CN107996522A - A kind of method for expanding numerous plant root-knot nematode using logical heart dish - Google Patents

A kind of method for expanding numerous plant root-knot nematode using logical heart dish Download PDF

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Publication number
CN107996522A
CN107996522A CN201711271341.2A CN201711271341A CN107996522A CN 107996522 A CN107996522 A CN 107996522A CN 201711271341 A CN201711271341 A CN 201711271341A CN 107996522 A CN107996522 A CN 107996522A
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China
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root
knot nematode
plant
knot
heart dish
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Inventor
赵志祥
严婉荣
肖敏
肖彤斌
陈圆
陈绵才
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Plant Protection Institute Hainan Academy Of Agricultural Sciences
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Plant Protection Institute Hainan Academy Of Agricultural Sciences
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/033Rearing or breeding invertebrates; New breeds of invertebrates
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00Botany in general
    • A01G7/06Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants

Abstract

The invention belongs to plant pathogeny line insect technical field, discloses a kind of method for expanding numerous plant root-knot nematode using logical heart dish, and using transparent polypots, alms bowl bottom burrows, and sows logical heart colza or directly plants logical heart dish seedling, accesses root-knot nematode pieces of an egg;Pieces of an egg are drawn, and separate root-knot nematode.The present invention uses transparent polypots, and alms bowl bottom makes a call to 45 thick duck eyes of thumb and is used to breathing freely and preventing waterflooding, sows and leads to heart colza or directly plant logical heart dish seedling, after seedling grows to root system prosperity enough, accesses line eggs block;Need not extract root using transparent polypots can easily observe the dynamic process of root knot formation at any time;When needing root-knot nematode, root of the part containing pieces of an egg, remaining continued growth can be cut, and substantial amounts of new root can be grown and be used to expand numerous root-knot nematode;New plant need not be planted again, saved trouble, time saving, saving of labor, material saving.

Description

A kind of method for expanding numerous plant root-knot nematode using logical heart dish
Technical field
The invention belongs to plant pathogeny line insect technical field, more particularly to a kind of utilize to lead to the numerous plant root-knot nematode of heart dish expansion Method.
Background technology
Root-knot nematode mainly includes Meloidogyne incognita (M.incognita), javanese root knot nematode (M.javanica), flower Raw root-knot nematode (M.arenaria), M hapla (M.hapla) and Meloidogyne enterolobii (M.enterolobii) etc. 5 kinds.Screening utilizes the disease-resistant variety of anti-plant root-knot nematode, identification resistant gene, the clonal expression of functional gene and plant The research etc. of the agricultural of root-knot nematode, physics, biological and chemical control method is required for mass propgation, expands numerous and separating plant root Tie lines worm.At present, plant root-knot nematode is typically biogenous, mainly utilizes plant of Solanaceae (such as tobacco, tomato, eggplant, peppery Green pepper etc.), cucurbitaceous plant (cucumber, sponge gourd, balsam pear etc.) seedling it is relatively narrow as host, host range.The reason for causing defect Mainly have at 3 points:First, these crops are larger by seasonal constraint.The torrid areas such as Hainan, summer temperature canopy medium temperature spend height, Capsicum, eggplant, tomato and some melon crops should not be planted, even if plantation, takes root also less, be unfavorable for infecting for root-knot nematode And breeding;Secondly, the acquisitions of root-knot nematode pieces of an egg must be on the premise of its root system be destroyed, and Solanaceae, cucurbitaceous plant root system It is undeveloped, be damaged can not normal growth, it is necessary to seedling again;Thirdly, time for being needed from seedling stage to Adult plant of above-mentioned crop It is very long, root system aging after nematode infection so that plant can not normal growth influence root-knot nematode and largely expand numerous undergone time It is again shorter.Thus, it is impossible to substantial amounts of pieces of an egg and second instar larvae are obtained in a short time, influence disease-resistant variety screening and root-knot nematode Progress is studied in prevention and control etc..And logical heart dish growth potential, root activity, yield, growth cycle, to expanding after the tolerance of high temperature and inoculation The ability of numerous nematode is stronger;It is stronger to the adaptability of environment and the cultural method of logical heart dish is simple, the huge and lasting root of tool System, and the ability of regenerated root, long-term breeding and conservation after being inoculated with for root-knot nematode provide more preferable basis.For experiment Room is used for for preserving root-knot nematode germ plasm resource for a long time, has important researching value and realistic meaning.
In conclusion problem existing in the prior art is:Plant root-knot nematode live body parasitism method existed by season at present The constraint of property is larger, takes root less;It is original expand numerous root-knot nematode root system of plant it is undeveloped, be damaged can not normal growth, Need seedling again;And the time needed from seedling stage to Adult plant is very long, root system aging can not be just down to plant after nematode infection It is frequently grown;In addition, the shortcomings of also regenerated root ability is poor, it is unfavorable for infecting and breeding for root-knot nematode, influences disease-resistant variety Screening and root-knot nematode prevention and control.
The content of the invention
In view of the problems of the existing technology, the present invention provides a kind of side for expanding numerous plant root-knot nematode using logical heart dish Method.
The present invention is achieved in that a kind of method for expanding numerous plant root-knot nematode using logical heart dish, described to utilize the logical heart The method that dish expands numerous plant root-knot nematode uses transparent polypots, and alms bowl bottom burrows, and sows logical heart colza or directly plantation is logical Heart dish seedling, accesses root-knot nematode pieces of an egg;Pieces of an egg are drawn, and separate root-knot nematode.
Further, the method for expanding numerous plant root-knot nematode using logical heart dish comprises the following steps:
Step 1, javanese root knot nematode, enterolobium cyclocarpum root knot line are separated from planting and being inoculated with the tomato seedling of more than 30d respectively Worm and the pieces of an egg of Meloidogyne incognita;
Step 2, leads to heart colza with after 1% liquor natrii hypochloritis's soaking disinfection 3min, aseptic water washing, and directly broadcasts Kind is in transparent plastic alms bowl;The plant planted is put into nematode resource conservation garden, and 40 alms bowls are planted per plant;Treat that leaf growth arrives During 51 core of leaf, javanese root knot nematode, Meloidogyne enterolobii and Meloidogyne incognita, every kind of root-knot nematode inoculation 10 are inoculated with respectively Alms bowl;
Step 3, when growing to 51 core of leaf, the cave of moon 2cm is down dug with sterile small iron rod along plant stalk;Meanwhile 1mL pipette tips cut off tip, and the pipette tips of tip are set up with 1mL pipettors, draw 1mL pieces of an egg suspension, are added to logical heart dish In cave, it has been inoculated with and has been filled and led up cave with the soil of surrounding.
Step 4, after leading to heart dish inoculation root-knot nematode 30d, through homemade transparent plastic alms bowl outer wall, can clearly observe To root knot and pieces of an egg, root system of the clip part with pieces of an egg, the pieces of an egg on picking root system, hatch second instar larvae, in subsequent chamber Bioassay and molecular biology etc. are tested, and remaining part continues to plant, and can grow substantial amounts of new root, for expanding numerous three kinds of roots Tie lines worm, avoids the trouble for planting new talent again.
Further, heart colza is led in the step 2 with after 1% liquor natrii hypochloritis's soaking disinfection 3min, sterile water rushes Wash, the logical heart dish stalk of 3-5 sections with 4-6 piece leaves is directly planted in transparent plastic alms bowl, and 3-5 are sowed per alms bowl.
Another object of the present invention is to provide a kind of method for utilizing the logical heart dish of the utilization to expand numerous plant root-knot nematode The plant root-knot nematode of breeding.
The present invention uses transparent polypots, and alms bowl bottom beats the thick duck eye of 4-5 thumb and is used to breathing freely and preventing waterflooding, sows Lead to heart colza or directly plant logical heart dish seedling, after seedling grows to root system prosperity enough, access line eggs block;Using transparent Polypots need not extract root can easily observe at any time root knot formation dynamic process;, can be with when needing root-knot nematode Root of the part containing pieces of an egg, remaining continued growth are cut, and substantial amounts of new root can be grown and be used to expand numerous root-knot nematode, it is not necessary to Again new plant is planted.Meanwhile by the comparative test with cucumber and tomato, study the effect that logical heart dish expands numerous root-knot nematode Fruit.Concrete scheme is as follows:
Retain logical 3 plants of heart dish, tomato seedling and cucumber seedling per alms bowl, when it grows to 51 core of leaf, with sterile small iron rod (diameter The cave of moon 2cm or so 3mm) is down dug along plant stalk, the root until that can see plant.Meanwhile 1mL pipette tips are cut off into point Head (in order to avoid pipette tips are blocked by soil, the grains of sand when hindering pieces of an egg or inoculation), next sets up the rifle of tip with 1mL pipettors Head, draws 1mL pieces of an egg suspension (containing 3~5, pieces of an egg), is added in the cave near logical heart dish, tomato and Cucumber root, connects Finish and filled and led up cave with the soil of surrounding, observe the growing state of plant root in transparent plastic alms bowl, root knot after inoculation daily Nematode infection forms the feature such as root knot situation and breeding worm amount.Often handle when 15d and 30d, take out 3 alms bowls and be used to investigate root It is the indexs such as length, quality and the quantity of level-one root and two level root.Often handle in remaining 4 alms bowl plant, 1 alms bowl 50d after inoculation Continue to plant after cutting off the root system of half, relatively more logical heart dish, tomato and the growing state of cucumber, the growing state of new root and ovum The numerous situation of expansion of block;Remaining 3 alms bowls, in the experiment of root-knot nematode inoculation reproduction, after nematode 50d is inoculated with, observes, take pictures, and unite Count root knot number, pieces of an egg number, second instar larvae number and root system line insect number.
What the present invention was realized in:(1) upgrowth situation for leading to heart dish, tomato and cucumber root compares.The logical heart dish of plantation, After tomato seedling and cucumber seedling 15d, 30d, it is preferable to lead to heart dish, tomato and the root system growing way of cucumber.Root length is followed successively by logical Heart dish > cucumber > tomatoes;Root system quality and the quantity of level-one root system are followed successively by cucumber > and lead to heart dish > tomatoes;Two level root system Quantity is followed successively by logical heart dish > cucumber > tomatoes, and the two level root quantity for leading to heart dish and cucumber during 30d is close, and difference is not shown Write.The two level root quantity that tomato produces is minimum, all the time no more than 50.After 30d, with the extension of implantation time, lead to heart dish Two level root system quantity in rapidly ascendant trend;And the quantity of cucumber and tomato two level root system tends towards stability.Thus, lead to heart dish It is more advantageous to infecting and expanding numerous for later stage various root-knot nematodes.Compare root length, root system quality, two level root quantity and the logical heart The time of the good host growth of the root-knot nematodes such as dish, tomato, cucumber.Logical heart dish is grown with root length, two level root quantity More, it is strong to produce new root ability, existence, the advantages that time-to-live is long.
(2) lead to heart dish, cucumber and tomato to compare the tolerance and growth cycle of summer high temperature.Tomato and cucumber exist respectively Plant starts Huang and withers during plantation 90d or so, and root system part occurs and rots;And cucumber needs built, root system is more if not built Undeveloped, the time that its Huang withers can further shorten;Meanwhile tomato and cucumber belong to warm season vegetable, growth thermophilic 20-30 DEG C, intolerant to summer high temperature, more than 35 DEG C growths and abnormal seeding.Planted by temperature control means in nematode garden cucumber and tomato with Expand numerous root-knot nematode, human and material resources cost will be artificially increased.And water spinach belongs to heat-resisting vegetables, 30 DEG C or so growths Preferably, normal growth is remained to for 35-40 DEG C;And root system just starts to decay after the time of plantation 300d or so, although overground part therebetween Divide cauline leaf to turn yellow, but after basal part of stem cuts off the cauline leaf of strong yellow, new cauline leaf, while also new root system can be grown from stem foot Produce;In addition, after needing clip part root because of experiment, plant back in polypots, and can be grown from basal part of stem substantial amounts of new again Root, ensure that the normal growth of plant.This has absolutely proved the logical heart dish for examination, in 3 kinds of plants of tomato and cucumber, water spinach root System is maximum, growth cycle is most long, preferable to the tolerance of high temperature, may be more advantageous to the amount reproduction of root-knot nematode and long-term protect Deposit.
(3) cut off lead to heart dish, tomato and the growth of cucumber after the root system of half, new root produces and the numerous feelings of expansion of pieces of an egg Condition.
Inoculation 50d continues to plant after cutting off the root system of half, and when 65d is investigated, and as a result leads to heart dish well-grown, moreover it is possible to after Continuous to grow substantial amounts of new root, its root length 26.3cm, (25.6cm) has been grown during compared with 50d, and root system quality, which returns to, cuts Level before going, is 8.7g/ plants, level-one root quantity is 65/plant, and two level root quantity is 2470/plant, than cutting off It is much more after half, illustrate that logical heart dish growth adaptability is stronger;Cut off after half that tomato and cucumber growth are poor, plant is short Small, the blade chrysanthemum of more than half is withered, root length and indifference before cutting off, respectively 7.6cm and 22.3cm, root It is that the quantity of quality, the quantity of level-one root system and two level root system is only the half before cutting off, almost without new root.
Investigated during 65d, three kinds of nematodes can infect logical heart dish, tomato and the root system of cucumber well.Due to leading to the heart Dish can rapidly grow substantial amounts of new root, be conducive to further infecting for root-knot nematode.Thus, in the root system of these three plants On, it is inoculated with root knot number caused by three kinds of root-knot nematodes, second instar larvae number, pieces of an egg number, ripe female borer population and root system and rhizosphere soil The total amount of root-knot nematode is logical heart dish > cucumber > tomatoes in earth.And three ages and four-age larva number are then on the contrary, tomato > Cucumber > leads to heart dish, wherein, cucumber is slightly larger than on tomato, difference is not notable.
Inoculation reproduction effect of (4) three kinds of root-knot nematodes on logical heart dish, cucumber and tomato.Logical heart dish inoculation enterolobium cyclocarpum root , there is the time of the visible root knot of naked eyes earliest in three kinds of different nematodes such as tie lines worm, Meloidogyne incognita and javanese root knot nematode And the quantity for forming root knot has certain difference.Directly observed by transparent polypots outer wall, be inoculated with three kinds of different roots Lead to the macroscopic root knots of the visible diameter about 1.2mm or so in heart dish root after tie lines worm between 25~33d, wherein seeing earliest It is Meloidogyne enterolobii to observe to form root knot, is 25d;Secondly it is Meloidogyne incognita, is 28d;At the latest it was observed that being formed Root knot is javanese root knot nematode, is 33d.And compare the time that cucumber " No. bis- green cucumbers of Nan You " forms the visible root knot of three kinds of naked eyes Respectively 28d, 31d and 35d;The time of the visible root knot of control tomato variety " expire Fructus Lycopersici esculenti in Guangdong " three kinds of naked eyes of formation is respectively 29d, 32d and 35d.
The time speed for forming the visible root knot of naked eyes has certain correlation with the quantity for forming root knot, i.e., logical heart dish, Huang Time required for being observed visually root knot after melon and tomato inoculation nematode is more early, and the root knot number that it is formed is also more, this is also to see The main reason for three kinds of nematode root knot numbers on the logical heart dish observed are much larger than cucumber and tomato.
In general, host crop of the same race, the infecting to cause harm of Meloidogyne enterolobii is higher than Meloidogyne incognita and pawl Root-knot nematode, the degree that causes harm are Meloidogyne enterolobii > Meloidogyne incognita > javanese root knot nematodes;For root knot of the same race For nematode, on the root system of this 3 kinds of host plants, root knot number, second instar larvae number, ripe female borer population, pieces of an egg number and root system Line insect number is logical heart dish > cucumber > tomatoes, and difference reaches the pole level of signifiance;And third-instar larvae and four-age larva number are then It is that tomato > cucumber > leads to heart dish, difference is extremely notable, and after this may be infected with tomato and cucumber by root-knot nematode, root system is easily old Change so as to which the time for shortening entrance ripe larval phase is related.In addition, after logical heart dish is inoculated with three kinds of root-knot nematodes, growth cycle is still Up to 10 months or so, and after cucumber and tomato are inoculated with three kinds of root-knot nematodes, grow 3 months or so at most, root system is just complete Complete rotten or aging.
Brief description of the drawings
Fig. 1 is the method flow diagram provided in an embodiment of the present invention for expanding numerous plant root-knot nematode using logical heart dish.
Fig. 2 is the comparison schematic diagram of logical heart dish provided in an embodiment of the present invention, cucumber and Tomato Root System length.
Fig. 3 is the comparison schematic diagram of logical heart dish provided in an embodiment of the present invention, cucumber and Tomato Root System quality.
Fig. 4 is the quantity of logical heart dish, cucumber and tomato planting provided in an embodiment of the present invention rear stage root system for a period of time Comparison schematic diagram.
Fig. 5 be logical heart dish, cucumber and tomato planting provided in an embodiment of the present invention for a period of time after two level root system quantity Comparison schematic diagram.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.
Logical heart Lepidium can be planted in leaf vegetables, by weaker, its well developed root system of seasonal constraint, be adapted to throughout the year Planted in sand soil, sand soil good permeability;Meanwhile although root also aging after nematode infection, can be in a short time Substantial amounts of new root is grown, does not interfere with the normal growth of logical heart dish, and the new root grown is conducive to root-knot nematode population again Expand numerous.
The application principle of the present invention is explained in detail below in conjunction with the accompanying drawings.
As shown in Figure 1, provided in an embodiment of the present invention expand the method for numerous plant root-knot nematode including following using logical heart dish Step:
S101:From having planted and being inoculated with " gold is holy " tomato seedling of more than 30d javanese root knot nematode is separated respectively, as ear The pieces of an egg of beans root-knot nematode and Meloidogyne incognita are used to be inoculated with;
S102:Logical heart colza is with after 1% liquor natrii hypochloritis's soaking disinfection 3min, aseptic water washing, and directly sows (or the logical heart dish stalk of 3-5 sections with 4-6 piece leaves is directly planted) is made by oneself in transparent plastic alms bowl in a diameter of 20cm, is sowed per alms bowl 3-5;The plant planted is put into nematode resource conservation garden, and 40 alms bowls are planted per plant;Treat leaf growth to 5 leaf, 1 core (about 25d or so) when, 3 kinds of root-knot nematodes are inoculated with respectively, and every kind of root-knot nematode is inoculated with 10 alms bowls;
S103:When growing to 51 core of leaf, down dig moon 2cm's or so along plant stalk with sterile small iron rod (diameter 3mm) Cave, the root until that can see plant;Meanwhile by 1mL pipette tips cut off tip (in order to avoid hinder pieces of an egg or inoculation when pipette tips it is native Earth, the grains of sand are blocked), the pipette tips of tip are next set up with 1mL pipettors, 1mL pieces of an egg suspension is drawn and (contains pieces of an egg 3~5 It is a), it is added in the cave of logical heart dish, has been inoculated with and has been filled and led up cave with the soil of surrounding.
S104:After logical heart dish inoculation root-knot nematode 30d, through homemade transparent plastic alms bowl outer wall, it can be clearly observed Root knot and pieces of an egg, root system of the clip part with pieces of an egg, the pieces of an egg on picking root system, hatch second instar larvae, for raw in subsequent chamber The experiment such as thing measure and molecular biology, remaining part continues to plant, and can grow substantial amounts of new root, for expanding numerous three kinds of root knots Nematode, avoids the trouble for planting new talent again.
The application principle of the present invention is further described with reference to specific embodiment.
The method provided in an embodiment of the present invention for expanding numerous plant root-knot nematode using logical heart dish specifically includes following steps:
(1) lead to heart dish as host by the use of leaf vegetables plant, establish the new method of the numerous plant root-knot nematode of indoor expansion, lead to Heart vegetable kind " Hainan green grass or young crops bone willow leaf water spinach " and control cucumber variety " No. bis- green cucumbers of Nan You " are purchased from the beautiful kind cervical orifice of uterus in Hainan Jinhua Portion of city.It is " expiring Fructus Lycopersici esculenti in Guangdong " to compare tomato variety, is purchased from the prosperous excellent kind of industry research and development Co., Ltd in Guangzhou.Root-knot nematode sample Meloidogyne incognita (M.incognita), javanese root knot nematode (M.javanica) and Meloidogyne enterolobii (M.enterolobii) preserve by this research institute nematode resource garden, gather in the wild.
(2) it is used for the acquisition of three kinds of root-knot nematode pieces of an egg being inoculated with:First from having planted before and be inoculated with more than 30d's The pieces of an egg for separating javanese root knot nematode, Meloidogyne enterolobii and Meloidogyne incognita on " gold is holy " tomato seedling respectively are used to connect Kind, specific method is:The seed of " gold holy " tomato variety is through hypochlorite disinfectant, and after being handled with sterile water wash, 22~ Vernalization 3d under the conditions of 28 DEG C of lucifuges, after it is broken in vain, moves into the red plastic flowerpot of 10~20cm of diameter and plants, per basin kind 1 ~3 young plants.The growth conditions and the logical heart dish of follow-up plantation of soil (through 121 DEG C, high-temperature sterilization 30min) and tomato seedling in flowerpot It is consistent with cucumber.The tomato seedling for transplanting 25d conventionally (i.e. by the sterile pipette tips of 1ml, after cutting off tip, is drawn 1ml and is contained 1-3 Meloidogyne incognita (M.incognita), javanese root knot nematode (M.javanica) or Meloidogyne enterolobii (M.enterolobii) 200-300 second instar larvaes are contained after pieces of an egg, or hatching) the above-mentioned three kinds of root-knot nematodes of inoculation.Tomato Compound fertilizer nutrition liquid is poured per basin 1 time within the 28th day after transplantation of seedlings.Then, tomato noodle growing way is observed every 2d, and watered in due course, prevented Antecedent soil moisture and hardened, so as to influence tomato seedling growth.Be inoculated with 49d (7 weeks) and extract tomato seedling afterwards, with behind sterile water root again With the nematode pieces of an egg on dissecting needle or transfer needle picking root, it is put into the transparent glass culture dish for filling sterile water.
(3) growth of heart dish seedling is led to:Logical heart colza and control tomato seeds, cucumber seeds are respectively with 1% sodium hypochlorite After solution soaking disinfection 3min, aseptic water washing, and directly sow (or the logical heart dish stalk of 3-5 sections with 4-6 piece leaves is directly planted Kind) made by oneself in a diameter of 20cm in transparent plastic alms bowl, 3-5 are sowed per alms bowl.Soil is by river sand and clay with 1:1 proportional arrangement Form, prepare rear moist hear heat test sterilizing (121 DEG C, 30min).The plant planted is put into nematode resource conservation garden, every kind of Plant plants 40 alms bowls, wherein every 30 alms bowl is used to be inoculated with root-knot nematode, in addition 10 alms bowls are spare, defence is short of seedling, few seedling, midway dead seedling or Other uncertain factors there is a situation where when need mend alms bowl.25~28 DEG C of its temperature is adjusted, pours suitable water daily, and spray on a small quantity Mist is to ensure the humidity in nematode garden as more than 80%, when its leaf growth is to 5 leaf, 1 core (about 25d or so), respectively in inoculation 3 kinds of root-knot nematodes are stated, every kind of root-knot nematode is inoculated with 10 alms bowls.Control tomato seedling, the method for cucumber seedling and propagating root-knot nematode are similar to The direct method of sowing of logical heart colza, watering daily, ensures ground moistening, same every kind of beneficial to the sprouting and growth of seedling Root-knot nematode is inoculated with 10 alms bowls.
(4) root-knot nematode inoculation and expand it is numerous:Retain logical 3 plants of heart dish, tomato seedling and cucumber seedling per alms bowl, treat that it grows to 5 leaves 1 During core, the cave of moon 2cm or so is down dug along plant stalk with sterile small iron rod (diameter 3mm), until that can see plant Root.Meanwhile 1mL pipette tips are cut off into tip (in order to avoid pipette tips are blocked by soil, the grains of sand when hindering pieces of an egg or inoculation), next use 1mL pipettors set up the pipette tips of tip, draw 1mL pieces of an egg suspension (containing 3~5, pieces of an egg), be added to logical heart dish, tomato and In cave near Cucumber root, it has been inoculated with and has been filled and led up cave with the soil of surrounding, has been observed daily in transparent plastic alms bowl after inoculation The growing state of plant root, root-knot nematode infect to form the feature such as root knot situation and breeding worm amount.Often handle respectively at 15d and During 30d, take out 3 alms bowls and be used to investigate the indexs such as root length, quality and the quantity of level-one root and two level root.Often processing remainder 4 In alms bowl plant, 1 alms bowl 50d after the inoculation continues to plant after cutting off the root system of half, relatively more logical heart dish, tomato and the growth feelings of cucumber The numerous situation of expansion of condition, the growing state of new root and pieces of an egg;Remaining 3 alms bowls, in the experiment of root-knot nematode inoculation reproduction, in inoculation line After worm 50d, observe, take pictures, and count root knot number, pieces of an egg number, second instar larvae number and root system line insect number.Nematode population in root The method using tissue staining is counted, root-knot nematode uses the graceful funnel method of improvement shellfish to be counted after separating in soil.With DPS numbers Statistical analysis is carried out to test data according to processing system and SPSS19.0 statistics softwares.
It is explained in detail with reference to the application effect to comparing the present invention.
1st, the upgrowth situation for leading to heart dish, tomato and cucumber root compares
Shown in below figure 2, Fig. 3, Fig. 4, Fig. 5, after the logical heart dish of plantation, tomato seedling and cucumber seedling 15d, 30d, logical heart dish, The root system growing way of tomato and cucumber is preferable.Root length is followed successively by logical heart dish > cucumber > tomatoes;Root system quality and level-one root The quantity of system is followed successively by cucumber > and leads to heart dish > tomatoes;The quantity of two level root system is followed successively by logical heart dish > cucumber > tomatoes, and 30d The two level root quantity of Shi Tongxin dishes and cucumber is close, and difference is not notable.The two level root quantity that tomato produces is minimum, all the time not More than 50.After 30d, with the extension of implantation time, lead to the quantity of two level root system of heart dish in rapidly ascendant trend;And cucumber Tend towards stability with the quantity of tomato two level root system.Thus, logical heart dish is more advantageous to infecting and expanding numerous for later stage various root-knot nematodes. Compare the good host growth of the root-knot nematode such as root length, root system quality, two level root quantity and logical heart dish, tomato, cucumber Time.Logical heart dish is grown with root length, and two level root quantity is more, and generation new root ability is strong, and existence, time-to-live length etc. are excellent Point, therefore, it is ideal plant root-knot nematode culture and propagating materials to be not difficult to find out logical heart dish.
2nd, lead to heart dish, cucumber and tomato to compare the tolerance and growth cycle of summer high temperature
It has been observed that the plant when planting 90d or so starts Huang and withers respectively for tomato and cucumber, there is part and rots in root system; And cucumber needs built, root system is more undeveloped if not built, and the time that its Huang withers can further shorten;Meanwhile tomato and Cucumber belongs to warm season vegetable, grows 20-30 DEG C of thermophilic, intolerant to summer high temperature, more than 35 DEG C growths and abnormal seeding.Pass through control Warm means plant cucumber and tomato to expand numerous root-knot nematode in nematode garden, will artificially increase human and material resources cost.It is and empty Heart Lepidium is in heat-resisting vegetables, and 30 DEG C or so growths are preferable, and 35-40 DEG C remains to normal growth;And the time of plantation 300d or so Root system just starts to decay afterwards, although aerial part cauline leaf turns yellow therebetween, after basal part of stem cuts off the cauline leaf of strong yellow, from stem foot New cauline leaf can be grown, while also new root system produces;In addition, after needing clip part root because of experiment, modeling is planted back again Expect in alms bowl, and substantial amounts of new root can be grown from basal part of stem, ensure that the normal growth of plant.This has absolutely proved the logical heart for examination In 3 kinds of dish, tomato and cucumber plants, water spinach root system is maximum, growth cycle is most long, preferable to the tolerance of high temperature, Ke Nenggeng Be conducive to the amount reproduction of root-knot nematode and long-term preservation.
3rd, cut off lead to heart dish, tomato and the growth of cucumber after the root system of half, new root produces and the numerous situation of expansion of pieces of an egg
Shown in table 1, inoculation 50d continues to plant after cutting off the root system of half, and when 65d is investigated, and it is good as a result to lead to the growth of heart dish It is good, moreover it is possible to which that continued growth goes out substantial amounts of new root, its root length 26.3cm, and (25.6cm) has been grown during compared with 50d, root system quality The level before cutting off is returned to, is 8.7g/ plants, level-one root quantity is 65/plant, and two level root quantity is 2470/plant, It is more much more after half than cutting off, illustrate that logical heart dish growth adaptability is stronger;Cut off after half tomato and cucumber growth compared with Difference, plant is short and small, it is existing more than half blade chrysanthemum it is withered, root length and indifference before cutting off, be respectively 7.6cm and 22.3cm, the quantity of root system quality, the quantity of level-one root system and two level root system are only the half before cutting off, and are given birth to almost without new root It is long, illustrate that tomato and cucumber growth adaptability are poor, expanding numerous aspect in root-knot nematode squamous subculture needs to supplement new talent incessantly, To meet worm sources needed for subsequent experimental.
65d leads to heart dish, tomato and the growing state of cucumber after table 1 cuts off the root system of half
Note:Data are the average value of 3 repetitions in table.With the identical person of lowercase is marked after column data, represent in P= 0.05 horizontal upper difference is not notable (DMRT methods).Conversely, then significant difference, following table are same.
By being inoculated with Meloidogyne enterolobii, Meloidogyne incognita and javanese root knot nematode (table 2), when 65d, is investigated, and three Kind nematode can infect logical heart dish, tomato and the root system of cucumber well.Since logical heart dish can rapidly be grown largely New root, is conducive to further infecting for root-knot nematode.Thus, on the root system of these three plants, it is inoculated with three kinds of root-knot nematode institutes The total amount of root-knot nematode is in the root knot number of generation, second instar larvae number, pieces of an egg number, ripe female borer population and root system and rhizosphere soil Logical heart dish > cucumber > tomatoes.And three ages and four-age larva number be then on the contrary, tomato > cucumber > leads to heart dish, wherein, tomato Upper to be slightly larger than cucumber, difference is not notable.It is probably the root system that tomato and cucumber cut off half the main reason for such case occur Afterwards, it is impossible to grow substantial amounts of new root, accelerate the aging of plant, nematode can not absorb the nutriment of small and tender root systems, shorten Its time to come to the ripening period.And nematode generates substantial amounts of pieces of an egg and follow-up molecular biosciences after the new root of logical heart dish is infected Learn experiment and the second instar larvae needed for Biological Control Test.
The breeding situation of 2 three kinds of root-knot nematodes of table 65d on logical heart dish, cucumber and the Tomato Root System for cut off half
4th, inoculation reproduction effect of three kinds of root-knot nematodes on logical heart dish, cucumber and tomato
" Hainan green grass or young crops bone willow leaf water spinach ", control cucumber variety " No. bis- green cucumbers of Nan You " and control tomato variety " Yue Manhong Equal successful reproduction root-knot nematode after tomato " inoculation Meloidogyne enterolobii, Meloidogyne incognita and javanese root knot nematode pieces of an egg. Three kinds of different nematodes such as logical heart dish inoculation Meloidogyne enterolobii, Meloidogyne incognita and javanese root knot nematode, occur earliest Visually the time of visible root knot and the quantity for forming root knot have certain difference (table 3).It is straight by transparent polypots outer wall Observation is connect, is inoculated with after three kinds of different root-knot nematodes and leads to visible diameter about 1.2mm or so the meat in heart dish root between 25~33d The visible root knot of eye, is 25d wherein observing that form root knot is Meloidogyne enterolobii earliest;Secondly it is Root Knot line Worm, is 28d;It is 33d at the latest it was observed that form root knot is javanese root knot nematode.And compare cucumber " No. bis- green cucumbers of Nan You " shape Time into the visible root knot of three kinds of naked eyes is respectively 28d, 31d and 35d;Compare tomato variety " expiring Fructus Lycopersici esculenti in Guangdong " and form three kinds of meat The time of the visible root knot of eye is respectively 29d, 32d and 35d.
The time speed for forming the visible root knot of naked eyes has certain correlation with the quantity for forming root knot, i.e., logical heart dish, Huang Time required for being observed visually root knot after melon and tomato inoculation nematode is more early, and the root knot number that it is formed is also more, this is also to see The main reason for three kinds of nematode root knot numbers on the logical heart dish observed are much larger than cucumber and tomato;And form the visible root knot of naked eyes Non-correlation passes through micro- sight between time speed and the pieces of an egg number for separating acquisition and the number for the line insect number for separating acquisition Examine, the root-knot nematode for separating acquisition is ceaselessly moving, and has vigorous vigor.
Table 3 leads to the earliest time of heart dish, cucumber and each self-forming root knot of tomato
(table 4~9) is observed after being inoculated with 50d, it is all a degree of on logical heart dish, cucumber and Tomato Root System to receive enterolobium cyclocarpum Root-knot nematode, Meloidogyne incognita and javanese root knot nematode infect.It infects cause harm degree and root-knot nematode species and host Variety type has certain relation.In general, host crop of the same race, the infecting to cause harm of Meloidogyne enterolobii is higher than south Root-knot nematode and javanese root knot nematode, the degree that causes harm are Meloidogyne enterolobii > Meloidogyne incognita > javanese root knot nematodes; For root-knot nematode of the same race, on the root system of this 3 kinds of host plants, root knot number, second instar larvae number, ripe female borer population, ovum Block number and root system line insect number are logical heart dish > cucumber > tomatoes, and difference reaches the pole level of signifiance;And third-instar larvae and Four-age larva number is then that tomato > cucumber > leads to heart dish, and difference is extremely notable, after being infected with tomato and cucumber by root-knot nematode, root system Easily aging is related so as to shorten time into ripe larval phase.In addition, after logical heart dish is inoculated with three kinds of root-knot nematodes, growth Cycle still up to 10 months or so, and after cucumber and tomato are inoculated with three kinds of root-knot nematodes, grows 3 months or so at most, and root system is just Completely rotten or aging.
The breeding situation of 4 Meloidogyne enterolobii of table inoculated and cultured 50d on logical heart dish, cucumber and Tomato Root System
The breeding situation of 5 Meloidogyne incognita of table inoculated and cultured 50d on logical heart dish, cucumber and Tomato Root System
The breeding situation of 6 javanese root knot nematode of table inoculated and cultured 50d on logical heart dish, cucumber and Tomato Root System
7 Meloidogyne enterolobii of table, Meloidogyne incognita and the javanese root knot nematode inoculated and cultured on logical heart dish root system The breeding situation of 50d
8 Meloidogyne enterolobii of table, Meloidogyne incognita and javanese root knot nematode the inoculated and cultured 50d on cucumber root Breeding situation
9 Meloidogyne enterolobii of table, Meloidogyne incognita and javanese root knot nematode the inoculated and cultured 50d on Tomato Root System Breeding situation
The logical heart dish of the present invention expands numerous host as plant root-knot nematode and is better than cucumber and tomato, is successfully expanded using logical heart dish Numerous Meloidogyne enterolobii, Meloidogyne incognita and javanese root knot nematode, enrich host's material of current breeding root-knot nematodes Material, overcomes and is usually used as host by the use of the tobacco, tomato and capsicum of plant of Solanaceae;Or utilize cucurbitaceous plant cucumber, hardship The problem of melon and sponge gourd etc. are used as host, and host range is more single.In addition, usually used ceramics are substituted using transparent plastic alms bowl With red, black plastic basin alms bowl breeding root-knot nematodes, the dynamic of root knot formation can be easily observed at any time without extracting root Process;Also, therefore, avoid to water to plant and breed management process without permeable hole using the transparent plastic alms bowl bottom of customization The middle pollution problem that may cause nematode escape to surrounding soil.The present invention under the hot and humid weather conditions of Hainan Tropical, Using logical heart dish substitute cucumber and tomato, three kinds of plant root-knot nematodes of successful reproduction, provide to breed and separating root-knot nematode One new way, also provides beneficial basis for the identification of anti-root-knot nematode crop varieties and the prevention and control research of root-knot nematode. Since cucumber and Tomato Root System growth period are shorter, the time of general 3 months or so begins to rot, and leads to heart dish not only with huger Big root system, and growth cycle is very long, up to 10 months or so., can be with when needing root knot or pieces of an egg are used for follow-up test Part root system is first cut, remaining part continues to plant, and it is numerous for the expansion of root-knot nematode to grow substantial amounts of root system;When logical heart dish After aerial part cauline leaf turns yellow, it can be cut off from basal part of stem by the overground part of strong yellow, new cauline leaf then can be grown from stem foot, together When also have new root system and produce, unlike tomato is as cucumber, without plant again new talent be used for follow-up nematode expand it is numerous;It is logical Heart dish growth potential, root activity, yield, growth cycle, to root knot number, pieces of an egg number, two ages after the tolerance of high temperature and inoculation Larva number etc. is significantly better than that tomato and cucumber.In addition, the cultural method of logical heart dish is simple, it is stronger to the adaptability of environment;And Under the high temperature conditions, cucumber root is easily caused harm by subterranean pest-insect and pathogen, more perishable;Tomato Root System growing state is worst, Growth cycle is also shorter.Therefore, high temperature resistant, logical heart dish stronger to environmental suitability have huge and lasting root system, and The ability of regenerated root, long-term breeding and conservation after being inoculated with for root-knot nematode provide more preferable basis.It is inoculated with root-knot nematode In reproduction test, compared with ground family crop cucumber and solanaceous crops tomato, logical heart dish, which breeds three kinds of root-knot nematodes, can produce more More pieces of an egg and root knot, reproductive effect are more preferable.Meanwhile after logical heart dish is inoculated with three kinds of root-knot nematodes, growth cycle can reach 10 Month or so;And cucumber and tomato grow most 3 months or so after being inoculated with three kinds of root-knot nematodes, root system begins to rot or aging is withered Extremely.Therefore, it is a kind of method of both economical, practical amount reproduction root-knot nematode to lead to heart dish breeding root-knot nematodes method, for For root-knot nematode germ plasm resource is preserved for a long time, the present invention has substantially use for laboratory compared with cucumber and tomato culture root-knot nematode Advantage.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.

Claims (4)

  1. A kind of 1. method for expanding numerous plant root-knot nematode using logical heart dish, it is characterised in that described to expand numerous plant using logical heart dish The method of root-knot nematode uses transparent polypots, and alms bowl bottom burrows, and sows logical heart colza or directly plants logical heart dish seedling, Access root-knot nematode pieces of an egg;Pieces of an egg are drawn, and separate root-knot nematode.
  2. 2. the method as claimed in claim 1 for expanding numerous plant root-knot nematode using logical heart dish, it is characterised in that described using logical The method that heart dish expands numerous plant root-knot nematode comprises the following steps:
    Step 1, from plant and be inoculated with the tomato seedling of more than 30d separate respectively javanese root knot nematode, Meloidogyne enterolobii and The pieces of an egg of Meloidogyne incognita;
    Step 2, leads to heart colza with after 1% liquor natrii hypochloritis's soaking disinfection 3min, aseptic water washing, and is directly seeded in Transparent plastic alms bowl;The plant planted is put into nematode resource conservation garden, and 40 alms bowls are planted per plant;Treat leaf growth to 5 leaves 1 During core, javanese root knot nematode, Meloidogyne enterolobii and Meloidogyne incognita are inoculated with respectively, and every kind of root-knot nematode is inoculated with 10 alms bowls;
    Step 3, when growing to 51 core of leaf, the cave of moon 2cm is down dug with sterile small iron rod along plant stalk;Meanwhile 1mL rifles Head cuts off tip, and the pipette tips of tip are set up with 1mL pipettors, draws 1mL pieces of an egg suspension, is added to the cave of logical heart dish In, it has been inoculated with and has been filled and led up cave with the soil of surrounding;
    Step 4, after leading to heart dish inoculation root-knot nematode 30d, through homemade transparent plastic alms bowl outer wall, can be clearly observed root Knot and pieces of an egg, root system of the clip part with pieces of an egg, the pieces of an egg on picking root system, hatch second instar larvae, for follow-up indoor biological The experiment such as measure and molecular biology, remaining part continues to plant, and can grow substantial amounts of new root, for expanding numerous three kinds of root knot lines Worm.
  3. 3. the method as claimed in claim 2 for expanding numerous plant root-knot nematode using logical heart dish, it is characterised in that the step 2 In lead to heart colza with after 1% liquor natrii hypochloritis's soaking disinfection 3min, aseptic water washing, the logical heart dish of 3-5 sections with 4-6 piece leaves Stalk is directly planted in transparent plastic alms bowl, and 3-5 are sowed per alms bowl.
  4. What 4. a kind of method for leading to the numerous plant root-knot nematode of heart dish expansion using being utilized described in 3 any one of claims 1 to 3 was bred Plant root-knot nematode.
CN201711271341.2A 2017-12-05 2017-12-05 A kind of method for expanding numerous plant root-knot nematode using logical heart dish Pending CN107996522A (en)

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