CN113777237A - Method for quantitatively detecting infection capability of sclerotinia sclerotiorum - Google Patents

Method for quantitatively detecting infection capability of sclerotinia sclerotiorum Download PDF

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CN113777237A
CN113777237A CN202111052604.7A CN202111052604A CN113777237A CN 113777237 A CN113777237 A CN 113777237A CN 202111052604 A CN202111052604 A CN 202111052604A CN 113777237 A CN113777237 A CN 113777237A
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sclerotinia sclerotiorum
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CN113777237B (en
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吴沿友
张承
苏跃
吴沿胜
方蕾
童成英
周英
罗亮
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Institute of Geochemistry of CAS
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Abstract

The invention discloses a method for quantitatively detecting the infection capacity of sclerotinia sclerotiorum. Using C-derived sources with large differences in stable carbon isotope values3Sugar beet of plants and C4The method comprises the steps of establishing a bidirectional isotope tracing culture system for sugarcane sucrose of plants, forming to-be-detected plants and sclerotinia sclerotiorum which are cultured on sugarcane sucrose and sugar beet culture media, infecting sclerotinia sclerotiorum which is cultured on the sugarcane sucrose and sugar beet culture media alternately by the to-be-detected plants which are cultured on the sugarcane sucrose and sugar beet culture media, establishing the bidirectional isotope tracing culture system, obtaining the share of fungus blocks in a fungus-leaf system by using the bidirectional isotope tracing culture system, and calculating the multiplication coefficient f of the sclerotinia sclerotiorum on leaves of the to-be-detected plants at different momentsniAnd the stable carbon isotope fraction value delta of the fungus-leaf systemiAccording to fniAnd deltaiQuantitatively judging the infection condition of the plant to be detected on sclerotinia sclerotiorum at different moments; and (4) quantitatively grading the infection conditions of the plants to be detected on the sclerotinia sclerotiorum at different moments.

Description

Method for quantitatively detecting infection capability of sclerotinia sclerotiorum
Technical Field
The invention relates to a method for quantitatively detecting the infection capability of sclerotinia sclerotiorum, belonging to the detection technology of plant pathological information.
Background
Sclerotinia is a plant fungal disease caused by sclerotinia, and has wide host range and strong destructiveness. Sclerotinia can infect more than 400 plants of 278 genus of 75 families such as cruciferae, solanaceae, leguminosae and the like, and is the most serious disease on orychophragmus violaceus and rape. Sclerotinia easily occurs in the whole growth period of orychophragmus violaceus and rape, the sclerotinia is particularly serious after the flowering period, and the sclerotinia can be damaged to the mature period. Sclerotinia seriously affects rape yield and vegetable oil quality; the annual incidence rate is 10-30%, the annual incidence rate is more than 80% in serious cases, the yield reduction rate is 10-70%, and the oil content is reduced by 1-5%. Therefore, the method has important significance for high-efficiency, safe and green prevention and control of the plant sclerotinia sclerotiorum by quickly and accurately acquiring the infection information of the plant sclerotinia sclerotiorum.
Tissue culture of plants is the most basic technology and means in current biotechnology and is now widely used in horticulture, agriculture and forestry production. It is a technique for quickly breeding plants under the condition of artificially providing a certain temp., light, humidity, nutrients and hormone, etc.
Both sclerotinia sclerotiorum and plants can be cultured on sucrose. Natural sucrose differs in its stable carbon isotopes because of differences in origin. In plant tissue culture, the tissue culture seedling is usually provided with sugar as an organic carbon source. C3Plant delta13The variation range of C is-20 to-35 per mill, C4Plant delta13The variation range of C is-9 to-17 per mill. Thus, derived from C3Sugar beet of plants and C4The stable carbon isotope value of sugarcane sucrose of plants is greatly different, and the delta of the culture cultured by the corresponding sucrose is13The difference in C is also large.
The bidirectional isotope labeling culture technology is a method for respectively culturing the same biological materials on the same culture medium with larger isotope value difference under the same condition and acquiring material source information according to the isotope value difference change of the biological materials. Organic substances secreted by sclerotinia sclerotiorum and produced by susceptible tissues are important pathogenic factors in pathogenic process. Whether they are secreted by sclerotinia or pathogenic factors produced by pathogenic tissues, is still difficult to distinguish. The analysis of the source of the pathogenic factor can provide basic information for the pathogenesis of the sclerotinia sclerotiorum and provide scientific data for understanding the pathogenesis of the sclerotinia sclerotiorum. The invention is to quantitatively detect the capability of the sclerotinia sclerotiorum infected plant by using isotope tracing culture and bidirectional isotope technology for a plurality of times.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for quantitatively detecting the infection capacity of the sclerotinia sclerotiorum, so as to overcome the defects that the infection capacity of the sclerotinia sclerotiorum is judged only by the apparent result of the size of a scab in the prior art, on one hand, the infection degree of the sclerotinia sclerotiorum is difficult to quantitatively compare, on the other hand, the information of the plant infection process of the sclerotinia sclerotiorum is difficult to obtain, and the like.
The invention adopts the following technical scheme: it comprises the following steps:
first, respectively select C4Representative sucrose sugarcane sucrose and C of plants3Representative sucrose beet sucrose of plants as organic carbon source, and delta was measured separately13C value, Delta of cane sugar13The value of C is δC1Delta of sugar beet13The value of C is δC2；
Secondly, preparing a culture medium for culturing a plant to be tested by using the sugarcane sucrose and the beet sucrose respectively, culturing a tissue culture seedling of the plant to be tested, and culturing for a period of time for a subsequent sclerotinia sclerotiorum infection experiment; the cultured tissue culture seedlings are clones from the same source, and multiple times of propagation culture are needed in the culture process, so that the obtained material can meet the subsequent requirements;
thirdly, at the same time, a medium for culturing sclerotinia sclerotiorum is prepared by using the sugar cane sugar and the sugar beet sugar, the medium is made into a flat plate, sclerotinia sclerotiorum is cultured, and after a period of culture, a part of the medium is used for delta of sclerotinia sclerotiorum block13Determination of C value, part for subsequent invasionAn experiment of staining a plant to be tested; the method for obtaining sclerotinia sclerotiorum block cultured by culture medium prepared from cane sugar and beet sugar comprises activating and culturing sclerotinia sclerotiorum strain on culture medium containing cane sugar and beet sugar for 7 days, cutting 6mm sclerotium block, contacting mycelium with culture medium downwards, inoculating to plate center of new corresponding culture medium, culturing for 7 days, and applying part of cultured material to delta of sclerotinia sclerotiorum block13C value determination, wherein one part of the C value is used for a subsequent experiment for infecting the plant to be detected;
fourthly, preparing a sucrose culture medium with high-abundance stable carbon isotopes for culturing the following sclerotinia sclerotiorum infected plant to be detected; the preparation method of the sucrose culture medium with high abundance and stable carbon isotopes comprises the following steps of13C/12C standard and cane sugar are configured into delta13PDB culture medium with C value of 30.0 ‰;
fifthly, using sclerotinia sclerotiorum blocks cultured by culture mediums prepared by cane sugar and beet sugar respectively to alternately infect plants to be tested which have consistent growth vigor and are cultured on the culture mediums prepared by the cane sugar and the beet sugar, and culturing the plants in a cane sugar culture medium with high abundance and stable carbon isotopes; the method for infecting the plant to be tested alternately with sclerotinia sclerotiorum blocks comprises the steps of transferring the plant to be tested which grows consistently and is cultured in a sugar beet sucrose culture medium into a sucrose culture medium with high-abundance stable carbon isotopes, then cutting 6mm sclerotinia sclerotiorum blocks which are cultured in the sugar beet sucrose culture medium for 7 days, enabling hypha of the sclerotinia sclerotiorum blocks to downwards contact with leaves, inoculating the sclerotinia sclerotiorum blocks to the plant leaves to be tested in the system, and enabling the inoculation positions of the leaves to be the central positions of the leaves; similarly, transferring the plant seedlings to be tested which are grown in the same way and cultured in cane sugar into a cane sugar culture medium with high-abundance stable carbon isotopes, then cutting 6mm fungus blocks of sclerotinia sclerotiorum cultured in the cane sugar culture medium for 7 days, wherein hyphae of the fungus blocks downwards contact leaves and are inoculated on the plant leaves to be tested in the system, and the inoculation position of each leaf is the central position of each leaf;
sixthly, the delta of sclerotinia sclerotiorum pieces cultured with a medium prepared from cane sugar and beet sugar was measured separately13C value, wherein the culture is performed in a sugarcane sucrose mediumDelta of sclerotinia sclerotiorum block of (1)13The value of C is δB1Delta. of sclerotinia sclerotiorum mass cultured in sugar beet sucrose medium13The value of C is δB2；
Seventh, the delta of the germ-leaf system at different infestation times is subsequently determined13C value, delta of sclerotinia sclerotiorum block cultured by using culture medium prepared from cane sugar to infect fungus-leaf system of plant to be tested cultured on culture medium prepared from beet sugar13The value of C is recorded as delta TAiUsing sclerotinia sclerotiorum block cultured by culture medium prepared by sugar beet sucrose to remove delta infecting fungus-leaf system of plant to be tested cultured on culture medium prepared by sugar beet sucrose13The value of C is recorded as delta TBiWhere i is the number of samples taken at different times, δ TA0And δ TB0δ T at 0 of the start of the experimentAiAnd δ TBi；
Eighth, according to δB1、δB2、δC1、δC2、δTA0And δ TB0Obtaining the share f of the bacterium block in a bacterium-leaf system and the delta of the leaf of the plant to be detected cultured on a culture medium prepared from cane sugar and beet sucrose13C value, wherein; delta of leaf of plant to be tested cultured on culture medium prepared from cane sugar13The value of C is δADelta of leaf of plant to be tested cultured on a culture medium prepared from sugar beet13The value of C is δB(ii) a The calculation formula for obtaining the share f of the bacterium block in the bacterium-leaf system is as follows:
Figure BDA0003253435250000031
then f is substituted into the formula:
Figure BDA0003253435250000032
obtaining delta of the leaf of the plant to be tested cultured on the culture medium prepared by cane sugar13C value deltaAAnd delta of leaf of plant to be tested cultured on culture medium prepared from beet sucrose13C value deltaB；
Ninth, according to δ TA0、δTB0、δC1、δC2、δTAiAnd δ TBiObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniThe calculation method comprises the following steps: will delta TA0、δTB0、δC1、δC2、δTAiAnd δ TBiSubstituting the equation:
Figure BDA0003253435250000033
where i is the number of samples taken at different times;
tenth, according to δ TA0、δB、δTAiAnd f, fniOr δ TB0、δA、δTBiAnd f, fniObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiThe method comprises the following steps:
Figure BDA0003253435250000034
or
Figure BDA0003253435250000035
Where i is the number of samples taken at different times;
eleventh, according to fniAnd deltaiQuantitatively judging the infection condition of the plant to be detected on sclerotinia sclerotiorum at different moments; the method for quantitatively judging the infection condition of the plant to be detected to sclerotinia sclerotiorum at different moments comprises the following steps: comparing different plants at the same time, or f of the same plant at different times, respectivelyniAnd deltai，fniAnd deltaiA larger value indicates more infection, and more severe infection of the plant sclerotinia sclerotiorum, whereas a smaller value indicates less infection, and less infection of the plant sclerotinia sclerotiorum;
twelfth, combined use of fniAnd deltaiQuantitatively grading the infection conditions of the plants to be detected on sclerotinia sclerotiorum at different moments; the quantitative grading method for the infection condition of the plant to be tested on the sclerotinia sclerotiorum at different moments comprises the following steps: si＝10fni+166.67δiThis isWhere i is the number of samples taken at different times, SiThe series of the sclerotinia sclerotiorum infected by the plant to be detected at different moments.
The invention has the following advantages:
1) the method can be used for determining the share f of the bacterium block in a bacterium-leaf system when the sclerotiniose infects the plant leaves and the delta of the plant leaves to be detected cultured on a culture medium prepared from cane sugar and beet sucrose13C value, and proliferation coefficient f of sclerotinia sclerotiorum on plant leaf to be detected at different timeniMost importantly, the method can also quantify the process of infecting the plant to be detected by sclerotinia sclerotiorum and quantitatively classify the infection condition of the plant to be detected to sclerotinia sclerotiorum at different moments;
2) the method can quantitatively compare the processes of the sclerotinia sclerotiorum infecting different plants, provide basic data for the research of the disease resistance mechanism of the sclerotinia sclerotiorum of the plants and provide scientific and technological support for the prevention and treatment of the sclerotinia sclerotiorum of the plants;
3) the method can predict and quantitatively grade the plant sclerotinia sclerotiorum disease infection condition, and provides a basis for accurate drug application of sclerotinia sclerotiorum disease.
The basic principle of the invention is as follows:
C3plant delta13The variation range of C is-20 to-35 per mill, C4Plant delta13The variation range of C is-9 to-17 per mill. Therefore, the C-derived carbon has a large difference in stable carbon isotope value3Sugar beet of plants and C4The method comprises the steps of establishing a bidirectional isotope tracing culture system for sugarcane sucrose of plants, forming to-be-detected plants and sclerotinia sclerotiorum which are cultured on sugarcane sucrose and sugar beet culture media, infecting the sclerotinia sclerotiorum cultured on the sugarcane sucrose and sugar beet culture media alternately by the to-be-detected plants cultured on the sugarcane sucrose and sugar beet culture media, establishing the bidirectional isotope tracing culture system, and obtaining the share f of a mycorrhiza in a fungus-leaf system by using the bidirectional isotope tracing culture system. The isotope mixture model of the two-terminal elements of the bidirectional isotope labeling culture system in the initial culture of the present invention can be expressed as:
δTA0＝fδB1+(1-f)δB (1)
δTB0＝fδB2+(1-f)δA (2)
δTA0in order to infect the sclerotinia sclerotiorum block cultured in the medium prepared from cane sugar at 0 moment of the beginning of the experiment, the delta of the fungus-leaf system of the plant to be tested cultured in the medium prepared from beet sugar is removed13C value, δ TB0For sclerotinia sclerotiorum blocks cultured by a culture medium prepared by sugar beet, the delta of a fungus-leaf system of a plant to be tested cultured on the culture medium prepared by sugar beet is removed13C value, δB1Delta for sclerotinia sclerotiorum mass cultured in sugarcane sucrose medium13C value, δB2Delta for sclerotinia sclerotiorum mass cultured in sugar beet sucrose medium13C value, f is the proportion of the bacterial mass in the bacterial-leaf system at 0 moment of the start of the experiment, deltaADelta of leaf of plant to be tested cultured on a Medium formulated with sugarcane sucrose13C value, δBDelta of leaves of plants to be tested cultured on a Medium formulated with sugar beet13And C value.
Combining the formulas (1) and (2), and obtaining the share f of the bacterium blocks in the bacterium-leaf system by the calculation formula:
Figure BDA0003253435250000041
then substituting f into formula (1) or (2) to obtain deltaAAnd deltaB：
Figure BDA0003253435250000042
Similarly, the fungus-leaf system under different culture time also satisfies the isotope mixed model of two end elements of the bidirectional isotope tracing culture system, which can be respectively expressed as:
fδB1+fni(δB+δi)+(1-f)(δB+δi)＝δTAi (3)
fδB2+fni(δA+δi)+(1-f)(δA+δi)＝δTBi (4)
where f isniThe proliferation coefficients of sclerotinia sclerotiorum on the plant leaves to be detected at different infection moments are obtained; deltaiIs the stable carbon isotope fractionation value, delta T, of the fungus-leaf system at different momentsAiFor sclerotinia sclerotiorum blocks cultured by a culture medium prepared from cane sugar, delta of a fungus-leaf system infecting a plant to be tested cultured on a culture medium prepared from beet sugar is removed13C value, δ TBiFor sclerotinia sclerotiorum blocks cultured by a culture medium prepared by sugar beet, the delta of a fungus-leaf system of a plant to be tested cultured on the culture medium prepared by sugar beet is removed13The value of C, where i is the number of samples taken at different times, and the other symbols represent the same meanings as described above.
By combining the formulas (1), (2), (3) and (4), f can be obtainedni
Figure BDA0003253435250000051
According to delta TA0、δB、δTAiAnd f, fniOr δ TB0、δA、δTBiAnd f, fniObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiThe method comprises the following steps:
Figure BDA0003253435250000052
or
Figure BDA0003253435250000053
Where i is the number of samples taken at different times.
Comparing different plants at the same time, or f of the same plant at different times, respectivelyniAnd deltai，fniAnd deltaiA larger value indicates a more severe infection of the plant sclerotinia sclerotiorum, whereas a less severe infection indicates a less severe infection of the plant sclerotinia sclerotiorum.
And the infection condition of the sclerotinia sclerotiorum is quantitatively graded, so that comparison of different plants is facilitated. Combined use of fniAnd deltaiThe infection condition of the plant to be detected to sclerotinia sclerotiorum at different momentsCarrying out quantitative grading on the conditions; the quantitative grading method for the infection condition of the plant to be tested on the sclerotinia sclerotiorum at different moments comprises the following steps: the infection condition of the sclerotinia sclerotiorum on the plant is divided into 10 grades, and the multiplication coefficient f of the sclerotinia sclerotiorum on the plant leaves to be detectedniStable carbon isotope fractionation value delta of Hei fungus-leaf SystemiAre each set to 0.5. Multiplication coefficient f of sclerotinia on plant leaves to be detectedniIs 50% (0.5) and the stable carbon isotope fraction value delta of the fungus-leaf systemiAnd if the grade is 30 per mill (PDB), determining 10 grades, and quantitatively grading the infection condition of the plant to be tested on sclerotinia sclerotiorum at different moments by the following steps: si＝10fni+166.67δiWhere i is the number of samples taken at different times, SiThe series of the sclerotinia sclerotiorum infected by the plant to be detected at different moments.
Detailed Description
Examples of the invention: it comprises the following steps:
first, respectively select C4Representative sucrose sugarcane sucrose and C of plants3Representative sucrose beet sucrose of plants as organic carbon source, and delta was measured separately13C value, Delta of cane sugar13The value of C is δC1Delta of sugar beet13The value of C is δC2；
Secondly, preparing a culture medium for culturing a plant to be tested by using the sugarcane sucrose and the beet sucrose respectively, culturing a tissue culture seedling of the plant to be tested, and culturing for a period of time for a subsequent sclerotinia sclerotiorum infection experiment; the cultured tissue culture seedlings are clones from the same source, and multiple times of propagation culture are needed in the culture process, so that the obtained material can meet the subsequent requirements;
thirdly, at the same time, a medium for culturing sclerotinia sclerotiorum is prepared by using the sugar cane sugar and the sugar beet sugar, the medium is made into a flat plate, sclerotinia sclerotiorum is cultured, and after a period of culture, a part of the medium is used for delta of sclerotinia sclerotiorum block13C value determination, wherein one part of the C value is used for a subsequent experiment for infecting the plant to be detected; the method for obtaining sclerotinia sclerotiorum blocks cultured by a culture medium prepared from cane sugar and beet sugar comprises the steps of respectively carrying out activation culture on sclerotinia sclerotiorum strains on the culture medium containing cane sugar and beet sugar for 7 days,cutting 6mm of the mycelia, contacting the mycelia with the culture medium downwards, inoculating to the center of a new plate of the corresponding culture medium, culturing for 7 days, and applying a part of the cultured material to delta of sclerotinia sclerotiorum13C value determination, wherein one part of the C value is used for a subsequent experiment for infecting the plant to be detected;
fourthly, preparing a sucrose culture medium with high-abundance stable carbon isotopes for culturing the following sclerotinia sclerotiorum infected plant to be detected; the preparation method of the sucrose culture medium with high abundance and stable carbon isotopes comprises the following steps of13C/12C standard and cane sugar are configured into delta13PDB culture medium with C value of 30.0 ‰;
fifthly, using sclerotinia sclerotiorum blocks cultured by culture mediums prepared by cane sugar and beet sugar respectively to alternately infect plants to be tested which have consistent growth vigor and are cultured on the culture mediums prepared by the cane sugar and the beet sugar, and culturing the plants in a cane sugar culture medium with high abundance and stable carbon isotopes; the method for infecting the plant to be tested alternately with sclerotinia sclerotiorum blocks comprises the steps of transferring the plant to be tested which grows consistently and is cultured in a sugar beet sucrose culture medium into a sucrose culture medium with high-abundance stable carbon isotopes, then cutting 6mm sclerotinia sclerotiorum blocks which are cultured in the sugar beet sucrose culture medium for 7 days, enabling hypha of the sclerotinia sclerotiorum blocks to downwards contact with leaves, inoculating the sclerotinia sclerotiorum blocks to the plant leaves to be tested in the system, and enabling the inoculation positions of the leaves to be the central positions of the leaves; similarly, transferring the plant seedlings to be tested which are grown in the same way and cultured in cane sugar into a cane sugar culture medium with high-abundance stable carbon isotopes, then cutting 6mm fungus blocks of sclerotinia sclerotiorum cultured in the cane sugar culture medium for 7 days, wherein hyphae of the fungus blocks downwards contact leaves and are inoculated on the plant leaves to be tested in the system, and the inoculation position of each leaf is the central position of each leaf;
sixthly, the delta of sclerotinia sclerotiorum pieces cultured with a medium prepared from cane sugar and beet sugar was measured separately13C value, wherein delta of sclerotinia sclerotiorum mass cultured in sugarcane sucrose medium13The value of C is δB1Delta. of sclerotinia sclerotiorum mass cultured in sugar beet sucrose medium13The value of C is δB2；
Seventh, thenThe delta of the fungus-leaf system at different infection times was determined13C value, delta of sclerotinia sclerotiorum block cultured by using culture medium prepared from cane sugar to infect fungus-leaf system of plant to be tested cultured on culture medium prepared from beet sugar13The value of C is recorded as delta TAiUsing sclerotinia sclerotiorum block cultured by culture medium prepared by sugar beet sucrose to remove delta infecting fungus-leaf system of plant to be tested cultured on culture medium prepared by sugar beet sucrose13The value of C is recorded as delta TBiWhere i is the number of samples taken at different times, δ TA0And δ TB0δ T at 0 of the start of the experimentAiAnd δ TBi；
Eighth, according to δB1、δB2、δC1、δC2、δTA0And δ TB0Obtaining the share f of the bacterium block in a bacterium-leaf system and the delta of the leaf of the plant to be detected cultured on a culture medium prepared from cane sugar and beet sucrose13C value, wherein; delta of leaf of plant to be tested cultured on culture medium prepared from cane sugar13The value of C is δADelta of leaf of plant to be tested cultured on a culture medium prepared from sugar beet13The value of C is δB(ii) a The calculation formula for obtaining the share f of the bacterium block in the bacterium-leaf system is as follows:
Figure BDA0003253435250000061
then f is substituted into the formula:
Figure BDA0003253435250000062
obtaining delta of the leaf of the plant to be tested cultured on the culture medium prepared by cane sugar13C value deltaAAnd delta of leaf of plant to be tested cultured on culture medium prepared from beet sucrose13C value deltaB；
Ninth, according to δ TA0、δTB0、δC1、δC2、δTAiAnd δ TBiObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniThe calculation method is: will delta TA0、δTB0、δC1、δC2、δTAiAnd δ TBiSubstituting the equation:
Figure BDA0003253435250000071
where i is the number of samples taken at different times;
tenth, according to δ TA0、δB、δTAiAnd f, fniOr δ TB0、δA、δTBiAnd f, fniObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiThe method comprises the following steps:
Figure BDA0003253435250000072
or
Figure BDA0003253435250000073
Where i is the number of samples taken at different times;
eleventh, according to fniAnd deltaiQuantitatively judging the infection condition of the plant to be detected on sclerotinia sclerotiorum at different moments; the method for quantitatively judging the infection condition of the plant to be detected to sclerotinia sclerotiorum at different moments comprises the following steps: comparing different plants at the same time, or f of the same plant at different times, respectivelyniAnd deltai，fniAnd deltaiA larger value indicates more infection, and more severe infection of the plant sclerotinia sclerotiorum, whereas a smaller value indicates less infection, and less infection of the plant sclerotinia sclerotiorum;
twelfth, combined use of fniAnd deltaiQuantitatively grading the infection conditions of the plants to be detected on sclerotinia sclerotiorum at different moments; the quantitative grading method for the infection condition of the plant to be tested on the sclerotinia sclerotiorum at different moments comprises the following steps: si＝10fni+166.67δiWhere i is the number of samples taken at different times, SiThe series of the sclerotinia sclerotiorum infected by the plant to be detected at different moments.
Detailed implementation and content
Respectively selectWith C4Representative sucrose sugarcane sucrose (δ) of plantsC1，δ13C value-14.45 ‰ (PDB)) and C3Representative of plants sucrose beet sucrose (. delta.) (C2，δ13C value-26.73 per mill (PDB)) as organic carbon source. The plants to be tested were: brassica napus and Brassica rapa. Cabbage type rape seeds of Baoyouhan 12 variety are provided by Guizhou Heming luck seeds Co. The Orychophragmus violaceus seeds are collected from the Lao institute of geochemistry of Chinese academy of sciences. The rape and the orychophragmus violaceus are respectively cultured in the culture media prepared by cane sugar and beet sugar through the steps of seed germination, multiplication and the like, and the obtained material is used for the subsequent sclerotinia sclerotiorum infection experiment. The seed germination culture medium is 1/2MS culture medium, then the culture medium after aseptic seeding is placed in a culture room for 2 weeks, then inoculated in a proliferation culture medium for proliferation culture, after cultured in the proliferation culture medium for 4 weeks, the cluster buds of the plant to be tested are selected to be connected to a sucrose culture medium (delta) of high-abundance stable carbon isotope13C value of 30 per mill (PDB)) was tested for sclerotinia infection.
The pathogenic bacteria are sclerotinia sclerotiorum. The sclerotinia sclerotiorum is cultured by respectively activating and culturing the strains on an MS culture medium containing cane sugar and beet sugar for 7 days, respectively activating and culturing the sclerotinia sclerotiorum strains on the culture medium containing cane sugar and beet sugar for 7 days, cutting 6mm strain blocks, enabling mycelia to downwards contact the culture medium, inoculating the mycelia to a new plate center point of a corresponding culture medium for culturing, and after culturing for 7 days, using a part of cultured materials for delta of the sclerotinia sclerotiorum blocks13C value determination, wherein one part of the C value is used for a subsequent experiment for infecting the plant to be detected; the delta of sclerotinia sclerotiorum pieces cultured in sugarcane sucrose culture medium in the experiment is determined13C value of-15.51 ‰ (PDB), and delta of sclerotinia sclerotiorum mass cultured in beet sucrose medium13The C value is-24.06 ‰ (PDB).
The experiment that sclerotinia sclerotiorum blocks are alternately infected with the plant to be detected: sclerotinia sclerotiorum blocks cultured by culture media prepared by cane sugar and beet sugar respectively are used for alternately infecting plants to be detected which have consistent growth vigor and are cultured on the culture media prepared by the cane sugar and the beet sugar, and are cultured in a cane sugar culture medium with high abundance and stable carbon isotopes; sclerotinia sclerotiorumThe method for alternately infecting the plant to be tested by the blocks comprises the steps of transferring the plant to be tested which grows consistently and is cultured in a sugar beet sucrose culture medium into the sucrose culture medium with high-abundance stable carbon isotopes, then cutting 6mm sclerotinia sclerotiorum blocks which are cultured in the sugar beet sucrose culture medium for 7 days, enabling hypha of the sclerotinia sclerotiorum blocks to downwards contact with leaves, inoculating the sclerotinia sclerotiorum blocks to the plant leaves to be tested in the system, and enabling the inoculation part of each leaf to be the central part of each leaf; similarly, plant seedlings to be tested which grow uniformly and are cultured in sugarcane sugar are transferred to a sugarcane culture medium with high-abundance stable carbon isotopes, then 6mm fungus blocks of sclerotinia sclerotiorum cultured for 7 days in the sugarcane culture medium are cut, hypha of the fungus blocks downwards contact leaves and are inoculated to plant leaves to be tested in the system, and the inoculation position of each leaf is the central position of each leaf. Delta measured in the bacterial-leaf system of the bacterial block and the disease-causing leaf part at 0h, 24h, 48h, 60h and 72h after inoculation13And C value. Using sclerotinia sclerotiorum block cultured by a culture medium prepared by cane sugar to infect delta of a fungus-leaf system of a plant to be tested cultured on a culture medium prepared by beet sugar13The value of C is recorded as delta TAiUsing sclerotinia sclerotiorum block cultured by culture medium prepared by sugar beet sucrose to remove delta infecting fungus-leaf system of plant to be tested cultured on culture medium prepared by sugar beet sucrose13The value of C is recorded as delta TBiWhere i is the number of samples taken at different times, δ TA0And δ TB0δ T at 0 of the start of the experimentAiAnd δ TBi(ii) a The results are shown in Table 1.
According to deltaB1、δB2、δC1、δC2、δTA0And δ TB0Obtaining the share f of the bacterium block in a bacterium-leaf system and the delta of the leaf of the plant to be detected cultured on a culture medium prepared from cane sugar and beet sucrose13C value, wherein; delta of leaf of plant to be tested cultured on culture medium prepared from cane sugar13The value of C is δADelta of leaf of plant to be tested cultured on a culture medium prepared from sugar beet13The value of C is δB(ii) a The calculation formula for obtaining the share f of the bacterium block in the bacterium-leaf system is as follows:
Figure BDA0003253435250000081
then f is substituted into the formula:
Figure BDA0003253435250000082
obtaining delta of the leaf of the plant to be tested cultured on the culture medium prepared by cane sugar13C value deltaAAnd delta of leaf of plant to be tested cultured on culture medium prepared from beet sucrose13C value deltaB(ii) a As in table 2.
TABLE 1 alternate infection of sclerotinia sclerotiorum blocks with delta of fungus-leaf system at different times in plant experiment to be tested13C value (‰PDB)
Figure BDA0003253435250000083
Figure BDA0003253435250000091
TABLE 2 Sclerotinia sclerotiorum block alternately infecting the fraction f of the block in the inoculated strain-leaf system of the plant to be tested and the delta of the leaf of the plant to be tested cultured on the culture medium prepared from cane sugar and beet sugar13C value (‰PDB)
Fraction f of clumps Delta of sucrose cultured plant leaf13C value
Sugarcane sucrose culture bacterium inoculated beet sucrose culture rape 0.57 -30.49
Sugar beet and cane sugar cultivation bacteria and sugar cane sugar inoculation rape cultivation 0.57 -18.16
Sugarcane sucrose cultivation bacteria inoculated beet sucrose cultivation orychophragmus violaceus 0.44 -26.68
Sugar beet and sucrose cultivation bacteria and sugarcane and sucrose inoculation cultivation orychophragmus violaceus 0.44 -14.88
According to delta TA0、δTB0、δC1、δC2、δTAiAnd δ TBiObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniThe calculation method comprises the following steps: will delta TA0、δTB0、δC1、δC2、δTAiAnd δ TBiSubstituting the equation:
Figure BDA0003253435250000092
where i is the number of samples taken at different times; the results are shown in Table 3.
TABLE 3 multiplication coefficient f of sclerotinia on plant leaf to be tested in different time bacterial-leaf system of sclerotinia block alternate infection test for plant to be testedni
Figure BDA0003253435250000093
According to delta TA0、δB、δTAiAnd f, fniOr δ TB0、δA、δTBiAnd f, fniObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiThe method comprises the following steps:
Figure BDA0003253435250000094
or
Figure BDA0003253435250000095
Where i is the number of samples taken at different times; the calculation results are shown in Table 4.
TABLE 4 Stable carbon isotope fractionation values delta of fungus-leaf systems at different times in experiments of alternate infection of sclerotinia sclerotiorum blocks on plants to be testedi(‰PDB)
Figure BDA0003253435250000101
Combined use of fniAnd deltaiQuantitatively grading the infection conditions of the plants to be detected on sclerotinia sclerotiorum at different moments; the quantitative grading method for the infection condition of the plant to be tested on the sclerotinia sclerotiorum at different moments comprises the following steps: si＝10fni+166.67δiWhere i is the number of samples taken at different times, SiThe series of the sclerotinia sclerotiorum infected by the plant to be detected at different moments. The calculation results are shown in Table 5.
TABLE 5 series of infection of Sclerotinia sclerotiorum by rape and orychophragmus violaceus at different times
Mode of infestation 0h 24h 48h 60h 72h
Brassica napus 0 1.432 1.677 1.718 1.735
Radix Brassicae Rapae 0 0.602 1.078 2.085 3.077
The implementation effect of the invention is as follows:
as can be seen from Table 2, the sugarcane-sucrose-cultured plant leaves had higher delta values for both Brassica campestris and Brassica rapa13C value, while the leaves of the beet sucrose-cultured plants all have lower delta13The C value and the difference between the C value and the PDB value reach 10 per mill, the method is suitable for bidirectional isotope tracing culture conditions, and simultaneously, the fraction f of the bacterial block in an initial culture bacterial-leaf system is also obtained, which provides a basis for later calculation, and simultaneously, the delta cultured by different cane sugars can be accurately obtained13C value, avoiding the culture delta at different times13The C value does not truly represent the delta of the plant material to be tested cultured by cane sugar under the same experimental conditions13And the C value greatly reduces experimental errors.
As can be seen from Table 3, the proliferation coefficient f of Sclerotinia sclerotiorum on different plant leaves in the fungus-leaf system at different timesniAre significantly different. The rape multiplication coefficient is reached in the first day0.067, while the proliferation coefficient of orychophragmus violaceus is only 0.029 on the first day and is only 0.050 on the second day; from the proliferation coefficients of rape and orychophragmus violaceus, the rape diseases are fast, the disease is basically stable by the next day, the orychophragmus violaceus is slow, and the disease is rapidly worsened in the next 48 hours. Similar results can be seen in the stable carbon isotope fractionation values of the germ-leaf system of different plants at different times in table 4.
In table 5, we can also see that the onset of rape is fast, the disease grade number reaches 1.432 on the first day, and the orychophragmus violaceus is only 0.602 on the first day, and only 1.078 on the second day, but reaches 3.077 on the third day; the number of the rape diseases in the next day is 1.677, and the number of the rape diseases in the third day is only 1.733. From the above results, it can be seen that rape, although susceptible quickly, has an "immune" function the next day; the orychophragmus violaceus is slow in disease, but the subsequent disease condition is fast in growth, and if the orychophragmus violaceus can be found early and prevented and treated early, the effect of achieving twice the result with half the effort can be achieved. In addition, the relation between the disease grade number y (disease grade) and the time x (hour) is simulated, and the orychophragmus violaceus infection is found to be suitable for an exponential growth model (y is 0.302 e)0.033x) And rape susceptibility is suitable for a 2-parameter single-rectangle hyperbolic model (the Mie equation y is 1.964x/(8.765+ x)), so that the susceptibility of rape and rape can be predicted and prevented.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.

Claims (10)

1. A method for quantitatively detecting the infection capability of sclerotinia sclerotiorum is characterized in that:
first, respectively select C4Representative sucrose sugarcane sucrose and C of plants3Representative sucrose beet sucrose of plants as organic carbon source, and delta was measured separately13C value, Delta of cane sugar13The value of C is δC1Delta of sugar beet13The value of C is δC2；
Secondly, preparing a culture medium for culturing a plant to be tested by using the sugarcane sucrose and the beet sucrose respectively, culturing a tissue culture seedling of the plant to be tested, and culturing for a period of time for a subsequent sclerotinia sclerotiorum infection experiment;
thirdly, at the same time, a medium for culturing sclerotinia sclerotiorum is prepared by using the sugar cane sugar and the sugar beet sugar, the medium is made into a flat plate, sclerotinia sclerotiorum is cultured, and after a period of culture, a part of the medium is used for delta of sclerotinia sclerotiorum block13C value determination, wherein one part of the C value is used for a subsequent experiment for infecting the plant to be detected;
fourthly, preparing a sucrose culture medium with high-abundance stable carbon isotopes for culturing the following sclerotinia sclerotiorum infected plant to be detected;
fifthly, using sclerotinia sclerotiorum blocks cultured by culture mediums prepared by cane sugar and beet sugar respectively to alternately infect plants to be tested which have consistent growth vigor and are cultured on the culture mediums prepared by the cane sugar and the beet sugar, and culturing the plants in a cane sugar culture medium with high abundance and stable carbon isotopes;
sixthly, the delta of sclerotinia sclerotiorum pieces cultured with a medium prepared from cane sugar and beet sugar was measured separately13C value, wherein delta of sclerotinia sclerotiorum mass cultured in sugarcane sucrose medium13The value of C is δB1Delta. of sclerotinia sclerotiorum mass cultured in sugar beet sucrose medium13The value of C is δB2；
Seventh, the delta of the germ-leaf system at different infestation times is subsequently determined13C value, delta of sclerotinia sclerotiorum block cultured by using culture medium prepared from cane sugar to infect fungus-leaf system of plant to be tested cultured on culture medium prepared from beet sugar13The value of C is recorded as delta TAiUsing sclerotinia sclerotiorum block cultured by culture medium prepared by sugar beet sucrose to remove delta infecting fungus-leaf system of plant to be tested cultured on culture medium prepared by sugar beet sucrose13The value of C is recorded as delta TBiWhere i is the number of samples taken at different times, δ TA0And δ TB0δ T at 0 of the start of the experimentAiAnd δ TBi；
Eighth, according to δB1、δB2、δC1、δC2、δTA0And δ TB0Obtaining the share f of the bacterium block in a bacterium-leaf system and the delta of the leaf of the plant to be detected cultured on a culture medium prepared from cane sugar and beet sucrose13C value, wherein; delta of leaf of plant to be tested cultured on culture medium prepared from cane sugar13The value of C is δADelta of leaf of plant to be tested cultured on a culture medium prepared from sugar beet13The value of C is δB；
Ninth, according to δ TA0、δTB0、δC1、δC2、δTAiAnd δ TBiObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniWhere i is the number of samples taken at different times;
tenth, according to δ TA0、δB、δTAiAnd f, fniOr δ TB0、δA、δTBiAnd f, fniObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiWhere i is the number of samples taken at different times;
eleventh, according to fniAnd deltaiQuantitatively judging the infection condition of the plant to be detected on sclerotinia sclerotiorum at different moments;
twelfth, combined use of fniAnd deltaiThe infection condition of the plant to be detected to sclerotinia sclerotiorum at different moments is quantitatively graded.
2. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: in the second step, when the plant tissue culture seedling to be detected is cultured in a culture medium prepared from cane sugar or beet sugar, the cultured tissue culture seedling is a clone with the same source, and multiple multiplication culture is needed in the culture process, so that the obtained material can meet the subsequent requirements.
3. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: in a third step, sugar cane sucrose and sugar beet sucrose formulations are obtainedThe method for culturing sclerotinia sclerotiorum block with the culture medium comprises the steps of respectively carrying out activation culture on the sclerotinia sclerotiorum strain on the culture medium containing cane sugar and beet sugar for 7 days, then cutting the sclerotinia sclerotiorum block with the diameter of 6mm, enabling the mycelium to downwards contact the culture medium, inoculating the sclerotinia sclerotiorum block to a new plate center point of the corresponding culture medium for culture, and after 7 days of culture, using a part of cultured materials for delta of the sclerotinia sclerotiorum block13And C value determination, and a part of the C value is used for subsequent experiments of infecting the plant to be tested.
4. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: the sucrose culture medium with high abundance and stable carbon isotopes in the fourth step is prepared by mixing the raw materials according to the mass conservation law13C/12C standard and cane sugar are configured into delta13C value is 30.0 per mill PDB culture medium.
5. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: in the fifth step, the method for infecting the plant to be tested alternately with sclerotinia sclerotiorum clumps is that the plant to be tested which is cultured in the sugar beet sucrose culture medium and has consistent growth vigor is transferred to the sucrose culture medium with high-abundance stable carbon isotopes, then the sclerotinia sclerotiorum clumps which are cultured in the sugar beet sucrose culture medium for 7 days are cut, the hypha of the clumps downwards contact the leaves and are inoculated on the plant leaves to be tested in the system, and the inoculation position of the leaves is the central position of each leaf; similarly, plant seedlings to be tested which grow uniformly and are cultured in sugarcane sugar are transferred to a sugarcane culture medium with high-abundance stable carbon isotopes, then sclerotinia sclerotiorum blocks cultured for 7 days in the sugarcane culture medium are cut, hypha of the sclerotinia sclerotiorum blocks downwards contact with leaves and are inoculated to plant leaves to be tested in the system, and the inoculation position of each leaf is the central position of each leaf.
6. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: in the eighth step, according to δB1、δB2、δC1、δC2、δTA0And δ TB0The calculation formula for obtaining the share f of the bacterium block in the bacterium-leaf system is as follows:
Figure FDA0003253435240000021
then f is substituted into the formula:
Figure FDA0003253435240000022
obtaining delta of the leaf of the plant to be tested cultured on the culture medium prepared by cane sugar13C value deltaAAnd delta of leaf of plant to be tested cultured on culture medium prepared from beet sucrose13C value deltaB。
7. The method of claim 1, wherein in the ninth step, the infectivity of sclerotinia sclerotiorum is quantitatively determined according to δ TA0、δTB0、δC1、δC2、δTAiAnd δ TBiObtaining the multiplication coefficient f of sclerotinia sclerotiorum on the plant leaves to be detected at different momentsniThe calculation method comprises the following steps: will delta TA0、δTB0、δC1、δC2、δTAiAnd δ TBiSubstituting the equation:
Figure FDA0003253435240000023
where i is the number of samples taken at different times.
8. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: in the tenth step, said basis is δ TA0、δB、δTAiAnd f, fniOr δ TB0、δA、δTBiAnd f, fniObtaining the stable carbon isotope fractionation value delta of the fungus-leaf system at different momentsiThe method comprises the following steps:
Figure FDA0003253435240000031
or
Figure FDA0003253435240000032
Where i is the number of samples taken at different times.
9. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: according to f in the eleventh stepniAnd deltaiThe method for quantitatively judging the infection condition of the plant to be detected to sclerotinia sclerotiorum at different moments comprises the following steps: comparing different plants at the same time, or f of the same plant at different times, respectivelyniAnd deltai，fniAnd deltaiA larger value indicates a more severe infection of the plant sclerotinia sclerotiorum, whereas a less severe infection indicates a less severe infection of the plant sclerotinia sclerotiorum.
10. The method of claim 1, wherein the ability to detect a sclerotinia sclerotiorum infection is determined by: combined use of f as described in the twelfth stepniAnd deltaiThe quantitative grading method for the infection condition of the plant to be tested on the sclerotinia sclerotiorum at different moments comprises the following steps: si＝10fni+166.67δiWhere i is the number of samples taken at different times, SiThe series of the sclerotinia sclerotiorum infected by the plant to be detected at different moments.
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