CN105638268B - Rubber tree red root disease inoculation method - Google Patents
Rubber tree red root disease inoculation method Download PDFInfo
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- CN105638268B CN105638268B CN201511016106.1A CN201511016106A CN105638268B CN 105638268 B CN105638268 B CN 105638268B CN 201511016106 A CN201511016106 A CN 201511016106A CN 105638268 B CN105638268 B CN 105638268B
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- 244000043261 Hevea brasiliensis Species 0.000 title claims abstract description 52
- 201000010099 disease Diseases 0.000 title claims abstract description 41
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 41
- 238000011081 inoculation Methods 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000013479 Amaranthus retroflexus Nutrition 0.000 title abstract description 12
- 240000005674 Ceanothus americanus Species 0.000 title abstract description 12
- 235000014224 Ceanothus americanus Nutrition 0.000 title abstract description 12
- 235000001904 Ceanothus herbaceus Nutrition 0.000 title abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 239000002054 inoculum Substances 0.000 claims abstract description 12
- 238000012360 testing method Methods 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 229920001971 elastomer Polymers 0.000 claims description 44
- 230000001717 pathogenic effect Effects 0.000 claims description 14
- 244000052769 pathogen Species 0.000 claims description 10
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- 240000008042 Zea mays Species 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 238000003780 insertion Methods 0.000 claims description 5
- 230000037431 insertion Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 4
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 claims description 2
- 229960001225 rifampicin Drugs 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 abstract description 8
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 208000035240 Disease Resistance Diseases 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 244000052616 bacterial pathogen Species 0.000 abstract 2
- 239000003814 drug Substances 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 240000008397 Ganoderma lucidum Species 0.000 description 16
- 235000001637 Ganoderma lucidum Nutrition 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 14
- 230000007918 pathogenicity Effects 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 7
- 231100000518 lethal Toxicity 0.000 description 7
- 230000001665 lethal effect Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 4
- 241001389734 Ganoderma philippii Species 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 240000003553 Leptospermum scoparium Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- ZDXLFJGIPWQALB-UHFFFAOYSA-M disodium;oxido(oxo)borane;chlorate Chemical compound [Na+].[Na+].[O-]B=O.[O-]Cl(=O)=O ZDXLFJGIPWQALB-UHFFFAOYSA-M 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 235000015097 nutrients Nutrition 0.000 description 1
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- 239000004576 sand Substances 0.000 description 1
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- 208000024891 symptom Diseases 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Mushroom Cultivation (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of biology, and discloses a rubber tree red root disease inoculation method, which comprises the steps of culturing rubber tree red root disease pathogenic bacteria on a culture medium in a test tube, taking mycelium in an exponential phase as an inoculum, inserting the root of a rubber tree sterile tissue culture seedling into the test tube in which the inoculum is cultured, and culturing until the rubber tree sterile tissue culture seedling is infected with rubber tree red root disease, wherein part of the root is positioned below the surface of the culture medium. The rubber tree red root disease inoculation method can directly observe the infection condition of pathogenic bacteria of the red root disease and the disease condition of plants in a test tube, solves the problems that strains are easy to pollute, the inoculation success rate is large in fluctuation and the disease condition is difficult to directly observe, is favorable for promoting the rubber tree germplasm resource red root disease resistance identification technology, and quickens researches such as screening of excellent disease-resistant stocks and prevention and control medicaments.
Description
Technical field
The present invention relates to field of biotechnology, particularly relate to a kind of Rubber Red Root Disease inoculation method.
Background technique
Rubber Red Root Disease be Para rubber tree (Hevea brasiliensis) China's hazard area it is most wide, influence most
Big root contagious disease, pathogen are rubber Ganoderma Lucidum Ganoderma pseudoferreum (Wakef) Over.et
Steinm.Pathogen invades from rubber tree root system, destroys the function that root system absorbs moisture and nutrient, reduces transporting function, causes
Rubber tree root system necrosis, branch leaf drop, final whole strain is dead, brings huge economic loss to Rubber Industry.Rubber tree red root
Host range is wide for sick pathogen, can infect ten various crop such as citrus, lichee, tea tree, while root disease pathogen is in soil
Incubation period is very long, and the pathogen of diameter 3cm old complaint can survive 2 years in soil, and annual sprawling amount is high after meeting suitable host
Up to 1.83 meters.Red root disease generally occurs in each area Zhi Jiao, China at present, and lesion is almost distributed in each plant glue farm, and morbidity is serious
Woods section disease incidence reach 40%, and do not carry out prevention processing treelet area can be up to 60%, if without handling the death rate in time
It is 100%, brings massive losses to Rubber Industry.
Studies at home and abroad show that the reason of infecting gesture and environmental condition because of germ, in the enterprising pedestrian's work of rubber tree root living
Inoculation germ is difficult success.Report that effective inoculation method has at present: rubber tree sawdust root segment inocalation method, rubber-woodflour bacteria stick connect
The methods of kind method, rubber tree old complaint root segment inocalation method and maize culture medium inocalation method, although these methods can be successfully inoculated with,
But it is easy in implementation process contaminated, success rate fluctuation is larger and be difficult to directly observe incidence.With rubber tree gene
The successful incubation of the development of engineering, rubber tree tissue-cultured seedling provides new approach to the inoculated identification of Rubber Red Root Disease.
Summary of the invention
The present invention proposes a kind of Rubber Red Root Disease inoculation method, and success ratio of inoculation greatly improves, convenient for directly observation hair
State of an illness condition.
The technical scheme of the present invention is realized as follows:
A kind of Rubber Red Root Disease inoculation method, comprising the following steps:
(1) it takes Pathogenic Fungi of Rubber Red Root Disease strain inoculated on culture medium, cultivates, taken in index under dark condition
The mycelium of phase is as inoculum；
(2) the sterile tissue-cultured seedling of rubber tree is taken, the root insertion culture of the sterile tissue-cultured seedling of rubber tree is had to the test tube of inoculum
Interior, the part root of the sterile tissue-cultured seedling of rubber tree is located under media surface, is placed under Light-Dark alternation condition and cultivates, until
The sterile tissue-cultured seedling of rubber tree infects Rubber Red Root Disease.
Further, in step (1), the pathogen strain culturing condition is 25-30 DEG C, cultivates 5-7d.
Further, in step (1), the composition of the culture medium are as follows: contain tender juice 20- of rubber tree in every 1L culture medium
25g, corn flour 20-25g, glucose 15-20g, agar 12-15g, rifampin 50-100mg, water surplus.
Further, in step (2), the sterile tissue-cultured seedling of rubber tree is the seedling for growing a paulin leaf.
Further, in step (2), the sterile tissue-cultured seedling of rubber tree is to be bred by embryoid.
Further, in step (2), the sterile tissue-cultured seedling height of rubber tree is 10-13cm.
Further, in step (2), the sterile tissue-cultured seedling root of rubber tree is located at 1-2cm under media surface.
Further, in step (2), the condition of culture of the sterile tissue-cultured seedling of rubber tree is, at a temperature of 25-30 DEG C, 6-10h light
According to -14-18h dark alternate culture, relative humidity 80-90%.
Beneficial effects of the present invention:
A kind of Rubber Red Root Disease inoculation method of the present invention, pathogen invade from tissue-cultured seedling root, spread to stem
Bar can directly observe the colonization status of red root disease pathogen and the upgrowth situation of plant in test tube.Pass through experiment, discovery
Pathogenic Fungi of Rubber Red Root Disease mycelia rate of propagation is 2mm/d or so in test tube；Infect rear blade and cotyledon slow growth extremely
It stops growing, blade is withered to wither and fall, until whole strain is dead.The method of Pathogenic Fungi of Rubber Red Root Disease inoculation of the present invention,
By directly carrying out inoculation experiments in test tube, solve in soil inoculation, bacterial strain vulnerable to pollution, success ratio of inoculation fluctuation
Greatly, it is difficult to the problem of directly observing incidence, rubber tree kind root disease Resistance Identification is may advantageously facilitate, accelerates excellent resistant anvil
The research such as screening of wood and pesticide control.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is rubber Ganoderma Lucidum strain inoculated result；
In figure, A is weak Pathogenicity Strains, and B is medium Pathogenicity Strains, and C is High pathogenicity bacterial strain, and D is that tissue-cultured seedling is dead
When symptom；
Fig. 2 is the pathogenicity disparity map after 11, Hainan Province rubber Ganoderma Lucidum strain inoculated tissue-cultured seedling；
Fig. 3 is the pathogenicity disparity map after 10, Yunnan Province rubber Ganoderma Lucidum strain inoculated tissue-cultured seedling；
Fig. 4 is the basidiospore of rubber Ganoderma Lucidum；
Fig. 5 is the rubber Ganoderma Lucidum of purifying culture；
Fig. 6 is the sterile tissue culture plant inoculation rubber Ganoderma Lucidum pathogenic process figure of rubber tree；
In figure, A: inoculation 1 day；B: inoculation 2 days；C: inoculation 10 days；D: inoculation 25 days；E: inoculation 35 days；F: inoculation 50 days.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
A kind of Rubber Red Root Disease inoculation method, comprising the following steps:
(1) mycelia of the Pathogenic Fungi of Rubber Red Root Disease rubber ganoderma lucidum bacteria strain isolated and purified is taken to transfer in invisible spectro training
It supports on base, culture culture 5d, takes the mycelium in exponential phase as inoculum under 25 DEG C of dark conditions；The culture medium
Composition are as follows: contain tender juice of 20g rubber tree, 25g corn flour, 15g glucose, 15g agar powder, 50mg benefit good fortune in every 1L culture medium
Flat, water surplus；
(2) the sterile tissue-cultured seedling heat of rubber tree that selection is bred by embryoid grinds 7-33-97, to the sterile tissue-cultured seedling of rubber tree
Grow a paulin leaf, highly for 10cm when, the root insertion culture of the sterile tissue-cultured seedling of rubber tree is had in the test tube of inoculum, root
Portion is located at about 1cm under media surface, is placed at 25 DEG C, cultivates under 6h illumination -18h dark alternation condition, relative humidity is
80%, until the sterile tissue-cultured seedling of rubber tree infects Rubber Red Root Disease.
Embodiment 2
A kind of Rubber Red Root Disease inoculation method, comprising the following steps:
(1) mycelia of the Pathogenic Fungi of Rubber Red Root Disease rubber ganoderma lucidum bacteria strain isolated and purified is taken to transfer in invisible spectro training
It supports on base, the culture culture 6d under 27 DEG C of dark conditions, using the mycelium in exponential phase as inoculum；The culture medium
Composition are as follows: contain tender juice of 22g rubber tree, 22g corn flour, 17g glucose, 13g agar powder, 70mg benefit good fortune in every 1L culture medium
Flat, water surplus；
(2) the sterile tissue-cultured seedling heat of rubber tree that selection is bred by embryoid grinds 7-33-97, to the sterile tissue-cultured seedling of rubber tree
Grow a paulin leaf, highly for 12cm when, the root insertion culture of the sterile tissue-cultured seedling of rubber tree is had in the test tube of inoculum, root
Portion is located at about 1.5cm under media surface, is placed at 27 DEG C, cultivates under 8h illumination -16h dark alternation condition, relative humidity is
85%, until the sterile tissue-cultured seedling of rubber tree infects Rubber Red Root Disease.
Embodiment 3
A kind of Rubber Red Root Disease inoculation method, comprising the following steps:
(1) mycelia of the Pathogenic Fungi of Rubber Red Root Disease rubber ganoderma lucidum bacteria strain isolated and purified is taken to transfer in invisible spectro training
It supports on base, the culture culture 7d under 30 DEG C of dark conditions, using the mycelium in exponential phase as inoculum；The culture medium
Composition are as follows: contain tender juice of 25g rubber tree, 20g corn flour, 20g glucose, 12g agar powder, 100mg benefit in every 1L culture medium
Good fortune is flat, water surplus；
(2) the sterile tissue-cultured seedling heat of rubber tree that selection is bred by embryoid grinds 7-33-97, to the sterile tissue-cultured seedling of rubber tree
Grow a paulin leaf, highly for 13cm when, the root insertion culture of the sterile tissue-cultured seedling of rubber tree is had in the test tube of inoculum, root
Portion is located at about 2cm under media surface, is placed at 30 DEG C, cultivates under 10h illumination -14h dark alternation condition, relative humidity is
90%, until the sterile tissue-cultured seedling of rubber tree infects Rubber Red Root Disease.
Inoculation method of the present invention is inoculated with interpretation of result to Pathogenic Fungi of Rubber Red Root Disease
Isolate and purify 21 rubber ganoderma lucidum bacteria strains are allocated as 3 groups at random, take its mycelia respectively according to 3 realities of the invention
The inoculation method provided in example is provided and carries out inoculated and cultured, infection rate is recorded and lethal number of days, each processing sets three repetitions, tie
Fruit is averaged.The infection rate of 21 rubber ganoderma lucidum bacteria strains and lethal number of days are carried out with SPSS software (version 13.0)
Pearson correlation coefficient is examined.The results are shown in Table 1 for it.
Distance (mm)/time (d) that infection rate S=mycelium is spread in tissue-cultured seedling root；
Lethal number of days D=mycelium time (d) needed for dead day from inoculation day to tissue-cultured seedling.
1 rubber Ganoderma Lucidum strain inoculated interpretation of result of table
As shown in table 1, after 21 rubber Ganoderma Lucidum strain inoculateds, mycelium is invaded from tissue-cultured seedling root, spreads to stem,
Mycelia is averaged infection rate as 2.14mm/d；It infects rear blade to stop growing, blade starts yellow, wilts, falls off；Root is formed
White mycoderm, until whole strain is dead, median lethal number of days is 63.6d.Choose 21 rubber ganoderma lucidum bacteria strains infection rate and
Lethal number of days carries out Pearson correlation coefficient inspection.Inspection result shows that the infection rate of bacterial strain and lethal number of days are in extremely aobvious
Negative correlation is write, related coefficient is -0.84, P < 0.001.
The pathogenic analysis of table 2G.pseudoferreum bacterial strain
21 rubber Ganoderma Lucidum G.pseudoferreum strain inoculated heat are ground with the cause of the sterile tissue-cultured seedling root 7-33-97
Characteristic of disease measurement result shows that they start to fall ill after inoculation, carries out significant difference analysis to it in conjunction with software, bacterial strain
Infection rate and lethal number of days are in extremely significant negative correlation, according to pathogenic analysis result by the rubber in China Hainan, two province of Yunnan
Gum red root disease bacterial strain divides are as follows: High pathogenicity bacterial strain (S >=2.5mm/d), medium Pathogenicity Strains (2.5mm/d > S >
1.5mm/d) and weak Pathogenicity Strains (S≤1.5mm/d), as shown in Figure 1.As shown in table 2, the criteria for classifying, this research divide in due order
From 21 G.pseudoferreum bacterial strains in, High pathogenicity bacterial strain 6, wherein 2, Hainan Province, 4, Yunnan Province；Medium cause
Sick power bacterial strain 12, wherein 7, Hainan Province, 5, Yunnan Province；Weak Pathogenicity Strains 3, wherein 2, Hainan Province, 1, Yunnan Province.
11 bacterial strains of glue point are planted to Hainan difference and 10 bacterial strains that Yunnan difference plants glue point carry out virulence analysis, from
Analysis result can be seen that each point Virulence is relatively large, and the strongest bacterial strain of pathogenicity comes from Yunnan Meng Han, and pathogenicity is most
Weak bacterial strain comes from Hainan Danzhou.There are certain relationship, white sand, the Bao Ting in Hainan Province, Yunnan Province in pathogenicity power and ground section
River mouth, Menla, Meng Han separation strain pathogenic strength it is stronger, as shown in Figure 2 and Figure 3.
Above data shows, by using inoculation method of the present invention, due to whole experiment process in test tube into
Row, convenient for control experiment condition, Rubber Red Root Disease success ratio of inoculation is high and result is stablized, and is not easily susceptible to pollute, is easy to observe
Incidence is conducive to the pathogenicity for determining pathogen, for rubber tree germ plasm resource red root disease Resistance Identification and excellent anti-
The researchs such as the screening of sick stock and pesticide control have great importance.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (1)
1. a kind of Rubber Red Root Disease inoculation method, which comprises the following steps:
(1) it takes Pathogenic Fungi of Rubber Red Root Disease strain inoculated on culture medium, cultivates, taken in exponential phase under dark condition
Mycelium is as inoculum；The composition of the culture medium are as follows: contain tender juice 20-25g of rubber tree, corn flour in every 1L culture medium
20-25g, glucose 15-20g, agar 12-15g, rifampin 50-100mg, water surplus；The pathogen strain culturing condition is
25-30 DEG C, cultivate 5-7d；
(2) the sterile tissue-cultured seedling of rubber tree for growing a paulin leaf is taken, the sterile tissue-cultured seedling of rubber tree is to be bred by embryoid,
The root insertion culture for the sterile tissue-cultured seedling of rubber tree that height is 10-13cm is had in the test tube of inoculum, sterile group of rubber tree
The part root of training seedling is located at 1-2cm under media surface, is placed at a temperature of 25-30 DEG C, and 6-10h illumination -14-18h dark is handed over
It replaces, relative humidity is cultivated under conditions of being 80-90%, until the sterile tissue-cultured seedling of rubber tree infects Rubber Red Root Disease.
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| 巴西橡胶树红根病病菌液体培养条件优化研究;林仕恺等;《热带农业科学》;20141031;第34卷(第10期);71-74 * |
| 橡胶树几种根病的人工接种方法;高秀兵等;《热带作物学报》;20100430;第31卷(第4期);626-630 * |
| 橡胶树红根病病原菌生物学培养特性;张贺等;《热带作物学报》;20081031;第29卷(第5期);632-635 * |
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