of light and darkness condition or dark culture condition after mycelium inoculation to culture medium described in synthesis
Length is more vigorous, and pycnidia generates less.And after being inoculated into culture medium with conidium, still regardless of alternation of light and darkness condition
Dark culture condition, pycnidia yield are all much higher than mycelium inoculation test (see Fig. 2 and Fig. 3).12h/12h alternation of light and darkness item
Under part, conidium be inoculated with every ware average sporulation quantity and dark culture under the conditions of quite, and under the conditions of 16h/8h alternation of light and darkness, point
The average sporulation quantity of the raw every ware of spore inoculating is 6 times or more under other condition of culture.So in 25 DEG C of alternation of light and darkness of constant temperature
(L//D=16h/8h) under the conditions of, most conidiums can be obtained with conidium inoculation.
Embodiment 3, the conidial suspension inoculated identification different peanut varieties disease resistance using various concentration
On superclean bench, the pycnidia on culture medium is gently scraped off, is collected into the ball milling added with sterile water
Instrument is drawn a design in device, is drawn a design broken with 30hz shake frequency progress 40s, is filtered with 3 layers of sterile gauze, remove agar medium and mycelia,
Pure spore suspension is obtained, Tween-20 is added, is mixed, it is 1 × 10 that concentration, which is made,7The conidial suspension of a/ml,
5,10,15,20 and 25 times are diluted again, and obtaining concentration is 2 × 106、1×106、6.7×106、5×105With 4 × 105A/ml
Dilution, for use.
Peanut varieties to be seeded totally 5, respectively Zheng 8903, Henan spend No. 4, Henan spend No. 22, Henan spend No. 15 and
NC94022.It tests seed used strictly to be selected, seed size, color accomplish neat and consistent, and sterilization is handled.It will
Peanut seed, which is placed in the culture dish for fill aqua sterilisa, carries out vernalization, and culture dish is placed in 30 DEG C of incubator during vernalization.
After vernalization about 48h, when the root long of peanut is to about 5cm, chitting piece is transplanted to the sterilizing vermiculite that fills ready in advance
In 10cm alms bowl basin, make peanut plant normal growth.Alms bowl basin is placed in illumination box/phjytotron, climate box temperature is set
25 DEG C are set to, relative humidity is 90%~95%, illumination 16h, dark 8h.
The experiment sets 6 inoculum densities altogether, and the kind of inoculation sets 3 repetitions, every 5 basin of repetition, 1 plant of every basin.Select growing way
Consistent peanut seedling (6~8 leaf age) carries out tether label to the compound leaf being newly unfolded and carries out sprinkling inoculation to whole strain, compares
Spray sterile water.After the completion of processing, the disease incidence under different disposal after each peanut varieties inoculation is observed and recorded within every 3 days, to disease
After feelings are stablized (12d or so), tether on stem and two compound leaves below (totally 3 compound leaves) are adopted down and scanned, blade is utilized
Lesion area analysis system (Wan Shen) analyzes leaf spot lesion area and calculates disease index.
Variance analysis is carried out to the lesion area of 5 kinds of inoculation various concentration conidial suspension, the results showed that
Difference is not significant (P=0.793) between 3 repetitions of each kind, illustrates that the concentration uniformity of conidial suspension is higher, sprays
The conidium for being applied to blade is evenly distributed, and the scab difference generated after infecting is not significant.And variance is carried out to 6 inoculum densities
Analysis, the result of multiple comparisons are as follows: 4 × 105A/ml and 5 × 105With kind lesion area difference under the two inoculum densities of a/ml
It is not significant；1×106A/ml and 6.7 × 106It is not significant with kind lesion area difference under the two inoculum densities of a/ml；And 2
×106A/ml and 1 × 107The two inoculum densities of a/ml and four inoculum density difference above-mentioned are extremely significant (P < 0.01).With
The increase of inoculum density, lesion area become larger, illustrate conidial suspension inoculation effect stability.To the disease of 5 kinds
Spot area carries out variance analysis, and difference is extremely significant (P < 0.01) between each kind under identical inoculum density, illustrates that conidium is outstanding
Supernatant liquid is inoculated with the Resistance Identification (Fig. 4) that can be used for different cultivars.
The disease index of lower 5 kinds of different vaccination concentration is calculated according to lesion area and inoculation plant quantity.To inoculation
The disease index of 5 kinds of various concentration conidial suspension carries out variance analysis, difference between 3 repetitions of each kind
Not significant (P=0.275), it is consistent with lesion area analysis result, illustrate conidium number in quantitative conidial suspension
It is high to measure consistency.Difference is extremely significant (P < 0.01) between each kind under same inoculum density, consistent with lesion area analysis result,
And it is extremely significant (P < 0.01) with difference between each inoculum density of kind, there are some differences compared with lesion area analysis, illustrates to count
Resistant Difference can be more precisely presented by calculating disease index.With the raising of conidium inoculum density, the disease index of each kind
It increases, illustrates that the stability of conidium morbidity and homogeneity are higher, the conidium of inoculation is more, the scab face of blade
Product is bigger, and disease index is higher (Fig. 5).The disease resistance of 5 kinds shows as 8903 > Henan of Zheng and No. 15 > NC94022 > Henan is spent to spend 22
Number > Henan spends No. 4, wherein Zheng 8903 is disease-resistant variety, Henan spend No. 15 be in anti-kind, NC94022 is susceptible variety, Henan spend No. 4,
No. 22 are spent to feel kind to be high in Henan.
Embodiment 4 utilizes the disease resistance of conidial suspension inoculated identification recombination self-fertilization family (sff)
It selects Zheng 8903 at random and No. 4 hybridization F is spent in Henan11The recombination self-fertilization family (sff) material 98 in generation, with concentration for 2 × 106
The disease resistance of these materials of the conidial suspension inoculated identification of a/ml.Wherein disease index is less than or equal to 20% resistance
Family accounts for 59.18%, and disease index accounts for 20.41% in the family greater than 20% less than or equal to 50%, and disease index is being greater than
50% family less than or equal to 80% accounts for 17.35%, and disease index is accounted in the family being less than or equal between 100% greater than 80%
3.06% (Fig. 6).So conidium inocalation method can apply to the net blotch Resistance Identification of more materials.
Embodiment 5, conidial suspension inocalation method are compared with hyphal suspension inocalation method
By on Peanut Web Blotch Disease pathogen mycelium inoculation to PDA solid medium, in alternation of light and darkness, (16h illumination/8h is black
7-10d secretly) is cultivated at 25 DEG C of constant temperature, until mycelia confluent cultures ware.The mycelia on culture dish is gently scraped, weighs 0.5g in disinfection
Cooking machine in, be added 200ml sterile water carry out mycelia fracture, microscopy Hyphal length is about 100-200 μm, and addition is spat
Temperature -20 make its final concentration of 1%, final concentration of the 2.5 × 10 of hyphal suspension at this time-3g/ml.It is 2.5 × 10 by concentration- 3The hyphal suspension and concentration of g/ml is 2 × 106The conidial suspension of a/ml is inoculated with 5 kinds, each kind respectively
If 3 repetitions, every 5 basin of repetition, 1 plant of every basin.Blade after inoculation 12d is collected, lesion area analysis is carried out and simultaneously calculates the state of an illness
Index.
Variance analysis is carried out to the disease index of 5 kinds of hyphal suspension inoculation, difference is extremely significant between each kind
(P<0.01).This result is consistent with the results of analysis of variance for the disease index that conidial suspension is inoculated with, the disease of 5 kinds
Feelings index be followed successively by from high in the end Henan spend No. 4, Henan spend No. 22, NC94022, Henan spend No. 15, Zheng 8903, illustrate two kinds of inoculation sides
Method can be used for the net blotch Resistance Identification (Fig. 7) of peanut material.But the duplicate three times of each kind is inoculated with to two methods
The coefficient of variation is analyzed, and the coefficient of variation duplicate three times of the discovery each kind of hyphal suspension inocalation method is all higher than mitogenetic spore
Sub- inocalation method (Fig. 8), illustrates that the stability of mycelium inoculation and consistency are relatively poor.Mycelia is not allowed due to structure etc.
It easily scatter, so being unevenly distributed when hyphal suspension is inoculated on blade, the more place of mycelia distribution, scab generates more.
And the mycelia of agglomerating distribution keeps hyphal suspension concentration homogeneity poor, and conidium inocalation method perfectly overcomes mycelia
These disadvantages of inocalation method.Conidial suspension concentration is easily quantitative, and the conidium for being inoculated into blade is evenly distributed, and scab generates
Homogeneity it is preferable, so conidium inocalation method has stronger advantage compared with mycelium inoculation.
Claims (7)
1. a kind of method using conidium inoculated identification Peanut Web Blotch Disease disease resistance, which comprises the following steps:
(1) it is inoculated with using the conidium of net blotch pathogen, cultivates pycnidia under the conditions of alternation of light and darkness；
(2) it is crushed pycnidia, conidial suspension is made；
(3) by conidial suspension spray inoculation peanut seedling, leaf spot lesion areal analysis network analysis leaf spot lesion is utilized
Area simultaneously calculates disease index, evaluates the net blotch disease resistance of different peanut materials.
2. the method according to claim 1, wherein point in step (1) for the net blotch pathogen of inoculation
It is sporogenic the preparation method comprises the following steps:
1. picking net blotch pathogen mycelium inoculation is cultivated under alternation of light and darkness on OA culture medium, point being sporadicly distributed is generated
Raw spore device；
2. picking pycnidia is crushed on new OA culture medium, is cultivated under alternation of light and darkness, obtain the mitogenetic of dense distribution
Spore device；
3. collecting, pycnidia is broken to be made conidial suspension, is inoculated with for conidium.
3. the method according to claim 1, wherein the conidium of net blotch pathogen is to connect in step (1)
Kind is in OA culture medium.
4. method according to claim 1-3, which is characterized in that alternation of light and darkness is first to carry out illumination cultivation 16h,
Then dark culturing 8h, such cycle alternation carry out.
5. method according to claim 1-3, which is characterized in that cultivation temperature is 25 DEG C, incubation time 7-
10d。
6. the method according to claim 1, wherein step (2) method particularly includes: pycnidia is collected,
The ball milling instrument added with sterile water is put into draw a design in device, with 30hz shake frequency carry out 40s draw a design it is broken, after being filtered with 3 layers of sterile gauze,
Tween-20 is added, mixes to obtain the final product.
7. the method according to claim 1, wherein in step (3) conidial suspension concentration be 1 ×
106-1×107A/ml.
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| CN111280008A (en) * | 2020-02-21 | 2020-06-16 | 云南省农业科学院粮食作物研究所 | Comprehensive resistance identification method for broad bean rust disease |
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