CN1664094A - Pathogenic bacteria of gummy stem blight of melon, induction method for its conidiophore and use thereof - Google Patents
Pathogenic bacteria of gummy stem blight of melon, induction method for its conidiophore and use thereof Download PDFInfo
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Abstract
The invention discloses a musk melon cucurbit wilt pathogenic bacteria and the conidiospore evoked method and application. The name of the pathogenic bacteria strain is STw-1, carrying out artificial inoculation with the conidiospore can estimate the musk melon species resource cucurbit wilt resistant level; sifting antigen from these and carrying out further research about these antigens can boost the development of thick-shelled musk melon anti cucurbit wilt breeding, which plays a positive part in effectivly controlling the hazard made by cucurbit wilt. The conidiospore evoked method comprises inoculating the pathogenic bacteria mycelia on the plate of the potato saccharose culture medium, culturing with continuous illumination, culturing with alternative light and dark after the ultraviolet irradiation or thermal stress when mycelia is full of the plate, inducing highly effective production pycnidium; then inoculating single pycnidium on the corn culture medium or on potato oat dextrose medium, producing conidiospore with highly effective.
Description
Technical field
The present invention relates to a strain gummy stem blight of melon pathogenic bacteria and conidial induction method and application, particularly relate to a strain gummy stem blight of melon pathogenic bacteria and conidial induction method thereof and the application in estimating the climing rot resistance level of melon variety resource.
Background technology
Muskmelon is the fruit that liked by the people all over the world.Raising along with the national economic development and living standards of the people; demand to the good high-grade muskmelon after the meal of flavor of fine quality increases day by day; recent two decades comes, and China's thick-skinned melon outdoor cropping and protection ground cultivation have obtained significant progress, have improved muskmelon ultimate production and year-round supply level significantly.Yet, when thick-skinned melon is flourish, also have many problems.
Continuous cropping continuous cropping obstacle is the subject matter of restriction muskmelon production Sustainable development.Because the continuous cropping continuous cropping causes soil-borne disease aggravations such as climing rot, blight, eqpidemic disease and root nodule oxyuriasis, has influenced the raising of fruit output and quality.
Climing rot (Didymella bryoniae (Auersw.) Rehm) is one of important soil-borne disease of global muskmelon.Because cultivation needs the whole leaf of training, has increased the chance of pathogenic bacteria from wound infection.In whole growing, each one all can be injured on the ground, but the main harm basal part of stem.The seedling base portion is injured, and water stain shape point originally occurs, after expansion up and down rapidly, complete stool is soft rotten dead soon.Later stage to the fruit expanding period of blooming is the morbidity Sheng phase, mainly infects the above basal part of stem of collar and main climing, also infects lateral bine sometimes.Begin to take place water stain shape, and then secrete drop, be transformed into dark redly, make the atrophy of plant base portion when serious, finally cause the wilting death of complete stool to dark red by yellowish.In China, no matter be the thick-skinned melon producing region, northwest of outdoor cropping, or the east of heliogreenhouse or booth protection ground cultivation move the thick-skinned melon producing region, climing rot generally takes place.
In order to prevent soil-borne disease, the method for pharmacy disinfection commonly used in the cultivation, cost height not only, weak effect is difficult to thorough elimination, and sterilization can exert an adverse impact to ecotope, fruit and HUMAN HEALTH.In China; climing rot is one of important disease of harm thick-skinned melon open country and the cultivation of protection ground; the pathogenic variation of research cause of disease; extensively collect variety source; identify anti-source gene; improving improved variety or the resistance level of graft stock and the persistence of resistance, is most economical, effective, a safe disease defense pathway.
The conidium of didymella bryoniae is difficult to induce.(Keinath A.P. such as Keinath, M.W.Farnham, andT.A.Zitter 1995 Morphological, pathological, and genetic differentiationof Didymella bryoniae and Phoma spp.isolated from Cucurbits.PhytopathologyVol.85 (3): 364-369) on the potato dextrose agar of V8 vegetables juice nutrient agar or 1/4, light dark (each 12h) was alternately cultivated for 2 weeks or is produced up to pycnidium in 22 ℃ of-24 ℃ of incubators, although mycelial growth is very fast, but the pycnidium that produces seldom, restricted the application of didymella bryoniae aspect the relevant research with pathology of muskmelon thremmatology.
Summary of the invention
The objective of the invention is a strain gummy stem blight of melon pathogenic bacteria and conidial highly effective revulsion induction method and application.
Gummy stem blight of melon pathogenic bacteria provided by the present invention (Didymella bryoniae) strain name is STw-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 01 12nd, 2004, preserving number is C6MCC № 1089.
Gummy stem blight of melon pathogenic bacteria STw-1 of the present invention is located away from the national vegetables Engineering Technical Research Centre muskmelon booth of continuous cropping continuous cropping for many years according to Koch's Postulates.Utilize the efficient inductive conidium of bacterial isolate environment stress to carry out artificial inoculation, estimated the resistance level of the climing rot of the local variety source of Chinese muskmelon, the result has therefrom screened a collection of anti-source, further investigation to these anti-source genes, with promoting the development of China's thick-skinned melon anti didymella breeding, active effect is played in the harm of effective control gummy stem blight of melon.
The conidial induction method of gummy stem blight of melon pathogenic bacteria, be that mycelium inoculation with the gummy stem blight of melon pathogenic bacteria is on the potato sucrose culture medium flat plate, continuous light is cultivated, after treating that mycelia is covered with flat board, carry out alternation of light and darkness and cultivate after uv irradiating or heat shock are coerced, inducement efficient produces pycnidium; On corn culture medium or potato oat dextrose culture-medium, inoculate single pycnidium then, efficiently expand numerous generation conidium.
In said process, the compound method of described potato sucrose substratum (PSA) is that the potato 180-220 gram section with peeling adds 1000 ml distilled waters, boils the elimination residue; Add 15-20 gram agar and 18-22 gram sucrose in the filtrate, be settled to 1000 milliliters; The Semen Maydis powder 280-320 that consists of of described corn culture medium (MA) restrains, agar 15-20 gram, and water is settled to 1000 milliliters; The compound method of described potato oat dextrose culture-medium (POGA) is with the potato 18-22 gram section of peeling, adds 1000 ml distilled waters, boils the elimination residue; Add oat 3-6 gram, glucose 18-22 gram and agar 18-20 gram in the filtrate, be settled to 1000 milliliters.
Described mycelia is on the potato sucrose culture medium flat plate, and general cultivation is 7 days under 25 ℃ of conditions.In the environment stress process, uv irradiating generally carried out under the 30W condition 0.5-4 minute, and heat shock is general the processing 1-4 days under 30-35 ℃ of condition; Alternation of light and darkness after coercing is cultivated and was generally carried out 2-15 days under 25 ℃ of conditions.
The present invention utilizes environment stress (uv irradiating and heat shock) dexterously, after the gummy stem blight of melon mycelium grows into certain phase, nourish and grow and be unlikely to kill under the mycelial prerequisite influencing mycelium, make mycelium by vegetative growth phase excessively to generative growth phase, promote pycnidial formation, produce a large amount of conidiums, can fully satisfy the needs of in pathology and the work of thremmatology practical study the didymella bryoniae conidium being measured.
Description of drawings
Figure 1A and Figure 1B have shown the upgrowth situation of mycelia on the PSA flat board.
Fig. 2 A and Fig. 2 B have shown the upgrowth situation of mycelia on the OA flat board.
Fig. 3 has shown the upgrowth situation of mycelia on the MA flat board.
Fig. 4 has shown the upgrowth situation of mycelia on the POGA flat board.
Fig. 5 shows the mycelia shape.
Fig. 6 shows pycnidial microscopic morphology feature.
Fig. 7 shows that pycnidium splits and discharges conidium.
Fig. 8 shows the conidium form.
Fig. 9 shows the diseased plant of pathogenic inoculation experiments.
Figure 10 is the climing rot resistance of a muskmelon variety source distribution histogram.
Embodiment
The separation of embodiment 1, gummy stem blight of melon pathogenic bacteria (Didymella bryoniae) STw-1 strain
1, surface sterilization:
Morbidity place of diseased plant basal part of stem is cleaned, be cut into about 4 * 4 * 2mm
3Fritter, with 70% alcohol sterilization 3-5S, aseptic water washing 3 times; Divide time gradient 4,8,12,16min sterilization, aseptic water washing 3 times with 5% NaClO again.
2, mycelia is induced in inoculation
To be inoculated on the PSA flat board through the diseased tissues piece of surface sterilization, place 25 ℃ of incubator continuous lights to cultivate.Behind the inoculation 2d, the surface of indivedual tissue block produces the mycelia of white on the PSA flat board.
3, purifying expands numerous mycelia
Above-mentioned PSA flat board is picking edge mycelia behind inoculation 5d, is inoculated on the PSA flat board, treat that bacterium colony forms after, again from colony edge picking mycelia, be inoculated on the PSA flat board and continue to cultivate, with purifying with expand numerous mycelia.
4, the observation of colonial morphology, upgrowth situation and mycelia microscopic morphology feature
With the mycelium inoculation of purifying in PSA, OA (oat medium, compound method is that rolled oats 30g places 1h in 60 ℃ of water-baths, the double gauze filter and remove residue is got supernatant liquor, adds agar 20g, be settled to 1000 milliliters), on the different flat boards of MA with POGA, cultivate 7d at 25 ℃ of growth case continuous lights, mycelial growth is vigorous, all covers with flat board, positive color is relevant with cell age, and the colonial morphology performance differs on different substratum.Shown in Figure 1A (culture dish front) and Figure 1B (the culture dish back side), on the PSA flat board, colony edge is thin, and is middle dense, slightly swells, and positive color is relevant with cell age, and back side yellow is to brown, and the later stage becomes black, and tangible concentric wheel stripe is arranged.Can smell when opening substratum ware lid " dirt flavor ".
Shown in Fig. 2 A (culture dish front) and Fig. 2 B (the culture dish back side), dense in the middle of the bacterium colony on the OA flat board, protuberance slightly, the edge is thin, and obvious ripples shape concentric wheel stripe is arranged, the back side just be yellow, after fade to brown to black, it is especially obvious, yellowish-brown alternate to take turns line.
As shown in Figure 3, dense in the middle of the bacterium colony on the MA flat board, obviously swell the circle of a diameter 4cm, the flat crawl of mycelia on every side is in media surface, and the back side just be yellow, and the later stage becomes black, and concentric wheel stripe is arranged, but not obvious.
As shown in Figure 4, on the MPGA flat board, mycelia is distributed in media surface more uniformly, does not have protuberance, and the back side just is yellow, and the later stage becomes black, no concentric wheel stripe.
The microscopic morphology feature of mycelia as shown in Figure 5, mycelia is thinner, colourless, have every, diameter 2.5-12.5 μ m.
5, the difference of STw-1 strain and other gummy stem blight of melon pathogenic bacteria diseased plant
Didymella bryoniae can infect all cucurbitaceous plants, but the pathogenic (Farr that there are differences of different fungus strains, D.F., Bills, G.F.et al 1989 Fangi on plants plant products in Unite State.American Phytopathological Society, St.Paul, MN; Punithalingam, E., andHolliday, P.1972 Didymella bryoniae.CMI Descriptions of Pathogenic Fungi andBacteria No.332; Chiu, W.F.and walker, J.C.1949 Physiology andpathogenicity of the cucubit black-rot fungus.J.Agric.Res.78:589-615; Lee, D.H.Mathur, S.B., and Neergaard, P.1984 Detection and location of seedbrone inoculum of Didymella bryoniae and its transmission in seedlings ofcucumber and pumpkin.Phytpathol.Z.109:301-308; Van Steekelenburg .A.M.1982 Factors influencing external fruit rot of cucumber caused by Didymellabryoniae.Neth.J.Plant Pathol.88:47-56).(Keinath A.P. such as Keinath, M.W.Farnham, and T.A.Zitter 1995 Morphological, pathological, and geneticdifferentiation of Didymella bryoniae and Phoma spp.isolated from Cucurbits.Phytopathology Vol.85 (3): 364-369) studied the area, eastern united states and derived from different cucurbitaceous plant thick-skinned melons, watermelon, summer squash, 22 polymorphisms that didymella bryoniae is a genomic dna random amplification fragment length of pumpkin and cucumber are found to have genotypic difference between different fungus strains in the didymella bryoniae colony.However, do not find also that so far there is the specialization of " kind " in didymella bryoniae to different cucurbitaceous plants, even do not find to exist " microspecies " differentiation on any therein plant such as muskmelon or the watermelon yet.
The gummy stem blight of melon bacterial strain STw-1 that the present invention announced is isolating at Vegetable Research center, Beijing, and it causes a disease to thick-skinned melon variety resource height, genotype with pathogenic aspect and other bacterial strain of crossing of bibliographical information have difference.
Embodiment 2, induce conidium
One, culture medium preparation
1, potato sucrose substratum:
Potato slices 200 grams of peeling are put into pot, add 1000 ml distilled waters, boil 30min, with gauze elimination residue; Filtrate is put back in the pot, adds 15-20 gram agar and 20 gram sucrose, is settled to 1000 milliliters.
2, corn culture medium:
Semen Maydis powder 300 grams, agar 15-20 gram, 1000 milliliters in water.
3, potato oat dextrose culture-medium
Potato slices 20 grams of peeling are put into pot, add 1000 ml distilled waters, boil 30min, with gauze elimination residue; Filtrate is put back in the pot, adds oat 3-6 gram, glucose 20 grams and agar 18-20 gram, is settled to 1000 milliliters.
More than 3 kinds of substratum be nature pH, autoclave sterilization (121 ℃, 0.103Mpa, 20min) back making sheet.
Two, environment stress is efficiently induced the melon didymella bryoniae conidium
1, with melon didymella bryoniae STw-1 mycelium inoculation on the potato sucrose culture medium flat plate, cultivate 7d at 25 ℃ of growth case continuous lights, mycelia is covered with flat board.
2, environment stress is handled
Environment stress is handled can take uv irradiating or heat shock dual mode, below narration respectively:
1) uv irradiating
The flat board 0.5,1,2 or the 4min that obtain in the irradiating step 1 respectively with the UV-light of 30W intensity in Bechtop place flat board then and carry out alternation of light and darkness between 25 ℃ of tissue culture and cultivate.4 processing all produce pycnidium after 15 days, and produce pycnidial amount and increase with the prolongation of handling the time.
2) heat shock
Placing incubator to carry out heat shock respectively with time (1d, 2d, 3d and 4d) gradient the flat board branch temperature (30 ℃ and 35 ℃) that obtains in the step 1 handles, place then and carry out the alternation of light and darkness cultivation between 25 ℃ of cultivations, behind the 2d: 30 ℃ of a large amount of pycnidiums that induce of handling 2d and 3d, 1d and 4d induce a spot of pycnidium; 35 ℃ each handle and all induce a large amount of pycnidium generations, especially handles 1d and 2d and induce pycnidial better effects if.
Environment stress inductive pycnidium is tawny to the brown point on the potato sucrose culture medium flat plate, diameter is 54.5-175.5 μ m, and average out to 87 μ m partly bury and are born in the mycelia clump.At microscopically, pycnidium as shown in Figure 6, spherical in shape or spheroid has the aperture, wall is membranous, tawny is to Vandyke brown, meets water crack and opens and expose conidium (as shown in Figure 7).
3, conidial efficient expansion is numerous
Induce the pycnidium that obtains to be inoculated in respectively on potato sucrose substratum, corn culture medium and the potato oat dextrose culture-medium environment stress, behind the 5-7d, produce a large amount of pycnidiums on corn culture medium and the potato oat dextrose culture-medium, and mycelial growth is vigorous on the potato sucrose substratum, does not produce pycnidium all the time.Explanation is on corn culture medium and potato oat dextrose culture-medium, and the inoculation pycnidium can efficiently expand numerous conidium.As shown in Figure 8, conidium ovalize or round shape, colourless, monospore, size is 2.5-15 μ m * 2.5-6.25 μ m, average 7.3 μ m * 3.6 μ m.
The pathogenic evaluation of embodiment 3, gummy stem blight of melon pathogenic bacteria STw-1 strain
Conidium is configured to the conidial suspension of 1 * 106 concentration with distilled water.At plastic greenhouse, dip in pin and to get conidial suspension, at stab inoculation in seedling stage muskmelon plants stems base portion, inoculate and observe incidence after 7 days, sickness rate is up to 100%, as shown in Figure 9 as a result, the acupuncture position is tangible water stain shape scab, the average 2cm of scab length, part plant disease portion rots, and is the classical symptom of muskmelon children phase basal part of stem morbidity.
Cause of disease is carried out in the sick strong junction of diseased plant separate, obtained the pure culture of pathogenic bacteria again, proterties is identical with the initial isolate that obtains.
Embodiment 4, the climing rot evaluation of resistance of the local variety source of Chinese muskmelon
1, state of an illness grading index and resistance classification
State of an illness grade scale: 1 grade (no scab), typical value is 0; 2 grades (having secretory product and scab not to have expansion), typical value is 0.5; 3 grades (scab length 0～1), typical value is 1; 4 grades (scab length 1～2), typical value is 2; 5 grades (scab length 〉=2), typical value is 3; 6 grades (diseased plant is withered), typical value are 4.
Carry out the resistance classification by disease index (DI): high anti-(HR), DI≤10%; Disease-resistant (R), 10-20%; In anti-(MR), 20-50%; Middle sense (MS), 50-80%; Susceptible (S), 80-90%; High sense (HR), DI 〉=90%.
2, the climing rot evaluation of resistance of the local variety source of Chinese muskmelon
The climing rot of the nearly all high sense of thick-skinned melon.China is the secondary center of origin of muskmelon and snake melon, for this reason, from domestic introduction with collected 100 melon varieties, utilize gummy stem blight of melon pathogenic bacteria STw-1 strain at stab inoculation in seedling stage muskmelon plants stems base portion, they have been carried out climing rot evaluation of resistance, the result is as shown in table 1, and 100 parts of melon variety resources are normal distribution, wherein 2 kind 3X basically to the resistance performance of climing rot
38And 3X
22Disease index be respectively 7.1 and 7.1, show as highly anti-, the disease index of other 9 kinds shows as disease-resistant between 10%-20%.These anti-sources have important use and are worth in the seed selection of breeding for disease resistance and graft stock.
The climing rot evaluation of resistance of the Chinese melon variety resource of table 1
| The material disease index | ??3X 11??19.6 | ??3X 107??57.8 | ??3X 38??7.5 | ??3X 22??7.1 | ??3X 27??18.8 | ??3X 124??33.9 | ??3X 97??50 | ??3X 90??21.4 | ??3X 118??57.1 | ??3X 129??48.2 |
| The material disease index | ??3X 100??35.7 | ??3X 131??28.1 | ??3X 75??56.9 | ??3X 3??35.4 | ??3X 119??52.1 | ??3X 50??41.7 | ??3X 29??12.5 | ??3X 21??20.8 | ??3X 31??25 | ??3X 112??23.1 |
| The material disease index | ??3X 113??37.5 | ??3X 37??46.9 | ??3X 6??42.2 | ??3X 76??31.7 | ??3X 33??25 | ??3X 132-5??68.8 | ??3X 8??21.4 | ??3X 65??25 | ??3X 54??40.6 | ??3X 4??77.5 |
| The material disease index | ??3X 126??30 | ??3X 96??70.8 | ??3X 48??31.3 | ??3X 16??32.5 | ??3X 105??31.9 | ??3X 121??58.3 | ??3X 2??63.8 | ??3X 98??54.2 | ??3X 79??25 | ??3X 57??14.3 |
| The material disease index | ??3X 73??73.8 | ??3X 17??53.8 | ??3X 10??46.6 | ??3X 44??37.5 | ??3X 7??23.4 | ??3X 49??42.5 | ??3X 78??43.8 | ??3X 25??45 | ??3X 68??87.5 | ??3X 117??33.3 |
| The material disease index | ??3X 39??78.1 | ??3X 85??81.3 | ??3X 132-4??70.8 | ??3X 132-1??70.8 | ??3X 34??44.2 | ??3X 60??41.7 | ??3X 5??19.6 | ??3X 91??42.9 | ??3X 22??50 | ??3X 24??46.9 |
| The material disease index | ??3X 28??33.8 | ??3X 36??41.3 | ??3X 14??35.7 | ??3X 114??35.7 | ??3X 88??48.4 | ??3X 95??35 | ??3X 99??32.5 | ??3X 43??70.8 | ??3X 53??76.4 | ??3X 35??75 |
| The material disease index | ??3X 70??22.9 | ??3X 1??52.8 | ??3X 101??53.1 | ??3X 67??62.5 | ??3X 75??15 | ??3X 106??18.8 | ??3X 12??71.8 | ??3X 132-3??18.8 | ??3X 81??17.9 | ??3X 42??51.8 |
| The material disease index | ??3X 16??41.7 | ??3X 111??68.8 | ??3X 332??58.3 | ??3X 41??35.7 | ??3X 113??60.7 | ??3X 59??58.3 | ??3X 26??91.7 | ??3X 103??78.1 | ??3X 123??67.5 | ??3X 55??32.8 |
| The material disease index | ??3X 13??29.7 | ??3X 84??22.9 | ??3X 93??45.3 | ??3X 73??69.4 | ??3X 20??57.8 | ??3X 9??25 | ??3X 92??29.7 | ??3X 99??46.9 | ??3X 64??67.9 | ??3X 69??25 |
Claims (9)
1, melon didymella bryoniae (Didymella bryoniae) STw-1 CGMCC № 1089.
2, the conidial induction method of gummy stem blight of melon pathogenic bacteria, be that mycelium inoculation with the gummy stem blight of melon pathogenic bacteria is on the potato sucrose culture medium flat plate, continuous light is cultivated, after treating that mycelia is covered with flat board, carry out alternation of light and darkness and cultivate after uv irradiating or heat shock are coerced, inducement efficient produces pycnidium; On corn culture medium or potato oat dextrose culture-medium, inoculate single pycnidium then, efficiently expand numerous generation conidium.
3, method according to claim 2 is characterized in that: described potato sucrose culture medium preparation method is that the potato 180-220 gram section with peeling adds 1000 ml distilled waters, boils the elimination residue; Add 15-20 gram agar and 18-22 gram sucrose in the filtrate, be settled to 1000 milliliters; The Semen Maydis powder 280-320 that consists of of described corn culture medium restrains, agar 15-20 gram, and water is settled to 1000 milliliters; The compound method of described potato oat dextrose culture-medium is with the potato 18-22 gram section of peeling, adds 1000 ml distilled waters, boils the elimination residue; Add oat 3-6 gram, glucose 18-22 gram and agar 18-20 gram in the filtrate, be settled to 1000 milliliters.
4, according to claim 2 or 3 described methods, it is characterized in that: described mycelia was cultivated 7 days under 25 ℃ of conditions on the potato sucrose culture medium flat plate.
5, according to claim 2 or 3 described methods, it is characterized in that: described uv irradiating is to carry out under the 30W condition 0.5-4 minute.
6, according to claim 2 or 3 described methods, it is characterized in that: described heat shock is to handle 1-4 days under 30-35 ℃ of condition.
7, according to claim 2 or 3 described methods, it is characterized in that: the alternation of light and darkness after described the coercing is cultivated and was carried out 2-15 days under 25 ℃ of conditions.
8, the application of melon didymella bryoniae (Didymella bryoniae) STw-1 CGMCC № 1089 in the climing rot evaluation of resistance of melon variety.
9, the application of melon didymella bryoniae (Didymella bryoniae) STw-1 CGMCC № 1089 in the muskmelon resistance breeding.
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